#21

Here’s a pretty good basic summary of how rapamycin works.

https://mmabrasil.localizer.co/t/former-whole-foods-execs-open-love-life-a-longevity-medical-club/15644/17?u=ng0rge

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#22

It’s interesting to know that Rapamycin cannot inhibit mTOR by itself but it must first bind a protein called FKBP12. The result is the so-called FRB complex (FKBP-Rapamycin Binding domain of mTOR). When part of this complex, mTOR is switched off.

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#23

FKBPs are involved in diverse cellular functions including protein folding, cellular signaling, apoptosis and transcription. They elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches.

2.1.1. FKBP12 as an Immunophilin

FKBP12 was originally identified as the target of FK506 and rapamycin [1]. Both drugs bind noncovalently to FKBP12 and inhibit its PPIase activity [3]. However, the inhibition of FKBP12 per se does not contribute to their immunosuppressive activity. Instead, the binding with FKBP12 allows the drugs to subsequently interact with the mechanistic targets of their action in immunosuppression. The FK506-FKBP12 complex specifically interacts with calcineurin (CaN), a Ca2±dependent serine-threonine phosphatase [11–14], whereas the rapamycin-FKBP12 complex targets mammalian target of rapamycin (mTOR) [15–17].

FK506-Binding Proteins and Their Diverse Functions - PMC (nih.gov)

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#24

We don’t fully understand. At best we have hints, but there’s always info coming out that seems hard to fit into the prevailing hypothesis. Here’s an example, where aging in different organs happens at different rates. It is the rapid turnover tissue that ages slower, while the slow turnover tissue is the one that ages faster. Example of rapid turnover, youthful tissues: intestines, skin - rapa slows cell division, yet seems to have good impact on skin and intestines. Meawhile the liver is a rapidly aging organ, because the cell turnover is slower - yet rapa slows cell division, which in this scenario should lead to senescence and error accumulation… made worse by rapa further slowing down cell turnover? How does all of this fit together?

The paper:

https://www.cell.com/cell/fulltext/S0092-8674(24)00963-2

A pop sci writeup:

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#25

@CronosTempi Interesting. I wonder if it’s a triage effect as the body has to prioritize the fast turnover cells. In a resource constrained situation, the slow turnover cells don’t get enough resources to age more slowly. The resource constraints could be in energy, minerals, vitamins, amino acids…

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#26

Possibly. But the whole growth vs antiproliferation axis is highly confusing. There are a number of very slowly, possibly not at all aging molluscs and crustacean species, lobsters and the like, surprisingly long lived for their size - and one thing that these animals do is they never stop growing, as they age, they just become bigger and bigger, and could go on for who knows how long (eventually they get eaten by predators, die accidentally etc.). You wonder - how… but think of it this way, you start aging/decline when you reach maturity, a developing baby/toddler has not yet reached maturity so it is not in aging decline. Now, what if you could prolong that state indefinitely, always growing, never reaching the “end state” from which a decline can start, so like that lobster, you just keep growing. A very different model of aging/antiaging than the mtor/rapa/CR pathway of “let’s hunker down, not grow or proliferate” - note how the IGF-1 inhibited specimens age slower, and in fact grow much smaller as seen in mice etc. (in humans the Laron dwarves) - so the opposite of the lobster, i.e. grow as little as possible and stop as soon as possible.

These two models are opposites, which tells us that there are different ways to skin the cat of aging. This, btw., we see over and over again - example are the slow aging because of slow metabolism, low core temperature, low heart rate, seen often in very long lived reptiles (which btw. often keep growing as they age, like crocks and giant turtles!), but at the other end you have the close relatives of reptiles, birds, which have extremely high metabolism and core body temperature and can be super long lived, especially for their size (parrot species that live longer than humans, being much smaller, or tiny hummingbirds living astonishing 8-10 years with a thermonuclear metabolism turnover, heartbeat and energy output of the sun!), high energy output birds in general don’t display overt signs of aging - what a contrast to the low energy output of the also slow aging reptiles, or mammals subject to CR, where energy output is diminished and conserved.

