I knew it was a good idea to invite you to join the party 
"NR oral supplements will be no different than taking straight niacinamide "
I read the paper twice to see if we could escape from that conclusion. There is no escaping from it. The stomach and intestine takes care of the following conversion:
NAD-->NMN-->NR-->NAM, bummer....
This raises a couple of questions:
- Why is there a NR-NMN-NAD pathway in mammals if no NR can get in?
- Why is there NR in cows milk?
- If no dietary source can deliver NR to the blood. How is it formed extracellularly?
To complicate matters more:
Qprt, Nampt, Naprt1, and Nrk1,2 are the committed enzymes in the synthesis of NAD+ from Trp, Nam, Na, and NR.
Different tissues express more of one type of enzyme than the other. Because of this, one tissue type uses Trp as the preferred or exclusive main precursor for NAD, the other NAM the other NA and finally NR.
As far as we know:
Nrk2 is present in heart, brain, and skeletal muscle, and is notably absent in kidney, liver, lung, pancreas, and placenta, giving the former tissues a preference for NR
Naprt1 is expressed in intestine, liver, kidney, and heart. In addition, human kidney cell lines are able to use Na to increase intracellular NAD+ concentration in a manner that depends on the NAPRT1 gene, giving these tissues a preference for NA
And so on…
So why so much NrK2 in so many cell types? Why do they prefer NR?
Note also that nasal delivery of NAD+ is neuroprotective. Is this because of conversion to NR?
http://www.futuremedicine.com/doi/pdf/10.2217/14796708.3.1.1?cookieSet=1
Also according to brenner:
"Nrk2 mRNA levels following axonopathy are induced approximately 20-fold, indicating a preferential use of NR as a precursor in maintaining intracellular NAD+ levels in DRGneurons"
The NR saga continues... Nasal delivery or sublingual could be a way out?
Those are great questions and points. Obviously, this issue is a lot more complex than it appeared at first glance...
I can answer the first question fairly easily. Most of those mechanisms are likely salvage pathways. Conversion of NAD into NR only requires the removal of ADP, which one can imagine can occur in a variety of ways, possibly even spontaneous hydrolysis by water?
This paper is one you've seriously, seriously gotta read, as it really looks over this whole issue in depth and addresses most of it. It really is an amazing paper for trying to figure all this out. Just to give everyone a taste of some of the important bits, we can see from a figure on human NAD metabolism from that paper:
NR is by far the most adventageous molecule as it goes into NMN and lacks the negative effects of nicotinic acid (NA). However, NA is still a better option than niacinamide, it seems! Here's a quote from that paper to show what I mean:
"Nicotinamide is a potent biochemical inhibitor and a biological regulator of sirtuins.36,57,58 The inhibitory constants for nicotinamide have been determined to be in the range of 30 to 200μM for a variety of different sirtuin enzymes.58,69,70 Physiological nicotinamide concentrations are not widely reported, but measurements from our lab, to be described in the next section, establish that intracellular nicotinamide concentrations are typically within this range for both yeast and mammalian cells, consistent with the proposal that nicotinamide is a relevant negative regulator of sirtuin biochemical function in cells."and:
"Although a nicotinamide derepression strategy has yet to be proven pharmacologically effective in activating human sirtuin biological functions, our recent measurements of nicotinamide concentrations in mammalian cells and tissues (typically in the 50-150 μM range) indicate that SIRT1 and other mammalian sirtuins are likely to be inhibited by intracellular nicotinamide. We are currently exploring the effectiveness of small molecules related to isonicotinamide in activating mammalian sirtuins in vitro and in vivo."
This means that niacinamide levels are already high enough to inhibit sirtuins in our cells, naturally, as it is right now. Now this is really starting to get deep.
