In the recent blog entry SIRT1, mTOR, NF-kappaB and resveratrol, I pointed out how “three different theories of longevity seem to be collapsing into one: 1. suppression of mTOR signaling, 2. activation of SIRT1, and 3. inhibition of expression of NF-kappaB.  Activating SIRT1 does all of these things, and this seems to be accomplishable to some extent by taking resveratrol supplements.”  Amplifying on that blog post here, I point out three additional theories of longevity that can be added to the list: 4. hypoxic signaling, 5. autophagy and 6. hormesis.   SIRT1 can activate the pathways of each of these and can in turn be activated by resveratrol

Hypoxic signaling

One my early blog entries was Another longevity-related biochemical pathway – the hypoxic response and I have listed The Hypoxic Response in my treatise as a fifth additional candidate theory of aging. “Another cross-species pathway has been discovered that allows interventions to lengthen life in primitive organisms, C. elegans nematode worms in this case.  The pathway is related to the hypoxic response, how cells respond to protect themselves when there is insufficient oxygen.  It turns out that if the hypoxic response can be turned on when normal oxygen is present, nematodes live significantly longer.  A recent research report indicates that this was experimentally accomplished by breeding nematodes that could not produce the protein VHL-1which destroys another protein called HIF which keeps the hypoxic response turned off when oxygen is present.  Also, it appears that the cells in such long-lived nematodes are relatively free of lipofuscin and toxic age-related protein aggregations such as seen in Alzheimer’s, Huntington’s and other age-related diseases(ref). — As of yet, however, just how HIF works downstream to extend longevity is still unclear.  The hypoxic response appears to operate in higher animals as well, including humans.”

Quoting the June 2009 publication Regulation of Hypoxia-Inducible Factor 2alpha Signaling by the Stress-Responsive Deacetylase Sirtuin 1:  “To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2 ) during hypoxia. The effect of Sirt1 on HIF-2 required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2 that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2 target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.”

Autophagy

“In cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes(ref).”  Efficiency of autophagy tends to decline with aging and increasing effective autophagy is seen as a possible anti-aging intervention. According to the 2009 publication Regulation of the aging process by autophagy “During aging, the efficiency of autophagic degradation declines and intracellular waste products accumulate. In Caenorhabditis elegans, there is clear evidence that lifespan is linked to the capacity to regulate autophagy. Recent studies have revealed that the same signaling factors regulate both aging and autophagocytosis, thus highlighting the role of autophagy in the regulation of aging and age-related degenerative diseases.”

The September 2009 publication SIRT1: Regulation of longevity via autophagy reports: “Recent studies have emphasized the importance of SIRT1, a mammalian homolog of Sir2 longevity factor, in the regulation of metabolism, cellular survival, and organismal lifespan. The signaling network interacting with SIRT1 continues to expand as does the number of functions known to be regulated by SIRT1. Autophagy is also an emerging field in longevity studies. Autophagocytosis is a housekeeping mechanism cleaning cells from aberrant and dysfunctional molecules and organelles. The extension of lifespan has been linked to the efficient maintenance of autophagic degradation, a process which declines during aging. Interestingly, recent observations have demonstrated that SIRT1 regulates the formation of autophagic vacuoles, either directly or indirectly through a downstream signaling network. — The interactions of SIRT1 with the FoxO and p53 signaling can also regulate both the autophagic degradation and lifespan extension emphasizing the key role of autophagy in the regulation of lifespan.”

Hormesis

My blog posting Hormesis and age retardation describes hormesis as a process of “challenging cells and body systems by mild stress resulting in them becoming stronger and resistant to aging(ref).  The stress can be physical, chemical and even possibly psychological.”  Exercise is an example.  That blog entry reviews the science behind hormesis and some of its demonstrable anti-aging effects.  Also, see my blog entry Stress and longevity for further discussion of how moderate stresses confer longevity.

Calorie restriction activating SIRT1 is a prime example of hormesis.  The article Sirt1 Regulates Aging and Resistance to Oxidative Stress in the Heart.  “– moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.”  And SIRT1 is important for cellular response to other forms of genotoxic stress(ref).  The heat shock response is a well-studied example of hormesis, involving heat shock proteins.  The 2009 publication Stress-inducible regulation of heat shock factor 1 by the deacetylase SIRT1 states “Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA-binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.”

So, when researchers like Leonard Guarante or David Sinclair talk about the sirtuin SIRT1 affecting multiple longevity-related pathways, there is a lot of emerging science behind what they are saying.  And there is a lot more to say about SIRT1 than I have covered so far.   Up to this point I have talked about how six of the 21 theories and candidate theories of aging/longevity described in my treatise are implicated.  But SIRT1 also links to several more of the aging theories, links I will discuss in future blog entries.

A few days ago I visited Leonard Guarante, Director of the Glenn Laboratory for the Science of Aging at MIT and pioneer in the investigation of sirtuins and their longevity properties.  The lead line of the Laboratory’s web site is “We work on mechanisms of aging so that people may lead healthier lives,” an intention that generates a lot of empathy in me.  We talked about some of the current investigations going on in the laboratory and some of Leonard’s views on aging science.  I cover high points of our discussion here, in the processes reviewing some of Leonard’s and the laboratory’s past achievements.  I also cite selected literature references pertinent to the science involved. 

Leonard started investigating aging genes in yeast back in 1991 and his 2003 book Ageless Quest: One Scientist’s Search for Genes That Prolong Youth presents a personal and very readable view of his investigations up to the time of its writing.  His studied the roles of sirtuins in yeast complexes and among his first major contributions over the years was identifying that the genetic pathways activated by the sirtuin SIR2 are the same ones activated by calorie restriction, an evolutionary-conserved pathway known to be capable of reliably conveying longevity across a number of species.  The 1999 publication co-authored by Guarante The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms has been cited by hundreds of subsequent publications and helped to prime the pump leading to what is now a steady river of publications related to sirtuins.  Leonard told me that a year or so ago, one new article relating to sirtuins  appeared just about every day and now about two such articles are appearing daily.  (I would love to cover those here but the task would clearly be impossible.)

Guarante’s 2004 publication The Sir2 family of protein deacetylases tells the story of actions of sirtuins in different organisms.  “We summarize the current knowledge of the Sir2 homologs from different organisms, and finally we discuss the role of Sir2 in caloric restriction and aging.”  This article is cited by 87 others in Pubmed Central.  A short video in which Guarante emphasizes how he believes the benefits of calorie restriction can be realized through activating sirtuins can be found here.  For discussions of pathways involved in calorie restriction and its roles in cancers, see my December 2009 blog entries Calorie restriction research roundup – Part I and Calorie restriction research roundup – Part II. 

The Glenn lab started to focus on sirtuin proteins about a dozen years ago.  Currently, about half of the activity in the lab is focused on the mammalian gene and sirtuin SIRT1, and the rest of the research relates to the other 6 members of the mammalian sirtuin family. SIRT1 is a mammalian homolog gene corresponding to the SIR2 yeast gene.  The working hypothesis with respect to longevity has been that the SIRT genes mediate the pathways of longevity related to calorie restriction, and that better understanding of the actions of the sirtuins could lead to practical interventions that postpone aging.   

The same interventions, it turns out, are likely to be highly useful for prevention or management of many late-onset diseases and diseases that flare with advancing age such as diabetes, Parkinson’s, Huntington’s  and Alzheimer’s.  In fact, Guarante points out that delaying aging is the same as delaying onset of the major diseases of aging, and I heartily agree with him.

About sirtuins, “Sir2 is active as an NAD+-dependent deacetylase, which is broadly conserved from bacteria to higher eukaryotes(ref).”  NAD+ dependency means essential involvement in metabolism.  The discovery of NAD dependency of sirtuins was in Guarante’s lab. Being a deacetylase (or an HDAC) means a capability for transcriptional silencing of gene expression.  See the blog entry Histone acetylase and deacetylase inhibitors for an explanation. “In mammalian cells, Sir2 proteins also deacetylate non-histone proteins such as the p53 tumour suppressor protein, alpha-tubulin and forkhead transcription factors to mediate diverse biological processes including metabolism, cell motility and cancer(ref).”

Guarante has very recently co-authored a review Ten years of NAD-dependent SIR2 family deacetylases: implications for metabolic diseases, a March 10, 2010 e-publication ahead of print. “Since the discovery of NAD-dependent deacetylase activity of the silent information regulator-2 (SIR2) family (‘sirtuins’), many exciting connections between protein deacetylation and energy metabolism have been revealed. The importance of sirtuins in the regulation of many fundamental biological responses to various nutritional and environmental stimuli has been firmly established. Sirtuins have also emerged as critical regulators for aging and longevity in model organisms. Their absolute requirement of NAD has revived an enthusiasm in the study of mammalian biosynthesis of NAD. Sirtuin-targeted pharmaceutical and nutriceutical interventions against age-associated diseases are also on the horizon. This review summarizes the recent progress in sirtuin research (particularly in mammalian sirtuin biology) and re-evaluates the connection between sirtuins, metabolism, and age-associated diseases (e.g., type-2 diabetes) to set a basis for the next ten years of sirtuin research. Copyright © 2010 Elsevier Ltd. All rights reserved.” 

Earlier papers of relevance authored or co-authored by Guarante include Genetics and the specificity of the aging process (2003), Calorie restriction extends life span by lowering the level of NADH (2004), Mammalian SIRT1 represses forkhead transcription factors (2004), Calorie restriction and SIR2 genes—Towards a mechanism (2005), and Calorie restriction–the SIR2 connection (2005).

