Therapeutic potential of dihydronicotinamide riboside (NRH) on obesity and glucose intolerance in mice

Domain: biology
Published: 2026-03-25
Editorial outcome: major_revision

Abstract
NAD+ is a crucial metabolic cofactor whose intracellular levels can influence the progression of multiple metabolic and age-related complications. There is therefore a strong interest in using NAD+ precursors (vitamin B3s) as therapeutic tools, but most current precursors exhibit either poor bioavailability or adverse effects. This study examines the metabolic impact of chronic dietary supplementation with a newly described NAD+ precursor, dihydronicotinamide riboside (NRH), in mice using a comprehensive approach including phenotyping tests, RNA sequencing in different tissues and microbiome analyses. We show that chronic NRH administration at 100 mg/(kg*day) is well tolerated, yet has minimal metabolic effects in mice on a regular diet. However, NRH mitigates high-fat diet-induced metabolic complications when used as a preventive or as a treatment strategy, including improvements in glucose tolerance, increased hepatic expression of lipid catabolism genes and fat redistribution. These results highlight the potential of NRH as a therapeutic agent, although further studies are needed to optimize its use, as higher doses reveal signs of toxicity.

Introduction
Nicotinamide adenine dinucleotide (NAD+) acts as a degradation substrate for diverse enzymatic activities next to its participation in redox reactions1. Sirtuins and ADP-ribosyl transferase enzymes (mono or poly-ADP ribose polymerases) can cleave NAD+ in order to transfer ADP-ribosyl moieties into proteins or to deacylate them, in a reaction that releases nicotinamide (NAM) as a product1–3. Other enzymes can use NAD+ to generate cyclic ADP-ribose (cADPr), which can act as a secondary messenger in the cell4. The fact that NAD+ is consumed by many cellular activities implies that the cell needs to constantly synthesize NAD+ to maintain its intracellular levels. To do so, cells are equipped with enzymatic activities aimed to regenerate NAD+ levels by recycling intracellularly produced NAM5,6. As these recycling systems are not 100% efficient, dietary NAD+ precursors are required to maintain NAD+ levels5,7. These dietary NAD+ precursors are popularly known as vitamin B3.

In mammals, a severe dietary deficiency in vitamin B3 causes pellagra, which can lead to death if left untreated7. However, more subtle declines in NAD+ levels are a hallmark for multiple metabolic and age-related complications5. Multiple lines of evidence suggest that impaired NAD+ levels might strongly influence the progression of diet and age-related metabolic complications, at least in rodents8–15. In humans, age-associated changes in NAD+ levels have been found in the liver16, brain17, muscle18 and skin cells19.

Circulating NAD+ precursors exist as pyridine bases such as nicotinic acid (NA) or NAM, or as pyridine nucleosides including nicotinamide riboside (NR), nicotinic acid riboside (NAR) and 1,4-dihydronicotinamide riboside (NRH)20 (Supplementary Fig. 1a). While NA has been used clinically to treat dyslipidemia, its application has been largely discontinued due to side effects unrelated to its role as a NAD+ precursor21. NAM has shown only modest clinical success so far, which could either be due to a largely enigmatic biodistribution22–24, its ability to act as an end-product inhibitor of NAD+ consuming enzymes1, or its metabolism into methylated and oxidized species25. The interest in the therapeutic use of NAD+ precursors was revitalized with the discovery of the nicotinamide riboside kinase (NRK) pathway, which allowed the direct use of NR as a NAD+ precursor26 and potentially avoiding the limitations of NA and NAM. However, NR suffers from strong degradation to NAM or NA in the gut27,28 and in circulation29, which largely curtails its possible advantages compared to classic NAD+ precursors. The discovery of NRH as a NAD+ precursor30,31 provided yet another new way to generate NAD+ that could be therapeutically exploited. NRH has a unique metabolic path towards NAD+ synthesis, characterized by the initial phosphorylation of NRH into 1,4-dihydronicotinamide mononucleotide (NMNH) by the adenosine kinase (AK)30,32. The oral administration of NRH in mice leads to detectable amounts of NRH in circulation and increased NAD+ hepatic content30, indicating that NRH could be used therapeutically as a NAD+ precursor.

However, no study to date has evaluated the effects of chronic NRH administration and thus some critical information is still missing before considering NRH for clinical therapeutic purposes. Here, we aimed to characterize the biodistribution and chronic effects of NRH in mice. Given the reported benefits of NAD+ precursors in diet-induced obesity8–10,13,14, we also explored if NRH could prevent or treat diet-induced metabolic damage. Our results illustrate that NRH can be detected in multiple tissues after oral administration. The chronic administration of NRH did not lead to any major metabolic effect in mice when fed a standard rodent diet, yet prevented body weight gain, hepatic steatosis and glucose intolerance in mice fed a high-fat diet (HFD). In addition, NRH improved glucose tolerance when administered to obese mice, coupled with an increase in the expression of fatty acid catabolism genes in the liver and a whole-body fat redistribution. The therapeutic window of NRH, however, might be narrower than other NAD+ precursors, as higher doses (>300 mg/(kg*day)) led to decreased levels of circulating alkaline phosphatase and altered behavioral patterns, which deserve further attention before taking NRH into clinical consideration.

Results
NRH can be detected in organs, tissues and body fluids after oral administration

Previous studies have indicated that NR is poorly bioavailable after oral administration. For example, Liu et al. failed to detect isotopically labelled NR in circulation up to 135 minutes after oral administration27. Their results indicated that intact NR marginally incorporated into the hepatic NAD+ pool27. Instead, most NR was degraded to NAM and downstream metabolites, either in the gut28, or in circulation29. These observations made us wonder whether NRH, which is structurally very similar to NR, could suffer from the same limitations. We have previously shown that NRH can be detected in circulation after oral administration in rodents30. However, little is known on whether it can then reach different tissues. To gain a more complete picture of the biodistribution of NRH upon oral intake, we used a version of NRH that is isotopically labelled (M+7) on both the nicotinamide (18O, M+2) and ribose (13C, M+5) moieties (Fig. 1a). Two hours after an oral dose of 250 mg/kg of NRH, we could observe how the NRH (M+7) was distributed in all tissues we analyzed, including plasma, urine, liver, lung, pancreas, kidney, heart, muscle and brain (Fig. 1b-c). High relative values of NRH were found in urine, plasma and kidney; brain was the tissue with the poorest distribution, as expected due to the blood-brain barrier. Residual amounts of other isotopologues could be detected (M+5, M+6, M+8 and M+9), associated with the isotope purity of our administered NRH (M+7) tracer (Fig. 1b). We next wondered whether the uptake of NRH in the tissue would be coupled to its use as a NAD+ precursor. For this, we monitored if NRH (M+7) led to the generation of NAD+ (M+7). Our analyses revealed that indeed NRH led to labelling of liver NAD+ both through direct label incorporation (M+7), as well as via NAM (M+2), as evident from the isotopologue distribution (Supplementary Fig. 1b-c).

The influence of chronic NRH treatment in young, healthy mice

Most in vivo studies to date using NRH have been very short in nature, lasting, at maximum, a few days30,33,34. While these short-term studies have reported therapeutic benefits, the possibility of a long-term administration needs to be balanced with evidence in cultured cells suggesting that NRH can promote a pro-inflammatory phenotype in macrophages35 and suppress replication in glioma cells36. To evaluate the metabolic impact of chronic NRH treatment, we randomly distributed 8-week-old C57BL/6NTac mice into two groups: a control group and a group that received a dose of 100 mg/(kg*day) of NRH in their drinking water for 16 weeks (Supplementary Fig. 2a). NRH treatment did not alter body weight (Fig. 2a), tissue weight (Supplementary Fig. 2b) or body composition (Supplementary Fig. 2c-d) in male or female mice. Food and water intake were similar between groups (Fig. 2b-c). Similarly, no alterations were seen in their daily spontaneous activity (Fig. 2d) or in the distance they managed to run when placed on a treadmill (Fig. 2e). Indirect calorimetry experiments did not detect any major alteration in energy expenditure (Fig. 2f) or respiratory exchange ratios (Fig. 2g), neither during the light or dark phase (Supplementary Fig. 2e-f). NRH treatment did not affect fasting blood glucose (Supplementary Fig. 2g) or glucose excursions after intraperitoneally administering glucose (Fig. 2h) or insulin (Fig. 2i). While a tendency towards higher insulin response was observed in male mice (Fig. 2i), this tendency did not reach statistical significance when the whole glucose excursion was measured (Supplementary Fig. 2h). Finally, basal body temperature was slightly, but significantly, lower in NRH-supplemented male mice (Fig. 2j). This difference did not alter the ability of mice to sustain body temperature when exposed to cold (Supplementary Fig. 2i). Collectively, these results suggest that 100 mg/(kg*day) NRH does not lead to major alterations in mouse behavior, energy metabolic profiles and physical performance.

Effects of chronic NRH administration on NAD+ and the hepatic transcriptome

Orally administered NRH is subject to hepatic first-pass, so we next decided to evaluate the impact of NRH on liver metabolism. Our previous results illustrated that oral NRH administration leads to NRH incorporation in the liver (Fig. 1b-c) and increased NAD+ levels after 1 hour30. In contrast to its acute effects, however, chronic NRH administration did not significantly alter steady-state hepatic NAD+ levels in male or female mice (Fig. 2k). However, it increased levels of NADH in male mice (Fig. 2k) and led to a decrease in the NAD+/NADH ratio (Supplementary Fig. 2j), which fits the concept that NRH-driven increases in NAD+ are secondary to the prior synthesis of NADH30,31.

To validate these observations in another tissue, we evaluated the impact of NRH on NAD+ and NADH levels in skeletal muscle (Tibialis anterior). Consistent with NRH reaching muscle tissue (Fig. 1b-c), we observed a modest but significant reduction in the NAD+/NADH ratio in males (Supplementary Fig. 2j), despite minimal changes in total NAD+ or NADH levels across sexes and treatment groups (Fig. 2l).

Collectively, these observations suggest that chronic NRH administration does not substantially alter steady state NAD+ levels but tends to decrease NAD+/NADH ratios. However, given the small magnitude of this change and the absence of overt phenotypes in NRH-treated mice, its physiological relevance remains uncertain. Also, these changes are very different in amplitude and direction from those described after acute administration, suggesting that the administration route and the time after administration are critical when evaluating the impact of NRH on NAD+ and NADH levels.

Transcriptomic analyses in liver samples from our mice illustrated that chronic NRH treatment also had little impact on hepatic gene expression, in line with the minor phenotypic impact. PCA plots did not display any clear clustering of the control vs. NRH-treated groups (Fig. 2m). Our analyses did reveal 53 and 45 differentially expressed genes (DEGs, adjusted p-value < 0.05, |log2 fold change| > 1) in the livers of respectively female and male NRH-treated compared to control mice (Fig. 2n-o). However, there was virtually no overlap between the genes changing in males and females, with only one gene in common (Col1a1), which changed in opposite directions in female and male mice (Fig. 2n-p). Altogether, the limited global transcriptomic changes, together with the discordant nature of these changes in males and females, suggest that NRH does not elicit major alterations in hepatic gene expression signatures when administered to healthy, young mice.

