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Alternative methods to extend telomeres

telomeres nad nampt ampk resveratrol allicin methylene blue nmn sirtuins statin

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#1 QuestforLife

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Posted 04 September 2018 - 03:18 PM


NAD-TERT
Using NAD+ and Resveratrol for Telomerase activation

This thread is to draw together mine and others’ research on resveratrol, Sirtuins, NAD and telomeres. I now believe we can rejuvenate cells and possibly ourselves by combining NAD uplift (using NAD precursors, NAMPT and AMPK) and telomerase activation via SIRT4 using a bioavailable form of resveratrol.

I have placed this thread in under the Telomeres Subforum because I believe that it is telomerase that is key to rejuvenating cells beyond that achieved by raising NAD alone. It could equally be placed under resveratrol or NAD subforums, however.  It is my decision to place it here however, partly to draw attention back to the importance of telomeres, whilst arguing that extending telomeres, particularly using this method (via Sirtuins) is not a route to cancer but to youth.

The protocol is still very much under development. But early signs are promising.

Rejuvenation is via NAMPT, NAD, SIRT 4 and HTERT

The key finding of this thread is that increasing NAD+ (either directly or via NAMPT) is a necessary but not sufficient step for cellular rejuvenation. A combination of a NAD and/or NAMPT booster with resveratrol however permits SIRT4 to use elevated NAD to re-activate telomerase in cells.

We know that if you raise NAD, this is used by the body for various processes to repair and protect our DNA and cells. It is the specific hypothesis of this thread that SIRT4 (and to a lesser extend SIRT1) can use NAD to reactivate telomerase when prompted by Resveratrol, and that this offers benefits beyond increasing NAD alone.

Here is the research evidence followed by my personal experiences with this protocol, which I call NAD-TERT

References

Resveratrol has been shown to be able to rejuvenate cells in vivo at low doses via modulation of splicing factors and re-extension of telomeres.
Ref: https://bmccellbiol....2860-017-0147-7

Resveratrol has been shown to reduce endothelial progenitor cell senescence through HTERT (telomerase) activation
Ref: https://bpspubs.onli...38/bjp.2008.272

Resveratrol has been shown to have different effects on early and late passage mesenchymal stem cells, and this is mediated by the presence or absence of SIRT1
Ref: https://www.scienced...006291X1530721X

Resveratrol appears to exert its effects on HTERT via SIRT4
Ref: http://www.oncotarge...580&path[]=7251

This last paper shows that SIRT4 is dependent on NAMPT. But lower doses of resveratrol can raise telomerase levels without raising NAMPT, suggesting background levels of NAMPT can be used up by this process. This suggests that the short term upregulation of HTERT seems to be due to the non-sustainable rise of NAMPT produced by dosing with resveratrol alone.

Other supplements can more sustainably increase NAMPT, for example Hydrogen sulphide (which can be induced in cells by allicin supplementation).
Ref: https://journals.plo...al.pone.0164710

Note H2S was more effective at boosting NAMPT than Resveratrol but less effect at boosting HTERT, probably because it acts primarily through SIRT1 not SIRT4

Sinclair et al. has also recently shown that the benefits of a H2S donor are additive in mouse studies using NMN
Ref: https://www.cell.com...?code=cell-site

Linking this back up to splicing factors, H2S donors alone also were able to achieve partial rejuvenation of old cells
Ref: https://www.ncbi.nlm...les/PMC6075431/

and that this is achieved by upregulating SRSF2 (and one other) splicing factor, which is interesting as this splicing factor is within 10MB of the telomere on chromosome 17 and likely affected by the shortening of the telomere via the Telomere Position Effect over long distance (TPE-OLD)

https://onlinelibrar...1111/acel.12646

https://journals.plo...al.pbio.2000016

Methylene Blue can postpone cellular senescence via boosting NAD (but not NAMPT), so this might also be a synergist pathway with the action of resveratrol and Allicin.
Ref: https://www.scienced...213231715001159

(Note: other NAD precursors may also be as or more effective, but in my experience this has not been the case).

Personal experience

I am a 39 year old male with no major health issues.

 

All the findings reported or conclusions drawn below are to be considered highly provisional.

I took 2 drops (~1mg) of Methylene blue for the first time in 2 months. This was in addition to my normal AM supplements that include Allicin, Vitamin C and Ashwaghanda. Within 4 hours I felt unusually fatigued and had a headache. The experience was similar to using Turnbuckle’s mitochondrial fission Nicotinamide and Ribose protocol for the first time (https://www.longecit...drial-dynamics/ ), but which has subsequently and even recently failed to elicit the same effect, even when combined with the same supplements. I have not experienced this fatigue when using MB alone.

