• Log in with Facebook Log in with Twitter Log In with Google      Sign In    
  • Create Account
  LongeCity
              Advocacy & Research for Unlimited Lifespans

Photo
- - - - -

The NNT Mutation: A Fly in the NAD Precursor Ointment

nnt mutation c57bl/6j nicotinamide riboside

  • This topic is locked This topic is locked
51 replies to this topic

#1 Michael

  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 17 August 2017 - 10:42 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.

 

My posts on this are scattered in bits and pieces in the [Curated] thread, so I'm putting it all together in a more coherent and easily-citable post here.

 

The mitochondrial NAD(P) transhydrogenase (NNT) gene encodes a protein that  is embedded in the inner mitochondrial membrane and transfers reducing equivalents between NADH in the cytosol and NADP(+) in the mitochondrial matrix. There, it provides the reducing power needed to detoxify reactive oxygen species (ROS) generated in the mitochondria, both directly and by regenerating thioredoxin and glutathione: it's necessary to maintain redox homeostasis and efficient ATP synthesis in every single cell in the body. "NNT expression differs between cell types, being highest in the heart and kidney. Approximately half of the mitochondrial NADPH in the brain is believed to depend on the action of the NNT, and its inhibition causes significant oxidative stress."(1)
 
The C57BL/6J mouse strain carries a mutation in this gene, which in turn impairs the function of this protein. Accordingly, C57BL/6J mice exhibit abnormalities in oxidative stress, impaired first-phase insulin secretion following a glucose challenge, and impaired glucose tolerance, and also alters its tendency to weight gain under different diets. And it does a lot of other less-than-obvious things as well:
 

The key role of the NNT and the network of interactions taking place in cytosolic glucose metabolism highlight that the pathways involved in maintenance of the NAD and NADP pools in their separate redox states are highly interconnected. Indeed, in addition to the malate-asparate and citrate-α-ketoglutarate shuttles providing separate transmission of NAD and NADP redox state between cytosol and mitochondria, a pyruvate-malate shuttle in which the redox state of the cytosolic NADP pool is coupled to that of the mitochondrial NAD pool has also been observed {109}.

Additional complexity in these redox networks also arises from the reversibility of a number of the reactions. For example, during ischaemia, the citric acid cycle may reverse and consume NADH {39}, the NNT may oxidise NADPH to produce NADH when the membrane potential is collapsed {104} or lactate dehydrogenase may reverse, using lactate as a metabolic substrate, producing NADH in the cytosol alongside pyruvate for aerobic ATP production {110}. Indeed, it has been suggested that lactate secreted by astrocytes may serve as the primary energy source for neurons in the brain {111}. Thus, the highly contrasting intracellular roles of the NAD and NADP pools and their separate redox states are supported by a complex and interconnected network of pathways.


Because of all of this, when you knock out the mitochondrial form of SOD, most mouse strains use NNT to partially make up for it; because they lack functional NNT, giving C57Bl/6J the same MnSOD mutation leads to much more severe effects. The more you then back-cross them with strains of mouse with functional NNT, the more they're able to cope with the MnSOD mutation:
 

congenic Sod2−/− mice on a C57BL/6J background (B6 Sod2−/−) develop a fetal form of dilated cardiomyopathy, and most of them die about day 15 (ED15) of gestation. On the other hand, Sod2−/− mice generated on a DBA/2J (D2 Sod2−/−) background develop normally through gestation and do not have dilated cardiomyopathy. However, these mice develop severe metabolic acidosis and have an average lifespan of 8 days. F1 mice (B6D2F1 Sod2−/−) generated from the two parental strains have a cardiac phenotype similar to that of D2 Sod2−/− mice, but with a milder form of metabolic acidosis. Consequently, these mice are able to survive for up to three weeks without any pharmacological intervention (13). Consistent with our observation, a different Sod2 mutant strain (SOD2m1BCM) generated on a B6/129 mixed genetic background was shown to survive for up to 3 weeks after birth and had a phenotype similar to that of B6D2F1 Sod2−/−tm1Cje (14).(2)

 
After a series of exciting-looking reports of megadose NR or NMN supplementation in mice, a new study(3) carried out in a different C57BL substrain that does not carry the NNT mutation(3) came as a great surprise. In a previous study,(4) Cantó and Auwerx had shown that megadose NR improved exercise performance and metabolic flexibility (ie, the ability to switch between fatty acids and glucose as fuel in response to changes in fuel availability) in C57BL6J mice. In this newer study,(3) they provided NR as the sole source of vitamin B3 across a wide range of doses (either 5, 15, 30, 180 or 900 mg NR per kg diet) plus the mouse "RDA" of tryptophan, because mammals can synthesize B3 from Trp, to see which dose would give the animals the best metabolic flexibility.

