Cannabinoid receptor 1 (CB1) antagonism enhances glucose utilisation and activates brown adipose tissue in diet-induced obese mice
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We examined the physiological mechanisms by which cannabinoid receptor 1 (CB1) antagonism improves glucose metabolism and insulin sensitivity independent of its anorectic and weight-reducing effects, as well as the effects of CB1 antagonism on brown adipose tissue (BAT) function.
Three groups of diet-induced obese mice received for 1 month: vehicle; the selective CB1 antagonist SR141716; or vehicle/pair-feeding. After measurements of body composition and energy expenditure, mice underwent euglycaemic–hyperinsulinaemic clamp studies to assess in vivo insulin action. In separate cohorts, we assessed insulin action in weight-reduced mice with diet-induced obesity (DIO), and the effect of CB1 antagonism on BAT thermogenesis. Surgical denervation of interscapular BAT (iBAT) was carried out in order to study the requirement for the sympathetic nervous system in mediating the effects of CB1 antagonism on BAT function.
Weight loss associated with chronic CB1 antagonism was accompanied by increased energy expenditure, enhanced insulin-stimulated glucose utilisation, and marked activation of BAT thermogenesis. Insulin-dependent glucose uptake was significantly increased in white adipose tissue and BAT, whereas glycogen synthesis was increased in liver, fat and muscle. Despite marked weight loss in the mice, SR141716 treatment did not improve insulin-mediated suppression of hepatic glucose production nor increase skeletal muscle glucose uptake. Denervation of iBAT blunted the effect of SR141716 on iBAT differentiation and insulin-mediated glucose uptake.
Chronic CB1 antagonism markedly enhances insulin-mediated glucose utilisation in DIO mice, independent of its anorectic and weight-reducing effects. The potent effect on insulin-stimulated BAT glucose uptake reveals a novel role for CB1 receptors as regulators of glucose metabolism.
KeywordsBrown adipose tissue CB1 receptors Diet-induced obesity Euglycaemic–hyperinsulinaemic clamp Glucose utilisation Obesity Rimonabant SNS denervation SR141716
Cannabinoid receptor 1
Brown adipose tissue
Vehicle-treated and pair-fed with the average amount of food ingested by the SR group during the previous day
Sympathetic nervous system
SR141716-treated with unlimited access to food
Vehicle-treated with unlimited access to food
White adipose tissue
The endocannabinoid system has emerged as a key player in both the central and peripheral control of energy balance and glucose metabolism. Chronic pharmacological interventions with selective inhibitors of cannabinoid receptor 1 (CB1), such as rimonabant (SR141716), leads to decreased adiposity as the result of both reduced food intake and increased energy expenditure. The latter mechanism plays a larger role in the weight-loss effects of these drugs, as demonstrated by pair-feeding studies [1, 2]. Mice null for Cb1 (also known as Cnr1) display reduced body weight, adiposity, resistance to diet-induced obesity (DIO), and improvement in glucose regulation . These results implicate the CB1 receptors and its intracellular signalling as critical modulators of energy balance and glucose metabolism.
Despite clear evidence that pharmacological CB1 antagonism improves insulin sensitivity [1, 4, 5, 6, 7], the specific mechanisms are still under investigation. In particular, it is still unclear how chronic CB1 antagonism improves insulin sensitivity and what tissues are its target of action. In this regard, the brown adipose tissue (BAT) has emerged as an important modulator of energy expenditure and glucose utilisation [8, 9]. The notion that adult humans do not retain functional BAT has been dispelled by recent evidence, which points to BAT as an important player in the pathogenesis of obesity and diabetes [10, 11, 12]. Notably, pharmacological blockade or genetic ablation of CB1 has been linked to the activation of the BAT, which further underscores the role of this tissue as a potential target for obesity and diabetes therapy .
In this report, we examine the effects of CB1 antagonism on glucose metabolism and insulin sensitivity, independent of its effects on food intake and adiposity. Since CB1 antagonism activates BAT thermogenesis and glucose uptake, we investigated whether these effects require an intact sympathetic nervous system (SNS).
