989 replies to this topic

[RWhigham]

Bigger image of L.Reuteri 6475 effects on mice

L. reuteri effects on hormone levels and thymus size of Swiss mice. L. reuteri consumption leads to statistically significant changes including (A) increased blood levels of oxytocin (Untreated N = 10, L. reuteri N = 10), (B) decreased levels of circulating corticosterone (N = 10 per group), © increased thymus weight, (N = 10 per group) and (D) lower circulating neutrophil counts (N = 10 per group). Numbers on the y-axis of bar graphs correspond to the mean ± SEM of the parameter assessed; **p < 0.001, ***p < 0.0001.

Edited by RWhigham, 11 February 2020 - 04:48 PM.

[QuestforLife]

I’ve been trialling a new protocol and have a few things to report. Generally, I’ve been feeling great, good energy, good sleep, but on the downside, I have been experiencing occasional fast heart beats. These are not flutters. They are proper, strong heartbeats that send the blood round my body. Imagine getting a small shock of adrenaline. That is what they feel like. I get these from time to time and to begin with it was unclear whether or not the protocol was making this worse. But it now appears that it is making it worse.

I built the protocol up one supplement at a time in the AM to determine effects, positive or negative.

• First, I introduced phloretin at 30-50mg/day to determine if this made me fatigued (see previous report). At this level it does not make me fatigued. I did notice my fat percentage creeping up again on the machine.
• After 2 days of phloretin this I added Boswellia extract back in.
• After 2 days of Boswellia (on the 2nd day I doubled the dose) I introduced forskolin.
• Forskolin affected my bowel movements and made me very alert for a few days but then things went pretty much back to normal. I noticed it also seems to counter the fat increasing effects of the phloretin.
• I then added artichoke extract to increase the effectiveness of the forskolin.
• After another couple of days I then added Tongkat Ali back in as a second ROCK inhibitor (in addition to phloretin).
• I then changed to taking all the supplements in the evening instead of the morning. Both seem to work equally well and no obvious side effects immediately occurred.

My diastolic blood pressure dropped about 10 points. My systolic blood pressure is inconsistent. My heart rate variability results are good (much better than population average for my age), although I have not been doing this monitoring for long, so I don’t have a previous baseline. I have now noticed during and after exercise that I am experiencing some sharp, spikey heartbeats that the monitor is removing as artifacts. But I know they are real heartbeats as I can feel them. This has not affected my exercise performance in any way, but it is a little disturbing.

 

• Last night I injected about 300ug of oxytocin and 10mg of epitalon.

I immediately felt flushed and hot in the face and to a lesser extent on the rest of my skin. This effect diminished after a few minutes. I know this is due to the oxytocin, as I’ve used epitalon lots of times before without this effect. I was quite excited and apprehensive injecting oxytocin so although I was monitoring my heart rate, which was elevated, it was impossible to say whether this was due to the oxytocin or just my excitement.
The next morning I felt normal, maybe a little more fatigued than usual given I hadn’t exercised the night before (although this could be due to the epitalon). Later in the day I took a blood pressure measurement. My diastolic had dropped by another 5 points. The first came back at 143/59. My normal levels are 120/74. The second reading was 107/58.

My best guess is that the combination of the supplements I am using is affecting my heart (and probably other muscles) by inhibiting the actin cytoskeleton and myosin fibres. This might explain the erratic heart beats and wildly varying systolic pressure (the lowered diastolic is expected from my protocol based on previous results). I may be jumping to conclusions but there is some support for the heart to be negatively affected by tgf-b inhibition: 

http://basictranslat.../content/4/1/41

 

The role of the transforming growth factor (TGF)-β pathway in myocardial fibrosis is well recognized. However, the precise role of this signaling axis in cardiomyocyte (CM) biology is not defined. In TGF-β signaling, SMAD4 acts as the central intracellular mediator. To investigate the role of TGF-β signaling in CM biology, the authors deleted SMAD4 in adult mouse CMs. We demonstrate that CM-SMAD4–dependent TGF-β signaling is critical for maintaining cardiac function, sarcomere kinetics, ion-channel gene expression, and cardiomyocyte survival. Thus, our findings raise a significant concern regarding the therapeutic approaches that rely on systemic inhibition of the TGF-β pathway for the management of myocardial fibrosis.

Even if the above paper is the extreme case (i.e. deleting SMAD4), it does indicate a fundamental limit to this method of rejuvenation and indicate I may need to moderate my protocol in either dose or duration. Nothing like this was reported in the Conboy study; their protocol was done for 7 days.

 

Edited by QuestforLife, 12 February 2020 - 02:22 PM.

[Oakman]

I’ve been trialling a new protocol and have a few things to report. Generally, I’ve been feeling great, good energy, good sleep, but on the downside, I have been experiencing occasional fast heart beats. These are not flutters. They are proper, strong heartbeats that send the blood round my body. Imagine getting a small shock of adrenaline. That is what they feel like. I get these from time to time and to begin with it was unclear whether or not the protocol was making this worse. But it now appears that it is making it worse.

I built the protocol up one supplement at a time in the AM to determine effects, positive or negative.

