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The NNT Mutation: A Fly in the NAD Precursor Ointment

nnt mutation c57bl/6j nicotinamide riboside

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#31 able

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Posted 17 October 2017 - 09:14 PM

 

[Michael,] you mentioned long term studies on NR - is there some 12 months or longer like the NMN study?

 
Not quite 12 months, but there are certainly long-term studies: notably, the Zhang et al late-life NR survival study  (PMID: 27127236; see analysis here) lasted nearly 10 months. Also, the Cantó/Auwerx NR vs. diet-induced obesity study (PMID: 22682224), which was the first study to really show promise for NR proper (as opposed to all the borrowed press from Sinclair's and Imai's earlier NMN work), did last 3 mo, which ain't peanuts; so did the later Trammell/Brenner NR diabetic neuropathy study (PMID 27230286).

 

 

 

Thanks Michael,

 

I don't know if I'm missing something, or you linked to wrong paper or what.

 

I see different experiments mention in the Zhang study you linked, but were a 6 week, and a 10 week intervention:

 

"Mdx mice received a dietary supplement with NR for 10 weeks "

"C57BL/6J mice following 6 weeks of chow or NR diets "



#32 Michael

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Posted 17 October 2017 - 10:33 PM

 

 

[Michael,] you mentioned long term studies on NR - is there some 12 months or longer like the NMN study?

 
Not quite 12 months, but there are certainly long-term studies: notably, the Zhang et al late-life NR survival study  (PMID: 27127236; see analysis here) lasted nearly 10 months.  [...]

 

 
Thanks Michael,
 
I don't know if I'm missing something, or you linked to wrong paper or what.
 
I see different experiments mention in the Zhang study you linked, but were a 6 week, and a 10 week intervention:
 
"Mdx mice received a dietary supplement with NR for 10 weeks "
"C57BL/6J mice following 6 weeks of chow or NR diets "

 


The survival study is briefly described in the main body of the paper, so I can see how you'd miss it if you're just skimming or using the "find" function; they briefly say "NR treatment of C57BL/6J mice slightly increased lifespan [...] with the NR treatment commencing late in life at 24 months." In the Supplementary Info, they add:
 

Sixty 24-months-old C57BL/6JRj mice were randomly divided into two groups.  [...] fed with a control chow diet with or without NR (400 mg/kg/day) supplementation. Mice were observed daily to record their survival – i.e. when they were found dead or were determined to be moribund by an experienced veterinary physician.

 
... so they don't specify a predetermined length. But you can see from the survival and mortality hazard curves (Fig. 6G and H) that the last NR mice were indeed on-therapy for >300 d. Of course, the mice who died earlier than that were not ;)  .

 

(And, in case anyone's wondering: yes, the C57BL/6JRj substrain has the NNT mutation).


Edited by Michael, 17 October 2017 - 10:35 PM.


#33 able

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Posted 18 October 2017 - 07:27 PM

Thanks again Michael, that certainly was easy to miss.

 

It is good to know that long term there were no negative side effects, and a slight increase in lifespan, even though started very late in life.

 

But what I was really wondering about is, the long term benefits on metabolic parameters such as were demonstrated for 12 months on nmn.  Insulin, glucose, etc.  Looks like all they did was check for end of life. Guess I'll study the 2-3 month studies for now.

 

Just trying to see if they both have similar benefits, one is better than the other for certain conditions, or if I might try  both together.


Edited by able, 18 October 2017 - 07:28 PM.


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#34 hav

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Posted 11 April 2018 - 07:46 PM

TL/DR: Nearly all the in vivo work on nicotinamide riboside and nicotinamide mononucleotide has been done using C57BL/6J mice, a substrain of the widely-used C57BL/6 strain that carries the C57BL/6J NNT mutation. This fact may mean that most or all of the very promising rodent data we've seen — particularly on outcomes involving metabolic health, oxidative stress, or exercise performance — are actually showing only that these nutrients help to compensate for the deleterious effects of this mutation: animals that don't carry such a mutation — including most other rodents and (importantly) humans — will not get similar benefits, because there's no defect there to compensate.