Effects of NRH on clinical biomarkers and on the gut microbiome

In line with the absence of major phenotypes induced by NRH, no alterations in basic blood biochemistry markers were observed, including markers of liver damage (alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP)), bone (ALP), kidney (creatinine), or muscle (creatine kinase (CK)) (Supplementary Fig. 3a). This suggests that, at this concentration, NRH is well-tolerated and does not lead to observable toxicity. Other blood biochemistry parameters, such as triglyceride or cholesterol levels, were also comparable between control and NRH-treated mice (Supplementary Fig. 3a).

Orally administered NAD+ precursors can also be used by the gut microbiome for their own biosynthetic purposes37,38. It is therefore plausible that dietary supplementation with NAD+-related compounds might influence bacterial growth and, therefore, the proportional abundance of different bacterial phyla. Several studies have recently highlighted how NAD+ precursors, such as NR, can alter the gut microbiome composition39,40, yet there is no information on the impact of NRH. We therefore evaluated whether oral supplementation of NRH for 4 weeks in mice altered fecal microbial content by collecting feces from the cages housing individual control or NRH-treated mice. PCA analyses of β-diversity based on the Bray-Curtis distance matrix41 revealed that the differences between the groups were subtle, especially since sex seemed to play a larger role than the treatment in determining between-group differences (Supplementary Fig. 3b). When assessing the within-sample diversity (alpha-diversity) using the Shannon index42, we observed that NRH treatment had no influence on females, yet it did increase the diversity in the male gut microbiome (Supplementary Fig. 3c). These changes in males were probably related to very mild proportional differences among different bacterial phyla, including increased Bacteroidetes and Verrucomicrobia, while decreased Firmicutes proportions. However, none of these changes in individual phyla reached statistical significance due to a notable variability across the individual mice (Supplementary Fig. 3d). Collectively, these results suggest that chronic NRH administration has a very subtle impact on the gut microbiome and that other variables, such as biological sex, have a larger impact on its composition.

Administration of a high NRH dose induces toxicity

Over these 16 weeks of intervention, we also supplemented a group of mice with a higher dose of NRH, 400 mg/(kg*day), to rule out the possibility that the lack of phenotypic impact was due to insufficient NRH dosage. We found that also at this higher dose, body weight or composition were not affected (Fig. 3a-c), as neither did their daily water or food intake (Fig. 3d-e). Unexpectedly, we observed that male mice in the NRH 400 mg/kg group showed a higher amount of daily spontaneous activity during the light phase (Fig. 3f). This was largely driven by spikes of activity occurring every 3-4 hours (Fig. 3g-h), which were not observed at 100 mg/(kg*day). These measures were carried out in 8 mice separated in two batches, all of them showing this abnormal behavioral pattern. This altered behavior in males was also observed in the treadmill test, revealing a lower performance (Fig. 3i) that did not necessarily stem from fatigue per se, but from a failure to properly engage into the running test, reaching the criteria for exclusion much quicker. Finally, we also observed that high NRH doses led to increased circulating ALT and creatinine levels both in males and females, which may be reflective of hepatic and renal damage, as well as decreased levels of ALP (Fig. 3j-l).

To gain further insights into the potential mechanisms behind the toxic events observed at 400 mg/(kg*day), and based on previous reports indicating that NRH can lead to DNA damage in hepatocytes36,43, we examined γH2AX-positive nuclei in the livers of mice treated with either 100 or 400 mg/(kg*day) of NRH. Immunofluorescence analyses revealed a significant increase in the number of γH2AX positive nuclei, but only in male mice treated with 400 mg/(kg*day) (Fig. 3m-n), in line with deleterious alterations observed in the treadmill performance and daily activity profiles. In addition, poly-ADPribosylation (PARylation) levels were notably higher in liver homogenates from male mice treated with 400 mg/(kg*day), compared to those in 100 mg/(kg*day) of NRH or control mice (Fig. 3o).

The above results from our long-term intervention illustrate that, despite being well-tolerated at 100 mg/(kg*day), NRH leads to hepatic toxicity in mice when used at higher doses. Nevertheless, these results were observed in a long-term, 16-week intervention, which makes it difficult to determine the initial events that trigger toxicity. To gain further insights into the early effects behind NRH toxicity and better define the range of doses that could drive them, we next performed a detailed dose-response experiment in which we administered 0, 100, 200, 300, or 400 mg/(kg*day) of NRH to mice through the drinking water for 2 weeks. We reasoned that this experimental paradigm would allow us to better differentiate early toxic events from late responses. After 2 weeks of treatment, NRH did not cause alterations in body weight or in the size of liver or adipose tissues at any of the doses tested (Supplementary Table 1). At this stage, blood biochemistry analyses did not yet reveal any significant change in hepatic (AST, ALT) or renal (creatinine) damage markers, but a clear dose-dependent decrease in circulating ALP levels was evident, reaching significant (p<0.05) effects at 300 mg/(kg*day) (Supplementary Fig. 4a and Supplementary Table 1). Transcriptomic analyses of liver tissues after 2 weeks of NRH treatment revealed significant gene expression changes across all tested doses (Supplementary Fig. 4b), including at 100 mg/(kg*day), a dose that showed no effect in the chronic supplementation study. This confirms the value of this experimental design, as the short-term exposure allowed us to capture early transcriptional responses before homeostasis is restored over time (Fig. 2m-o). Notably, the transcriptomic changes were dose-dependent with limited overlap in DEGs between doses (Supplementary Fig. 4c-d). This was evident in the top 50 DEGs, of which most exhibited gradual, dose-related increases or decreases in expression (Supplementary Fig. 4d), mirroring the phased effects seen in circulating ALP levels. To identify key pathways affected at the toxic NRH dose (400 mg/(kg*day)), we performed pathway and gene ontology analyses. “Circadian rhythms” emerged as the most significantly altered category (Supplementary Fig. 4e), in line with the altered daily activity patterns observed in male mice at this dose in our previous long-term intervention (Fig. 3g-h). Other enriched terms included cell cycle regulation, radiation response and glucose homeostasis, suggestive of DNA damage/replication and metabolic disruption. To further support the detrimental effects of acute dosing with high NRH concentrations, we moved to a cultured cell model. We used AML12 hepatocytes treated with submaximal (0.01 mM) and maximal (0.5 mM) NRH concentrations, which resulted in a dose-dependent increase in intracellular NAD+ levels (Supplementary Fig. 4f). Consistent with our in vivo data, DNA damage occurred only at the higher dose (Supplementary Fig. 4g-h). In addition, 24h exposure to NRH 0.5 mM impaired both the respiration capacity and glycolytic rate of AML12 cells (Supplementary Fig. 4i), suggesting that treated cells suffered a metabolic breakdown. These effects were reversed by adenosine kinase inhibition with 5-iodotubercidin (5-IT) (Supplementary Fig. 4i), indicating that toxicity arises from excessive NAD+ synthesis rather than NRH itself. Together, all these independent interventions demonstrate that high NRH doses induce toxic effects, likely driven by metabolic dysfunction and DNA damage.

NRH prevents diet-induced body weight gain in mice

Previous studies have shown how NAD+ precursors have little effect on young healthy mice, yet can protect against obesity-related complications8,10. To evaluate if this could also be the case for NRH, we placed mice on a HFD for 8 weeks with or without NRH (100 mg/(kg*day)) in the drinking water. We focused on the 100 mg/(kg*day) dose, given the undesirable effects observed at 400 mg/(kg*day). Mice supplemented with NRH showed a robust protection against diet-induced body weight gain (Fig. 4a). The difference in body weight could be attributed to changes in fat mass, as there was a marked decrease in the weight of fat depots, as well as a slight decrease in liver mass (Fig. 4b). In contrast, other tissues, such as the heart and the brain, were similar in weight between control and NRH-treated mice (Fig. 4b). NRH also protected against the HFD-induced increase in fasting glycemia and insulinemia observed (Fig. 4c-d), suggesting improvements in glycemic control. Confirming this point, NRH-treated mice showed better glucose clearance during glucose tolerance tests (Fig. 4e). This was not due to a higher insulin release, as insulin secretion was attenuated in NRH-treated mice (Fig. 4f). Collectively, these results suggest that NRH treatment protects against the development of glucose intolerance and insulin resistance triggered by HFDs. Additional parameters further supported that NRH protected against HFD-induced metabolic damage. Most notably, the circulating levels of ALT, creatinine and LDL-cholesterol largely increased in HFD-fed mice, and this increase was prevented by NRH (Fig. 4g-h). Finally, NRH also counteracted the effects of HFD on the circulating levels of some critical metabolic hormones, such as adiponectin or leptin (Fig. 4i-j).

Metabolic influence of chronic NRH treatment on different tissues

LC-MS-based analyses44 showed that NRH could be detected in the plasma of NRH-treated mice, in contrast to control plasma samples, where it was below detection limits (Fig. 5a). NAM, one of the main products of NAD+ degrading enzymes, along with its related metabolites N-methyl nicotinamide (meNAM), N-methyl-2-pyridone-5-carboxamide, and N-methyl-4-pyridone-3-carboxamide (both referred to here as MeXPY), increased significantly with HFD but not when the mice were supplemented with NRH (Fig. 5a). Chronic NRH treatment did not alter NAD+ baseline levels in the liver, kidney, or muscle albeit prevented the decrease in NAD+ observed in brown adipose tissue (BAT) in response to HFD (Fig. 5b).

While the effect of NRH in the drinking water on tissue NAD+ might be transient, the chronic effects on tissues were clearly manifested at the histological level. Liver and BAT from NRH treated mice showed a remarkable decrease in lipid accumulation compared to HFD mice, as observed through Oil-RedO and hematoxylin-eosin staining (Fig. 5c). The decrease in hepatic triglyceride content was further confirmed via enzymatic tests (Supplementary Fig. 5a). We also examined whether NRH influenced hepatic fibrosis, but neither HFD nor NRH treatment led to differences compared to control mice (Supplementary Fig. 5b), which is consistent with the fact that the fibrotic signatures seen using our HFD are very mild, and only detectable after much more protracted treatments than the ones used in this study45,46.

Finally, and given the lower lipid accumulation observed in liver and BAT tissues, we also evaluated whether NRH treatment influenced mitochondrial respiratory capacity in these tissues. Confirming this possibility, NRH prevented the decrease in mitochondrial respiratory capacity triggered by HFD in liver and BAT (Fig. 5d). In contrast, NRH treatment did not alter the respiratory capacity of permeabilized EDL muscle, suggesting tissue-specific effects (Supplementary Fig. 5c).

NRH prevents HFD-induced transcriptomic alterations

To acquire molecular insight into the effects of NRH, we performed transcriptomic analyses of the liver, BAT, epididymal white adipose tissue (eWAT), subcutaneous adipose tissue (scWAT) and kidney derived from control, HFD-fed and HFD+NRH-fed mice. Except for the kidney (Supplementary Fig. 6b-c), we found that HFD led to drastic changes in the tissues’ transcriptome, leading to hundreds of DEGs in most examined tissues (Fig. 5e-f and Supplementary Fig. 6a-c). HFD had the highest impact on gene expression in scWAT and eWAT, where it led to thousands of genes being differentially expressed (Supplementary Fig. 6b-c). Gene set enrichment analyses revealed that the main changes prompted by HFD in the liver, BAT, scWAT and eWAT were related to mitochondrial and lipid metabolism (Supplementary Fig. 6a). Showing the consistency of the effect, some of the most significant gene ontology (GO) terms overlapped across 2 or more tissues (PPAR signaling pathway, biosynthesis of unsaturated fatty acids, fatty acid elongation, fatty acid biosynthesis, pantothenate and CoA biosynthesis) (Supplementary Fig. 6a).

