Absence of cannabinoid 1 receptor in beta cells protects against high-fat/high-sugar diet-induced beta cell dysfunction and inflammation in murine islets
The cannabinoid 1 receptor (CB1R) regulates insulin sensitivity and glucose metabolism in peripheral tissues. CB1R is expressed on pancreatic beta cells and is coupled to the G protein Gαi, suggesting a negative regulation of endogenous signalling in the beta cell. Deciphering the exact function of CB1R in beta cells has been confounded by the expression of this receptor on multiple tissues involved in regulating metabolism. Thus, in models of global genetic or pharmacological CB1R blockade, it is difficult to distinguish the indirect effects of improved insulin sensitivity in peripheral tissues from the direct effects of inhibiting CB1R in beta cells per se. To assess the direct contribution of beta cell CB1R to metabolism, we designed a mouse model that allows us to determine the role of CB1R specifically in beta cells in the context of whole-body metabolism.
We generated a beta cell specific Cnr1 (CB1R) knockout mouse (β-CB1R−/−) to study the long-term consequences of CB1R ablation on beta cell function in adult mice. We measured beta cell function, proliferation and viability in these mice in response to a high-fat/high-sugar diet and induction of acute insulin resistance with the insulin receptor antagonist S961.
β-CB1R−/− mice had increased fasting (153 ± 23% increase at 10 weeks of age) and stimulated insulin secretion and increased intra-islet cAMP levels (217 ± 33% increase at 10 weeks of age), resulting in primary hyperinsulinaemia, as well as increased beta cell viability, proliferation and islet area (1.9-fold increase at 10 weeks of age). Hyperinsulinaemia led to insulin resistance, which was aggravated by a high-fat/high-sugar diet and weight gain, although beta cells maintained their insulin secretory capacity in response to glucose. Strikingly, islets from β-CB1R−/− mice were protected from diet-induced inflammation. Mechanistically, we show that this is a consequence of curtailment of oxidative stress and reduced activation of the NLRP3 inflammasome in beta cells.
Our data demonstrate CB1R to be a negative regulator of beta cell function and a mediator of islet inflammation under conditions of metabolic stress. Our findings point to beta cell CB1R as a therapeutic target, and broaden its potential to include anti-inflammatory effects in both major forms of diabetes.
Microarray data have been deposited at GEO (GSE102027).
KeywordsBeta cell Beta cell proliferation Beta cell viability Cannabinoid 1 receptor Diabetes Inflammation Insulin Insulin resistance Islet of Langerhans Mouse
Cannabinoid 1 receptor knockout
Cannabinoid 1 receptor
Beta cell specific cannabinoid 1 receptor knockout
Cannabinoid 2 receptor
Comprehensive Lab Monitoring System
Extracellular acidification rate
Fasting blood glucose
Gastric inhibitory polypeptide
High glucose and palmitate
Mitogen-activated protein kinase
NLR family, pyrin domain containing 3
Oxygen consumption rate
Respiratory exchange rate
Reactive oxygen species
Standard diet (assignment group)
Insulin secretion is tightly controlled as dysregulation has life-threatening consequences. When chronically stimulated, beta cell function deteriorates, and insufficient insulin secretion, especially when coupled with insulin resistance, causes glucose elevation and type 2 diabetes. Glucose metabolism is the primary trigger for insulin secretion, while other factors such as gastric inhibitory polypeptide (GIP) and glucagon-like peptide (GLP-1), cholecystokinin, amino acids (leucine and arginine) and acetylcholine enhance glucose-mediated insulin secretion [1, 2]. Negative regulators exist, with adrenaline (epinephrine), noradrenaline (norepinephrine) and somatostatin being the most studied [1, 3]. The endocannabinoid system (ECS) also has a regulatory role in beta cells. In humans and rodents, beta cells express the cannabinoid 1 receptor (CB1R, encoded by Cnr1), synthesise endocannabinoids (ECs) in a glucose-dependent manner [4, 5] and contain the enzymes necessary for EC degradation [4, 6, 7, 8]. In obesity, the ECS becomes overactive [8, 9].
