52 replies to this topic

[Iporuru]

A summary:

 

Aging correlates with increasing methylation. Epigenetic clocks measure age based on DNA methylation levels. Less methylation is associated with a younger phenotype.

 

Question: can we decrease our DNA methylation by following a regimen involving (some of) these demethylating agents:

• Procaine (or procainamide) – Gerovital H3?
• AKG alpha-ketoglutarate
• EGCG (plus possibly quercetin, resveratrol, curcumin, and sulforaphane)
• Vitamin C
• rapamycin
• reduced IGF (fasting, calorie restriction)

 

Research: 

 

Progress on the role of DNA methylation in aging and longevity

Environmental signals have a widespread influence on the aging process. Epigenetic modification, e.g. DNA methylation, represents a link between genetic and environmental signals via the regulation of gene transcription. An abundance of literature indicates that aberrant epigenetic change occurs throughout the aging process at both the cellular and the organismal level. In particular, DNA methylation presents globally decreasing and site-specific increasing in aging. (…)

Intriguingly, abundant evidence has demonstrated that DNA methylation has a close association with aging, age-related diseases and longevity (…)

More importantly, accumulated studies suggest that age-dependent DNA methylation changes could be inversed by certain interventions, such as dietary control and chemicals, presenting the great potential of DNA methylation as a therapeutic target in preventing age-related diseases and promoting healthy aging.

 

DNA hypermethylation as a chemotherapy target.

It was found that accompanying DNA demethylation is a dramatic reactivation of the silenced genes and inhibition of cancer cell proliferation, promotion of cell apoptosis, or sensitization of cells to other chemotherapeutic reagents.

 

Dynamic DNA Methylation During Aging: A “Prophet” of Age-Related Outcomes

Currently, a growing number of studies have shown that dynamic DNA methylation throughout human lifetime exhibits strong correlation with age and age-related outcomes. Indeed, many researchers have built age prediction models with high accuracy based on age-dependent methylation changes in certain CpG loci. For now, DNA methylation based on epigenetic clocks, namely epigenetic or DNA methylation age, serves as a new standard to track chronological age and predict biological age. Measures of age acceleration (Δage, DNA methylation age – chronological age) have been developed to assess the health status of a person. In addition, there is evidence that an accelerated epigenetic age exists in patients with certain age-related diseases (e.g., Alzheimer’s disease, cardiovascular disease). (…)

Research has shown that semi-supercentenarians and their offspring have a relatively younger biological age as reflected by their decreased DNA methylation age (Horvath et al., 2015b).

 

Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock

Epigenetic regulation can occur by the direct methylation and demethylation of DNA bases, so called “cis-epigenetics” (Bonasio et al., 2010).

 

Demethylating Agents as Epigenetic Anticancer Therapeutics

Demethylating agents are a class of anti-cancer drugs which reduce cytosine methylation, promoting transcriptional activation of genes by virtue of reducing methylation in their promoter regions. Most compounds that inhibit methylation are inhibitors of DNA methyltransferases (DNMTs) that are responsible for methylating cytosine residues on DNA. Azacitidine and Decitabine are two such demethylating agents that are approved for use in myelodysplastic syndromes

 

Demethylating agents can be divided into two major structural groups: Nucleoside and nonnucleoside analogs.

 

Nucleoside analogs are structurally similar to cytosine and are currently being investigated in clinic. They are also known as azanucleosides, because they have a nitrogen instead of carbon at position 5 of the cytosine ring (Fig. 1A). Azacitidine, Decitabine and Zebularine are drugs of this group that are being tested for efficacy in clinical trials.

 

Nonnucleoside DNMT Inhibitors

Although it does not inhibit DNMTs directly in vitro, hydralazine, a vasodilator, can decrease the levels of DNMT1 and 3A protein in vivo [33]. It has also been shown that hydralazine interacts directly with DNMT1 DNA binding sites [34]. It can induce hypomethylation of the APC gene promoter leading to its reactivation in cervical cancer cell lines.

Unlike hydralazine, procainamide, a Na+ channel blocker, can inhibit DNMT activity in vitro. Its inhibition activity is relatively specific to DNMT1, leading to global loss of methylation. Also, a new derivative of procainamide (IM25) has been recently identified as a potent demethylating agent, which has greater demethylating activity than azacitidine and exhibits less cytotoxicity.

 

The antiarrhythmic drug procainamide has been known as an inhibitor of DNA methylation in human T cells. Its demethylating effect on T cells led to the over-expression of lymphocyte function associated antigen 1 that makes T cells autoreactive. Initially proposed as a perturbative of the interactions between DNMTs and CpG-rich sequences, procainamide was reported to specifically inhibit the maintenance methyltransferase activity of DNMT1 and to demethylate hypermethylated genes. Procainamide causes global DNA hypomethylation and restores the expression of the detoxifier gene glutathione S-transferase P1 (GSTP1).

Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1

Procainamide Inhibits DNA Methylation and Alleviates Multiple Organ Dysfunction in Rats with Endotoxic Shock

 

Tea catechins, particularly epigallocatechin-3-gallate (EGCG) has been reported to have DNA demethylating activity. Not only can it reduce the expression levels of DNMTs, but it also has inhibitory effects on HDACs resulting in increased histone acetylation, as described in one study[39].