Surprisingly, these guys found that metabolic flexibility is maxed out at 2 x the mouse "RDA" of B3 as NR, and may even get worse when NR is administered at the megadose levels used in previous studies. Fasting metabolic flexibility, maxΔRERCHO1→FAO, was better at 30 mg/kg than at higher or lower doses, although only statistically significantly better as compared to 5NR; "Refeeding metabolic flexibility, maxΔRERFAO→CHO2, was significantly greater in 30NR than in 5NR, 15NR, or 900NR ... No differences were seen in blood glucose, serum TG or NEFA among NR doses (Fig. 3A-C). Serum insulin, leptin, adiponectin, leptin/adiponectin ratio and HOMA-IR index exhibited a tendency towards a dose-response curve, without reaching statistical significance (Fig. 3D-H). In all cases, except adiponectin which shows opposite behaviour, the measured value decreased and then increased, with 30NR being the turning point" — that is, the dose-response curve on markers of insulin resistance were U-shaped, with the nominal insulin resistance lowest at 30 NR and then rising again at higher doses. The suggestion in the trend is that mice given high-dose NR forced their pancreases to pump out more insulin just to maintain the same glucose level.(3)
 
This is in stark contrast to Cantó and Auwerx, (4) who found megadose NR to improve metabolic flexibility over and above the 30 mg/kg diet of vitamin B3 (as nicotinamide) in the background diet — and the similar findings with NMN in age- and obesity-related diabetes, again in C57BL/6J mice.(10)
 
Because nearly all the in vivo work on NR and NMN has been done using C57BL/6J mice, most or all of the very promising rodent data we've seen — particularly on outcomes involving metabolic health, oxidative stress, or exercise performance — may actually showing only that NR and NMN help to compensate for the fact that the mutation impairs their ability to transfer reducing equivalents from NADH to NADP+ in the mitochondria. The same applies to the core of the muscle stem cell and lifespan study in Science;(5)* the report that "Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice;"(6) the  Sinclair study on NMN restoring more youthful muscle bioenergetics in aging mice that initially got people excited around NR (on the assumption that it would have similar effects);(7) the Sinclair study on NMN restoring PARP1 DNA repair in aging mouse liver;(8) the finding that NMN protected against age- and obesity-related diabetes(10) and a mouse model of Alzheimer's disease;(11) etc etc.

This would in turn predict that contrast, animals that don't carry such a mutation — including the strain of mouse used in (3), most other rodents, and (most importantly) humans — will not get similar benefits, because there's no defect there to compensate.  Indeed, in such an organism, increasing the level of NAD+ via megadose NR might instead disrupt optimal NADP:NADPH redox coupling, leading to functional impairments. Consistent with this, "The NAD+ precursor nicotinamide riboside decreases exercise performance in rats,"(9) even though it's been reported to improve exercise performance mice in Canto and Auwerx's (obese, diabetic) mice bearing the NNT mutation.(4)

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.
 
*The initial in vitro muscle stem cell study in the Zhang et al Science report(5) used cells from the C57BL/10SnJ substrain, which does not appear to carry the mutation, but they used the Nnt-mutant C57BL/6JRj substrain both for the in vivo study on mitochondrial function and stem cell aging, and also for the lifespan study, and possibly for other parts that are not explicitly spelled out.
 
Reference
1: Blacker TS, Duchen MR. Investigating mitochondrial redox state using NADH and NADPH autofluorescence. Free Radic Biol Med. 2016 Nov;100:53-65. doi: 10.1016/j.freeradbiomed.2016.08.010. Epub 2016 Aug 9. Review. PubMed PMID: 27519271; PubMed Central PMCID: PMC5145803.

2: Huang TT, Naeemuddin M, Elchuri S, Yamaguchi M, Kozy HM, Carlson EJ, Epstein CJ. Genetic modifiers of the phenotype of mice deficient in mitochondrial superoxide dismutase. Hum Mol Genet. 2006 Apr 1;15(7):1187-94. Epub 2006 Feb 23. PubMed PMID: 16497723.
 
3: Shi W, Hegeman MA, van Dartel DA, Tang J, Suarez M, Swarts H, van der Hee B, Arola L, Keijer J. Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet. Mol Nutr Food Res. 2017 Feb 16. doi: 10.1002/mnfr.201600878. [Epub ahead of print] PubMed PMID: 28211258.
 
4: Cantó C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, Fernandez-Marcos PJ, Yamamoto H, Andreux PA, Cettour-Rose P, Gademann K, Rinsch C, Schoonjans K, Sauve AA, Auwerx J. The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metab. 2012 Jun 6;15(6):838-47. doi: 10.1016/j.cmet.2012.04.022. PubMed PMID: 22682224; PubMed Central PMCID: PMC3616313.
 
5: Zhang H, Ryu D, Wu Y, Gariani K, Wang X, Luan P, D'Amico D, Ropelle ER, Lutolf MP, Aebersold R, Schoonjans K, Menzies KJ, Auwerx J. NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science. 2016 Jun 17;352(6292):1436-43. doi: 10.1126/science.aaf2693. Epub 2016 Apr 28. PubMed PMID: 27127236.
 
6: Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, Redpath P, Migaud ME, Apte RS, Uchida K, Yoshino J, Imai SI. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. Cell Metab. 2016 Dec 13;24(6):795-806. doi: 10.1016/j.cmet.2016.09.013. Epub 2016 Oct 27. PubMed PMID: 28068222.