While NRH did not lead to any major change in the liver transcriptome in mice fed a control diet (Fig. 2n-p), it largely attenuated the transcriptomic changes induced by HFD (Fig. 5e-f), and for all examined tissues, NRH reduced the number of DEGs by 90% or more (Fig. 5e-f and Supplementary Fig. 6b-c). For example, DEG analyses revealed 184 and 352 DEGs in the liver and BAT, respectively, from HFD-fed compared to control mice (Fig. 5e-f). In contrast, supplementation of NRH to HFD-fed mice resulted in only 19 and 32 DEGs in the liver and BAT respectively (Fig. 5e-f). In all examined tissues, most DEGs in the HFD+NRH group overlapped with those in the HFD group alone (Fig. 5e-f; Supplementary Fig. 6b-c), suggesting that NRH does not drive a distinct transcriptional response under HFD conditions. Rather, NRH appears to attenuate or even counteract many of the transcriptomic alterations induced by HFD across multiple tissues.

We also wondered whether NRH’s impact on HFD-induced phenotypes could involve changes to microbial composition. Notably, NRH had only very modest effects on the fecal microbiome in control diet-fed mice. However, we hypothesized that under HFD conditions, the microbiome might modulate NRH’s beneficial effects on weight gain and glucose intolerance. As expected, HFD induced notable alterations in the fecal microbiome composition (Supplementary Fig. 7a-b). This was characterized by an increased firmicutes to bacteroidetes ratio (Supplementary Fig. 7b), as previously reported47,48. However, HFD-fed mice showed similar microbiome phylum profiles, irrespective of NRH treatment (Supplementary Fig. 7b). This confirms that NRH has marginal effects on the composition of the microbiome and suggests that diet, rather than body weight, was the key determinant in our experimental setting.

NRH treats glucose intolerance in HFD-induced obese mice

The above results indicate that NRH can counteract the transcriptional and metabolic effects of HFD feeding. To test whether NRH can also reverse established obesity-related pathophysiology, we conducted an intervention study in which mice were fed a HFD for 8 weeks, after which half received NRH supplementation (100 mg/(kg*day)) for an additional 4 weeks, while the rest remained on HFD alone as controls (Fig. 6a). Over the 4-week treatment period, NRH-treated mice gained weight at the same rate as HFD controls (Fig. 6b-c), indicating that NRH does not induce weight loss once obesity is established. However, fat distribution was altered: NRH-treated mice had significantly larger fat depots, while liver, kidney, and heart weights remained unchanged (Fig. 6d), suggesting a shift in fat partitioning.

We next assessed glucose homeostasis. Fasting glucose and insulin levels were comparable between groups (Fig. 6e and 6f, respectively), but NRH-treated mice showed improved glucose clearance, especially in the early phase (Fig. 6g), as reflected by a significantly reduced glucose excursion (Fig. 6h). NRH also tended to lower circulating AST and ALP activity while significantly increasing HDL-C and decreasing creatinine levels (Fig. 6i). The latter suggests improved kidney function, consistent with higher renal NAD+ levels (Fig. 6j). We could also observe a trend towards an increase in baseline NAD+ content in white adipose tissue (Fig. 6k), yet no differences were seen in muscle, liver or BAT (Fig. 6j).

Interestingly, the increased size of the fat pads in NRH-treated mice occurred in parallel to a reduction in muscle and liver triglyceride content (Fig. 6l-m). While overall lipid droplet profiles in the liver appeared unchanged, a subtle, non-significant shift toward smaller lipid droplets was also noted (Fig. 6n). In adipose tissues, adipocyte size was similar between groups, indicating that the increased fat mass was not due to exacerbated adipocyte hypertrophy (Fig. 6o). Collectively, our results are consistent with a scenario of lipid redistribution upon NRH treatment that would favor accumulation of fat in adipose tissues while reducing ectopic fat deposition.

NRH treatment in obese mice increases fat oxidation programs in the liver

Given the short duration of the intervention (4 weeks) and absence of body weight differences, this model provided an opportunity to assess direct transcriptional effects of NRH without major confounding by adiposity. Transcriptomic analysis of liver tissue revealed that NRH treatment partially shifted the expression profile, as seen by PCA (Fig. 7a), with 1481 DEGs (631 upregulated and 850 downregulated; Fig. 7b). Upregulated genes were enriched in pathways related to lipid catabolism and steroid biosynthesis (Fig. 7c-d), including cholesterol 7 alpha-hydroxylase (Cyp7a1), the cytochrome P450 oxidoreductase (Por) and the farnesoid X receptor (Fxr, also known as Nr1h4), key regulators of cholesterol and bile acid biosynthesis. Other notably upregulated pathways were related to xenobiotic metabolism and detoxification (Fig. 7d). In agreement with the nature of the treatment, genes with increased expression also fell in the category of “NADP+ metabolic process”, and, consistent with the phenotype caused by NRH, “response to insulin” also appeared as an upregulated term (Fig. 7d). Downregulated genes were associated with ER stress, protein translation and trafficking (Fig. 7e). Together, these results suggest that NRH promotes lipid breakdown and detoxification pathways while mitigating ER stress, potentially explaining the reduction in hepatic fat and the rise in circulating HDL-C.

NRH regulates adipogenesis in adipose stromal cells

We next explored how NRH treatment could promote systemic lipid redistribution by focusing on the effects of NRH on adipose tissue. Gene expression analyses in adipose tissue revealed that NRH triggered distinct transcriptional programs from those observed in the liver. When using similar thresholds as used in the liver, we identified fewer DEGs in adipose tissue (Fig. 8a-b), and the overlap between adipose tissue and hepatic DEGs was very limited (Fig. 8a), underscoring potential differences in the response to NRH across tissues. Transcriptional programs related to de novo lipid anabolism, such as triglyceride metabolic process and fatty acyl-CoA biosynthesis, including genes such as Aacs, Acca and Accb, were downregulated upon NRH treatment (Fig. 8c-d), suggesting an adaptive response of adipocytes to limit excessive lipid accumulation and hypertrophy. In addition, genes related to the GO term “insulin resistance” were also downregulated by NRH (Fig. 8d), consistent with the improved glucose tolerance observed in NRH-treated mice. However, neither these downregulated GO terms, nor the upregulated ones (Fig. 8d) provide a clear path to explaining the enlarged adipose tissue depots observed in response to NRH. One limitation of bulk analyses in adipose tissue stems from this tissue’s large cellular heterogeneity, which might mask changes in specific cell type populations. Hence, we wondered whether the adipose tissue expansion observed in NRH-treated mice could be linked to changes occurring at the level of adipose progenitor cells and, more precisely, their ability to generate new adipocytes. To test this hypothesis, we isolated mouse adipose stromal cells (mASCs), which comprise adipogenic precursors, from the inguinal scWAT of wild-type mice. NRH dose-dependently increased NAD+ in cultured mASCs (Supplementary Fig. 8a), an effect that was notably larger than that triggered by NR, while NA and NAM failed to alter NAD+ levels at the doses and time tested (Supplementary Fig. 8b). We next evaluated how the presence of NAD+ precursors in the adipogenic media would influence the differentiation of mASCs, evaluated as the ratio of lipid accumulation per number of cells (from here on, referred to adiposcore49). While NA, NAM or NR did not alter the adiposcore of mASCs, NRH prompted a dose-dependent increase, reaching its maximum around 0.05-0.1 mM NRH (Fig. 8e and Supplementary Fig. 8c). It is also worth mentioning that NRH impaired adipocyte differentiation at higher concentrations, to the point of fully blunting it at 1 mM (Fig. 8e), consistent with the toxic effects previously observed at high doses in AML12 cells (Supplementary Fig. 4g-i). Interestingly, the increase in the adiposcore triggered by submaximal concentrations (0.1 mM) of NRH was largely derived from a larger number of cells committing to adipogenesis, rather than a higher lipid accumulation per cell (Fig. 8e-f). In fact, lipid droplets were smaller in NRH-treated cells (Fig. 8g). Importantly, the effect of NRH on the adiposcore was blunted when cells were simultaneously treated with the adenosine kinase inhibitor 5-IT (Fig. 8h), indicating that the phosphorylation of NRH to engage NAD(H) synthesis was required for the adipogenic effect. Collectively, these observations suggest that NRH promotes adipogenesis in mASCs.

We next performed RNA sequencing analyses to study the impact of NRH on mASCs. We found that, among the different NAD+ precursors, NRH promoted the larger changes in gene expression (Supplementary Fig. 8d). We detected more >1,000 DEGs upon NRH treatment (Supplementary Fig. 8e). The most prominent GO terms in upregulated genes referred to the regulation of angiogenesis, which also emerged as an upregulated program in eWAT, and lipid metabolism and fat cell differentiation (Fig. 8i), which are consistent with the observed cellular phenotype. Downregulated genes evoked GO terms related to the biosynthesis of ribosomes and translation factors, amongst others, which echoed our observations in the liver and collectively suggest that NRH attenuates translation processes (Fig. 8j). It was also interesting to see that, in addition to alterations in lipid metabolism, both the liver and mASCs displayed a prominent signature of genes related to detoxification and the regulation of oxidative stress (Fig. 8i,k). Given that the metabolism of reactive oxygen species (ROS) can have multiple, and even opposing, roles during adipocyte differentiation and adipose tissue metabolic function50, we next evaluated whether NRH influenced oxidative stress. Lipid peroxidation is a critical manifestation of oxidative damage, and one of its major end-products is 4-hydroxynonenal (4-HNE)50. NRH significantly reduced 4-HNE levels in mASCs, compared to vehicle treatment (Fig. 8l). Importantly, a reduction in 4-HNE levels was also observed in eWAT and liver tissues from obese mice upon NRH treatment (Fig. 8m). Together, these findings support a model in which NRH alters the expression of adipogenic- and redox-related genes in mASCs, promoting de novo adipogenesis while limiting oxidative damage, which could prevent the metabolic complications associated with adipose tissue expansion.

Methods
Animal care and phenotyping

All animal experiments were carried out according to national Swiss and EU ethical guidelines and approved by the local animal experimentation committee under licenses VD2770.1, VD3647 and VD3841. Male and female C57Bl/6NTac were purchased from Taconic. All mice were kept in a standard temperature- and humidity-controlled environment with a 12h:12h light-dark cycle. Mice had nesting material and ad libitum access to water and commercial diets. For the studies in Figures 1-3, female and male mice were fed a regular mouse housing diet (Safe®150). For the HFD studies (Figures 4-7), only male mice were used, as female C57Bl/6NTac mice rarely develop HFD-induced metabolic complications. Male mice were fed with D12492 from Research Diets, while using D12450J as a sucrose matched control. NRH was provided to mice in the drinking water, at a concentration of 0.1% or 0.4%, which approximately leads to a dose of 100 mg/(kg*day) or 400 mg/(kg*day) assuming mice drink around 3 ml per day and weigh around 30 grams. Water bottles were renewed twice a week with a fresh solution. For the HFD study, the dose of NRH in the water bottles was adjusted weekly according to the average body weight of the cage.

