The first discussion on Nrf2 in this blog was in the October 2009 entry Nrf2 and cancer chemoprevention by phytochemicals.  Since then hundreds of research papers that deal with aspects of Nrf2 have been published.  In these three blog entries, I seek to characterize some of the most important findings.   This first blog entry deals with the general mechanisms of operation of Nrf2, with positive effects of Nrf2 in preventing or treating a number of pathological conditions via its ability to turn on genes for the body’s own antioxidant system and genes for combating stress – hundreds of such genes. A second blog entry deals with how a number of substances that are incidentally antioxidants, plant-derived phyto-substances in particular, actually exercise their benefits through promoting the expression of Nrf2 which in turn activates the body’s own antioxidant and hormetic defense systems: The pivotal role of Nrf2 Part 2 – foods, phyto-substances and other substances that turn on Nrf2.  The third entry explores whether supplementation with substances that promote Nrf2 might be life-extending: Nrf2 Part 3 – is promoting expression of Nrf2 life extending?.

What does Nrf2 do?

Nrf2 is a stress-sensing genetic transcription factor that is part of the cap’n’collar family.  As such it appears to be a master regulator of cellular responses to oxidative damage and other stressful conditions.  In simplified form, the main theory of how it works is as shown in the diagram(ref):

“Nrf2 is normally bound in the cell’s cytosol in a complex with the cytoskeletal protein Keap1 (Kelch-like ECH-associated protein 1).  Under normal conditions, as Nrf2 accumulates it is polyubiquitinated and sent off for destruction by the cell’s proteolysis machinery where it is broken down into simple molecules for recycling.  Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. If metabolic stress signals are present such as strong change in redox state, proteolysis is blocked, Nrf2 translocates into the cell’s nucleus where it accumulates.  “After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5′-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes(ref).”  Hundreds of antioxidant and stress response genes may be affected.  “These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. (ref)” 

To complete the picture, there are many substances including certain phytochemicals that can block ubiquination and proteolysis as illustrated in the following diagram.  Sulforaphane is is an isothiocyanate found in cruciferous vegetables like broccoli.  Electrophiles are chemical species that are electron deficient.  Electrophilic xenobiotics, statins, and cancer chemopreventive agents are among the substances that block ubiquination and proteolysis of Nrf2.

“Fig. 2

The above-described process is basic to all of the positive effects of Nrf2 and is described in a great many publications.  For example,the Biocart Pathways article Oxidative Stress Induced Gene Expression Via Nrf2relates “Reactive oxygen species (ROS) can damage biological macromolecules and are detrimental to cellular health. Electrophilic compounds, xenobiotics and antioxidants are sources of reactive oxygen species, creating oxidative stress that can harm cells. Enzymes are involved in the Phase II detoxification of xenobiotics to reduce cellular stress include glutathione transferases, quinone reductase, epoxide hydrolase, heme oxygenase, UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase. Expression of these genes protects cells from oxidative damage and can prevent mutagenesis and cancer. Transcription of these enzymes is coordinately regulated through antioxidant response elements (AREs). Nrf2 (NF-E2-related factor 2) and Nrf1 are transcription factors that bind to AREs and activate these genes. Inactive Nrf2 is retained in the cytosol by association a complex with the cytoskeletal protein Keap1. Cytosolic Nrf2 is phosphorylated and translocates into the nucleus in response to protein kinase C activation and Map kinase pathways. In the nucleus, Nrf2 activate genes through AREs by interacting with transcription factors in the bZIP family, including CREB, ATF4 and fos or jun. Nrf2 activation of genes is opposed by small maf proteins, including MafG and MafK, maintaining a counterbalance to Nrf2 and balancing the oxidation level of the intracellular environment.”

Put yet again in different terms “Oxidative stress promotes anti-oxidative gene expression via nuclear factor (erythroid-derived 2)-like 2 activation. In a basal state, free Nrf2 level is very low because it forms a complex with Keap1 and the E3 ligase Cul3-Rbx-1, leading to its proteasome degradation. Under the stimulation of oxidative stress, the level of free Nrf2 increases as it is dissociated with Keap1. Free Nrf2 molecules will then enter the nuclei, bind to the cis-element ARE and stimulate the expression of Nrf2 target genes[7]. ARE: Antioxidant response element; Cul3: Cullin 3; E3: Ubiquitin ligase; Keap1: Kelch-like ECH-associated protein 1; Nrf2: Nuclear factor (erythroid-derived 2)-like 2; Rbx-1: RING box protein 1(ref).”

Nrf2 provides a response to stress.

From the 2010 publication Nrf2: friend or foe for chemoprevention? – “Health reflects the ability of an organism to adapt to stress. Stresses–metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses–not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whilst disruption of the pathway exacerbates these outcomes.”

NRF2 is protective against multiple types of disorders associated with oxidative insults, for example airways disorders.

The same general pattern of protection appears to apply in the cases of multiple kinds of pathological conditions.  For example, the 2010 publication Nrf2 protects against airway disorders reports: “Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master transcription factor that regulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. In the unstressed condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and drives its proteasomal degradation. Nrf2 can be activated by diverse stimuli including oxidants, pro-oxidants, antioxidants, and chemopreventive agents. Nrf2 induces cellular rescue pathways against oxidative injury, abnormal inflammatory and immune responses, apoptosis, and carcinogenesis. Application of Nrf2 germ-line mutant mice has identified an extensive range of protective roles for Nrf2 in experimental models of human disorders in the liver, gastrointestinal tract, airway, kidney, brain, circulation, and immune or nerve system. In the lung, lack of Nrf2 exacerbated toxicity caused by multiple oxidative insults including supplemental respiratory therapy (e.g., hyperoxia, mechanical ventilation), cigarette smoke, allergen, virus, bacterial endotoxin and other inflammatory agents (e.g., carrageenin), environmental pollution (e.g., particles), and a fibrotic agent bleomycin. Microarray analyses and bioinformatic studies elucidated functional AREs and Nrf2-directed genes that are critical components of signaling mechanisms in pulmonary protection by Nrf2. Association of loss of function with promoter polymorphisms in NRF2 or somatic and epigenetic mutations in KEAP1 and NRF2 has been found in cohorts of patients with acute lung injury/acute respiratory distress syndrome or lung cancer, which further supports the role for NRF2 in these lung diseases. In the current review, we address the role of Nrf2 in airways based on emerging evidence from experimental oxidative disease models and human studies.”

Another important form of stress responded to by Nrf2 expression is nitrosative stress.

Although much discussion of Nrf2 is couched in terms of oxidative stress, Nrf2 responds to many other forms of stress including nitrosative stress.  The 2009 publication Role of Nrf2-mediated heme oxygenase-1 upregulation in adaptive survival response to nitrosative stress reports: “Nitrosative stress caused by reactive nitrogen species such as nitric oxide and peroxynitrite overproduced during inflammation leads to cell death and has been implicated in the pathogenesis of many human ailments.   However, relatively mild nitrosative stress may fortify cellular defense capacities, rendering cells tolerant or adaptive to ongoing and subsequent cytotoxic challenges, a phenomenon known as ‘preconditioning’ or ‘hormesis’.  One of the key components of cellular stress response is heme oxygenase-1 (HO-1), the rate limiting enzyme in the process of degrading potentially toxic free heme into biliverdin, free iron and carbon monoxide. HO-1 is upregulated by a wide array of stimuli and has antioxidant, anti-inflammatory and other cytoprotective functions. This review is intended to provide readers with a well-documented account of the research done in the area of cellular adaptive survival response against nitrosative stress with special focus on the role of HO-1 upregulation, especially through activation of the transcription factor, Nrf2.”

Nuclear levels of Nrf2 and its expression generally decline with age

This has been known for some time.  The 2004 publication Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acidrelated “Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively (P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% (P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an approximately 50% age-related loss in total (P < 0.001) and nuclear (P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with (R)-alpha-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo. LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.

The theme of age-related decline has been picked up in subsequent publications.  For example, the December 2011 publication Identification of age-specific Nrf2 binding to a novel antioxidant response element locus in the Gclc promoter: a compensatory means for the loss of glutathione synthetic capacity in the aging rat liver?  “NFE2-related factor 2 (Nrf2) transcriptionally governs the cellular response to harmful electrophiles, xenobiotics, and reactive oxygen species. Its nuclear levels decline with age (Suh et al., 2004a), which in part explains the age-related loss of phase II detoxification. However, little work has yet characterized how age affects Nrf2 DNA binding or the role that alterations to the Nrf2 transcriptional apparatus plays in modulating Nrf2-mediated gene expression. In this study, we used immunoprecipitation assays to show that Nrf2bound to the active antioxidant response element (ARE) of the catalytic subunit of glutamate cysteine ligase (GCLC) is significantly lower in hepatic chromatin from aged vs. young rats. Moreover, the activity at this ARE locus is diminished during aging because of the presence of Bach1 and the absence of CREB-binding protein (CBP), a transcriptional repressor and co-activator, respectively. Further analysis reveals that Nrf2 occupies an alternate ARE site located -2.2 kb downstream from the normally active ARE binding site in livers of old rats, indicating an age-specific adaptation to maintain gene expression. Our results, thus, show that the conversion of Nrf2 binding from an active ARE to an alternative ARE element is not adequate to maintain basal expression of hepatic Gclc in old rats, which provides a potential mechanism for the age-related loss of glutathione synthetic and other phase II enzymes.”

Some important positive biological effects of AMPK such as its anti-apoptotic capabilities  are modulated by Nrf2.

Both Victor and I have discussed AMPK, “The Master Metabolic Sensor and Regulator” in previous blog entries.  See for example Victor’s latest post Circadian Regulation, NMN, Preventing Diabetes, and Longevity. “In the end, we see that AMPK, once again, plays the primary role of sensing metabolic status and regulating cellular and organismal responses through multiple pathways. In fact, AMPK regulates circadian function by direct phosphorylation of clock components completely independent of sirt1.”  Among the multiple pathways affected by AMPK expression, some are mediated by AMPK’s effects on Nrf2.  The December 2011 publication AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network reports: “Efficient control of energy metabolic homeostasis, enhanced stress resistance, and qualified cellular housekeeping are the hallmarks of improved healthspan and extended lifespan. AMPK signaling is involved in the regulation of all these characteristics via an integrated signaling network. Many studies with lower organisms have revealed that increased AMPK activity can extend the lifespan. Experiments in mammals have demonstrated that AMPK controls autophagy through mTOR and ULK1 signaling which augment the quality of cellular housekeeping. Moreover, AMPK-induced stimulation of FoxO/DAF-16, Nrf2/SKN-1, and SIRT1 signaling pathways improves cellular stress resistance.  Furthermore, inhibition of NF-κB signaling by AMPK suppresses inflammatory responses. Emerging studies indicate that the responsiveness of AMPK signaling clearly declines with aging.  The loss of sensitivity of AMPK activation to cellular stress impairs metabolic regulation, increases oxidative stress and reduces autophagic clearance. These age-related changes activate innate immunity defence, triggering a low-grade inflammation and metabolic disorders.Metf”

The January 2011 publication Activation of AMPK stimulates heme oxygenase-1 gene expression and human endothelial cell survivalreports “In conclusion, AMPK stimulates HO-1 (heme oxygenase) gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.”

Nrf2 appears to stimulate the same pathways that extend life via calorie restriction or alternative day fasting.

From the 2011 publication The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation: “Recent evidence identifies Nrf2 signaling as a mediator of the salutary effects of caloric restriction. — CR, i.e. restriction of food intake without malnutrition, is a regimen that has been shown to extend the lifespan of organisms across the evolutionary spectrum. In addition to its effect on longevity, CR can confer diverse health benefits, which include decreased risk of cancer, lower blood pressure, higher insulin sensitivity, and improved neuronal function (reviewed in [19]. These broad health effects might be perceived as a result of the switch from “reproduction mode” to “longevity mode”, discussed above. At least some of the benefits of CR correlate with increased resistance against oxidative stress and may involve a system like the Nrf2 pathway, which is known for its antioxidant, cancer preventive and lifespan-extending functions. For example, aging is associated with a decline in the abundance of antioxidant proteins; this has been shown to be reversible with pharmacological activation of Nrf2 [20,21]. — Evidence for a function of Nrf2 as a CR effector has been provided by a number of studies in mice and worms. Pearson et al. [22] showed that CR induces antioxidant gene expression in mice and that this response is suppressed in nrf2^−/− animals, arguing for an upregulation of Nrf2 function in response to CR. Importantly, the previously described cancer protective function of CR [23,24] was diminished in nrf2^−/− mice. Thus, CR decreased the cancer incidence and tumor load elicited by a chemical carcinogenesis regimen in an Nrf2-dependent manner. These experiments established that CR increased Nrf2-dependent gene expression, and that Nrf2 was a required factor for several of the health benefits afforded by CR. — Some CR-mediated health benefits have also been reported in humans [26,27]. However, adhering to a strict CR regimen (as low as 50 % of ad libitum food intake) is not a realistic option for the vast majority of the population. Much effort has therefore been devoted to identify “CR mimetics”, drugs that induce metabolic and physiological changes akin to the shift to “longevity mode” induced by CR without the discomfort of a stringent diet [28]. Several such drugs are being evaluated, and two of the best known among them are resveratrol and metformin.”

 Regarding calorie restriction and alternative day fasting, see Victor’s recent blog entries Alternate-day Fasting – a better alternative and Mechanisms and Effects of Dietary Restriction.

There appears to be significant cross-talk between the insulin signaling, NF-kappaB, and Nrf2 pathways.

The February 2012 publication Redox-regulating role of insulin: The essence of insulin effect reports: “It is well-known that insulin acts as an important hormone, controlling energy metabolism, cellular proliferation and biosynthesis of functional molecules to maintain a biological homeostasis. Over the past few years, intensive insulin therapy has been believed to be benefit for the outcome of diabetic patients, in which the suppression of oxidative stress plays a role. Moreover, insulin is accepted as a key component of glucose-insulin-potassium, a treatment which has been believed to exert significant cardiovascular protective effect via the reduction of oxidative stress. Furthermore, accumulating evidence has suggested that insulin exerts important redox-regulating actions in various insulin-sensitive target organs, implying the systematic antioxidative role of insulin as a hormone. It is time for us to revisit insulin effects, through summarizing and evaluating the novel functions of insulin and their mechanisms. This review focuses on the antioxidative effect of insulin and highlights insulin-induced regulation of various antioxidant enzymes via insulin signaling pathways and the cross talk between key transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NF-κB) which are responsible for the transcription of antioxidant enzymes, leading to reduced generation of reactive oxygen species (ROS) and the enhancement of the elimination of ROS.”

Associated with its interactions with insulin, treatment of diabetes may be based on targeting Nrf2.

The April 2011 report Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle reported: “Long-standing diabetes and complications thereof particularly, neuropathy stands for one of the major causes of morbidity across the globe. It is postulated that excessive production of reactive oxygen species is a key component in the development and progression of diabetic neuropathy. Oxidative damage is the most common concluding pathway for various pathogenetic mechanisms of neuronal injury in diabetic neuropathy. However despite optimistic preclinical data, it is still very ambiguous that why antioxidants have failed to demonstrate significant neuroprotection in humans. A growing body of evidences now suggests that strategies utilizing a more targeted approach like focusing on Nrf2 (a transcription factor modulating oxidative stress) may provide an enthralling avenue to optimize neuroprotection in diabetes and diabetic neuropathy.”

The January 2012 publication Role of nuclear factor (erythroid-derived 2)-like 2 in metabolic homeostasis and insulin action: A novel opportunity for diabetes treatment?reports: “Redox balance is fundamentally important for physiological homeostasis. Pathological factors that disturb this dedicated balance may result in oxidative stress, leading to the development or aggravation of a variety of diseases, including diabetes mellitus, cardiovascular diseases, metabolic syndrome as well as inflammation, aging and cancer. Thus, the capacity of endogenous free radical clearance can be of patho-physiological importance; in this regard, the major reactive oxygen species defense machinery, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system needs to be precisely modulated in response to pathological alterations. While oxidative stress is among the early events that lead to the development of insulin resistance, the activation of Nrf2 scavenging capacity leads to insulin sensitization. Furthermore, Nrf2 is evidently involved in regulating lipid metabolism. Here we summarize recent findings that link the Nrf2 system to metabolic homeostasis and insulin action and present our view that Nrf2 may serve as a novel drug target for diabetes and its complications.”

The February 2011 publication Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo reported: “Objective: Oxidative stress is implicated in cardiac insulin resistance, a critical risk factor for cardiac failure, but the direct evidence remains missing. This study explored a causal link between oxidative stress and insulin resistance with a focus on a regulatory role of redox sensitive transcription factor NF-E2-related factor 2 (Nrf2) in the cardiac cells in vitro and in vivo.  Conclusions: ERK-mediated suppression of Nrf2 activity leads to the oxidative stress-induced insulin resistance in adult cardiomyocytes and downregulated glucose utilization in the diabetic heart.”

P62 protein appears to play an important role in oxidative defense through facilitating release of Nrf2 from Keap1.

P62 is involved in autophagy. “Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway(ref).”

Also, “Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulated p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-kappaB regulation and gene expression and to promote tumorigenesis. Thus, defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis(ref).”

The 2012 publication p62 at the Interface of Autophagy, Oxidative Stress Signaling, and Cancer reports: “ Significance: Sequestosome 1 (p62/SQSTM1) is a multifunctional adapter protein implicated in selective autophagy, cell signaling pathways, and tumorigenesis. Recent Advances: Recent evidence has revealed that p62/SQSTM1 has a critical role in an oxidative stress response pathway by its direct interaction with the ubiquitin ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), which results in constitutive activation of the transcription factor NF-E2-related factor 2 (NRF2). Critical Issues: Both NRF2 and KEAP1 are frequently mutated in cancer. The findings just cited uncover a link between p62/SQSTM1, autophagy, and the KEAP1-NRF2 stress response pathway in tumorigenesis and shed light on the interplay between autophagy and cancer. Future Directions: Here, we review the mechanisms by which p62/SQSTM1 implements its multiple roles in the regulation of tumorigenesis with emphasis on the KEAP1-NRF2 stress response signaling pathway.”

Decline in levels of p62 and disturbances in signaling of Nrf2 and other pathways can possibly contribute to the pathology of Alzheimer’s disease.

The January 2012 publication Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease reports: “The p62/sequestosome-1 is a multifunctional protein containing several protein-protein interaction domains. Through these interactions p62 is involved in the regulation of cellular signaling and protein trafficking, aggregation and degradation. p62 protein can bind through its UBA motif to ubiquitinated proteins and control their aggregation and degradation via either autophagy or proteasomes. p62 protein has been reported to be seen in association with the intracellular inclusions in primary and secondary tauopathies, α-synucleinopathies and other neurodegenerative brain disorders displaying inclusions with misfolded proteins. In Alzheimer’s disease (AD), p62 protein is associated with neurofibrillary tangles composed primarily of hyperphosphorylated tau protein and ubiquitin. Increasing evidence indicates that p62 has an important role in the degradation of tau protein. The lack of p62 protein expression provokes the tau pathology in mice. Recent studies have demonstrated that the p62 gene expression and cytoplasmic p62 protein levels are significantly reduced in the frontal cortex of AD patients. Decline in the level of p62 protein can disturb the signaling pathways of Nrf2, cyclic AMP and NF-κB and in that way increase oxidative stress and impair neuronal survival.”  

Interventions affecting Nrf2 may also play key roles in control if inflammatory diseases.

The 2010 publication A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders reports: “Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor that plays a central role in cellular defense against oxidative and electrophilic insults by timely induction of antioxidative and phase-2 detoxifying enzymes and related stress-response proteins. The 5′-flanking regions of genes encoding these cytoprotective proteins contain a specific consensus sequence termed antioxidant response element (ARE) to which Nrf2 binds. Recent studies have demonstrated that Nrf2-ARE signaling is also involved in attenuating inflammation-associated pathogenesis, such as autoimmune diseases, rheumatoid arthritis, asthma, emphysema, gastritis, colitis and atherosclerosis. — Thus, disruption or loss of Nrf2 signaling causes enhanced susceptibility not only to oxidative and electrophilic stresses but also to inflammatory tissue injuries. During the early-phase of inflammation-mediated tissue damage, activation of Nrf2-ARE might inhibit the production or expression of pro-inflammatory mediators including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases, cyclooxygenase-2 and inducible nitric oxide synthase. It is likely that the cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and also repress the induction of pro-inflammatory genes. This review highlights the protective role of Nrf2 in inflammation-mediated disorders with special focus on the inflammatory signaling modulated by this redox-regulated transcription factor.”

Nrf2 plays an important role in detoxification of drugs and other xenobiotics via upregulating expression of Phase 2 drug metabolizing enzymes.

“Xenobiotic metabolism (from the Greek xenos “stranger” and biotic “related to living beings”) is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism’s normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects(ref).”

The 2009 publication Role of phase II drug metabolizing enzymes in cancer chemoprevention reports: “Chemical insults, such as environmental or occupational carcinogenic agents, play a major role in the pathogenesis of many cancers. Many carcinogens exert genotoxic and cytotoxic effects via bioactivation into electrophilic species, a process catalyzed primarily by phase I drug metabolizing enzymes, typically cytochrome P450s. These reactive intermediates can induce DNA and RNA damage, and formation of protein adducts. The reactive species are often detoxified by phase II drug metabolizing enzymes, such as glutathione S-transferases (GSTs), UDP-glucuronosyl transferases (UGTs), sulfotransferase (ST) and N-acetyltransferase (NAT). Phase II enzymes classically conjugate these hydrophobic intermediates to a water-soluble group, thus masking their reactive nature, and allowing subsequent excretion. Therefore, strategies that modulate the levels of phase II enzymes by either pharmacological or nutritional means can lead to enhanced elimination of reactive species. Agents that preferentially activate phase II over phase I enzymes can be beneficial as chemopreventives. Compounds, such as isothiocyanates and dithiolthiones have been shown to act as transcriptional activators of phase II enzymes. A consensus enhancer element, known as antioxidant response element (ARE), in the regulatory domains of many phase II genes and an ARE-binding transcription factor nuclear factor E2-related factor 2 (Nrf2) have been implicated in the action of many chemopreventive agents. In this review, we will discuss the mechanisms of regulation of phase II enzymes, including the signal transduction events elicited by chemopreventive agents. We will also summarize the data available for these agents in preclinical models of tumorigenesis. Some chemopreventive agents have progressed to various stages of clinical trials, e.g. biomarker studies in healthy volunteers or in susceptible populations.”

The 2009 publication Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters reports: “The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play pivotal roles in preventing xenobiotic-related toxicity and carcinogen-induced carcinogenesis. These protective roles of Nrf2 have been attributed in part to its involvement in the induction of Phase II drug conjugation/detoxification enzymes as well as antioxidant enzymes through the Nrf2-antioxidant response element (ARE) signaling pathways. This review summarizes the current research status of the identification of Nrf2-regulated drug metabolism enzymes (DMEs), especially Phase II DMEs, and Phase III drug transporters. In addition, the molecular mechanisms underlying the coordinated regulation of Phase II DMEs and Phase III transporters will also be discussed based on findings published in the literature.”

Nrf2 is protective against the oxidative stress induced by drinking alcohol and mediated by CYP2E1.

As reported in the 2006 publication Cytochrome P450 2E1-dependent oxidant stress and upregulation of anti-oxidant defense in liver cells, “Induction of cytochrome P450 2E1 (CYP2E1) is a central pathway by which ethanol generates oxidative stress. Cytochrome P450 2E1 metabolizes many other toxicologic compounds. Toxicity of these agents is enhanced by ethanol, due to induction of CYP2E1. — These results suggest that Nrf2 is activated and its levels are increased when CYP2E1 is elevated. It is suggested that Nrf2 plays a key role in the adaptive response against increased oxidative stress caused by CYP2E1.”  See also (2009) Nrf2 and antioxidant defense against CYP2E1 toxicity, and (2011) Proteasome inhibitor up regulates liver antioxidative enzymes in rat model of alcoholic liver disease.

Nrf2 and Parkinson’s disease

There seems to be a substantial interest in developing therapeutics for Parkinson’s disease that work via activating Nrf2 expression.  I mention three relevant 2011 publications. The July 2011 publication NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson’s disease relates:  “BACKGROUND: Without appropriate cellular models the etiology of idiopathic Parkinson’s disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson’s disease.  RESULTS: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson’s Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson’s disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment.  CONCLUSIONS: Our results confirmed NRF2 as a potential therapeutic target for Parkinson’s disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not coordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.”

The Sept-Oct 2011 article Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson’s disease reports:  “Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.”

The article The Nrf2/ARE Pathway: A Promising Target to Counteract Mitochondrial Dysfunction in Parkinson’s Disease reports: “Mitochondrial dysfunction is a prominent feature of various neurodegenerative diseases as strict regulation of integrated mitochondrial functions is essential for neuronal signaling, plasticity, and transmitter release. Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson’s disease (PD). Several PD-associated genes interface with mitochondrial dynamics regulating the structure and function of the mitochondrial network. Mitochondrial dysfunction can induce neuron death through a plethora of mechanisms. Both mitochondrial dysfunction and neuroinflammation, a common denominator of PD, lead to an increased production of reactive oxygen species, which are detrimental to neurons. The transcription factor nuclear factor E2-related factor 2 (Nrf2, NFE2L2) is an emerging target to counteract mitochondrial dysfunction and its consequences in PD. Nrf2 activates the antioxidant response element (ARE) pathway, including a battery of cytoprotective genes such as antioxidants and anti-inflammatory genes and several transcription factors involved in mitochondrial biogenesis. Here, the current knowledge about the role of mitochondrial dysfunction in PD, Nrf2/ARE stress-response mechanisms, and the evidence for specific links between this pathway and PD are summarized. The neuroprotection of nigral dopaminergic neurons by the activation of Nrf2 through several inducers in PD is also emphasized as a promising therapeutic approach.”

Nrf2 and cancers

A great deal of research related to Nrf2 has been conducted in the context under the umbrella of cancer research.  I present a number of key findings.

Certain cancers work to epigenetically silence the expression of Nrf2.  Such may be the case, for example, in prostate cancers. 

The 2010 publication Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Micereported “Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells.  Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. — Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.”

The 2010 review publication Targeting NRF2 signaling for cancer chemoprevention relates: “Modulation of the metabolism and disposition of carcinogens through induction of cytoprotective enzymes is one of several promising strategies to prevent cancer. Chemopreventive efficacies of inducers such as dithiolethiones and sulforaphane have been extensively studied in animals as well as in humans. The KEAP1-NRF2 system is a key, but not unilateral, molecular target for these chemopreventive agents. The transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of the expression of a subset of genes, which produce proteins responsible for the detoxication of electrophiles and reactive oxygen species as well as the removal or repair of some of their damage products. — It is believed that chemopreventive enzyme inducers affect the interaction between KEAP1 and NRF2 through either mediating conformational changes of the KEAP1 protein or activating phosphorylation cascades targeting the KEAP1-NRF2 complex. These events in turn affect NRF2 stability and trafficking. Recent advances elucidating the underlying structural biology of KEAP1-NRF2 signaling and identification of the gene clusters under the transcriptional control of NRF2 are facilitating understanding of the potential pleiotropic effects of NRF2 activators and discovery of novel classes of potent chemopreventive agents such as the triterpenoids. Although there is appropriately a concern regarding a deleterious role of the KEAP1-NRF2 system in cancer cell biology, especially as the pathway affects cell survival and drug resistance, the development and the use of NRF2 activators as chemopreventive agents still holds a great promise for protection of normal cells from a diversity of environmental stresses that contribute to the burden of cancer and other chronic, degenerative diseases.”

The 2011 publication NRF2, cancer and calorie restriction speaks about the interest in Nrf2 from a cancer therapeutic viewpoint: “The transcription factor NF-E2-related factor (NRF2) is a key regulator of several enzymatic pathways, including cytoprotective enzymes in highly metabolic organs. In this review, we summarize the ongoing research related to NRF2 activity in cancer development, focusing on in vivo studies using NRF2 knockout (KO) mice, which have helped in defining the crucial role of NRF2 in chemoprevention. The lower cancer protection observed in NRF2 KO mice under calorie restriction (CR) suggests that most of the beneficial effects of CR on the carcinogenesis process are likely mediated by NRF2. We propose that future interventions in cancer treatment would be carried out through the activation of NRF2 in somatic cells, which will lead to a delay or prevention of the onset of some forms of human cancers, and subsequently an extension of health- and lifespan.”

Nrf2 activators may be of benefit to patients with chronic renal failure.

