989 replies to this topic

[Learner056]

So Interesting.  If I may join you folks in this Resveratrol conversation.  Appreciate criticism of my understanding below:

 

Benefits:

1. Durig my personal trial: I liked its effects (when in proper form), e.g. lowered BP, skin glow, until I paired it with NMN. 

2. Increase of undifferentiated HSCs i.e. Immuno-senescence.  "induced a significant increase in T helper cells (CD4(+)" Ref: Dietary intake of resveratrol enhances the adaptive immunity of aged rats - PubMed (nih.gov)

 

Disadvantages: 

1. All the ongoing rumors. 

2. It does so many things, that for an informed consumer (or call it biohacker), I find it difficult to place it in a proper "model/combination" that allows variations to fit unique physiology/needs of an individual (i.e. what works for me may not work for you).  A good model enables us to work around these variations, but for Resveratrol, I am finding this impossible 'for now'). 

 

// That said, long-term understanding these variations, I believe is vital, as they reveal pathological conditions in a dormant and at worse primed state (knowing them before hand, enables pro-activeness).  For e.g. using Resveratrol stack enabled me to understand that my mitochondrial "fission" is malfunctioning.  I have not figured out yet (a matter of time) whether it is hyper/hypo/lacking key enzyme or what.  

 

 

[Castiel]

So Interesting. If I may join you folks in this Resveratrol conversation. Appreciate criticism of my understanding below:

Benefits:
1. Durig my personal trial: I liked its effects (when in proper form), e.g. lowered BP, skin glow, until I paired it with NMN.
2. Increase of undifferentiated HSCs i.e. Immuno-senescence. "induced a significant increase in T helper cells (CD4(+)" Ref: Dietary intake of resveratrol enhances the adaptive immunity of aged rats - PubMed (nih.gov)

Disadvantages:
1. All the ongoing rumors.
2. It does so many things, that for an informed consumer (or call it biohacker), I find it difficult to place it in a proper "model/combination" that allows variations to fit unique physiology/needs of an individual (i.e. what works for me may not work for you). A good model enables us to work around these variations, but for Resveratrol, I am finding this impossible 'for now').

// That said, long-term understanding these variations, I believe is vital, as they reveal pathological conditions in a dormant and at worse primed state (knowing them before hand, enables pro-activeness). For e.g. using Resveratrol stack enabled me to understand that my mitochondrial "fission" is malfunctioning. I have not figured out yet (a matter of time) whether it is hyper/hypo/lacking key enzyme or what.

Wellness Messiah on youtube has had several videos covering benefits of resveratrol.

It seems lower dose under 500mg potentially just a few 10s to 100s of mg may be optimal.

The article on cell rejuvenation with resveratrol suggests prolonged exposure may be of benefit. But resveratrol is rapidly cleared from system. Which is why Im thinking perhaps small doses every few hours to maintain prolonged exposure sounds promising.

[dlewis1453]

Spermidine is an interesting molecule that appears to slow telomere attrition, upregulate gdf11, and increase autophagy partly through an MTOR independent mechanism. 

 

 

Spermidine is able to slow telomere attrition, according to the paper "Novel aspects of age-protection by spermidine supplementation are associated with preserved telomere length" https://www.ncbi.nlm...les/PMC8110654/

 

 

 

Here, we show that a 6-month administration of the natural autophagy inducer spermidine in the drinking water to aged mice is sufficient to significantly attenuate distinct age-associated phenotypes. These include modulation of brain glucose metabolism, suppression of distinct cardiac inflammation parameters, decreased number of pathological sights in kidney and liver and decrease of age-induced hair loss. Interestingly, spermidine-mediated age protection was associated with decreased telomere attrition, arguing in favour of a novel cellular mechanism behind the anti-ageing effects of spermidine administration.

 

Spermidine also upregulates GDF11, according to the paper, "Spermidine inhibits high glucose-induced endoplasmic reticulum stress in HT22 cells by upregulation of growth differentiation factor 11"   https://www.ingentac...000018/art00006  GDF11's benefits for telomeres has already been discussed multiple times in this thread.

 

 

 

Hyperglycemia-induced neuronal endoplasmic reticulum (ER) stress is particularly important for the pathogenesis of diabetic encephalopathy. Spermidine (Spd) has neuroprotection in several nervous system diseases. Our current study to explore the potential protective role of Spd in hyperglycemia-induced neuronal ER stress and the underlying mechanisms....These results indicated that Spd prevents HG-induced neurotoxicity and ER stress through upregulation of GDF11. Our findings identify Spd as a potential treatment for diabetic encephalopathy as well as ER stress-related neurologic diseases.

 

Spermidine increases autophagy overall, partly through an MTOR-independent mechanism, but also appears to modulate  mTORC1 by increasing its activity in some tissues and decreasing it in others, according to the paper "The Autophagy Inducer Spermidine Protects Against Metabolic Dysfunction During Overnutrition." https://www.ncbi.nlm...les/PMC8436989/

 

 

Unlike CR or rapamycin, however, spermidine has been shown to induce autophagy by means of a mTORC1-independent pathway, as spermidine did not alter the phosphorylation status of mTOR or its downstream substrate, ribosomal protein S6 kinase (19). Interestingly, our results showed spermidine modulates mTORC1 activity, a conserved regulator of autophagy, and autophagy activity in age-, diet-, and tissue-dependent fashion. For instance, spermidine increased mTORC1 activity, indicated by phosphorylation of rpS6 (p-S6 S240/244), as well as Beclin-1 protein levels in subcutaneous fat of young mice, whereas these effects were reversed in mice fed a HFD. Since a HFD is reported to induce adipose-specific autophagy, it is possible that spermidine directly counteracts this response (24). In liver, HFD suppresses autophagy (24), which was not reversed by spermidine. This age- and tissue-dependent autophagic response was also observed (which was not limited to spermidine) in cardiac tissue where CR suppressed autophagy, indicated by Beclin-1 protein, in young mice, while it accelerated autophagy in middle-aged and old mice (25). As a catabolic process, it is worth noting that autophagy levels have to be tightly regulated and dynamic in response to aging and energetic demands (26). Furthermore, although mTORC1 activity is generally associated with antagonizing the induction of autophagy, it is interesting to note that induction of autophagy can occur through both mTORC1-dependent and -independent pathways (27).

 

I believe this research shows that Spermidine is an interesting molecule with complex effects, and that it is much more than just an endogenous MTOR suppressor. I would be interested to see further research that addresses to what extent Spermidine interacts with TERC and TERT and whether Spermidine is capable of upregulating Klotho. 

 

On the anecdotal front, a member of a longevity facebook group mentioned that he met a doctor who claimed to have reversed his epigenetic age by 19 years by taking GDF11, Klotho, and Spermidine simultaneously. I have asked this person to provide more information. Hopefully they respond. 

Edited by dlewis1453, 01 December 2022 - 08:45 PM.

[QuestforLife]

Spermidine is an interesting molecule that appears to slow telomere attrition, upregulate gdf11, and increase autophagy partly through an MTOR independent mechanism. 

 

 

Spermidine is able to slow telomere attrition, according to the paper "Novel aspects of age-protection by spermidine supplementation are associated with preserved telomere length" https://www.ncbi.nlm...les/PMC8110654/

 

 

 

 

 

 

Very odd. Paper has frequency distribution analysis of telomere length of cardiac tissue extracted from mice, and spermidine seems to arrest the slide of the distribution towards the shorter end (top three images).  But the analysis of the percentage of cells in the bottom 50% is very strange; spermidine treatment appears to squash all variability (bottom image), which seems to directly contradict the distribution. The only way I can think of is if spermidine was removing very long telomeres (not seen in the distribution), so as to move the mean length down and reduce the % of telomeres below the mean. Or acted as a senolytic and destroyed cells with very short telomeres, perhaps too short to be seen in the distribution. Either that, or they looked at a much smaller sample size for the aged+spermidine treated cells. 