For the phenotyping interventions, animals were randomized into the different groups upon arrival. Body composition was determined by Echo-MRI (Echo Medical Systems). Indirect calorimetry, food intake, water intake and activity were monitored using the Promethion Core High-Resolution Metabolic and Behavioral Phenotyping System from Sable Systems during a 48-hour period. Glucose tolerance was analyzed by measuring blood glucose after an intraperitoneal injection of 2 g/kg glucose after an overnight fast. Insulin tolerance was measured by injecting 0.3 U/kg insulin after 6 h fasting. Cold test experiments were performed by placing mice in a cold chamber kept at 6°C and measuring rectal body temperature every hour. Treadmill tests were performed as described before75. At the end of the phenotyping, animals were euthanized after a 2 h fast, in order to stabilize systemic parameters and to allow the measurement of blood biochemistry in the fasting state. For that, mice were initially anesthetized with isoflurane. Once they were fully anesthetized, blood samples were collected via cardiac puncture, after which terminal euthanasia was executed via cervical dislocation and tissue collection. Blood was placed in EDTA-coated tubes, and plasma was isolated after centrifugation (2000 g, 15 min, 4°C). The tissues collected upon sacrifice were flash-frozen in liquid nitrogen.

NRH synthesis

NRH was custom synthesized as described before30,76.

NAD+ and NAD+-related metabolites measurements

The NAD+ and NADH measurements in Fig. 3a and cultured cells were performed as previously described76. For the measurements in Fig. 5a, NAD+-related metabolites were measured using a liquid chromatography (LC)- triple quadrupole (QQQ)-mass spectrometry (MS) approach following a previously described assay44.

For the analysis of NRH distribution after oral administration, mice were administered 250 mg/kg of isotopically labelled NRH (Nam-18O, ribose-13C) by oral gavage. Two hours later, tissues were collected, quickly washed in PBS and dried from excess liquid and blood before freezing them. Then LC-high resolution MS was used to evaluate the relative presence of NRH, following a recently described assay77. Protein layers from tissue samples were quantified using a bicinchoninic acid (BCA) assay (ThermoFisher Scientific) for normalization of metabolite concentrations across tissues. Given that mice were not perfused before tissue collection, we cannot fully rule out that part of the labelling detected in the different tissues derives from remaining blood cells.

For all other NAD+ measurements in tissue and cellular samples, NAD+ was extracted and quantified using an EnzyChrom NAD/NADH Assay Kit (BioAssay Systems) according to the manufacturer's instructions.

Histology

H&E and Oil Red O stainings were performed using the fully automated Ventana Discovery XT (Roche Diagnostics, Rotkreuz, Switzerland). All steps were performed on the machine with Ventana solutions. A total of 3 control and 3 NRH-treated mice were compared for histology analyses, by imaging and quantifying approximately 18 sections per mouse tissue. DNA damage in the liver was assessed by immunofluorescence staining for γH2AX (Cell Signaling, 9718; 1:400) on OCT-embedded tissue sections. For quantification, the proportion of γH2AX-positive nuclei relative to the total number of nuclei (DAPI-stained) was determined using QuPath software78.

Triglyceride measurements

Triglyceride content in liver and muscle samples was measured enzymatically using a commercial kit (MAK266; Sigma-Aldrich).

High-resolution respirometry in mouse tissues

Respirometry studies in liver and brown adipose tissue homogenates, as well as in permeabilized EDL muscle, were performed using high-resolution respirometry (Oroboros Oxygraph-2k, Oroboros Instruments) as described previously79. Briefly, freshly isolated liver and BAT were homogenized in amino acid-depleted respirometry medium (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 10 mM KH2PO4, 20 mM HEPES, and 110 mM sucrose, pH 7.1) and 2 mg of wet tissue were added to the experimental chamber. In the case of muscle, fiber bundles from EDL muscle were separated as previously described79 and, after permeabilization with saponin (50 μg/ml) for 30 minutes, 1-2 mg of tissue were used for respirometry assays. O2 flux through Complex I (CI) was measured by adding malate (2 mM), pyruvate (5 mM), glutamate (10 mM) and ADP (5 mM for liver and muscle; 0.5 mM for BAT). This was followed by the addition of succinate (10 mM) for electron flow through both complex I and II (‘’CI+II’’). Subsequently, carbonylcyanide-4-(trifluoromethoxy)-phenyl-hydrazone (FCCP) was titrated to achieve maximum flux through the electron transfer system (‘’ETS’’). Finally, electron transport through complex I and complex III was inhibited by the addition of rotenone (0.1 μM) and antimycin A (2.5 μM), respectively. The remaining O2 flux after inhibition with antimycin A (O2 flux independent of the electron transfer system) was subtracted from all the values from the previous steps. O2 flux values are expressed relative to tissue wet weight per second (pmol O2 x mg−1 s−1).

AML12 cell culture

All common cell culture media and reagents were obtained from Gibco (Thermo Fisher Scientific Inc. (Waltham, MA, USA)). AML12 cells (ATCC Cat #: CRL-2254) were cultured in Dulbecco’s modified Eagle’s medium/Nutrient mixture F-12 (DMEM:F-12), supplemented with 10% fetal bovine serum (FBS), 1× Insulin/Transferrin/Selenium solution from Gibco (ITS-G) and 100 nM dexamethasone.

Seahorse analyses in cultured cells

Cellular O2 consumption was measured using a Seahorse bioscience XF96 analyzer (Seahorse bioscience Inc., North Billerica, MA, USA). AML12 hepatocytes were seeded in Seahorse XF96 Cell Culture Microplates (Agilent) at a density of 10’000 cells per well and left for 24 hours. Immediately before the measurement, cells were washed with PBS and 150 μl of non-buffered (sodium carbonate free) pH 7.4 DMEM medium supplemented with 10% fetal calf serum was added to each well. After a 15 min equilibration period, successive 2 min measurements were performed at 5 min intervals with inter-measurement mixing to homogenize O2 concentrations in the medium and each condition was measured in five replicates in at least 15 independent wells. Oligomycin (10 μM; to evaluate non-coupled respiration) and FCCP (2 μM) were sequentially injected, using 10x concentrated solutions, to each well and successive 2 min measurements were performed at 5 min intervals with inter-measurement mixing. Finally, a solution containing rotenone and Antimycin A was injected into each well (to reach a final concentration of 2 μM and 2 μg/ml, respectively) to block mitochondrial respiration and evaluate residual O2 consumption. After that, cells were lysed in RIPA buffer to quantify the amount of protein in each well for correction.

mASCs isolation and culture

To evaluate the effect of NRH and other NAD+ precursors on mASCs NAD+ levels, differentiation capacity and transcriptional profiles, we isolated the stromal vascular fraction of inguinal subcutaneous fat, cultured it and differentiated it as previously described49. Briefly, inguinal subcutaneous adipose tissue depots were dissected from 8–16-week-old wild‐type C57BL/6JRj mice and placed into PBS. Male and female mice were used indistinguishably at an approximate ratio of 1:1. The isolation of the adipose stromal cells was performed as previously described49 and then grown in 4.5 g/L glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (growth media, GM). Then, the GM media was changed 24 hours later to remove all the non-attached cells (which include immune cells). The remaining attached cells were then expanded in GM for at maximum 2 passages before being used for different purposes, as described in other sections below.

In vitro adipogenic differentiation of mASCs

mASCs were plated in flat‐bottom microscopy‐adapted cell culture plates (Corning #353219) and grown until confluence in GM. Once confluent, cells were exposed to an adipocyte differentiation induction media, which is composed of GM supplemented with 0.5 μM 3‐isobutyl‐1‐methylxanthine (IBMX, Sigma #15879), 1 μM dexamethasone (Sigma #D2915) and 170 nM insulin (Sigma #19278) for 72 hours. Then, cells were kept for 4 additional days in GM supplemented with 170 nM insulin. At this point (7 days of differentiation), cells were then either stained for imaging or collected for RNA extraction.

Imaging and quantification of in vitro adipogenesis

After 7 days of differentiation, cells were fixed with PFA 4% and then stained with fluorescence dyes: Bodipy (boron‐dipyrromethene, Invitrogen #D3922) for lipids and Hoechst for nuclei. Given a substantial variation in the extent of lipid accumulation by cells within the same well, the imaging was optimised to cover the largest surface of the well possible. Then, images were obtained using the automated platform Operetta (Perkin Elmer), as previously described49. The images, supported by Harmony software, were exported as TIFF files. Image analysis was performed in ImageJ/Fiji, lipid droplets (yellow) and nuclei (blue) images were filtered using a Gaussian blur (sigma equal to 2 and 3, respectively) before an automatic thresholding. The automatic thresholding algorithm selections were chosen based on visual inspection of output images. The areas corresponding to the thresholded lipid and nuclei signals were next used to calculate the normalized lipid accumulation (Adiposcore). In Fig. 8e, Fig. 8h and Supplementary Fig. 8c, tiled (25 images) and thresholded images are shown. In addition, lipid droplet size was measured using an adapted version of StarDist80.

BRB-seq library generation and data analysis

The extraction of RNA and generation of libraries for bulk RNA barcoding and sequencing (BRB-seq) from mouse cells or tissues were performed as described before81. Briefly, BRB-seq libraries were generated using a MERCURIUS BRB-seq kit (Alithea Genomics, #10813) according to the manufacturer’s instructions. Libraries were pooled per mouse organ and sequenced on an Illumina Novaseq 6000. RNA-seq reads obtained through the BRB-seq protocol were aligned to the reference mouse mm10 genome (GRCm38, Ensembl release 102) using STARsolo (Version 2.7.10a)82. Reads mapping to multiple positions were excluded. STARsolo also generated the UMI count matrix. The raw read count matrix was generated using FastReadCounter (Version 1.1) (https://github.com/DeplanckeLab/FastReadCounter) run on the tagged bam file generated by STARsolo, and lowly expressed genes (less than average 50 reads per sample) were filtered out. Raw count data were normalized using DESeq2 (version 1.42.1)83 with Benjamini-Hochberg correction (FDR < 0.05). Gene set enrichment analysis was performed using the R package EnrichR (version 3.2)84 and Metascape85 applying a hypergeometric test with Benjamini-Hochberg correction (FDR < 0.05). All the DEGs for each analysis can be found in the Source Data File.

Microbiome analyses

DNA from mouse feces samples was obtained using a Quick-DNA Fecal/Soil Microbe Miniprep Kit from Zymo (#D6010). Libraries were generated using a "16S library prep with ZYMO Quick-16S_Plus" library prep kit according to manufacturer’s instructions. Libraries were pooled prior to sequencing and quantified by a qubit DNA High Sensitivity assay (Thermo, #Q32851). The pooled library was sequenced on an Illumina MiSeq using 300 PE sequencing chemistry. Data preprocessing was performed using DADA286. Downstream analysis was performed using Phyloseq87. Alpha diversity was quantified with the Shannon Index and group differences were assessed by Wilcoxon rank-sum tests.

Protein analyses

Proteins were extracted from AML12 cells or liver tissue using RIPA buffer (Millipore, 20-188) supplemented with protease inhibitors (cOmplete, Roche) and PJ34 (10 μM). Proteins were quantified using a BCA assay (Pierce). For western blotting, proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes before incubation with primary antibodies against GAPDH (#2118; Cell Signaling), PARP1 (sc-1561; SantaCruz) or poly(ADP-ribose) (ALX-210–899; Enzo) at a 1:1000 dilution. Primary antibodies were used at 1:5,000 dilution for GAPDH and 1:1000 dilution for others. Antibody detection reactions were developed by enhanced chemiluminescence (Amersham) after incubation with the appropriate peroxidase-linked secondary antibodies (Jackson Immunoreagents) at a 1:20000 dilution.