• First, I introduced phloretin at 30-50mg/day to determine if this made me fatigued (see previous report). At this level it does not make me fatigued. I did notice my fat percentage creeping up again on the machine.
• After 2 days of phloretin this I added Boswellia extract back in.
• After 2 days of Boswellia (on the 2nd day I doubled the dose) I introduced forskolin.
• Forskolin affected my bowel movements and made me very alert for a few days but then things went pretty much back to normal. I noticed it also seems to counter the fat increasing effects of the phloretin.
• I then added artichoke extract to increase the effectiveness of the forskolin.
• After another couple of days I then added Tongkat Ali back in as a second ROCK inhibitor (in addition to phloretin).
• I then changed to taking all the supplements in the evening instead of the morning. Both seem to work equally well and no obvious side effects immediately occurred.

My diastolic blood pressure dropped about 10 points. My systolic blood pressure is inconsistent. My heart rate variability results are good (much better than population average for my age), although I have not been doing this monitoring for long, so I don’t have a previous baseline. I have now noticed during and after exercise that I am experiencing some sharp, spikey heartbeats that the monitor is removing as artifacts. But I know they are real heartbeats as I can feel them. This has not affected my exercise performance in any way, but it is a little disturbing.

 

• Last night I injected about 300ug of oxytocin and 10mg of epitalon.

I immediately felt flushed and hot in the face and to a lesser extent on the rest of my skin. This effect diminished after a few minutes. I know this is due to the oxytocin, as I’ve used epitalon lots of times before without this effect. I was quite excited and apprehensive injecting oxytocin so although I was monitoring my heart rate, which was elevated, it was impossible to say whether this was due to the oxytocin or just my excitement.
The next morning I felt normal, maybe a little more fatigued than usual given I hadn’t exercised the night before (although this could be due to the epitalon). Later in the day I took a blood pressure measurement. My diastolic had dropped by another 5 points. The first came back at 143/59. My normal levels are 120/74. The second reading was 107/58.

My best guess is that the combination of the supplements I am using is affecting my heart (and probably other muscles) by inhibiting the actin cytoskeleton and myosin fibres. This might explain the erratic heart beats and wildly varying systolic pressure (the lowered diastolic is expected from my protocol based on previous results). I may be jumping to conclusions but there is some support for the heart to be negatively affected by tgf-b inhibition: 

http://basictranslat.../content/4/1/41

 

Even if the above paper is the extreme case (i.e. deleting SMAD4), it does indicate a fundamental limit to this method of rejuvenation and indicate I may need to moderate my protocol in either dose or duration. Nothing like this was reported in the Conboy study; their protocol was done for 7 days.

 

I have not been following your experiments at all Questforlife, I just thought I'd read a post added to this thread - and yours was it - but I'll offer you a couple observations, perhaps helpful to you, perhaps not, and based on only this post of yours. Please feel free to ignore or not at your pleasure. And a reply is not needed, I'll not continue with any of this, it's just an observation from a dis-interested bystander stopping by.

 

1) You sound desperate for unknown reasons, trying this and that based on I'm not sure what reasoning and for no particular purpose.

2) You are changing your protocols way too often, too fast, as stated, "after 2 days this, after 2 days that, etc.

3) When something unwanted/strange happens you continue changing/adding, rather than stop or remove substances to reassess.

4) A day or two is not enough time to assess any results from something added/removed, short of it poisoning yourself/making you sick

5) Injecting yourself? Honestly you sound like a drug addict, if I didn't hope you were not.

6) If something is affecting your heart beat/HR and blood pressure...stop! Wait, remove these substances altogether. You should not be playing around with substances in a way that can kill you.

7) Your reasoning for this and that happening sound off the cuff / unfounded conjecture, not scientific justification or research based.

 

From one self experimenter to another - Please be safe, don't hurt yourself, be careful and methodical, do things slowly over longer periods of time. Cheers!

[QuestforLife]

A very interesting recent paper on telomere lengths in mice given an antioxidant product. I’m not so interested in the telomere length results, which weren’t very impressive when started in middle age (although cell numbers did seem to be sustained with treatment), and annoyingly they didn’t do a lifespan study or look at telomere lengths in very young mice for comparison, but – and this is a big one – they looked at telomere lengths in eggs and BONE MARROW cells. The results showed a precipitous drop in the telomere lengths of bone marrow with age; far greater than the (absolute) loss in the eggs cells (female mice). Mice in middle age had bone marrow telomere lengths of 1228 (units) (compared to 33 in eggs cells). But by old age bone marrow telomere lengths had dropped to 121 (compared to 7.3 in egg cells).
In humans the average telomere length lost per year in leukocytes (supplied from the bone marrow) is quite small (~50 bp/year) and more akin to the changes in egg cell telomeres in this study. To me this is clear evidence that bone marrow is constantly dividing to repair the body. This is leading to a small but steady loss of telomeres in replaced tissue but a fast, alarming (even) loss in the bone marrow. This must be a primary cause of aging and death both in mice and humans.

It is not clear to me whether any telomerase activator can reach the bone marrow to address this, or whether the benefits to telomere lengths come from slower attrition of telomeres in peripheral tissues and therefore preservation of bone marrow telomeres (slower use). It is clear that the product used in this study is acting via only the latter method, unfortunately. It should also be noted that mice have telomerase activated in every cell, unlike humans, albeit at an insufficient level to counteract aging, and therefore are more likely to benefit from antioxidants than humans.

