Teresa Teruel

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3‐kinase‐dependent but p70S6‐kinase‐independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin‐induced glucose uptake by the use of a chemical compound, ML‐9, as well as by transfection with a dominant‐negative form of Akt (ΔAkt). Pretreatment with ML‐9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML‐9 did not affect insulin‐stimulated PI3‐kinase nor PKCζ activities. In consequence, ML‐9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, ΔAkt impaired insulin stimulation of glucose uptake and GFP‐tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML‐9 treatment for 6 h down‐regulated insulin‐induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co‐transfection of brown adipocytes with ΔAkt precluded the transactivation of GLUT4‐CAT promoter by insulin in a similar fashion as a dominant‐negative form of PI3‐kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.

Tumor Necrosis Factor-α Causes Insulin Receptor Substrate-2-Mediated Insulin Resistance and Inhibits Insulin-Induced Adipogenesis in Fetal Brown Adipocytes1

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.

Inhibition of PI 3-kinase and RAS blocks IGF-I and insulin-induced uncoupling protein 1 gene expression in brown adipocytes.

Fetal brown adipocytes expressed uncoupling protein 1 (UCP1) mRNA, this expression being blunted throughout culture for 24 h in a serum‐free medium. At physiological doses, either insulin‐like growth factor I (IGF‐I) or insulin turned out to be as potent as dibutyryl cAMP (dbcAMP) in increasing UCP1 gene transcription rate (1 h) and also UCP1 mRNA accumulation (3 h), their maximal effect (15‐fold increase) reached upon treatment for 24 h. Upon treatment with either IGF‐I or insulin for 48 h, a 7‐fold increase in the UCP1 protein content relative to levels in the control cells was found, this induction being abolished in the presence of cycloheximide. Moreover, either IGF‐I or insulin transactivates the UCP1‐chloramphenicol acetyl transferase (CAT) fusion gene after transient transfection of primary brown adipocytes, these effects being tissue‐specific. Transient transfection of dominant‐negative form of phosphatidylinositol (PI) 3‐kinase completely blocked the transactivation of the fusion gene UCP1‐CAT induced by either IGF‐I or insulin, although inhibition of p70S6kinase with rapamycin does not preclude transactivation of the UCP1 promoter by insulin. Furthermore, transient transfection of dominant‐negative form of p21‐ras or treatment of cells with a mitogen‐activated protein kinase kinase (MEK‐1) inhibitor (PD098059) completely abolished insulin‐induced UCP1‐CAT transactivation. Cotransfectionwith dominant‐negative p85 or with dominant‐negative Ras also produced down‐regulation of the insulin or IGF‐I‐induced 12‐O‐tetradecanoylphorbol‐13‐acetate response element (TRE)‐CAT (five AP‐1, activating protein‐1, binding sites arranged in tandem) transactivation. In addition, insulin induced AP‐1 DNA binding activity, this effect being totally prevented in the presence of MEK‐1 inhibitor. These results strongly suggest that either IGF‐I or insulin induced thermogenic‐differentiation through AP‐1 activity in a PI 3‐kinase and Ras/MAPK dependent manner in brown adipocytes. J. Cell. Physiol. 176:99–109, 1998.

Ceramide mediates TNF-α-induced insulin resistance on GLUT4 gene expression in brown adipocytes

