BACKGROUND & AIMS Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB1) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB1 in insulin resistance and inhibition of insulin signaling pathways. METHODS Wild-type mice and mice with disruption of CB1 (CB1−/− mice) or with hepatocyte-specific deletion or transgenic overexpression of CB1 were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB1 in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS The HFD induced hepatic insulin resistance in wild-type mice, but not in CB1−/− mice or mice with hepatocyte-specific deletion of CB1. CB1−/− mice that overexpressed CB1 specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB1, the HFD or CB1 activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB1 activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB1 was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB1-mediated inhibition of insulin signaling and clearance.

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 M glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 M) on CGNs markedly reduced glutamate-induced apoptosis in dose-and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 M bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-␤-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-D-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca 2؉ imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca 2؉ increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [ 3 H]MK-801 with an IC 50 value of 0.763 M in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.

Hepatocellular carcinoma (HCC) has high mortality and no adequate treatment. Endocannabinoids interact with hepatic CB1 receptors (CB1R) to promote hepatocyte proliferation in liver regeneration via inducing cell-cycle proteins involved in mitotic progression, including Forkhead Box M1 (FOXM1). Because FOXM1 is highly expressed in HCC and contributes to its genesis and progression, we analyzed the involvement of the endocannabinoid/CB1R system in murine and human HCC. Postnatal diethylnitrosamine (DEN) treatment induced HCC within 8 months in wild-type mice, but fewer and smaller tumors in CB1R−/− mice or in wild-type mice treated with the peripheral CB1R antagonist JD5037, as monitored in vivo by serial magnetic resonance imaging. Genome-wide transcriptome analysis revealed CB1R-dependent, tumor-induced upregulation of the hepatic expression of CB1R, its endogenous ligand anandamide, and a number of tumor promoting genes, including the GRB2 interactome as well as FOXM1 and its downstream target the tryptophan-catalyzing enzyme indoleamine 2,3-dioxygenase (IDO2). Increased IDO2 activity and consequent induction of immunosuppressive Treg cells in tumor tissue promote immune tolerance. Conclusion The endocannabinoid/CB1R system is upregulated in chemically induced HCC resulting in the induction of various tumor promoting genes, including IDO2, and attenuation of these changes by blockade or genetic ablation of CB1R suppresses the growth of HCC and highlights the therapeutic potential of peripheral CB1R blockade.