Thanks for writing this up. A question for anyone: Why are all the scientists working with NR ignoring this if an important factor? I suspect because it isn't.

 

I have the impression that current thinking is that it's something that might justify further study for certainty (it always is) but not a red flag.  Because there are studies that show no significant insulin secretion difference between b6/j and wild-type mice.

 

The deletion variant of nicotinamide nucleotide transhydrogenase (Nnt) does not affect insulin secretion or glucose tolerance. (2010)
 

The presence of a truncated Nnt did not affect insulin secretion or glucose tolerance on the C57BL/6 background. We suggest that low or normal levels of NNT (regardless of truncation) have little effect on insulin secretion. Rather, it is the increase in expression of Nnt that regulates and enhances insulin secretion. Our data confirm that B6J is a reasonable control strain for diabetes research; this is especially important considering that it is the strain commonly used to generate genetically modified animals.

That study, however, only compared animals fed a standard diet. 

All animals were fed a standard rodent control chow diet (74% carbohydrate, 20% protein, and 3% fat by weight).

 

Animals on a high fat diet do seem to show higher glucose levels and weight gain with the nnt deficiency.  Should not be a show stopper, however, if we know both types have a response in the same direction to diet, one merely being larger than the other... I suppose the advantage being that b6/j diet study mice might show larger and more significant responses to diet for statistical purposes.  But again, best to verify the same pertains to treatment.
 

Diet-induced obesity in two C57BL/6 substrains with intact or mutant nicotinamide nucleotide transhydrogenase (Nnt) gene. (2010)
 

Potential contributions of the wildtype Nnt gene were demonstrable on a lower fat diet (10% of calories) where a significantly greater weight gain over time by B6/NJ males was correlated with a significantly higher serum insulin. In conclusion, DIO [diet induced obesity] developed in response to 60% fat feeding regardless of Nnt allele status. Contribution of the B6/J-unique Nnt mutation was most evident in response to 10% fat feeding that resulted in reduced serum insulin and weight gain compared to B6/NJ males.

 

Howard

 


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#35 LawrenceW

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Posted 11 April 2018 - 11:56 PM

Hey guys, how did we get from:

 

The mouse "RDA" for B3 is 15 mg/kg chow, and the Dietary Reference Intake RDA for B3 in humans is 16 mg/day, so this dose-response study(3) would suggest that optimal NR intake for humans is a mere 32 mg daily, and brings into further question whether there is any real advantage for NR over other forms of the vitamin.

 

in post #1 of this thread to post #16?: 

 

- below 30 no obvious effects to use NR

- low dose NR for people 30- 40 for example 250mg. The audience would be health focussed individuals that believe they can ward off aging

- medium dose NR for people over 40 for example 250-500mg. With an objective of maintaining health and youthfullness

- high dose NR for people struggling with the aging process for example 750 -1000mg. With an objective to remove some aging complaints

- Extreme dose NR to tackle specific conditions for example 2-3g per day. This would be done under doctors supervision. Likely such dose has minor negative impacts but those are outweighed by the healing capability for the condition at hand


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#36 Michael

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Posted 13 April 2018 - 06:05 PM

TL/DR: Nearly all the in vivo work on nicotinamide riboside and nicotinamide mononucleotide has been done using C57BL/6J mice, a substrain of the widely-used C57BL/6 strain that carries the C57BL/6J NNT mutation. This fact may mean that most or all of the very promising rodent data we've seen — particularly on outcomes involving metabolic health, oxidative stress, or exercise performance — are actually showing only that these nutrients help to compensate for the deleterious effects of this mutation: animals that don't carry such a mutation — including most other rodents and (importantly) humans — will not get similar benefits, because there's no defect there to compensate.

Thanks for writing this up. A question for anyone: Why are all the scientists working with NR ignoring this if an important factor? I suspect because it isn't.

I have the impression that current thinking is that it's something that might justify further study for certainty (it always is) but not a red flag.  Because there are studies that show no significant insulin secretion difference between b6/j and wild-type mice.
 
