Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Abstract Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R −/−) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R −/− mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.


| INTRODUCTION
Insulin-producing pancreatic b cells have to secrete insulin in a way that maintains the blood glucose level in a narrow range at all times.
Glucose is the primary stimulus for insulin secretion, a process that requires glucose sensing. 1 Pancreatic b cells can sense and respond to changing blood glucose concentrations with the help of glucose transporter 2 (GLUT2) and glucokinase (GCK), which are the glucosesensing machinery. 1 Blood glucose is transported into the b cells through GLUT2, and GCK traps glucose in the cytoplasm by immediate phosphorylation. Glucose metabolism in b cells generates ATP that inhibits potassium efflux through K ATP channels, resulting in b-cell membrane depolarization and entry of extracellular Ca 2+ through voltage-dependent Ca 2+ channels, which then initiate insulin release. The glucose dependency of insulin secretion is greatly enhanced by increased adenylyl cyclase (AC) activity and in the fed state, enhanced AC activity is generally controlled by the incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP).
The binding of GLP-1 to its specific receptors (GLP-1Rs) on b cells results in activation of AC, cAMP production and subsequent activation of PKA and the Epac family. This sequence of events leads to enhanced glucose-dependent exocytosis of insulin from insulin-secretory vesicles 2,3 and other downstream effects such as improved insulin biosynthesis and increased pro-insulin, GLUT2 and GCK expressions. [4][5][6] Accordingly, altered glucose-sensing machinery of b cells leads to defects in glucose-stimulated insulin secretion (GSIS).
Indeed, islets from diabetic Zucker rats have impaired GSIS due to defective glucose sensing associated with a reduction in the expressions of GLUT2 and GCK. 7 Moreover, b-cell-specific knockout of GLUT2 or GCK led to severe hyperglycaemia and infant death due to impaired GSIS and, conversely, re-expression of GLUT2 in b cells rescued GLUT2-null mice from infant death and restored normal GSIS. 8,9 b cells synthesize and secrete insulin as well as the known endogenous cannabinoids (ECs). [10][11][12] The ECs, 2-arachidonoylglycerol  and anandamide (AEA), are lipid transmitters that are not pre-stored in granules, but are synthesized and released from cells only "on demand" by Ca 2+ -dependent enzymes in the brain, fat, macrophages, and liver, in addition to islets, following membrane depolarization. [10][11][12][13][14][15] Thus, both insulin secretion and EC synthesis are controlled by cell depolarization and Ca 2+ mobilization. The biological effects of ECs are mediated by two G protein-coupled receptors (CB1R and CB2R), which, when activated, inhibit AC activity and cAMP accumulation. 15,16 CB1Rs are expressed in neurons and peripheral tissues such as liver, adipose tissue and skeletal muscle and contribute to food intake and peripheral metabolic regulation including lipid and glucose homoeostasis and insulin sensitivity. [17][18][19] In fact, as ECs and CB1Rs are overactive in obesity and type 2 diabetes (T2D), many scientists have been interested in blockade of CB1R as a new therapeutic approach for the treatment of obesity-related diseases. In addition, the majority of reports, including our previous study, [20][21][22] found that rodent and human b cells express CB1Rs, whereas CB2Rs were not expressed in b cells 11,20,23,24 ; however, there are also reports to the contrary. [10][11][12]23,25 EC treatment of isolated mouse islets was reported to inhibit GSIS and lower intracellular Ca 2+ levels 24 ; there are similar findings from another group. 25,26 However, the published literature is conflicting as regards the coupling of CB1Rs with Gai or Gas as well as their effects on insulin secretion. It has been reported that in certain circumstances, CB1R stimulation leads to Gas coupling, AC activation and increased GSIS. 11,16,[27][28][29][30][31] In addition, there are reports of EC treatment of mouse islets leading to decrease in cAMP, 16,28 while increasing intracellular calcium and insulin secretion. 16 Here, we evaluated human islets, several b-cell lines and CB1R-null (CB1R À/À ) mice in order to outline what the relevance of b-cell-derived ECs might be to AC activity and insulin secretion. Moreover, we also investigated the effect of CB1Rs on the expression of pro-insulin, GLUT2 and GCK.

