• Clinical and Experimental Pediatrics
• /
• v.46 no.8
• /
• pp.795-802
• /
• 2003

Purpose : We investigated the hormonal control of OB gene expression and leptin secretion in cultured human visceral adipose tissue. Methods : Visceral adipose tissues were cultured for up to 48 hrs in modified Eagle's medium with varying concentration of hormones : Control(no hormone), bovine insulin(100 nM), Dexamethasone(DEX, 100 nM), growth hormone(GH, 40 ng/mL), insulin+DEX(100 nM each), insulin+DEX+GH(100 nM insulin and DEX, 40 ng/mL GH). Quantitative analysis of leptin mRNA was performed by competitive reverse transcription polymerase chain reaction, and leptin secretion in culture medium was measured by IRMA using a commercial kit. Results : The addition of dexamethasone to the medium significantly increased OB gene expression and leptin secretion(P<0.05). Unlike dexamethasone, insulin did not affect OB gene expression and leptin secretion. Both insulin and dexamethasone, at high concentration, significantly stimulated leptin secretion compared with basal values(P<0.05). Leptin gene expression was not significantly increased by GH treatment alone, however GH, in combination with high concentrations of insulin and dexamethasone, attenuated the stimulatory effects of high concentrations of insulin and dexamethasone. Conclusion : Insulin cannot increase leptin secretion without the presence of dexamethasone. The mechanism suggested is that insulin may increase leptin secretion in cytoplasm only after dexamethasone increases the expression of OB gene. Further studies are necessary to elucidate the mechanism of the action of insulin on leptin secretion after increasing OB gene expression by dexamethasone.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.4
• /
• pp.430-436
• /
• 2010

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.