20‐HETE Interferes with Insulin Signaling Through GPR75

20-Hydroxyeicosatetraenoic acid (20-HETE) is the omega-hydroxylated metabolite of arachidonic acid produced by the Cytochrome P450 (CYP) 4A and 4F family of enzymes. Clinical studies have demonstrated that the urinary and/or plasma levels of 20-HETE are elevated in obese and diabetic individuals. Studies have shown that 20-HETE stimulates adipogenesis, contributes to pathogenesis of diabetes, induces hyperglycemia and impedes the cellular actions of insulin. A recent study from our lab revealed that mice overexpressing the Cyp4a12-20-HETE synthase, when fed a high fat diet, rapidly develop obesity, hyperglycemia, hyperinsulinemia and impairment of glucose tolerance. These animals also developed insulin resistance in skeletal muscle, liver and adipose tissue, evident by impaired tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate. Based on these results, we hypothesize that 20-HETE interferes with insulin signaling at a cellular level and contributes to insulin resistance by a yet to be explored mechanism. In this study, we used Human Umbilical vein endothelial cells (HUVECs) in which 20-HETE signaling has been extensively studied and differentiated 3T3L1 adipocytes, which is an established model for in vitro screening of insulin sensitivity. Treatment with 10nM 20-HETE for 5 minutes resulted in a 50% inhibition of insulin-mediated phosphorylation of the insulin receptor (IR) at Tyr972 in both HUVECs (3.4±0.1 vs 7.7±0.2 fold change in vehicle treated, p<0.05) and differentiated 3T3L1 adipocytes (2.4±0.3 vs 4.7±0.3 fold change in vehicle treated, p<0.05). Knockdown of the 20-HETE receptor GPR75 using GPR75 SiRNA significantly attenuated the 20-HETE-mediated inhibition of insulin-stimulated IR phosphorylation at Tyr972. GPR75 is a Gq-coupled Class-A orphan receptor whose activation is associated with an increase in intracellular calcium and IP-1 accumulation. 20-HETE treatment resulted in a dose-dependent accumulation of IP-1 in differentiated 3T3L1 adipocytes. This effect was not observed in cells treated with GPR75 SiRNA (40±2 vs 112±6 nM IP-1 for control SiRNA, p<0.05) indicating that 20-HETE-mediated 1P-1 accumulation is GPR75-dependent. Moreover, 20-HETE-mediated inhibition of IR phosphorylation at Tyr972 was significantly attenuated in the presence of a specific protein tyrosine phosphatase 1b (PTP1B) inhibitor (6.3±0.9 vs 4.3±0.6 fold change, p<0.05). PTP1B is a negative regulator of the insulin signaling pathway which strongly dephosphorylates the insulin receptor (IR) and insulin receptor substrate (IRS). This study, for the first time, demonstrates that 20-HETE interferes with insulin signaling by an effect mediated through activation of its receptor GPR75, which then triggers Gq/IP3/DAG pathway and increase in intracellular calcium. Support or Funding Information Supported by NIH grants PO1034300 and HL139793 20-HETE interferes with insulin signalling. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.