The Glenn lab works basically with mice, either mice where a gene such as SIRT1 is knocked out in selected tissues or in mice where an extra copy of the gene has been added to the genome, super-sirtuin mice.  In typical past experiments, mice might be challenged, say by feeding them with high-calorie high-fat diets to attempt to induce diabetes in them.  Or a mouse might be genetically modified both to increase susceptibility to Alzheimer’s disease and to express increased amounts of SIRT1.   

Another area of research being pursued in Guarante’s lab involves the roles of sirtuins in the brain.  Guarante thinks sirtuins play important roles in brain aging and repair. In the 2009 publication Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction, Guarante and his colleagues reported “Since the somatotropic axis is controlled by the brain, we created mice lacking Sirt1 specifically in the brain and examined the impacts of this manipulation on somatotropic signaling and the CR response. These mutant mice displayed defects in somatotropic signaling when fed ad libitum, and defects in the endocrine and behavioral responses to CR. We conclude that Sirt1 in the brain is a link between somatotropic signaling and CR in mammals.”    In a late 2008 publication by some of Guarante’s counterpart colleagues at Harvard they reported “Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.”

Among the few interventions that demonstrably extend lifespans across multiple species besides calorie restriction are 1. inhibition of the mTOR pathway, 2. the activation of sirtuins such as via calorie restriction or substances such as resveratrol, and 3. Inhibition of the expression of a cell nuclear factor, NF-kappaB. For a long time these pathways were thought to be independent.  However, recent research reviewed here shows that these aging and longevity-related  pathways are very closely related.   

Background 

The ability to extend lives of organisms from yeast cells to mammals via inhibition of the mTOR (mammalian target of rapamycin) pathway is discussed in my previous blog entries Longevity genes, mTOR and lifespan, Viva mTOR! Caveat mTOR!, and More mTOR links to aging theories.  In my treatise I discuss Aberrant mTOR as one of the Additional Candidate Theories of Aging. Among other actions, Rapamycin fed late in life extends lifespan in mice(ref). 

The mammalian sirtuin SIRT1 activates the same pathway that conveys longevity via calorie restriction, and SIRT1 in turn can be activated by resveratrol and other substances being developed by Sirtris Pharmaceuticals.  I have a long post on this subject in the works based on a recent visit to the Glenn Laboratory for the Science of Aging at MIT and its key leader Leonard Guarante.  A current review of research on the sirtuins over the last 10 years can be found here and discussions of calorie restriction can be found in my December 2009 blog entries Calorie restriction research roundup – Part I and Calorie restriction research roundup – Part II.   

Both laboratory and small-animal studies strongly implicate expression of NF-kappaB in aging and suggest anti-aging interventions based on the inhibition of NF-kappaB.   In my Anti-Aging Firewalls treatise the role of NF-kappaB in aging is discussed under the Programmed epigenomic changes theory of aging.  Also, see the blog posts Updates on NF-kappaB and A further update on NF-kappaB.    

The links between SIRT1 activation, mTOR signaling suppression, and inhibition of NF-kappaB   

A 2007 publication linked the two pathways in the yeast Saccharomyces cerevisiae: MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae.  “Here we show that TOR inhibition extends lifespan by the same mechanism as CR: by increasing Sir2p activity and stabilizing the rDNA locus. Further, we show that rDNA stabilization and lifespan extension by both CR and TOR signaling is due to the relocalization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1. These findings suggest that TOR and sirtuins may be part of the same longevity pathway in higher organisms, and that they may promote genomic stability during aging.”   It was already believed that SIRT1 activation extends lifespan by the same mechanism as CR(see the video).  But, it was yet to be established that a clear link exists in mammals between SIRT1 and mTOR signaling. 

A 2007 doctoral thesis revealed more about the link: Regulation Of Translation And Transcription By Sirt1: Potential Novel Mechanisms For Regulating Stress Response And Aging.  “Both SIRT1 and the target of rapamycin (TOR) are involved in age related diseases and lifespan. We demonstrate for the first time that these two pathways are interconnected. We show that SIRT1 null mouse embryonic fibroblasts (MEFs) have larger cell morphology and upregulated mTOR signaling. Furthermore, SIRT1 activator reduces, whereas SIRT1 inhibitor nicotinamide activates the mTOR pathway.  Rapamycin is effective in inhibiting mTOR activity in both SIRT1 positive and deficient cells. Finally, we show that SIRT1 physically associates with TSC2 in HeLa cells. These observations demonstrate that SIRT1 negatively regulates mTOR pathway upstream of mTOR complex-1 (TORC1), potentially, by regulating the TSC1/2 complex.” 

The same dissertation relates the expression of SIR1 to the suppression of gene activation by NF-kappaB.  “TLE1 is co-repressor for several transcriptional factors including NF-κB. We demonstrate that SIRT1 and TLE1 repress NF-κB activity and that the catalytic activity of SIRT1 may not be critical for this. Using knock-out cell lines, we further demonstrate that both SIRT1 and TLE1 are required for the down-regulation of NF-κB activity. Our results suggest that the interaction between SIRT1 and TLE1 is important for mediating repression of NF-κB activity, potentially through a deacetyalse independent mechanism.”   In the blog post A further update on NF-kappaB, I mention the role of histone deacetylation in preventing the expression of NF-kappaB.  “it involves coiling up the DNA in the neighborhood of genes so that those genes are not accessible for activation by the NF-kappaB. This appears to be the main mechanism used by curcumin, resveratrol and other dietary polyphenols for inhibition of gene activation by NF-kappaB(ref).”   Not surprisingly, SIRT1 is a powerful deacetylase and is activated by resveratrol.

The 2010 publication SIRT1 Negatively Regulates the Mammalian Target of Rapamycin is co-authored by Hiyaa Singhee Ghosh, the author of the aforementioned dissertation, and rounds out our understanding of the linkages even more completely.  “We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex.”  This publication is rich in detail and I suggest it as a good read for those of you interested in digging further.  Among the observations in this publication are:

·        “Resveratrol suppressed mTOR signaling regardless of stress or growth conditions, suggesting that inducing the catalytic activity of SIRT1 negatively regulates mTOR signaling,”

·        “Both SIRT1 and mTOR have been linked to age-associated diseases with SIRT1 activation having a protective effect, whereas inhibition of mTOR conferring a beneficial effect. For example, SIRT1 activation confers a therapeutic effect in type 2 diabetes, obesity and neurodegenerative diseases such as Alzheimer’s and amyotrophic lateral sclerosis, whereas inhibition of mTOR is protective against cardiovascular and neurological diseases, diet-induced obesity and cancer [31], [32], [33], [34], [35], [36], [37], [38]. Autophagy, a mechanism important in regulating stress response and aging is negatively regulated by mTOR [39], [40], whereas SIRT1 has been reported to activate autophagy by deacetylating several essential components of the autophagy machinery [41], ”

·        “The inverse relationship between the roles of SIRT1 and mTOR in aging-associated diseases and lifespan extension suggests a functional interrelationship between these two proteins. Our results demonstrate that SIRT1 and mTOR signaling pathways are indeed interconnected in a way that promotes stress sensing pro-survival signals, where the regulation of mTOR is mediated potentially through an interaction of SIRT1 with the TSC1-TSC2 complex,” and

·        “Resveratrol has been reported to affect insulin signaling through SIRT1 independent pathways. Consistent with these reports, our data demonstrated that at lower doses, resvetratrol regulated the mTOR pathway in a SIRT1 dependent manner. However, at higher doses, reveratrol likely activated SIRT1 independent pathways in parallel, to inhibit mTOR activity.”

Summary

So, three different theories of longevity seem to be collapsing into one: suppression of mTOR signaling, activation of SIRT1, and inhibition of expression of NF-kappaB.  Activating SIRT1 does all of these things, and this seems to be accomplishable to some extent by taking resveratrol supplements.  As time goes on, even more powerful activators of SIRT1 are likely to become available.

Why not nicotinamide? 

As a side to this discussion, I have often been asked why I do not include nicotinamide, one of the two principal forms of the B-complex vitamin niacin, in my anti-aging supplement regimen.  In fact, nicotinamide was recommended by my physician for control of cholesterol levels but I don’t take it and effectively control my cholesterol in other ways.  The answer is simple: nicotinamide is a powerful inhibitor of SIRT1 and its anti-aging activities, not only capable of negating any benefits from taking resveratrol but also stopping natural expression of SIRT1 in the body.  As stated above “The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner(ref).”  That is, nicotinamide is a pro-aging substance so I avoid it.

Mechanisms for getting stuff into and out of cells are of great importance.  A new item of research came to my attention related to substance-trafficking that goes on within cells and across cell membrane barriers.  It has relevancy in terms of several disease processes and aging.  I review the research in the context of the fascinating cell biology involved.   

