Sven's Science Column

Geroprotector review: Rapamycin

Tue, 18 Jul 2017 20:50:00 +0000

Rapamycin and other mTOR inhibitors as geroprotectors
This article is solely for information purposes, not a substitute for professional medical or dietary advice. The provisos of the LongeCity user agreement apply.

What: A drug to prevent rejection of transplanted kidneys. Also used in cancer therapy. Various related compounds (‘-olimus’ /’rapalogs’)
Lifespan: Increase in yeast, roundworm, fruit flies, and rodents,
Cancer: Potentially reduced risk in nonmelanoma skin cancers. Maybe increased risk for prostate cancers.
Heart disease: Prevention of atherosclerosis, reduction in stroke severity, possible improvement in heart function.
Alzheimer’s disease: Protective in a mouse model of Alzheimer’s disease.
Mechanism: Inhibition of mTORC1 leading to upregulation of authophagy and downregulation of protein and lipid synthesis and metabolism. Attenuation of cellular senescence and senescence-associated secretory phenotype. May prevent progerin accumulation, age related changes in the epigenome and improve hematopoietic stem cell function.
Side effects: Main side effects are insulin resistance, glucose intolerance and hyperlipidemia, decreased immune function, delayed wound healing and mouth ulcers. However, it has been argued that the insulin resistance, glucose intolerance and hyperlipidemia may be benign. Improved immune response to vaccination in elderly humans but worsens infection risk and outcome of acute infections.

Introduction

Rapamycin, also known as Sirolimus, is an immunomodulatory compound used to prevent rejection of transplanted kidneys. It was first isolated in 1972 by Surendra Nath Sehgal and colleagues from a strain of the bacterium Streptomyces hygroscopicus isolated from soil samples from Easter Island. The native name for Easter Island is Rapa Nui hence the name rapamycin. It was originally developed as an antifungal drug. When the immunosuppressive effects were discovered, it opened up a new line of research in preventing the rejection of transplanted organs (Arriola Apelo and Lamming, 2016). Since then, many derivatives of rapamycin have been developed including temsirolimus, everolimus, ridaforolimus, zotarolimus, umirolimus and others, collectively known as rapalogs (Figure 1).

(click to enlarge) Figure 1 Some examples of rapalogs. The difference is highlighted in yellow.

Dose and dosage schedule

We still have not discovered the optimal rapamycin dose for lifespan extension. For example, in a dose-response study (4.7, 14 or 42 ppm) it was found that the highest dose resulted in the largest lifespan extension (Miller et al., 2014). So, it may be that many published studies have used rapamycin well below the optimal dose (Kaeberlein, 2014). One study that found that rapamycin improved survival in a mouse model of Leigh syndrome (see below) used a dose of rapamycin leading to a blood concentration of rapamycin at least 20-fold higher than what has been observed in ‘normal aging’ studies with rapamycin (Johnson et al., 2013).

In contrast , it has been found that giving rapamycin less frequently (for example once a week rather than every day) prevented some of the side effects. For example, while daily rapamycin treatment results in significant higher blood sugar levels in an oral glucose tolerance test, mice treated with rapamycin once a week had a similar blood glucose profile as those in the control group. Once a week rapamycin treatment also had a significant reduced impact on immune function (as measured by the abundance of various immune cells) compared to daily rapamycin. However, immune markers at once a week rapamycin treatment were still different from controls (Arriola Apelo et al., 2016).

The rapalogs, everolimus and temsirolimus, have a reduced impact on glucose tolerance and immune function compared to rapamycin (Arriola Apelo et al., 2016). The search for rapalogs that have an even better safety profile is still ongoing (see below).

In animal studies, rapamycin is typically administered in an micro-encapsulated formulation (EUDRAGIT®) that is mixed into the diet. Encapsulation prevents the degradation of the rapamycin by the acidic conditions of the stomach (WO 2015103447 A1). Furthermore, it is believed that the microencapsulation of rapamycin could help to prevent mouth ulcers, a common side effect observed with use of non-encapsulated rapamycin (Tardif et al., 2015).

Lifespan extension

Rapamycin has been found to increase lifespan in yeasts (Powers et al., 2006; Medvedik et al., 2007; Rallis et al., 2013), worms (Robida-Stubbs et al., 2012), and fruit flies (Bjedov et al., 2010; Moskalev and Shaposhnikov, 2010). Furthermore, the rapalog everolimus extends lifespan in fruit flies (Spindler et al., 2012).

(click to enlarge) Figure 2 Lifespan extension by rapamycin in non-transgenic rodents.

In 2009 a landmark paper was published in Nature. In this paper the results from the Interventions Testing Program (ITP) with rapamycin in middle-aged mice were reported. Rapamycin significantly extended the mean and maximal lifespan in both male and female mice. Since multiple studies have confirmed that rapamycin extends lifespan in rodents (see figure 2).

Not all studies have published data that allowed me to include them in Figure 2. Chen et al. (2009) fed rapamycin every other day to old (22-24 month) mice for 6 weeks and observed a significant reduction in mortality rate. Similarly, Neff et al. (2013) observed a significant lifespan extension upon rapamycin treatment in male C57BL/6J Rj mice.

Neff et al. (2013) measured a wide array of age-related phenotypes and found that rapamycin improved some but not others (see table 1). From this the authors concluded that rapamycin has little effect on aging. However can we really expect that a single life extending drug would beneficially impact all age-related phenotypes?

Rapamycin has been tested in various transgenic mouse models (at least 16 models so far). Lifespan was extended in most transgenic models except in a mouse model of type two diabetes and in two models of amyotrophic lateral sclerosis (ALS) (Richardson et al., 2015; Arriola Apelo and Lamming, 2016). Most impressive are the results from two studies with mice harboring mutations in APC. APC mutations lead to colorectal cancer. In these mice lifespan was extended by 140-220% (Fujishita et al., 2008) and 280-440% (Hasty et al., 2014). Of course, mice harboring mutations in APC have a strongly reduced lifespan compared to wild type mice. Interestingly, Hasty et al. (2014) observed that the APC mutant mice fed rapamycin at the highest dose (42 ppm) lived longer than the typical median lifespan reported in the literature for wild type mice! Rapamycin also increases lifespan of mice who lack one or both copies of the p53 tumor suppressor gene (Comas et al., 2012; Komarova et al., 2012).

Combination with metformin

The combination of metformin with rapamycin in the Interventions Testing Program (ITP) lead to a larger lifespan extension than either drug in isolation (Strong et al., 2016). Although, to compare rapamycin + metformin with rapamycin alone the data from the rapamycin alone group was derived from previous ITP studies. The fact that the two groups were not studied simultaneously introduces the risk that a change in some unknown variable could make this comparison invalid. In worms the combination of rapamycin with metformin each at optimal dose does not lead to any additional lifespan extension compared to either alone. However when used at suboptimal doses then the combination lead to a bigger increase in maximal but not mean lifespan compared to either alone (Dessale et al., 2017). Metformin is also a well known potential life extension drug (http://www.longecity.org/forum/blog/201/entry-3593-geroprotector-review-metformin/) and inhibition of mTORC1 (the target of rapamycin, see below) is one of the mechanisms that has been proposed to explain its effects.

Human data are scarce but one study of kidney transplant patients found that mortality was increased in those who had received a transplant from a deceased donor while mortality was unchanged in those receiving a transplant from a living donor (Knoll et al., 2014). Another meta-analysis also observed no effect from rapamycin on mortality in kidney transplant patients (Liu et al., 2017). However, these data should be interpreted with great care as mortality in the years after a transplant is more dependent on factors such as rejection of the transplanted organ than from age-related diseases.

Effect on aging phenotypes and diseases

Rapamycin treatment in mice improved some but not all phenotypes of aging (Wilkinson et al., 2012; Neff et al., 2013).

(click to enlarge) Table 1 Age-related phenotypes in rapamycin treated mice. Data from (Wilkinson et al., 2012; Neff et al., 2013; Bitto et al., 2016).

Rapalogs have been found to be protective against atherosclerosis in mouse models (Pakala et al., 2005; Mueller et al., 2008; Ma et al., 2013). Furthermore, rapamycin protected animals against experimentally-induced stroke. Rapamycin treated animals had a smaller infarct area, were protected from ischemia-induced cell death, and motoric impairment was attenuated (Sheng et al., 2010; Chauhan et al., 2011; Yang et al., 2015). Rapamycin attenuates load-induced cardiac hypertrophy in mice (Shioi et al., 2003). Rapamycin did not improve heart function as measured by echocardiography in mice (Neff et al., 2013). In contrast, ten weeks of rapamycin treatment was found to improve heart function as measured by echocardiography in companion dogs (Urfer et al., 2017). However, this was a small proof-of-principle trial with just 24 dogs. The researchers now prepare to scale up this trial and possibly run it in multiple centers around the US.

