Endocannabinoids in adipocytes during differentiation and their role in glucose uptake

Abstract

The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-gamma, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake.

Characterization of the Endocannabinoid System in Human Neuronal Cells and Proteomic Analysis of Anandamide-induced Apoptosis

“…After 6 h, 10 M AEA was added to the medium, alone or in the presence of 1 M SR141716, and 24 h later cells were harvested and subjected to either apoptosis quantitation or total protein extraction for Western blotting analysis. CBR and TRPV1 Receptor Binding-For cannabinoid receptor studies, SH-SY5Y, SK-NBE, or LAN-5 cells were resuspended in 2 mM Tris/EDTA, 320 mM sucrose, 5 mM MgCl 2 , pH 7.4, and then they were homogenized in a Potter homogenizer and centrifuged as reported (18). The resulting pellet was resuspended in assay buffer (50 mM Tris-HCl, 2 mM Tris/EDTA, 3 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, pH 7.4) to a protein concentration of 1 mg/ml.…”

“…The membrane preparation was divided into aliquots that were stored at Ϫ80°C for no longer than 1 week. These membrane fractions (100 g of protein/test) were used in rapid filtration assays with the synthetic cannabinoid [ 3 H]CP55940 (400 pM), as described (18). Also binding of the TRPV1 agonist [ 3 H]RTX (500 pM) to SH-SY5Y cells was evaluated by rapid filtration assays (18).…”

“…These membrane fractions (100 g of protein/test) were used in rapid filtration assays with the synthetic cannabinoid [ 3 H]CP55940 (400 pM), as described (18). Also binding of the TRPV1 agonist [ 3 H]RTX (500 pM) to SH-SY5Y cells was evaluated by rapid filtration assays (18). Unspecific binding was determined in the presence of cold agonists (1 M CP55940 or 1 M RTX) and was further corroborated by selective antagonists (0.1 M SR141716 for CB1R, 0.1 M SR144528 for CB2R, and 1 M I-RTX for TRPV1), as reported (18).…”

The Role of the Endocannabinoid System in the Pathogeny of Type 2 Diabetes

“…Because adiponectin stimulates AMPK activity, the CB1-mediated inhibition of adiponectin expression in adipocytes might reinforce this effect (Matias et al 2006). Another way for ECS-enhanced lipogenesis in adipocytes might be through their ability to stimulate glucose uptake by these cells via the enhancement of both basal and insulininduced translocation to the plasma membrane of the glucose transporter 4 (GLUT4) (Gasperi et al, 2007;Pagano et al, 2007). In mouse adipocytes this effect seems to be mediated by CB1-induced stimulation of nitric oxide synthesis (Gasperi et al, 2007), while in human adipocytes CB1 activates the phosphatidylinositol-3-kinase cascade (Pagano et al, 2007).…”

“…Another way for ECS-enhanced lipogenesis in adipocytes might be through their ability to stimulate glucose uptake by these cells via the enhancement of both basal and insulininduced translocation to the plasma membrane of the glucose transporter 4 (GLUT4) (Gasperi et al, 2007;Pagano et al, 2007). In mouse adipocytes this effect seems to be mediated by CB1-induced stimulation of nitric oxide synthesis (Gasperi et al, 2007), while in human adipocytes CB1 activates the phosphatidylinositol-3-kinase cascade (Pagano et al, 2007). Increased glucose uptake and, subsequently, glycolysis might be the way for de novo fatty acid biosynthesis, and this effect might underlie part of the pro-lipogenetic effect of CB1 receptor agonists in these cells (Matias et al, 2008).…”