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E. Pailhoux, A. Martinez, Ch. Jean-Faucher, G. Veyssière, and Cl. Jean


We have previously characterized an androgen-inducible secretory protein from the mouse vas deferens (MVDP), and a cDNA to its mRNA has been obtained. This report describes altered MVDP gene expression after neonatal exposure to oestrogens. As shown by immunohistochemistry and Western blot analysis, MVDP was missing in the vas deferens from adult mice neonatally exposed to oestrogens. Northern blot analysis showed that the expression of MVDP mRNA was also suppressed. Exogenous testosterone was unable to stimulate MVDP production (either message or protein) in neonatally oestrogenized males. The results suggest that the alterations in gene expression in the oestrogen-exposed vas deferens reflect changes in the programme of differentiation of the organ itself.

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GD Jahnke, MJ Miller, A Martinez, L Montuenga, and F Cuttitta

Adrenomedullin (AM) is a recently identified amidated peptide produced by a variety of tissue types. We have investigated the involvement of AM and its receptor (AM-R) in developing mouse mammary glands and have examined what influence ovarian hormones have on AM and AM-R expression in this system. Tissues from ductal morphogenesis, virgin adult, pregnancy, and lactation stages were assessed for AM and AM-R by molecular, biochemical and immunohistochemical techniques. Results from these studies indicated that messenger RNA for AM and AM-R and immunoreactivity for AM were expressed in the luminal epithelium of small and large ducts and in terminal end buds. Immunoreactive AM was identified as a cytoplasm component of ductal cells, with some cells also having nuclear staining. Western blot analysis of mammary gland tissues yielded two molecular mass species (M(r) 14,000 and 18,500) of AM immunoreactivity in the mammary gland for the above developmental stages, consistent with processed intermediate and prohormone forms respectively. Ovariectomy alone or followed by hormonal treatments did not alter the expression pattern for these two proteins. By Western blot, the fully processed AM form (M(r) 6000) was identified in milk extracts from lactating glands. These data suggest a potential role for AM and its receptor in the maintenance of mammary gland homeostasis and suggests a potential role for AM in development of the newborn.

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A Martinez, DL Hodge, M Garayoa, HA Young, and F Cuttitta

The adrenomedullin (AM) gene codifies for two bioactive peptides, AM and proAM N-terminal 20 peptide (PAMP). We have found two forms of the AM mRNA. Form A is devoid of introns and results in a prohormone containing both peptides. Form B retains the third intron, which introduces a premature stop codon, producing a shorter prohormone with only PAMP. Tissues with a higher B/A ratio were more immunoreactive for PAMP than for AM. The form B message was found in the cytoplasmic compartment, thus excluding that the longer message was a result of contaminating nuclear mRNA. Form B was found in cells that express PAMP but not AM. mRNA expression in a variety of cell lines was investigated by ribonuclease protection assay and form B was found in significant amounts in two of them. Treatments that modify AM expression, such as exposure to hypoxia, were shown to change the B/A ratio and the relative secretion of AM and PAMP, indicating that the splicing mechanism for AM can be modulated and is physiologically relevant. Analysis of the sequence of the third intron and the fourth exon of the AM gene found motifs compatible with a highly regulated alternative splicing mechanism.

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N Martínez-Micaelo, N González-Abuín, A Ardévol, M Pinent, E Petretto, J Behmoaras, and M Blay