All animal protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Nine-week-old C57BL/6 J male mice (Jackson Laboratories, Bar Harbor, ME, USA) were housed in temperature-controlled rooms (21–23°C) with a 12 h light–dark cycle, and fed with a 60% high-fat diet (D12492, Research Diets, New Brunswick, NJ, USA) for 1 month.
Energy balance and euglycaemic–hyperinsulinaemic clamp studies
DIO mice were randomised into three groups (n = 11/group): vehicle-treated with unlimited access to food (VEH); SR141716-treated with unlimited access to food (SR); and vehicle-treated and pair-fed with the average amount of food ingested by the SR group during the previous day (PF). Mice were injected daily i.p. and fed 1 h before lights out for 7 weeks. Body composition by quantitative magnetic resonance (Echo MRI Whole Body Composition Analyzer, Echo Medical Systems, Houston, TX, USA) and indirect calorimetry (TSE Systems, Chesterfield, MO, USA) were performed after 4 weeks of treatment. Euglycaemic–hyperinsulinaemic clamp experiments were carried out after 5–6 weeks of treatment.
BAT denervation studies
The interscapular BAT (iBAT) of DIO mice was surgically denervated or sham-operated. After recovery, each group was randomised to receive SR or vehicle (n = 13–15/group). Mice were injected daily i.p. 1 h before lights out for 8 weeks. All mice had unlimited access to food and water. Food intake and body weight were recorded daily. Body composition analysis by MRI and indirect calorimetry were performed after 6 weeks of treatment and glucose uptake after 8–9 weeks of treatment.
BAT thermogenesis studies
DIO mice were implanted with temperature probes (see methods in the electronic supplementary material [ESM]) or sham-operated. After recovery, the mice received vehicle or SR (n = 6/group). Mice were injected i.p. daily 4 h before lights out. BAT temperature was measured every 30 min from 11:00 hours to 19:00 hours for 12 days. Afterwards, mice were implanted with a jugular catheter and, upon recovery, were injected with a bolus of 2-deoxy-d-[U-14 C]glucose (370 kBq) to measure basal glucose uptake in BAT. All mice had unlimited access to food and water.
Food-restricted euglycaemic–hyperinsulinaemic clamp studies
Ten-week-old C57bl/6 mice from Jackson Laboratories were fed a low-fat standard chow or a 60% high-fat diet (D12492, Research Diets) for 5–6 weeks. DIO mice were divided into two groups receiving a high-fat diet that was either unlimited or restricted to 85% of the unlimited food intake. After 5 weeks, mice underwent surgical implantation of jugular catheters and, after recovery, underwent euglycaemic–hyperinsulinaemic clamp studies (n = 5/group).
SR141716 was obtained from the NIMH Chemical Synthesis and Drug Supply Program (www.nimh-repository.rti.org), and administered i.p. at 10 mg/kg daily in all studies. This dose has been previously used i.p. and by mouth to similar effect [9, 13, 14]. A 1 mg/ml emulsion of SR141716 was made by dissolving 100 mg of SR141716 in 1 ml DMSO. Next, 2 ml of Tween 80 was added while vortexing. Finally, 97 ml of saline was slowly added while vortexing.
Euglycaemic–hyperinsulinaemic clamp studies and glucose uptake in skeletal muscle
Euglycaemic–hyperinsulinaemic clamp studies were performed in conscious, chronically catheterised mice, as described by Okamoto et al. and Lo et al. [15, 16]. Briefly, during the clamp studies, which lasted for 90 min, we infused a solution of glucose (20% wt/vol.) at a variable rate as required to maintain euglycaemia. Mice received primed-constant infusions of [3-3H]glucose (bolus of 74 kBq followed by 3.7 kBq/min; NET-331 C, Perkin Elmer, Boston, MA, USA) and insulin (bolus of 88 mU/kg followed by 5 mU min−1 kg−1; Humulin R, Eli Lilly, Indianapolis, IN, USA).
We collected plasma samples to determine glucose concentrations and tracer-specific activities at times 0, 30, 50, 60, 70, 80 and 90 min. Additional sample volumes were taken at 0, 60 and 90 min to determine basal and clamped insulin levels and NEFA. To measure tissue-specific glucose uptake, we injected a bolus (370 kBq) of 2-deoxy-d-[U-14 C]glucose at time = 45 min. At the end of the clamp studies, mice were killed and tissue samples were quickly harvested and stored at −80°C for further analysis.
