Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD + depletion

Wellcome Open Research

article

Alpesh Thakker¹  Oliver D. K. Maddocks²  Tong Zhang²  Daniel A. Tennant¹  Christian Ludwig¹  Gareth G. Lavery¹  Lucy A. Oakey¹  Rachel S. Fletcher¹  Yasir S. Elhassan¹  David M. Cartwright¹  Craig L. Doig¹  Antje Garten¹ 

[1] Institute of Metabolism and Systems Research, University of Birmingham;Institute of Cancer Sciences, University of Glasgow

关键词: NAD+;    NAMPT;    NR;    metabolism;    skeletal muscle;    isotopic tracing;    aspartate;   

DOI  :  10.12688/wellcomeopenres.14898.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown.Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis.Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD+, which was rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation.Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.

【 授权许可】

CC BY