The first step of this project was the technical development of an in-vivo MRS technique to monitor NAD+ and NADH non-invasively. To this end, a novel 31P-MRS sequence to selectively suppress the alpha ATP spin system was developed and implemented. Alpha ATP resonances are usually overlapping with the NAD metabolites in vivo in human muscle, hampering the quantification of NAD.
For validation of the newly developed MR sequence, we performed phantom measurements and a study in healthy volunteers to test reproducibility and test whether we could detect physiological changes in NAD+ and NADH. It is well known that ischemia leads to an accumulation of NADH, at the expense of NAD+ in muscle. Therefore, we included 8 young healthy lean participants and determined NAD+/NADH levels at rest and during ischemia (applying a cuff on the upper leg). The newly developed MR sequence allowed us to determine changes in NAD+ and NADH during ischemia in young healthy lean participants and indeed, we found that NADH was increased and NAD+ decreased upon ischemia. A hallmark within this ERC project since the new developed MR sequence was able to detect physiological changes in these metabolites in vivo.
To test whether the newly developed MRS sequence can detect differences in NAD+ and NADH between groups, we assessed NAD+ and NADH content in skeletal muscle of normally active and exercise-trained older adults, anticipating a higher NAD+ content in exercise-trained group. Indeed, we found higher relative NAD+ concentrations in the more active group. We also determined the agreement of the MRS method with biopsy values. A scientific paper highlighting this new MR sequence and the validation steps taken has been written and after positive reviewer comments is in the process of resubmission. Furthermore, the description of the novel technique and validation measurements were presented on multiple national and international conferences.
We next performed a clinical intervention study to investigate the effect of oral supplementation with nicotinic riboside (NR) (a precursor of NAD+) on metabolic health. Earlier studies did not show very pronounced effects in humans, but it was suggested that NR supplementation may only be effective if the need for NAD+ is augmented, which may be the case with exercise training. Therefore, we tested whether NR combined with exercise leads to greater improvements on skeletal muscle mitochondrial respiratory capacity as compared to exercise alone. We also investigated if oral NR supplementation amplifies the effects of exercise on parameters related with metabolic health and functional markers of physical function. Thirty participants (13 male and 17 female, aged between 65-80 years, BMI: 25-35 kg/m2) were included and we investigated whether oral NR supplementation combined with exercise augments the NAD+ levels in blood and modifies the skeletal muscle NAD+ metabolome. Furthermore, we tested whether oral NR supplementation amplified the exercise-induced improvements on skeletal muscle mitochondrial respiratory capacity, increased the exercise-induced improvements on sleeping metabolic rate, augmented the exercise effects on intrahepatic lipid content and composition, and amplified the benefits of exercise on physical function. We currently are finalizing the manuscript for submission. These findings have important implications for successful supplementation with NAD precursors, as such supplementation seems to be more successful when combined with exercise training.