Bottom line, the proliferation/growth vs nonproliferation/non growth axis vs aging is very complicated.

6 Likes
#27

This is me exactly. The absence of illness and hay fever takes time to notice, but 2 years is enough time to convince me even with n=1.

4 Likes
#28

:rofl: :rofl: :rofl: :rofl: :rofl: :rofl: :rofl: :rofl: :rofl: Yes very simple. :rofl: :rofl: :rofl: :rofl: :rofl: :rofl: :rofl:

#29

The same is true for me. I user to get down once or twice a year with someyhing nasty.

After rapa, I either don’t catch anything (or have discernible symptoms) or have a very mild case, despite having 3 kids at school age.

Shitty imunossupressor…

1 Like
#30

Take it everyday and get back to us on that one.

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#31

What about this?

Is the liver resilient to the process of ageing?

https://www.sciencedirect.com/science/article/pii/S1665268124003636

The liver’s unique regenerative capacity, immunotolerant feature, and polyploidy status distinguish it as a metabolic organ unlike any other in the body. Despite aging, the liver generally exhibits fewer pathological abnormalities than other organs (such as the kidney), maintaining its functions near-normal balanced manner.

Notably, a recent report suggests that the liver is a youthful organ, with hepatocytes averaging an age of only 3 years.

Despite the liver’s impressive regenerative capabilities and cellular reserve, a lingering question persists: how does the liver maintain its youthful characteristic amidst the chronological aging of the entire organism?

#32

SLC13A5 - that I think is the answer.

#33

The SLC13A5 gene codes for a sodium dependent citrate transporter (NaCT) that brings citrate, a key substrate involved in energy production, into the cell.

High-affinity sodium/citrate cotransporter that mediates the entry of citrate into cells, which is a critical participant of biochemical pathways.
May function in various metabolic processes in which citrate has a critical role such as energy production (Krebs cycle), fatty acid synthesis, cholesterol synthesis, glycolysis, and gluconeogenesis.
Involved in the regulation of citrate levels in the brain (By similarity).

About SLC13A5, a gene responsible for Citrate Transport

Citrate is a small molecule that is found in many types of food and throughout a person’s body. It is an important part of how a cell makes energy. The protein that moves citrate from outside a cell into the inner part of the cell is called a citrate transporter.

In people with seizures and neurologic problems related to the functioning of the citrate transporter, there are changes in the amino acids that make up the transporter protein. The changes in the amino acids are likely to decrease the amount of citrate that is transported into the cell.

Reduced expression of this gene is associated with longer lifespan in many organisms, including some non-human primates. Increased expression is associated with type 2 diabetes and non-alcoholic fatty liver disease. A sugary diet upregulates the expression of the gene, and so does Interleukin 6 signaling.

SLC13A5 (solute carrier family 13, member 5) encodes sodium/citrate cotransporter, which mainly localizes in cellular plasma membranes in the frontal cortex, retina, and liver.

The transporter is widely expressed in neurons, localized in the plasma membrane of various cell types, including hepatocytes in the liver, spermatozoa in the testis, and mostly astrocytes and neurons in the brain (3). Citrate is vital in cellular metabolism and neurotransmitter biogenesis (3). It is known to have an important role in the tricarboxylic acid cycle, where the molecule represents the starting point for generating reducing equivalents nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (reduced form) (FADH2), which in turn enter the electron transport chain to generate ATP (4). The brain cannot produce citrate independently; hence, it depends on citrate uptake via NaCT. Thus, the carrier has a pivotal role in mediating the uptake of circulating citrate for metabolism (4), preferably in the trivalent form rather than the divalent form.

