Dear GRG Member,
Here’s a comprehensive message from James P. Watson, M.D.
Forget everything you learned about Caloric Restriction (CR)!
Forget half of what you learned about Cellular Stress Resistance Pathways.
Just when I thought we were starting to figure out how things work, along comes a disruptive new pathway – one that I had never heard of before.
It is called the “Transsulfration Pathway” (aka TSP) and requires sulfur amino acid restriction.
The TSP generates hydrogen sulfide from methionine and cysteine amino acids, but the paradoxic finding is that the TSP only does this if you are restricting these amino acids!
If you restrict methionine and cysteine, you do not have to restrict calories.
Moreover, the hydrogen sulfide produced mediates stress resistance and longevity!
Who knew! Here is the contrasts between the “Old View” and the “New View” of CR
OLD VIEW of CR – There are many CR pathways that overlap and convey longevity.
The “drivers” of these pathways are AMPK and SIRT1&3. The “brakes” are Insulin/IGF and mTOR
Major CR Pathways:
- Insulin/IGF-1 pathway (must be inhibited)
- mTOR pathway (must be inhibited)
- AMPK pathway (must be activated)
- Sirtuin enzymes (SIRT1, SIRT3, and maybe SIRT6)
Minor (or additional) CR pathways:
- Klotho protein (needs to be activated via Vit D3)
- Methionine restriction (works via GCN2 and ATF)
- Hormesis pathways (work via low level ROS and RNS stress, which confers longevity via Nrf2, FOXOs, etc.)
- p66 (must be inhibited via ATR1 blockade)
- GSK-3beta (must be inhibited via Lithium)
- Nrf2 pathway (and the inhibition of NF-kB, by reciprocal action)
- Adiponectin signaling
- Glucagon signaling
NEW VIEW of CR – The “New View” states that regardless of what the upstream pathways are (Insulin/IGF, mTOR, AMPK, SIRT, Klotho, Nrf2, p66, GSK-3beta, etc.), there is one final common pathway for the “longevity component” of CR and that is the synthesis of H2S by the TSP
This is a completely new concept that completely upsets conventional wisdom (i.e. the “Apple Cart” is overturned)
This new view does not negate or contradict the old views (i.e. that there are multiple “upstream mechanisms” of CR).
Instead, this paper suggests that the final common pathway is the production of H2S via the Transsulfuration pathway (TSP).
The paper is very convincing! Since there are other benefits of CR besides longevity, there are other “end points” with CR pathways, such as the induction of phase II enzymes by NRF2 which reduces carcinogen-induced cancer.
The following paper shows that all of the CR pathways and Stress Resistance Interventions all produce H2S!
The only “brake” to the Transsulfuration Pathway (TSP) is the mTOR pathway and the dietary intake of calories, protein, or specifically sulfur-containing amino acids.
If this is true, then the production of H2S may be the “Grand Unified Theory” (GUT) of how CR works!
Here are two links to the article, which came out in January, 2015
Here are some key facts illustrated with diagrams that help explain how CR, methionine and cysteine restriction, H2S are all linked.
- CR, PR, or Sulfur-containing amino acid restriction induces the transulfuration pathway (TSP) which process H2S
Original Theory: – Methionine restriction => 30-40% longevity without calorie restriction
Methionine restriction (0.17% – 0.86%) was first proved to extend longevity by 30% in normal rats way back in 1992 by Cold Springs Harbor researchers (1st ref below)
This was repeated in 1994 with 0.17% MetR and shown to increase longevity by 40% without caloric restriction by researchers in Valhalla, New York (2nd ref below)
Unfortunately, no one really took this research seriously, since at that time there was a very strong “Dogma” that essential amino acids were “essential” and should not be restricted. In fact, this same “Dogma” was used to explain why plant-base proteins were inferior to meat-based proteins and that not eating meat was harmful.
Moreover, the Valhalla researchers claimed that the molecular mechanism of action was the increase in blood glutathione, which was a paradoxical finding, since sulfur amino acids are needed to make glutathione (i.e. it does not make sense that decreasing methionine, a sulfur containing amino acid, would increase levels of the sulfur containing GSH. They also documented that the GSH was coming from the liver. They did not document or discover that H2S was involved, however.
Subsequent mechanisms – decrease ROS, decrease electron leak, increase in insulin sensitivity, decrease in lipoproteins/cholestero, decrease in IGF-1, mTOR inhibition, etc.
