The Depot-specific Regulations Of 11β-HSD1 Expression By Steroids In Adipose Tissue From Children

Obesity, particularly visceral (central ) obesity is associated with metabolic syndrome, such as insulin resistance, dyslipidemia, type 2 diabetes, hypertension, cardiovascular diseases. It is well established that, children obesity may persist into adulthood and increases the risk of metabolic syndrome, This clearly emphasizes the importance of studying the factors that are involved in body fat accumulation and distribution for prevention and treatments of obesity and its related diseases during childhood.Steroids, including glucocorticoids (GCs) and sex hormones, play important roles in body fat accumulation and distribution. However, the underlying pathogenetic mechanism is poorly understood. It has been confirmed that excessive GC is associated with metabolic syndrome and visceral obesity. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the key enzyme that is responsible for activation of glucocorticoid (GC) by converting inactive cortisone to active cortisol. 11β-HSD1 displays oxo-reductase activity depending on reduced nicotinamide adenine dinucleotide phosphate (NADPH) provided by hexose-6-phosphate dehydrogenase (H6PDH), and amplifies the local effects of GC in adipose tissue. It strongly suggests that depot-specific regulation of 11β-HSD1 may be the crucial link between visceral obesity and metabolic syndrome. Therefore, 11β-HSD1 becomes a hot topic in the mechanisms of visceral obesity and metabolic disorders in recent studies.However, previous studies about 11β-HSD1 are mainly focused on adults or animal models. Childhood is a pivotal stage for the rapid development and redistribution of adipose tissue with sharp changes in endocrine hormones, especially sex hormones. It is not clear whether 11β-HSD1 expression is regulated by various hormones already during childhood and related with increasing risk of developing visceral adiposity or related metabolic disorders. In order to confirm the hypothesis, we investigated the effect of hormone (GC and sex hormones) treatment on 11β-HSD1 expression and the influence related with adipocyte differentiation and adipokines secretion in culture of both subcutaneous and omental adipose tissue, and analyze if there are the depot-specific differences in regulation of gene expression and adipokines secretion. Finally, a pilot study was carried out to investigate the possible receptor pathways that steroids might regulate 11β-HSD1. Therefore, understanding of the expression and regulation of genes described above and identified risk factors during childhood that can lead to visceral obesity and metabolic syndromes later on are essential in order to find practical biologic target for future prevention and treatment strategies.Part I The effect of steroid hormones treatment on adipose tissue11β-HSD1 expression in a depot-specific manner in children Objective: To investigate the effect of hormone (GC, androgen and estrogen) treatment on 11β-HSD1 gene expression and activity in abdominal subcutaneous (SC) and omental (OM) adipose tissue in children and its relationship with H6PDH, thus to illustrate the effect of depot-specific regulation of 11β-HSD1 on visceral fat accumulation.Subjects and methods: Paired fat biopsies (SC and OM) were obtained from children (19 boys, age 6.0～14.8 years, BMI 14.6～25.3 kg/m2, BMI SDS -1.6～3.1; and 14 girls, age 4.3～12.0 years, BMI 13.0～20.4 kg/m2, BMI SDS -2.0～1.1) undergoing abdominal surgery. Pieces of adipose tissue were incubated with cortisol (100nM,1μM,10μM), testosterone (5nM,50nM,500nM) or estradiol (5nM,50nM,500nM) for 24 h. The expression of 11β-HSD1 and H6PDH mRNA were measured by real-time PCR and 11β-HSD1 enzyme activity was determined by radioimmunoassay. Results: 1, Dose effects showed that the most optimal effects on 11β-HSD1 mRNA expression in OM adipose tissue from both boys and girls were obtained by cortisol at 100nM (P<0.05) or testosterone at 50nM (P<0.05), while the effects were disappeared when increasing the concentration of hormones; 11β-HSD1 mRNA expression was increased in OM adipose tissue from girls by estradiol treatment at the concentrations of 50nM and 500nM(P<0.05); 2, Cortisol or testosterone treatment increased 11β-HSD1 mRNA expression in OM but not SC adipose tissue from both boys and girls (P<0.001), while estradiol just increased 11β-HSD1 mRNA expression in OM adipose tissue from