Lipid peroxidation measurements

The detection of 4-HNE in mASCs, liver and eWAT was performed with commercial kit (ab238538, ABCAM) using 100 mg of protein for cellular lysates and 200 mg for eWAT and liver lysates.

Statistical Analyses

Statistical analyses were performed with GraphPad Prism version 7.02 for Windows (La Jolla, CA, USA). Differences between two groups were analyzed using Student's two-tailed Student’s t-test. One-way ANOVA analysis was used when comparing more groups by applying Tukey's post-hoc test. Group variances were similar in all the cases. Unless otherwise specified, all the data are expressed as mean ± SEM.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes work conducted to determine effects of oral supplementation of NRH in mice. While only minor\neffects were noted in healthy mice treated with NRH, this compound was able to prevent high-fat diet (HFD)-induced weight\ngain. No specific mechanism for the effects of NRH in mice fed the HFD is provided, but it is noteworthy that these effects\noccur at a significant lower dose than those used in previous studies on various NAD precursors, as also pointed out by the\nauthors.\nThe manuscript is really well-written and the data are presented in a clear and convincing way. This is the first study with\nNRH where it is supplemented over a longer period of time. Thus, this study provides an important first step towards\ndelineating the potential of NRH treatment to affects NAD metabolism and metabolic health.\nThere are a few key things that could have been better addressed. While the HFD study is designed to be able to prevent\nHFD-induce obesity, and given the potent effect of this compound, it would have been a great addition to the study if data\nfrom a treatment-type study could had been included. This would be a fairly simple experiment to conduct and well within the\ncapabilities of the laboratory. Data from such an experiment would allow us to better evaluate the potential of NRH to\nmitigate metabolic conditions/diseases related to surplus energy intake.\nFigure 1 shows how the labelled compound is present in different tissues. However, if this compound is taken up by those\ntissues, it should produce NAD (NAD+/NADH). How much of the NAD pool in those tissues is from the labelled NRH? Can\nyou detect labelled NAD in those samples? It is not obvious if the mice were perfused before tissues were dissected, but this\nwas probably not the case. Thus, could the labelled NRH detected simply be from what is present in the blood stream in\nthose tissues?\nIt is not clear to me why the authors chose not to include an analysis of the gut microbiome from fecal samples obtained in\nthe HFD study. Data from such an analysis would be meaningful given the slight changes observed in the control diet, and it\nwould enable the authors to be less speculative in the discussion where these data are discussed.\nFinally, I would appreciate if the authors could provide water and feed intake in the NRH 400 experiment. Is water or feed\nintake different between the experimental groups? Has the authors tested if any taste aversion is associated with the higher\ndoses of NRH in the drinking water?", "reviewer_2": "In this report, Canto and colleagues expand on their previous work to evaluate, in exquisite detail, the impact of chronic\nexposure of mice to the NAD+ precursor dihydronicotinamide riboside (NRH). As shown, low dose chronic exposure was\nwell tolerated and protected from high-fat diet (HFD) induced glucose intolerance, among other HFD-induced effects,\nincluding transcriptional changes in several tissues. Overall, this is an outstanding study that is well designed and well\ncontrolled.\nSome minor concerns include:\nWhy does Figure 5 lack an “NRH only” control? It would seem to be critical. It is noted that some data on such an “NRH only”\ntreatment is shown in Figure 3 and elsewhere. It would be of value to show alongside the data in Figure 5 as well and to\ninclude the same analysis if not shown elsewhere.\nRegarding the section on “NRH prevents HFD-induced transcriptomic alteration” – there are likely many studies on HFD-\ninduced transcriptomic alterations in mouse tissue and maybe even human studies published elsewhere. It would be of\nvalue to include an analysis of other published studies to see how these results compare – are they in-line with previous\nanalysis?\nSample or mouse numbers are not indicated in all figure legends.", "reviewer_3": "This paper by Rumpler et al tested the therapeutic potential of NRH on obesity and related metabolic outcomes in mice. The\ndata is interesting but mostly descriptive in nature. The potential toxicity of NRH even at doses lower than 400 mg per kg is\nalso a major concern that needs to be clarified in further initial studies.\nSpecific concerns\n1) Figures 2 and 3 are just showing lack of metabolic effects in young lean mice and should be transferred to supplement\nmaterials. Very small effects on NAD+/NADH ratios in Fig 3 are not physiologically relevant to metabolic outcomes in these\nmice\n2) Apparent Effect of 100 mg dose on ALP in healthy mice and HF fed mice is worrying. Ext fig3 also suggests some\nincreased impact on creatinine and CKIA. This calls for looking at the dose dependent effect of NRH on tox measures to\nassess potential toxicity at 200 and 300 mg/kg in both lean and obese mice.\n3) Fig4 should show all tissues and especially skeletal muscles.\n4) the liver histology data is of poor quality and should be quantified for all mice. Fibrotic markers should be determined and\nstéatose also measured biochemically.\nSo overall the work remains preliminary and primarily descriptive. No mechanism put forward to explain effects of NRH and\nthe transcriptional analyses are interesting but remains exploratory without further work"}, "author_response": "Response to the reviewers - NCOMMS-24-52397-T:\nTherapeutic potential of dihydronicotinamide riboside (NRH) as a NAD+ precursor and\nits effects on obesity and glucose intolerance in mice\nRumpler et al.\nWe would like to thank the reviewers and the editorial team for their dedicated time and effort\nin evaluating our work, as well as for their insightful comments. In this resubmission, we have\npaid attention to their valuable feedback and incorporated multiple new elements which\nundoubtedly enhanced the overall quality of the manuscript.\nThis revision includes multiple new figures and additional discussion elements that provide a\nbetter contextualization of the implications of the study. A detailed point-by-point answer to\nthe reviewers’ queries can be found below:\n-------\nReviewer 1:\n1.1 “There are a few key things that could have been better addressed. While the HFD study\nis designed to be able to prevent HFD-induced obesity, and given the potent effect of this\ncompound, it would have been a great addition to the study if data from a treatment-type study\ncould have been included. This would be a fairly simple experiment to conduct and well within\nthe capabilities of the laboratory. Data from such an experiment would allow us to better\nevaluate the potential of NRH to mitigate metabolic conditions/diseases related to surplus\nenergy intake.”\nWe thank the reviewer for the constructive criticism and excellent suggestion. As\nsuggested, we have now performed an intervention study in which mice were made obese\nand insulin resistant by feeding them a high-fat diet (HFD) for 8 weeks. Then, mice were\nrandomly divided in two groups, one in which mice remained on a HFD, and a second in which\nthe HFD treatment was complemented with NRH (100 mg/kg*day, through the drinking water)\nfor 4 additional weeks.\nThe results indicate that NRH treatment in already obese mice did not lead to body weight\nloss, yet improved glucose tolerance and led to a reduction in circulating biochemical markers\nof tissue damage, most notably creatinine levels. Interestingly, while mice treated with NRH\ndid not lose weight, we observed a redistribution in their fat deposition. This was most notably\nmanifested in fat mass being predominantly accumulated in the fat tissue depots rather than\nin the liver. Accordingly, the transcriptomic profiles in the livers of NRH-treated mice pointed\ntowards NRH increasing hepatic fat catabolism.\nOverall, our results suggest that at 100 mg/kg*day, NRH can lead to benefits in the context\nof diet-related metabolic complications, either using prevention or intervention strategies.\nNevertheless, our results also highlight that, while the preventive effects might rely on blunting\nbody weight gain, the intervention effects rather rely on fat redistribution leading to decreased\nectopic fat deposition. The results from this new intervention are described in Pages 15-16\nand constitute Figs. 6 and 7 in the manuscript.\n1.2 “Figure 1 shows how the labelled compound is present in different tissues. However, if this\ncompound is taken up by those tissues, it should produce NAD (NAD+/NADH). How much of\nthe NAD pool in those tissues is from the labelled NRH? Can you detect labelled NAD in those\nsamples? It is not obvious if the mice were perfused before tissues were dissected, but this\nwas probably not the case. Thus, could the labelled NRH detected simply be from what is\npresent in the bloodstream in those tissues?”\nFollowing the suggestion of the reviewer, we have evaluated the incorporation of labelled\nNRH (M+7) in the hepatic NAD+ pool. The results can be found in Page 6 and in the\nSupplemental Fig. 1b-c. Briefly, the generation of NAD+ (M+7) could be detected after oral\nadministration of labelled NRH, supporting that intact NRH is utilized for NAD+ synthesis in\nthis tissue. We could also see an increase in the M+2 form of NAD+, which likely originates\nfrom internal recycling of NAM+2 produced after NAD+ M+7 breakdown by NAD+-consuming\nenzymes. However, we cannot rule out the possibility that the M+2 form of NAD+ originates\nfrom the M+2 dihydronicotinamide molecule resulting from the cleavage of the N-glycosidic\nbond of the NRH M+7 form. This said, to our knowledge, NAD+ synthesis from\ndihydronicotinamide has not been previously characterized.\nDespite the fact that the above results indicate that intact NRH is directly used to\nsynthesize NAD+ in the liver, we would like to clarify that our original tracing study was primarily\ndesigned to detect the uptake of labelled NRH in the tissue, but not to evaluate its downstream\ncellular metabolism. In order to consistently and comparatively evaluate the incorporation of\nNRH (or any other NAD+ precursor) in the NAD+ pool, longer times are desirable. In agreement\nwith our results (Supplementary Fig.1b-c), work from the Rabinowitz lab has shown that less\nthan 10% of the hepatic NAD+ pool is labelled after the intravenous infusion of labelled NAD+\nprecursors for two hours1,2. For this reason, most studies exploring the incorporation of NAD+\nprecursors in the NAD+ pool use timings above three hours1–4.\nTo complement the above findings, we would like to mention that we recently performed\ntracing experiments using a longer time (6 hours) and a different NRH labelling strategy. More\nspecifically, we used a M+9 form of NRH where the nicotinamide ring was labelled as\n13C=18O15NH and the ribose labelled as 13C (Figure for the Reviewers 1a). This new\n2 5\nlabelling strategy allows us to monitor additional NRH downstream fates by better\ndiscriminating deamidation events. Consistent with the results presented in this manuscript\n(Supplementary Figure 1b-c), a raise in hepatic NAD+ levels upon M+9 NRH oral\nadministration was also observed in the new intervention (Figure for the Reviewers 1b). In\naddition, the label incorporation distributed over multiple NAD+ isotopologues, including M+9\nNAD+ resulting from direct incorporation of M+9 NRH, as well the as M+4 NAD+ derived from\nM+4 NAM (Figure for the Reviewers 1c). Hence, the preliminary data from our new\nintervention confirms the conclusions presented in this manuscript. We hope the reviewer\nunderstands and agrees with us that a full exploration of the results of our new M+9 NRH form,\nincluding a thorough scrutiny of NAD+ turnover and an extensive characterization of the label\ndistribution, is out of the scope of this manuscript and will be the topic of a separate, upcoming\nwork.