The January 2012 publication Dietary and synthetic activators of the antistress gene response in treatment of renal disease reports: “Renal failure is associated with increased vascular inflammation, oxidative stress and dicarbonyl stress linked to development of cardiovascular disease, and other complications. The endogenous defense to inflammatory, oxidative, and dicarbonyl challenge to vascular function is coordinated by nuclear factor E2-related factor 2 (nrf2), kelch-related erythroid cell-derived protein with CNC homology (ECH) protein 1 (keap1), and antioxidant response element-linked gene expression in the antistress gene response. Intervention trials of the synthetic nrf2 activator, bardoloxone methyl, in patients with advanced diabetic nephropathy, showing improvement of renal function and decreased inflammation, suggest that nrf2 activators may have therapeutic benefit in chronic renal failure. Activators of nrf2 are of both synthetic and dietary origin. The aim of this review is to describe the “nrf2/keap1/antioxidant response element” transcriptional system and studies of this system in renal failure, and to assess the current status and future prospects that dietary nrf2 activators may be of benefit to patients with chronic renal failure.”

Protection against the negative effects of strokes is another of the many medical benefits that might be possible through endogenous stimulation of Nrf2.

The September 2011 publication Targeting the Nrf2-Keap1 antioxidant defence pathway for neurovascular protection in stroke reports: “Endogenous defence mechanisms by which the brain protects itself against noxious stimuli and recovers from ischaemic damage are a key target of stroke research. The loss of viable brain tissue in the ischaemic core region after stroke is associated with damage to the surrounding area known as the penumbra. Activation of the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the cellular defence against oxidative stress via transcriptional upregulation of phase II defense enzymes and antioxidant stress proteins.  Although recent evidence implicates Nrf2 in neuroprotection, it is not known whether activation of this pathway within the neurovascular unit protects the brain against blood-brain barrier breakdown and cerebrovascular inflammation. Targeting the neurovascular unit should provide novel insights for effective treatment strategies and facilitate translation of experimental findings into clinical therapy. This review focuses on the cytoprotective role of Nrf2 in stroke and examines the evidence that the Nrf2-Keap1 defence pathway may serve as a therapeutic target for neurovascular protection.”

Steady laminar blood flow (s-flow) results in activation of Nrf2 and consequent inhibition of oxidative stress and inflammation in the vessel wall and is atheroprotective.  By contrast, disturbed blood flow (d-flow) results in the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF-κB), inflammation, and predisposes to the development of plaques.

The September 2011 publication  Flow shear stress and atherosclerosis: a matter of site specificity reports: “It is well accepted that atherosclerosis occurs in a site-specific manner especially at branch points where disturbed blood flow (d-flow) predisposes to the development of plaques. Investigations both in vivo and in vitro have shown that d-flow is pro-atherogenic by promoting oxidative and inflammatory states in the artery wall. In contrast, steady laminar blood flow (s-flow) is atheroprotective by inhibition of oxidative stress and inflammation in the vessel wall. The mechanism for inflammation in endothelial cells (ECs) exposed to d-flow has been well studied and includes redox-dependent activation of apoptosis signal-regulating kinase 1 (ASK1) and Jun NH2-terminal kinase (JNK) that ultimately lead to the expression of adhesive molecules. In contrast, s-flow leads to the activation of the mitogen extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) pathway that prevents pro-inflammatory signaling. Important transcriptional events that reflect the pro-oxidant and pro-inflammatory condition of ECs in d-flow include the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NFκB), whereas in s-flow, activation of Krüppel-like factor 2 (KLF2) and nuclear factor erythroid 2-like 2 (Nrf2) are dominant. Recent studies have shown that protein kinase c zeta (PKCζ) is highly activated under d-flow conditions and may represent a molecular switch for EC signaling and gene expression. The targeted modulation of proteins activated in a site-specific manner holds the promise for a new approach to limit atherosclerosis.”

Overexpression of Nrf2 is not always necessarioly a good thing.  It can result in protection of cancer cells.

The April 2011 publication Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy relates: “Background: Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.  Results: Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).  Conclusions: Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.”

Circadian Regulation is a fascinating topic with ties to many issues involved in antiaging research, including the regulation of stem cell populations.(See The circadian molecular clock creates epidermal stem cell heterogeneity, Biological Clock Controls Activation of Skin Stem Cells).  Studies have shown that 10-15% of all cellular transcripts oscillate in a circadian manner, including 50% of all nuclear receptors.  That is a huge number of genes involved in virtually all complex cellular processes in all of the cells of the body.  Decline of circadian function (rhythmicity and amplitude) appears to be associated with many age-related pathologies.  Resetting or re-synchronizing the clocks may be responsible for many of the benefits of calorie restriction (CR).  The peripheral clocks are entrained by metabolic factors, like feeding and physical activity, while the central clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is entrained by ambient conditions, such as light and to a lesser extent temperature.  Clearly, in modern lifestyles these so called “zeitgebers” are often not synchronized.  The central importance of synchronicity between the peripheral and central clocks is very clearly illustrated by the dramatically elevated risks of a variety of disorders in night-shift workers.  These include depression, cognitive decline, obesity, diabetes, heart disease, and cancer, mostly the same disorders associated with aging.  One mystery has always been, how do circadian clocks sense and respond to metabolic factors, if they are purely transcriptional feedback oscillators?

Epigenetics and Circadian Regulation

It has been discovered that circadian oscillations are not just a matter of transcriptional feedback loops, but that epigenetic mechanisms play a crucial role.  Recent research has begun to elucidate the epigenetic pathways involved in circadian regulation, such as the link between metabolism and circadian clocks.(ref, ref)  Actually, a decade ago it was discovered that the central clock in the SCN is activated by an epigenetic mechanism.  A single pulse of light to the optic nerve was shown to induce phosphorylation of serine 10 in H3, which directly results in circadian transcriptional activity in the hypothalamus.  It also results in a 10-fold increase in acetylation of lysine-14 of H3 (H3K14).(ref)  It turns out that one of the components of the canonical transcriptional clock, CLOCK, is also the HAT (histone acetyl transferase) responsible for acetylation of histones, as well as many important non-histone proteins.(ref)  Acetylation is an important “switch”, which alters the activity of many proteins (p53 is one clear example of this.).  If there is a cyclic writer (HAT), then there must also be an oscillating eraser, or histone deacetylase (HDAC).  The natural question then is what is the corresponding eraser, or HDAC, to CLOCK?  The surprising answer to this question also reveals the link between the metabolic state of a cell and circadian oscillations, elucidating how peripheral clocks can be regulated by metabolic factors.

The Missing Link

The mysterious link between metabolism and circadian regulation turns out to be the HDAC, sirt1.(ref,ref)  One very surprising thing about this is that sirt1 does not oscillate in a circadian manner.  However, sirtuins depend upon the essential coenzyme NAD (nicotinamide dinucleotide), which does oscillate in a very clear circadian pattern.(ref)  One of the pathways by which NAD levels are regulated is the salvage pathway, which is an enzymatic feedback loop, see: Fig 2.  Sirt1 links these two feedback loops, one enzymatic and the other transcriptional, and acts as a metabolic rheostat, controlling circadian function.  Why exactly do levels of NAD respond to metabolic changes? 

The Master Metabolic Sensor and Regulator – AMPK

NAD is the passive product of enzymatic pathways.  In the salvage pathway, illustrated in the Fig2, we see that two key enzymes are involved in the production of NAD, NAMPT, which converts nicotinamide into nicotinamide mononucleotide (NMN) and NMNAT which then converts NMN into NAD.  NAMPT (which is interestingly also an adipokine known as “visfatin”) is the rate-limiting step in this process; and in fact, NAD levels correlate extremely well with NAMPT levels.  Moreover, NMNAT does not oscillate as does NAMPT.  AMPK (AMP-activated protein kinase), which is known as the “master metabolic sensor” and plays a central role in mediating the effects of both exercise and nutrient restriction, determines the level of NAMPT.  Transgenic models have demonstrated that NAMPT does not oscillate in tissues lacking AMPK.  In the end, we see that AMPK, once again, plays the primary role of sensing metabolic status and regulating cellular and organismal responses through multiple pathways.  In fact, AMPK regulates circadian function by direct phosphorylation of clock components completely independent of sirt1.  See:  AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner. 

For more information on sirt1, see:  SIRT1-dependent Regulation of Chromatin and Transcription: Linking NAD Metabolism and Signaling to the Control of Cellular Functions.  I have also commented on AMPK and SIRT1 in earlier blog entries.(ref, ref)

Anti-aging Implications

Why do any of these technical details about molecular pathways matter?  Although, I have focused on the role of sirt1/NAD as a link between metabolism and circadian function, sirt1 and the other sirtuins (humans have a total of 7 orthologs with distinct functions) play a key role in many physiologic processes.  Decreased sirtuin activity is thought to be associated with many age-related disorders.  Although some have recently questioned the direct association with increased longevity, it remains clear that sirt1 mimics some of the effects of CR, and that sirt1 is necessary for many metabolic adaptations to CR.  A search of posts in this blog will find many entries related to the sirtuin SIRT1, its epigenetic actions and its health and putative life-extending properties.  Pharmaceutical companies have invested billions of dollars in developing sirtuin-activating compounds. 

Understanding the pathways involved in sirtuin activity suggests another approach, identifying nutriceutical compounds that naturally increase sirtuin bioactivity.  All sirtuins depend absolutely upon the adequate presence of NAD for their function.  However, this critical cofactor is frequently not readily available.  For example, NAD depletion and reduced sirtuin activity are the direct cause of cell death during acute heart failure(ref).  However, NAMPT-generated NAD can protect heart tissue from damage during stress(ref),  NAD protects cells from genotoxic stress by activating the mitochondrial sirtuins, sirt3 and sirt4(ref).  Although NAD can be synthesized de novo from tryptophan, the salvage pathway requiring NAMPT, previously mentioned, is much more direct, and accounts for the majority of NAD.  Despite its important role in NAD production, it would not be advisable to increase NAMPT activity, because of its inflammatory effects.  NAMPT is a proinflammatory adipokine, which increases the activity of other proinflammatory mediators, including COX enzymes, TNFalpha.(ref)  Unfortunately, direct NAD administration is also not a viable option, since doing so has been shown to cause rapid glycogen breakdown and severe hyperglycemia.  An ideal solution would be to find a way to increase activation of sirtuins by promoting natural NAD biosynthesis, without increasing NAMPT activity.(ref)

Bypassing NAMPT

Although NAMPT has many other functions, its role in the maintenance of NAD levels is crucial for life and health.  There are two forms of NAMPT, intracellular (iNAMPT) and extracellular (eNAMPT).  Both forms are involved in NAD production.  Certain tissues are more susceptible to the harmful effects of NAD depletion than others.  These include cardiac tissue due to its high energy demands, as well as other tissues, which do not produce iNAMPT (in detectable quantities) such as the brain and pancreas.(ref)  Such tissues are dependent upon eNAMPT for their NAD needs.  One recent study has questioned whether eNAMPT increases plasma levels of NAD, which would imply an even greater vulnerability of pancreas and brain tissues to NAD shortages.(ref) 

Recent genomic studies indicate that pancreatic beta cell dysfunction may be the primary cause of type-2 diabetes.  It is likely that the age-related decline in systemic NAD plays an important role in the pathology of type-2 diabetes.(ref)  The same is true for brain tissue.  Once neurological and metabolic deterioration begin, a vicious cycle of physiological decline would ensue.  As one researcher has observed:  “Once pancreatic β cells and brain start having functional problems due to insufficient NAD biosynthesis, other peripheral tissues/organs would also be affected through insulin secretion and central metabolic regulation, resulting in the gradual deterioration of the physiological robustness through the entire body.  Therefore, in the concept of the NAD World, aging is considered as the process in which organismal robustness gradually breaks down according to a functional hierarchy determined by the susceptibility to systemic NAD biosynthesis.  Additionally, tissues that control the NAD-dependent branch of circadian clock feedback regulations throughout the body could be considered as “the aging clock”, and any imbalance in this fine-tuning system, which could be imposed by long-term nutritional and environmental perturbations, might initiate the process of organismal robustness breakdown (Fig. 4).  Although further investigation will be necessary, tweaking “the aging clock”, possibly by supplementing NAD intermediates to systemic NAD biosynthesis, might be an effective way to make all of our clocks tick robustly in this otherwise inevitable process of aging.”(ref)

The suggestion is that direct supplementation of NMN, which is the rate-limiting step in NAD production, could have therapeutic potential, while effectively bypassing the need for NAMPT and its potentially harmful, proinflammatory effects.  Moreover, NAMPT requires adequate supplies of ATP in order to produce NMN.  This means that critical shortages NAD may result, whenever ATP becomes depleted.  It is likely that NAD deficiency is a primary cause of tissue death resulting from hypoxia, following cerebral or cardiac ischemia.  (Hypoxia is oxygen shortage which directly reduces the cellular production of ATP.)  Another advantage of direct NMN supplementation is that by bypassing NAMPT and its need for ATP, NMN supplementation has the potential to maintain NAD levels in critical times of reduced oxygen/ATP supply.

Animal Studies of NMN Supplementation

What do we know about the effects of NMN?  To-date no human trials of NMN have been conducted.  However, several animal studies have shown impressive results.

Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice

In this first study, despite overexpressing sirt1, sirt1 activity decreases with age resulting in decreased GSIS (glucose-stimulated insulin secretion).  NMN administration completely reversed this pathology restoring youthful pancreatic function.  NMN also improved GSIS in normal (wild-type) mice.  “Interestingly, plasma levels of nicotinamide mononucleotide (NMN), an important metabolite for the maintenance of normal NAD biosynthesis and GSIS in β cells, are significantly reduced in aged BESTO mice. Furthermore, NMN administration restores enhanced GSIS and improved glucose tolerance in the aged BESTO females, suggesting that Sirt1 activity decreases with advanced age due to a decline in systemic NAD biosynthesis. These findings provide insight into the age-dependent regulation of Sirt1 activity and suggest that enhancement of systemic NAD biosynthesis and Sirt1 activity in tissues such as β cells may be an effective therapeutic intervention for age-associated metabolic disorders such as type 2 diabetes.”

NMN, a Key NAD Intermediate, Treats the Pathophysiology of Diet- and Age-Induced Diabetes in Mice

Highlights

• NAMPT-mediated NAD+ biosynthesis is compromised in metabolic organs by HFD  (high fat diet)
• NMN ameliorates defects in NAD+ biosynthesis and glucose metabolism in T2D mice
• NMN enhances hepatic insulin sensitivity by reversing gene expression caused by HFD
• NMN also ameliorates defects in glucose and lipid metabolism in age-induced T2D mice

“Summary: Type 2 diabetes (T2D) has become epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis, and the NAD+-dependent protein deacetylase SIRT1. Here, we show that NAMPT-mediated NAD+ biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD+ intermediate, ameliorates glucose intolerance by restoring NAD+ levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD+ and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a potential nutriceutical intervention against diet- and age-induced T2D.”

Graphical abstract is from the publication.

See also Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function.  “CONCLUSIONS/INTERPRETATION:  Chronic fructose feeding causes severe islet dysfunction in mice. [Another of the many hazards of fructose.] Onset of beta cell failure in FRD-fed mice may occur via lowered secretion of eNAMPT, leading to increased islet inflammation and impaired beta cell function. Administration of exogenous NMN to FRD-fed mice corrects inflammation-induced islet dysfunction. Modulation of this pathway may be an attractive target for amelioration of islet dysfunction associated with inflammation.”

Wrapping it up-

Circadian clock functions are ubiquitous and impacted by aging, dietary, lifestyle and environmental conditions.  They play important roles with respect to metabolism, health and disease susceptibilities.  A key link between circadian regulation and metabolism appears to be the sirtuin SIRT1.  Age or disease related-dysregulation of circadian metabolic control can lead to multiple kinds of havoc including type 2 diabetes.  It is possible that supplementation of humans with NMN might work to prevent or serve as a therapy for diet or age-induced Type 2 diabetes.  It would do that, if mouse-model results carry over to humans, by enhancing natural biosynthesis of NAD, and thereby increasing SIRT1 activity.  Further, some researchers have argued that enhanced activity of SIRT1 could precipitate the same life-extending pathways that act in the case of calorie restriction or alternative day fasting.  See my previous blog entries:   Alternate-day Fasting – a better alternativeandMechanisms and Effects of Dietary Restriction

For humans who wish to live long lives, alternative-day fasting may be a better approach than following a restricted calorie diet.  The approach avoids premature induction of frailty most likely by periodically inhibiting myogenesis which encourages replenishment of progenitor satellite cell pools.  The argument for this proposition is presented here.  For background, see my earlier post Mechanisms and Effects of Dietary Restriction.

The body uses metabolic sensors to monitor and regulate anabolic and catabolic processes in response to various factors such as energy demands, nutrient availability, etc.  In most cells throughout the body cellular energy depends upon the availability of the critical cellular energy molecule, ATP.  When levels of ATP drop in response to increased energy demands from exercise, decreased nutrient availability from dietary restriction, or other factors the ratio of AMP to ATP increases, signaling the activation of AMPK (AMP-activated protein kinase), frequently referred to as “the master metabolic sensor”.  AMPK then switches on catabolic pathways which generate ATP, such as lipid and glucose oxidation; AMPK also switches off catabolic processes including synthesis of lipids, glucose, and proteins. 

AMPK activation produces very important changes in transcriptional activity by phosphorylating critical transcription factors.  (Kinases, like AMPK, work by a process called phosphorylation, see Kinase Inhbition for a more detailed explanation.)  AMPK can also be activated itself by direct phosphorylation, as well as certain signaling molecules, including adipokines, such as leptin.(ref)  For an earlier discussion of AMPK by Dr. Giuliano, please see AMPK and longevity

Sirt1 is one of seven human orthologs of the yeast silent information regulator 2 (sir2).  Sirt1 is a deacetylase which removes acetyl groups from other proteins including histones, various transcription factors, etc.  Sirt1 has been thought to be a molecular link between the dietary restriction and longevity.  Although recently this has been called into question, see the November 2011 publication An unSIRTain role in longevity.  Like AMPK, sirt1 is activated by nutrient restriction and exercise.  It also appears to be activated by AMPK through several mechanisms.  Directly, by AMPK-induced phorphorylation, and indirectly by AMPK phosophorylation of FoxO transcription factors (ref), and by AMPK-induced alterations in levels of NAD+, which is an essential coenzyme for sirt1 activity (ref).

Both AMPK and sirt1 have many of the same down-stream molecular targets.  Although specific effects vary depending upon tissue type, usually their effects are complementary, and very often interdependent (ref).  One example of divergent effects is the secretion of insulin by pancreatic beta cells.  Sirt1 increases insulin release, while AMPK actually inhibits it (ref,ref).  This, interestingly, explains why the anti-diabetic drug, metformin, paradoxically lowers plasma insulin levels.(ref)  Although this insulin-lowering effect is often erroneously explained as the result of increased peripheral insulin uptake, it is actually a direct result of the fact that metformin is an AMPK activator.(ref)  AMPK increases muscle glucose uptake, but it does so by directly activating the transportor, GLUT4 completely independent of insulin.(ref).  The is the same mechanism by which another AMPK activator, AICAR, increases muscle energy uptake.(ref). 

Sirt1 also activates GLUT4.  In fact, a recent transgenic mouse study demonstrates that sirt1 is necessary for AMPK to activate GLUT4, further demonstrating their frequent interdependence.(ref)  Together these results explain the mechanism of the observed improved insulin sensitivity, despite decreased insulin levels, that accompany caloric restriction.

Since AMPK and sirt1 both increase nutrient uptake in muscle tissue, it would be logical to conclude that activators of these compounds would increase muscle growth.  The ability of both AICAR and metformin to increase muscle glucose uptake has been confirmed in a recent animal study.(ref)  Of particular interest is the fact that this effect appears to be limited to muscle tissue; fortunately, for example, they do not increase uptake of glucose into liver tissue.

Sirt1 prevents myoblast differentiation.

Muscular growth is dependent upon the activation and differentiation of quiescent muscle satellite cells, a process known as “myogenesis”.  AMPK and sirt1 have been shown to inhibit this process, see:  Glucose Restriction Inhibits Skeletal Myoblast Differentiation by Activating SIRT1 through AMPK-Mediated Regulation of Nampt. 

This was found to be the case in glucose restriction and under normal caloric  conditions with the use of AMPK activators including AICAR and metformin.

“AICAR promoted AMPK and ACC phosphorylation in normocaloric (NC) conditions (Figure 1F), and cells exposed to AICAR in NC conditions failed to appropriately differentiate (Figures 1G and 1H). In addition to AICAR, two other AMPK activators—the furancarboxylic acid derivative D942 (Kosaka et al., 2005) and the hypoglycemic drug metformin (Zhou et al., 2001) —also inhibited cell differentiation in a dose-dependent manner (Fig. S1E, and F).”

These results are consistent with the concept of AMPK sirt1 acting as metabolic switches which turn off anabolic processes when activated either by dietary restriction or by the use of synthetic activators such as AICAR, metformin including putative activators of sirt1, like resveratrol.  In this case, sirt1 appears to play the key role by deacetylating MyoD, a transcription factor essential for satellite cell activation which is necessary for both muscle growth (ref) and repair (ref).  In the experimental model, activation of AMPK failed to prevent myogenesis, when sirt1 was inhibited.

A problem with calorie restriction diets

Such catabolic effects have been commonly reported by practitioners of calorie restriction.  One such longevity enthusiast, Michael Young, who was featured on a recent BBC program, after consuming a reduced calorie diet for several years, found that he was suffering from premature frailty at the age of 42.  He was six feet tall, but only weighed 58k (about 138 lbs), and had underdeveloped bones.  See the video War against ageing.  This is one compelling reason that caloric restriction may not be advisable in humans.  Even under normal dietary conditions, most elderly eventually suffer from sarcopenia to some degree, along with accompanying risks and increased morbidity/mortality.  For many, age-related frailty can be as debilitating as cognitive decline, greatly restricting mobility, independence of living, and quality of life.

AMPK and Sirt1 Activity without Muscle Catabolism

Is there any way to have the benefits of caloric restriction without its unwanted catabolic effects?  Evidence suggests the answer is Yes, using a strategy known as “intermittent fasting.”  In particular, alternate day fasting (ADF) has been shown, in both animal and human studies, to reproduce the many of the same beneficial results as continuous caloric restriction, even though overall caloric intake need not be reduced.  ADF improves lipid and glucose metabolism, reduces inflammation, improves cardiovasucular, renal, and hepatic function.(ref)  It also improves the adikpokine secretion profile, as well as body fat distribution patterns.(ref)  ADF has been shown to increase longevity in both animal (ref, ref) and humans.(ref)  ADF has also been shown to prevent neurological aging, in part  by increasing expression of brain-derived neurotrophic factor (BDNF)(ref), and by dramatically increasing neuronal autophagy.(ref)

For review articles, see:

Alternate-day fasting and chronic disease prevention

Impact of caloric and dietary restriction regimens on markers of health and longevity in humans and animals

ADF Improves Muscle Function

In contrast to caloric restriction, a recent study found that ADF was able to improve mobilization of nutrients during exercise, and increase lipid utilization, preventing muscle damage and oxidative stress even following strenuous exercise.  Muscle mass was not decreased, and physical strength and endurance were both increased by ADF.  See Muscle Physiology Changes Induced by Every Other Day Feeding and Endurance Exercise in Mice.  These results may reflect the beneficial effects of periodically inhibiting myogenesis, in order to allow for replenishment of progenitor satellite cell pools.  In this respect, it is interesting to note that sirt1-knockout mice have very small, underdeveloped muscles.  Both AMPK and sirt1 activity decrease with age, and could contribute to the age-related loss of satellite cell pools, ultimately leading to sarcopenia and frailty.(ref, ref)

Like the previous two blog entries Dietary factors and dementia – Part 1: important recent researchandDietary factors and dementia Part 2: possible interventions, this blog entry is focused on research during the last two years relating dietary substances and supplements to late-onset dementias including Alzheimer’s disease, and to the potential roles of such substances for prevention or treatment of dementia.  The Part 1 blog entry deals with research on a variety of subtopics such as the value of the relationship of dementia to diabetes, the role of oxidative stress in AD and, generally when and how diet can make a difference.  The Part 2 blog entry describes research on possible interventions that could delay, prevent or cure dementia or Alzheimer’s disease including ingesting fatty acids and following a Mediterranean diet.  This present blog entry describes research during the last three years on how sixteen different plant-derived substances have been shown in-vitro and in transgenic mouse models to inhibit the formation of or enhance the clearance of beta amyloid or to reverse other symptoms of Alzheimer’s disease.   It also describes how supplementation with a specific combination of such supplements has been shown to clear up symptoms of Alzheimer’s disease in a transgenic mouse model.

At least sixteen plant-derived substances have been shown in-vitro and in transgenic mouse models to inhibit the formation of or enhance the clearance of beta amyloid or to reverse other symptoms of Alzheimer’s disease, including fermented papaya, L-3-n-butylphthalide, olive oil, tetrahydrocurcumin, aged garlic extract, green tea, cinnamon extract , the mushroom Hericium erinaceus, olive bark, piperine, bacopa monnieri, purple rice berry, oroxylin A, silymarin, silibinin, and resveratrol.

Fermented papaya

The 2010 publication Applications and bioefficacy of the functional food supplement fermented papaya preparation reports: “Fermented papaya preparation (FPP) (a product of yeast fermentation of Carica papaya Linn) is a food supplement. Studies in chronic and degenerative disease conditions (such as thalassemia, cirrhosis, diabetes and aging) and performance sports show that FPP favorably modulates immunological, hematological, inflammatory, vascular and oxidative stress damage parameters. Neuroprotective potential evaluated in an Alzheimer’s disease cell model showed that the toxicity of the β-amyloid can be significantly modulated by FPP. Oxidative stress trigger apoptotic pathways such as the c-jun N-terminal kinase (JNK) and p38-mitogen activated protein kinase (MAPK) are preferentially activated by pro-inflammatory cytokines and oxidative stress resulting in cell differentiation and apoptosis. FPP modulated the H₂O₂-induced ERK, Akt and p38 activation with the reduction of p38 phosphorylation induced by H₂O₂. FPP reduces the extent of the H₂O₂-induced DNA damage, an outcome corroborated by similar effects obtained in the benzo[a]pyrene treated cells. No genotoxic effect was observed in experiments with FPP exposed to HepG2 cells nor was FPP toxic to the PC12 cells. Oxidative stress-induced cell damage and inflammation are implicated in a variety of cancers, diabetes, arthritis, cardiovascular dysfunctions, neurodegenerative disorders (such as stroke, Alzheimer’s disease, and Parkinson’s disease), exercise physiology (including performance sports) and aging. These conditions could potentially benefit from functional nutraceutical/food supplements (as illustrated here with fermented papaya preparation) exhibiting anti-inflammatory, antioxidant, immunostimulatory (at the level of the mucus membrane) and induction of antioxidant enzymes.”

L-3-n-butylphthalide

The 2011 publication L-3-n-butylphthalide improves cognitive impairment and reduces amyloid-beta in a transgenic model of Alzheimer’s disease reports “Alzheimer’s disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. L-3-n-butylphthalide (L-NBP), an extract from seeds of Apium graveolens Linn (Chinese celery), has been demonstrated to have neuroprotective effects on ischemic, vascular dementia, and amyloid -beta (Abeta)-infused animal models. —  In the current study, we examined the effects of L-NBP on learning and memory in a triple-transgenic AD mouse model (3xTg-AD) that develops both plaques and tangles with aging, as well as cognitive deficits. Ten-month-old 3xTg-AD mice were given 15 mg/kg L-NBP by oral gavage for 18 weeks. L-NBP treatment significantly improved learning deficits, as well as long-term spatial memory, compared with vehicle control treatment. L-NBP treatment significantly reduced total cerebral Abeta plaque deposition and lowered Abeta levels in brain homogenates but had no effect on fibrillar Abeta plaques, suggesting preferential removal of diffuse Abeta deposits. Furthermore, we found that L-NBP markedly enhanced soluble amyloid precursor protein secretion (alphaAPPs), alpha-secretase, and PKCalpha expression but had no effect on steady-state full-length APP. Thus, L-NBP may direct APP processing toward a non-amyloidogenic pathway and preclude Abeta formation in the 3xTg-AD mice. The effect of l-NBP on regulating APP processing was further confirmed in neuroblastoma SK-N-SH cells overexpressing wild-type human APP(695) (SK-N-SH APPwt). L-NBP treatment in 3xTg-AD mice also reduced glial activation and oxidative stress compared with control treatment. L-NBP shows promising preclinical potential as a multitarget drug for the prevention and/or treatment of Alzheimer’s disease.”

