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Might NAD+ Precursors Exacerbate Inflammatory Disease?

nicotinamide riboside nicotinamide nampt autoimmune disease colitis

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#1 Michael

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Posted 12 September 2017 - 03:47 AM


NAD metabolism fuels human and mouse intestinal inflammation

 

In mammals, the salvage pathway is the predominant source of NAD due to its high adaptability. In this pathway, nicotinamide (Nam...) is enzymatically converted into nicotinamide mononucleotide (NMN) by intracellular nicotinamide phosphoribosyltransferase (iNAMPT), which catalyses this first and rate-limiting step of the NAD salvage pathway. ...
 
NAMPT ... has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD.22 23
 
The small molecule inhibitor FK866 potently inhibits NAMPT enzymatic activity in a competitive fashion with little toxicity24 and may represent an attractive target in various inflammatory disorders.19 25 26 Based on these observations, we hypothesised that blockage of the NAD salvage pathway by the NAMPT inhibitor FK866 may be effectively limiting intestinal inflammation.
 
Here, we demonstrate that FK866 protects against experimental colitis and colitis−associated tumorigenesis by suppression of activated leukocytes particularly macrophages, inflammatory monocytes and T cells. NAMPT blockage influenced macrophage differentiation processes, suppressing inflammatory characteristics of M1 and promoting anti-inflammatory properties of M2 macrophages. ...
 
[They used the Dextran Sulfate Sodium (DSS)-induced colitis in mice].

[Suppression of NAD+ Synthesis via] FK866 impairs mucosal NAD recycling resulting in a dampened PARP/SIRT-mediated inflammatory response and alters macrophage polarisation [from pro-inflammatory to anti-inflammatory, repair-oriented polarization]
Nampt expression correlates well with intracellular NAD levels29 and inhibition by FK866 results in cytosolic NAD depletion particularly in inflammation. ... FK866 did not alter the concentrations of NAD, Nam and NMN in the steady state, yet strongly reduced its abundances in the context of DSS-induced inflammation (figure 4A). ...

NAD serves as substrate for NAD-cleaving enzymes such as PARPs and SIR .... Therefore, we investigated the effect of FK866 on the[se] NAD-consuming enzymes ... Indeed, we noted increased PARP1 abundance in mucosal scrapings of WT mice upon DSS exposure (figure 4B). While FK866 had no effect on PARP1 abundances in the steady state, FK866 strongly reduced PARP1 in DSS-exposed mice (figure 4B). ...Increased PARP1 immunoreactivity in DSS-treated mice localised to infiltrating leukocytes and [intestinal epithelial cells] ...

 

As SIRT and PARPs have been linked to the regulation of inflammation, we next investigated whether FK866 directly modulated inflammatory responses. Indeed, FK866 minimised the release of proinflammatory cytokines such as IL-1β, IL-6, TNFα and Lcn2 after LPS treatment of RAW 264.7 cells (online supplementary figure 3B). In line with our in vivo data, we observed a strong reduction in nuclear expression of PARP1 and Sirt6 in FK866-treated, LPS-stimulated RAW cells. PARP1 and Sirt6 have been shown to interfere with nuclear factor kappa B (NF-κB) signalling.33 34 Notably, FK866 treatment strongly reduced NF-κB phosphorylation consequent to LPS treatment ...

 

These results indicate that FK866’s anti-inflammatory capacity involves a reduction in the activity of NAD-dependent enzymes including PARP1 and Sirt6 thereby suppressing NF-κB-dependent immune responses and skewing macrophage differentiation and polarisation towards M2-like macrophages.

 

FK866 treatment protects mice from colitis-associated cancer [still in mice!]

 

Importantly, FK866 also reduced inflammatory responses of lamina propria mononuclear cells (LPMNC) from colonic biopsies of [human] patients with IBD to a comparable extent as dexamethasone. ...

 

Thus, we suggest FK866 as a promising novel compound for the treatment of IBD.


Edited by Michael, 12 September 2017 - 03:50 AM.

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#2 Harkijn

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Posted 12 September 2017 - 09:00 AM

Hi Michael, what do they mean by 'the steady state'?



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#3 MikeDC

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Posted 12 September 2017 - 02:38 PM

It appears that NAMPT is critical for NAD+ synthesis and too much of it causes inflammation. So NAMPT is not a good target to increase NAD+.

 

 


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#4 able

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Posted 12 September 2017 - 03:29 PM

NAMPT IS critical for NAD+ synthesis.  

 

I don't see that this study tells us if it is inhibition of NAMPT, or, the resulting decrease in NAD+ that results in decreased PARP and inflammation.

 

Would be good if they supplemented NR, NMN, and/or NAD+ to the same mouse model to see what effect it has on the inflammation, both before, and after fk866 NAMPT inhibition.

 

IF inflammation was less after fk866, then returned  (or not) after NAD+ replenishment,  then we'd know if it was the NAMPT, or NAD+ itself.

