目的:胰高血糖素通过促进糖原分解在肝脏葡萄糖生成中起重要作用,诱导糖异生,抑制糖原生成。此外,胰高血糖素增加环磷酸腺苷(cAMP)水平,从而激活蛋白激酶A(PKA)和cAMP鸟嘌呤核苷酸交换因子(也称为Epac)。尽管PKA在肝脏中的功能已被广泛研究,肝Epac的功能知之甚少。这项研究的目的是阐明Epac在介导胰高血糖素对肝细胞的作用中的作用。
方法:EpacmRNA和蛋白表达,本地化,通过逆转录聚合酶链反应分析肝细胞中的活性,西方印迹,免疫荧光染色,和Rap1活性测定,分别。此外,我们研究了Epac特异性激活剂的作用,8-CPT,和Epac特异性抑制剂,ESI-05,对分离的大鼠肝细胞中糖原代谢的影响。通过检查葡萄糖激酶(GK)易位和糖异生酶的mRNA表达来评估糖原代谢的进一步机制。
结果:Epac2而非Epac1主要在肝脏中表达。此外,8-CPT抑制糖原积累和GK易位,并增强糖异生酶的mRNA表达。ESI-05未能逆转胰高血糖素诱导的糖原储存抑制,并部分抑制胰高血糖素诱导的GK易位和糖异生酶的mRNA表达。
结论:Epac信号在介导胰高血糖素在肝细胞中的降糖作用中起作用。
OBJECTIVE: Glucagon plays an essential role in hepatic glucogenesis by enhancing glycogen breakdown, inducing gluconeogenesis, and suppressing glycogenesis. Moreover, glucagon increases cyclic adenosine monophosphate (cAMP) levels, thereby activating protein kinase A (PKA) and cAMP guanine nucleotide exchange factor (also known as
Epac). Although the function of PKA in the liver has been studied extensively, the function of hepatic
Epac is poorly understood. The aim of this study was to elucidate the role of Epac in mediating the action of glucagon on the hepatocytes.
METHODS: Epac mRNA and protein expression, localization, and activity in the hepatocytes were analyzed by reverse transcription polymerase chain reaction, western blotting, immunofluorescence staining, and Rap1 activity assay, respectively. Additionally, we investigated the effects of an
Epac-specific activator, 8-CPT, and an
Epac-specific inhibitor, ESI-05, on glycogen metabolism in isolated rat hepatocytes. Further mechanisms of glycogen metabolism were evaluated by examining glucokinase (GK) translocation and mRNA expression of gluconeogenic enzymes.
RESULTS: Epac2, but not Epac1, was predominantly expressed in the liver. Moreover, 8-CPT inhibited glycogen accumulation and GK translocation and enhanced the mRNA expression of gluconeogenic enzymes. ESI-05 failed to reverse glucagon-induced suppression of glycogen storage and partially inhibited glucagon-induced GK translocation and the mRNA expression of gluconeogenic enzymes.
CONCLUSIONS: Epac signaling plays a role in mediating the glucogenic action of glucagon in the hepatocytes.