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

Tumour necrosis factor (TNF)-alpha 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-alpha could be mediated by the generation of ceramide, since TNF-alpha 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-alpha. In consequence, TNF-alpha-induced insulin resistance on glucose uptake was completely alleviated. In addition, TNF-alpha 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-alpha construct transactivates GLUT4-CAT construct. Our results indicate that de novo ceramide produced by TNF-alpha-induced insulin resistance on GLUT4 gene expression in brown adipocytes by interfering C/EBP-alpha expression, a transcription factor essential for the expression of GLUT4.

Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner.

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.

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.

Rosiglitazone and retinoic acid induce uncoupling protein-1 (UCP-1) in a p38 mitogen-activated protein kinase-dependent manner in fetal primary brown adipocytes.

Brown adipose tissue expresses the thermogenic uncoupling protein-1 (UCP-1), which is positively regulated by peroxisome proliferator-activated receptor (PPAR) agonists and retinoids through the activation of the heterodimers PPAR/retinoid X receptor (RXR) and retinoic acid receptor (RAR)/RXR and binding to specific elements in the ucp-1 enhancer. In this study we show that in fetal rat brown adipocyte primary cultures the PPARgamma agonist rosiglitazone (Rosi), as well as retinoic acids 9-cis-retinoic acid and all-trans-retinoic acid also have "extragenic" effects and induce p44/p42 and p38 mitogen-activated protein kinase (p38MAPK) activation. The latter is involved in UCP-1 gene expression, because inhibition of p38MAPK activity with PD169316 impairs the ability of Rosi and retinoids for UCP-1 induction. The inhibitory effects of PD169316 are mimicked by the antioxidant GSH, suggesting a role for reactive oxygenated species (ROS) generation in the increase of UCP-1 expression in response either to Rosi or 9-cis-retinoic acid. Thus, we propose that Rosi and retinoids act as PPAR/RXR and RAR/RXR agonists and also activate p38MAPK. These two coordinated actions could result in a high increase of transcriptional activity on the ucp-1 enhancer and hence on thermogenesis. PPARalpha and gamma agonists but not retinoids also increase UCP-3 expression in fetal brown adipocytes. However, the regulation of UCP-3, which is not involved in thermogenesis, seems to differ from UCP-1 given the fact that is not affected by p38MAPK inhibition.

PLCgamma participates in insulin stimulation of glucose uptake through activation of PKCzeta in brown adipocytes.

Based on recent studies showing that PLCgamma associates to insulin receptor, we investigated its role in insulin stimulation of glucose transport in brown adipocytes. Insulin stimulation induced rapid PLCgamma association to phosphorylated insulin receptor, and activation of PLCgamma, as assessed by the mobilization of Ca(2+) from intracellular stores and by the production of the second messenger DAG. Both events are dependent on activation of PI3-kinase. Inhibition of PLCgamma activity either with the chemical compound U73122 or with an inhibitor peptide precluded insulin stimulation of glucose uptake, GLUT4 translocation, and actin reorganization, as wortmannin did. In contrast, the inactive analog U73343 did not have an inhibitory effect. Furthermore, translocation of GLUT4-GFP in response to insulin was completely abolished by cotransfection with a PLCgamma-inactive mutant in HeLa cells, a cell model sensitive to insulin that express PLCgamma. U73122 did not affect PI3-kinase nor Akt activation, but impaired PKCzeta activation by insulin, as wortmannin did. PLC activity renders two products, IP(3) and DAG, and DAG can be metabolized to PA by the action of DAG-kinase. Using the compound R54494, a DAG-kinase inhibitor, insulin-induced PKCzeta activation was also suppressed, this activity being restored by addition of PA. In summary, these data indicate that PLCgamma, activated at least partially by PI3-kinase, is a link between insulin receptor and PKCzeta through the production of PA and could mediate insulin-induced glucose uptake and GLUT4 translocation.

Ceramida como mediador de la resistencia a insulina producida por el factor de necrosis tumoral alfa en adipocitos marrone / Ceramide mediates insulin resistance by tumor necrosis factor alpha in brown adipocytes

La resistencia a la acción de la insulina es la característica fundamental de la Diabetes tipo 2, que afecta al 6 por ciento de la población, siendo la Obesidad el factor de riesgo más importante para el desarrollo de la misma. El nexo de unión entre ambas patologías puede ser el factor de necrosis tumoral (TNF) alfa, una citoquina antilipogénica secretada por el propio tejido adiposo. En estudios previos hemos demostrado que el TNF-alfa produce resistencia a la insulina en adipocitos marrones fetales en cultivo primario. TNF-alfa activa varias cascadas de señalización incluyendo la estimulación de esfingomielinasas y la producción de ceramidas tras 30 min de tratamiento. Hemos estudiado el efecto de un análogo de ceramida permeable, de cadena corta (C2-ceramida) añadido exógenamente, sobre los efectos metabólicos de la insulina y su señalización. Este compuesto completamente impide la estimulación del transporte de glucosa por insulina, impidiendo la translocación de GLUT4 a la membrana, tanto determinada por Western blot o por la localización immunofluorescente de GLUT4. El pretratamiento de las células con C2-ceramida previno la fosforilación de Akt total y de las dos isoformas expresadas en los adipocitos marrones, Aktl y Akt2, pero no inhibió la actividad PI3-quinasa total ni la activación de PKCzeta. La ceramida está activando una fosfatasa implicada en defosforilar Akt, como se desprende de las observaciones siguientes: I) el tratamiento con ceramida o con TNF-alfa aumenta la actividad fosfatasa PP2A, 2) el tratamiento con ácido okadaico junto con ceramida restaura completamente la fosforilación de Akt por insulina y 3) la transfección transitoria de una forma constitutivamente activa de Akt no restablece la fosforilación de Akt. Estos resultados indican que la ceramida producida por el TNF-alfa induce un estado de resistencia a insulina en los adipocytos marrones, manteniendo Akt en un estado defosforilado e inactivo. (AU)