NAD+ boosting by oral nicotinamide mononucleotide administration regulates key metabolic and immune pathways through SIRT1 dependent and independent mechanisms to mitigate diet-induced obesity and dyslipidemia in mice

Reviewer #1 (Public review):

Summary:

This manuscript investigates the effects of oral supplementation with nicotinamide mononucleotide (NMN) on metabolism and inflammation in mice with diet-induced obesity, and whether these effects depend on the NAD⁺-dependent enzyme SIRT1. Using control and inducible SIRT1 knockout mice, the authors show that NMN administration mitigates high-fat diet-induced weight gain, enhances energy expenditure, and normalizes fasting glucose and plasma lipid profiles in a largely SIRT1-dependent manner. However, reductions in fat mass and adipose tissue expansion occur independently of SIRT1. Comprehensive plasma proteomic analyses (O-Link and mass spectrometry) reveal that NMN reverses obesity-induced alterations in metabolic and immune pathways, particularly those related to glucose and cholesterol metabolism. Integrative network and causal analyses identify both SIRT1-dependent and -independent protein clusters, as well as potential upstream regulators such as FBXW7, ADIPOR2, and PRDM16. Overall, the study supports that NMN modulates key metabolic and immune pathways through both SIRT1-dependent and alternative mechanisms to alleviate obesity and dyslipidemia in mice.

Strengths:

Well-written manuscript, and state-of-the-art proteomics-based methodologies to assess NMN and SIRT1-dependent effects.

Weaknesses:

Unfortunately, the study design, as well as the data analysis approach taken by the authors, are flawed. This limits the authors' ability to make the proposed conclusions.

Reviewer #2 (Public review):

Summary:

Majeed and colleagues aimed to evaluate whether the metabolic effects of NMN in the context of a high-fat diet are SIRT1 dependent. For this, they used an inducible SIRT1 KO model (SIRT1 iKO), allowing them to bypass the deleterious effects of SIRT1 ablation during development. In line with previous reports, the authors observed that NMN prevents, to some degree, diet-induced metabolic damage in wild-type mice. When doing similar tests on SIRT1 iKO mice, the authors see that some, but not all, of the effects of NMN are abrogated. The phenotypic studies are complemented by plasma proteomic analyses evaluating the influence of the high-fat diet, SIRT1, and NMN on circulating protein profiles.

Strengths:

The mechanistic aspects behind the potential health benefits of NAD+ precursors have been poorly elucidated. This is in part due to the pleiotropic actions of NAD-related molecules on cellular processes. While sirtuins, most notably SIRT1, have been largely hypothesized to be key players in the therapeutic actions of NAD+ boosters, the proof for this in vivo is very limited. In this sense, this work is an important contribution to the field.

Weaknesses:

While the authors use a suitable methodology (SIRT1 iKO mice), the results show very early that the iKO mice themselves have some notable phenotypes, which complicate the picture. The actions of NMN in WT and SIRT1 KO mice are most often presented separately. However, this is not the right approach to evaluate and visualize SIRT1 dependency. Indeed, many of the "SIRT1-dependent" effects of NMN are consequent to the fact that SIRT1 deletion itself has a phenotype equivalent to or larger than that induced by NMN in wild-type mice. This would have been very evident if the two genotypes had been systematically plotted together. Consequently, and despite the value of the study, the results obtained with this model might not allow for solidly established claims of SIRT1 dependency on NMN actions. The fact that some of the effects of SIRT1 deletion are similar to those of NMN supplementation also makes it counterintuitive to propose that activation of SIRT1 is a major driver of NMN actions. Unbiasedly, one might as well conclude that NMN could act by inhibiting SIRT1. The fact that readouts for SIRT1 activity are not explored makes it also difficult to test the influence of NMN on SIRT1 in their experimental setting, or whether compensations could exist.

A second weak point is that the proteomic explorations are interesting, yet feel too descriptive and disconnected from the overall phenotype or from the goal of the manuscript. It would be unreasonable to ask for gain/loss-of-function experiments based on the differentially abundant peptides. Yet, a deeper exploration of whether their altered presence in circulation is consistent with changes in their expression - and, if so, in which tissues - and a clearer discussion on their link to the phenotypes observed would be needed, especially for changes related to SIRT1 and NMN.