 

(see attachment 1; for some reason longecity is no longer allowing image addresses)

 

We get a similar result looking at the number of detectable telomere signals per cell; as expected this decreases from young to old as telomeres are reduced below the threshold of florescent detectability. But for the aged+spermidine treated cells once again there is an improvement, but also a reduction in variability, even compared to young controls.

 

(see attachment 2; for some reason longecity is no longer allowing image addresses)

 

I can find almost no mention in the literature of spermidine and telomerase. There is one paper that posits autophagy is important for the release of NO and so may mediate telomerase production in the vasculature (see https://doi.org/10.1161/ATVBAHA.120.314944), which is not the first I've heard of NO (or indeed H2S2) stimulating telomerase. If this is true, then we are looking at an indirect effect. It may also be of limited applicability to humans, given mice have the TERT gene turned on in every cell by default, and loss of this with aging may be the cause of any telomere loss they experience.

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Edited by QuestforLife, 06 December 2022 - 03:43 PM.

[QuestforLife]

Is it time to abandon methylation based aging tests?

 

 

I have attached a document because it includes pictures I can't get Longecity to accept within the main window. 

 

In it I discuss my recent epigenetic aging acceleration in comparison to my traditional biomarkers.

 

 

Attached Files

•  Is it time to abandon methylation based aging tests.docx   793.59KB   97 downloads

[dlewis1453]

I thought the below portion of your writeup was very interesting. Volatility of a time series can sometimes contain information about the future value of the time series, and it is interesting that this phenomenon seems to hold true here as well. 

 

 

 

I also did an analysis of variance for the 3 month period in question compared to the previous 3 months (not shown in plots). It might be that deterioration (or improvements) show up more quickly as a change in variance than they do in the actual biomarker itself. 

 

I think it is clear that you are super healthy and becoming healthier, despite your increasing epigenetic age. I would be willing to bet money on you becoming younger rather than older by pursuing your telomere experiment. I like the idea of using yourself as an experiment to disprove the theory that younging must always equal epigenetic age reduction.

 

You have stated that epigenetics may be a consequence of, but not a cause of, aging. However in this thread you have also explained how the methylation and demethylation of specific genes may accelerate the aging process. Do you think AKG + GDF11 (maybe + Klotho) is sufficient to handle these specific genes? Or will these genes handle themselves as telomeres lengthen and cells are replaced from the stem cell pool? I wonder if we need an epigenetic age test that looks at just a few sites that have been confirmed in the research to play a causative role in aging? 

 

 

At this point I am highly tempted to continue my supertelomerase protocol (ref) in conjunction with the occasional Turnbuckle protocol or rock-inhibitor, to see quite how much I can increase my epigenetic age without any real decline in function. Either way it should prove something.

 

As you continue with this approach, do you think at some point you will see a decrease in epigenetic age, as your increasingly healthy stem cells differentiate into somatic cells? Didn't Bill Lawrence state in his video that he eventually experienced epigenetic age reduction from long term use of epitalon? 

Edited by dlewis1453, 14 December 2022 - 02:42 PM.

[QuestforLife]

I thought the below portion of your writeup was very interesting. Volatility of a time series can sometimes contain information about the future value of the time series, and it is interesting that this phenomenon seems to hold true here as well.

 

 

It may be a canary in the coal mine - if my biomarkers become more scattered, then change is afoot, even if the average is relatively steady. 

 

 

I think it is clear that you are super healthy and becoming healthier, despite your increasing epigenetic age. I would be willing to bet money on you becoming younger rather than older by pursuing your telomere experiment. I like the idea of using yourself as an experiment to disprove the theory that younging must always equal epigenetic age reduction.

 

I could be wrong and I would admit that, if continuing epigenetic age acceleration began to cause biomarker deterioration. That is why I think the experiment is worth doing. Either we find epigenetic aging tests are worthless, or we find out epigenetic age acceleration is actually harmful in the context of a health intervention.  

 

You have stated that epigenetics may be a consequence of, but not a cause of, aging. However in this thread you have also explained how the methylation and demethylation of specific genes may accelerate the aging process. Do you think AKG + GDF11 (maybe + Klotho) is sufficient to handle these specific genes? Or will these genes handle themselves as telomeres lengthen and cells are replaced from the stem cell pool? I wonder if we need an epigenetic age test that looks at just a few sites that have been confirmed in the research to play a causative role in aging?

 

AKG is most likely acting on a pathway that links mitochondrial health to gene regulation, which seems to give a fixed 5-6 years of epigenetic age reduction, but without stopping the overall process (it advances upwards from this lower level). GDF11 can elongate telomeres via TERC upregulation, which is why it is part of my protocol, but it no doubt has other effects too. It might be that even my supertelomerase protocol is insufficient for telomere elongation, in which case I will just be slowing shortening rate, and keeping cell lines going for longer. So it is very hard to draw definite conclusions about what is happening.   

 

As you continue with this approach, do you think at some point you will see a decrease in epigenetic age, as your increasingly healthy stem cells differentiate into somatic cells? Didn't Bill Lawrence state in his video that he eventually experienced epigenetic age reduction from long term use of epitalon?

 

 

The epigenetic age reduction with epitalon might be due to a slow rejuvenation of the telomeres of stem cells, leading to a younger epigenetic profile of the downstream cells, because stem cells are able to replace them more often. Epigenetic age acceleration (with epitalon) only seems to occur when it is combined with stimulators of symmetric cell division (SCD). SCD is required for rejuvenation because this is the pathway by which stem cells are freed from the matrix and proliferate freely before differentiating. My theory is that SCD prevents a cell from regulating its epigenetic state because it can't control which side of the DNA (original or new) goes into which cell.  For now I am operating on the assumption that epigenetic age acceleration is just a consequence of greater SCD of stem cells, and that this will benefit health more than it harms it. But that is the point of the experiment.

 

I am expecting one of the following results:

1. epigenetic age acceleration continues upward due to my continued combination of SCD and telomerase activators, but health is not affected. In which case we can ignore methylation aging tests

2. epigenetic age acceleration continues upward due to my continued combination of SCD and telomerase activators, but health is adversely affected. In which case we have learnt that methylation aging tests are useful.

3. Or perhaps epigenetic age acceleration plateaus or even falls again, with continued use of SCD and TAs,  as stem cells become more able to replace downstream cells. This would be an ideal result, particularly is health biomarkers remained good. It would tell us epigenetic acceleration due to health interventions is a transitory or minor effect, and methylation aging tests would still be useful in this context.

4. There might be detrimental effects from my protocol that are unrelated to epigenetic age acceleration, but mean I have to modify or discontinue the protocol. For example if my BP gets too low, etc. 

It will be interesting to find out!

 

I should note that I am taking everolimus x1/week separated from the protocol as far as possible; this is my ‘insurance’. But this has been the case on and off throughout this year, and has not impacted the epigenetic aging results already reported.

[QuestforLife]

Can ultrasound overcome replicative senescence?

Rejuvenating Senescent Cells and Organisms with Only Ultrasound
https://doi.org/10.1...22.12.08.519320

In this preprint, the authors render cells senescent by various methods, including
(supposedly) replicative senescence, and then rejuvenate those cells with Ultrasound. The cells return to normal (smaller) size and proliferate normally. The purported mechanism is mitochondrial fission and clearance of excess lysosomal activity, as well as growth factors secreted by still healthy cells.

