4 replies to this topic

[Michael]

Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance
Sheila R. Costford, Bram Brouwers, Meghan E. Hopf, Lauren M. Sparks, Mauro Dispagna, Ana P. Gomes, Heather H. Cornnell, Chris Petucci, Peter Phelan, Hui Xie, Fanchao Yi, Glenn A. Walter, Timothy F. Osborne, David A. Sinclair, Randall L. Mynatt, Julio E. Ayala, Stephen J. Gardell, Steven R. Smith
DOI: http://dx.doi.org/10...met.2017.10.012
Publication stage: In Press Uncorrected Proof
Published online: November 1, 2017
 
Highlights

• Skeletal muscle NAMPT overexpression increases NAD⁺ via elevated NAMPT activity.
• Elevated NAMPT partially protects against very-high-fat-diet-induced weight gain.
• Elevated NAMPT amplifies exercise-induced improvements in exercise endurance.

Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+) ... We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding.

Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD ... (2016, Harlan Teklad, Indianapolis, IN [apparently this one: 12% fat]). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal).

NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice.  ... NMN, the product of NAMPT activity, was ∼2-fold higher in skeletal muscle from NamptTg versus WT mice ... There was a concomitant 1.6-fold elevation of skeletal muscle NAD+ ... This relatively modest increase of NAD⁺ is comparable to other published studies that used various strategies to boost tissue NAD⁺ levels [27-30]. Activation of NAD⁺ consuming pathways or the contribution of other homeostatic mechanisms probably serves to place a ceiling on the maximal NAD⁺ level. [That's consistent also with the larger effect on NMN vs. NAD⁺-MR]. For instance, physiological levels of NAD⁺ can inhibit NAMPT activity, thus posing a powerful feedback inhibition loop that blunts the impact of elevated NAMPT activity [31]. ...
 
No difference in body weight or fat mass percentage was observed between NamptTg and WT mice fed CD [normal rodent chow: 12% fat] ... NamptTg and WT mice exhibited similar increases in body weight and fat mass percentage when fed HFD [45% fat] or LFD [10% fat] (data not shown). ... NamptTg mice fed VHFD [60% fat] were partially protected against body weight gain ... with 9.0% lower body weight (p < 0.001, Figure 4A) and 8.5% lower fat mass percentage (p < 0.001) ...
 
[However, NAMPTtg did not protect] against insulin resistance. ... Average glucose infusion rate, insulin stimulated glucose disposal rate (Rd), 24 h oxygen consumption (VO2), and respiratory exchange ratio (RER) was not different between NamptTg and WT mice on the same diet (p > 0.050, Figure 4C–F). Daily food intake was not different between mice fed the same diet (p > 0.050, Figure S2A). Daily water intake and daily ambulatory activity was similar across groups (p > 0.050, Figures S2B–C). ... The Baur group reported that skeletal muscle NAMPT overexpression did not protect from metabolic consequences after 24 weeks of VHFD feeding [28], and temporal overexpression of SIRT1 in skeletal muscle had no effect on insulin sensitivity in adult mice [33]. In contrast, increasing whole-body NAD⁺ levels in rodents using NAD⁺-precursors such as NMN and NR [%5B27%5D, %5B30%5D, %5B34%5D] attenuated the glucose and insulin intolerance, which developed in response to HFD feeding. Thus, the exclusive elevation of NAD⁺ in skeletal muscle in the NamptTg mouse might not be enough to improve the metabolic profile. On the other hand, NR or NMN administration will increase the NAD⁺ concentrations in other organs (such as adipose tissue and liver) that might play a pivotal role in preserving metabolic health. ...
 
Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice ... despite similar duration and intensity of the recorded voluntary (running wheel) exercise activity. ... . VO₂max significantly decreased over time in sedentary WT mice (p < 0.050, Figures 5C and S3A). Sedentary NamptTg mice did not decrease VO₂max significantly, suggesting that skeletal muscle NAMPT overexpression might provide some protection (p > 0.050, Figures 5C and S3A). [MR: if you actually look at the figures in question, I'd suggest their interpretation relies on leaning too heavily on a nominally NS p-value: it seems reasonably clear that sedentary NAMPT-TG suffered the same age-related decline in exercise capacity as WT].
 