Impact on the field and further significance of the work:

Despite the fact that, in my opinion, the authors might not have conclusively achieved their main aim, there are multiple valuable aspects in this manuscript:

(1) It provides independent validation for the potential benefits of NAD+ boosters in the context of diet-induced metabolic complications. Previous efforts using NR or NMN itself have provided contradicting observations. Therefore, additional independent experiments are always valuable to further balance the overall picture.

(2) The metabolic consequences of deleting SIRT1 in adulthood have been poorly explored in previous works. Therefore, irrespective of the actions of NMN, the phenotypes observed are intriguing, and the proteomic differences are also large enough to spur further research to understand the role of SIRT1 as a therapeutic target.

(3) Regardless of the influence of SIRT1, NMN promotes some plasma proteomic changes that are very well worth exploring. In addition, they highlight once more that the in vivo actions of NMN, as those of other NAD+ boosters, are pleiotropic. Hence, this work brings into question whether single gene KO models are really a good approach to explore the mechanisms of action of NAD+ precursors.

Author response:

Reviewer #1 (Public review):

Summary:

This manuscript investigates the effects of oral supplementation with nicotinamide mononucleotide (NMN) on metabolism and inflammation in mice with diet-induced obesity, and whether these effects depend on the NAD⁺-dependent enzyme SIRT1. Using control and inducible SIRT1 knockout mice, the authors show that NMN administration mitigates high-fat diet-induced weight gain, enhances energy expenditure, and normalizes fasting glucose and plasma lipid profiles in a largely SIRT1-dependent manner. However, reductions in fat mass and adipose tissue expansion occur independently of SIRT1. Comprehensive plasma proteomic analyses (O-Link and mass spectrometry) reveal that NMN reverses obesity-induced alterations in metabolic and immune pathways, particularly those related to glucose and cholesterol metabolism. Integrative network and causal analyses identify both SIRT1-dependent and -independent protein clusters, as well as potential upstream regulators such as FBXW7, ADIPOR2, and PRDM16. Overall, the study supports that NMN modulates key metabolic and immune pathways through both SIRT1-dependent and alternative mechanisms to alleviate obesity and dyslipidemia in mice.

Strengths:

Well-written manuscript, and state-of-the-art proteomics-based methodologies to assess NMN and SIRT1-dependent effects.

We thank the reviewer for highlighting that state-of-the-art proteomic research methods used, and we report for the first time on significant changes in plasma proteomics in mice after NMN supplementation in both wild-type and SIRT1-KO mice using a combination of DIA mass spectrometry and Olink.

Weaknesses:

Unfortunately, the study design, as well as the data analysis approach taken by the authors, are flawed. This limits the authors' ability to make the proposed conclusions.

We agree that the administration of tamoxifen, along with the associated weight loss, could affect the obesity phenotype. For this reason, we ensured that both Cre-positive and Cre-negative mice received tamoxifen. Importantly, after the tamoxifen 'washout', the two groups weighed essentially the same. Going forward, we plan to address this comment by performing additional statistical tests on all six experimental groups to gain insights into dependencies. Based on your suggestions, we will clarify the limitations of the study design and improve the data analysis approaches to provide stronger support for our conclusions in the revised version of the paper.

Reviewer #2 (Public review):

Summary:

Majeed and colleagues aimed to evaluate whether the metabolic effects of NMN in the context of a high-fat diet are SIRT1 dependent. For this, they used an inducible SIRT1 KO model (SIRT1 iKO), allowing them to bypass the deleterious effects of SIRT1 ablation during development. In line with previous reports, the authors observed that NMN prevents, to some degree, diet-induced metabolic damage in wild-type mice. When doing similar tests on SIRT1 iKO mice, the authors see that some, but not all, of the effects of NMN are abrogated. The phenotypic studies are complemented by plasma proteomic analyses evaluating the influence of the high-fat diet, SIRT1, and NMN on circulating protein profiles.

Strengths:

The mechanistic aspects behind the potential health benefits of NAD+ precursors have been poorly elucidated. This is in part due to the pleiotropic actions of NAD-related molecules on cellular processes. While sirtuins, most notably SIRT1, have been largely hypothesized to be key players in the therapeutic actions of NAD+ boosters, the proof for this in vivo is very limited. In this sense, this work is an important contribution to the field.