Since then, many other researchers have corroborated their findings, including researchers at universities in Lleida and Madrid, Spain, as well as researchers at University of Michigan in Ann Arbor, and others who suggested other mechanisms as follows:
- a) a decrease in mitochondrial ROS generation – see 3rd reference below (2006)
- b) a decrease in mitochondrial “electron leak” – see 3rd reference below (2006)
- c) a decrease in visceral fat mass and increase in insulin sensitivity – see 4th reference below (2006)
- d) a decrease in LDL, triglycerides, total cholesterol and an increase in HDL – see 4th reference below (2006)
- e) a 40% decrease in IGF-1 that is sustained throughout life – see 4th reference below (2006)
- f) an increase in mitochondrial biogenesis – see 5th reference below (2007)
- g) an inhibition of mTOR (phosphorylation of mTORC1) and inhibition of 4EBP1 (mTOR’s substrate) – see 6th reference below (2009)
- h) an increase in fatty acid oxidation in obese adults with metabolic syndrome – see 7th reference below (2011)
- i) mitohormesis – these researchers claimed that 40% Met restriction induced mitochondrial ROS generation, thereby inducing a hormesis effect whereby Nrf2-mediated and FoxO-mediated increases in antioxidant enzymes occurred – see 8th reference (2009)
- j) increase in adiponectin, decrease in leptin, and increase in UCP-1 gene expression via beta-adrenergic receptors – see reference 9 (2010)
- k) decrease in CpG hypermethylation in gene promoters, lowers oxidative damage to mitochondrial DNA, etc. – see reference 10 (2011)
- l) decreases serum homocysteine and reduces atherosclerosis due to homocysteinemia – see reference 11 (1970)
- m) decreases inflamation – see reference 12 (2013)
The “New Theory”: – The main molecular mechanism of all CR pathways involves the Transsulfuration pathway activation, which produces H2S
The 2015 paper by Hene and colleagues suggests that severe methionine and cysteine restriction must be done to activate the “Transsulfuration Pathway” which involves the conversion of methionine to SAM, SAM to SAH, SAH to homocysteine, homocysteine cystathionine, and cystathionine to cysteine.
Here is a picture of this pathway, which is activated by methionine and cysteine restriction:
Illustration reference: http://www.livewell-bioscience.com/uploads/2/9/4/0/29407049/2014_12_24_c.pdf
The 2015 paper above does not refute or negate any of the above findings about what methionine restriction does. Instead, it supports them.
However, it points out that either methionine or cysteine will inhibit the Transsulfuration pathway (TSP) and thereby negates the health benefits of this pathway.
A simpler illustration of how the TSP works is shown below (PAG is an inhibitor of the enzyme cystathionine gamma-lyase, which makes the H2S)
Illustration reference: http://www.livewell-bioscience.com/uploads/2/9/4/0/29407049/2014_12_24_c.pdf
Explanation: Protein restriction (PR) is a longevity mechanism that occurs with 50% Calorie Restriction or with only specific amino acid restriction (SAA) including methionine and cysteine amino acid restriction. These subtypes trigger the transsulfuration pathway (TSP) to create hydrogen sulfide from enzymatic reactions in the TSP via the enzyme cystathionine gamma-lyase (CGL).