 

Procaine Is a DNA-demethylating Agent with Growth-inhibitory Effects in Human Cancer Cells 

Following this need to find new demethylating agents, we have tested the potential use of procaine, an anesthetic drug related to procainamide. Using the MCF-7 breast cancer cell line, we have found that procaine is a DNA-demethylating agent that produces a 40% reduction in 5-methylcytosine DNA content as determined by high-performance capillary electrophoresis or total DNA enzyme digestion. Procaine can also demethylate densely hypermethylated CpG islands, such as those located in the promoter region of the RARβ2 gene, restoring gene expression of epigenetically silenced genes. This property may be explained by our finding that procaine binds to CpG-enriched DNA. Finally, procaine also has growth-inhibitory effects in these cancer cells, causing mitotic arrest. Thus, procaine is a promising candidate agent for future cancer therapies based on epigenetics.(…)

Its long-established and safe use as a local anesthetic, with well-known pharmacological characteristics, may stimulate its prompt transition to preclinical and early clinical trials for epigenetics-based cancer treatments.

 

Most of you will have heard about Gerovital (GH3), a preparation developed by the Romanian scientist Ana Aslan and heavily promoted from the 1950s to 1980s. In the 1970s, the National Institute on Aging commissioned a thorough evaluation of the studies and claims surrounding Gerovital H3. The conclusion of that work was that, except for a possible mild monoamine oxidase (MAO) inhibitor effect that would potentially ameliorate depression, there was no scientifically credible evidence supporting the claims that procaine is beneficial in treating age‐related diseases or syndromes. But on the other hand in the 1970s nobody heard of epigenetic clocks and TETs, so perhaps if they had measured the patients’ epigenetic age they would have found some benefit to taking GH3…

 

 

Alpha‐ketoglutarate

Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice

AKG administration up‐regulated Prdm16 expression, which was correlated with an increase of DNA demethylation in the Prdm16 promoter. In summary, AKG supplementation promotes beige adipogenesis and alleviates HFD‐induced obesity in middle‐aged mice, which is associated with enhanced DNA demethylation of the Prdm16 gene (…)

Ten–eleven translocation family of proteins (TET) catalyze hydroxylation of 5mC to 5hmC, a key step in active DNA demethylation, which requires α‐ketoglutarate (AKG) as a cofactor (Tahiliani et al., 2009). Moreover, AKG integrates key pathways in cellular metabolism.(…)

We found that AKG is a rate‐limiting factor controlling DNA demethylation in the Prdm16 promoter, and its deficiency in progenitor cells profoundly attenuates brown adipogenesis.(…)

Our results suggested that dietary AKG up‐regulated Prdm16 expression and beige/brown adipogenesis partially through facilitating active DNA demethylation. Dietary supplementation of AKG increased intracellular levels of AKG and enhanced beige adipogenesis, which improved metabolic health of aged mice challenged with HFD. Because active DNA demethylation is not only limited to brown/beige adipogenesis but also presents in the differentiation of progenitor cells in other tissues, the dietary AKG intervention might have preventive effects in the senescence of other tissues as well, which warrant further studies.

 

AMPK/α-ketoglutarate axis dynamically mediates DNA demethylation in the Prdm16 promoter and brown adipogenesis

 

Active DNA demethylation is mediated by the ten-eleven translocation hydroxylases (TETs), including TET1, 2 and 3. Importantly, TET catalytic reaction requires α-ketoglutarate (αKG), a key metabolite of the Krebs cycle, linking metabolism to epigenetic modifications  and stem cell differentiation.(…)

Consistent with increased Prdm16 expression, the DNA methylation in these three regions was reduced during differentiation (Figure 3F), showing the occurrence of DNA demethylation.

The expression of all Tets, which catalyze 5hmC formation, was pronouncedly enhanced during brown adipogenic differentiation (Figure 3I), suggesting the importance of DNA demethylation during brown adipogenesis.

 

Dietary Compounds as Epigenetic Modulating Agents in Cancer

It has been reported that a diet rich in vegetables and fruits can significantly reduce the risk of cancer development, due to the action of phytochemicals which may regulate the expression of oncogenes and tumor suppressor genes. Remarkably, phytochemicals may act through epigenetic mechanisms such as modulation of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) activities. In general, cancer treatments involve the use of chemo-radio therapeutic agents, kinase inhibitors, personalized antibodies as well as compounds that stimulate the immune system. In particular, HDAC inhibitors and demethylating drugs modified gene expressions by reversing the aberrant epigenetic alterations acquired during tumorigenesis (Luczak and Jagodziński, 2006).(…)

 

Recent reports indicate that dietary supplements and natural compounds may restore the normal epigenetic marks which are altered during carcinogenesis. The phytochemicals most studied in cancer are epigallocatechin-gallate (EGCG), quercetin, resveratrol, curcumin, and SFN [sulforaphane]. (…) These effects are mediated, in part, by the modulation of epigenetic machinery which included the regulation of DNMTs and HDACs activities.(…)

 

Remarkably, EGCG is a potential epigenetic modifier of DNMTs and HDACs and restores epigenetically silenced genes in skin and cervical cancers. For instance, in skin cancer cells, EGCG significantly decreased the proteins levels of DNMT1, DNMT3a, and DNMT3b and modulated the HDAC activities allowing the transcriptional activation of tumor suppressor genes such as p16 INK4a and Cip1/p21. In esophageal cancer, EGCG induced apoptosis and inhibited cell growth of ECa109 cells through p16 gene demethylation. Moreover, EGCG reactivated the expression of WIF-1 (Wnt inhibitory factor-1) through promoter demethylation and inhibited cell growth by downregulating the Wnt canonical pathway in H460 and A549 lung cancer cell lines. (…)

 

Several studies indicate that curcumin has antioxidant, anti-inflammatory, anti-proliferative, anti-angiogenic, and anti-cancer properties. Moreover, this natural compound has been considered as an excellent non-toxic hypomethylating agent for breast cancer therapy. For instance, curcumin inhibited DNMT1 expression and restored the function of RASSF1A by promoter hypomethylation in estrogen positive MCF-7 breast cancer cell line.