In the part of the paper most interesting to us, they grow human fetal fibroblasts until they no longer divide and then treat them. They do not give much detail on the Ultrasound itself, other than to say it is low power (compared to what has been approved for human use) and low frequency.

Do I think this is real? I don't dismiss it out of hand. But they give me reason for doubt.

Firstly they never measure telomeres. Might they have caused another sort of reversible arrest? Secondly, they don't passage the cells when they have filled the plate, but every 48 hours, regardless of growth rate. With this method they manage about 15 passages before cell division has slowed right down. But this makes it very difficult for us to assess how many divisions they achieved. Any less than 50 and I would be suspicious we haven't actually reached the hayflick limit.

Conclusion: it is possible that Ultrasound works in a similar manner to large doses of nicotinamide to extend out the replicative limit of cells [1], by reducing mitochondrial mass and reducing the rise in ROS that comes with continued division. This leads to a reduction in telomere shortening rate. It is surprising that Ultrasound can actually reverse replicative senescence, which is supposedly irreversible without telomerase. They do also reverse senescence induced by chemical agents, and also show some physical benefits to old mice by Ultrasound treatment. They speculate exercise may work via similar mechanical stress on cells. And they show it may synergise with rapamycin. All in all a very interesting paper. Perhaps the final published version will be more complete.

[1] Kang HT, Lee HI, Hwang ES. Nicotinamide extends replicative lifespan of human cells. Aging Cell. 2006 Oct;5(5):423-36. doi: 10.1111/j.1474-9726.2006.00234.x. Epub 2006 Aug 25. PMID: 16939485.

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Edited by QuestforLife, 18 December 2022 - 10:14 AM.

[QuestforLife]

A reanalysis of some epitalon studies and a new take on the DNA mutation theory of aging

In this study (1) passage 10 ('young') human lung fibroblasts - passaged once per week - were compared to passage 34 ('old') fibroblasts, which had stopped dividing, as well as to another cell line, dosed with epitalon at 0.05ug/ml from passage 28 and examined at a very old passage 38. This line continued dividing beyond passage 44.

I am taking the paper on trust at this point, fully replicated (in the West) or not.

The telomeres of the 'old' cells were much shorter than the 'young' ones: 21 vs 11 (arbitrary units), but the epitalon old treated cells were much longer at 26, which agrees with their continual proliferation. The distribution of these lengths was rather odd however, with treated cells having a range of telomere lengths of roughly equal likelihood rather than being clustered around some peak value. There seem to be less cells overall. Perhaps epitalon is rescuing a subset of cells, not all.

Additionally, old and older (epitalon) treated cells both had cells with extra DNA compared to the younger cells (compared by staining). You'd expect a few cells to be stuck with dividing chromosomes and hence extra DNA. But when this becomes more prevalent it is associated with aneuploidy, when there are extra copies of chromosomes present. Note I also found this extra chromosome effect in a paper on the liver, an organ whose cells are replaced unfailingly throughout life (2). Extra DNA might be expected with eroding telomeres unable to prevent chromosomal fusion. Going back to the epitalon studies, the authors hypothesize that starting treatment at a relatively late 28 passages, the chromosomal damage had already been done. Two other studies (by some of the same authors) back up the ability of epitalon to prevent additional chromosomal abnormalities, but possibly not repair those already present (3)(4). I am not altogether convinced of this argument however, and I have long been preoccupied with what else might go wrong in cells supplied with sufficient telomerase.

What if there was another source of extra DNA that could contaminate cells? I think that there is and this is a contributing factor in aging (and cancer). From (1), young cells average DNA content is 13 vs 19 in old cells (arbitrary units again,) with very old treated cells at 21 (ish). To me it looks like the process is continuing in spite of epitalon. What I think is happening is that when cells die as they inevitably do in both the petri dish and the body, some of their DNA survives and is taken up by other cells. It then gets treated like legitimate chromosomal DNA, stitched together, repaired, maybe even merged with chromosomal DNA when double strand breaks occur. If true, then this gives new life to the old DNA mutation theory of aging. It also explains why cancer is so hard to get rid of: kill cancerous cells and they just spew their DNA into the bloodstream for other healthy cells to take up.

I looked and remarkably there are papers looking into this very issue: cell free chromatin particles as an underlying cause of aging (4). They even have a treatment for it - a combination of low dose resveratrol and very low dose copper to create reactive oxygen species that cause cleavage of the cell free DNA. They've even tested this on mice and find it clears the DNA in the blood, without damaging healthy cells. A detailed review of that paper can wait for another time. Suffice it to say, I have now expanded my protocol to include cell free DNA clearance, as I have reason to believe it will synergies with telomerase activation.

References

(1)Khavinson VKh, Bondarev IE, Butyugov AA, Smirnova TD. Peptide promotes overcoming of the division limit in human somatic cell. Bull Exp Biol Med. 2004 May;137(5):503-6. doi: 10.1023/b:bebm.0000038164.49947.8c. PMID: 15455129.
(2)Heinke P, Rost F, Rode J, Trus P, Simonova I, Lázár E, Feddema J, Welsch T, Alkass K, Salehpour M, Zimmermann A, Seehofer D, Possnert G, Damm G, Druid H, Brusch L, Bergmann O. Diploid hepatocytes drive physiological liver renewal in adult humans. Cell Syst. 2022 Jun 15;13(6):499-507.e12. doi: 10.1016/j.cels.2022.05.001. Epub 2022 May 31. PMID: 35649419.
(3)Rosenfeld SV, Togo EF, Mikheev VS, Popovich IG, Khavinson VKh, Anisimov VN. Effect of epithalon on the incidence of chromosome aberrations in senescence-accelerated mice. Bull Exp Biol Med. 2002 Mar;133(3):274-6. doi: 10.1023/a:1015899003974. PMID: 12360351.
(4)Dzhokhadze TA, Buadze TZh, Rubanov KD, Kiriia NA, Lezhava TA. [Genome instability in pulmonary tuberculosis before and after treatment]. Georgian Med News. 2013 Nov;(224):77-81. Russian. PMID: 24323970.
(5)Pal K, Raghuram GV, Dsouza J, Shinde S, Jadhav V, Shaikh A, Rane B, Tandel H, Kondhalkar D, Chaudhary S, Mittra I. A pro-oxidant combination of resveratrol and copper down-regulates multiple biological hallmarks of ageing and neurodegeneration in mice. Sci Rep. 2022 Oct 14;12(1):17209. doi: 10.1038/s41598-022-21388-w. PMID: 36241685; PMCID: PMC9568542.

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Edited by QuestforLife, 23 December 2022 - 10:40 AM.

[dlewis1453]

Wishing a merry Christmas and happy new year to QuestforLife and everyone else who follows this thread! Much thanks to QuestforLife for diligently and selflessly sharing his detailed research and valuable insights with this community. Hopefully this thread will see some big longevity breakthroughs in 2023. 

[dlewis1453]

 

 

I looked and remarkably there are papers looking into this very issue: cell free chromatin particles as an underlying cause of aging (4). They even have a treatment for it - a combination of low dose resveratrol and very low dose copper to create reactive oxygen species that cause cleavage of the cell free DNA. They've even tested this on mice and find it clears the DNA in the blood, without damaging healthy cells. A detailed review of that paper can wait for another time. Suffice it to say, I have now expanded my protocol to include cell free DNA clearance, as I have reason to believe it will synergies with telomerase activation.

 

I did some searching on this topic and found a newer paper from the same authors which linked cell free chromatin particle dispersion to mitochondrial aging as well. 

