Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells.

AIMS/HYPOTHESIS: Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C2-ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis. METHOD: L6 muscle cells were pre-incubated with C2-ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined. RESULTS: Incubation of L6 muscle cells with ceramide (100 micromol/l) for 2 h led to a complete loss of insulin-stimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositide-dependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P3) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide. CONCLUSIONS/INTERPRETATION: These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in PtdIns(3,4,5)P3 is necessary but not sufficient for protein kinase B activation in this system.

L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport.

Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3, and this is likely to have important regulatory implications for translation initiation.

Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways.

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.