Indirect calorimetry was performed as described by Pfluger et al. . Mice were individually housed in metabolic chambers (TSE Systems) in which fluid, food intake, locomotor activity and gas exchanges can be monitored. Following 48 h of acclimatisation, O2 consumption, CO2 production and locomotor activity were measured every 6 min for a total of 72 h to measure the gas exchange, respiratory quotient and energy expenditure. Energy expenditure values were normalised to the body weight of the animals. Home-cage locomotor activity was determined using a tri-dimensional infrared light beam system and expressed as beam breaks/24 h.
Tissue was homogenised using the Qiagen TissueLyser bead mill (Qiagen, Valencia, CA, USA). Proteins were extracted with RIPA buffer (150 mmol/l NaCl, 1.0% [vol./vol.] Triton X-100, 0.5% [wt/vol.] sodium deoxycholate, 0.1% [vol./vol.] SDS, 50 mmol/l Tris, pH 8.0) and then centrifuged for 10 min at 10,000 g. A 30 μg sample of protein was separated on a 10% Tris-HCl gel and transferred to polyvinylidene-fluoride membrane for 1 h at 4°C at 100 V. The membrane was blocked with 5% (wt/vol.) non-fat dry milk in Tris-buffered saline with Tween 20 for 1 h at room temperature, followed by 1:1,000 dilution of tyrosine hydroxylase antibody (catalogue identifier ab152, Millipore, Billerica, MA, USA) overnight at 4°C with a 1:10,000 dilution of goat anti-rabbit secondary antibody (Abcam, Cambridge, MA, USA) for 1 h at room temperature. Bands were visualised using the Pierce ECL western blotting substrate (catalogue identifier 32106, Thermo Scientific, Rockford, IL, USA).
Analytical procedures and calculations
Plasma glucose was measured by the glucose oxidase method in a GM7 Analyser (Analox Instruments USA, Lunenburg, MA, USA). The rate of glycolysis was determined by measuring the tritium on the C-3 position of our glucose tracer that is lost into water during glycolysis . Thus, the plasma tritium is present in two chemical forms: 3H-labelled water or [3-3H]glucose. To measure both forms, we counted radioactivity in plasma samples deproteinised with Ba(OH)2 and ZnSO4, before and after each sample was evaporated to dryness. The dry counts represent plasma [3-3H]glucose, whereas the difference between the wet and dry counts is a measure of the tritiated water.
Under steady-state conditions for plasma glucose, the rate of glucose disposal (R d) is assumed to be equal to the rate of glucose appearance (R a). We determined R d by dividing the infusion rate for [3-3H]glucose (disintegrations/min) by the specific activity of plasma [3-3H]glucose (disintegrations min–1 [mg glucose]–1). The rate of glucose production is calculated as the difference between R d and the rate of glucose infusion.
The tissue-specific rate of glycogen synthesis was quantified by measuring the rate of incorporation of [3-3H]glucose into glycogen. Tissue glycogen concentrations were determined after digestion with amyloglucosidase, as described by Massillon et al. and Rossetti et al. [18, 19]. The glycogen synthetic rate was obtained by dividing the [3-3H]glucose radioactivity in glycogen (disintegrations min–1 [g tissue]–1) by the mean specific activity of [3-3H]glucose in plasma during the insulin clamp (disintegrations min–1 [μg plasma glucose]–1).
To measure the tissue-specific rate of glucose uptake we weighed and dissolved tissues in 0.5 ml of 1 mol/l NaOH, and incubated them in a shaking water bath at 60°C for 1 h. After neutralisation with 0.5 ml of 1 mol/l HCl, two aliquots were taken. One was deproteinised with Ba(OH)2 and ZnSO4 and the other with a 6% solution of HClO4. The HClO4 supernatant fraction contained both phosphorylated and unphosphorylated 2-deoxyglucose, whereas the Ba(OH)2 and ZnSO4 supernatant fraction contained only the unphosphorylated form. The difference in counts between the two supernatant fractions is a measure of the tissue content of 2-deoxy-[U-14 C]glucose 6-phosphate.