 

"Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells"

https://www.biorxiv....0.474529v1.full

________________________________________________________________________

I am a little disturbed by the discovery of what appears to me to be an entirely distinct, previously unknown mechanism of dna damage, but I am also comforted by the fact that a simple protocol of resveratrol + copper appears to ameliorate this pathway. Some questions that come to my mind are: (1) just how effective is the resveratrol + copper protocol? (2) what is the optimal dosing amount and frequency? (3) How does cell free chromatin particle dispersion induced dna damage interact with other aging pathways? (4) Does the body possess a greater ability to resist damage from cell free chromatin particle induced dna damage in youth, as with other forms of dna damage? (5) are there other interventions to treat this aging pathway besides resveratrol + copper that could be more effective?

________________________________________________________________________

 

In addition to the copper + resveratrol protocol, perhaps we could benefit from upregulating dna repair capabilities throughout the body by inducing mild mitochondrial uncoupling? DNP is powerful inducer of mitochondrial uncoupling that, although dangerous and potentially fatal at higher doses, appears to be very beneficial at low doses. The paper "Mild mitochondrial uncoupling protects from ionizing radiation induced cell death by attenuating oxidative stress and mitochondrial damage" https://www.scienced...005272820301754 applied low dose DNP to cells and then blasted the cells with ionizing radiation. the DNP treated cells were better able to resist the radiation-induced damage to mitochondria and to DNA, see below abstract. 

 

 

Ionizing radiation (IR) induced mitochondrial dysfunction is associated with enhanced radiation stimulated metabolic oxidative stress that interacts randomly with intracellular bio-macromolecules causing lethal cellular injury and cell death. Since mild mitochondrial uncoupling emerged as a valuable therapeutic approach by regulating oxidative stress in most prevalent human diseases including ageing, ischemic reperfusion injury, and neurodegeneration with comparable features of IR inflicted mitochondrial damage. Therefore, we explored whether mitochondrial uncoupling could also protect from IR induced cytotoxic insult. Our results showed that DNP, BHT, FCCP, and BAM15 are safe to cells at different concentrations range depending on their respective mitochondrial uncoupling potential. Pre-incubation of murine fibroblast (NIH/3T3) cells with the safe concentration of these uncouplers followed by gamma (γ)-radiation showed significant cell growth recovery, reduced ROS generation, and apoptosis, compared to IR treatment alone. We observed that DNP pre-treatment increased the surviving fraction of IR exposed HEK-293, Raw 264.7 and NIH/3T3 cells. Additionally, DNP pre-treatment followed by IR leads to reduced total and mitochondrial oxidative stress (mos), regulated calcium (Ca2+) homeostasis, and mitochondrial bioenergetics in NIH/3T3 cells. It also significantly reduced macromolecular oxidation, correlated with the regulated ROS generation and antioxidant defence system. Moreover, DNP facilitated DNA repair kinetics evidenced by reducing the number of γ-H2AX foci formation and fragmented nuclei with time. DNP pre-incubation restrained the radiation induced pro-apoptotic factors and inhibits apoptosis. Our findings raise the possibility that mild mitochondrial uncoupling with DNP could be a potential therapeutic approach for radiation induced cytotoxic insult associated with an altered mitochondrial function.

 

________________________________________________________________________

There is some new, compelling research regarding the use of low-dose DNP as a pro-longevity agent, with benefits to telomere maintenance and epigenetic remodeling. A few self-experimenters have also used low-dose DNP with great results. I am compiling research on low-dose DNP and will be sharing it in a post soon. 

Edited by dlewis1453, 26 December 2022 - 05:59 PM.

[QuestforLife]

I did some searching on this topic and found a newer paper from the same authors which linked cell free chromatin particle dispersion to mitochondrial aging as well.

"Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells"
https://www.biorxiv....0.474529v1.full
________________________________________________________________________
I am a little disturbed by the discovery of what appears to me to be an entirely distinct, previously unknown mechanism of dna damage, but I am also comforted by the fact that a simple protocol of resveratrol + copper appears to ameliorate this pathway. Some questions that come to my mind are: (1) just how effective is the resveratrol + copper protocol? (2) what is the optimal dosing amount and frequency? (3) How does cell free chromatin particle dispersion induced dna damage interact with other aging pathways? (4) Does the body possess a greater ability to resist damage from cell free chromatin particle induced dna damage in youth, as with other forms of dna damage? (5) are there other interventions to treat this aging pathway besides resveratrol + copper that could be more effective?
________________________________________________________________________

In addition to the copper + resveratrol protocol, perhaps we could benefit from upregulating dna repair capabilities throughout the body by inducing mild mitochondrial uncoupling? DNP is powerful inducer of mitochondrial uncoupling that, although dangerous and potentially fatal at higher doses, appears to be very beneficial at low doses. The paper "Mild mitochondrial uncoupling protects from ionizing radiation induced cell death by attenuating oxidative stress and mitochondrial damage" https://www.scienced...005272820301754 applied low dose DNP to cells and then blasted the cells with ionizing radiation. the DNP treated cells were better able to resist the radiation-induced damage to mitochondria and to DNA, see below abstract.



________________________________________________________________________
There is some new, compelling research regarding the use of low-dose DNP as a pro-longevity agent, with benefits to telomere maintenance and epigenetic remodeling. A few self-experimenters have also used low-dose DNP with great results. I am compiling research on low-dose DNP and will be sharing it in a post soon.

Thanks for the paper about the interactions between mitochondrial dysfunction and cell free chromatin particles. I've read the authors' 'back catalogue' but not this one. I'll give it a proper read soon.

Although I am firmly of the opinion that extending human life significantly will require telomerase, some things didn't add up. For a while I thought methylation was the culprit. But no longer. This fits the bill as an independent (of telomeres) source of aging, leading to mosaic gene expression.

Upregulating DNA repair is accomplished by longer telomeres, but DNA repair is no panacea against extracellular DNA, as this is one mechanism by which it can be mistakenly integrated within chromosomes.

In terms of optimal dosing, that is not yet established - suffice it to say, I don't agree with their recommendations, as I think their copper dose is too low. It is difficult to know how often to dose; but your own antioxidant response has to be upregulated in response, so take it easy. I've been doing this since the start of November and I haven't experienced any dramatic changes positive or negative, besides some short lived headaches early on.

[QuestforLife]

Should we take SAMe for telomeres?

It would take a huge article to discuss the various actions of SAMe (S-adenosyl-L-methionine), which are legion. This French paper (1) highlights its antioxidant contribution, which would not be my first thought, although the methionine cycle is certainly important for the folate cycle, and consequently for glycine import to mitochondria, for example. SAMe is also vital to support methylation of the genome in new cells. So adding it to a telomere stack does make sense. Which is what this paper champions.

26 volunteers took a mix including astragalus extract and SAMe and after a year of dosing showed increased leukocyte telomere lengths relative to healthy age matched controls. In a further experiment SAMe, astragalus extract or a mix of both was added to leukocytes from a 28 year old individual. Each increased telomere length relative to untreated cells, and decreased the percentage of telomeres less than 5k base pairs long, with the order of telomere length being astragalus extract+SAMe>astragalus extract>SAMe>controls.

(1) M'kacher R, Breton L, Colicchio B, Puget H, Hempel WM, Al Jawhari M, Jeandidier E, Frey M. Benefit of an association of an antioxidative substrate and a traditional chinese medicine on telomere elongation. Cell Mol Biol (Noisy-le-grand). 2019 Dec 31;65(8):54-58. PMID: 32133978.

[QuestforLife]

Can NO (nitric oxide) increase telomerase activity?

There is a thread of evidence suggesting that the answer is, in fact, yes.

