Sharing mechanisms with turmeric and resveratrol …

Carnosine May Mimic Rapamycin
… and provide some of the same benefits of the mTOR inhibitor

By Will Block

N ot an ordinary peptide,* carnosine (β-alanyl-L-histidine) may be considered a multitasking molecule. It’s an antioxidant that also has anti-aging, anti-glycation, chelation, and anti-proliferation properties. Moreover, it has been demonstrated to play an anti-tumorigenic role in certain types of cancer. Carnosine is not a drug and is available as an over-the-counter food supplement.

Seeking to clarify its role in human gastric carcinoma, a new study examined the effect of carnosine on cell proliferation and its underlying mechanisms in the cultured human gastric carcinoma cells.1

Carnosine Inhibits mTOR

In the study, mTOR signaling axis molecules were analyzed in carnosine treated cells, and the results showed that treatment with carnosine led to proliferation inhibition, cell cycle arrest in the quiescent phase, apoptosis increase, and inhibition of mTOR signaling activation.


* A compound consisting of two or more amino acids linked in a chain.


This was accomplished by decreasing the phosphorylation of Akt, mTOR, and p70S6K, suggesting that proliferation inhibition by carnosine in human gastric carcinoma was through the inhibition of Akt/mTOR/p70S6K pathway, and that carnosine is likely to be a mimic of rapamycin.

Carnosine is traditionally used to increase athletic and exercise performance, and has preventive and therapeutic benefits in obesity, insulin resistance, type 2 diabetes, and diabetic microvascular and macrovascular conditions (cardiovascular disease and stroke) as well as number of neurological and mental health conditions.


Treatment with carnosine led to
proliferation inhibition, cell cycle
arrest in the quiescent phase,
apoptosis increase, and inhibition of
mTOR signaling activation.


Life Extension by Inhibiting Growth

When cells become senescent, they no longer proliferate, but that doesn’t mean they don’t grow. In fact, a very interesting recent review2 explains how cellular senescence involves both blocked cell cycling (discontinuation of replication) as well as excessive growth-promoting pathways. (See Durk Pearson & Sandy Shaw’s Life Extension News, Volume 13 No. 1 • February 2010 in the April 2010 issue of Life Enhancement.)

When the cell cycle is arrested, a continuation of cellular-mass growth results in senescent morphology. In fact, in another paper3 it is noted that older cells are indeed larger. An increase in cell size is a hallmark of senescent fibroblasts. Their cell volume is several-fold greater compared with proliferating cells. Cell size is progressively increased in cell culture as cells progress toward senescence. In other words, when the cell cycle is blocked in the presence of growth-promoting signaling, then cells increase in size.


LEM1608mTOR-PATHWAY274.jpg
(click on thumbnail for full sized image)

A major growth-promoting pathway includes TOR (target of rapamycin) along with its upstream regulators and downstream effectors. The TOR gene is structurally and functionally conserved from yeast to humans (including worms, flies, plants, and mice), acting as a cell growth regulator. Excessive growth is a driving force for aging.2 Indeed, inhibition of TOR signaling increases lifespan in worms, flies, yeast, and possibly mammals. In mice, decreased signaling through the insulin/insulin-like growth factor (IGF-1) pathway in adipose tissue results in less mTOR (mammalian TOR) signaling and increases lifespan.3 Reduced caloric intake (as in dietary restriction) also reduces signaling through mTOR and is a well-known method of increasing lifespan in many species, including monkeys.


Inhibition of TOR signaling increases
lifespan in worms, flies, yeast, and
possibly mammals.


Fig. 1 Possible effects of carnosine on maintenance of proteolysis: (1) scavenging reactive carbonyl species and reactive oxygen species; (2) mTOR inhibition; (3) stimulationof proteolysis. mTOR: mechanistic target of rapamycin, RCC: reactive carbonyl species, ROS: reactive oxygen species, DNA: deoxyribonucleic acid.
LEM1608Carnosine_Fig1_274.jpg
(click on thumbnail for full sized image)

TOR (target of rapamycin) is inhibited by rapamycin, a natural metabolite produced by soil bacteria to inhibit growth of fungal competitors. Interestingly, rapamycin is a prescription drug in clinical practice; it is administered to renal (kidney) transplant patients to prevent organ rejection.3 Results in these patients include the prevention of cancer and even cures of some pre-existing cancers. Moreover, two years after renal transplantation, the body-mass index of patients treated with rapamycin was significantly lower than the patients treated with cyclosporine,3 another immunosuppressant. In a study of 11 healthy men treated with 6 mg of rapamycin, (insulin resistance that accompanies the large increase of nutrients that ordinarily induce mTOR signaling) was prevented.3 At present, rapamycin is being investigated in clinical trials as a treatment for cancer.


