Transcriptional Regulation of Melanocortin 4 Receptor by Nescient Helix-Loop-Helix-2 and its Implications in Peripheral Energy Homeostasis

Mutations in the melanocortin 4 receptor (MC4R) are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation that underly the melanocortin pathway are far from being fully understood. The transcription factor Nescient Helix Loop Helix 2 (Nhlh2), is known to influence the melanocortin pathway. It regulates the transcription of genes by binding to the E-Box binding sites present in the promoter region. Here in this dissertation, Nhlh2's role as a transcriptional regulator of Mc4r and the effects of deletion of Nhlh2 on peripheral energy expenditure, glucose homeostasis and fatty acid oxidation are reported. To investigate the transcriptional mechanisms of Mc4r and the involvement of Nhlh2, gene expression analysis, DNA-protein binding, transactivation assays, and SiRNA induction were used. We show that Nhlh2 regulates the transcription of Mc4r by binding to the three E-Boxes present on the promoter at -553, -361 and +47. Further, SiRNA knockdown of Nhlh2 in the N29/2 cell line depresses Mc4r expression which suggests the requirement of Nhlh2 for Mc4r transcription.

Development of adult onset obesity in the absence of evident hyperphagia questions the ability of mice which lacks Nhlh2 (N2KO) to utilize energy substrate efficiently. To test the effect of deletion of Nhlh2 in N2KO, body composition analysis, tissue specific characterization, fatty acid oxidation and glucose and insulin homeostasis were assessed. N2KO mice have a higher fat content than WT at the age of 12 weeks. There are architectural differences in adipose tissue of N2KO. White adipose tissue (WAT) shows infiltration of macrophages, and increased mRNA and serum levels of interleukin 6 which suggests the presence of a systemic inflammatory state in the N2KO mice. Sympathetic nervous system tone is reduced in both brown adipose tissue (BAT) and WAT, as evidenced by gene expression analysis, and this may be because of overall reduced melanocortinergic tone in N2KO mice. N2KO mice have an impaired glucose tolerance on the basis of their late glucose clearance on glucose (non-significant) and insulin (significant) challenges. Fatty acid oxidation (FAO) is higher in red fibers of skeletal muscle, and the respiratory exchange ratio (RER) is lower in N2KO, which is indicative of using fat as a preferential energy source. Increased expression of genes involved in the lipid metabolism in skeletal muscle and liver supports the RER and FAO, and are indicative of high turnover of lipids in N2KO.

Findings from these studies implicate Nhlh2 as a transcriptional regulator of Mc4r which has a direct relevance to the ever increasing epidemic of obesity. Characterization of N2KO mice sheds light on the adult onset obesity phenotype. Knowledge gained from these findings will help us understand the monogenetic form of obesity more completely and could lead to the design of improved pharmacological therapies that target Nhlh2 or Mc4r or modify physical activity behavior.