If we want to reverse human aging in a meaningful and long-lasting way, i.e. aging proceeds after treatment at the normal and not an accelerated rate, then we not only need powerful telomerase activators, but will need to target the bone marrow directly, or else somehow replace it.

https://www.mdpi.com/2076-3921/9/2/144

Abstract: In recent decades much attention has been paid to how dietary antioxidants may positively affect the human health, including the beneficial effects of fermented foods and beverages. Fermented Papaya Preparation (FPP®) has been shown to represent a valuable approach to obtain systemic antioxidants effect. In this study, we wanted to verify whether FPP® had a clear and scientifically supported in vivo anti-aging effect together with the induction of a systemic antioxidant reaction. To this purpose we daily treated a mouse model suitable for aging studies (C57BL/6J) with FPP®-supplemented water from either the 6th weeks (early treatment) or the 51th weeks (late treatment) of age as compared to mice receiving only tap water. After 10 months of FPP® treatment, we evaluated the telomerase activity, antioxidants and Reactive Oxygen Species ROS plasmatic levels and the telomeres length in the bone marrow and ovaries in both mice groups. The results showed that the daily FPP® assumption induced increase in telomeres length in bone marrow and ovary, together with an increase in the plasmatic levels of telomerase activity, and antioxidant levels, with a decrease of ROS. Early treatment resulted to be more effective, suggesting a potential key role of FPP® in preventing the age-related molecular damages.

The product used for those that are interested (UK Amazon)
https://www.amazon.c...uct_top?ie=UTF8

[QuestforLife]

 

 

From one self experimenter to another - Please be safe, don't hurt yourself, be careful and methodical, do things slowly over longer periods of time. Cheers!

 

Thanks for your concern. 

 

You'd have to read more of the thread to understand the various additions to the protocol. All have been used alone for some period beforehand, and for the majority I know what effect they have in isolation. The effects on blood pressure are not unexpected. The effects on heart rate are concerning and seem to be due to the specific combination. I do agree that side effects of this nature warrant backing off and reassessing. 

 

ps - I should clarify that 'injecting' means subcutaneous.

Edited by QuestforLife, 12 February 2020 - 04:08 PM.

[QuestforLife]

Another interesting article that considers telomerase therapy (of various sorts) for CV disease. 

 

https://www.scienced...301779?via=ihub

 

Nothing particularly new for readers of this thread, but more confirmation that statins, sartans, glitazones, danazol and TA-65 - all mentioned at various times here - are under consideration as a therapy.

 

 

PHARMACEUTICAL INTERVENTIONS FOR

TELOMERES AND TELOMERASE ACTIVITY
Because of the pertinence of telomerase in antiaging
gene therapy in mice models, several studies
focused on the therapeutic interventions for telomerase modulations in humans. Several cardiovascular
medications, which have been used for decades and
have been shown to have significant survival benefits
in patients, possess the effects of telomere length
maintenance and senescence prevention.
Statins (3-hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors) exert various pleiotropic effects
to prevent the development of atherosclerotic plaque
(46). A cross-sectional analysis of 3,496 subjects from
the U.S. National Health and Nutrition Examination
Survey showed that telomere length appeared to be
longer with a longer duration of statin usage (47).
Statin therapy was associated with higher telomerase
activity independently of multiple covariates, such as
age, sex, smoking, lipid profile, and inflammation
(48). Statins can enhance telomerase activity and
protect telomeres through upregulation of the telomere repeat-binding factor (TRF)-2 in endothelial
cells and endothelial progenitor cells (49). A more
specific analysis of human T-lymphocytes showed
that atorvastatin in pharmacologically relevant doses
led to a transient increase in telomerase activity in
T-cells. This effect, which could be blocked by
inhibitors of Akt and phosphatidylinositol-4,5-
bisphosphate 3 (PI3)-kinase, was more pronounced
in the CD4þ than in the CD8þ T-cell subsets (50). In
addition, it also prevented telomere shortening by
accelerating DNA repair through Nijmegen breakage
syndrome-1 protein stabilization and telomere maintenance in vascular smooth muscle cells (51).
The crosstalk between angiotensin II and telomere
systems are noteworthy. Overexpression of TERT
in vivo modified the angiotensin IIinduced
microvascular endothelial dysfunction (52). Angiotensin II induces oxidative stress and senescence in
vascular smooth muscle cells with telomeraseindependent oxidative stress-induced senescence
and telomerase-dependent replicative senescence
(53). Acute exposure of vascular smooth muscle cells
to angiotensin II results in vascular smooth muscle
senescence, which is not associated with telomerase
activity changes and cannot be reversed by TERT
overexpression. However, long-term exposure of
vascular smooth muscle cells to angiotensin II
induced reduction in proliferation and replicative
senescence with telomere shortening (54). Angiotensin II receptor blockers (e.g., losartan) and
angiotensin-converting enzyme inhibitors (e.g.,
captopril) were both shown to protect endothelial
progenitor cells from senescence and dysfunction
through telomerase cross-talk (55,56). However, some
studies showed that captopril and losartan had no
effect on telomere attrition caused by cardiac hypertrophy after abdominal aortic constriction in rats (57).
Therefore, the clinical use of angiotensin-converting
enzyme inhibitors or angiotensin II inhibitors for the
modification of telomere systems requires further
clinical studies.
Peroxisome proliferator-activated receptor agonists (e.g., pioglitazone) can increase the activity of
telomerase and expression of TRF-2 in mice aorta and
in mononuclear cells. Pioglitazone-treated mice were
shown to possess the reduced senescence markers,
p16, cell-cycle checkpoint kinase 2, and p53 (58).
Angiotensin IIinduced endothelial progenitor cells
senescence was significantly reversed by pioglitazone
through telomerase activity enhancement (59).
Moreover, pioglitazone was able to increase the TERT
and TRF-2 expression in the hearts of diabetic rats
(60).
The low potency telomerase activator TA-65, a
bioactive molecule extracted from Astragalus membranaceus, has been historically used in Chinese
traditional medicine as an antiaging drug and has
been shown to have effects on telomere lengthening
in mice. TA-65 treatment induces telomerasedependent elongation of short telomeres and reverses DNA damages in fibroblasts (61) and human T
cells (62). Randomized, double-blind, and placebocontrolled clinical trials showed that TA-65 treatment increased high-density lipoprotein cholesterol
and reduced C-reactive protein in patients with
metabolic syndrome (63) and was also found to
elongate telomeres (64).
In addition, sex hormones were also reported to
activate TERT transcription. For decades, androgen
therapy was considered as the first treatment choice
for aplastic anemia, without a clear understanding of
the underlying mechanism. A recent study showed
that the upregulated telomerase activity is responsible for the effectiveness of the androgen treatment
effect in aplastic anemia. In mice with aplastic anemia induced by short telomeres, testosterone therapy
halted telomere attrition and prevented subsequent
death, by enhancing telomerase expression and
lengthening telomeres (65). Moreover, a synthetic
androgen, danazol, which was used in the treatment
of human telomeropathies, was shown to elongate
telomeres in circulating leukocytes and improve hematological parameters (66).
Although specific telomere-lengthening effects of
telomerase activation affect cardiovascular health
and aging, noncanonical, extracellular, and non-
telomere-lengthening functions of telomerase were
recently described (67). The off-target effects of
these telomerase activating or telomere-lengthening
compounds, including those in mitogen signaling
and oncogenesis, should be considered before clinical
usage.