This is an exciting finding as it
suggests that it might be possible to
suppress mTOR activation even under
conditions of full feeding by using
appropriate (safe and effective) doses
of certain powerful reducing agents.


Pharmaceutical companies are seeking to develop rapamycin derivatives to inhibit mTOR for possible treatment of cancer, autoimmune disorders, type 1 and type 2 diabetes, and obesity. For example, in relation to type 2 diabetes, chronic hyperglycemia can lead to chronic activation of mTOR in pancreatic beta cells.4 Rapamycin (which reduces the activity of mTOR) induces autophagy, a process of programmed self-digestion which, for example, helps to clear aggregated proteins in neurodegenerative diseases such as Alzheimer’s disease.

The mTOR Pathway is Sensitive to Redox State

A complex containing mTOR and the regulatory protein raptor is a key component of a nutrient-sensitive signaling pathway that regulates cell size by controlling the accumulation of cellular mass. HEK293T cells treated with potent oxidants activated the raptor-mTOR [regulatory-associated protein of mTOR] pathway even under nutrient-deprived conditions, when this pathway is ordinarily suppressed (see Figure 1).

If the oxidizing compounds are mimicking an endogenous oxidant that normally activates the raptor-mTOR pathway, the reducing reagent should inhibit pathway activation caused by nutrients. Indeed, the authors found this to be the case; incubating cells with a reducing agent called BAL (2,3-dimercapto-1-propanol) significantly reduced the phosphorylation of S6K1 (an effector of the raptor-mTOR pathway) and was associated with an increase in the amount of raptor recovered with mTOR as is seen in cells in nutrient-deprived conditions.

This is an exciting finding as it suggests that it might be possible to suppress mTOR activation even under conditions of full feeding by using appropriate safe and effective doses of certain powerful reducing agents. We haven’t seen any further work on this (though it may be that such research is being done but is being kept proprietary).


Two natural products that have been
reported to be possible inhibitors of
mTOR are curcumin and resveratrol.


Natural Products That Inhibit mTOR

Two natural products that have been reported to be possible inhibitors of mTOR are curcumin and resveratrol. Curcumin disrupts the mTOR-raptor complex (mTORC1) that results from the activation of the mTOR pathway, and thus may represent a new class of mTOR inhibitor.5 Curcumin, along with possibly active (in inhibiting mTOR) molecularly related curcuminoids, can be obtained by supplementing with turmeric root powder.

Resveratrol inhibits the mitogenic signaling (growth promoting) by mTOR that causes smooth muscle cells to proliferate in response to oxidized LDL.6 This could be a very important protective effect of resveratrol since the proliferation of smooth muscle cells is a major part of atherosclerotic development. Rapamycin dose-dependently inhibited the DNA synthesis (marker of cellular proliferation) and cell proliferation of smooth muscle cells in culture, with complete inhibition taking place at 10-100 nM, indicating that the smooth muscle cell proliferation was under the control of mTOR.

This effect was not due to cytotoxicity of rapamycin because in cells treated with oxidized LDL (50μg apoB/ml), rapamycin was not toxic up to 100 nM. Since resveratrol has been reported to have inhibitory effects on smooth muscle cell (SMC) proliferation, the authors tested it for its effects on mTOR and SMC proliferation. Dose-response experiments showed that DNA synthesis and cell proliferation were significantly inhibited by 25 μM resveratrol without any significant apoptotic effects [indicative of toxicity] at this concentration. It should be noted that 50 μM resveratrol exhibited a slight toxic effect in the presence of oxLDL [oxidized LDL]. This strongly suggests that resveratrol acts on an upstream target in the PI3K/Akt/mTOR signaling pathway.


Durk & Sandy take their turmeric
root powder (2 capsules four times a
day) rather than taking only curcumin
due to the possible additional benefits
of the curcuminoids. ­­­­


Resveratrol Dose Limited by Toxicity

Although resveratrol can inhibit mTOR and thus suppress cellular senescence, the concentration required is close to the high dose at which resveratrol is toxic to cells.7 At lower doses, 8-25 μM, resveratrol was reported to “slightly but detectably” prevent the loss of proliferative activity (e.g., senescence) of the cells in which it was tested. Still, 6.25 - 12.5 μM resveratrol was shown to block the cell cycle and 25 μM caused apoptosis in vascular smooth cells in another study (cited in paper #7).

A “low dose” of dietary resveratrol (4.9 mg/kg) partially mimics caloric restriction and retards aging parameters in mice on a non-calorically restricted diet.


