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)-␣ 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-␣ 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-␣. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-␣ was still produced. Moreover, TNF-␣ produced inhibitor B kinase (IKK)-␤ activation and inhibitor B-␤ and -␣ degradation in a p38 MAPKdependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-␣ 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-␣, 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.

Tumor necrosis factor (TNF)-␣ causes insulin resistance on glucose uptake in fetal brown adipocytes. We explored the hypothesis that some effects of TNF-␣ could be mediated by the generation of ceramide, given that TNF-␣ treatment induced the production of ceramide in these primary cells. A short-chain ceramide analog, C2-ceramide, completely precluded insulin-stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane, as determined by Western blot or immunofluorescent localization of GLUT4. These effects were not produced in the presence of a biologically inactive ceramide analog, C2-dihydroceramide. Analysis of the phosphatidylinositol (PI) 3-kinase signaling pathway indicated that C2-ceramide precluded insulin stimulation of Akt kinase activity, but not of PI-3 kinase or protein kinase C-activity. C2-ceramide completely abolished insulin-stimulated Akt/protein kinase B phosphorylation on regulatory residues Thr 308 and Ser 473, as did TNF-␣, and inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in PAGE. Moreover, C2-ceramide seemed to activate a protein phosphatase (PP) involved in dephosphorylating Akt because 1) PP2A activity was increased in C2-ceramide؊ and TNF-␣؊ treated cells, 2) treatment with okadaic acid concomitantly with C2-ceramide completely restored Akt phosphorylation by insulin, and 3) transient transfection of a constitutively active form of Akt did not restore Akt activity. Our results indicate that ceramide produced by TNF-␣ induces insulin resistance in brown adipocytes by maintaining Akt in an inactive dephosphorylated state.

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Highlights Optimal liver stiffness thresholds for community-based screening in populations with metabolic risk factors and alcoholic is between 9.1 and 9.5 kPa for the diagnosis of significant fibrosis (stages ≥F2)  Transient elastography is a cost-effective intervention for identifying patients with liver fibrosis in primary care. Healthcare systems would need to invest between 2,500 (at-risk population) to 6,500 (general population) purchasing power parity-adjusted euros to gain an extra year of life, adjusted per quality of life. The survival effect of screening is most pronounced for the identification of significant (≥F2) fibrosis.

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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 PPAR␥ 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. PPAR␣ and ␥ 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.

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 (v vAkt). 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 PKCj j 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, v vAkt 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 insulininduced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with v vAkt 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. ß

Baseline PCT levels were higher in patients who had febrile neutropenia with bacteremia compared with patients who had clinical infections or fever of unknown origin. PCT helped to identify patients who had microbiologic infections and patients who were at high risk of treatment failure, and PCT may constitute a complementary tool in the initial assessment of such patients.