TRANSCRIPTIONAL CONTROL OF CARBOHYDRATE RESPONSIVE GENES

这个项目的总体目标是增加我们对调节碳水化合物反应基因的转录机制的理解。葡萄糖的动态平衡依赖于许多酶和吸收途径的严格控制。作为一个模型，我们一直在研究小肠高亲和力Na+/葡萄糖共转运体(SGLT1)，其活性具有昼夜周期性，并受碳水化合物的诱导。我们已经证明SGLT1的表达在mRNA丰度和转录方面都表现出日周期。大鼠SGLT1启动子包含一个HNF-1(肝细胞核因子1)元件，它与肠上皮细胞核提取物形成复合体，不同于HNF-1β亚型的存在，取决于一天中核被分离的时间。在另外两个碳水化合物反应基因--肝丙酮酸激酶和蔗糖酶-异麦芽糖酶的启动子中，在碳水化合物反应元件中存在同源的HNF-1位点，我们也证明了这两个基因具有昼夜周期性，这表明HNF-1可能既介导了日常转录程序，也可能介导了饮食碳水化合物的诱导。恒河猴肠道SGLT1 mRNA水平的周期性表明，人类也存在类似的机制。有人认为，SGLT1的节律性转录是由与SGLT1启动子相互作用的转录因子的日常激活(或合成)控制的。确定负责的因素并确定它们的功能是描述这些动态平衡机制的重要步骤。为此，我们将检验两个假设：(1)SGLT1启动子中的“昼夜节律”元件驱动其日常转录；(2)碳水化合物通过调节SGLT1和其他碳水化合物响应基因的日常表达程序来诱导它们。由于参与碳水化合物代谢的广泛蛋白质--消化酶、转运蛋白、糖酵解酶和糖异生酶--都是碳水化合物反应的，了解这一类成员表达的共同和独特的途径将有助于更好地理解碳水化合物耐受性的变化。突显阐明这一调控的重要性的是，最近在HNF-1α和β二聚体伙伴中都发现了突变，这些突变是人类一种或多种形式糖尿病的病因。最后，预计对SGLT1调节机制的阐明可能揭示肥胖和糖尿病等条件下碳水化合物吸收的药物控制靶点。

The general goal of this project is to increase our understanding of the transcriptional mechanisms regulating carbohydrate- responsive genes. Glucose homeostasis depends on the strict control of numerous enzymatic and absorptive pathways. As a model, we have been studying the small intestinal high-affinity Na+/glucose cotransporter (SGLT1), the activity of which shows diurnal periodicity and induction by carbohydrate. We have shown that SGLT1 expression exhibits diurnal periodicity in both mRNA abundance and transcription. The rat SGLT1 promoter contains an HNF-1 (hepatocyte nuclear factor 1) element that forms complexes with intestinal epithelial cell nuclear extracts differing in the presence of the HNF-1beta isoform depending on the time of day nuclei were isolated. The presence of homologous HNF-1 sites in the carbohydrate-response elements in the promoters of two other carbohydrate-responsive genes--liver pyruvate kinase and sucrase- isomaltase, which we have also shown to exhibit diurnal periodicity--indicates that HNF-1 may mediate both the diurnal transcription program as well as induction by dietary carbohydrate. Periodicity in the intestinal SGLT1 mRNA levels in rhesus monkeys suggests that a similar mechanism is also present in humans. It is proposed that rhythmic SGLT1 transcription is controlled by the diurnal activation (or synthesis) of transcription factors interacting with the SGLT1 promoter. Identifying the factors responsible and defining their functions are important steps toward delineating these homeostatic mechanisms. To these ends, two hypotheses will be tested: (1) "Circadian" elements in the SGLT1 promoter drive its diurnal transcription; and (2) Carbohydrate induces SGLT1 and other carbohydrate-responsive genes by modulating their diurnal expression program. Because a wide range of proteins involved in carbohydrate metabolism--digestive enzymes, transporters, glycolytic enzymes, and gluconeogenic enzymes--are carbohydrate- responsive, knowledge of the shared and distinctive pathways regulating the expression of members of this class will lead to a better understanding of changes in carbohydrate tolerance. Underscoring the importance of elucidating this regulation, mutations have recently been identified in both HNF-1alpha and beta dimerization partners that are etiologic in one or more forms of diabetes in humans. Finally, it is anticipated that elucidation of the mechanisms regulating SGLT1 may reveal targets amenable to pharmaceutical control of carbohydrate absorption in conditions such as obesity and diabetes mellitus.