ABSTRACT
Cytochrome P450 aromatase (P450arom), a product of cyp19a1 gene, plays pivotal roles in vertebrate steroidogenesis and reproduction. In this study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female rohu, Labeo rohita and investigated the regulation of cyp19a1a by gonadotropin and SF-1. The cyp19a1a and cyp19a1b were expressed predominantly in the ovary and brain respectively, with quantity of the former attuned to reproductive cycle. To elucidate gonadotropin regulation of cyp19a1a mRNA expression and P450 aromatase activity for 17ß-estradiol (E2) biosynthesis in vitro by the vitellogenic ovarian follicles, time- and dose-dependent studies were conducted with HCG and porcine FSH. Results demonstrated that HCG stimulated significantly higher expression of cyp19a1a mRNA and aromatase activity leading to increased biosynthesis of E2 than FSH. To understand the involvement of SF-1 to in the regulation of cyp19a1a and aromatase activity, ovarian follicles were incubated with increasing concentrations of HCG and expression of sf1gene and activation of SF-1 protein were measured. Results demonstrated that HCG significantly induced expression of sf-1 gene and activation of SF-1 protein suggesting a link between SF-1 and P450 aromatase activation in this fish ovary during gonadotropin-induced steroidogenesis.
Subject(s)
Aromatase/genetics , Chorionic Gonadotropin/metabolism , Cyprinidae/genetics , Oocytes/physiology , Steroidogenic Factor 1/metabolism , Animals , Aromatase/metabolism , Brain/physiology , Chorionic Gonadotropin/pharmacology , Cyprinidae/growth & development , Cyprinidae/metabolism , Estradiol/metabolism , Female , Fish Proteins/genetics , Fish Proteins/metabolism , Follicle Stimulating Hormone/pharmacology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Oocytes/drug effects , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Ovary/physiology , Phylogeny , Steroidogenic Factor 1/geneticsABSTRACT
It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p <0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKCepsilon was also inhibited. Surprisingly, phosphorylation of cytosolic PKCepsilon was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKCepsilon translocation inhibitor peptide, epsilonV1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKCepsilon migration to implement palmitate effect. Experimental evidences indicate that phospho-PKCepsilon adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.
