While reading up on the UCP2 protein uncoupler an intuitive counter-argument surfaced against Turnbuckle's theorised role of c60 molecules acting as UCP2 pore blockers triggering the activation of stem cells. The criticism, though, is not mathematically robust and could be blunted through the scale of c60 present.
The position as I understand it, is that c60 blocks the pores of UCP2 and so the prevents the emission of photons whose emission regulate ROS production through negative feedback mechanism.
The c6O is then redundant, presumably, as the protein is taken out of the game and the embedded c60 molecules with it. The C60 though is a finite exogenous resource, UCP2 an unlimited endogenous supply - so levels of UCP2 could be ramped up in spite of the c60 to keep ROS at bay. And UCP2 production is extremely dynamic with a half life of 30 minutes (compared to 30 hours of UCP1, say) - so it can and does adjust rapidly to changes in ROS levels.
Of course it may well be that c60 is ubiquitous long enough for ROS to reach critical levels to wake up the stem cell but for how long before it returns to quiescence? And of course it seems that less with c60 according to some accounts might be more, and regardless certainly powerful at low levels, which would certainly seem to be a problem with this model.
C60 would seem more uniquely defined by its antioxidant property than its size, and in its role as an antioxidant, reducing the concentration of the molecule wouldn’t seem to matter when it can be used indefinitely, (limited by its half life presumably) - one c60 molecule could do the job of 10 or 100 in the antioxidant model, albeit more slowly, not so in the UCP2 pore model, presumably.
More to the point there is plenty of evidence that demonstrate C60 can act as an SOD2 (Mn-SOD) mimic, and of SOD2's role in regulating stem cell activity and indeed UCP2 molecules, though controlling superoxide levels.
There is plenty of literature documenting C60 as an SOD2 mimic perhaps, one rather powerful demonstration, with a c60 derivative, where life is extended in the short-lived SOD2 knockout mice by 300%.
A natural pursuit would seem to be look for evidence in the much more studied SOD2 interactions in order to speculate how SOD2 upregulation might account for c60oo's role in ramping up stem cell differentation.
While to date, I haven't over recent days found a study showing upregulation of SOD2 to increase stem cell differentation, there is clear evidence of SOD2's involvement in stem cell regulation:
“Manganese superoxide dismutase (MnSOD) is a nuclear-encoded and mitochondria-matrix-localized oxidation-reduction (redox) enzyme that regulates cellular redox homeostasis. Cellular redox processes are known to regulate proliferative and quiescent growth states. Therefore, MnSOD and mitochondria-generated reactive oxygen species (ROS) are believed to be critical regulators of quiescent cells' entry into the cell cycle and exit from the proliferative cycle back to the quiescent state.”
“In general, loss of MnSOD activity results in aberrant proliferation.”
Though, this would seem to suggest the opposite effect, supercharging SOD2 (MnSOD) would likely encourage stem cell quiescence over differentation, however, nevertheless there is clear regulation of stem cell differentation through SOD2 activity and so too with it, we should expect, c60oo to an exert an augmented influence, acting as a ramped up expression of SOD2, some influence of stem cell activity with c60oo.
One possibility could be that c60oo downregulates endogenous SOD2 production and maybe there is a window where ‘aberrant proliferation’ occurs as SOD2 production upregulates. That would presumably depend on the depletion rate of c60 from the mitochondrial membranes and the upregulating rate of SOD2.
There are several mechanisms which regulate stem cell activity (citation) other than UCP2 and SOD2, which will muddy the waters further.
Also, on neural stem cell quiescence,
“We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life.”
Again, we have the opposite, where c60 as an SOD2 mimic would seem likely to maintain quiescence, rather disrupt it. Note too, that fission signals quiescence. This wasn’t if I recall something mentioned in the protocols by TB, though it may have been, and its opposite fusion - if I recall - was associated with increasing the stem cell store, rather than too for differentiation into progenitor cells (though this may have been implcitly expressed).
Fusion would seem to encourage both the increase in stem cell pools through increased symmetric division but also they are likely woken up too, if the above logic is consistent in reverse, by fusion - not necessarily with c60oo, though c60oo might just amplify the effect, by waking up those stem cells in deeper sleep, through the method Turnbuckle has described and others, experienced with considerable success.
This would be expected during fasting, where fusion is expressed, and more symmetric division amongst stem cells, an increased stem cell pool and the subsequent utilisation of those stem cells as the body enters 'repair mode'. As an aside, interestingly, Longo has mentioned the refeeding phase to be where much of the benefit of fasting can be found, observing, for example, that white blood cells plummet during autophagy, as they are metabolised in an energy scarce environment, however, on refeeding, presumably a fission expressed state, white blood cells increase above the pre-fast baseline.
But again a clear indication that SOD2 as a mitochondrial is antioxidant strongly involved regulating stem cell quiescence and so too potentially as a theorised powerful SOD2 mimic, c60.
There appear to be many regulators of stem cell quiescence, one of them UCP2, regulates ROS. As previously mentioned, UCP2 has a half-life of 30 minutes and is over expressed during stem cell quiescence and disappears during differentiation (at least in neuronal cells). It has been suggested in other cells there is likely interplay (link required) between SOD2 and UCP2 and that the concentration of UCP2 in quiescent stem cells needs to be fluid to regulate ROS and so facilitate the waking and sleeping of stem cells. Again, it could be conjectured that as a dramatically overexpressed form of SOD2, c60 might lead to the rapid down regulation of UCP2. On the one hand ROS in the presence of c60 would decrease rapidly (though H202 would increase as a byproduct of the superoxide reaction) presumably locking down the quiescent state, but UCP2 levels would rapidly diminish to a state associated with differentiated stem cells. How is the differentiation signalled: is it UCP2 levels or ROS?
