Mireille Vasseur-Cognet

Glucose Regulation of Gene Transcription

Nutrient gene regulation is an important adaptation allowingsurvival on intermittent food supplies. This adaptative processexists in all species from yeast to mammals. Glucose, the mostabundant monosaccharide in nature, provides a very good exampleof how organisms have developed regulatory mechanisms to copewith a fluctuating level of nutrient supply. In yeast, glucose facil-itates its own use by inducing expression of genes involved in itsmetabolism while repressing that of those involved in the utiliza-tion of alternative carbon sources (for review, see Ref. 1). Themechanisms by which glucose affects gene expression in yeast arenow relatively well understood.In mammals the response to dietary glucose is more complexbecause it combines effects related to glucose metabolism itself andeffects secondary to glucose-dependent hormonal modifications,mainly pancreatic stimulation of insulin secretion and inhibition ofglucagon secretion. In the pancreatic bcells, glucose is the primaryphysiological stimulus for the regulation of insulin synthesis andsecretion. In the liver, glucose, in the presence of insulin, inducesexpression of genes encoding glucose transporters and glycolyticand lipogenic enzymes,

Chicken Ovalbumin Upstream Promoter-Transcription Factor II, a New Partner of the Glucose Response Element of the L-type Pyruvate Kinase Gene, Acts as an Inhibitor of the Glucose Response

Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.

Expression of COUP-TFII in metabolic tissues during development

In mammals, the COUP-TF-family consisting of two structurally related proteins, COUP-TFI and COUP-TFII belongs to the orphan member of the steroid/thyroid hormone receptor superfamily. In an attempt to gain insights into the role of COUP-TFII, we examined developmental expression pattern of the mouse COUP-TFII focusing our studies on endoderm-derived tissues, pancreas and liver in particular. Independent lines of transgenic mice expressing Escherichia coli beta-galactosidase driven by the COUP-TFII promoter were generated. Embryonic expression of the beta-gal protein at day 9 of gestation was detected in the notochord, the ventral neural tube and, interestingly, in the gut endoderm, a site where COUP-TFII has not been detected previously. Between 9.5 and 11.5 dpc, beta-gal expression pattern that was established earlier persisted and sections revealed a staining of the common atrial chamber of the heart. At 15.5 dpc, beta-gal activity was found in all endoderm-derived tissues. We found that COUP-TFII mRNA and protein were present in fetal and adult hepatocytes. Finally, COUP-TFII expression was detected in pancreas, as judged by co-expression of the beta-gal in some of the glucagon and PDX1 positive-cells at 12.5 dpc and co-expression with insulin positive-cells at 15.5 dpc. In adult pancreas, COUP-TFII protein was present in the endocrine islet cells.

The Transcription Factor COUP-TFII Is Negatively Regulated by Insulin and Glucose via Foxo1- and ChREBP-Controlled Pathways

ABSTRACT COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.

The MODY1 gene for hepatocyte nuclear factor 4alpha and a feedback loop control COUP-TFII expression in pancreatic beta cells.

ABSTRACT Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4α (HNF4α) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4α activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4α with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4α transactivation of COUP-TFII. The dominant negative suppression of HNF4α function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4α mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4α by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4α mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4α/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.

COUP-TFII Controls Mouse Pancreatic β-Cell Mass through GLP-1-β-Catenin Signaling Pathways

Background The control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined. Methodology/Principal Findings Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat β-cells providing a feedback loop. Conclusions/Significance Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.

Negative cyclic AMP response elements in the promoter of the L‐type pyruvate kinase gene

L‐type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP‐dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site‐dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA‐dependent phosphorylation sites have been mutated still conferred cAMP‐sensitive transactivation of a L3‐dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4‐dependent transactivation but this effect remained sensitive to cAMP inhibition.

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.

Glucose-Dependent Regulation of NR2F2 Promoter and Influence of SNP-rs3743462 on Whole Body Insulin Sensitivity

Background The Nuclear Receptor 2F2 (NR2F2/COUP-TFII) heterozygous knockout mice display low basal insulinemia and enhanced insulin sensitivity. We previously established that insulin represses NR2F2 gene expression in pancreatic β-cells. The cis-regulatory region of the NR2F2 promoter is unknown and its influence on metabolism in humans is poorly understood. The present study aimed to identify the regulatory regions that control NR2F2 gene transcription and to evaluate the effect of NR2F2 promoter variation on glucose homeostasis in humans. Methodology/Principal Findings Regulation of the NR2F2 promoter was assessed using gene reporter assays, ChIP and gel shift experiments. The effects of variation at SNP rs3743462 in NR2F2 on quantitative metabolic traits were studied in two European prospective cohorts. We identified a minimal promoter region that down-regulates NR2F2 expression by attenuating HNF4α activation in response to high glucose concentrations. Subjects of the French DESIR population, who carried the rs3743462 T-to-C polymorphism, located in the distal glucose-responsive promoter, displayed lower basal insulin levels and lower HOMA-IR index. The C-allele at rs3743462 was associated with increased NR2F2 binding and decreased NR2F2 gene expression. Conclusions/Significance The rs3743462 polymorphism affects glucose-responsive NR2F2 promoter regulation and thereby may influence whole-body insulin sensitivity, suggesting a role of NR2F2 in the control of glucose homeostasis in humans.

The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway

Background The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. Methodology/Principal Findings Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. Conclusions/Significance These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.