Background

We humans may have around 100 trillion eukaryotic cells, complex entities consisting of many components and constantly engaged in many internal activities.   Some of these activities involve transportation of substances either within cells or from the cell’s outer membrane surface into the cell or visa-a-versa.  For those of you not familiar with cell biology I need to identify a number of cell entities that are important for understanding how substance-trafficking in cells works.  I will try to keep things as simple as possible for the purpose of this discussion.  Vesicles are little bubbles of fluid in cells surrounded by lipid bilayers that serve a number of functions including molecular cargo transportation.  Among other things, they can transport needed substances into a cell or unwanted ones out.  “Vesicles store, transport, or digest cellular products and waste. The membrane enclosing the vesicle is similar to that of the plasma membrane, and vesicles can fuse with the plasma membrane to release their contents outside of the cell. Vesicles can also fuse with other organelles within the cell.”  “ — an endosome is a membrane bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the Golgi and either continue to lysosomes or recycle back to the Golgi. — Early Endosomes Consist of a dynamic tubular-vesicular network (vesicles up to 1 µm in diameter with connected tubules of approx. 50 nm diameter). Markers include RAB5 and RAB4, Transferrin and its receptor and EEA1(ref).“  “Lysosomes are spherical organelles that contain enzymes (acid hydrolases). They break up food so it is easier to digest. — Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria(ref).”  Finally, “the Gogli apparatus (also Golgi body)^[1] is an organelle found in most eukaryotic cells.  The primary function of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the cellular endomembrane system(ref).”  OK, enough biological distinctions for now.

The new research

The February 2010 paper The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes deals with signaling that tells vesicles how fast they have to move when they are transporting substances to and from the surfaces of cells.  According to ScienceDaily (Mar. 18, 2010) “Defects in this trafficking pathway can have severe consequences, leading to numerous diseases such as high cholesterol, neuropathies, sterility and complications in immune response.– Dr. Peter McPherson and Dr. Brigitte Ritter and their colleagues have discovered how a molecule called Rab35, which acts as a switch is turned on in order to activate the fast-track recycling pathway — in which cargo that needs to be recycled back to the surface of the cell is rapidly selected and transported. — “The cells that make up our bodies are like a busy city,” says Dr. McPherson, neuroscientist at The Neuro and the co-principal investigator for the study. “The cell surface is defined by a membrane that separates its interior from the external world, like the walls or borders of a city. Within this environment, there are simultaneous trafficking pathways that transport vital nutrients, receptors and other components required for cells to function, within cargo vehicles called ‘vesicles.’ Like traffic in a city, these ‘cargo’ vesicles travel at different speeds to numerous destinations within the cell with different purposes. For example, the receptors on the cell surface that bind to cholesterol are on the fast track pathway, so that once they deliver the cholesterol inside the cell, they move back to the surface quickly so that they can pick up some more. It is therefore crucial to understand the controls and switching mechanisms of trafficking inside cells, as this system is of vital importance to the proper functioning of the body.”  

The ScienceDaily article continues “The Rab35 molecule is the trafficking switch for the fast-track or high-priority recycling pathway signaling the quick return of cargo to the cell surface membrane. It is known that Rab35 exists in two forms, ‘on’ (GTP- bound) or off (GDP- bound). When Rab35 is turned ‘on’, it allows the cargo to go back up to the cell surface. What Dr. McPherson and Dr. Ritter and colleagues have discovered is the switch that turns Rab35 on. — “In this study we identified that a particular region of the vesicle-bound protein connecden, called the DENN domain, is the ‘finger’ that flips the switch,” says Dr. Ritter. “The DENN domain connects with the Rab35 molecule and through enzymatic activity converts Rab35 from the inactive to the active form, in essence, turning on the switch.”  You can find articles relevant to the DENN domain here.  According to the new publication the DENN domain is a lipid-binding module with enzymatic guanine nucleotide exchange factor (GEF) activity for Rab35, and Rab35 controls cargo-specific recycling from early endosomes.  “The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.” 

A second February 2010 relevant e-publication ahead of print is The connecdenn family: Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.  ‘We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35 whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C-termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2.”  Clathrin is a protein which plays a major role in the formation of coated vesicles. 

The ScienceDaily article points to the importance of the new results in helping us understand healthy cell cargo transportation:  “If the finger or the switch itself is mutated or missing, cargo can’t recycle, which has dire consequences,” adds Dr. McPherson. “For example a very important cargo transported by this specific fast track recycling pathway, controlled by Rab35 is the MHC class I receptor involved in the immune system response. If a cell becomes infected by a virus, the MHC receptor is loaded with fragments of the virus that have infected the inside of a cell. The MHC receptor needs to be taken back to the cell surface quickly so that so that it can act as a signpost indicating to circulating immune cells that this particular cell has been infected by a virus and needs to be destroyed, preventing viral infection to other cells.”  If the cargo trafficking system can’t work well the immune system won’t be able to do its job well and the result will be disease and premature aging.   

So, some diseases and aging processes are consequent to problems associated with cell substance-trafficking.  New insights being developed related to the control mechanisms for the cells’ trafficking system are likely to prove very useful.

An important new research article appeared yesterday, in the online edition of Future Medicine: Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming. The study is important because it contradicts an important earlier assumption about induced pluripotent stem cells (iPSCs).  It contradicts the assumption that reverting a cell to iPSC status restores the expression of telomerase and telomere length to equivalent embryonic stem cell length. 

(Note: hiPSCs stands for human iPSCs, hESCs stands for human embryonic stem cells)

In my February 2010 blog post IPSCs, telomerase, and closing the loop in the stem cell supply chain I related “The 2009 study Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripotent Stem Cells was important in that it showed that reversion of cells to iPSC status fully restores telomerase activity to iPSCs, equivalent to that in embryonic stem cells (ESCs). “We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals.  We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Finally, reprogramming efficiency of cells derived from increasing generations of telomerase-deficient mice shows a dramatic decrease in iPS cell efficiency, a defect that is restored by telomerase reintroduction.”  The study related to mouse cells. Further, the 2009 publication Balancing Out the Ends during iPSC Nuclear Reprogramming discusses how “telomere length maintenance and long-term proliferative capacity of iPSCs is dependent on telomerase,” and concludes “Although a number of hurdles must still be cleared before iPS-based cell therapy becomes practical, the results (cited above) suggest that reprogramming of telomerase and telomeres may not be one them.” Finally, this 2009 study concluded “While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age. While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.”  All of this seems now to be largely thrown out of the window.

The new study, based on carefully contrived experimental procedures, concludes the opposite is true for human iPSC cells: they generally end up with shorter telomeres than hESCs, telomeres of lengths commensurate with those of the adult cells which were originally reverted to make the iPSCs . “Aim: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. Materials & methods: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. — Results: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09–21.1 kb) but markedly shorter TRF lengths (6.4–12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines.” 

The new study says restoring a cell to pluripotency and providing the cell with youth are two quite different things and the former does not necessarily imply the latter.  Pluripotency of an iPSC implies that, like an embryonic stem cell, the iPSC can differentiate into any somatic cell type.  Absence of youth in this case means that the restored iPSC cell could probably not lead to many generations of descendents like an embryonic stem cell could,  because its telomere lengths are short like those in old cells.   In fact, iPSC cells can be near senescent. 

As related in the new study report “– recently, the focus of reprogramming research has shifted to defined transcriptional methods, that is, the exogenous expression of transcription factors critical to germline gene expression such as MYC, KLF4, OCT4 and SOX2 [29], or LIN28, NANOG, OCT4 and SOX2 [30]. When introduced into somatic cells, varied combinations of these genes or complementing small molecules are capable of altering the differentiated state of somatic cells, leading to induced pluripotent stem (iPS) cells similar to hES cells. This facile, cost-effective and ethically nonproblematic means of potentially manufacturing a host of transplantable patient-specific cells has led to numerous studies of iPS cell pluripotency.  However, there is little research on the effects of transcriptional reprogramming on cellular aging, in particular on telomere length regulation.”   

In other words, the iPSC researchers and the telomerase researchers were paying insufficient attention to each other.  Telomerase expression and iPSC stem cells are each central to a theory of aging treated in my treatise, Telomere Shortening and Damage and Stem Cell Supply Chain Breakdown.  I have reviewed hundreds of papers related to these topics and my impression is that not paying attention to what the other guys are researching is more the general rule than the exception.  

The researchers in the new study used cells from a single genetic source to sort through what could be a very confusing situation.  “ — In the case of human cells, there are contradictory reports as to the aged status of reprogrammed cells [32–34]. This confusion may be due, in part, to the genetic variability in the subtelomeric ‘X’ region of telomere restriction fragments (TRFs) [35,36], often complicating comparisons of TRF length in differing genotypes. “We therefore undertook an analysis of telomere dynamics during transcriptional reprogramming in an isogenic background of the hES-derived clonal embryonic progenitor cell line EN13, such that TRF length can be measured and compared with both the starting somatic cells (EN13) and the normal hES cells with embryonic TRF length from which EN13 was obtained.” 

“We observed variable but relatively long TRF lengths in hES cell lines of 16.1–21.1 kb, but markedly shorter TRF lengths ranging from 6.4 to 12.6 kb in all of the iPS cell lines studied.  In serially passaged iPS cell lines (iPS[IMR90]‑1, iPS[IMR90]‑4 and iPS[foreskin]‑1), TRF length progressively shortened during propagation.” — iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines.” On the other hand, the researchers discovered that one of six cloned lines of iPSC cells they generated expressed telomerase and that in that particular cell line telomeres got longer in successive cell generations until they got to be comparable to the lengths in hES cells. “In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES‑derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells.  Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. Conclusion: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.” It is interesting that for mouse iPSCs the same thing is observed to happen.  The iPSCs start out with short telomeres like the mature cells that were reverted had, but they express telomerase and grow longer telomeres in succeeding cell-division generations.   See the 2009 publication Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells . 