 

 

 


Edited by able, 12 September 2017 - 03:32 PM.

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#5 MikeDC

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Posted 12 September 2017 - 04:34 PM

50,000 papers has proven the importance of NAD+. Reducing NAD+ and getting health benefit is unbelievable.
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#6 Michael

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Posted 12 September 2017 - 06:41 PM

Hi Michael, what do they mean by 'the steady state'?

 
Without the inflammatory stimulus. Ie, NAMPT (and elevated NAD+) doesn't simply cause inflammation.
 

NAMPT IS critical for NAD+ synthesis.  
 
I don't see that this study tells us if it is inhibition of NAMPT, or, the resulting decrease in NAD+ that results in decreased PARP and inflammation.
 
Would be good if they supplemented NR, NMN, and/or NAD+ to the same mouse model to see what effect it has on the inflammation, both before, and after fk866 NAMPT inhibition.
 
IF inflammation was less after fk866, then returned  (or not) after NAD+ replenishment,  then we'd know if it was the NAMPT, or NAD+ itself.


That's good thinking. Strictly speaking you're right that it would be better if they'd gone to more trouble to nail down the attribution to NAD+ vs. to some noncanonical activity of NAMPT.
 
However, we do already know that PARP1 and SIRT6 are NAD+-dependent enzymes (that, along with disruption of the PARP1-DBC1 complex, is why NMN supplementation restored PARP1-mediated DNA repair in aging mice, you'll remember), and there's no known function of intracellular function of NAMPT other than synthesis of NAD+ (though there are now largely-discounted claims of a non-NAD+-synthesizing function for extracellular NAMPT ("visfatin") ). And SIRT6 and PARP1 have known roles in inflammation, and PARP1-knockout mice are protected from inflammation and mortality in the DSS model, consistent with this as well. So there's a pretty strong case from the known biology that the observed effect is because FK866 is inhibiting NAMPT and thereby reducing the induction of NAD+ salvage in response to inflammation.


Edited by Michael, 12 September 2017 - 06:46 PM.

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#7 MikeDC

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Posted 12 September 2017 - 07:01 PM

Sirt1 down regulate inflammation.

This paper shows purple sweet potato reduces inflammation through increasing NAD+.

Calorie restriction also increases NAD+ and reduces inflammation.

https://www.ncbi.nlm...ad inflammation

Edited by MikeDC, 12 September 2017 - 07:07 PM.

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#8 Michael

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Posted 12 September 2017 - 07:20 PM

Sirt1 down regulate inflammation.

This paper shows purple sweet potato reduces inflammation through increasing NAD+.

Calorie restriction also increases NAD+ and reduces inflammation.

https://www.ncbi.nlm...ad inflammation

 

There's evidence for that — although not in the study you link here, and all of the evidence to that effect that I've seen has been in vitro (got an in vivo study?). However, in this model, inhibition of NAMPT inhibited NAD+ synthesis and reduced inflammation via reducing NAD+-dependent PARP1 and SIRT6.



#9 stefan_001

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Posted 12 September 2017 - 07:43 PM

The only thing this study shows is that in an experimental dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer and in human with the real disease depleting NAD+ is usefull. Seems to me that is hardly evidence that can be extrapolated to healthy tissue.


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#10 Michael

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Posted 12 September 2017 - 10:39 PM

The only thing this study shows is that in an experimental dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer and in human with the real disease depleting NAD+ is usefull. Seems to me that is hardly evidence that can be extrapolated to healthy tissue.

 
Sure: that's why I titled the thread "Might NAD+ Precursors Exacerbate Inflammatory Disease?", not "Might NAD+ Precursors Cause Inflammatory Disease?", and I explained above to Harkijn that when the investigators report that "FK866 did not alter the concentrations of NAD, Nam and NMN in the steady state, yet strongly reduced its abundances in the context of DSS-induced inflammation," they meant a lack of effect in the absence of inflammatory stimulus.

 

That said, this clearly has implications beyond mice administered DSS or even human IBD: the authors cite several previous studies  in mice and humans (most of which summary I quoted in my opening post) showing NAMPT hyperactivation and/or NAD+-dependent inflammatory signaling in a range of other inflammatory diseases, and in diabetes, which is now well-understood to be in many cases driven in part by inflammation resulting from visceral fat accumulation.



#11 stefan_001

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Posted 12 September 2017 - 11:52 PM

The only thing this study shows is that in an experimental dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer and in human with the real disease depleting NAD+ is usefull. Seems to me that is hardly evidence that can be extrapolated to healthy tissue.


Sure: that's why I titled the thread "Might NAD+ Precursors Exacerbate Inflammatory Disease?", not "Might NAD+ Precursors Cause Inflammatory Disease?", and I explained above to Harkijn that when the investigators report that "FK866 did not alter the concentrations of NAD, Nam and NMN in the steady state, yet strongly reduced its abundances in the context of DSS-induced inflammation," they meant a lack of effect in the absence of inflammatory stimulus.

