Therapeutic potential of nicotinamide adenine dinucleotide for nonalcoholic fatty liver disease
Authors: Philippe Gual, Catherine Postic
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 1190 Nonalcoholic fatty liver disease (NAFLD) represents a major health problem worldwide and concerns around 30% of the general population. NAFLD represents a wide spectrum of severity, ranging from benign steatosis without any inflammation or no tendency to progress to nonalcoholic steatohepatitis (NASH) with inflammation and hepatocyte damage. Approximately 5%‐20% of NAFLD patients develop NASH, which may transition to higher‐grade fibrosis in 10%‐20% of cases. Fibrosis may progress to cirrhosis, and around 2% of patients with cirrhosis per year develop hepatocellular carcinoma. No pharmacological treatment is currently available to limit the disease progression, and physicians can only recommend lifestyle changes and exercise to reduce body weight. Mitochondrial dysfunction has emerged as a major trigger of NASH.1 Abnormal morphological changes in liver mitochondria are observed in patients and animal models with NASH. These mitochondrial abnormalities include ultrastructural lesions, depletion of mitochondrial DNA, and impaired mitochondrial β‐oxidation. Recently, impaired respiratory capacity, proton leakage, and elevated oxidative stress were also reported in obese patients with NASH.2 In this context, strategies dedicated at improving mitochondrial activity/respiratory rates may represent appealing therapeutic approaches for NASH treatment. Potential interesting targets include sirtuins (SIRTs), desacetylases representing cellular energy sensors requiring nicotinamide adenine dinucleotide (NAD)+ for their enzymatic activity.3 Mammalian sirtuins all have different subcellular distributions, with a subset of sirtuins residing predominantly in nuclear, cytosolic (SIRT1), or mitochondrial (SIRT3) compartments. Indeed, SIRT1 and SIRT3 improve both mitochondrial biogenesis and function, and defects in their pathways result in various metabolic alterations, including NAFLD.4 Interestingly, a study published in this issue of Hepatology demonstrates that NAD+ repletion, by counteracting mitochondrial dysfunction, is able to fully prevent and reverse fatty liver onset, inflammation, and fibrosis in two different NAFLD mouse models (Fig. 1). In their study, Gariani et al.5 first reveal the existence of a positive correlation between gene patterns involved in NAD+ biosynthesis and the β‐oxidation pathway in two separate data sets of normal human liver samples. This supports the role of NAD+ as a coenzyme in important metabolic pathways and suggests that NAD+ limitations could affect metabolic efficiency. Indeed, NAD+ levels dynamically respond to a number of physiological processes, including caloric restriction, exercise, circadian rhythms, and senescence. The link between NAD+ and metabolism is particularly underlined by the observation that tissue NAD+ concentrations decrease with high‐fat diets in rodents, and that enhancements in NAD+ levels are linked with improved mitochondrial function under stress, leading to protection against dietary and age‐related metabolic complications.6 Interestingly, before the study of Gariani et al.,5 the contribution of NAD+ to NAFLD onset and/or prevention had not been defined.Figure 1: NAD+ boosting strategies in HFHS‐fed mice prevent and reverse NAFLD. Two protocols were used to rescue tissue and mitochondrial NAD+ concentrations: (1) a preventive protocol (NR‐Prev) was applied, for which C57BL6/J mice were fed for 18 weeks with a HFHS diet supplemented or not with NR (400 mg/kg/day) and (2) a therapeutic protocol (NR‐Ther) for which NR supplementation started 9 weeks after the onset of the HFHS diet. As a result of the NAD+ boost, the activity of SIRT1 and SIRT3, two NAD+ requiring desacetylases, increased and triggered an adaptive pathway in mitochondria (UPRmt), leading to enhanced hepatic β‐oxidation and increased mitochondrial complex content and activity. Interestingly, both NR‐Prev and NR‐Ther treatments were associated with a significant reduction in hepatic steatosis, inflammatory and fibrosis markers, as well as indicators of liver damage. Although not reported in the study, the NAD+‐mediated activation of SIRT1 may have also induced PPARα‐dependent β‐oxidation through the potential desacetylation of the coactivator, PGC‐1α.To experimentally corroborate the link between NAD+ and NAFLD, the investigators generated a nutritional mouse model of NAFLD by feeding C57BL6/J mice with a high‐fat high‐sucrose (HFHS) diet. HFHS fed mice exhibited key features of NAFLD, including marked hepatic steatosis, increased markers of inflammation and fibrosis, and enhanced indicators of liver damage (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). Interestingly, a progressive loss in liver NAD+ concentrations was observed concomitantly to