I think it is difficult because of the timing issues and combinatory issues, but I understand the idea.
Citrate has actually been tried with fruit flies. It found a life extension, but it was not a subtle detailed experiment.
The balance between acetyl-CoA issues and mitochondrial efficiency will vary from species to species.
Joseph: Great if you can make interviews, and even get guidance on risks of cancelling-out effects between specific substances. I mentioned some I am/was taking, but you will have your own list.
This is awesome, very proactive approach!
You may have mentioned this before, but how much citrate supplementation is effective?
Can anyone get this?
Combinatorial interventions in aging
I think you will see effects at 4g per day. Currently I alternate between 20 and 40g per day taken in 5g quantities at Least 30 mins apart.
Move along, nothing to see here.
Olafurpall:
I would be very relieved if most roads do not lead to mTOR so I can continue taking newly purchased highest quality supps!
What I found on those substances made me think that their influence on mTOR could be significant. However, the links are mainly in vitro and I take it you mean that those can be disregarded, if you have found evidence of no or only limited in vivo effects that contradict those.
On a few I believe there is in vivo evidence of links (zinc, taurine, tadalafil and reishi). But perhaps the links are weak?.
It is very long but for FWIW I post the links I found between the substances I was (am) taking and mTOR (taurine I already posted on). Some such as Quercetin lit up like a Christmas tree when I searched, others e.g. CoQ10 I had to hunt before finding the links. Vitamin K2 was about the only substance that I didn´t find a link on.
CURCUMIN
As the first study shows, mTOR appears to be a key target of curcumin but there may be other central targets.
From Hitting the Golden TORget: Curcumin’s Effects on mTOR Signaling | Bentham Science
“mTOR has emerged as an exciting and novel molecular target for curcumin, particularly in cancer cell lines. It appears that curcumin inhibits both mTORC1 and mTORC2”
“These studies also suggest that curcumin possesses amazing molecular versatility as evidenced by the large number of proposed drug receptors for the compound, including plasma membrane-bound receptors, proteases, transporters, apoptotic factors, kinases, transcription factors, and adhesion molecules [2, 12]. While it remains a possibility that curcumin is in fact interacting with this seemingly large range of molecules, a more plausible explanation is that curcumin predominately targets only a few key cell regulators, and these actions spill over to affect many different pathways, factors, and processes within the cell. Identifying these key cellular regulators, however, remains challenging due to the seemingly diverse molecular promiscuity of the compound. Recently, the mammalian target of rapamycin (mTOR) has been identified as a novel molecular target of curcumin, which may in fact represent one of these central targets due to the fact that mTOR stands at the center of numerous key cellular processes (cell growth/proliferation, survival and motility), almost all of which are affected to some degree by curcumin”.
From other studies “Curcumin has been suggested to regulate several cellular signaling pathways including mTOR signaling and functions as a new class of mTOR inhibitor”
“ Curcumin is termed as a multifunctional targeting therapy drug that regulates the mTOR signaling pathway in the treatment of numerous diseases”.
QUERCETIN
Inhibition of mTOR signaling by quercetin in cancer treatment and prevention - PubMed (nih.gov)
CoQ10
Coenzyme Q10 Ameliorates Pancreatic Fibrosis via the ROS-Triggered mTOR Signaling Pathway - PubMed (nih.gov) “In primary PSCs, expression levels of p-PI3K, p-AKT, and p-mTOR were upregulated with CoQ10. A rescue experiment using specific inhibitors of the PI3K-AKT-mTOR pathway demonstrated that the PI3K-AKT-mTOR signaling pathway was the underlying mechanism by which CoQ10 ameliorated fibrosis”.
Coenzyme Q10 Inhibits the Aging of Mesenchymal Stem Cells Induced by D-Galactose through Akt/mTOR Signaling - PMC (nih.gov) “These data indicate that the Akt/mTOR signaling plays a critical role in MSCs senescence inhibited by CoQ10”. “The results suggested that CoQ10 could inhibit D-gal-activated Akt/mTOR signaling in MSCs”……” Therefore, we hypothesized that Akt/mTOR signaling inactivation might be the mechanism by which CoQ10 inhibited MSC aging induced by D-gal. Our result showed that the expression of phosphorylated Akt and mTOR could be deceased by CoQ10. Finally, after overexpression of constitutively active Akt (CA-Akt), the number of SA-β -gal–positive cells was increased and the level of p53, p21, and p16 was also elevated in the CoQ10 treatment group; these results hint that the Akt/mTOR signaling may be the main mediator of MSC aging regulated by CoQ10”.