Rapamycin was found to improve memory in two mouse models of Alzheimer’s disease (Caccamo et al., 2010; Spilman et al., 2010; Majumder et al., 2011; Lin et al., 2013). Rapalog treatment may improve several of the key pathophysiological factors observed in Alzheimer’s disease.

-- rapalogs reduce tau tangles and tau hyperphosphorylation (Ozcelik et al., 2013; Jiang et al., 2014a).
-- rapalogs promote the autophagic clearance of amyloid-beta leading to lower amyloid-beta levels in the brain (Spilman et al., 2010; Jiang et al., 2014b). However, one contrasting study observed increased amyloid-beta levels in the-- brain of a mouse model of Alzheimer’s disease (Zhang et al., 2010).
-- rapalogs reduce neuroinflammation (Wang et al., 2017a).
-- rapamycin restores cerebral blood flow and vascular function in a mice model of Alzheimer’s disease (Lin et al., 2013).

Rapamycin has also been found to be neuroprotective in mice models of Parkinson’s disease (Malagelada et al., 2010; Jiang et al., 2013).

We have already discussed the potential effect of rapamycin on colon cancer (see APC mice above). Now what about other cancers? Rapamycin has been found to lower spontaneous cancer incidence in mice (Neff et al., 2013). Everolimus and temsirolimus have received FDA and EMA approval for the use as monotherapy against various cancers (Eiden et al., 2016). In a meta-analysis of human randomized, controlled clinical trials it was found that rapamycin use in people who had undergone a kidney transplant was associated with a reduced risk of cancer. However, this decrease was caused by a strong reduction in the incidence of nonmelanoma skin cancer. When this type of cancer was excluded then rapamycin no longer influenced cancer risk except for a lower risk for kidney cancer and a higher risk for prostate cancer (Yanik et al., 2015). In another meta-analysis of liver transplant recipients rapamycin was found to lower both nonmelanoma skin cancer as well as other cancers (Knoll et al., 2014).

Rapalogs are known immunosuppressive drugs and hence it would be expected that their use was associated with worse outcomes during infections. Unexpectedly, rapalogs improves the response to vaccination in mice (Jagannath et al., 2009; Jagannath and Bakhru, 2012; Keating et al., 2013) and in elderly humans (Mannick et al., 2014). Rapamycin is however deleterious during acute infections (Goldberg et al., 2014).

Other diseases

Rapamycin is beneficial in mouse models of several autoimmune diseases including (Donia et al., 2009; Esposito et al., 2010; Prevel et al., 2013).
In mice it was also found that rapamycin worsened cataract severity (Wilkinson et al., 2012).
Daily injection of rapamycin more than doubled lifespan in a mouse model of Leigh syndrome. Furthermore disease symptoms were absent in nearly half of the mice injected with rapamycin (Johnson et al., 2013).
Rapamycin is FDA approved for the treatment of the rare, progressive and systemic disease lymphangioleiomyomatosis that results in cystic destruction of the lung(link).

Mechanism of action

Rapamycin works by inhibiting the mTORC1 complex. Consistent with the fact that rapamycin extends lifespan is the observation that mutations that downregulate mTORC1 signaling extend lifespan in yeast, worms, fruit flies, and mice (Wu et al., 2013; Kaeberlein, 2014). mTORC1 is a protein complex composed of mTOR (mechanistic target of rapamycin) with several other proteins that integrates a large set up input signals such as energy status, amino acid levels, growth factors, stress, oxygen availability and when activated stimulates cell growth and proliferation by promoting protein synthesis, lipid synthesis, and metabolism while reducing autophagy (Laplante and Sabatini, 2013; Saxton and Sabatini, 2017).

The contribution of the various downstream effectors of mTORC1 on lifespan is not fully understood so far. Reducing protein synthesis by knocking out the downstream effector of mTORC1, S6K, increases lifespan in worms (McQuary et al., 2016) and female mice (Selman et al., 2009). It is also known that autophagy induction is necessary for lifespan extension by various interventions such as calorie restriction (Madeo et al., 2015). Rapamycin fails to extend lifespan in fruit flies in which the downstream targets of mTORC1 (S6K, 4E-BP and autophagy) have been made to be insensitive to mTORC1 signalling (Bjedov et al., 2010). Hence, both protein synthesis and autophagy may be necessary for rapamycin’s life-extending effects.

(click to enlarge) Figure 3 mTORC1 and mTORC2 signaling. The blunted arrows indicate inhibition. For simplicity: both complexes are drawn as monomers, in reality they form homodimers; not all upstream and downstream effectors have been listed.

mTORC1 is strongly activated by the branched-chain amino acids (leucine, isoleucine, and valine). Leucine directly inhibits Sestrin2 thereby abrogating its inhibitory action on GATOR2 leading to the activation of mTORC1 (see figure 4). The amino acid sensors that signal for mTORC1 activation have been reviewed by Goberdhan et al. (2016). New drugs to inhibit mTORC1 have been developed that make use of this upstream amino acid sensing mechanism (see below).

(click to enlarge) Figure 4 A simplified diagram illustrating the regulation of mTORC1 by leucine and growth factors. Notice that mTORC1 and Rheb are bound to the surface of the late endosome or lysosome. The intralysosomal breakdown of proteins releases free amino acids. Various amino acids (proline, arginine, glycine, and alanine) in the lysosome can signal for the activation of mTORC1 (not depicted). In addition to leucine other amino acids in the cytosol can signal for the activation of mTORC1 through mechanisms not depicted in this image.

Rapamycin suppresses cell senescence (Demidenko et al., 2009; Pospelova et al., 2012) and reduces the senescence-associated secretory phenotype in cells that are already senescent (Laberge et al., 2015; Wang et al., 2017b). Furthermore, pan-mTOR inhibitors (compounds that block both mTORC1 and mTORC2) have been shown to prevent cell senescence (Leontieva and Blagosklonny, 2016). Rapamycin treatment reversed the cellular phenotype of Hutchinson-Gilford progeria syndrome in cell culture. Furthermore, it prevented the accelerated onset of cell senescence and increased the degradation of progerin in cells from Hutchinson-Gilford progeria syndrome patients (Cao et al., 2011). Progerin accumulation, the molecular defect responsible for Hutchinson-Gilford progeria syndrome, has also been observed during ‘normal’ aging although at much lower amount (Graziotto et al., 2012). It has also been shown that rapamycin attenuates age-related changes in DNA methylation (Cole et al., 2017). Finally, rapamycin treatment restores hematopoietic stem cell function in old mice (Chen et al., 2009).

However chronic use of rapamycin may also lead to an inhibition of mTORC2 in certain tissues probably as a result of an inability of rapamycin-bound mTOR to be incorporated into new mTORC2 complexes (Schreiber et al., 2015). The inhibition of mTORC2 by chronic rapamycin treatment is a possible explanation for the glucose intolerance observed in several studies. mTORC2 is needed for full activation of Akt which in turn is needed for glucose uptake in the cell. Hence when mTORC2 is inhibited glucose uptake is also reduced.

Side effects of rapamycin

In several but not all strains of mice rapamycin treatment causes glucose intolerance and insulin resistance (Liu et al., 2014; Kennedy and Lamming, 2016). Human data similarly indicate that rapamycin causes glucose intolerance but we have to be cautious in interpretation of the human data as all of it comes from seriously ill people who are often treated with other drugs known to induce metabolic disturbances (e.g. steroids to prevent organ rejection). Surprisingly enough in a mouse study it was found that short term (2 or 6 weeks) of rapamycin had negative effects on insulin resistance and blood triglyceride levels while these spontaneously reversed after 20 weeks treatment. Furthermore, after 20 weeks of rapamycin treatment some parameters (such as insulin resistance) were even better than the control group (Fang et al., 2013). Other studies find that the glucose intolerance caused by rapamycin is quickly reversible upon cessation of treatment, although not strong enough to return to baseline levels (Schindler et al., 2014). Rapamycin improved insulin sensitivity in three out of five mouse models of type 2 diabetes (Reifsnyder et al., 2016). Fourteen months of rapamycin treatment did not cause glucose intolerance in marmoset monkeys (Ross et al., 2015). All these contradictory data make it very hard to come to any solid conclusions concerning rapamycin’s effect on glucose intolerance and insulin sensitivity. Also, not everyone agrees that the glucose intolerance and insulin resistance caused by rapamycin is harmful. Mikhail Blagosklonny argues that rapamycin mimics “starvation diabetes” (Blagosklonny, 2011; 2012; 2014).

