Hepatic NAD Research Articles

ACMSD inhibition corrects fibrosis, inflammation, and DNA damage in MASLD/MASH

Recent findings reveal the importance of tryptophan-initiated de novo nicotinamide adenine dinucleotide (NAD+) synthesis in the liver, a process previously considered secondary to biosynthesis from nicotinamide. The enzyme α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), primarily expressed in the liver and kidney, acts as a modulator of de novo NAD+ synthesis. Boosting NAD+ levels has previously demonstrated remarkable metabolic benefits in mouse models. In this study, we aimed to investigate the therapeutic implications of ACMSD inhibition in the treatment of metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH). Invitro experiments were conducted in primary rodent hepatocytes, Huh7 human liver carcinoma cells and induced pluripotent stem cell-derived human liver organoids (HLOs). C57BL/6J male mice were fed a western-style diet and housed at thermoneutrality to recapitulate key aspects of MASLD/MASH. Pharmacological ACMSD inhibition was given therapeutically, following disease onset. HLO models of steatohepatitis were used to assess the DNA damage responses to ACMSD inhibition in human contexts. Inhibiting ACMSD with a novel specific pharmacological inhibitor promotes de novo NAD+ synthesis and reduces DNA damage exvivo, invivo, and in HLO models. In mouse models of MASLD/MASH, de novo NAD+ biosynthesis is suppressed, and transcriptomic DNA damage signatures correlate with disease severity; in humans, Mendelian randomization-based genetic analysis suggests a notable impact of genomic stress on liver disease susceptibility. Therapeutic inhibition of ACMSD in mice increases liver NAD+ and reverses MASLD/MASH, mitigating fibrosis, inflammation, and DNA damage, as observed in HLO models of steatohepatitis. Our findings highlight the benefits of ACMSD inhibition in enhancing hepatic NAD+ levels and enabling genomic protection, underscoring its therapeutic potential in MASLD/MASH. Enhancing NAD+ levels has been shown to induce remarkable health benefits in mouse models of metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH), yet liver-specific NAD+ boosting strategies remain underexplored. Here, we present a novel pharmacological approach to enhance de novo synthesis of NAD+ in the liver by inhibiting α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), an enzyme highly expressed in the liver. Inhibiting ACMSD increases NAD+ levels, enhances mitochondrial respiration, and maintains genomic stability in hepatocytes exvivo and invivo. These molecular benefits prevent disease progression in both mouse and human liver organoid models of steatohepatitis. Our preclinical study identifies ACMSD as a promising target for MASLD/MASH management and lays the groundwork for developing ACMSD inhibitors as a clinical treatment.

The Effectiveness of Four Nicotinamide Adenine Dinucleotide (NAD+) Precursors in Alleviating the High-Glucose-Induced Damage to Hepatocytes in Megalobrama amblycephala: Evidence in NAD+ Homeostasis, Sirt1/3 Activation, Redox Defense, Inflammatory Response, Apoptosis, and Glucose Metabolism.

The administration of NAD+ precursors is a potential approach to protect against liver damage and metabolic dysfunction. However, the effectiveness of different NAD+ precursors in alleviating metabolic disorders is still poorly elucidated. The current study was performed to compare the effectiveness of four different NAD+ precursors, including nicotinic acid (NA), niacinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) in alleviating high-glucose-induced injury to hepatocytes in a fish model, Megalobrama amblycephala. An in vitro high-glucose model was successfully established to mimic hyperglycemia-induced damage to the liver, which was evidenced by the reduced cell viability, the increased transaminase activity, and the depletion of cellular NAD+ concentration. The NAD+ precursors all improved cell viability, with the maximal effect observed in NR, which also had the most potent NAD+ boosting capacity and a significant Sirt1/3 activation effect. Meanwhile, NR presented distinct and superior effects in terms of anti-oxidative stress, inflammation inhibition, and anti-apoptosis compared with NA, NAM, and NMN. Furthermore, NR could effectively benefit glucose metabolism by activating glucose transportation, glycolysis, glycogen synthesis and the pentose phosphate pathway, as well as inhibiting gluconeogenesis. Moreover, an oral gavage test confirmed that NR presented the most potent effect in increasing hepatic NAD+ content and the NAD+/NADH ratio among four NAD+ precursors. Together, the present study results demonstrated that NR is most effective in attenuating the high-glucose-induced injury to hepatocytes in fish compared to other NAD+ precursors.

