Deficiency of CB2 cannabinoid receptor in mice improves insulin sensitivity but increases food intake and obesity with age
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The endocannabinoid system has a key role in energy storage and metabolic disorders. The endocannabinoid receptor 2 (CB2R), which was first detected in immune cells, is present in the main peripheral organs responsible for metabolic control. During obesity, CB2R is involved in the development of adipose tissue inflammation and fatty liver. We examined the long-term effects of CB2R deficiency in glucose metabolism.
Mice deficient in CB2R (Cb2 −/− [also known as Cnr2]) were studied at different ages (2–12 months). Two-month-old Cb2 −/− and wild-type mice were treated with a selective CB2R antagonist or fed a high-fat diet.
The lack of CB2R in Cb2 −/− mice led to greater increases in food intake and body weight with age than in Cb2 +/+ mice. However, 12-month-old obese Cb2 −/− mice did not develop insulin resistance and showed enhanced insulin-stimulated glucose uptake in skeletal muscle. In agreement, adipose tissue hypertrophy was not associated with inflammation. Similarly, treatment of wild-type mice with CB2R antagonist resulted in improved insulin sensitivity. Moreover, when 2-month-old Cb2 −/− mice were fed a high-fat diet, reduced body weight gain and normal insulin sensitivity were observed.
These results indicate that the lack of CB2R-mediated responses protected mice from both age-related and diet-induced insulin resistance, suggesting that these receptors may be a potential therapeutic target in obesity and insulin resistance.
KeywordsEndocannabinoids Food intake Glucose uptake Insulin resistance Obesity
Protein kinase B
Brown adipose tissue
Endocannabinoid receptor 1
Endocannabinoid receptor 2
Glycogen synthase kinase 3 beta
Insulin receptor substrate 1
Mitogen-activated protein kinase
White adipose tissue
The endocannabinoid system plays an important role in the control of food intake and metabolism and participates in the pathophysiology of obesity and type 2 diabetes [1, 2]. Two different endocannabinoid receptors, CB1R and CB2R [3, 4], have been identified. Peripheral CB1R is located in adipocytes, hepatocytes, skeletal muscle and pancreas [1, 5]. Overactivation of the endocannabinoid system has been reported in rodent models of obesity [1, 6, 7] and in obese and type 2 diabetic patients . Moreover, overactivity of the endocannabinoid system in obese patients has been associated with enhanced visceral rather than subcutaneous fat mass [8, 9, 10]. These findings suggest that increased activity of the endocannabinoid system in visceral fat may be a key component in the development of insulin resistance. Furthermore, overactivation of CB1R in skeletal muscle promotes insulin resistance ; in this regard, pharmacological activation of CB1R activity diminishes mitogen-activated (MAP) kinase- and protein kinase B (PKB)-directed signalling . In the liver, overactivation of CB1R enhances hepatic insulin resistance and lipogenesis , while the selective disruption of CB1R in hepatocytes protects mice fed a high-fat diet from liver steatosis and insulin resistance . Moreover, CB1R hyperactivity in adipocytes facilitates fat accumulation and insulin resistance . In cultured pancreatic islets exposure to endocannabinoids increases insulin release , suggesting that overactivity of the endocannabinoid system may contribute to the hyperinsulinaemia associated with insulin resistance.
CB2R is mainly located in the cells of the immune system and participates in the modulation of immune responses [16, 17]. In both mice and humans, CB2R is also present in the main peripheral organs responsible for the control of metabolism, including the liver , adipose tissue [5, 19, 20, 21], skeletal muscle  and pancreatic islets . Obesity leads to increased expression of the cannabinoid receptor 2 gene (Cb2 [also known as Cnr2]) both in adipose tissue and liver in high fat fed and ob/ob mice . However, this expression is predominantly located in the stromal vascular fraction of adipose tissue, while in liver CB2R is present only in the non-parenchymal cell fraction. CB2R deficiency decreases body weight gain during the feeding of a high-fat diet and prevents obesity-associated inflammation, insulin resistance and fatty liver . Nevertheless, the role of long-term CB2R deficiency in the control of whole body metabolism has not been clarified. Here we used Cb2 knockout mice (Cb2 −/−)  to further evaluate the contribution of CB2R to the regulation of glucose metabolism in young and old mice.