Olive oil

“Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats. It is an odorless, colourless oil, although commercial samples may be yellowish. In chemical terms, oleic acid is classified as a monounsaturatedomega-9fatty acid. — The term “oleic” means related to, or derived from, oil or olive, the oil of which is predominantly derived from oleic acid(ref).” The May 2011 publication Oleic acid ameliorates amyloidosis in cellular and mouse models of Alzheimer’s disease reports: “Several lines of evidence support protective as well as deleterious effects of oleic acid (OA) on Alzheimer’s disease (AD) and other neurological disorders; however, the bases of these effects are unclear. Our investigation demonstrates that amyloid precursor protein (APP) 695 transfected Cos-7 cells supplemented with OA have reduced secreted amyloid-beta (Aβ) levels. — An early-onset AD transgenic mouse model expressing the double-mutant form of human APP, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings when they were fed a high-protein, low-fat (18% reduction), cholesterol-free diet enriched with OA. These mice exhibited an increase in Aβ40/Aβ42 ratio, reduced levels of beta-site APP cleaving enzyme (BACE) and reduced presenilin levels along with reduced amyloid plaques in the brain. The decrease in BACE levels was accompanied by increased levels of a non-amyloidogenic soluble form of APP (sAPPα). Furthermore, the low-fat/+OA diet resulted in an augmentation of insulin-degrading enzyme and insulin-like growth factor-II. These results suggest that OA supplementation and cholesterol intake restriction in a mouse model of AD reduce AD-type neuropathology.”

Another relevant publication is the August 2011 item Tyrosol and hydroxytyrosol, two main components of olive oil, protect N2a cells against amyloid-β-induced toxicity. Involvement of the NF-κB signaling which relates: “Alzheimer’s disease (AD) is the most common form of dementia. Recently, a number of epidemiological studies have evidence that some dietary factors such as low antioxidants and vitamins intake could increase the risk of AD. In the opposite, diets rich in unsaturated fatty acids, in polyphenols, vitamins and antioxidants were identified as preventive factors. Several studies have reported that adherence to the Mediterranean diet (MeDi) was associated with a reduction in incident of dementia. The beneficial effect of MeDi may be the result of the association of some individual and non-identified food components and high consumption of olive oil. In this study we have investigated the protective effects of two components of olive oil, tyrosol (Tyr) and hydroxytyrosol (OH-Tyr), against Aβ-induced toxicity. In cultured neuroblastoma N2a cells, we found that Aβ(25-35) (100 µg/ml) treatment induced a decrease of glutathione (GSH) and the activation of the transcription factor NF-κB and cell death. Our results demonstrated that the number of cell death decreased when cells were co-treated with Aβ and Tyr or OH-Tyr. However, neither of these phenolic compounds was able to prevent the decrease of GSH induced by H(2)O(2) or Aβ. We found that the increase in the nuclear translocation of the NF-κB subunits after Aβ exposure was attenuated in the presence of Tyr or OH-Tyr. These results identified two individual food components of the MeDi as neuroprotective agent against Aβ and their potential involvement in the beneficial effect of the MeDi for the prevention of AD.”

The May 2011 report Olive oil reduces oxidative damage in a 3-nitropropionic acid-induced Huntington’s disease-like rat modelrelates: “Free radicals contribute to altered neuronal functions in neurodegenerative diseases and brain aging, by producing lipid- and other molecule-dependent modifications. The Mediterranean diet has been associated with a reduced risk of neurodegenerative disease. This study sought to verify whether extra-virgin olive oil (EVOO) exerted a brain antioxidant effect, protecting the brain against the oxidative stress caused by 3-nitropropionic acid (3NP). 3NP was administered intraperitoneally (i.p.) at a dose of 20 mg/kg body weight over four consecutive days. EVOO (representing 10% of calorie intake in the total standard daily diet of rats) and hydroxytyrosol (HT; 2.5 mg/kg body weight) were administered for 14 days. In all studied samples, 3NP caused a rise in lipid peroxides (LPO) and a reduction in glutathione (GSH) content. While the results showed that EVOO and HT reduces lipid peroxidation product levels and blocks the GSH depletion prompted by 3NP in both striatum and rest of the brain in Wistar rats. In addition, EVOO blocks and reverses the effect of 3NP on succinate dehydrogenase activity. In brief, the data obtained indicate that EVOO and HT act as a powerful brain antioxidant.”

Curcumin

The January 2011 publication Tetrahydrocurcumin confers protection against amyloid β-induced toxicity reports: “Amyloid plaques and neurofibrillary tangles are the hallmarks of Alzheimer’s disease. Amyloid β, a primary component of the amyloid plaques, is neurotoxic. Considerable attention has been directed toward identifying compounds with neuroprotective properties. Using rat primary hippocampal cultures, we show that tetrahydrocurcumin (THC), a metabolite of curcumin, shows a protective effect against oligomeric amyloid-β-induced toxicity. We further show that THC reduces amyloid-β-induced (i) increase in the level of reactive oxygen species, (ii) decrease in mitochondrial membrane potential, and (iii) caspase activation. In addition, we show that THC protects human neurons from oligomeric amyloid-β-induced toxicity as well. Thus, THC confers protection against amyloid-β-induced toxicity, and the antioxidant activity may contribute to its protective effect.”

The August 2010 blog entry Neurogenesis, curcumin and longevity is specifically concerned with the impact of the dietary supplement curcumin on neurogenesis in the hippocampus and the impact of curcumin neural plasticity.  A number of research reports quoted there relate to the neural protective effects of curcumin and how curcumin can increase neurogenesis under conditions of stress.  

A January 2011 publication Revisiting dietary antioxidants, neurodegeneration and dementia comments editorially “Epidemiological studies suggest there is marginal benefit that dietary antioxidants reduce risk of Alzheimer type of dementias. Yet cumulative biological evidence indicates oxidative and nitrosative stresses are precursors of neurodegenerative and neurovascular processes. Different dietary flavonoids and polyphenols found in fruits, vegetables, and spices such as curcumin offer neuroprotection through different mechanisms. A study in this volume shows that tetrahydrocurcumin confers protection against amyloid [beta]-induced toxicity by reducing reactive oxygen species and retaining mitochondrial membrane potential. Alzheimer’s disease is a complex disorder. A single target through use of antioxidants may be effective in some but multiple approaches for its control seem to be necessary.”

Aged garlic extract

The May 2011 publication Oxidative insults to neurons and synapse are prevented by aged garlic extract and S-allyl-L-cysteine treatment in the neuronal culture and APP-Tg mouse model reports: “Alzheimer’s disease (AD) is one of the most common forms of dementia in the elderly. In AD patients, β-amyloid peptide (Aβ) plaques and neurofibrillary tangles are common features observed in the CNS. Aβ deposition results in the production of reactive oxygen species (ROS) leading to the hyperphosphorylation of tau that are associated with neuronal damage. Cholinesterase inhibitors and a partial NMDA receptor antagonist (memantine) have been identified as potential treatment options for AD. However, clinical studies have found that these drugs fail to prevent the disease progression. From ancient times, garlic (Allium sativum) has been used to treat several diseases. By ‘aging’ of garlic, some adverse reactions of garlic can be eliminated. Recent findings suggest that ‘aged garlic extract’ (AGE) may be a therapeutic agent for AD because of its antioxidant and Aβ lowering properties. To date, the molecular properties of AGE have been sparsely studied in vitro or in vivo. The present study tested specific biochemical and molecular effects of AGE in neuronal and AD rodent models. Furthermore, we identified S-allyl-L-cysteine (SAC) as one of the most active chemicals responsible for the AGE-mediated effect(s). We observed significant neuroprotective and neurorescue properties of AGE and one of its ingredients, SAC, from ROS (H(2)O(2))-mediated insults to neuronal cells. Treatment of AGE and SAC were found to protect neuronal cells when they were independently co-treated with ROS. Furthermore, a novel neuropreservation effect of AGE was detected in that pre-treatment with AGE alone protected ∼ 80% neuronal cells from ROS-mediated damage. AGE was also found to preserve pre-synaptic protein synaptosomal associated protein of 25 kDa (SNAP25) from ROS-mediated insult. For example, treatment with 2% AGE containing diet and SAC (20 mg/kg of diet) independently increased (∼70%) levels of SNAP25 and synaptophysin in Alzheimer’s amyloid precursor protein-transgenic mice, of which the latter was significantly decreased in AD. Taken together, the neuroprotective, including preservation of pre-synaptic proteins by AGE and SAC can be utilized in future drug development in AD.”

Green tea

The January 2012 publication The importance of the multiple target action of green tea polyphenols for neuroprotection reports: “–Brain aging and neurodegenerative diseases of the elderly are characterized by oxidative damage, dysregulation of redox metals homeostasis and inflammation. Thus, it is not surprising that a large amount of drugs/agents in therapeutic use for these conditions are antioxidants/metal complexing, bioenergetic and anti-inflammatory agents. Natural plant polyphenols (flavonoids and non-flavonoids) are the most abundant antioxidants in the diet and as such, are ideal nutraceuticals for neutralizing stress-induced free radicals and inflammation. — Human epidemiological and new animal data suggest that green and black flavonoids named catechins, may help protecting the aging brain and reduce the incidence of dementia, AD and PD. This review will present salient features of the beneficial multi-pharmacological actions of black and green tea polyphenols in aging and neurodegeneration, and speculate on their potential in drug combination to target distinct pathologies as a therapeutic disease modification approach.”

Cinnamon extract.

The January 2011 report Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer‘s disease animal models reports: “An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimer’s disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.”

The mushroom Hericium erinaceus

The February 2011 publication Effects of Hericium erinaceus on amyloid β(25-35) peptide-induced learning and memory deficits in micereports:  “The mushroom Hericium erinaceus has been used as a food and herbal medicine since ancient times in East Asia. It has been reported that H. erinaceus promotes nerve growth factor secretion in vitro and in vivo. Nerve growth factor is involved in maintaining and organizing cholinergic neurons in the central nervous system. These findings suggest that H. erinaceus may be appropriate for the prevention or treatment of dementia. In the present study, we examined the effects of H. erinaceus on amyloid β(25-35) peptide-induced learning and memory deficits in mice. Mice were administered 10 µg of amyloid β(25-35) peptide intracerebroventricularly on days 7 and 14, and fed a diet containing H. erinaceus over a 23-d experimental period. Memory and learning function was examined using behavioral pharmacological methods including the Y-maze test and the novel-object recognition test. The results revealed that H. erinaceus prevented impairments of spatial short-term and visual recognition memory induced by amyloid β(25-35) peptide. This finding indicates that H. erinaceus may be useful in the prevention of cognitive dysfunction.”

Olive bark

The January 2011 publication Neuroprotective effects of Eucommia ulmoides Oliv. Bark on amyloid beta(25-35)-induced learning and memory impairments in mice reports: “In the present study, we examined whether aqueous extract of Eucommia ulmoides Oliv. Bark (EUE) with graded doses exerted its neuroprotective effects on amyloid beta(25-35) (Aβ(25-35))-induced learning and memory impairments in mice. Mice received a single intracerebroventricular (i.c.v.) injection of Aβ(25-35) 6 nmol as the critical factor in Alzheimer’s disease (AD), cognition was evaluated using Y-maze, passive avoidance, and Morris water maze tests. EUE significantly improved the Aβ(25-35)-induced memory deficit in the Y-maze test. Also, EUE increased step-through latency time with Aβ(25-35)-induced learning and memory deficits in the passive avoidance test. In addition, EUE decreased the escape latencies with Aβ(25-35)-induced cognitive impairments in the Morris water maze test. In the probe trial session, EUE increased time spent in the target quadrant. In the in vitro study, EUE was found to inhibit acetylcholinesterase (AChE) activity in a dose-dependent manner (IC50 value; 172 μg/ml). Ex vivo study, EUE significantly inhibited AChE activity in the hippocampus and frontal cortex. These results demonstrate that EUE possesses potent neuroprotective effects and that its beneficial effects are mediated, in part, by AChE inhibition, and therefore, might be a potential candidate in neurodegenerative diseases such as AD.”

Piperine

The 2010 publication Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model of cognitive deficit like condition of Alzheimer’s disease reports: “Recently, numerous medicinal plants possessing profound central nervous system effects and antioxidant activity have received much attention as food supplement to improve cognitive function against cognitive deficit condition including in Alzheimer’s disease condition. Based on this information, the effect of piperine, a main active alkaloid in fruit of Piper nigrum, on memory performance and neurodegeneration in animal model of Alzheimer’s disease have been investigated. Adult male Wistar rats (180-220 g) were orally given piperine at various doses ranging from 5, 10 and 20mg/kg BW at a period of 2 weeks before and 1 week after the intracerebroventricular administration of ethylcholine aziridinium ion (AF64A) bilaterally. The results showed that piperine at all dosage range used in this study significantly improved memory impairment and neurodegeneration in hippocampus. The possible underlying mechanisms might be partly associated with the decrease lipid peroxidation and acetylcholinesterase enzyme. Moreover, piperine also demonstrated the neurotrophic effect in hippocampus. However, further researches about the precise underlying mechanism are still required.”

Bacopa monnieri

The 2010 publication Cognitive enhancement and neuroprotective effects of Bacopa monnieri in Alzheimer’s disease model reports: “ETHNOPHARMACOLOGICAL RELEVANCE: Bacopa monnieri (L.) Wettst., a plant belonging to the family Scrophulariaceae, has been used in the traditional system of Ayurvedic medicine to improve intelligence and memory for a long time. Therefore, the potential of this plant to protect against Alzheimer’s disease has been raised but less supported document is available.  AIM OF THE STUDY: To determine the effect of alcoholic extract of Bacopa monnieri on cognitive function and neurodegeneration in animal model of Alzheimer’s disease induced by ethylcholine aziridinium ion (AF64A).  MATERIALS AND METHODS: Male Wistar rats were orally given the alcoholic extract of Bacopa monnieri at doses of 20, 40 and 80 mg/kg BW via feeding needle for a period of 2 weeks before and 1 week after the intracerebroventricular administration of AF64A bilaterally. Rats were tested for spatial memory using Morris water maze test and the density of neurons and cholinergic neurons was determined using histological techniques 7 days after AF64A administration.  RESULTS: Bacopa monnieri extract improved the escape latency time (p<.01) in Morris water maze test. Moreover, the reduction of neurons and cholinergic neuron densities were also mitigated.  CONCLUSION: These findings suggest that Bacopa monnieri is a potential cognitive enhancer and neuroprotectant against Alzheimer’s disease.”

Purple rice berry

The July 2011 publication Purple rice berry is neuroprotective and enhances cognition in a rat model of Alzheimer’s disease reports: “Alzheimer’s disease, a neurodegenerative disease characterized by progressive memory loss and cognitive impairment, is the most common type of dementia in aging populations due to severe loss of cholinergic neurons in a specific area. Oxidative stress is known to be involved in the pathogenesis of this condition. Therefore, the cognition-enhancing and neuroprotective effects of rice berry (Oryza sativa), a purple-pigmented rice that is rich in antioxidant substances, was evaluated. Young adult male Wistar rats, weighing 180-220 g, were orally given rice berry once daily at doses of 180, 360, and 720 mg/kg of body weight for a period of 2 weeks before and 1 week after the induction of memory deficit and cholinergic lesions with AF64A, a specific cholinotoxin, via bilateral intracerebroventricular administration. One week following AF64A administration the rats were evaluated for spatial memory, neuron density, acetylcholinesterase activity, and hippocampal lipid peroxidation products. Our results showed that rice berry could significantly prevent memory impairment and hippocampal neurodegeneration in hippocampus. Moreover, it also decreased hippocampal acetylcholinesterase activity and lipid peroxidation product formation. These results suggest that rice berry has potential as an effective agent for neurodegeneration and memory impairment in Alzheimer’s disease.”

Oroxylin A

Going back to 2008 we can find the publication The effects of acute and repeated oroxylin A treatments on Abeta(25-35)-induced memory impairment in mice. “Oroxylin A is a flavonoid that is found in the roots of Scutellaria baicalensis Georgi. The aim of this study was to characterize the effects of oroxylin A on the memory impairments and pathological changes induced by Abeta(25-35) peptide in mice. The ameliorating effect of oroxylin A on memory impairment was investigated using passive avoidance and Y-maze tasks and pathological changes were identified by immunostaining and western blotting. Abeta(25-35) peptide (5nmol) was administered by intracerebroventricular injection. In the acute treatment study, a single dose of oroxylin A (5mg/kg, p.o.) treated 1h before behavioral tests was found to significantly reverse Abeta(25-35)-induced cognitive impairments based on passive avoidance and Y-maze task findings (P<0.05). Moreover, these acute effects of oroxylin A were blocked by diazepam (1mg/kg, i.p.), a GABA(A)/benzodiazepine binding site agonist (P<0.05). On the other hand, our subchronic studies revealed that oroxylin A (1 or 5mg/kg/day, p.o.) for 7 days ameliorated the memory impairment induced by Abeta(25-35) peptide. Moreover, Abeta(25-35)-induced increases in GFAP (an astroglia marker) and OX-42 (a microglia marker), and increases in iNOS positive cells in the hippocampus were found to be attenuated by subchronic oroxylin A (1 or 5mg/kg/day, i.p., P<0.05). In addition, reductions in the immunoreactivity and protein level of ChAT (a cholinergic neuronal cell marker) in the CA3 hippocampal area induced by Abeta(25-35) peptide were also attenuated by oroxylin A. Furthermore, lipid peroxidation induced by Abeta(25-35) was also reduced by oroxylin A. These results suggest that the amelioration of Abeta(25-35) peptide-induced memory impairment by oroxylin A is mediated via the GABAergic neurotransmitter system after a single administration, or by reductions in Abeta(25-35) peptide-induced astrocyte and microglia activations, iNOS expression, lipid peroxidation, and increased cholinergic neurotransmission after subchronic administration.”

Silymarin

The November 2010 publication Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer’s disease mouse model reports: “Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.”

Silibinin

“Silibinin (INN), also known as silybin, is the major active constituent of silymarin, standardized extract of the milk thistle seeds, containing mixture of flavonolignans consisting of among others of silibinin, isosilibinin, silicristin and silidianin. Silibinin itself is mixture of two diastereomers Silibinin A and Silybinin B in approximately equimolar ratio(ref).” The 2008 publication Silibinin prevents amyloid beta peptide-induced memory impairment and oxidative stress in micerelates: “BACKGROUND AND PURPOSE: Accumulated evidence suggests that oxidativestress is involved in amyloid beta (Abeta)-induced cognitive dysfunction. Silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), has been shown to have antioxidative properties; however, it remains unclear whether silibinin improves Abeta-induced neurotoxicity. In the present study, we examined the effect of silibinin on the memory impairment and accumulation of oxidativestress induced by Abeta(25-35) in mice.  EXPERIMENTAL APPROACH:  Aggregated Abeta(25-35) (3 nmol) was intracerebroventricularly administered to mice. Treatment with silibinin (2, 20 and 200 mg.kg(-1), once a day, p.o.) was started immediately after the injection of Abeta(25-35). Locomotor activity was evaluated 6 days after the Abeta(25-35) treatment, and cognitive function was evaluated in a Y-maze and novel object recognition tests 6-11 days after the Abeta(25-35) treatment. The levels of lipid peroxidation (malondialdehyde) and antioxidant (glutathione) in the hippocampus were measured 7 days after the Abeta(25-35) injection.  KEY RESULTS: Silibinin prevented the memory impairment induced by Abeta(25-35) in the Y-maze and novel object recognition tests. Repeated treatment with silibinin attenuated the Abeta(25-35)-induced accumulation of malondialdehyde and depletion of glutathione in the hippocampus.  CONCLUSIONS AND IMPLICATIONS: Silibinin prevents memory impairment and oxidative damage induced by Abeta(25-35) and may be a potential therapeutic agent for Alzheimer‘s disease.”  It could of course be the case that the actions of silymarin against AD are mainly due to its silibinin component.

Resveratrol

When I compiled my list of potentially relevant publications in early January prior to generating these blog entries, I expected to find resveratrol on the list of phytosubstances shown to be active against AD. But surprisingly there were no citations to that effect in the long list I had compiled.  Sure enough though, a bit of last-minute searching turned up two very-recent publications indicating that my hunch was correct.  The December 2011 publicationResveratrol Protects Rats from Aβ-induced Neurotoxicity by the Reduction of iNOS Expression and Lipid Peroxidation reports: “Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β-amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.”

A report dated January 2012 Resveratrol, a neuroprotective supplement for Alzheimer’sdisease relates: “The polyphenolic compound resveratrol (3,4′,5-trihydroxystilbene) is a naturally occurring phytochemical which has been found in more than 70 plant species, including herbs and human food products such as grapes, berries, and peanuts. Resveratrol was first isolated in 1940; however, little attention was paid to it until its benefits in coronary heart disease were studied in 1992. Since then, increasing evidence has indicated that resveratrol may be useful in treating cardiovascular diseases, cancers, pain, inflammation, tissue injury, and in reducing the risk of neurodegenerative disorders, especially Alzheimer’sdisease (AD). AD is characterized by a progressive dementia, and is one of the most common neurodegenerative disorders in the elderly. It has been reported that resveratrol exhibits neuroprotective benefits in animal models of AD. Resveratrol promotes the non-amyloidogenic cleavage of the amyloid precursor protein, enhances clearance of amyloid beta-peptides, and reduces neuronal damage. Despite the effort spent trying to understand the mechanisms by which resveratrol functions, the research work in this field is still incomplete. Many concerns such as bioavailability, biotransformation, synergism with other dietary factors, and risks inherent to its possible pro-oxidant activities still need to be addressed. This review summarizes and discusses the neuroprotective effects of resveratrol on AD, and their potential mechanisms.”

A combination of dietary supplements in the anti-aging firewall ameliorates the symptoms and neuropathy of Alzheimer’s disease in a transgenic mouse model.

The November 2010 publication  Formulation of a medical food cocktail for Alzheimer’s disease: beneficial effects on cognition and neuropathology in a mouse model of the disease reports: ”BACKGROUND: Dietary supplements have been extensively studied for their beneficial effects on cognition and AD neuropathology. The current study examines the effect of a medical food cocktail consisting of the dietary supplements curcumin, piperine, epigallocatechin gallate, α-lipoic acid, N-acetylcysteine, B vitamins, vitamin C, and folate on cognitive functioning and the AD hallmark features and amyloid-beta (Aβ) in the Tg2576 mouse model of the disease.  PRINCIPAL FINDINGS: The study found that administering the medical food cocktail for 6 months improved cortical- and hippocampal- dependent learning in the transgenic mice, rendering their performance indistinguishable from non-transgenic controls. Coinciding with this improvement in learning and memory, we found that treatment resulted in decreased soluble Aβ, including Aβ oligomers, previously found to be linked to cognitive functioning.  CONCLUSION: In conclusion, the current study demonstrates that combination diet consisting of natural dietary supplements improves cognitive functioning while decreasing AD neuropathology and may thus represent a safe, natural treatment for AD.”

In my treatise ANTI-AGING FIREWALLS  - THE SCIENCE AND TECHNOLOGY OF LONGEVITY, I suggest a number of supplemental substances likely to mitigate against aging or key phenomena of aging.  It is interesting that all of the mentioned substances in the “medical food cocktail” are in my combined anti-aging supplement firewall.  The big question of course is whether the cocktail works to halt or neutralize human Alzheimer’s disease.  If so, that would be very big news.   The study report has to say “The current study provides evidence that a combination diet of dietary supplements, individually known to be beneficial, can not only improve cognitive functioning in a transgenic mouse models of AD but also decreases Aβ levels and oligomerization. As yet there is an unmet need for effective treatments and preventative strategies for AD, and the fastest route to human patients involves the use of either existing medications, or the formulation of known safe remedies. Given that human AD is far more complex than we can effectively model in mice, which develop AD related pathology and cognitive decline but lack extensive neuronal loss, we must formulate treatments that attack not just the symptoms seen in these mice, but also those which we predict will show benefits downstream of pathology that occur in humans. Our formulation here has been designed to alter APP processing through reductions in both Aβ production, as well as aggregation, but also to prevent downstream pathologies such as excessive oxidative damage and inflammation. It is our hope that such a strategy will slow disease progression in humans. — Our rationale is that the individual components of the medical food cocktail work synergistically to produce cognitive and pathological benefits, and together have larger effects than any single component alone. In order to take the step from formulation to human administration we have tested the medical food cocktail in a well-described transgenic mouse model of AD. Serving as a proof of principal we saw cognitive recovery, as well as reduction of Aβ. Our results here show that combination approaches to the treatment of AD are effective in mouse models of AD, and have high translation potential for the human disorder(ref).”

We don’t know if this particular combination of supplements would work in humans as it does in the TG mice.  Only a clinical trial would tell us convincingly, but who would spend the millions necessary to fund such a clinical trial?

Commentary

The field of Alzheimer’s disease research is very large and very messy with many dark corridors and corners.  Much of this research happens in pharma companies and is yet unpublished.  These blog entries cover only a fraction of the research that is going on.  On the other hand, Part 2 of this three-part blog series identified a half-dozen pharmacological agents shown in vitro or small-animal models to be capable of halting or reversing symptoms of dementia or Alzheimer’s disease.  Above in this blog entry, sixteen plant-derived substances are identified that can do the same.  These include several of the “usual suspect” supplement substances that act against cancers and have other positive qualities, like curcumin, green tea and resveratrol.  And without doubt there are many more.  And one dietary supplement combination of such substances appears to reverse the AD phenotype in a transgenic mouse model of AD.  Yet the conventional wisdom in clinical practice is that nothing can be done either to avert or treat AD. 

Something appears to be seriously wrong with this picture.  Given the costs to our society of AD of billions or trillions of dollars, why are there not massive efforts to take some of the substances known to be safe and efficacious with small animals, try them in larger more systematic controlled experiments, and rush the best of these through clinical trials?  Why are there not public education campaigns on the roles of exercise and diet in averting dementia?  Why is there such a persistent fog about what can be done about dementia despite all the research that is out there?

Perhaps the answer lies in part in the nature of our healthcare system.  In medical practice, substances are not regarded to be legitimate treatments unless they have gone through clinical trials and are certified by the FDA as being pharmaceuticals that are safe, efficacious and controlled. But what drug company would be willing to fund a clinical trial of a combination of off-the-shelf plant-derived substances?  One viewpoint is that they can’t make money that way. 

On the other hand the clinicaltrials.gov database on Alzheimer’s disease clinical trials does show 853 studies in various stages of completion.  And a few of these are concerned with testing phytosubstances, albeit in many cases proprietary combinations of these.  Perhaps there is hope.

The first blog entry in this series of three Dietary factors and dementia – Part 1: important recent research dealt with research on a variety of subtopics such as the value of the relationship of dementia to diabetes, and, generally when and how diet can make a difference.  This second blog entry Dietary factors and dementia – Part 2: possible interventions describes research on possible interventions that could delay, prevent or cure dementia or Alzheimer’s disease including fatty acids and following a Mediterranean diet.  I seek to focus on topics not covered in previous blog entries and save much discussion related specifically to phytosubstances for the third blog entry Dietary factors and dementia – Part 3: plant-derived substances that can make a difference. That post describes research during the last two years on how sixteen different plant-derived substances have been shown in-vitro and in transgenic mouse models to inhibit the formation of or enhance the clearance of beta amyloid pr to reverse other symptoms of Alzheimer’s disease.   It also describes how supplementation with a specific combination of such supplements has been shown to clear up symptoms of Alzheimer’s disease in a mouse model.

Exponents of the Mediterranean diet express confidence that adherence to it reduces risk of dementia as well as risk of several other age-related diseases. 

The March 2011 publiction Mediterranean diet in healthy lifestyle and prevention of stroke reports “Several studies demonstrated the beneficial and preventive role of Mediterranean diet in the occurrence of cardiovascular diseases, chronic neurodegenerative diseases and neoplasms, obesity and diabetes. In randomized intervention trials, Mediterranean diet improved endothelial function and significantly reduced waist circumference, plasma glucose, serum insulin and homeostasis model assessment score in metabolic syndrome. Several studies support favorable effects of Mediterranean diet on plasma lipid profile: reduction of total and plasma LDL cholesterol levels, plasma triglyceride levels, and apo-B and VLDL concentrations, and an increase in plasma HDL cholesterol levels. This effect is associated with increased plasma antioxidant capacity, improved endothelial function, reduced insulin resistance, and reduced incidence of the metabolic syndrome. The beneficial impact of fish consumption on the risk of cardiovascular diseases is the result of synergistic effects of nutrients in fish. Fish is considered an excellent source of protein with low saturated fat, nutritious trace elements, long-chain omega-3 polyunsaturated fatty acids (LCn3PUFAs), and vitamins D and B. Fish consumption may be inversely associated with ischemic stroke but not with hemorrhagic stroke because of the potential antiplatelet aggregation property of LCn3PUFAs. Total stroke risk reduction was statistically significant for fish intake once per week, while the risk of stroke was lowered by 31% in individuals who ate fish 5 times or more per week. In the elderly, moderate consumption of tuna/other fish, but not fried fish, was associated with lower prevalence of subclinical infarcts and white matter abnormalities on MRI examination. Dietary intake of omega-3 fatty acids in a moderate-to-high range does not appear to be associated with reduced plaque, but is negatively associated with carotid artery intima-media thickness. Greater adherence to Mediterranean diet is associated with significant reduction in overall mortality, mortality from cardiovascular diseases and stroke, incidence of or mortality from cancer, and incidence of Parkinson’s disease and Alzheimer‘s disease and mild cognitive impairment.”