As my headache grew worse I decided to try and trigger mitochondrial fusion by ingesting 20g of fish oil (ref: https://www.ncbi.nlm...les/PMC3963938/ ). I gave it two hours to work but the effect was minimal. I lay down for a rest. Letting my mind wonder over various papers I’d read recently the potential synergy of resveratrol with high NAMPT/NAD occurred to me. I took 10ml (180mg) of liposomal resveratrol from Actinovo that I had lying about. This liposomal resveratrol supplement had not previously produced any significant positive results, and I had taken it at various doses from 180mg to 4 g a day.

Within 10 minutes not only did I feel better, but I felt euphoric. I ran down the stairs and played with my kids in the garden with a renewed and fresh energy. I continued to feel fantastic for the rest of the day. That night I vigorously made love to my partner (we are sexually active but this an unusual enough event to comment on).

The following day I again took 1mg of MB with my normal AM supplements. I again grew fatigued by early afternoon, but only had the beginnings of a headache. I lay down for a rest, intending to take resveratrol as soon as a headache set in, but I fell asleep for several hours. When I woke I remembered to take the 180mg liposomal resveratrol. I again felt restored and fresh but the effect was less pronounced than on the previous day. Perhaps the sleep had already restored me, or the NAD/NAMPT levels had begun to fall.

The following and third day of treatment I once again took MB with my morning regime. Because I was to be out all day I took the liposomal resveratrol only 1 hour later; I had not yet begun to feel fatigued. I did not experience the fatigue or the headache induced previously, but neither did I experience an obvious boost or euphoria. I was able to drive for 4 hours that day without more than slight fatigue, and I did note that my skin looked unusually good that evening, in particular some small but annoying cysts that had appeared over the previous year on my left cheek were reduced in size.

After a week’s break I intend to repeat the protocol with various changes to determine to best combinations. I will continue to report back in this thread.

 

Many others here have been experimenting with various NAD, NAMPT and AMPK boosters, with and without resveratrol, whether liposomal or not, with various timings. It would be great to see if we can work out what the best combinations are.

 


Edited by QuestforLife, 04 September 2018 - 03:27 PM.

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#2 Phoebus

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Posted 05 September 2018 - 05:02 PM

Interesting!

 

now what i noticed is that you had the greatest effect following 20 g fish oil followed by liposomal resveratrol

 

Resv.'s problem has always been bioavailability. So what I am wondering is if you found a way to create maximum resv bioavailability using mega dosing of fish oil + lipid soluble resv. 

 

maybe it was that combo that created the energy rush? 

 

also...20 grams? thats a crazy high amount of fish oil. if I did that i would be weeping fish oil from my facial pores for the next three weeks! ha! 

 

anyway would be interesting to try just that combo again and see what kind of result you get. 

 

 

 

Due to its chemical characteristics, resveratrol can interact with fatty acids. Recent studies in vitro show that more than 90% of freetrans-resveratrol binds to human plasma lipoproteins. This binding is also found in vivo, as shown by the presence of dietary polyphenolic compounds detected in isolated LDL in blood samples of healthy human volunteers [3132].

Fatty acids facilitate a lipophilic environment, which favors resveratrol binding [33]. Normally they are employed as vectors because of their high affinity for the liver and their efficient cellular uptake, resulting from specific interactions with transmembrane transporters.

https://www.ncbi.nlm...les/PMC4499410/


Edited by Phoebus, 05 September 2018 - 05:14 PM.

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#3 QuestforLife

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Posted 05 September 2018 - 10:07 PM

That's possible Phoebus, and another thing for me to look into. It's liposomal resveratrol so it should be fairly bioavailabile anyway, but I'm going to trial this protocol again starting tomorrow, but without fish oil.

Incidentally, 20g is only about 20ml of Seven Seas fish oil.

