An ectopic synthesis of the melanin in the adipocytes of the morbidly obese subjects

AN ECTOPIC SYNTHESIS OF THE MELANIN IN THE ADIPOCYTES OF THE MORBIDLY OBESE SUBJECTS Manpreet Randhawa, PhD. George Mason University, 2008 Dissertation Director: Dr. Ancha Baranova Melanocortins produced from the post-translational processing of proopiomelanocortin (POMC) are regulators of pigmentation in the hair and skin and are also critical for proper maintenance of energy balance. Particularly, α-MSH is a melanocortin ligand involved in the control of both pigmentation in skin and energy homeostasis through various subtypes of the melanocortin receptors present both in the skin and the adipose tissue. Expression profiling of 40,173 individual cDNA clones with RNA from visceral adipose samples showed statistically significant overexpression of genes encoding tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT/TYRP2), melanosome transport protein RAB27a and Melan-A (MLANA). These findings lead to the following two hypotheses: 1) Melanin biosynthesis pathway is functional in adipose tissue and is excessively stimulated in morbid obesity. 2) Melanogenesis may be stimulated in morbidly obese individuals due to high levels of α-MSH. Fontana-Masson staining performed on the adipose tissue registered the presence of a black pigment in the periphery of the adipocytes. Granules of the pigment were found in higher quantities in visceral adipocytes from obese individuals as compared to samples from lean subjects. An LC/MS/UV analysis of the chemical composition of the pigment proved that these granules contain melanin. The expression of the TYR, TYRP1, TYRP2, MITF and MC1R genes involved in melanogenesis was studied by real time PCR in adipose tissue samples obtained from morbidly obese and from lean subjects. The expression level of TYR, encoding the ratelimiting enzyme required for the conversion of L-DOPA to dopachrome, was found to be expressed at much higher levels in obese subjects as compared to lean subjects whereas no expression was registered in liver and gastric tissues. The other genes showed the same pattern as TYR, but the differences were less pronounced as compared to TYR. The expression of TYR was further localized to the adipocytes as determined by in situ hybridization of adipose tissue slides, where TYR was found only in the periphery of the cell. The study of the expression of tyrosinase protein in adipose tissue by Western blotting revealed properly folded and mature tyrosinase homodimer of 140kDa. The presence of the tyrosinase as well as TYRP1 and TYRP2 proteins were confirmed in the human adipocytes by immunohistochemistry. A substantial difference has been seen between adipose samples of obese and lean subjects, with more tyrosinase in adipocytes from obese samples. The biological activity of TYR was evaluated by C assay that showed increased enzyme activity in the adipose tissue from morbidly obese subjects as compared to lean subjects, whereas no activity was found in gastric and liver tissue samples. Both murine 3T3-L1 adipocytes and primary human adipocytes at different stages of differentiation were exposed to different concentrations of α-MSH for different time periods. Real-time PCR performed on mRNA extracts obtained from murine 3T3-L1 cells and human adipocytes provided no consistent expression data for melanogenesis related genes. The enzymatic activity of a tyrosinase from protein extracts obtained from 3T3-L1 adipocytes was evaluated by L-dopa assay. A gradual decrease in the rate of Ldopa oxidation was observed spectrophotometrically during the differentiation of adipocytes. C assays indicated the presence of minimal residual activity of tyrosinase in cultured human cells. Western blotting performed on extracts from human adipocytes showed the presence of a specific band characterized by a smaller molecular weight than normal tyrosinase. The glycosidase digestions confirmed that this band corresponds to an inactive, nonglycosylated form of tyrosinase. These collective findings indicate that the melanin synthesis pathway is functional in intact human adipose tissue while further work on the appropriate cellular model of the adipocytic melanogenesis is warranted.