As mentioned before, mouth ulcers are a common side effect of rapamycin treatment but these might be preventable by using an enteric-coated formulation such as EUDRAGIT® (Tardif et al., 2015). Rapamycin has also been found to delay wound healing (Kahn et al., 2005; Ekici et al., 2007; Willems et al., 2010).

Newer mTOR inhibitors

Rapamycin does not directly bind to the mTOR protein rather it first binds to a protein from the FKBP family and then the rapamycin-FKBP complex binds to mTORC1 leading to its inhibition (see figure 3). Many of the 14 known family members can form complexes with rapamycin and inhibit mTOR activity (März et al., 2013). Different cell types express different FKBP family members (Baughman et al., 1997; Rulten et al., 2006). Rapamycin-bound FKBP51 inhibits only mTORC1 and not mTORC2. In contrast rapamycin-bound FKBP12 can inhibit mTORC1 but after chronic exposure to rapamycin it also inhibits mTORC2 (see figure 3).

Delos Pharmaceuticals and Buck Institute for Research on Aging have recently filed a patent on new rapalogs that are more selective towards mTORC1 (WO 2017040341 A1).

Rather than trying to block mTOR directly Navitor Pharmaceuticals is testing an alternative approach in which a drug stimulates the inhibitory effect of Sestrin2 on GATOR2 leading ultimately to an inhibition of mTORC1. Navitor Pharmaceuticals has recently filed a patent on Sestrin2/GATOR2 modulators (WO 2017044720 A1).

However, not everyone agrees with this reasoning. Mikhail Blagosklonny argues that the hyperglycemia and hyperlipidemia caused by rapamycin treatment are benign. He has in fact taken the complete opposite strategy and is looking at pan-mTOR inhibitors that are designed to block both mTORC1 and mTORC2. Using a reversible model of senescence he was able to demonstrate that pan-mTOR inhibitors prevent geroconversion (Leontieva and Blagosklonny, 2016).

Rapamycin is currently being tested in marmoset monkeys. Marmoset monkeys have a shorter lifespan than other monkey species. Data from these experiments have confirmed that rapamycin reduces mTORC1 activity (Tardif et al., 2015) and have not observed any glucose intolerance or increase in blood lipids after 14 month treatment (Ross et al., 2015). The NIA has awarded the researchers a $2.7 million grant to investigate the effect of rapamycin on the lifespan of these monkeys (https://www.sciencedaily.com/releases/2016/02/160209105352.htm).

Conclusions
Like many potential life extension promoters, rapamycin comes from a different field of clinical activity and testing it in ‘normal aging’ is challenging and likely to reveal new benefits and long term toxicities. While there is hope for rapamycin as a broad-spectrum drug, it is unlikely to be is the groundbreaking anti-aging intervention in humans that it was sometimes made out to be, but a better understanding of the mTOR pathway that rapamycin and its derivatives inhibit is likely to play an important role in developing effective life extension strategies.

