#1

Summary

Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase ι and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated β-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.

Full paper (PDF) here: https://www.cell.com/cell-reports/pdf/S2211-1247(24)00066-4.pdf

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#2

I find it strange that the above paper did not get more attention here. Or is it noice? Collagen comes to my mind, as a rich source of glycine but also a rich source of proline.

Highlights:

• Senescent MSCs exhibit reduced PYCR1 and PYCR2 expression

• Proline induces Parkin-mediated mitophagy leading to improved mitochondrial function

• Proline ameliorates several aging hallmarks, including SA-β-Gal and DNA damage

• Proline improves myogenic differentiation capacity of senescent MSCs

" It is worth noting that although proline treatment for 7 days activated mitophagy and basal autophagy, it was only longer proline treatment for 14 days that resulted in improved mitochondrial function and reversal of aging hallmarks"

“Previously, we reported that senescent stem cells rewired their metabolism to use glutamine as their carbon source to maintain their cellular bioenergetics. Excessive glutamine catabolism led to accumulation of intracellular urea, resulting in impaired mitochondrial function, DNA damage, and increased expression of inflammatory cytokines. Inhibition of glutaminolysis led to decreased urea level in aged cells and in the heart and skin tissues of progeria mice. This was accompanied by partially restored mitochondrial function and amelioration of aging hallmarks. Interestingly, in this study, we observe that supplementation of an alternative amino acid, proline, reduced intracellular urea accumulation and led to decreased DNA damage as well as decreased expression of inflammatory cytokines, suggesting that more than one pathway may be playing a role in proline-mediated improvement of cellular bioenergetics and mitigation of senescence hallmarks”

Proline restores mitochondrial function and reverses aging hallmarks in senescent cells: Cell Reports

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#3

Yes. Very interesting

“ Our investigations have revealed that the use of amino acids, glutamine, and methionine by senescent cells exacerbated cellular senescence, while inhibiting glutamine or methionine catabolism reversed aging hallmarks in the heart, skin, and muscle and increased muscle strength in mice. Similarly, dietary restriction of isoleucine improved metabolic health and even extended lifespan in mice. In contrast, here, we report that proline supplementation significantly improved mitochondrial function and mitigated various aging-related senescence hallmarks.”

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#4

Thomas Seyfried has hypothesized that the use of glutamine by mitochondria is a major factor in cancer. Now we see a link to aging as well. Would using DON while taking supplemental proline be even more effective?

#5

I don’t know how these findings will hold up when it comes to human physiology, but evidence/indications build up that food sources and supplements that are low in methionine and isoleucine and at the same time high in Glycine and proline are useful to incorporate in the diet.

I already use Collagen,

Specific Amino Acid Content (per 100 grams)

While exact values can vary based on the source and processing methods, here is a general overview:

Amino Acid Collagen (approx.) Gelatin (approx.)
Glycine 28-30% 25-30%
Proline 12-17% 10-15%
Hydroxyproline 10-15% 10-15%
Alanine 7-10% 8-10%
Arginine 8-9% 8-9%
Aspartic Acid 6-7% 5-7%
Glutamic Acid 10-12% 9-11%
Isoleucine Low (approx. 1%) Low (approx. 1%)
Methionine Low (approx. 0.5%) Low (approx. 0.5%)
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#6

A fun comparison:

Rapamycin is from a soil bacterium.
Rapamycin blocks the response to leucine lowering mtorc1. Rapamycin has pro longevity and anti cancer properties.
DON is from a streptomyces bacterium. DON blocks the response to glutamine. DON has anti cancer properties. Pro longevity too?

We often lump macronutrients together. Fat is fat, carbs are carbs, and protein is protein. Of course the various fats saturated, omega 3, mono unsaturated, etc. affect the body differently. Protein is not so simple either. Each of the amino acids has a different effect.

A periodic mitochondria renewal protocol might include proline and taurine supplements, Rapamycin, and DON. It needs to be periodic since we do need leucine and glutamine for normal growth processes some of the time.

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#7

I can’t remember the source, perhaps it was Valter Longo, but I had heard methionine was not a positive for longevity. That is why I felt just having straight pea protein as my protein powder seemed like a fine idea (high in leucine but not enough methionine, which is why, if I recall, many powders mix pea with rice protein)

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#8

Yes, Isoleucine restriction and methionine restriction are prolongevity. Pea protein, soy protein and collagen are good protein sources. Some propose that Glycin can reduce the negative effects of metionine.

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#9
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#10

Hi @Ray1 :wave:

Do you know how I could source DON (6-diazo-5-oxo-l-norleucine)?

Thank you :blush:

#11

No idea. It has been around since the 1950s

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#12

Another radical propodal to reverse mitochondrial dysfunction, this time targeting epigenetic mutations in mitochondrial DNA.

A way to determine if this ailment occurs is proposed, but that may be superfluous given that it is commonly associated with aging.

The condition is claimed to be cured with a single dose of 100 mg PQQ plus 1 g of AKG, AAKG or any AKG salt (CaAKG is said to be less effective in this protocol).

Could anybody weigh in on this proposal? I assume it would at best be a very long-shot chance, but the prize is high.

