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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