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 t hat intracellular free Ca2+ concentration ([Ca2+](i)) is elevated in v iable yellow mice. Because [Ca2+](i) has a key role in the pathogenesi s of insulin resistance, obesity, and hypertension, the role of the pu rified agouti gene product in regulating [Ca2+](i) was evaluated in a number of cell types. Purified murine agouti induced slow, sustained i ncreases in [Ca2+](i) in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agout i stimulated an increase in [Ca2+](i) with an apparent concentration e liciting 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 regu lation of [Ca2+](i), we examined the effect of melanocortin peptides a nd agouti in cells stably transfected with human melanocortin receptor s. Human embryonic kidney cells (HEK-293 cells) transfected with eithe r 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 d id 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 a gouti antagonism of I-125-Nle,D-Phe-alpha-melanocyte-stimulating hormo ne binding in the same cell line. This direct effect of agouti on stim ulating increases in [Ca2+](i) suggests a potential mechanism for agou ti-induced insulin resistance.