Let's look at two more snipits, first about NAD+ levels and what they take to activate sirtuins:
"Perturbations of NAD+ metabolism alter sirtuin catalytic activity in yeast and in human cells and implicate NAD+ and related metabolites as regulators of genetic events in the cell nucleus. NAD+ is an abundant metabolite, and recent data indicate that concentrations of this metabolite are normally in the range of 400 to 700μM in human cells.61Km's for NAD+ of human and yeast sirtuins are in the range of 100 to 300μM.26,39 Given that the average cellular NAD+ concentration exceeds this
Km range, it is not clear that fluctuations in NAD+ concentrations would be expected to significantly affect intracellular sirtuin biochemical function. On the other hand, a large number of proteins bind NAD+ and NADH such that the unbound NAD+ concentration in cells could be significantly lower than the determined intracellular NAD+ concentrations."And then let's look at activators of sirtuins (resveratrol, quercetin, and potentially this thing called isonicatinamide):
"The activity identified for sirtuins in upregulating stress adaptation pathways and in reducing cell sensitivity to apoptosis has led to attempts to increase the catalytic rate of these enzymes in cells. Howitz et al73 identified a family of molecules from a library screen designed to detect compounds causing sirtuin activation. The screen identified a family of the well-known plant polyphenols, such as quercetin and the trans-stilbenoid resveratrol.73 These sirtuin-activating compounds (STACs) increase sirtuin biochemical functions by decreasing the apparent Km for the peptide substrate.73 These compounds have potent effects in decreasing apoptosis caused by ionizing radiation, trauma, or genotoxicity.29,73 The published biochemical activation effects of STACs appear to require the presence of a nonphysiologic fluorophore, complicating the understanding of the molecular mechanism by which these compounds provide sirtuin activation in cells.74
An alternative activation strategy pioneered by Sauve et al focuses on derepression of nicotinamide inhibition of sirtuins.59 Nicotinamide inhibition of sirtuins is caused by a chemical process, called base exchange, that occurs at the active site of sirtuins. This base exchange mechanism competes for an enzyme-bound intermediate called the peptidyl-imidate, which is formed from the acetylated substrate and NAD+. The imidate intermediate links the base exchange and the deacetylation reaction pathways.38,68 We found that nicotinamide exchange and deacetylation reaction mechanisms compete for this intermediate, leading to nicotinamide inhibition of deacetylation. We hypothesized that a small molecule that could bind to the nicotinamide binding pocket within the active site would cause antagonism of nicotinamide inhibition of deacetylase activity.59 Accordingly, a small-molecule isostere of nicotinamide called isonicotinamide (pyridine-4-carboxamide) that binds competitively with nicotinamide to inhibit base exchange was identified. This competitive effect does not inhibit deacetylation and causes antagonism of nicotinamide inhibition of Sir2p deacetylation catalysis. Consequently, isonicotinamide can reduce nicotinamide inhibition to increase the deacetylation reaction rate.59"This is very very interesting. Resveratrol and quercetin can obviously overcome niacinamide inhibition of sirtuins, and apparently this may occur by increasing the effectiveness of sirtuin binding (and thus catalysis) of its substrate, speeding up the reaction so that niacinamide does not have time to inhibit sirtuins. What this means is that resveratrol and quercetin win out against niacinamide when competing - they will stop niacinamide from inhibiting sirtuins whatever the level of niacinamide present. Therefore, if one took niacinamide and resveratrol/quercetin at the same time (especially resveratrol), the inhibitory effects of niacinamide could be completely abolished and NAD levels still boosted :D
Another interesting thing we find is that NAD levels are already way above the level needed to activate sirtuins. That is, niacinamide wins out against NAD to inhibit sirtuins. This is obvious from the mechanism by which niacinamide does the inhibition: competing with the intermediate from the start of the reaction to hault deactylation. Consequently, even at very high NAD levels, niacinamide will win out, as I had guessed, and stop NAD alone from activating sirtuin activity. Hence why resveratrol and quercetin are so amazing.
One last interesting point is this:
"Exposure to a genotoxin (methyl methane sulfonate [MMS] 0.01%) causes rapid loss of up to 80% of cellular NAD+ in mouse embryonic stem cells within 4 hours after treatment (A.A. Sauve and T. Yang, unpublished data, July 2005). Surprisingly, nicotinamide concentrations in cells do not increase as a result of NAD+ degradation but rather decrease along with NAD+ (A.A. Sauve and T. Yang, unpublished data, July 2005). This decline suggests that nicotinamide is either degraded to 1-methylnicotinamide and its downstream metabolites or converted to an intermediary metabolite such as nicotinamide mononucleotide. We are currently attempting to address these possibilities experimentally."
Why this is so interesting is that it supports that hypothesis I meantioned where NAD and niacinamide levels are at some equilibrium ratio with eachother (since niacinamide is the degredation product of NAD). Therefore, even though one can use large doses of niacinamide to increase the levels of NAD (since it's an equilibrium, this must happen as long as the enzymes are there), the levels of niacinamide will also be above basal levels until break down pathways restore
both NAD and niacinamide back to normal levels. So then, if this is so, taking niacinamide to boost NAD will allow sirtuins to remain inhibited.