Nuclear Nicotinamide Adenine Dinucleotide Deficiency by Nmnat1 Deletion Impaired Hepatic Insulin Signaling, Mitochondrial Function, and Hepatokine Expression in Mice Fed a High-Fat Diet

Metabolic syndrome (MetS) is a worldwide challenge that is closely associated with obesity, nonalcoholic liver disease, insulin resistance, and type 2 diabetes. Boosting nicotinamide adenine dinucleotide (NAD+) presents great potential in preventing MetS. However, the function of nuclear NAD+ in the development of MetS remains poorly understood. In this study, hepatocyte-specific Nmnat1 knockout mice were used to determine a possible link between nuclear NAD+ and high-fat diet (HFD)-induced MetS. We found that Nmnat1 knockout significantly reduced hepatic nuclear NAD+ levels but did not exacerbate HFD-induced obesity and hepatic triglycerides accumulation. Interestingly, loss of Nmnat1 caused insulin resistance. Further analysis revealed that Nmnat1 deletion promoted gluconeogenesis but inhibited glycogen synthesis in the liver. Moreover, Nmnat1 deficiency induced mitochondrial dysfunction by decreasing mitochondrial DNA (mtDNA)-encoded complexes Ⅰ and Ⅳ, suppressing mtDNA replication and mtRNA transcription and reducing mtDNA copy number. In addition, Nmnat1 depletion affected the expression of hepatokines in the liver, particularly downregulating the expression of follistatin. These findings highlight the importance of nuclear NAD+ in maintaining insulin sensitivity and provide insights into the mechanisms underlying HFD-induced insulin resistance.

Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats.

Nicotinamide riboside (NR) is an NAD+ precursor capable of regulating mammalian cellular metabolism. Phycocyanin oligopeptide (PC), a phytonutrient found in blue-green algae, has antioxidant and anti-inflammatory properties. This study explored the effects of NR, PC, and their combination on the telomere length as well as inflammatory and antioxidant status of rats under chronic stress conditions (CS). Forty-nine rats were allocated into seven groups: control, chronic stress (CS), CS with NR (26.44 mg/kg), a low dose of 2.64 mg/kg of PC (PC-LD), or a high dose of 26.44 mg/kg PC (PC-HD), NR + PC-LD, and NR + PC-HF. The rats were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. NR and PC supplementation, particularly NR plus PC, increased the serum antioxidant enzyme activities, hepatic nicotinamide adenine (NAD+) content, and telomere length (p < 0.001 for all) compared to the CS group. The levels of serum malondialdehyde (MDA), liver interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), IL-1β, and IL-8 were reduced under the CS condition (p < 0.001). In addition, CS decreased the levels of hepatic telomere-related proteins and sirtuins (SIRT1 and 3), whereas administration of NR and PC or their combination to CS-exposed rats increased the levels of telomere-related proteins (e.g., POT1b, TRF1 and TRF2), SIRT3 and NAMPT (p < 0.05). In conclusion, NR and PC, especially their combination, can alleviate metabolic abnormalities by enhancing hepatic cytokines, SIRT3, NAMPT, and NAD+ levels in CS-exposed rats. More research is needed to further elucidate the potential health effects of the combination of NR and PC in humans.