 

Prolongevity interventions, including reduced growth hormone (GH) and insulin-like growth factor (IGF) signaling, CR, and rapamycin, also slow down ticking of the presumptive biological clock.

Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions

 

Epigenetic aging signatures in mice livers are slowed by dwarfism, calorie restriction and rapamycin treatment.

 

Caloric restriction:

Caloric restriction attenuates age-related changes of DNA methyltransferase 3a in mouse hippocampus.

Progress on the role of DNA methylation in aging and longevity

 

 

TETs

Role of TET enzymes in DNA methylation, development, and cancer

Until recently, DNA methylation was believed to be an irreversible epigenetic event associated with gene repression, which could only be alleviated through DNA replication. Thus, it was remarkable when ten eleven translocation protein 1 (TET1) was discovered and shown to be able to modify methylcytosine and potentially erase DNA methylation (…)

Disruption of epigenetic landscapes, including DNA methylation patterns, is a hallmark of cancer. Somatic mutations in genes (e.g., in DNMT3A) that encode for the machinery that establishes DNA methylation have been causally linked to malignant transformation. Interestingly, the activity of TET enzymes, which is involved in removing this epigenetic mark, has also emerged as an important tumor suppressor mechanism in cancer.(…)

Thus, precise regulation of DNA methylation patterns, which is partly mediated by TET enzymes, is important for normal development and provides a fundamental protection against cellular transformation.

 

Increasing the levels of vitamin C (ascorbic acid) has been shown to stimulate TET protein enzymatic activity in cultured cells as well as mouse tissues. This can be detected as increased levels of the cytosine oxidation products 5hmC, 5fC, and 5caC as well as a small reduction of global DNA methylation in the absence of changes in TET expression levels. Although the precise mechanism is unknown, it is likely that vitamin C interacts directly with the catalytic domain of TET proteins and provides a local reducing environment that increases recycling efficiency of the Fe(II) cofactor (Yin et al. 2013).

 

Vitamin C

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells

Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. (…)

Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

 

Chromatin Dynamics during Cellular Reprogramming

Nutrients and cofactors present in the extracellular environment represent a final class of molecules that influence the epigenome and cellular reprogramming. A point in case is ascorbic acid (vitamin C), which has been shown to strongly enhance the efficiency and kinetics of reprogramming and to increase the quality of mouse iPSCs by preventing aberrant hypermethylation. Ascorbic acid presumably functions both as an antioxidant and as a cofactor for specific epigenetic modifiers such as the H3K36 HDMs Jmjd1a/1b. Furthermore, ascorbic acid was suggested to be a cofactor for H3K9 HDMs and Tet enzymes according to recent studies, which reported a global decrease of the repressive H3K9me2/3 marks and genome-wide DNA hypomethylation, respectively, in nascent iPSCs exposed to this compound. Together, these observations provide compelling new evidence for the tight communication between reprogramming-associated signaling molecules and TFs in order to rewire epigenetic regulatory circuits.

 

TET enzymes, TDG and the dynamics of DNA demethylation

Although the exact function of the TET proteins in ES cells needs further study, several recent publications are supportive of a role for TET in reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs). For example, at the early stage of transduction with the transcription factors Oct4, Klf4, Sox2 and c-Myc (collectively referred to as OKSM), Tet2 is recruited to the Nanog and Esrrb loci to activate their transcription. In addition, both Tet1 and Tet2 can associate with Nanog and facilitate iPSC generation in an enzymatic activity dependent manner. Remarkably, Tet1 overexpression can not only enhance reprogramming efficiency by promoting demethylation and reactivation of Oct4, but can also replace Oct4 in the iPSC reprogramming cocktail. Furthermore, beyond reprogramming mediated by OKSM, Tet1 and Tet2 seem to have distinct roles in reprogramming mediated by fusion of somatic cells to pluripotent cells.

 

 

David Sinclair: Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

 

Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage. Changes to DNA methylation patterns over time form the basis of an 'ageing clock’, but whether old individuals retain information to reset the clock and, if so, whether this would improve tissue function is not known.(…)

Having previously found evidence for epigenetic noise as an underlying cause of ageing, we wondered whether mammalian cells might retain a faithful copy of epigenetic information from earlier in life, analogous to Shannon's "observer" system in Information Theory, essentially a back-up copy of the original signal to allow for its reconstitution at the receiving end if information is lost or noise is introduced during transmission.(…)

 

Ten-Eleven-Translocation (TET) dioxygenases are known for their ability to remove DNA demethylation at CpG sites. Because Yamanaka factors promote in vitro reprogramming by upregulating Tet1 and Tet2, but have no effect on Tet3, we tested whether Tet1 and Tet2 were required for the beneficial effects of OSK on RGCs. Four weeks of OSK AAV expression significantly decreased DNA methylation age, and this was Tet1- and Tet2-dependent (Fig. 4i). Together, these results demonstrate that Tet-dependent in vivo reprogramming can restore youthful gene expression patterns, reverse the DNA methylation clock, and restore the function and regenerative capacity of a tissue as complex as the retina.(…)

 

In this study, we show that in vivo reprogramming of aged neurons can reverse DNA methylation age and allow them to regenerate and function as though they were young again. The requirement of the DNA demethylases Tet1 and Tet2 for this process indicates that altered

DNA methylation patterns may not just a measure of age but participants in ageing. These data lead us to conclude that mammalian cells retain a set of original epigenetic information, in the same way Shannon's observer stores information to ensure the recovery of lost information.