Relatedly, when citrate transport and metabolism are disrupted, intracellular citrate levels fall, resulting in neuronal energy failure, which is thought to be one of the explanations behind epileptic symptoms (3). In other words, a lack of cellular citrate results in energy deficiency in the brain, thus possibly contributing to the pathogenesis of epilepsy and delayed brain development.

Just a brief collection of explanatory info.

1 Like
#34

Yes, this is the problem, when using terms like “youthful organ” - it depends on what you measure. Like in that notorious paper that claimed brain antiaging/youthful profile of 6 years in monkeys based on a handful of markers. Well, all that says is what it says about those specific markers, it doesn’t say that about the whole organ (brain), as other markets may differ. If I administer a drug that turns gray hair back to its original color, have I made that person younger? Gray hair is one marker of aging, but if my drug does nothing for anything else, have I made an anti-aging drug? What if the intervention gives you a result as measured by a marker, but it means nothing, like my intervention is to simply color your hair at the hairdresser? Have I made you younger? Don’t laugh - a ton of drugs and interventions, even FDA approved, work on the marker but do nothing for the disease - like that notorious Alzheimer’s drug that was approved by the FDA despite widespread opposition from scientists, the drug focused on lowering a marker amyloid beta, while doing nothing tangible for AD, and having dangerous side effects to boot.

And that’s the other side of the coin, where I might reverse gray hair, but destroy your heart and shorten your life.

So showing a handful of markers and claiming 6 years age difference in the whole monkey brain, I find highly suspicious.

Back to youthful vs rapidly aging liver - by carefully picking your markers you could argue either case. One way to test would be to say “never mind if it looks/measures as young or old - how does it function, like a young output, or old output?” If as you age, the liver function deteriorates faster than other organs, I say it’s a rapidly aging organ. That would be my criterion. Hearing and vision is an example of rapidly aging organs - you could be firing on all cylinders, but your eyesight deteriorates much more than for example kidney function (comparatively, eyesight is worse compared to its peak sooner than the same for kidneys), or the case of hearing where you lose the very high frequencies already past teenage years, while your brain still keeps getting better until your mid twenties etc. So that’s MY way of judging youthfulness of organs, through function, but others may have different criteria for what a rapidly aging organ is. YMMV.

2 Likes
#35

I was joking about it OFC. But I am not sure a low daily dose would be detrimental.

#36

Came across this on liver age:

Liver cells age differently depending on where they are in the organ, study shows

The location of the liver cells has a strong influence on the aging process. In the region of the liver where the liver cells use oxygen for energy production in their mitochondria, this process deteriorates significantly with age. In the central, oxygen-deprived area of the liver, however, the researchers found no change in the mitochondria, but a change in the cells’ fat metabolism.

The liver is largely made up of a single type of cell, the hepatocyte. Depending on where they are in the liver, they have different roles. Near the portal vein, where fresh, oxygen-rich blood enters the liver, hepatocytes use the oxygen to process fats in their mitochondria and produce energy. In contrast, carbohydrates are broken down in the less oxygen-rich regions of the liver.

https://medicalxpress.com/news/2023-11-liver-cells-age-differently.html

In experiments using mice and liver tissue from humans, the researchers identified how the aging process prompts certain liver cells to die off. They were then able to reverse the process in the animals with an investigational drug.

The finding, which appears in the journal Nature Aging, holds high promise for the millions of people who have some degree of liver damage—livers that are essentially old due to the metabolic stresses of high cholesterol, obesity, diabetes or other factors.

Studying the livers of mice, the researchers identified a genetic signature distinct to old livers. Compared to young livers, the old organs had an abundance of genes that were activated to cause degeneration of hepatocytes, the main functioning cells of the liver.

“We found that aging promotes a type of programmed cell death in hepatocytes called ferroptosis, which is dependent on iron,” Diehl said. “Metabolic stressors amplify this death program, increasing liver damage.”

Armed with their genetic signature of old livers, the researchers analyzed human liver tissue and found that the livers of people diagnosed with obesity and MASLD carried the signature, and the worse their disease, the stronger the signal.

