192-OR: Lipid-Induced Insulin Resistance in Brown Adipose Tissue Is Mediated by the sn-1,2 DAG-PKCe-IRKT1150 Phosphorylation Pathway Free

While there is substantial evidence that brown adipose tissue (BAT) is insulin-responsive, it remains unclear whether BAT becomes insulin resistant with high fat diet (HFD) feeding. Here, we examined the hypothesis that the sn-1,2-diacylglycerol (DAG)/Protein Kinase C epsilon (PKCε)/insulin receptor kinase (IRK)^T1150 phosphorylation pathway may trigger HFD-induced BAT insulin resistance. To address this hypothesis we first studied two groups of male Sprague Dawley rats fed either a regular chow diet (RC) or a HFD. HFD-fed rats manifested BAT insulin resistance as reflected by reduced insulin-stimulated phosphorylation of IRK^T1158 (p=0.03), AKT^T308 (p=0.003), PDH^S232 (p=0.0002), and PDH^S300 (p=0.0004) compared to RC rats during a hyperinsulinemic-euglycemic clamp (HEC). These reductions in insulin signaling occured at the level of the IRK and were associated with increased sn-1,2 diacylglycerols (p=0.03) and increased PKCε translocation (p<0.004). These defects in insulin signaling were also associated with a 35% (p<0.009) reduction in insulin-stimulated BAT mitochondrial pyruvate dehydrogenase/citrate synthase flux (V_PDH/V_CS) as assessed by LC-MS/MS and GC-MS measurements of C4C5 glutamate (m+2)/pyruvate (m+3) during a HEC where [¹³C₆]glucose was the infusate. In order to assess the role of PKCε-induced IRK^T1150 phosphorylation in this process we next studied the effects of HFD feeding in: 1) wild type mice fed a regular chow diet (WT), 2) wild type mice fed a HFD (WT-HFD) and 3) IRK^T1150A knockin mice fed a HFD (IRK^T1150A-HFD) and found that IRK^T1150A-HFD mice have preserved levels of insulin-stimulated AKT^S473 phosphorylation compared to WT-HFD mice (p=0.04).

Conclusion: These results demonstrate a critical role of the sn-1,2-DAG-PKCε-IRK^T1150 phosphorylation pathway in mediating HFD-induced insulin resistance and mitochondrial dysfunction in BAT.