Antioxidants proved a bust for life extension almost 25 years ago, but glutathione stands out as an exception. We lose glutathione as we age, and supplementing to increase glutathione levels has multiple benefits, possibly on lifespan.

Glutathione is manufactured in the body via an ancient mechanism taking as input cysteine, glutamic acid, and glycine. Supplementing N-Acetyl Cysteine (NAC) and glycine are independently associated with health benefits, and possibly increased lifespan. Glutamine seems to be in adequate supply for most of us.

Each cell manufactures its own glutathione. (GSH is an abbreviation for the reduced form of glutathione.) Concentrations of GSH within a cell a typically 1,000-fold higher than in blood plasma. When we look for glutathione deficiency, we measure the blood level, because that is convenient. It is much harder to measure intracellular levels of GSH. These two studies [2011, 2013] demonstrated that intracellular levels decline with age more consistently and more severely than blood levels. People in their 70s have less than ¼ the glutathione (in red blood cells) that they had when they were in their 20s. The same study also found that intracellular levels of cysteine and glycine but not glutamate decline with age.

Supplementing with NAC is already known to boost glutathione levels. But here is a motivation to try a combination of glycine and NAC, dubbed “GlyNAC” to see if we can do even better. This work has been spearheaded by Rajagopal Sekhar.

In humans, “Supplementing GlyNAC for a short duration of 2 wk corrected the intracellular deficiency of glycine and cysteine, restored intracellular GSH synthesis, corrected intracellular GSH deficiency, lowered OxS, improved MFO, and lowered insulin resistance.” [Sekhar] Most of these benefits are theoretical. Lowering oxidation levels is a double-edged sword. MFO=mitochondrial fatty acid oxidation, and this benefit is on firmer footing. Membranes are made of fatty acids, and mitochondrial efficiency, like most everything in the body, depends on highly selective membranes. The crowning benefit is improved insulin sensitivity, and we can be fairly confident this leads to longer healthspan.

The two recent studies, in humans and mice, are indeed impressive.

The small human study found that “GlyNAC supplementation for 24 weeks in OA corrected RBC-GSH deficiency, OxS, and mitochondrial dysfunction; and improved inflammation, endothelial dysfunction, insulin-resistance, genomic-damage, cognition, strength, gait-speed, and exercise capacity; and lowered body-fat and waist-circumference.” Though they didn’t measure methylation age, this constellation of improvements gives us confidence that people were looking and acting younger.

In older (71-80 yo) subjects 24 weeks of GlyNAC supplementation raised intracellular GSH levels from 0.4 mmol to 1.2, compared to 1.8 in young adults. (Levels were measured in red blood cells.)

Two central players in aging are inflammation and insulin resistance; both showed excellent response.

Inflammation decreased markedly: Average C-reactive protein (CRP) dropped from 4.9 to 3.2 (compared to 2.4 for young people). IL-6 dropped from 4.8 to 1.1 (ref 0.5 for young). TNFα dropped from 98 to 59 (ref 45).

Insulin resistance fell just as dramatically, along with fasting glucose and plasma insulin.

Cognitive performance improved markedly! as did grip strength, endurance, and gait speed.

GlyNAC subjects lost a lot of weight — 9% of body weight in 24 weeks. This is both very good news and a hint that some of the benefits of GlyNAC may be caloric restriction mimetic effects, indirectly due to suppression of appetite or of food absorption.

Is all this evidence of a decrease in biological age?

But the effects faded weeks after the treatment stopped. This, I believe, is different from resetting methylation age. There is not a lot of data yet to test this, but I believe that methylation is close to the source of aging; in other words, the body senses its age by its epigenetic state, and adjusts repair and protection levels accordingly. Thus changing epigenetics to a younger state, IMO, effectively induces an age change in the body.

If this is correct, then my guess is that GlyNAC does not set back methylation age, based on the fact that the effects must be continually renewed by daily doses of glycine and NAC. On the other hand, mitochondria are such a central player in expressing multiple symptoms of aging that it may well be that continuous treatment with GlyNAC leads to longer lifespan.