 

 

So, the question is: can we affect our epigenetic age by a taking a combination of the demethylating agents described above? If yes, what would the protocol be: doses, duration, etc.? What do you think?

 

 

 

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[Andey]

Thanks, thats a very nice investigation you done! 

This idea is floating in the air, I wrote a question about this to an upcoming D/ Sinclair podcast https://www.longecit...ok/#entry881500

 

What do you think could be a simple protocol to have a feel for it? AKG + VitC + EGCG? 

What could be a biomarker/metric to see if there any effect from it?

[Iporuru]

I think the best way to test fhe effectiveness of this protocol is to do DNA methylation age test before and after the treatment

[Turnbuckle]

Epigenetic age is associated with generally random changes to the epigenome, and to fix that you can't just add or remove methylation randomly, as that will make you older yet. Think of a computer code of 1's and 0's. If you've lost some 1's, you can't just add 1's randomly, you have to add them in the right place.

Edited by Turnbuckle, 17 November 2019 - 05:03 PM.

[Iporuru]

Epigenetic age is associated with generally random changes to the epigenome, and to fix that you can't just add or remove methylation randomly, as that will make you older yet. Think of a computer code of 1's and 0's. If you've lost some 1's, you can't just add 1's randomly, you have to add them in the right place.

You're right and it's a reasonable concern. However, most of these agents demethylate DNA globally, not randomly

[Andey]

Epigenetic age is associated with generally random changes to the epigenome, and to fix that you can't just add or remove methylation randomly, as that will make you older yet. Think of a computer code of 1's and 0's. If you've lost some 1's, you can't just add 1's randomly, you have to add them in the right place.

 

Sinclair speculates that there exists a master copy of a methylation pattern and Yamanaka factors reset the methylation pattern.  As lporuru cited reprogramming involves a TET family of enzymes and Vit C could be a cofactor there.

  Another way of looking at it is that generally when a cell is young it has very little methylation on DNA and reducing DNA methylation overall could lead to a younger phenotype.(I dont know what are histones methylation dynamic in that picture though) Maybe Yamanaka works as - reducing methylation to switch cells to a reprogramming mode, then different tissue microenvironments leads to DNA getting the right methylation pattern.

[Iporuru]

Such global changes will be random. The epigenetic code is different for each of the 200 cell types -- it's how one type is distinguished from another, as the code tells the cells what balance of proteins to make. Without those epigenetic differences, a skin cell might act like a liver cell, or it might act like protoplasm. So while changes due to aging are random and widely distributed (noise), you can't apply global methods via supplements to reverse that, as you would only increase the noise. The only way to do it is to reprogram the cell (inducing pluripotency), or by replacing an epigenetically old cell with a stem cell that matures into the desired somatic cell. The programing you get going through a stem cell phase is de novo, and thus has a very low epigenetic age.

 

Turnbuckle, you obviously know more than me about these things. Yet, all of the demethylating agents I listed are known to confer numerous benefits (or at least be harmless) to healthspan and/or lifespan. Many people in the antiaging community do a combination of some of these interventions and it's somehow hard to believe the net effect of them would result in a higher epigenetic age.

 

Also, if you look at the results of G. Fahy's TRIIM study - they also used a combination of three common substances and achieved a decrease of 2.5 years in the participants' epigenetic age. Did the drugs affect global (de)methylation patterns?

 

What's your take on this?
 

[Turnbuckle]

Turnbuckle, you obviously know more than me about these things. Yet, all of the demethylating agents I listed are known to confer numerous benefits (or at least be harmless) to healthspan and/or lifespan. Many people in the antiaging community do a combination of some of these interventions and it's somehow hard to believe the net effect of them would result in a higher epigenetic age.

 

Also, if you look at the results of G. Fahy's TRIIM study - they also used a combination of three common substances and achieved a decrease of 2.5 years in the participants' epigenetic age. Did the drugs affect global (de)methylation patterns?

 

What's your take on this?
 

 

2.5 years is peanuts. I saw an 11 year drop by enlarging endogenous stem cell pools and using them to replace senescent cells (using mito fusion and a UCP2 blocker).  Another approach is reprogramming, taking cells back to a pluripotent state with Yamanaka factors. In one trial, mice lived 33-50% longer. See this 4 minute video from Youthereum Genetics. In both cases cells are getting de novo programing.

Edited by Turnbuckle, 18 November 2019 - 03:54 PM.

[Iporuru]

2.5 years is peanuts. I saw an 11 year drop by enlarging endogenous stem cell pools and using them to replace senescent cells (using mito fusion and a UCP2 blocker).  Another approach is reprogramming, taking cells back to a pluripotent state with Yamanaka factors. In one trial, mice lived 33-50% longer. See this 4 minute video from Youthereum Genetics. In both cases cells are getting de novo programing.

