Agouti regulation of intracellular calcium: Role of melanocortin receptors

Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+](i)) is elevated in viable yellow mice. Because [Ca2+](i) has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+](i) was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+](i) in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+](i) with an apparent concentration eliciting 50% of the maximal response (EC(50)) Of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+](i), we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+](i), whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC(50) of 18 nM, which is comparable to that for agouti antagonism of I-125-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+](i) suggests a potential mechanism for agouti-induced insulin resistance.