Durk & Sandy do not know what the
optimal amount of resveratrol is for
the purpose of decreasing cellular
senescence and inhibiting mTOR,
though they do drink moderate
amounts of red wine and also take
resveratrol supplements.


Rats fed a standard diet plus 6 mg of resveratrol/liter of drinking water had a reduced ratio of GSH/GSSG (reduced glutathione/oxidized glutathione) and enhanced GSSG, indicative of increased oxidative stress, in liver cells; in the same study, rats on a high fat diet receiving the same amount of resveratrol in their drinking water had reduced GSSG with GSH/GSSG not significantly different from controls on a standard diet, indicative of less oxidative stress. Though this dose of resveratrol (6 mg/liter of water) is, the authors say, below the maximal tolerated dose, the study suggests that the dose ingested by the standard diet fed rats (an average of a total of 48.2 mg/kg of body weight of resveratrol over 45 days or about 1 mg of resveratrol/kg body weight/day) had toxic effects, particularly (as noted above) increased oxidative stress in the liver. Meanwhile, the total amount of resveratrol, 14.8 mg/kg, ingested over 15 days (about 1 mg/kg body weight per day) by the high fat diet fed rats had protective effects.

Further research is needed to understand the effects of different doses of resveratrol in rats (and, indeed, in humans) fed different diets to determine optimal doses. It has already been found that dietary composition may affect the degree of life extension resulting from caloric restriction in fruit flies.

The amount of resveratrol in red wine is reportedly about 90 µg of resveratrol/fluid ounce of red wine.

The authors of paper #7 speculate that “even transient inhibition of mTOR is already sufficient to slightly suppress senescence.” They also suggest that “a combination of non-toxic doses of resveratrol with rapamycin would also extend life span in animals on a standard diet.” Resveratrol has already been shown to extend the lifespan of mice on a high-fat diet. They, of course, would like to see a test of non-toxic doses of resveratrol along with curcumin for its effects on mTOR and on life extension in animals on a standard diet. They would also be interested in the effects on mTOR of the curcumin-related curcuminoids found in turmeric root powder.

Durk & Sandy take their turmeric root powder (2 capsules four times a day) rather than taking only curcumin due to the possible additional benefits of the curcuminoids. They do not know what the optimal amount of resveratrol is for the purpose of decreasing cellular senescence and inhibiting mTOR, though they do drink moderate amounts of red wine and also take resveratrol supplements.


Justly, it can be argued that any
effective anti-aging agent should be
pluripotent in order to counteract the
various molecular changes, which
underlie age-related cellular
dysfunction.


Carnosine Is an mTOR inhibitor, Too

Both the cause and outcome of aging are usually regarded as multifactorial. Accordingly, more effective control might be achieved by intervention at multiple sites.

The endogenous dipeptide carnosine may have anti-aging properties due to its reputed pluripotency. The pluripotency of biological compounds refers to the ability of these substances to produce several distinct biological responses. There are three anti-aging mechanisms of carnosine (see Figure 1).8

1. Inhibition of the mTOR pathway (see “Resveratrol’s Second Life Extension Mechanism” in the May 2010 issue)

2. Inhibition of the TGF-β (Transforming growth factor-β)/Smad3 pathway (Aging and aging related chronic diseases are associated with an increase of TGF-β/Smad3 signaling and expression)

3. Suppression of the effects of reactive carbonyl compounds

Aging is a Multifactorial in its Causality and Final Outcome

It is generally assumed that aging is not a single process, but is the result of various persistent deleterious effects which eventually compromise cellular and organism homeostasis. Physiologically, homeostatic dysfunction characterizes cellular and whole animal aging, ultimately resulting in reproductive failure. When analyzed from a biochemical perspective, aging is usually regarded as multifactorial in both its causality and ultimate outcome. Macromolecular dysfunction, in particular deleterious changes in nucleic acids, proteins and lipids appear to accumulate in aged tissues. For example, aging is associated with increased somatic mutation, progressive homeostatic dysfunction, accompanied by protein modification and lipid peroxidation, which may be attributed to the effects of either exogenous agents or/and interaction with endogenous but potentially deleterious metabolites.

Justly, it can be argued that any effective anti-aging agent should be pluripotent in order to counteract the various molecular changes, which underlie age-related cellular dysfunction.

The endogenous dipeptide carnosine is synthesized in muscle and by astrocytes in the brain. In muscle, carnosine is found in higher concentrations in glycolytic (fast- twitch) fibers than in mitochondria-enriched aerobic muscle; it is degraded back to its constituent amino acids by carnosinases present in a variety of tissues, including plasma and kidney.