 

Yes, I'm aware of Belmonte's experiments with Yamanaka factors and I have also followed your experiments (congratulations!). I just thought my idea of DNA demethylating agents could be used to at least slightly reverse the aging clock before epigenetic reprogramming with Yamanaka factors becomes available for humans.

 

Anyway, coming back to my question from my previous post - don't you think it's hard to believe that a combination of the interventions I listed in the opening post could be deleterious and increase epigenetic age rather than decrease it?
 

[Turnbuckle]

 

Anyway, coming back to my question from my previous post - don't you think it's hard to believe that a combination of the interventions I listed in the opening post could be deleterious and increase epigenetic age rather than decrease it?
 

 

I don't think anyone yet knows how the 200 epigenetic programs are stored, but the information is surely in DNA somewhere. So when the cell is methylated de novo, it goes back to a clean slate and reprograms the epigenetic marks. The epigenetic age is thus close to zero, and many such cells will substantially lower the average epigenetic age. So unless the intervention you propose stimulates the proliferation of stem cells or is able to reprogram somatic cells somehow, it's difficult to see how this could do anything but make cells epigenetically older.

 

Bottom line, the cells supply the code. These interventions just stimulate cells to perform inbuilt functions.

 

Using growth hormones as in the TRIIM study would presumably lower epigenetic age by increasing stem cell activity. You can come at this from the other direction and use senolytics to lower epigenetic age by killing off the epigenetically oldest cells (which have the shortest telomeres on average). And you can make yourself older by using telomerase stimulants, which give old cells more years and thus raise the average age.

[Iporuru]

I don't think anyone yet knows how the 200 epigenetic programs are stored, but the information is surely in DNA somewhere. So when the cell is methylated de novo, it goes back to a clean slate and reprograms the epigenetic marks. The epigenetic age is thus close to zero, and many such cells will substantially lower the average epigenetic age. So unless the intervention you propose stimulates the proliferation of stem cells or is able to reprogram somatic cells somehow, it's difficult to see how this could do anything but make cells epigenetically older.

 

Bottom line, the cells supply the code. These interventions just stimulate cells to perform inbuilt functions.

 

Using growth hormones as in the TRIIM study would presumably lower epigenetic age by increasing stem cell activity. You can come at this from the other direction and use senolytics to lower epigenetic age by killing off the epigenetically oldest cells (which have the shortest telomeres on average). And you can make yourself older by using telomerase stimulants, which give old cells more years and thus raise the average age.

 

What’s your opinion on David Sinclair’s tweet, especially the two bolded questions at the end: „Pretty amazed by what alpha-keto glutarate, an activator of TET enzymes that demethylate DNA, can do. Here at 1% in drinking water turning white fat in healthier beige fat via Prdm16. Wonder what else is reprogrammed. Is biological age reversed?”

 

 

[Iporuru]

Retinol increases cellular levels of TET proteins

 

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

 

Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity.
RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate
potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously.
We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance
the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative

medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.

 

Significance
Naïve embryonic stem cells are characterized by genome-wide low levels of cytosine methylation, a property that may be
intrinsic to their function. We found that retinol/retinoic acid (vitamin A) and ascorbate (vitamin C) synergistically diminish
DNA methylation levels and in doing so enhance the generation of naïve pluripotent stem cells. This is achieved by two
complementary mechanisms. Retinol increases cellular levels of TET proteins (which oxidize DNA methylation), whereas ascorbate
affords them greater activity by reducing cellular Fe3+ to Fe2+. This mechanistic insight is relevant for the production of
induced pluripotent stem cells used in regenerative medicine, and contributes to our understanding of how the genome is
connected to extrinsic and intrinsic signals.

 

[TMNMK]

Here's another interesting one: TET-2 up-regulation is associated with the anti-inflammatory action of Vicenin-2 which I haven't purchased because it is so expensive, but apparently one can get meaningful quantities from extracts of various plants:

 

For example:

          - Ocimum Sanctum Linn or Tulsi (holy basil)

          - Ethanol extract of the aerial parts of Urtica circularis

          - Perilla frutescens

          - Cyclopia subternata (honeybush - tea from South Africa)

          - Lychnophora ericoides Mart, Asteraceae, leaves

          - Lychnophora salicifolia (arnicão) (leaves)

          - Cayaponia tayuya

 

Note that the drink cachaça that you can get down in Brazil is flavored with arnicão (also used for anti-inflammatory purposes there).

 

EDIT: ah, as you mentioned Iporuru in the first post: "Tea catechins, particularly epigallocatechin-3-gallate (EGCG)"

Edited by TMNMK, 18 November 2019 - 06:43 PM.

[Turnbuckle]

What’s your opinion on David Sinclair’s tweet, especially the two bolded questions at the end: „Pretty amazed by what alpha-keto glutarate, an activator of TET enzymes that demethylate DNA, can do. Here at 1% in drinking water turning white fat in healthier beige fat via Prdm16. Wonder what else is reprogrammed. Is biological age reversed?”

 

 

I can't make heads or tails of it, as I don't see the connection to the paper.

[TMNMK]

Yeah he had the wrong link in there it looked like to me, but here:

 

One from 2019: Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice

 

And one from 2016: AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis

 

 

 

Edited by TMNMK, 18 November 2019 - 07:18 PM.

