Insulin Action and Characterization of Insulin Receptors in Rat Luteal Cells*

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Abstract
The effect of insulin (Ins) on luteal cell function was assayed and Ins binding was characterized in isolated collagenase-dispersed rat luteal cells. Ins stimulated progesterone production at concentrations over 10-8 M, whereas human chorionic gonadotropin [hCG] produced maximal stimulation at 10-11 M. Ins had an additive effect when it was added to the luteal cells in the presence of hCG in concentrations that produced submaximal stimulation. At 10-9 M Ins the additive effect on hCG response became apparent (80% of maximal progesterone production). The maximal hCG response cannot be exceeded, even in the presence of high amounts of both hormones. hCG maximally stimulated cAMP production at 10-10 M, whereas Ins neither stimulated cAMP production nor enhanced hCG response. Ins binding to luteal cells attained highest values after 30 min of incubation at 20.degree. C. A curvilinear Scatchard plot was obtained and it was analyzed using a 2-binding site model. The affinity constant values were 2.1 .times. 108 M-1 and 5.2 .times. 106 M-1 and the number of binding sites were 35,000 and 900,000/cell, respectively. Bound Ins was not displaceable by hCG, human growth hormone, human luteinizing hormone, epidermal growth factor, or ovine prolactin. A protein moiety was essential for the receptor activity, since after trypsin digestion marked loss of binding was verified. Subcellular fractionation was performed and the highest binding was observed in the 105,000 .times. g pellet fraction. Ins binds specifically to rat luteal cells and stimulates steroidogenesis, adding support for the hypothesis that insulin acts directly upon the ovary.