\nIn relation to the final point, we must clarify that the mice were not perfused in our\nexperiments. Tissues were quickly washed in PBS and the excess liquid was dried before\nfreezing. Previous efforts evaluating blood contamination in freshly collected mouse tissues\n(by measuring the presence of serum albumin)5, found the highest contamination levels in the\nliver (3.6% of the total protein). Other tissues, such as the kidney, showed much lower\ncontamination values (1.2%). However, the signal for labelled NRH that we observed in the\nkidney was higher than that in the liver. Therefore, while we cannot fully rule out an influence\nof blood contamination in our analyses, the readouts are not consistent with blood NRH levels\nbeing a critical driver of the differences observed across tissues. The fact that we did not\nperfuse our animals and that this could be a confounding element in our results, however, is\nacknowledged now in the Methods section (Page 21).\nFigure for the Reviewers 1. Orally administered NRH is directly incorporated into the hepatic NAD+ pool.\n(a) Chemical structure of the M+9 form of NRH. (b) 8 week-old male (M) and female (F) mice were orally gavaged\neither water or the M+9 form NRH (250 mg/kg). Then, 6 hours later, the liver was collected and total NAD+ levels\nwere evaluated. The results are shown as the mean +/- SD for n=4 mice per group. (c) The isotopologue distribution\nof NAD+ was evaluated in the experiment described in (b).\n1.3 “It is not clear to me why the authors chose not to include an analysis of the gut microbiome\nfrom fecal samples obtained in the HFD study. Data from such an analysis would be\nmeaningful given the slight changes observed in the control diet, and it would enable the\nauthors to be less speculative in the discussion where these data are discussed.”\nWe have now analyzed the effects of NRH on the gut microbiome in the context of HFD.\nThe results, shown in the Supplementary Fig.7, illustrate that the dietary change to high-fat\nfeeding largely overrides the very subtle changes triggered by NRH on the gut microbiome. In\nparticular, high-fat feeding triggered a large increase in the abundance of firmicutes, at the\nexpense of bacteroidetes. These observations are in agreement with the published literature6\nand emphasize our previous conclusion (“...these results suggest that chronic NRH\nadministration had a very subtle impact on the gut microbiome and that other variables, such\nas biological sex, have a larger impact on its composition”). Based on this, we also decided to\nrephrase the discussion to prevent that modest changes in the gut microbiome caused by\nNRH would be interpreted as causal for the health benefits of the compound.\n1.4 “Finally, I would appreciate if the authors could provide water and feed intake in the NRH\n400 experiment. Is water or feed intake different between the experimental groups? Has the\nauthors tested if any taste aversion is associated with the higher doses of NRH in the drinking\nwater?”\nWe thank the reviewer for the suggestion. We observed no significant changes in food or\nwater intake at any of the tested NRH concentrations. The data in relation to the NRH 400\nmg/kg*day group is now provided in Figs.3d-e.\nReviewer 2:\n2.1 “Why does Figure 5 lack an “NRH only” control? It would seem to be critical. It is noted\nthat some data on such an “NRH only” treatment is shown in Figure 3 and elsewhere. It would\nbe of value to show alongside the data in Figure 5 as well and to include the same analysis if\nnot shown elsewhere.”\nThe results from our original Figs. 2 and 3 (as well as the original Supplementary Figs. 2\nand 3) were generated in different cohorts than those in Figs. 4-5. Unfortunately, due to\ninternal ethical considerations aiming to minimize experimental mouse use, the “NRH only”\ngroup was excluded from the experiments constituting Figs. 4-5, as (1) the main goal was to\ncompare the effects of NRH in the context of high-fat feeding, and (2) the previous results from\nour low-fat diet intervention already showed that NRH itself had little to no effects on any of\nthe parameters measured.\n2.2 “Regarding the section on “NRH prevents HFD-induced transcriptomic alteration” – there\nare likely many studies on HFD-induced transcriptomic alterations in mouse tissue and maybe\neven human studies published elsewhere. It would be of value to include an analysis of other\npublished studies to see how these results compare – are they in-line with previous analysis?”\nFollowing the reviewer’s suggestion, we have compared the transcriptomic effects of HFD\nin our study with those observed in previous works. The results indicate that our outcomes\nlargely overlap with the published literature.\nPublished studies show a large variability in relation to the specific C57Bl/6 strains used,\nthe % of fat in the diet, the length of the exposure to the diet and transcriptomic approaches.\nFor this reason, we initially compared the main pathways affected by high-fat feeding in the\ndifferent studies. Our results in the liver (Supplementary Fig. 5a) identified PPAR signalling,\nfatty acid biosynthesis and elongation and cholesterol metabolism as critical gene ontology\n(GO) terms in the differentially expressed genes prompted by high-fat feeding. These terms\nlargely overlap with GO terms or KEGG pathways reported in previous studies using B6 mice\nand approximately similar times of exposure to HFD7,8, supporting the consistency of our\nintervention with the published literature. We then took a deeper look at these similitudes using\na publicly available RNA-seq dataset closely resembling our study, in which C57BL/6 wild-\ntype male mice were fed on a 60% kcal HFD for 18 weeks9 (Figure for the Reviewers 2).\nAfter normalization of the data and correction for batch effect, we performed standard principal\ncomponent analysis (PCA), which revealed the tendency of the samples from Matsushita et\nal. and our study to cluster by diet (chow vs. HFD). Neither the control nor the HFD groups\nfrom the 2 studies totally merged in the PCA. This is very much expected, considering that\nthese experiments were done in different animal houses and that the RNA-seq approaches\nwere slightly different. Unsupervised differential expression analysis further confirmed\ntranscriptomic similarities between the respective control groups and HFD groups in the two\ndatasets. Finally, the terms identified in their GO analyses9 also largely resembled those in\nour study, including pathways related to fatty acid biosynthesis, lipid transport and\nsteroid/cholesterol metabolism.\nFigure for the Reviewers 2. Computational comparison of our HFD dataset with HFD RNAseq dataset from\nMatsushita et al. (a) PCA plot (b) Heatmap representing the top 50 genes\nIn relation to adipose tissues, our results also reflect data and ontology terms identified in\ncomparable interventions, highlighting alterations in lipids and amino acid metabolism10,11.\nInterestingly, transcriptomic studies in subcutaneous biopsies from obese people, either using\nmicroarrays12 or RNA sequencing-based techniques13, also identified as top pathways\nalterations in lipid and amino acid metabolism.\nGiven all the above elements, we consider that the changes elicited by HFD in our study\nare in line with those reported in the literature. Considering that the manuscript focuses on the\neffects of NRH rather than HFD, we opted to present these results as a Figure for the\nReviewers, yet if the reviewers/editor consider it necessary, we would be happy to include this\nin the revised manuscript.\n“2.3 Sample or mouse numbers are not indicated in all figure legends.”\nWe thank the reviewer for noticing this unintended omission. The mouse numbers\ncomposing the different figure panels are now specified in the figure legends.\nReviewer 3:\n3.1 “Figures 2 and 3 are just showing lack of metabolic effects in young lean mice and should\nbe transferred to supplement materials. Very small effects on NAD+/NADH ratios in Fig 3 are\nnot physiologically relevant to metabolic outcomes in these mice.”\nFollowing the suggestion from the reviewer, we have now restructured old Figures 2 and\n3 into a single figure (the new Fig.2, complemented by the new Supplemental Fig.2). In\naddition, we also now clarify that the physiological impact of the modest changes observed in\nthe NAD+/NADH ratio is uncertain (See Page 8).\n3.2 “Apparent Effect of 100mg dose on ALP in healthy mice and HF fed mice is worrying. Ext\nfig3 also suggests some increased impact on creatinine and CKIA. This calls for looking at the\ndose dependent effect of NRH on tox measures to assess potential toxicity at 200 and 300\nmg/kg in both lean and obese mice.”\nWe share the curiosity of the reviewer on having a deeper understanding of the nature of\nNRH-driven toxicity. As the reviewer surely understands, detailed animal toxicity studies are\nlarge and very expensive in nature, which makes them unaffordable for academic laboratories.\nIn addition, NRH is not yet commercially sourced, and the costs associated with the custom\nsynthesis of the amounts of NRH required for a fully developed toxicity study would be very\nsignificant. For the above reasons, we opted to perform a short-term experiment which\nprovided already valuable and conclusive insights into the toxicity of NRH.\nAs suggested by the reviewer, our intervention was designed as a dose response\nexperiment in which we treated mice for 2 weeks with either 0, 100, 200, 300 or 400\nmg/(kg*day) of NRH. Given that the toxicity was more markedly observed in males, we only\nused male mice for these experiments. We also reasoned that a relatively short treatment\nwould allow us to identify the primary early events of toxicity rather than the downstream\nconsequences of a protracted exposition to NRH.\nOur results illustrate that after 2 weeks of NRH treatment, most clinical blood markers for\ntissue damage remained still unchanged irrespectively of the dose tested, with the exception\nof alkaline phosphatase (ALP) activity, which dose-dependently decreased in response to\nNRH treatment (Supplementary Fig.4a). This decrease in circulating ALP activity relative to\nthe untreated group is consistent with our previous observations and was significant already\nat the 300 mg/kg*day dose in our experimental setup. We also explored the hepatic\ntranscriptional response to a high dose of NRH. The results highlighted that, at 400 mg/kg*day,\nNRH triggered transcriptomic signatures related to alterations in cell cycle, energy stress and\ncircadian rhythms. This last point is interesting, as one peculiar feature observed in our mice\nchronically treated at 400 mg/kg*day was an alteration in their diurnal locomotion pattern,\nshowing activity spikes every 3-4 hours. These results are now shown in Pages 10-11,\nSupplementary Table 1 and in the Supplementary Figs. 4a-e.\nAs also suggested by the reviewer, we performed similar dose-response experiments in\nthe context of a HFD (Figure for the Reviewer 3). For this, we fed mice with a HFD for two\nweeks while being supplemented with different doses of NRH. The results were very similar\nto those on a low fat diet. Namely, after two weeks of treatment, none of the doses led to\nsignificant changes in body weight or in the mass of the liver or the main adipose tissue depots.\nIn terms of blood biochemistry, no changes were seen in ALT or AST levels, yet a decrease\nin ALP levels was detectable at doses of 300 and 400 mg/kg*day.\nFigure for the Reviewers 3. Dose-response experiment of NRH in high-fat fed mice. 8-week-old male mice\nwere placed on a HFD and provided 0, 100, 200, 300 or 400 mg/kg*day of NRH in the drinking water. 2 weeks\nlater, the body weight was measured (a), as well as the weight of different tissues (b) and blood biochemistry\nparameters (c). All values are presented as mean +/- SEM of n=8 mice per group. * and *** indicate p<0.05 or\np<0.001, respectively, between the indicated groups.