Large Supplements of Nicotinic Acid and Nicotinamide Increase Tissue NAD+ and Poly(ADP-Ribose) Levels but Do Not Affect Diethylnitrosamine-Induced Altered Hepatic Foci in Fischer-344 Rats

Poly(ADP-ribose) is a homopolymer of ADP-ribose units synthesized from NAD+ on nuclear acceptor proteins and is known to be involved in DNA repair. It is not known whether large oral doses of the clinically utilized NAD precursors nicotinic acid or nicotinamide affect poly(ADP-ribose) metabolism or the cellular response to DNA damage. In our first study, using Fischer-344 rats, 2 wk of dietary nicotinic acid supplementation (500 and 1000 mg/kg diet) caused elevated levels of NAD+ in the blood, liver, heart and kidney, while nicotinamide caused elevated levels only in the blood and liver, compared with controls fed a diet containing 30 mg/kg nicotinic acid. Both nicotinic acid and nicotinamide, at 1000 mg/kg diet, caused elevations in liver NAD+, by 44 and 43%, respectively. Only nicotinamide, however, elevated liver poly(ADP-ribose) (63% higher than control group). Following treatment with the hepatocarcinogen diethylnitrosamine, higher levels of hepatic NAD+ were observed in rats fed both nicotinic acid and nicotinamide at 1000 mg/kg diet, but only nicotinic acid supplementation caused a greater accumulation of hepatic poly(ADP-ribose) (61% higher than control group). Neither of the dietary treatments significantly affected the proportion of the liver occupied by placental glutathione-S-transferase positive foci. These results show that poly(ADP-ribose) synthesis is not directly responsive to hepatic NAD+ levels during niacin supplementation, and that the mechanisms of action of nicotinic acid and nicotinamide are different. The observed changes in poly(ADP-ribose) metabolism do not appear to cause any change in susceptibility to chemically induced carcinogenesis in this organ.

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

Hepatic NAD+ homeostasis is essential to metabolic flexibility upon energy balance challenges. The molecular mechanism is unclear. This study aimed to determine how the enzymes involved in NAD+ salvage (Nampt, Nmnat1, Nrk1), clearance (Nnmt, Aox1, Cyp2e1), and consumption pathways (Sirt1, Sirt3, Sirt6, Parp1, Cd38) were regulated in the liver upon energy overload or shortage, as well as their relationships with glucose and lipid metabolism. Male C57BL/6N mice were fed ad libitum with the CHOW diet, high-fat diet (HFD), or subjected to 40% calorie restriction (CR) CHOW diet for 16 weeks respectively. HFD feeding increased hepatic lipids content and inflammatory markers, while lipids accumulation was not changed by CR. Both HFD feeding and CR elevated the hepatic NAD+ levels, as well as gene and protein levels of Nampt and Nmnat1. Furthermore, both HFD feeding and CR lowered acetylation of PGC-1α in parallel with the reduced hepatic lipogenesis and enhanced fatty acid oxidation, while CR enhanced hepatic AMPK activity and gluconeogenesis. Hepatic Nampt and Nnmt gene expression negatively correlated with fasting plasma glucose levels concomitant with positive correlations with Pck1 gene expression. Nrk1 and Cyp2e1 gene expression positively correlated with fat mass and plasma cholesterol levels, as well as Srebf1 gene expression. These data highlight that hepatic NAD+ metabolism will be induced for either the down-regulation of lipogenesis upon over nutrition or up-regulation of gluconeogenesis in response to CR, thus contributing to the hepatic metabolic flexibility upon energy balance challenges.

Poly(ADP-Ribose) Polymerase Activity and DNA Strand Breaks Are Affected in Tissues of Niacin-Deficient Rats