ZINC
The mTOR target is primarily inhibited by the activation mechanism of AMP-activated protein kinase (AMPK) [1]. Yun et al. found that activation of phospho-mTOR inhibited autophagy, whereas the inhibition of phospho-mTOR induced autophagy [29]. Cao et al. found that activation of autophagy increased AMPK phosphorylation with a reduction in phosphorylation of the S6 kinase P70 subtype (P70S6K) [4].
It was shown that zinc significantly induced the level of Beclin1 and LC3B by activating adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway.
Moreover, recent studies have demonstrated that zinc is the regulation of autophagy.
MELATONIN
Melatonin significantly increased expression of p-AMPK and p-ULK1, and decreased mTOR expression. Treatment with compound C (an inhibitor of AMPK) or MHY1485 (an agonist of mTOR) ablated the observed benefits of melatonin treatment. Melatonin protects VSMCs against calcification by activating autophagy via the AMPK/mTOR/ULK1 pathway.
The last sentence shows a nice additive effect with rapa: “Our results show that melatonin activates autophagy in both wild-type (WT) and GCD2-homozygous (HO) corneal fibroblast cell lines via the mammalian target of rapamycin (mTOR)-dependent pathway. Melatonin treatment also led to increased levels of beclin 1, which is involved in autophagosome formation and maturation. Furthermore, melatonin significantly reduced the amounts of mutant- and WT-TGFBIp. Treatment with melatonin counteracted the autophagy-inhibitory effects of bafilomycin A1, a potent inhibitor of autophagic flux, demonstrating that melatonin enhances activation of autophagy and increases degradation of TGFBIp. Cotreatment with melatonin and rapamycin, an autophagy inducer, had an additive effect on mutant-TGFBIp clearance compared to treatment with either drug alone**”.**
TADALAFIL
“ Furthermore, TAD tadalafil and BG boosted hippocampal levels of cGMP, PKG, Wnt3a, and AMPK and reduced expression of β-catenin and mTOR by 74% and 51%, respectively. TAD and BG also halted neuroinflammation by reducing IL-23 and IL-27 levels, as well as protein expression of NF-κB by 62% & 61%, respectively. In conclusion, this study offers novel insights on the neuroprotective effects of TAD or BG in the management of SAD as evidenced by improved cognitive function and histological architecture. This could be attributed to modulation of the crosstalk among PI3K/Akt/GSK-3 β , PP2A, mTOR/autophagy, cGMP/PKG, and Wnt/ β -catenin signaling cascades and mitigation of neuroinflammation”.
The expression of p-mTOR was significantly decreased in mice with FGR on 13 days post coitum (d.p.c.) but recovered to the same level as that of the control on 17 d.p.c. following tadalafil treatment. The results were similar for 4E-binding protein 1 (4E-BP1) and S6 ribosomal (S6R) protein, which act downstream in the mTOR signaling pathway. We demonstrate that the tadalafil treatment of FGR in mice improved placental mTOR signaling to facilitate fetal growth.
CARNOSINE
Carnosine inhibits glioblastoma growth independent from PI3K/Akt/mTOR signaling - PubMed (nih.gov)
REISHI
An in vivo study on mice injected with inflammatory breast cancer (IBC) and treated with the commercial extract ReishiMax GLpTM (carpophore and cracked spores) highlighted a selective action on gene and protein expression, with smaller tumor size and weight and reduced expression of E-cadherin, mammalian target of rapamycin (mTOR),
VITAMIN D
“We have shown that 1,25(OH)2D is able to regulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway by stimulating expression of DNA damage-inducible transcript 4 (DDIT4), also known as regulated in development and DNA damage response 1 (REDD1), a potent suppressor of mTOR activity.2 Given the role of mTOR as a “master regulator” of cell function,3 it seems likely that DDIT4-mediated inhibition of this pathway will also play a pivotal role in mediating cellular responses to 1,25(OH)2D, as well as provide new strategies for its use in disease therapy”.
