β-羟基丁酸对饥饿牦牛脂肪细胞脂联素表达的调控机理研究

Yak is mainly distributed in the Qinghai-Tibetan Plateau, approximately 92% of the world's total yak population live in China. The primary factor responsible for body weight loss and death in cold season is starvation. Body fat mobilization is the main way for yak to deal with starvation stress, and adiponectin is an important adipocytokines for animals to cope with starvation stress and fat metabolism regulation. β-hydroxybutyric acid is the main ketone body of hungry animals, participating in body metabolism as energy substances and precursors. Recently, it was reported that β-hydroxybutyric acid acted as a signal molecule to bind with GPR109A receptor to regulate adiponectin expression in mice. Our team found that β-hydroxybutyric acid could affect adiponectin expression and gluconeogenesis in fasted yak, but the regulation mechanism was still unclear. Therefore, we hypothesize that β-hydroxybutyric acid can act as a signal molecule to bind with the GPR109A receptor, phosphorylate the receptor Gα and Gβγ subunits, activate downstream signal pathways, and finally regulate adiponectin expression in fasted yaks. In this project, the primary culture of adipocytes and yak starvation model will be established, and through overexpression and inhibition of GPR109A receptor, blocking and activating the signal pathways, in the aim of revealing the signal pathways of β-hydroxybutyric acid acting as a signal molecule to manipulate adiponectin expression in fasted yak adipocytes, exploring new targets to regulate adiponectin expression, perfecting regulation theory of yak fat metabolism, promoting the yak’s scientific breeding.

牦牛主要分布于青藏高原，我国牦牛约占世界牦牛总量的92%，冷季饥饿是牦牛掉膘、死亡的首要因素。体脂动员是牦牛应对饥饿胁迫的主要方式，脂联素是动物应对饥饿胁迫和调节脂肪代谢的重要脂肪因子。β-羟基丁酸是饥饿动物主要的酮体，作为能源物质和前体参与机体代谢。近年小鼠研究表明β-羟基丁酸可作为信号分子与GPR109A受体结合调控脂联素表达。本课题组研究发现β-羟基丁酸可影响饥饿牦牛糖异生和脂联素表达，但调控机制不清楚。因此，本项目假设β-羟基丁酸可作为信号分子与GPR109A受体结合，使受体Gα和Gβγ亚基磷酸化，激活下游信号通路，调控饥饿牦牛脂联素表达。项目拟建立牦牛饥饿模型和脂肪细胞原代培养，通过对GPR109A受体过表达和抑制、对信号通路阻断和激活，揭示β-羟基丁酸作为信号分子调控饥饿牦牛脂肪细胞脂联素的信号通路，探明调控脂联素表达的新靶点，丰富牦牛脂肪代谢调控理论，促进牦牛科学饲养。

牦牛是青藏高原的优势畜种，我国牦牛约占世界牦牛总量的92%，冷季缺草料饥饿是牦牛掉膘、死亡和限制牦牛产业发展的首要原因，体脂动员是牦牛应对饥饿胁迫的主要方式，脂联素是动物饥饿条件下调控脂肪代谢的重要脂肪因子，脂肪降解产物β-羟基丁酸（BHBA）可作为信号分子调控脂联素分泌和脂肪代谢。本项目通过牦牛饥饿试验，牦牛脂肪细胞原代培养实验等，主要从牦牛饥饿模型的建立与评价、β-羟基丁酸对饥饿牦牛脂肪细胞GPR109A受体及脂联素表达的量效关系、β-羟基丁酸对饥饿牦牛脂肪细胞脂联素分泌的调控机理等方面进行研究，主要研究结果与结论：.（1）建立牦牛饥饿模型。饥饿后牦牛瘤胃总挥发性脂肪酸、微生物蛋白浓度显著降低，营养供给不足，血糖浓度降低，机体脂解代谢关键酶活性增加，血液中脂解产物β-羟基丁酸、NEFA等增加，脂肪降解供能增加，机体掉膘失重。 .（2）β-羟基丁酸缓解饥饿导致的牦牛脂肪分解。β-羟基丁酸增加饥饿牦牛脂肪中ACC、DGAT等脂肪合成代谢关键酶活性，降低ACOX和HSL等脂肪降解代谢关键酶活性，增加饥饿牦牛脂肪细胞面积，降低血清脂解产物NEFA浓度，缓解饥饿牦牛掉膘失重。.（3）β-羟基丁酸浓度与饥饿牦牛脂肪细胞GPR109A受体基因蛋白表达以及脂联素分泌均呈显著三次相关，适宜浓度BHBA可促进脂肪细胞脂联素分泌。.（4）β-羟基丁酸主要通过激活GPR109A受体及其下游MEK/ERK1/2通路，促进饥饿牦牛脂肪细胞脂联素分泌，并可通过激活GPR109A受体抑制下游AC/PKA/CREB通路参与促进饥饿牦牛脂肪细胞脂联素分泌。.综上，本研究构建了牦牛饥饿动物模型，建立了牦牛脂肪细胞原代培养方法和饥饿细胞模型，发现β-羟基丁酸与饥饿牦牛脂肪细胞GPR109A基因蛋白表达量和脂联素分泌均呈显著三次相关关系，β-羟基丁酸通过激活GPR109A受体激活MEK/ERK1/2通路促进脂联素分泌，并可抑制AC/PKA/CREB通路参与促进脂联素分泌，促进脂肪合成代谢，抑制脂肪降解，缓解饥饿牦牛机体掉膘失重。本研究揭示了β-羟基丁酸对饥饿牦牛脂肪细胞脂联素分泌以及脂肪代谢的调控机理，对促进牧区牦牛冷季有效补饲和丰富牦牛营养代谢理论提供了基础。