Dissecting the tissue-specific role of the NADP(H) phosphatase, Nocturnin - Nocturnin (NOCT) is a NADP(H) phosphatase with highly rhythmic expression peaking in the early dark phase (ZT12). Notably, Noct-/- (Noct-KO) mice are resistant to obesity and hepatic steatosis on a high-fat diet. Given the widespread role of NADP(H) as an essential cofactor in numerous metabolic reactions, it is likely that NOCT has distinct tissue-specific effects, making it difficult to understand NOCT’s role in metabolism using a global KO model. In fact, previous studies have observed contradictory changes in lipid and lipoprotein metabolism across different tissues in Noct-KO mice. Specifically, while previous studies have found that Noct- KOs have delayed chylomicron transit into circulation following olive oil gavage, other studies have found significantly higher circulating VLDL, triglycerides, and cholesterol at certain times of day in the plasma of chow- fed Noct-KO mice as compared to controls. In further contradiction to the lean phenotype seen under high-fat diets, chow-fed Noct-KOs have significantly higher expression of hepatic enzymes involved in fatty acid synthesis during their active phase. Given these seemingly paradoxical phenotypes, this proposal will use novel adipose- , liver-, muscle-, and intestine-specific conditional Noct-KO (cKO) mice, generated via the Cre-loxP system, to better understand NOCT’s role in metabolism. Both male and female cKO and control mice will be subject to a high-fat diet for twenty weeks, with body weight and food intake recorded on a weekly basis, to determine if loss of NOCT in one tissue is sufficient to confer resistance to diet-induced obesity. A separate cohort of cKO mice will be placed in metabolic cages for one week to discern any differences in energy expenditure. At the end of the high-fat diet challenge, body composition measurements will be taken to determine differences in fat and lean mass. Plasma, adipose, liver, muscle, and intestinal tissues will be collected from each cKO line and their respective controls at two circadian phases for further analyses. Plasma lipoprotein classes will be fractionated and characterized to determine how tissue-specific loss of NOCT impacts lipid mobilization and trafficking. Plasma adipokine levels will also be measured to identify any differences in adipocyte metabolism and health. To confirm the mechanism by which any observed phenotype originates, NAD(H) and NADP(H) levels will be measured in each cKO tissue. Further, RNA-seq will be performed on all collected tissues to investigate how tissue-specific loss of NOCT impacts transcriptional networks. Tissues will also be stained with hematoxylin and eosin to define any gross morphological changes. Lastly, lipogenic flux will be measured in both liver-cKO and global KO mice to determine the direct impact on de novo lipogenesis. The experiments outlined in this proposal will define how tissue-specific loss of NOCT, and thus how tissue-specific increases in NADP(H) levels, impact metabolism. More specifically, this proposal will aid in understanding how loss of NOCT protects against diet- induced obesity and hepatic steatosis, which, in turn, can aid in the development of treatments for obesity and obesity-related diseases.