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#4 jorgy72

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Posted 09 September 2018 - 07:21 PM

How about NAC for H2S production ?
https://www.cell.com...9456(18)30033-3
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#5 QuestforLife

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Posted 10 September 2018 - 01:09 PM

How about NAC for H2S production ?
https://www.cell.com...9456(18)30033-3

 

Certainly NAC helps, or if you can afford it, (liposomal) glutathione, as this boosts H2S very effectively, atleast in vitro (and my experiences suggest also in vivo)

 

http://www.pnas.org/...nt/104/46/17977

 

I've yet to recreate the initial dramatic effects of taking liposomal resveratrol to alleviate the effects of methylene blue. I am certain it was mitochondrial fission that caused the headache/tiredness -as I've experienced this before with N+R, and I expect MB penetrates the blood-brain barrier much better, which is why the headache was so bad and more noticeable that with N+R. And I also have to assume that my various previous efforts with mitochondrial fusion were successful, in that a sudden fission of mutated mitos caused me to feel so bad. At this point the feeling of mitochondrial fission is a useful symptom rather than the point of this thread - but I'd like to recreate it. A good bet for the culprit is my repeated doses of allicin plus liposomal glutathione over the summer, so I might try this for a few weeks and then repeat the MB and resveratrol experiment.

 

Taking allicin, MB and resveratrol daily for 4 days caused the initial fresh, energetic feeling to be replaced with early afternoon tiredness, though I have to say my skin, hair and eyes look very good.  I definitely feel I need to take a break from this protocol though, so it's probably something to cycle.


Edited by QuestforLife, 10 September 2018 - 01:10 PM.


#6 QuestforLife

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Posted 17 October 2018 - 10:59 AM

Another way to raise NAMPT is troxerutin.

 

https://www.ncbi.nlm...pubmed/25026599

 

(full text on sci-hub)

 

Bear in mind that any oxidative stress (that activates DNA repair) will increase PARP and use up NAD and NAMPT. So a nice antioxidant will always help and this study showed troxerutin could cancel out the negative effects of having lots of oxidized lipids in the blood and liver of rats.



#7 QuestforLife

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Posted 17 October 2018 - 12:41 PM

I've continued this line of research (Resveratrol appears to exert its effects on HTERT via SIRT4) with the finding that SIRT4 reinforces cellular arrest during DNA repair via inhibition of glutamine synthesis. Interesting SIRT4 is activated by rapamycin and deactivated by mTOR. There is more detail here (https://www.longecit...ns/#entry859169), but I'm not sure whether or not this ability of SIRT4 is causally related to its activation of HTERT, which is not a known target of rapamycin.

One possible link is the requirement for glutamine to create a protein required by telomerase to fasten onto the DNA strand it is elongating:

https://www.ncbi.nlm...les/PMC2744565/

Whether or not this increases telomerase via activation of HTERT or just increases the affinity of telomerase for DNA is not clear to me. In either case we’d expect glutamine users to have longer telomeres. Do we see this in bodybuilders that use it?

 

 



#8 QuestforLife

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Posted 18 October 2018 - 07:48 AM

For a while I’ve been pursuing several telomerase activation leads. One is via resveratrol and the Sirtuins. The other is via statins and inhibition of the mevalonate pathway. These two lines of investigation have finally converged somewhere in the mysterious signalling mechanisms of the mitochondria.

 

The potential of statins (and sartans) to activate telomerase (in vivo!) and repair the cardiovascular system was first brought to my attention by this paper:

https://www.ncbi.nlm...pubmed/26214555

 

Subsequent research revealed that this was a fairly well known pleitrophic affect, independent of the cholesterol lowering effects of statins and seems to be related to melavonate pathway inhibition and consequent AKT activation. The upshot is, people on statins have longer telomeres: 

https://www.ncbi.nlm...pubmed/25127175

https://www.ncbi.nlm...les/PMC5043056/

https://www.ncbi.nlm...pubmed/12676819

 

Further work strengthened the link to mitochondria. Inhibiting the mevalonate pathway leads to mitochondria fusion:

https://www.ncbi.nlm...les/PMC5104693/

 

This next paper draws a tentative link between mitochondrial fusion, low ROS and AKT (and therefore telomerase upregulation)

https://www.ncbi.nlm...pubmed/29378182

 

So how does this link us to the SIRTUINS and resveratrol? Well we know SIRT3 – SIRT5 are mitochondrial sirtuins. And Sirt4, the ‘culprit’ supposedly behind resveratrol’s telomerase activation, I have now found upregulates Opa1 – an important inner membrane fusion protein.

https://www.ncbi.nlm...ing-09-2163.pdf

 

(This might, just might, explain my massive energy boost when I took resveratrol after 20g of fish oil, as fish oil also upregulates Opa1 – so maybe the combination is synergistic after all, see https://www.ncbi.nlm...pubmed/24663492 Fig 4d. Could this also explain why those who eat fish generally have longer telomeres, too?)