CB1R is coupled to the G protein type Gi/o, Gαi, which inhibits adenylyl cyclase (AC) and cAMP–protein kinase A signalling, activates mitogen-activated protein kinases (MAPKs) and stimulates ceramide synthesis in many tissues [10, 11, 12, 13]. It also inhibits voltage-gated L-, N- and P/Q-type Ca2+ channels, inwardly rectifying K+ channels and leading to inhibition of signal transmission and release of secretory products [14, 15]. These complex but connected signalling pathways impact on many physiological processes, including exocytosis, cell survival and differentiation, metabolism and immune cell responses [11, 16]. Coupling CB1R to the Gαi protein indicates a potential negative regulatory role in beta cells, where AC positively regulates insulin secretion and beta cell function. We and others have dissected the actions of CB1R in metabolically active tissues using whole-body Cnr1 (CB1R) knockout (CB1KO) rodents [17, 18] and pharmacological approaches [8, 19, 20, 21, 22, 23, 24]. Nonetheless, the complexity of CB1R’s action, its presence in numerous tissues and the potential off-target effects of pharmacological approaches have confounded the study of CB1R in beta cells. The effects of specifically targeting CB1R in pancreatic beta cells and whether this has direct effects on beta cell function and/or whole-body metabolism have not been studied.
To further interrogate the functions of CB1R in adult beta cells, and to build upon previous work on CB1R blockers as therapeutic agents for obesity-related disorders, we generated an inducible beta cell specific CB1R knockout (β-CB1R−/−) mouse and studied the implications of CB1R ablation in beta cells under conditions of acute and chronic insulin resistance in vivo and ex vivo.
For detailed methods, please refer to the electronic supplementary material (ESM) Methods.
Animal care and procedures were approved by the National Institute on Aging Animal Care and Use Committee. Mice were housed in groups of four using 12 h dark/light cycles, provided with water and fed ad libitum. Cnr1flox/flox mice were generated as described in the ESM Methods. Cnr1flox/flox mice were mated with MIP-Cre/ERT mice (University of Chicago, Chicago, IL, USA) and injected daily for 5 days with i.p. tamoxifen to obtain β-CB1R−/− mice (Cnr1flox/flox:MIP-Cre/ERT+), β-CB1R+/+ control littermates (Cnr1flox/flox:MIP-Cre/ERT−) and MIP-Cre/ERT mice (Cnr1wt/wt:MIP-Cre/ERT+). CB1KO mice (NIH, Bethesda, MD, USA) were bred as described in the ESM Methods. Male mice (n = 6–7 mice/group) were aged to 25 weeks and body weights and metabolic variables were analysed. Body composition was analysed using NMR. Pancreases were fixed and processed for immunohistochemistry (anti-insulin [1:100; Dako], anti-glucagon [1:500; Sigma-Aldrich], anti-BrdU [1:100; Accurate Chemicals]). Islet size was quantified using Pancreas++ . Hormones were quantified using ELISA. Methanol–chloroform-extracted ECs from plasma were analysed using LC-MS/MS, as described in the ESM Methods.
Induction of acute insulin resistance
Male mice (n = 8–11 mice/group) were randomised to receive vehicle or S961 (0.05 nmol/h) via ALZET miniosmotic pumps. Blood glucose and insulin levels were measured from tail bleed at 0–6 days; mice were injected with BrdU (0.1 nmol/g) and euthanised 12 h later.
