Lipid-induced insulin resistance in tissues such as liver and skeletal muscle is strongly associated with the accumulation of lipid intermediates such as ceramide and diacylglycerol, which in turn activate inhibitory signalling pathways and interfere with normal insulin action. Diacylglycerol activates isoforms of the protein kinase C family, especially the novel PKC isoforms delta, epsilon and theta, which are generally assumed to inhibit insulin signal transduction at the level of IRS-1 tyrosine phosphorylation. Through detailed studies of mice deficient in specific PKC isoforms, we have shown that these kinases do not merely act as mediators of the inhibitory effects of lipids, but that they in fact play contrasting roles in the regulation of lipid accumulation itself. Thus PKC epsilon deletion promotes triglyceride accumulation in the liver whereas PKC delta deletion reduces lipid storage, even though in each case absence of the kinase protects against insulin resistance. Further proteomic and metabolomic analysis of livers from PKC epsilon-deficient mice reveals changes in enzymes and metabolites involved in cholesterol synthesis, ketogenesis and gluconeogenesis, and has enabled us to discover marked differences in the physiological responses of these mice to different metabolic stresses such as fasting. These observations indicate that the role of PKC epsilon has been oversimplified, and place it in a key regulatory position in the control of hepatic nutrient metabolism.