the increase in hepatic triglyceride concentrations. To determine the direct contribution of NAD+ loss to NAFLD, the investigators applied in vivo protocols of NAD+ repletion: (1) a preventive protocol (NR‐Prev), in which C57BL6/J mice were fed for 18 weeks with the HFHS diet supplemented or not with 400 mg/kg/day of nicotinamide riboside (NR), a robust precursor of NAD+,7 and (2) a therapeutic protocol (NR‐Ther) in which NR supplementation was started 9 weeks after the onset of the HFHS diet (Fig. 1). Both treatments were equally efficient in rescuing NAD+ concentrations in liver lysates and mitochondria. As a result, NR‐Prev and NR‐Ther treatments were associated with a striking decrease in liver weight and in hepatic steatosis and correlated with reduced expression in inflammatory and fibrosis markers. Importantly, indicators of liver damage remain low in NR‐treated HFHS mice and comparable to those measured in plasma from mice fed a control diet. Whereas these beneficial effects occurred independently of a change in food intake, NR‐therapies were also associated with a modest reduction in body weight gain and a 30% decrease in fat mass (epidydimal fat pad), suggesting that the decrease in proinflammatory cytokine concentrations (tumor necrosis factor alpha) observed may be linked to a combination of beneficial effects in liver and adipose tissues. In this study, the improvement in hepatic mitochondrial function was instrumental in driving the beneficial effects of NR. Increases in citrate cycle activity and oxidative phosphorylation (OXPHOS) in response to NR led to a recovery in adenosine triphosphate concentrations compared to mice fed only on HFHS diet. Importantly, induction in both SIRT1 and SIRT3 triggered mitochondrial unfolded protein response (UPRmt), leading to adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity (Fig. 1). The UPRmt is a stress response that activates transcription of nuclear‐encoded mitochondrial chaperone genes to promote protein homeostasis within the organelle.8 In agreement, expression of two known UPRmt biomarkers, caseinolytic peptidase P, which encodes a mitochondrial matrix protease and the heat shock protein 10 chaperone, was up‐regulated upon NR‐treatment, suggesting that UPRmt was turned on to protect against HFHS‐mediated mitochondrial stress. Loss‐of‐function studies in Sirt1 or Sirt3 knockout hepatocytes revealed that the NR‐mediated induction of UPRmt was significantly attenuated in the absence of SIRT1 and, to a lesser extent, in the absence of SIRT3. Although not studied, NR‐therapy could have also affected muscle and brown adipose tissue function because it was previously reported that chronic NR treatment increased NAD+ levels, sirtuin activity, and oxidative capacity as a result of enhanced mitochondrial function in muscle and brown adipose tissue under high‐fat diet conditions.6 Last, the investigators reported that NR partially lost its ability to enhance β‐oxidation and mitochondrial function when the nuclear receptor, peroxisome proliferator‐activated receptor alpha (PPARα) was silenced. PPARα is a key ligand‐activated transcription factor involved in the regulation of lipid homeostasis. However, the direct contribution of PPARα to the beneficial effects of NR would require further analysis. In particular, it would have been interesting to measure acetylation levels of peroxisome proliferator‐activated receptor‐gamma coactivator 1 alpha (PGC‐1α), an important coactivator of PPARα, and a direct target of SIRT19 (Fig. 1). An important question is whether NAD+ boosting therapies can be feasible for NASH treatment in patients. Of particular interest is the fact that the NR‐Ther was as effective as the NR‐Prev in reducing hepatic steatosis, inflammation, and fibrosis, suggesting that even in a setting of established NAFLD, NAD+ repletion therapies could be efficient. Arguments in favor of a potential easy and affordable translation to human therapy is that daily requirement for NAD+ biosynthesis can be met with consumption of less than 20 mg of vitamin B3 (niacin) a day. However, niacin, already used for the treatment of several metabolic diseases, is also reported to exert some side effects. Results from the current clinical trial evaluating the effect of NR on substrate metabolism, insulin sensitivity, and body composition in obese patients will be informative. Therefore, clinical trials dedicated at testing the bioavailability and effectiveness of known (the amino acid, tryptophan, nicotic acid, and NR)7 or potential newly identified NAD+ boosters will be needed before establishing relevance for NASH treatment in patients. Future exciting research in the field of NAD+ metabolism will most likely unravel whether NAD+ boosting strategies can be therapeutically applied for other pathologies characterized by tissue and mitochondrial NAD+ depletion, such as alcoholic liver disease, as recently proposed.10