Both prolonged high-fat diet consumption and calorie restriction boost hepatic NAD+ metabolism in mice

Caloric intake dramatically influences the health and diseases in humans and animals. Excess energy intake often leads to the metabolic syndrome and associated complications. In contrast, calorie restriction (CR) without malnutrition and other interventions has been reported to extend the lifespan and delay the onset of aging-related conditions, such as obesity, type 2 diabetes, and cardiovascular diseases [1,2]. The liver has a central role in the regulation of systemic glucose and lipid metabolism and is sensitive to dietary challenges. The liver maintains normal glucose homeostasis during feeding and fasting by controlling glycogenesis, glycogenolysis, glycolysis, and gluconeogenesis [3]. Long-term high-fat diet (HFD) feeding may induce nonalcoholic fatty liver disease (NAFLD), in some cases, hyperglycemia [4]. Meanwhile, CR reduces hepatic lipid content and meliorates hepatic glucose regulation in obese humans [5,6]. Thus, understanding the molecular mechanisms of hepatic responses to energy balance challenges will help to develop new effective strategies to reverse obesity-associated metabolic complications.

Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme that participates in hundreds of energy metabolism processes, including glycolysis, fatty acid β-oxidation, and oxidative phosphorylation [7]. Endogenous NAD+ bioavailability is affected by the biosynthetic, consumption, and clearance pathways, as shown in Figure 1. The majority of NAD+ is generated from a salvage pathway where the nicotinamide split from NAD+-consuming reactions is recycled into nicotinamide mononucleotide (NMN) via the rate-limiting enzyme nicotinamide phosphoribosyl transferase (NAMPT) [8]. NMN is subsequently converted to NAD+ by nicotinamide mononucleotide adenylyltransferases (NMNAT1-3) [9]. NAD+ is utilized in various enzymatic reactions by three major classes of NAD+ consumers, sirtuins, poly (ADP-ribose) polymerases (PARPs), and cyclic ADP ribose hydrolase (e.g., CD38) [10]. The NAD+ clearance pathway involves three enzymes, nicotinamide N-methyltransferase (NNMT), cytochrome P450 2E1 (CYP2E1), and aldehyde oxidase 1 (AOX1). Excessive nicotinamide is methylated to N-methyl nicotinamide (MNAM) by NNMT, or directly oxidized by CYP2E1 [11]. MNAM can be subsequently oxidized by AOX1 into pyridones and excreted through the urine.

Numerous studies have shown that hepatic NAD+ levels decline with aging and obesity [12], [13], [14]. Dietary supplementation of NAD+ precursors, such as NMN, nicotinamide riboside (NR), could boost NAD+ levels and have proved beneficial in the context of NAFLD, implicating the significance of adequate NAD+ homeostasis [15,16]. Studies about the hepatic NAD+ metabolism in response to HFD feeding reported conflicting results, showing the increased [17,18], decreased [14,19,20], or constant NAMPT and/or NAD+ levels [21] in livers of rodents or humans. Besides the conflicting data, it is also unknown how the key enzymes in the NAD+ biosynthesis, consumption, and clearance pathways are regulated in mice challenged with long-term HFD feeding.

Transcriptional regulation of the hepatic NAD+ metabolism was less pronounced in HFD-fed mice compared with CR mice. The majority of studies focused on the role of hepatic sirtuins in the beneficial effects of the CR diet. For example, CR promotes mammalian cell survival by inducing the SIRT1 [22], and overexpression of SIRT1 can mitigate metabolic syndrome, which is similar to the effect of CR [23]. Reciprocally, genetic ablation of SIRT1, SIRT3, and SIRT6 would block the benefits of CR [24], [25], [26]. Chen et al. [27] reported that NAD+/NADH ratio and SIRT1 protein level were significantly decreased in the livers of 3-month CR mice, but increased in adipose tissue and muscle, suggesting the tissue-specific regulation of NAD+ metabolism by CR. Consistently, our previous work confirmed that both NAD+/NADH ratio and SIRT1 protein levels in white adipose tissue and brown adipose tissue were increased in the CR mice [28]. However, the hepatic NAD+ homeostasis under long-term CR as well as the expression of the specific components related to NAD+ salvage, consumption, and clearance pathways. has not yet been documented. Therefore, it is necessary to clarify the dynamic changes in hepatic NAD+ metabolism during energy balance challenges.

In the present study, we have examined the expression of specific molecular components of NAD+ metabolism in the liver of mice challenged with long-term HFD feeding or CR. Since AMPK and SIRT1 are both fuel-sensing enzymes and comprise a reciprocal positive regulating loop where NAD+ is involved [29], we have also evaluated the AMPK and SIRT1 activity as well as the expression of their common target genes involved in gluconeogenesis, fatty acid oxidation, and lipogenesis. Finally, we have investigated the correlations between gene expressions involved in NAD+ metabolism and multiple metabolic parameters as well as gluconeogenesis- and lipogenesis-related gene expression to explore the potential of NAD+ metabolism in developing strategies to improve the metabolic adaption upon energy balance in livers.

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