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Molecular clocks in the periphery coordinate tissue-specific daily biorhythms by integrating input from the hypothalamic master clock and intracellular metabolic signals. One such key metabolic signal is the cellular concentration of NAD + , which oscillates along with its biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT). NAD + levels feed back into the clock to influence rhythmicity of biological functions, yet whether this metabolic fine-tuning occurs ubiquitously across cell types and is a core clock feature is unknown. Here, we show that NAMPT-dependent control over the molecular clock varies substantially between tissues. Brown adipose tissue (BAT) requires NAMPT to sustain the amplitude of the core clock, whereas rhythmicity in white adipose tissue (WAT) is only moderately dependent on NAD + biosynthesis, and the skeletal muscle clock is completely refractory to loss of NAMPT. In BAT and WAT, NAMPT differentially orchestrates oscillation of clock-controlled gene networks and the diurnality of metabolite levels. NAMPT coordinates the rhythmicity of TCA cycle intermediates in BAT, but not in WAT, and loss of NAD + abolishes these oscillations similarly to high-fat diet-induced circadian disruption. Moreover, adipose NAMPT depletion improved the ability of animals to defend body temperature during cold stress but in a time-of-day-independent manner. Thus, our findings reveal that peripheral molecular clocks and metabolic biorhythms are shaped in a highly tissue-specific manner by NAMPT-dependent NAD + synthesis.

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD + . As low hepatic NAD + levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD + , and liver injury was prevented by supplementation with NAD + precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD + levels. Our data suggest a critical threshold level of hepatic NAD + that determines the predisposition to liver injury and supports that NAD +  precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD + -synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD + levels, but the metabolic phenotype of Nampt- deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD + levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD + levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD + , suggesting that the mitochondrial NAD + pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [ 15 N]glutamine, HNKO hepatocytes had a higher [ 15 N]NAD + enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD + synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD + levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver., (© 2019 Dall et al.)

Dietary supplementation of nicotinamide adenine dinucleotide (NAD + ) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD + is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD + have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD + salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD + levels, but caused a decrease in NADP + and NADPH levels. Decreased NADP + content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD + salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD + pool in HFD-challenged C57BL/6JBomTac male mice., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: The ability of adipose tissue to expand and contract in response to fluctuations in nutrient availability is essential for the maintenance of whole-body metabolic homeostasis. Given the nutrient scarcity that mammals faced for millions of years, programs involved in this adipose plasticity were likely evolved to be highly efficient in promoting lipid storage. Ironically, this previously advantageous feature may now represent a metabolic liability given the caloric excess of modern society. We speculate that nicotinamide adenine dinucleotide (NAD + ) biosynthesis exemplifies this concept. Indeed NAD + /NADH metabolism in fat tissue has been previously linked with obesity, yet whether it plays a causal role in diet-induced adiposity is unknown. Here we investigated how the NAD + biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) supports adipose plasticity and the pathological progression to obesity., Methods: We utilized a newly generated Nampt loss-of-function model to investigate the tissue-specific and systemic metabolic consequences of adipose NAD + deficiency. Energy expenditure, glycemic control, tissue structure, and gene expression were assessed in the contexts of a high dietary fat burden as well as the transition back to normal chow diet., Results: Fat-specific Nampt knockout (FANKO) mice were completely resistant to high fat diet (HFD)-induced obesity. This was driven in part by reduced food intake. Furthermore, HFD-fed FANKO mice were unable to undergo healthy expansion of adipose tissue mass, and adipose depots were rendered fibrotic with markedly reduced mitochondrial respiratory capacity. Yet, surprisingly, HFD-fed FANKO mice exhibited improved glucose tolerance compared to control littermates. Removing the HFD burden largely reversed adipose fibrosis and dysfunction in FANKO animals whereas the improved glucose tolerance persisted., Conclusions: These findings indicate that adipose NAMPT plays an essential role in handling dietary lipid to modulate fat tissue plasticity, food intake, and systemic glucose homeostasis., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD + ) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT in maintaining NAD + levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express Cre recombinase in tibialis anterior muscle of floxed Nampt mice. In sh Nampt KD C2C12 myoblasts, Nampt and NAD + levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55%, and 2-deoxyglucose uptake increased by 25% in sh Nampt KD cells. Treatment with the NAD + precursor nicotinamide riboside restored NAD + levels in sh Nampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh Nampt KD cells, respectively. Expression of Cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NAD + levels by 38% and 43%, respectively. Glucose uptake increased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxia-inducible factor (HIF)-1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known sirtuin 6 (SIRT6) target, were increased in shNampt KD cells. Thus, we propose that the shift toward glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NAD + levels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.

Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)