The niacin cofactor, NAD, is the substrate for poly(ADP-ribose) polymerase, an enzyme associated with DNA repair. We investigated, therefore, whether hepatic poly(ADP-ribose) polymerase activity was altered and DNA strand breaks in lymphocytes and liver were greater in niacin-deficient rats. A niacin deficiency was established in weanling rats with diets containing 1.5 mg/kg of niacin. Based on lower growth rates and NAD concentrations in blood, liver and skeletal muscle, this diet maintained rats in a deficient state for 1 mo, and, when the dietary niacin was reduced to 0.5 mg/kg, rats remained deficient for an additional month. The hepatic poly(ADP-ribose) polymerase activity was decreased in one experiment when mean hepatic NAD concentrations were 0.60 and 0.51 mumol/g at d 34 and d 60, respectively, compared with 0.77 and 0.80 mumol/g in pair-fed controls. Enzyme activity, however, was greater than in controls when hepatic NAD concentrations were < 0.30 mumol/g. Strand breaks in DNA did not accumulate except after tissues were exposed to hypoxanthine-xanthine oxidase, a free radical-generating system. Exposure to this system caused more DNA strand breaks in lymphocytes and hepatic nuclei from niacin-deficient rats compared with the same tissues from controls. The results suggest that, in rats, although hepatic poly(ADP-ribose) polymerase activity can be elevated, a severe niacin deficiency may increase the susceptibility of DNA to oxidative damage, likely due to a lower availability of NAD.

The mitochondrial NAD+ transporter SLC25A51 is a fasting-induced gene affecting SIRT3 functions

IntroductionNicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD+. What is the mechanism, then, to transport more NAD+ into mitochondria to sustain enhanced SIRT3 activity during fasting? ObjectiveSLC25A51 is a recently discovered mitochondrial NAD+ transporter. We tested the hypothesis that, during fasting, increased expression of SLC25A51 is needed for enhanced mitochondrial NAD+ uptake to sustain SIRT3 activity. Because the fasting-fed cycle and circadian rhythm are closely linked, we further tested the hypothesis that SLC25A51 is a circadian regulated gene. MethodsWe examined Slc25a51 expression in the liver of fasted mice, and examined its circadian rhythm in wild-type mice and those with liver-specific deletion of the clock gene BMAL1 (LKO). We suppressed Slc25a51 expression in hepatocytes and the mouse liver using shRNA-mediated knockdown, and then examined mitochondrial NAD+ levels, SIRT3 activities, and acetylation levels of SIRT3 target proteins (IDH2 and ACADL). We measured mitochondrial oxygen consumption rate using Seahorse analysis in hepatocytes with reduced Slc25a51 expression. ResultsWe found that fasting induced the hepatic expression of Slc25a51, and its expression showed a circadian rhythm-like pattern that was disrupted in LKO mice. Reduced expression of Slc25a51 in hepatocytes decreased mitochondrial NAD+ levels and SIRT3 activity, reflected by increased acetylation of SIRT3 targets. Slc25a51 knockdown reduced the oxygen consumption rate in intact hepatocytes. Mice with reduced Slc25a51 expression in the liver manifested reduced hepatic mitochondrial NAD+ levels, hepatic steatosis and hypertriglyceridemia. ConclusionsSlc25a51 is a fasting-induced gene that is needed for hepatic SIRT3 functions.

Nicotinamide riboside supplementation exerts an anti–obesity effect and prevents inflammation and fibrosis in white adipose tissue of female diet-induced obesity mice

Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor. We previously reported that NR supplementation prevented the development of liver fibrosis in male mice. However, whether NR exerts a similar effect in females is unknown. Therefore, we determined whether NR supplementation can prevent obesity-induced inflammation and fibrosis in the liver and white adipose tissue (WAT) by providing NAD+ in obese female mice. Female C57BL/6J mice at the age of 8 weeks (young) and 16 weeks (old) were fed a high-fat/high-sucrose/high-cholesterol diet (HF) or HF diet supplemented with NR at 400 mg/kg/d for 20 weeks. While NR had minor effects in young female mice, it significantly reduced body weight gain, fat mass, glucose intolerance, and serum cholesterol levels compared to the HF group in old females. Hepatic NAD+ level tended toward an increase in the NR group (P=.054), but NR did not attenuate serum alanine aminotransferase levels, steatosis, and liver fibrosis in old female mice. However, NR decreased weight and adipocyte size in gonadal WAT (gWAT) of old females. NR also reduced the number of crown-like structures and the expression of inflammatory genes, along with decreases in fibrogenic gene expression and collagen accumulation in gWAT compared with the HF group. Also, old mice fed NR showed increased metabolic rates, physical activity, and energy expenditure compared with the HF. Thus, our results indicated that NR supplementation exerted an anti–obesity effect and prevented the development of inflammation and fibrosis in the WAT of old, but not young, female mice with diet-induced obesity.