 

So there we have it: resveratrol via NAMPT and SIRT4* and Statins via melvonate pathway inhibition, lead to increases in mitochondrial fusion. And somehow this is leading to an upregulation in AKT and telomerase.

The research continues.

 

* in fact all the mitochondrial sirtuins seem to upregulate fusion and downregulate fission (Sirt3 and 5 do it via the other mito fusion proteins), which makes sense – as the sirtuins are supposed to resist stress (which generally causes mito fission and an increase in ROS).


Edited by QuestforLife, 18 October 2018 - 07:56 AM.

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#9 MikeDC

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Posted 20 October 2018 - 04:26 PM

NAD+/Sirt1 appears to be a powerful telomerase activator. H2S also increases NAMPT/NAD+/Sirt1.
Look at the big jump in htert at the right H2S level. NR/NMN should have bigger effect.

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#10 QuestforLife

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Posted 07 November 2018 - 02:56 PM

Just a quick update.

 

I am now pursuing the low dose, intermittent statin-sartan protocol outlined in these papers 

 

 

https://www.ncbi.nlm...pubmed/26214555

 

https://www.ncbi.nlm...les/PMC4533138/

 

With addition of Vit D and Q10, which according to my research should not interfere with the desired arterial wall rejuvenation effects.

 

So the topic title should probably be changed to 'Methods of Activating Telomerase'. Perhaps the moderators will permit me to change it (it will still remain in the Telomeres subforum)?

 

I am not currently pursing the NAD-Resveratrol angle, mainly because any persistent (or intermittent large dose) use of resveratrol or pterostilbene destroys my sex drive and makes me feel generally low. Happy the statin-sartan protocol does not have this effect! But I believe that the mechanism that activates telomerase is probably the same, i.e. a low ROS environment (sartan or antioxidant) combined with mitochondrial fusion (statin via the mevalonate pathway or SIRT3/4/5). 


Edited by QuestforLife, 07 November 2018 - 03:00 PM.

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#11 Fafner55

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Posted 14 November 2018 - 02:39 AM

That's possible Phoebus, and another thing for me to look into. It's liposomal resveratrol so it should be fairly bioavailabile anyway, ...

 

Resveratrol is hydrophobic and doesn't lend itself to liposomal encapsulation. My attempts at making liposomal resveratrol have all failed. I suspect that what is marketed as liposomal resveratrol are actually emulsions. You could verify this by diluting a sample with 8 or 10 times the amount of water and placing it in a refrigerator for a few days. Excess water will break down an emulsion and the resveratrol will precipitate to the bottom.



#12 QuestforLife

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Posted 15 November 2018 - 03:58 AM

Resveratrol is hydrophobic and doesn't lend itself to liposomal encapsulation. My attempts at making liposomal resveratrol have all failed. I suspect that what is marketed as liposomal resveratrol are actually emulsions. You could verify this by diluting a sample with 8 or 10 times the amount of water and placing it in a refrigerator for a few days. Excess water will break down an emulsion and the resveratrol will precipitate to the bottom.


I will do this experiment.

#13 QuestforLife

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Posted 15 November 2018 - 04:16 AM

Another update. My statin-sartan experiment is going well. I feel good on the protocol and I am almost 3 weeks into the 4 week cycle.

I have continued to research the link with fusion and believe that C18 (stearic acid) is signalling in a similar manner to a statins to trigger fusion. I believe this is done via a PPAR agonist pathway, which makes the body think that it should burn fats and not store or manufacture them. In this way it is similar to a ketogenic diet. With statins it is via blockade of SREBP (creation of chloresterol). With ingestion of C18 it is stimulation via direct fatty acid oxidation. The connection with a ketogenic diet and PPAR suggests that one should limit intake of carbohydrates, particularly when taking the stearic acid or statins, to avoid the conflicting signals from PPAR and SREBP.

REFERENCES:

https://www.nature.c...467-018-05614-6
(Previously posted reference in the stem cell protocol thread regarding human in vivo fusion with stearic acid. Note the mention of fatty acid B oxidation, which doesn't occur with ingestion of palmitic acid, only with stearic).

https://www.liebertp...journalCode=rej
(Excellent overview on PPAR and SREBPs by Karen Kurtak. Need sci-hub).