Male mice (6–8 weeks old; n = 6–7/group) were randomised to a standard diet (16.7% kJ fat and 12.4% kJ sugar wt/wt) or high-fat/high-sugar diet (HFHS; 49.2% kJ fat and 32.2% kJ sugar wt/wt) for 15 weeks. Body weight and food consumption were measured weekly. At the end of the study, GTTs and metabolic measurements were performed. Metabolic measurements were measured using a Columbus Instruments Comprehensive Lab Monitoring System (CLAMS; Columbus Instruments), as described in ESM. Tissues (pancreas, liver and white adipose tissue) were dissected, weighed and flash frozen or fixed for immunohistochemistry (anti-CD3, anti-CD68, anti-thioredoxin-interacting protein [TXNIP] and anti-phospho [p][S536]-p65 [1:100; Abcam], anti-ceramide [1:10; Enzo Life Sciences]). Immunoprecipitated insulin receptor (IR) or IRS-2 from liver protein extracts (anti-IRS2 [Cell Signaling], anti-IR [Santa Cruz Biotechnology]; 1 μg) were subjected to Tris-glycine PAGE, immunoblotted with anti-IRS2 or anti-p-Tyr (1:1000; Santa Cruz Biotechnology), visualised by enhanced chemiluminescence and quantified using ImageJ.
Ex vivo analysis of viability and metabolism of isolated islets
Freshly isolated islets were cultured in non-stimulatory conditions (2 mmol/l glucose) to quantify basal cAMP (n = 100 islets/mouse) and resting insulin secretion (n = 40 islets/mouse). Islets were cultured with exendin-4 (Ex-4) and 3-isobutyl-1-methylxanthine (IBMX; 25 μmol/l; Sigma-Aldrich), and intra-islet cAMP levels and insulin secretion were quantified. Islets (n = 100–120 islets/mouse) were infused with glucose (7.5 mmol/l) in a perifusion system and insulin secretion was quantified. Freshly isolated islets (n = 30 islets/group) were used to measure the oxygen consumption rate (OCR), extracellular acidification rate (ECAR) using an XFe24 Seahorse Analyzer (Agilent) and intra-islet levels of reactive oxygen species (ROS) as described in ESM. Islets (n = 30–50 islets/group) were cultured in the presence or absence of 500 μmol/l palmitate and 16.5 mmol/l glucose for 24 h. Islet viability, cAMP levels, mRNA expression (Il1b, Nlrp3, Tnfa [also known as Tnf], Ldha), cytokine secretion (IL-1β, TNF-α), protein phosphorylation (p-Erk1/2, p-SAPK/JNK, p-p38, p-p53, p-Stat1, p-HSP27) and cleavage (caspase 3, PARP) were measured. Islets (n = 15 islets/group) were incubated with a mixture of cytokines (10 ng/ml IL-1β, 50 ng/ml TNF-α and 50 ng/ml IFN-γ) or vehicle for 18 h and cytotoxicity was assayed. There were at least three replicates in all experiments.
Pancreatic lymphocytes were isolated after pancreas perfusion and cells were stained using PE anti-mouse CD8a and FITC anti-mouse CD69 (Biolegend), as detailed in the ESM Methods. Populations were determined by using BD FACSCanto II and BD FACSDiva software (BD Biosciences).
Microarray experiments and analysis were performed as previously described .
Method of randomisation
Age and sex-matched littermate mice were randomly assigned to vehicle, S961, standard diet or HFHS.
Quantification and statistical analysis
Data are presented as means ± SEM. No statistical method was used to predetermine the sample size. Differences between mean values for variables within individual experiments were compared statistically using Student’s t test or ANOVA, as appropriate. Comparisons were performed using GraphPad Prism version 6.0. p ≤ 0.05 was considered statistically significant.