Review publications may offer sound perspectives though they offer no new evidence.  An example is the August 2011 publication Mediterranean diet in predementia and dementia syndromeswhich relates: “There is a critical need to potentially individualize new strategies able to prevent and to slow down the progression of predementia and dementia syndromes. Only recently higher adherence to a Mediterranean-type diet was associated with decreased cognitive decline although the Mediterranean diet (MeDi) combines several foods, micro- and macronutrients already separately proposed as potential protective factors against dementia and predementia syndromes. In fact, elevated saturated fatty acids could have negative effects on age-related cognitive decline and mild cognitive impairment (MCI). Furthermore, at present, epidemiological evidence suggested a possible association among fish consumption, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA) (particularly, n-3 PUFA) and reduced risk of cognitive decline and dementia. Light to moderate alcohol use may be associated with a reduced risk of incident dementia and Alzheimer’s disease (AD), while for vascular dementia, cognitive decline, and predementia syndromes the current evidence is only suggestive of a protective effect. Finally, the limited epidemiological evidence available on fruit and vegetable consumption and cognition generally supported a protective role of these macronutrients against cognitive decline, dementia, and AD. Moreover, recent prospective studies provided evidence that higher adherence to a Mediterranean-type diet could be associated with slower cognitive decline, reduced risk of progression from MCI to AD, reduced risk of AD, and decreased all-causes mortality in AD patients. These findings suggested that adherence to the MeDi may affect not only the risk for AD, but also for predementia syndromes and their progression to overt dementia. Nonetheless, at present, no definitive dietary recommendations are possible. However, high levels of consumption of fats from fish, vegetable oils, non-starchy vegetables, low glycemic fruits, and diet low in foods with added sugars and with moderate wine intake should be encouraged. In fact, this dietary advice is in accordance with recommendations for lowering the risk of cardiovascular disease, obesity, diabetes, and hypertension and might open new ways for the prevention and managemnt of cognitive decline and dementia.”

The 2010 publication Mediterranean diet, inflammatory and metabolic biomarkers, and risk of Alzheimer’s disease reports: “We aimed to investigate the association between adherence to the Mediterranean diet (MeDi) and Alzheimer’s disease (AD) risk in a prospective study. Specifically, we analyzed reduced inflammation and improved metabolic profile as a potential medium through which the MeDi reduced the risk of AD. During a 4-year follow-up, 118 incident AD cases were identified among the 1219 non-demented elderly (age ≥ 65) subjects who provided dietary information and blood samples at baseline. We used high-sensitivity C-reactive protein (hsCRP) as an index of systemic inflammation, and fasting insulin and adiponectin as indexes of metabolic profile. We investigated whether there was a change in the association between MeDi and incident AD risk when the biomarkers were introduced into multivariable adjusted COX models. Better adherence to MeDi was associated with lower level of hsCRP (p =0.003), but not fasting insulin or adiponectin. Better adherence to MeDi was significantly associated with lower risk for AD: compared to those in the lowest tertile of MeDi, subjects in the highest tertile had a 34% less risk of developing AD (p-for-trend =0.04). Introduction of the hsCRP, fasting insulin, adiponectin, or combinations of them into the COX model did not change the magnitude of the association between MeDi and incident AD. Ultimately, the favorable association between better adherence to MeDi and lower risk of AD did not seem to be mediated by hsCRP, fasting insulin, or adiponectin. Other aspects of inflammatory and metabolic pathways not captured by these biomarkers, or non-inflammatory or non-metabolic pathways, may be relevant to the MeDi-AD association.”

Among elderly, adherence to a Mediterranean diet can lead to reduced incidences of cerebrovascular infarcts.

The February 2011 publication Mediterranean diet and magnetic resonance imaging-assessed cerebrovascular disease reports: “OBJECTIVE: Cerebrovascular disease is 1 of the possible mechanisms of the previously reported relationship between Mediterranean-type diet (MeDi) and Alzheimer’s disease (AD). We sought to investigate the association between MeDi and MRI infarcts.  METHODS: High-resolution structural MRI was collected on 707 elderly 65 years or older community residents of New York with available dietary assessments administered an average of 5.8 years (3.22 standard deviations [SDs]) before the MRI. Participants were divided into 3 groups of adherence to MeDi (low, middle, and high tertiles). We examined the association of increasing adherence to MeDi with presence of infarcts on MRI. Models were run without adjustment, adjusted for basic demographic and clinical factors, and adjusted for vascular risk factors.  RESULTS: A total of 222 participants had at least 1 infarct. In the unadjusted model, compared to the low adherence group, those in the moderate MeDi adherence group had a 22% reduced odds of having an infarct (odds ratio [OR], 0.78; 95% confidence interval [CI], 0.55-1.14), while participants in the highest MeDi adherence group had a 36% reduced odds (OR, 0.64; 95% CI, 0.42-0.97; p for trend = 0.04). In adjusted models, the association between MeDi adherence and MRI infarcts remained essentially unchanged. The association of high MeDi adherence with infarcts was comparable to that of hypertension (40% reduced probability), did not vary by infarct size or after excluding patients with dementia (n = 46) or clinical strokes (n = 86). There was no association between MeDi and white matter hyperintensities.  INTERPRETATION: Higher adherence to the MeDi is associated with reduced cerebrovascular disease burden.”

The September 2011 report The Mediterranean Diet is Not Related to Cognitive Change in a Large Prospective Investigation: The PATH Through Life Study is among the most negative with respect to the Mediterranean diet.  “OBJECTIVE: To determine whether the Mediterranean diet and other dietary variables are predictors of transition from healthy cognitive aging to mild cognitive impairment and cognitive decline.  DESIGN: Longitudinal.  PARTICIPANTS: We assessed 1528 individuals, aged 60-64 years, who were participating in a prospective epidemiological study of mental health and aging. We tested participants at two time points, 4 years apart, for mild cognitive impairment using either the International Consensus Criteria, impairment on the Clinical Dementia Rating scale (Clinical Dementia Rating: 0.5), or any of a suite of criteria sets (any mild cognitive disorder). We used logistic regression to assess the dietary predictors of conversion to clinical diagnoses and multiple regression to identify the predictors of cognitive decline (change in global cognition) in healthy participants.  RESULTS: Of the 1528 participants with no cognitive impairment in the first wave of assessment and complete data, 10 participants were diagnosed with mild cognitive impairment, 19 with Clinical Dementia Rating 0.5, and 37 participants presented with any mild cognitive disorder at follow-up. Adherence to Mediterranean diet was not found to be protective against cognitive decline but excessive caloric intake, and high intake of monounsaturated fats was predictive of mild cognitive impairment.  CONCLUSIONS: In this large longitudinal investigation of generally healthy individuals Mediterranean diet was not found to be protective of cognitive decline.”

Observing a Mediterranean diet can lead to higher plasma values of EPA but only in apoE-ɛ4 non-carriers.

The July 2011 report Adherence to a Mediterranean diet and plasma fatty acids: data from the Bordeaux sample of the Three-City study relates: “Higher adherence to a Mediterranean diet (MeDi) and n-3 PUFA may both contribute to decreased dementia risk, but the association between MeDi adherence and lipid status is unclear. The aim of the present study was to analyse the relationship between plasma fatty acids and MeDi adherence in French elderly community dwellers. The study population (mean age 75·9 years) consisted of 1050 subjects from Bordeaux (France) included in the Three-City cohort. Adherence to the MeDi (scored as 0-9) was computed from a FFQ and 24 h recall. The proportion of each plasma fatty acid was determined. Cross-sectional analysis of the association between plasma fatty acids and MeDi adherence was performed by multi-linear regression. After adjusting for age, sex, energy intake, physical activity, smoking status, BMI, plasma TAG and apoE-ɛ4 genotype, plasma palmitoleic acid was significantly inversely associated with MeDi adherence, whereas plasma DHA, the EPA+DHA index and total n-3 PUFA were positively associated with MeDi adherence. The n-6:n-3 PUFA, arachidonic acid (AA):EPA, AA:DHA and AA:(EPA+DHA) ratios were significantly inversely associated with MeDi adherence. Plasma EPA was positively associated with MeDi adherence only in apoE-ɛ4 non-carriers. There was no association between MeDi adherence and SFA and total MUFA. The present results suggest that the protective effect of the MeDi on cognitive functions might be mediated by higher plasma DHA and lower n-6:n-3 PUFA ratios.”

Several studies have been concerned with how dietary components work to affect dementia or Alzheimer’s disease, in particular polyunsaturated fatty acids (PUFAs)

The 2010 publication Nutraceutical properties of Mediterranean diet and cognitive decline: possible underlying mechanisms speculates on why a Mediterranean diet may exercise its effects.  “Recent prospective studies provided evidence that higher adherence to a Mediterranean-type diet could be associated with slower cognitive decline, reduced risk of progression from mild cognitive impairment to Alzheimer’s disease (AD), reduced risk of AD, and decreased mortality in AD patients. Furthermore, the Mediterranean diet (MeDi) combines several foods, micro- and macronutrients already separately proposed as potential protective factors against dementia and predementia syndromes. At present, epidemiological evidence suggests a possible association between fish consumption, monounsaturated fatty acids, and polyunsaturated fatty acids (PUFA) (particularly, n-3 PUFA), and reduced risk of cognitive decline and dementia. Light to moderate alcohol use may be associated with a reduced risk of incident dementia and AD, while for vascular dementia, cognitive decline, and predementia syndromes, the current evidence is only suggestive of a protective effect. Finally, the limited epidemiological evidence available on fruit and vegetable consumption and cognition generally support a protective role of these macronutrients against cognitive decline, dementia, and AD. We reviewed evidence on the possible mechanisms underlying the suggested protective role of MeDi against age-related changes in cognitive function, predementia syndromes, and dementia, examining the possible role of macronutrients and food nutrients of the MeDi and their nutraceutical properties in modulating the risk of cognitive decline. Although vascular variables are likely to be in the causal pathway between MeDi and dementia syndromes and should be considered as possible mediators, other nonvascular biological mechanisms (i.e., metabolic, oxidative, and inflammatory) may be invoked to explain the complex epidemiological association between MeDi and cognitive decline.”

The June 2011 report Omega-3 polyunsaturated fatty acids in the brain: metabolism and neuroprotectionexplains: “Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are a group of essential fatty acids that serve as energy substrates and integral membrane components, and therefore play crucial roles in the maintenance of normal neurological function. Recent studies show that n-3 PUFAs display neuroprotective properties and exert beneficial effects on the cognitive function with aging. The brain’s need of n-3 PUFAs is predominantly met by the blood delivery due to their limited synthesis in the brain. The present review focuses on the metabolism of n-3 PUFAs in the brain, including their accumulation and turnover. We also highlight the current understanding of the neuroprotective effects of n-3 PUFAs against cerebral ischemia and neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease.”

The 2011 publication Structural insight into the differential effects of omega-3 and omega-6 fatty acids on the production of Abeta peptides and amyloid plaquesreports: “Several studies have shown the protective effects of dietary enrichment of various lipids in several late-onset animal models of Alzheimer Disease (AD); however, none of the studies has determined which structure within a lipid determines its detrimental or beneficial effects on AD. High-sensitivity enzyme-linked immunosorbent assay (ELISA) shows that saturated fatty acids (SFAs), upstream omega-3 FAs, and arachidonic acid (AA) resulted in significantly higher secretion of both Aβ 40 and 42 peptides compared with long chain downstream omega-3 and monounsaturated FAs (MUFA). Their distinct detrimental action is believed to be due to a structural template found in their fatty acyl chains that lack SFAs, upstream omega-3 FAs, and AA. Immunoblotting experiments and use of APP-C99-transfected COS-7 cells suggest that FA-driven altered production of Aβ is mediated through γ-secretase cleavage of APP. An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein (APP); Swedish (K670N/M671L) and Indiana (V717F), corroborated in vitro findings by showing lower levels of Aβ and amyloid plaques in the brain, when they were fed a low fat diet enriched in DHA. Our work contributes to the clarification of aspects of structure-activity relationships.”

The September 2011 publication Effects of n-3 fatty acids, EPA v. DHA, on depressive symptoms, quality of life, memory and executive function in older adults with mild cognitive impairment: a 6-month randomised controlled trial relates: “Depressive symptoms may increase the risk of progressing from mild cognitive impairment (MCI) to dementia. Consumption of n-3 PUFA may alleviate both cognitive decline and depression. The aim of the present study was to investigate the benefits of supplementing a diet with n-3 PUFA, DHA and EPA, for depressive symptoms, quality of life (QOL) and cognition in elderly people with MCI. We conducted a 6-month double-blind, randomised controlled trial. A total of fifty people aged >65 years with MCI were allocated to receive a supplement rich in EPA (1·67 g EPA+0·16 g DHA/d; n 17), DHA (1·55 g DHA+0·40 g EPA/d; n 18) or the n-6 PUFA linoleic acid (LA; 2·2 g/d; n 15). Treatment allocation was by minimisation based on age, sex and depressive symptoms (Geriatric Depression Scale, GDS). Physiological and cognitive assessments, questionnaires and fatty acid composition of erythrocytes were obtained at baseline and 6 months (completers: n 40; EPA n 13, DHA n 16, LA n 11). Compared with the LA group, GDS scores improved in the EPA (P = 0·04) and DHA (P = 0·01) groups and verbal fluency (Initial Letter Fluency) in the DHA group (P = 0·04). Improved GDS scores were correlated with increased DHA plus EPA (r 0·39, P = 0·02). Improved self-reported physical health was associated with increased DHA. There were no treatment effects on other cognitive or QOL parameters. Increased intakes of DHA and EPA benefited mental health in older people with MCI. Increasing n-3 PUFA intakes may reduce depressive symptoms and the risk of progressing to dementia. This needs to be investigated in larger, depressed samples with MCI.”

Only non-carriers of the ε4 allele of the ApoE gene ApoE can be expected to benefit from the protective effects of fish oil/EPA supplements (long-chain n-3 PUFAs) against cognitive decline

The August 2011 publication Dietary omega 3 polyunsaturated fatty acids and Alzheimer‘s disease: interaction with apolipoprotein E genotype reports: “Epidemiological studies suggest a protective role of omega-3 poly-unsaturated fatty acids (n-3 PUFA) against Alzheimer’s disease (AD). However, most intervention studies of supplementation with n-3 PUFA have yielded disappointing results. One reason for such discordant results may result from inadequate targeting of individuals who might benefit from the supplementation, in particular because of their genetic susceptibility to AD. The ε4 allele of the apolipoprotein E gene (ApoE) is a genetic risk factor for late-onset AD. ApoE plays a key role in the transport of cholesterol and other lipids involved in brain composition and functioning. The action of n-3 PUFA on the aging brain might therefore differ according to ApoE polymorphism. The aim of this review is to examine the interaction between dietary fatty acids and ApoE genotype on the risk for AD. Carriers of the ε4 allele tend to be the most responsive to changes in dietary fat and cholesterol. Conversely, several epidemiological studies suggest a protective effect of long-chain n-3 PUFA on cognitive decline only in those who do not carry ε4 but with inconsistent results. An intervention study showed that only non-carriers had increased concentrations of long-chain n-3 PUFA in response to supplementation. The mechanisms underlying this gene-by-diet interaction on AD risk may involve impaired fatty acids and cholesterol transport, altered metabolism of n-3 PUFA, glucose or ketones, or modification of other risk factors of AD in ε4 carriers. Further research is needed to explain the differential effect of n-3 PUFA on AD according to ApoE genotype.”

DHA consumption may impact on various forms of cognitive impairment.  However, DHA supplementation does not slow the rate of progress of Alzheimer’s disease.

A Chapter from a book published in 2010 relates to  What Is the Link between Docosahexaenoic Acid, Cognitive Impairment, and Alzheimer’s Disease in the Elderly?  An excerpt is: “Cognitive impairment in the elderly, particularly in the form of Alzheimer’s disease (AD), has emerged in the past 20 years as a major challenge to the quality of life for the elderly and their caregivers, and to healthcare resources. AD is the most common form of dementia and the primary neurodegenerative disorder in the elderly. Once it is clinically diagnosed, there is little prospect of improving the prognosis of AD. Cognitive deficits can progress gradually over many decades before reaching the clinical threshold for the diagnosis of AD (Petersen et al., 2001; Jorm et al., 2007). As the population ages, the prevalence of cognitive impairment leading to dementia and AD is expected to increase. Most of the subjects with mild cognitive impairment will progress to AD at a rate of 10%–15% per year compared with healthy control subjects who convert at a rate of 1%–2% per year (Petersen et al., 2001; Solfrizzi et al., 2006). The cause of the progression of cognitive impairment to dementia and AD is not established. Genetic factors have been implicated and the apolipoprotein E ɛ4 allele is the genetic risk factor most associated with AD (Mahley et al., 2006). It is plausible that genetic factors, especially genes involved in lipid metabolism and transport, interact with environmental factors for lowering or increasing the risk of AD. Since aging is unavoidable and there is not yet a cure for AD, strategies to identify environmental factors lowering risk of AD are essential. Therefore, research on potentially modifiable risk factors for cognitive impairment, such as diet, is of great relevance. Several studies showed that cognitive impairment in the elderly is associated with deficiencies of micronutrients and macronutrients (Rosenberg and Miller, 1992; Grant, 1999; Dye et al., 2000; Gonzalez-Gross et al., 2001; Gillette Guyonnet et al., 2007). Among macronutrients, there is increasing interest in the possible impact of dietary fatty acids on cognitive impairment and dementia. One class of dietary fatty acids closely associated with the function of the brain is the ω3 polyunsaturated fatty acids (PUFA), particularly docosahexaenoic acid (DHA), which is a major component of the membrane phospholipids in the brain. Fish and seafood (shellfish and crustacean) consumption is the main dietary source of preformed DHA. Most epidemiological studies, but not all, suggest that fish and seafood consumption might protect the elderly from developing cognitive impairment or dementia including AD (reviewed in Gillette Guyonnet et al., 2007). Whether ω3 PUFA from fish and seafood, especially DHA, might be the principal contributors in preventing cognitive impairment and dementia in the elderly is presently debated. Previous reviews describing the relationship between ω3 PUFA and cognitive decline reported an inconclusive association (Maclean et al., 2005; Gillette Guyonnet et al., 2007; Plourde et al., 2007). Therefore, this chapter examines the possible link between fish and seafood or DHA intakes and cognitive impairment and dementia including AD with emphasis on three types of human studies—evaluation of epidemiological studies on fish and seafood or DHA intake, analysis of DHA levels in blood or brain tissues, and clinical trials of supplementation with DHA-enriched oils in cognitively impaired nondemented (CIND) elderly and AD patients. In view of the literature as it stands presently, we sought to answer the following questions: (1) Does the intake of fish and seafood protect against cognitive impairment and its progression to dementia such as AD in the elderly?, (2) What is the biological evidence from tissue fatty acid analyses that DHA plays a significant role in the protective effect of fish and seafood consumption?, and (3) is DHA alone effective in the treatment of cognitive impairment and AD?”

The November 2010 publication Docosahexaenoic acid supplementation and cognitive decline in Alzheimer disease: a randomized trialreports: “CONTEXT: Docosahexaenoic acid (DHA) is the most abundant long-chain polyunsaturated fatty acid in the brain. Epidemiological studies suggest that consumption of DHA is associated with a reduced incidence of Alzheimer disease. Animal studies demonstrate that oral intake of DHA reduces Alzheimer-like brain pathology.  OBJECTIVE: To determine if supplementation with DHA slows cognitive and functional decline in individuals with Alzheimer disease.  DESIGN, SETTING, AND PATIENTS: A randomized, double-blind, placebo-controlled trial of DHA supplementation in individuals with mild to moderate Alzheimer disease (Mini-Mental State Examination scores, 14-26) was conducted between November 2007 and May 2009 at 51 US clinical research sites of the Alzheimer’s Disease Cooperative Study.  INTERVENTION: Participants were randomly assigned to algal DHA at a dose of 2 g/d or to identical placebo (60% were assigned to DHA and 40% were assigned to placebo). Duration of treatment was 18 months.  MAIN OUTCOME MEASURES: Change in the cognitive subscale of the Alzheimer’s Disease Assessment Scale (ADAS-cog) and change in the Clinical Dementia Rating (CDR) sum of boxes. Rate of brain atrophy was also determined by volumetric magnetic resonance imaging in a subsample of participants (n = 102).  RESULTS: A total of 402 individuals were randomized and a total of 295 participants completed the trial while taking study medication (DHA: 171; placebo: 124). Supplementation with DHA had no beneficial effect on rate of change on ADAS-cog score, which increased by a mean of 7.98 points (95% confidence interval [CI], 6.51-9.45 points) for the DHA group during 18 months vs 8.27 points (95% CI, 6.72-9.82 points) for the placebo group (linear mixed-effects model: P = .41). The CDR sum of boxes score increased by 2.87 points (95% CI, 2.44-3.30 points) for the DHA group during 18 months compared with 2.93 points (95% CI, 2.44-3.42 points) for the placebo group (linear mixed-effects model: P = .68). In the subpopulation of participants (DHA: 53; placebo: 49), the rate of brain atrophy was not affected by treatment with DHA. Individuals in the DHA group had a mean decline in total brain volume of 24.7 cm(3) (95% CI, 21.4-28.0 cm(3)) during 18 months and a 1.32% (95% CI, 1.14%-1.50%) volume decline per year compared with 24.0 cm(3) (95% CI, 20-28 cm(3)) for the placebo group during 18 months and a 1.29% (95% CI, 1.07%-1.51%) volume decline per year (P = .79).  CONCLUSION: Supplementation with DHA compared with placebo did not slow the rate of cognitive and functional decline in patients with mild to moderate Alzheimer disease.”

Some review studies report negatively on associations of diet or supplement interventions with reduced risk of dementia or AD.

For example, the 2010 publication B-vitamins and fatty acids in the prevention and treatment of Alzheimer’s disease and dementia: a systematic review reports: “The increasing worldwide prevalence of dementia is a major public health concern. Findings from some epidemiological studies suggest that diet and nutrition may be important modifiable risk factors for development of dementia. In order to evaluate the strength of the available evidence of an association of dietary factors with dementia including Alzheimer’s disease (AD), we systematically searched relevant publication databases and hand-searched bibliographies up to end July 2007. We included prospective cohort studies which evaluated the association of nutrient levels with the risk of developing dementia and randomized intervention studies examining the treatment effect of nutrient supplementation on cognitive function. One hundred and sixty studies, comprising ninety one prospective cohort studies and sixty nine intervention studies, met the pre-specified inclusion criteria. Of these, thirty-three studies (19 cohort and 14 randomized controlled trials) investigated the effects of folate, B-vitamins, and levels of homocysteine (a biomarker modifiable through B-vitamin supplementation) or fish/fatty acids and are the focus of the present report. Some observational cohort studies indicated that higher dietary intake or elevated serum levels of folate and fish/fatty acids and low serum levels of homocysteine were associated with a reduced risk of incident AD and dementia, while other studies reported no association. The results of intervention studies examining the effects of folic acid or fatty acid supplementation on cognitive function are inconsistent. In summary, the available evidence is insufficient to draw definitive conclusions on the association of B vitamins and fatty acids with cognitive decline or dementia, and further long-term trials are required.”

This is the first of three blog entries focusing on research during the last two years relating diet, dietary substances and supplements to late-onset dementias including Alzheimer’s disease (AD), and to the potential roles of such substances for prevention or treatment of dementia.  This first blog entry deals with research on a variety of subtopics such as the value of the relationship of dementia to diabetes, the role of oxidative stress in AD and, generally when and how diet can make a difference.  The second blog entry Dietary factors and dementia – Part 2: possible interventions describes research on possible interventions that could delay, prevent or cure dementia or Alzheimer’s disease including ingesting fatty acids and following a Mediterranean diet.  I seek to focus on topics not covered in previous blog entries and save much discussion related specifically to phytosubstances for the third blog entry Dietary factors and dementia – Part 3: plant-derived substances that can make a difference.  That post describes research during the last two years on how thirteen different plant-derived substances have been shown in-vitro and in transgenic mouse models to inhibit the formation of or enhance the clearance of beta amyloid pr to reverse other symptoms of Alzheimer’s disease.   It also describes how supplementation with a specific combination of such supplements has been shown to clear up symptoms of Alzheimer’s disease in a mouse model.

Over the three year lifespan of this blog I I have written rather extensively on age-related cognitive decline, Alzheimer’s disease and dietary and supplement interventions.  For example, see the July 2011 blog entry Age-related cognitive decline: focus on interventions which also contains links to relevant earlier blog entries. 

Focus in these three blog entries is both on newer discoveries and on studies that cast new light on older theories and viewpoints.  In preparing this and the following blog entry, I have reviewed some 150 publications, mainly  dated 2011 and 2010 and looked at many others.  I found so much relevant material that I split my writeup up into three blog entries

Background on age-related loss of cognitive ability dementia

As background, age-related loss of cognitive ability and dementia tends to be characterized by a number of factors.  From the blog entry Age-related memory and brain functioning – focus on the hippocampus:

• There are several important changes in human brains that typically start in middle age and that accelerate with advancing age: hippocampus size decreases, BDNF expression decreases, there is significant shrinkage of gray matter; there is a decrease in neurogenesis and often but not always decrease in cognitive capability and loss of memory.
• Shrinking of the hippocampus, the prefrontal cortex, entorhinal cortex, and caudate nucleus in late adulthood are thought to contribute to the patterns of cognitive and memory decline often observed in older adults.
• Age-related loss of neurogenesis is thought to be a main factor leading to age-related decline. Neurogenesis in the brain is a tightly controlled lifelong process. It primarily takes place in neurogenic niches in parts of the hippocampus. New neurons migrate to their destination locations.
• Disorders in BDNF gene expression are implicated in many aberrant mental conditions and Alzheimer’s disease. BDNF expression decreases with age and age-related loss in BDNF expression is thought to lead to hippocampal shrinking with age.
• Maintaining neuronal and cognitive plasticity is important for averting age-related memory decline and cognitive aging.
• Cognitive and memory decline with age is not inevitable and can be influenced by many factors. Neurogenesis, BDNF expression and synaptic plasticity are highly dynamic processes in healthy individuals. They can be upregulated with physical and mental exercise, good lifestyle patterns, via good diet and via taking certain supplements including resveratrol, curcumin and omega3 fatty acids.
• Epigenetic regulation of brain aging is a new topic that I expect will attract significant attention as time progresses, revealing the behavior-driven gene-activation mechanisms that affect brain aging and the mechanisms that inhibit such aging.

From the blog entry Alzheimer’s Disease Update – March 2011:

The major mechanisms of AD pathology that have been studied intensely over the recent years are the intercellular accumulation of beta-amyloid protein and the intra-cellular buildup of tau tangles. As background, I briefly characterize both of these phenomena which characterize AD.“Amyloid beta (Aβ or Abeta or beta amyloid) is a peptide of 36–43 amino acids that appears to be the main constituent of amyloid plaques in the brains of Alzheimer’s disease patients. Similar plaques appear in some variants of Lewy body dementia and in inclusion body myositis, a muscle disease. Aβ also forms aggregates coating cerebral blood vessels in cerebral amyloid angiopathy(ref).” Generally, the amount of amyloid plaques in the brain is used as a measurement of the severity of AD.

Tau tangles [also known as Neurofibrillary tangles (NFTs)] are tangles of misfolded tau protein that occur in nerve cells in AD patients. Tau tangles are “aggregates of hyperphosphorylation tau that are most commonly known as a primary marker of Alzheimer’s Disease. Their presence is also found in numerous other diseases known as Tauopathies(ref).” Tau proteins play important roles in healthy nerve tissues. The normal function of tau is to support microtubules, physical scaffold structures within nerve cells. “Tau proteins are proteins that stabilize microtubules. They are abundant in neurons in the central nervous system and are less common elsewhere. When tau proteins are defective, and no longer stabilize microtubules properly, they can result in dementias, such as Alzheimer’s disease(ref).” The presence of amyloid beta is known to lead to tau tangles. “The pathologic hallmarks of Alzheimer’s disease (AD) include senile plaque, neurofibrillary tangles (NFTs), synaptic loss, and neurodegeneration. Senile plaque and NFTs are formed by accumulation of amyloid-β (Aβ) and hyperphosphorylated tau, respectively(ref).”

