OBJECTIVE-Obesity is associated with an overactive endocannabinoid (EC) system. The mechanisms responsible for increased ECs in obese individuals are poorly understood. Therefore, we examined the role of adipocyte insulin resistance in intracellular EC metabolism. METHODS-We used 3T3-L1 adipocytes and diet-induced obese (DIO) mice to examine the role of obesity and insulin resistance in the regulation and/or dysregulation of intracellular ECs. RESULTS -For the first time, we provide evidence

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... t insulin is a major regulator of EC metabolism. Insulin treatment reduced intracellular ECs (2-arachidonylglycerol [2-AG] and anandamide [AEA]) in 3T3-L1 adipocytes. This corresponded with insulinsensitive expression changes in enzymes of EC metabolism. In insulin-resistant adipocytes, patterns of insulin-induced enzyme expression were disturbed in a manner consistent with elevated EC synthesis and reduced EC degradation. Expression profiling of adipocytes from DIO mice largely recapitulated in vitro changes, suggesting that insulin resistance affects the EC system in vivo. In mice, expression changes of EC synthesis and degradation enzymes were accompanied by increased plasma EC concentrations (2-AG and AEA) and elevated adipose tissue 2-AG. CONCLUSIONS-Our findings suggest that insulin-resistant adipocytes fail to regulate EC metabolism and decrease intracellular EC levels in response to insulin stimulation. These novel observations offer a mechanism whereby obese insulin-resistant individuals exhibit increased concentrations of ECs. Diabetes 57:1262-1268, 2008 RESEARCH DESIGN AND METHODS Cell culture. The mouse fibroblast cell line 3T3-L1 (ATTC, Manassas, VA) was maintained and differentiated as previously described (19) . Briefly, differentiation was induced in high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), 5 g/ml insulin, 0.25 mol/l dexamethasone, and 0.5 mmol/l 3-IBMX (isobutyl-1-methylxanthine). After 3 days, insulin, dexamethasone, and IBMX were removed, and cells were maintained in 10% FBS media until the day of experiments (days 7-8). To induce insulin resistance in adipocytes, media was supplemented with 100 nmol/l insulin or vehicle (pH-equivalent distilled water) control from day 3 to days 7-8. All cells were subjected to the same manipulation, and media was changed every 2 days with fresh treatment. Similarly, for shorter duration From the Diabetes and Metabolism Disease Area,