I have previously experimented with adipocyte generation using pioglitazone, a potent PPAR agonist. Now I think this could be a good addition to a future fusion protocol.
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#14 QuestforLife

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Posted 15 November 2018 - 09:06 AM

Further evidence my hypothesis is correct:

https://www.ncbi.nlm...ubmed/11226238/

This paper discusses how PPAR is activated by liver fatty acid binding protein (L-FABP), upregulated by statins (or fatty acids).

The only missing piece of the puzzle is how does this then lead to upregulated mitofusin and fused mitochondria? My bet is it is to do with the PPAR switch to burning fats means they are not available for making chloresterol to go into cell or mitochondrial walls, hence the mitochondria need to reduce overall surface area. Just a guess though. It could equally just be an adaptation to starvation, which is when your body would normally start burning fats

#15 QuestforLife

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Posted 19 November 2018 - 08:58 AM

I will do this experiment.

Two days in the fridge at 5C and a significant amount of resveratrol powder has separated out and been deposited in the bottom of the shot glass I put it in. This is Actinovo Resveratrol. It is 3 months old, but it is in date.

There was no separation in my home made vitamin C liposomal liquid.

So it appears lipophilic molecules are an issue when trying to encapsulate.

It's a pity as resveratrol still fits in well with this protocol, and I've recently found it is a PPAR agonist

https://www.ncbi.nlm...les/PMC4104805/

We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα

So it also acts using the same pathway as Statins and a ketogenic diet and this probably explains it's benefits on the heart and endothelium more than any anti oxidant effect. SIRT1 also has an effect along the same pathway but it appears to be in the other direction.

Moreover, the SIRT1-dependent repression of visceral white adipocyte genes involves deacetylation of PPARγ


Edited by QuestforLife, 19 November 2018 - 09:00 AM.

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#16 HaplogroupW

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Posted 24 November 2018 - 03:01 AM

So it appears lipophilic molecules are an issue when trying to encapsulate.

 

 

Maybe I'm the one missing something here, but my understanding was that liposomal formulations are for water-soluble compounds or nutrients that would otherwise get sent to the liver. Anything fat-soluble, taken with long chain fats, will get packaged into chylomicrons and thence to lymph and to adipocytes. So there's no need to do liposomal formulation of fat-soluble resveratrol. Just take it with fat.

 

Another thing though is that my understanding is that once delivered to adipocytes, it doesn't get burned or released as free fatty acids right away. Rather, the fat (and fat-soluble things such as resveratrol, vit A, D, E)  that one ingests becomes part of the lipid droplets in adipocytes. As and when fatty acids are released from adipocyte lipid stores, it is mostly from what was previously accumulated. So one way of looking at it is fat is a first-in first-out queuing process. Or maybe more accurately a mixing problem: there is a reservoir of lipids in adipocytes, and newly-ingested fat gets incorporated and mixed into the lipid droplets. It is from this lipid reservoir from which fat is released and ultimately beta-oxidized elsewhere for energy. So I'd expect utilization and availability of any such fat soluble-thing one eats gets smoothed out over a longer window of time. One might need to "charge up" the adipocytes for a while before it is substantially available.

 

Caveat: this is all based on my reading; I'm not researcher or expert about this. It's possible I've speculated or misunderstood so if I've gotten this wrong maybe someone can correct me.


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#17 QuestforLife

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Posted 25 November 2018 - 02:29 PM

Maybe I'm the one missing something here, but my understanding was that liposomal formulations are for water-soluble compounds or nutrients that would otherwise get sent to the liver. Anything fat-soluble, taken with long chain fats, will get packaged into chylomicrons and thence to lymph and to adipocytes. So there's no need to do liposomal formulation of fat-soluble resveratrol. Just take it with fat.

Another thing though is that my understanding is that once delivered to adipocytes, it doesn't get burned or released as free fatty acids right away. Rather, the fat (and fat-soluble things such as resveratrol, vit A, D, E) that one ingests becomes part of the lipid droplets in adipocytes. As and when fatty acids are released from adipocyte lipid stores, it is mostly from what was previously accumulated. So one way of looking at it is fat is a first-in first-out queuing process. Or maybe more accurately a mixing problem: there is a reservoir of lipids in adipocytes, and newly-ingested fat gets incorporated and mixed into the lipid droplets. It is from this lipid reservoir from which fat is released and ultimately beta-oxidized elsewhere for energy. So I'd expect utilization and availability of any such fat soluble-thing one eats gets smoothed out over a longer window of time. One might need to "charge up" the adipocytes for a while before it is substantially available.

