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 K 2 CrO 4 , 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.

Autophagic proteolysis in rat liver is under the control of the cellular hydration state. Because the morphological site of swelling-dependent proteolysis regulation has not yet been identified, the formation of autophagosomes was investigated with transmission electron microscopy in slices from perfused livers. In livers from fed rats, hypo-osmotic exposure (185mosmol/l) led within 30min to a decrease in fractional cytoplasmic autophagosome volume that was sensitive to colchicine and p38 MAPK inhibition. Similarly, the decrease in autophagosome volume, but not the increase in cell volume caused by insulin or glutamine/glycine, was strongly inhibited by colchicine and SB 203580, an inhibition of p38 MAPK activation. Immune complex assays from perfused liver showed that hypo-osmotic activation of p38 MAPK was not inhibited by colchicine. Further, experiments using confocal laser microscopy in cultivated hepatocytes incubated with mouse-derived anti-(α-tubulin) showed that microtubular structures were not influenced by the inhibition of p38 MAPK by SB 203580. It is concluded that the sequestration of autophagic vacuoles is a major site of proteolysis regulation by cell hydration. Swelling-induced activation of p38 MAPK is required for this process and occurs upstream of the putative microtubule regulation site.

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI3-kinase) in cultured hepatocytes. Ras activation was dependent on PI3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.

The influence of hypo-osmotic cell swelling on the activity of the mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 (where Erk stands for extracellular signal-regulated protein kinase) was studied in cultured rat astrocytes. Hypo-osmotic treatment led within 10 min to an increased activity of Erk-1 and Erk-2, which became maximal at 20 min and returned to the basal level within 60 min. Moreover, exposure to hypo-osmotic conditions induced a biphasic increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ): a rapid peak-like increase was followed by a sustained plateau. The absence of extracellular Ca 2+ completely abolished Erk activation as well as the plateau of the [Ca 2+ ] i response after hypo-osmotic stimulation. Application of wortmannin and agents to elevate intracellular cAMP levels also completely blocked Erk activation but were without effect on the biphasic [Ca 2+ ] i response to hypo-osmotic treatment of the cells, suggesting a role of PtdIns 3-kinase and the Ras/Raf pathway downstream of the calcium signal. Protein kinase C (PKC) and Ca 2+ /calmodulin (CaM)-dependent kinases are unlikely to play a role in the hypo-osmolarity-induced signalling towards MAP kinases, as revealed by the blockage of PKC and CaM kinases. Inhibition of tyrosine kinases, pertussis-toxin- or cholera-toxin-sensitive G-proteins and phospholipase C had no effect on the [Ca 2+ ] i response; the Erk response to hypo-osmolarity was also largely unaltered. This is different from the swelling-induced MAP kinase activation in hepatocytes, which was shown to occur via a calcium-independent but G-protein- and tyrosine kinase-dependent mechanism. Thus osmo-signalling towards MAP kinases might exhibit cell-type-specific features.