孕酮对脂肪细胞促酰化蛋白受体和下游信号蛋白表达的作用

Role of progesterone in acylation stimulating protein-receptor C5L2 pathway in adipocytes and preadipocytes

目的观察孕酮对3T3-L1（前）脂肪细胞促酰化蛋白（ASP）受体C5L2 mRNA和细胞表面C5L2蛋白表达的影响，以及孕酮对ASP下游信号蛋白的作用。方法体外培养3T3-L1细胞，诱导细胞分化，不同浓度孕酮作用于3T3-L1（前）脂肪细胞，孵育过夜后收获细胞，分别采用RT-PCR和流式细胞仪检测ASP受体mRNA和蛋白表达情况；采用Western印迹法检测基础状态和ASP激发后Gαq／11，GB，p-PKCα和p-PKCζ蛋白表达。结果孕酮最大抑制成熟脂肪细胞14％C5L2 mRNA（P〉0．05）和蛋白表达22％（36％±15％vs46％±12％，P〈0．01），高浓度孕酮（1×10^-6mol／L）能显著性抑制前脂肪细胞66％C5L2 mRNA（0．17±0．11vs0．50±0．18，P〈0．01）和29％C5L2蛋白表达（36％±16％vs51％±20％，P〈0．05）。高浓度孕酮在一定程度上抑制ASP激发后成熟脂肪细胞Gαq／11，Gβ，p-PKCα和p-PKCζ的表达，各蛋白表达分别减少了41％（0．71±0．21vs1．20±0．24，P〈0．05），63％（0．55±0．32vs1．48±0．40，P〈0．05），49％（0．53±0．20vs1．04±0．19，P〈0．01）和32％（0．36±0．10vs0．53±0．20，P〉0．05）。在前脂肪细胞，高浓度孕酮显著性抑制ASP刺激的59％Gαq／11（0．42±0．18vs1．04±0．28，P〈0．01），43％Gβ（0．77±0．09vs1．35±0．27．P〈0．05），51％p—PKCα（0．44±0．15vs0．90±0．25，P〈0．05）和30％P-PKC（（0．27±0．08vs0．39±0．12，P〈0．05）蛋白表达。结论孕酮诱导ASP抵抗的发生，ASP抵抗参与了高浓度孕酮引起的脂肪细胞胰岛素抵抗状态的病理生理过程。

Objective 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. Methods Preadipocytes of the line 3T3-LI were cultured and induced to differentiate. Progesterone of the doses 0-1 × 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 μmol/L ASP for 4 hours, then the cell protein was extracted and the expressions of G protein （ including Gαq/11 and Gβ, ） and phosphated protein kinase C （ including p-PKCα and p- PKCζ） were measured by Western blotting. Results The C512 protein expression level of the mature adipocytes stimulated by progesterone 1× 10^-6 mol/L for 18 h was 36% ± 15% , significantly downregnlated 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 × 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 Gαq/11, Gβ, p-PKCα, and p-PKCζ expression levels of the mature adipocytes after overnight exposure to progesterone 1 × 10^-8 and 1 × 10^-6 mol/L were suppressed dose-dependently. For example, the ASP-stimulated Gαq/11, Gβ, and p-PKCα expression levels of the progesterone 1 × 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 Gα/q11,Gβ, and p-PKCζ expression levels were observed at the concentration of progesterone as low as 1× 10^-8 mol/L, and all the four proteins were inhibited significantly at the 1 × 10^-6 mol/L progesterone concentration. Conclusion Progesterone induces ASP resistance in adipocytes and preadipocytes. ASP resistance may contribute to the physiological abnormalities associated with insulin resistance induced by progesterone.