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

Abstract

The activation of phosphoinositide 3-kinase (PI3K) by insulin represents a key signalling event in the hormonal stimulation of diverse cellular responses including glucose transport and glycogen synthesis. The activation of PI3K increases the production of 4,5 trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4 bisphosphate [PtdIns(3,4)P 2 ]), which act as important signalling intermediates in the downstream activation of the serine/threonine kinase, Protein Kinase B (PKB/Akt). Activation of PKB depends upon its phosphorylation on two key amino acid residues, Thr 308 and Ser 473, with full activation requiring the phosphorylation of both [1]. The Nterminal domain of PKB contains a pleckstrin homology (PH) domain, which is thought to be critical in allowing the kinase to interact with 3-phospho- Diabetologia (2001) Abstract 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, C 2 -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 C 2 -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 mmol/l) for 2 h led to a complete loss of insulinstimulated 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-phosphoinositidedependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P 3 ) 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)P 3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173±183]

Skeletal muscle lipid metabolism with obesity

“…The accumulation of IMTG is not believed to be the direct cause of the development of insulin resistance but more of a marker for the presence of other lipid intermediates, which have been directly linked to defects in insulin signaling (8,17,25,32,37). Intramyocellular long-chain fatty acyl-CoA accumulation has been implicated in the pathogenesis of insulin resistance in skeletal muscle (4,8,32,37).…”

“…It is believed, however, that the accumulation of IMTG is not the direct cause of the development of insulin resistance but that IMTG is an inert marker for the presence of other lipid intermediates (diacylglycerol, fatty acyl-CoAs, or ceramide, etc. ), which have been directly linked to defects in insulin signaling (8,17,25,32,37).To date, the mechanism(s) responsible for the accretion of IMTG and intermediates of lipid metabolism in intact skeletal muscle are not evident. Two possibilities include an increase in lipid synthesis and/or a reduction in fatty acid oxidation, both of which may result in the accumulation of IMTG and other intermediates of lipid metabolism.…”

“…It is believed, however, that the accumulation of IMTG is not the direct cause of the development of insulin resistance but that IMTG is an inert marker for the presence of other lipid intermediates (diacylglycerol, fatty acyl-CoAs, or ceramide, etc. ), which have been directly linked to defects in insulin signaling (8,17,25,32,37).…”

Cell-permeable ceramides increase basal glucose incorporation into triacylglycerols but decrease the stimulation by insulin in 3T3-L1 adipocytes

“…22 Work in other cell types has also demonstrated TNFa and ceramides can activate PKB downstrem of PI 3-K. 45,46,55 However, ceramides can also prevent the translocation of proteins that bind 3-phosphoinositides despite their provision by PI 3-K. 56 The lack of PKB recruitment to plasma membranes in ceramide treated cells may explain the inhibition of insulin-stimulated glucose uptake. 27 It was reported that treatment of 3T2-L1 adipocytes with C 2 -ceramide lowered PKB activity in the presence of insulin. 28 In this work, C 2 -ceramide alone doubled basal glucose uptake as predicted from our previous 22 and present work.…”

“…26 In L6 myocytes, ceramides inhibit insulin-stimulated glucose uptake, but this did not depend upon decreased IRS-1 phosphorylation, or PI 3-K activity. 27 Ceramides appear to inhibit insulin-stimulated glucose uptake downstream of PI 3-K at the level of protein kinase B (PKB). 28 In contrast to these effects on insulin-stimulated glucose uptake, TNFa and ceramides stimulate insulin-independent glucose uptake in the long-term in 3T3-L1 adipocytes through PI 3-K activation.…”