Control of cAMP Mediated Glucagon Response by Bile Acids

描述（由申请人提供）：慢性肝病，特别是肝硬化，是美国第12大死亡原因，也是45至54岁人群的第4大死亡原因。预后差，通常不可逆，并以肝细胞的进行性破坏为标志。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。这种胰高血糖素诱导的肝细胞葡萄糖产生的减少是由于对胰高血糖素的显著肝抗性和胰高血糖素诱导的cAMP产生的减弱。我们的早期研究表明，胰高血糖素的反应性显着减弱，从胆汁淤积仓鼠分离的肝细胞中，这种效果是模仿亚胶束浓度的胆汁酸，包括鹅去氧胆酸（CDCA）从对照仓鼠分离的肝细胞。我们的数据表明胆汁酸诱导的减弱的cAMP反应是PKCalpha和/或PKCdelta介导的。我们的总体假设是，在肝细胞中，CDCA对PKC的磷酸化导致该激酶的激活，进而磷酸化胰高血糖素受体并减弱胰高血糖素反应性，这是胆汁淤积期间与肝脏疾病进展相关的事件之一。本研究的目的是：1）通过蛋白质组学方法体外鉴定在CDCA存在下磷酸化的PKC残基，以及磷酸化对PKC稳定性和活性的影响。2)在体内确定负责PKC磷酸化的位点和机制以及对该激酶的定位、稳定性和活性的影响。3)在胆汁淤积中，这种激酶的激活对胰高血糖素受体功能的影响。我们提出的研究结果将具有直接的临床意义，并为胆汁淤积性肝病中胰高血糖素反应性减弱的理解提供新的见解。这些研究将确定胆汁酸调节PKC和胰高血糖素受体活化的分子机制。此外，PKC的磷酸化和活化之间的分子平衡的描绘可能有额外的好处，确定合理的药物设计在治疗胆汁淤积性肝胆疾病，以及糖尿病的新的分子靶点。公共卫生相关性：这项研究将突出新的机制，其中信号转导的生理控制是衰减胆汁淤积。从这些研究中获得的知识反过来可能影响胆汁淤积性肝胆疾病以及糖尿病的诊断和治疗。

DESCRIPTION (provided by applicant): Chronic liver disease and cirrhosis in particular, is the 12th leading cause of mortality in the United States and the 4th leading cause of death for individuals aged 45 to 54 years. The prognosis is poor, generally irreversible, and marked by progressive destruction of the liver cells. Around 50% of patients with liver disease and 80% of cirrhotic patients display glucose intolerance associated with a decreased gluconeogenic response to glucagon. This decreased glucagon-induced hepatocellular glucose production is due to a significant hepatic resistance to glucagon and attenuation of glucagon-induced cAMP production. Our early studies showed glucagon responsiveness to be significantly attenuated in hepatocytes isolated from cholestatic hamsters, and this effect was mimicked by submicellar concentrations of bile acids including chenodeoxycholic acid (CDCA) in hepatocytes isolated from control hamsters. Our data suggested the attenuated cAMP response induced by bile acids to be PKCalpha and/or PKCdelta-mediated. Our overarching hypothesis is that in hepatocytes, phosphorylation of PKC by CDCA leads to activation of this kinase, which in turn phosphorylates the glucagon receptor and attenuates glucagon responsiveness, which is one of the events associated to the progression of liver disease during cholestasis. The aims of the proposed study are: 1) Identification in vitro by a proteomic approach of the PKC residues that are phosphorylated in the presence of CDCA, and the consequences that the phosphorylation has on PKC stability and activity. 2) Determination in vivo the sites and the mechanism responsible for the PKC phosphorylation and the consequences on the localization, stability and activity of this kinase. 3) The impact that the activation of this kinase has on glucagon receptor function in cholestasis. Findings from our proposed studies will have direct clinical significance and add new insight to the understanding of the attenuation of glucagon responsiveness in cholestatic liver diseases. These studies will define molecular mechanisms regulating both PKC and glucagon receptor activation by bile acids. Furthermore, the delineation of the molecular balance between phosphorylation and activation of PKC may have the added benefit of identifying novel molecular targets for rational drug design in the treatment of cholestatic hepatobiliary disorders, as well as diabetes. Public Health Relevance: This study will highlight novel mechanisms by which physiological control of signal transduction is attenuated in cholestasis. The knowledge gained from these studies could in turn impact both the diagnosis and treatment of cholestatic hepatobiliary disorders, as well as diabetes.