Although the effect of genetic background on obesity-related phenotypes is well established, the main objective of this study is to determine the phenotypic responses to cafeteria diet (CAF) of two genetically distinct inbred rat strains and give insight into the molecular mechanisms that might be underlying. Lewis (LEW) and Wistar–Kyoto (WKY) rats were fed with either a standard or a CAF diet. The effects of the diet and the strain in the body weight gain, food intake, respiratory quotient, biochemical parameters in plasma as well as in the expression of genes that regulate leptin signalling were determined. Whereas CAF diet promoted weight gain in LEW and WKY rats, as consequence of increased energy intake, metabolic management of this energy surplus was significantly affected by genetic background. LEW and WKY showed a different metabolic profile, LEW rats showed hyperglycaemia, hypertriglyceridemia and high FFA levels, ketogenesis, high adiposity index and inflammation, but WKY did not. Leptin signalling, and specifically the LepRb-mediated regulation of STAT3 activation and Socs3 gene expression in the hypothalamus were inversely modulated by the CAF diet in LEW (upregulated) and WKY rats (downregulated). In the present study, we show evidence of gene-environment interactions in obesity exerted by differential phenotypic responses to CAF diet between LEW and WKY rats. Specifically, we found the leptin-signalling pathway as a divergent point between the strain-specific adaptations to diet.

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R Serrano, M Villar, C Martínez, J M Carrascosa, N Gallardo, and A Andrés

The insulin receptor (IR) occurs as two alternatively spliced isoforms, IR-A (exon 11−) and IR-B (exon 11+), which exhibit functional differences and are expressed in a tissue-specific manner. The IR substrate (IRS) proteins 1, 2 and 3 also differ in function and tissue distribution. Here we show the differential gene expression of IRs and IRSs in several rat target tissues of insulin action. IR-B is significantly higher than IR-A in epididymal white adipose tissue and adipogenesis induces a shift in the alternatively spliced species of IR from the A to the B isoform. Moreover, since aging in the rat is associated with the development of insulin resistance we looked for alterations of expression of these proteins in adipocytes from old rats. Our results reveal that there is a specific decrease in the expression of the IR-B isoform, as well as both mRNA and protein levels of IR, IRS-1 and IRS-3 being significantly decreased, in epididymal adipose tissue from old compared with adult rats. It is concluded that the down-regulation of early components of the insulin transduction pathway in a primary insulin target tissue could be related to the insulin resistance of aging.

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R González-Fernández, F Gaytán, E Martínez-Galisteo, P Porras, C A Padilla, J E Sánchez Criado, and J A Bárcena

Glutaredoxins (Grxs) are low-molecular-weight proteins which participate in redox events in association with glutathione (GSH) and are involved in a variety of cellular processes. It is known that oxidative stress plays important physiological roles within the ovary. In the present study, we have prepared specific antibodies against rat Grx and have used them to localize the protein in the ovaries of rats during postnatal development and during the oestrous cycle, by immunohistochemical methods. We have also performed a quantitative analysis of Grx by ELISA and Western blotting in homogenates of whole ovaries of cycling and pseudopregnant rats. We have found a prominent presence of Grx in the oocytes and in corpora lutea (CL) during developmental and oestrous cycle changes. Grx was absent from the oocytes in the first days of postnatal life when marked oocyte degeneration takes place, but its presence was very conspicuous in the cytoplasm of oocytes in healthy and attretic follicles in rats from 10 days of age onward, independently of the day of oestrous cycle. Follicular cells were negative. Grx immunostaining in the CL was strong in infiltrating macrophages and in a population of steroidogenic cells that survived the apoptotic burst in regressing CL and in CL remnants, but was faint or absent in young CL of the current cycle and in CL during pseudopregnancy. Grx content and oxidoreductase activity in whole ovaries increased significantly during the phase transition from proestrous to oestrous along the cycle. These results support a role of Grx in the maintenance of functional oocytes and in luteal cells surviving the regression process, probably as a consequence of the demonstrated deglutathionylating function of this protein in an antioxidant and antiapoptotic context.