There is a general lack of agreement as to whether age-related human dementia can likely be delayed or prevented using available interventions

The picture may seem confusing because it is confusing.  A number of the publicationsI reviewed express contradictory opinions.  E.g.  “Greater adherence to Mediterranean diet is associated with significant reduction in overall mortality, mortality from cardiovascular diseases and stroke, incidence of or mortality from cancer, and incidence of Parkinson’s disease and Alzheimer’s disease and mild cognitive impairment (ref).” Also “Better adherence to MeDi was significantly associated with lower risk for AD: compared to those in the lowest tertile of MeDi, subjects in the highest tertile had a 34% less risk of developing AD (p-for-trend =0.04)(ref).”  In contrast: “In this large longitudinal investigation of generally healthy individuals Mediterranean diet was not found to be protective of cognitive decline(ref).”

To what extent can dietary factors postpone onset of age-related dementia or Alzheimer’s disease or slow, halt or reverse its progression?

Opinions tend to vary strongly according to background and focus of researchers.  Some look mainly to pharmaceutical interventions and in this domain so far there is a tremendous amount of research but indeed nothing in medical practice now that slows the progress of Alzheimer’s Disease once it is started.  See for example the blog entries Key roles of glia and microglia in age-related neurodegenerative diseases, Alzheimer’s Disease Update – March 2011,and New views of Alzheimer’s disease and new approaches to treating it.  So, many pharma-oriented researchers and physicians may be hopeful for therapies in the research pipeline but do not think that there is sufficient evidence that any known generally-available  interventions will make a significant difference.  Other scientists look to lifestyle factors such as exercise and cognitive and human engagement as capable of making a significant difference right now.  .  Some look to diet as a key factor that makes a tremendous difference.   See the blog entries Alzheimer’s disease studies validate anti-aging firewalls suggestions, and Age-related cognitive decline: focus on interventions.  In those later domains as previously reported, there does seem to be evidence from large population studies as well a molecular biology studies that the interventions can indeed act to delay or prevent the onset of dementias.  

In this triad of blog entries, I report on individual publications and the facts and opinions stated in them, and you as readers may come to your own conclusions.  Those of you who regularly read this blog know that I am of the opinion that lifestyle and dietary interventions can indeed drastically reduce the probability of contracting age-related dementia.  Not all researchers agree with me.  In my opinion, the research described in the following blog entry Part 3– plant-derived substances that can make a difference makes a compelling case that the probability of late-onset dementia and perhaps Alzheimer’s disease can be drastically reduced through supplementation with dietary phytosubstances.  And perhaps the progress of Alzheimer’s disease itself can be halted or reversed.  We don’t know this for sure because most AD research is looking in different directions.

I start here with a November2010 publication that lays out the landscape and offers a conservative view Is dementia preventable? Focus on Alzheimer’s disease “The prevention of dementia, and particularly of Alzheimer’s disease, is a major challenge for researchers and clinicians. In this article, the mixture of evidence, observations and hypotheses in the current literature is categorized into four avenues for possible preventive interventions, as suggested by the NIH State-of-the-Science Conference. The main categories are: antihypertensive medications; nutrition; cognitive engagement; and physical activity. There is, as yet, no conclusive evidence, but each category may hold promise for the prevention of dementia. The robust findings are as follows: cognitive engagement and regular physical activity may reduce the risk of Alzheimer’s disease; the Mediterranean diet and consumption of omega-3 fatty acids deserves further elucidation; and the meticulous management of risk factors, and especially hypertension, is the infrastructure of Alzheimer’s disease prevention.”

The May 2011 publication Diet and Alzheimer’s disease risk factors or prevention: the current evidencereports: “Preventing or postponing the onset of Alzheimer’s disease (AD) and delaying or slowing its progression would lead to a consequent improvement of health status and quality of life in older age. Elevated saturated fatty acids could have negative effects on age-related cognitive decline and mild cognitive impairment (MCI). Furthermore, at present, epidemiological evidence suggests a possible association between fish consumption, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA; in particular, n-3 PUFA) and a reduced risk of cognitive decline and dementia. Poorer cognitive function and an increased risk of vascular dementia (VaD) were found to be associated with a lower consumption of milk or dairy products. However, the consumption of whole-fat dairy products may be associated with cognitive decline in the elderly. Light-to-moderate alcohol use may be associated with a reduced risk of incident dementia and AD, while for VaD, cognitive decline and predementia syndromes, the current evidence is only suggestive of a protective effect. The limited epidemiological evidence available on fruit and vegetable consumption and cognition generally supports a protective role of these macronutrients against cognitive decline, dementia and AD. Only recently, higher adherence to a Mediterranean-type diet was associated with decreased cognitive decline, although the Mediterranean diet (MeDi) combines several foods, micro- and macro-nutrients already separately proposed as potential protective factors against dementia and predementia syndromes. In fact, recent prospective studies provided evidence that higher adherence to a Mediterranean-type diet could be associated with slower cognitive decline, reduced risk of progression from MCI to AD, reduced risk of AD and a decreased all-cause mortality in AD patients. These findings suggested that adherence to the MeDi may affect not only the risk of AD, but also of predementia syndromes and their progression to overt dementia. Based on the current evidence concerning these factors, no definitive dietary recommendations are possible. However, following dietary advice for lowering the risk of cardiovascular and metabolic disorders, high levels of consumption of fats from fish, vegetable oils, nonstarchy vegetables, low glycemic index fruits and a diet low in foods with added sugars and with moderate wine intake should be encouraged. Hopefully this will open new opportunities for the prevention and management of dementia and AD.”

Nutrients biomarkers identify important differences in cognitive functioning depending on nutrition.

The December 2011 publication Nutrient biomarker patterns, cognitive function, and MRI measures of brain aging reports: “OBJECTIVE: To examine the cross-sectional relationship between nutrient status and psychometric and imaging indices of brain health in dementia-free elders.  METHODS: Thirty plasma biomarkers of diet were assayed in the Oregon Brain Aging Study cohort (n = 104). Principal component analysis constructed nutrient biomarker patterns (NBPs) and regression models assessed the relationship of these with cognitive and MRI outcomes.  RESULTS: Mean age was 87 ± 10 years and 62% of subjects were female. Two NBPs associated with more favorable cognitive and MRI measures: one high in plasma vitamins B (B1, B2, B6, folate, and B12), C, D, and E, and another high in plasma marine ω-3 fatty acids. A third pattern characterized by high trans fat was associated with less favorable cognitive function and less total cerebral brain volume. Depression attenuated the relationship between the marine ω-3 pattern and white matter hyperintensity volume.  CONCLUSION: Distinct nutrient biomarker patterns detected in plasma are interpretable and account for a significant degree of variance in both cognitive function and brain volume. Objective and multivariate approaches to the study of nutrition in brain health warrant further study. These findings should be confirmed in a separate population.”

Diet throughout life can affect the chances of late-onset dementia.

The November 2010 publication Neurodevelopment and neurodegeneration: are there critical stages for nutritional intervention? reports: “Rather than being an inevitable consequence of age, cognitive decline can occur with marked variation among individuals. In this context, nutrition is one factor that is believed to be influential. When considering the potential role of diet, two factors need to be considered. First, cognitive or brain reserve is said to decrease the incidence of dementia; that is, it has been suggested that those with larger brains and better intellectual functioning have a greater capacity to resist the effects of the biological changes that define dementia. As such, the adequacy of nutrition before birth and in the early formative years may have long-term consequences. Second, shrinkage of the brain begins in young adulthood, suggesting that any insidious influence of diet will take place from that time onward over a period of many decades. The marked decline in the weight of the brain associated with advanced dementia suggests it will be easier to slow that decline than to repair the brain. If this model is accurate, diet is influential throughout the entire lifespan, and this has substantial methodological implications for the study of the topic.”

Nature of diet at midlife is likely to affect the probability of later onset of dementia or Alheimer’s disease

The January 2011 publication Midlife healthy-diet index and late-life dementia and Alzheimer’s disease reported: “AIM: To study long-term effects of dietary patterns on dementia and Alzheimer‘s disease (AD).  METHODS: Of 525 subjects randomly selected from population-based cohorts surveyed at midlife, a total of 385 (73%) subjects were re-examined 14 years later in the CAIDE study. A healthy-diet index (range 0-17) was constructed including both healthy and unhealthy dietary components.  RESULTS: Persons with a healthy diet (healthy-diet index >8 points) had a decreased risk of dementia (OR 0.12, 95% CI 0.02-0.85) and AD (OR 0.08, 95% CI 0.01-0.89) compared with persons with an unhealthy diet (0-8 points), adjusting for several possible confounders.  CONCLUSIONS: Healthy diet at midlife is associated with a decreased risk of dementia/AD in late life. These findings highlight the importance of dietary patterns and may make more effective measures for dementia/AD prevention or postponement possible.”

Key biomarker reactions in humans to a high-fat diet and a low-fat diet were different for healthy adults and for adults with amnestic mild cognitive impairment.

The June 2011 oublication Diet intervention and cerebrospinal fluid biomarkers in amnestic mild cognitive impairment reports: “OBJECTIVE: To compare the effects of a 4-week high-saturated fat/high-glycemic index (HIGH) diet with a low-saturated fat/low-glycemic index (LOW) diet on insulin and lipid metabolism, cerebrospinal fluid (CSF) markers of Alzheimer disease, and cognition for healthy adults and adults with amnestic mild cognitive impairment (aMCI).  DESIGN: Randomized controlled trial.  SETTING: Veterans Affairs Medical Center clinical research unit.  PARTICIPANTS: Forty-nine older adults (20 healthy adults with a mean [SD] age of 69.3 [7.4] years and 29 adults with aMCI with a mean [SD] age of 67.6 [6.8] years).  INTERVENTION: Participants received the HIGH diet (fat, 45% [saturated fat, > 25%]; carbohydrates, 35%-40% [glycemic index, > 70]; and protein, 15%-20%) or the LOW diet (fat, 25%; [saturated fat, < 7%]; carbohydrates, 55%-60% [glycemic index, < 55]; and protein, 15%-20%) for 4 weeks. Cognitive tests, an oral glucose tolerance test, and lumbar puncture were conducted at baseline and during the fourth week of the diet.  MAIN OUTCOME MEASURES: The CSF concentrations of β-amyloid (Aβ42 and Aβ40), tau protein, insulin, F2-isoprostanes, and apolipoprotein E, plasma lipids and insulin, and measures of cognition.  RESULTS: For the aMCI group, the LOW diet increased CSF Aβ42 concentrations, contrary to the pathologic pattern of lowered CSF Aβ42 typically observed in Alzheimer disease. The LOW diet had the opposite effect for healthy adults, ie, decreasing CSF Aβ42, whereas the HIGH diet increased CSF Aβ42. The CSF apolipoprotein E concentration was increased by the LOW diet and decreased by the HIGH diet for both groups. For the aMCI group, the CSF insulin concentration increased with the LOW diet, but the HIGH diet lowered the CSF insulin concentration for healthy adults. The HIGH diet increased and the LOW diet decreased plasma lipids, insulin, and CSF F2-isoprostane concentrations. Delayed visual memory improved for both groups after completion of 4 weeks of the LOW diet.  CONCLUSION: Our results suggest that diet may be a powerful environmental factor that modulates Alzheimer disease risk through its effects on central nervous system concentrations of Aβ42, lipoproteins, oxidative stress, and insulin.”

Some cases of neurodegenerative diseases may be due to earlier-life exposure to neurotoxins.

The October 2011 publication Is neurodegenerative disease a long-latency response to early-life genotoxin exposure?Reports: “Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, a disappearing neurodegenerative disease linked to use of the neurotoxic cycad plant for food and/or medicine, is intensively studied because the neuropathology (tauopathy) is similar to that of Alzheimer’sdisease. Cycads contain neurotoxic and genotoxic principles, notably cycasin and methylazoxymethanol, the latter sharing chemical relations with nitrosamines, which are derived from nitrates and nitrites in preserved meats and fertilizers, and also used in the rubber and leather industries. This review includes new data that influence understanding of the neurobiological actions of cycad and related genotoxins and the putative mechanisms by which they might trigger neurodegenerative disease.” 

I mention that the blog entry Nitrates and nitrites – Part 1: bad for youdescribes nitrosamines.  From that blog entry: ”The professionally-worded title of this 2009 publication conceals a strong underlying message: Epidemilogical trends strongly suggest exposures as etiologic agents in the pathogenesis of sporadic Alzheimer’s disease, diabetes mellitus, and non-alcoholic steatohepatitis: “Nitrosamines mediate their mutagenic effects by causing DNA damage, oxidative stress, lipid peroxidation, and pro-inflammatory cytokine activation, which lead to increased cellular degeneration and death. However, the very same pathophysiological processes comprise the “unbuilding” blocks of aging and insulin-resistance diseases including, neurodegeneration, diabetes mellitus (DM), and non-alcoholic steatohepatitis (NASH). Previous studies demonstrated that experimental exposure to streptozotocin, a nitrosamine-related compound, causes NASH, and diabetes mellitus Types 1, 2 and 3 (Alzheimer (AD)-type neurodegeneration). Herein, we review evidence that the upwardly spiraling trends in mortality rates due to DM, AD, and Parkinson’s disease typify exposure rather than genetic-based disease models, and parallel the progressive increases in human exposure to nitrates, nitrites, and nitrosamines via processed/preserved foods.”

Obesity as well as high-fat diets appear to be significant risk factors for development of both type-2 diabetes and dementia. Insulin resistance is a key mediating factor.

The 2011 publication Central insulin and insulin-like growth factor-1 signaling – implications for diabetes associated dementia reports: “Patients with type 2 diabetes (T2DM) have a two- to three-fold increased risk for Alzheimer‘s disease (AD), the most common form of dementia. Vascular complications might explain partially the increased incidence of neurodegeneration in patients with T2DM. Alternatively, neuronal resistance for insulin/insulin-like growth factor-1 (IGF- 1) might represent a molecular link between T2DM and AD, characterizing AD as “brain-type diabetes”. According to this hypothesis, brains from AD patients showed substantially downregulated expression of the Insulin receptor (IR), the IGF-1 receptor (IGF-1R), and the insulin receptor substrate (IRS) proteins. Similar changes in insulin/IGF-1 signaling (IIS) have been described in animals fed a high fat diet and human T2DM, suggesting that decreased IIS might be involved in the pathogenesis of both T2DM and AD.  In contrast, type 2 diabetic patients suffering from AD accumulate less β-amyloid (Aβ) compared to non-diabetic AD patients raising the question, whether the changes in IIS are cause, consequence, or compensatory counterregulation to neurodegeneration. Recent data in C. elegans showed that reducing IIS decreases Aβ toxicity. This effect is accomplished via two transcription factors downstream of IIS, DAF-16 and HSF- 1: The first detoxification path leads to degradation of the toxic misassemblies and is mediated via HSF-1. The second mechanism mediates the formation of low toxic, high molecular weight aggregates from highly toxic small molecular weight aggregates regulated by DAF-16 suggesting that Insulin/IGF-1 transmitted signals influence Aβ proteotoxicity. —.”

Chronic high-fat consumption is likely to lead to diabetes as well as dementia. 

The February 2011 publication High fat feeding promotes simultaneous decline in insulin sensitivity and cognitive performance in a delayed matching and non-matching to position task reports: “Obesity is the single greatest risk factor for the development of Type 2 diabetes mellitus (T2DM), with the prevalence of both dramatically increasing in recent years. These conditions are associated with medical complications such as hypertension, neuropathy and cardiovascular disease. Recent evidence also suggests a greater risk of developing dementia including Alzheimer’s disease. The molecular mechanisms governing these changes remain obscure, although epidemiological evidence suggests that reduced insulin sensitivity (a characteristic of T2DM) is an independent risk factor for Alzheimer’s disease. Here we examine the effects of diet-induced insulin resistance on cognitive ability in an animal model not predisposed to develop Alzheimer’s pathology. Following 12 weeks on a high fat diet (45% of calories as crude fat) male Wistar rats were overweight and insulin resistant but not frankly diabetic. High fat fed animals were consistently poorer in all aspects of an operant based delayed matching to position task, yet were not impaired in spatial working memory as judged by the open field watermaze test. The cognitive deficit of the HF fed animals was most apparent when the task was switched from matching to non-matching to position, suggestive of an inability to change contingency. Performance in this task was negatively correlated with whole body insulin sensitivity but not weight gain. In conclusion this study has shown that insulin resistant animals exhibit impairments in an operant measure of behavioural flexibility which precede the development of diabetes.”

Susceptibility to body weight gain induced by high fat diet and to the associated glucose intolerance and insulin resistance is increased by the presence of Alzheimer’s disease .

The August 2011 publication Susceptibility to diet-induced obesity and glucose intolerance in the APP (SWE)/PSEN1 (A246E) mouse model of Alzheimer‘s disease is associated with increased brain levels of protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4), and basal phosphorylation of S6 ribosomal protein reports: “AIMS/HYPOTHESIS: Obesity is a major risk factor for development of insulin resistance, a proximal cause of type 2 diabetes and is also associated with an increased relative risk of Alzheimer’s disease. We therefore investigated the susceptibility of transgenic mice carrying human mutated transgenes for amyloid precursor protein (APP (SWE)) and presenilin 1 (PSEN1 (A246E)) (APP/PSEN1), or PSEN1 (A246E) alone, which are well-characterised animal models of Alzheimer’s disease, to develop obesity, glucose intolerance and insulin resistance, and whether this was age- and/or diet-dependent.  METHODS: We analysed the effects of age and/or diet on body weight of wild-type, PSEN1 and APP/PSEN1 mice. We also analysed the effects of diet on glucose homeostasis and insulin signalling in these mice.  RESULTS: While there were no body weight differences between 16-17- and 20-21-month-old PSEN1 mice, APP/PSEN1 mice and their wild-type controls on standard, low-fat, chow diet, the APP/PSEN1 mice still exhibited impaired glucose homeostasis, as investigated by glucose tolerance tests. This was associated with increased brain protein tyrosine phosphatase 1B protein levels in APP/PSEN1 mice. Interestingly, short-term high-fat diet (HFD) feeding of wild-type, PSEN1 and APP/PSEN1 mice for a period of 8 weeks led to higher body weight gain in APP/PSEN1 than in PSEN1 mice and wild-type controls. In addition, HFD-feeding caused fasting hyperglycaemia and worsening of glucose maintenance in PSEN1 mice, the former being further exacerbated in APP/PSEN1 mice. The mechanism(s) behind this glucose intolerance in PSEN1 and APP/PSEN1 mice appeared to involve increased levels of brain retinol-binding protein 4 and basal phosphorylation of S6 ribosomal protein, and decreased insulin-stimulated phosphorylation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 in the brain.  CONCLUSIONS/INTERPRETATION: Our results indicate that Alzheimer’s disease increases susceptibility to body weight gain induced by HFD, and to the associated glucose intolerance and insulin resistance.”

B-vitamin deficiency may modulate the one-carbon metabolism pathway so as possibly to lead to Alzheimer’s disease.

I described the One-carbon metabolism.pathway in the February 2011 blog entryThe many faces of folic acidand in that blog entry I pointed to research suggesting that the epigenetics of one-carbon (folate) metabolism is likely implicated in Alzheimer’s disease. The 2010 publicationOne-carbon metabolism alteration affects brain proteome profile in a mouse model of Alzheimer’s disease relates: “Late Onset Alzheimer’s Disease (LOAD) can be associated to high homocysteine level and alteration of one-carbon metabolism. We previously demonstrated in the TgCRND8 mice strain, over-expressing human amyloid-β protein precursor, that B vitamin deficiency causes alteration of one-carbon metabolism, together with unbalance of S-adenosylmethionine/S-adenosylhomocysteine levels, and is associated with AD like hallmarks as increased amyloid-β plaque deposition, hyperhomocysteinemia, and oxidative stress. The same model of nutritional deficit was used here to study the variation of the brain protein expression profile associated to B vitamin deficiency. A group of proteins mainly involved in neuronal plasticity and mitochondrial functions was identified as modulated by one-carbon metabolism. These findings are consistent with increasing data about the pivotal role of mitochondrial abnormalities in AD patho-physiology. The identified proteins might represent new potential biomarkers of LOAD to be further investigated.”

Several reports view dietary interventions ineffective for preventing or delaying the progress of Alzheimer’s disease.

The September 2011 publication Risk factors and preventive interventions for Alzheimer disease: state of the sciencetakes a negative stance as to whether there are any interventions that will reliably work to avert or lessen the impact of AD.   “BACKGROUND: Numerous studies have investigated risk factors for Alzheimer disease (AD). However, at a recent National Institutes of Health State-of-the-Science Conference, an independent panel found insufficient evidence to support the association of any modifiable factor with risk of cognitive decline or AD.  OBJECTIVE: To present key findings for selected factors and AD risk that led the panel to their conclusion.  DATA SOURCES: An evidence report was commissioned by the Agency for Healthcare Research and Quality. It included English-language publications in MEDLINE and the Cochrane Database of Systematic Reviews from 1984 through October 27, 2009. Expert presentations and public discussions were considered. STUDY SELECTION: Study inclusion criteria for the evidence report were participants aged 50 years and older from general populations in developed countries; minimum sample sizes of 300 for cohort studies and 50 for randomized controlled trials; at least 2 years between exposure and outcome assessment; and use of well-accepted diagnostic criteria for AD.  DATA EXTRACTION: Included studies were evaluated for eligibility and data were abstracted. Quality of overall evidence for each factor was summarized as low, moderate, or high.  DATA SYNTHESIS: Diabetes mellitus, hyperlipidemia in midlife, and current tobacco use were associated with increased risk of AD, and Mediterranean-type diet, folic acid intake, low or moderate alcohol intake, cognitive activities, and physical activity were associated with decreased risk. The quality of evidence was low for all of these associations.  CONCLUSION: Currently, insufficient evidence exists to draw firm conclusions on the association of any modifiable factors with risk of AD.” 

Note that the authors of a great many of these studies indeed stated that such associations exist.  The “independent panel” based its finding on “low quality of evidence” which I surmise mainly to mean lack of controlled clinical trials.

Healthy diet does not appear to affect the progression of Alzheimer’s disease.

The October 2011 publication Health and Nutrition Promotion Program for Patients with Dementia (NutriAlz): Cluster Randomized Trial reported: “Objective: To assess the effectiveness of health and nutrition program (NutriAlz) versus usual care on functional level in elderly people with dementia living at home, as well as on clinical practice related to nutrition and on the caregiver’s burden. Design: Cluster randomized multi-centre study with one-year follow-up. Setting: 11 Alzheimer outpatients and day care centres (Barcelona, Spain). Participants: Nine hundred and forty six home-living Alzheimer patients with identified caregiver were consecutively recruited (intervention group: 6 centres, 448 patients vs control group: 5 centres, 498 patients). Intervention: The intervention was a teaching and training intervention on health and nutrition program, NutriAlz, directed both to physician and main caregiver, as well as persons affected by Alzheimer‘s disease or other dementias, including a standardised protocol for feeding and nutrition. Main Outcome Measures: The main outcome measure was the reduction in the loss of autonomy (Activities of daily living (ADL/IADL) scales) assessed at 6 and 12 months. Secondary outcomes measures were Improvement in nutritional status (Mini Nutritional Assessment (MNA), BMI, and weight changes), and caregiver burden (Zarit scale). Results: The one-year assessment was completed for 293 patients (65.4%) in the intervention group and 363 patients (72.9%) in the control group (usual care). The annual rate of ADL change was -0.83 vs -0.62 (p=0.984), and the caregiver’s subjective burden 0.59 vs 2.36 (p=0.681) in intervention and control group, respectively. MNA, however, showed an improvement (+0.46 vs -0.66, p=0.028), suggesting an effective nutritional behaviour. Conclusion: The NutriAlz program had no effect on functional decline in Alzheimer disease patients living at home over one year, but reduced the risk for malnutrition, as recommendations concerning diet and exercise were provided.”

Some researchers see modulation of oxidative stress to be important for controlling AD.

A paper focusing on the oxidative stress aspects of AD pathology is the July 2011 review publication Contribution of genetic and dietary insulin resistance to Alzheimer phenotype in APP/PS1 transgenic mice:  “In this review, we emphasize studies on the connection between oxidative stress and AD pathology, recent approaches to the prevention and treatment of AD. — Due to its elevated levels of peroxidizable fatty acids, high request for oxygen, and relative paucity of antioxidant systems, the brain is extremely sensitive to oxidative stress. Altered mitochondrial function, Aβ peptides, and the presence of trace metal ions such as iron and copper, have been identified as potential sources of oxidative stress (10–12). It is now understood that these three areas are not mutually exclusive. For example, Aβ may induce the production of ROS in the mitochondrial membrane causing subsequent oxidative damage in the early stages of disease progression. This has been shown in studies of AD patients as well as in transgenic mice overexpressing AβPP (11, 13–16). Suprisingly, redox-active transition metals collect in AD susceptible neurons (10) and, along with Aβ, can locally produce higher levels of ROS when around cytoplasmic H₂O₂ (17–19) leading to lipid and RNA oxidation (20). There are likely numerous mechanisms which cause oxidative stress to occur leading to dysfunctional neuronal responses in AD and the progression of the AD (21–23).”

Another publication, the August 2011 report Nutritional approaches to modulate oxidative stress in Alzheimer’s disease relates: “Alzheimer’s disease (AD) brain is characterized by amyloid β-peptide (Aβ) deposits, neurofibrillary tangles, synapse loss, and extensive oxidative stress. Aβ-induced oxidative stress is indexed by protein oxidation, lipid peroxidation, free radical formation, DNA oxidation and neuronal cell death. Oxidative stress is combated by antioxidants. Antioxidants and nutrition have long been considered as an approach to slow down AD progression. In this review, we focus on antioxidants that have been shown to protect against Aβ-induced oxidative stress, particularly vitamin E, ferulic acid, various polyphenols, including quercetin and resveratrol, α-lipoic acid, N-acetyl-L-cysteine (NAC), curcumin, epigallocatechin gallate (EGCG), and γ-glutamylcysteine ethyl ester (GCEE). Brain-accessible antioxidants with both radical scavenging properties and ability to induce protective genes are hypothesized to be helpful in treatment for AD.” 

Readers of this blog will note that I believe that several of the mentioned substances have important properties independent of their antioxidant potential, that oxidative processes are complex and intrinsic to metabolism, and that antioxidants by themselves are unlikely to provide a key to longevity.  See Victor’s blog entry End of the free radical theory of aging and negative consequences of indiscriminante antioxidant supplementation.

The 2010 publication Oxidative Stress and its Implications for Future Treatments and Management of Alzheimer Diseasereported: “Oxidative imbalance is one of the earliest manifestations of Alzheimer disease (AD) actually preceding the classic pathology of amyloid β deposits and neurofibrillary tangles. Clinical trials examining antioxidant modulation by a number of global interventions show efficacy, while simple supplementation has limited benefit suggesting complexity of multiple contributing factors. In this review, we highlight new insights regarding novel approaches to understanding and treating AD based on holistic views of oxidative balance including diet.”

Antioxidant supplementation does not appear to slow progression of Alzheimer’s disease in a mouse model

The 2011 publication Memory function in a mouse genetic model of Alzheimer’s diseasereports: “The E4 allele of the apolipoprotein E (ApoE) gene has been identified as a major risk factor for the development of late onset Alzheimer’s disease (AD). However, the mechanisms by which this gene affects AD are not fully understood. Studies of ApoE knock-out (ApoE KO) mice have revealed an exacerbation of two major pathologies that are diagnostic of AD: neurofibrillary tangles and senile plaques. However, evidence as to whether these mice have cognitive deficits is not yet conclusive. This ambiguity may arise partly from confounds associated with reliance on limited memory models, primarily, the Morris water maze task. An 8-arm radial maze task was therefore used to measure spatial memory in the ApoE KO mice, compared to controls over time. Furthermore, the effectiveness of a combination antioxidant therapy (CAT), designed to slow down the progression of AD based on concepts of oxidative stress and inflammatory processes underlying the pathology, was tested on memory ability. A significant strain difference was observed with the ApoE KO mice performing better than controls in terms of reference memory and corrects entries. No significant strain difference was observed for performance in terms of working memory errors. No significant effect of the CAT supplementation was observed.”