The rate of tissue glucose uptake was obtained by dividing the tissue content of 2-deoxy-[U-14 C]glucose 6-phosphate by the specific activity of 2-deoxy-[U-14 C]glucose in plasma . Plasma insulin and leptin were measured with a MILLIPLEX MAP Mouse Serum Adipokine Panel (catalogue identifier MADPK-71 K, Linco Research, St Charles, MO, USA). Plasma adiponectin was measured with MILLIPLEX MAP Mouse Adipocyte Panel (catalogue identifier MADPCYT-72 K, Millipore). NEFA in plasma was measured with a kit from Waco Pure Chemical Industries (Osaka, Japan).
Mice were anaesthetised with isofluorane and a transverse incision was made in the skin anterior to the iBAT fat pads. The pads were identified and separated from the underlying muscle by blunt dissection. Four intercostal nerve bundles entering each fat pad were identified, and a section of each nerve bundle was removed bilaterally. Extra care was taken to ensure that the blood supply was not disrupted.
Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). One-way ANOVA with Bonferroni post hoc test was used to determine significance at (p ≤ 0.05) for studies involving pair-feeding. Denervation studies were analysed using a two-way ANOVA with Bonferroni post hoc test for significance (p ≤ 0.05). All time course studies were analysed by two-way ANOVA with repeated measures and Bonferroni post hoc test (p ≤ 0.05).
CB1 antagonism reduces adiposity and increases energy expenditure in DIO mice
Plasma measurements during fasting and clamp studies in animals treated with vehicle or 10 mg/kg SR i.p., or pair fed for 8 weeks (n = 11/group)
11.5 ± 0.44
9.00 ± 0.50*
9.8 ± 0.39*
136.5 ± 24.5
64.8 ± 8.8*
78.8 ± 12.3
1.07 ± 0.05
1.33 ± 0.09*
1.16 ± 0.08
0.659 ± 0.03
0.676 ± 0.04
0.715 ± 0.03
7.6 ± 1.5
1.1 ± 0.3*,†
5.0 ± 1.4
21.8 ± 1.4
18.4 ± 1.5
18.7 ± 1.6
8.7 ± 0.33
8.5 ± 0.22
8.4 ± 0.39
434.0 ± 50.75
325.5 ± 43.75
330.8 ± 33.25
0.76 ± 0.05
0.49 ± 0.04*
0.67 ± 0.06
CB1 antagonism increases whole-body insulin-dependent glucose utilisation
SR-induced weight loss was accompanied by a reduction in fasting plasma glucose and insulin levels (Table 1), suggesting improved insulin sensitivity. Fasting levels of adiponectin, triacylglycerols and NEFA were not changed. However, insulin-dependent suppression of plasma NEFA was restored selectively in the SR group (Table 1).
In order to identify the target tissues for an SR141716-induced increase in glucose utilisation, we measured in vivo glucose uptake in fat and skeletal muscle under hyperinsulinaemic conditions. Glucose uptake in white adipose tissue (WAT; Fig. 2e) and BAT (Fig. 2f) was significantly stimulated by SR141716. In particular, CB1 antagonism stimulated the uptake of glucose in BAT by more than threefold in the SR group compared with the VEH and PF groups (Fig. 2f). In a separate cohort of mice treated with SR141716 or vehicle, we measured in vivo BAT glucose uptake under basal conditions and found no significant difference (31.9 ± 3.9 mg kg−1 min−1 and 45.1 ± 7.0 mg kg−1 min−1 for the VEH and SR groups, respectively; n = 6/group, p > 0.05). The lack of an increase in BAT glucose uptake under basal conditions suggests that the effect of SR141716 on BAT glucose uptake is mediated by improved insulin sensitivity.
To determine whether chronic CB1 antagonism improved insulin sensitivity via its effect on fat mass reduction, we induced weight loss in DIO mice by food restriction to match the weight reduction achieved with SR141716. We then subjected these mice to euglycaemic–hyperinsulinaemic clamp studies (ESM Fig. 1). Despite reduced adiposity, food-restricted DIO mice did not have improved insulin sensitivity compared with DIO mice that had unrestricted access to food (ESM Fig. 1). Thus, CB1 antagonism improves insulin sensitivity independent of its ability to reduce adiposity.
