脂联素（adiponectin）对饥饿牦牛肝脏糖异生代谢的影响及其调控途径研究

Yak is mainly distributed in Tibetan-Plateau in China. It is the predominant productive subsistence that herdsman heavily rely on. The main cause of off-fat and death in cold season is resulted from starvation. Gluconeogenesis is a distinctive difference between ruminant and monogastric animals. With normal feed intake, the propionic acid is the main precursor for gluconeogenesis in the yak liver. With the condition of starvation, the rumen fermentative pattern changes while the body fat storage is decomposed, and the main precursors for gluconeogenesis and hormone secretion changes. All of the causes mentioned above would affect gluconeogenesis in the yak liver. Adiponectin is the only adipose cytokines which is negative correlated with fat deposition that secreted by adipose tissue, can affect secretion and sensitivity of insulin. The key enzymes in the process of gluconeogenesis are its potential targets. Therefore, we hypothesize that the adiponectin and its receptors may be the key regulatory factors in hepatic gluconeogenesis of yak with starvation condition. In this study, a hungry yak model will be established and primary hepatocytes of yak will be cultured, and the RT-PCR and metabolomics techniques will be applied to investigate how the changes of precursors for hepatic gluconeogenesis in yak will affect gluconeogenesis efficiency. And how the adiponectin will affect key enzymes such as PEPCK, PC, HSL and in glucose and lipid metabolism and key factors such as PPARα and AMPK in yak hepatocytes will also be determined. Through this research, the mechanisms of adiponectin to regulate hepatic gluconeogenesis of yak with starvation condition will be elucidated, and new regulatory targets will be identified. Finally, this work will provide theoretical guidance for scientific supplementary feeding of yak in the cold season.

牦牛主要分布于我国青藏高原地区，是牧民的生产和生活资料，冷季饥饿是牦牛掉膘、死亡的首要因素。糖异生是反刍动物碳水化合物代谢的显著特点，正常采食时，主要由丙酸等经肝脏糖异生合成葡萄糖。饥饿状态下，牦牛瘤胃发酵改变，体脂分解、主要生糖前体物质和激素水平改变，将影响牦牛肝脏糖异生。脂联素由脂肪组织分泌，是唯一与脂肪沉积呈负相关的脂肪细胞因子，可影响胰岛素的分泌和敏感性，糖异生关键酶是其潜在调控靶点。推测脂联素及其受体可能是饥饿状态下牦牛肝脏糖异生的关键调控因子。本项目拟通过建立牦牛饥饿模型和肝细胞原代培养，采用RT-PCR等方法研究饥饿条件下生糖前体物的改变对牦牛肝脏糖异生效率的影响，测定脂联素对牦牛肝细胞中PEPCK、PC、HSL等糖脂代谢关键酶和PPARα、AMPK等关键因子的影响，阐明脂联素对饥饿牦牛肝脏糖异生的调控机制，探明新的调控靶点，为冷季牦牛科学补饲提供理论指导。

牦牛是青藏高原地区的特有牛种，是牧民赖以生存的生活、生产资料。青藏高原枯草期长，冷季牦牛失重可达25%以上，冷季饥饿成为牦牛产业亟待解决的问题。糖异生是反刍动物典型的代谢特点，饥饿会导致肝脏糖异生前体物质和生糖效率变化。脂联素能够调控血糖稳态，在调节能量代谢和糖异生等方面都有重要作用。本项目通过牦牛饥饿实验、屠宰实验、肝脏细胞原代培养实验等，主要从牦牛饥饿模型的建立与评价、饥饿对牦牛肝脏糖异生代谢的影响、脂联素对饥饿牦牛肝脏糖异生途径的调控等方面进行研究，主要研究结果与结论：.（1）建立牦牛饥饿模型，饥饿后牦牛显著失重，血糖含量显著降低，血清中非酯化脂肪酸和甘油三酯含量显著升高，脂联素和胰高血糖素含量显著升高，胰岛素含量显著降低，肌肉蛋白质和脂肪含量显著降低，肝糖原和肌糖原含量显著降低，说明饥饿使牦牛大量动用体储维持血糖平衡。.（2）饥饿后牦牛机体脂肪动员增强，脂肪合成前体物浓度显著降低，饥饿提高了脂肪降解酶ACOX等的表达，降低了脂肪合成关键酶的表达，以促进脂肪降解，且主要消耗肌肉中SFA和PUFA，而对MUFA的利用较少，PUFA中Σｎ-3 PUFA 消耗量大于Σｎ-6 PUFA。.（3）饥饿后牦牛糖异生关键酶基因表达量与活性显著升高，肝细胞糖异生量显著升高，糖异生作用增强。同时，糖异生前体物质来源改变，食物来源的糖异生前体物质丙酸、乳酸含量显著降低，体储降解的甘油、丙氨酸和β-羟丁酸含量显著升高。.（4）饥饿后牦牛糖异生前体物质生糖效率发生变化，丙酸在正常饲喂牦牛肝细胞内生糖效率最高，β-羟基丁酸在饥饿牦牛肝细胞内生糖效率最高。.（5）饥饿可上调组织中脂联素及其受体的浓度，脂联素及其受体与脂肪合成关键酶呈负相关，与脂肪降解关键酶呈正相关。.（6）正常饲喂下，脂联素通过其受体AdipoR2促进AMPK磷酸化进而调控下游因子FoxO1、HNF4α表达抑制糖异生；而在饥饿状态下，AMPK的下游通路因子转变为了PGC-1α、TORC2、CREB。