Several review studies have been recently published relating nutrition to cognitive aging and dementia.  Many review publications on the topic stick largely to generalizations.

For example the November 2011 publication Nutritional determinants of cognitive aging and dementia reports: “The objective of this review is to provide an overview of nutritional factors involved in cognitive aging and dementia with a focus on nutrients that are also important in neurocognitive development. Several dietary components were targeted, including antioxidant nutrients, dietary fats and B-vitamins. A critical review of the literature on each nutrient group is presented, beginning with laboratory and animal studies of the underlying biological mechanisms, followed by prospective epidemiological studies and randomised clinical trials. The evidence to date is fairly strong for protective associations of vitamin E from food sources, the n-3 fatty acid, DHA, found in fish, a high ratio of polyunsaturated to saturated fats, and vitamin B12 and folate. Attention to the level of nutrient intake is crucial for interpreting the literature and the inconsistencies across studies. Most of the epidemiological studies that observe associations have sufficient numbers of individuals who have both low and adequate nutrient status. Few of the randomised clinical trials are designed to target participants who have low baseline status before randomising to vitamin supplement treatments, and this may have resulted in negative findings. Post-hoc analyses by some of the trials reveal vitamin effects in individuals with low baseline intakes. The field of diet and dementia is a relatively young area of study. Much further work needs to be done to understand dietary determinants of cognitive aging and diseases. Further, these studies must be particularly focused on the levels of nutrient intake or status that confer optimum or suboptimal brain functioning.”

For older men, being overweight does not increase the chance of dementia. 

The March 2001 publication Body adiposity in later life and the incidence of dementia: the health in men study relates: “OBJECTIVE: To determine if adiposity in later life increases dementia hazard.  METHODS: Cohort study of 12,047 men aged 65-84 years living in Perth, Australia. Adiposity exposures were baseline body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR). We used the Western Australian Data Linkage System (WADLS) to establish the presence of new cases of dementia between 1996 and 2009 according to the International Classification of Diseases (ICD). Crude and adjusted hazard ratio (HR, 95% confidence interval, 95%CI) of dementia for each adiposity marker was calculated using Cox regression models. Other measured factors included age, marital status, education, alcohol use, smoking, diet, physical activity, and prevalent hypertension, diabetes, dyslipidaemia and cardiovascular disease.  RESULTS: Compared with men with BMI<25, participants with BMI between 25-30 had lower adjusted HR of dementia (HR = 0.82, 95% CI = 0.70-0.95). The HR of dementia for men with BMI ≥ 30 was comparable to men with BMI<25 (HR = 0.82, 95%CI = 0.67-1.01). Waist circumference showed no obvious association with dementia hazard. Men with WHR ≥ 0.9 had lower adjusted HR of dementia than men with WHR <0.9 (HR = 0.82, 95%CI  =  0.69-0.98). We found a “J” shape association between measures of obesity and the hazard of dementia, with the nadir of risk being in the overweight range of BMI and about 1 for WHR.  CONCLUSIONS: Higher adiposity is not associated with incident dementia in this Australian cohort of older men. Overweight men and those with WHR ≥ 0.9 have lower hazard of dementia than men with normal weight and with WHR<0.9.

The discussion goes on in the following blog entry Dietary factors and dementia – Part 2: possible interventions.

Salvia miltiorrhiza Bunge (Danshen root) is an important staple of traditional Chinese medicine that has long been used to treat a variety of illnesses including type-2 diabetes, cardiovascular and cerebrovascular diseases.  Strong research evidence such as that cited here indicates that the health-producing properties of this substance are based on the molecular biological activities of its component substances.  Some important properties of the remarkable substance are highlighted in the publications cited here. 

About Denshen root and its components – a traditional Chinese medicine and a possible new wonder substance

Danshen and its major chemical components have undergone significant modern research scrutiny in recent years using the powerful current tools of molecular biology, genomics, etc.  The Pubmed listingfor danshen or tanshinone or salvia miltiorrhiza bunge shows 1684 research publication.  Many of these are very recent.  Going through these as summarized here, it appears that the substance has amazing curative powers and that it may become the basis for anticancer therapies.  The research appears to be of the highest quality.  However, a matter that strikes me strongly is that all of the research authors appear to have Chinese or Korean names, whether they are in China itself, Taiwan, Korea or elsewhere in the world.  Unlike astragalus root and certain other traditional Chinese cures, the substance seems not ever to have come to the attention of non-Asian researchers.

Basics about Danshen

“Salvia miltiorrhiza (simplified Chinese: 丹参; traditional Chinese: 丹參; pinyin: dānshēn), also known as red sage, Chinese sage, tan shen, or danshen, is a perennial plant in the genus Salvia, highly valued for its roots in traditional Chinese medicine.^[2] Native to China and Japan, it grows at 90 to 1,200 m (300 to 3,900 ft) elevation, preferring grassy places in forests, hillsides, and along stream banks. The specific epithet miltiorrhiza means “red juice extracted from a root”.^{[3] “}The oldest documented record of danshen as a medical agent is found in Shen Nun Ben Cao (The Divine Husbandman’s Classic of the Materia Medica), dated at about 200 CE.⁴(ref).

One of the most important active phytochemical components of salviamiltiorrhiza bunge is tanshinone IIA (Tan IIA; 14,16-epoxy-20-nor-5(10),6,8,13,15-abietapentaene-11,12-dione).  Research described below indicates that tanshinone IIA is capable of anti-angiogenic, anti-oxidant, anti-inflammatory, anti-microbial and apoptotic activities.  Moreover, it appears to act strongly against a variety of cancer types.  The Denshen plant contains a number of other important phytochemicals “Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7).(Ref)” Regarding the health benefits of two of the mentioned substances, see the blog entries Rosmarinic acid and Phytochemicals – focus on caffeic acid.

Anti-microbial properties of Danshen

The March 2011 publication Diterpenoid tanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhizaBunge and their antimicrobial activitiesreports “ — tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7 –  were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.”

Danshen suppresses inflammatory cytokines and promotes the expression of anti-inflammatory cytokines.

The October 2011 publication Role of Salvia miltiorrhiza for Modulation of Th2-derived Cytokines in the Resolution of Inflammation reports: “Background: Salvia miltiorrhiza (SM) has been used to treat inflammatory diseases including edema and arthritis; however, the anti-inflammatory mechanism of SM action remains unresolved.  Methods: The effects of an ethanol extract of SM (ESM) on pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and NO, on anti-inflammatory cytokines including IL-4, IL-10, TGF-β, and IL-1Ra have been studied in an attempt to elucidate the anti-inflammatory mechanism in murine macrophages. Results: ESM inhibited the production of pro-inflammatory cytokines via down-regulation of gene and protein expression whereas it increased the anti-inflammatory cytokines. Furthermore, ESM inhibited the expression of the chemokines, RANTES and CX3CL1, as well as of inflammatory mediators such as TLR-4 and 11β-HSD1.  Conclusion: These results indicated that the regulatory effects of ESM may be mediated though the suppression of pro-inflammatory cytokines as well as the induction of anti-inflammatory cytokines. Consequently, we speculate that ESM has therapeutic potential for inflammation-associated disorders.”

Danhen is cytoprotective against oxidative stress

The December 2011 publication Extract of Salvia miltiorrhiza (Danshen) induces Nrf2-mediated heme oxygenase-1 expression as a cytoprotective action in RAW 264.7 macrophagesreports: Ethnopharmacological Relevance: Danshen (Salvia miltiorrhiza) is widely used in traditional herbal medicines for relief of a variety of symptoms related to complications arising from vascular diseases such as hypertension, diabetes, and atherosclerosis. Induction of heme oxygenase-1 (HO-1) expression protects against oxidative stress-induced cell damage, which plays an important role in cytoprotection in a variety of pathological models.  Aim: In the present study, we investigated the influence of Danshen on the up-regulation of HO-1, an inducible and cytoprotective enzyme in RAW 264.7 macrophages. Materials And Methods: Danshen induced HO-1 mRNA expression and protein production, and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophages. Pharmacological inhibitors of PI3K/Akt and MEK1 attenuated HO-1 induction in Danshen-stimulated RAW 264.7 macrophages. Furthermore, Danshen pretreatment reduced intracellular production of reactive oxygen species after stimulation with hydrogen peroxide; this effect was reversed by the HO-1 inhibitor ZnPP.  Conclusion: Danshen induced HO-1 expression through PI3K/Akt-MEK1-Nrf2 pathway and reduced intracellular production of reactive oxygen species via induction of HO-1 expression. The results support a role of HO-1 in the cytoprotective effect of Danshen.”

Mechanisms of action of danshen for prevention and treatment of coronary artery and heart disease are being discovered.

The June 2011 publicationCardiovascular actions and therapeutic potential of tanshinone IIA relates: “Tanshinone IIA (TS), a pharmacologically active component isolated from the rhizome of the Chinese herb Salvia miltiorrhizaBunge (Danshen), has been clinically used in Asian countries for the prevention and treatment of coronary heart disease. Recently, the pharmacological properties of TS in the cardiovascular system have attracted great interest. Emerging experimental studies and clinical trials have demonstrated that TS prevents atherogenesis as well as cardiac injury and hypertrophy. In atherosclerosis, TS acts by inhibiting LDL oxidation, monocyte adhesion to endothelium, smooth muscle cell migration and proliferation, macrophage cholesterol accumulation, proinflammatory cytokine expression and platelet aggregation. TS has some activity and potential to stabilize atherosclerotic plaques. The cardioprotective effects of TS are mainly related to its anti-oxidant and anti-inflammatory actions. In this review, we focus on the protective effects and the mechanism of action of TS in the cardiovascular system, and provide a novel perspective on clinical use of TS.”

The November 2011 document Tanshinone II-A attenuates and stabilizes atherosclerotic plaques in apolipoprotein-E knockout mice fed a high cholesterol diet reports: “Tanshinone II-A (Tan), a bioactive diterpene isolated from Salvia miltiorrhizaBunge (Danshen), possesses anti-oxidant and anti-inflammatory activities. The present study investigated whether Tan can decrease and stabilize atherosclerotic plaques in Apolipoprotein-E knockout (ApoE(-/-)) mice maintained on a high cholesterol diet (HCD). Six week-old mice challenged with a HCD were randomly assigned to 4 groups: (a) C57BL/6J; (b) ApoE(-/-); (c) ApoE(-/-)+Tan-30 (30 mg/kg/d); (d) ApoE(-/-)+Tan-10 (10mg/kg/d). After 16 weeks of intervention, Tan treated mice showed decreased atherosclerotic lesion size in the aortic sinus and en face aorta. Furthermore, immunohistochemical analysis revealed that Tan rendered the lesion composition a more stable phenotype as evidenced by reduced necrotic cores, decreased macrophage infiltration, and increased smooth muscle cell and collagen contents. Tan also significantly reduced in situ superoxide anion production, aortic expression of NF-κB and matrix metalloproteinase-9 (MMP-9). In vitro treatment of RAW264.7 macrophages with Tan significantly suppressed oxidized LDL-induced reactive oxygen species production, pro-inflammatory cytokine (IL-6, TNF-α, MCP-1) expression, and MMP-9 activity. Tan attenuates the development of atherosclerotic lesions and promotes plaque stability in ApoE(-/-) mice by reducing vascular oxidative stress and inflammatory response. Our findings highlight Tan as a potential therapeutic agent to prevent atherosclerotic cardiovascular diseases.”

The 2011 publication Tanshinone IIA inhibits angiotensin II-induced cell proliferation in rat cardiac fibroblasts reports: “Tanshinone IIA extracted from danshen, a popular medicinal herb used in traditional Chinese medicine, exhibits cardio-protective effects. However, the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II, cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation, and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effect of tanshinone IIA on nitric oxide (NO) production, and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation, and ERK phosphorylation. In addition, tanshinone IIA was found to increase the NO generation, and eNOS phosphorylation. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.”

The December 2010 publication Salvianolic acid B inhibits SDF-1α-stimulated cell proliferation and migration of vascular smooth muscle cells by suppressing CXCR4 receptorreports: “Salvianolic acid B (Sal B), a bioactive compound from Salvia miltiorrhiza, widely used to treat cardiovascular diseases, and stromal cell-derived factor-1α (SDF-1α)/CXCR4 pathway has been correlated with balloon angioplasty-induced neointimal formation. The purposes of the present study were to investigate whether Sal B can inhibit SDF-1α/CXCR4-mediated effects on the cell proliferation and migration of vascular smooth muscle cells (VSMCs) and to examine its possible molecular mechanisms. Under 0.5% FBS medium, all of the cellular studies were investigated on VSMCs (A10 cells) stimulated with 10ng/ml SDF-1α alone or co-treated with 0.075mg/ml Sal B. Our results showed that SDF-1α markedly stimulated the cell growth and migration of A10 cells, whose effects can be significantly reversed by co-incubation of Sal B. Similarly, Sal B also obviously down-regulated the SDF-1α-stimulated up-regulation of CXCR4 (total and cell-surface levels), Raf-1, MEK, ERK1/2, phospho-ERK1/2, FAK and phospho-FAK as well as an increase of the promoter activity of NF-κB. Besides, Sal B also effectively attenuated balloon angioplasty-induced neointimal hyperplasia. In conclusion, suppressing the expression levels of CXCR4 receptor and downstream molecules of SDF-1α/CXCR4 axis could possibly explain one of the pharmacological mechanisms of Sal B on prevention of cell proliferation, migration and subsequently neointimal hyperplasia.”

Mechanisms of action of danshen for prevention and treatment of diabetes are being discovered.

The November 2011 publication Antidiabetic Effect of the Total Polyphenolic Acids Fraction from Salvia miltiorrhiza Bunge in Diabetic Ratsreports: “An investigation was made to evaluate the therapeutic potential of the total polyphenolic acids fraction (PAF) from Salvia miltiorrhiza Bunge in the type 2 diabetes mellitus rats model with an oral dose of 187 mg/kg for 28 days. The results showed that PAF induced a significant decrease in fasting blood glucose (FBG), fasting blood insulin (FINS), total cholesterol (TC), triglyceride (TG) and blood urea nitrogen (BUN), and an obvious increase in insulin sensitivity index (ISI) in diabetic rats induced by a high fat diet and a low dose of streptozocin (STZ). These results suggested that PAF has antidiabetic potential in vivo.”

Danshen may help prevent anaphylaxis reactions.

“Anaphylaxis is defined as “a serious allergic reaction that is rapid in onset and may cause death”.^[1] It typically results in a number of symptoms including an itchy rash, throat swelling, and low blood pressure. Common causes include insect bites, foods, and medications. — On a pathophysiologic level, anaphylaxis is due to the release of mediators from certain types of white blood cells triggered either by immunologic or non-immunologic mechanisms(ref).”  The 2010 publication Tanshinones isolated from the rhizome of Salvia miltiorrhiza inhibit passive cutaneous anaphylaxis reaction in mice reports: “Aim Of The Study: This study aimed to elucidate anti-allergic effects of the root of Salvia miltiorrhiza Bunge (SM, family Labiatae) and its main constituents, tanshinones, against passive cutaneous anaphylaxis (PCA) reaction. Materials And Methods: PCA reaction was induced by IgE-antigen complex (IAC) in ICR mice. Protein expression of IL-4 and TNF-α in rat basophilic leukemia (RBL)-2H3 cells was performed by enzyme-linked immunosorbent assay and NF-κB and c-jun (AP-1) activation assayed by immunoblot.  Results: Tanshinones inhibited the PCA reaction and reduced IL-4 and TNF-α production in mice as well as in IAC-stimulated RBL-2H3 cells. Tanshinones also inhibited NF-κB and AP-1 activation in RBL-2H3 cells stimulated with IAC. Among tested tanshinones, tanshinone I exhibited the most potent inhibition, followed by 15,16-dihydrotanshinone I, tanshinone IIA and cryptotanshinone. Conclusions: SM and tanshinones may ameliorate the PCA reaction by inhibiting the allergic cytokines IL-4 and TNF-α via NF-κB and AP-1 pathways.”

Tanshinone IIA induces apoptosis and inhibits the proliferation, migration, and invasion of a number of cancer cell types including hepatoma, osteosarcoma, leukemia, prostate cancer, gastric cancer, breast cancer, colon cancer, small-cell lung cancer and gliaoma.

This conclusion appears to be supported by a number of cell-level and mouse model studies all of which show rather similar results.

Gastric cancer

The February 2011 publication Tanshinone IIA induces growth inhibition and apoptosis in gastric cancer in vitro and in vivo reports “As a phytochemical derived from the roots of Salvia miltiorrhiza Bunge, Tanshinone IIA has been reported to possess anti-inflammatory and antioxidant activity. Studies in breast, colon, prostate and lung cancer indicate that Tanshinone IIA may exhibit a promising antitumor activity. However, systemic studies of the cytotoxic effects of Tanshinone IIA on gastric cancer have not been described. The present study offers a comprehensive evaluation of the antitumor effects of Tanshinone IIA in gastric cancer cells in vitro and in a mouse xenograft model. Cell viability and apoptosis in vitro were evaluated through the MTT assay and flow cytometry analysis. The results indicate that Tanshinone IIA can induce gastric cancer cell growth inhibition and apoptosis in a time- and concentration-dependent manner. Furthermore, we investigated the mechanism of the apoptotic effects induced by Tanshinone IIA. We found that Tanshinone IIA can not only cause cell cycle arrest in the G2/M phase, but also trigger the intrinsic apoptotic signaling pathway. The results suggest that Tanshinone IIA may serve as an effective adjunctive reagent in the treatment of gastric cancer.”

Osteosarcoma

The November 2011 publication Tanshinone IIA induces apoptosis and inhibits the proliferation, migration, and invasion of the osteosarcoma MG-63 cell line in vitroreports “Tanshinone IIA (Tan IIA) is an active ingredient extracted from the widely used Danshen root (Salvia miltiorrhiza Bunge), a traditional Chinese medicine. Recent studies have indicated that Tan IIA may play important roles in anticancer treatment. However, its effects on the most common primary malignant bone tumor, osteosarcoma (OS), are unknown.  – Here, we report that Tan IIA may be an efficacious anti-OS drug as it could induce cell apoptosis and inhibit proliferation, migration, and invasion in vitro. Furthermore, we detected possible molecular mechanisms for Tan IIA activity by examining the levels of Bcl-2, Bax expression, and caspase-3, caspase-8, and caspase-9 activities that regulate apoptosis, matrix metalloproteinase (MMP)-2, and MMP-9 involved in regulating migration and invasion. In this study, we find that Tan IIA inhibits proliferation and induces apoptosis in the human OS cell line MG-63 in a time-dependent and dose-dependent manner. In addition, Tan IIA displays inhibitory activity on OS cell migration and invasion. Mechanistic studies have shown that Tan IIA activity is mediated by caspase activation. Tan IIA was also shown to reduce antiapoptotic Bcl-2, MMP-2, and MMP-9 levels, whereas it increased proapoptotic Bax levels. These data suggest that Tan IIA may be a novel, efficient candidate agent for OS treatment.”

Leukemia

The September 2011 publication Cytotoxic effect and apoptotic mechanism of tanshinone A, a novel tanshinone derivative, on human erythroleukemic K562 cells reports “Tanshinone A is a novel derivative of phenanthrene-quinone extracted from Salvia miltiorrhizaBUNGE, a traditional herbal medicine. Cytotoxic effect of tanshinone A was observed in this study. Additionally its mechanism of promoting apoptosis was also investigated. MTT and SRB assays were applied to measure the effects of tanshinone A on the cell viability, the cell cycle distribution and cell apoptosis were measured by flow cytometry using PI staining and Annexin V/PI double staining method respectively. The changes of mitochondrial membrane potential were also detected by flow cytometry. Spectrophotometric method was used to detect the changes of caspase-3 activity. Western blotting assay was used to evaluate the expression of bcl-2, bax and c-Myc proteins. Results indicated that tanshinone A displayed a significant inhibitory effect on the growth of K562 cells in a dose- and time-dependent manner, and showed obvious minor damage to LO2 cells. Tanshinone A could arrest K562 cells in the G(0)/G(1) phase and induce apoptosis, decrease the mitochondrial transmembrane potential, decrease the expressions of bcl-2 and c-Myc proteins, increase the expression of bax protein and the activity of caspase-3. Accordingly, it was presumed that the apoptosis induction maybe through the endogenous pathway. Subsequently, tanshinone A could be a promising candidate in the development of a novel antitumor agent.”

Hepatoma

The January 2012 publication Tanshinone IIA activates calcium-dependent apoptosis signaling pathway in human hepatoma cells reports “Tanshinone IIA (Tan IIA), a natural product from herb Salvia miltiorrhizaBunge, has potential anti-tumor activity. The aim of this study was to pinpoint the molecular mechanisms underlying Tan IIA-induced cancer cell apoptosis. Human hepatoma BEL-7402 cells treated with Tan IIA underwent assessment with MTT assay for cell viability, 10-day culture for colony formation, flow cytometry and fluorescence microscopy for apoptosis and cell cycle analysis. Changes in intracellular [Ca(2+)] and mitochondrial membrane potential (∆ψ) reflected the calcium-dependent apoptosis pathway. RT-PCR was used to detect gene expression of Bad and metallothionein 1A (MT 1A). Cytotoxicity of Tan IIA was tested in human amniotic mesenchymal stem cells (HAMCs). Tan IIA exhibited dose-dependent and time-dependent anticancer effects on BEL-7402 cells through apoptosis and G(0)/G(1) arrest. Cells treated with Tan IIA increased their intracellular calcium, decreased their mitochondrial membrane potential and induced Bad and MT 1A mRNA expression. No adverse effects of Tan IIA were found in HAMCs. In conclusion, these results indicate that Tan IIA-induced cancer cell apoptosis acts via activation of calcium-dependent apoptosis signaling pathways and upregulation of MT 1A expression.”

A prior 2009 study report Tanshinone II-A inhibits invasion and metastasis of human hepatocellular carcinoma cells in vitro and in vivo reported “The aim of this study was to investigate the effects of tanshinone II-A on tumor invasion and metastasis in human hepatocellular carcinoma (HCC) and its possible mechanism of action. — Treatment with tanshinone II-A had inhibitory effects on the migration and invasion of HCC cells. Increasing doses resulted in enhanced inhibitory effects. At 0.5 mg/L, the inhibitory effect was noticeable. At 1 mg/L, the inhibitory rate was 53.15%. The inhibitory effect became stronger with time; among 24, 48, 72 and 96 hours of treatment, the most significant effects were observed at 72 hours. Tanshinone II-A also significantly inhibited in vivo metastasis of HepG2 cells. Tanshinone II-A inhibited in vitro and in vivo invasion and metastasis of HCC cells by reducing the expression of the metalloproteinases MMP2 and MMP9 and by blocking NF-kappa B activation. — Tanshinone II-A effectively inhibited invasion and metastasis of HCC cells in vitro and in vivo, partly by inhibiting the activity of MMP2 and MMP9, and partly via the NF-kappa B signal transduction pathway.”

Prostate cancer

The 2010 publication Tanshinone IIA induces mitochondria dependent apoptosis in prostate cancer cells in association with an inhibition of phosphoinositide 3-kinase/AKT pathway reports “Tanshinone IIA (Tan IIA; 14,16-epoxy-20-nor-5(10),6,8,13,15-abietapentaene-11,12-dione), a phytochemical derived from the roots of Salvia miltiorrhizaBUNGE, has been reported to posses anti-angiogenic, anti-oxidant, anti-inflammatory and apoptotic activities. However, the cancer growth inhibitory/cytocidal effects and molecular mechanisms in prostate cancer cells have not been well studied. In the present study, we demonstrate that Tan IIA significantly decreased the viable cell number of LNCaP (phosphate and tensin homolog (PTEN) mutant, high AKT, wild type p53) prostate cancer cells more sensitively than against the PC-3 (PTEN null, high AKT, p53 null) prostate cancer cells. Tan IIA significantly increased TdT-mediated dUTP nick-end labeling (TUNEL) positive index and sub-G1 DNA contents of treated cells, consistent with apoptosis. Tan IIA treatment led to cleavage activation of pro-caspases-9 and 3, but not pro-caspase-8, and cleavage of poly (ADP ribose) polymerase (PARP), a caspase-3 substrate. Additionally, Tan IIA treatment induced cytochrome c release from the mitochondria into the cytosol and reduced mitochondrial membrane potential and suppressed the expression of mitochondria protective Bcl-2 family protein Mcl-1(L). Tan IIA reduced the expression of phosphoinositide 3-kinase (PI3K) p85 subunit, and the phosphorylation of AKT and mammalian target of rapamycin (mTOR) in a concentration-dependent manner. Moreover, the combination of Tan IIA and LY294002, a specific PI3K inhibitor, enhanced PARP cleavage of LNCaP and PC-3, but not in MDA-MB-231 breast cancer cells which do not contain detectable active AKT. The findings suggest that Tan IIA-induced apoptosis involves mitochondria intrinsic caspase activation cascade and an inhibition of the PI3K/AKT survival pathway.”

The December 2011 publication Antiandrogenic, maspin induction and anti-prostate cancer activities of tanshinone IIA and its novel derivatives with modification in ring A reports” “Expression of metastatic suppressor maspin is lost in advanced prostate cancer. Clinically-relevant mutations in androgen receptor (AR) convert antiandrogens into AR agonists, promoting prostate tumor growth. We discovered tanshinone IIA (TS-IIA) is a potent antagonist of mutated ARs and induces maspin expression through AR. TS-IIA suppressed AR expression and induced apoptosis in LNCaP cells. Syntheses of TS-IIA derivatives (1-9) revealed the 4,4-dimethyl group at ring A is important for TS-IIA’s antiandrogenic and maspin induction activities.”

The October 2011 publication Activation of p53 Signaling and Inhibition of Androgen Receptor Mediate Tanshinone IIA Induced G1 Arrest in LNCaP Prostate Cancer Cells reports: “Our group previously reported that tanshinone IIA induced apoptosis via a mitochondria dependent pathway in LNCaP prostate cancer cells. In the present study, the roles of androgen receptor (AR) and p53 signaling pathways were investigated in tanshinone IIA-induced G1 arrest in LNCaP cells. Tanshinone IIA significantly inhibited the growth and proliferation of LNCaP cells by colony formation and BrdU incorporation assays, respectively. Tanshinone IIA induced cell cycle arrest at G1 phase and down-regulated cyclin D1, CDK2 and CDK4. Furthermore, tanshinone IIA activated the phosphorylation of p53 at Ser 15 residue and its downstream p21 and p27. Additionally, tanshinone IIA suppressed the expression of AR and prostate specific antigen (PSA). Conversely, silencing p53 using its specific siRNA reversed cyclin D1 expression inhibited by tanshinone IIA. However, knockdown of AR had no effect on the p53/p21/p27 signaling pathway activated by tanshinone IIA in LNCaP cells. In AR siRNA-transfected cells, tanshinone IIA did not cause cell cycle arrest and reduce cyclin D1, implying that AR is essential to induce G1 arrest by tanshinone IIA in LNCaP cells. Taken together, the findings suggest that tanshinone IIA induces G1 arrest via activation of p53 signaling and inhibition of AR in LNCaP cells.”

5,16-dihydrotanshinone I (DHTS), another component of Denshen, is effective in killing prostate cancer cells via a different mechanism than that employed by tanshinone IIA.  The 2011 document 15,16-Dihydrotanshinone I, a Compound of Salvia miltiorrhiza Bunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells reports: “5,16-dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhizaBunge (tanshen root) and was found to be the most effective compound of tanshen extracts against breast cancer cells in our previous studies. However, whether DHTS can induce apoptosis through an endoplasmic reticular (ER) stress pathway was examined herein. In this study, we found that DHTS significantly inhibited the proliferation of human prostate DU145 carcinoma cells and induced apoptosis. DHTS was able to induce ER stress as evidenced by the upregulation of glucose regulation protein 78 (GRP78/Bip) and CAAT/enhancer binding protein homologous protein/growth arrest- and DNA damage-inducible gene 153 (CHOP/GADD153), as well as increases in phosphorylated eukaryotic initiation factor 2α (eIF2α), c-jun N-terminal kinase (JNK), and X-box-binding protein 1 (XBP1) mRNA splicing forms. DHTS treatment also caused significant accumulation of polyubiquitinated proteins and hypoxia-inducible factor (HIF)-1α, indicating that DHTS might be a proteasome inhibitor that is known to induce ER stress or enhance apoptosis caused by the classic ER stress-dependent mechanism. Moreover, DHTS-induced apoptosis was reversed by salubrinal, an ER stress inhibitor. Results suggest that DHTS can induce apoptosis of prostate carcinoma cells via induction of ER stress and/or inhibition of proteasome activity, and may have therapeutic potential for prostate cancer patients.”

