A contribution of intracellular dehydration to insulin resistance has been established in human subjects and in different experimental systems. Here the effect of hyperosmolarity (405mosmol/l) on insulin-induced mitogen-activated protein (MAP) kinase phosphatase (MKP)-1 expression was studied in H4IIE rat hepatoma cells. Insulin induces robust MKP-1 expression which correlates with a vanadate-sensitive decay of extracellular-signal-regulated kinase (Erk-1/Erk-2) activity. Hyperosmolarity delays MKP-1 accumulation by insulin and this corresponds to impaired MKP-1 synthesis, whereas MKP-1 degradation remains unaffected by hyperosmolarity. Rapamycin, which inhibits signalling downstream from the mammalian target of rapamycin (mTOR) and a peptide inhibiting protein kinase C (PKC) ζ/λ abolish insulin-induced MKP-1 protein but not mRNA expression, suggesting the involvement of the p70 ribosomal S6 protein kinase (p70S6-kinase) and/or the eukaryotic initiation factor 4E-binding proteins (4E-BPs) as well as atypical PKCs in MKP-1 translation. Hyperosmolarity induces sustained suppression of p70S6-kinase and 4E-BP1 hyperphosphorylation by insulin, whereas insulin-induced tyrosine phosphorylation of the insulin receptor (IR) β subunit and the IR substrates IRS1 and IRS2, recruitment of the phosphoinositide 3-kinase (PI 3-kinase) regulatory subunit p85 to the receptor substrates as well as PI 3-kinase activation, and Ser-473 phosphorylation of protein kinase B and Thr-410/403 phosphorylation of PKC ζ/λ are largely unaffected under hyperosmotic conditions. The hyperosmotic impairment of both, MKP-1 expression and p70S6-kinase hyperphosphorylation by insulin is insensitive to K2CrO4, calyculin A and vanadate, and inhibition of the Erk-1/Erk-2 and p38 pathways. The suppression of MKP-1 may further contribute to insulin resistance under dehydrating conditions by allowing unbalanced MAP kinase activation.

Abbreviations used: CHX, cycloheximide; DTT, dithiothreitol; eIF-4E, eukaryotic initiation factor 4E; 4E-BP; eIF-4E-binding protein; Erk, extracellular-signal-regulated kinase; IR, insulin receptor; IRS, IR substrate; MAP, mitogen-activated protein; MKP, MAP kinase phosphatase; m7GTP–Sepharose, 7-methyl-GTP–Sepharose; mTOR, mammalian target of rapamycin; NP-40, Nonidet P-40; p70S6-kinase, p70 ribosomal S6 protein kinase; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; TCA, trichloroacetic acid.

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