Wen Yu, Wang Hong-Wei, Hu Xiu-Fen, Cianflone Katherine, Wei Jun, Xia Zhi, Li Rui-Zhen
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Yi Xue Za Zhi. 2008 Jan 8;88(2):114-8.
To evaluate the effects of progesterone on the mRNA expression of acylation stimulating protein (ASP)-receptor C5L2 in adipocytes and preadipocytes and the C5L2 protein expression on the cell surface.
Preadipocytes of the line 3T3-L1 were cultured and induced to differentiate. Progesterone of the doses 0 - 1 x 10(-6) mol/L was added into the cultured fluid of the mature 3T3-L1 adipocytes and preadipocytes overnight. RT-PCR and flow cytometry were used to detect the mRNA and protein expression of ASP receptor C5L2. Both non-progesterone treated and progesterone-treated 3T3-L1 cells were cultured with 5.0 micromol/L ASP for 4 hours, then the cell protein was extracted and the expressions of G protein (including Galphaq/11 and Gbeta) and phosphated protein kinase C (including p-PKCalpha and p-PKCzeta) were measured by Western blotting.
The C5L2 protein expression level of the mature adipocytes stimulated by progesterone 1 x 10(-6) mol/L for 18 h was 36% +/- 15%, significantly downregulated compared with that of the adipocytes stimulated by progesterone 0 mol/L (46% +/- 12%, P < 0.01), with a inhibition rate of 22%. The C5L2 mRNA and protein expression levels of the preadipocytes stimulated by progesterone 1 x 10(-6) mol/L for 18 h were 0.17 +/- 0.11 and 36% +/- 16% respectively, both significantly lower than those of the preadipocytes stimulated by progesterone 0 mol/L (0.50 +/- 0.18 and 51% +/- 20% respectively, P < 0.01 and P < 0.05) with the inhibition rates of 66% and 29%respectively. The ASP-stimulated Galphaq/11, Gbeta, p-PKCalpha, and p-PKCzeta expression levels of the mature adipocytes after overnight exposure to progesterone 1 x 10(-8) and 1 x 10(-6) mol/L were suppressed dose-dependently. For example, the ASP-stimulated Galphaq/11, Gbeta, and p-PKCalpha expression levels of the progesterone 1 x 10(-6) mol/L group were significantly lower than those of the progesterone 0 mol/L group by 41%, 63%, and 49% respectively (P < 0.05 to P < 0.01. In the preadipocytes the reduction of ASP-induced Galphaq/11, Gbeta, and p-PKCzeta expression levels were observed at the concentration of progesterone as low as 1 x 10(-8) mol/L, and all the four proteins were inhibited significantly at the 1 x 10(-6) mol/L progesterone concentration.
Progesterone induces ASP resistance in adipocytes and preadipocytes. ASP resistance may contribute to the physiological abnormalities associated with insulin resistance induced by progesterone.
评估孕酮对脂肪细胞和前脂肪细胞中酰化刺激蛋白（ASP）受体C5L2的mRNA表达以及细胞表面C5L2蛋白表达的影响。
培养3T3-L1细胞系的前脂肪细胞并诱导其分化。将0 - 1×10⁻⁶ mol/L剂量的孕酮加入成熟的3T3-L1脂肪细胞和前脂肪细胞的培养液中孵育过夜。采用逆转录-聚合酶链反应（RT-PCR）和流式细胞术检测ASP受体C5L2的mRNA和蛋白表达。未用孕酮处理和用孕酮处理的3T3-L1细胞均用5.0 μmol/L的ASP培养4小时，然后提取细胞蛋白，通过蛋白质免疫印迹法检测G蛋白（包括Gαq/11和Gβ）和磷酸化蛋白激酶C（包括p-PKCα和p-PKCζ）的表达。
用1×10⁻⁶ mol/L孕酮刺激成熟脂肪细胞18小时后，C5L2蛋白表达水平为36%±15%，与用0 mol/L孕酮刺激的脂肪细胞（46%±12%）相比显著下调（P<0.01），抑制率为22%。用1×10⁻⁶ mol/L孕酮刺激前脂肪细胞18小时后，C5L2 mRNA和蛋白表达水平分别为0.17±0.11和36%±16%，均显著低于用0 mol/L孕酮刺激的前脂肪细胞（分别为0.50±0.18和51%±20%，P<0.01和P<0.05），抑制率分别为66%和29%。成熟脂肪细胞在过夜暴露于1×10⁻⁸和1×10⁻⁶ mol/L孕酮后，ASP刺激的Gαq/11、Gβ、p-PKCα和p-PKCζ表达水平呈剂量依赖性抑制。例如，孕酮1×10⁻⁶ mol/L组的ASP刺激的Gαq/11、Gβ和p-PKCα表达水平分别比孕酮0 mol/L组显著降低41%、63%和49%（P<0.05至P<0.01）。在前脂肪细胞中，在孕酮浓度低至1×10⁻⁸ mol/L时即可观察到ASP诱导的Gαq/11、Gβ和p-PKCζ表达水平降低，在孕酮浓度为1×10⁻⁶ mol/L时所有这四种蛋白均受到显著抑制。
孕酮诱导脂肪细胞和前脂肪细胞产生ASP抵抗。ASP抵抗可能导致与孕酮诱导的胰岛素抵抗相关的生理异常。
