Regulation of rat Liver Glucokinase Gene Expression by Sterol Regulatory Element Binding Protein-1a and Forkhead box classO1 Transcription factors

Abstract

Glucokinase (GK) also known as hexokinase IV catalyzes phosphorylation of glucose to glucose-6-phosphate. In contrast to other hexokinases, GK has a low affinity for glucose, is not inhibited by its reaction product and, although existing as monomer, displays sigmoidal kinetics. Defects in the GK gene lead to maturity-onset diabetes of the young type 2 (non-insulin-dependent [MODY-2]). Thus, GK plays an important role for maintenance of glucose homeostasis. GK is predominantly expressed in hepatocytes of the liver, pancreatic -cells and some neuroendocrine cells of the gastrointestinal tract and the brain. Insulin and glucagon are the major hormones regulating expression of GK in hepatocytes. Thereby, insulin acts mainly via the PI3K/PKB pathway and modulates the activity of several transcription factors such as sterol regulatory element binding protein-1 (SREBP-1) and FoxO/forkhead transcription factors (FoxO). Recent reports from cell culture experiments and transgenic mice indicated that both SREBP-1 and FoxO1 may act in an antagonistic fashion on GK expression and thus on hepatic glucose/lipid metabolism. However, the complete details of the SREBP-1 and FoxO1 regulated GK gene expression are not yet known. Therefore, it was the aim of this study to investigate, the insulin-dependent SREBP-1- and FoxO1-mediated GK gene expression at the molecular level in primary rat hepatocytes and HepG2 hepatoma cells. Stimulation of primary hepatocytes with insulin induced GK and SREBP-1 expression. Similarly SREBP could be induced by treatment with the LXR agonist TO901317 which in turn induced GK mRNA levels. Likewise, overexpression of SREBP-1 in hepatocytes induced GK mRNA levels. Computer analysis of the liver-specific GK promoter revealed three putative SREBP-1 binding sites (SREs). Transfection experiments in hepatocytes and HepG2 cells with luciferase gene constructs driven by serially deleted GK promoter fragments indicated that a sequence known as the footprint B site is critically involved in SREBP-1-dependent regulation. Further analysis of the footprint B site which could be divided in part 1 and part 2 showed that part 2 rather than part 1 is necessary for the SREBP-1 effect. In addition, two other sequences termed SRE2 and SRE3 were identified by mutation analyses of GK promoter. Interestingly, transfection data in primary hepatocytes and HepG2 cells implicated that these elements are utilized in a cell-specific manner. While both SRE2 and SRE3 are important for the SREBP-1-mediated GK promoter activity in primary hepatocytes, only SRE2 contributed to the SREBP-1 effect in HepG2 cells. Moreover, the SREBP-1-mediated activation of the GK promoter was lost upon mutation of the HNF-4 binding element indicating that full induction of GK gene expression by SREBP-1 requires interaction of these transcription factors. Insulin has a dynamic effect on FoxO transcription factors which are mediated by PKB-dependent phosphorylation which lead to inactive FoxO by nuclear exclusion. Hepatocytes transfected with FoxO1 expression vectors down regulated GK mRNA and GK promoter activity and the repression was lost when hepatocytes were stimulated with insulin. When rats were fasted for 48 h the GK protein levels were nearly undetectable, whereas FoxO1 protein levels were induced. In addition to insulin, transcriptional activity of FoxO proteins is known to be regulated by NAD+-dependent SIRT1 deacetylases. Resveratrol down regulated GK mRNA and protein levels and reversed the inducing effects of insulin. Similar results were observed with the GK enzyme activity. Computational analysis of the GK promoter predicted two FoxO1 binding elements (FBEa and FBEb). Overexpression of FoxO1 suppressed GK promoter activity in primary hepatocytes and HepG2 cells. Mutations in the FoxO1 binding element FBEb abolished the FoxO1-mediated repression of GK promoter. Further, treatment of hepatocytes with the SIRT1 activator resveratrol deacetylated and activated FoxO1. Resveratrol also down regulated GK promoter activity in transfected hepatocytes; it was unable to repress promoter activity when FBEb was mutated. Interestingly, the FoxO1 effect was also lost when the HNF-4 binding site was mutated. This suggested that FoxO1 interacts physically with HNF-4 to mediate its action. Indeed, coimmunoprecipitation assays revealed that FoxO1 physically interacts with HNF-4. Together, the present study showed that SREBP-1 could activate GK gene expression via the footprint B2 site, SRE2 and interaction with HNF-4. Although interaction with HNF-4 is also important for the FoxO1-dependent GK promoter regulation, two additional binding sites were identified. The FoxO1 activity was shown for the first time to be regulated by resveratrol and SIRT1, and the resveratrol-mediated down regulation of GK expression was due to either binding of FoxO1 to the binding elements or via interaction with HNF-4.