Davies G F, Crosson S M, Khandelwal R L, Roesler W J
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Arch Biochem Biophys. 1995 Nov 10;323(2):477-83. doi: 10.1006/abbi.1995.0070.
Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis. This metabolically important enzyme is unique in that it has no known allosteric modifiers, and all of the regulation of its activity is exerted at the level of gene expression. The expression of the PEPCK gene in liver is elevated in most forms of diabetes, and plays a major contributory role in the hyperglycemia characteristic of this disease. In this study, we initiated studies to determine the molecular basis for the increased PEPCK gene expression in diabetes. RNase protection assays of RNA isolated from control, streptozotocin-induced diabetic, and insulin-treated diabetic rat liver indicated that PEPCK mRNA levels are elevated two- to threefold in diabetic rat liver compared to controls. Nuclear run-on assays indicated that the increased PEPCK mRNA levels can be fully accounted for by changes in the transcription rate of the gene. We next initiated characterization of the cAMP response element binding protein (CREB) in diabetic rat liver, since it is known to play a major role in mediating the it is known to play a major role in mediating the basal transcriptional activity of the PEPCK gene as well as the cAMP-dependent stimulation of PEPCK gene transcription, the latter through the phosphorylation of serine 133 of CREB. Western blot analysis of nuclear lysates prepared from rat livers indicated that CREB protein levels in diabetic rat liver nuclei were similar to those of controls. However, using an antibody which specifically recognizes the serine 133-phosphorylated form of CREB, we found that the levels of phospho-CREB were significantly decreased in diabetic rat liver, an effect which insulin treatment reversed. This observation suggests that overexpression of the PEPCK gene in diabetes is not linked to the cAMP signaling system in liver.
磷酸烯醇式丙酮酸羧激酶（PEPCK）是糖异生的限速酶。这种在代谢方面具有重要意义的酶很独特，因为它没有已知的变构调节因子，其活性的所有调节都在基因表达水平发挥作用。在大多数糖尿病类型中，肝脏中PEPCK基因的表达都会升高，并且在该疾病的高血糖特征中起主要作用。在本研究中，我们开始研究以确定糖尿病中PEPCK基因表达增加的分子基础。对从对照、链脲佐菌素诱导的糖尿病以及胰岛素治疗的糖尿病大鼠肝脏中分离出的RNA进行核糖核酸酶保护分析表明，与对照相比，糖尿病大鼠肝脏中PEPCK mRNA水平升高了两到三倍。细胞核连续转录分析表明，PEPCK mRNA水平的升高完全可以由该基因转录速率的变化来解释。接下来，我们开始对糖尿病大鼠肝脏中的环磷酸腺苷反应元件结合蛋白（CREB）进行表征，因为已知它在介导PEPCK基因的基础转录活性以及PEPCK基因转录的环磷酸腺苷依赖性刺激中起主要作用，后者是通过CREB丝氨酸133的磷酸化实现的。对大鼠肝脏制备的细胞核裂解物进行的蛋白质免疫印迹分析表明，糖尿病大鼠肝细胞核中的CREB蛋白水平与对照相似。然而，使用一种特异性识别丝氨酸133磷酸化形式的CREB的抗体，我们发现糖尿病大鼠肝脏中磷酸化CREB的水平显著降低，胰岛素治疗可逆转这种效应。这一观察结果表明，糖尿病中PEPCK基因的过表达与肝脏中的环磷酸腺苷信号系统无关。
