Heart disease is the leading cause of mortality in patients with type 2 diabetes. While coronary disease and atherosclerosis are the primary reason for this, accumulating evidence suggests that alterations in substrate metabolism may play a role in cardiac dysfunction in the insulin resistant/diabetic state¹. Our work investigating the role of Heat-shock-protein-72 (HSP72) in cardiac metabolism has identified that cardiac insulin resistance is dissociated from cardiac lipid accumulation and impaired insulin signaling. HSP72 wild-type (WT), heterozygous (+/-) and knockout (-/-) mice were fed a chow (5% fat) or high fat diet (HFD;42% fat) for 12-weeks. Using intravenous insulin, combined with [3H]-2-deoxy-D-glucose tracers we observed severe defects in cardiac insulin-stimulated glucose uptake (p=<0.001). Interestingly, despite the severe insulin resistance, we did not identify a corresponding defect in AKT phosphorylation. Using liquid chromatography, electrospray ionisation-tandem mass spectrometry (LC-ESI MS/MS) we observed no changes in diacylglycerol or triacylglycerol, accumulation in the heart compared with chow but a significant increase in cardiac ceramide in WT mice (p=<0.05). Interestingly, in HSP72-/+ and -/- mice, ceramide accumulation was not increased in response to HFD. However, this prevention of ceramide accumulation did not improve cardiac insulin resistance as HSP72KO-/+ and -/- cardiac glucose uptake was the same as WT mice following a HFD, suggesting that reduction of ceramide levels did not impact cardiac glucose uptake. Coinciding with these findings, in studies performed in isolated cardiac mitochondria and assessed for mitochondrial function, we observed increased maximal oxidative capacity in the HSP72KO+/- and -/- mice compared with WT mice (p=<0.05). Despite this higher oxidative capacity this did not coincide with improved cardiac insulin resistance. We hypothesize that the proposed mechanisms for the induction of cardiac insulin resistance associated with obesity, those being lipid accumulation, insulin signaling impairment and defective oxidative capacity are disassociated from cardiac insulin resistance in our models.