The deletion variant of nicotinamide nucleotide transhydrogenase (Nnt) does not affect insulin secretion or glucose tolerance. (2010)
 
That study, however, only compared animals fed a standard diet. 
 
Animals on a high fat diet do seem to show higher glucose levels and weight gain with the nnt deficiency.  Should not be a show stopper, however, if we know both types have a response in the same direction to diet, one merely being larger than the other... I suppose the advantage being that b6/j diet study mice might show larger and more significant responses to diet for statistical purposes.  But again, best to verify the same pertains to treatment.
 
Diet-induced obesity in two C57BL/6 substrains with intact or mutant nicotinamide nucleotide transhydrogenase (Nnt) gene. (2010)
 
 
Howard

 
That's true, but I'm not sure why you think that's relevant to the specific question at hand, which is not about insulin resistance or obesity per se, but about the core molecular biology of the NNT mutation — transfer of redox-equivalents between NADH in the cytosol and NADP(+) in the mitochondrial matrix, with ensuing effects on mitochondiral function and redox tone, including cytosolic and mitochondrial NAD+:NADH and NADP:NADPH — and whether this implies that the effects of NR and NMN (and presumably NAM, where it phenocopies the two) on mice bearing this mutation (which have been used in nearly all the NAD+ precursor rresearch) are in whole or in large part artifacts of their genetics.
 
It's also worth noting that a lot of the research on NR and NMN  is exactly in HFD-induced diabetes and metabolic syndrome.
 

Hey guys, how did we get from:
 
The mouse "RDA" for B3 is 15 mg/kg chow, and the Dietary Reference Intake RDA for B3 in humans is 16 mg/day, so this dose-response study(3) would suggest that optimal NR intake for humans is a mere 32 mg daily, and brings into further question whether there is any real advantage for NR over other forms of the vitamin.
 
in post #1 of this thread to post #16?: 
 
- below 30 no obvious effects to use NR
- low dose NR for people 30- 40 for example 250mg. The audience would be health focussed individuals that believe they can ward off aging
- medium dose NR for people over 40 for example 250-500mg. With an objective of maintaining health and youthfullness
- high dose NR for people struggling with the aging process for example 750 -1000mg. With an objective to remove some aging complaints
- Extreme dose NR to tackle specific conditions for example 2-3g per day. This would be done under doctors supervision. Likely such dose has minor negative impacts but those are outweighed by the healing capability for the condition at hand


Well, you'll see if you read the intervening posts ;) . The 32 mg/day comes from extrapolation from PMID: 28211258, one of the few studies done in mice that (like humans) do not have the NNT mutation, which found that NR doses higher than this showed trends toward worse metabolic outcomes, rather than better as seen in studies with NNT-mutation-bearing mice. Again, we similarly saw worse exercise performance in rats given NR (PMID: 27489522) — and again, rats — like humans — do not bear the NNT mutation.
 
Stefan then suggested that an alternative explanation for these two studies showing suggestions of deleterious effects of "moderate megadose" NR is that both of them were carried out in quite young animals. Young animals still have, er, youthful levels of NAD+, so maybe the dysregulation was not what happens when you push NR into a system with a functioning NNT gene, but what happens when you push high levels of NAD+ precursors into an animal that already has youthful NAD+ levels. This got some important support from Able, who pointed out that the long-term middle-age NMN mouse study (PMID: 28068222) used C57Bl6N mice (not 6J), which do not have the NNT mutation. (As I noted here, however, that even in that study, some outcomes seemed to come out better at "merely" 100 mg/kg than at 300 mg/kg, and reasonably suggests that different tissues might respond differently to a given dose).
 
IF that's correct, then the issue is at what age to begin supra-RDA NAD+ precursor supplementation in order to avoid, at least, making things worse — and at what dose. Presumably, you'd start at a lower dose initially, and build up as you age and your unsupplemented NAD+ levels began to fall. From that premise, Stefan threw out the numbers you quote (which he clarified were purely hypothetical).

