Molecular physiological studies on health effects of dietary nicotinamide riboside, a vitamin B3

Scope: Vitamin B3 serves as a precursor of NAD+, a multifunctional molecule that is involved in various cellular processes, such as energy metabolism, inflammation and insulin signalling. Adequate intake of vitamin B3 is essential to maintain health, especially in aging and obese subjects. In addition, vitamin B3 supplementation in certain high doses have been considered as a therapeutic strategy to counteract the decline of metabolic health that occurs in some diseases, such as type II diabetes. Nicotinamide riboside (NR) is a novel dietary vitamin B3, thus having large potential as dietary component, in fortification or as supplement. However, it is not known how NR as an exclusive source of vitamin B3 affects metabolic health. A better understanding of these effects and the relevant underlying molecular mechanisms will contribute to the proper use of this vitamin B3 in dietary and clinical approaches for health improvement and disease prevention.

Objective: The aim of this thesis was to elucidate the effects of dietary NR on whole body metabolic health and to identify the associated molecular and physiological responses.

Material and methods: Dietary intervention studies were performed in a C57BL/6JRcc mouse strain, an animal model with an intact nicotinamide nucleotide transhydrogenase (NNT) gene as is present in humans. To exclude the interference of other forms of vitamin B3, we employed a semi-purified rodent diet and used NR as an exclusive source of vitamin B3. To minimize the interaction effects of Trp on NR metabolism, we reduced dietary Trp by a low, but sufficient level. To compromise metabolic health, a mildly obesogenic diet was used. To understand effects on the whole body level, MRI was employed to monitor changes in body composition over time as well as a standardized indirect calorimetry based challenge tests to assess metabolic flexibility and oral glucose tolerance tests (OGTT) to assess glucose tolerance. Both indirect calorimetry and OGTT were used to determine the capacity to respond to perturbation of metabolic homeostasis. To explore molecular mechanisms, we harvested blood and tissue samples and analysed circulating lipids, cholesterols and proteins using enzymatic reaction kits, ELISA kits and multiplex assay; analysed tissue histological changes using immunohistochemistry and immunofluorescence; and analysed molecular changes in tissues using real-time qRT-PCR, transciptomics, western blot as well as metabolomics. At the tissue level, the focus was mainly on morphology and molecular regulation in white adipose tissue (WAT), although transcriptional responses in skeletal muscle and brain were also examined.

Main results: 30 mg NR per kg diet was most beneficial for metabolic health, based on metabolic flexibility, WAT morphology and gene expression. High dose of NR (9000 mg NR per kg diet) impaired metabolic flexibility and glucose tolerance, and induced WAT dysfunction including a decreased insulin sensitivity and aggravated inflammation, which may be molecularly explained by a mechanism of SIRT1-mediated PPARγ repression due to an enhanced NAD+/NADP+ pool. Vitamin B3 withdrawal resulted in a mild vitamin B3 deficiency, showing a reduced insulin sensitivity and a downward tendency of metabolic flexibility. The downregulation of Anp32a,Tnk2 and the upregulation of Mapk1, Map2k1, Mthfs, Mthfsl and Qdpr were proposed as a WAT transcriptional signature marker for mild vitamin B3 deficiency. Dietary NR affects motor performance in a dose-dependent pattern, with 30 mg NR per kg diet being best. Vitamin B3 deficient diet (5 mg NR per kg diet) led to the upregulation of Mapk1 and Mthfs in hypothalamus, while high dose of NR (900 mg NR per kg diet) induced the  upregulation of hypothalamic Tdo2. The regulation of these genes indicated that tetrahydrofolate (THF) metabolism and Trp metabolism may be affected in the brain by dietary vitamin B3.

Conclusions and implications: 30 mg NR per kg diet, or 2.3 mg NR per kg body weight per day, to be optimal for health of male adult mice fed a moderate high-fat diet containing a reduced, but sufficient, Trp level. Both inadequate intake and excessive intake of vitamin B3 can negatively affect metabolic health. In both conditions metabolic flexibility and insulin sensitivity were impaired, but the underlying mechanisms were distinct. We also proposes molecular biomarkers for mild vitamin B3 deficiency. When validated in humans it can be used to establish the prevalence of subclinical vitamin B3 deficiency in humans. Overall, this thesis underlines the importance of an optimal vitamin B3 intake to support health.