The 2010 publication Embryonic Stem Cells/Induced Pluripotent Stem Cells points out what can be important differences between hESCs and hiPSCs, probably because of the fact that the hiPSCs were, in fact, old cells with short telomeres.  “we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells and hematopoietic cells with phenotypic and morphological characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast to the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, as well as a substantially decreased hematopoietic colony forming capability. After further differentiation into erythroid cells, >1000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs.” 

What I get out of this is: 

·        Reversion of cells to hiPSC status in many cases results in cells that do not express telomerase and have short telomeres.  They are very different in these respects from hESCs.  Such cells may be approaching senescence despite their pluripotency, cannot reliably be depended on for such tasks as organ renewal, and cannot be used to close the loop in the stem cell supply chain(ref)(ref). 

·        A strain of iPSCs could express telomerase in which case the telomeres in succeeding generations could get longer.  How to reliably produce such strains remains unclear.  Telomerase can be exogenously applied in the process of generating iPSCs to provide them with longer initial telomeres but, without inherent capability to express telomerase, such iPSCs are apt to be of limited use. 

·        It appears that much more is to be learned before iPSCs with long replicative life spans can be reliably produced and used for regenerative purposes.  We need black-belt iPSC and telomerase researchers to put their heads together to figure out what can be done.

This blog entry reviews five very-recent clinical trials related to diabetes treatments, three that have failed and two that have succeeded.  I conclude by commenting on what I think are some underlying messages. 

FAILED DIABETES-RELATED CLINICAL TRIALS

I picked these items up from a March 14 New York Times story pointing to studies just published on March 14 in the New England Journal of Medicine, studies that have invalidated three favorite strategies for treating diabetics. As the article points out “An estimated 21 million Americans have Type 2 diabetes, the kind once known as adult-onset, and they are at enormous risk for heart disease. The only measures proved to reduce their chances — avoiding cigarettes and taking medication to lower bad cholesterol and blood pressure — still leave diabetics with a heart attack risk equivalent to that of a nondiabetic who has already had a heart attack.  —  So doctors began trying other strategies they hoped would help: getting blood pressure to a normal range; raising levels of good cholesterol and lowering levels of dangerous triglycerides; or modulating sharp upswings in blood sugar after a meal.”  In large-scale clinical trials, all three strategies failed.

Strategy 1: Reducing high blood pressure in diabetics will reduce incidence of heart attacks and heart attack deaths.

“Because cardiovascular risk in patients with diabetes is graded and continuous across the entire range of levels of systolic blood pressure, even at prehypertensive levels, the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) recommended beginning drug treatment in patients with diabetes who have systolic blood pressures of 130 mm Hg or higher, with a treatment goal of reducing systolic blood pressure to below 130 mm Hg(ref).^1,2,3”

According to the March 14 2010 NEJM report Effects of Intensive Blood-Pressure Control in Type 2 Diabetes Mellitus  Background There is no evidence from randomized trials to support a strategy of lowering systolic blood pressure below 135 to 140 mm Hg in persons with type 2 diabetes mellitus. We investigated whether therapy targeting normal systolic pressure (i.e., <120 mm Hg) reduces major cardiovascular events in participants with type 2 diabetes at high risk for cardiovascular events. ^— Methods A total of 4733 participants with type 2 diabetes were randomly assigned to intensive therapy, targeting a systolic pressure of less than 120 mm Hg, or standard therapy, targeting a systolic pressure of less than 140 mm Hg. The primary composite outcome was nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular causes. The mean follow-up was 4.7 years.  ^— Results After 1 year, the mean systolic blood pressure was 119.3 mm Hg in the intensive-therapy group and 133.5 mm Hg in the standard-therapy group. The annual rate of the primary outcome was 1.87% in the intensive-therapy group and 2.09% in the standard-therapy group (hazard ratio with intensive therapy, 0.88; 95% confidence interval [CI], 0.73 to 1.06; P=0.20). The annual rates of death from any cause were 1.28% and 1.19% in the two groups, respectively (hazard ratio, 1.07; 95% CI 0.85 to 1.35; P=0.55). The annual rates of stroke, a prespecified secondary outcome, were 0.32% and 0.53% in the two groups, respectively (hazard ratio, 0.59; 95% CI, 0.39 to 0.89; P=0.01). Serious adverse events attributed to antihypertensive treatment occurred in 77 of the 2362 participants in the intensive-therapy group (3.3%) and 30 of the 2371 participants in the standard-therapy group (1.3%) (P<0.001).

Conclusions In patients with type 2 diabetes at high risk for cardiovascular events, targeting a systolic blood pressure of less than 120 mm Hg, as compared with less than 140 mm Hg, did not reduce the rate of a composite outcome of fatal and nonfatal major cardiovascular events. (ClinicalTrials.gov number, NCT00000620 [ClinicalTrials.gov] .)  

Strategy 1 is shot down.  In fact, the Times article points out that many of those taking the blood pressure reducing medication were worse off because of side effects and that “A second, less rigorous study, involving 6,400 patients with Type 2 diabetes and heart disease, asked whether getting systolic blood pressure lower than 130 was any better than getting it to 130 to 140. It found that patients actually were worse off: those with the lower blood pressure ended up with a 50 percent greater risk of strokes, heart attacks or deaths.”

Strategy 2: Raising levels of good cholesterol and lowering levels of dangerous triglyceride in patients with diabetes will reduce incidence of heart attacks and heart attack deaths.

Another March 14 NEJM report is Effects of Combination Lipid Therapy in Type 2 Diabetes Mellitus.  “Background: We investigated whether combination therapy with a statin plus a fibrate, as compared with statin monotherapy, would reduce the risk of cardiovascular disease in patients with type 2 diabetes mellitus who were at high risk for cardiovascular disease.  ^— Methods We randomly assigned 5518 patients with type 2 diabetes who were being treated with open-label simvastatin to receive either masked fenofibrate or placebo. The primary outcome was the first occurrence of nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular causes. The mean follow-up was 4.7 years.

“The hypothesis that we tested in ACCORD Lipid was that in high-risk patients with type 2 diabetes, combination treatment with a fibrate (both to raise HDL cholesterol levels and to lower triglyceride levels) and a statin (to reduce LDL cholesterol levels) would reduce the rate of cardiovascular events, as compared with treatment with a statin alone.”

“By the end of the study, the mean LDL cholesterol level fell from 100.0 to 81.1 mg per deciliter (2.59 to 2.10 mmol per liter) in the fenofibrate group and from 101.1 to 80.0 mg per deciliter (2.61 to 2.07 mmol per liter) in the placebo group (Figure 1, and Section 16 in Supplementary Appendix 1). Mean HDL cholesterol levels increased from 38.0 to 41.2 mg per deciliter (0.98 to 1.07 mmol per liter) in the fenofibrate group and from 38.2 to 40.5 mg per deciliter (0.99 to 1.05 mmol per liter) in the placebo group. Median plasma triglyceride levels decreased from 189.0 to 147.0 mg per deciliter (2.13 to 1.66 mmol per liter) in the fenofibrate group and from 186.2 to 170.0 mg per deciliter (2.10 to 1.92 mmol per liter) in the placebo group.” 

“Conclusions The combination of fenofibrate and simvastatin did not reduce the rate of fatal cardiovascular events, nonfatal myocardial infarction, or nonfatal stroke, as compared with simvastatin alone. These results do not support the routine use of combination therapy with fenofibrate and simvastatin to reduce cardiovascular risk in the majority of high-risk patients with type 2 diabetes. (ClinicalTrials.gov number, NCT00000620 [ClinicalTrials.gov] .)” 

Strategy 2 is shot down. 

Strategy 3: Modulating sharp upswings in blood sugar after a meal will reduce incidence of diabetes, heart attacks and heart attack deaths among persons with impaired glucose tolerance and established cardiovascular disease or cardiovascular risk factors.“

A final study investigated the popular hypothesis that rapid rises in blood glucose after a meal were dangerous and could lead to heart disease. Many doctors were giving drugs assuming the hypothesis was correct, Dr. Nathan said(ref).”A third March 14 2010 NEJM publication Effect of Nateglinide on the Incidence of Diabetes and Cardiovascular Events reports “Background The ability of short-acting insulin secretagogues to reduce the risk of diabetes or cardiovascular events in people with impaired glucose tolerance is unknown. ^— Methods In a double-blind, randomized clinical trial, we assigned 9306 participants with impaired glucose tolerance and either cardiovascular disease or cardiovascular risk factors to receive nateglinide (up to 60 mg three times daily) or placebo, in a 2-by-2 factorial design with valsartan or placebo, in addition to participation in a lifestyle modification program. We followed the participants for a median of 5.0 years for incident diabetes (and a median of 6.5 years for vital status). We evaluated the effect of nateglinide on the occurrence of three coprimary outcomes: the development of diabetes; a core cardiovascular outcome that was a composite of death from cardiovascular causes, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure; and an extended cardiovascular outcome that was a composite of the individual components of the core composite cardiovascular outcome, hospitalization for unstable angina, or arterial revascularization.  ^— Results After adjustment for multiple testing, nateglinide, as compared with placebo, did not significantly reduce the cumulative incidence of diabetes (36% and 34%, respectively; hazard ratio, 1.07; 95% confidence interval [CI], 1.00 to 1.15; P=0.05), the core composite cardiovascular outcome (7.9% and 8.3%, respectively; hazard ratio, 0.94, 95% CI, 0.82 to 1.09; P=0.43), or the extended composite cardiovascular outcome (14.2% and 15.2%, respectively; hazard ratio, 0.93, 95% CI, 0.83 to 1.03; P=0.16). Nateglinide did, however, increase the risk of hypoglycemia.  ^—Conclusions Among persons with impaired glucose tolerance and established cardiovascular disease or cardiovascular risk factors, assignment to nateglinide for 5 years did not reduce the incidence of diabetes or the coprimary composite cardiovascular outcomes. (ClinicalTrials.gov number, NCT00097786 [ClinicalTrials.gov] .)