#13

Found that on a post in a long thread at longecity that spans 77 pages. Took me two weekends to read through all pages.

That protocol was developed by a self experimenter posting as “Turnbuckle”.

The two posts above are the latest two iterations of a protocol that evolved since the first post - April 2017.

I’m doing the penultimate iteration (my first link above). Alternate days of fission and fusion supplementing, as follows:

  • Fission/biogenesis/demethylase
    Niacinamide/20 mg PQQ/1 g AKG

  • Fusion/biogenesis/demethylase
    DHM/20 mg PQQ/1 g AKG

Have done only three cycles of fission/fusion. So far, no noticeable changes. Haven’t done the barbell curl test.

The second link is the latest iteration. Am still researching if 100 mg PQQ is safe.

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#14

Apologies if this has already been posted. This paper shows the impact of various amino acids on the lifespan of C. elegans. Proline has the best lifespan extension. I’m not sure how to convert the concentrations to something comparable for humans, and whether results on C. elegans are in any way a strong indicator of what to expect in people. If methionine extends lifespan in C. elegans, it likely means these results can’t be carried over to mammals.

image

Artigo completo: https://pmc.ncbi.nlm.nih.gov/articles/PMC4328591/

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#15

The no observed effect level (NOAEL) for PQQ has been determined to be 400 mg/kg bw/day in a sub chronic toxicity study in rats. By applying a safety margin of 100, it can be concluded that doses up to 4 mg/kg BW/day or 240 mg/person/day would be safe in adult humans weighing 60 kg.

Do you think the second, shorter protocol of 100 mg PQQ + 1 mg AKG is superior?

#16

From Pyrroloquinoline-Quinone Is More Than an Antioxidant: A Vitamin-like Accessory Factor Important in Health and Disease Prevention - PMC

#17

The author has been revising his protocol through the years, trying to improve it, based on his experience, and feedback of the followers at longecity. So I believe the latest version is the best.

I am sticking to the penultimate version because I like the idea of cycling between fission and fusion.

It seems the only two advantages of the new version are speed and less need for repetition.

I previously posted a method of alternating fission/fusion (plus PQQ and AKG) to eliminate statin damage to mitochondria, however, it now appears that this can be done far faster, in as little as one treatment. Such epigenetic damage can be substantially removed in two hours or less by combining PQQ and AKG as shown in ellipse A in the plot below. It is only required that sufficient PQQ be used — i.e., 100mg instead of the 20mg used previously. Fission and fusion can then be dispensed with.

The author refers to the introduction of lactate. I do not know how to get that.

The 100mg PQQ of the PQQ/AAKG dose in A was sufficient to promote biogenesis of substantially all mtDNA, and it was done three times, presumably producing a far higher mtDNA count and component density in the matrix. The maximum ATP proxy output did not change however, until lactate was introduced at day 71, then the ATP output increased dramatically in a stepwise or ratcheting fashion, as shown in B in the plot below.

The new version also uses AAKG. I am wary of AAKG, because of the few reported instances of heart palpitations. With AKG, none have been reported, and I have not experienced any.

https://examine.com/supplements/alpha-ketoglutarate/#what-are-alpha-ketoglutarates-main-drawbacks

Adverse effects, including heart palpitations and dizziness, have been reported by those using AAKG, which is a combination of arginine with AKG. This combination is commonly used by athletes.[16][4]

Correction.

In the comments on linkedin, the author was asked about AAKG. He said “You can use AAKG, AKG, or any AKG salt. (AKG and some salts can be hard on the stomach. I haven’t found any problem with AAKG.”

Read the comments (on the right) at linkedin (my second link). The author answered a few queries from followers.

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#18

FWIW, the excerpt below from AI appears to refute Dischler’s assertion that most damage to mitochondria is caused by methylation / epigenetic modifications.

But never mind, getting rid of one type of damage is good on its own. 100 mg of PQQ one time is low-risk but I would like to check the merits of the case more perhaps there is more in the link you sent.

I happen to have CaAKG on hand but Dischler questioned its efficacy.

“Most mitochondrial damage during aging is not primarily caused by epigenetic changes, such as methylation of mitochondrial DNA (mtDNA). While mtDNA methylation and other epigenetic modifications do occur with age and can contribute to mitochondrial dysfunction, the predominant causes of damage are reactive oxygen species (ROS)-induced oxidative stress, mutations in mtDNA, and impaired mitochondrial quality. Epigenetic changes, such as increased methylation, can exacerbate mitochondrial dysfunction by altering gene expression related to energy production and biogenesis.However, they represent one of several contributors to mitochondrial aging”

(Mitochondrial epigenetics in aging and cardiovascular diseases - PMC)[
(Frontiers | Mitochondrial epigenetics in aging and cardiovascular diseases).

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#19

It is interesting that at the 10 mM level some aminos that were in the top 3 turned into a negative, and serine jumped up 9 places to the lead.

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#20

Did the promotion of biogenesis of almost all mtDNA from the 100 mg PQQ happen in humans, perhaps in Dischler himself, as opposed to a cell study in the lab? It´s not quite clear to me.

What I can find is the inner membrane latching study with accopanying dialogue. I understand there is a lot of prior research.