7: Gomes AP, Price NL, Ling AJ, Moslehi JJ, Montgomery MK, Rajman L, White JP, Teodoro JS, Wrann CD, Hubbard BP, Mercken EM, Palmeira CM, de Cabo R, Rolo AP, Turner N, Bell EL, Sinclair DA. Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell. 2013 Dec 19;155(7):1624-38. doi: 10.1016/j.cell.2013.11.037. PubMed PMID: 24360282; PubMed Central PMCID: PMC4076149.
 
8: Li J, Bonkowski MS, Moniot S, Zhang D, Hubbard BP, Ling AJ, Rajman LA, Qin B, Lou Z, Gorbunova V, Aravind L, Steegborn C, Sinclair DA. A conserved NAD(+) binding pocket that regulates protein-protein interactions during aging. Science. 2017 Mar 24;355(6331):1312-1317. doi: 10.1126/science.aad8242. PubMed PMID: 28336669; PubMed Central PMCID: PMC5456119.
 
9: Kourtzidis IA, Stoupas AT, Gioris IS, Veskoukis AS, Margaritelis NV, Tsantarliotou M, Taitzoglou I, Vrabas IS, Paschalis V, Kyparos A, Nikolaidis MG. The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats. J Int Soc Sports Nutr. 2016 Aug 2;13:32. doi: 10.1186/s12970-016-0143-x. eCollection 2016. PubMed PMID: 27489522; PubMed Central PMCID: PMC4971637.
 
10: Yoshino J, Mills KF, Yoon MJ, Imai S. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab. 2011 Oct 5;14(4):528-36. doi: 10.1016/j.cmet.2011.08.014. PubMed PMID: 21982712; PubMed Central PMCID: PMC3204926.
 
11: Long AN, Owens K, Schlappal AE, Kristian T, Fishman PS, Schuh RA. Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model. BMC Neurol. 2015 Mar 1;15:19. doi: 10.1186/s12883-015-0272-x. PubMed PMID: 25884176; PubMed Central PMCID: PMC4358858.


  • WellResearched x 5
  • Well Written x 4
  • Informative x 2
  • Disagree x 1

#2 bluemoon

  • Guest
  • 723 posts
  • 86
  • Location:south side
  • NO

Posted 18 August 2017 - 03:14 AM

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.

 


  • Disagree x 3
  • Agree x 1

Click HERE to rent this ad spot for the NAD+ forum to support LongeCity (this will replace the google ad above).

#3 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 18 August 2017 - 03:30 AM

Why are all the scientists working with NR ignoring this if an important factor? I suspect because it isn't.


The C57BL/6J  is a very widely-used mouse, and studies you're seeing published now were completed many months ago, and some of them took years to complete. You'll note that the first study suggesting this only came out earlier this year. You can't undo research you've already done, and you're not going to abandon a project that's funded and half-done, either — particularly as more research will be needed to confirm that the NNT mutation is really what's responsible for these discordant results.

 

EDIT: Moreover, I wouldn't assume that the fact that many scientist ignore some important scientific procedure means it's not important. Ever since Weindruch and Spindler's breathrough study showing CR was still effective when implemented in mid-life,(0) scientists have been drafting editorials warning their colleagues of the distorting effects introduced into toxicology, carcinogenesis, and above all lifespan studies by following the unfortunately widespread protocol of giving experimental access unlimited access to food, 24 hours a day, leading to obesity, metabolic disease, and altered responses to toxins(1-5) — yet the practice continues to be widespread, and is likely the reason why the majority of lifespan studies wind up having short-lived controls and an intervention group that lives longer than controls, but shorter than a properly-fed lean control mice in labs that know what they're doing.

 

ADDITIONAL EDIT: Similarly, scientists have known for more than 50 years that cancer and other cell lines are widely contaminated with HeLa cells, so that scientists who think and report that they're studying liver cancer cells or normal lung cells are actually studying cervical cancer cells. And HeLa isn't the only problem: There are a variety of resources available to make sure that one has the kind of cell one thinks one has (see here , here, and also here), yet the problem persists.

 

 

References

0: Weindruch R, Walford RL. Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence. Science. 1982 Mar 12;215(4538):1415-8. PMID: 7063854 [PubMed - indexed for MEDLINE]

 

1: Carey GB, Merrill LC. Meal-feeding rodents and toxicology research. Chem Res Toxicol. 2012 Aug 20;25(8):1545-50. doi: 10.1021/tx300109x. Epub 2012 Jun 13. PubMed PMID: 22642213.

 

2: Spindler SR. Review of the literature and suggestions for the design of rodent survival studies for the identification of compounds that increase health and life span. Age (Dordr). 2012 Feb;34(1):111-20. doi: 10.1007/s11357-011-9224-6. Epub 2011 Mar 22. Review. PubMed PMID: 21424790; PubMed Central PMCID: PMC3260350.