A little background: Telomerase has two components, TERC and TERT. TERC is the template part, allowing the complete telomerase protein to link to the TTAGGG DNA sequence on the existing telomere. The TERT part adds new TTAGGG sequences to the telomere.

TERC associates with TERT more efficiently in the presence of HUR, an RNA binding protein, via methylation of TERC, and this increases telomerase activity in telomerase positive cells. (1)

Nitric oxide upregulates HUR both in human and mouse fibroblasts. (2)

Finally, nitric oxide upregulates telomerase and delays senescence in human Endothelial cells. (3)

My take on this is that nitric oxide will increase telomerase, by increasing the combination of TERC and TERT into the active telomerase protein. In much the same way as I've previously theorized that epitalon acts to increase protein synthesis (4), NO may act as a catalyst for telomerase creation and synergise with telomerase activators (that increase TERT gene expression). It is expected that this would have benefits in endothelial cells and fibroblasts (benefiting vasculature and skin)and possibly other cell types (like WBCs) too. Therefore NO activators such as arginine and citrulline should be considered as part of a telomerase stack.

References

(1)Tang H, Wang H, Cheng X, Fan X, Yang F, Zhang M, Chen Y, Tian Y, Liu C, Shao D, Jiang B, Dou Y, Cong Y, Xing J, Zhang X, Yi X, Songyang Z, Ma W, Zhao Y, Wang X, Ma J, Gorospe M, Ju Z, Wang W. HuR regulates telomerase activity through TERC methylation. Nat Commun. 2018 Jun 7;9(1):2213. doi: 10.1038/s41467-018-04617-7. Erratum in: Nat Commun. 2018 Jul 10;9(1):2721. PMID: 29880812; PMCID: PMC5992219.

(2)Kuwano Y, Rabinovic A, Srikantan S, Gorospe M, Demple B. Analysis of nitric oxide-stabilized mRNAs in human fibroblasts reveals HuR-dependent heme oxygenase 1 upregulation. Mol Cell Biol. 2009 May;29(10):2622-35. doi: 10.1128/MCB.01495-08. Epub 2009 Mar 16. PMID: 19289500; PMCID: PMC2682029.

(3)Vasa M, Breitschopf K, Zeiher AM, Dimmeler S. Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res. 2000 Sep 29;87(7):540-2. doi: 10.1161/01.res.87.7.540. PMID: 11009557.

(4) My epitalon post: https://www.longecit...-26#entry915113

Edited by QuestforLife, 03 January 2023 - 02:14 PM.

[QuestforLife]

Are we being poisoned?

We can't successfully apply rejuvenation therapies if we're being poisoned at the same time. For some time now I've been concerned that this is the case. The prime candidate for the poison is cell free chromatin particles (cfChP). These are particles consisting of small lengths of DNA still wrapped around histones. They are left over from the DNA in cell nuclei, which have been chopped up when the cell dies (apoptosis). The idea is they are eaten by immune cells, broken down by DNAses or removed by the liver. This process is imperfect however, and as we lose billions of cells a day, this is a constant onslaught on the body. CfChPs are readily taken up by other cells and enter the nucleus, where normal DNA repair will join small pieces into longer lengths and can also during double strand breaks of chromosomal DNA, be integrated within the legitimate DNA of the chromosomes. It is even claimed cfChPs themselves can cause double strand breaks (1).

The long term consequences of this is mosaic gene expression; different cells have different genomes. I've heard Aubrey de Grey state that random genetic mutations will not be a meaningful driver of aging, outside of cancer. I think this is wrong, as there is evidence that this genetic mutation is not random and leads to common outcomes. How? Most of the genome is regulatory not protein producing, so it stands to reason that corruption of the genome will in most cases affect gene regulation. As longer proteins require more splicing, it is most likely longer proteins will be affected with age. And that is what we see, a global reduction in the longer proteins (2). It is now believed, according to recent discussions on Josh Mittledorf's site, that adding longer length blood proteins is the basis of Harold Katcher's magic potion.

Replacement of proteins can only be a stop gap if the genome is compromised, however. And this brings us back to cell replacement and my telomerase activation stack.

First, some advice on reducing cfChP load. The authors of the first reference identified the pro oxidant combination of resveratrol and copper, which can bind and destroy DNA. They tested it in several settings including aging mice (3), humans suffering with covid (4) and humans undergoing chemotherapy (5), all with promising results. Indeed it seems one of the most toxic things to the human body is DNA! It should also be noted that this will also be a good antiviral treatment.

I've not gone into any detail on these papers, as I wanted this to be an introductory piece, but I'd encourage you to at least skim read the referenced papers.

Their dosing is guided by an earlier in vitro study (6). This is the weakest part of their argument. Based on my reading of this paper, I'd say the best ratio of Resveratrol to Copper would be 100:1, but in their later studies they use 10,000:1. I've no idea how they come to that conclusion. I'm taking 10
mg resveratrol powder (as near to 100% as possible) and 100ug copper (1 drop of 200ug liquid copper sulphate) morning and night, mixing them together in my mouth before swallowing, separate from food. But if you want to follow their dosing recommendations you can dilute the copper sulphate 100 times in distilled water.

For replacement of cells I've improved on my telomerase stack with the addition of a bonafide Rho kinase inhibitor, but I'll post on that another time.

References
(1) Raghuram GV, Chaudhary S, Johari S, Mittra I. Illegitimate and Repeated Genomic Integration of Cell-Free Chromatin in the Aetiology of Somatic Mosaicism, Ageing, Chronic Diseases and Cancer. Genes (Basel). 2019 May 28;10(6):407. doi: 10.3390/genes10060407. PMID: 31142004; PMCID: PMC6628102.
(2) preprint: https://www.biorxiv....0.1101/691154v1
(3) Pal K, Raghuram GV, Dsouza J, Shinde S, Jadhav V, Shaikh A, Rane B, Tandel H, Kondhalkar D, Chaudhary S, Mittra I. A pro-oxidant combination of resveratrol and copper down-regulates multiple biological hallmarks of ageing and neurodegeneration in mice. Sci Rep. 2022 Oct 14;12(1):17209. doi: 10.1038/s41598-022-21388-w. PMID: 36241685; PMCID: PMC9568542.
(4) pre print: https://www.medrxiv....7.21.20151423v1
(5) Agarwal A, Khandelwal A, Pal K, Khare NK, Jadhav V, Gurjar M, Punatar S, Gokarn A, Bonda A, Nayak L, Kannan S, Gota V, Khattry N, Mittra I. A novel pro-oxidant combination of resveratrol and copper reduces transplant related toxicities in patients receiving high dose melphalan for multiple myeloma (RESCU 001). PLoS One. 2022 Feb 4;17(2):e0262212. doi: 10.1371/journal.pone.0262212. PMID: 35120140; PMCID: PMC8815866.
(6) Subramaniam S, Vohra I, Iyer A, Nair NK, Mittra I. A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA. F1000Res. 2015 Oct 27;4:1145. doi: 10.12688/f1000research.7202.2. PMID: 27134724; PMCID: PMC4833056.

[rarefried]

Hi Questforlife, if you don't mind sharing the information, what brand of liquid copper sulphate are you using (if you are using a branded supplement)?

[QuestforLife]

Hi Questforlife, if you don't mind sharing the information, what brand of liquid copper sulphate are you using (if you are using a branded supplement)?

 

'Good State' where 1 drop corresponds to about 200ug of copper sulphate.

 

There is also the possibility of using pure (99.9%) colloidal copper, which is also in liquid form, but whose concentration is much lower (~30ug/L), or copper biglycinate, which only seems to exist in capsules of a much too high dose. 