Caveat: this is all based on my reading; I'm not researcher or expert about this. It's possible I've speculated or misunderstood so if I've gotten this wrong maybe someone can correct me.


Very well put. I think you are probably right, especially as it chimes with my past personal experience of resveratrol only having an effect on me after a delay when I have been taking it for some time. I am now on a Ketogenic diet, with energy mostly generated through fat burning, so I'd expect all the PPAR pathway triggering supplements to be potentiated on this diet. We shall see.

#18 QuestforLife

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Posted 29 November 2018 - 02:33 PM

This week I completed the low dose statin-sartan protocol.

 

I will be continuing the Vit D+Q10 this week and maybe next as well (but without the statin and sartan) to see if I can discern any difference in how I feel or look. I will then discontinue each in turn.  I will also be continuing the low carb diet that I have been following throughout the majority of this protocol.

 

I feel extremely good, and to myself at least, appear healthier and younger. I have had no side effects from this 1 month treatment. Contrary to some reports I have heard from those on statins, my sex drive has been undeniably increased. I have also felt calmer overall.

 

My blood pressure is healthy within its long term range, so the sartan has remained sub therapeutic, as intended. I intend to do a full blood panel in the new year. This should hopefully confirm the low dose statin has not reduced chloresterol. 

 

I also intend to re-run the statin-sartan protocol in around 6 months (as recommended in the Janic papers) and then in the Summer take another Life Length Telomere test. This will be 1 year after I took my first one.


Edited by QuestforLife, 29 November 2018 - 02:39 PM.


#19 QuestforLife

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Posted 02 December 2018 - 08:42 AM

I also noticed some skin improvements.

I had a random bald patch on my beard (about 2cm X 1 cm) for approx 3 months, which I realised had started to fill in (may be related or unrelated to the protocol). So I took a photo to compare the bald patch to one I took a few months ago.

The photo included the eye and cheek area and I was surprised to see my skin is now much improved in terms of moisture and general complexion.

Skin takes up to a month to turn over and I would expect peak Telomerase activation from the statin-sartan protocol at the 1 month mark (when it completes), so any skin improvements, if any, will be most obvious over the month after the end of the protocol.

Edited by QuestforLife, 02 December 2018 - 08:44 AM.


#20 QuestforLife

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Posted 06 December 2018 - 11:23 AM

Some further research that I beleive is pertinent.

 

I've discovered a link between telomerase activation and ROCK inhibitors:

 

https://www.ncbi.nlm...76/pdf/main.pdf

 

 

Abstract

We demonstrate that a Rho kinase inhibitor (Y27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 106 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the
CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host. (Am J Pathol 2012, 180: 599–607; DOI: 10.1016/j.ajpath.2011.10.036)

 

Further Statins possibly exert their pleitrophic effects via the same pathway.

 

https://www.ncbi.nlm.../nihms84969.pdf

 

 

 

Role of ROCK in Cardiovascular Disease Many cholesterol-independent or so-called “pleiotropic” effects 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors or statins are due to their ability to block the synthesis of isoprenoid intermediates, which serve as important lipid attachments for a variety of intracellular signaling molecules.In particular, the inhibition of small GTP-binding proteins Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation.

 

 


Edited by QuestforLife, 06 December 2018 - 11:26 AM.


#21 QuestforLife

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Posted 11 December 2018 - 09:54 AM

Further research (and reading the first of the two papers above) suggests that in this experiment atleast, it is the senescent feeder cells that (mainly) supply the telomerase and the ROCK inhibition that permits the growth and spread of the progenitor cells.  

 

The following paper also shows that the expansion in the population of cultured (in this case epithelial) cells  correlates very closely with the production of apoptosis products from the irradiated feeder cells. 

 

https://ajp.amjpatho...0594-4/fulltext

 

 

The correlation between apoptosis of irradiated feeder cells and the release of feeder factors that promote conditional immortalization suggests that irradiation induces these factors and/or that the factors are released from dying cells.

 

This is huge! It means that the next time I do my statin-sartan protocol I should overlap it (near the end when telomerase and ROCK inhibition is at its highest) with a senolytic protocol to supply apoptosis products to aid in population expanstion of progenitor cells. The same might be true in Turnbuckle's Stem Cell Renewal Protocol - perhaps the senolytics and stem cell renewal portions of the protocol should in fact be merged for maximum efficiency.


Edited by QuestforLife, 11 December 2018 - 10:22 AM.