Strategy 3 is shot down.

SUCCESSFUL DIABETES-RELATED CLINICAL TRIALS

Not all the diabetes-related clinical trial results have been negative.  A Feb 11 2010 News Highlights report from Gen describes how “Roche Reports Positive Data from Five Phase III Trials of Type 2 Diabetes Therapy —  Roche says data from the first five Phase III trials with its once-weekly type 2 diabetes candidate taspoglutide showed that the GLP-1 analogue met its primary endpoints of reducing blood glucose levels. — “These Phase III studies have shown that treatment with once weekly taspoglutide leads to significantly improved blood glucose control, consistent weight loss, a minimal risk of hypoglycemia, and a manageable safety profile,” comments Hal Barron, Roche’s global head of product development. — The Phase III development program for taspoglutide includes eight studies that will enroll some 6,000 patients. Four of the studies have active comparators including exenatide, sitagliptin, insulin glargine, and pioglitazone.” 

A January 26 report in Gen indicates “Novo Nordisk’s Type 2 Diabetes Drug Green-Lighted by FDA — FDA sanctioned Novo Nordisk’s type 2 diabetes drug Victoza® (liraglutide) as a monotherapy in second-line treatment and in combination with other commonly prescribed oral diabetes therapeutics. U.S. approval comes less than a week after Japanese regulatory authorities gave Victoza the go-ahead. — U.S. approval of Victoza came with a Risk Evaluation and Mitigation Strategy consisting of a medication guide and a communication plan. Novo Nordisk aims to introduce the drug in the U.S. within weeks. — Victoza is a once-daily human GLP-1 (glucagon-like peptide-1) analogue and is given as an injection. “This is the first direct competition to the Byetta franchise,” according to Canaccord Adams life sciences analyst Adam Cutler. Amylin Pharmaceuticals’ Byetta and Victoza belong to the same family of drugs. “Byetta sales and prescription trends have remained flat, and we expect this news to further weaken them.” Byetta brought in $503.9 million through U.S. sales during the first three quarters of last year. ==Victoza is intended to help lower blood sugar levels along with diet, exercise, and other selected diabetes medicines. It is not recommended as initial therapy in patients who have not achieved adequate diabetes control on diet and exercise alone. –In five clinical trials involving more than 3,900 people, pancreatitis occurred more often in patients who took Victoza than in patients taking other antidiabetics, the FDA points out. The agency also advices that the therapy should be used with caution in people with a history of pancreatitis.”

COMMENTS

First, I acknowledge that these clinical trials have contributed to our incremental knowledge about what diabetes is and treatments that do and do not work.  Taspoglutide which has to be taken only once a week has the potential of becoming a blockbuster drug.  Exenatide, an existing drug in the same class, has to be taken twice daily.

Second, the clinical trial failures indicate how much we still are in an extremely expensive trial-and-error approach to treating diabetes, as I believe we are in for multiple other diseases.  Enormous amounts of money, effort and time have been poured into these clinical trials, each involving thousands of participants.  Taspoglutide alone has been scheduled for eight Phase III trials.  Ultimately these costs of hundreds of millions of dollars are reflected in the prices of approved drugs and contribute to the cost of health care.  Further, time required for the tests can keep needed drugs off the market for years.

Third, all the treatments mentioned relate to management of diabetic conditions or cardiovascular risk factors.  Such management is extremely important but lifestyle and dietary factors can be equally or even more important in preventing onset of diabetes as well as its management.  I wonder whether spending the amount of money required for these clinical trials instead on anti-obesity education might have resulted in a much greater overall positive impact.

Fourth, as time progresses, basic scientific approaches may obviate the necessity for such large-scale trial-and-error clinical trial approaches and allow smaller, faster and more focused clinical trials.  As more individual genomes are decoded and more and more diabetes whole-genome association studies are performed(ref)(ref), “one size fits all” therapies will give way to personalized ones.  (See the recent blog entry on Genome-wide association studies.) Emphasis will hopefully shift from management of diabetes to its prevention(ref).  Genomic knowledge may allow determining susceptibility of an individual to diabetes early in life and establishing a lifestyle program to assure that diabetes does not emerge in that person(ref)(ref)(ref). 

Once upon a time, an emphasis in medical treatment of people with polio was to have them live their lives in iron lung machines.  Then, basic scientific research allowed us virtually to wipe polio out.  It is time for us to do the same with Type 2 diabetes, and perhaps to let up on looking for better iron-lung type disease-management approaches.

Every once in a while I review a natural substance supplement.  This time its fucoidan, a complex sulfated polysaccharide (multi-sugar substance) found mainly in various species of brown seaweed.  Known for about 40 years for multiple health-giving biological activities, fucoidan has been the subject of hundreds of research studies.  Yet, my impression is that there is still much to be learned about this substance.  In this post I attempt to clarify what is known about fucoidan and what is still to be learned.

The species of brown seaweed containiong fucoidan include kombu, limu moui, bladderwrack, wakame, mozuku, and hijiki.  Also, variant forms of fucoidan have also been found in animal species, including the sea cucumber.  Seaweed containing fucoidan have traditionally been consumed in places like Okinawa,Hawaii andTonga, locations known for the health and longevity of their populations.  It is interesting that much of the research on fucoidan appears to have been done in Asia.  Of the hundreds of publications on fucoidan, I can cite only a small sample of representative ones here.

From a web page of the Sloan Kettering Memorial Cancer Center:

“Clinical Summary:   Fucoidan is a sulfated polysaccharide found in the cell walls of many species of Brown seaweed. Preliminary data show that fucoidan has antitumor and antiangiogenic ^{(2) (3) (4) (5) (6) (7)} effects in vitro. These effects are brought about by stimulating natural killer cells and by down regulating AP-I involved in cellular proliferation. Fucoidan also exhibited neuroprotective effects ^{(11) (12)}, but human data is lacking.   In other studies, fucoidan demonstrated anticoagulant ^{(8) (9)} and antithrombotic ⁽¹⁰⁾ activities, and can have additive effects when taken with anticoagulants.”

“Mechanism of Action:  Fucoidan has been shown to inhibit metastasis by preventing adhesion of tumor cells to the extracellular matrix. This is achieved by blocking the fibronectin cell-binding domain, necessary for formation of adhesion complexes ⁽⁴⁾. Fucoidan was also shown to induce apoptosis of human T-cell leukemia virus type I (HTLV-1) that causes Adult T-cell leukemia. It does so by inactivating NF-kB that regulates antiapoptotic proteins. It suppresses AP-I, a transcription factor involved in cellular proliferation and transformation ⁽³⁾. An vitro study showed that Fucoidan can suppress angiogenesis induced by Sarcoma 180 cells in mice ⁽⁵⁾. Fucoidan has immunomodulating effects and enhanced the activity of NK cells, which play a crucial role in mediating tumor cell death ⁽²⁾. The neuroprotective effects of fucoidan are attributed to its ability to suppress tumor necrosis factor-alpha (TNF-alpha)- and interferon-gamma (IFN-gamma)-induced NO production in C6 glioma cells ⁽¹¹⁾ and to its antioxidative effects ⁽¹²⁾.”

Fucoidan and cancers

The 2005 paper Fucoidan extracted from Cladosiphon okamuranus Tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells is a cell and mouse-level study.  “Further analysis showed that fucoidan inactivated NF-kappaB and activator protein-1 and inhibited NF-kappaB-inducible chemokine, C-C chemokine ligand 5 (regulated on activation, normal T expressed and secreted) production, and homotypic cell-cell adhesion of HTLV-1-infected T-cell lines. In vivo use of fucoidan resulted in partial inhibition of growth of tumors of an HTLV-1-infected T-cell line transplanted subcutaneously in severe combined immune deficient mice. Our results indicate that fucoidan is a potentially useful therapeutic agent for patients with ATL.”.

The 2009 paper Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12 described yet-another cell-level study.   “The aim of this study is to assess whether fucoidan — exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC50) of 2.0 and 4.0 mg/ml, respectively.”

The 2009 paper Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells describes a pre-clinical study on colon cancer cells.  “These findings provide evidence demonstrating that the pro-apoptotic effect of fucoidan is mediated through the activation of ERK, p38 and the blocking of the PI3K/Akt signal pathway in HCT-15 cells. These data support the hypothesis that fucoidan may have potential in colon cancer treatment.”

