1147-P: Pseudo Insulin Resistance: Palmitate Inactivates Insulin Signaling but Stimulates Basal Glucose Uptake in 3T3-L1 Adipocytes

Abstract

Increased levels of circulating free fatty acids (FFA) is the major risk factor for type 2 diabetes in obesity. While the long-term effects of increased FFA on adipose tissue likely involve latent inflammation, the short-term effects on insulin-sensitive adipocytes are less clear. We treated fully differentiated 3T3-L1 adipocytes with increased concentrations (0.3-1 mM) of a typical FFA, palmitate, for 2 days, and measured insulin stimulation of 2-deoxyglucose (2-DG) uptake. The insulin cascade and AMPK activities, Glut-1 and Glut-4 levels were measured by western blots (WB). Glut-4 translocation was measured either by WB after cell membrane fractionation, or by fluorescence analysis after lentiviral transduction of adipocytes with Glut-4 construct containing C-terminal mCherry and outer Myc epitope insertion in the Glut-4 extracellular loop. While fluorescence of mCherry reported the total Glut-4, that of anti-Myc antibody revealed Glut-4 exposed on the plasma membrane. Palmitate dose dependently increased basal 2-DG uptake, whereas insulin-stimulated uptake and Glut-4 translocation to the plasma membrane were significantly reduced only by high (1 mM) palmitate. High palmitate did not alter Glut-1 and Glut-4 expression, but reduced IRS phosphorylation (Tyr612) and completely ceased that of Akt (Thr308/Ser473) and AS160 (Ser318) in the absence or presence of insulin, as well as phosphorylation of ACC (Ser-79) by AMPK. These results suggest that the short-term effects of palmitate on adipocytes are associated with “pseudo” insulin resistance, i.e., an increased basal glucose uptake likely due to the “pull” mechanism for increased triglyceride turnover, and the loss of insulin stimulation possibly due to fill-up of fat depots. Disclosure N. Podkuychenko: None. S. Michurina: None. I. Stafeev: None. A. Y. Khapchaev: None. V. P. Shirinsky: None. A. V. Vorotnikov: None. Funding Russian Foundation for Basic Research (20-015-00471)