[Iporuru]

I can't make heads or tails of it, as I don't see the connection to the paper.

 

Perhaps the connection is through Sinclair's paper from which I quoted in the first post: "Four weeks of OSK AAV expression significantly decreased DNA methylation age, and this was Tet1- and Tet2-dependent (Fig. 4i). Together, these results demonstrate that Tet-dependent in vivo reprogramming can restore youthful gene expression patterns, reverse the DNA methylation clock, and restore the function and regenerative capacity of a tissue as complex as the retina"
 

And AKG upregulates TETs (along with Vitamin C and Retinol)

[Iporuru]

Yeah he had the wrong link in there it looked like to me, but here:

 

One from 2019: Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice

 

And one from 2016: AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis

 

Yeah, he had the wrong link but I quoted both these papers in my opening post when describing AKG, so I thought Turnbuckle had a look at them
 

[TMNMK]

I should mention, I do get a very slight odd feeling that I can't quite describe when I drink a-KG (more specifically 2-ketoglutaric acid), I'm curious if anyone else has felt that or if it's just me. I'll put maybe 300mg in a glass dissolved in water, drink that down (tastes tart) and then follow that with 1-2 more glasses of water (because it is a bit acidic, I do this once in the morning and once in the eve before or after dinner typically). I'm out of town for the next three months but when I get home I'll figure out portions of vitamin C & A to add to the concoction based on those papers that you posted Iporuru and post thoughts if I wind up with any. My wife and I are getting DNA methylation age checks (along with other DNA tests) for the holidays for each other, though I don't have a start reference, I'll at least post what those show and maybe get one every year or so going forward provided the science keeps pointing in that direction.

 

(I keep anticipating questions from airport security, you wouldn't believe the things I bring through without even a glance!)

Edited by TMNMK, 18 November 2019 - 07:50 PM.

[Nate-2004]

I'd rather wait for a way to do it with the 3 out of 4 yamanaka factors that Sinclair talks about. Especially if we still have a backup of the original DNA in the nucleus. 

 

What I wonder is if the DNA in the hair sample my mom kept from when I had my very first haircut as a young kid is a good backup copy were it to ever be useful in some way.

[Iporuru]

I should mention, I do get a very slight odd feeling that I can't quite describe when I drink a-KG (more specifically 2-ketoglutaric acid), I'm curious if anyone else has felt that or if it's just me. I'll put maybe 300mg in a glass dissolved in water, drink that down (tastes tart) and then follow that with 1-2 more glasses of water (because it is a bit acidic, I do this once in the morning and once in the eve before or after dinner typically). I'm out of town for the next three months but when I get home I'll figure out portions of vitamin C & A to add to the concoction based on those papers that you posted Iporuru and post thoughts if I wind up with any. My wife and I are getting DNA methylation age checks (along with other DNA tests) for the holidays for each other, though I don't have a start reference, I'll at least post what those show and maybe get one every year or so going forward provided the science keeps pointing in that direction.

 

(I keep anticipating questions from airport security, you wouldn't believe the things I bring through without even a glance!)

 

That's the spirit TMNMK!

In the Interviews thread I proposed asking D. Sinclair what he thought of the regimen I have delineated in this thread. If he answers and is positive, I think I'll also give it a try next year with a DNA methylation test before and after the intervention. Good luck!
 

[TMNMK]

I'd rather wait for a way to do it with the 3 out of 4 yamanaka factors that Sinclair talks about. Especially if we still have a backup of the original DNA in the nucleus. 

 

What I wonder is if the DNA in the hair sample my mom kept from when I had my very first haircut as a young kid is a good backup copy were it to ever be useful in some way.

 

I assume that could be informative simply from a comparison perspective!

[dlewis1453]

The only way to do it is to reprogram the cell (inducing pluripotency), or by replacing an epigenetically old cell with a stem cell that matures into the desired somatic cell. The programing you get going through a stem cell phase is de novo, and thus has a very low epigenetic age.

 

 

Hi Turnbuckle and others, 

 

Apologies for the long post, but I have been wrestling with a concept concerning the topic at hand that hopefully you can shed some light on. I'll start with some assertions and follow up with questions (underlined) that are based on those assertions. 

 

"Replacing an epigenetically old cell with a stem cell that matures into the desired somatic cell" is a process that forms one of the foundations of your c60 stem cell protocol. Your epigenetic age tests show that your protocol has reversed your epigenetic age considerably. 

 

However, everyday in our bodies, old somatic cells are naturally replaced by new cells from our stem cell pools (Note: not to the same extent as with your protocol, and becoming progressively less efficient with age). 

 

These new somatic cells that are produced naturally by our bodies from our stem cell pools would also have a lower epigenetic age. 

 

Different cells have different lifetimes, ranging from a few days, to a few months, to years. 

 

Why then - if cells have different lifetimes, and our body is constantly replacing old cells with new cells produced from stem cells - is an epigenetic age test based entirely on short-lived cells taken from a blood/urine sample able to correlate so strongly with our chronological age? 

 

Is this result possible because the stem cells that produce the somatic cells are aging epigenetically as well?  This would result in new somatic cells being created already aged, so that the older the stem cells, the older the somatic cell that is born from the stem cell. 

 

If so, are the results of your protocol simply due to accelerating this naturally occurring cell replacement to an extent that the average epigenetic age of the blood/urine cells is decreased? 