The 2009 paper Fucoidan induces apoptosis through activation of caspase-8 on human breast cancer MCF-7 cells describes another cell-level study. “ — we investigated the effect of fucoidan on the induction of apoptosis in human breast cancer MCF-7 cells. Our data demonstrated that fucoidan reduced the viable cell number of MCF-7 cells in a dose- and time-dependent manner. In contrast, fucoidan did not affect the viable cell number of normal human mammary epithelial cells. Results from the apoptosis assay demonstrated that fucoidan induced internucleosomal DNA fragmentation, chromatin condensation, activation of caspase-7, -8, and -9, and cleavage of poly(ADP ribose) polymerase.”  Also see Intracellular signaling in the induction of apoptosis in a human breast cancer cell line by water extract of Mekabu

The 2009 paper Fucoidan-Vitamin C complex suppresses tumor invasion through the basement membrane, with scarce injuries to normal or tumor cells, via decreases in oxidative stress and matrix metalloproteinases, describes yet-another cell level study, this time involving fucoidan in combination with vitamin C.  “Invasion of human fibrosarcoma cells HT-1080 through the basement membrane was repressed by Fucdn-VC-IB of non-cytotoxic concentrations without significant inhibition to human skin dermal fibroblasts DUMS-16 cells. Fucdn-VC-IB suppressed the invasiveness-related gelatinases MMP-2/9, and diminished reactive oxygen species inside the cytoplasm around the nucleus, in HT-1080 cells as shown by electrophoretic zymography and the redox indicator NBT assay, respectively. Thus Fucdn-VC-IB markedly exhibits antioxidant and MMP-suppressing activities and preferentially inhibited tumor invasion without cytotoxicity to normal cells, and is suggested as a potent tumor-invasion suppressor.”

The 2007 paper Antitumor and antimetastatic activity of fucoidan, a sulfated polysaccharide isolated from the Okhotsk Sea Fucus evanescens brown alga describes a mouse-based study.  “Antitumor and antimetastatic activities of fucoidan, a sulfated polysaccharide isolated from Fucus evanescens (brown alga in Okhotsk sea), was studied in C57Bl/6 mice with transplanted Lewis lung adenocarcinoma. Fucoidan after single and repeated administration in a dose of 10 mg/kg produced moderate antitumor and antimetastatic effects and potentiated the antimetastatic, but not antitumor activities of cyclophosphamide. Fucoidan in a dose of 25 mg/kg potentiated the toxic effect of cyclophosphamide.”

Fucoidan, neurogenesis and neurological diseases

A 2010 Japanese paper Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus suggests the interesting result that fucoidan can promote neurogenesis from neural stem progenitor cells. “Our results showed that HS (haishen, a highly sulfated fucoidan) alone increased NSPC viability in a dose-dependent manner. Moreover, HS acted synergistically with fibroblast growth factor-2 (FGF-2) but not epidermal growth factor (EGF) to enhance the proliferation of NSPCs. Finally, HS did not induce apoptosis of NSPCs. Our findings suggest that HS can serve as an adjuvant for promoting the proliferation of NSPCs.”  Again, this is a cell-level study.

The 2009 publication Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK, JAK/STAT, AP-1 and IRF-1, and depends on up-regulation of scavenger receptor B1 expression in TNF-alpha- and IFN-gamma-stimulated C6 glioma cells is quite technical but interesting.  “In neurodegenerative disorders, activated glial cells overproduce nitric oxide (NO), which causes neurotoxicity. Inducible NO synthase (iNOS) is a potential therapeutic target in neurodegenerative diseases. Here, we examined the action of fucoidan, a high-molecular-weight sulfated polysaccharide, on tumor necrosis factor-alpha (TNF-alpha)- and interferon-gamma (IFN-gamma)-induced NO production in C6 glioma cells. Fucoidan suppressed TNF-alpha- and IFN-gamma-induced NO production and iNOS expression. In addition, fucoidan inhibited TNF-alpha- and IFN-gamma-induced AP-1, IRF-1, JAK/STAT and p38 mitogen-activated protein kinase (MAPK) activation and induced scavenger receptor B1 (SR-B1) expression. — The present results also suggest that fucoidan could be a potential therapeutic agent for treating inflammatory-related neuronal injury in neurological disorders.”

Fucoidan and Alzheimer’s disease

 

Fucoidan could conceivably play a role in the treatment of Alzheimer’s disease, as suggested by the paper Functional and physical interactions between Formyl-Peptide-Receptors and Scavenger Receptor MARCO and their involvement in Amyloid beta 1-42 (Abeta1-42)-induced signal transduction in Glial Cells.  “ — we have demonstrated a functional interaction between FPRL1 and scavenger receptors in fucoidan-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition, co-immunoprecipitation data and fluorescence microscopy measurements revealed a physical interaction between FPR, FPRL1 and MARCO. These results suggest that FPRL1 plays a pivotal role for Abeta1-42-induced signal transduction in glial cells and the interaction with MARCO (which is promoted by fuciodan)could explain the broad ligand spectrum of formyl peptide receptors.” Again, this is a cell-level study.

The 2005publication Fucoidan inhibits cellular and neurotoxic effects of β-amyloid (Aβ) in rat cholinergic basal forebrain neurons reports on a cell-level study.  The publication states “Fucoidan also attenuated Aβ-induced down-regulation of phosphorylated protein kinase C. Aβ1−42-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, Aβ activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block Aβ-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against Aβ-induced neurotoxicity in basal forebrain neuronal cultures.”

Fucoidan and Parkinson’s disease

 

The 2009 publication Fucoidan protects against dopaminergic neuron death in vivo and in vitro concludes  “– we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP(+)-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson’s disease via its antioxidative activity.”

 

Fucoidan and herpes simplex

 

The 2008 study Defensive effects of a fucoidan from brown alga Undaria pinnatifida against herpes simplex virus infection reports “Fucoidan, — was previously shown to be a potent inhibitor of the in vitro replication of herpes simplex virus type 1 (HSV-1). HSV-1 is a member of herpes viruses that cause infections ranging from trivial mucosal ulcers to life-threatening disorders in immunocompromised hosts. — The production of neutralizing antibodies in the mice inoculated with HSV-1 was significantly promoted during the oral administration of the fucoidan for 3 weeks. These results suggested that oral intake of the fucoidan might take the protective effects through direct inhibition of viral replication and stimulation of both innate and adaptive immune defense functions.”  Being a mouse study, the results are a step closer to having clinical relevancy than the results of cell-level studies such as cited above. 

Clinical trials related to fucoidan 

·        A pilot clinical trial testing fucoidan as an oral anti-coagulant was reported last year Pilot clinical study to evaluate the anticoagulant activity of fucoidan.  The outcome was negative. “Thus, fucoidan in the form used in this study does not seem to have an oral anticoagulant activity, but it has a very strong in-vitro anticoagulant activity.”

 

·         A tiny and highly specialized clinical trial is reported in the publication Fucoidan ingestion increases the expression of CXCR4 on human CD34+ cells. The conlusion was “Oral fucoidan significantly amplified the CXCR4+ HPC (hematopoietic progenitor stem cells) population. The ability to mobilize HPC using sulfated polysaccharides and mobilize more HPC with high levels of CXCR4 could be clinically valuable.”·          

·        There is a Phase 1 clinical trial now in the recruiting stage Phase 1 Dosing Study of BAX 513 in Healthy Volunteers.  Bax 513 is a fucoidan extract of Laminaria japonica.  “A Phase 1 Study to Evaluate the Effects of BAX 513 on Hemostatic Parameters in Healthy Volunteers.”  This is possibly the initial clinical trial in a series of them sponsored by big pharma companies

 

My take on fucoidan

 

·        Based on cell-level and some mouse studies the substance appears to have possible therapeutic potential in many dimensions.  Fucoidan has only recently become the subject of clinical investigations, however.  So, despite the long-familiarity with the substance its utility as a human therapy remains largely unexplored.

·        As conventional drug-discovery approaches become less productive, fucoidan is one of the natural substances pharmaceutical companies are starting to investigate.  We may expect soon to see clinical trials of proprietary fucoidan compounds for specific disease indications.

·        There appears to be an active market for fucoidan supplements with many sellers(ref).  As of this moment, however, I am unclear whether or not fucoidan belongs in an anti-aging supplement regimen. 

I expect we will be hearing a lot more about fucoidan as time progresses

My recent blog posting New views of Alzheimer’s disease and new approaches to treating it describes recent research relating to the disease and new understanding of what creates it.  This week the Alzheimer’s Association has released a new report that describes the social consequence of the disease: 2010 Alzheimer’s Disease Facts and Figures.  I cover a few highlights from that report in this blog entry, relate the facts and figures to what is known about the cause of the devastating disease,  and offer an opinion of what we should do as a society about Alzheimer’s disease and senile dementia.

Facts and Figures

• About 16.2 million Americans are now deeply affected by Alzheimer’s, 5.3 million who have the disease and another 10.9 million whose lives are wrapped up in caring for those who have the disease.

“In 2009, an estimated 10.9 million family members and friends provided unpaid care for a person with Alzheimer’s disease or another dementia.”  About 94 % of unpaid caregivers are family members; about 60% are women and about 60% are aged over 50.  The quality of the lives of many of these unpaid caregivers is seriously impacted by their caregiver responsibilities. 40% of caregivers of Alzheimer’s disease patients report high emotional stress compared to 28% of caregivers of other older people. “Because Alzheimer’s and other dementias usually progress slowly, most caregivers spend many years in the caregiving role. At any point in time, 32 percent of family and other unpaid caregivers of people with Alzheimer’s and other dementias have been providing help for five years or longer, including 12 percent who have been providing care for 10 years or longer. An additional 43 percent have been providing care for one to four years, and 23 percent have provided care for less than a year.”  (Quotes here are from the report)

• The total current economic impact of the disease is $316 billion, $172 of which is for paid care of those having the disease and $144 billion is for the effort of unpaid caregivers.  (Note that these figures do not take into account another very large amount for lost economic productivity of those having the disease.)

