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Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.

ABSTRACT:

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With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD(+) ) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 knockout mice (Sirt1(hep-/-) ), whereas apolipoprotein E-deficient mice (Apoe(-/-) ) challenged with a high-fat high-cholesterol diet affirmed the use of NR in other independent models of NAFLD.

Conclusion

Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD.

SUBMITTER: Gariani K

PROVIDER: S-EPMC4805450 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nicotinamide adenine dinucleotide (NAD+) and related coenzymes play critical roles in liver function. Although hepatic alcohol metabolism depresses NAD+, current understanding of the NAD+ metabolome in alcohol-related liver disease (ArLD) is based on animal models. We used human liver samples to quantify the NAD+ metabolome in ArLD with samples obtained at the time of liver transplantation or resection at University Hospitals Birmingham National Health Service Foundation Trust. The severity of steatohepatitis in liver from patients with ArLD was assessed with standard liver function tests and histology. NAD-targeted quantitative metabolomic analysis of liver tissue was performed by liquid chromatography-tandem mass spectrometry. Seventy-two human liver specimens were analyzed, including 43 with ArLD. The NAD+ metabolome differed significantly between different types of liver disease (two-way analysis of variance [ANOVA], P = 0.001). ArLD liver tissue showed markedly depressed concentrations of NAD+ (432 μM vs. 616 μM in normal liver) and precursor molecules nicotinic acid and nicotinamide riboside. There was a significant overall difference in the NAD+ metabolome between ArLD samples with and without steatohepatitis (two-way ANOVA, P = 0.018). After correcting for multiple comparisons, a significant difference for individual components of the metabolome was observed for the concentration of NAD+ (mean, 462 μM vs. 322 μM; P < 0.01 in nonsevere vs. severe alcoholic steatohepatitis, respectively). NAD+ concentration was inversely related to serum bilirubin concentration (r 2 = -0.127; P = 0.04) and positively correlated with myeloperoxidase activity (r 2 = 0.31; P = 0.003). The concentration of NAD+ and its precursor molecules are significantly reduced in ArLD and are associated with disease activity. Conclusion: Liver samples from people with ArLD show depressed NAD+ and precursor levels as well as depressed myeloperoxidase activity.

Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

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Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.

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