We thank the reviewer for acknowledging the importance of this work to the field. In this report, we provide in vivo evidence of the action of NAD+ boosting, and hope to delineate the action of Sirt1, as well as the pleiotropic effects of NAD-related molecules on cellular and metabolic processes.

Weaknesses:

While the authors use a suitable methodology (SIRT1 iKO mice), the results show very early that the iKO mice themselves have some notable phenotypes, which complicate the picture. The actions of NMN in WT and SIRT1 KO mice are most often presented separately. However, this is not the right approach to evaluate and visualize SIRT1 dependency. Indeed, many of the "SIRT1-dependent" effects of NMN are consequent to the fact that SIRT1 deletion itself has a phenotype equivalent to or larger than that induced by NMN in wild-type mice. This would have been very evident if the two genotypes had been systematically plotted together. Consequently, and despite the value of the study, the results obtained with this model might not allow for solidly established claims of SIRT1 dependency on NMN actions. The fact that some of the effects of SIRT1 deletion are similar to those of NMN supplementation also makes it counterintuitive to propose that activation of SIRT1 is a major driver of NMN actions. Unbiasedly, one might as well conclude that NMN could act by inhibiting SIRT1. The fact that readouts for SIRT1 activity are not explored makes it also difficult to test the influence of NMN on SIRT1 in their experimental setting, or whether compensations could exist.

We thank the reviewer for raising this point and acknowledge the limitations of using Sirt1 iKO mice. However, inducing Sirt1 KO in adulthood is a better alternative than using a homozygous Sirt1 KO mouse model, as the latter leads to embryonic lethality and many other developmental defects (1, 2). The proteomics analysis can provide insight into the effects of SIRT1 deletion under chow and high-fat diet (HFD) conditions, as well as the effects of diet in the presence or absence of nicotinamide mononucleotide (NMN). We will discuss these limitations and present the results for the two genotypes together, as suggested.

A second weak point is that the proteomic explorations are interesting, yet feel too descriptive and disconnected from the overall phenotype or from the goal of the manuscript. It would be unreasonable to ask for gain/loss-of-function experiments based on the differentially abundant peptides. Yet, a deeper exploration of whether their altered presence in circulation is consistent with changes in their expression - and, if so, in which tissues - and a clearer discussion on their link to the phenotypes observed would be needed, especially for changes related to SIRT1 and NMN.

First, we presented the data in this manner as a proof of concept, to demonstrate the effect of the diet on the plasma proteome and corroborate our findings with those published in the literature. We then investigated the effects of NAD boosting and Sirt1 KO in order to identify significant changes. We agree with the reviewer that it would be unreasonable to validate all the differentially abundant proteins. However, we will choose key proteins and assess their expression in different tissues, such as the liver, white adipose tissue (WAT) and muscles, and attempt to connect these changes with the phenotypes.

Impact on the field and further significance of the work:

Despite the fact that, in my opinion, the authors might not have conclusively achieved their main aim, there are multiple valuable aspects in this manuscript:

(1) It provides independent validation for the potential benefits of NAD+ boosters in the context of diet-induced metabolic complications. Previous efforts using NR or NMN itself have provided contradicting observations. Therefore, additional independent experiments are always valuable to further balance the overall picture.

(2) The metabolic consequences of deleting SIRT1 in adulthood have been poorly explored in previous works. Therefore, irrespective of the actions of NMN, the phenotypes observed are intriguing, and the proteomic differences are also large enough to spur further research to understand the role of SIRT1 as a therapeutic target.

(3) Regardless of the influence of SIRT1, NMN promotes some plasma proteomic changes that are very well worth exploring. In addition, they highlight once more that the in vivo actions of NMN, as those of other NAD+ boosters, are pleiotropic. Hence, this work brings into question whether single gene KO models are really a good approach to explore the mechanisms of action of NAD+ precursors.

We thank the reviewer for their analysis in highlighting the valuable aspects of the manuscript and we hope the revised manuscript will further strengthen the key results.

References:

(1) McBurney MW, Yang X, Jardine K, Hixon M, Boekelheide K, Webb JR, Lansdorp PM, Lemieux M. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. Mol Cell Biol 2003; 23:38–54.

(2) Cheng HL, Mostoslavsky R, Saito S, Manis JP, Gu Y, Patel P, Bronson R, Appella E, Alt FW, Chua KF. Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc Natl Acad Sci U S A 2003; 100:10794–10799.