Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation, Insulin Resistance and Hepatic Steatosis

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 disorder

CB2 receptors promote the development of hepatic steatosis.

<p>A, <i>Cnr2</i> knock-out blunts steatosis. Representative liver tissue sections (magnification ×100), mean steatosis score and hepatic triglycerides in WT and <i>Cnr2 −/−</i> mice fed a HFD for 15 weeks. B, Hepatic triglycerides in HFD-fed WT or <i>Cnr2−/−</i> 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 <i>vs</i> vehicle or for HFD-fed <i>Cnr2 −/− vs</i> HFD-fed WT mice.</p

<i>Cnr2</i> knock-out reduces the inflammatory response in the adipose tissue and the liver.

<p>WT and <i>Cnr2</i> −/− 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 <i>Emr1</i>, <i>Ccl2</i> and <i>Tnf</i> expression over control conditions (ND-fed WT mice). C, Fold induction of hepatic <i>Emr1</i>, <i>Ccl2</i> and <i>Tnf</i> expression over control conditions (ND-fed WT mice). D, Body weight progression over time of WT and <i>Cnr2</i> −/− mice fed a HFD for 15 weeks (p<0.05 by two way ANOVA). *p<0.05 for HFD-fed <i>Cnr2</i> −/− <i>vs</i> HFD-fed WT mice. #p<0.05 in HFD-fed WT <i>vs</i> ND-fed WT mice.</p

Metabolic parameters of HFD WT and <i>Cnr2</i> −/− mice.

*<p>p<0.05 <i>vs</i> HFD WT.</p>**<p>p<0.01 <i>vs</i> HFD WT.</p>***<p>p<0.001 <i>vs</i> HFD WT.</p

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

<p>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 <i>Emr1</i>, <i>Tnf</i> and <i>Ccl2</i> 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 <i>Tnf and Ccl2</i> expression in explants isolated from <i>ob/ob</i> mice fat pads. D, Impact of JWH-133 on hepatic inflammation. Fold induction of hepatic <i>Emr1</i>, <i>Tnf</i> and <i>Ccl2</i> expression over control conditions (ND-fed mice injected with vehicle). *p<0.05 for JWH-133 <i>vs</i> vehicle. #p<0.05 for HFD-fed <i>vs</i> ND-fed mice.</p

CB2 receptors potentiate insulin resistance in HFD-fed mice.

<p>A–C, <i>Cnr2</i> knock-out reduces obesity-induced insulin resistance. WT and <i>Cnr2</i> −/− 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 <i>Cnr2</i> −/− <i>vs</i> HFD-fed WT mice. # p<0.05 in HFD-fed WT <i>vs</i> ND-fed WT mice.</p

Selective distribution of <i>Cnr2</i> in the adipose tissue and regulation during obesity.

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