肌肉腌制过程中食盐介导PLIN1蛋白磷酸化调控ATGL和HSL水解肌内甘油三酯机制研究

Intramuscular lipolysis is the important pathway of flavour precursors formation in dry-cured meat products, and is also the important research area of meat products. Our previous studies have found that the endogenous HSL activity was significantly increased and the fat hydrolyzed markedly. But it is not clear about its specific mechanism. So, the present project intends to take the muscle dry salting as research object, and carry out following research works from tissue, cellular and molecular levels by using the technical means of electron microscopy, phosphorylated proteomics, Western blot immunoblotting and immunofluorescence analysis etc. 1)the variation of muscle microstructure, intramuscular fat distribution and the morphology of intramuscular adipocytes and lipid droplets during the salting process of different conditions; 2)the changes of PKA, AMPK, alkaline phosphatase activity and overall protein phosphorylation in intramuscular adipocytes during the salting process of different conditions; 3)the effect of PLIN1 phosphorylation on the translocation and activity changes of ATGL and HSL in the intramuscular adipocytes during the salting process of different conditions; 4)the effect of PLIN1 phosphorylation on the interaction of ATGL with ABHD5 and G0S2 during the salting process of different conditions. Finally, by analyzing the correlation between these changes and intramuscular triglyceride hydrolysis and salting condition, the mechanism of salt induced intramuscular lipid droplet morphological changes and the regulation of PLIN1 phosphorylation on ATGL and HSL hydrolysis of intramuscular triglycerides were elucidated. This will provide theoretical guidance for the regulation of lipolysis in dry cured meat products during processing.

肌内脂肪水解是干腌肉制品风味前体物形成的重要途径，也是肉品研究的重要领域。课题组前期研究发现腌制过程中肌内HSL活性显著升高、脂肪显著水解，但其具体机理还不清楚。因此，本项目拟以肌肉腌制为研究对象，借助电镜技术、磷酸化蛋白组学、Western blot、免疫荧光等技术手段，从组织、细胞、分子水平研究不同条件腌制过程中：1）肌肉微观结构、肌内脂肪分布以及脂肪细胞和脂滴形态变化规律；2）肌内脂肪细胞中PKA、AMPK、碱性磷酸酶活性及蛋白质整体磷酸化规律；3）脂肪细胞内PLIN1磷酸化对ATGL、HSL在细胞内转位及活性变化的影响；4）PLIN1磷酸化对ABHD5、G0S2与ATGL互作影响。通过分析这些变化与肌内甘油三酯水解以及腌制条件的相关性，阐明腌制过程中食盐诱导肌内脂滴形态变化以及PLIN1蛋白磷酸化调控ATGL、HSL水解甘油三酯机制，为干腌肉制品加工过程中脂质水解调控提供理论指导。

为探明肉品腌制过程中食盐对肌内TG水解的作用机制，本项目以屠宰新鲜猪后腿股二头肌肌肉作为实验材料，用不同浓度食盐（0%、1%、3%、5%）在4℃下进行腌制，对腌制过程中食盐浓度变化对肌内ATGL和HSL脂酶活性变化、TG水解规律、脂滴形态变化、脂滴蛋白质组及相关调控蛋白PLIN1、ABHD5和G0S2磷酸化的影响及相关性展开系统研究。主要获得如下结论：. （1）腌制会促进肌内TG的水解，随着盐浓度的增加，促进效果先增强后减弱，3%食盐腌制时，肌内总TG水解酶活性最高，TG水解程度最高，其次是5%食盐处理组和1%食盐处理组，3%是腌制促进肌内TG水解的拐点浓度。免疫印迹结果表明，随着盐浓度的增加，ATGL、HSL 及其磷酸化蛋白含量变化与脂酶活性及TG的水解变化规律高度正相关。微观结构分析进一步证实随着盐浓度的增加，肌内脂滴由大脂滴裂解生成小脂滴，更有利于脂酶对其水解。. （2）腌肉脂肪细胞中PKA、AMPK的活性显著高于对照组，且随腌制用盐量增加先升高后降低，3%腌制组活性最高。Pro-Q Diamond荧光染色SDS-PAGE电泳结果证明，腌制过程中细胞内蛋白整体磷酸化水平与PKA活性变化高度正相关，3%腌制组细胞内蛋白整体磷酸化水平最高。Western Blotting检测到细胞内ATGL、HSL磷酸化蛋白表达量在腌制样品中均有显著上调；3%浓度食盐腌制3天时，细胞内PLIN1和ABHD5及其磷酸化蛋白表达量最高，G0S2的表达量最低，更有利于ATGL的激活，促进细胞内甘油酯发生水解。. （3）脂滴多与线粒体接触，且在对照组中有较多大脂滴。腌制处理，尤其是3%盐浓度腌制后的肌肉组织中线粒体发生损伤，有较多溶酶体和自噬体样的结构存在。且食盐腌制处理以及盐浓度的增加促使脂滴平均直径变小。腌制处理后，主导脂滴新生的FITM2和参与脂质水解的脂酶以及PLIN1蛋白表达下调，调控脂肪酸降解的CPT2、SCD等蛋白和调控脂噬的RAB7A、RAB10蛋白表达上调。3%盐浓度腌制使PLIN1、ATGL、HSL蛋白序列中特定位点的磷酸化水平发生不同程度的上下调。