 

3: Martin B, Ji S, Maudsley S, Mattson MP. "Control" laboratory rodents are metabolically morbid: why it matters. Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6127-33. doi: 10.1073/pnas.0912955107. Epub 2010 Mar 1. PubMed PMID: 20194732; PubMed Central PMCID: PMC2852022.

 

4: Festing MF. Fat rats and carcinogenesis screening. Nature. 1997 Jul 24;388(6640):321-2. PubMed PMID: 9237745.

 

5: Keenan KP, Laroque P, Ballam GC, Soper KA, Dixit R, Mattson BA, Adams SP, Coleman JB. The effects of diet, ad libitum overfeeding, and moderate dietary restriction on the rodent bioassay: the uncontrolled variable in safety assessment. Toxicol Pathol. 1996 Nov-Dec;24(6):757-68. Review. PubMed PMID: 8994307.


Edited by Michael, 18 August 2017 - 10:14 PM.

  • Well Written x 1
  • WellResearched x 1
  • Agree x 1

#4 Evan Yang

  • Guest
  • 23 posts
  • 8

Posted 18 August 2017 - 11:34 AM

You have your points. But human trials like Colorado trial has shown NAD+ precursors improves health even though it has not been published yet. People who are taking Niagen (over 200,000) are reporting good results from it.  There is no doubt for either rats or humans that sufficient levels of NAD+ is crucial to our health at middle to old age. There is no doubt that Niagen increases NAD+ in humans. 

 

4) What are the consequences of defective Nnt in C57BL/6J mice?

B6/J mice do manifest altered redox-related imbalances (Ronchi et al. 2013).   By and large, however, B6/J mice are long-lived mice – indeed, B6/J mice are known for their longevity compared to other inbred strains. The NntC57BL/6J mutation appears to minimally impact the overall health of B6/J mice.


Edited by Evan Yang, 18 August 2017 - 11:38 AM.

  • Ill informed x 2
  • Needs references x 1
  • Enjoying the show x 1
  • Good Point x 1

#5 bluemoon

  • Guest
  • 723 posts
  • 86
  • Location:south side
  • NO

Posted 18 August 2017 - 12:06 PM

You have your points. But human trials like Colorado trial has shown NAD+ precursors improves health even though it has not been published yet. People who are taking Niagen (over 200,000) are reporting good results from it.  There is no doubt for either rats or humans that sufficient levels of NAD+ is crucial to our health at middle to old age. There is no doubt that Niagen increases NAD+ in humans. 

 

 

NR clearly increases NAD+ but how do you know anything about the Colorado trial?


Edited by bluemoon, 18 August 2017 - 12:07 PM.

  • Good Point x 5

#6 Oakman

  • Location:CO

Posted 18 August 2017 - 02:56 PM

Summarizing this in lay terms to what I believe, and that I call the "pothole theory" of supplementation (of NAD+ and most other intrinsic biologic factors), that could encompass many of these findings.

 

If you don't have a 'hole' in your metabolism, meaning a diminished ability to produce something due to age or malfunction, or lacking it altogether, supplementation isn't likely to help. Plus, you only need enough to fill the 'hole' not more (i.e., a bump is as bad as a hole). 


Edited by Oakman, 18 August 2017 - 02:57 PM.

  • Good Point x 4
  • Agree x 2

#7 bluemoon

  • Guest
  • 723 posts
  • 86
  • Location:south side
  • NO

Posted 18 August 2017 - 06:12 PM

  Plus, you only need enough to fill the 'hole' not more (i.e., a bump is as bad as a hole). 

 

I doubt this. Since NR is a vitamin and safe up to quite high doses, shouldn't any extra the body doesn't need just get discarded so that there is never a "bump"?  


  • unsure x 1
  • Ill informed x 1
  • Good Point x 1
  • Disagree x 1

#8 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 18 August 2017 - 06:40 PM

There is no doubt for either rats or humans that sufficient levels of NAD+ is crucial to our health at middle to old age. There is no doubt that Niagen increases NAD+ in humans.


There is no doubt for either rats or humans that sufficient levels of selenium crucial to our health at middle to old age. There is no doubt that selenium supplementation increases selenium levels in humans. None the less, high-dose selenium raises the risk of diabetes (and possibly some cancers) except in people with outright selenium deficiency.
 

4) What are the consequences of defective Nnt in C57BL/6J mice?
B6/J mice do manifest altered redox-related imbalances (Ronchi et al. 2013).   By and large, however, B6/J mice are long-lived mice – indeed, B6/J mice are known for their longevity compared to other inbred strains. The NntC57BL/6J mutation appears to minimally impact the overall health of B6/J mice.

 
That's all true — until you subject them to redox challenge, upon which they suffer more mortality than other strains (see my reference 2, excerpted in my opening post). The question is whether megadose NAD+ precursor use could pose a similar redox challenge in mice and people without the NNT mutation.
 

 

Plus, you only need enough to fill the 'hole' not more (i.e., a bump is as bad as a hole). 

 
I doubt this. Since NR is a vitamin and safe up to quite high doses, shouldn't any extra the body doesn't need just get discarded so that there is never a "bump"?