Skeletal muscle gene expression of several genes related to mitochondrial biogenesis (NRF2, TFAM) (Figure 3A), oxidative phosphorylation (COX5b, ATP5a1, ND1, COX1, ATP6) (Figure 3B), oxidative stress modulation (MnSOD2, TXN2) (Figure 3C), and fuel selection (Cpt1b) (Figure 3D) was significantly higher in NamptTg versus WT mice ... especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice. ...SIRT1 gene expression was not different across groups in red quadriceps muscle (Figure 6A). In white quadriceps muscle, SIRT1 gene expression was lower in exercised NamptTg mice versus sedentary WT and sedentary NamptTg mice ...

 

 
All of this has implications for the potential benefits of supplementation with NAD⁺ precursors — particularly the homeostatic ceiling on NAD⁺, and the lack of effect on insulin resistance or weight gain in animals fed normal or moderately obesogenic chow (although as the authors note, other studies suggest that there is an effect after supplementation when consuming a moderately obesogenic diet, perhaps because NAD⁺ levels (and fuel switching and oxidative capacity) are elevated in multiple tissues, not just the muscles.
 
The better resistance agains age-related decline in exercise capacity despite similar levels of voluntary exercise is also notable. Equally, however, it seems clear that such a benefit only manifests as an enhancement of the effect of exercise: it's not an "exercise-mimetic" that would let you preserve exercise capacity while watching Olympic Track & Field at home.

[stefan_001]

This is an interesting study. The homeostatic ceiling on NAD+ of 1.6x poses some questions though. The study used young mice e.g. HFD was fed for 30 weeks from weaning. So is this ceiling factor age dependent or is it a "constant"? NAD+ levels fall far more than a factor 1.6 as we age so it could we be different at a later age. Would have been more interesting when they would have done it with older mice. At a young age I dont think more NAD+ will help you compensate for a HFD unhealty diet, there is only so much abuse that can be compensated.

Edited by stefan_001, 07 November 2017 - 09:42 PM.

[able]

Excellent!  I find that very encouraging.

 

That the test mice maintained their VO2 Max  and was 2x the control mice as they aged is impressive, and the 3x endurance even more so. 

 

These mice had extra Nampt in muscle, which increased NMN and NAD+ in muscle, but not systemic NAD+.  So I'm not surprised or discouraged at all that it didn't improve insulin resistance or other liver health parameters.  

 

Actually I am surprised the authors thought it might help metabolic health outside of muscle.  

 

I do see that even without exercise, the  increased muscle NAD+ had a slight benefit in fat mass as they aged, which is also encouraging.

 

I know studies show the benefits from exercise decline in older humans.

 

Michael, is the assumption that  NAD+  boosting supplements could allow older subjects to benefit more from exercise? Perhaps maintaining VO2 max and endurance as we age (if we exercise)  like the mice in this study?

 

Also, this study was different in that they did not just boost NAD+ back up in tissue that was deficient in NAD+, but they show a benefit from having MORE Nampt/NAD+   than "normal" mice.    

 

Are there others that have shown a benefit from EXTRA NAD+?

 

 

Edited by able, 08 November 2017 - 05:23 PM.

[Michael]

This is an interesting study. The homeostatic ceiling on NAD+ of 1.6x poses some questions though. The study used young mice e.g. HFD was fed for 30 weeks from weaning. So is this ceiling factor age dependent or is it a "constant"?

 
Good question. I suspect the absolute ceiling (µmol/kg and redox ratio) will be a constant, in the sense that you're likely to plateau as you get closer and closer to a young, otherwise-healthy organism of a given genetics and activity level and consuming a "healthy AL" diet. However, the room for a relative increase in levels might expand in older organisms, since part of the low steady-state in older organisms is due to the "excessive" demand on NAD+-consumers (PARP1, SIRT1, and possibly CD38, or other SIRTs or consumers). Once that "excessive" demand is saturated, there might be additional leeway to get closer to healthy-young NAD+ levels.
 
On the other hand, there are several pieces to the puzzle, and they seem unlikely to all move in the same direction at once. I still haven't posted on this, but despite Chini & Sinclair's report (claiming that the age-related change in NAD+ is almost entirely attributable to increased CD38 activity, with no age-related change in NAMPT, SIRT1, or PARP1), most studies find that an age-related decline in NAMPT expression and/or activity is a major driver of the age-related decline in NAD+, and my hunch is that that's going to be harder to counteract: taking more precursors certainly won't increase its expression or activity, and as levels of NMN rise you might reasonably expect exactly the same negative feedback on its activity seen in young animals at a higher baseline activity. That's a situation where alternative precursors that don't depend on NAMPT are more plausibly going to sidestep the problem, or where tgNAMPT might lead to a greater relative increase —tho' you can also imagine that as you "sidestep" NAMPT with NMN or NR for NAD+ synthesis, the presence of NMN and/or NAD still perversely suppreses NAMPT activity just as NAMPT-derived NMN would, with the net result that the bypass is still counterbalanced by reduced NAMPT activity.
 