That said, this clearly has implications beyond mice administered DSS or even human IBD: the authors cite several previous studies in mice and humans (most of which summary I quoted in my opening post) showing NAMPT hyperactivation and/or NAD+-dependent inflammatory signaling in a range of other inflammatory diseases, and in diabetes, which is now well-understood to be in many cases driven in part by inflammation resulting from visceral fat accumulation.

Ah okay then we actually agree. The elevated NAMPT could be part of the disease pattern and targetting inhibition may be a usefull strategy. It would have been great to understand how much extra NAD+ is generated in the cell in order to understand whether NAD+ precurser boosting would make a difference in term of extra NAD+ that comes from that.

#12 MikeDC

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Posted 13 September 2017 - 12:00 AM

https://www.ncbi.nlm...9109034/related

NAMPT has multiple roles. NAD+ synthesis is just one of them.
Nampt plays an important role in the regulation of insulin secretion insulin secretion in pancreatic β-cells. Nampt also functions as an immunomodulatory cytokine cytokine and is involved in the regulation of inflammatory responses.

Edited by MikeDC, 13 September 2017 - 12:02 AM.

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#13 MikeDC

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Posted 16 September 2017 - 10:34 PM

This is from personal experience thread.

"I gave a bottle of HPN NR to a woman, 40 y.o, with severe arthritis (excruciating pain on wrist, ankle, and feet/hand fingers), hoping she felt some relief.

15 days after, she told me she´s not feeling any pain and sleeping like a baby (she had insomnia).

Truly, I don´t know what is happening ´cause I never read anything about NR helping arthritis, and I only gave her the bottle ´cause she insisted a lot."
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#14 Michael

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Posted 24 September 2017 - 03:40 AM

Now, here's a surprising twist ....

 

Recall from the opening post that NAMPT, the rate-limiting enzyme for NAD+ synthesis via the dominant salvage pathway, "has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD." The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation in animal models of IBD and in vulnerable tissues from human IBD patients, with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND  CD38 (both previously implicated in inflammation)  as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly.

 

Because the canonical function of NAMPT is NAD+ biosynthesis, and because PARP1  and CD38 are NAD+-dependent enzymes, I speculated that NAD+ precursors might similarly exacerbate inflammatory disease by providing additional substrate for these enzymes

 

This group of Chinese researchers claims (a bit prematurely, based on very preliminary evidence) that contrary to what you might expect, NMN actually "alleviates diabetic nephropathy inflammatory‑fibrosis by inhibiting endogenous Nampt."

 

That significantly overstates what they've actually shown: most of it is just gene-expression studies, showing that expression of NAMPT and inflammation- and fibrosis-associated genes are suppressed upon NMN treatment in cells in high glucose and diabetic mice, without either showing effects on protein levels or activity, or actually showing any effect in vivo on actual inflammation and renal disease. Product inhibition of NAMPT by its product seems plausible, at least, and that would (all things being equal) reduce NAD+ availability and activity of NAD+-consuming enzymes — but of course, NMN itself is a precursor of NAD+, which is what got me speculating in the opening post.

 

They additionally note that in the diabetic milieu, high oxidative stress also lowers the  NAD+:NADH ratio (which is a known fact), but how exactly that is supposed to fit in with the broader research on NAMPT in inflammation or the (possible) anti-inflammatory, anti-fibrotic effect of NMN suggested by their study is not clear.

 

 



#15 stefan_001

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Posted 24 September 2017 - 10:02 AM

Now, here's a surprising twist ....

Recall from the opening post that NAMPT, the rate-limiting enzyme for NAD+ synthesis via the dominant salvage pathway, "has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD." The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation in animal models of IBD and in vulnerable tissues from human IBD patients, with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND CD38 (both previously implicated in inflammation) as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly.

Because the canonical function of NAMPT is NAD+ biosynthesis, and because PARP1 and CD38 are NAD+-dependent enzymes, I speculated that NAD+ precursors might similarly exacerbate inflammatory disease by providing additional substrate for these enzymes

This group of Chinese researchers claims (a bit prematurely, based on very preliminary evidence) that contrary to what you might expect, NMN actually "alleviates diabetic nephropathy inflammatory‑fibrosis by inhibiting endogenous Nampt."

That significantly overstates what they've actually shown: most of it is just gene-expression studies, showing that expression of NAMPT and inflammation- and fibrosis-associated genes are suppressed upon NMN treatment in cells in high glucose and diabetic mice, without either showing effects on protein levels or activity, or actually showing any effect in vivo on actual inflammation and renal disease. Product inhibition of NAMPT by its product seems plausible, at least, and that would (all things being equal) reduce NAD+ availability and activity of NAD+-consuming enzymes — but of course, NMN itself is a precursor of NAD+, which is what got me speculating in the opening post.