…and indeed that is what was just reported in a mouse study. 16 mice lived 24% longer with GlyNAC supplementation, compared to 16 controls. 24% is impressive (see table below). For example, rapamycin made headlines a decade ago with an average lifespan increase of 14%. (In other studies, rapamycin was associated with even greater life extension.) The winner in this table is a Russian pineal peptide, which claims 31% increase in lifespan. I have previously bemoaned the fact that this eye-popping work from the St Petersburg laboratories of Anisimov and Khavinson has not been replicated in the West (though Russian peptides are now commercialized in he West). 

Table source: https://genomics.senescence.info/drugs/browse.php#details-549
(This is a sample — not a complete list.)

An asterisk must be placed next to the new 24% life extension from GlyNAC. Eleven years ago, Flurkey, found the same 24% life extension with NAC alone. NAC supplementation without glycine is known to increase glutathione production. Do we need glycine in addition, or is cysteine the bottleneck? Levels of both free glycine and cysteine decline with age. This would suggest that supplementation of both should be more effective than supplementing NAC alone. But I was unable to find any study that asked whether GSH levels are raised to a greater extent by GlyNAC than by NAC alone.

Glycine supplementation in large amounts mimics methionine restriction, which is a known but impractical life extension strategy.

If you decide to take glycine, it should be at bedtime, and in large amounts, a teaspoon or two. (I did this for awhile using glycine as a sweetener in hot chocolate soymilk, until I decided it ruined the taste of the chocolate drink. Whether this is a sound reason for tailoring an anti-aging agenda I’ll leave you to decide.)

All this work comes out of the laboratory of Rajagopal V. Sekhar at Baylor College of Medicine in Texas. It’s time that a broader life extension community joined in the action. I’m grateful to Dr Sekhar for commenting on earlier drafts of this article.

A methylation clock is an empirical construct. There is no understanding of physiology or metabolism built into the process. The clock is engineered to do the best job predicting (in the case of the GRIM-Age clock, for example) future mortality and morbidity based on methylation patterns. The whole process is agnostic about biological mechanism.

It is a legitimate question whether a drug or diet that sets back the methylation clock has actually increased life expectancy. Maybe methylation is a downstream consequence of aging, like grey hair or wrinkled skin. We would hardly expect a skin cream or hair dye to increase life expectancy.

For me, personally, this is an easy question. I have devoted much of my professional career since 1996 to opposing the “selfish gene” version of evolution and promoting multilevel selection. I have collected evidence that aging is a systemic phenomenon, centrally controlled, and that epigenetics (including methylation) is the primary way in which aging is enforced on the body. I was poised to believe that methylation clocks measure something real and important even before the first clocks appeared [2013].

Many other scientists looking at anti-aging interventions have been happy to take a practical approach, not invoking theory at all, but accepting the impressive correlations of aging clocks with other measures of biological age as good enough reason to trust that any intervention able to sett back the methylation clocks is probably setting back biological age.

Morgan Levine is a biostatistician par excellence. As a post-doc working with Steve Horvath, she developed what I consider to be the best, most usefull methylation clock..With her own research group at Yale, she has continued to innovate, with a promising approach based on the mathematics of principal component analysis [my write-up last September]

In a recent preprint, Morgan Levine has deeply questioned whether methylation clocks can be trusted in the way that so many of us have trusted them. Although young and just at the beginning of her career, she has done more than anyone except Horvath himself to advance methylation clock technology. For her to question the foundational value of her own work is a gesture of courage and deep intellectual honesty.

Before the post-doctoral work with Horvath that created the PhenoAge clock, Levine studied evolutionary biology of aging with the incomparable authority, Caleb Finch. She has her own ideas about the evolutionary origins of aging, and they are rooted in classical evolutionary theory. She sees the cause of aging as somatic evolution and accumulation of damage. She is deeply influenced by Peter Medawar’s [1952] hypothesis that what happens late in life is outside the influence of natural selection.

And so she raises the deep question: how much of epigenetic change associated with age is a driver of aging, and how much is a response to the body’s increasingly damaged state?

