Cannabinoid CB2 receptor potentiates obesity-associated inflammation, insulin resistance and hepatic steatosis

Abstract

Background: Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver.

Methodology: Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 -/-).

Principal findings: In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 -/- mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 -/- mice.

Conclusion/significance: These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorders.

Figure 1. Selective distribution of Cnr2 in the adipose tissue and regulation during obesity.

A, Time course of Cnr2 and Emr1 expression in the epididymal adipose tissue of obese ob/ob and HFD-fed WT mice and corresponding body weight. B, Cnr2 expression was quantified in the SVF and adipocyte fraction of ob/ob and ob+/ob mice. C, Time course of Cnr2 expression in the liver of obese ob/ob and HFD-fed WT mice. D, Cnr2 expression was quantified in the non parenchymal and parenchymal liver cell fractions of ob/ob and ob+/ob mice. *p<0.05 for ob/ob vs age-matched ob+/ob mice or for HFD-fed vs age-matched ND-fed mice.

Figure 2. Activation of CB2 receptors by JWH-133 enhances fat inflammation in HFD-fed mice.

WT mice were fed a HFD or a ND for 6 weeks, and treated with JWH-133 (3 mg/kg) or vehicle i.p daily during the last 15 days. A, Body weight progression during JWH-133 treatment. B, JWH-133 increases adipose tissue inflammation. Fold induction of adipose tissue Emr1, Tnf and Ccl2 expression over control conditions (ND-fed mice injected with vehicle). C, Activation of CB2 receptors with 1 µM JWH-133 for 48 hours up-regulates Tnf and Ccl2 expression in explants isolated from ob/ob mice fat pads. D, Impact of JWH-133 on hepatic inflammation. Fold induction of hepatic Emr1, Tnf and Ccl2 expression over control conditions (ND-fed mice injected with vehicle). *p<0.05 for JWH-133 vs vehicle. #p<0.05 for HFD-fed vs ND-fed mice.

Figure 3. Pharmacological inactivation of CB2 receptors reduces the inflammatory response in the adipose tissue.

ob/ob and ob+/ob mice were daily treated for 15 days with an intraperitoneal injection of AM-630 (1 mg/kg) or vehicle. A, Body weight progression of AM-630 and vehicle-treated ob/ob mice over time. B, C AM-630 reduces adipose tissue inflammation, as assessed by quantification of fat F4/80 stained cells/total cells and fold induction of fat Emr1, Tnf and Ccl2 expression over control conditions (ob+/ob mice injected with vehicle). *p<0.05 for AM-630 vs vehicle. #p<0.05 for ob/ob vs ob+/ob mice.

Figure 4. Cnr2 knock-out reduces the inflammatory response in the adipose tissue and the liver.

WT and Cnr2 −/− mice were fed a HFD or a ND for 15 weeks. A, Macrophage infiltration into epididymal fat was assessed by immunohistochemical detection of F4/80 (magnification ×400) and quantification of F4/80 stained cells/total cells. B, Fold induction of fat Emr1, Ccl2 and Tnf expression over control conditions (ND-fed WT mice). C, Fold induction of hepatic Emr1, Ccl2 and Tnf expression over control conditions (ND-fed WT mice). D, Body weight progression over time of WT and Cnr2 −/− mice fed a HFD for 15 weeks (p<0.05 by two way ANOVA). *p<0.05 for HFD-fed Cnr2 −/− vs HFD-fed WT mice. #p<0.05 in HFD-fed WT vs ND-fed WT mice.

Figure 5. CB2 receptors potentiate insulin resistance in HFD-fed mice.

A–C, Cnr2 knock-out reduces obesity-induced insulin resistance. WT and Cnr2 −/− mice were fed a ND or a HFD for 15 weeks A, Fasting glycemia, insulinemia, HOMA-IR; B, Blood glucose level during insulin tolerance tests in HFD-fed mice (p<0.05 by two way ANOVA). C, Hyperinsulinemic-euglycemic clamp in HFD-fed mice, as assessed by glucose turnover rate, whole body glycolysis and glycogen synthesis, following insulin stimulation. D, CB2 receptor activation increases insulin resistance. Blood glucose level during insulin tolerance tests in WT mice fed a HFD for 6 weeks and treated daily with an i.p injection of JWH-133 (3 mg/kg) or vehicle during the last 15 days of HFD (p<0.05 by two way ANOVA). * p<0.05 for HFD-fed Cnr2 −/− vs HFD-fed WT mice. # p<0.05 in HFD-fed WT vs ND-fed WT mice.

Figure 6. CB2 receptors promote the development of hepatic steatosis.

A, Cnr2 knock-out blunts steatosis. Representative liver tissue sections (magnification ×100), mean steatosis score and hepatic triglycerides in WT and Cnr2 −/− mice fed a HFD for 15 weeks. B, Hepatic triglycerides in HFD-fed WT or Cnr2−/− mice for 15 weeks and in vehicle or JWH-133-treated (3 mg/kg) HFD-fed WT mice for 6 weeks. *p<0.05 for JWH-133 vs vehicle or for HFD-fed Cnr2 −/− vs HFD-fed WT mice.