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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.

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) spectroscopy offers high resolution data in terms of resolving metabolic pathway utilisation. Despite this, NMR spectroscopy is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting of 13C signals due to homonuclear 13C,13C or heteronuclear 13C,15N J-coupling in 1H,13C-HSQC NMR spectra. Together, these allow the rapid collection of NMR spectroscopy data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ. In addition, we show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) offers high resolution data, yet is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting due to J-coupling in 1H,13C-HSQC NMR spectra, which allow the rapid collection of NMR data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these novel techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ, and also show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.