Male knockout mice deficient in CB2R (Cb2 −/−) and wild-type littermates (Cb2 +/+)  on a C57Bl/6J background of different ages (2, 6 and 12 months) and maintained under a 12 h light–dark cycle (lights on at 08:00 hours) were used. Mice were fed ad libitum with a standard diet (Basal Purified Diet with 12% energy from fat; Test Diet, Richmond, IN, USA) or, when stated, a high-fat diet (Basal Purified Diet with 60% energy from fat; Test Diet). An additional group of C57Bl/6J wild-type mice were treated with the CB2R inverse agonist SR 144528 (Sanofi Recherche, Montpellier, France; for more information, see Electronic supplementary methods [ESM]). These mice received once daily i.p. injections, on consecutive days, for 28 days of either vehicle (1.8% DMSO, 0.0004% Tween 80 in distilled water) or vehicle plus antagonist (3 mg/kg body weight). Where stated, mice were fasted for 16 h. Body temperature was assessed anally with a thermometer in the morning either at room temperature or at 4°C (cold exposure). Animal procedures were conducted in accordance with the guidelines of the European Communities Directive 86/609/EEC regulating animal research and were approved by the local ethics committee.
Hormone and metabolite determination
Serum insulin and leptin were evaluated with ELISA kits (Crystal Chem, Chicago, IL, USA). Serum adiponectin levels were evaluated by radioimmunoassay (Linco Research, St Charles, MO, USA). Non-sterified fatty acids (NEFA) were measured by enzymatic colorimetric assay (NEFA C; Wako Chemicals, Neuss, Germany).
Food intake and leptin-induced anorexigenic effects
Mice were housed two per cage. Baseline food intake was measured for 4 weeks. Mice were habituated to the injection procedure (saline, i.p.) for 4 days. Leptin (Prepotech, London, UK) was administered twice a day at a dose of 0.5 μg/g for each injection (i.p.). The decrease in food intake was expressed as a percentage with respect to the baseline and as grams per mouse per day.
To study the inflammatory response after the acute administration of an external antigen, mice received an acute i.p. injection of lipopolysaccharide (LPS; 0.5 mg/kg in saline). Blood samples were obtained 3 and 12 h after treatment and TNF-α (3 h after injection) and IL-6 (12 h after injection) concentrations were measured by ELISA (Assay Designs, Ann Arbor, MI, USA).
Insulin and glucose tolerance tests
To test insulin tolerance, insulin was administered i.p. (0.75 U/kg; Humilin Regular; Eli Lilly, Indianapolis, IN, USA) to fed mice and tail blood was sampled 0, 15, 30, 45, 60 and 75 min later. For the glucose tolerance test, mice were food-deprived overnight (16 h) and then glucose (1 g/kg i.p.) was administered. Blood glucose was measured from the tail vein 0, 15, 30, 60, 90 and 120 min after glucose injection. Blood glucose was measured with a Glucometer Elite (Bayer Diagnostics, Tarrytown, NY, USA).
Insulin secretion from isolated islets
Pancreatic islets were isolated from Cb2 +/+ and Cb2 −/− mice by intraductal injection of collagenase P (1 mg/ml; Roche Molecular Chemicals, Mannheim, Germany) in Hank’ balanced salt solution (HBSS) for 10 min at 37°C. Digestion was stopped by washing three times with cold HBSS. Islets were picked by hand. Insulin secretion was measured during a 1 h incubation of islets at 37°C in 1 ml HBSS/0.5% BSA in the presence of 2.8 or 15 mmol/l glucose. After this time, insulin levels in media and insulin content in islets (after homogenisation in ethanol/HCl [10%] solution) were determined with an RIA kit (Linco Research, St Charles, MO, USA).
Gene expression analysis
Total RNA was obtained from skeletal muscle, white and brown adipose tissue and liver samples. To synthesise cDNA, 1 μg RNA was used (Omniscript kit; Qiagen, Hilden, Germany). Random primers (Invitrogen, Carlsbad, CA, USA) were used for the reaction in the presence of Protector RNase Inhibitor (Roche Molecular Biochemicals, Mannheim, Germany). RT-PCR was performed in a SmartCycler II (Cepheid, Maurens-Scopont, France) using the QuantiTect SYBR Green PCR Kit (Qiagen). Data were normalised to ribosomal protein S26 (RBS) gene values. Primer sequences are shown in ESM Table 1.