 

Or perhaps the drastic results of your protocol is indeed due to proliferating and mobilizing very small embryonic like stem cells (VSELs), which have zero epigenetic age? If so, do we know if VSELs age epigenetically? 

 

 

Thanks! 

[Iporuru]

An admittedly small study of healthy volunteers claimed that the DNA methylation age of blood cells is reversed within a week and, astoundingly, only ten hours after taking a single 850 mg pill of metformin

 

Significantly altered peripheral blood cell DNA methylation profile as a result of immediate effect of metformin use in healthy individuals

 

 

Abstract

Background

Metformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin’s therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design.

 

Results

Twelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases.

 

Conclusions

Here we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin.

 

 

Other corroborating studies:

 

"This has been supported by data showing that metformin promotes global methylation by decreasing S-adenosylhomocysteine (SAH) intracellular levels in various cell types, including non-cancerous": Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism

 

„One of the latest studies have specifically shown metformin’s effect on lowering the methylation levels at the metformin transporter genes, resulting in higher expression levels in liver tissue”: Diabetes medication associates with DNA methylation of metformin transporter genes in the human liver

 

 

„Studies describing other epigenetic effects of metformin have shown its impact on various histone modifications via multiple mechanisms, mostly AMPK dependent, and effect on expression levels of numerous miRNAs through increase in DICER protein levels as well”:

Epigenetic effects of metformin: From molecular mechanisms to clinical implications

 

[Turnbuckle]

Hi Turnbuckle and others, 

 

Apologies for the long post, but I have been wrestling with a concept concerning the topic at hand that hopefully you can shed some light on. I'll start with some assertions and follow up with questions (underlined) that are based on those assertions. 

 

"Replacing an epigenetically old cell with a stem cell that matures into the desired somatic cell" is a process that forms one of the foundations of your c60 stem cell protocol. Your epigenetic age tests show that your protocol has reversed your epigenetic age considerably. 

 

However, everyday in our bodies, old somatic cells are naturally replaced by new cells from our stem cell pools (Note: not to the same extent as with your protocol, and becoming progressively less efficient with age). 

 

These new somatic cells that are produced naturally by our bodies from our stem cell pools would also have a lower epigenetic age. 

 

Different cells have different lifetimes, ranging from a few days, to a few months, to years. 

 

Why then - if cells have different lifetimes, and our body is constantly replacing old cells with new cells produced from stem cells - is an epigenetic age test based entirely on short-lived cells taken from a blood/urine sample able to correlate so strongly with our chronological age? 

 

Is this result possible because the stem cells that produce the somatic cells are aging epigenetically as well?  This would result in new somatic cells being created already aged, so that the older the stem cells, the older the somatic cell that is born from the stem cell. 

 

If so, are the results of your protocol simply due to accelerating this naturally occurring cell replacement to an extent that the average epigenetic age of the blood/urine cells is decreased? 

 

Or perhaps the drastic results of your protocol is indeed due to proliferating and mobilizing very small embryonic like stem cells (VSELs), which have zero epigenetic age? If so, do we know if VSELs age epigenetically? 

 

 

Thanks! 

 

 

In most tissues, especially those that renew themselves frequently, stem cells don't differentiate directly into somatic cells, but into transit amplifying cells (TACs). Those intermediate cells do most of the dividing, and most of the epigenetic aging will occur there.

Edited by Turnbuckle, 19 November 2019 - 08:15 PM.

[dlewis1453]

In most tissues, especially those renew themselves frequently, stem cells don't differentiate directly into somatic cells, but into transit amplifying cells (TACs). Those intermediate cells do most of the dividing, and most of the epigenetic aging will occur there.

 

Ah yes! Transit amplifying cells. I forgot about them.  Yes that makes sense and explains the gap in my reasoning. Thanks

 

 

What about stem cells and VSELs? Do we have an understanding of to what extent they are/maybe subject to epigenetic aging? 

[dlewis1453]

An admittedly small study of healthy volunteers claimed that the DNA methylation age of blood cells is reversed within a week and, astoundingly, only ten hours after taking a single 850 mg pill of metformin

 

Significantly altered peripheral blood cell DNA methylation profile as a result of immediate effect of metformin use in healthy individuals

 

 

Abstract

Background

Metformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin’s therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design.

 

Results

Twelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases.

 

Conclusions

Here we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin.

 

 

Other corroborating studies:

 

"This has been supported by data showing that metformin promotes global methylation by decreasing S-adenosylhomocysteine (SAH) intracellular levels in various cell types, including non-cancerous": Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism

 

„One of the latest studies have specifically shown metformin’s effect on lowering the methylation levels at the metformin transporter genes, resulting in higher expression levels in liver tissue”: Diabetes medication associates with DNA methylation of metformin transporter genes in the human liver

 

 

„Studies describing other epigenetic effects of metformin have shown its impact on various histone modifications via multiple mechanisms, mostly AMPK dependent, and effect on expression levels of numerous miRNAs through increase in DICER protein levels as well”:

Epigenetic effects of metformin: From molecular mechanisms to clinical implications

 

This is very interesting Iporuru. There is some evidence that Rapamycin has some positive effects on epigenetics as well. This may be additional benefit to combining metformin and rapamycin.

[Iporuru]

This is very interesting Iporuru. There is some evidence that Rapamycin has some positive effects on epigenetics as well. This may be additional benefit to combining metformin and rapamycin.