References

Anisimov VN et al. (2010). Rapamycin extends maximal lifespan in cancer-prone mice. Am J Pathol 176(5): 2092-2097.
Anisimov VN et al. (2011). Rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice. Cell Cycle 10(24): 4230-4236.
Arriola Apelo SI, Lamming DW (2016). Rapamycin: An inhibiTOR of aging emerges from the soil of Easter Island. J Gerontol A Biol Sci Med Sci 71(7): 841-849.
Arriola Apelo SI et al. (2016). Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell 15: 28-38.
Bitto A et al. (2016). Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. eLife 5: e16351.
Bjedov I et al. (2010). Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab 11: 35-46.
Blagosklonny M (2011). Rapamycin-induced glucose intolerance: hunger or starvation diabetes. Cell Cycle 10: 4217-4224.
Blagosklonny M (2012). Once again on rapamycin-induced insulin resistance and longevity: despite of or owing to. Aging (Albany NY) 4(5): 350-358.
Blagosklonny M (2014). Koschei the immortal and anti-aging drugs. Cell Death Dis 5: e1552.
Baughman G et al. (1997). Tissue distribution and abundance of human FKBP51, and FK506-binding protein that can mediate calcineurin inhibition. Biochem Biophys Res Commun 232: 437-443.
Caccamo A et al. (2010). Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and tau: effects on cognitive impairments. J Biol Chem 285(17): 13107-13120.
Chauhan A et al. (2011). Rapamycin protects against middle cerebral artery occlusion induced focal cerebral ischemia in rats. Behav Brain Res 225(2): 603-609.
Chen C et al. (2009). mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal 2(98): ra75.
Cole JJ et al. (2017). Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions. Genome Biol 18: 58.
Comas M et al. (2012). New nanoformulation of rapamycin Rapatar extends lifespan in homozygous p53-/- mice by delaying carcinogenesis. Aging 4: 715-722.
Demidenko ZN et al. (2009). Rapamycin decelerates cellular senescence. Cell Cycle 8(12): 1888-1895.
Dessale T et al. (2017). Slowing ageing using drug synergy in C. elegans. bioRxiv: 153205. http://biorxiv.org/content/early/2017/06/23/153205
Donia M et al. (2009). Treatment with rapamycin ameliorates clinical and histological signs of protracted relapsing experimental allergic encephalomyelitis in Dark Agouti rats and induces expansion of peripheral CD4+CD25+Foxp3+ regulatory T cells. J Autoimmun 33(2): 135-140.
Eiden AM et al. (2016). Molecular Pathways: Increased Susceptibility to Infection Is a Complication of mTOR Inhibitor Use in Cancer Therapy. Clin Cancer Res 22(2): 277-283.
Ekici Y et al. (2007). Effect of rapamycin on wound healing: an experimental study. Transplant Proc 39(4): 1201-1203.
Esposito M et al. (2010). Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation. J Neuroimmunol 220(1-2): 52-63.
Fang Y et al. (2013). Duration of rapamycin treatment has differential effects on metabolism in mice. Cell Metab 17: 456-462.
Fok WC et al. (2014). Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome. PLoS One 9: e83988.
Fujishita T et al. (2008). Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcDelta716 mice. Proc Natl Acad Sci USA 105: 13544-13549.
Goberdhan DC et al. (2016). Amino acid sensing by mTORC1: intracellular transporters mark the spot. Cell Metab 23: 550-589.
Goldberg EL et al. (2014). Immune memory-boosting dose of rapamycin impairs macrophage vesicle acidification and curtails glycolysis in effector CD8 cells, impairing defense against acute infections. J Immunol 193(2): 757-763.
Graziotto JJ et al. (2012). Rapamycin activates autophagy in Hutchinson-Gilford progeria syndrome: Implications for normal aging and age-dependent neurodegenerative disorders. Autophagy 8(1): 147-151.
Harrison DE et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460(7253): 392-395.
Hasty P et al. (2014). eRapa restores a normal life span in a FAP mouse model. Cancer Prev Res (Phila) 7: 169-178.
Jagannath C et al. (2009). Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med 15(3): 267-276.
Jagannath C, Bakhru P (2012). Rapamycin-induced enhancement of vaccine efficacy in mice. Methods Mol Biol 821: 295-303.
Jiang J et al. (2013). Rapamycin protects the mitochondria against oxidative stress and apoptosis in a rat model of Parkinson's disease. Int J Mol Med 31(4): 825-832.
Jiang T et al. (2014a). Temsirolimus attenuates tauopathy in vitro and in vivo by targeting tau hyperphosphorylation and autophagic clearance. Neuropharmacology 85: 121-130.
Jiang T et al. (2014b). Temsirolimus promotes autophagic clearance of amyloid-β and provides protective effects in cellular and animal models of Alzheimer's disease. Pharmacol Res 81: 54-63.
Johnson SC et al. (2013). mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. Science 342: 1524-1528.
Kaeberlein M (2014). Rapamycin and ageing: When, for how long, and how much? J Genet Genomics 41(9): 459-463.
Kahn D et al. (2005). The effect of rapamycin on the healing of the ureteric anastomosis and wound healing. Transplant Proc 37(2): 830-831.
Keating R et al. (2013). The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection with influenza virus. Nat Immunol 14: 1266-1276.
Kennedy BK, Lamming DW (2016). The mechanistic target of rapamycin: The grand conducTOR of metabolism and aging. Cell Metab 23: 990-1003.
Knoll GA et al. (2014). Effect of sirolimus on malignancy and survival after kidney transplantation: systematic review and meta-analysis of individual patient data. BMJ 349: g6679.
Komarova EA et al. (2012). Rapamycin extends lifespan and delays tumorigenesis in heterozygous p53+/- mice. Aging 4: 709-714.
Laberge RM et al. (2015). MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nat Cell Biol 17: 1049-1061.
Laplante M, Sabatini DM (2013). Regulation of mTORC1 and its impact on gene expression at a glance. J Cell Sci 126(Pt 8): 1713-1719.
Lelegren M et al. (2016). Pharmaceutical inhibition of mTOR in the common marmoset: effect of rapamycin on regulators of proteostasis in a non-human primate. Pathobiol Aging Age Relat Dis 6: 31793.
Leontieva OV, Blagosklonny MV (2016). Gerosuppression by pan-mTOR inhibitors. Aging (Albany NY) 8(12): 3535-3549.
Lin AL et al. (2013). Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer’s disease. J Cereb Blood Flow Metab 33:1412-1421.
Liu Y et al. (2014). Rapamycin-induced metabolic defects are reversible in both lean and obese mice. Aging (Albany NY) 6(9): 742-754.
Liu J et al. (2017). Efficacy and Safety of Everolimus for Maintenance Immunosuppression of Kidney Transplantation: A Meta-Analysis of Randomized Controlled Trials. PLoS ONE 12(1): e0170246.
Ma KL et al. (2013). Increased mTORC1 activity contributes to atherosclerosis in apolipoprotein E knockout mice and in vascular smooth muscle cells. Int J Cardiol 168(6): 5450-5453.
Madeo F et al. (2015). Essential role for autophagy in life span extension. J Clin Invest 125(1): 85-93.
Majumder S et al. (2011). Inducing autophagy by rapamycin before, but not after, the formation of plaques and tangles ameliorates cognitive deficits. PLoS One 6: e25416.
Malagelada C et al. (2010). Rapamycin Protects Against Neuron Death In in vitro and in vivo Models of Parkinson's Disease. J Neurosci 30(3): 1166-1175.
Mannick JB et al. (2014). mTOR inhibition improves immune function in the elderly. Sci Trans Med 6(268): 268ra179.
März AM et al. (2013). Large FK506-binding proteins shape the pharmacology of rapamycin. Mol Cell Biol 33(7): 1357-1367.
McQuary PR et al. (2016). C. elegans S6K Mutants Require a Creatine Kinase-Like Effector for Lifespan Extension. Cell Rep 14(9): 2059-2067.
Medvedik O et al. (2007). MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLoS Biol 5: e261.
Miller RA et al. (2011). Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. J Gerontol A Biol Sci Med Sci 66: 191-201.
Miller RA et al. (2014). Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell 13: 468-477.
Moskalev AA, Shaposhnikov MV (2010). Pharmacological inhibition of phosphoinositide 3 and TOR kinases improves survival of Drosophila melanogaster. Rejuvenation Res 13(2-3): 246-247.
Mueller MA et al. (2008). Prevention of atherosclerosis by the mTOR inhibitor everolimus in LDLR-/- mice despite severe hypercholesterolemia. Atherosclerosis 198: 39-48.
Neff F et al. (2013). Rapamycin extends murine lifespan but has limited effects on aging. J Clin Invest 123(8): 3272-3291.
Ozcelik S et al. (2013). Rapamycin attenuates the progression of tau pathology in P301S tau transgenic mice. PLoS One 8(5): e62459.
Pakala R et al. (2005). Rapamycin attenuates atherosclerotic plaque progression in apolipoprotein E knockout mice: inhibitory effect on monocyte chemotaxis. J Cardiovasc Pharmacol 46: 481-486.
Pospelova TV et al. (2012). Suppression of replicative senescence by rapamycin in rodent embryonic cells. Cell Cycle 11(12): 2402-2407.
Prevel N et al. (2013). Beneficial role of rapamycin in experimental autoimmune myositis. PLoS One 8(11): e74450.
Rallis C et al. (2013). TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast. Aging Cell 12(4): 563-573.
Reifsnyder PC et al. (2016). Rapamycin treatment benefits glucose metabolism in mouse models of type 2 diabetes. Aging (Albany NY) 8(11): 3120-3130.
Richardson A et al. (2015). How longevity research can lead to therapies for Alzheimer’s disease: The rapamycin story. Exp Gerontol 68: 51-58.
Powers 3rd RW et al. (2006). Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev 20: 174-184.
Robida-Stubbs S et al. (2012). TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. Cell Metab 15: 713-724.
Ross C et al. (2015). Metabolic consequences of long-term rapamycin exposure on common marmoset monkeys (Callithrix jacchus). Aging (Albany NY) 7(11): 964-973.
Rulten SL et al. (2006). The human FK506-binding proteins: characterization of human FKBP19. Mamm Genome 17(4): 322-331.
Santini E et al. (2009). Inhibition of mTOR Signaling in Parkinson’s Disease Prevents l-DOPA–Induced Dyskinesia. Sci Signal 2(80): ra36.
Saxton RA, Sabatini DM (2017). mTOR signaling in growth, metabolism, and disease. Cell 168: 960-976.
Schindler CE et al. (2014). Chronic rapamycin treatment causes diabetes in male mice. Am J Physiol Regul Integr Comp Physiol 307(4): R434-R443.
Schreiber et al. (2015). Rapamycin-mediated mTORC2 inhibition is determined by the relative expression of FK506-binding proteins. Aging Cell 14(2): 265-273.
Selman C et al. (2009). Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan. Science 326(5949): 140-144.
Sheng R et al. (2010). Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning. Autophagy 6(4): 482-494.
Shioi T et al. (2003). Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation 107(12): 1664-1670.
Spilman P et al. (2010). Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease. PLoS One 5(4): e9979.
Spindler SR et al. (2012). Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila. PLoS ONE 7(2): e29782.
Strong R et al. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α‐glucosidase inhibitor or a Nrf2‐inducer. Aging Cell 15: 872-884.
Tardif S et al. (2015). Testing Efficacy of Administration of the Antiaging Drug Rapamycin in a Nonhuman Primate, the Common Marmoset. J Gerontol A Biol Sci Med Sci 70(5): 577-588.
Urfer SR et al. (2017). A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs. GeroScience 39: 117-127.
Yang X et al. (2015). Inhibition of mTOR Pathway by Rapamycin Reduces Brain Damage in Rats Subjected to Transient Forebrain Ischemia. Int J Biol Sci 11(12): 1424-1435.
Yanik EL et al. (2015). Sirolimus effects on cancer incidence after kidney transplantation: a meta-analysis. Cancer Med 4(9): 1448-1459.
Wang AC et al. (2017a). Rapamycin as a Novel Therapeutic for Alzheimer’s Disease: Prevention Assessed Through Neuroimaging. FASEB J 31(Suppl. 1): 814.6.
Wang R et al. (2017b). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism. Aging Cell 16(3): 564-574.
Wilkinson JE et al. (2012). Rapamycin slows aging in mice. Aging Cell 11(4): 675-682.
Willems MC et al. (2010). Persistent effects of everolimus on strength of experimental wounds in intestine and fascia. Wound Repair Regen 18(1): 98-104.
Wu JJ et al. (2013). Increased mammalian lifespan and a segmental and tissue-specific slowing of aging after genetic reduction of mTOR expression. Cell Rep 4(5): 913-920.
Zhang S et al. (2010). Rapamycin promotes beta-amyloid production via ADAM-10 inhibition. Biochem Biophys Res Commun 398: 337-341.
Zhang Y et al. (2014). Rapamycin extends life and health in C57BL/6 mice. J Gerontol A Biol Sci Med Sci 69: 119-130.

NOTE: The information contained in this article is solely intended for information purposes and must not be held as substitute for professional medical or dietary advice. The provisos of the LongeCity user agreement apply. Neither the author nor LongeCity have financial interests in the sale or promotion ofrapamycin.

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Geroprotector review: Metformin

Tue, 18 Jul 2017 19:50:27 +0000

Geroprotector review: Metformin

Sven Bulterijs

This article
- is a summary - The full version can be downloaded here:
- is a 2017 (spring) update - See here for a 2011 article on metformin by the Author.
- is solely for information purposes, not a substitute for professional medical or dietary advice. The provisos of the LongeCity user agreement apply.

Introduction

Metformin (1,1-dimethylbiguanide) is an oral drug used for the treatment of type 2 diabetes and polycystic ovary syndrome (PCOS). It belongs to a category of drugs known as biguanides but metformin is the only one of this class that remains in clinical use. Metformin is, unless contra-indicated, the first line treatment for type 2 diabetes to which other drugs can be added if needed to achieve the desired level of blood sugar control. Metformin has been included in the World Health Organization’s (WHO) list of essential medicines. Nearly 120 million metformin prescriptions are issued worldwide each year making metformin one of the most sold drugs. Metformin is so popular because it’s relatively safe, efficient, and costs only cents per dose.

