The reduced body weight gain of NR-fed mice upon HFD was not due to reduced food intake, as NR-fed mice actually had a tendency to eat more, especially on HFD conditions (Fig.3C). Similarly, NR did not affect the activity pattern of mice (Fig.3D), indicating that the lower BW on HFD was not consequent to different physical activity. Rather, the phenotype was due to enhanced energy expenditure (EE). Mice on CD had a marked tendency to display higher O2 consumption rates when fed with NR, and this tendency became clearly significant under HFD conditions (Fig.3E). Of note, NR-fed mice became more flexible in their use of energy substrates, as reflected in the higher amplitude of the changes in RER between feeding and fasting periods (Fig.S2C) in CD conditions. Altogether, these results indicate that NR lowers HFD-induced BW gain by enhancing EE.
From a metabolic perspective, NR- and vehicle-fed mice had similar fasting blood glucose levels in either CD or HFD conditions (Fig.3F). However, fasting insulin levels were much lower in NR-supplemented mice (Fig.3G). This lower insulin/glucose ratio is indicative of insulin sensitization after NR administration. This speculation was further supported by glucose tolerance tests. NR promoted a slight, albeit not significant, improvement in glucose tolerance (Fig.3H) in mice fed a HFD, accompanied by a robust reduction in insulin secretion (Fig.3I). Therefore, NR-fed mice on HFD display a better glucose disposal with lower insulin levels. In order to conclusively establish whether NR fed mice were more insulin sensitive, we performed insulin tolerance tests and hyperinsulinemic-euglycemic clamps on CD and CD-NR mice. We chose not to perform this analysis on the HFD groups in order to avoid the possible influence of differential BW. Glucose disposal upon insulin delivery was largely enhanced in NR-fed mice (Fig.3J). In agreement, mice supplemented with NR required an almost 2-fold higher glucose infusion rate to maintain euglycemia in hyperinsulinemic-euglycemic clamps (Fig.3K). Together, these observations unequivocally demonstrate that NR-fed mice are more insulin-sensitive. Furthermore, NR partially prevented the increase in total (Fig.3K) and LDL cholesterol levels (Fig.S2D) induced by HFD, even though HDL-cholesterol levels were unaffected (Fig.S2E). The amelioration of cholesterol profiles is fully in line with previous observations from the use of other NAD+ precursors, such as NA (Houtkooper et al., 2010).
NR enhances the oxidative performance of skeletal muscle and brown adipose tissue
NR-fed mice had a clear tendency to display a better endurance performance than vehicle fed mice (Fig.S3A). This tendency was significantly accentuated upon HFD (Fig.4A), suggesting an enhanced muscle oxidative performance. Similarly, NR-fed mice, both on CD and HFD, showed enhanced thermogenic capacity, as manifested in the ability to maintain body temperature during cold exposure (Fig,S3B and Fig.4B). The latter observation hints toward an improvement in brown adipose tissue (BAT) oxidative performance. To gain further insight into the ability of BAT and muscle to enhance their oxidative performance, we performed some histological analysis. Gastrocnemius muscles from NR mice displayed a more intense SDH staining than their vehicle-fed littermates, indicating a higher oxidative profile (data not shown). Electron microscopy revealed that mitochondria in BAT of NR-fed mice, despite not being significantly larger, had more abundant cristae (Fig.4C), which has been linked to increased respiratory capacity (Mannella, 2006). Altogether, the above results suggest that NR supplemented mice display a higher oxidative capacity due to enhanced mitochondrial function.