In this paper superoxide in the matrix activates UCP2 and low concentrations of membrane targeted antioxidants abolished uncoupling.
If c60 were involved in suppressing UCP2 concentration in mitochondria, then this might provide an explanation for some of the other effects of c60. UCP2, for example, is upregulated in cancer cells and is considered to be a possible therapeutic target - Wistar rats, iirc, were cancer free and the recent c60oo study with Thiomacenamide suggests cancer protective effects at lower levels. However, "UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia". (the high dose c60oo group in Thiomacenamide had raised levels and was consistent with another study).
One of the rather puzzling anecdotes which unsurprisingly was not too often reported was the consumption of c60 while inebriated. Sensei, who previously noted the increased alcohol tolerance and hangover prevention effects of c60oo, reported the opposite hangover effect when taking c60 after consuming a significant though not unfamiliar volume of alcohol. I recall once applying some c60 to sore, broken skin with alcohol in the system and was surprisingly heavy headed the next day.
“In UCP2 overexpressing mice, sensitivity to ethanol was decreased compared to that of wild-type animals, while UCP2 knockouts had increased ethanol sensitivity. In addition, UCP2 expression was inversely correlated with the impairment of pain and temperature sensation induced by ethanol. Taken together, these results indicate that UCP2, a mitochondrial uncoupling protein previously associated with peripheral energy expenditure, is involved in the mediation of acute ethanol exposure on the central nervous system. Enhancement of UCP2 activation after acute alcohol consumption might decrease the time of recovery from intoxication, whereas UCP2 inhibition might decrease the tolerance to ethanol.”
This might provide some explanation to the apparent effect of introducing such a powerful SOD2 mimic if it rapidly shuts down UCP2 production under intoxication.
If true, this creates something of a confusing picture, if c60 is active in the mitochondria as an SOD2 mimic increasing tolerance to alcohol through faster metabolism and removal of ROS, while improving hangover, then why isn’t also inhibiting UCP2 at the time of intoxication? If UCP2 is there to inhibit runaway ROS, then why the problem with wiping it out when introducing such a powerful SOD2 mimic that should do the job as better?
So pre-treating with c60 lessens the hangover and alchohol tolerance, while treating c60 in ebrio, appears to substantially worsen the hangvoer. Perhaps, there is a different UCP2 pathway responding to ethanol, expressable when c60oo is present for some time, but which is disrupted when c60 is dramatically introduced - disrupting a vital role for UCP2 in the alcohol ethanol state. Perhaps over a period of several hours as some c60-ROS homeostasis develops within the cell, with the introduction of ethanol a greater SOD2 reponse is required, which isn't perhaps easily compensated by c60 since it cannot be upregulated, though can be deployed indefinitely. And it is much slower in reducing superoxide, 100 times in fact, than SOD2.
C60 gives the impression of being a rapid responder, perhaps because of the scale of antixoidant cover it provides, as in this study on nano particle restoration of blood flow within minutes in the case of brain injury. The endogenous antioxidants may be rapid, but are quickly exhausted. So perhaps SOD2 and UCP2 are upregulated during ethanol exposure with c60 present but wiped out on its introduction.
A role for UCP2 which is distinct from uncoupling:
"This effect of UCP2 occurred in the absence of stimulation of its uncoupling activity. Therefore, although the exact mechanism (transport activity of UCP2) remains to be determined, this report represents a landmark in understanding the importance of UCP2 for cellular metabolism."
If C60 were suppressing UCP2, then it might have some desired and undesired responses and inhibiting non-ROS regulating roles.
High levels of UCP2 has been linked with NLFD, with one individual on longecity curing NFLD on the c60oo stem cell protocol.
"In summary, it is an intriguing possibility toconsider UCP2, a negative regulator of ROS production, toact as a modulator of apoptosis and a factor in the adaptation of cancer cells to oxidative stress. Furtherstudies are needed to establish the role of UCP2 in cancerin general, and in NAFLD-related HCC formation inparticular."
Baati's rats were notable in addition to life-extension, in both the lack of tumours and the reported preserved liver condition. So it could be worth viewing the surprising and consistent anecdotal effects of c60oo as possible correlators of low UCP2 levels and perhaps explore UCP2 low level/knockout studies as possible risk factors.
Also, intriguingly from a paper on stem cell aging:
“In addition, ectopic induction of SIRT3 expression improves the function of aged HSCs by enhancing the antioxidant activity of SOD2. Together, these studies suggest that aging of stem cells may be reversible by modulation of their metabolic and redox state, which in turn can influence intracellular accumulation of ROS.”
where, referencing the following paper:
“The more surprising finding of our study is that upregulation of SIRT3 rescues functional defects of aged HSCs, providing direct evidence that physiological stem cell aging can be an acute casualty of high levels of oxidative stress and that oxidative stress-induced physiological stem cell aging and tissue degeneration are reversible. Although we do not rule out the possibility that chronic oxidative damage to cellular components contributes to the functional decline of aged stem cells, our data suggest that ROS-initiated signaling events are the likely regulators of physiological stem cell aging, providing the basis for reducing oxidative stress to rejuvenate aged stem cells and improve tissue regeneration.”
And so the ramping up SOD2 expression, via SIRT3, could both prevent rescue damage to stem cells which offers another possible route for c60 to improve stem cell function, bypassing sirtuin expression, and likely a far more powerful actor than an upregulated SOD2.
So C60's role as a powerful SOD2 mimic could see it as an a regulator of stem cell differentation and quiescence, as a potential rate limiter of UCP2 production, and too stem cell rejuvenation and repair.
Apologies, there a couple of references missing, of which I read but cannot retrieve, and maybe useful circumstantial omitted evidence which would need to be "refound" - and there will be conceptual errors but it is no more than a discussion starting point.