“Though the connection between risk and time may appear probabilistic on the surface, the emerging pathology is rooted in the molecular and cellular remodeling of the organismal system over its lifetime. Such changes likely result from accumulated damage, selection pressures at the level of cells, compensatory mechanisms, and/or the unintended consequences of a biological program. However, alterations to a complex system must abide by a hierarchical structure2, initiating at lower levels of biological organization (e.g. molecules) prior to manifesting at the higher levels in which they are typically observed (e.g. tissue and organ dysregulation and failure, and eventually death)3. Thus, to delay, prevent, or even reverse the maladies currently awaiting us in late life, we must discover how to decipher and remodel the molecular fingerprint of aging.”

Here, Levine is raising the most basic questions about the origin and meaning of methylation changes with age. Levine proceeds from her strongest skill—She is master of a wide array of sophisticated statistical tools.

Part of the classification has to do with Yamanaka programming, which is about stem cell vs differentiated cell. Another part comes from Framingham Heart study patients, and which CpGs change with age in FHS subjects. She distinguishes sites that increase methylation with age from others that decrease methylation with age. She associates some CpGs with cancer.

She maps Shores, Islands, and Open Seas. CpG islands are promoter regions of the genome that have lots of CpGs in close proximity. Shores are regions on the boundary of CpG islands, and Open Seas are regions in which CpGs exist as isolated, disconnected units.

Having divided 4779 CpGs into 12 groups, she can ask, How much of each group is represented in each of the most commonly used methylation clocks?

And which modules are performing best and most consistently across clocks?

Conclusions

Levine entertains the idea of “epigenetic drift” as part of the story, however she recognizes that the changes that underpin the most reliable clocks are not “drift” but clearly directional. She asks, to what extent do methylation changes cause aging and to what extent are they responses to various, incidental results of the aging process?

“If DNAm changes were purely reflecting entropic alterations or epigenetic drift, we would expect to see a bias against changes in CpGs that start around 0.5 (corresponding to random chance of methylation at a given site) . However, what we observe is actually a regression away from the mean, in which these heterogeneous populations of cells are systematically losing DNAm with time. This suggests that the green-yellow module’s notable pattern of epigenetic aging is unlikely to stem from noise or aberrant DNAm changes with age. Instead, DNAm changes may reflect cellular selection pressure or clonal expansion in which the cells without DNAm at these CpGs are able to outcompete (proliferate more than) the ones with DNAm . Alternatively, it could reflect a regulated compensatory mechanism that gets initiated with aging, or a continuation of a developmental program that is not turned-off . These scenarios have different implications for our understanding of epigenetic changes. The first would suggest that individual cells are not changing DNAm patterns with age, but rather the changes that are observed in bulk data are happening at the level of cell populations, shifting prevalence of cells with heterogenous states. The second and third scenarios, on the other hand, would suggest within cell DNAm changes, perhaps as a response to extracellular environment or signaling changes with aging (e.g. integrated stress response (ISR) ), or as an extended developmental program that fails to be extinguished—somewhat aligned with the hyper-function theory of aging . In moving forward, single-cell DNAm data may help distinguish individual vs. population changes.”

Here she references “Integrated Stress Response” as a theory of the aging metabolism. She also refers to Mikhail Blagosklonny’s idea that developmental programs have a momentum that spills over into aging phenotypes.

Morgan Levine is a brilliant scientist, facing the harshest possible self-criticism of her work. Her conclusions are tentative and open-ended.

A more definitive, empirical approach

The big, interesting question may not require theoretical analysis. What we want to know is whether we have been justified in using methylation clocks to indicate whether aging has been slowed or reversed. The most direct answer to that question comes from Harold Katcher’s rats, — the most successful example of setting back methylation age in a whole animal. Currently, Katcher has two sets of 8 rats that are the same chronological age; one group has been treated with E5 and has a much lower methylation age. He is waiting to see how long each group lives. So far, 3 of the untreated rats have died, and 1 of the 8 treated rats. Of course, the treated rats look and act much younger, and have physiological characteristics of younger rats. Over the coming months, the survival test will produce an answer to the important question whether a younger methylation age implies a younger biological age, in a form that is independent of theory.