β-CB1R−/− mice have increased beta cell proliferation and improved glucose homeostasis
CB1R ablation in beta cells reduced hyperglycaemia during acute insulin resistance
β-CB1R−/− mice fed an HFHS diet gained more weight and became more glucose intolerant than β-CB1R+/+ mice
CB1R ablation in beta cells leads to enhanced function in isolated islets
CB1R ablation in beta cells leads to a metabolic shift and reduces ROS production
CB1R ablation in beta cells protects islets from metabolic stress and inflammation
Our study describes the role of CB1R specifically in pancreatic beta cells and its relevance to whole-animal physiology. By genetically ablating CB1R in adult beta cells, we conclude that CB1R is a negative regulator of beta cell function, proliferation and viability, and is a key component of the inflammatory response of islets. ECs orchestrate the organisation of pancreatic islets in perinatal development . Ablating CB1R in adult mice avoids potential alterations in islet structure. Previous reports related to the ECS in beta cells have been conflicting due to the influence of CB1R in other tissues involved in glucose homeostasis and off-target effects of pharmacological approaches; other non-CB1R cannabinoid receptors have been described in islets [5, 47]. While whole-body genetic or pharmacological CB1R blockade has been reported to result in increased insulin secretion [4, 24], enhanced beta cell function may be a consequence of reduced insulin resistance in liver and muscle [10, 11], producing a favourable environment for beta cells. Our model definitively describes the importance of CB1R in beta cell biology and function by eliminating these confounding factors and, for the first time, describes the direct effects of CB1R on beta cell mitochondria. CB1R ablation in beta cells resulted in a metabolic shift towards reduced mitochondrial respiration that protected islets from oxidative stress and improved beta cell functional capacity. Unexpectedly, ablation of CB1R in beta cells was sufficient to prevent the inflammatory response in islets under the metabolic stress of an HFHS diet.
Ablation of CB1R, a constitutively active Gαi protein-coupled receptor and AC inhibitor, resulted in increased intra-beta cell cAMP and earlier onset of in vivo and ex vivo insulin secretion. We propose that increased insulin secretion led to increased levels of circulating insulin in β-CB1R−/− mice, eventually resulting in insulin resistance. Isolated β-CB1R−/− islets confirmed increased insulin secretion as a primary event, and our mouse model provides insight into the response of the whole body to primary hyperinsulinaemia. Excessive unremitting insulin secretion due to a primary beta cell event, such as overexpression of the Ins gene or chronic exogenous administration of insulin, results in insulin resistance in the liver, weight gain, increased adiposity, ectopic adipose deposition and loss of the ability to increase glucose-stimulated insulin secretion [48, 49]. Primary hyperinsulinaemia with normal blood glucose levels occurs with inactivating mutations of the SUR1 subunit of the K+-ATP channel in mice; however, these mice lose their glucose-stimulated insulin secretion capacity time . Therefore, insulin resistance in HFHS-β-CB1R−/− mice is probably a homeostatic response to prevent hypoglycaemia. However, unlike in previous genetic models of primary hyperinsulinaemia, beta cell dysfunction was not observed in HFHS-β-CB1R−/− mice. This is due to a myriad of beneficial intracellular events, an increased beta cell number and a reduced intracellular inflammatory environment. These data underline the potential of CB1R blockade as a treatment for type 2 diabetes compared with classic treatments that elevate insulin secretion, such as sulfonylureas, which do not integrate other signals in beta cells.
An HFHS diet led to greater weight gain in β-CB1R−/− mice. SD- or HFHS-CB1KO mice do not gain weight due to reduced food intake because of CB1R ablation in the brain [51, 52]. Chronic hyperinsulinaemia per se may drive fat deposition in HFHS-β-CB1R−/− mice as it stimulates insulin signalling in white adipose tissue and increases body weight [49, 53, 54]. This is substantiated by our observation that HFHS-β-CB1R−/− mice had a lower RER and decreased activity relative to HFHS-β-CB1R+/+ mice, resulting in less glucose uptake by muscle and increased fat deposition. A lower RER in HFHS-β-CB1R−/− mice during daylight probably indicates that the two strains are not using the same stored fuel source. HFHS-β-CB1R−/− mice have more subcutaneous adipose tissue, which may alter the ‘accessibility’ to fuel in the resting state compared with HFHS-β-CB1R+/+ mice.