“Results demonstrated that the addition of Vitamin D to insulin and leucine significantly enhanced the activity of the mTOR pathway and protein synthesis. The authors conclude that Vitamin D has the potential to directly alter protein synthesis in muscle cells”
BETA GLUCAN
I only find glimpses of possible direct action from beta glucan on mTor:
mTOR/HIF1α-mediated aerobic glycolysis as metabolic basis for trained immunity - PMC (nih.gov) The gene expressing mTOR and the glycolytic genes that are targets of the transcription factor HIF1 α were also enhanced by β-glucan
But the indirect links appear clear, from reading both the following link extracts
β- glucan plays a significant role in regulating the PI3K/Akt biochemical pathway via IRS1, PI3K-p85, and phosphorylated Akt, which increases insulin sensitivity and reduces the associated risks.
“PI3K binds to the PH domain of AKT and induces conformational changes and the subsequent phosphorylation of AKT, and activated AKT moves from the cytoplasm to the cell membrane. This cascade directly or indirectly activates its downstream molecular proteins, such as NF-κB and mTOR21”.”
My view is that mTOR is important because it is a route towards autophagy/mitophagy which is a route towards improved mitochondrial efficiency.
COQ10 and Vitamin K2 (menaquninone-7) are both routes towards improved mitochondrial efficiency whether or not they have any effect on mTOR. (Melatonin helps as well).
I think HDAC inhibitors have a separate important role that includes: Quercetin, Curcumin, etc
Vitamin D is a route toward the VDR being active and that has an important cellular function.
Thanks! This will make for interesting reading.
Yes I disregard all in vitro studies unless the concentration used in vivo can reasonably be reached in vivo after consumption by humans, which is almost never the case. You mentioned curcumin and quercetin. They are prime examples. They are poorly absorbed and heavily metabolized. If someone shows me a study where curcumin or quercetin inhibit mTOR at some tiny nanomolar concentration and that such concentrations are seen in the blood after ingestion, then we’re talking. But this is highly unlikely.
Whenever I see something having a particular effect in vitro, I take note of the concentration and then look at what concentrations are seen in the blood of humans after ingestion of that compound and the latter is almost always far too low to expect the effect to be seen in vivo.
The same is true for other pathways besides mTOR. If you look up supplements that may influence some pathway, e.g. IGF-1, AMPK or what not, you will find tons of studies on various supplements being connected to the pathway in some in vitro conditions, but at least 95% of them won’t have any significant effect on that pathway in vivo at reasonably ingested doses. That’s why my deffault assumption is always that in vitro doesn’t translate to in vivo. Exceptions are likely seen when the concentration tested in vitro was exceptionally low, like low nanomolar, or when the compound is highly bioavailable after ingestion.
Olafurpall:
Your point that the concentrations tested in vitro are almost never reached after consumption by humans seems most relevant and I am gladly open to changing my view.
You write: “That’s why my default assumption is always that in vitro doesn’t translate to in vivo. Exceptions are likely seen when the concentration tested in vitro was exceptionally low, like low nanomolar, or when the compound is highly bioavailable after ingestion”.
Let me take a dive into the example of curcumin. The lowest in vitro concentration I found on curcumin affecting mTOR is 2,5 micromolar, i.e. 2500 nanomolar, much higher than the level you indicate should be a concern.
But what about a curcumin formulation developed for high bioavailability, showing inhibition of mTOR signalling in vivo, with the study highlighting the mTOR inhibition effect? Is there no or little risk of (negative) interaction with e.g. rapamycin? Potential therapeutic effect of curcumin, a natural mTOR inhibitor, in tuberous sclerosis complex - ScienceDirect From the study:
“Recent studies suggest that curcumin inhibit mTOR activity in vitro , which prompts us to investigate curcumin function as a new class of mTOR inhibitor suitable for tuberous sclerosis complex (TSC) treatment”.
“Solid lipid curcumin particle (SLCP), a novel curcumin formulation, was used to treat TSC related manifestations in Tsc2 knockout mice”.
“First, we evaluated the effect of curcumin on AMPK and mTOR pathways in vivo . Eight week-old WT male mice were i.p. injected once daily for 7 days with the vehicle or 300 μg of solubilized solid lipid curcumin particle (Longvida®)”.
