Effect Of Sex Hormones To The Secretion Of Leptin And Adiponectin By Human Omental Preadipocytes

Objective:The aim of this study is to investigate the effect of sex hormones to the secretion of leptin and adiponectin during the process of cell differentiation when culturing human omental preadipocytes primarily and induced them to maturity in vitro.Methods:1. Omental adipose tissue were obtained from patients undergoing elective open-abdominal surgery (BMI < 25kg/m² ,age40±8). None of the patients had diabetes or severe systemic illness. None was taking medications known to affect glycometabolism or lipid metabolism.2. Using collagenase -digesting method, human preadipocytes were separated from omental adipocyte tissue. The morphological changes of the cultured cells were observed and identified by extracting stained intracytoplasmic lipid with oil red O. And then we investigated the processes of proliferation and differentiation of human omental preadipocytes primarily in vitro.3. Collecting culture supernatants of the foundation group,the differentiation group and sex hormones intervention group every 2 days during proliferation and differentiation, the contents of leptin and adiponectin were detected by enzyme linked immunosorbent assay (ELISA).4. After the preadipocytes differentiated to maturity,using three different concentrations of estradiol and testosterone to interfere 24 hours and collecting culture supernatants, the contents of leptin and adiponectin were detected by enzyme linked immunosorbent assay (ELISA).5. Statistical analysis: Values were given as means±SE .Differences were considered significant at a value of P<0.05.Results:1. Human preadipocytes were primary cultured successfully. The cultured preadipocytes were fibroblast-like cells and after they were being induced into differentiation phase, lipid droplets could be seen on the 4^th day and were increased with the differentiation process going on. Most preadipocytes became mature adipocytes about on the 16^st day.2. In proliferation: The low level of leptin could be detected in the collected culture supernatants of preadipocytes. In the differentiation of the preadipocytes,the volume of leptin increased and arrived to peak on the 16^th day of differentiation. Then the secretion of leptin kept a high rate until the differentiation process was completed.The level of leptin in estradiol intervention group in every time section had increased obviously compared with the differentiation group,whereas the level of leptin in testosterone intervention group had decreased obviously. In proliferation: the adiponectin could not be detected in the culture supernatants. The adiponectin could be detected in the culture supernatants on the 5^th day of differentiation,while the adipocytes contained lipid droplets could be observed under inverted microscope. The max secretion was detected on about the 11^th day. An obvious descent of the adiponectin level could be observed on the 16^st day. The level of adiponectin in both estradiol testosterone intervention groups had decreased obviously in every time section compared with the differentiation group (P<0.01).3. Estradiol may increase the leptin secretion of the differentiated fat cell,whereas higher concentration of testosterone may reduce the leptin secretion of the differentiated fat cell (P<0.05).4. Estradiol and testosterone can reduce the adiponectin secretion of the differentiated fat cell(P<0.05).Conclusion:1. Human preadipocytes are primary cultured successfully. The feasibility and practicability of the method have been verified by our experiments. This study provided a cytology platform for further probing into correlation between obesity and adipokines.2. In vitro, a certain concentration of estradiol may increase the leptin secretion whereas testosterone may reduce the leptin secretion.Estradiol and testosterone can reduce the adiponectin secretion.It can offer laboratory evidence for investigate on sex differences of obesity and metabolic diseases.