[dlewis1453]

Since apoptosis is the source of the cfChP, should we expect Rapamycin to benefit us on the cfChP front by reducing cell turnover and by extension the amount of apoptosis we are undergoing at any particular time? Maybe this is an additional, unexplored mechanism whereby Rapamycin enhances longevity. 

 

The existence of cfChP also makes lengthening the telomeres of somatic cells more attractive, as a tool to reduce apoptosis. 

 

If the copper + resveratrol protocol mops up much, but not all, of the free-floating DNA, then using telomere treatments and Rapamycin to reduce the rate of generation of free-floating DNA could reduce the workload of the copper + resveratrol protocol and enable it to get most of the free-floating DNA in circulation. 

Edited by dlewis1453, 09 January 2023 - 04:46 PM.

[QuestforLife]

Since apoptosis is the source of the cfChP, should we expect Rapamycin to benefit us on the cfChP front by reducing cell turnover and by extension the amount of apoptosis we are undergoing at any particular time? Maybe this is an additional, unexplored mechanism whereby Rapamycin enhances longevity.  [/quote]

 

 

 

As rapamycin is a immunosuppressant that reduces WBC, it should reduce the death rate of these cells. It would be an easy experiment for someone to do, and since rapamycin seems to slow most aspects of aging, it may well reduce cfChP load as well.

 

 

 

The existence of cfChP also makes lengthening the telomeres of somatic cells more attractive, as a tool to reduce apoptosis. [/quote[

 

Possibly, although the fact that I came across this mechanism of aging by studying DNA accumulation in non senescing epitalon treated human cells suggests that telomerase is no magic bullet for stopping cfChPs. On the other hand, fully immortalised human cells seem to have stable gene expression, so with enough telomerase who knows?

 

If the copper + resveratrol protocol mops up much, but not all, of the free-floating DNA, then using telomere treatments and Rapamycin to reduce the rate of generation of free-floating DNA could reduce the workload of the copper + resveratrol protocol and enable it to get most of the free-floating DNA in circulation.

 

I see telomerase treatment as a complement to anti-cfChP therapy; some cells will inevitably have already accumulated illegitimate DNA within their chromosomes, something we want to remove. This can only be done by replacing those cells from an uncontaminated (hopefully) stem cell pool, and we can encourage this via the appropriate protocol. As we are also reducing cfChP load at the same time, these new cells should have a better time of it. It is a bit like trying to rebreed an extinct dog breed. The genes you want are in the current animals but so are other genes you don't want, so you have to select for traits. 

 

If you wanted to further the effectiveness of res+cu in removing cfChPs, which may or may not be needed, you'd need to look at other things that you have reason to believe would increase oxidative stress in the blood, without rising to such a level it damages or kills cells. Exercise springs to mind. Alcohol or fasting are other possibilities. 

Edited by QuestforLife, 10 January 2023 - 12:18 PM.

[dlewis1453]

A study just released at the start of this year shows that lifelong DHA ingestion ameliorates telomere aging and mitochondrial aging. The effects of DHA in a human would be modest, but given the safety and low cost of DHA in the form of fish/krill oil supplements, this seems like an easy, low risk intervention. 

 

"Lifelong docosahexaenoic acid intervention ameliorates aging in the telomere-DNA-mitochondria axis in telomerase-deficient mice"

https://www.scienced...955286322002704

 

 

The health benefits of n-3 polyunsaturated fatty acids (PUFAs) in multiple age-related diseases are associated with telomere length. Telomerase is intimately related to inflammation and oxidative stress, but whether the underlying function of n-3 PUFAs on telomere maintenance is based on telomerase activation or related mechanisms remains unclear. Herein, we utilized late-generation (G4) telomerase-deficient (Terc−/−) mice to perform a lifelong docosahexaenoic acid (DHA) intervention to determine the potential of DHA in telomere maintenance and health promotion. Unfortunately, DHA failed to prolong mouse longevity in either intrinsic or premature aging. However, intriguingly, lifelong dietary DHA intervention slowed the aging phenotypes and profoundly attenuated telomere attrition in blood leukocytes and multiple tissues, consistent with decreased β-galactosidase activity and other senescence hallmarks with no observed sex differences. Notably, DHA intervention alleviated telomere attrition-induced γ-H2AX accumulation dependent on poly (ADP-ribose) polymerase 1 (PARP1) recruitment, and further regulated mitochondrial dysfunction critically involved in the DNA damage response. Together with the improvement of mitochondria function, the blocked reactive oxygen species (ROS) accumulation and suppression of the nuclear factor-κB (NF-κB)/nucleotide-binding domain-like receptor protein 3 (NLRP3)/caspase-1 pathways partially indicated anti-oxidative and anti-inflammatory effects of DHA. These data revealed a regulatory paradigm involving DHA in the telomere-DNA-mitochondria feedback loop mediated by DNA damage response and inflammation in alleviating senescence, which may hold potential as a translatable intervention in telomere-related diseases during aging.

 

The quote below and accompanying picture provide a nice summary of the interactions between mitochondria, ROS, and telomeres. 

 

Proposed model for accelerated aging and reactive oxygen species (ROS)-mediated exacerbation of telomere attrition in G4 Terc−/− mice and the impact of lifelong docosahexaenoic acid (DHA) treatment. Telomere attrition, shown as chromosomal instability, accelerates the DNA damage response and recruits poly (ADP-ribose) polymerase 1 (PARP1), leading to transcriptional signaling from the nucleus to mitochondria during the aging process. Thus, DNA damage and mitochondrial dysfunction, including decreased mitochondrial DNA (mtDNA) copy number and disturbed homeostasis, synergistically lead to increased  oxidative stress with the inactivation of superoxide dismutase (SOD), which ultimately generate elevated ROS levels. In addition to the role of PARP1 in DNA repair, the interaction between PARP1 and the nuclear factor-κB (NF-κB) transcriptional pathway subsequently triggers inflammation and mitochondria damage-induced apoptosis via the nucleotide-binding domain-like receptor protein3 (NLRP3)/caspase-1 pathway, converging on increased ROS to accelerate telomere attrition and DNA damage. DHA treatment exerts DNA damage repair and anti-inflammatory effects through regulation of PARP1 and mitochondrial homeostasis recovery via the NLRP3/caspase-1 pathway. The black arrows indicate the telomere attrition-induced aging process, while the red arrows indicate the effect of DHA treatments potentially depends on the telomere-DNA-mitochondria axis to restore the vicious cycle in the telomere attrition-induced aging process.

Edited by dlewis1453, 19 January 2023 - 05:18 PM.

[QuestforLife]

 

 

I am expecting one of the following results:

1. epigenetic age acceleration continues upward due to my continued combination of SCD (symmetric cell division) and telomerase activators, but health is not affected. In which case we can ignore methylation aging tests

2. epigenetic age acceleration continues upward due to my continued combination of SCD and telomerase activators, but health is adversely affected. In which case we have learnt that methylation aging tests are useful.

3. Or perhaps epigenetic age acceleration plateaus or even falls again, with continued use of SCD and TAs,  as stem cells become more able to replace downstream cells. This would be an ideal result, particularly is health biomarkers remained good. It would tell us epigenetic acceleration due to health interventions is a transitory or minor effect, and methylation aging tests would still be useful in this context.

4. There might be detrimental effects from my protocol that are unrelated to epigenetic age acceleration, but mean I have to modify or discontinue the protocol. For example if my BP gets too low, etc. 

 

 

 

Results are in!

 

You may recall I decided to spit in the face of my epigenetic age acceleration (7.5 years in 5 months) by continuing my telomerase activation stack along with possible symmetric cell division stimulation (a combination that has been reported to cause epigenetic age acceleration in, to my knowledge, 3 people). 

