AMPK-SIRT1信号通路在肉牛脂肪细胞凋亡中的作用

Apoptosis of adipose cells, which means the exhausted of energy storage, has opened up a new way to treat human obesity and control animal fat deposition. Sirtuin type 1 (SIRTl), as an important member of energy metabolism network, has been considered to have inherent contact with AMP activated protein kinase (AMPK). SIRTl and AMPK play an important role in regulating energy metabolism, and both of them can regulate apoptosis of adipose cells. However, the coordination relationship between SIRTl and AMPK in the whole signal network level remain largely unknown. Previous study showed that inhibition of SIRT1 expression could activate Forkhead box O family 1 (FoxOl) apoptosis pathway. We speculated that the regulation of apoptosis of bovine subcutaneous preadipocytes(BSP) and intramuscular preadipocytes(BIP) of SIRT1 maybe in a different way. In this study, BSP and BIP are used to detect apoptosis of adipose cells and related genes and protein expression, by using fluorescence microscope，flow cytometry, RNA interference, Real-time PCR (RT-PCR), and Western blot. The mechanisms of SIRT1/AMPK signal pathway in regulating apoptosis of adipose cells will be further investigated in cell and molecular level, in order to decrease bovine subcutaneous fat deposition, increase intramuscular fat and therefore improve livestock meat quality through nutritional control technology.

脂肪细胞凋亡，即能量储存的死亡，为治疗人类肥胖、控制动物脂肪沉积开辟了新的解决途径。组蛋白去乙酰化酶1（SIRT1），作为能量代谢网络重要成员，与“机体能量感受器”激活蛋白激酶（AMPK）存在内在联系，在调节能量代谢过程中起重要作用，两者均可调控凋亡。但在整体信号网络水平两者之间如何协调仍不清楚。前期研究中发现抑制SIRT1可激活FoxO1凋亡通路；并推测SIRT1对肉牛肌内和皮下两种前体脂肪细胞的凋亡调控存在不同途径。本课题以肉牛肌内和皮下两种前体脂肪细胞为材料，利用荧光显微镜、流式细胞仪、RNA干扰、RT-PCR、Western-blot等技术手段，检测脂肪细胞凋亡情况、相关基因及蛋白表达，在细胞和分子水平上探讨SIRT1/AMPK信号通路对脂肪细胞凋亡的影响机制，为丰富其调控机体能量代谢网络体系，通过营养调控技术降低牛皮下脂肪沉积、提高肌内脂肪含量和改善畜禽肉品质奠定一定的理论基础。

脂肪细胞凋亡，即能量储存的死亡，和增殖分化共同维持机体脂肪细胞数目的恒定。组蛋白去乙酰化酶1 (SIRT1)与腺苷一磷酸激活的蛋白激酶（AMPK）在调节能量代谢过程中起着重要的调控作用，均参与细胞增殖、分化、衰老、凋亡和代谢过程。本研究采用流式细胞分析仪、荧光定量PCR、Western-blot等技术方法，利用白藜芦醇（Resveratrol，SIRT1激活剂）、Sirtinol（SIRT1抑制剂）、AICAR（AMPK激活剂）、Compound C（AMPK抑制剂）处理牛皮下脂肪细胞和肌内脂肪细胞，并利用RNA干扰慢病毒载体侵染细胞，研究AMPK-SIRT1信号通路在牛脂肪细胞凋亡和脂质代谢中的作用，同时比较分析了两种细胞的差异机制。结果表明：（1）AMPK-SIRT1信号通路上的关键因子SIRT1和AMPK激活，可显著增加HSL、ATGL、Caspase-3和 Bax等的mRNA和蛋白表达，显著降低PPARγ、HMGCR、FAS和Bcl-2等的mRNA和蛋白表达，细胞TG含量减少，凋亡率增加，抑制脂肪合成，促进脂解和凋亡；而在AMPK-SIRT1通路被干扰抑制的基础上，加入RES和AICAR可部分恢复脂质代谢相关基因和凋亡相关基因的mRNA和蛋白表达。（2）HMGCR基因可以负调控AMPK-SIRT1信号通路，并通过AMPK- SIRT1信号通路对脂质代谢过程进行调控。（3）皮下脂肪细胞中TG和胆固醇含量均比肌内脂肪细胞高，而AMPK和SIRT1的表达均低于肌内脂肪细胞，说明皮下脂肪细胞比肌内脂肪细胞具有更强的分化能力；AMPK-SIRT1信号通路激活时，皮下脂肪细胞的凋亡率高于肌内脂肪细胞，说明皮下脂肪细胞受AMPK-SIRT1信号通路的影响更大。本研究结果有助于从脂肪细胞凋亡的角度调控脂肪沉积，也可为通过饲喂植物提取物等营养技术调控肉牛脂肪沉积提供理论依据。