Again turning to mice, the researchers fed young and old mice diets that caused them to develop MASLD. They then gave half the animals a placebo drug and the other half a drug called Ferrostatin-1, which inhibits the cell death pathway.

Upon analysis after treatment, the livers of the animals given Ferrostatin-1 looked biologically like young, healthy livers—even in the old animals that were kept on the disease-inducing diet.

https://medicalxpress.com/news/2024-06-liver-stress-aging-reversible.html

2 Likes
#37

And a new study on longevity gene S6K1 and rapamycin. First how rapamycin and S6K are related:

Anti-ageing drug rapamycin improves immune function through endolysosomes

Understanding how rapamycin extend lifespan is important, as it helps to prevent unwanted side effects. “We know that rapamycin extends lifespan via two mechanisms: increased autophagy and decreased activity of a protein called S6K. It has been shown that mice with altered S6K live longer. But the mechanism by which S6K extends lifespan is unclear,” says Sebastian Grönke, co-author of the study.

Better immune function in old age thanks to endolysosomes

The researchers were able to show that an altered activity of S6K influences the endolysosomes. These break down material in the cells and play an important role in regulating various cellular processes, such as inflammatory reactions.

https://www.age.mpg.de/392233/240228_pm_rapamycin_endolysosomes?c=19391

An article on the new study:

Blocking the longevity gene S6K1 extends lifespan by reducing inflammation

S6K1 is a protein involved in the regulation of ageing and age-related diseases. Blocking this protein in mice makes them live longer and mimics the health benefits of reducing calorie intake, such as reduced body fat, stronger bones and resistance to diabetes, though the underlying mechanisms were not previously understood.

S6K1 is a key target of the mTOR signalling pathway, which regulates growth and metabolism in response to nutrients and stress. The pathway also influences cellular senescence. As we age, senescent cells accumulate and release of high levels of inflammatory proteins – a phenomenon known as the senescence-associated secretory phenotype (SASP).

Elucidating the relationship between S6K1, senescence and the SASP will advance our understanding of ageing and holds potential for treating age-related diseases.

This includes complementary work in fruit flies showing that the S6K protein controls inflammation, a study showing that simulating an excess of nutrients in mice increased inflammation and shortened lifespan, as well as work from Professor Stuart Cook, Head of the Cardiovascular Disease Mechanisms Group at the LMS showing that [inhibiting an inflammatory protein, IL-11, led to health benefits and increased lifespan](Scientists find “master switch” which could hold the key to longer, healthier lives.).

Now that the principles of S6K1 deletion have been established in liver tissue, the team plans to carry out follow-up studies to determine whether the same thing is happening in other tissues in the body.

https://lms.mrc.ac.uk/blocking-the-longevity-gene-s6k1-extends-lifespan-by-reducing-inflammation/

And the study:

Inhibition of S6 kinase 1 (S6K1) extends lifespan and improves healthspan in mice, but the underlying mechanisms are unclear. Cellular senescence is a stable growth arrest accompanied by an inflammatory senescence-associated secretory phenotype (SASP). Cellular senescence and SASP-mediated chronic inflammation contribute to age-related pathology, but the specific role of S6K1 has not been determined. Here we show that S6K1 deletion does not reduce senescence but ameliorates inflammation in aged mouse livers. Using human and mouse models of senescence, we demonstrate that reduced inflammation is a liver-intrinsic effect associated with S6K deletion.

The mammalian target of rapamycin (mTOR) pathway plays a key role in integrating hormone and nutrient signaling and stress responses with both cellular and organismal growth and metabolism1. Furthermore, mTOR signaling plays an evolutionarily conserved role in regulating longevity and healthspan2,3. For example, pharmacological inhibition of mTOR by rapamycin extends lifespan in yeast4, flies5 and mice6. A key effector of mTOR signaling is ribosomal protein S6 kinase 1 (S6K1), which plays several roles in regulating the translational machinery and controlling cellular energy levels and has feedback effects on insulin signaling7,8. S6K1 itself has been shown to regulate aging and different age-related processes9,10. Deletion of S6K1 (Rps6kb1) extends lifespan and healthspan in mice and also regulates longevity in flies and worms9,11.