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A Cote-Vélez, L Pérez-Martínez, M Y Díaz-Gallardo, C Pérez-Monter, A Carreón-Rodríguez, J-L Charli, and P Joseph-Bravo

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

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M J Moreno-Aliaga, M M Swarbrick, S Lorente-Cebrián, K L Stanhope, P J Havel, and J A Martínez

We have previously demonstrated that insulin-stimulated glucose metabolism, and not insulin per se, mediates the effects of insulin to increase the transcriptional activity of the leptin promoter in adipocytes. Here, we sought to identify the specific cis-acting DNA elements required for the upregulation of leptin gene transcription in response to insulin-mediated glucose metabolism. To accomplish this, 3T3-L1 cells and primary rat adipocytes were transfected with a series of luciferase reporter genes containing portions of the mouse leptin promoter. Using this method, we identified an element between −135 and −95 bp (relative to the transcriptional start site) that mediated transcription in response to insulin-stimulated glucose metabolism in adipocytes. This effect was abolished by incubation with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism. Gel shift electrophoretic mobility shift assays confirmed that the stimulatory effect of insulin-mediated glucose metabolism on leptin transcription was mediated by a previously identified Sp1 site. Consistent with these findings, incubation of primary rat adipocytes with WP631, a specific inhibitor of specificity protein (Sp)1-dependent transcription, inhibited glucose- and insulin-stimulated, but not basal, leptin secretion. Together, these findings support a key role for Sp1 in the transcriptional activation of the leptin gene promoter by insulin-mediated glucose metabolism.

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D F Garcia-Diaz, J Campion, F I Milagro, N Boque, M J Moreno-Aliaga, and J A Martinez

Antioxidant-based treatments are emerging as an interesting approach to possibly counteract obesity fat accumulation complications, since this is accompanied by an increased systemic oxidative stress. The aim of this study was to analyze specific metabolic effects of vitamin C (VC) on epididymal primary rat adipocytes. Cells were isolated and incubated for 72 h in culture medium, in the absence or presence of 1.6 nM insulin, within a range of VC concentrations (5–1000 μM). Glucose- and lipid-related variables as well as the secretion/expression patterns of several obesity-related genes were assessed. It was observed that VC dose dependently inhibited glucose uptake and lactate production, and also reduced glycerol release in both control and insulin-treated cells. Also, VC caused a dramatic concentration-dependent fall in leptin secretion especially in insulin-stimulated cells. In addition, VC (200 μM) induced Cdkn1a and Casp8, partially inhibited Irs3, and together with insulin drastically reduced Gpdh (listed as Gpd1 in the MGI database) gene expressions. Finally, VC and insulin down-regulatory effects were observed on extracellular and intracellular reactive oxygen species production respectively. In summary, this experimental assay describes a specific effect of VC in isolated rat adipocytes on glucose and fat metabolism, and on the secretion/expression of important obesity-related proteins.

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J G Miquet, J F Giani, C S Martinez, M C Muñoz, L González, A I Sotelo, R K Boparai, M M Masternak, A Bartke, F P Dominici, and D Turyn

Acromegaly is associated with cardiac hypertrophy, which is believed to be a direct consequence of chronically elevated GH and IGF1. Given that insulin is important for cardiac growth and function, and considering that GH excess induces hyperinsulinemia, insulin resistance, and cardiac alterations, it is of interest to study insulin sensitivity in this tissue under chronic conditions of elevated GH. Transgenic mice overexpressing GH present cardiomegaly and perivascular and interstitial fibrosis in the heart. Mice received an insulin injection, the heart was removed after 2 min, and immunoblotting assays of tissue extracts were performed to evaluate the activation and abundance of insulin-signaling mediators. Insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was conserved in transgenic mice, but the phosphorylation of IR substrate 1 (IRS1), its association with the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and the phosphorylation of AKT were decreased. In addition, total content of the glucose transporter GLUT4 was reduced in transgenic mice. Insulin failed to induce the phosphorylation of the mammalian target of rapamycin (mTOR). However, transgenic mice displayed increased basal activation of the IR/IRS1/PI3K/AKT/mTOR and p38 signaling pathways along with higher serine phosphorylation of IRS1, which is recognized as an inhibitory modification. We conclude that GH-overexpressing mice exhibit basal activation of insulin signaling but decreased sensitivity to acute insulin stimulation at several signaling steps downstream of the IR in the heart. These alterations may be associated with the cardiac pathology observed in these animals.