Breast cancer

The October 2008 publication Tanshinone I suppresses growth and invasion of human breast cancer cells, MDA-MB-231, through regulation of adhesion molecules reported: “The role of cell adhesion molecules has been studied extensively in the process of inflammation, and these molecules are critical components of carcinogenesis and cancer metastasis. This study investigated the effect of tanshinone I derived from the traditional herbal medicine, Salvia miltiorrhiza Bunge, on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells. Furthermore, this study investigated the effect of tanshinone I on cancer growth, invasion and angiogenesis on human breast cancer cells MDA-MB-231, both in vitro and in vivo. Tanshinone I dose dependently inhibited ICAM-1 and VCAM-1 expressions in human umbilical vein endothelial cells (HUVECs) that were stimulated with TNF-α for 6 h. Pretreatment with tanshinone I significantly reduced adhesion of either monocyte U937 or MDA-MB-231 cells to HUVECs. Interestingly, the inhibitory effect of tanshinone I on monocyte and cancer cell adhesion to HUVECs was mimicked by transfection with ICAM-1 and VCAM-1 small interfering RNA. In addition, tanshinone I effectively inhibited TNF-α-induced production of vascular endothelial growth factor (VEGF) and VEGF-mediated tube formation in HUVECs. Tanshinone I also inhibited TNF-α-induced VEGF production in MDA-MB-231 cells and migration of MDA-MB-231 cells through extracellular matrix. Additionally, reduction of tumor mass volume and decrease of metastasis incidents by tanshinone I were observed in vivo. In conclusion, this study provides a potential mechanism for the anticancer effect of tanshinone I on breast cancer cells, suggesting that tanshinone I may serve as an effective drug for the treatment of breast cancer.”

Lung cancer

The 2008 publication Anticancer effects of tanshinone I in human non-small cell lung cancer reported: “Tanshinones are the major bioactive compounds of Salvia miltiorrhiza Bunge (Danshen) roots, which are used in many therapeutic remedies in Chinese traditional medicine. We investigated the anticancer effects of tanshinones on the highly invasive human lung adenocarcinoma cell line, CL1-5. Tanshinone I significantly inhibited migration, invasion, and gelatinase activity in macrophage-conditioned medium-stimulated CL1-5 cells in vitro and also reduced the tumorigenesis and metastasis in CL1-5-bearing severe combined immunodeficient mice. Unlike tanshinone IIA, which induces cell apoptosis, tanshinone I did not have direct cytotoxicity. Real-time quantitative PCR, luciferase reporter assay, and electrophoretic mobility shift assay revealed that tanshinone I reduces the transcriptional activity of interleukin-8, the angiogenic factor involved in cancer metastasis, by attenuating the DNA-binding activity of activator protein-1 and nuclear factor-κB in conditioned medium-stimulated CL1-5 cells. Microarray and pathway analysis of tumor-related genes identified the differentially expressed genes responding to tanshinone I, which may be associated with the Ras-mitogen-activated protein kinase and Rac1 signaling pathways. These results suggest that tanshinone I exhibits anticancer effects both in vitro and in vivo and that these effects are mediated at least partly through the interleukin-8, Ras-mitogen-activated protein kinase, and Rac1 signaling pathways. Although tanshinone I has a remarkable anticancer action, its potential anticoagulant effect should be noted and evaluated.”

Glioma

The 2010 publication Tanshinone IIA inhibits constitutive STAT3 activation, suppresses proliferation, and induces apoptosis in rat C6 glioma cells reported: “Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Thus, STAT3 may be a promising target for treatment of tumor cells. Recently, Tanshinone IIA (Tan IIA), a major active constituent from the root of Salvia miltiorrhiza Bunge, was reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, we evaluate the anti-proliferation and apoptosis inducing effects of Tan IIA on C6 glioma cells. Cell growth and proliferation were measured by MTT assay, cell apoptosis was observed by flow cytometry and DNA-fragmentation analysis. Further more, we investigated inhibitory effects of Tan IIA on STAT3 activity and its downstream targets: Bcl-XL, cyclin D1. Alteration of STAT3 activity was examined by measuring their DNA binding activity and tyrosine phosphorylation. Changes in the expression levels of Bcl-XL and cyclin D1 were examined by Western blot analysis. We found that the cellular growth were inhibited and cell apoptosis were observed after the treatment with Tan IIA. The STAT3 activity was significantly reduced by Tan IIA parallel with a significant attenuation of expression of Bcl-XL and cyclin D1. These results suggest that Tan IIA may serve as an effective adjunctive reagent in the treatment of glioma for its targeting of constitutive STAT3 signaling.”

Colon cancer

A 2009 publication Inhibitory effects of tanshinone II-A on invasion and metastasis of human colon carcinoma cells reports much the same story for colon cancer:  “Aim: To investigate the effects and possible mechanisms of tanshinone II-A, an alcohol extract of the root of Salvia miltiorrhizaBunge, on tumor invasion and metastasis of human colon carcinoma (CRC) cells.  Methods: The effects of tanshinone II-A on invasion and metastasis of CRC cell lines HT29 and SW480 were evaluated by in vitro and in vivo assays. Western blotting was used to investigate possible molecular mechanisms of tanshinone II-A anti-cancer actions. Results: Tanshinone II-A inhibited migration and invasion of CRC cells in a dose-dependent manner. The inhibitory effect also depended on time, with the most significant effects observed at 72 h. Tanshinone II-A also significantly inhibited in vivo metastasis of colon carcinoma SW480 cells. It inhibited in vitro and in vivo invasion and metastasis of CRC cells by reducing levels of urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMP)-2 and MMP-9, and by increasing levels of tissue inhibitor of matrix metalloproteinase protein (TIMP)-1 and TIMP-2. Tanshinone II-A was also shown to suppress the nuclear factor-kappaB (NF-kappaB) signal. Conclusion: Tanshinone II-A inhibited in vitro and in vivo invasion and metastasis of CRC cells. The effect resulted from changes in the levels of uPA, MMP-2, MMP-9, TIMP-1 and TIMP-2, and apparent inhibition of the NF-kappaB signal transduction pathway.”

Tanshinone II-A inhibits angiogenesis in cancers.

The November 2011 publication Anti-angiogenic effect of Tanshinone IIA involves inhibition of matrix invasion and modification of MMP-2/TIMP-2 secretion in vascular endothelial cells reports: “Tanshinone IIA (Tan IIA) is one of the major lipophilic components of Salvia miltiorrhizaBunge reported to exhibit anti-carcinogenic effect. In the present study, we further evaluated the anti-angiogenic effect of Tan IIA using the chorioallantoic membrane (CAM) in chicken embryos and human umbilical vascular endothelial cells (HUVECs). Tan IIA was confirmed to inhibit in vivo angiogenesis by CAM assay. Tan IIA also exhibited in vitro anti-angiogenic effects as demonstrated by tube formation assay, transwell migration assay and TNF-α-induced matrix invasion assay. The mRNA expressions of matrix metalloproteinase-2, -3, -9, -14 (MMP-2, -3, -9, -14), tissue inhibitor of metalloproteinase-2 (TIMP-2) and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were not affected by Tan IIA as analyzed by reverse transcription polymerase chain reaction (RT-PCR). However, the extracellular matrix metalloproteinase-2 (MMP-2) activity was found to be reduced dose-dependently by Tan IIA as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the dose-dependent decrease of MMP-2 and increase of TIMP-2 secretion from cytosol of vascular endothelial cells simultaneously after Tan IIA treatment. Together, the present study confirmed the anti-angiogenic effects of Tan IIA both in vivo and in vitro. Our results also demonstrated that Tan IIA could modulate the secretion of MMP-2 and TIMP-2 in an opposite way and resulted in the decreased MMP-2 activity of vascular endothelial cells.”

Cryptotanshinone, another component of danshen, is a potent stimulator of ER stress, leading to apoptosis in many cancer cell lines.

The November 2011 publication Cryptotanshinone induces ER stress-mediated apoptosis in HepG2 and MCF7 cellsreports: “The endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that functions in protein synthesis and maturation, and also functions as a calcium storage organelle. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress can activate apoptotic pathways in damaged cells. For this reason, pharmacological interventions that effectively enhance tumor death through ER stress have been the subject of a great deal of attention for anti-cancer therapy. Cryptotanshinone, the major active constituent isolated from the root of Salvia miltiorrhiza Bunge, has been recently evaluated for its anti-cancer activity, but the molecular mechanisms underlying these activities remain poorly understood. In particular, it remains completely unknown as to whether or not cryptotanshinone can induce ER stress. Herein, we identify cryptotanshinone as a potent stimulator of ER stress, leading to apoptosis in many cancer cell lines, including HepG2 hepatoma and MCF7 breast carcinoma, and also demonstrate that mitogen-activated protein kinases function as mediators in this process. Reactive oxygen species generated by cryptotanshinone have been shown to play a critical role in ER stress-induced apoptosis. Cryptotanshinone also evidenced sensitizing effects to a broad range of anti-cancer agents including Fas/Apo-1, TNF-α, cisplatin, etoposide or 5-FU through inducing ER stress, highlighting the therapeutic potential in the treatment of human hepatoma and breast cancer.”

Tanshinone IIA can possibly help prevent liver fibrosis

The 2010 publication Tanshinone II A induces apoptosis and S phase cell cycle arrest in activated rat hepatic stellate cells reports: “Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, improves blood circulation and treats chronic hepatitis and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is the predominant event in liver fibrosis. The therapeutic goal in liver fibrosis is the reversal of fibrosis and selective clearance of activated HSCs. We used rat HSCs transformed by Simian virus 40 (t-HSC/Cl-6) to overcome the limitations inherent in studying subcultures of HSCs. Treatment of t-HSC/Cl-6 cells with tanshinone IIA inhibited cell viability in a dose- and time-dependent manner. Tanshinone IIA induced apoptosis as demonstrated by DNA fragmentation, poly(ADP-ribose) polymerase and caspase-3 cleavage, increased Bax/Bcl-2 protein ratio, and depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol. Furthermore, this compound markedly induced S phase cell cycle arrest, and down-regulated cyclins A and E, and cdk2. Thus, tanshinone IIA induces apoptosis and S phase cell cycle arrest in rat HSCs in vitro.”

Danshen downregulates telomerase expression

The December 2011 publication  Ortho-Quinone tanshinones directly inhibit telomerase through an oxidative mechanism mediated by hydrogen peroxide reports: “The tanshinone natural products possess a variety of pharmacological properties including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic activity. The molecular basis of these effects, however, remains largely unknown. In the present study, we explored the direct effect of tanshinones on the enzyme telomerase. Telomerase is up-regulated in the majority of cancer cells and is essential for their survival, making it a potential anti-cancer drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger catalase protected telomerase from inactivation. These findings demonstrate that ortho-quinone containing tanshinones can inhibit telomerase owing to their ability to generate reactive oxygen species. The results also provide evidence that telomerase is directly and negatively regulated by reactive oxygen species.”

Danshen-based drugs have entered the clinical trials mill.

A search in clinicaltrials.gov reveals two clinical trials involving danshen, one completed.  An October 2010 “Herbal Egram” reported  “A patented Chinese herbal medicine has successfully completed Phase II clinical trials in the United States and will soon begin Phase III investigations, raising the possibility that it could become the first Traditional Chinese Medicine (TCM) product to obtain drug approval from the US Food and Drug Administration (FDA).^{1 —} The product, Compound Danshen Dripping Pill (also referred to as Cardiotonic Pill), is produced by Tianjin Tasly Pharmaceutical Co. Ltd. in Tianjin, China. It contains the extract of the root of Chinese salvia (Salvia miltiorrhiza; known as danshen in Chinese), the extract of the root of notoginseng (Panax notoginseng; known as sanchi or tien-chi ginseng), and synthetic borneol, an active ingredient that replaces the more expensive natural borneol found in cardamom (Elettaria cardamomum var. cardamomum), ginger (Zingiber officinale), and other spices.^1,2,3 According to Tasly’s website, the pill is sold as a prescription drug in China, Vietnam, Pakistan, South Korea, India, and the United Arab Emirates and reportedly is taken by about 10 million people every year to treat angina and coronary heart diseases.³ Last year, its international sales brought in a reported $148 million. “

A few comments

There is much more to the literature of danshen than I have been able to cite here.  Near as I know, salvia miltiorrhiza Bunge is not employed in Western medical practice.  Although Chinese and Korean researchers have been working to close the gap between Chinese traditional medical practice and what goes on in Western Medicine, there is still a long way to go.  The studies reported here are mostly in-vitro cell-level studies with limited in-vivo support that can be done by a small number of researchers with conventional laboratory tools like western blotting, microscopic, flow cytometry, colony formation and DNA-fragmentation analysis and a few mice or rats.  Though very promising, the studies still leave a large gap between cell-level knowledge and the anecdotal positive disease outcomes with humans observed in traditional Chinese Medicine practice.  Does danshen treatment slow down or stop cancers in mice?  Which ones?  How effectively? We don’t know for sure.  Still to be done are animal model disease outcome studies and then more clinical trials. 

Seriously, in going through hundreds of literature abstracts to create this blog entry, I have not come a single non-Chinese or non-Korean researcher-author name.  Perhaps it is time for a big Western pharma companies to pick this substance up and run with it.

History and where we are

I started my career as a longevity scientist some four years ago in January 2008 by writing and publishing a major online treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY.  My approach was to characterize the 14-odd major theories of aging and then ask for each theory “If this theory is correct, then what can be done now to slow or stop aging according to that theory?”  As 2008 proceeded I kept updating the treatise every week or so to reflect current developments.  But I soon came to realize that far too much was happening in research related to longevity to cram into a single treatise..  So, I decided to supplement the treatise with a blog.  The blog is now my major communication vehicle although I continue to update the treatise every few weeks.

In January 2009, I started this blog with the initial idea that it would be a medium to report and comment on science news related to longevity.  I thought this approach would lead to insights about what is known concerning human longevity and the possibility for longer healthier lives.  However, soon thereafter I largely abandoned this plan and started to evolve the blog into a medium for serious review articles covering key areas of science related to longevity.  This shift followed from realization that: 1.  Both the general press and responsible science-reporting media like Science Daily  do a good job on reporting on one-off new developments,  and 2.  Such simple reporting on new developments does not really yield the desired insights  and often can leave readers with incorrect perceptions.  This is because without already-knowing a great deal about the context of a new research development, say one related to induced pluripotent stem cells, the P53 protein or the SIR-1 pathway, it becomes nearly impossible to grasp the significance or absence of significance of the development. 

Even among responsible reporters of science in the general press, incremental developments are too-often reported as breakthroughs when they are actually just more bricks in a large rambling but still-mostly-incomplete edifice.  With very rare exceptions, important research discoveries are incremental.  To understand them requires understanding the context of prior work, so I soon recognized that it is import to provide my readers with contextual backgrounds.  See the blog entry When reading press releases and newspaper articles about research discoveries, beware!.  

In August 2009 I wrote the post What motivates me to write this blog?  More than two years later my motivations remain basically unchanged.

As time progressed, the blog entries have tended to become review articles with large numbers of citations, similar in many respects to major journal articles.  I have kept coming back to certain themes highly relevant to longevity including:

• The major theories of aging, what is being learned about them and how they can be linked up
• Stem cell developments, both stem cell therapies and progress with induced pluripotent stem cells and therapies based on adult stem cells.
• Key genes, proteins and biological pathways known to be related to longevity in animals, including BFNF, CETP, Klotho,P53, FOXO, NRG-1, IGF-1 mTOR, the Sirtuins, PGC1-alpha, Fas and P16(Ink4a)
• Epigenetic effects and their relationship to disease processes and aging
• Multiple foods, phytosubstances and dietary supplements having health and possible longevity benefits strongly supported by research.
• Theories of biological evolution that affect views on longevity, like antagonistic pleiotropy
• Key biological processes related to health and longevity such as oxidative damage, inflammation and tissue glycation
• Alternative theories of aging such as micronutrient triage, increasing mTOR signaling or decline in expression of Klotho.
• DNA damage and its repair
• Micro RNAs and their roles
• Cell senescence; its relationships to aging, cancers and telomeres.
• Roles of glia and microglia and their relationship to neurological diseases
• The immune system, immunotherapies, roles of B, T, NK cells,
• Heat shock proteins, protein folding and hormesis
• Telomeres and telomerase
• Animal models of aging including nematodes, pythons, zebrafish, naked mole rats, salamanders, fruit flys, mice, rats and humans.
• Cancers and cancer therapies
• Cognitive function, Alzheimer’s disease and cognitive decline
• Other key diseases such as spinal cord injury, Parkinson’s disease, autoimmune disorders, lymphomas, Lyme disease and glioblatoma
• Novel therapeutic approaches to aging-related diseases such as kinase inhibition
• Disease biomarkers and future trends in predictive personalized preventative participatory medicine
• Demographic populations known to be related to longevity
• Lifestyle factors related to longevity including mental attitude, exercise, stress, nature of diet, social relationships
• Novel viewpoints such as systems biology and quantum biology
• Mitochondrial metabolism
• Prospects for breaking through the 123 year known human age limit

Some blog entries contain brief video segments, many from the film To Age or Not to Age, in which I was interviewed by the producer-director Robert Kane Pappas.

Other than for an occasional opinion piece or message about the blog itself I have above all striven to focus on reputable published research.  This research can be of several types ranging from in-vitro studies of pathways using tools of molecular biology to large multi-year population studies.  See the blog entry About longevity research.  I strive to be meticulous in providing citations to back up key assertions.

Once in a great while I will publish a spoof with a message.  One favorite is P38, P39 and P40 channel receptor functions inhibit activities of BF-110, HE111 and HE177 leading to reduced expression of (SC)1000 in BOB.

The intention of my blog discussions is to convey current research findings and opinions, not to give medical advice.  See the Medical Disclaimer for this blog.  I avoid any linkage with commercial interests.  See Scientific integrity and advertising on my sites. 

Some current blog statistics are:

• Total number of posts to date: 393
• Total number of comments to date: 2,243
• Number of unique daily visitors (typical): 2,500
• Visitors view average of 1.8 posts per visit
• Number of registered users: 4,553
• Typical number of new blog registrants per day: 20-30
• Guess estimated number of total occasional readers: 20.000 plus
• More than 50% of usage appears to be from outside US

Shifts in my viewpoint over the 4 years of the blog

About twice a year, I write a blog piece about where I currently stand with respect to aging science.  My most recent was the February 2011 post The evolution of my perspective as a longevity scientist.  That post outlines some of my personal history with respect to longevity science and describes a number of perceptions I still stand by today.  Some of the most important shifts for me are:

• Telomeres/telomerase - I started out thinking that keeping telomeres long by activating telomerase via ingestion of substances like astragaloside IV or cycloastragenol could contribute to our longevity.  I now see telomere length as a downstream effect rather than itself as a possible cause of longevity.  I have written a series of blog entries on this topic.  You can start with the March 2011 blog entry The epigenetic regulation of telomeres and link from there to other telomere-related blog entries and the related discussion in my treatise.
• Oxidative damage - I also started out thinking, with many other researchers, that oxidative damage is a major cause of aging and that inhibiting such damage by taking antioxidants might be life extending.  I now think these views are too simplistic and see oxidative damage in a much more nuanced perspective.  See Victor’s September 2011 blog post End of the free radical theory of aging and negative consequences of indiscriminante antioxidant supplementation and my editorial A shift in a key aging sciences paradigm.
• Complexity – Having some academic background in theoretical physics, I started out thinking that compared to quantum physics, relativity theory, quantum electrodynamics, gauge field theories, fractal, chaos and string theories, etc., biology would turn out to be relatively simple and easy to grasp.  Instead I now see biology as at least an order of magnitude more complex than physics.  Compared to biology, studying origins of the universe is relatively simple stuff where widely applicable principles apply.  This is not just a crazy idea of my own.  Two prominent theoretical physicists (Martin A. Burcher and David N.Spergel) recently initiated their article in the special edition of Scientific American on The Cosmic Life Cycle by writing “Cosmology has a reputation as a difficult science, but in many ways explaining the whole universe is easier than understanding a single-celled animal.”  And they wrote “The unfolding of the cosmos, it seems, is almost completely insensitive to the details of its contents.  Unfortunately for biologists, the same principle does not apply to even the simplest organism.”
• Breaking the 123 year age limit – I started out four years ago with the viewpoint that extending human lifespans beyond the normal limit will probably become possible in time and would probably be based on some kind of telomerase- based intervention.  Now I still think that extending human lifespans beyond the normal limit will become possible but that the intervention(s) most likely to do the trick will either involve induced pluripotent stem cells or epigenetic modifications of gene promoter areas.  See Closing the loop in the stem cell supply chain – presented graphicallyand my presentation Towards a Systems View of Aging at the American Aging Society 2010 annual meeting.
• Need for new societal views of aging – There is little recognition that this 21st century is undergoing a revolution in health and longevity with ultimate consequences more profound than the electronics and information revolution had in the 20th century.  We are already living much longer than recognized by our institutions like Social Security, and science soon will probably allow us to extend our lives by a dozen or more healthy years, perhaps longer.  This change is being driven by advances in the life sciences and our basic understanding of the relationship between disease and aging.  And it is being driven even more by societal forces operating in the background.  Everything else will change as a consequence.  We could well have an older more wisdom-oriented population   The 20th century saw immense gains in productivity connected with electronics, computers and worldwide networking.  The 21st century is likely to see even greater gains through reduction in health care costs coupled with the preservation, expansion and utilization of our pool of human capital.   The ramifications will become more and more profound, and a public dialog is needed to bring them into public awareness.  What this watershed may ultimately lead to in terms of the human condition is anyone’s guess, but the more the awareness the more we may be able consciously to shape our future.
• Scientific breakthroughs versus a complex social process – Four years ago I was looking for a single blockbuster breakthrough in scientific knowledge that would allow us to live much longer in a healthy condition.  I was looking for science to unlock the secret of the fountain of youth.  Now I still anticipate the same result of longer healthier lives.  But I expect it to happen as a complex social process involving many actors and much more than scientific knowledge.  Massive applications of engineering, new companies, investments of money, trial-and-error and risk taking will be required.  Not one big secret but a combination of hundreds of smaller ones will be involved.  The computer and information revolution of the last century was not based on a single discovery but emerged as a very complicated and messy process involving elements of science, engineering, commerce, finance, entrepreneurship, government and public policy.  I expect the longevity revolution to happen in the same way.  It is the only way profound things can happen.

Increase in complexity of blog articles, decline in their numbers

As time progresses my associates and I have been writing repeated updates on specific topics, both to explore deeper and to keep up with the literature.  This turns out to be a very challenging task, particularly when research in the area concerned is is going on at a furious pace.  For example, between October 2010 when I generated the blog entry Klotho anti-aging gene in the news and the December 7 2011 update entry More about Klotho – spinner of the thread of lifeover 100 serious research publications related to Klotho appeared.  And I attended a multi-speaker session on Klotho at the November meeting at the Gerontological Society of America.  Digesting all that material prior to publishing the new blog entry was a challenging task requiring many days.

So, as time progresses I am tending to publish longer and more comprehensive treatise-like review studies that describe the state-of-the-art for specific topics.   But it is taking me a lot longer to do them so the number of new entries per month has been declining. 

Continuously growing readership of the blog suggests that the blog has already proven itself to be useful.  Yet, to keep up with the pace as the research expands I believe it will be necessary to publish items more frequently.  The need is to do this while maintaining full integrity and quality in the blog.  This brings me back to the topic of Associate Researchers/Blog writers.  To increase the rate of publication and flow of blog materials I have invited others to apply to join me as associate authors, in April 2011 putting out Call for associate researcher-writers.  I now have two excellent associate researcher-writers, Victor and Brendan Hussey.  They have each produced some excellent articles.  But, like myself, they have are living full lives and can devote only limited resources to reading or writing.  We need a few additional key colleagues, so I am repeating my “casting call” here, slightly expanding the original version.

Casting call II for blog researchers/writer associates

I am looking for a few Associate Researchers/Writers to join me in generating interesting and quality posts for this blog. I am initially looking for people who can cover relatively broad swaths of the longevity sciences in depth as I have been doing. Later, it may make sense to add more researchers/writers focusing on specialized areas. In such a case, the initial Associates could possibly graduate to becoming Area Editors. If you satisfy the following, please communicate with me by e-mail:

• You have a passionate interest in the longevity sciences or in an applied area related to longevity, perhaps with a focus in a certain key area.
• You are good at perusing the scientific literature to see what is going on,
• You can express yourself well in writing and would like that writing to have targeted exposure that generates feedback.
• You have sufficient training, background and patience to scan key areas of the emerging literature, identify important new trends, and generate blog entries with the sophistication and degree of literature documentation typically found in this blog. A number of readers have commented that this blog is the best source of scientific information related to aging on the web. I want to keep it that way.
• You are aligned with the Mission of this blog and willing to respect the blog guidelines outlined here. The Mission of the blog is to present information clearly on key current research developments pertaining to aging and information on related technological and social topics. Secondarily, it is to share informed opinions on the same topic. The target audience includes professionals and students in the aging science community, health and geriatric practitioners, and informed individuals with a substantial interest in aging and aging sciences.  Editorial commentary and informed opinion may also be of great value provided it is identified as such.
• You would like to get your signed writings out to a growing International community which I estimate currently to be more than 20,000 regular readers. Currently, an average of 2,500 different readers visit the blog daily.  And 150 t0 200 new registrations come in weekly from all over the world. 
• You are willing to research and write at least one blog entry every two months.  Documented mini-reviews of key topics are particularly welcome. The history of previous blog entries illustrates what I consider acceptable topics and blog formats, the degree of citation documentation, and the quality of the writing I will be looking for.
• You are willing to follow the general guidelines of this blog such as no commercial product promotions, no giving of medical advice and staying broadly “on topic” with respect to longevity. While individual blog entries can be concerned with specific theories of aging or anti-aging interventions, the blog itself is inclusive of a wide variety of viewpoints provided that they are informed and anchored in respectable research. Opinions, when offered, will be identified as such. And I will also entertain occasional humor pieces.
• You are willing to work now for the same compensation I am getting – zero pay but significant public exposure, acknowledgement and opportunity to learn.
• You are willing to have me review and possibly lightly edit your blog entries before they are published on the blog.  And I will reserve the right to not publish any writings if I feel they don’t meet quality or other standards of the blog.

My e-mail is vegiuliano@comcast.net. Please use the e-mail heading BLOG WRITER so I can easily sort your message out from junk. Tell me about yourself, your background, what you know/don’t know, your academic and work status, any institutional affiliation you may have or had, why you want to contribute and what you have to offer. Also please point me to online examples of your relevant writings or attach same to your e-mails. Finally, you might wish to identify the subject of any initial blog entries you might want to write.

If I invite you to join me as an Associate, I will do an editorial review of your blog entries before they go online and everything you write will be attributable to your authorship.

Please do not let your age or academic or institutional status be seen as a barrier here if you have what it takes to do the job. Whether you are a vital oldster like me or a young graduate student, this could be an opportunity to get yourself out to a big audience of readers and make a difference.

Discovered in 1997, the Klotho gene has attracted increasing attention.  In small animals, insufficient or knocked-down Klotho expression leads to short-lived disease-ridden animals while forced over-expression of Klotho can produce mice that live 20% to 30% longer than their wild type counterparts.  And the same pathways appear to work in humans.  More-recently, research has been decoding the relationship of Klotho expression to several important disease processes. 