 

My telomerase stack includes TAM818, cycloastragenol, ginseng, epitalon and GDF11, with the majority of these components taken 3 times per week (epitalon and GDF11 subQ injection approx. once/week)

I also used a rho kinase inhibitor (netasudil eye drops), which can be taken sublingually (3 times a week initially PM then increased to AM and PM). 

I did the C60/stearic acid cookies combination a few times also in this period, with no effort to separate from my TA stack.

I took AAKG only very occasionally (mostly forgot it).

 

During this period my biomarkers were stable or improved and the day-day variance in these biomarkers also went down from the previous period (although the period was shorter): see attachment 'BiomarkersUpdateJan23'. 

To my surprise my epigenetic age decreased dramatically, dropping by 8.45 years in 3 months: see attachment 'TruMeJan23'

 

We might tentatively conclude that (3) is correct and that epigenetic acceleration due to my stack was a transitory effect. You could argue that the test is unreliable, but given the number of tests I have done, with epiAge acceleration occurring twice, in similar circumstances, to me suggests that this is unlikely.

 

 

Attached Thumbnails

• 
• 

[dlewis1453]

Great result! Really happy to see this reduction in epigenetic age alongside some improvement in biomarkers. 

 

To what extent do you think the netasudil eye drops contributed to your epigenetic age reduction?

 

Were you also taking the copper + resveratrol protocol during this time?  

[QuestforLife]

Great result! Really happy to see this reduction in epigenetic age alongside some improvement in biomarkers.

To what extent do you think the netasudil eye drops contributed to your epigenetic age reduction?

Were you also taking the copper + resveratrol protocol during this time?

Good point. I did add Cu+res at the end of October and that may have had an impact. I have stopped Netasudil now (may restart again in a few months), so my next test should be able to distinguish between the benefits of this and Cu+res.

During my TA days I normally cut Cu+res down from twice to once a day as I wanted to reduce oxidative stress. May or may not have been the right thing to do.

[kurt9]

Where are you getting the Cu+Res supplements? All the ones I find on the net and through lef are much too high of dosage. 

 

I want to try the Cu+Res along with Turbuckle's C60 protocol.

[JamesPaul]

'Good State' where 1 drop corresponds to about 200ug of copper sulphate.

 

There is also the possibility of using pure (99.9%) colloidal copper, which is also in liquid form, but whose concentration is much lower (~30ug/L), or copper biglycinate, which only seems to exist in capsules of a much too high dose. 

Be sure to get Good State's 8 oz. bottle product which is different from Good State's other, nano copper technology product.  Their nano product has a higher concentration.  Also, a German reviewer on amazon.com wrote that nano copper is toxic to kidneys and other organs.  I do not know whether nano copper is toxic or not.

[dlewis1453]

Results are in!

....

 

We might tentatively conclude that (3) is correct and that epigenetic acceleration due to my stack was a transitory effect. You could argue that the test is unreliable, but given the number of tests I have done, with epiAge acceleration occurring twice, in similar circumstances, to me suggests that this is unlikely.

 

A data point in support of your theory that epigenetic age acceleration during telomerase activation is a transitory phenomenon is the experience of Dr. Bill Lawrence. Earlier in this thread you shared one of Dr. Lawrence's videos in which he described how he had reduced his epigenetic age by a considerable amount after a prolonged period of taking the telomerase activator epitalon. 

[QuestforLife]

A data point in support of your theory that epigenetic age acceleration during telomerase activation is a transitory phenomenon is the experience of Dr. Bill Lawrence. Earlier in this thread you shared one of Dr. Lawrence's videos in which he described how he had reduced his epigenetic age by a considerable amount after a prolonged period of taking the telomerase activator epitalon. 

here's a theory for why the effect is transitory:

• extra telomerase may result in an increase in the number of downstream cells rather than an increase in their telomere length
• This is beneficial in the case where more is better, for example total lymphocyte count in children is 9k/uL but only 4k/uL in adults
• My numbers are 5k/uL +, which explains why I never got covid, when all those around me did (until I had a prolonged period off TAs)
• Every time a cell divides, each chromotid gets a fresh DNA daughter strand, without methylation. The process of de novo methylation plus subsequent demethylation is error prone
• Hence extra cell divisions are recorded in methylation age measures 
• As lymphocytes lifespan is in months (up to 200 days for some types), this measure is dynamic on those time scales(same as telomere length); this suggests how often testing should be done to assess changes
• Telomerase is also a powerful mitochondrial antioxidant, therefore it also reduces lymphocyte attrition.
• This means a larger pool can be maintained with less turnover, less divisions, and therefore less methylation aging
• Therefore the cost in increase methylation age only occurs when you are increasing cell numbers, not maintaining them
• It is apparent then, why stem cell symmetric division plus TAs maximises this effect: you get a huge pulse of extra cells moving down through the hierarchy
• Other predictions from this theory: children have epigenetic age accelaration; TAs may make children grow bigger; any substance that reduces telomere loss/division would have a similar effect, i.e., effective in vivo antioxidants, methylene blue, NMN, etc (notwithstanding their other effects). 

[QuestforLife]

 

• Telomerase is also a powerful mitochondrial antioxidant, therefore it also reduces lymphocyte attrition.
• This means a larger pool can be maintained with less turnover, less divisions, and therefore less methylation aging
• Therefore the cost in increase methylation age only occurs when you are increasing cell numbers, not maintaining them

 

 

* It is also worth noting that even without reduced attrition on lymphocytes due to the mitochondrial protection of telomerase, attrition is likely to be a function of viral/microbial assaults on the body, which will be roughly averaged out over time. Hence having more lymphocytes doesn't necessarily mean you will lose more in a given time period. 

 

This is not to suggest that more is always better, you might encounter auto immune conditions with very elevated levels, or deplete the body's resources - I myself got low in glutamine, which I reported on previously. The point is that evolutionarily speaking, lymphocyte levels in adults are a compromise between available resources and protection. I just think we do better with higher lymphocyte counts, given inputs to the body aren't really a constraint anymore.

[QuestforLife]

What is the optimal strategy for slowing telomere loss? It is not telomerase activators

 

It is with great reluctance that I make this post. Although I remain convinced that telomere shortening with cellular replication imposes a limit on our lifespan, and that an effective telomerase therapy would be an ideal treatment for this, I have come to realise that telomerase activation is not (currently) a valid or cost effective strategy. 

 

There are various molecules purported to activate telomerase, and these have been extensively covered in this thread. I have even tried combining them in various clever ways for maximum synergy. I even took a Lifelength test and found that epitalon was somewhat effective. Nevertheless I have serious concerns about whether telomeres are actually getting longer. I believe that they are not, that we are only reducing the shortening rate (in leukocytes) and that this being the case (it is), there are more effective and cheaper ways of doing this.  

 

First let me cover the point about whether telomeres are actually getting lengthened. This is a oft misunderstood point, and probably the most common question I get from people. If a telomerase activator can increase telomerase to 16% of the level required to maintain telomere length indefinitely, then this should lead to a reduction in the shortening rate by 16%, not to lengthening. Now there is a slight complication to this, as short telomeres are easier to lengthen than long ones (probably because the reduced telomere overhang doesn’t impeded HTERT expression - Shay and Wright showed this can happen on human chromosome 5 [1],[2] - or because a longer overhang somehow impedes the telomerase protein docking with the telomere (my speculation)), so there may be an averaging out effect, and therefore some benefit of telomerase activators offsetting the harm of the shortest telomeres. But we should still see the average telomere length decline. In cell culture this is indeed what is seen (Bill Andrew’s has confirmed this in various talks). And yet people who take TAM818 or cycloastragenol or epitalon report increases in leukocyte telomere length (LTL). How can this be? The answer is rather simple. 