Summary: If you do not restrict sulfur-containing amino acids, you will limit the benefits of CR. Moreover, the health benefits of CR may be enhanced with exogenous hydrogen sulfide gas, sodium hydrogen sulfide salts in water, or synthetic compounds that slowly release H2S like GYY4137. Here is the list of references for the above paragraphs:
- The production of H2S with CR pathways is evolutionarily conserved and essential for the longevity effects of CR
Illustration reference: http://www.cell.com/cell/abstract/S0092-8674(14)01525-6?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867414015256%3Fshowall%3Dtrue
Explanation: Calorie restriction is an evolutionarily conserved longevity mechanism that is seen in yeast, nematodes, fruit flies, and rodents. Regardless of the organism or the subtype of CR, all CR pathways in all organisms produce hydrogen sulfide (H2S). Hydrogen sulfide increases lifespan and increases cellular/organismic stress resistance in all of these organisms in a tissue-specific and CR subtype-specific format. For instance,in mice, 50% CR increased H2S production in the kidney, spleen, and carotid artery but not in the aorta, brain, and skeletal muscle. Methionine and cysteine restriction (which is more specific than 50% CR) increased H2S production in the aorta but not in the brain. Fasting (water only) produced H2S in the brain. The exact mechanism by which hydrogen sulfide extends longevity and stress resistance involves a general mechanism (protein sulfhydration of cysteine thiols) and a specific mechanism (SQR – sulfide quinone oxidoreductase). (See #3 below)
III. Hydrogen Sulfide exerts its effects via sulfhydration of cystein thiols, including the sulfhydration of mitochondrial SQR (sulfide quinone oxidoreductase)
- General Molecular Mechanism on how H2S works:
The general molecular mechanism by which H2S works is by reacting with the free sulfur group on the side chain of cysteine amino acids. This sulfur group is called a “cysteine thiol” and when it reacts with H2S, it forms a “sulfhydryl group”. (In the illustration below, this is depicted as the yellow hydrogen sulfide molecule (H2S) interacting with the free thiol to form the “SSH” molecule in the center of the picture. This “SSH” form is called “sulfhydration” and protects the protein from ROS induced damage. Thus the interaction of H2S with cysteine “free thiols” forms sulfhydryl groups, which protects the proteins from oxidation damage. This is essentially a “gas-induced post-translational modification” of proteins and can occur with thousands of cysteine thiols in proteins throughout the cells. The number of proteins that can be protected by this form of “sulfhydration” by H2S is limitless and can occur with practically all proteins with exposed cysteine amino acid side chains.
Ref for illustration: http://openi.nlm.nih.gov/detailedresult.php?img=3757701_gr4&req=4#
- Specific Molecular Mechanisms on how H2S induces longevity
- The Mitochondrial Electron Transport Mechanisms – SQR is a renewable, rechargeable “mitochondrial scavenger of leaking electrons” which uses H2S sulfhydration to “catch” the leaking electrons and put them on a molecule called “S2O3”, or thiosulfate.
– SQR is a “catcher’s mit” for a baseball “infielder” that catches fly balls before they get out of the infield.
There is a specific protein target of H2S that produces longevity. This is the mitochondrial protein, sulfide quinone oxidoreductase (aka SQR).
SQR allows the transfer of electrons from H2S to Coenzyme Q and to the electron transport chain (ETC). Electrons that would have “escaped” the mitochondria are effectively “scavenged” by SQR. If SQR is fully sulfhydrated by H2S, then these sylfhydryl groups can be oxidized by the electrons “leaking” out of the mitochondrial. The sulfhydryl bonds in SQR are oxidized by ROS, forming thiosulfate. This is an enzyme catalyzed reaction involving two enzymatic reactions, sulfur dioxygenase (SD) and sulfur transferase (ST). Because this is an enzyme-catalyzed reaction, it is very efficient and renewable (i.e. the cysteine thiols can be recharged by more H2S gas). In essence, this is an evolutionarily conserved enzyme catalyzed “electron scavenger” system that catches “fly balls” (ROS) before they “leak out” of the mitochondria (infield) to damage the entire cell (outfield)
Illustration reference: http://www.livewell-bioscience.com/uploads/2/9/4/0/29407049/2014_12_24_c.pdf
Explanation: Inside the mitochondria, electrons must be shuttled from
Conclusion: This is NOT a mitohormesis phenomena. There is no ROS being produced here. SQR is simply a “catchers mit” on a “baseball infielder” that efficiency catches free radicals before they “get out of the outfield”.
- H2S has a Anti-Inflammatory Effect at low levels, blocking TLR-4 mediated inflammation and NF-kB signaling; whereas H2S has a Pro-Inflammatory Effect at high levels, activating SFK and IkBalpha mediated signaling.
There is increasing evidence that oxidative stress alone cannot account for the phenotype of aging. Instead, inflammatory signaling appears to be as important as ROS damage and exerts its effects in an independent mechanism that overlaps with ROS, but has components that are unique (Ex: pro-inflammatory cytokines, STAT1 signaling, STAT3 signaling, and NF-kB signaling). It now appears that H2S also counteracts inflammatory signaling at low doses and augments inflammatory signaling at high doses.