| Materials and reagents
The Rat/Mouse Insulin ELISA Kit was from Linco Research, and the cAMP EIA kit was from Assay Designs. WIN55,212-2, CP 55,940, arachidonyl-2 0 -chloroethylamide (ACEA) and AM251 were obtained from Cayman Chemical. Exendin-4 (Ex-4) was obtained from Bachem. The human CB1R cDNA was amplified by RT-PCR from human pancreatic RNA (Stratagene), with oligo-dT (18 bp) for the reverse transcription. The CB1R cDNA was incorporated into a mCerulean-N1 vector for CB1R-cerulean with the cerulean epitope.

| Mice
CB1R À/À mice and their wild-type littermates were developed and backcrossed to a C57Bl/6J background as previously described. 34 Two-to three-month-old male CB1R +/+ and CB1R À/À mice were used for this study. All animal care and experimental procedures followed National Institutes of Health guidelines and were approved by the National Institute on Aging Animal Care and Use Committee.

| Islet isolation and measurement of insulin content and secretion in islets
Freshly isolated islets of Langerhans from human cadaveric donors were obtained from the Islet Cell Resource Center. Mouse islets were isolated from CB1R À/À and age-matched CB1R +/+ mice using collagenase digestion as previously described. 35 For insulin secretion assays, we picked 10 size-matched islets per tube in Krebs buffer containing 4 mmol/L glucose followed by 30-minutes incubation at 37°C. We pelleted the islets and replaced the buffer with Krebs solution containing the indicated glucose concentration. After 10-minutes incubation at 37°C, we collected the supernatant for insulin measurement and measured insulin content or protein concentration for normalization.
Insulin levels were determined from three independent experiments performed in triplicate. Intra-islet insulin content was measured using ice-cold acid-alcohol as we previously described. 6

| Immunostaining
For frozen sections, mice were anaesthetized, and pancreata were rapidly dissected, fixed in 4% paraformaldehyde, immersed in 20% sucrose before freezing and then sectioned at a thickness of 7 lm.
Using LSM Image Browser software (Carl Zeiss), multiple sections from three mice per genotype, separated by at least 200 lm from each section, were assessed for quantification of GCK and GLUT2.

| Quantitative real-time PCR analysis
Total RNAs were isolated from frozen mouse islets or bTC6 cells using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's protocol. All extractions were followed by DNase I treatment (Invitrogen). After reverse transcription (RT) using random hexamers and reverse transcriptase (Toyobo, Osaka, Japan), the mRNA abundance was assessed using a CFX Connect TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the SYBR green PCR master mix (Kapa Biosystems, Wilmington, MA, USA). The 18S gene was used for normalization to quantify the relative mRNA expression levels. Gene-specific primer sequences were summarized in Table S1.

| Immunoblotting
Protein samples were extracted from cells using RIPA buffer Homogenates were solubilized by end-over-end mixing at 4°C for 60 minutes and subjected to centrifugation. Total protein was determined using Bradford assay (Bio-Rad).   Figure 2B). Using human islets, we further confirmed that the insulinotropic effects of Ex-4 were enhanced by blocking CB1Rs with AM251, a CB1R antagonist ( Figure 2C). These results prove that ECs antagonize incretin-mediated effects in b cells.

| Statistical analysis
We then examined the effect of CB1R on GSIS. As expected, pharmacological activation of CB1R by WIN55,212-2 inhibited GSIS from bTC6 cells in a concentration-dependent manner, and this was counteracted by pre-incubation of the cells with AM251 ( Figure 2D). Furthermore, ACEA and 2-AG inhibited GSIS from human islets ( Figure 2E). Overall, these results favour the conclusion that ECs are inhibitory mediators of AC activity and insulin secretion in b cells.