\nTo complement the above mouse studies, we performed parallel experiments in AML12\nhepatocytes, which highlighted how maximal NRH doses (0.5 mM) lead to a significant\nincrease in DNA damage. In agreement with the mouse experiments, this did not take place\nat submaximal doses (0.01 mM), despite an effective increase in cellular NAD+ levels. Further,\n0.5 mM NRH led to a metabolic collapse, in which cells showed both decreased respiratory\nand glycolytic rates. This observation fits very nicely with a recent publication that tested the\neffects of dihydronicotinamide mononucleotide (NMNH), where the authors also observed a\ndecline in glycolytic and tricarboxylic acid cycle metabolites in cultured hepatocytes at doses\nabove 0.1 mM14. Interestingly, the metabolic effects of 0.5 mM NRH were completely blocked\nby inhibiting adenosine kinase, indicating that NRH requires engaging its transformation into\nNAD(H) in order to prompt a metabolic collapse. These results in AML12 cells are now\npresented in Page 11 and Supplementary Fig. 4f-h. Collectively, our observations suggest\nthat (1) low NRH doses can increase NAD+ and provide therapeutic effects without triggering\nDNA damage, and (2) the toxicity exerted by high doses is linked to DNA damage and a\nmetabolite ‘crisis’, which requires NRH metabolism into NAD(H). It is still unclear, however,\nhow the effects of high-dose NRH lead to a decrease in circulating ALP levels.\n3.3 “Fig4 should show all tissues and especially skeletal muscles.”\nWe are a bit unsure on what the reviewer means by “all tissues”, especially in the context\nof the old Figure 4, where no tissue-specific effects were evaluated. In relation to skeletal\nmuscle, we now also present respirometry data (Page 13 and Supplementary Fig.5c) and\nqPCR assessment of mRNA levels for multiple transcriptional regulators of oxidative\nmetabolism (Figure for the Reviewers 4). The results illustrate that none of the parameters\ntested in muscle displayed significant differences between control and NRH-treated mice.\nUnfortunately, we did not fix muscle tissue for histology. However, and given the results from\nthe gene expression and respirometry analyses, we believe it is unlikely that we would see\nany major alterations in tissue structure.\nFigure for the Reviewers 4. NRH treatment does not alter muscle mRNA levels of transcriptional regulators\nof oxidative metabolism. Quadriceps muscle was collected from mice fed either a control diet (Control), a high-\nfat diet (HFD) or a high fat diet while supplemented with NRH (100 mg/kg*day) in the drinking water (HFD+NRH)\nfor 8 weeks. Then, total mRNA was extracted and the expression of the indicated genes was evaluated through\nquantitative real-time PCR. All values are presented as mean +/- SEM from n=6-8 mice per group. None of the\ngenes evaluated showed significantly different expression across the experimental groups.\n3.4 “The liver histology data is of poor quality and should be quantified for all mice. Fibrotic\nmarkers should be determined and steatose also measured biochemically.”\nWe believe this was an artifact from the .pdf conversion during the submission. We now\nverified that the submitted images are of higher-resolution. As suggested by the reviewer, we\nhave now analyzed hepatic lipid accumulation through enzymatic assays, which confirmed the\nlower lipid accumulation suggested by our histology readouts (Page 13 and Supplementary\nFig.5a). In addition, and as also suggested by the reviewer, we performed Sirius Red staining\nin our liver histology samples to examine fibrosis. NRH did not lead to any apparent difference\nin the Sirius Red staining (Page 13 and Supplementary Fig.5b), yet it must be mentioned\nthat, beyond the expected perivascular staining, no obvious signs of fibrosis were observed in\nour HFD intervention. This is not surprising, as it is well documented in the literature that high-\nfat diets, such as the one used in our study (RD12492), weakly induce steatohepatitis and its\nassociated fibrosis in B6 mice, unless accompanied by high fructose or high cholesterol\nlevels15–18.\n3.5 “So overall the work remains preliminary and primarily descriptive. No mechanism put\nforward to explain effects of NRH and the transcriptional analyses are interesting but remain\nexploratory without further work.”\nAs the reviewer probably knows, initial efforts to unveil the mechanistic links driving the\nmetabolic benefits of NAD+ precursors focused on NAD+-consuming enzymes. However, the\nimplementation of unbiased approaches, including transcriptomic and metabolomic analyses,\nhave revealed that the actions of these molecules impact a large variety of cellular\nprocesses19,20. These far-ranging actions can be explained by multiple factors, but most\nnotably, that (1) NAD+ precursors are metabolized into multiple molecules beyond NAD+,\nwhose functional impact is very poorly understood, and (2) beyond NAD-consuming enzymes,\nalterations in NAD+ can also influence a plethora of enzymes coupling their catalytic activity to\nNAD(P)+/NAD(P)H.\nFor this reason, we opted to explore the mechanistic aspects of NRH actions by evaluating\nthe transcriptomic signatures triggered by NRH treatment. Our previous transcriptomic\ndatasets from the protracted interventions (now Fig.5 and Supplementary Fig.6) failed to\nprovide clear NRH-driven signatures. This was probably due to the long-term nature of the\npreventive intervention (16 weeks), allowing for homeostatic responses, and to the fact that\nthe large differences on body weight probably masked the primary effects of NRH. Similar\nproblems have been faced in transcriptomic datasets from preventive studies using NR or\nNMN20. Hence, we took advantage of our new treatment intervention, which we used to\ngenerate new hepatic transcriptomic datasets. In that intervention, mice were made obese\nusing a high-fat diet for 8 weeks and then were treated for 4 weeks with NRH (Fig.6a). NRH\ntreatment led to improvements in glucose tolerance (Figs.6g-h) compared to their non-\nsupplemented counterparts, despite having no impact on body weight (Figs.6b-c). Therefore,\nthis new dataset benefits from a shorter length in the treatment and prevents the bias of\ndifferences in body weight, which would increase the likelihood of capturing the genuine\neffects triggered by NRH.\nOur new transcriptomic dataset revealed 1481 differentially expressed genes (DEGs) in\nthe livers of NRH-treated mice, 631 of which were upregulated and 850 were downregulated.\nWe then interrogated the main paths and gene ontology terms represented in the upregulated\nand downregulated genes.\n● Upregulated: Among the upregulated genes, there was a large enrichment for\npathways related to lipid metabolism, most notably catabolic processes, and steroid\nmetabolism, as well as to the metabolism of vitamins and xenobiotics. This is\nconsistent with the changes observed in cholesterol levels (Fig.6i), as well as the\ntendency towards lower hepatic lipid accumulation and lipid droplet size in NRH-\ntreated mice (Fig.6k). Since results also showed that the weight of adipose tissues\nwas slightly increased in the NRH supplemented group (Fig.6d), in the absence of\nchanges in total body weight (Fig.6b-c), these results suggest the possibility that NRH\ncould promote fat deposition towards adipose tissues, rather than the liver.\n● Downregulated: As for the downregulated genes, the main pathways affected\nseemed to be related to protein translation, protein folding and protein processing in\nthe endoplasmic reticulum (ER), suggesting that NRH might relieve ER stress. Notably,\nER stress has been proposed as one of the main drivers of hepatic insulin resistance\nin situations of obesity21,22. Therefore, a reduction in these markers is consistent with\nthe benefits of NRH on glucose metabolism in obese mice.\nCollectively, our results suggest that NRH influences multiple pathways, increasing lipid\ncatabolism and cholesterol metabolism, while decreasing markers related to proteostatic\nresponses. These transcriptomic results and analyses can now be found in Page 16 and Fig.7.\nREFERENCES\n1. Liu, L. et al. Quantitative Analysis of NAD Synthesis-Breakdown Fluxes. Cell Metab. 27,\n1067-1080.e5 (2018).\n2. McReynolds, M. R. et al. NAD+ flux is maintained in aged mice despite lower tissue\nconcentrations. Cell Syst. 12, 1160-1172.e4 (2021).\n3. Yaku, K. et al. BST1 regulates nicotinamide riboside metabolism via its glycohydrolase and\nbase-exchange activities. Nat. Commun. 12, 6767 (2021).\n4. Yaku, K. et al. Nicotinamide riboside and nicotinamide mononucleotide facilitate NAD+\nsynthesis via enterohepatic circulation. Sci. Adv. 11, eadr1538 (2025).\n5. De Campeneere, D., Baurain, R., Slachmuylder-Otte, C. & Trouet, A. Immunological\nevaluation of blood contamination in tissue distribution studies. Pharmacol. Res. 21, 19–26\n(1989).\n6. Siersbæk, M. S. et al. C57BL/6J substrain differences in response to high-fat diet\nintervention. Sci. Rep. 10, 14052 (2020).\n7. Siersbæk, M. et al. High fat diet-induced changes of mouse hepatic transcription and\nenhancer activity can be reversed by subsequent weight loss. Sci. Rep. 7, 40220 (2017).\n8. Nishikawa, S., Sugimoto, J., Okada, M., Sakairi, T. & Takagi, S. Gene Expression in Livers\nof BALB/C and C57BL/6J Mice Fed a High-Fat Diet. Toxicol. Pathol. 40, 71–82 (2012).\n9. Matsushita, M. et al. An antisense transcript transcribed from Irs2 locus contributes to the\npathogenesis of hepatic steatosis in insulin resistance. Cell Chem. Biol. 29, 680-689.e6\n(2022).\n10. Caputo, T. et al. Anti-adipogenic signals at the onset of obesity-related inflammation in\nwhite adipose tissue. Cell. Mol. Life Sci. 78, 227–247 (2021).\n11. Nasias, D. et al. Transcriptome analysis of the adipose tissue in a mouse model of\nmetabolic syndrome identifies gene signatures related to disease pathogenesis. Genomics\n112, 4053–4062 (2020).\n12. Henegar, C. et al. Adipose tissue transcriptomic signature highlights the pathological\nrelevance of extracellular matrix in human obesity. Genome Biol. 9, R14 (2008).\n13. Honecker, J. et al. Transcriptome and fatty-acid signatures of adipocyte hypertrophy\nand its non-invasive MR-based characterization in human adipose tissue. eBioMedicine 79,\n104020 (2022).\n14. Liu, Y. et al. Reduced Nicotinamide Mononucleotide (NMNH) Potently Enhances\nNAD+ and Suppresses Glycolysis, the TCA Cycle, and Cell Growth. J. Proteome Res. 20,\n2596–2606 (2021).\n15. Fonseca, C. S. M. et al. Distinct Influence of Hypercaloric Diets Predominant with Fat\nor Fat and Sucrose on Adipose Tissue and Liver Inflammation in Mice. Molecules 25, 4369\n(2020).\n16. Ishimoto, T. et al. High-fat and high-sucrose (western) diet induces steatohepatitis that\nis dependent on fructokinase. Hepatology 58, 1632–1643 (2013).\n17. Torres-Villalobos, G. et al. Combined high-fat diet and sustained high sucrose\nconsumption promotes NAFLD in a murine model. Ann. Hepatol. 14, 540–546 (2015).\n18. Baiges-Gaya, G. et al. Hepatic metabolic adaptation and adipose tissue expansion are\naltered in mice with steatohepatitis induced by high-fat high sucrose diet. J. Nutr. Biochem.\n89, 108559 (2021).\n19. Elhassan, Y. S. et al. Nicotinamide Riboside Augments the Aged Human Skeletal\nMuscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures.\nCell Rep. 28, 1717-1728.e6 (2019).\n20. Zhang, J. & Chen, F. Integrated transcriptome and metabolome study reveal the\ntherapeutic effects of nicotinamide riboside and nicotinamide mononucleotide on\nnonalcoholic fatty liver disease. Biomed. Pharmacother. 175, 116701 (2024).\n21. Özcan, U. et al. Endoplasmic Reticulum Stress Links Obesity, Insulin Action, and Type\n22. Hotamisligil, G. S. Inflammation and metabolic disorders. Nature 444, 860–867 (2006)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for addressing my comments.", "reviewer_2": "The authors have very effectively addressed the concerns raised.\nI did have one minor clarification – Figure 1B shows levels in several tissues, including blood whereas in Figure 1C NRH\nlevels are listed in Plasma. Was plasma measured in both panels - why the different labels?", "reviewer_3": "My main issues with this paper was 1) the lack of mechanistic insights to explain the effect of NRH and 2) the need to better\naddress the potential toxicity of NHR which is claimed in the title and absract to represent a potential therapeutic agent for\nobesity and type 2 diabetes. Unfortunately, these major concerns were not appropriately adressed in the revised paper. Most\nof the study remains very descriptive in nature and the potential toxicity of NRH also remains a major concern and\nprecluding to propose that NRH is a potential new therapeutic molecule.