 

Yes, I mentioned rapa in my first post:

 

 

Prolongevity interventions, including reduced growth hormone (GH) and insulin-like growth factor (IGF) signaling, CR, and rapamycin, also slow down ticking of the presumptive biological clock.

Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions

 

Epigenetic aging signatures in mice livers are slowed by dwarfism, calorie restriction and rapamycin treatment.

 

 

[Turnbuckle]

Ah yes! Transit amplifying cells. I forgot about them.  Yes that makes sense and explains the gap in my reasoning. Thanks

 

 

What about stem cells and VSELs? Do we have an understanding of to what extent they are/maybe subject to epigenetic aging? 

 

 

SCs can age as well. They can suffer telomere shortening, DNA mutations, and to the extent they have epigenetic marks, they could suffer epigenetic aging, though this would be considerably more restricted than with somatic cells. VSELs would have the least problem with epigenetic aging, as their epigenetic coding as pluripotent cells would minimal. Thus the stem cell pools may not decline in absolute numbers with chronological aging, but the numbers of viable SCs will decline. 

 

Adult stem cells, also known as somatic stem cells, are found throughout the body in every tissues and organs after development and function as self-renewing cell pools to replenish dying cells and regenerate damaged tissues throughout life. However, adult stem cells appear to age with the person. As stem cells age, their functional ability also deteriorates. Specifically, this regenerative power appears to decline with age, as injuries in older individuals heal more slowly than in childhood. For example, healing of a fractured bone takes much longer time in elderly than in young individuals. There is a substantial amount of evidence showing that deterioration of adult stem cells in the adult phase can become an important player in the initiation of several diseases in aging

https://www.ncbi.nlm...les/PMC5316899/

 

 

 

My feeling is that the biggest problem is the build up of the non-dividing fraction of the SC pools. Insofar as there are homeostatic mechanisms to maintain SC pool size, increasing SC numbers from viable SCs will, at a minimum, reduce the number of non-dividing SCs by those homeostatic mechanisms, and thus return the pools to a more youthful state.

[Iporuru]

Turnbuckle, sorry to bother you, but I really value and respect your opinion – did you have time to have a look at the 2 papers about AKG that TMNMK linked in a post above? (Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis). If yes, what’s your opinion on David Sinclair’s tweet, mentioned above: „Pretty amazed by what alpha-keto glutarate, an activator of TET enzymes that demethylate DNA, can do. Here at 1% in drinking water turning white fat in healthier beige fat via Prdm16. Wonder what else is reprogrammed. Is biological age reversed?”

 

Also, how about these fragments from Sinclair’s recent book?:  

 

we may be on the verge of understanding what makes biological time tick and how to wind it back. We know from our experiments that the biological information correcting device requires enzymes called ten-eleven translocation enzymes, or TETs, which clip off methyl tags from DNA, the same chemical tags that mark the passage of the Horvath aging clock. This is no coincidence, and points to the DNA methylation clock as not just an indicator of age but a controller of it. It’s the difference between a wristwatch and physical time. In their role as a component of the correcting device, the TETs cannot just strip off all the methyls from the genome, for that would turn a cell into a primordial stem cell. We would not have old mice that can see better: we would have blind mice with tumors. How the TETs know to remove only the more recent methyls while preserving the original ones is a complete mystery. (…)

 

I have little doubt that cellular reprogramming is the next frontier in aging research. One day it might be possible to reprogram cells via pills that stimulate the activity of the OSK factors or the TETs. This may be simpler than it sounds. Natural molecules stimulate the TET enzymes, including vitamin C and alpha-ketoglutarate, a molecule made in mitochondria that is boosted by CR and, when given to nematode worms, extends their lifespan, too.(…)

 

[about NMN] We also know that the way it does this, in terms of the epigenetic landscape, is by creating the right level of stress—just enough to push our longevity genes into action to suppress epigenetic changes to maintain the youthful program. In doing so, NMN and other vitality molecules, including metformin and rapamycin, reduce the buildup of informational noise that causes aging, thus restoring the program.(…) the increased activity of the sirtuins may prevent Waddington’s marbles from escaping their valleys. And even if they have started to head out of the valley, molecules such as NMN may push them back down, like extra gravity. In essence, this would be age reversal in some parts of the body—a small step, but age reversal nonetheless.(…) Armies of chemists are now working to create and analyze natural and synthetic molecules that have the potential to be even better at suppressing epigenomic noise and resetting our epigenetic landscape.(…)

 

[QuestforLife]

What’s your opinion on David Sinclair’s tweet, especially the two bolded questions at the end: „Pretty amazed by what alpha-keto glutarate, an activator of TET enzymes that demethylate DNA, can do. Here at 1% in drinking water turning white fat in healthier beige fat via Prdm16. Wonder what else is reprogrammed. Is biological age reversed?”

 

There is evidence that some super quiescent, possibly pluripotent stem cells are held that way by methylation (basically they are what's left over from the embryo - stem cells that resisted differentiation), and sufficient demethylation agents might cause them to re-enter circulation.

 

https://www.ahajourn...SAHA.118.314287

 

Of course demethylating your body long term will screw you up due to effects on various vital metabolic processes like the one carbon cycle, and probably kill your liver because of the increased demand for methylating agents. So I speculate that any intentional body wide demethylation will have to be for short bursts only, unless you can somehow make it selective.