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岁人群第4大死亡原因。预后很差，通常是不可逆的，以肝细胞的进行性破坏为特征。大约50%的肝病患者和80%的肝硬化患者表现出葡萄糖不耐受，并伴有对胰高血糖素糖异生反应的降低。这种胰高血糖素诱导的肝细胞葡萄糖生成的减少是由于肝脏对胰高血糖素的显著抵抗和胰高血糖素诱导的cAMP生成的衰减。我们的早期研究表明，从胆汁淤积的仓鼠分离的肝细胞中，胰高血糖素的反应性显著减弱，这种效应被从对照仓鼠分离的肝细胞中添加亚胶束浓度的胆汁酸（包括鹅去氧胆酸（CDCA））模拟。我们的数据表明胆汁酸诱导的cAMP减弱反应是PKCalpha和/或pkcdelta介导的。我们的主要假设是，在肝细胞中，CDCA磷酸化PKC导致该激酶活化，进而磷酸化胰高血糖素受体并减弱胰高血糖素的反应性，这是与胆汁淤积期间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在体外鉴定在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)确定体内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)该激酶的激活对胰高血糖素受体在胆汁淤积症中的作用。我们提出的研究结果将具有直接的临床意义，并为理解胰高血糖素反应性在胆汁淤积性肝病中的衰减提供新的见解。这些研究将确定胆汁酸激活PKC和胰高血糖素受体的分子机制。此外，对PKC磷酸化和激活之间的分子平衡的描述可能有额外的好处，即为治疗胆汁淤积性肝胆疾病和糖尿病的合理药物设计确定新的分子靶点。公共卫生相关性：本研究将强调在胆汁淤积症中信号转导的生理控制减弱的新机制。从这些研究中获得的知识反过来可以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.