Immunohistochemical and morphometric analysis
White and brown adipose tissue, liver and pancreas were fixed for 24 h in formalin, embedded in paraffin and sectioned. To determine white and brown adipose tissue and liver morphology, sections were stained with haematoxylin/eosin. For detection of macrophages, white adipose tissue sections were incubated either with a rabbit anti-F4/80 antibody (Acris, Hidenhausen, Germany) or with a rabbit anti-macrophage galactose-specific lectin-2 (MAC-2) antibody (Cedarlane Laboratories, Burlington, ONT, Canada). For detection of insulin, pancreatic sections were incubated with a guinea pig anti-insulin antibody (Sigma, St Louis, MO, USA). Secondary antibodies were: peroxidase-conjugated goat anti-guinea pig (Dako, Glostrup, Denmark); biotinylated goat anti-rabbit (Pierce Biotechnology, Rockford, IL, USA); horseradish peroxidase-conjugated streptavidin (Molecular Probes, Leiden, the Netherlands). Morphometric analysis is described in the ESM.
2-[18F]Fluoro-2-deoxy-d-glucose (FDG) was used as a radiotracer and was synthesised by Barnatron (Barcelona, Spain) in accordance with the standard procedure . Imaging was performed using a small animal PET scanner (rPET; Suinsa Medical Systems, Madrid, Spain). The methods used for the PET experiment are described in the ESM.
CB2R antagonist treatment in 3T3-L1 adipocytes and Oil Red O staining
Maintenance and differentiation of 3T3-L1 cells are described in the ESM. For treatment with the CB2R antagonist (SR 144528), stock solutions of drugs were prepared in DMSO at 10 mmol/l and stored at −20°C. The concentration of solvent in an assay never exceeded 0.1% (vol./vol.). This final concentration was without effect on assays. Cells were cultured in the presence of a final concentration of 100 nmol/l of SR 144528 or the same volume of DMSO. For Oil Red O staining, after being washed twice with PBS buffer, cells were fixed with 10% formaldehyde for 10 min. After two washes in PBS, cells were stained for 20 min with Oil Red O solution (0.18% in 60% isopropanol). Oil Red O was then removed and the cells were washed twice with PBS.
Hepatic and 3T3L1 cell triacylglycerol content
The triacylglycerol content of liver and 3T3L1 cells was determined by extracting total lipids from liver samples or cultured cells with chloroform–methanol (2:1 vol./vol.) as described in . Triacylglycerols were quantified spectrophotometrically in the supernatant fractions using an enzymatic assay kit (GPO-PAP; Roche Diagnostics, Basel, Switzerland).
Western blot analysis
Tibialis muscles were homogenised in protein lysis buffer. Proteins (50 μg) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes and probed with primary antibodies against insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphorylated-Tyr, phosphoSer473-protein kinase B (phosphoSer473-AKT), AKT, phosphoSer9-GSK3B and GSK3 (Cell Signaling Technology, Danvers, MA, USA) and GLUT4 (Chemicon, Billerica, MA, USA) overnight at 4°C. Detection was performed using horseradish peroxidase-labelled anti-goat IgG or horseradish peroxidase-labelled anti-rabbit IgG (Dako) and ECL Plus Western Blotting Detection Reagent (Amersham, Arlington Heights, IL, USA).
All values are expressed as mean ± SEM. Differences between groups were compared by Student’s t test. A p value <0.05 was considered statistically significant.