They additionally note that in the diabetic milieu, high oxidative stress also lowers the NAD+:NADH ratio (which is a known fact), but how exactly that is supposed to fit in with the broader research on NAMPT in inflammation or the (possible) anti-inflammatory, anti-fibrotic effect of NMN suggested by their study is not clear.

Hi Michael, interesting study indeed. Wrt SIRT1 I understood the study to suggest that increased NAMPT leads to sgnalling causing SIRT1 down regulation:
-Increased Nampt and NF‐κB p65 expression and decreased Sirt1 expression in response to HG conditions over time.
And that NMN not only activates SIRT1 via more NAD+ as is usually shown but also:
-The results indicated that NMN was able to activate Sirt1 by inhibiting the Nampt pathway.

Or?

I have read couple studies indicating that during aging the salvage effectives goes down and there is compensation via increased NAMPT. So perhaps we are seeing here a NMN specific anti aging effect via direct signalling pathway leading to more sirt1 apart fom the nad+ boosting.

Edited by stefan_001, 24 September 2017 - 10:03 AM.


#16 stefan_001

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Posted 24 September 2017 - 11:28 AM

then again that only makes sense if the NMN adds more NAD+ than is reduced by the NAMPT inhibition. Unless the path of action is still different and that NMN blocks the NAMPT pathway that inhibits SIRT1 without actually inhibiting NAMPT. Mmmm...

Edited by stefan_001, 24 September 2017 - 11:59 AM.


#17 MikeDC

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Posted 24 September 2017 - 11:46 AM

Great study!
Diabetes causes inflammation which lowers NAD+. This up regulates NAMPT and other bad genes and down regulate Sirt1.
Supplementing NMN or NR increase NAD+ and suppress NAMPT and other bad genes through the negative feed back loop
And up regulate Sirt1.

So keeping NAD+ high with external NMN and NR will suppress NAMPT and bad genes and increase Sirt1
Expression.
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#18 MikeDC

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Posted 24 September 2017 - 01:00 PM

They also did in vivo study on NMN supplementation.

"To explore the effect of these results in the whole animal, severely diabetic rats were treated with NMN in the present study, and mRNA was isolated and measured from kidney tissues of the rats. The results indicated that the mRNA expression levels of Nampt, NF‐κB p65 and vimentin were signi cantly decreased in the NMN‐treated tissue, whereas Sirt1 expression was significantly increased compared with the untreated groups, respectively. The results further indicated that the Nampt‐NF‐κB p65 signaling pathway is likely inhibited via NMN‐induced inhibition of Nampt in a negative feedback loop. This suggests that endogenous Nampt overexpression may be closely involved in the pathogenesis of glomeruli brosis of DN"

#19 MikeDC

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Posted 24 September 2017 - 01:09 PM

to reconcile the papers, NAMPT up regulation is pro inflammatory and down regulation is anti inflammatory.
Low NAD+ up regulate NAMPT.
External NMN and NR increase NAD+ and down regulate NAMPT

Any process that decreases NAD+ create a vicious cycle of inflammation.
NAD+ precursors can put a stop to it by creating a positive feed back loop.
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#20 MikeDC

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Posted 24 September 2017 - 01:15 PM

Since use of NAM as NAD+ precursor depends on NAMPT, there is a check and balance that prevented NAD+ and NAD+/ NADH ratio
From going high enough to start a positive feed back loop. That is why NAM and Niacin can increase NAD+, but
Don't have the same anti aging effects that NMN and NR have.
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#21 MikeDC

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Posted 24 September 2017 - 01:55 PM

NAMPT IS critical for NAD+ synthesis.

I don't see that this study tells us if it is inhibition of NAMPT, or, the resulting decrease in NAD+ that results in decreased PARP and inflammation.

Would be good if they supplemented NR, NMN, and/or NAD+ to the same mouse model to see what effect it has on the inflammation, both before, and after fk866 NAMPT inhibition.

IF inflammation was less after fk866, then returned (or not) after NAD+ replenishment, then we'd know if it was the NAMPT, or NAD+ itself.

The paper did what you said in a different way. They showed increasing NAD+ from NMN that doesn't need NAMPT reduced inflammation and increased Sirt1 expression. So NAD+ is not the cause of inflammation.

Edited by MikeDC, 24 September 2017 - 01:56 PM.


#22 MikeDC

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Posted 24 September 2017 - 02:20 PM

Now, here's a surprising twist ....

Recall from the opening post that NAMPT, the rate-limiting enzyme for NAD+ synthesis via the dominant salvage pathway, "has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD." The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation in animal models of IBD and in vulnerable tissues from human IBD patients, with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND CD38 (both previously implicated in inflammation) as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly.

Because the canonical function of NAMPT is NAD+ biosynthesis, and because PARP1 and CD38 are NAD+-dependent enzymes, I speculated that NAD+ precursors might similarly exacerbate inflammatory disease by providing additional substrate for these enzymes

This group of Chinese researchers claims (a bit prematurely, based on very preliminary evidence) that contrary to what you might expect, NMN actually "alleviates diabetic nephropathy inflammatory‑fibrosis by inhibiting endogenous Nampt."