Inhibition of NAD kinase elevates the hepatic NAD+ pool and alleviates acetaminophen-induced acute liver injury in mice

Acetaminophen (APAP) overdose induces acute liver injury (ALI), even acute liver failure (ALF). There is a significant unmet need to furtherly elucidate the mechanisms and find new therapeutic target. Recently, emerging evidence indicates that nicotinamide adenine dinucleotide (NAD+) plays a crucial role in APAP-induced ALI. Herein, we firstly investigated the protein expression of NAD kinase (NADK), as the rate-limiting enzyme converting NAD+ to nicotinamide adenine dinucleotide phosphate (NADP+), and found it was positively correlated with APAP-induced ALI in a dose- and time-dependent manner. Additionally, supplementation of N-acetylcysteine (NAC), known as an antidote of APAP, mitigated the ALI and downregulated the expression of NADK which was also in a dose-dependent manner. Moreover, pretreatment with methotrexate (MTX), the inhibitor of NADK, attenuated the levels of transaminases, alleviated morphological abnormalities, and improved oxidative stress triggered by APAP overdose, which was attributed to elevated hepatic NAD+ pool. Subsequently, the increased NAD+ upregulated the expression of Sirt1, SOD2 and attenuated DNA damage. Collectively, elevated expression of NADK is related to APAP-induced ALI, and inhibition of NADK alleviates the ALI through elevating liver NAD+ level and improving antioxidant capacity.

NAMPT-mediated NAD+ biosynthesis suppresses activation of hepatic stellate cells and protects against CCl4-induced liver fibrosis in mice.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD+) biosynthesis. Through its NAD+-biosynthetic activity, NAMPT is able to regulate the development of hepatic steatosis and inflammation induced by diet or alcohol. However, the roles NAMPT plays in the development of liver fibrosis remain obscure.Purpose: To investigate the roles of NAMPT-mediated NAD+biosynthesis in hepatic stellate cell (HSC) activation and liver fibrosis.Research Design: Realtime RT-PCR and western blot analyses were performed to analyze the expression of profibrogenic genes. Sirius red staining was conducted to examine the fibrosis in liver. Mouse liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) 2 times a week for 6 weeks. Adenovirus-mediated NAMPT overexpression or nicotinamide mononucleotide (NMN) administration was carried out to study the effects of elevation of NAD+levels on protecting CCl4-induced liver fibrosis in mice. LX2 cells or primary HSCs were used to study the role of NAMPT overexpression or NMN treatment in reducing profibrogenic gene expression in vitro.ResultsCCl4administration suppresses NAMPT expression in liver and reduces hepatic NAD+content. Tgfβ1 treatment decreases intracellular NAD+levels and NAMPT expression in LX2 cells. Adenovirus-mediated NAMPT overexpression augments liver NAD+levels, inhibits HSC activation and alleviates CCl4-induced liver fibrosis in mice. Administration of NMN also suppresses HSC activation and protects against CCl4-induced liver fibrosis in mice.Conclusions: NAMPT-mediated NAD+biosynthesis inhibits HSC activation and protects against CCl4-induced liver fibrosis.

Hepatocyte-specific perturbation of NAD+ biosynthetic pathways in mice induces reversible nonalcoholic steatohepatitis–like phenotypes

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.