\n- Figures 1-3 remains very descriptive and I still think most of these data should have been transferred to supplementary\nmaterials in order to focus the work on the obese animals where NRH did exert some meaningful effects. Moreover, the bulk\nof the study represents prevention or treatment intervention that, while showing interesting effects on several metabolic\noutcomes, actually fails to provide evidence for some causally plausible mechanisms. The transcriptomic dataset revealed\nmany differentially expressed genes in the livers of NRH-treated mice, and gene ontology analyses revealed the enrichment\nfor some interesting pathways related to hepatic pathways that are consistent with some of the observed outcomes. But this\nremains descriptive hypothesis-driven association studies that are generally used to inform on potential mechanisms to\nfocus on, and that should be next tested experimentally to provide evidence for their causal implications.\n- Moreover, the authors claim that TOX studies should not be performed by academic labs. I understand this comment but\nthis paper is about evaluating the “therapeutic potential of NRH” as the title emplies. It is not only to determine how NRH\nmay act on host tissues and the microbiome. Evaluating the toxicity of a molecule is criticially important to determine its\ntherapeutic potential and clinical relevance. The authors elect to further test for the potential toxic effects of NRH using only a\nvery short term (2 weeks) protocol and, not surprinsingly, they found little evidence for toxic effects. In my view, they should\nhave looked after 16 weeks of treatment, as they did in the actual animal prevention studies. At the very least they should\nhave look at 4 weeks in mice already made obese as they have performed in their treatment intervention studies."}, "author_response": "RESPONSE TO THE REVIEWERS\nReviewer #1 (Remarks to the Author):\n“Thank you for addressing my comments.”\nWe thank the reviewer for his/her constructive comments during the revision process.\nReviewer #2 (Remarks to the Author):\n“The authors have very effectively addressed the concerns raised.\nI did have one minor clarification – Figure 1B shows levels in several tissues, including blood\nwhereas in Figure 1C NRH levels are listed in Plasma. Was plasma measured in both panels\n- why the different labels?”\nWe thank the reviewer for his/her help during the revision process. In relation to Fig1.b-c, all\nanalyses were done on plasma. The figure has been changed accordingly, and we apologize\nfor this unintended mistake.\nReviewer #3 (Remarks to the Author):\n“My main issues with this paper was 1) the lack of mechanistic insights to explain the effect of\nNRH and 2) the need to better address the potential toxicity of NHR which is claimed in the\ntitle and absract to represent a potential therapeutic agent for obesity and type 2 diabetes.\nUnfortunately, these major concerns were not appropriately adressed in the revised paper.\nMost of the study remains very descriptive in nature and the potential toxicity of NRH also\nremains a major concern and precluding to propose that NRH is a potential new therapeutic\nmolecule.”\nWe thank the reviewer for his/her time and critical evaluation of our manuscript. We addressed\nthe concerns of the reviewer in the following point-by-point response:\n“Figures 1-3 remains very descriptive and I still think most of these data should have been\ntransferred to supplementary materials in order to focus the work on the obese animals where\nNRH did exert some meaningful effects.”\nWe acknowledge this suggestion, but we consider that Figures 1-3 contain three distinct\nconcepts that are key to understanding the therapeutic spectrum of NRH: Fig.1 demonstrates\nthat intact NRH reaches multiple tissues after oral administration. Fig.2 demonstrates that the\nchronic (16 weeks) administration of NRH is well-tolerated in lean healthy mice when used at\n100 mg/kg*day. Fig.3 demonstrates that, at higher doses, NRH leads to toxicity, which we\nconsider to be a key message of the paper, in line with one of the reviewer’s following\ncomments.\n“Moreover, the bulk of the study represents prevention or treatment intervention that, while\nshowing interesting effects on several metabolic outcomes, actually fails to provide evidence\nfor some causally plausible mechanisms. The transcriptomic dataset revealed many\ndifferentially expressed genes in the livers of NRH-treated mice, and gene ontology analyses\nrevealed the enrichment for some interesting pathways related to hepatic pathways that are\nconsistent with some of the observed outcomes. But this remains descriptive hypothesis-\ndriven association studies that are generally used to inform on potential mechanisms to focus\non, and that should be next tested experimentally to provide evidence for their causal\nimplications.”\nThe criticism from the reviewer is well taken. However, the health benefits of boosting NAD+\nin mice arise from very pleiotropic and context-dependent processes that are unlikely to rely\non a single molecular mediator. This is illustrated by our transcriptomic analyses, now\nextended also to adipose tissue and cultured cells (New Fig.8a-c, Fig.8i-j and\nSupplementary Fig.8d-e), which demonstrate that NRH influences diverse and strongly\ntissue-specific transcriptional programs.\nIt is for the above reasons that we decided to focus on solidifying plausible physiological\nmechanisms that could explain the health benefits of NRH in obese mice. Our hypothesis\ncenters around the possibility that NRH improves glucose tolerance in obese mice by at least\ntwo interconnected mechanisms: a reduction in ectopic fat deposition and an increased ability\nof the adipose tissue to safely store fat. As the reviewer understands, a classic gain-/loss of\nfunction of these processes cannot be performed without major whole-body metabolic\nconsequences that would largely override the effects of NRH.\nHence, to support the physiological actions of NRH, we performed a series of new\nexperiments, namely:\n● Additional support for a reduction in ectopic fat deposition: Complementing our previous\nobservations in the liver, we now provide data from the same intervention demonstrating\na significantly lower lipid content in skeletal muscle (Fig.6l), supporting the notion that\nNRH reduced ectopic fat deposition.\n● Demonstration that NRH favors de novo adipogenesis: Our previous results suggested\nthat the reduction in ectopic fat deposition was coupled to an enlargement of fat depots\n(Fig.6d), yet devoid of adipocyte hypertrophy (Fig.6o). We therefore speculated that\nNRH might promote the generation of new adipocytes. To test this possibility, we\nperformed in vitro experiments on mouse adipose stromal cells, which showed that NRH\neffectively increased their adipogenic potential (Fig.8e). Transcriptomic analyses further\nhighlighted an upregulation of adipogenesis-related programs (Fig.8i), amongst others,\nin response to NRH treatment. These adipogenic effects of NRH were not observed\nwhen using other NAD+ precursors (Supplementary Fig.8c) and could be reversed by\npreventing NRH metabolism towards NAD(H) (Fig.8h).\n● Further exploration of the responses to NRH treatment: When evaluating downstream\npaths affected by NRH, we identified a significant upregulation of redox-associated\ngenes in adipose stromal cells and liver. Therefore, we evaluated if NRH influenced\noxidative damage by measuring lipid peroxidation (4-HNE) levels. Our new results\nindicate that NRH decreased lipid peroxidation levels in adipose stromal cells, adipose\ntissue and liver (Figs.8l-m). This highlights how the physiological changes promoted by\nNRH are consistent with a reduction in oxidative stress, counteracting diet-induced\nmetabolic damage.\nAltogether, our results provide support for the notion that NRH decreases ectopic fat\ndeposition by increasing the expression of lipid catabolism-related genes in the liver while\nfacilitating lipid storage in adipose tissue. The increased lipid storage in fat tissues was not\naccompanied by adipocyte hypertrophy or oxidative damage, probably linked to the ability of\nNRH to increase de novo adipogenesis in adipose stromal cells and to stimulate transcriptional\nprograms aimed to decrease oxidative stress. These combined effects of NRH would allow for\na safer storage of lipids and the prevention of lipotoxicity, which is consistent with the\nimprovement of whole-body glucose tolerance.\n“Moreover, the authors claim that TOX studies should not be performed by academic labs. I\nunderstand this comment but this paper is about evaluating the “therapeutic potential of NRH”\nas the title emplies. It is not only to determine how NRH may act on host tissues and the\nmicrobiome. Evaluating the toxicity of a molecule is criticially important to determine its\ntherapeutic potential and clinical relevance. The authors elect to further test for the potential\ntoxic effects of NRH using only a very short term (2 weeks) protocol and, not surprinsingly,\nthey found little evidence for toxic effects. In my view, they should have looked after 16 weeks\nof treatment, as they did in the actual animal prevention studies. At the very least they should\nhave look at 4 weeks in mice already made obese as they have performed in their treatment\nintervention studies.”\nWe would like to clarify that our statement was that “detailed animal toxicity studies are large\nand very expensive in nature, which makes them unaffordable for academic laboratories” not\nthat “TOX studies should not be performed by academic labs”. This said, we fully agree that\nexamining potential signs of toxicity in our interventions is an important readout. In our opinion,\nthis is exactly what we have done in this manuscript: Fig.2 and Fig.3 provide the results of\nour 16-week studies at 2 doses of NRH (100 and 400 mg/kg*day), reporting toxicity at the\nhigher dose. Our analyses comprise morphometric measurements, behavioral studies, blood\nbiochemistry markers and molecular readouts of DNA damage (γH2AX and PARylation\nlevels). As a follow-up experiment, we used the 2-week study in Supplementary Fig.4 to\nuncover the early events explaining this toxicity and to better pinpoint the doses at which\ntoxicity could be observed. Thanks to this intervention, we also obtained key information on\nearly transcriptomic events linked to NRH supplementation. We would also like to mention that\nblood biochemistry markers for toxicity were included in the prevention and intervention\nstudies on obesity (Fig.4g and Fig.6i).\nThe fact that toxic events could limit the therapeutic spectrum of NRH is indeed one of the\nmain conclusions of our work, and statements related to it explicitly appear multiple times in\nkey sections of the text, such as:\nAbstract (last sentence):\n“These results highlight the potential of NRH as a therapeutic agent, although further studies\nare needed to optimize its use, as a higher dose (400 mg/(kg*day)) revealed signs of toxicity.”\nIntroduction (last section):\n“The therapeutic window of NRH, however, might be narrower than other NAD+ precursors,\nas high doses (400 mg/(kg*day)) led to decreased levels of circulating alkaline phosphatase\nand altered behavioral patterns, which deserve further attention before taking NRH into clinical\nconsideration.”\nDiscussion (Page 19 and final statement):\n“However, we could detect apparent signs of toxicity and altered behavior when mice were\ndosed with NRH concentrations above 300 mg/kg*day. This suggests that the tolerance\nwindow of NRH has a lower amplitude than that of other NAD+ precursors, (..)”\n“Collectively, our study suggests that NRH administration can treat and prevent obesity-\nrelated metabolic complications, therefore constituting a valuable alternative to other less\nstable and bioavailable precursors. Nevertheless, our data also pinpoint how NRH at high\ndoses might lead to adverse effects and, therefore, its therapeutic window might be narrower\nthan other NAD+ boosting strategies. “\nAltogether, we consider that this body of evidence strongly supports our conclusion that NRH\ncan lead to toxic effects when used at high doses (at least >300 mg/kg*day) and that,\ntherefore, its therapeutic value should always be counterbalanced by its potential side-effects."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have very effectively addressed the concerns that I raised in the previous review."}, "author_response": "RESPONSE TO THE REVIEWERS\nReviewer #2 (Remarks to the Author):\n“The authors have very effectively addressed the concerns that I raised in the previous review.”\nWe would like to thank the reviewer for the constructive comments and suggestions during the reviewing process.\nReviewer #3 (Remarks to the Author):\n“none”\nWe thank reviewer 3 for the many insights and points of improvement provided during the revision process."}]

Decision letter