The “dose” of H2S as well as the rate that it is released appears to determine if H2S is “good” or “bad”. At low levels (50-100 ppm or 50-200 mM), H2S seems to mainly have an anti-inflammatory effect. However at higher doses (300-500 ppm or 500-1,000 mM) hydrogen sulfide is pro-inflammatory and can actually kill cells. Likewise, molecules that release H2S slowly appear to be more effective than H2S or the salt form, NaHS. NaHS is very easy to administer orally, but has such a short half life that in the Illustration reference: http://www.pancreapedia.org/sites/www.pancreapedia.org/files/hydrogen_sulfide_fig_2.png
- H2S is an “ER stress reliever”
For those of you who don’t remember the endoplasmic reticulum unfolded protein response (aka ER UPR or ER stress), this is a pathway that is activated by proteotoxicity, the kind that occurs with heat (protein unfolding), with Alzheimer’s (amyloid beta), with diabetes (many proteins including Insulin and Amylin), and all of the amyloidoses. With ER stress, the PERK/elF2alpha/ATF4 pathway is activated, which turns on the gene for CSE. CSE is the enzyme that makes H2S. H2S can prevent or relieve ER stress by sulfhydrating cysteine “free thiols” on several enzymes, such as PTP1B, NF-kB, etc. Here is an illustration of protein sulfhydration by H2S:
- H2S is a “vasodilator”
The first effect that was discovered for H2S was that of vasodilation. At that time, it was thought that “endothelial-derived relaxing factor” (i.e. EDRF) was just nitric oxide (NO). Now we know that there are 3 EDRFs: NO, CO, and H2S. All three of these gases relax blood vessels by inhibiting vascular smooth muscle contraction. The molecular mechanism by which H2S induces vascular smooth muscle relaxation is hypoxia-dependent and is illustrated as follows:
Under basal conditions when the muscarinic acetylcholinergic receptors are not stimulated, ATP-sensitive potassium channels (Katp) on the muscle cell membrane are bound to ATP. The ATP maintains these potassium channels in the closed conformation. With cholinergic activation, calmodulin is activated by Ca++. Ca++/Calmodulin then binds to cystathionine gamma-lyase (CSE, aka CGL), stimulating CGL to make H2S. H2S sulfhydrates the potassium channel, preventing it from binding to ATP. This opens the channel and allows K+ influx into the cell. As a result, vasodilation occurs.
In the brain, the effects of H2S are dependent on whether there is hypoxia or normoxida. Under conditions of normoxia, neurons secrete carbon monoxide (CO) which then diffuses into the nearby astrocytes. In the astrocytes, this CO inhibits the enzyme cystathionine beta-synthase (CBS) in the astrocytes. CBS is the enzyme in the brain that makes H2S (whereas CSE aka CGL is the enzyme that makes H2S in the rest of the body). The inhibition of CBS in the brain under conditions of normoxia results in a decrease in H2S and vasoconstriction. Under conditions of hypoxia, the opposite happens. Neurons make less carbon monoxide (CO) and this then lowers levels of CO in the nearby astrocytes. As a result, astrocytes make more H2S by the enzyme CBS. The H2S then vasodilates the cerebral blood vessels. This is essentially an “auto regulatory system” whereby the brain ensures that it always gets enough oxygen and blood flow, even under conditions of hypoxia.