 

[QuestforLife]

What is ‘healthy aging’?
(other than an oxymoron).
One of the mysteries of aging is how aging leads to decreased cellular turnover/decreased cellular turnover leads to aged tissues, but that various interventions that increase cellular turnover seem to be beneficial in the long as well as short run, even though they should (in theory) result in accelerated aging. Take for example tretinoin, increased skin turnover results in younger looking skin as expected, but does not seem to lead to a delayed but sudden, ‘catch-up’ aging of the skin later, as might be expected (people have been using it for 20 years). Exercise that loads the skeleton increases bone turnover and bone density – a free meal, it seems, as at no point does this suddenly seem to accelerate osteoporosis. This is not to say there are not limits, clearly this is a hormetic effect. We wouldn’t recommend ripping your skin off each day or taking a hammer to your bones. Yet still, the effect requires some explanation, particularly for a telomere enthusiast such as I.
I think we can understand the phenomena of hormesis by looking at the distribution of the health of cells. Take the argument often advanced by the advocates of senolytics – a treatment that seems to increase mean but not max lifespan – that a few unhealthy cells can cause harm (via inflammation) in the bulk of healthy tissue. This has been shown to occur in skin for example, due to the comparatively early senescence of melanocytes, which then slow the turnover of the otherwise healthy keratinocytes. If we were able to effectively remove the few bad cells, the rest of the tissue would then behave normally again, albeit after some division to replace the lost cells. In this way repeated senolytic treatments would in theory gradually adjust the distribution of health from mostly excellent with a few bad cells to all cells being pretty ‘good’. Now before we move on we need to address the confusion in the community about cellular senescence as a result of telomere shortening versus stress induced. They are not really a distinct phenomenon. The nearer the length of telomere to its original (young) length, the more stress resistant and the less likely the cell is to succumb to stress induced senescence. This does not mean stress immunity, as already stated hormesis has limits and even a young cell can be shunted into senescence in bad enough conditions. Telomerase therapy can be seen as the reverse of senolytics. Whilst senolytics remove the cells at the bottom of the health distribution then causing a small shift down in the remainder –  still a net gain, at least in the short run – telomerase therapy preferentially rescues the least healthy cells, pushing up the entire bulk ever so slightly. It is an open question whether telomerase can rescue senescent cells. As we should know by now, cells exist on a continuum from fully healthy to fully senescent; not all fully senescent cells are really senescent, they have a range of markers – p21 vs p16, etc. But there is no need to belabour the point anymore. We have done enough to understand that healthy aging is a lifestyle or regime causing sufficient harm or stress to the body to either remove the worst cells, or keep the remainder dividing despite their presence. This strategy creates healthier - but still older - people. The strategy is akin to the game of Tetris, where the blocks constantly build up but do not end the game so long as none reach the top of the screen. The game of healthy aging also becomes progressively harder as the bulk of cells move further towards the unhealthy end of the spectrum. In the end this leads to a quick death without long sickness – the definition to my mind, of healthy aging.

[QuestforLife]

 

A visual representation of where my protocol currently stands. Refs to follow. 

 

You could also add an arrow from 'proliferation and repair' to 'telomere shortening', and from 'telomere shortening' to 'tgf-b' (and vice versa). But as a simplification I think it works okay as it stands.

Edited by QuestforLife, 14 February 2020 - 04:10 PM.

