Periodic Reporting for period 4 - MRS in diabetes (Novel methods in Magnetic Resonance Spectroscopy to investigate mechanisms underlying metabolic disease)
Berichtszeitraum: 2022-09-01 bis 2023-07-31
The current high prevalence of overweight and obesity and the low levels of physical activity results in a high prevalence of type 2 diabetes and other metabolic diseases. Studies investigating the underlying molecular mechanisms have suggested that NAD+ may play a crucial role in preserving metabolic health, however studies were mostly performed in animals, because investigation in humans requires invasive procedures (muscle biopsies) where repeated sampling is problematic. To identify and monitor the mechanisms responsible for the development of metabolic disease, non-invasive imaging methods are needed to investigate metabolism dynamically. Magnetic Resonance Spectroscopy (MRS) has great potential as it can non-invasively yield metabolic information in many tissues, with high time resolution and without radioactive tracers.
To benefit from the full potential of MRS, we need to put more effort into designing novel sequences. Within the first objective of this project we have successfully set-up, optimized and validated a novel MRS- based method to quantify NAD+ on a clinical scanner at 3T. In a second objective, it was tested whether skeletal muscle NAD+ levels are related to insulin resistance and metabolic disease. To this end, we have investigated the concentration of NAD+ in volunteers differing in metabolic health andhave shown that NAD+ in muscle of physically very active elderly volunteers is elevated when compared to normally active elderly. Finally, the novel methodology was used to investigate the effect of supplementation of NAD precursors in humans and we showed that the combination of high intensity exercise and NAD precursor supplementation had beneficial effects on mitochondrial function and physical fitness when compared to exercise alone.
To benefit from the full potential of MRS, we need to put more effort into designing novel sequences. Within the first objective of this project we have successfully set-up, optimized and validated a novel MRS- based method to quantify NAD+ on a clinical scanner at 3T. In a second objective, it was tested whether skeletal muscle NAD+ levels are related to insulin resistance and metabolic disease. To this end, we have investigated the concentration of NAD+ in volunteers differing in metabolic health andhave shown that NAD+ in muscle of physically very active elderly volunteers is elevated when compared to normally active elderly. Finally, the novel methodology was used to investigate the effect of supplementation of NAD precursors in humans and we showed that the combination of high intensity exercise and NAD precursor supplementation had beneficial effects on mitochondrial function and physical fitness when compared to exercise alone.
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.
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.
The first significant achievement was the development of a novel MR sequence, as we are the first to develop a MRS-based NAD+/NADH quantification protocol in skeletal muscle, which allows for completely non-invasive quantification of NAD+ and NADH.
The second significant achievement is that we showed that the developed technique is sensitive enough to observe physiological changes in NADH and NAD+ concentrations. This was shown in an proof of principle study, investigating NAD+ and NADH during normoxia and ischemia and documenting the expected increase in NADH (and decrease in NAD+) during ischemia.
The third substantial achievement is the demonstration of physiological relevant differences in NAD+ and NADH between groups, varying in metabolic health, with the new MR sequence. Our results demonstrate that NAD metabolites in muscle are sensitive to the metabolic health status and therefore represent a valuable target for intervention studies and the current technique provides a tool to monitor changes and therefore efficiency of treatments.
A fourth major achievement is the development of fitting algorithms for spectral analysis that incorporates both, time domain and frequency domain fitting strategies. This was implemented in MATLAB for the spectral analysis of the current project, but the superiority of this fitting routine could also be shown for analysis of 1H-MRS data of the liver and 31P-MRS saturation transfer data. The fitting routine is extremely flexible and adjustable for various applications and can be used in the future for many more applications. This can be considered as break through in data analysis. The transfer to other applications was not expected.
The fifth major achievement covers the clinical intervention study where we investigated whether oral NR supplementation combined with exercise leads to greater improvements in skeletal muscle mitochondrial respiratory capacity in overweight healthy people. This finding is of importance for future strategies to improve metabolic health and promote healthy aging.
The second significant achievement is that we showed that the developed technique is sensitive enough to observe physiological changes in NADH and NAD+ concentrations. This was shown in an proof of principle study, investigating NAD+ and NADH during normoxia and ischemia and documenting the expected increase in NADH (and decrease in NAD+) during ischemia.
The third substantial achievement is the demonstration of physiological relevant differences in NAD+ and NADH between groups, varying in metabolic health, with the new MR sequence. Our results demonstrate that NAD metabolites in muscle are sensitive to the metabolic health status and therefore represent a valuable target for intervention studies and the current technique provides a tool to monitor changes and therefore efficiency of treatments.
A fourth major achievement is the development of fitting algorithms for spectral analysis that incorporates both, time domain and frequency domain fitting strategies. This was implemented in MATLAB for the spectral analysis of the current project, but the superiority of this fitting routine could also be shown for analysis of 1H-MRS data of the liver and 31P-MRS saturation transfer data. The fitting routine is extremely flexible and adjustable for various applications and can be used in the future for many more applications. This can be considered as break through in data analysis. The transfer to other applications was not expected.
The fifth major achievement covers the clinical intervention study where we investigated whether oral NR supplementation combined with exercise leads to greater improvements in skeletal muscle mitochondrial respiratory capacity in overweight healthy people. This finding is of importance for future strategies to improve metabolic health and promote healthy aging.