Increased intracellular Ca concentrations prevent membrane localization of PH domains through the formation of Ca-phosphoinositides.

Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca acts as a negative regulator of insulin signaling. Chronic intracellular Ca overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P, PI(4,5)P, and PI(3,4,5)P Finally, thermodynamic analysis of the binding interaction showed that Ca-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca rather than PH domains to PIPs forming Ca-PIPs. Thus, Ca-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca-PIPs. Our findings provide a mechanistic link between intracellular Ca dysregulation and Akt inactivation in insulin resistance.