I've just given preliminary evidence to suggest that there is such a bump. Moreover, we don't actually know that NR is safe up to quite high doses: aside from studies in C57BL/6 mice (reference to which is, in this context, petitio principii), we have only subacute toxicity studies in rats, and extremely short-term studies in humans.


Edited by Michael, 18 August 2017 - 06:41 PM.

  • Good Point x 2
  • Informative x 1
  • Agree x 1

#9 Oakman

  • Location:CO

Posted 18 August 2017 - 07:24 PM

 

  Plus, you only need enough to fill the 'hole' not more (i.e., a bump is as bad as a hole). 

 

I doubt this. Since NR is a vitamin and safe up to quite high doses, shouldn't any extra the body doesn't need just get discarded so that there is never a "bump"?  

 

 

Well, I have my own anecdotal evidence that you can have too much NR as described here.  Now that's just me, but I know what I experienced, I know that reducing NR reduced the symptoms almost completely.  I have no doubt in my mind currently that it IS possible to get a NR 'bump' that is NOT pleasant.


  • Good Point x 1
  • Informative x 1

#10 stefan_001

  • Guest
  • 1,070 posts
  • 222
  • Location:Munich

Posted 18 August 2017 - 08:24 PM

The rat study (9) used 4 month old rats and dosing of 300mg / kg of bodyweight. That is the same as feeding teenagers 3.5 gram NR / day. No idea what that study means except confirming that you dont need NAD+ boosting when its at peak level.....

The NNT parts is interesting. I guess the question there is whether aging overtakes the impacts of the defect.
  • Good Point x 1

#11 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 18 August 2017 - 11:18 PM

The rat study (9) used 4 month old rats and dosing of 300mg / kg of bodyweight. That is the same as feeding teenagers 3.5 gram NR / day.


A 4-month-old rat is actually roughly equivalent to a 23-year-old human, not a teenager (Figure V.3 is actually for mice, but mice and rats have broadly similar life courses). And 300mg/kg bodyweight is the same dose or lower than is used in all the mouse studies that have gotten people excited about NR (altho' it's a somewhat lower human dose after allometric scaling than the same mg/kg for rats).
 

No idea what that study means except confirming that you dont need NAD+ boosting when its at peak level.....

 
... which is kinda the issue about which I'm warning ;) .
 

The NNT parts is interesting. I guess the question there is whether aging overtakes the impacts of the defect.


Right: that is a good question. (And if so, "when," not just "whether"). Of course, most of these studies have been done (appropriately!) in middle-aged or older mice.


  • Good Point x 3

#12 stefan_001

  • Guest
  • 1,070 posts
  • 222
  • Location:Munich

Posted 19 August 2017 - 10:47 AM

I thought a 6 month rat is equivalent to an 18 year old human:
https://www.ncbi.nlm...29/#!po=64.2857
  • Informative x 1

#13 Phoebus

  • Guest
  • 660 posts
  • 142
  • Location:Upper Midwest, US

Posted 20 August 2017 - 11:02 PM

 

 

 

 

ADDITIONAL EDIT: Similarly, scientists have known for more than 50 years that cancer and other cell lines are widely contaminated with HeLa cells, so that scientists who think and report that they're studying liver cancer cells or normal lung cells are actually studying cervical cancer cells. And HeLa isn't the only problem: There are a variety of resources available to make sure that one has the kind of cell one thinks one has (see here , here, and also here), yet the problem persists.


 

 

what the FUCK kind of BS is that???

 

50 years they knew their cell lines were contaminated and they just...ignore it??

 

science has serious corruption issues no one talks about. holy shit.
 


  • Pointless, Timewasting x 1
  • Agree x 1

#14 DJS

  • Member
  • 5,797 posts
  • 10

Posted 21 August 2017 - 01:46 AM

Wow, just wow. If Michael's assertions are proven correct there's going to be a lot of Noble prize winners with egg on their faces. SPLAT!

#15 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 21 August 2017 - 03:00 AM

I thought a 6 month rat is equivalent to an 18 year old human:
https://www.ncbi.nlm...29/#!po=64.2857

 
I see from this and other references that you're quite correct — thanks. OK, so these were indeed very young rats. That's still, of course, consistent with a redox imbalance from NAD+ overload (though your point about age of redox transition is well-taken).
 

 

Similarly, scientists have known for more than 50 years that cancer and other cell lines are widely contaminated with HeLa cells, so that scientists who think and report that they're studying liver cancer cells or normal lung cells are actually studying cervical cancer cells. And HeLa isn't the only problem

 
what the FUCK kind of BS is that???
 
50 years they knew their cell lines were contaminated and they just...ignore it??
 
science has serious corruption issues no one talks about. holy shit.

 
To be clear, while there is of course corruption in science as there is in every field of human endeavor, this is not a corruption problem, but a mixture of lack of due diligence and ignorance. When I said "Scientists have known for more than 50 years ..." what I should probably have said was "some scientists have known, and have tried to make the entire community aware ...". And, people tend to just trust reputable suppliers, and assume that their labs' protocols are sufficient to prevent cross-contamination, etc.
 