Figure 1 Some of the molecules mentioned in this article. Notice that all molecules share the same guanidine moiety.

During the Middle Ages physicians prescribed Galega officinalis, better known as goat’s rue, the French lilac, Italian fitch, Spanish sanfoin or false indigo, to treat the intense urination in people suffering from type 2 diabetes (Fig. 2).

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Figure 2 Galega officinalis. Credit JoJan on Wikimedia Commons

The active ingredient in this plant is galegine or isoamylene guanidine but this is too toxic for therapeutic use. In fact the name goat’s rue refers to the fact that this plant can be deadly when eaten by grazing sheep or goats. In 1926 two synthetic molecules were discovered that have chemical similarity to the active ingredient of G. officinalis, termed synthalins A and B. These two synthetic molecules were better tolerated and more efficient but still had some toxicity. The discovery of insulin eventually lead to a discontinuation of the synthalins in the early 1930s. In 1929 several biguanides were synthesised including metformin but it was not until 1956 until the antidiabetic properties of these compounds would be investigated by French researcher Jean Sterne. Sterne proposed the name ‘Glucophage’, which is still a brand name of metformin until this day. In the following years two more biguanides were developed: buformin and phenformin. Buformin and phenformin have been withdrawn from the market due to concerns about the increased risk of lactic acidosis (see below). The concern about lactic acidosis kept metformin from the US market until it was finally approved in 1995.

The first paper in Pubmed that contains the search term “metformin” was published in 1959. Remarkably, it took until 1991 before the milestone of 50 papers/year was reached. Just 5 years later this had grown to 100 papers a year and from then on it kept growing, reaching 1717 papers in 2016 (Fig. 3). That’s almost 5 new papers every day!

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Figure 3 Metformin citations in Pubmed. Search conducted on 1st April, 2017.

Metformin’s effects on lifespan

In the full version (see PDF) all available data on lifespan have been included in three tables. Here we just present a short summary of the most important findings.

simple organisms: Metformin extended chronological lifespan in two yeast studies but not in a third one. Three studies report lifespan extension by metformin in the roundworm and one reports lifespan extension by buformin. Metformin also improved several markers of healthspan in worms such as locomotory activity and age-related degradation of the cuticle (= the ‘skin’ of the worm). One interesting result in worms was the finding that metformin actually reduced lifespan in worms grown in bacteria-free conditions. The authors demonstrated that metformin influenced the metabolism of bacteria on which these worms live resulting in a decrease in dietary methionine. No effect of metformin was found on lifespan in fruit flies - at high dosages lifespan actually tended to decrease. However metformin rescued the shortened lifespan of amyloid-β overexpressing flies. Amyloid-β is an aggregation-prone protein that forms the plaques found in the brain of Alzheimer’s disease patients. Furthermore, metformin reduced mortality in 'obese' flies infected with a mold (R. oryzae). Metformin however did not improve survival in 'obese' non-infected flies even though it did induce weight loss. Finally, metformin extended lifespan in crickets.

mammals: Phenformin extended lifespan in female mice and female rats. One study found that buformin extended lifespan in female rats. Nine studies have been published examining the effect of metformin on lifespan in rodents. As can be seen in figure 4 metformin largely had a positive effect on mean or median lifespans. Though one study found a significant decrease of 13.4% in male 129/Sv mice. Maximum lifespan was also extended in several studies. Anisimov and colleagues have also demonstrated that metformin reduced the rate of aging in multiple studies. Metformin extended median lifespan of male UM-HET3 mice by 7% (although this was not significant) in the Interventions Testing Program of the NIA. Metformin had no effect on lifespan in this study.

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Figure 4 Mean or median changes in lifespan by metformin in rodents. The asterisks indicate significance by whatever criteria applied in the original paper. 1-15 refers to the order in which the metformin data have been tabled in the full text version. One datapoint, a 14.4% decrease in lifespan in male C57BL/6 mice was censored because the dose used (1% of diet) was toxic while lifespan was extended in that same study by the low (0.1%) dose.

The effect of metformin on lifespan may be age-, gender-, strain-, and dose-dependent (see full text). The combination of metformin and rapamycin may extend lifespan more than either drug alone. Interestingly, long-term rapamycin treatment causes some side effects (glucose intolerance and hyperlipidemia) which could be improved by metformin treatment.

Calorie restriction (CR) is the most robust experimental method to increase lifespan. It has been demonstrated to increase lifespan in a wide variety of model organisms from yeast to monkeys. Stephen Spindler and colleagues tested the effect of metformin on gene expression in the livers of mice. Eight weeks of metformin treatment reproduced 75% of the gene expression changes observed in long term calorie restriction (CR). In comparison eight weeks of CR only reproduced 71% of gene expression changes observed in long term CR. These data support the idea that metformin may work as a CR mimetic.

Multiple trials show that metformin lowers all cause mortality in type 2 diabetes patients when compared to other interventions (see full text, table 3).
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Table: metformin trials & mortality

Surprisingly enough, Bannister and colleagues found that mortality in type 2 diabetes patients treated with metformin was lower than in non-diabetic controls.

The Targeting Aging with Metformin (TAME) trial will enrol roughly 3,000 elderly (65-79 years old). This placebo-controlled, randomized clinical trial will investigate the effect of metformin on a composite outcome that includes cardiovascular events, cancer, dementia, and mortality.

The Me.Me.Me trial is a phase III randomized controlled trial in which the effect of metformin-treatment on the risk for age-related non-communicable chronic diseases will be investigated in people who suffer from metabolic syndrome but are otherwise healthy.

Protective effects of metformin on diseases

Cancer

Various animal studies have shown that metformin reduces spontaneous and induced (such as by exposure to carcinogens) cancers as well as reducing cancer incidence in animals that are genetically susceptible to cancer. Furthermore, metformin sensitizes cancer cells to radiation- or chemotherapy-induced cell death and may simultaneously protect healthy cells from damage from the cancer treatment (see full text).

Multiple cohort and case-control studies in humans show that metformin use by diabetics is associated with a lower risk of cancer. Indeed, when a meta-analysis is done only including cohort and case-control studies than metformin use is associated with a lower cancer risk. However, randomized placebo-controlled clinical trials generally fail to find any benefit from metformin and when they are included in meta-analyses then the beneficial effect of metformin against cancer disappears. Multiple clinical studies are currently ongoing that will hopefully provide better evidence about metformin’s effectiveness in treating cancer.

Lewis Cantley, director of the Cancer Center at Beth Israel Deaconess Medical Center, told Gary Taubes in an interview that “Metformin may have already saved more people from cancer deaths than any drug in history”.

Cardiovascular disease

Metformin treatment improved several classical measures of cardiovascular health including reductions in triglycerides, total cholesterol, very low-density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, lipoprotein(a), and Apo B levels (see full text).

Metformin treatment significantly reduced the progression of aortic atherosclerosis in a rabbit model. Multiple rodent studies have demonstrated that metformin reduces infarct size and ameliorates heart failure after an experimentally-induced heart attack (see full text). Most famously, the UK Prospective Diabetes Study (UKPDS) found a significant reduction in myocardial infarction in type 2 diabetes patients using metformin.

Multiple rodent studies also demonstrate significant benefit from metformin therapy on experimental-induced stroke including reductions in infarct size, neurological symptoms and improvements in the formation of new blood vessels (see full text). Cheng and colleagues used data from the Taiwan National Health Research Institute database to investigate the effect of metformin use on stroke risk in diabetic patients. After a 4-year follow up, metformin use was associated with a significant decrease in the risk for stroke (Hazard Ratio: 0.468). Mima and colleagues found that neurological severity of stroke was lower in type 2 diabetes patients treated with metformin compared to those on other treatments.

Other diseases

Finally, there’s some evidence for a beneficial effect from metformin in various autoimmune diseases (rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and lupus), eye diseases (open-angle glaucoma and cataracts), allergic diseases (psoriasis and asthma), anti-fibrotic (lung and liver), non-alcoholic fatty liver disease, infectious diseases (tuberculosis and Clostridium difficile), intervertebral disc degeneration, Huntington’s disease, cyclic edema, and seizures (see full text).

Metformin had a negative effect on disease onset and progression in an amyotrophic lateral sclerosis (ALS) mouse model.