 

To get a grip on all of this, we need more studies in a wider age range, with dose-ranging, and above all in animals free of the NNT mutation.


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#37 hav

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Posted 13 April 2018 - 09:30 PM

Thanks for writing this up. A question for anyone: Why are all the scientists working with NR ignoring this if an important factor? I suspect because it isn't.

That's true, but I'm not sure why you think that's relevant to the specific question at hand, which is not about insulin resistance or obesity per se, but about the core molecular biology of the NNT mutation — transfer of redox-equivalents between NADH in the cytosol and NADP(+) in the mitochondrial matrix, with ensuing effects on mitochondiral function and redox tone, including cytosolic and mitochondrial NAD+:NADH and NADP:NADPH — and whether this implies that the effects of NR and NMN (and presumably NAM, where it phenocopies the two) on mice bearing this mutation (which have been used in nearly all the NAD+ precursor rresearch) are in whole or in large part artifacts of their genetics.
 
It's also worth noting that a lot of the research on NR and NMN  is exactly in HFD-induced diabetes and metabolic syndrome.
 


Well, you'll see if you read the intervening posts ;) . The 32 mg/day comes from extrapolation from PMID: 28211258, one of the few studies done in mice that (like humans) do not have the NNT mutation, which found that NR doses higher than this showed trends toward worse metabolic outcomes, rather than better as seen in studies with NNT-mutation-bearing mice. Again, we similarly saw worse exercise performance in rats given NR (PMID: 27489522) — and again, rats — like humans — do not bear the NNT mutation.

 

I was speaking to bluemoon's question of why they use NNT deficient mice in glucose and NR/NMN studies. I thought you found the studies I expounded upon relevant because both of those studies were cited as relevant in the 6-things list you posted... they were #5 and number #6.  And they seem relevant to mouse glucose measurements generally and provide a good basis of comparability to glucose mouse studies with and without NR.

 

I thought you already settled the question in your own mind of possible NNT mutation explaining observed glucose impacts from NR and NMN on glucose levels by reason of the PMID: 28211258 study.  I'm not sure it stands for demonstration of worse metabolic outcomes from NR supplementation.  It seems more reflective of a possible ceiling on observed effects as dosage is increased.  I have issues with that study's dosing methodology so I'm not even sure that conclusion is necessarily supported.  And observe it seems to be refuted for humans by the Elysium study showing a clear dosage dependent response.  At most, in my mind, it proves that there is some observed effect even at low doses with sub-optimal administration.

 

I think the (PMID: 27489522) rat study does pose a strong question as to whether NR might be positive or negative to muscle health of humans in it's showing of the reduced swimming endurance of rats.  Wow, reads like they swam them just short of drowning. The human clinical that did the sit-to-stand reps seems to rebut the negative inference on muscle health... someone earlier calculated it to be roughly a 7% improvement. That study did note no significant changes in human aerobic performance however, which might be closer to what was measured with the rats.  The only specific human tests I saw that had lower NR results were the heel rise and rate of torque development tests, with all others being equal or marginally better. Fwiw, I judge these tests more reflective of muscle health than a simple work out till you drop metric. Still, definitely something to think about, particularly for those that do high endurance activities.

 

I hesitate to bring up red blood cells again, but... <helmet-on> maybe if NR is entering red blood cells it's somewhat of a double-edged sword.  Improving a1c and cell life at the expense of high endurance oxygen transfer capacity.  If it binds to the hemoglobin protein, it may be displacing oxygen in doing so. <helmet-off>.

 

Howard


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#38 bluemoon

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Posted 14 April 2018 - 02:12 PM

The human clinical that did the sit-to-stand reps seems to rebut the negative inference on muscle health... someone earlier calculated it to be roughly a 7% improvement. That study did note no significant changes in human aerobic performance however, which might be closer to what was measured with the rats.  

 

The Elysium results showed at 500 mg healthy people walked 8% further for 6 minutes. Why wouldn't that be an aerobic improvement?