Among the statements documented in the above-cited blog entries or known for some time are:

• There are two forms of Klotho: alpha which is a circulating hormone and beta which relates to cell surface receptors.
• Mutation of the Klotho gene can produce a syndrome that resembles aging and Klotho expression generally declines with age.
• Klotho knockout mice have only half to one-third the body weight of normal mice, live only a median of 60 days, have arteriosclerosis, emphysema, osteoporosis, skin atrophy, cognitive impairment, markers of oxidative damage, ectopic calcification, hypervitaminosis D, hypercalcemia, and hyperphosphatemia.
• “Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1 (IGF-1); and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety(ref).”.
• One of the aging mechanisms that may be accelerated by insufficient expression of Klotho is the buildup of advanced glycation endproducts (AGEs).
• One viewpoint is that Klotho derives much of its anti-aging capability from the protein acting “by increasing the cell’s ability to detoxify harmful reactive oxygen species. Over-expression of Klotho can protect against oxidative stress.
• Klotho gene delivery in mice increases natural SOD antioxidant protection in aorta, liver and kidneys.
• Klotho overexpression suppresses insulin and inhibits the IGF-1 channel; its deficiency leads to expression of IGF-1.
• Inhibition of the IGF-1 channel signaling partially ameliorates aging and age-related problems in Klotho knockout mice.
• Klotho deficiency leads to overexpression of vitamin D3 which leads to premature aging.  The mechanism is associated with Klotho’s function in regulating FGF23.
• Secretion of Klotho appears to be partially regulated by insulin.
• Klotho appears to regulate nitric oxide production in the endothelium.  Insufficient Klotho leads to diminished angiogenesis and vasculogenesis..
• Defects in Klotho expression can lead to underexpression of FGF23 and accumulation of phosphates.  Accumulated phosphates can accelerate aging.  Phosphate retention can lead to an aging phenotype.
• FGF23 and its relationship to Klotho are linked to a number of bone and joint diseases.
• Klotho is a regulator of oxidative stress and cell senescence.
• Klotho inhibits growth and promotes apoptosis in some cancer lines.
• Klotho protein protects against endothelial dysfunction and hypertension.
• Control of Klotho expression comes about through epigenetic mechanisms; some cancers can silence Klotho expression.
• Consuming cola drinks rich in phosphoric acid when coupled with Klotho insufficiency may exert a pro-aging effect.
• In humans, studies suggest that Klotho KL-VS gene polymorphisms may be associated with stroke, coronary artery disease and longevity.
• Klotho pathways might be targets for anti-aging interventions.

Unless you are already thoroughly familiar with Klotho, I strongly suggest you review the above-mentioned two blog entries as background before reading this one. In generating this blog entry I found there was a very large amount of information to digest and see in context.

Additional observations gleaned from more-recent publications

Plasma Klotho level is inversely associated with cardiovascular disease.

The August 2011 publication Plasma Klotho and Cardiovascular Disease in Adultslooked at “One thousand twenty-three men and women aged 24 to 102 participating in the Invecchiare in Chianti (InCHIANTI) study.”  – Anthropometric measures, plasma klotho, fasting plasma total, high-density lipoprotein cholesterol (HDL-C), triglycerides, glucose, creatinine, C-reactive protein (CRP). Clinical measures: medical assessment, diabetes mellitus, hypertension, coronary heart disease, heart failure, stroke, peripheral artery disease, cancer, chronic kidney disease. Logistic regression models were used to examine the relationship between plasma klotho and prevalent CVD. — RESULTS: Of 1,023 participants, 259 (25.3%) had CVD. Median (25th, 75th percentile) plasma klotho concentrations were 676 pg/mL (530, 819 pg/mL). Plasma klotho was correlated with age (correlation coefficient (r)=-0.14, P<.001), HDL-C (r=0.11, P<.001), and CRP (r=-0.10, P<.001) but not systolic blood pressure, fasting plasma glucose, or renal function. Plasma klotho age-adjusted geometric means were 626 pg/mL (95% confidence interval (CI)=601-658 pg/mL) in participants with CVD and 671 pg/mL (95% CI=652-692 pg/mL) in those without CVD (P=.001). Adjusting for traditional cardiovascular risk factors (age, sex, smoking, total cholesterol, HDL-C, systolic blood pressure, and diabetes mellitus), log plasma klotho was associated with prevalent CVD (odds ratio per 1 standard deviation increase=0.85, 95% CI=0.72-0.99). — CONCLUSION: In community-dwelling adults, higher plasma klotho concentrations are independently associated with a lower likelihood of having CVD.”

The InChianti study established in a substantial population that many of the Klotho effects observed in mice apply also to humans; for example, low circulating Klotho is associated with shorter lifespans, greater incidents of cardiovascular diseases. 

An interesting association is reported in the July 2011 publication Relationship of low plasma klotho with poor grip strength in older community-dwelling adults: the InCHIANTI study.  “Handgrip strength is a strong indicator of total body muscle strength and is a predictor of poor outcomes in older adults. The aging suppressor gene klotho encodes a single-pass transmembrane protein that is secreted as a circulating hormone. In mice, disruption of klotho expression results in a syndrome that includes sarcopenia, atherosclerosis, osteoporosis, and shortened lifespan, and conversely, overexpression of klotho leads to a greater longevity. The objective was to determine whether plasma klotho levels are related to skeletal muscle strength in humans. We measured plasma klotho in 804 adults, ≥65 years, in the InCHIANTI study, a longitudinal population-based study of aging in Tuscany, Italy. Grip strength was positively correlated with plasma klotho at threshold <681 pg/mL. After adjusting for age, sex, education, smoking, physical activity, cognition, and chronic diseases, plasma klotho (per 1 standard deviation increase) was associated with grip strength (beta = 1.20, standard error = 0.35, P = 0.0009) in adults with plasma klotho <681 pg/mL. These results suggest that older adults with lower plasma klotho have poor skeletal muscle strength.’

The August 2011 report  Relationship of serum fibroblast growth factor 23 with cardiovascular disease in older community-dwelling womenlooks at the relationship of FGF23to cardiovascular disease in a cohort of 659 women, aged 70–79.  “Conclusion Elevated serum FGF23 concentrations are independently associated with prevalent cardiovascular disease in older community-dwelling women.”

See also (January 2011) Klotho: An Elixir of Youth for the Vasculature?

Klotho expression is a key factor in phosphate metabolism, which in turn can be an important factor in cardiovascular, kidney and bone diseases

A number of different studies point to this conclusion. 

The 2010 publication Overview of the FGF23-Klotho axis sets the scene.  “Recent studies have identified a novel bone-kidney endocrine axis that maintains phosphate homeostasis. When phosphate is in excess, fibroblast growth factor-23 (FGF23) is secreted from bone and acts on the kidney to promote phosphate excretion into urine and suppress vitamin D synthesis, thereby inducing negative phosphate balance. One critical feature of FGF23 is that it requires Klotho, a single-pass transmembrane protein expressed in renal tubules, as an obligate coreceptor to bind and activate FGF receptors. Several hereditary disorders that exhibit inappropriately high serum FGF23 levels are associated with phosphate wasting and impaired bone mineralization. In contrast, defects in either FGF23 or Klotho are associated with phosphate retention and a premature-aging syndrome. The aging-like phenotypes in Klotho-deficient or FGF23-deficient mice can be rescued by resolving hyperphosphatemia with dietary or genetic manipulation, suggesting a novel concept that phosphate retention accelerates aging. Phosphate retention is universally observed in patients with chronic kidney disease (CKD) and identified as a potent risk of death in epidemiological studies. Thus, the bone-kidney endocrine axis mediated by FGF23 and Klotho has emerged as a novel target of therapeutic interventions in CKD.”

The October 2011 publication Phosphate Metabolism in Cardiorenal Metabolic Disease reports “Hyperphosphatemia is a major risk factor for cardiovascular disease, abnormalities of mineral metabolism and bone disease, and the progression of renal insufficiency in patients with chronic renal disease. In early renal disease, serum phosphate levels are maintained within the ‘normal laboratory range’ by compensatory increases in phosphaturic hormones such as fibroblast growth factor-23 (FGF-23). An important co-factor for FGF-23 is Klotho; a deficiency in Klotho plays an important role in the pathogenesis of hyperphosphatemia, renal tubulointerstitial disease, and parathyroid and bone abnormalities. Clinical hyperphosphatemia occurs when these phosphaturic mechanisms cannot counterbalance nephron loss. Hyperphosphatemia is associated with calcific uremic arteriolopathy and uremic cardiomyopathy, which may explain, in part, the epidemiologic connections between phosphate excess and cardiovascular disease. However, no clinical trials have been conducted to establish a causal relationship, and large, randomized trials with hard endpoints are urgently needed to prove or disprove the benefits and risks of therapy. In summary, hyperphosphatemia accelerates renal tubulointerstitial disease, renal osteodystrophy, as well as cardiovascular disease, and it is an important mortality risk factor in patients with chronic kidney disease.”

See also (August 2011) Downregulation of NaPi-IIa and NaPi-IIb Na-coupled phosphate transporters by coexpression of Klotho,  (Mar 2011) Phosphate levels and cardiovascular disease in the general population, (March 2011) Derangements in phosphate metabolism in chronic kidney diseases/endstage renal disease: therapeutic considerations, and (July 2005) Fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease, (April 2011) Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate.  “Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification.”

Klotho-deficient mice and FGF23-deficient mice exhibit identical phenotypes.

Several publications mention this observation including the June 2011 publication Klotho and the aging process.  This publication treats a number of topics discussed elsewhere in this blog entry and includes a discussion of the roles of secreted Klotho in promoting longevity.  “Secreted Klotho regulates the activity of multiple growth factors, including insulin/insulin-like growth factor-1 (IGF-1) [2], Wnt [58], and transforming growth factor (TGF)-β1 [59]. Because adequate suppression of insulin/IGF-1 signaling pathway has been identified as an evolutionarily conserved mechanism for extending life span [60], the anti-aging properties of Klotho may stem partly from its ability to suppress insulin/IGF-1 signaling. In fact, transgenic mice that overexpress Klotho are long-lived and slightly resistant to insulin and IGF-1 without overt diabetes [2]. The mechanism by which secreted Klotho suppresses insulin/IGF-1 signaling remains to be determined. However, secreted Klotho inhibits Wnt signaling by directly binding to Wnt ligands and preventing them from binding to their receptors. Wnt signaling is enhanced in Klotho-deficient mice, which results in exhaustion of stem cells in highly proliferative tissues such as skin and intestine and may partly contribute to atrophy of these tissues in Klotho-deficient mice [58]. — We recently found that secreted Klotho suppresses TGF-β1 signaling; secreted Klotho directly binds to type-II TGF-β receptor (TGFβR2) on the cell surface and prevents TGF-β1 binding to TGFβR2 [59]. TGF-β1 is the most potent inducer of the epithelial-to-mesenchymal transition (EMT) [61]. EMT is a cellular process whereby epithelial cells lose epithelial characters and undergo a phenotypic transition to acquire mesenchymal characters, including the ability to migrate and proliferate. EMT is essential for tissue repair in response to injury but can result in fibrosis under pathological conditions in the kidney as well as in many other tissues, including the liver, lung, and heart [62]. Furthermore, cancer cells undergo EMT and acquire the ability to migrate and proliferate, leading to metastasis [62]. Thus, the ability of secreted Klotho to inhibit TGF-β1 activity may counteract EMT and prevent tissue fibrosis and cancer metastasis. In fact, injecting secreted Klotho prevents renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts in mice [59]. These activities of secreted Klotho may also contribute to life span extension by Klotho overexpression in mice [2].”

Besides serving as cofactor for FGF23, the Klotho gene family encodes proteins that are cofactors for regulation of tissue-specific metabolic activities of the endocrine fibroblast growth factors (FGF19, FGF21, and FGF23).

See the 2008 publication The Klotho gene family and the endocrine fibroblast growth factors “In addition to the Klotho-FGF23 axis, recent studies have shown that betaKlotho, a Klotho family protein, also functions as a cofactor required for FGF19 and FGF21 signaling and determines tissue-specific metabolic activities of FGF19 and FGF21.” And see the 2010 publicationRelevant use of Klotho in FGF19 subfamily signaling system in vivo. “Alpha-Klotho (alpha-Kl) and its homolog, beta-Klotho (beta-Kl) are key regulators of mineral homeostasis and bile acid/cholesterol metabolism, respectively. FGF15/ humanFGF19, FGF21, and FGF23, members of the FGF19 subfamily, are believed to act as circulating metabolic regulators. Analyses of functional interactions between alpha- and beta-Kl and FGF19 factors in wild-type, alpha-kl(-/-), and beta-kl(-/-) mice revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of alpha-Kl, FGF23, and 1,25(OH)(2)D and an analogous regulatory network composed of beta-Kl, FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Contrary to in vitro data, beta-Kl is not essential for FGF21 signaling in adipose tissues in vivo, because (i) FGF21 signals are transduced in the absence of beta-Kl, (ii) FGF21 could not be precipitated by beta-Kl, and (iii) essential phenotypes in Fgf21(-/-) mice (decreased expressions of Hsl and Atgl in WAT) were not replicated in beta-kl(-/-) mice. These findings suggest the existence of Klotho-independent FGF21 signaling pathway(s) where undefined cofactors are involved. One-to-one functional interactions such as alpha-Klotho/FGF23, beta-Klotho/FGF15 (humanFGF19), and undefined cofactor/FGF21 would result in tissue-specific signal transduction of the FGF19 subfamily.”

Low levels of Klotho may serve as an early warning biomarker for kidney disease and cardiovascular complications

More than 26 million people in the U.S. are affected by chronic kidney disease.   The November 2010 publication Klotho Deficiency Causes Vascular Calcification in Chronic Kidney Disease reports “Soft-tissue calcification is a prominent feature in both chronic kidney disease (CKD) and experimental Klotho deficiency, but whether Klotho deficiency is responsible for the calcification in CKD is unknown. Here, wild-type mice with CKD had very low renal, plasma, and urinary levels of Klotho. In humans, we observed a graded reduction in urinary Klotho starting at an early stage of CKD and progressing with loss of renal function. Despite induction of CKD, transgenic mice that overexpressed Klotho had preserved levels of Klotho, enhanced phosphaturia, better renal function, and much less calcification compared with wild-type mice with CKD. Conversely, Klotho-haploinsufficient mice with CKD had undetectable levels of Klotho, worse renal function, and severe calcification. The beneficial effect of Klotho on vascular calcification was a result of more than its effect on renal function and phosphatemia, suggesting a direct effect of Klotho on the vasculature. In vitro, Klotho suppressed Na⁺-dependent uptake of phosphate and mineralization induced by high phosphate and preserved differentiation in vascular smooth muscle cells. In summary, Klotho is an early biomarker for CKD, and Klotho deficiency contributes to soft-tissue calcification in CKD. Klotho ameliorates vascular calcification by enhancing phosphaturia, preserving glomerular filtration, and directly inhibiting phosphate uptake by vascular smooth muscle. Replacement of Klotho may have therapeutic potential for CKD.”

The September 2011 publication Cross talk between the renin-angiotensin-aldosterone system and vitamin D-FGF-23-klotho in chronic kidney disease provides additional insight related to Klotho and renal disease.  “There is increasingly evidence that the interactions between vitamin D, fibroblast growth factor 23 (FGF-23), and klotho form an endocrine axis for calcium and phosphate metabolism, and derangement of this axis contributes to the progression of renal disease. Several recent studies also demonstrate negative regulation of the renin gene by vitamin D. In chronic kidney disease (CKD), low levels of calcitriol, due to the loss of 1-alpha hydroxylase, increase renal renin production. Activation of the renin-angiotensin-aldosterone system (RAAS), in turn, reduces renal expression of klotho, a crucial factor for proper FGF-23 signaling. The resulting high FGF-23 levels suppress 1-alpha hydroxylase, further lowering calcitriol. This feedback loop results in vitamin D deficiency, RAAS activation, high FGF-23 levels, and renal klotho deficiency, all of which associate with progression of renal damage. Here we examine current evidence for an interaction between the RAAS and the vitamin D-FGF-23-klotho axis as well as its possible implications for progression of CKD.”

The negative health consequences of hypercalcemia due to underexpression of Klotho can be very serious. 

Expression of Klotho is highly relevant to maintenance of calcium homeostasis.  The 2011 publication Calcium metabolism & hypercalcemia in adults reports “Calcium is essential for many metabolic process, including nerve function, muscle contraction, and blood clotting. The metabolic pathways that contribute to maintain serum calcium levels are bone remodeling processes, intestinal absorption and secretion, and renal handling, but hypercalcemia occurs when at least 2 of these 3 metabolic pathways are altered. Calcium metabolism mainly depends on the activity of parathyroid hormone (PTH). Its secretion is strictly controlled by the ionized serum calcium levels through a negative feed-back, which is achieved by the activation of calcium-sensing receptors (CaSRs) mainly expressed on the surface of the parathyroid cells. The PTH receptor in bone and kidney is now referred as PTHR1. The balance of PTH, calcitonin, and vitamin D has long been considered the main regulator of calcium metabolism, but the function of other actors, such as fibroblast growth factor-23 (FGF-23), Klotho, and TPRV5 should be considered. Primary hyperparathyroidism and malignancy are the most common causes of hypercalcemia, accounting for more than 90% of cases. Uncontrolled hypercalcemia may cause renal impairment, both temporary (alteration of renal tubular function) and progressive (relapsing nephrolithiasis), leading to a progressive loss of renal function, as well as severe bone diseases, and heart damages. Advances in the understanding of all actors of calcium homeostasis will be crucial, having several practical consequences in the treatment and prevention of hypercalcemia. This would allow to move from a support therapy, sometimes ineffective, to a specific and addressed therapy, especially in patients with chronic hypercalcemic conditions unsuitable for surgery.”

Klotho suppresses renal fibrosis.

The February 211 publication Phosphate and Klothorelates “Klotho is a putative aging suppressor gene encoding a single-pass transmembrane co-receptor that makes the fibroblast growth factor (FGF) receptor specific for FGF-23. In addition to multiple endocrine organs, Klotho is expressed in kidney distal convoluted tubules and parathyroid cells, mediating the role of FGF-23 in bone–kidney–parathyroid control of phosphate and calcium. Klotho^–/– mice display premature aging and chronic kidney disease-associated mineral and bone disorder (CKD-MBD)-like phenotypes mediated by hyperphosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter). CKD can be seen as a state of hyperphosphatemia-induced accelerated aging associated with Klotho deficiency. Humans with CKD experience decreased Klotho expression as early as stage 1 CKD; Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases, and hypovitaminosis D. Secreted Klotho protein, formed by extracellular clipping, exerts FGF-23-independent phosphaturic and calcium-conserving effects through its paracrine action on the proximal and distal tubules, respectively. We contend that decreased Klotho expression is the earliest biomarker of CKD and the initiator of CKD-MBD pathophysiology. Maintaining normal phosphate levels with phosphate binders in patients with CKD with declining Klotho expression is expected to reduce mineral and vascular derangements.”

Hyperphosphatemia associated with to reduced Klotho expression may also play a key role in obesity.

The November 2011 publicationGenetic induction of phosphate toxicity significantly reduces the survival of hypercholesterolemic obese mice reports: “OBJECTIVE: The adverse effects of metabolic disorders in obesity have been extensively studied; however, the pathologic effects of hyperphosphatemia or phosphate toxicity in obesity have not been studied in similar depth and detail, chiefly because such an association is thought to be uncommon. Studies have established that the incidence of obesity-associated nephropathy is increasing. Because hyperphosphatemia is a major consequence of renal impairment, this study determines the in vivo effects of hyperphosphatemia in obesity.  METHODS AND RESULTS: We genetically induced hyperphosphatemia in leptin-deficient obese (ob/ob) mice by generating ob/ob and klotho double knockout [ob/ob-klotho(-/-)] mice. As a control, we made ob/ob mice with hypophosphatemia by generating ob/ob and 1-alpha hydroxylase double knockout [ob/ob-1α(OH)ase(-/-)] mice. Compared to the wild-type mice, all three obese background mice, namely ob/ob, ob/ob-klotho(-/-), and ob/ob-1α(OH)ase(-/-) mice developed hypercholesterolemia. In addition, the hyperphosphatemic, ob/ob-klotho(-/-) genetic background induced generalized tissue atrophy and widespread soft-tissue and vascular calcifications, which led to a shorter lifespan; no such changes were observed in the hypophosphatemic, ob/ob-1α(OH)ase(-/-) mice. Significantly, in contrast to the reduced survival of the ob/ob-klotho(-/-) mice, lowering serum phosphate levels in ob/ob-1α(OH)ase(-/-) mice showed no such compromised survival, despite both mice being hypercholesterolemic.  CONCLUSION: These genetic manipulation studies suggest phosphate toxicity is an important risk factor in obesity that can adversely affect survival.”

Inflammatory cytokines inhibit expression of Klotho via  NF-kappaB.

The July 2011 publication The inflammatory cytokines TWEAK and TNFα reduce renal klotho expression through NFκB reports: “Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNFα, and siRNA-mediated knockdown of IκBα all activated NFκB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFκB with parthenolide prevented TWEAK- or TNFα-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAK-induced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNFα, downregulate Klotho expression through an NFκB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.”

The vitamin D receptor (VDR) is a complicit partner in the feedback loops involving FGF23 and Klotho signaling.  The VDR upregulates FGF23 and Klotho.

The October 2011 publication Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells reports: “Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand.  The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. — Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.”

Klotho and FGF23 expression, via vitamin D3 vitamin D responsive elements (VDREs) in target genes, can be affected by dietary supplements including vitamin D3, omega3/omega6 polyunsaturated fatty acids (PUFAs) and curcumin.

This inference can be arrived at by combining the previously mentioned results with those mentioned in the 2007 publication Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands.  “The vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)(2)D(3) is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real-time PCR and reporter gene transfection assays were used to probe VDR-mediated transcriptional control by 1,25(OH)(2)D(3). Reporter gene and mammalian two-hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)(2)D(3) induces FGF23 78-fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)(2)D(3) synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)(2)D(3)-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)(2)D(3)-FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)(2)D(3)-PTH axis that regulates calcium. 1,25(OH)(2)D(3) also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)(2)D(3) supports a cloverleaf model, whereby VDR-RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co-modulators and chromatin remodeling enzymes. VDR also selectively binds certain omega3/omega6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR-RXR complexes. Moreover, the turmeric-derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)(2)D(3)-independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR-containing tissues.”

The September 2011 publication The role of vitamin D in the FGF23, klotho, and phosphate bone-kidney endocrine axis amplifies on the above.  “1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.”

Also relevant here is the September 2010 publication Reciprocal control of 1,25-dihydroxyvitamin D and FGF23 formation involving the FGF23/Klotho system.  “Fibroblast growth factor 23 (FGF23) is a circulating hormone that is synthesized by osteocytes and osteoblasts. This glycosylated peptide controls phosphate balance by modulating urinary phosphate excretion and indirectly intestinal phosphate absorption by reducing expression of the renal and intestinal sodium phosphate transporters. In a feedback loop, 1,25-dihydroxyvitamin D and phosphate intake control FGF23 production. FGF23 is inactivated by cleavage by a still unidentified enzyme. FGF23 cleavage occurs within cells and probably in the circulation. Klotho, a protein expressed at the cell surface of few organs, forms complexes with FGF receptors, which increases their affinity for FGF23. Klotho is also released into the plasma and urine by an enzymatic cleavage. FGF23 plays a central role in vitamin D metabolism: It inhibits calcitriol synthesis in the kidney and stimulates the catabolism of active vitamin D sterols. In turn, calcitriol stimulates FGF23 and Klotho expression. In chronic kidney diseases, FGF23 concentration increases as GFR declines, whereas Klotho tissue expression decreases. The modifications of FGF23 and Klotho expression are probably involved in the genesis of hyperparathyroidism and the resistance to vitamin D receptor (VDR) activation in chronic kidney disease. Low vitamin D, high FGF23 concentrations, and defects in VDR activation are associated with similar risks, which evoke the possibility that potential FGF23 toxicity might be partly mediated by FGF23-induced decrease in calcitriol or 25-hydroxyvitamin D. Conversely, VDR activators could be used to modulate Klotho or FGF23 expression.”

See also (2007) Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands, (2007)1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism, .and (2005)  1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts.

In fact, it well be that some of the health-producing affects of some dietary polyphenols may be mediated by its effects on VDEEs and expression of Klotho and FGF23.

The December 2010 publication Curcumin: a novel nutritionally derived ligand of the vitamin D receptor with implications for colon cancer chemoprevention states: “Numerous studies have shown chemoprotection by CM (curcumin) against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDR-expressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.”  I (Vince) think we have enough evidence to go the next step and hypothesize “Curcumin facilitates expression of Klotho and its health benefits via activation of vitamin D receptor elements.”

Klotho expression can be modulated by dehydration.

See the October 2011 items  Dehydration: a new modulator of klotho expression and Downregulation of Klotho expression by dehydration.  “Klotho, a transmembrane protein, protease, and hormone mainly expressed in renal tissue counteracts aging. Overexpression of Klotho substantially prolongs the life span. Klotho deficiency leads to excessive formation of 1,25(OH)₂D₃, growth deficit, accelerated aging, and early death. Aging is frequently paralleled by dehydration, which is considered to accelerate the development of age-related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR, and Klotho protein abundance was detected by Western blotting in renal tissue from hydrated and 36-h-dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase in plasma osmolarity as well as plasma ADH, aldosterone, and 1,25(OH)₂D₃ levels. Antidiuretic hormone (ADH; 50 nM) and aldosterone (1 μM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.”  

There are more publications on how cancers epigenetically silence the expression of Klotho.

The October 2011 publication Epigenetic silencing of the tumor suppressor klotho in human breast cancer. Reports “We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.”

The November 10, 2011 publication Klotho is silenced through promoter hypermethylation in gastric cancer reports “As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. — Demethylation with 5-aza-2′-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.”

Age-related decline in Klotho may be related to promoter methylation.

The September 2011 publication Promoter methylation and age-related downregulation of Klotho in rhesus monkeyrelates “While overall DNA methylation decreases with age, CpG-rich areas of the genome can become hypermethylated. Hypermethylation near transcription start sites typically decreases gene expression. Klotho (KL) is important in numerous age-associated pathways including insulin/IGF1 and Wnt signaling and naturally decreases with age in brain, heart, and liver across species. Brain tissues from young and old rhesus monkeys were used to determine whether epigenetic modification of the KL promoter underlies age-related decreases in mRNA and protein levels of KL. The KL promoter in genomic DNA from brain white matter did not show evidence of oxidation in vivo but did exhibit an increase in methylation with age. Further analysis identified individual CpG motifs across the region of interest with increased methylation in old animals. In vitro methyl modification of these individual cytosine residues confirmed that methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5′ regulatory region.“

Though this has been a long blog entry, the literature related to Klotho during the last 14 months has more things to say than I have been able to report here.  Klotho is one of the three Greek Morie fates, moon goddesses.  Klotho spins the thread of life, so she is quite important from the viewpoint of longevity.  Her two sister fates are important too: Lachesis measures the thread of life.  She decides just how long each person will live.  “Lachesis is the measurer of the thread woven by Clotho’s spindle, and in some texts, determines Destiny, or thread of life.^”  Atropos cuts the thread of life.  She is the killer.  “It was Atropos who chose the mechanism of death and ended the life of each mortal by cutting their thread with her abhorred shears.”  They are the daughters of night and Erebus.  My writings in this blog are all about their work.

There is current excitement about a new approach to using a person’s own immune system to fight and defeat otherwise incurable cancers: Adoptive immunotherapy, a technology that is currently the subject of multiple clinical trials.  Here, I cover selected current research in this area and summarize where we are. 

I have written about immunotherapies before.  See, for exampe, the posts Skin Cancer immunotherapies  and Dendritic cell cancer immunotherapy.

Adoptive immunotherapy, in its simplest terms is “a form of immunotherapy used in the treatment of cancer in which an individual’s own white blood cells are coupled with a naturally produced growth factor to enhance their cancer-fighting capacity(ref).”  There are multiple strategies for pursuing adoptive immunotherapy including a classical approach using IL-2 that can be effective but involves very high toxicity(ref)  and a newer approach which is based on modifying chimeric antigen receptors on T cells so that they recognize CD-19 markers on tumor cells.  

Background on stem cell therapies

The use of stem cell therapies to treat leukemias and selected congenital disorders is far from new.  The November 2011 publication Pediatric Hematopoietic Stem Cell Transplantation traces the history back 50 years.  “Hematopoietic stem cell transplantation was first performed more than 50 years ago. The earliest work in the field was performed using animal models in the mid 1950s. During the 1960s, the first few successful hematopoietic stem cell transplants used in the treatment of congenital immunodeficiency disorders and end-stage leukemia were reported. The success of these early attempts was compromised by high morbidity and mortality, in large part due to toxicity related to the chemotherapy (called conditioning) administered prior to bone marrow transplantation , post-transplant infectious complications, and graft versus host disease .”  The newer approach described below being autologous (using patient’s own stem cells) avoids the immune response of graft versus host disease.  And risk of infectious complications is also less.

CARS – Chimeric antigen receptors

Enhancing T cells with chimeric  antigen receptors (CARs) has been of particular interest with respect to developing therapies for otherwise-refractory leukemias.  “Using gene transfer technologies, T cells can be genetically modified to stably express antibody binding domains on their surface that confer novel antigen specificities that are major histocompatibility complex (MHC)–independent. Chimeric antigen receptors (CARs) are an application of this approach that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-ζ chain or FcγRI protein into a single chimeric protein(ref).”  See ref and ref for additional background.   

The potential advantages of a CAR-based therapy are outlined in the 2010 publication Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells. “Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.”