Mechanisms of metformin

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Figure 5 Some of the mechanisms through which metformin could promote longevity. The blunt-ended arrows indicate inhibition.

Metformin activates many pathways that have been linked to increased longevity including SIRT1 and FOXO and decreases pro-aging pathways like mTOR, insulin/IGF-1 signaling, and the proinflammatory transcription factor NF-κB. Metformin inhibits the formation of advanced glycation end products (AGEs) by scavenging the reactive intermediates (glyoxal and methylglyoxal) involved in AGE formation. Metformin also changes the metabolism of the microbiome leading to a decrease in dietary methionine content, which is known to extend lifespan in rodents. Metformin stimulates cell senescence in some experiments while reducing it in others. Interestingly, metformin may prevent the senescence-associated secretory phenotype (SASP) which is known to cause damage to surrounding tissues. Metformin reduced progerin expression and restored nuclear morphology in cells from Hutchinson-Gilford progeria patients. Metformin was also found to extend the lifespan of worms by inducing a mild stress (ROS production) in the mitochondria activating a protective response known as “mitohormesis”. However many studies find that metformin decreases ROS production, decreases oxidative damage, and upregulates the expression of antioxidant enzymes. Metformin may also lead to a slight decrease in body weight. However, the decrease observed in most studies in so small that it’s unlikely to have significant effects on health. The effects of metformin on autophagy, cell death, and DNA damage are discussed in the full text.

Side effects of metformin

Minor side effects

Metformin has several non-serious side effects such as gastrointestinal problems (diarrhea, flatulence, vomiting, upset stomach, abdominal bloating, anorexia, and nausea), taste disturbances including a metallic taste in the mouth, and dermatological problems (erythema, pruritus, and urticaria) (Product monograph: GlucophageⓇ, 2009).

Lactic acidosis

The most serious concern with biguanide therapy is the development of lactic acidosis. Lactic acidosis has a mortality rate of about 30-50%. The two other biguanides (phenformin and buformin) have been withdrawn because of the risk for lactic acidosis.

Salpeter and colleagues conducted a large meta-review of 347 studies and found no increased risk for lactic acidosis in metformin-treated patients compared to those on other diabetes medications. Furthermore blood lactate levels were not significantly different between both groups. The incidence of lactic acidosis is estimated at 5-9 cases per 100,000 patient years of metformin use. The lack of lactic acidosis cases in published trials probably reflects the fact that trials typically exclude patients at risk for lactic acidosis and that trial participants receive standard of care. Lactic acidosis is a risk especially in patients with contraindications for the use of metformin or in people who take an overdose.

Decrease in vitamin B12 and folate levels

Many studies have observed that metformin users have lower serum vitamin B12 levels compared to nonusers. Several studies also observed a decrease in serum folate levels in patients treated with metformin. However, not all studies observe a decline in folate levels.

In a meta-analysis of 29 studies with a total of over 8,000 patients it was demonstrated that metformin use was associated with lower serum vitamin B12 levels and a higher incidence of B12 deficiency.

Sadly many long-term users are never tested for vitamin B12 status. In fact current clinical guidelines do not make any recommendations on vitamin B12 testing or prevention in metformin users. In a recent study it was found that only 37% of long-term metformin users had their vitamin B12 status tested.

Decrease in testosterone levels

Multiple studies observe a decline in testosterone levels in women treated with metformin for either PCOS syndrome or breast cancer. There’s a surprising lack of male studies. One study found that two weeks of metformin treatment lead to a significant decline in total and free testosterone levels in normal males. Another study that combined metformin with a hypocaloric diet for three months found a decline in total testosterone levels in diabetic males and a decline in free testosterone in non-diabetic males.

Cognitive decline

Several studies report that metformin increases amyloid-beta production in cell culture. Leading many people to be concerned that metformin may promote Alzheimer’s disease. However there are two limitations to this conclusion. First, the dosages of metformin used in these studies greatly exceed those found in the brain after metformin administration. Secondly, when insulin was added to the cell culture metformin no longer increased amyloid-beta levels. In fact, amyloid-beta levels in cultured neurons exposed to metformin plus insulin were lower than in non-treated neurons. Insulin is present in the human brain.

Several human studies found that metformin use was associated with a higher risk for neurodegenerative diseases. Except in the Singapore Longitudinal Aging Study in which long term metformin use was actually associated with a reduced risk for cognitive decline.

One highly speculative explanation for the increased risk for Alzheimer’s disease found in diabetic patients is that diabetes patients develop neuronal insulin resistance. As discussed above metformin decreases amyloid-beta production in neurons in the presence of insulin while increasing it when insulin is absent. The insulin resistant brain state mimics the absence of insulin and in this condition metformin leads to an increase in amyloid-beta production and hence Alzheimer’s disease risk. In contrast, in the insulin sensitive (normal) state metformin might lower amyloid-beta levels and could hence maybe decrease Alzheimer’s disease risk. Though, I should stress again that this is speculation as no data exist on Alzheimer’s disease risk in non-diabetic metformin users.

Beta-cell function and apoptosis

Contradictory data exist on metformin's effect on pancreatic beta-cells. Some studies show that metformin causes beta-cell dysfunction and death while others find protective effects. However, all these studies have been conducted in cultured cells. To the best of my knowledge no data exist on metformin's effect on pancreatic beta-cell viability in whole organisms.

SUMMARY:

Based on current studies, the prospect of using metformin as part of a pro-longevity regimen merits further attention.
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Summary slide: Metformin

NOTE: The information contained in this article is solely intended for information purposes and must not be held as substitute for professional medical or dietary advice. The provisos of the LongeCity user agreement apply. Neither the author nor LongeCity have financial interests in the sale or promotion of metformin.

CORRECTION: In the full text on page 32 in the first paragraph of the testosterone section the sentence "To the best of my knowledge no intervention study has tested the effect of metformin supplementation on lifespan." should say testosterone instead of metformin.

Extended version of this article:

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Interview with Aschwin de Wolf

Sun, 19 Feb 2017 14:34:43 +0000

Interview with Aschwin de Wolf (February, 2017)

Recently an English judge ruled that a 14-year old terminally-ill girl had the right to choose cryonics. While the girl received support for her choice from her mother, her estranged father had initially objected. As the girl was under age she needed the permission of both parents and hence the case appeared in front of the High Court’s Family Division in England.

The girl wrote a moving letter to the court saying:

“I am only 14-years-old and I don't want to die but I know I am going to die. I think being cryo-preserved gives me a chance to be cured and woken up - even in hundreds of years' time. I don't want to be buried underground. I want to live and live longer and I think that in the future they may find a cure for my cancer and wake me up. I want to have this chance. This is my wish”.

Justice Peter Jackson ruled that allowing the mother to make the decision was in the best interest of the girl. The girl has since died and was successfully cryopreserved by Cryonics Institute (CI), one of the three facilities in the world that offer this service.

The news made headlines around the world and many news agencies voiced disbelief in the feasibility of cryonics.

Hence I have asked cryonics researcher Aschwin de Wolf for an ⇒ interview about cryonics.

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The gut microbiome in health and disease

Thu, 29 Dec 2016 01:58:25 +0000

We live in a microbial world. Microorganisms are everywhere, from the depths of the ocean to hot, acidic volcanic lakes. They are on and inside us. Our own ancient ancestor was a single free living cell and the tiny power-plants inside our cells (the mitochondria) were once free-living bacteria that invaded our single celled ancestor.

Our bodies are inhabited by a large number of micro-organisms that live in many distinct habitats: the skin, mouth, airways, gut, oesophagus, genitals, external auditory channel, hair on head, and nostrils (Spor et al., 2011; Marsland et al., 2013). While it was often cited that the number of bacterial cells outweighs the human ones by a ratio of 10:1 new estimates place the ratio rather on a 1:1 (Sender et al., 2016) - still a huge number of 38 trillion bacterial cells.

The composition, density, and species-richness of the microbial communities varies greatly between these different habitats. In the intestine the density of microorganisms increases from the start point to anus (Schroeder and Bäckhed, 2016). There’s a large diversity in the classes of microorganisms that make up our gut microbiome. It consists of bacteria, viruses, archaea, fungi, and other maybe even other Eukaryota. In the gut the bacteria and viruses dominate in numbers and both groups may be about equally abundant (Ogilvie and Jones, 2015). Yet of all classes of microorganisms present in the gut we by know by far the most about bacteria and hence most of the discussion in this article will be focused on bacteria.

