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F Goglia, A Lanni, C Horst, M Moreno, and R Thoma


In this study we have demonstrated that specific binding sites for 3,5-di-iodo-l-thyronine (3,5-T2) can be detected in rat liver mitochondria. After incubation with the homogenate, liver mitochondria bound only a small portion of [3,5-125I]T2. The addition of a 100-fold excess of unlabelled 3,5-T2 caused the displacement of on average 50-60% of the [3,5-125I]T2 bound. Specific binding of 3,5-T2 to rat liver mitochondria occurred rapidly; a maximum was achieved after 5 min. Maximal binding was obtained at 37 °C, while at 0 °C and 20 °C the values were only slightly lower. Binding was maximal at pH 70; mean (±s.e.m.) values for the apparent association constant and the binding capacity (calculated at pH 7·0, 0 °C and after 30min of incubation) were 0·5±0·04×108 m -1 and 0·4±0·04 pmol/mg mitochondrial protein respectively. The specificity of binding, examined in competition studies, followed the order: 3,5-T2>3,3′-di-iodo-l-thyronine>3′,3,5-tri-iodo-l-thyronine>thyroxine. Other iodothyronines (3′,5′-di-iodo-l-thyronine, 3,5-di-iodo-d-thyronine, 3,3′, 5′-tri-iodo-l-thyronine, 3-iodo-l-thyronine and 3,5-di-iodothyroacetic acid) showed little or no competition. This suggests that the specific 3,5-T2 binding sites could be of biological relevance with respect to the understanding of the mechanism of physiological action of thyroid hormones at the cellular level.

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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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M V Legorreta-Haquet, K Chávez-Rueda, E Montoya-Díaz, L Arriaga-Pizano, R Silva-García, L Chávez-Sánchez, M Moreno-Lafont, E Zenteno-Galindo, and F Blanco-Favela

Among its many functions, prolactin (PRL) participates in immune responses and promotes the activation, differentiation and proliferation of T cells. However, the mechanisms by which PRL regulates regulatory T (Treg) cells are still unknown. Our goal was to determine whether PRL plays a role in Treg function. We measured the expression of PRL and its receptor in Treg and effector T (Teff) cells from 15 healthy individuals. We also evaluated the functional activity of Treg cells by examining proliferation and cytokine secretion in cells activated with anti-CD3/CD28 in the presence or absence of PRL. We report that Treg cells constitutively expressed PRL receptor, whereas Teff cells required stimulation with anti-CD3/CD28 to induce PRL receptor expression. Expression of PRL was constitutive in both populations. We found that the addition of PRL inhibited the suppressor effect (proliferation) mediated by Treg cells in vitro, reducing suppression from 37.4 to 13% when PRL was added to co-cultures of Treg and Teff cells (P<0.05). Cultures treated with PRL favoured a Th1 cytokine profile, with increased production of TNF and IFNγ. We report for the first time that PRL receptor expression was constitutive in Treg cells but not in Teff cells, which require stimulation to induce PRL receptor expression. PRL inhibited the suppressive function of Treg cells, apparently through the induced secretion of Th1 cytokines.