That significantly overstates what they've actually shown: most of it is just gene-expression studies, showing that expression of NAMPT and inflammation- and fibrosis-associated genes are suppressed upon NMN treatment in cells in high glucose and diabetic mice, without either showing effects on protein levels or activity, or actually showing any effect in vivo on actual inflammation and renal disease. Product inhibition of NAMPT by its product seems plausible, at least, and that would (all things being equal) reduce NAD+ availability and activity of NAD+-consuming enzymes — but of course, NMN itself is a precursor of NAD+, which is what got me speculating in the opening post.

They additionally note that in the diabetic milieu, high oxidative stress also lowers the NAD+:NADH ratio (which is a known fact), but how exactly that is supposed to fit in with the broader research on NAMPT in inflammation or the (possible) anti-inflammatory, anti-fibrotic effect of NMN suggested by their study is not clear.

Hi Michael, interesting study indeed. Wrt SIRT1 I understood the study to suggest that increased NAMPT leads to sgnalling causing SIRT1 down regulation:
-Increased Nampt and NF‐κB p65 expression and decreased Sirt1 expression in response to HG conditions over time.
And that NMN not only activates SIRT1 via more NAD+ as is usually shown but also:
-The results indicated that NMN was able to activate Sirt1 by inhibiting the Nampt pathway.

Or?

I have read couple studies indicating that during aging the salvage effectives goes down and there is compensation via increased NAMPT. So perhaps we are seeing here a NMN specific anti aging effect via direct signalling pathway leading to more sirt1 apart fom the nad+ boosting.

No. NMN increases NAD+ which causes down regulation of NAMPT through the negative feed back loop between NAD+ and NAMPT.

Also note that even though NAMPT is increased greatly during inflammation, the NAD+ and NAD/ NADH ratio still are below controls.

#23 stefan_001

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Posted 24 September 2017 - 05:38 PM

Now, here's a surprising twist ....

Recall from the opening post that NAMPT, the rate-limiting enzyme for NAD+ synthesis via the dominant salvage pathway, "has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD." The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation in animal models of IBD and in vulnerable tissues from human IBD patients, with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND CD38 (both previously implicated in inflammation) as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly.

Because the canonical function of NAMPT is NAD+ biosynthesis, and because PARP1 and CD38 are NAD+-dependent enzymes, I speculated that NAD+ precursors might similarly exacerbate inflammatory disease by providing additional substrate for these enzymes

This group of Chinese researchers claims (a bit prematurely, based on very preliminary evidence) that contrary to what you might expect, NMN actually "alleviates diabetic nephropathy inflammatory‑fibrosis by inhibiting endogenous Nampt."

That significantly overstates what they've actually shown: most of it is just gene-expression studies, showing that expression of NAMPT and inflammation- and fibrosis-associated genes are suppressed upon NMN treatment in cells in high glucose and diabetic mice, without either showing effects on protein levels or activity, or actually showing any effect in vivo on actual inflammation and renal disease. Product inhibition of NAMPT by its product seems plausible, at least, and that would (all things being equal) reduce NAD+ availability and activity of NAD+-consuming enzymes — but of course, NMN itself is a precursor of NAD+, which is what got me speculating in the opening post.

They additionally note that in the diabetic milieu, high oxidative stress also lowers the NAD+:NADH ratio (which is a known fact), but how exactly that is supposed to fit in with the broader research on NAMPT in inflammation or the (possible) anti-inflammatory, anti-fibrotic effect of NMN suggested by their study is not clear.

Hi Michael, interesting study indeed. Wrt SIRT1 I understood the study to suggest that increased NAMPT leads to sgnalling causing SIRT1 down regulation:
-Increased Nampt and NF‐κB p65 expression and decreased Sirt1 expression in response to HG conditions over time.
And that NMN not only activates SIRT1 via more NAD+ as is usually shown but also:
-The results indicated that NMN was able to activate Sirt1 by inhibiting the Nampt pathway.

Or?

I have read couple studies indicating that during aging the salvage effectives goes down and there is compensation via increased NAMPT. So perhaps we are seeing here a NMN specific anti aging effect via direct signalling pathway leading to more sirt1 apart fom the nad+ boosting.
No. NMN increases NAD+ which causes down regulation of NAMPT through the negative feed back loop between NAD+ and NAMPT.

Also note that even though NAMPT is increased greatly during inflammation, the NAD+ and NAD/ NADH ratio still are below controls.
as long as the NAD+ is not rate limited by the increased NAMPT the feedback loop doesnt bring the NAD+ generation down. Would be interesting to see some in vivo data on that where they use a NAMPt inhibitor.

Edited by stefan_001, 24 September 2017 - 05:41 PM.


#24 MikeDC

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Posted 24 September 2017 - 05:47 PM

It is quite established that NAMPT is the rate limiting step of NAD+ recycling.
Otherwise there won't be a negative feed back loop. They did the study in vivo and proved
That NMN brought down NAMPT expression in vivo.