Whether higher and higher doses might eventually overcome this is even more speculative — and even if you did, you might then expect negative feedback on NMNAT.
 
And I don't have any informed idea of how CD73 levels or activity change with age or might respond to higher substrate or product levels.
 

NAD+ levels fall far more than a factor 1.6 as we age so it could we be different at a later age. Would have been more interesting when they would have done it with older mice. At a young age I dont think more NAD+ will help you compensate for a HFD unhealty diet, there is only so much abuse that can be compensated.

 
I don't think that's the right way to read these results — especially that last phrase (underlined), and especially since they did see a small benefit in the VHFD group. You might reasonably expect more benefit in animals  subject to some NAD+consuming stress when they're young, as they have a high basal capacity for NAD+ synthesis and a low basal activity of consumers, and the only reason their levels are low is higher consumption. Older animals have less homeostatic dynamic range and a lower steady-state levels that, as discussed, are subject to feedback.
 

These mice had extra Nampt in muscle, which increased NMN and NAD+ in muscle, but not systemic NAD+.  So I'm not surprised or discouraged at all that it didn't improve insulin resistance or other liver health parameters.  
 
Actually I am surprised the authors thought it might help metabolic health outside of muscle.

A couple of things. First, it might be reasonable to expect some functional improvements outside of muscle due to a mixture of intercellular signaling (myokines, exosomes, inflammatory factors, etc), and also because of muscle-derived circulating NAD+ or intermediates (hence, systemic NAD+).
 
More importantly, the way you put these sentences together suggests you think either that the liver is the only place where insulin sensitivity matters, or that they had expected that TgNAMPT in muscle was going to improve hepatic insulin sensitivity, or both. Rather, they had expected that the increased NAD+ in muscle would lead to increased muscle insulin sensitivity—and thereby, an increase in the total glucose metabolic capacity of the organism (whole-body insulin sensitivity). Hepatic insulin sensitivity is most important to fasting glycemia, but muscle sensitivity is very important for postprandial insulin sensitivity and of course activity-induced glucose disposal. Alas, there was no such effect (which is consistent wtih previous findings from Frederick & Baur).
 

Excellent!  I find that very encouraging.
 
That the test mice maintained their VO2 Max  and was 2x the control mice as they aged is impressive, and the 3x endurance even more so. 
 
I know studies show the benefits from exercise decline in older humans.
 
Michael, is the assumption that  NAD+  boosting supplements could allow older subjects to benefit more from exercise? Perhaps maintaining VO2 max and endurance as we age (if we exercise)  like the mice in this study?
 
I do see that even without exercise, the  increased muscle NAD+ had a slight benefit in fat mass as they aged, which is also encouraging.

It's important to remember that this study was done for a relatively brief period starting from when the animals were first weaned: it's premature to extrapolate that this might do something about exercise capacity or fat mass gain during aging.
 

Able wrote: Also, this study was different in that they did not just boost NAD+ back up in tissue that was deficient in NAD+, but they show a benefit from having MORE Nampt/NAD+   than "normal" mice.    
 
Are there others that have shown a benefit from EXTRA NAD+?

 
None clearly so far; as we discussed, it seems biologically quite plausible that normalizing the NAD+ deficit in aging or otherwise-stressed tissues would be beneficial, but much less so to NAD+ levels higher than those in young, healthy organisms. And as noted, we've seen a couple of studies where higher-than-RDAish doses in very young animals has apparently led to functional and metabolic deficits instead of improvements.

[Harkijn]

Some days ago in the News section John Ross already pointed to this study (thanks for that John) but I wonder if it attracted much attention there:

 

Mitochondrial dysfunction in aging and disease, and Aβ aggregation in the brain during AD, can be reversed by NAD+ replenishment approaches, such as dietary supplementation with NR and treatment with PARP inhibitors, such as olaparib (AZD) 

 

https://www.cell.com...91?showall=true