Control of cAMP-mediated glucagon response by bile acids

胆汁淤积性肝病，特别是肝硬变，是美国第九大主要死亡原因。预后很差，通常不可逆的情况以肝细胞的进行性破坏为标志。大约50%的肝病患者和80%的肝硬变患者表现出葡萄糖不耐受，这与对胰高血糖素的糖异生反应降低有关。胰升糖素对肝细胞代谢的调节部分是通过增加cAMP合成来实现的。中心假说是某些胆汁酸通过磷酸化/去磷酸化机制改变胰高血糖素受体-刺激性G蛋白(Gs)偶联，这些改变是导致胆汁淤积症中胰高血糖素反应性减弱和延迟恢复的原因。我们已经证明，胆汁酸在生理浓度下抑制肝脏高血糖素诱导的cAMP合成。这种作用是在受体-Gs偶联的水平上进行的，很可能是通过磷酸化，并由钙依赖的PKC介导的。我们已经报道，在结扎胆总管(BDL)诱导的胆汁淤积中，肝脏高血糖素介导的cAMP产生减少。胆汁酸对胆汁淤积无影响或减少，提示胆汁淤积时cAMP合成级联改变的部位与胆汁酸改变的部位相同。具体目标将检验假说：1)肝细胞与胆汁酸短期孵育通过涉及PKC的磷酸化/去磷酸化机制导致胰高血糖素受体-Gs偶联减少；2)胰高血糖素受体-Gs偶联和受体去磷酸化的改变分别导致胆汁淤积症中胰高血糖素反应性的减弱和延迟恢复。在表达胰高血糖素受体的HEK293克隆中，以及在BDL仓鼠的肝细胞中，我们将使用多方面的方法来研究生理/病理生理胆汁酸浓度和胆汁淤积对受体/Gs偶联和磷酸化的影响。我们将研究蛋白磷酸酶在胆汁淤积症中胰高血糖素反应恢复的时间进程中的作用。从这些研究中获得的知识将对胆汁淤积性肝胆疾病的诊断和治疗产生影响。

Cholestatic liver disease with cirrhosis in particular, is the 9th leading cause of death in the USA. Prognosis is poor, with a generally irreversible condition marked by progressive destruction of liver cells. Around 50 percent of patients with liver disease and 80 percent of cirrhotic patients display glucose intolerance associated with decreased gluconeogenic response to glucagon. Regulation of helpatocellular metabolism by glucagon is in part through increased cAMP synthesis. The central hypothesis is that certain bile acids alter the glucagon receptor-stimulatory G protein (Gs) coupling through a phosphorylation/dephosphorylation mechanism and that these alterations are responsible for attenuation and delayed recovery of glucagon responsiveness in cholestasis. We have shown that bile acids inhibit hepatic glucagon-induced cAMP synthesis at physiologic concentrations. The effect was at the level of receptor-Gs coupling, most likely through phosphorylation, and was mediated by a calcium-dependent PKC. We have reported that hepatic glucagon-mediated cAMP production was attenuated in cholestasis in hamster induced by ligation of the common bile duct (BDL). Bile acids were either without or with reduced effects after BDL suggesting that the site of cAMP synthesis cascade altered in cholestasis is the same as that altered by bile acids. Specific aims will test the hypotheses: 1)that short-term incubation of hepatocytes with bile acids leads to decreased glucagon receptor-Gs coupling through a phosphorylation/dephosphorylation mechanism involving PKC; 2)that alteration of both glucagon receptor-Gs coupling and receptor dephosphorylation are responsible for the respective attenuation and delayed recovery of glucagon responsiveness in cholestasis. In HEK293 clones expressing glucagon receptor, and in hepatocytes from BDL hamsters we will study the respective effect of physiologic/pathophysiologic bile acid concentrations and cholestasis on receptor/Gs coupling and phosphorylation using a multifaceted approach designed to determine the protein phosphorylation target. We will study the role of protein phosphatases on the time course of glucagon response recovery in cholestasis. Knowledge gained from these studies will have bearing on both diagnosis and treatment of cholestatic hepatobiliary disorders.