[QuestforLife]

It is increasingly apparent to me that there can be no lasting rejuvenation of the body without rejuvenation of the bone marrow. If a therapy was to return all the cells in circulation to youth, as measured by whatever biomarker, still the rejuvenation would be temporary - as the BM compartment stem cells are responsible for replenishing the rest of the tissues of the body. So we should specifically look for ways to keep the BM healthy, for example by stopping or reversing osteoporosis and fatty bone marrow (which undergoes the same deleterious changes as the thymus, albeit later). Interestingly, ROCK inhibitors (and phloretin specifically[1]) are known to prevent osteoporosis, and oxytocin has been shown to reverse bone aging, including bone marrow adipocity [2].

[1]Https://www.ncbi.nlm.nih.gov/m/pubmed/24932975/?i=17&from=phloretin%20in%20serum

[2]https://www.frontier...2015.00079/full

[QuestforLife]

An absolutely fascinating recent paper that finally fills in some of the missing pieces I've been seeking for a while. I've always known changing the structure and shape of cells influences their gene expression, including telomerase expression, and this is somehow mediated through the cytoskeleton. But this paper fills in some of the blanks. It turns out disturbing the cytoskeleton is like tweaking a spider's web. The extracellular mechanical changes are transmitted through the cell's interior by the cytoskeletal fibres to the lamina of the nucleus, and from there (I speculate) via the telomeres that attach to the lamina. This paper reports how this is accomplished via TERRA, the non coding RNA of the telomeres, which can be broken off and then communicate via an intermediate histone protein to influence gene transcription. Also very interesting is how the ECM can profoundly affect cell shape and hence differentiation, which then affects TERRA abundance. It explains how the matrix encourages differentiation, locking cells into a somatic rather than stem state, which makes them more sensitive, amongst other things to ROS, and on a related note - how cells on a rigid surface, say like a Petri dish, will tend to differentiate more. This probably explains why no telomerase activity is reported in adult stem cells by authorative sources like Mike West and Bill Andrews, whose data comes exclusively from cell culture experiments. And also how interventions that reduce or mitigate ROS on cells in a Petri dish always seem to work out well in a dish, but not in an actual body. I expect that TERRA activation is linked to the antioxidant response genetic element, but I'd need to do further research to prove this. If proven it would explain how antioxidants end up doing more harm than good (in general) if they interfer with this natural antioxidant mechanism.

Other ways to activate TERRA is via ROS (I assume this is a hormetic mechanism), and this is via PKA, which is also the target of cAMP, so forskolin would also do the job, which is probably part of why ROCK inhibitors and Forskolin are synergistic (in my protocol).

So maybe, and this is a big maybe, much of the aging we see in cells is extrinsic, I.e. communicated from the outside, and can be reversed by appropriate extracellular signalling. This is partly supported by the in vitro experiments with ROCK inhibitors, which turn somatic cells back into progenitors, and allow them to proliferate instead of senesce.

https://doi.org/10.1...mcr.2020.118643

Taken together, our results suggest that a delicate mechanotransduction mechanism may
influence TERRA expression, in which deviation from the basal cytoskeleton organization, either due
to disruption of microtubules (H2O2, colcemid), increased stability of microtubule fibers (paclitaxel) or
a generalized reorganization of the cells’ cytoarquitecture (hydrogel cultures, C2C12 myoblast
differentiation), results in differential TERRA expression.

Edited by QuestforLife, 16 February 2020 - 01:44 PM.

[QuestforLife]

 

A visual representation of where my protocol currently stands. 

 

 

 

Updated.

Attached Thumbnails

• 

[QuestforLife]

1cc OxyLuv subq + TGF-b inh + Rho Kinase inh in the morning dropped my Elite HRV (heart rate variability) from 75 to 49 an hour later, but it fully rebounded the next day. A week ago I repeated this 4 days in a row without noticing much.  Starting today I'm going to do it every other day for a week or so. (I'm trying every other day because Dr. Harold Katcher rejuvenated old rats with an injection given every other day for a total of 4 injections. His rats continued to rejuvenate for a full month, ending as young adults per a number of measurements. He has not revealed the content of the injections pending patent applications). It's been pointed out that a day in the life of a rat corresponds to about a month in the life of a human, so that's an argument for repeating every other month for about 7 months. Rejuvenation that took a month in rats might take 30 months in humans. On the other hand reprogramming cells in culture with Yamanaka factors takes about 20 days, during which their epigenetic age gradually reverses, slowly at first and then faster near the end.

 

Any updates, are you still trying this?

 

I did a week with x2 oxytocin doses in that time, then took a break. Will repeat.

Main observations are blood pressure reductions (some systolic but mainly diastolic) with ROCK-inhibitor and tgf-b inhibitor. A slow, cumulative reduction in HRV over the week but generally feeling great with good sleep and strong sex drive. 

Felt more drained the day after the oxytocin dose. But also recovered rapidly. 

I had some concerns with occasional fast heartbeats on the HRV monitor, but issue was pre-existing has subsided somewhat.   

[RWhigham]

Metformin is a Rho Kinase inhibitor like Statins.  REF: Metformin enhances nitric oxide production and diminishes Rho Kinase activity in Rats with Hyperlipidemia.   "After 4 weeks of medical intervention, CRP level and Rho kinase activity were profoundly diminished while NO production was significantly enhanced in the atorvastatin and metformin groups" The rats were given 50mg/kg per day of metformin. The "human equivalent dose" (divide by 6.2) would only be 8mg/kg.

 

Epigenetic age was reduced in the TRIiM thymus rejuvenation study by Metformin, HGH, and DHEA.  REF: 1st-age-reversal-results-is-it-hgh-or-something-else/

 

The life expectancy of diabetics on metformin is greater than that of non-diabetics.

