Abstract:
Melanocortins produced from the post-translational processing of pro-opiomelanocortin
(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 rate-limiting
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 C14 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. C14 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.