#39 hav

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Posted 14 April 2018 - 03:24 PM

The Elysium results showed at 500 mg healthy people walked 8% further for 6 minutes. Why wouldn't that be an aerobic improvement?

 

I don't think they intended to do aerobic testing and only cited mobility in their report.  I've taken aerobic tests myself and it was on a treadmill while hooked up to bp and heart rate monitors with technicians watching the readings and me like a hawk.  For both state calibration and my safety.  As I recall they got me going at a pace that gave them the readings they wanted then slowly started ramping up the incline to see how much it took till I showed signs of exhaustion, mainly to test my ability to tolerate it.  I survived without incident or alarm. :) The Colorado study seemed to do that kind of thing with more markers being monitored.  I'm guessing the Elysium folks didn't want to go to all that trouble or risk any subjects collapsing at the far end of the course.
 

Howard


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#40 bluemoon

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Posted 14 April 2018 - 04:26 PM

I don't think they intended to do aerobic testing and only cited mobility in their report.  

Howard

 

Actually, both are in there and around 7.6% and 7.8% increases for a 6 min walk and the chair balance. 


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#41 Michael

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Posted 14 April 2018 - 07:28 PM

I thought you already settled the question in your own mind of possible NNT mutation explaining observed glucose impacts from NR and NMN on glucose levels by reason of the PMID: 28211258 study.


I'm surprised you would think that: I think I've been pretty clear (in my original post and even more so in followups with Stefan and Able) that I haven't come to any clear conclusions on these questions.
 

I'm not sure it stands for demonstration of worse metabolic outcomes from NR supplementation.  It seems more reflective of a possible ceiling on observed effects as dosage is increased.

 
Sure: I don't think I've ever suggested that NR/NMN is "unsafe at any dose" ;) . As I said in my opening post, if the PMID: 28211258 study is to be taken at face extrapolative value, it would suggest that the optimum dose is somewhere around twice the RDA — way lower than is being used in the rest of the rodent studies, or than the company is recommending, or than anyone is taking AFAIK, and more suggestive of  avoidance of deficiency, with the suggestion that we may need a slight upward bump to the RDA at an upcoming NAM Food and Nutrition Board meeting, rather than a pharmacological effect at megadose — and also more likely to suggest that any NAD+ precursor will do. (Cf. this study of RDAish NMN vs. NAM).
 

I think the (PMID: 27489522) rat study does pose a strong question as to whether NR might be positive or negative to muscle health of humans in it's showing of the reduced swimming endurance of rats.  Wow, reads like they swam them just short of drowning. The human clinical that did the sit-to-stand reps seems to rebut the negative inference on muscle health... someone earlier calculated it to be roughly a 7% improvement. That study did note no significant changes in human aerobic performance however, which might be closer to what was measured with the rats.  The only specific human tests I saw that had lower NR results were the heel rise and rate of torque development tests, with all others being equal or marginally better. Fwiw, I judge these tests more reflective of muscle health than a simple work out till you drop metric. Still, definitely something to think about, particularly for those that do high endurance activities.

 
 Well, the sit-stand test doesn't really enter into it: that's a muscle strength and balance test, not an aerobic endurance test.
 

The Elysium results showed at 500 mg healthy people walked 8% further for 6 minutes. Why wouldn't that be an aerobic improvement?

 
 Aerobic function is certainly involved to a degree in the 6-minute walk test, but it's not really intended for that, and certainly isn't the human equivalent of the rat swim test: as Howard says, the cardiac treadmill stress test is a closer analogy. The six-minute walk is a more integrated test for functional problems in motility. Eg, per this guidance statement from teh American Thoracic Society:
 

The strongest indication for the 6MWT is for measuring the response to medical interventions in patients with moderate to severe heart or lung disease. The 6MWT has also been used as a one-time measure of functional status of patients, as well as a predictor of morbidity and mortality (see Table 1 for a list of these indications).