A mouse model of the regression of alcoholic hepatitis: Monitoring the regression of hepatic steatosis, inflammation, oxidative stress, and NAD+ metabolism upon alcohol withdrawal

This study aimed to develop a well-characterized mouse model of alcoholic hepatitis (AH) regression. Male C57BL/6J mice were fed a Lieber-DeCarli (LD) control diet or LD containing 5% ethanol for ten days followed by one binge, which is the chronic-binge model of AH developed by the National Institute on Alcohol Abuse and Alcoholism. To determine AH regression, mice previously exposed to ethanol were put on LD control diet and metabolic and inflammatory features were monitored weekly for three weeks. Serum alcohol, total cholesterol, and alanine transaminase levels were increased in ethanol-fed mice, which declined to those of no ethanol controls within one and three weeks after ethanol withdrawal, respectively. Serum malondialdehyde was increased with ethanol feeding, but it was restored to no ethanol control levels within one week. Ethanol-induced changes in the hepatic expression of genes involved in lipogenesis, fatty acid oxidation, ethanol metabolism, and antioxidant response were restored to those of no ethanol controls after 3 weeks of ethanol withdrawal. Also, ethanol-induced hepatic inflammation was gradually decreased during the 3 weeks of ethanol withdrawal. Hepatic nicotinamide adenine dinucleotide (NAD+) levels and the expression of enzymes involved in the NAD+ salvage pathway were decreased by ethanol feeding, which was mitigated after ethanol withdrawal. Ethanol significantly lowered hepatic sirtuin 1 expression, but its levels were restored with ethanol cessation. This study established a mouse model of AH regression, which can be used as a preclinical model to study the potential of dietary bioactives or therapeutic agents on AH regression.

Silybin Restored CYP3A Expression through the Sirtuin 2/Nuclear Factor κ-B Pathway in Mouse Nonalcoholic Fatty Liver Disease.

Silybin is widely used as a hepatoprotective agent in various liver disease therapies and has been previously identified as a CYP3A inhibitor. However, little is known about the effect of silybin on CYP3A and the regulatory mechanism during high-fat-diet (HFD)-induced liver inflammation. In our study, we found that silybin restored CYP3A expression and activity that were decreased by HFD and conditioned medium (CM) from palmitate-treated Kupffer cells. Moreover, silybin suppressed liver inflammation in HFD-fed mice and inhibited nuclear factor κ-B translocation into the nucleus through elevation of SIRT2 expression and promotion of p65 deacetylation. This effect was confirmed by overexpression of SIRT2, which suppressed p65 nuclear translocation and restored CYP3A transcription affected by CM. The hepatic NAD+ concentration markedly decreased in HFD-fed mice and CM-treated hepatocytes/HepG2 cells but increased after silybin treatment. Supplementing nicotinamide mononucleotide as an NAD+ donor inhibited p65 acetylation, decreased p65 nuclear translocation, and restored cyp3a transcription in both HepG2 cells and mouse hepatocytes. These results suggest that silybin regulates metabolic enzymes during liver inflammation by a mechanism related to the increase in NAD+ and SIRT2 levels. In addition, silybin enhanced the intracellular NAD+ concentration by decreasing poly-ADP ribosyl polymerase-1 expression. In summary, silybin increased NAD+ concentration, promoted SIRT2 expression, and lowered p65 acetylation both in vivo and in vitro, which supported the recovery of CYP3A expression. These findings indicate that the NAD+/SIRT2 pathway plays an important role in CYP3A regulation during nonalcoholic fatty liver disease. SIGNIFICANCE STATEMENT: This research revealed the differential regulation of CYP3A by silybin under physiological and fatty liver pathological conditions. In the treatment of nonalcoholic fatty liver disease, silybin restored, not inhibited, CYP3A expression and activity through the NAD+/ sirtuin 2 pathway in accordance with its anti-inflammatory effect.

Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice

ObjectiveThroughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). MethodsUsing targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies. ResultsExogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia. ConclusionsKetone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.