A substantial part of the viruses present in the gut are bacteriophages (or phages for short) (Abeles and Pride, 2014). Phages are viruses that prey on bacteria. Phages have been found in the bloodstream of healthy humans and it is believed that phages can cross mucosal surfaces (such as the lining of the gut) (Abeles and Pride, 2014). ((Even more surprising is the fact that bacteria, probably in dormant form, have been observed in the human bloodstream (Damgaard et al., 2015; Païsse et al., 2016; Potgieter et al., 2015))

Every individual harbors a combination of approximately 160 bacterial species in their gut. In total around 1,200 bacterial species have been identified in the human gut, hence each human harbors only a small set of the potential bacterial diversity (Schroeder and Bäckhed, 2016).
While everyone has this different composition of bacteria in their gut we can identify three large groups, the so called enterotypes. The three enterotypes are characterized by the relative abundance of bacterial species belonging to one of the following bacterial ‘groups’ (correct term is genera): Bacteroides (type 1), Prevotella (type 2), Ruminococcus (type 3) (Arumugam et al., 2011). This classification has been questioned (Knights et al., 2014). These three groups are likely not as discrete as the original work suggested but this classification remains useful to help us thinking about this complex bacterial community. This enterotype system also points out that certain species of bacteria co-occur in the human gut. The reason for that is that certain species of bacteria rely on each others metabolic activity while being in competition with other species that share the same niche (Schroeder and Bäckhed, 2016).

The composition of our gut microbiome is determined by various factors including host genetics, age, diet, lifestyle, smoking status, hormonal cycles, delivery mode (birth), use of specific drugs (e.g. antibiotics), hygiene, cold exposure, pro- and prebiotics (D’Argenio and Salvatore, 2015; Falony et al., 2016; Sommer and Bäckhed, 2013; Schroeder and Bäckhed, 2016; Zhernakova et al., 2016).

Is was conventionally thought that fetuses are sterile and that colonization with bacteria starts during birth. However, recent studies suggest that bacteria may be present in the womb (Schroeder and Bäckhed, 2016). Babies born in the natural vaginal delivery mode are coated with a microbiome from the mother that is absent in those born through cesarean section (Neu and Rushing, 2011). Breast milk feeding also has a big impact on the gut microbiome of babies because human breast milk contains specific oligosaccharides, different from those present in cow milk, that influence the microbiome composition (Barile and Rastall, 2013).

Family members tend to have similar microbiomes, which could reflect either similar environment or the effect of host genetics on microbiome composition (Spor et al., 2011). People who consume a mostly animal protein-based diet tend to have a Bacteroides enterotype (type 1) while people who consume a lot of carbohydrates tend to have a Prevotella enterotype (type 2) (Wu et al., 2011).

Interestingly, the effect of diet on gut microbiome can be age-dependent. In a recent study published in BMC Microbiology researchers fed chicken protein to young and middle aged rats and observed that in young rats chicken protein caused an increase in the probiotic bacteria belonging to the Lactobacillus group while in middle aged rats the relative abundance of Lactobacillus species was decreased by the chicken protein (Zhu et al., 2016a).

Normal physiological role of our microbiome

Our gut microbiome has various roles in health and disease. Some of these have been known for a long time (for example the role in digestion), while others are more recent discoveries (such as the role in various diseases of other organ systems). The gut microbiome has been described as the “forgotten organ” owing to the large role it plays in normal physiology (O’Hara and Shanahan, 2006).

The roles of the normal gut microbiome

Aiding digestion (e.g. complex carbohydrates)
Vitamin biosynthesis (e.g. folate, riboflavin, B12, vitamin K,...)
Production of numerous natural products (short-chain fatty acids, secondary bile acids, conjugated linoleic acids, neurotransmitters, hydrogen sulfide,...)
Suppress the growth of pathogenic bacteria
Metabolizing drugs and environmental toxins
Modulating immunity
Influence the development and homeostasis of host tissues (e.g. bone and gut wall)

(Jandhyala et al., 2015; O’Hara and Shanahan, 2006; Ohlsson and Sjögren, 2015; Round and Mazmanian, 2009; Schroeder and Bäckhed, 2016)

Collectively, the bacteria in our guts harbor more than 150-fold the number of genes present in the human genome (Lepage et al., 2013). This ‘extra genome’ greatly increases the metabolic capacity of the human body. Bacteria inside our guts help us to digest our foods, detoxify drugs and environmental toxins, produce vitamins, and a whole host of other beneficial metabolites. One surprising example of detoxification by our gut microbiome is that of dietary advanced glycation products (AGEs) (Bui et al., 2015). Studies performed in the last two decades illustrate that dietary AGEs may promote the development of a range of diseases including diabetes, dementia, and heart disease (Vlassara et al., 2016).

The gut microbiome may be very important in the development of our immune system. In a recent study the researchers observed that pet store mice had a much higher survival rate than laboratory mice after bacterial infection. Interestingly, laboratory mice were protected from the infection by co-housing them with pet store bought mice indicating that the immunity was (Beura et al., 2016). The "hygiene hypothesis" has long proposed that the lack of exposure to infections early in life leads to immune system abnormalities such as allergies and autoimmune diseases (Willis-Karp et al., 2001). More recently with the realization that the normal microbiome plays a larger role the “old friends hypothesis” and “disappearing microbiota hypothesis” suggest that our modern lifestyle leads to the loss of “old friend” microorganisms that are needed to properly educate our immune system making us more vulnerable to allergies and autoimmune disorders (Candela et al., 2012).

The bacteria in our gut can be classified in four groups based on their relation with their human host: mutualists, commensals, pathobionts, and pathogens. Mutualism is when both partners (the bacterium and its human host) benefit from the association. Commensalism is when one partner benefits from the association and the other is not harmed. Pathobionts are symbiotic bacteria that can turn pathogenic under certain genetic or environmental conditions. And finally, pathogenic bacteria cause disease . Researchers use the term “dysbiosis” to indicate that there’s an unbalance in the microbiome that is capable of initiating or propagating disease (Round and Mazmanian, 2009).

Gut microbiome in aging and disease

The father of immunology and gerontology Elie Metchnikoff conducted, among his extensive diversity of research, also studies on the gut microbiome in bats, horses, birds and humans (Cavaillon and Legout, 2016). Metchnikoff suggested that aging was caused by the toxic effects over a lifetime of putrefaction products produced in the colon (autotoxins). He also observed that people in some Southern European countries where long lifespans are common tend to consume a lot of sour milk and yogurt containing lactic acid bacteria. He suggested that lactic acid bacteria could prevent putrefaction and hence promote longevity (Korenchevsky, 1961). While Metchnikoff’s bacterial autointoxication theory is now considered dead, the idea that our microbiome is involved in human disease is very much alive (Bested et al., 2013).

A large number of chronic diseases have been linked to the gut microbiome including cardiovascular disease (Wang et al., 2011), rheumatoid arthritis (Scher and Abramson, 2011; Zhang et al., 2015), obesity (Turnbaugh et al., 2006), certain cancers (Schwabe and Jobin, 2013), asthma (Huang and Boushey, 2015), inflammatory bowel disease (Kostic et al., 2014), and psychological problems (such as anxiety and depression) (Cryan and Dinan, 2012). In many cases we are not certain if a change in the microbiome is causing the disease or if the disease is causing the change in the microbiome - but for certain diseases interventions that change the microbiome improve the disease (at least in animal models).

I still remember when I looked through the table of content of the April 7th, 2011 issue of Nature and saw a paper titled “Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease”. Before that day I had not really given any thought about a possible role for microorganisms in aging or non-communicable age-related diseases. I assumed that the 100-year old bacterial autointoxication theory of Metchnikoff had long been disproven and hence bacteria didn’t deserve my time. Then this Nature paper pointed out that dietary choline was transformed by the gut microbiome into the vascular toxin trimethylamine (TMA). When mice were fed choline they developed atherosclerosis but this was prevented by treating the mice with antibiotics (Wang et al., 2011). Trimethylamine is later by the liver oxidized to trimethylamine oxide (TMAO) and to my knowledge we still don’t know if the harmful effects come from TMA, TMAO or both. TMAO levels are higher in omnivores than in vegan/vegetarian people and challenge with a 250mg dose of carnitine (another dietary molecule that is converted into TMA by the gut microbiome) increases plasma TMAO levels in omnivores but not in vegan/vegetarians (Koeth et al., 2013). Possibly a vegan/vegetarian diet shifts the microbiome away from TMA-producers. People with higher plasma TMAO levels have more atherosclerosis (Randrianarisoa et al., 2016) are at greater risk for a cardiovascular event (Tang et al., 2013; Zhu et al., 2016b).

