Hypertension is a frequently observed complication among obese patients. Recently, undetermined adipocyte-derived factor(s) have been recognized as one of the causes of the obesity-related hypertension independent of angiotensin (A) II. In this study, we aim to identify the undetermined adipocyte-derived factor(s) that stimulate aldosterone synthase gene (CYP11B2) expression aldosterone secretion that may induce hypertension.
We first differentiated mouse fibroblast 3T3-L1 cells into adipocytes and collected their supernatants. The supernatants were incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by quantitative PCR with reverse transcription (qRT-PCR). Thereafter, the supernatants were fractionated by ammonium sulfate precipitation to purify adipocyte-derived factor(s) that stimulate CYP11B2 expression. The precipitates were collected in 30%, 60% and 90% saturated concentration of ammonium sulfate. Each of precipitates was concentrated using 100 kDa ultrafiltration membrane and incubated with H295R cells. CYP11B2 mRNA expression in H295R cells was measured by qRT-PCR. Then, we purified the precipitation of the supernatants that obtained in 60% ammonium sulfate by anion-exchange chromatography. Each of the fractions was incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by qRT-PCR.
The supernatants obtained from 3T3-L1 adipocytes were demonstrated to stimulate CYP11B2 expression. Fractionation of the supernatants by ammonium sulfate precipitation demonstrated that the ability to stimulate CYP11B2 expression was detected in fractions precipitated in 60% saturated concentration. Fractionation of the supernatants by anion-exchange chromatography demonstrated that the ability to stimulate CYP11B2 expression was detected in two specific fractions. Specific fractions were analyzed by mass spectrometry, resulting in the identification of 39 specific proteins. We are now investigating that the relationship between CYP11B2 gene expression and 39 proteins.