Cb2 −/− mice showed increased food intake, adipose tissue hypertrophy and obesity with age
Twelve-month-old obese Cb2 −/− mice did not develop adipose tissue inflammation
Obesity and type 2 diabetes are associated with chronic inflammation in WAT, characterised by increased production of cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and chemokine (C-C motif) ligand 2 (CCL2) . In addition, CB2R, which is expressed in immune cells [32, 33], plays a major role in the modulation of obesity-associated inflammatory responses . Despite adipose hypertrophy, no macrophage infiltration in WAT from obese 12-month-old Cb2 −/− mice was observed after immunohistochemical analysis with F4/80 (Fig. 1j). In agreement, quantitative PCR analysis of WAT revealed similar expression levels of Tnfa, Il6 and Ccl2 in Cb2 −/− obese mice compared with Cb2 +/+ mice (Fig. 1k). We also evaluated the inflammatory response of Cb2 −/− mice after the acute administration of an external antigen. Cb2 +/+ and Cb2 −/− mice were treated with an acute i.p. injection of LPS (0.5 mg/kg). Blood samples were obtained 3 and 12 h after treatment and TNF-α (3 h after injection) and IL-6 (12 h after injection) concentrations were measured by ELISA. However, no differences were observed between Cb2 +/+ and Cb2 −/− mice (TNF-α: wild type, 646 ± 101.85 vs Cb2 −/−, 1033 ± 483.3 pg/ml; IL-6: wild type, 6048 ± 1686 vs Cb2 −/−, 8822 ± 1269 pg/ml), indicating that Cb2 −/− mice were not protected from acute antigen-induced systemic inflammation.
Twelve-month-old Cb2 −/− obese mice revealed enhanced insulin sensitivity and increased glucose uptake
Therefore, in spite of weight gain with age, Cb2 −/− mice showed improved insulin sensitivity, which agreed with the lack of adipose tissue inflammation and the normal circulating NEFA levels. The lack of CB2R in Cb2 −/− mice also led to a 60% decrease in the expression of Cnr1 in skeletal muscle (Fig. 2j), which could contribute to enhanced insulin sensitivity . This decrease in Cnr1 expression was also observed in 6-month-old non-obese Cb2 −/− mice (ESM Fig. 2i).
CB2R antagonism resulted in improved insulin sensitivity
Cb2 −/− mice fed a high-fat diet showed reduced body weight gain and increased insulin sensitivity
Cb2 −/− mice fed a standard diet displayed increased body weight gain and adipose tissue hypertrophy with age (older than 6 months), which was associated with increased food intake and hyperleptinaemia. In contrast, 2-month-old high fat fed Cb2 −/− mice displayed reduced body weight gain and decreased fat pad mass compared with high fat fed wild-type mice. However, during high-fat feeding, no differences in food intake were observed, as described previously . These results may suggest that while differences in food intake could explain long-term enhanced body weight during standard diet feeding, the lack of CB2R protected Cb2 −/− mice from body weight gain during high fat feeding in young animals. Both CB1R and CB2R are present in white adipose tissue  and upregulation of CB1R activity in adipose tissue has been associated with increased lipogenesis and decreased lipolysis in obese humans and rodents [9, 10, 36]. Two- to 4-month-old Cb2 −/− mice presented normal Cnr1 expression in adipose tissue in both the standard and the high-fat feeding condition, suggesting that improved body weight in high fat fed animals was probably not due to decreased CB1R activity. However, adipose tissue of old Cb2 −/− mice (older than 6 months) showed a gradual overexpression of Cnr1 that increased in parallel to epididymal fat pad weight, suggesting that enhanced CB1R levels may be a consequence of the progressive adipose tissue hypertrophy. Treatment with a CB2R antagonist neither altered Cnr1 expression in white adipose tissue nor increased fat pad mass, and these mice showed normal food intake. Moreover, the CB2R antagonist did not alter lipogenesis in isolated adipocytes, whereas the CB1R antagonist did . This suggests that adipose tissue hypertrophy in Cb2 −/− mice resulted from a long-term adaptive response to the constitutive lack of CB2R, which involved increased food intake. In addition, although standard-fed Cb2 −/− mice showed greater food intake, Cnr1 expression was not increased in the hypothalamus, suggesting that lack of CB2R may directly induce signals that increase appetite. This phenotype was opposite to that of cb1 −/− mice, which are leaner than cb1 +/+ mice and show reduced food intake .