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#22 QuestforLife

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Posted 11 December 2018 - 03:50 PM

From the same paper, the attached image suggests the ideal time for stimulation of progenitor cells is 2 days after the irradiation of feeder cells. Note the bottom image is the activation of the caspases that initiate cell self destruction and the top two images are culture cell numbers.

 

 Senolytics might be similar to irradiation, or perhaps involve an additional delay for the action of the senolytic to cause cell death. So 3 days might be optimal.

 

 

 

 

 

Attached Thumbnails

  • feeder cells.png


#23 QuestforLife

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Posted 20 December 2018 - 11:26 AM

Another interesting paper using ROCK and mTOR inhibitors to reprogram brain cancer cells into neurons.

https://jeccr.biomed...3046-018-0857-5

We screened a kinase inhibitor library and found that a combination of two inhibitors, Rho-associated protein kinase (ROCK) and mammalian target of rapamycin (mTOR), could substitute for transcription factors and convert human GBM cells into neural-like cells. The induced neural cells were subsequently trans-differentiated into functional neurons. The induced neurons lost their tumorigenicity and reverted to the ‘normal state’.



#24 QuestforLife

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Posted 20 December 2018 - 11:34 AM

It appears that statins are actually quite potent ROCK inhibitors

https://iovs.arvojou...ticleid=2359716

Proliferation of CECs (corneal endothelial cells) was enhanced by administration of 10 µM Y-27632, 2 µM H-1152, and 10 µM lovastatin compared to control


Edited by QuestforLife, 20 December 2018 - 11:34 AM.


#25 QuestforLife

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Posted 02 January 2019 - 08:04 AM

An interesting update from over on the Aging Biomarkers thread:

So far we have both Droplet and Bobolander with exceptionally young epigenetic age (and long telomeres in Bobolander's case, Droplet hasn't tested). Both have also shared they are on long term, low dose Statins.

Statins are Rho kinase (ROCK) inhibitors and known to inhibit differentiation, which would explain the positive effects on epigenetic age and telomere length.

Is anyone else on Longecity on Statins long term? It would great if we could test my theory that ROCK inhibition is responsible with more than n=2!
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#26 NeilR

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Posted 06 January 2019 - 12:49 AM

What’s your protocol routine? Could you please post the doses?

#27 QuestforLife

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Posted 06 January 2019 - 08:39 AM

What’s your protocol routine? Could you please post the doses?

Thank you for the opportunity to spell out my protocol as it stands. In November I took 10mg of atorvastatin and 25mg of losartan along with 100mg of Q10, all once per day in the morning, for 1-month.

Since then I have taken almost no supplements with the intention of repeating the protocol in February or March. In the original Janic etc al. work posted upthread the interval was 6 months (or in with the diabetics study 3 months). I believe this is based on the time for the progenitor cells to fully differentiate back into normal cells. I am considering a different interval however, based on 2 weeks on and 6 weeks off. This is based on the following paper where they used ROCK and mTOR inhibitors to reprogram glioblastoma cells (in 3-7days), and differentiate then into normal neurons (in 2-3 weeks). I've doubled their timescales because I know detectable ROCK inhibition takes about 2 weeks (in Vivo).

https://jeccr.biomed...3046-018-0857-5

After induction for 3–7 days, about 70–90% of these GBM cells converted into neural-like cells ....In 2–3 weeks, 70–90% of GBM U118 cells converted to neurons with mature neuronal morphology.

I am also considering using rapamycin during the protocol as well to enhance the reprogramming efficiency. If I go with a 2 week protocol rather than 1-month doses will also be higher than what I used previously. I will post these once I finalise them.

I believe the benefits are largely lost once the progenitor cells differentiate because the somatic cells go back to being the age they were before de-differentiation, with the same length telomeres. For this reason I will include cyclastragneol in the protocol to try and lengthen telomeres whilst the progenitor cells are active.
https://www.ncbi.nlm...ubmed/24774536/

We showed previously that, despite high telomerase ex-
pression, the telomeres erode with passage but finally
stabilize at a defined length [1]. We speculate that the
time to senescence after removal of Y-27632 is deter-
mined by the remaining length of the telomeres

As you can tell the protocol is still in flux, but it is very promising.

One other note, I believe that some of the benefits of the protocol overlap with those of a ketogenic diet, because Statins cause upregulation of fatty acid oxidation. Since late November I've been ketogenic. This is not strictly necessary (Janic etc al. didn't do this), but I believe it may help. Just something to consider.