REF: Study Shows Type 2 Diabetics on Metformin Can Live Longer Than People Without the Disease

 

It was suggested that those with lowest reported epigenetic age in the Longecity funded epigenetic age testing appear to be long term users of Statins (and perhaps metformin)

 

If "epigenetic age" turns out to be a valid indicator of biological age (to be determined), it might be advantageous to take a Rho Kinase inhibitor--either daily, or on some yet to be determined schedule.

 

Edited by RWhigham, 20 February 2020 - 01:44 AM.

[QuestforLife]

Metformin is a Rho Kinase inhibitor like Statins.  REF: Metformin enhances nitric oxide production and diminishes Rho Kinase activity in Rats with Hyperlipidemia.   "After 4 weeks of medical intervention, CRP level and Rho kinase activity were profoundly diminished while NO production was significantly enhanced in the atorvastatin and metformin groups" The rats were given 50mg/kg per day of metformin. The "human equivalent dose" (divide by 6.2) would only be 8mg/kg.

 

Epigenetic age was reduced in the TRIiM thymus rejuvenation study by Metformin, HGH, and DHEA.  REF: 1st-age-reversal-results-is-it-hgh-or-something-else/

 

The life expectancy of diabetics on metformin is greater than that of non-diabetics.

REF: Study Shows Type 2 Diabetics on Metformin Can Live Longer Than People Without the Disease

 

It was suggested that those with lowest reported epigenetic age in the Longecity funded epigenetic age testing appear to be long term users of Statins (and perhaps metformin)

 

If "epigenetic age" turns out to be a valid indicator of biological age (to be determined), it might be advantageous to take a Rho Kinase inhibitor--either daily, or on some yet to be determined schedule.

 

There is a difference between blocking the action of rho kinase directly, and diminishing its effects in a sick animal model. I am not sure that metformin is acting on rho kinase directly or indirectly. I suspect it is the latter. That doesn't mean it is not helpful partly through its effects on rho kinase, but at this stage I am reticent to add it to the list of compounds that clearly work directly on rho kinase, such as statins, phloretin and tongat ali. 

 

Given the TRIM study used growth hormone, I suspect the reversal in epigenetic age was more due to the increased action of stem cells, perhaps synergised with metformin.

 

I do agree with your broader point about many beneficial substances being rho kinase inhbitors and /or partial de-differentiators.  

Edited by QuestforLife, 20 February 2020 - 10:44 AM.

[QuestforLife]

Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues

 

https://www.jmmc-onl...1259-8/fulltext

 

An interesting paper on using ROCK inhibitors on an engineered connective tissue model. Shows ROCK inhibition blocks the effects of tgf-b on tissue contraction and stiffness.

These results demonstrate that ROCK inhibition with Fasudil or H1152P strongly interferes with the ability of cardiac fibroblasts to form compact and stiff tissues.

And…

We therefore generated rat engineered heart muscle (rEHM) which were cultured for the last 7 days in the presence of either 10 μM Fasudil or 3 μM H1152P. Similar to the ECT experiments, both ROCK inhibitors significantly increased the cross-sectional areas of rEHM. Next, we performed isometric force measurements and found that ROCK inhibition reduced the resting tension of the rEHM reflecting a decline in tissue stiffness. Interestingly, there was a trend towards an increase in twitch tension of the rEHM

This may explain why my protocol seems to be causing me some irregular heartbeats. I eliminated oxytocin as the cause – it causes short lived flushing, drop in heart rate variability and a rise in heart rate, but doesn’t cause the heart to jump about erratically.
More generally the use of 3D substrates in this study, rather than an overly stiff 2D petri dish echoes earlier comments I made about the inappropriateness of 2D cell cultures for studying the body, and also suggests ROCK inhibitors will have a stronger (or at least different) effect in the body that in a dish.

These results indicate that TGF-β signalling compensates for the effect of ROCK inhibition on tissue compaction, and that ROCK inhibition reduces the pro-fibrotic effect of TGF-β as demonstrated by the reduction in tissue stiffness.

This suggests that inhibiting tgf-b and ROCK together is worthwhile, and that a ROCK inhibitor in a high tgf-b environment might be less effective.

Fasudil displays a difference in its inhibitory potency between ROCK and PKA of only 10- to 20-fold and thus partial PKA inhibition is likely when Fasudil is used.

Also mentioned in the paper, Fausidil is a partial PKA inhibitor, meaning in too high a concentration it will inhibit cAMP – the opposite of what we want to do.  I have no information on whether phloretin would do the same, it seems unlikely at my dose and the boost to alertness I have got from forskolin, but it is something to bear in mind. ROCK inhibitors also appear to be ATP competitors, so side effects are almost guaranteed if you take too much.

In any case I intend to terminate my herbal protocol this week, give a final report on it, and do my blood tests. I am doing a DNAge methylation test as well as a standalone CRP test.

 

[dlewis1453]

Will you be doing a telomere length test as well? 

 

 

[QuestforLife]

Will you be doing a telomere length test as well? 

 

Unfortunately not.

 

Based on the cost of the Lifelength telomere test and the time to travel to the clinic for the blood drawer, I can only do one per year, and I last did it in November 2019.

 

Ideally aging biomarker tests would be cheap and convenient. 

 

In the interim we need to get more longecity members testing interventions, rather than relying on a small group of members.  

