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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 that 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 insulin-sensitive 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. [ABSTRACT FROM AUTHOR]

Ghrelin acts through the ghrelin receptor (GhrR) to play an important role in the control of energy balance. Genetic ablation of both ghrelin and GhrR results in resistance to diet-induced obesity and increased insulin sensitivity. Ghrelin is believed to act primarily through central mechanisms to induce food intake and promote adiposity. It is unclear whether there are direct effects on peripheral tissues and, in particular, whether GhrR is expressed by adipocytes. The aim of our work was to explore the possibility that ghrelin may have a direct action on adipocytes via GhrR. To examine ghrelin signaling, we first assessed insulin sensitivity in the murine 3T3-L1 adipocyte cell-line treated with ghrelin for 4 days. Insulin sensitivity was assessed by examining Akt phosphorylation after a 5 minute insulin treatment. We found that 10nM and 100nM ghrelin caused a repression of insulin-induced Akt phosphorylation in response to 1nM insulin (3-fold and 22-fold, respectively) or 10nM insulin (3-fold and 5-fold, respectively). Secondly, we compared GhrR mRNA expression in adipose tissue, isolated adipucytes from C57B16 mice and 3T3-L1 adipocytes, using quantitative RT-PCR with pituitary and hypothalamus as positive controls. Data are expressed as mean ± SEM relative to pituitary (= 1.0). As expected, pituitary and hypothalamus had the highest expression (1.00 ± 0.11,1.06 ± 0.13, respectively). GhrR mRNA was clearly detectable in freshly isolated adipocytes (0.030±0.004). However, GhrR mRNA was barely detectable in epididymal adipose tissue. Because adipocytes comprise only 20% of adipose tissue, it is probable that the adipocyte signal is diluted in the total tissue mRNA. 3T3-L1 cells expressed GhrR at higher levels than the isolated adipocytes (0.23 ± 0.04). This level remained constant throughout adipocyte differentiation. In summary, chronic ghrelin treatment reduces insulin signaling in adipocytes and the detection of GhrR in isolated adipocytes and 3T3-L1 cells suggests ghrelin acts directly through GhrR on adipocytes to regulate energy homeostasis. [ABSTRACT FROM AUTHOR]