Recognition memory deficit began as early as 4 weeks of age in both male and female Tsc2+/− mice. Oral administration with SLCP activates AMPK activity and inhibits mTOR activity in the brain tissue of Tsc2+/− mice, and can rescue the electrophysiological abnormality and object recognition memory loss in the mice”.
Our results suggest that SLCP could be an effective treatment for TSC patients”.
Or take the formulation I have, NovaSol A1 Vitality, with 185 times the bioavailability of unformulated curcumin based on blood measurements according to Consumer Lab and based on an independent analysis of bioavailability studies (which were mostly done by the manufacturers). https://pubmed.ncbi.nlm.nih.gov/30006023/).
The reasons for NovaSol´s superiority are given in Increasing Post‐Digestive Solubility of Curcumin Is the Most Successful Strategy to Improve its Oral Bioavailability: A Randomized Cross‐Over Trial in Healthy Adults and In Vitro Bioaccessibility Experiments (wiley.com) But drawing conclusions from the effects of molecular concentrations written here on mTOR interaction with e.g. rapamycin Is beyond my capacity.
I study the effects of HDAC inhibitors (such as curcumin) rather than mTOR inhibitors (other than Rapamyin).
Most of the HDAC inhibitors popularly used for longevity (of which there are quite a few) have IC50 levels in the tens of micro molar.
The inhibitors used often for their anti cancer effects run at the nano molar level, but in fact the HDAC inhibitor most known for its epigenetic effects is 10-hydroxy-2-decenoic acid or 10-HDA found in Royal Jelly and it acts to create the Queen Bee phenotype (not in isolation, but it is a key part of the process).
What I have found with the micromolar ones that if you take a combination in sufficiently high quantity you can get a dose dependent result.
If it were to be that the only thing that matters is mTOR then the same approach would be possible.
This is an interesting paper discussing studies of combinations of interventions on worms, flies and yeasts resulting in compounded increases (and decreases) in lifespan. I assume all the positive studies are listed but most of the negative studies never got published. The big effects seem to be combinations using gene alterations. Other combinations using cancer drugs and antibiotics are interesting.
The more I think about taking additional serious chemicals the more I am convinced that I need more time in health to allow me to wait to benefit from anti-aging interventions that get past studies on larger mammals (vs worms, flies, mice). The key is to do the basics right and not injure myself in the meantime.
The basics:
Sleep (& circadian rhythm)
Diet (calories, gut health, protein, nutrients, timing, fasting)
Exercise (strength, cardio, mobility, balance)
Stress (social, nature, fun, dogs, family)
Other (hydration, sunshine, purpose, accidents)
What am I missing?
As you probably know at the core of this I think it is an issue about reducing senescence (hence having enough acetyl-coa in the nucleus, but you can exogenously increase acetyl-CoA levels) and having efficient mitochondria (which has an impact on acetyl-CoA levels, but also affects the ATP response to the ATP/ADP gradient).
At a cellular level micronutrients are also important for cellular function.
I’m beginning to come around to thinking mitochondria are the key. Sufficient energy to run (and maintain) the body properly without excessive energy production related wastes products. Perhaps you’ve said this before. I’ve been hearing it in multiple places. I continue to think re-establishing the normal cycle of growth / replacement of proteins vs scavenging of old proteins is important. I don’t understand how these are connected but perhaps you do. Rapamycin plus exercise plus not doing stupid shit. Plus what?
Caloric restriction?
A key part of cellular activity is producing proteins. This takes a lot of energy. It has two key stages, the transcription of DNA producing mRNA and then the Ribosome takes this and produces proteins.
At the transcription stage it can produce splicing variants if there is not enough acetyl-CoA or some other impact on RNA Pol II.
At the translation stage if a protein production goes wrong it goes to recycling (RQC). I think this relates at times to a shortage of ATP causing a delay in protein produciton.
New mitochondria are efficient and respond to the ATP/ADP gradient by producing more ATP, but as they get harmed they get less efficient and don’t respond.
Hence efficient mitochondria are needed to make sure that proteins are produced properly.
CR is a route to autophagy.
Thanks. Good explanation.