[RWhigham]

QuestforLife on Feb 25  "In any case I intend to terminate my herbal protocol this week"

Our first pass was to try a combination of known herbal supplements that are known to bind with the targets we’d identified.  We gave them to rats, and at first nothing seemed to be happening. But after two months (about 4 years in human terms) the rats showed signs of rejuvenation -- Harold Katcher  https://joshmitteldo...-fraction-cure/  [Ed: two months bolded for emphasis] Don't give up too soon

Edited by RWhigham, 06 March 2020 - 06:13 AM.

[QuestforLife]

Superior de-differentiation to pluripotent state achieved via a cocktail of GSK and MEK inhibitors. Phloretin is a GSK inhibitor (although fairly fatiguing after a few days dosing with this aim). Myricetin is a MEK inhibitor, typical poor bioavailability (~10% as with phloretin), but might be another worthwhile addition for those interested in this sort of thing.

https://www.nature.c...467-020-14764-5

Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1

https://www.ncbi.nlm...ubmed/17693661/

Edited by QuestforLife, 14 March 2020 - 12:05 PM.

[QuestforLife]

Apologies for the brain dump, but I needed to park some ideas here regarding the current direction of my research.

 

Following on from the interesting telomerase activation of gotu kola triterpene extracts (https://www.ncbi.nlm...les/PMC6755196/) , I have zeroed in on asiaticoside (and its metabolite asiatic acid) as an interesting compound, probably responsible for the benefits (not withstanding that madecassoside and madecassic acid may also contribute).
It appears that there is a link with tgf-b inhibition and smad7 upregulation.
https://pubmed.ncbi....id-fibroblasts/
https://www.ijbs.com/v09p1032.htm
Effects seem to translate in vivo
https://journals.plo...al.pone.0031350
and have been used in wound healing
https://www.hindawi....am/2012/848093/
Just from a tgf-b suppression point of view, this looks well worth adding to the protocol. But still I’d like to find what the link is to telomerase activation.
We already know tgf-b suppresses telomerase, as elucidated here
https://pubmed.ncbi....the-htert-gene/
It looks almost certain from the above work that Smad7 blocks Smad3 and hence tgf-b blockade of TERT. Therefore, anything that upregulates Smad7 should upregulate telomerase. Having said that, the below paper shows smad7 removal enhances stable expression of c-myc in cancer cells – it’s unclear how this relates to normal cells.
https://jcs.biologis...ntent/127/2/411
But the following paper takes the crown with a lovely demonstration of bone marrow insufficiency caused by depletion of smad7 and consequent rise in tgf-b triggered arrest of the hematopoietic progenitors. This was consequently reversed by… an Alk5 inhibitor! This work (2010) predates the Conboy’s work with rats.
https://cancerres.aa...0-2933.full.pdf
This confirms that tgf-b suppression and/or smad7 upregulation is vital to protect the bone marrow niche and that this is related to the TERT promoter.
It is perhaps unsurprising that Smad7 upregulates pluripotency
https://www.pnas.org...nt/114/38/10113
Is there a connection to ROCK inhibition via KLF4, perhaps?
https://www.ncbi.nlm...les/PMC5444978/

[Rorororo]

How does your stack look so far?  I would love to get an update!

[QuestforLife]

How does your stack look so far?  I would love to get an update!

 

This diagram on post 221 outlines some of the things I've tried.

 

The statin-sartan protocol was succesful in reducing my epigenetic age by a couple of years (see below).

 

Since then I've been looking for herbs that may be able to replace the statin and sartan. This has not to date been successful. Phloretin seemed like it could be a powerful ROCK inhibitor, but it also blocked glucose transporters (so made me very tired if I took to much). It may have also given me heart palpitations. Forskolin seemed to increase my mental acuity but I have some concerns related to mitochondrial turnover that I need to research further. Boswellia definitely has powerful anti-inflammatory properties. Oxytocin probably is one to explore more in the future. Unfortunately I've been side tracked by another issue. 

 

My most recent test, after about 3 cycles of epitalon (which reversed my telomere age by a couple of years) led to an increase of my epigenetic age by a couple of years.

 

March 2020: biological age 42 years, chronological age 41

April 2019: biological Age 38, chronological Age 40

 

The results are disappointing but not altogether surprising given the literature from Horvath and others supporting a role for telomerase in epigenetic age acceleration:
https://pubmed.ncbi....h.gov/29667924/

https://www.ncbi.nlm...les/PMC6224244/

https://www.nature.c...467-017-02697-5

And reports from Turnbuckle of something similar:
https://www.longecit...-30#entry871972

 

In any case I've clearly overdone it with the epitalon. I still think telomere shortening can kill you and is probably the limit on human maximum lifespan, but there are clearly limits  - probably to do with impeding natural somatic cell line replacement.

 

I have plans for a trial to reverse epigenetic aging with several substances and see whether this can counteract simultaneous telomerase activation, but this will await this years Longecity Biomarkers program.  

[dlewis1453]

Thanks for updating us on the results of your latest test. That's unfortunate that this latest set of results did not turn out how you hoped.

Do you think switching out the statin and sartan for the herbal extracts is to blame for this increase in epigenetic age? Or maybe too much telomere elongation with epitalon? Or a bit of both?

[MikeDC]

This diagram on post 221 outlines some of the things I've tried.

The statin-sartan protocol was succesful in reducing my epigenetic age by a couple of years (see below).

