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

EXPRESSION ET FONCTION DES IDS, FACTEURS DE TRANSCRIPTION DE LA FAMILLE HELICE-BOUCLE-HELICE (LEUR ROLE DANS L'ADIPOGENESE DES PREADIPOCYTES 3T3 (DOCTORAT : ENDOCRINOLOGIE ET INTERACTIONS CELLULAIRES))

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Molecular Cloning of a Major mRNA Species in Murine 3T3 Adipocyte Lineage

International audienceIn an effort to identify novel mRNAs modulated during the course of adipose conversion, we have used a simplified differential display technique and have isolated a cDNA encoding an amine oxidase tremendously expressed in the adipocyte, the semicarbazide-sensitive amine oxidase (SSAO). The predicted amino acid sequence (765 amino acids) is likely to be the homologue of the human placental amine oxidase and of the partially known sequence of the rat adipocyte membrane amine oxidase. SSAO mRNAs are present in several tissues, but strikingly, the highest levels of gene expression are found in adipose tissue and aorta. Enzyme transcript levels are barely detectable in preadipocytes but are induced several hundred-fold during the adipocyte differentiation of 3T3-L1 or 3T3-F442A cells and of rat precursor primary cultures. These changes in transcript levels parallel a sharp increase in SSAO enzyme activity. The biochemical properties of the SSAO present in 3T3-L1 or 3T3-F442A adipocytes closely resemble the features of the SSAO activity previously described in white and brown adipose tissues. Interestingly, SSAO mRNA levels and enzyme activity drop in response to effectors of the cAMP pathway and to the cytokine tumor necrosis factor-alpha, indicating that two major signaling molecules of adipose tissue development and metabolism can control SSAO function. Moreover, the expression of SSAO transcripts and activity are clearly down-regulated in white adipose tissue from obese Zücker rats. Because of its known stimulatory effect on glucose transport, its biochemical properties and its pattern of expression and regulation, SSAO could play an important role in the regulation of adipocyte homeostasis

Semicarbazide-Sensitive Amine Oxidase in Vascular Smooth Muscle Cells

International audienceCultured vascular smooth muscle cells (VSMCs) derived from rat aortic media were used to examine semicarbazide-sensitive amine oxidase (SSAO) expression during their differentiation process. In a defined serum-free medium permissive for in vitro VSMC differentiation, there was a large increase in SSAO mRNA and protein levels and in the related enzyme activity during the course of cell culture. This pattern of expression was concomitant with that of some smooth muscle–specific mRNA markers of differentiation. mRNAs in differentiated cultured VSMCs were comparable to those detected in total aorta and media. Pharmacological properties of SSAO present in VSMCs were similar to enzyme activities previously described in the aortic wall. In this model, we also demonstrated that methylamine, a physiological substrate of SSAO, activated 2-deoxyglucose transport in a time- and dose-dependent manner. This methylamine effect was reproduced by other SSAO substrates and was prevented by the SSAO inhibitor semicarbazide. It was antagonized in the presence of catalase, suggesting that SSAO-activated glucose transport was mediated through H 2 O 2 production. In addition, methylamine promoted glucose transporter 1 accumulation at the cell surface. Thus, we demonstrate for the first time the differentiation-dependent expression of SSAO in VSMCs and its role in the regulation of VSMC glucose uptake

WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity OPEN

International audienceWISP1 (Wnt1-inducible signaling pathway protein-1, also known as CCN4) is a member of the CCN family able to mediate cell growth, transformation and survival in a tissue-specific manner. Here, we report that WISP1 expression was highly increased in preadipocytes and decreased during adipocyte differentiation. Moreover, we observed an increase in WISP1 gene expression in adipose tissue from both diet-induced and leptin-deficient ob/ob obese mice, suggesting that WISP1 could be involved in the pathophysiological onset of obesity. Interestingly, overexpression of WISP1 in 3T3-F442A cells prevented adipocyte differentiation via downregulation of peroxisome proliferator-activated receptor (PPARγ) transcriptional activity thereby attenuating the expression of adipogenic markers. Conversely, silencing of WISP1 enhanced adipocyte differentiation. We further show that the inactivation of PPARγ transcriptional activity was mediated, at least in part, by a direct physical association between WISP1 and PPARγ, followed by proteasome-dependent degradation of PPARγ. These results suggest for the first time that WISP1 interacts with PPARγ and that this interaction results in the inhibition of PPARγ activity. Taken together our results suggest that WISP1 functions as a negative regulator of adipogenesis. WISP1 (Wnt1-inducible signaling pathway protein-1, also known as CCN4) is a member of the connective tissue growth factor/cystein-rich 61/nephroblastoma overexpressed (CCN) family 1. The CCN family is composed of six members, Cyr61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1/CCN4, WISP2/CCN5 and WISP3/CCN6, referred to as CCN1-6, based on the unified nomenclature 2. Three of these proteins, WISP1-3, were initially identified in C57MG cells, a mouse mammary epithelial cell line with Wnt-1 expression, and subsequently shown to be Wnt-1-induced genes 3. WISP1/CCN4 (hereafter referred to as WISP1) is both an intracellular and a secreted protein found in the extracellular matrix (ECM) and, like many other matricellular proteins able to modulate cellular responses such as cell growth, differentiation and survival 4. WISP1 is expressed in various normal tissues including heart, kidney, lung, pancreas, placenta, ovary, small intestine and spleen. Interestingly, elevated WISP1 expression has also been observed in a variety of cancers including hepatocellular carcinoma 5 , colon adenocarci-noma 3 , lung carcinoma 6 , breast cancer 7 and cholangiocarcinoma 8. Wnt signaling pathway plays a key role for maintaining the cells in an undifferentiated state 9. The Wnt proteins are secreted signaling factors that affect cell fate and differentiation, including adipogenesis, myogenesis and mammary development. When Wnt signaling is active, GSK-3β is inhibited, allowing β-catenin to accumulate in the nucleus where it binds to TCF-LEF transcription factors and activates Wnt-target genes. The Wnt-1 protein has been described as an inhibitor of adipocyte differentiation 9. Interestingly, ectopic expression of Wnt-1 in 3T3-L1 murine preadipocytes induced the expression of several downstream genes including WISP1 10 , thereby suggesting a potential role for WISP1 in adipocyte differentiation. Adipogenesis is characterized by dynamic changes in gene expression 11–13. It is well accepted that both peroxi-some proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPs) function as critical regulators of adipogenesis 14, 15. PPARs are members of the Nuclear Receptor family, comprising a subgroup of three homologous genes. Among them, PPARγ acts primarily as a master regulator of adipocyte differentiation and metabolism 15, 16. In particular, PPARγ is sufficient and necessary for fat cell differentiation. Followin

Tumor Necrosis Factor-α-induced Adipose-related Protein (TIARP), a Cell-surface Protein That Is Highly Induced by Tumor Necrosis Factor-α and Adipose Conversion

International audienceTumor necrosis factor-alpha (TNF alpha) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNFalpha on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF alpha-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF alpha exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF alpha effects on the fat cell as a channel or a transporter