Hepatic NADH reductive stress underlies common variation in metabolic traits

Abstract

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of ‘causal metabolism’.

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Fig. 1: LbNOX alters compartment-specific free NADH/NAD+ ratio in hepatocytes.
Fig. 2: In vivo manipulation of the hepatic cytosolic NADH/NAD+ ratio alters hepatic and circulating αHB levels.
Fig. 3: A common GCKR variant is associated with plasma αΗΒ levels in humans.
Fig. 4: Direct oxidation of free hepatic cytosolic NADH improves glucose tolerance and hepatic insulin sensitivity in vivo.
Fig. 5: Many GCKR-associated metabolic traits lie downstream of hepatic NADH reductive stress.

Data availability

All data generated and used in this study are either included in this article (and its Supplementary Information) or are available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. Avruch and members of the Mootha laboratory for valuable discussions and feedback. R.P.G. was supported by the US National Institutes of Health (NIH) grant F32DK111132 and NIH K08DK1158811. R.P.G. and R.M. were both supported by a Pinnacle Research Award from the American Association for the Study of Liver Diseases (AASLD). O.S.S. was supported by NIH F32GM133047.  Y.-H.H.H. was supported by NIH T32DK110919. Part of this study was performed at the National Mouse Metabolic Phenotyping Center at University of Massachusetts supported by NIH 5U2C-DK093000 (to J.K.K.) This work was supported in part by a gift from the Marriott Mitochondrial Disorders Collaborative Research Network and grants from the NIH (R35GM122455 and TR01GM099683). V.K.M. is an investigator of the Howard Hughes Medical Institute.

Author information

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Authors

Contributions

R.P.G. and V.K.M. designed the study, which was supervised by V.K.M. R.P.G. and R.M. performed the Peredox experiments, which were supervised by G.Y. R.P.G., A.L.M., A.P. and O.G. performed the in vivo mouse experiments, tissue analyses and hepatocyte experiments. R.P.G., R.S., O.S.S., H.S., C.B.C. and A.D. processed and analysed in vitro and in vivo mouse metabolomics data. H.L.N., S.S. and J.K.K. processed and analysed the hyperinsulinaemic–euglycaemic clamp experiments. Y.-H.H.H. and J.N.H. contributed to the human genetic data analyses. R.P.G. wrote the initial manuscript, which was further edited by V.K.M.

Corresponding author

Correspondence to Vamsi K. Mootha.

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Competing interests

V.K.M. is an inventor on the patent PCT/US2016/045015 filed by Massachusetts General Hospital on the use of the LbNOX technology as protein prosthesis for mitochondrial diseases or conditions. V.K.M. and R.P.G. are inventors on a patent provisionally filed by Massachusetts General Hospital on modulating hepatic reductive stress with chemicals.

Additional information

Peer review information Nature thanks Joshua D. Rabinowitz, Charles M. Brenner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 The effects of LbNOX expression in primary hepatocytes.

a, Dose-dependent adenovirus-mediated expression of LbNOX in primary hepatocytes at 24 h. A representative western blot from two independent experiments is shown. b, Effect on basal or antimycin + rotenone-insensitive respiration with LbNOX or mitoLbNOX. n = 3. c, Effect of LbNOX on free cytosolic NADH/NAD+ as measured by Peredox with increasing alcohol concentrations. dg, Whole cell NAD+ (d), NADH (e), NADH/NAD+ (f) and NADH + NAD+ (g). n = 6 independent hepatocyte isolations. Nominal P values were determined using paired, two-sided Student’s t-tests between hepatocyte isolations (b, dg) or unpaired, two-sided Student’s t-test for Peredox experiments (c). Data are mean  ± s.e.m. Source Data

Extended Data Fig. 2 NR and LbNOX have distinct effects on pyridine dinucleotide pool sizes and redox ratios.

af, Relative total cellular NAD+ (a), NADH (b), NADH/NAD+ (c), secreted lactate/pyruvate ratio (d), secreted βHB/AcAc ratio (e) and αHB levels (f) in primary hepatocytes with or without NR supplementation and LbNOX. g, h, Effect of pyruvate (Pyr), LbNOX expression or NR on the inhibition of HeLa cell proliferation (g) and total NAD+ levels (h) by piericidin 4 days after seeding. Data are mean ± s.e.m. from n = 7 (ac) or 10 (df) independent hepatocyte isolation, or 3 independent HeLa cell experiments (g and h). Nominal P values were determined using paired (between hepatocyte isolations; af) or unpaired (HeLa cells; g and h) two-sided Student’s t-tests. Source Data

Extended Data Fig. 3 αHB levels and the hepatic NADH/NAD+ ratio are elevated in the Ndufs4-KO mouse model of mitochondrial disease.

a, Plasma αHB levels. b, Hepatic NADH/NAD+ ratio. n = 3 mice in each group for NADH/NAD+ measurements and n = 7 mice for αHB measurements. Data are mean ± s.e.m. P values were determined using one-sided Student’s t-test. WT, wild type. Source Data