Wow, just wow. If Michael's assertions are proven correct there's going to be a lot of Noble prize winners with egg on their faces. SPLAT!

 
No worries there. The main reason that it takes decades between a discovery and the awarding of a Nobel prize is that it takes that long for a discovery to be validated elsewhere and to build into something widely-recognized as a major contribution to science: a single sloppily-done experiment is not going to get you there (and, indeed, these days it's hard to imagine you'd get there from original work exclusively in any kind of cell model, and especially a cancer cell model).

 

The real problem instead is that it wastes resources and misleads the field, slowing progress and denying resources to projects of real merit.

 

If you're interested in these and other problems in biomedical science and attempts to solve them, check out Rigor Mortis: How Sloppy Science Creates Worthless Cures, Crushes Hope, and Wastes Billions.



#16 stefan_001

  • Guest
  • 1,070 posts
  • 222
  • Location:Munich

Posted 21 August 2017 - 06:23 AM

 

I thought a 6 month rat is equivalent to an 18 year old human:
https://www.ncbi.nlm...29/#!po=64.2857

 
I see from this and other references that you're quite correct — thanks. OK, so these were indeed very young rats. That's still, of course, consistent with a redox imbalance from NAD+ overload (though your point about age of redox transition is well-take

 

 

 

 

I think we will see a dosing scheme with more research with some categories emerging. For example:

 

- 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

 


Edited by stefan_001, 21 August 2017 - 06:25 AM.

  • Needs references x 2
  • Dangerous, Irresponsible x 2
  • Good Point x 1
  • unsure x 1
  • like x 1

#17 sthira

  • Guest
  • 1,966 posts
  • 392

Posted 21 August 2017 - 12:49 PM

I thought a 6 month rat is equivalent to an 18 year old human:
https://www.ncbi.nlm...29/#!po=64.2857


I see from this and other references that you're quite correct — thanks. OK, so these were indeed very young rats. That's still, of course, consistent with a redox imbalance from NAD+ overload (though your point about age of redox transition is well-take



I think we will see a dosing scheme with more research with some categories emerging. For example:

- 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

Wait, what? Where are you getting these specific human dosing strategies? Based on what?

This thread has gone from the assertion that the "very promising rodent" NR studies may be flawed due to the fact that researchers used mutated rodents to your idea that humans of different ages should use different dosages to do what? "Ward off aging"? For what purpose and why must I use expensive vitamin B3?

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

Kindly explain.
  • Good Point x 4
  • Well Written x 1
  • Agree x 1

#18 Evan Yang

  • Guest
  • 23 posts
  • 8

Posted 21 August 2017 - 12:52 PM

I would recommend the following:

30-40   125mg

40-50   250mg

50+      250-375mg

Cure Disease 500 - 1,000mg


  • Needs references x 3
  • Dangerous, Irresponsible x 2
  • unsure x 1
  • Enjoying the show x 1
  • Pointless, Timewasting x 1

#19 sthira

  • Guest
  • 1,966 posts
  • 392

Posted 21 August 2017 - 01:09 PM

I would recommend the following:
30-40 125mg
40-50 250mg
50+ 250-375mg
Cure Disease 500 - 1,000mg


Why?
  • like x 2
  • Good Point x 1
  • Agree x 1

#20 Harkijn

  • Guest
  • 634 posts
  • 177
  • Location:Amsterdam
  • NO

Posted 21 August 2017 - 01:18 PM

It is a bit early to try to determine human dosing. The study we are all waiting for will create more clarity about this. Stefan's conjecture does not exceed the safety assessment as described here:

https://www.ncbi.nlm...pubmed/26791540 but as it stands now I feel there is reason for conservatism.

If additional forms of NR appear a lot of testing will have to be repeated  :wacko:



#21 Harkijn

  • Guest
  • 634 posts
  • 177
  • Location:Amsterdam
  • NO

Posted 21 August 2017 - 01:25 PM

Sthira said:

Kindly explain.

 

 

 

Please  read here:

http://www.longecity...boside-curated/

 

and here:

http://aboutnr.com/


Edited by Harkijn, 21 August 2017 - 01:26 PM.

  • Enjoying the show x 1
  • Dangerous, Irresponsible x 1
  • Agree x 1

#22 sthira

  • Guest
  • 1,966 posts
  • 392

Posted 21 August 2017 - 01:52 PM

It is a bit early to try to determine human dosing. The study we are all waiting for will create more clarity about this. Stefan's conjecture does not exceed the safety assessment as described here:
https://www.ncbi.nlm...pubmed/26791540 but as it stands now I feel there is reason for conservatism.
If additional forms of NR appear a lot of testing will have to be repeated :wacko:


Not only will results, if any, need replication but those results will require interest in and funding from objective, non-industry profit unmotivated researchers who are disinterested in finding positive results which might enable them to get more funding, a better job, or even tenure. For the corporate science it may be motivation to make more money for shareholders by selling us another scam bolstered by flawed research.

As suggested, read this book:

If you're interested in these and other problems in biomedical science and attempts to solve them, check out Rigor Mortis: How Sloppy Science Creates Worthless Cures, Crushes Hope, and Wastes Billions.