The end point for my n=1 study is another (my third) Zymo epigenetic age test next summer.

Edited by QuestforLife, 06 January 2019 - 08:45 AM.


#28 marcobjj

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Posted 06 January 2019 - 09:21 AM

Making a drug is like trying to pick a lock at the molecular level. There are two ways in which you can proceed. You can try thousands of different keys at random, hopefully finding one that fits. The pharmaceutical industry does this all the time – sometimes screening hundreds of thousands of compounds to see if they interact with a certain enzyme or protein. But unfortunately it’s not always efficient – there are more drug molecule shapes than seconds have passed since the beginning of the universe.

Alternatively, like a safe cracker, you can x-ray the lock you want to open and work out the probable shape of the key from the pictures you get. This is much more effective for discovering drugs, as you can use computer models to identify promising compounds before researchers go into the lab to find the best one. Now a study, published in Nature, presents detailed images of a crucial anti-ageing enzyme known as telomerase – raising hopes that we can soon slow ageing and cure cancer.

The researchers behind the new study were not just able to obtain the structure of a proportion of the enzyme, but of the entire molecule as it was working. This was a tour de force involving the use of cryo-electron microscopy – a technique using a beam of electrons (rather than light) to take thousands of detailed images of individual molecules from different angles and combine them computationally.

 

 

http://theconversati...elomerase-95591

 

 

 


Edited by marcobjj, 06 January 2019 - 09:22 AM.

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#29 QuestforLife

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Posted 06 January 2019 - 09:56 AM


Making a drug is like trying to pick a lock at the molecular level. There are two ways in which you can proceed. You can try thousands of different keys at random, hopefully finding one that fits. The pharmaceutical industry does this all the time – sometimes screening hundreds of thousands of compounds to see if they interact with a certain enzyme or protein. But unfortunately it’s not always efficient – there are more drug molecule shapes than seconds have passed since the beginning of the universe.

Alternatively, like a safe cracker, you can x-ray the lock you want to open and work out the probable shape of the key from the pictures you get. This is much more effective for discovering drugs, as you can use computer models to identify promising compounds before researchers go into the lab to find the best one. Now a study, published in Nature, presents detailed images of a crucial anti-ageing enzyme known as telomerase – raising hopes that we can soon slow ageing and cure cancer.

The researchers behind the new study were not just able to obtain the structure of a proportion of the enzyme, but of the entire molecule as it was working. This was a tour de force involving the use of cryo-electron microscopy – a technique using a beam of electrons (rather than light) to take thousands of detailed images of individual molecules from different angles and combine them computationally.



http://theconversati...elomerase-95591



https://www.youtube....h?v=iySP8A5Yoxc


The fact they have now managed to discover the entire structure of telomerase is interesting, but I'm not sure how much it will help in activating telomerase in human cells. That's because what we really need to discover is the structure of the promoters and inhibitors of HTERT on the DNA. We know part of that inhibitory mechanism is the Telomere tail itself, looping back and associating with the DNA near HTERT by virtue of further curling of DNA around histones. And this inhibition is removed when telomeres are short, or during replication. Which is why cyclastragneol can more effectively repair short telomeres than extend them when they are longer. But why for example do progenitor cells have active telomerase while somatic cells do not? It's a complex net of different things no doubt involving histones, splicing factors and growth factors. In some of the ROCK inhibition studies I've posted upthread they used senescent mouse embryonic stem cells as 'feeder cells' to provide the Telomerase for the rest of the culture. This worked for progenitor cells (formed by the ROCK inhibition) but not on somatic cells (once ROCK was re established). So there is the possibility that we could use senolytics to cause the release of appropriate apoptosis factors, and simultaneously use ROCK inhibitors to cause de-differentiation. I'd love to find a study where they used senescent human feeder cells to confirm that this is viable. The nearest I could find was the study with senescent cultures and resveratrol, which caused a stir a few years ago (I think Nate started the thread).

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#30 marcobjj

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Posted 07 January 2019 - 05:15 AM

As far as I understood, Questforlife, I think the idea is to use cryo-electron microscopy to identify the structure of HTERT activators and repressors and later create bio-identicals to use respectively as longevity and anti-cancer medication. Scan for activators in cancer cells with telomerase activity. Scan for repressor proteins in regular somatic cells.







Also tagged with one or more of these keywords: telomeres, nad, nampt, ampk, resveratrol, allicin, methylene blue, nmn, sirtuins, statin

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