Everything fit nicely on the anti aging theory of NR and NMN.

#25 stefan_001

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Posted 24 September 2017 - 05:56 PM

Periodontitis study links increased NAMPT to SIRT2:

Involvement of SIRT2 in NAMPT-mediated PTGS2, MMP1 and MMP3 expression was verified by SIRT2 overexpression using Ad-SIRT2. Our results demonstrate that SIRT2 might be involved in NAMPT-induced gene expression in periodontal inflammation.

However, EX527, a SIRT1-specific inhibitor,28 had no effect on NAMPT overexpression-induced upregulation of MMP1, MMP3 and PTGS2.

They are howeveralso loosely linking to NAD+ in this disease case:

As expected, inhibition of SIRT activity with NIC significantly blocked IL-1β-induced or Ad-Nampt-induced upregulation of PTGS2, MMP1 and MMP3 (Figures 5c and d) in addition to the activities of these target genes (Figures 5e and f). E

NAMPT enzymatic activity is known to stimulate the synthesis of NAD+,14, 22 an essential cofactor of SIRT protein deacetylases. NAMPT overexpression or IL-1β treatment increased NAD+-dependent SIRT activity in human GF, and SIRT activity was inhibited by NIC, a broad SIRT inhibitor.27

Btw NIC = nicotinamide

NAMPT enzyme activity regulates catabolic gene expression in gingival fibroblasts during periodontitis
https://www.ncbi.nlm...10/#!po=71.6667

Edited by stefan_001, 24 September 2017 - 05:57 PM.


#26 stefan_001

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Posted 24 September 2017 - 06:00 PM

It is quite established that NAMPT is the rate limiting step of NAD+ recycling.
Otherwise there won't be a negative feed back loop. They did the study in vivo and proved
That NMN brought down NAMPT expression in vivo.

Everything fit nicely on the anti aging theory of NR and NMN.

But if NAMPT is also rate limiting even when overexpressed then NMN / NR have to compensate for the downregulation. Perhaps there is a ceiling for increased NAMPT leading to more NAD+ that gets hit?Did they measure NAD+ before and after NMN supplementation?

This may be a consideration for the dosing level of NR / NMN.

Edited by stefan_001, 24 September 2017 - 06:21 PM.


#27 MikeDC

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Posted 24 September 2017 - 07:12 PM

It is quite established that NAMPT is the rate limiting step of NAD+ recycling.
Otherwise there won't be a negative feed back loop. They did the study in vivo and proved
That NMN brought down NAMPT expression in vivo.

Everything fit nicely on the anti aging theory of NR and NMN.

But if NAMPT is also rate limiting even when overexpressed then NMN / NR have to compensate for the downregulation. Perhaps there is a ceiling for increased NAMPT leading to more NAD+ that gets hit?Did they measure NAD+ before and after NMN supplementation?

This may be a consideration for the dosing level of NR / NMN.

You probably didn't consider NAD+ consumption. When NAMPT is up regulated, more NAD+ is produced. But most of the increased NAD+ are consumed by CD38 and other genes. Obviously up regulation of NAMPT is not unlimited so NAD+ don't get back to normal level before inflammation. I don't think we need to revisit the ability of NR and NMN to increase NAD+.

Periodontitis study links increased NAMPT to SIRT2:

Involvement of SIRT2 in NAMPT-mediated PTGS2, MMP1 and MMP3 expression was verified by SIRT2 overexpression using Ad-SIRT2. Our results demonstrate that SIRT2 might be involved in NAMPT-induced gene expression in periodontal inflammation.

However, EX527, a SIRT1-specific inhibitor,28 had no effect on NAMPT overexpression-induced upregulation of MMP1, MMP3 and PTGS2.

They are howeveralso loosely linking to NAD+ in this disease case:

As expected, inhibition of SIRT activity with NIC significantly blocked IL-1β-induced or Ad-Nampt-induced upregulation of PTGS2, MMP1 and MMP3 (Figures 5c and d) in addition to the activities of these target genes (Figures 5e and f). E

NAMPT enzymatic activity is known to stimulate the synthesis of NAD+,14, 22 an essential cofactor of SIRT protein deacetylases. NAMPT overexpression or IL-1β treatment increased NAD+-dependent SIRT activity in human GF, and SIRT activity was inhibited by NIC, a broad SIRT inhibitor.27

Btw NIC = nicotinamide

NAMPT enzyme activity regulates catabolic gene expression in gingival fibroblasts during periodontitis
https://www.ncbi.nlm...10/#!po=71.6667


Maybe some assumptions were wrong. This article says sirt2 is anti inflammation.
https://www.ncbi.nlm...t2 inflammation

#28 Michael

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Posted 24 September 2017 - 07:27 PM

 

Now, here's a surprising twist ....