If you read it, you may learn that (bio)science has been often corrupted by market-driven competition for limited funding (our tax dollars) and researchers who are understandably seeking better science jobs, or even tenure. That's academia. The problems with profit motivated companies performing research (like some of these NR studies) seem worse.

To repair aging damages will require honest researchers who are unmotivated by career advancement or rising stock values. For this we look to disinterested institutions who hire disinterested scientists, and this is a political problem.
  • Well Written x 1
  • Good Point x 1

#23 stefan_001

  • Guest
  • 1,070 posts
  • 222
  • Location:Munich

Posted 21 August 2017 - 02:05 PM

It is a bit early to try to determine human dosing. The study we are all waiting for will create more clarity about this. Stefan's conjecture does not exceed the safety assessment as described here:

https://www.ncbi.nlm...pubmed/26791540 but as it stands now I feel there is reason for conservatism.

If additional forms of NR appear a lot of testing will have to be repeated  :wacko:

 

Agree, the eternal "soon to be released" study will help here.


Edited by stefan_001, 21 August 2017 - 02:07 PM.


#24 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 22 August 2017 - 06:23 PM

Sthira said:

Kindly explain.

 

Please  read here:

http://www.longecity...boside-curated/

 

and here:

http://aboutnr.com/

 

But, that doesn't address Sthira's question, which is where folks are getting these specific dose regimens from (which are themselves based on the speculation that an age-related decline might justify a rising dose with age despite the possible downside in youth in organisms, like humans, lacking the NNT mutation — which is, itself, still not a nailed-down problem). As you said in an earlier post. we need a lot more human data:  just showing how much it raises NAD+ will not address this question, but metabolic outcomes would help, as would the specific study in older adults.

 

Bluemoon: your last post, once again expressing frustration about the lack of release of study results, would probably fit better either in the FASEB Meeting thread or the NR Human Trials thread. I have a tiny bit of news on that front; may I move your post to one of those threads (your choice)?
 



#25 stefan_001

  • Guest
  • 1,070 posts
  • 222
  • Location:Munich

Posted 22 August 2017 - 07:39 PM

 

But, that doesn't address Sthira's question, which is where folks are getting these specific dose regimens from (which are themselves based on the speculation that an age-related decline might justify a rising dose with age despite the possible downside in youth in organisms, like humans, lacking the NNT mutation — which is, itself, still not a nailed-down problem). As you said in an earlier post. we need a lot more human data:  just showing how much it raises NAD+ will not address this question, but metabolic outcomes would help, as would the specific study in older adults.

 

Bluemoon: your last post, once again expressing frustration about the lack of release of study results, would probably fit better either in the FASEB Meeting thread or the NR Human Trials thread. I have a tiny bit of news on that front; may I move your post to one of those threads (your choice)?
 

 

 

Hey Michael, as I wrote the dosing scheme down I will comment your post. I would have expected that the context of the scheme was recognized, meaning there is likely no single dose and/or single age experiment that can be extrapolated across all cases all yielding the same results. It was not about the absolute values that is why it says "for example". Interesting you prefer to focus on "where do these folks get these dose regimens from" instead of the intended context.

 


  • Enjoying the show x 1
  • Good Point x 1

#26 bluemoon

  • Guest
  • 723 posts
  • 86
  • Location:south side
  • NO

Posted 22 August 2017 - 08:57 PM

But, that doesn't address Sthira's question, which is where folks are getting these specific dose regimens from...

 

I think people are basing those above doses on a couple of things but should be considered just speculation. First, 250 mg quickly became a standard dose by many vendors in part because Elysium suggested that for its Basis. But on what basis did they set that? I assume as a reasonable extrapolation from mouse studies and to a degree on what they think customers are willing to pay for an unproven pill in humans. People also see that some centers are running trials on specific conditions where the doses are higher, from 750 mg to 2,000 mg. There has also been the idea floating around that those under 40 don't feel any different so are guessing 125 mg is good for those between 30 and 40, which to me seems like pure speculation at this point although a possibility.

 

Guarente said a few weeks ago that people should take it "the sooner the better" which implies all the way down to age 18, where that is the cut off on Elysium's label. I have a hard time believing NR, even if effective, is necessary for anyone under 25 but always possible.   

 

I only wrote the post above to remind along with specifics that not just one but five studies have been completed with no signs of data being released this year apart from ChromaDex's repeated delays to release their results "sometime in the next several months." Ever since Elysium decided to not put up results apart from effects of NR on raising NAD+ levels at 250mg and 500 mg, I thought it was because of what it assumed would soon be direct competition with ChromaDex as well as the results no longer showing 250 mg was the best overall dose. Guarente also recently said that six more Basis trials will better show what different doses do and doubt he would announce that if Basis did almost nothing or nothing at all.

 

(Please move the previous post on the five trials over to the "FASEB meeting" thread if it makes more sense to have it there. Thanks.)  