Mice that have been engineered to develop rheumatoid arthritis (joint inflammation) have a much milder presentation when they are raised in germ-free conditions compared to normal (SPF, see above) conditions. Furthermore, the introduction of a single bacterial species (segmented filamentous bacteria) is enough to induce arthritis in germ-free mice (Scher and Abramson, 2011; Wu et al., 2010). In a recent study researchers wondered why autoimmune diseases (allergies and type 1 diabetes) are more prevalent in Finland and Estonia compared to Russia (Vatanen et al., 2016). The researchers discovered that the microbiome of children born in Finland and Estonia differs from Russian babies. Bacteria species present in Finnish and Estonian children have lipopolysaccharide (LPS) with a chemical structure that is different from the LPS found in the bacterial species common in Russian children. LPS is part of the bacterial cell wall and is a strong pro-inflammatory molecule. However repeated exposure to LPS will induce tolerance, meaning that further exposure to LPS will no longer induce inflammation. The researchers discovered that the LPS produced by bacteria from the Finnish and Estonian children does not induce tolerance. The researchers injected LPS from either Finnish/Estonian or Russian children into animals that were genetically engineered to develop autoimmune diabetes. Treatment with LPS-derived from Russian but not from Finnish/Estonian bacteria caused a delay in disease onset.

Given the high bacterial load in the inside (lumen) of the gut it is of vital importance that the gut barrier prevents the translocation of these bacteria to the bloodstream. This barrier is based on a mucus layer that keeps the gut content away from the wall, an epithelial cell layer, and active antimicrobial peptides and antibodies that are secreted near the surface (Ostaff et al., 2013). However in certain conditions this barrier may be broken and bacteria and bacterial products are then able to invade the gut tissue leading to inflammation. Several factors such as alcohol consumption, energy-dense diets, major trauma (hypoperfusion of the gut), toxins, changes in the microbiome, and virus infections are known to increase the permeability of the gut (Bischoff et al., 2014). Increased gut permeability has been observed in people with or at risk for type 1 diabetes (Vaarala et al., 2008; Li and Atkinson, 2015). Increased LPS levels (Creely et al., 2007) and gut bacteria (Sato et al., 2014) have been found in the blood of patients suffering from type 2 diabetes. Increased LPS levels in plasma have also been observed in old compared to young mice and this might be (partially) responsible for the chronic inflammation (inflammaging) that is observed to occur with age (Kim et al., 2016).

Roundworms grown on UV-killed bacteria live longer than worms grown on living bacteria (Garigan et al., 2002; Gems and Riddle, 2000; Win et al., 2013). However, the roundworm were exposed to the geroprotective drug metformin then their lifespan was actually reduced in the absence of bacteria! In contrast when living bacteria are present metformin-fed worms live longer. In worms metformin increases lifespan by influencing bacterial metabolism (Cabreiro et al., 2013). Interestingly, recent research shows that intestinal bacteria may also be involved in the therapeutic effect of metformin in humans (Forslund et al., 2015). Roundworms fed the lactic acid bacterium L. gasseri SBT2055 (Nakagawa et al., 2016) or Bacillus subtilis (Sánchez-Blanco et al., 2016) live longer than those grown on the traditional E. coli bacteria. Another study investigated the effect of bifidobacteria on lifespan in C. elegans (Komura et al., 2013). These authors observed a dose-dependent lifespan extension of bifidobacteria mixed with E. coli. However the question remains if worms grown on probiotic bacteria will also live longer than bacteria grown on UV-killed bacteria or on bacteria-free medium.

Germ-free mice live longer than conventionally raised mice (Gordon et al., 1966; Snyder et al., 1990; Tazume et al., 1991). Interestingly, calorie restriction does not further extend the lifespan of germ-free mice (Snyder et al., 1990; Tazume et al., 1991). Germ-free mice have reduced IGF-1 serum levels compared to conventionally raised mice (Schwarzer et al., 2016) and transplantation of the gut microbiome from conventionally raised mice into germ-free mice results in increased IGF-1 serum levels (Yan et al., 2016). The IGF-1 pathway is one of the best established targets in modulating longevity of laboratory animals. Animals with decreased IGF-1 pathway activity live longer (Junnila et al., 2013).

The gut microbiome changes with age in humans (Claesson et al., 2011; Biagi et al., 2016; Kong et al., 2016; Odamaki et al., 2016) and in fruit flies (Clark et al., 2015). The microbiome composition differs between centenarians and 70-year olds. Centenarians have higher levels of pathobionts and lower levels of symbiotic bacteria this is associated with an increased inflammatory state. The researchers also identified that Eubacterium limosum and its relatives are more than 10-fold increased in centenarians providing a ‘microbiome signature’ of exceptional longevity (Biagi et al., 2010). The loss of microbiome diversity has been linked to increased frailty and reduced cognitive performance (Claesson et al., 2012; Jackson et al., 2016). A recent study found that specific patterns of bacterial species that live on the teeth are associated with all-cause and diabetes-related mortality in humans (Chiu et al., 2016).

How can we influence the composition of the gut microbiome to treat diseases?

The most radical way is by a so called “fecal transplant” in which fecal matter from a healthy person is introduced in a sick person. Fecal transplants are successfully used as a last resort treatment for drug-resistant Clostridium difficile infections.

In a “fecal transplant” one transplants all micro-organisms from a donor to a host but likely only a limited number of them are really necessary to achieve the desired effect. So rather than transplanting a cocktail of hundreds of species we could just make pills that contain a few types of microorganisms. These are the so called probiotics that are for sale in many health food shops and typically contain one or more bacteria (often lactic acid bacteria) and sometimes also some yeasts. More advanced probiotics are being studied for the treatment of specific diseases including obesity. Last month researchers reported in the journal Nature Medicine that living Akkermansia bacteria and more surprisingly dead ones improved obesity, blood lipid profile, and insulin sensitivity in mice on a high-fat diet. The reason why dead bacteria have a physiological effect is because a heat-stable bacterial protein binds to the human Toll-like receptor 2. The authors also report the preliminary results from a human trial with Akkermansia showing that the use of this bacteria is safe. The trial is still ongoing and we will have to wait longer to see if the use of Akkermansia pills reduces obesity and improves metabolic health in the human research subjects (Plovier et al., 2016). Researchers are also working on genetically engineered bacteria that can be used in the treatment of human disease. For example, bacteria producing the anti-inflammatory protein interleukin-10 have been developed for the treatment of Crohn’s disease (Braat et al., 2006).

A third approach is to eat products that stimulate the growth of specific microorganisms. One such product are dietary fibers. Human breast milk oligosaccharides are currently being commercialized for infant nutrition (Barile and Rastall, 2013). Just last month the biotech company Inbiose that developed the technology to produce human breast milk oligosaccharides partnered up with DuPont to commercialize this technology. They plan to submit for regulatory approval in 2017 to bring infant nutrition supplemented with human breast milk oligosaccharides to the market (http://www.danisco.com/about-dupont/news/news-archive/2016/inbiose-partner-to-bring-novel-infant-nutrition-ingredients-to-market/).

Fourthly, we can kill off bacteria using antibiotics. Indeed, antibiotic treatment seems to have some beneficial effects on rheumatoid arthritis (Ogrendik, 2014) and can prevent choline-induced cardiovascular disease (Wang et al., 2011).

Finally, we can think of specific drugs to steer bacteria - such as those influencing quorum sensing (Hentzer and Givskov, 2003; Thompson et al., 2015).

Conclusion

The role of the microbiome in health has become a hot topic in recent times. For example, when one types "microbiome AND health" in PubMed one sees an exponential increase in papers published in the last 10 years from as little as 187 in 2009 to 2,612 this year. Nevertheless, we still have a lot of holes in our understanding of how the microbiome influences health and aging. For some diseases such as rheumatoid arthritis we now have evidence that suggests that the microbiome plays a causal role in the disease as the introduction of a single bacterial species can induce the disease in genetically susceptible animals. For other diseases such as psychological ones the role of the microbiome is less well established. It is also important to understand that the effect of the microbiome on health may be complicated. In the roundworm C. elegans the presence of living bacteria shortens lifespan while the presence of living bacteria is essential for metformin's life extension effect in the same species. While we have methods to influence the gut microbiome, the lack of understanding of how these changes may impact health currently restricts the use of these interventions. What we do know is that a healthy diet that contains enough fibers (which promote the growth of specific bacteria) improves health. More research will undoubtedly lead to insights in how microbiome manipulation methods can be used to improve our health.

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Nicotinamide riboside

Fri, 18 Nov 2016 18:28:40 +0000

Sven's Science Column

Fri, 18 Nov 2016 18:27:00 +0000

Sven Bulterijs discusses topics in life extension science