 

TABLE 1. Indications for the six-minute walk test
Pretreatment and posttreatment comparisons
Lung transplantation (9, 10)
Lung resection (11)
Lung volume reduction surgery (12, 13)
Pulmonary rehabilitation (14, 15)
COPD (16–18)
Pulmonary hypertension
Heart failure (19, 20)
Functional status (single measurement)
COPD (21, 22)
Cystic fibrosis (23, 24)
Heart failure (25–27)
Peripheral vascular disease (28, 29)
Fibromyalgia (30)
Older patients (31)
Predictor of morbidity and mortality
Heart failure (32, 33)
COPD (34, 35)
Primary pulmonary hypertension (10, 36)

 

It's also sometimes used for arthritis and Parkinson's.
 
As Howard notes, they did a proper test of aerobic function in the Colorado trial, with no effect.
 
It bears noting IAC that the 250 mg NR+PT group's 6MWT actually appeared to decline (nonsignificantly) relative to baseline and was lower than either placebo or 500 mg NR+PT (supplementary Table 6) — and this was in the subgroup that was on its face the most likely to benefit, having started with a lower baseline value. That's a very odd dose-response, and makes me suspect a false positive in the 500 mg NR group.



#42 hav

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Posted 14 April 2018 - 09:30 PM

Sure: I don't think I've ever suggested that NR/NMN is "unsafe at any dose" ;) . As I said in my opening post, if the PMID: 28211258 study is to be taken at face extrapolative value, it would suggest that the optimum dose is somewhere around twice the RDA — way lower than is being used in the rest of the rodent studies, or than the company is recommending, or than anyone is taking AFAIK, and more suggestive of  avoidance of deficiency, with the suggestion that we may need a slight upward bump to the RDA at an upcoming NAM Food and Nutrition Board meeting, rather than a pharmacological effect at megadose — and also more likely to suggest that any NAD+ precursor will do. (Cf. this study of RDAish NMN vs. NAM).
 

 

That rat study is so divergent in dosage response from the 2 human studies and all the mouse studies we've been talking about, the only thing I can make of it is that NR response in rats does not compare well to humans or mice. I guess we should feel blessed the clinical trials didn't get scared off by the rats.

 

I'm kind of tempted to ignore the anomalous rat study but I'd love to know why the difference.  I wonder if it's a digestive tract enzyme thing.  I suppose if similar results obtained from another rat study using ip injection, that might eliminate that as a suspect.  But I'm guessing researchers will just stick with mice and humans and call it a day.

 

Howard

 



#43 Michael

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Posted 14 April 2018 - 11:07 PM

That rat study is so divergent in dosage response from the 2 human studies and all the mouse studies we've been talking about, the only thing I can make of it is that NR response in rats does not compare well to humans or mice. I guess we should feel blessed the clinical trials didn't get scared off by the rats.
 
I'm kind of tempted to ignore the anomalous rat study but I'd love to know why the difference.  I wonder if it's a digestive tract enzyme thing.  I suppose if similar results obtained from another rat study using ip injection, that might eliminate that as a suspect.  But I'm guessing researchers will just stick with mice and humans and call it a day.
 
Howard

 
First, what do you mean here by "dosage response"? There's no dose-response data as such in the rat study: they only used one dose (300 mg/kg).
 
Second, as has animated this thread, it's not just the exercised rat NR study (PMID: 27489522): the PMID: 28211258 mouse study also found trends toward deleterious effects with NR above RDAish levels, and those results are at least loosely similar to some of the dose-response trends in the long-term middle-age NMN mouse study (PMID: 28068222). Again, one thing that distinguishes these 3 studies from all the other rodent studies is that they were conducted in animals that (like humans) do not have the NNT mutation — and I've put forward an hypothesis for why that might be, based on the molecular biology of the NNT gene.

 

Alternatively, again, as Stefan has pointed out, another thing that unites the first two at least is that they were conducted in much younger animals than most other studies, for which again there is a mechanistically-plausible hypothetical explanation.



#44 able

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Posted 15 April 2018 - 12:27 AM

Michael - I think I understand the concerns you raise about possible significance of these  3 studies on exercise performance.

 

But I don't see if you have really commented on the significance of the recent Sinclair study on increased endurance from 8 weeks of large dosages of NMN.

