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

Hypothalamic ventromedial COUP-TFII protects against hypoglycemia-associated autonomic failure

The nuclear receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is an important coordinator of glucose homeostasis through its function in different organs such as the endocrine pancreas, adipose tissue, skeletal muscle, and liver. Recently we have demonstrated that COUP-TFII expression in the hypothalamus is restricted to a subpopulation of neurons expressing the steroidogenic factor 1 transcription factor, known to play a crucial role in glucose homeostasis. To understand the functional significance of COUP-TFII expression in the steroidogenic factor 1 neurons, we generated hypothalamic ventromedial nucleus-specific COUP-TFII KO mice using the cyclization recombination/locus of X-overP1 technology. The heterozygous mutant mice display insulin hypersensitivity and a leaner phenotype associated with increased energy expenditure and similar food intake. These mutant mice also present a defective counterregulation to hypoglycemia with altered glucagon secretion. Moreover, the mutant mice are more likely to develop hypoglycemia-associated autonomic failure in response to recurrent hypoglycemic or glucopenic events. Therefore, COUP-TFII expression levels in the ventromedial nucleus are keys in the ability to resist the onset of hypoglycemia-associated autonomic failure

In vivo COUP-TFII expression in the hypothalamus during the fasting refeeding cycle.

<p>C57BL/6J mice are either fasted for 24 h (□) or fasted 24 h and refed on a regular diet (▪). (A) RT-qPCR analysis of POMC (upper left panel), AgRP mRNA levels (upper right panel), NPY mRNA (lower left panel), Neuro D mRNA levels (lower right panel) (B) RT-qPCR analysis of COUP-TFII mRNA levels. Data are expressed as mean ± SEM. (<i>n</i> = 5 mice/group) *P≤0.05; **P≤0.01 when compared to fasted conditions. (C) Autoradiogram of a western blot analysis of COUP-TFII protein expression in the hypothalamus. Upper panel: COUP-TFII protein, lower panel: β actin protein.</p

COUP-TFII protein expression in adult mouse ventromedial hypothalamus.

<p>(A) Identification of the hypothalamic structures by Kluver Barrera staining. Black square: Arcuate nucleus, red square: Ventromedial hypothalamic nucleus. (B–F) Immunostaining of brain sections from adult mice using mouse monoclonal antibodies directed against COUP-TFII (PP-H7147-00, dilution 1∶50). The secondary antibody is coupled with the horse radish peroxydase and revealed with diaminobenzidine leading to a brown precipitate. The sections shown are from the same experiment with the same exposure times. Magnification ×20. (C) Secondary antibody incubation control for COUP-TFII antibody specificity (D) Enlargement from the VMH region, magnification ×40. (E) Enlargement of the arcuate nucleus region (ARH), magnification ×40. (F) Enlargement from the LHA region, magnification ×40. These pictures are representative of 6 independent experiments. (G–J) Immunofluorescence staining of adult mice hypothalamic cryostat sections, magnification ×20. (G) DAPI staining of the ARH-VMH region (blue fluorescence) (H) Identification of COUP-TFII positive neurons in the VMH-ARH region of adult mice using the COUP-TFII monoclonal antibody (green fluorescence) (I) Identification of COUP-TFI positive neurons in the VMH-ARH region of the hypothalamus using a COUP-TFI monoclonal antibody (red fluorescence) (J) Identification of COUP-TFI positive neurons in the cortex region of the same cryostat section than (I) as a positive control for COUP-TFI antibody. Scale bars  = 100 µm.</p

Regulation of hypothalamic COUP-TFII mRNA levels in vivo by activation of the melanocortin pathway.

<p>(A-C) RT-qPCR analysis of mRNA levels from hypothalamus of <i>ad libitum</i> fed mice receiving a ICV injection of CSF (□), or 18 h fasting mice receiving a ICV injection of CSF (grey bar), or 18 h fasting mice with a ICV injection of MTII (▪). (A) BDNF mRNA levels (B) Neuro D mRNA levels (C) COUP-TFII mRNA levels. Data are expressed as mean ± SEM (n = 5) *P≤0.05 **P ≤0.01 when compared to control fed mice receiving a CSF injection. § P≤0.05 when compared to control fasting mice receiving a CSF injection. (D) Glucose and plasma insulin concentrations from the different mouse models.</p

COUP-TFII mRNA levels in the hypothalamus of STZ treated mice.

<p>(A) Glycemia (B) RT-qPCR analysis of mRNA levels. Upper left panel: POMC mRNA levels; upper middle panel: AgRP mRNA levels; upper right panel: NPY mRNA levels; lower left panel: Neuro-D mRNA levels; lower right panel: COUP-TFII mRNA levels. Citrate control mice (□), STZ treated mice (▪), STZ mice treated with insulin (grey square). Data are expressed as mean ± SEM. (<i>n</i> = 6 mice/group). *P≤0.05 **P≤0.01 compared to citrate treated mice. § P≤0.05 §§P≤0.01 compared to STZ treated mice.</p

COUP-TFII gene expression in response to the MC4-R activation in GT1-7 cell line.

<p>GT1-7 cells were cultured for 18 hours in the presence of either (A) insulin (100 nM) or increasing amount of (B) MTII or (C) DiBcAMP. Results are expressed as a fold increase over the value obtained without MTII (A) or without DiBcAMP (B). (D) Transient transfection of a minimal COUP-TFII promoter in response to DiBcAMP. Data are expressed as the mean ± SEM. (<i>n</i> = 4). *P≤0.05 **P≤0.01 when compared to cells cultured in the absence of MTII or DiBcAMP.</p

COUP-TFII protein co-localization studies with SF1 in the VMH.

<p>(A–G) Immunofluorescence staining of adult mice hypothalamic cryostat sections. (A–E) The sections shown are from the same experiment with the same exposure time (A) Identification of SF1 positive cells in the VMH by immunofluorescence using a SF1 polyclonal antibody (red fluorescence) (B) Identification of the COUP-TFII positive neurons in the VMH of adult mice using a COUP-TFII monoclonal antibody (green fluorescence) (C) Secondary antibody alone control for SF1 antibody specificity (D) Secondary antibody alone for COUP-TFII antibody specificity (E) Superimposed SF1 and COUP-TFII immunostaining. Magnification ×40. (F–G) The sections shown are from the same experiment and the same exposure time. (F) COUP-TFII immunostaining (red fluorescence) (G) Superimposed MC4-R and COUP-TFII immunofluorescences. Magnification ×100. These pictures are representative of 4 independent experiments. Scale bars  = 10 µm.</p