Control of cAMP-mediated glucagon response by bile acids

胆汁淤积性肝病特别是肝硬化，是美国第九大死亡原因。 预后很差，通常不可逆的情况下，肝细胞进行性破坏的标志。 大约50%的肝病患者和80%的糖尿病患者表现出与胰高血糖素致血管生成反应降低相关的葡萄糖耐受不良。 胰高血糖素对肝细胞代谢的调节部分是通过增加cAMP的合成。 中心假设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，并且这些改变是胆汁淤积中胰高血糖素反应性减弱和延迟恢复的原因。 我们已经表明，胆汁酸抑制肝胰高血糖素诱导的cAMP合成在生理浓度。 这种效应是在受体-Gs偶联水平上，最可能通过磷酸化，并由钙依赖性PKC介导。 我们曾报道，肝胰高血糖素介导的cAMP的生产减弱，在仓鼠胆汁淤积引起的结扎胆总管（BDL）。 胆汁酸在BDL后没有或具有降低的作用，这表明胆汁淤积中改变的cAMP合成级联的位点与胆汁酸改变的相同。 具体目的将检验以下假设：1）肝细胞与胆汁酸的短期孵育通过涉及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HEK 293克隆和BDL仓鼠肝细胞中，我们将使用旨在确定蛋白磷酸化靶点的多方面方法研究生理/病理生理胆汁酸浓度和胆汁淤积对受体/Gs偶联和磷酸化的各自影响。 我们将研究蛋白磷酸酶在胆汁淤积时胰高血糖素反应恢复的时间过程中的作用。 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.