According to the report, payments for health and long-term care services for people with Alzheimer’s will total $172 billion this year. Unpaid caregivers provided 12.5 billion hours of care in 2009, valued at $144 billion with care valued at $11.50 per hour.  It is pointed out that this is more than the federal government spends on Medicare and Medicaid combined for people with Alzheimer’s and other dementias.

• More than any other disease, Alzheimer’s and other dementias are signature diseases of old age. Death rates rise precipitously with age.

Death rate per 100,000Age                

                         2000                 2006

45–54            0.2                        0.2

55–64            2.0                        2.1

65–74            18.7                   20.2

75–84            139.6               175.6

85+                  667.7              848.3 

Other diseases of old age do not present such a steep rise in the death rate with increasing age.  “To put such age-related differences into perspective, for U.S. deaths in 2006, the differences in total mortality rates from all causes of death for those aged 65–74 and those aged 75–84 was 2.5-fold, and between the 75–84 age group and the 85 and older age group, 2.6-fold. For diseases of the heart, the differences were 2.8-fold and 3.2-fold, respectively.  For all cancers, the differences were 1.7-fold and 1.3-fold respectively. The corresponding differences for Alzheimer’s were 8.7-fold and 4.8-fold.  This is because Alzheimer’s is most likely caused by cell senescence of microglia, increasing cell senescence being a normal consequence of the aging process.  Getting Alzheimer’s is part of normal aging.  It is not necessarily caused by a bacterium or virus and does not necessarily require a gene defect although it could be triggered by such conditions.

·        There is currently no treatment for the disease 

“No treatment is available to slow or stop the deterioration of brain cells in Alzheimer’s disease. The U.S. Food and Drug Administration has approved five drugs that temporarily slow worsening of symptoms for about six to 12 months, on average, for about half of the individuals who take them.”  Puny results for expensive drugs is all the pharmaceutical industry has been able to provide at this point. 

·        While the risk of death due to other diseases continues to decrease with time, the risk of death due to Alzheimer’s disease is rapidly increasing. 

Between 3000 and 2006 causes of death % changes:

Alzheimer’s disease           +46.1%

Stroke                                    -18.2%

Prostate cancer                    -8.7%          

Heart disease                       -11.1%

HIV                                         -16.3%

The report projects 500,000 new cases of Alzheimer’s will be diagnosed this year. The report estimates that almost a million new cases of Alzheimer’s will be diagnosed annually by 2050.

• Medicare costs for Alzheimer’s patients are almost three times higher than for other older people and Medicaid costs are almost nine times higher.

“Hospital: In 2004, Medicare beneficiaries aged 65 and older with Alzheimer’s and other dementias were 3.1 times more likely than other Medicare beneficiaries in the same age group to have a hospital stay.  — Skilled nursing facility: In 2004, Medicare beneficiaries aged 65 and older with Alzheimer’s and other dementias were eight times more likely than other Medicare beneficiaries in the same age group to have a Medicare-covered stay in a skilled nursing facility.”

• The disease affects women more than men and blacks and Hispanics more than whites.
  • Blacks are roughly twice as likely to get the disease compared to whites.
  • Hispanics  are roughly 1.5 times as likely to get the disease compared to whites.
  • Women are more or less 1.7 times or more likely to get the disease then men, depending on the age group.
• Alzheimer disease patients are likely to have concurrent medical issues

Percentage withAlzheimer’s or OtherDementia and theCoexisting Condition       

Hypertension                                                    60%

Coronary heart disease                                  26%

Stroke—late effects                                         25%

Diabetes                                                             23%

Osteoporosis                                                     18%

Congestive heart failure                                16%

Chronic obstructive pulmonary disease     15%

Cancer                                                                 13%

Parkinson’s disease                                            8%

Data is for 2004 medicare beneficiaries aged 65 and older

What more can be done about Alzheimer’s disease?

I would like to see a few things shifted.

1.     I think it would be beneficial to stop viewing Alzheimer’s disease as yet another disease we are seeking to cure and start viewing AD as intrinsically wrapped up with aging, one of several aging processes that gets ahead of the other aging processes in AD patients.  The present find-a-specific-cure viewpoint has not worked and can’t work because AD is due to cell senescence of microglia. It is due to a process that is intrinsic to aging itself.  In fact, if we could delay all other age-related diseases, then AD would get virtually everybody by age 125.  In other words, curing AD and “curing aging” are likely to be part and parcel of the same thing.

2.     Major shifts in AD research are in order.  Efforts to find a chemical by trial-and-error that is a partial or entire cure, such as pharmaceutical companies have been pursuing for decades, are a modern form of alchemy rather than scientific research.  Any research that is going to move us forward has to look at the fundamental molecular biological and genetic-genomic processes involved. 

3.     I suspect that current research and clinical trials aimed at reducing or eliminating tau tangles and amyloid-beta plaques in AD patients(ref) may lead to therapies that slow the progress of AD but are unlikely to produce cures because they do not address the root cause of the disease.

4.     I think the research cited in the blog entry New views of Alzheimer’s disease and new approaches to treating it points its fingers at microglial cell senescence as a root cause for AD.  Additional research I have reviewed since writing that blog entry confirms this conclusion.  Therefore, any treatment that fundamentally addresses AD must address cell senescence and/or failure of cell replacement through stem cell differentiation.  If a treatment works to significantly delay or prevent AD it is likely to work to significantly delay or prevent many other diseases and processes of aging as well.  The answer may lie in a more effective form of telomerase activation(ref), in reversing epigenomic markers of aging such as DNA methylation at selected promoter sites(ref) or in manipulating the stem cell supply chain probably using induced pluripotent stem cells(ref) or embryonic stem cells(ref).  Perhaps activation of the BDNF gene could be involved(ref).  Those are the kinds of basic research that should be encouraged.

5.     Another shift in aging and Alzheimer’s research is that there should be lots more of it. The report says “for every $25,000 the government spends on care for people with Alzheimer’s and dementia, it spends only $100 for Alzheimer research.”  This is an order-of-magnitude too little.

6.     Finally it is important to reiterate that a number of approaches are available to ordinary people that can usually delay onset of Alzheimer’s disease, other forms of dementia and other diseases resulting from cell senescence.  These are the measures in the anti-aging firewalls in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.  Many of my blog entries are also relevant such as Warding off Alzheimer’s Disease and things in my diet and Seven Ps of health and longevity.  And a search of blog entries using the term dementia will turn up several additional relevant blog entries.

Vitamin D is much in the news nowadays.  Over the last 10 years it has become increasingly clear that vitamin D plays several important roles beyond those involved in bone health.  Vitamin D presence or absence is implicated in several disease processes including tuberculosis and cancer and an adequate level of vitamin D is required for immune system responsiveness.  A just-published research study reveals a critical way in which vitamin D is involved in the immune response.  This blog entry reviews a little background on vitamin D3, what is known about the vitamin D receptor (VDR) protein, the new research, and implications for health and longevity.

Vitamin D and the VDR

Vitamin D is mentioned several times in my treatise and is a central pillar of the combined anti-aging firewalls Supplement Regimen.    I have also discussed or mentioned it in many` blog posts.  The post Vitamin D – don’t fall for it  describes research on how vitamin D administration in nursing communities significantly reduces the number of falls and consequent hip fractures and other orthopedic injuries.  The blog post Klotho anti-aging gene in the news describes how “Klotho expression is also important for averting premature aging due to overexpression of Vitamin D,” citing a number of papers relevant to that topic.  And the post Hypervitaminosis D and premature aging indicates, among other topics, how mice with their VDR gene knocked out age prematurely.  “The publication Premature aging in vitamin D receptor mutant mice  (vitamin D receptor (VDR) knockout mice)  states “Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts.”  “Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D(3), our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D(3) homeostasis regulates physiological aging.”

Vitamin D3 (1alpha,25-dihydroxyvitamin D) is the active form of vitamin D taken in supplements and is of concern in this post.  It functions as a hormone.  D3 is also known as calcitriol.  A growing body of research publications point to the health importance of D3 in numerous biological pathways that go beyond bone health.  I cite only two as examples.  According to the 2008 publication The noncalciotropic actions of vitamin D: recent clinical developments, “RECENT FINDINGS: 1,25-Dihydroxyvitamin D stimulates the innate immune system, facilitating the clearance of infections such as tuberculosis. Hypovitaminosis D has been associated with several autoimmune disorders, various malignancies, and cardiovascular risk factors in a number of recent epidemiologic reports. Based on these observational reports, vitamin D and its analogues are being evaluated for the prevention and treatment of a variety of conditions, with early findings showing mixed results. SUMMARY: The broad tissue distribution of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme and the vitamin D receptor establish a role for 1,25-dihydroxyvitamin D in the pathophysiology of various disease states and provide new therapeutic targets for vitamin D and its analogues.”