A final note to make: caloric restriction (CR) activates the stress induced enzymes that convert niacinamide into other forms and NAD. These enzymes, when active, will break the equilibrium, greatly increasing NAD
and greatly decreasing the levels of niacinamide so that sirtuins can finally be uninhibited. This is one of the reasons why CR does what it does. If we could find a supplement that could activate these stress enzymes such as PBEF, it could be just as potent as CR, and if paired with resveratrol/quercetin's method of activation of sirtuins, could allow incredible benefits.
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Conclusions
It seems to me from all this that we have these set of conclusions:
1. NAD levels are naturally high enough (unless NAD is bound by so many proteins as to lower its free concentration extremely) to activate sirtuins. Also, sirtuin catalysis of NAD to deactylate targets
makes niacinamide as a byproduct. Thus the reaction is self limiting outside of any exogenous systems and molecules to modulate these pathways.
2. Niacinamide levels are naturally high enough to inhibit sirtuins, and this inhibition is potent, overriding NAD activation seemingly almost completely (always some very low basal rates of sirtuin activity I would bet, but not sure on).
3. Resveratrol/quercetin override niacinamide inhibition of sirtuins by speeding up the catalytic reaction with NAD such that niacinamide does not have time to inhibit the reaction (this mechanism is not fully known and still controversial, but seems to be how it is done). This also answers the issue with 1. above - in that, NAD levels are already high enough to activate sirtuins once resveratrol is added to free sirtuins from niacinamide activity since no extra niacinamide/NA/NMN/NR/NAD has to be added to cause the massive upregulation of sirtuin activity as seen by resveratrol administration. So yes, NAD levels are high enough in the cell already to activate sirtuins, it appears, but niacinamide is just that dang potent, and already high enough to do its job in crippling sirtuin activity. This also leads to the idea that taking resveratrol/quercetin at the same time as niacinamide would negate niacinamide's inhibition of sirtuins, activate sirtuins, and boost NAD levels considerably all at the same time - giving a person the best of all worlds at once.
4. Isonicotinamide may have the ability to directly block niacinamide binding to sirtuins and thus prevent inhibition by niacinamide. This should make isonicotinamide a potent activator of sirtuins (though not as much so as resveratrol which not only activates but speeds up sirtuin activity). Where one can get isonicotinamide and if it has other affects in the body, and if it can even enter the body without being metabolized and so forth I have no idea. But at least the idea is out there.
5. Any system that can change the equilibrium distribution of NAD and niacinamide towards NAD will also stop the inhibition of sirtuins and activate them. We see this with CR causing upregulation of PBEF, which may well be how CR activates sirtuins. It certainly makes sense to me.
6. NA and NR are the best forms to take to boost NAD while avoiding sirtuin inhibition, if that is one's goal, though NA has side effects so NR is by far the best system. This will only last for so long before NAD is broken down and the niacinamide equilibrium is reestablished, which will deactivate sirtuins. But the same is for resveratrol clearance eventually resulting in enhanced sirtuin activity ending. However, the question remains as to how much NR can enter the body orally, if its metabolized in the blood (only if niacinamide levels are low), and so forth. It seems the methods you listed at the end there, 100YearsToGo, may be the best ways to administer NR untill we know more.
I'm still trying to digest all the information myself, but we are now beginning to see a clearer (to me anyways) and testable picture as to how CR and resveratrol may activate sirtuins, and how this all relates to niacinamide's action to inhibit sirtuins. We also can see that boosting NAD levels without dropping niacinamide levels or otherwise stopping niacinamide induced sirtuin inhibition (by speeding up the reaction, or blocking niacinamide binding via another molecule) will most likely
not activate sirtuins, as the mode of niacinamide inhibition wins out, and equilibrium with niacinamide in the abscense of stress enzyme mediated scavenging pathways (PBEF) will cause niacinamide levels to rise according to some ratio determined by the NAD/niacinamide equilibrium constant (it's the same way as how niacinamide intake and increase leads to NAD increase).
Yeah.. so.. I'm going to go mull on this a lot more. Hopefully my information, findings, and ideas are helpful and right. The picture is now very interesting and clearer, but still theory and hypotheses. New discoveries could always change this picture radically, but so far this makes sense of all available evidence that I know of.
Edited by geddarkstorm, 14 December 2008 - 01:10 AM.