Mice lacking S6K1 display beneficial metabolic effects, including reduced adipose mass, resistance to the consequences of high-fat diet feeding and increased insulin sensitivity12,13, a constellation of phenotypes that aligns with the effects of calorie restriction (CR), a conserved longevity mechanism14. Different molecular mechanisms have been proposed to explain these effects. For example, loss of S6K1 leads to upregulation of the activity of AMP kinase, a key regulator of cellular energy homeostasis15, thus mimicking the effects of CR and motivating the use of metformin as a potential geroprotective drug.

Interestingly, mTOR influences different phenotypes associated with senescence14. Inhibition of mTOR prevents senescence by interfering with the establishment of an irreversible growth arrest23,24. On the other hand, treatment of already senescent cells with rapamycin inhibits the inflammatory SASP.

Given our incomplete understanding of the mechanisms by which loss of S6K1 signaling benefits aging and age-related pathologies, we undertook a series of studies in long-lived S6K1 −/− mice and other genetic and pharmacological models of S6K inhibition, including liver-specific and myeloid-specific S6K knockout (KO) mice. We explored the role of senescence, the SASP and inflammation in the liver, as this organ displays several age-related changes (including an increased inflammatory profile30,31) and shows beneficial metabolic phenotypes in mice lacking S6K1 (refs. 32,33). In these studies, we found that loss of S6K1 attenuates age-related liver pathology and does not influence senescence but reduces liver inflammation via effects on the pro-inflammatory SASP and immune surveillance. Thus, S6K signaling plays a key role in age-related inflammation (inflammaging34), and targeting this pathway may be a strategy for treating the diseases of aging.

Inhibition of mTOR signaling, including the key effector S6K1, extends lifespan in an evolutionarily conserved manner and increases healthspan in mice9. Underlying mechanisms include beneficial long-term effects on glucose homeostasis, adipose tissue biology and effects upon key hormone and energy-sensing signaling pathways9,12,13,17. In the current study, we demonstrate that loss of S6K signaling in the liver has marked anti-inflammatory effects and attenuates various age-related hepatic liver pathologies. This blockade of S6K signaling appears to act, at least in part, via changes in age-related inflammation (inflammaging) rather than altering senescence per se, with concomitant beneficial effects on associated phenotypes, such as age-related fibrosis.

Increased accumulation of senescent cells is a recognized feature of aging in the mouse liver, although its precise origin and its pathophysiological impact remain to be determined31,35. Work by us and others has shown that mTOR inhibitors prevent the induction of the pro-inflammatory SASP while not affecting senescence growth arrest, an effect in part mediated by 4EBP25,26. However, these studies did not specifically investigate the role of S6Ks, even though the loss of S6K1 has beneficial effects on aging and related liver phenotypes9,32. Our current studies demonstrate in vivo, and using multiple cellular models, that depletion of S6K1 and S6K2 (singly or in combination) does not alter the accumulation of senescent cells or the senescence response per se but, rather, selectively affects their pro-inflammatory properties.

We show that loss of S6K signaling has marked effects on the SASP, with a profound reduction in a subset of inflammatory markers. In the liver of old S6K1 KO mice, several inflammatory cytokines showed reduced expression, which was associated with lowered immune cell infiltration. Aging in the liver is associated with low-grade inflammation, which may, in part, be related to calorie and macronutrient intake31,35. Accumulation of senescent cells in the liver was reported to promote hepatic fat accumulation and steatosis features of liver aging, and ablation of these cells ameliorates this phenotype35. Our findings suggest that the primary driver of the beneficial effects of the removal of senescent cells upon late-life metabolic dysfunction in the liver stems from the abrogation of the pro-inflammatory profile engendered by these cells.