NAD+ and NAFLD - caution, causality and careful optimism.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide, and new treatments are sorely needed. Nicotinamide adenine dinucleotide (NAD+ ) has been proposed as a potential target to prevent and reverse NAFLD. NAD+ is an important redox factor for energy metabolism and is used as a substrate by a range of enzymes, including sirtuins (SIRT), which regulates histone acetylation, transcription factor activity and mitochondrial function. NAD+ is also a precursor for reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is an important component of the antioxidant defense system. NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are available as over-the-counter dietary supplements, and oral supplementation with these precursors increases hepatic NAD+ levels and prevents hepatic lipid accumulation in pre-clinical models of NAFLD. NAD+ precursors have also been found to improve hepatic mitochondrial function and decrease oxidative stress in pre-clinical NAFLD models. NAD+ repletion also prevents NAFLD progression to non-alcoholic steatohepatitis (NASH), as NAD+ precursor supplementation is associated with decreased hepatic stellate cell activation, and decreased fibrosis. However, initial clinical trials have only shown modest effects when NAD+ precursors were administrated to people with obesity. We review the available pre-clinical investigations of NAD+ supplementation for targeting NAFLD, and discuss how data from the first clinical trials can be reconciled with observations from preclinical research.

Moderate Exercise Inhibits Age-Related Inflammation, Liver Steatosis, Senescence, and Tumorigenesis.

Age-related chronic inflammation promotes cellular senescence, chronic disease, cancer, and reduced lifespan. In this study, we wanted to explore the effects of a moderate exercise regimen on inflammatory liver disease and tumorigenesis. We used an established model of spontaneous inflammaging, steatosis, and cancer (nfkb1-/- mouse) to demonstrate whether 3 mo of moderate aerobic exercise was sufficient to suppress liver disease and cancer development. Interventional exercise when applied at a relatively late disease stage was effective at reducing tissue inflammation (liver, lung, and stomach), oxidative damage, and cellular senescence, and it reversed hepatic steatosis and prevented tumor development. Underlying these benefits were transcriptional changes in enzymes driving the conversion of tryptophan to NAD+, this leading to increased hepatic NAD+ and elevated activity of the NAD+-dependent deacetylase sirtuin. Increased SIRT activity was correlated with enhanced deacetylation of key transcriptional regulators of inflammation and metabolism, NF-κB (p65), and PGC-1α. We propose that moderate exercise can effectively reprogram pre-established inflammatory and metabolic pathologies in aging with the benefit of prevention of disease.

Activation of AhR-NQO1 Signaling Pathway Protects Against Alcohol-Induced Liver Injury by Improving Redox Balance.

Background & AimsAryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD).MethodsChronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhRΔhep) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis.ResultsChronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD+)-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD+ depletion, thereby enhancing activities of NAD+-dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation.ConclusionsThis study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD+ depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD.

Exposure of Rats to Multiple Oral Doses of Dichloroacetate Results in Upregulation of Hepatic Glutathione Transferases and NAD(P)H Dehydrogenase [Quinone] 1.

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

Biomarkers of Broccoli Consumption: Implications for Glutathione Metabolism and Liver Health.

Diet and lifestyle choices contribute to obesity and liver disease. Broccoli, a brassica vegetable, may mitigate negative effects of both diet and lifestyle. Currently, there are no clinically relevant, established molecular biomarkers that reflect variability in human absorption of brassica bioactives, which may be the cause of variability/inconsistencies in health benefits in the human population. Here, we focused on the plasma metabolite profile and composition of the gut microbiome in rats, a relatively homogenous population in terms of gut microbiota, genetics, sex and diet, to determine if changes in the plasma metabolite profiles caused by dietary broccoli relate to molecular changes in liver. Our aim was to identify plasma indicators that reflect how liver health is impacted by dietary broccoli. Rats were fed a 10% broccoli diet for 14 days. We examined the plasma metabolite composition by metabolomics analysis using GC–MS and gut microbiota using 16S sequencing after 0, 1, 2, 4, 7, 14 days of broccoli feeding. We identified 25 plasma metabolites that changed with broccoli consumption, including metabolites associated with hepatic glutathione synthesis, and with de novo fatty acid synthesis. Glutamine, stearic acid, and S-methyl-L-cysteine (SMC) relative abundance changes correlated with changes in gut bacteria previously implicated in metabolic disease and with validated increases in expression of hepatic NAD(P)H dehydrogenase [quinone] 1 (NQO1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), associated with elevated hepatic glutathione synthesis. Circulating biomarkers following broccoli consumption reflect gut–liver axis health.