Recall from the opening post that NAMPT, the rate-limiting enzyme for NAD+ synthesis via the dominant salvage pathway, "has been implicated in a variety of inflammatory disorders such as sepsis, rheumatoid arthritis and diabetes.18–20 Furthermore, NAMPT overexpression has been associated with tumorigenesis.21 We have reported elevated NAMPT concentrations in colonic tissue and serum of patients with IBD [inflammatory bowel disease] and recently, NAMPT has been identified as a marker for severity in paediatric IBD." The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation in animal models of IBD and in vulnerable tissues from human IBD patients, with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND CD38 (both previously implicated in inflammation) as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly.

Because the canonical function of NAMPT is NAD+ biosynthesis, and because PARP1 and CD38 are NAD+-dependent enzymes, I speculated that NAD+ precursors might similarly exacerbate inflammatory disease by providing additional substrate for these enzymes

This group of Chinese researchers claims (a bit prematurely, based on very preliminary evidence) that contrary to what you might expect, NMN actually "alleviates diabetic nephropathy inflammatory‑fibrosis by inhibiting endogenous Nampt."


Hi Michael, interesting study indeed. Wrt SIRT1 I understood the study to suggest that increased NAMPT leads to sgnalling causing SIRT1 down regulation:
-Increased Nampt and NF‐κB p65 expression and decreased Sirt1 expression in response to HG conditions over time.

And that NMN not only activates SIRT1 via more NAD+ as is usually shown but also:
-The results indicated that NMN was able to activate Sirt1 by inhibiting the Nampt pathway.

 


You're misreading either my post, or the first study, or both: when I say, above, "The study in the opening post found that FK866, an inhibitor of NAMPT, inhibited inflammation [...] with evidence that it worked via inhibiting levels and activity of the NAD+-dependent enzymes PARP1 AND CD38 (both previously implicated in inflammation) as well as and multiple sirtuins (most profoundly SIRT6, but also a rather striking effect on SIRT1), surprisingly," note that I am referring to "the study in the opening post," which indeed did find:
 

FK866 impairs mucosal NAD recycling resulting in a dampened PARP/SIRT-mediated inflammatory response ... FK866 reduced mucosal abundance of various SIRT (online supplementary figure 3A) during intestinal inflammation with the most pronounced effect on Sirt6 (figure 4B,D). Again this was paralleled by a markedly decreased enzymatic function as measured by deacetylase activity (figure 4C).

 
Their supplementary figure 3A shows that SIRT1 protein levels are more than doubled in DSS-IBS mice vs. water-treated non-IBD controls, and FK866 reduced it down even below that control baseline. (Quantitation of immunoblots is a soft science, but that's a very large and obvious effect).

When you say:
 

And that NMN not only activates SIRT1 via more NAD+ as is usually shown but also:
-The results indicated that NMN was able to activate Sirt1 by inhibiting the Nampt pathway.


... those are results from the second, Chinese study, which does report to the contrary that NMN was able to increase gene expression of Sirt1 in association with decresed expression of NAMPT. (Despite their sloppy use of language, they provide no data on "activation" (increased activity) of SIRT1 — just increased gene expression). So that's another oddity in all of this. I would certainly trust protein levels more than gene expression readouts, which can always be nullified posttranslationally.

stefan_001 wrote: Or?

I have read couple studies indicating that during aging the salvage effectives goes down and there is compensation via increased NAMPT. So perhaps we are seeing here a NMN specific anti aging effect via direct signalling pathway leading to more sirt1 apart fom the nad+ boosting.

 
All the studies I've seen show either no effect of aging on NAMPT (Chini & Sinclair) or downregulation (Imai, Radak, and importantly Miao, which as you spotted the other day gives the first human data on this question in addition to more mouse data, finding a "decrease of NAD+ content in livers from elderly patients compared with those from middle-aged patients and found that the NAMPT-mediated NAD+ salvage pathway, but not indolamine 2,3-dioxygenase (IDO)-mediated NAD+ de novo pathway, was impaired in livers from elderly humans compared with those from middle-aged patients." One caution is that these are all "patients" with either hepatolithiasis or hepatocellular carcinoma, rather than otherwise-healthy aging people. Still, even among patients, the levels were down with age.

I am aware of no proposed mechanism or data to support a role for NMN in controlling SIRT1 expression, and the only role of which I'm aware for NMN in SIRT1 activity is via NAD+. Are you aware of any such?
 
stefan_001 wrote: then again that only makes sense if the NMN adds more NAD+ than is reduced by the NAMPT inhibition. Unless the path of action is still different and that NMN blocks the NAMPT pathway that inhibits SIRT1 without actually inhibiting NAMPT. Mmmm...
 
I agree with your first sentence; I'm not clear on what you're suggesting with the second, but their entire thesis is that NMN is providing product inhibition of the NAMPT enzyme, and support that with expression data.
 

Great study!
Diabetes causes inflammation which lowers NAD+. This up regulates NAMPT and other bad genes and down regulate Sirt1.
Supplementing NMN or NR increase NAD+ and suppress NAMPT and other bad genes through the negative feed back loop
And up regulate Sirt1.