 

https://www.longecit...-in-aging-mice/

 

Do you believe that demonstrates significant improvement, and might dispel some of the concern from these earlier studies?



#45 Michael

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Posted 15 April 2018 - 02:30 AM

Michael - I think I understand the concerns you raise about possible significance of these  3 studies on exercise performance.

 

But I don't see if you have really commented on the significance of the recent Sinclair study on increased endurance from 8 weeks of large dosages of NMN.

 

https://www.longecit...-in-aging-mice/

 

Do you believe that demonstrates significant improvement, and might dispel some of the concern from these earlier studies?

 

Well, they used C57Bl/6J mice for all parts of the paper (WT, SIRT1-TG, and SIRT1-KO/ESCO), so the whole thing is still confounded by the NNT mutation.



#46 bluemoon

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Posted 15 April 2018 - 05:08 AM

 . That's a very odd dose-response, and makes me suspect a false positive in the 500 mg NR group.

 

It was stastically significant, so you have a 5% probability that it is a false positive.



#47 Michael

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Posted 15 April 2018 - 04:59 PM

That's not quite what a P-value means, but quibbling aside: the simple threshold addresses only prior probability; once you look at the actual results, consideration of biological plausibility brings more active doubts to bear on the matter.



#48 bluemoon

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Posted 18 April 2018 - 01:55 AM

That's not quite what a P-value means, but quibbling aside: the simple threshold addresses only prior probability; once you look at the actual results, consideration of biological plausibility brings more active doubts to bear on the matter.

 

That was short hand for a p value meaning, and you are right to object to my short hand even if a quibble, but the point holds that it is quite unlikely that those 60 to 79 and healthy did not on average see an 8% increase in walking speed and balance.

 

What biological possibility? what active doubts? Those are in your mind. All they did was measure walking and had nothing to do with mice. 



#49 Michael

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Posted 18 April 2018 - 02:10 AM

the simple [p-value] threshold addresses only prior probability; once you look at the actual results, consideration of biological plausibility brings more active doubts to bear on the matter.
 

the point holds that it is quite unlikely that those 60 to 79 and healthy did not on average see an 8% increase in walking speed and balance.
 
What biological possibility? what active doubts? Those are in your mind. All they did was measure walking and had nothing to do with mice.

 

 
As I said in the post to which you were responding, "they did a proper test of aerobic function in the Colorado trial, with no effect[, and while they report an improvement in walking speed at 500 mg NR +PT], the 250 mg NR+PT group's 6MWT actually appeared to decline (nonsignificantly) relative to baseline and was lower than either placebo or 500 mg NR+PT (supplementary Table 6) — and this was in the subgroup that was on its face the most likely to benefit, having started with a lower baseline value. That's a very odd dose-response, and makes me suspect a false positive in the 500 mg NR group."


Edited by Michael, 18 April 2018 - 02:11 AM.

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#50 hav

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Posted 22 April 2018 - 03:05 AM

 
First, what do you mean here by "dosage response"? There's no dose-response data as such in the rat study: they only used one dose (300 mg/kg).

 

You're right about the rat study not comparing responses to different dosages.  It reported an endurance response in rats that does not seem to jive with either mobility or aerobic response observed in a pretty comprehensive human muscle-response study.

 

Howard

 


Edited by hav, 22 April 2018 - 03:06 AM.


#51 Michael

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Posted 08 November 2018 - 06:58 PM

A Direct Comparison of Metabolic Responses to NAD repletion in C57BL/6J and C57BL/6N diet-induced obesity mouse models
Endocrine Abstracts (2018) 59 P176 | DOI: 10.1530/endoabs.59.P176

Antje Garten1,2, David Cartwright2, Lucy Oakey2, Rachel Fletcher2, Daniela Nasteska2, David Hodson2, Dean Larner2, Craig Doig2, Christian Ludwig2, Katarina Kluckova2 & Gareth Lavery2

... Mice were fed a high fat diet (HFD, 60% fat) or standard chow with or without supplementation of 3 g/l NR in the drinking water for 8 weeks. [PMID 27809334 supplemented the drinking water of wild-type C57BL/6J mice with the same amount of NR, and said that this worked out to ~500 mg/kg; based on PMID  12467341, C57Bl/6J mice consume ~6.2 mL water per day and  weight 25g (though this is for very young mice), so this might be 744 mg/kg, or less in older mice -MR]. Body and organ weights, liver lipids as well as glucose tolerance were measured. ...