A 2007 publication Expanding role for vitamin D in chronic kidney disease: importance of blood 25-OH-D levels and extra-renal 1alpha-hydroxylase in the classical and nonclassical actions of 1alpha,25-dihydroxy vitamin D(3) reports “Recent advances in the understanding of vitamin D have revolutionized our view of this old nutritional factor and suggested that it has much wider effects on the body than ever believed before. In addition to its well-known effects on calcium/phosphate homeostasis, vitamin D, through its hormonal form, 1alpha,25-dihydroxyvitamin D(3) or calcitriol, is a cell differentiating factor and anti-proliferative agent with actions on a variety of tissues around the body (e.g., skin, muscle, immune system). By influencing gene expression in multiple tissues, calcitriol influences many physiological processes besides calcium/phosphate homeostasis including muscle and keratinocyte differentiation, insulin secretion, blood pressure regulation, and the immune response. The incidence of various diseases including epithelial cancers, multiple sclerosis, muscle weakness as well as bone-related disorders has been correlated with vitamin D deficiency/insufficiency and has led to a re-evaluation of recommended daily intakes both in the normal subject and CKD patient.”

Even back in the early 2000s it was clear that D3 was important for the functioning of the immune system, but how and why was unclear.  The 2004 publication Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system  reported “The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to inhibit the development of autoimmune diseases, including inflammatory bowel disease (IBD). Paradoxically, other immune system-mediated diseases (experimental asthma) and immunity to infectious organisms were unaffected by 1,25(OH)2D3 treatment. There are similar paradoxical effects of vitamin D deficiency on various immune system functions. Vitamin D and vitamin D receptor (VDR) deficiency resulted in accelerated IBD.

”The vitamin D3 receptor (VDR) gene generates a protein which is a transcription factor that is activated by the presence of D3. “The calcitriol receptor, also known as the vitamin D receptor (VDR) and also known as NR1I1 (nuclear receptor subfamily 1, group I, member 1), is a member of the nuclear receptor family of transcription factors.^[1] Upon activation by vitamin D, the VDR forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA resulting in expression or transrepression of specific geneproducts. In humans, the vitamin D receptor is encoded by the VDR gene.^[2]”  Exploration of the activities of VDR has been key to discovering the various roles of vitamin D3 in maintaining health.

Vitamin D3 in the immune response

The new 7 March 2010 publication Vitamin D controls T cell antigen receptor signaling and activation of human T cells reports an important finding – Presence of vitamin D3 in the bloodstream is required for T cells being activated and mounting an effective immune response.  As reported yesterday in Science Daily: “For T cells to detect and kill foreign pathogens such as clumps of bacteria or viruses, the cells must first be ‘triggered’ into action and ‘transform’ from inactive and harmless immune cells into killer cells that are primed to seek out and destroy all traces of a foreign pathogen. — The researchers found that the T cells rely on vitamin D in order to activate and they would remain dormant, ‘naïve’ to the possibility of threat if vitamin D is lacking in the blood.”  The sequence of events is “First when the naive T cell recognizes foreign invaders like bacteria or viruses with T cell receptor (TCR), it sends activating signals – to the vitamin D receptor gene. The VDR gene then starts producing DVR protein, which binds vitamin D in the T cell – and becomes activated. Then the vitamin D bound and activated DVR gets into the cell nucleus and activates the gene for PLC-gamma1 (5), which in turn produces PLC-gamma1 protein – and “the T cells can get started(ref).””

““Professor Carsten Geisler from the Department of International Health, Immunology and Microbiology explains that “when a T cell is exposed to a foreign pathogen, it extends a signaling device or ‘antenna’ known as a vitamin D receptor, with which it searches for vitamin D. This means that the T cell must have vitamin D or activation of the cell will cease. If the T cells cannot find enough vitamin D in the blood, they won’t even begin to mobilize. ” — T cells that are successfully activated transform into one of two types of immune cell. They either become killer cells that will attack and destroy all cells carrying traces of a foreign pathogen or they become helper cells that assist the immune system in acquiring “memory”. The helper cells send messages to the immune system, passing on knowledge about the pathogen so that the immune system can recognize and remember it at their next encounter. T cells form part of the adaptive immune system, which means that they function by teaching the immune system to recognize and adapt to constantly changing threats(ref).”

Implications

The new finding can explain a lot of the protective effects of D3,  ranging from reducing susceptibility to colds and flu(ref) to the role of D3 in chronic kidney disease(ref), reducing risk of asthma and respiratory infections(ref), its role in Graves’ hyperthyroidism(ref), how it fights placental infection(ref), bacterial vaginosis(ref) and pediatric infections(ref), and its protective effects against many other infections too numerous to catalog here.  And of course, vitamin D is essential for prevention and control of cardiovascular diseases(ref).  Unless someone is out in the sunlight for sustained periods every day, vitamin D3 supplementation is an absolute health necessity.

Please note the medical disclaimer for this blog.

A recent research report indicates that sestrin proteins can inhibit age-related pathologies in fruit flies and contribute to their longevity.  The genes and biological pathways involved exist also in humans.  This blog post reviews what the sestrin genes are, what the sestrin proteins do,  the new research describing the channels through which sestrins promote longevity, and how sestrins protect against cancer.

About sestrins

Sestrins are small protein molecules produced by three evolutionary conserved  genes in mammals, of which the sesn1 and sesn2 genes are activated by the p53 tumor-suppressor gene(ref)(ref).  Normally, when oxidative or other forms of stress activate p53, the target sesn1 and sesn2 genes are activated in turn.  These genes in turn activate AMP-dependent protein kinase (AMPK), which serves to inhibit  the Target of Rapamycin. E.g. inhibit  mTOR signaling(ref). Sestrins also provide a level of antioxidant defense in cells(ref). The 2007 publication p53 Target Genes Sestrin1 and Sestrin2 Connect Genotoxic Stress and mTOR Signaling provides ample detail for those of you who may wish to dig deeper.

Sestrins and longevity

The actions of sestrins are interesting from the viewpoint of longevity because for some time it has been known that inhibiton of mTOR signaling can lead to longer lifespans.  See the earlier blog post More mTOR links to aging theories “In my May 2009 blog post Longevity genes, mTOR and lifespan, I discussed the mTOR signaling pathway in mammals, its role in diseases, the relationship of mTOR to mitochondrial activity and how inhibiting mTOR could conceivably be a strategy for extending longevity.  In my Anti-Aging Firewalls treatise I subsequently added ABERRANT mTOR SIGNALLING as one of six additional candidate theories of aging to be considered.” See also the blog entry Viva mTOR! Caveat mTOR!.  Note that the m in mTOR stands for mammalian.“

The role of AMPK in regulating cellular energy charge places this enzyme at a central control point in maintaining energy homeostasis. More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients(ref).”  Exercise can increase AMPK activity as can taking certain polyphenol supplements like resveratrol(ref).  Another of the conditions that can activate AMPK is calorie restriction, a condition that slows down aging.  On the other hand, over-nutrition activates mTOR, accelerating aging.

The latest result appeared in the March 5 issue of Science: Sestrin as a Feedback Inhibitor of TOR That Prevents Age-Related Pathologies.  “We show that the abundance of Drosophila sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species that cause activation of c-Jun amino-terminal kinase and transcription factor Forkhead box O (FoxO).  Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids.”  In other words, if TOR gets ramped up, in the absence of genetic damage it turns on sestrin which ramps TOR down again before TOR activity creates major damage.

According to a Science Daily article about this latest research, “They also showed that Sestrin, whose structure and biochemical function are conserved between flies and humans, is needed for regulation of a signaling pathway that is the central controller of aging and metabolism. – The new study took advantage of the finding that the fruit fly Drosophila, whose AMPK-TOR signaling pathway functions in the same manner as its mammalian equivalent, contains a single Sestrin gene. Using a variety of genetic techniques, first author Jun Hee Lee inactivated the Sestrin gene of Drosophila and found that although Sestrin-deficient flies do not exhibit any developmental abnormalities, they suffer from under-activation of AMPK and over-activation of TOR — confirming that Sestrin is needed for keeping this pathway in check. Most importantly, the biochemical imbalance incurred by loss of Sestrin expression resulted in several age-related pathologies. — “Strikingly, the pathologies caused by the Sestrin deficiency included accumulation of triglycerides, cardiac arrhythmia and muscle degeneration that occurred in rather young flies,” said Karin. “These pathologies are amazingly similar to the major disorders of overweight, heart failure and muscle loss that accompany aging in humans.” — Lee and colleagues at UC San Diego and the Sanford-Burnham Institute in La Jolla, California, went on to demonstrate that feeding flies with drugs that either activate AMPK or inhibit TOR conferred protection against most of these early aging, degenerative symptoms. The researchers also found that over-activation of TOR is likely to accelerate aging of heart and skeletal muscles by disrupting an important “quality control” process called autophagy. Autophagy allows cells to rid themselves of and replace damaged mitochondria, the little power plants that provide all cells, especially muscles, with energy. However, when mitochondria get old, they produce high concentrations of reactive oxygen species (ROS), or free radicals, that can lead to tissue damage.”

I have previously discussed how an effective antiaging intervention might be inhibition of mTOR.  The new research goes on to say how this might be accomplished through activation of sestrins. 

Sestrins and cancer

In many cancers, oncogenic mutations in RAS genes result in inactivation of p53 genes and resulting failure of activation of sestrin genes which results in increased  ROS (reactive oxygen species) levels and further oncogenic mutations.  This scenario is depicted in the 2007 paper Repression of Sestrin Family Genes Contributes to Oncogenic Ras-Induced Reactive Oxygen Species Up-regulation and Genetic Instability.  “Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. — Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. — Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53.”