It would also be of value to determine if the loss of S6K signaling similarly impacts inflammaging beyond the liver, as this would point toward a broader effect and strengthen the idea that chronic inflammation is a key mechanism underlying the lifespan and healthspan benefits seen in global S6K1 KO mice. Future studies should also address how inhibiting S6K1 signaling contributes to the effects that different interventions targeting mTORC1 (such as rapamycin treatment, protein restriction or branched-chain amino acid restriction) have on senescence and lifespan.

In summary, our findings show that loss of S6K signaling in aging models both in vivo and in vitro attenuates senescence-associated inflammatory processes and may play an important role in the beneficial effects of attenuation of mTOR signaling on detrimental phenotypes, particularly in the aging liver.

https://www.nature.com/articles/s43587-024-00695-z

5 Likes
#38

That’s a very nice paper, thank you for bringing it to our attention, ng0rge.

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#39

Thanks. Very interesting. Reading this made me wonder how I ever got bold enough to start taking rapamycin. I’m nowhere near signing up for a gene therapy. It’s a bit scary how little is well understood. I suppose the key motivating factor was the ticking clock…I felt a need to do more than the basic pillars of a healthy lifestyle.

2 Likes
#40

As an add-on to ng0rge’s posts, I’m going to paste the relevant wiki voice. S6K1 and p70S6K is the same thing.

Ribosomal protein S6 kinase beta-1 (S6K1 ), also known as p70S6 kinase (p70S6K , p70-S6K ), is an enzyme (specifically, a protein kinase) that in humans is encoded by the RPS6KB1 gene.[5][6] It is a serine/threonine kinase that acts downstream of PIP3 and phosphoinositide-dependent kinase-1 in the PI3 kinase pathway.[7] As the name suggests, its target substrate is the S6 ribosomal protein.[8] Phosphorylation of S6 induces protein synthesis at the ribosome.

Function

[edit]

This gene encodes a member of the S6K family of serine/threonine kinases, which phosphorylate several residues of the S6 ribosomal protein. The kinase activity of this protein leads to an increase in protein synthesis and cell proliferation. Amplification of the region of DNA encoding this gene and overexpression of this kinase are seen in some breast cancer cell lines. Alternate translational start sites have been described and alternate transcriptional splice variants have been observed but have not been thoroughly characterized.

mTOR

[edit]

The p70S6 kinase is a downstream target of mTOR (mammalian target of rapamycin) signaling, specifically mTORC1, an mTOR-containing complex characterized by the inclusion of Raptor rather than Rictor (mTORC2). mTOR can be activated via an AND-gate-like mechanism at the lysosome, integrating signals about growth factors and bioavailability of important molecules. For instance, amino acids such as arginine and leucine can trigger lysosomal recruitment of mTORC1. Once at the lysosome, mTOR can be activated by Rheb, a small, lysosomal-resident GTPase, in its GTP-bound state. Rheb GTPase activity is stimulated (and therefore capacity to activate mTOR diminished) by the upstream TSC complex, which is inhibited by IGF signalling. Thus, the AND gate consists of proper localization by sufficiency of amino acids and activation by growth factors. Once mTOR has been properly localized and activated, it can phosphorylate downstream targets such as p70S6K, 4EBP, and ULK1 which are important for regulating protein anabolic/catabolic balance.

Physical exercise activates protein synthesis via phosphorylation (activation) of p70S6K in a pathway that is dependent on mTOR, specifically mTORC1. This has been demonstrated by using an inhibitor of mTOR, rapamycin, to block an increase in muscle mass, despite increases in load (e.g., exercise). Exercise has been shown to increase levels of IGF-1 in muscle, thus inducing the IGF-1/PI3K/Akt/p70S6K signaling pathway, and thereby increasing the protein synthesis is required to build muscle.

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