Results: NR supplementation had a slight positive effect on fasting blood glucose and on energy expenditure of B6/N mice on HFD. In B6/J mice, NR influenced substrate usage as determined by respiratory exchange ratio both in chow and HFD-fed mice.

 

Mitochondrial O2 flux and citrate synthase activity were significantly increased by NR supplementation specifically in heart muscle fibers of B6/N, but not B6/J mice on HFD. No effect on mitochondrial function was found in the other examined tissues.

 

The mitochondrial enzyme nicotinamide nucleotide transhydrogenase (Nnt) was found to be 2-fold upregulated in hearts of B6/N mice on HFD+NR, which was reflected by lower levels of the oxidative stress marker 4-hydroxynonenal.

Conclusion: The effect of NR supplementation in diet-induced obesity is influenced by mouse genotype and possibly related to cellular redox status.


 It would be nice to have a full paper, but it looks like they're saying that NR improved fasting glucose and increased energy expenditure  in B6/N mice (which do not have the NNT mutation) on HFD, but did not have such effects in nonobese mice on regular chow (since they mention effects in chow and HFD-fed mice for the C57BL/6J mice, which do have the mutation). I think that's in line with the "NNT hypothesis," since HFD subjects B6/N  mice to high oxidative stress and mitochondrial dysfunction, which NR then helps counteract; absent those conditions, NR was not beneficial.

 

I'm a bit surprised that "Mitochondrial O2 flux and citrate synthase activity were significantly increased by NR supplementation specifically in heart muscle fibers of B6/N, but not B6/J mice on HFD." The "NNT hypothesis" might have predicted that for B6/N, but maybe also for B6/J; you'd also predict an effect in B6/J based on prior studies in HFD animals of this strain.

 

I'm not sure what it'd've predicted in nonobese mice on regular chow, which they don't say anything about, which might imply there was no effect at all.

 

I'm also not sure what I'd've predicted for effects on NNT itself in either HFD+NR group (I'm assuming that they only looked at gene expression, not protein levels).

 

An improvement in oxidative stress in HFD+NR C57BL/6N animals is consistent with NNT, but I'm surprised that ex silentio they seem to be saying that it was not improved in any 6J group. And again ex silentio, they seem to be saying that oxidative stress was not improved in nonobese C57BL/6N on a chow diet; the "NNT hypothesis" would predict either this, or possibly an increase in ox. stress (which I'd expect them to report if they'd found).

 

You're right about the rat study not comparing responses to different dosages.  It reported an endurance response in rats that does not seem to jive with either mobility or aerobic response observed in a pretty comprehensive human muscle-response study.

 
What "aerobic response observed in a pretty comprehensive human muscle-response study"? As far as comparing the endurance exercise test here to the motility test in the Elysium Basis trial, see my comments above.


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#52 bluemoon

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Posted 08 November 2018 - 11:19 PM

 

 
It bears noting IAC that the 250 mg NR+PT group's 6MWT actually appeared to decline (nonsignificantly) relative to baseline and was lower than either placebo or 500 mg NR+PT (supplementary Table 6) — and this was in the subgroup that was on its face the most likely to benefit, having started with a lower baseline value. That's a very odd dose-response, and makes me suspect a false positive in the 500 mg NR group.

 

After your response today, I reread the above. Since the strange result with the 250 mg was non-significant, why would you suspect the unlikely chance that the 500 mg groups statistically significant result is a false positive?

 

By the way, in July 2017, Guarente said that Elysium was set to set up several more trails with Basis, but the only one I see listed is a small one for acute kidney failure. Does anyone know more about this?



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