Abstract Tumour necrosis factor (TNF)-α impaired insulin induction on GLUT4 mRNA in foetal brown adipocytes, as demonstrated by quantitative RT-PCR and Northern blot. We have explored the hypothesis that some effects of TNF-α could be mediated by the generation of ceramide, since TNF-α treatment induced the production of ceramide in these primary cells. A short-chain ceramide analogue, C2-ceramide, precluded insulin-induced GLUT4 mRNA accumulation and GLUT4-chloramphenicol acetyltransferase (CAT) full promoter activation. Moreover, inhibition of the ceramide biosynthesis with fumonisin B, which inhibits ceramide synthase, completely restored insulin-induced GLUT4 mRNA and protein accumulation as well as GLUT4-CAT transactivation in the presence of TNF-α. In consequence, TNF-α-induced insulin resistance on glucose uptake was completely alleviated. In addition, TNF-α down-regulated insulin-induced CCAAT/enhancer binding protein (C/EBP)-alpha gene expression and DNA binding activity, but fumonisin B precludes these effects. Furthermore, co-transfection with a wild-type C/EBP-α construct transactivates GLUT4-CAT construct. Our results indicate that de novo ceramide produced by TNF-α-induced insulin resistance on GLUT4 gene expression in brown adipocytes by interfering C/EBP-α expression, a transcription factor essential for the expression of GLUT4.

Insulin and dexamethasone induce GLUT4 gene expression in foetal brown adipocytes: synergistic effect through CCAAT/enhancer-binding protein alpha

Treatment of foetal brown adipocytes in primary culture with either dexamethasone or insulin, at physiological concentrations, for 24 h up-regulates the expression of the GLUT4 gene, producing a synergistic effect on mRNA accumulation (20-fold increase), in the amount of protein in the total membrane fraction (8-fold increase) and in the transactivation of a full-promoter GLUT4 -chloramphenicol acetyltransferase gene ( CAT ) construct (7-fold increase). However, GLUT1 expression remains essentially unmodified regardless of the presence of the hormones. As a consequence, exposure of brown adipocytes to dexamethasone and insulin results in a dramatic increase of glucose uptake (12-fold). Dexamethasone induces the expression of CCAAT/enhancer-binding protein (C/EBP) alpha, insulin promotes myocyte enhancer factor-2 DNA-binding activity and both combined produces a significant increase in C/EBPalpha DNA-binding activity. Moreover, co-transfection with a wild-type C/EBPalpha construct transactivates a full-promoter GLUT4 - CAT fusion gene, whereas a dominant-negative C/EBPalpha expression vector impairs the hormonal effects. Our results show that the synergism between insulin and glucocorticoids on glucose uptake is a consequence of the activation of the GLUT4 promoter by the transcription factor C/EBPalpha.

Transforming growth factorβ1 induces differentiation-specific gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes show a low number of transforming growth factorβ 1 (TGF‐β 1) binding sites of high affinity, revealing the presence of type I, II and IIITGF‐β 1 receptors and a minor‐labeled species of approximately 140 kDa. The culture of cells in the presence ofTGF‐β 1 induced the expression of the tissue‐specific gene uncoupling protein in a dose‐ and time‐dependent manner. In addition,TGF‐β 1 up‐regulates the expression of genes involved in adipogenesis such as fatty acid synthase, glycerol 3‐phosphate dehydrogenase, malic enzyme and glucose 6‐phosphate dehydrogenase, as well as induces the expression of fibronectin (specific target gene forTGF‐β 1). Our results suggest thatTGF‐β 1 is a major signal involved in initiating and/or maintaining the thermogenic and adipogenic differentiation of rat fetal brown adipocytes.

Transforming growth factor β1 induces mitogenesis in fetal rat brown adipocytes

The presence of transforming growth factor β1 (TGF‐β1) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G2/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF‐β1 (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF‐β1 for 48 h increased by 5‐fold the percentage of cells containing (3H)thymidine‐labeled nuclei as compared to quiescent cells. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (3H)thymidine‐labeled nuclei in response to TGF‐β1. Moreover, TGF‐β1 induced the mRNA expression of a complete set of proliferation‐related genes, such as c‐fos (30 min), c‐myc and β‐actin (2 h), and H‐ras, cdc2 kinase, and glucose 6‐phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF‐β1 for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6‐fold and p21‐ras by 2‐fold. Although our results demonstrate that TGF‐β1 induces the expression of very early genes related to cell proliferation, TGF‐β1 could be acting either as a mitogen or as a survival factor to induce proliferation in fetal brown adipocytes.