Several pieces of evidence suggest a high correlation between life expectancies of mammalian species and muscle carnosine concentration. For example, carnosine content in human muscle (20–30 mM) was twenty times higher than found in mice, ten times than in rabbits and three times than in cows—such differences approximately consistent with their different lifespans. In humans, lower levels of muscle carnosine were found in elderly individuals compared to younger adults.

To repeat, supplementation with carnosine has anti-inflammatory, antioxidant, antiglycation and chelating roles, and may act as a buffering agent in skeletal muscle and improve calcium handling. Although circulating carnosine levels are affected by the presence of plasma carnosinase in humans, long-term supplementation of carnosine results in improved health and/or behavioral outcomes.

Consequently, we suggest that chronic supplementation maintains a more constant plasma level of carnosine mainly due to saturation of carnosinase. Carnosine is considered to possess anti-aging properties because of its pluripotency, although the exact route or routes whereby carnosine achieves this remain(s) to be defined.

While few studies have investigated the effect of carnosine on aging, administration of carnosine to senescence-accelerated mice increased the mean lifespan by 20%, and 50% survival rate by 20%.


In humans, lower levels of
muscle carnosine were found in
elderly individuals compared to
younger adults.


­Possible areas in which carnosine could exert beneficial effects include suppression of telomere shortening, along with the already-mentioned anti-oxidant activity, anti-AGE activity (carbonyl scavenging), suppression of glycolysis, upregulation of mitochondrial activity, activation of proteolysis, inhibition of tumor cell growth, apoptosis, extension of Hayflick limit, rejuvenation of senescent cells, effects on phosphorylation of translation initiation factors, and effects. Carnosine has no known side effects.

Beware Rapamycin

Long term use of rapamycin, approved for use in several disease indications, has had side effects such as canker sores, impaired wound healing, weight gain and glucose insensitivity (which could lead to diabetes)—raising questions about its use to prevent the chronic diseases of aging. That said, it’s notable that researchers at the Buck Institute for Research on Aging have discovered new insights into how rapamycin inhibits the nutrient signaling pathway mTOR, a finding that could provide a way to avoid or eliminate side effects of the drug.

References

  1. Zhang Z, Miao L, Wu X, Liu G, Peng Y, Xin X, Jiao B, Kong X. Carnosine inhibits the proliferation of human gastric carcinoma cells by retarding Akt/mTOR/p70S6K signaling. J Cancer. 2014 Apr 24;5(5):382-9.
  2. Blagosklonny MV. Aging-suppressants. Aging-suppressants: cellular senescence (hyperactivation) and its pharmacologic deceleration. Cell Cycle. 2009; 8(12):1883-7.
  3. Blagosklonny MV, Hall MN. Growth and aging: a common molecular mechanism. Aging (Albany NY). 2009 Apr 20;1(4):357-62.
  4. Tsang CK, Qi H, Liu LF, Zheng XF. Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Discov Today. 2007 Feb;12(3-4):112-24. Epub 2006 Dec 15. Review. Erratum in: Drug Discov Today. 2008 Sep;13(17-18):824.
  5. Beevers CS, Chen L, Liu L, Luo Y, Webster NJ, Huang S. Curcumin disrupts the Mammalian target of rapamycin-raptor complex. Cancer Res. 2009 Feb 1;69(3):1000-8. doi: 10.1158/0008-5472.CAN-08-2367. Epub 2009 Jan 27. PubMed PMID: 19176385; PubMed Central PMCID: PMC4307947.
  6. Brito PM, Devillard R, Nègre-Salvayre A, Almeida LM, Dinis TC, Salvayre R, Augé N. Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells. Atherosclerosis. 2009 Jul;205(1):126-34.
  7. Barger JL, Kayo T, Vann JM, et al. A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS One. 2008 Jun 4;3(6):e2264. doi:10.1371/journal.pone.0002264. Erratum in: PLoS One. 2008;3(6). doi: 10.1371/annotation/c54ef754-1962-4125-bf19-76d3ec6f19e5. PLoS One. 2008;3(6). doi: 10.1371/annotation/8333176c-b08c-4dfb-a829-6331c0fc6064. PLoS One. 2008;3(6). doi: 10.1371/annotation/7d56e94e-3582-413d-b987-fccd0da79081. PubMed PMID: 18523577; PubMed Central PMCID: PMC2386967.
  8. Hipkiss AR, Baye E, de Courten B. Carnosine and the processes of ageing. Maturitas. 2016 Jun 22. pii: S0378-5122(16)30134-7.


Will Block is the publisher and editorial director of Life Enhancement magazine.

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