So keeping NAD+ high with external NMN and NR will suppress NAMPT and bad genes and increase Sirt1
Expression.


It's a bit silly to describe any gene as a "bad gene" (unless maybe you mean a disease-associated mutation), and certainly calling the most important gene in NAD+ biosynthesis a "bad gene" is a bit rich. FK866 inhibits NAMPT and thereby PARP1, SIRT1, SIRT3, and SIRT6; "Sirt6 regulated TNFα production in dendritic cells in a NAD and NAMPT-dependent manner39 and Sirt1 represents an important regulator of monocyte/macrophage polarisation towards M1.4" Are those 'bad genes' for their role in inflammation?

MikeDC wrote:
They also did in vivo study on NMN supplementation.

"To explore the effect of these results in the whole animal, severely diabetic rats were treated with NMN in the present study, and mRNA was isolated and measured from kidney tissues of the rats. The results indicated that the mRNA expression levels of Nampt, NF‐κB p65 and vimentin were significantly decreased in the NMN‐treated tissue, whereas Sirt1 expression was significantly increased compared with the untreated groups, respectively. The results further indicated that the Nampt‐NF‐κB p65 signaling pathway is likely inhibited via NMN‐induced inhibition of Nampt in a negative feedback loop. This suggests that endogenous Nampt overexpression may be closely involved in the pathogenesis of glomeruli fibrosis of DN"


Yes — as I noted in my post, they show "that expression of NAMPT and inflammation- and fibrosis-associated genes are suppressed upon NMN treatment in cells in high glucose and diabetic mice, without either showing effects on protein levels or activity, or actually showing any effect in vivo on actual inflammation and renal disease."

MikeDC wrote:to reconcile the papers, NAMPT up regulation is pro inflammatory and down regulation is anti inflammatory.
Low NAD+ up regulate NAMPT.
External NMN and NR increase NAD+ and down regulate NAMPT

Any process that decreases NAD+ create a vicious cycle of inflammation.
NAD+ precursors can put a stop to it by creating a positive feed back loop.


That just restates the paradox — it doesn't resolve it, unless (contrary to the implications of the presented evidence) there is some hitherto-unknown NAD+-independent pro-inflammatory effect of NAMPT. (Again, there are claims of a non-NAD+-synthesizing function for extracellular NAMPT ("visfatin"/PBEF), but those are now largely-discounted, and IAC they wouldn't explain the cell culture data). And most studies find NAMPT activity or expression is downregulated in aging, and CR protects against that decline, so certainly it would be prima facie unwise for otherwise-healthy aging people to be doing anything to downregulate NAMPT.

able wrote:
NAMPT IS critical for NAD+ synthesis.

I don't see that this study tells us if it is inhibition of NAMPT, or, the resulting decrease in NAD+ that results in decreased PARP and inflammation.

Would be good if they supplemented NR, NMN, and/or NAD+ to the same mouse model to see what effect it has on the inflammation, both before, and after fk866 NAMPT inhibition. IF inflammation was less after fk866, then returned (or not) after NAD+ replenishment, then we'd know if it was the NAMPT, or NAD+ itself.

 
Correct — that would be ideal. Again, however, from the known biology I can't think of a non-NAD+-based mechanism, and they provide evidence of effects via NAD+-consuming enzymes (albeit not conclusively).

MikeDC wrote: The paper did what you said in a different way. They showed increasing NAD+ from NMN that doesn't need NAMPT reduced inflammation and increased Sirt1 expression. So NAD+ is not the cause of inflammation.

That doesn't follow, absent a mechanism for an NAD+-independent effect of NAMPT in ignoring the dependence of PARP1 and the rest on NAD+.

MikeDC wrote:
NMN increases NAD+ which causes down regulation of NAMPT through the negative feed back loop between NAD+ and NAMPT.



That's not what they're suggesting: they're suggesting product inhibition by NMN itself. Did you misspeak, or do you have evidence for what you wrote? Their mechanism certainly is more prima facie plausible.

MikeDC wrote: Also note that even though NAMPT is increased greatly during inflammation, the NAD+ and NAD/NADH ratio still are below controls.

Yes, but that's because of reductive stress created by hyperglycemia leading to an excess of glycolytic and TCA cycle intermediates and activation of the polyol pathway, as well as increased consumption of NAD+ by PARP1, all of which alters the ratio of the NAD+:NADH couple, not a reduction in NAD+ biosynthesis (via salvage or otherwise).


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#29 MikeDC

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Posted 24 September 2017 - 07:36 PM

I am getting tired arguing. Each person has a different understanding based on the same data. If you keep chasing your own tails it will never ends. NR and NMN are the holy grails of anti aging. This statement will end my discussions about the validity of NR in this forum.
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#30 able

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Posted 24 September 2017 - 08:21 PM

Thanks for all your patient explanations Michael - much appreciated!







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