CB1 antagonism activates BAT
Baseline BAT temperatures were similar in both groups and displayed the typical circadian profile with higher BAT temperatures in the dark period (ESM Fig. 2). SR141716 injection promptly increased BAT temperature within 1 h from the first dose (Fig. 4a) compared with the same time at baseline or with vehicle (average increase of 1.5°C). VEH mice showed no change in BAT temperature compared with baseline. The difference in iBAT temperature between the SR and VEH groups was greater in the light than in the dark phase of the cycle, perhaps because of the time of SR141716 delivery (12:00 hours in the light cycle). Although the magnitude of SR141716 effect on iBAT temperature was attenuated by day 10 (Fig. 4b), the AUC of iBAT temperature for days 10 to 12 was still significantly elevated (Fig. 4c).
The strong thermogenic effect of SR141716 on BAT was accompanied by a significant increase in BAT expression of Ucp1, which encodes the uncoupling protein that mediates thermogenesis in BAT , and Pgc-1α (also known as Ppargc1a) and Pparδ (also known as Ppard), which encode the co-activator and transcription factor, respectively, that are implicated in the activation and differentiation of BAT  (Fig. 4d).
SNS-dependent effects of CB1 antagonism on iBAT
In agreement with previous reports, we find that chronic CB1 antagonism reduces body weight because of both transient anorexia and, more importantly, increased energy expenditure .
Our data show that chronic SR141716 administration results in a significant increase in insulin-dependent glucose utilisation, including an increase in the rates of glycolysis and glycogen synthesis. In our experimental model of DIO, the effects of CB1 antagonism show an unexpected pattern in the amelioration of specific components of insulin action. Although SR141716 treatment greatly increased insulin-dependent glucose utilisation, the rate of hepatic glucose production was not significantly changed, implying that CB1 antagonism cannot improve hepatic insulin resistance in DIO mice. This result is at odds with previous reports in which chronic SR141716 treatment elicited a significant improvement in hepatic insulin sensitivity [4, 5]. The effect of SR141716 on hepatic insulin sensitivity was shown to depend on increased levels of plasma adiponectin, whereas the effects of SR141716 on weight loss and glucose utilisation were not . Indeed, several reports have implicated the endocannabinoid system as a modulator of adiponectin production and secretion [4, 5, 23, 24, 25, 26]. This adipokine is known as a powerful inhibitor of hepatic glucose production [27, 28]. However, we and others could not detect changes in adiponectin levels (Table 1) [1, 29, 30, 31], which might explain why we did not detect an improvement in hepatic insulin sensitivity. The reasons for this lack of effect on adiponectin are not known and need further investigation.
An alternative explanation for the failure of SR141716 to decrease hepatic glucose production could be an inhibitory effect of dietary fat on insulin sensitivity. Indeed, we show that weight reduction by food restriction alone in our DIO mice fails to ameliorate in vivo insulin action, albeit these mice have a fat mass similar to that of SR141716-treated DIO mice (ESM Fig. 1). These results also suggest that the stimulatory effects of SR141716 on insulin-dependent glucose utilisation are specific to weight loss secondary to CB1 antagonism, as similar weight loss induced with food restriction does not restore insulin sensitivity (ESM Fig. 1). Since weight reduction does not always appear to lower endocannabinoid levels in obese individuals , we speculate that the beneficial effects of SR141716 on glucose metabolism are possibly secondary to the blockade of overstimulated CB1 receptors.
Another typical result of weight loss is an improvement in insulin-stimulated glucose uptake in skeletal muscle , which was not observed in our experiments with chronic administration of SR141716, nor was it observed in the food-restricted DIO mice. Our findings are in agreement with previous reports that did not observe a significant effect of SR141716 on insulin-stimulated glucose uptake in muscle of DIO mice [4, 5] However, our studies did show significantly enhanced insulin-stimulated glycogen synthesis in skeletal muscle to an extent large enough to contribute significantly to the whole-body increase in glucose utilisation promoted by SR141716.
