Robert K. Hall

Epigallocatechin Gallate, a Constituent of Green Tea, Represses Hepatic Glucose Production

Herbs have been used for medicinal purposes, including the treatment of diabetes, for centuries. Plants containing flavonoids are used to treat diabetes in Indian medicine and the green tea flavonoid, epigallocatechin gallate (EGCG), is reported to have glucose-lowering effects in animals. We show here that the regulation of hepatic glucose production is decreased by EGCG. Furthermore, like insulin, EGCG increases tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), and it reduces phosphoenolpyruvate carboxykinase gene expression in a phosphoinositide 3-kinase-dependent manner. EGCG also mimics insulin by increasing phosphoinositide 3-kinase, mitogen-activated protein kinase, and p70s6kactivity. EGCG differs from insulin, however, in that it affects several insulin-activated kinases with slower kinetics. Furthermore, EGCG regulates genes that encode gluconeogenic enzymes and protein-tyrosine phosphorylation by modulating the redox state of the cell. These results demonstrate that changes in the redox state may have beneficial effects for the treatment of diabetes and suggest a potential role for EGCG, or derivatives, as an antidiabetic agent.

Regulation of Phosphoenolpyruvate Carboxykinase and Insulin-like Growth Factor-binding Protein-1 Gene Expression by Insulin THE ROLE OF WINGED HELIX/FORKHEAD PROTEINS

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins,per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein.

Estrogen-related Receptor α Is a Repressor of Phosphoenolpyruvate Carboxykinase Gene Transcription

The orphan nuclear receptor estrogen-related receptor (ERR) α is a downstream effector of the transcriptional coactivator PGC-1α in the regulation of genes important for mitochondrial oxidative capacity. PGC-1α is also a potent activator of the transcriptional program required for hepatic gluconeogenesis, and in particular of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). We report here that the regulatory sequences of the PEPCK gene harbor a functional ERRα binding site. However, in contrast to the co-stimulating effects of ERRα and PGC-1α on mitochondrial gene expression, ERRα acts as a transcriptional repressor of the PEPCK gene. Suppression of ERRα expression by small interfering RNA leads to reduced binding of ERRα to the endogenous PEPCK gene, and an increase in promoter occupancy by PGC-1α, suggesting that part of the ERRα function at this gene is to antagonize the action of PGC-1α. In agreement with the in vitro studies, animals that lack ERRα show increased expression of gluconeogenic genes, including PEPCK and glycerol kinase, but decreased expression of mitochondrial genes, such as ATP synthase subunit β and cytochrome c-1. Our findings suggest that ERRα has opposing effects on genes important for mitochondrial oxidative capacity and gluconeogenesis. The different functions of ERRα in the regulation of these pathways suggest that enhancing ERRα activity could have beneficial effects on glucose metabolism in diabetic subjects by two distinct mechanisms: increasing mitochondrial oxidative capacity in peripheral tissues and liver, and suppressing hepatic glucose production.

Insulin Regulates Expression of Metabolic Genes through Divergent Signaling Pathways

The regulation of metabolic gene expression is a major mechanism by which insulin modulates glucose homeostasis. Defective transcription factors or signal transduction molecules that are required for insulin regulated gene expression could contribute to insulin resistance. The phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase II (HKII) genes are involved in metabolic processes that represent opposing facets of glucose homeostasis, namely gluconeogenesis and glucose utilization. The regulation of the PEPCK and HKII genes by insulin has been studied in great detail at the level of both transcription and signal transduction. Recent work on the insulin signaling pathways that lead to down-regulation of PEPCK gene expression and upregulation of HKII gene expression has shown that they both require activation of phosphatidylinositol 3-kinase (PI3K) for the transmission of the insulin signal. However, the pathways diverge after PI3K and lead to activation of different downstream effectors. In this paper we review the results of studies on the transcriptional regulation of these genes by insulin and the signal transduction pathways that mediate these responses.

Regulation of the Xenopus laevis transcription factor IIIA gene during oogenesis and early embryogenesis: Negative elements repress the O-TFIIIA promoter in embryonic cells

Expression of the Xenopus laevis transcription factor IIIA (TFIIIA) gene is developmentally regulated. In this study we have used defined nucleotide mutations to map cis-elements involved in transcriptional regulation of the promoter for oocyte-TFIIIA (O-TFIIIA) in stage II-IV oocytes, stage VI oocytes, and tail bud embryos. During oogenesis O-TFIIIA mRNA levels decline 5- to 10-fold, and during early embryogenesis O-TFIIIA mRNA levels decline approximately 10(6)-fold per cell. In stage II-IV oocytes we find evidence for at least three distinct positive-acting cis-elements that contribute to the efficient expression of O-TFIIIA. These elements are located between -1800 to -425, -280 to -235, and -235 to -220. The most distal cis-element(s) appears to be developmentally regulated during oogenesis, since deletion of nucleotide sequences from -1800 to -425 does not reduce O-TFIIIA expression in stage VI oocytes. However, the two cis-elements located between -280 to -235 and -235 to -220 are required for the efficient expression of O-TFIIIA in stage VI oocytes. In tail bud embryos we find evidence for several developmentally regulated positive and negative cis-elements involved in O-TFIIIA expression. The positive-acting cis-elements are located between -159 to -110 and -110 to -58. The negative-acting cis-elements are found at positions -425 to -350 and -200 to -159. In addition to the developmentally regulated elements controlling O-TFIIIA gene expression in tail bud embryos, the positive-acting cis-elements active during oogenesis (located between -280 to -235 and -235 to -220) are also active during early embryogenesis. Thus, transcription from the O-TFIIIA promoter appears to be regulated by a combination of constitutive positive factors and developmentally regulated positive and negative factors during oogenesis and early embryogenesis.

The Xenopus B2 factor involved in TFIIIA gene regulation is closely related to Sp1 and interacts in a complex with USF.

In the Xenopus laevis oocyte there is a million fold more transcription factor IIIA (TFIIIA) and its corresponding mRNA than in a somatic cell. These high levels of TFIIIA gene expression are achieved primarily by transcriptional regulation. The TATA box along with three positive cis-elements in the control region of the TFIIIA gene located at positions -269 to -264 (E1), -235 to -220 (E2), and -669 to -636 (E3) are required for this high level of expression in oocytes. The proteins that bind E1 and E3 of the TFIIIA gene have been identified as Xenopus USF (Xl-USF) and B3 (homolog of Vg1 RBP/VERA). In this study the B2 protein was found to bind E2 in a zinc-dependent fashion and anti-human Sp1 (but not Sp2, Sp3, nor Sp4) supershifted the B2:element 2 complex. The E2 binding protein was purified by DNA affinity chromatography. Based on supershift analysis, molecular weight estimation experiments, and purified human Sp1 DNA binding affinity tests the data strongly support the idea that the B2 protein is the Xenopus ortholog of Sp1, but not Sp2, Sp3, nor Sp4. Xl-USF binds to element 1 of the TFIIIA gene which is immediately adjacent to element 2. Coimmunoprecipitation experiments using crude whole oocyte extracts revealed that Xenopus Sp1 and USF or closely related factors are present together in a high-affinity complex. This structure contributes positively to the initiation of TFIIIA gene transcription in Xenopus oocytes.