围产期奶牛能量负平衡引发胰岛素抵抗的分子机制

In transition period, dairy cows with negative energy balance displayed insulin resistance. Insulin resistance could increase negative energy balance and then increase the incidence of infectious diseases in dairy cows. Negative energy balance could induce high blood concentration of nonesterified fatty acid (NEFA) and/or β-hydroxybutyric acid (BHBA). Therefore, we hypothesized that high blood concentration of NEFA and BHBA might be the main pathogenic factor for the incidence of insulin resistance in dairy cows. In this study, the effects of high concentration of NEFA and/or BHBA on the sensibility of insulin signaling pathway were detected, which revealed high concentration of NEFA and/or BHBA could induce insulin resistance and impair insulin signaling pathway in peripheral tissues and organs. Furthermore, the effects of high concentration of NEFA and/or BHBA on the changes of key molecules involved in TLR-medicated inflammatory signaling pathway, endoplasmic reticulum stress signaling pathway, and mitochondrial dysfunction signaling pathway in the hepatocytes and adipocytes were determined. Therefore, the mechanism of insulin resistance induced by high concentration of NEFA and/or BHBA in dairy cows with negative energy balance could be revealed. This study will provide a valuable theoretical base to reduce the incidence of energy metabolic diseases in the transition period of dairy cows.

围产期能量负平衡奶牛存在胰岛素抵抗，而胰岛素抵抗不仅能加剧能量负平衡，还增加围产期感染性疾病的发生。基于能量负平衡导致高非酯化脂肪酸（NEFA）血症和高β羟丁酸（BHBA）血症这一事实，提出高NEFA血症和高BHBA血症有可能是引发围产期能量负平衡奶牛胰岛素抵抗的重要因素的科学假设。据此，本项目观测高NEFA和/或高BHBA对脂肪组织和肝脏胰岛素敏感性及胰岛素信号通路的影响，明确高NEFA和/或高BHBA能引发外周组织器官胰岛素抵抗和干扰胰岛素信号转导；观测高NEFA和/或高BHBA对肝细胞和脂肪细胞Toll样受体介导的炎性信号通路、内质网应激信号通路和线粒体代谢通路的影响，探寻高NEFA和/或高BHBA对脂肪细胞和肝细胞炎症、应激等能影响胰岛素信号转导有关的信号通路的作用。阐明能量负平衡奶牛高NEFA和/或高BHBA血症诱发胰岛素抵抗的信号机制，为防治奶牛能量负平衡性疾病提供理论依据。

围产期奶牛由于干物质摄入减少但能量需求增加而使机体处于能量负平衡状态，进而启动脂肪动员，造成高非酯化脂肪酸（nonesterified fatty acid, NEFA）血症和高 β-羟丁酸（β-hydroxybutyric acid, BHBA）血症，而高浓度NEFA和BHBA具有明显的脂毒性。胰岛素抵抗（Insulin Resistance, IR）是指机体胰岛素作用的靶器官(肝脏和脂肪组织)对胰岛素作用的敏感性下降。能量负平衡的奶牛易于发生胰岛素抵抗，而且胰岛素抵抗会加剧能量负平衡而引发酮病和脂肪肝等能量负平衡性疾病。但是目前，尚不清楚能量负平衡是通过那种和那些途径引发胰岛素抵抗的。因此本研究的目的在于探究围产期能量负平衡奶牛胰岛素抵抗的诱发因素以及诱发机制。我们的结果显示，围产期发生能量负平衡的奶牛存在明显的高NEFA和BHBA血症，肝脏胰岛素信号通路相关蛋白如p-AKT、p-GSK3β的表达低于正常奶牛，NF-κB信号通路过度激活，内质网应激标志分子p-IRE1α、p-PERK、Cleaved-ATF6的表达高于正常奶牛，而且线粒体功能关键调节因子PGC-1α、Mfn2、TFAM、NRF1的表达均低于正常奶牛，肝脏ATP的含量却低于正常奶牛，这些结果说明能量负平衡奶牛的肝脏存在胰岛素抵抗，炎症，内质网应激以及线粒体功能紊乱。接下来，我们体外分离犊牛原代肝细胞和脂肪细胞，并用不同浓度的NEFA处理。我们发现高浓度的NEFA（NEFA≥1.2 mM）可以引起肝细胞和脂肪细胞的内质网应激，并进而导致胰岛素信号通路受损，而且抑制内质网应激可以缓解高NEFA诱发的胰岛素信号通路受损；高NEFA可以激活TLR4介导的NF-κB炎症通路；高NEFA也可以引起肝细胞的线粒体功能紊乱并进一步损伤胰岛素信号通路，而且通过过表达线粒体功能关键调节因子PGC-1α或Mfn2可以缓解高NEFA引起的线粒体功能紊乱和胰岛素信号通路受损。另外我们还发现围产期能量负平衡奶牛血液中miR-181a的表达高于正常奶牛，且高表达的miR-181a可以通过靶向SIRT1促进肝细胞脂积累抑制脂合成，促进脂肪肝的发生发展。这些结果明确了围产期能量负平衡奶牛胰岛素抵抗发生的原因以及机制，将有助于进一步揭示酮病和脂肪肝等围产期能量负平衡性疾病的发病机理，及针对胰岛素抵抗采取有效的防治措施。