 

Say the white blood cells whose telomeres we are measuring are the progeny of a stem cell with a telomere length of 9000 base pairs (bp). To populate the body with about 4000 WBCs/uL (typical adult level), it will need to split about 24 times. The stem cell itself may only split once, but then each downstream cell splits in turn until there are enough cells. 

 

The telomere length of the 4000 white blood cells in each microlitre of your blood depends on three things: 1) telomere length of the original progenitor, 2) number of divisions, 3) the telomere loss per division. In our example we’ve set the progenitor to have 9kbp, which is pretty long (pristine for a human adult stem cell is about 10k bp). The number of divisions is also fixed - in reality this will change depending on infection status - and this will in real life influence your LTL, which is why it is a good idea to take a telomere test in the summer, or at least the same time each year. But we don’t need to think about this too much in our example. The remaining factor we are left with is the telomere loss per division. 

 

An effective telomerase activator will naturally reduce this. But so will other things. For example Bill Lawrance used epithalamin tablets and over the course of seven years increased his LTL by 27% (6.41kbp to 8.15kbp; I posted about this previously upthread if you want to read more). Using our example stem cell telomere length of 9kbp and 24 divisions, we can work out that his initial 2012 result implied a loss of 107bp/division. Assuming he still has progenitors with 9kbp seven years later, and he has maintained 4000/uL concentration of WBCs in the blood, then the loss per division would have needed to  have dropped to 35bp/division to give the new result of 8.15kbp. If we decide his stem cell telomeres will also have gotten shorter in that time, then the loss would be even less than 35bp per division. 

 

And this isn’t an isolated example. Go and have a look at Defytime’s published results on their website (https://defytime.com...omere-analysis/). They’ve had similar results, but over a shorter timespan using TAM818 (admittedly they sometimes measure the change in the shortest 20% telomeres, not the average length, but still..).

 

So this explains why we can see telomere length increases in downstream cells, with telomerase activators that only slow telomere loss. It also explains why even other lifestyle interventions like meditation, diet or exercise can have mild effects on increasing telomere length with no effect on telomerase expression. It also explains why there is so much variation in the results of telomere tests. 

 

So now we come to the second part of the post. Why telomerase activators aren’t the optimal way to slow telomere loss. The reason is obvious. If all telomerase activators can do is slow loss, then we don’t actually need telomerase activators to do that. We can use other supplements. Let’s look at the evidence.

 

Nicotinamide increases the number of divisions in human cells by 50% [3]. This is an insane increase! (Bear in mind that Bill Andrews has said TAM818 only gave them about 5% more divisions). But with nicotinamide no telomerase was activated but even so the telomere loss was reduced from 24bp/division to only 9! Now the nicotinamide dose was rather high (5mM), more than we could get in vivo. But we also see similar results from Methylene Blue, using in vivo achievable concentrations (100nM): 20-60% increase in divisions depending on the level of oxygen[4], [5].   Once again, they showed that telomere loss was slowed right down. 

 

The mechanism for slowed telomere loss using either nicotinamide or methylene blue involves increasing the NAD+/NADH ratio, possibly only transiently, in order to improve the functioning of the electron transport chain and control the rise in ROS that normally occurs. 

 

Now before you object that this is all in vitro, we even have evidence that this works in vivo. The NAD+ donor NMN, at a relatively modest dose of 300mg/day increased the LTL of humans by 2-fold [6]! If we feed this into our earlier calculation, this would correspond to reducing the shortening rate by something like 5 times (depending on the actual telomere length values, which aren’t given). 

 

So there we have it. If you want to maintain your telomeres and live longer, the best way to do this is by raising your NAD+ levels. Might it be worth combining doing this with a telomerase activator? Possibly. If telomerase activators are reducing shortening of telomeres by telomerase acting on the telomere itself, then yes, as this is a completely different mechanism to increasing NAD+. But if telomerase activators are reducing shortening by being diverted to the mitochondria, as we know telomerase can do [7], then no.  

 

References

 

 [1] Robin JD, Ludlow AT, Batten K, Magdinier F, Stadler G, Wagner KR, Shay JW, Wright WE. Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances. Genes Dev. 2014 Nov 15;28(22):2464-76. doi: 10.1101/gad.251041.114. PMID: 25403178; PMCID: PMC4233240.

[2] Kim W, Shay JW. Long-range telomere regulation of gene expression: Telomere looping and telomere position effect over long distances (TPE-OLD). Differentiation. 2018 Jan-Feb;99:1-9. doi: 10.1016/j.diff.2017.11.005. Epub 2017 Nov 22. PMID: 29197683; PMCID: PMC5826875.

[3] Kang HT, Lee HI, Hwang ES. Nicotinamide extends replicative lifespan of human cells. Aging Cell. 2006 Oct;5(5):423-36. doi: 10.1111/j.1474-9726.2006.00234.x. Epub 2006 Aug 25. PMID: 16939485.

[4] Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 2008 Mar;22(3):703-12. doi: 10.1096/fj.07-9610com. Epub 2007 Oct 10. PMID: 17928358.

[5] Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue. Redox Biol. 2015 Dec;6:426-435. doi: 10.1016/j.redox.2015.09.004. Epub 2015 Sep 10. PMID: 26386875; PMCID: PMC4588422.

[6] Niu KM, Bao T, Gao L, Ru M, Li Y, Jiang L, Ye C, Wang S, Wu X. The Impacts of Short-Term NMN Supplementation on Serum Metabolism, Fecal Microbiota, and Telomere Length in Pre-Aging Phase. Front Nutr. 2021 Nov 29;8:756243. doi: 10.3389/fnut.2021.756243. PMID: 34912838; PMCID: PMC8667784.

[7] Ale-Agha N, Jakobs P, Goy C, Zurek M, Rosen J, Dyballa-Rukes N, Metzger S, Greulich J, von Ameln F, Eckermann O, Unfried K, Brack F, Grandoch M, Thielmann M, Kamler M, Gedik N, Kleinbongard P, Heinen A, Heusch G, Gödecke A, Altschmied J, Haendeler J. Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function. Circulation. 2021 Dec 7;144(23):1876-1890. doi: 10.1161/CIRCULATIONAHA.120.051923. Epub 2021 Oct 21. PMID: 34672678.

 

Edited by QuestforLife, 19 May 2023 - 01:23 PM.

[dlewis1453]

Thanks for this detailed and well researched write up, QuestforLife. I appreciate your willingness to share the latest developments in your protocols and theories, even when the outcomes are not favorable.

 

I have a few follow up questions for you: 

 

1. I'm curious, was there a particular piece of research or epiphany that led you to the conclusion that most telomerase activators were only slowing telomere loss? Or had you been contemplating this idea for a while? 

 

2. The mechanism you have described makes sense, but it seems we don't have much concrete evidence for whether telomerase activators are truly lengthening telomeres or just slowing their loss, because when measuring telomere length in living subjects we have to rely on white blood cells. We can't easily measure telomere length in stem cells at the moment. Is there no other test we could conduct to get a better sense of what is happening with our telomeres? 

 

3. If we assume that most telomerase activators are only slowing telomere loss, then it makes sense to increase NAD+ as you mentioned. Consuming the SOD mimetic Tempol would help as well. How do you currently feel about substances that increase TERC, like GDF11? Since stem cells already express some telomerase, maybe it would be easier to lengthen telomeres in stem cells via TERC targeted approaches 

 

 

Also, note that Klotho also plays a role in maintaining telomeres in stem cells, according to this paper "Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity"   https://pubmed.ncbi....h.gov/30452555/