- H2S is an “oxygen sensor”
H2S appears to mediate oxygen/hypoxia induced vasoconstriction vs vasorelaxation. In this molecular mechanism, hydrogen sulfide essentially becomes an “oxygen sensor”. When tissues are hypoxic, H2S increases. At room air or when hyperoxic conditions exist, H2S levels go down and H2S is consumed. Thus H2S may be the ultimate “antioxidant”, protecting the ETC from damage due to oxygen produced superoxide. This explains why patients undergoing hyperbaric oxygen treatment actually have transient vasoconstriction that occurs during the hyperemic period in the chamber. Here is an illustration of how H2S acts as an “oxygen sensor”:
Illustration reference: http://www.degruyter.com/view/j/cclm.2013.51.issue-3/cclm-2012-0551/cclm-2012-0551.xml
- More Details on this NEW VIEW of CR
Here is a summary of what I learned about the details of this new view of CR, which still includes the “upstream” pathways well studied in the old view, but has a different “end point”. The old “end points” were Sirtuin-mediated deacetylation of histones, Nf-kB, FoxOs, p53, and many other proteins; a FoxO-mediated increase in SOD, catalase, and up regulation of anti-apoptotic factors; up regulation of autophagy, an increase in Nrf2-mediated antioxidant enzymes and phase II detoxification enzymes; an AMPK-mediated increase in mitochondrial biogenesis; and an mTOR inhibition mediated increase in autophagy and mitophagy. This new view has 500 “end points” – the proteins that undergo sulfhydryl formation with H2S. That makes “aging” much more complex to understand, but may actually explain a fundamental mechanism that includes all of the above. Here are the details of this new view
- Hydrogen Sulfide (H2S) is necessary and sufficient to confer CR-mediated Stress Resistance
This was a surprise to me! I thought that mTOR inhibition, AMPK activation, and down regulation of the Insulin/IGF pathway were necessary to induce stress resistance. This study showed that H2S was required, but glucose restriction was not. Apparently, the two major ways by which H2S works to confer stress resistance is by
- FOXO transcription factor activation is needed for the stress resistance effects of CR but not the longevity effects. This is consistent with the studies that show that FoxO3 mediated SOD1 expression is not needed for longevity (and why antioxidant interventions did not work)
This was a real surprise to me! This recent paper showed that methionine + cysteine restriction (without food restriction) protected the animals from reperfussion injury without expression of FOXO target genes. Others have also questioned how important FOXO is as a “bona fide” longevity gene, in light of the fact that many of the polymorphisms found in FoxO3a may be in noncoding regions of the gene where FoxO3a gene interacts with enhancers or possibly that some of the affects are due to transcription of the FoxO3b pseudogene, located on a different chromosome (but that produces similar transcripts that may act as a “miRNA sink”)
Ref: page 2 in http://www.livewell-bioscience.com/uploads/2/9/4/0/29407049/2014_12_24_c.pdf
- NRF2 pathway is needed for the anti-cancer benefits of CR, but not the longevity benefits of CR
Mitohormesis was an alternative “Grand Unifying Theory” of how CR worked. This was promoted by Sinclair (2005), Calabrese and Mattson (2011).
Bishop and Guarente proved this to be true in worms (2007) but in mammals, Pearson and colleagues showed in 2008 that NRF2 was required for CR-mediated prevention of chemically induced cancers (via Phase II detoxification enzymes) but was not required for the longevity effects of CR.
Thus it was Pearson and colleagues work in 2008 that shed doubt on the longevity mechanisms of NRF2 in humans. This paper by Christopher Hines and colleagues, just published in January, 2015, showed clearly that Nrf2 knock out did not eliminate the benefits of CR on preventing ishemia/reperfusion injury or CR-mediated longevity. Nrf2 was clearly unregulated by CR, but was not the cause of the longevity effect.
If this new information is an earthquake, it is a “9.0 on the Richter scale”! H2S may be the fundamental final common pathway by which all CR pathways exert their effect. The triggering of the “Transsulfuration Pathway” (TSP) is required to produce H2S in response to CR, PR (general amino acid restriction), and SAA (Sulfur-amino acid restriction). It is really paradoxical that when you restrict sulfur-containing amino acids that you actually increase the enzyme that makes H2S! This does not make sense, but the data is incontrovertible! Less sulfur amino acids means more H2S. More H2S means more sulfhydration of free cysteine thiols on thousands of proteins, protecting them from the effects of ROS and RNS. The net effect is the following:
- Longevity – 30-40% more lifespan in normal rodents
- Stress resistance – resistance to heat shock, genotoxic stress, ROS stress, RNS stress, ischemia/reperfusion, etc.
- Less inflammation – sulfhydration of NF-kB subunits, IK-Balpha, etc.
- Less “ER stress” – amelioration of proteotoxicity in the ER
- Less mitochondrial ROS leak – via SQR, the “infielder’s catcher’s mit that catches baseballs before they get out of the infield”
- Less Alzheimer’s disease (I did not go over that one above)
- Vasodilation – less hypertension
- Lipid profile improvement
- mTOR inhibition – I left this off the email
- SIRT1 activation – I left this off the email
- Increase in Nrf2 activation (important in stress resistance, but not longevity)
- Increase in FoxO activation (important in stress resistance, but not longevity)
- Decrease in IGF-1
- Increase in adiponectin
- Decrease in leptin
- Decrease in visceral fat
- Decrease in CpG hypermethyation (probably of age-related CpG sites)
I think I am going to start doing methionine/cysteine restriction!
It does not require caloric restriction!
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