E. Hajduch et al., Ceramide impairs the insulin-dependent membrane recruitment of Protein Kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells, DIABETOLOG, 44(2), 2001, pp. 173-183
Aims/hypothesis. Increased cellular production of ceramide has been implica
ted in the pathogenesis of insulin resistance and in the impaired utilisati
on of glucose. In this study we have used L6 muscle cells to investigate th
e mechanism by which the short-chain ceramide analogue, C-2-ceramide, promo
tes a loss in insulin sensitivity leading to a reduction in insulin stimula
ted glucose transport and glycogen synthesis.
Method. L6 muscle cells were pre-incubated with C-2-ceramide and the effect
s 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 mu mol/l) for 2 h
led to a complete loss of insulin-stimulated glucose transport and glycoge
n synthesis. This inhibition was not due to impaired insulin receptor subst
rate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation bu
t was caused by a failure to activate protein kinase B. This defect could n
ot be attributed to inhibition of 3-phosphoinositide-dependent 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 inabilit
y to recruit protein kinase B to the plasma membrane. Expression of a membr
ane-targetted protein kinase B led to its constitutive activation and an in
crease 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 sensit
ivity of key cell responses such as glucose transport and glycogen synthesi
s 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 sy
stem.