ABSTRACT
Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4alpha (HNF4alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4alpha strongly activated the expression of a reporter gene in both directions. The HNF4alpha-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4alpha and GATA were essential for maximal synergism. We also show that HNF4alpha, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , GATA4 Transcription Factor/metabolism , GATA6 Transcription Factor/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Lipoproteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP Binding Cassette Transporter, Subfamily G, Member 8 , ATP-Binding Cassette Transporters/genetics , Adenoviridae/genetics , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Binding Sites , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line , Cell Line, Tumor , Consensus Sequence , Conserved Sequence , GATA4 Transcription Factor/genetics , GATA6 Transcription Factor/genetics , Gene Deletion , Genes, Reporter , Hepatocyte Nuclear Factor 4/chemistry , Humans , Lipoproteins/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Luciferases/metabolism , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , RNA Interference , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.
Subject(s)
DNA-Binding Proteins/metabolism , Down-Regulation , Glucose/pharmacology , High Mobility Group Proteins/metabolism , Homeodomain Proteins/antagonists & inhibitors , Hyperinsulinism/metabolism , Insulin/metabolism , Obesity/metabolism , Trans-Activators/antagonists & inhibitors , Transcription Factors/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , Cell Movement , Chromatin/metabolism , Diet , Dietary Fats/administration & dosage , Dietary Fats/pharmacology , Glucose/metabolism , Histones/metabolism , Homeodomain Proteins/metabolism , Hyperinsulinism/genetics , Insulin/genetics , Insulin Secretion , Islets of Langerhans/metabolism , Mice , Mice, Obese , Mitochondria/metabolism , Obesity/genetics , Protein Structure, Tertiary , RNA, Messenger/metabolism , Repressor Proteins/metabolism , SOXD Transcription Factors , Trans-Activators/metabolism , Transcription, GeneticABSTRACT
In this study, we defined the role of peroxisome proliferator-activated receptor beta/delta (PPARdelta) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPARdelta subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPARdelta controls fatty acid oxidation by regulating genes involved in fatty acid transport, beta-oxidation, and mitochondrial respiration. Similar PPARdelta-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid beta-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid beta-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob/ob mice. These data suggest that PPARdelta is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals.
Subject(s)
Fatty Acids, Nonesterified/metabolism , Insulin Resistance/physiology , Metabolic Syndrome/prevention & control , Muscle, Skeletal/physiology , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Animals , Dimethyl Sulfoxide/pharmacology , Enzymes/genetics , Lipid Metabolism , Liver/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Muscle, Skeletal/drug effects , Oxidation-Reduction , Rats , Receptors, Cytoplasmic and Nuclear/drug effects , Thiazoles/pharmacology , Transcription Factors/drug effectsABSTRACT
[reaction: see text] A newly developed strategy for eight-membered carbocycles via [3 + 4] annulation that involves the combination of beta-substituted acryloylsilanes and enolates of cycloheptenone is described. A unique feature of this annulative approach is its capacity to generate, in two steps, eight-membered ring systems containing useful functionalities for further synthetic elaboration from readily available three- and four-carbon components.