Notably, glycogen synthesis of skeletal muscle was increased in the absence of a significant stimulation of glucose uptake. Although the effect of insulin on glycogen stores is typically influenced by glucose transport, insulin can increase glycogen synthesis in muscle independently of an increase in glucose uptake . The effect of SR141716 on glycogen synthesis is not mediated by a change in glycogen phosphorylase or glycogen synthase (data not shown). Thus, CB1 antagonism may affect the activity of glycogen enzymes via alteration of transduction signalling or production of glycogen regulatory proteins. One possible explanation for the lack of a significant increase in glucose uptake in muscle is that CB1 antagonism increases fatty acid oxidation  which, in turn, can lead to inhibition of glucose uptake .
Unlike muscle, chronic CB1 antagonism strikingly increases insulin-stimulated glucose uptake of BAT. However, under basal conditions, chronic CB1 antagonism does not lead to an appreciable increase in BAT glucose uptake. This result indicates that chronic CB1 antagonism does not increase basal glucose uptake in BAT, suggesting that an SNS-dependent increase in BAT glucose uptake is not tonically stimulated. However, it reveals a novel role for CB1 receptors in the SNS as modulators of insulin action in BAT.
Denervation studies show that CB1-antagonist-mediated BAT differentiation and activation are strongly dependent on SNS innervation. Our data are in agreement with a report showing that CB1 antagonism in lean rats activates BAT in an SNS-dependent fashion . Moreover, Quarta et al. have shown that BAT denervation blunts the enhancement of BAT glucose uptake after administration of SR141716 or exposure to cold . Accordingly, we observed changes in histological, molecular and thermogenic variables after surgical denervation that indicate a substantial loss of function of BAT.
Interestingly, although the effects of SR141716 on BAT functions were blunted, iBAT denervation did not alter SR141716-mediated loss of body weight or fat mass, or energy expenditure. Previous studies on denervation or ablation of iBAT have yielded contrasting results with failure [36, 37, 38] and success  in altering body weight. Explanations for the failure are that the denervation of iBAT is limited to only 40% of total BAT mass. This may lead to compensatory activation of BAT in other fat depots, particularly as our studies were not conducted at thermoneutrality (ambient temperature 22°C). Nevertheless, these results point to a novel role for CB1 receptors in modulating BAT insulin sensitivity with SNS-dependent mechanisms. Although the complex interaction between the SNS and insulin action needs to be elucidated, we can speculate that CB1 signalling through the SNS is crucial for the modulation of BAT glucose and fat uptake. Indeed, the coordinated partitioning of these fuels plays an important role in BAT thermogenesis [39, 40].
It is important to note that the CB1 receptors responsible for modulating BAT function and thermogenesis may not be exclusively located in the central nervous system. In support of this notion, peripheral but not central administration of SR141716 induced food-independent weight loss in DIO rats . Also, peripherally restricted CB1 antagonists can cause substantial weight loss and increase energy expenditure . Taken together, these data suggest the thermogenic effects of CB1 antagonism could involve peripheral mechanisms, including BAT activation.
In conclusion, our data show that CB1 antagonism markedly improves insulin-mediated glucose utilisation in DIO mice independent of its anorectic properties, via mechanisms that are not reproduced by food restriction alone. BAT is the major target organ for the effect of CB1 antagonism on insulin-dependent glucose uptake via a mechanism that requires an intact SNS. These novel data point to a crucial role of SNS CB1 receptors in modulating body weight and glucose metabolism through activation of BAT function.
This work was supported by grants from the National Institutes of Health (DK078283), The American Diabetes Association (research grant 707RA11677), the Cincinnati Mouse Metabolic Phenotyping Center (MMPC DK59630) and Ethicon Endosurgery. Special thanks to S. Lipp and M. Fitzgerald for their outstanding technical assistance.
All authors substantially contributed to this work by participating in the design and analysis of the experiments, the drafting and revisions of the article and gave final approval of the published version.
Duality of interest
M. Tschöp is a consultant for Roche Pharma and for Ambrx Inc. The remaining authors declare that they have no duality of interest associated with this manuscript.
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