Since then I've been looking for herbs that may be able to replace the statin and sartan. This has not to date been successful. Phloretin seemed like it could be a powerful ROCK inhibitor, but it also blocked glucose transporters (so made me very tired if I took to much). It may have also given me heart palpitations. Forskolin seemed to increase my mental acuity but I have some concerns related to mitochondrial turnover that I need to research further. Boswellia definitely has powerful anti-inflammatory properties. Oxytocin probably is one to explore more in the future. Unfortunately I've been side tracked by another issue.

My most recent test, after about 3 cycles of epitalon (which reversed my telomere age by a couple of years) led to an increase of my epigenetic age by a couple of years.

March 2020: biological age 42 years, chronological age 41
April 2019: biological Age 38, chronological Age 40

The results are disappointing but not altogether surprising given the literature from Horvath and others supporting a role for telomerase in epigenetic age acceleration:
https://pubmed.ncbi....h.gov/29667924/
https://www.ncbi.nlm...les/PMC6224244/
https://www.nature.c...467-017-02697-5
And reports from Turnbuckle of something similar:
https://www.longecit...-30#entry871972

In any case I've clearly overdone it with the epitalon. I still think telomere shortening can kill you and is probably the limit on human maximum lifespan, but there are clearly limits - probably to do with impeding natural somatic cell line replacement.

I have plans for a trial to reverse epigenetic aging with several substances and see whether this can counteract simultaneous telomerase activation, but this will await this years Longecity Biomarkers program.

Your test results may just be noise. None of the herbs will elongate your telomere. A recent study showed a small molecule can elongate telomere length effectively. Wait for the human trial results. At your age telomere shortening is not a critical issue. NAD+ precursors such as NR/NMN is the way to go.

[QuestforLife]

Thanks for updating us on the results of your latest test. That's unfortunate that this latest set of results did not turn out how you hoped.

Do you think switching out the statin and sartan for the herbal extracts is to blame for this increase in epigenetic age? Or maybe too much telomere elongation with epitalon? Or a bit of both?

Yes to both. The epitalon definitely increases telomere length as per my LifeLength results in November, but it seems this might have been at the cost of additional aging as measured via methylation.

I will probably use alpha ketoglutarate (helps demethylases) for a while, maybe a statin-sartan cycle and recheck methylation in a few months.

[thenewwildone]

Actually epigenetic aging is irrelevant, it just measures the age of the cell, so an immortal cell through telomerase will be seen as older and so raise your epigenetic age without you actually becoming older.

Edited by thenewwildone, 25 May 2020 - 04:36 AM.

[QuestforLife]

Actually epigenetic aging is irrelevant, it just measures the age of the cell, so an immortal cell through telomerase will be seen as older and so raise your epigenetic age without you actually becoming older.

That is an interesting perspective, and one that I used to share - but am now less certain of. Yes it's certainly true in the Petri dish, but even there the cell will slowly gain slightly different gene expression even as it continues to replicate. In the human body a whole plethora of different cell types are expiring and being replaced from progenitor cells, who are in turn being replaced from stem cells in the bone marrow. This makes it harder to say with certainty what the net effect of telomere lengthening would be. No doubt if it affected all cells you would then have more replications left, but would also slow down replacement. This is what I think has happened in my case.

Now the methylation clock is another matter; it is not entirely clear how it relates to age related gene expression changes, though the fact demthylators are required to induce pluripotent suggests it is a causative rather than correlative link.

I still think reversing both the telomere and methylation clocks is possible, and that is what I'll be working on in 2020.

[Turnbuckle]

Actually epigenetic aging is irrelevant, it just measures the age of the cell, so an immortal cell through telomerase will be seen as older and so raise your epigenetic age without you actually becoming older.

 

What is ageing, anyway? It is the sum of billions of cellular dysfunctions. And when epimutations build up, cells make the wrong mix of proteins and become dysfunctional. Senescence due to telomere shortening also contributes to dysfunction, but as long as senescent cells are driven to apoptosis and are replaced, their contribution to ageing reaches a steady state. Thus epigenetic aging is the main contributor to overall aging, followed by the build up of senescent cells and the failure of mito QC. It is the essence of ageing.

 

To stop and even reverse ageing requires:

1. Healthy populations of stem cells,

2. Active apoptosis of senescent cells, and

3. An optimum rate of telomere shortening so that somatic cells do not live too far past their prime before replacement.

Edited by Turnbuckle, 25 May 2020 - 03:35 PM.

[kurt9]

What is ageing, anyway? It is the sum of billions of cellular dysfunctions. And when epimutations build up, cells make the wrong mix of proteins and become dysfunctional. Senescence due to telomere shortening also contributes to dysfunction, but as long as senescent cells are driven to apoptosis and are replaced, their contribution to ageing reaches a steady state. Thus epigenetic aging is the main contributor to overall aging. It is the essence of ageing.

 

I have to agree with this. This makes sense from a bio-engineering standpoint.

 

This is one reason (the other being that telomere shortening is a regulated process) why I've never bought into the idea that telomere shortening itself is a cause of aging and why I am not a fan of elongation efforts. Telomere shortening and apoptosis is a "house cleaning" mechanism.

Edited by kurt9, 25 May 2020 - 03:15 PM.

[Turnbuckle]

Telomere shortening and apoptosis is a "house cleaning" mechanism.

 

Precisely, and if one was assured of unlimited stem cell replacement, then telomere shortening would be the road to youth and vitality.