In spite of the development of obesity, consistent with the results in high fat fed mice, insulin sensitivity was enhanced in 12-month-old Cb2 −/− mice, as revealed by an improved insulin tolerance test. Moreover, PET imaging revealed an enhancement of skeletal muscle insulin-mediated glucose uptake in Cb2 −/− mice. Similarly, CB1R antagonists facilitate skeletal muscle insulin-induced glucose uptake  and NEFA oxidation  and CB1R agonists can decrease insulin-induced responses in muscle cells . Furthermore, a CB1R receptor inverse agonist increases glucose uptake both in muscle isolated from ob/ob mice and in cultured L6 muscle cells [38, 39]. The CB1R inhibition in muscle cells increases the signalling through the phosphatidyl inositol 3 (PI3) kinase pathway . However, in skeletal muscle from insulin-treated Cb2 −/− mice no further increase in either AKT or GSK3B phosphorylation levels were observed, suggesting that downstream signals may be involved in increasing insulin sensitivity. Thus, CB1R/CB2R activation may regulate the Ras/Raf/ERK cascade downstream of AKT while having little or no effect on components of the PI3 kinase/AKT pathway. Since skeletal muscle contains both CB1R and CB2R , improvement in insulin sensitivity could be related to the absence of CB2R and/or the downregulation of CB1R in skeletal muscle of Cb2 −/− mice. Moreover, mice treated with CB2R antagonist showed increased insulin sensitivity with no changes in body weight or Cnr1 expression. Thus, increased insulin sensitivity resulted from lack of CB2R in Cb2 −/− mice rather than decreased Cnr1 expression. Similarly, when fed a high-fat diet, Cb2 −/− mice did not develop insulin resistance. This may have resulted from the fact that, although high fat fed Cb2 −/− mice became obese, they showed lower body weight gain compared with high fat fed Cb2 +/+ mice. Indeed, high fat fed Cb2 −/− mice gained about 30% in body weight, whereas Cb2 +/+ mice gained around 50%. High fat fed Cb2 −/− mice displayed similar insulin sensitivity compared with standard-fed lean mice. Similarly, Deveaux et al.  have shown a reduction in body weight gain and improved insulin sensitivity in high-fat-fed Cb2 −/− mice. In addition, high-fat-fed Cb2 −/− mice showed reduced hepatic steatosis, which would also have contributed to improved insulin sensitivity. The absence of inflammatory responses in WAT could also be involved in the improvement of peripheral insulin sensitivity in Cb2 −/− mice . This agrees with the enhanced insulin sensitivity that was previously reported in obese mice with reduced WAT inflammation, such as Tnf-α (also known as Tnf) −/− , CCL2 or Mcp-1 −/−  and osteopontin−/− mice . The reduced adipose tissue macrophage infiltration and cytokine expression in high fat fed Cb2 −/− mice was not associated with any alteration of Cnr1 expression, suggesting that the lack of CB2R is responsible for the reduced inflammation. The increased glucose uptake described in Cb2 −/− mice, together with the similar food intake compared with wild-type mice, may result in decreased body weight gain during high fat feeding.
In summary, this study reveals that genetic ablation of CB2R led to improved insulin sensitivity during both age-related and diet-induced insulin resistance. Thus, we demonstrated that CB2R plays an important role in glucose metabolism by modulating skeletal muscle insulin sensitivity and the inflammatory response in the adipose tissue. We provide new data confirming the key role of CB2R in the pathophysiology of obesity and type 2 diabetes, which may open new therapeutic approaches to counteract insulin resistance by using CB2R ligands.
We thank S. Franckhauser and C. J. Mann for helpful discussions and L. Maggioni for technical support. This work was supported by grants from NIDA (5R01-DA016768 to R.M. and A. Zimmer), European Commission DG Research FP6 (GENADDICT #LSHM-CT-2004-05166 to R. Maldonado and A. Zimmer, PHECOMP #LSHM-CT-2007-037669 to R. Maldonado and EUGENE2, LSHM-CT-2004-512013 to F. Bosch), Ministerio de Ciencia e Innovación (SAF2007-64062 to R. Maldonado and SAF2005-01262 and SAF2008-00962 to F. Bosch), Instituto Salud Carlos III (RETICS-RTA #RD06/001/001 to R. Maldonado and CIBER de Diabetes y Enfermedades Metabólicas Asociadas to F. Bosch), the Deutsche Forschungsgemeinschaft (SFB645 to A. Zimmer) and Bundesministerium für Bildung und Forschung (NGFN2 to A. Zimmer).
Duality of interest
R. Maldonado has received research grants from sanofi-aventis, Laboratorios Esteve and Ferrer. None of the other authors have relevant financial interests to disclose, nor a conflict of interest of any kind associated with this manuscript.
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