girls but not boys (P<0.05). The combination treatments of cortisol and testosterone /estradiol showed no statistical significance compared with solo treatment; 3, Cortisol, testosterone or estradiol also regulated the enzyme activities of 11β-HSD1 in a depot-specific manner, and there was positive correlation between enzyme activity and mRNA expression of 11β-HSD1 (r=0.632, P<0.001); 4, Cortisol and testosterone had similar effects on regulating expression of H6PDH mRNA as well as 11β-HSD1.Conclusion: Steroids hormones, specially cortisol or testosterone significantly increased the 11β-HSD1 mRNA expression and enzyme activity in a depot-specific manner in OM adipose tissue of children, which was correlated with change of H6PDH mRNA expression. It suggests that GC and androgen may contribute to fat distribution and accumulation during childhood by depot-specific regulation of active GC. The mechanism remains unclear that estrogen effected on visceral adipose tissue in gender-specific manner.PartⅡThe effects of regulation of 11β-HSD1 on adipocytes differentiation and endocrine functionObjective: To investigate effects of 11β-HSD1 expression on adiposities differentiation, fat accumulation and adipokines in abdominal SC or OM adipose tissue.Subjects and methods: Paired abdominal SC and OM adipose tissue (n=23) were taken biopsied from children during operation, Cortisol /testosterone alone or combined with 11β-HSD1 inhibitor carbenoxolone (CBX) were incubated with adipose tissue for 24 hours in vitro. The mRNA expression of PPAR-γwas measured by real-time PCR. TNF-α, IL-6, resistin and leptin concentrations in the cultured supernatant were measured by ELISA. The morphology of adipose tissue was observed using hematoxylin and eosin (HE) stain.Results: 1, mRNA expression of PPAR-γin OM adipose tissue (P<0.05)was increased and was positively correlated with the mRNA expression of 11β-HSD1 (P<0.05) in treatment of cortisol or testosterone; 2, The level of TNF-α(P<0.05), IL-6 (P<0.05), resistin (P<0.05) were decreased but leptin (P<0.05) was increase in OM adipose tissue after treatment of cortisol while testosterone had only inhibited effect in TNF-α(P<0.05), IL-6 (P<0.05), resistin (P<0.05); 3, The size of adipocytes in OM but not SC adipose tissue was increased by the treatment of Cortisol or testosterone (P<0.05); 4,The expression of 11β-HSD1 in OM adipose tissue was decreased when cortisol or testosterone combined with CBX (P<0.05). At the same time, the PPAR-γmRNA expression was also decreased significantly (P<0.05) and the inhibition of TNF-αwas disappeared.Conclusion: The differentiation of adipocytes and the secretion of adipokines display a depot-specific manner in adipose tissue under treatment of cortisol or testosterone and these effects disappear with 11β-HSD1 inhibition. It is indicated that the effects of androgen and GC might have effects on adipocytes differentiation and adipokines secretion by excess active GC via the regulation of 11β-HSD1 in OM adipose tissue. PartⅢThe receptor-mediated pathway involved in the regulation of 11β-HSD1 by androgen in adipocytesObjective: To study the possible receptor-mediated pathway via that androgen regulates 11β-HSD1 mRNA expression in adipocytes.Materials and methods: 3-isobutyl-1-methyxanthine (MIX), dexamethasone, insulin and rosiglitazone were taken together to induce the differentiation of human preadipocyte-visceral (HPA-v) cell line. On the 7th day of preadipocyte differentiation, cortisol or testosterone alone or combined with GCs receptor inhibitor mifepristone (RU486) or androgen receptor inhibitor bicalutamide (CDX) were treated for 24 hours, after which adipocytes were collected. 11β-HSD1 mRNA expression from adipocytes was measured using real-time PCR.Results: 1, 11β-HSD1 mRNA expression in HPA-v was increased during adipocyte differentiation in tesosterone treatment (P<0.05); 2, Testosterone increased the mRNA expression of 11β-HSD1 mRNA in a"U"shaped curve, which indicated upregulation of 11β-HSD1 mRNA (P<0.001) in lower (0.5nM) or higher dosage (50-1000nM) but not in moderate dosage (1-5nM); 3, The upregulation of 11β-HSD1 mRNA expression by testosterone was blocked by RU486 and CDX (P<0.05).Conclusion: Androgen stimulates the expression of 11β-HSD1 mRNA but its effect can be blocked by androgen or GCs receptor inhibitor, which indicating that androgen regulates 11β-HSD1 mRNA expression by multiple receptor-mediated pathways.