Edited by Michael, 22 August 2017 - 09:49 PM.
Link to Post About 5 Unpublished Studies


#27 able

  • Guest
  • 722 posts
  • 325
  • Location:austin texas
  • NO

Posted 12 October 2017 - 09:57 PM

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


Edited by Michael, 16 October 2017 - 07:58 PM.
material from deleted thread


#28 able

  • Guest
  • 722 posts
  • 325
  • Location:austin texas
  • NO

Posted 12 October 2017 - 10:19 PM

I see the long-term NMN mouse study used c57bl-6n mice (not 6j) , which according to this article, do NOT have the NNT mutation.  If accurate, that would be somewhat comforting.... 

 

 

https://www.jax.org/...l-6n-substrains


Edited by able, 12 October 2017 - 10:22 PM.

  • Good Point x 2
  • WellResearched x 1

#29 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 16 October 2017 - 05:20 PM

I see the long-term NMN mouse study used c57bl-6n mice (not 6j) , which according to this article, do NOT have the NNT mutation.  If accurate, that would be somewhat comforting....
 
https://www.jax.org/...l-6n-substrains

 
Good find! Unlike in most studies, it's right there in the abstract, yet you're the first person to notice. In digging into this, I'd been double-checking the mice used in NR studies, but didn't look specifically for NMN studies. Yet I've now gone for a specific dig for more such reports, and this seems to be the only one: even the NMN diabetes study by the same authors used C57BL/6J.
 
There's nothing in the reasoning here that would make you think that NR would have this interaction with the mutation where NMN did not, so if additional studies replicate it, I think we can stop worrying about this gene x nutrient confounder. Instead, it might be an age x nutrient confounder:  as has been noted, both of the studies finding deleterious effects of megadose NR used very young mice, who have not yet undergone the age-related decline in NAD+, so a redox imbalance fom NR/NMN supplementation could still explain the finding as per the reasoning laid out above, but for different reasons. That would suggest NR/NMN supplementation would still be useful in aging humans, and perhaps only harmful in very young ones.
 
It will also be important to get more dose-ranging studies. Although the results are actually pretty ambiguous, Imai himself found some suggestion that their lower dose (100 mg/kg) was better than their higher dose (300 mg/kg) for some outcomes in the long-term NMN mouse study, and reasonably suggests that different tissues might respond differently to a given dose:
 

an optimal dose of NMN to maximize its efficacy appears to differ depending on physiological functions. For example, whereas the effects of NMN on body weight gain, insulin sensitivity, tear production, and bone mineral density were dose-dependent, 100 mg/kg/day of NMN improved oxygen consumption, energy expenditure, and physical activity better than 300 mg/kg/day. For rod and cone photoreceptor function, both doses had similar effects. Indeed, we found that expression of Ox2r and Prdm13, two downstream genes in the SIRT1-mediated signaling pathway in the hypothalamus, exhibited significant decreases in the hypothalami of 300 mg/kg/day NMN-treated mice (our preliminary finding), which could partly explain some of the observed differences in the effects of NMN, particularly those on physical activity, between two tested doses.  Additionally, the extent of age-dependent NAD+ decline or NMN uptake in each tissue or organ might determine an optimal dose of NMN to restore each physiological function.


I'll jump in here to add:
 

Distinct metabolic routes, starting from various precursors,are known to support NAD+ biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. ... Nam phosphoribosyltransferase (NamPRT [= NAMPT], EC 2.4.2.12) and NR kinase (NRK, EC 2.7.1.22), which catalyze NMN formation from Nam and NR, respectively, and quinolinic acid (QA) phosphoribosyltransferase (QAPRT, EC 2.4.2.19) and NA phosphoribosyltransferase (NAPRT, EC 6.3.4.21), which synthesize NAMN from QA and NA, respectively. ... Understanding the contribution of these enzymes to NAD+ levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD+ availability" (reference).

 
Returning to Imai:
 

Given that 100 mg/kg/day of NMN was able to mitigate most age-associated physiological declines in mice, an equivalent surface area dose for humans would be ∼8 mg/kg/day (Freireich et al., 1966 [ie, per the FDA human-equivalent dose (HED) calculation]), providing hope to translate our findings to humans. [An alternative method of allometric scaling suggests a human-equivalent dose of 1000-1100 mg depending on what age and weight you look at-MR].

 
And the fact that Imai's lowest dose was 100 mg/kg mouse leaves open the question of whether even lower, RDAish doses are just as good or even better than megadoses, as the headline dose-ranging metabolic study (PMID 28211258 — (3) in my opening post) suggests, or (as Stefan suggests) that the optimal dose would continue to rise with age from such a baseline, again with optimal doses also varying by tissue type and choice of precursor.


  • Informative x 3

Click HERE to rent this ad spot for the NAD+ forum to support LongeCity (this will replace the google ad above).

#30 Michael

  • Topic Starter
  • Advisor, Moderator
  • 1,234 posts
  • 1,695
  • Location:Location Location

Posted 17 October 2017 - 07:25 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).

 







Also tagged with one or more of these keywords: nnt mutation, c57bl/6j, nicotinamide riboside

0 user(s) are reading this topic

0 members, 0 guests, 0 anonymous users