Recently, CB1R was found in the mitochondrial membranes of neurons and muscle [32, 33], but its role in mitochondria has not been described in beta cells. Some reports have indicated that CB1R activation decreases mitochondrial respiration  while others have reported the opposite . We found a reduction in mitochondrial respiration in β-CB1R−/− islets and increased Ldha expression under the metabolically stressful condition of HGP. Decreased mitochondrial respiration possibly results from nullifying CB1R in beta cell plasma membranes. This could be the result of two effects: increased intracellular cAMP that induces Ldha expression  and/or increased efficiency of insulin secretion that requires less mitochondrial activation. Regardless, the reduction in mitochondrial respiration led to reduced ROS, thereby probably preventing oxidative stress damage.
In summary, CB1R exerts control over beta cell function, proliferation, viability and intracellular signalling (Fig. 8). We can now speculate why an autonomous ECS exists in beta cells. Many toxins that are no longer a threat to humans in the developed world because of vaccinations and antibiotics increase intra-beta cell cAMP, leading to possible death from hypoglycaemia because of hyperinsulinaemia. The presence of a robust intrinsic system, such as the ECS, to reduce intracellular cAMP and limit insulin secretion under duress probably provided a survival advantage. Simultaneously enhancing the ability of beta cells to attract immune cells to eliminate bacterial antigens, cell debris, islet amyloid polypeptide deposition, toxins and plasmids would also have been beneficial. We postulate that the ECS evolved in islets to serve a dual function: to diminish the effects of AC activators while increasing the ability of beta cells to protect themselves. Islets express all the machinery to synthesise ECs on demand [4, 5] and, similar to insulin, ECs (mainly 2-arachidonoylglycerol) are secreted from the islets upon glucose stimulation . Unfortunately our Westernised diets, high in fats and glucose, lead to a near-constant demand for insulin, increased synthesis of islet amyloid polypeptide and possibly increases in bacterial antigens from the gut, increased overall chemokine production, intra-islet inflammation, islet EC secretion, and over-activation of the islet ECS, eventually leading to beta cell dysfunction and hyperglycaemia. Pharmacological blockade of CB1R or hepatocyte CB1R nullification improves insulin action and reduces fat deposition in the liver [12, 19]. Furthermore, CB1R inhibition in the periphery lowers insulin resistance [12, 19]. We now report that CB1R blockade improves beta cell function and protects against HFHS-induced islet inflammation, and may represent a therapeutic strategy in diabetes and impaired glucose tolerance. It has recently been reported that peripheral CB1R blockade in mice with impaired glucose tolerance improved glucose handling and resulted in several anti-inflammatory and cytoprotective actions . Nonetheless our study has the limitation that the rodent and human islet ECS are not identical, and further studies in humans are therefore needed. Previously , we found that human beta cells and hepatocytes, but not brain, predominantly express an isoform of CB1R, CB1b. Pharmacological blockade or morpholino oligos  to block CB1b mRNA transcription may lead to novel therapies for type 2 diabetes.
MIP-Cre/ERT mice were kindly donated by L. Philipson (Department of Medicine, University of Chicago, Chicago, IL, USA). S961 was a donation from Novo Nordisk. JD-5037 was a donation from Jenrin Discovery (J. F. McElroy and R. Chorvat). The study sponsors were not involved in the design of the study, the collection, analysis or interpretation of data, writing the report, or the decision to submit for publication.
IGM conceptualised, designed and performed the majority of experiments, and analysed, interpreted and organised the data. JME conceptualised and designed the experiments, and interpreted the data. IGM and JME drafted the article. RAM performed and analysed immunohistochemistry staining. MED performed the islet perifusion. Q-RL, EL, YZ and KGB performed and analysed the microarray. SSC carried out immune cell isolation and profiling by flow cytometry. SG quantified ECs. All authors substantially contributed to acquisition, analysis and interpretation of data. All authors reviewed and approved the published version of the article. JME is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This work was supported by the Intramural Research Programs of the National Institute on Aging in the National Institutes of Health.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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