Extended Data Fig. 4 A common GCKR variant increases hepatic cytosolic NADH reductive stress.

a, Gene structure, variant location, variant linkage disequilibrium blocks and haplotype frequency for the GCKR risk haplotype from the 1000 Genomes Project50. AFR, African; EAS, East Asian; EUR, European; SAS, South Asian. b, Enrichment of redox-sensitive metabolites in reported metabolite associations by loci from ref. 5. P values were determined from one-tailed Fisher’s exact test as described in the Methods. c, Effects of overexpression of Gckr or Gckr p.P446L in mouse primary hepatocytes. Data are mean ± s.e.m. Nominal P values were determined using paired, two-sided Student’s t-tests between n = 5 independent hepatocyte isolations. Source Data

Extended Data Fig. 5 Effects of LbNOX expression on metabolic parameters during hyperinsulinaemic–euglycaemic clamp.

ak, Effects of LbNOX expression in HFD-fed mice using a 2.5 mU min−1 kg−1 insulin infusion during hyperinsulinaemic–euglycaemic clamp on body weight (a), basal glucose levels (b), clamp glucose levels (c), glucose infusion rate (d), whole-body glucose turnover (e), whole-body glycolysis (f), whole-body glycogen synthesis (g), lean mass (h), fat mass (i), WAT glucose uptake (j) and skeletal muscle glucose uptake (k). P values were determined using two-sided Student’s t-test. Data are reported as mean ± s.e.m. from n = 8 (luciferase) or n = 9 (LbNOX) mice. Source Data

Extended Data Fig. 6 Hepatic diacyl-glycerols and ceramides in DIO mice with LbNOX expression.

a, b, Effect of LbNOX expression of hepatic diacyl-glycerol (a) and ceramide (b) content in DIO mice. Data are reported as mean ± s.e.m. from n = 9 mice. P values were determined using two-sided Student’s t-test. Source Data

Extended Data Fig. 7 LbNOX improves hepatic insulin resistance in vivo independent of hepatic insulin signalling.

ac, Western blots of liver lysate from DIO mice 15 min after an intraperitoneal injection of saline or 2 U/kg insulin (a) with relative pS474 AKT/total AKT (b) and relative pT308 AKT/total AKT (c). n = 3 from representative western blots from 2 independent experiments. d, Transcriptional FOXO1 targets G6pc, Pepck1 and Pc in DIO mice with LbNOX or luciferase. n = 6. e, Western blots of liver lysates at the end of hyperinsulinaemic–eugylcaemic clamps. n = 3 representative of n = 8 (luciferase) and 9 (LbNOX). f, g, Relative pS474 AKT/total AKT and pT308 AKT/total AKT (f) and transcriptional FOXO1 targets G6pc, Pepck1 and Pc (g). n = 8 (luciferase) and 9 (LbNOX). h, Crossover analysis of relative abundance of gluconeogenic intermediates at the end of hyperinsulinaemic–euglycaemic clamps. Top, LbNOX versus Luc mice are compared. Bottom, samples are divided by high or low liver lactate/pyruvate (L/P) ratios and compared. *P < 0.05, **P < 0.01, using two-sided Student’s t-test. BPG, 1,3-bisphosphoglycerate; DAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; F1P, fructose 1-phosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; MAL, malate; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PYR, pyruvate. i, Western blots and relative protein levels of GAPDH and triosephosphate isomerase (TPI) at the end of the insulin clamp. n = 8 (luciferase) and 9 (LbNOX). Data are reported as mean ± s.e.m. Source Data

Extended Data Fig. 8 NAD(P)(H) levels in LbNOX versus luciferase livers at the end of hyperinsulinaemic–euglycaemic clamps.

af, The relative abundance of total NAD+ (a), total NADH (b), total NADH/NAD+ (c), total NADP+ (d), total NADPH (e) and total NADPH/NADP+ (f). n = 4 mice for each group. P values were determined by one-sided Student’s t-test. Data are reported as mean ± s.e.m. Source Data

Extended Data Fig. 9 Metabolic origins and fate of αHB.

α-AB, α-aminobutyrate; BCKDH, branched-chain α-keto acid dehydrogenase complex; CGL, cystathionine γ-lyase; PDH, pyruvate dehydrogenase; S/TDH, serine/threonine dehydratase; TCA, tricarboxylic acid.

Supplementary information

Supplementary Figure

Supplementary Figure 1: Original westerns blots.

Reporting Summary

Supplementary Table

Supplementary Table 1: Literature summary of traits associated with GCKR genetic variants.

Supplementary Table

Supplementary Table 2: Hepatocyte metabolite screen related to Figure 1f.

Supplementary Table

Supplementary Table 3: Hyperinsulinemic-euglycemic clamp of LbNOX mice on high-fat-diet.

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Goodman, R.P., Markhard, A.L., Shah, H. et al. Hepatic NADH reductive stress underlies common variation in metabolic traits. Nature 583, 122–126 (2020). https://doi.org/10.1038/s41586-020-2337-2

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