| Effects of CB1R ablation on gene expression
Next, we measured the abundance of intra-islet insulin and insulin mRNA in b cells. Knockdown of CB1Rs by siRNA in bTC6 cells enhanced insulin gene expression ( Figure 3A). Freshly isolated islets from CB1R À/À mice had increased insulin content after acid-alcohol extraction compared to those of age-matched wild-type (CB1R +/+ ) littermates ( Figure 3B), most likely as a direct result of up-regulation of insulin gene expression ( Figure 3C). Consistently, Western blot analysis confirmed that pancreas of CB1R À/À mice had significantly increased level of pro-insulin compared to those of age-matched CB1R +/+ mice ( Figure 3D). In addition, we measured protein levels of glucose-sensing apparatus involved in the early steps of GSIS and glucose metabolism in b cells. Western blot analysis showed that pancreas of CB1R À/À mice had significantly increased level of GCK and GLUT2 compared to those of CB1R +/+ mice ( Figure 3D). Consistently, levels of GCK ( Figure 3E) and GLUT2 ( Figure 3F) in fasting F I G U R E 2 Effects of CB1R agonists on insulin secretion. A and B, Relative intracellular cAMP (A) and insulin (B) concentrations secreted from bTC6 cells treated with the synthetic CB1R agonist WIN55,212-2 before the subsequent addition of Ex-4. C, Effects of blocking CB1R on Ex-4mediated insulin secretion in human islets. Human islets were pre-treated with a CB1R inverse agonist AM251 before the subsequent addition of Ex-4. D, Relative insulin concentrations secreted from bTC6 cells in response to glucose in the absence or presence of WIN55,212-2, AM251 or both. E, Relative insulin concentrations secreted from human islets in response to glucose in the absence or presence of ACEA or 2-AG. All values were normalized to protein concentration. Data are shown as the mean AE SEM from three independent experiments. *P < .05; **P < .01 state were significantly higher in b cells of CB1R À/À mice compared to those of CB1R +/+ mice.  Figure 4C), suggesting that CB1R regulates the expression of insulin, GCK and GLUT2 by inhibiting IR signalling ( Figure 4D). F I G U R E 3 Effects of blocking CB1R on intra-b-cell insulin content and glucokinase (GCK) and glucose transporter 2 (GLUT2) expressions. A, CB1R and Insulin (Ins2) mRNA levels in bTC6 cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. B, Intra-islet insulin content in islets isolated from CB1R +/+ and CB1R À/À mice. Insulin was extracted from islets freshly isolated from CB1R +/+ and CB1R À/À mice using acid-alcohol (n = 3 separate isolates). Size-matched 10 islets per tube were analysed, and data were normalized to protein concentration. C, Insulin (Ins1 and Ins2) mRNA levels in islets isolated from CB1R +/+ and CB1R À/À mice (n = 4 separate isolates). Data were normalized to 18S ribosomal RNA levels. D, Western blot analysis of preproinsulin, GCK and GLUT2 expressions in total lysates prepared from whole pancreata of overnight-fasted CB1R +/+ and CB1R À/À (n = 4 per genotype) mice. Signals on Western blots were quantified by densitometry and are shown on the right. E and F, Immunofluorescent staining for GCK (E) and GLUT2 (F) in islets of overnight-fasted CB1R +/+ and CB1R À/À mice. Scale bar, 50 lm. Relative signal intensity for the indicated proteins in islets is shown on the right (n = 3 per genotype). Quantification of GCK and GLUT2 intensities was shown on the right. Data are shown as the mean AE SEM from three independent experiments. *P < .05; **P < .01

| DISCUSSION
As mentioned in the introduction, there are many reports of CB1R expression on pancreatic b cells in mouse and human 10,11,16,23,24,38,39 and we concur. [20][21][22][23] Recent reports, including our own, 20 have also found that b cells contain the other components of EC system including the necessary enzymes for their biosynthesis and degradation, and have the capacity to generate ECs in response to glucose stimulation even when islets are isolated from the pancreas. [10][11][12]20 As EC synthesis and insulin secretion are controlled by membrane depolarization and Ca 2+ mobilization, [10][11][12]15,20,40 this supports the notion that the metabolically derived stimuli to insulin secretion also lead to EC generation and therefore should mean that insulin secretion and EC generation are proportional to one another.
We have now found that ECs inhibit AC activity and that they inhibit cAMP accumulation in b cells, which results in diminished insulin secretion. Therefore, it seems reasonable to conclude that ECs limit insulin secretion under physiological conditions. Activated CB1Rs are coupled to Gai class of heterotrimeric G proteins and can initiate signalling events including closure of Ca 2+ channels, opening of K + channels and inhibition of AC activity (with its consequent decrease in cytosolic cAMP concentrations), resulting in inhibition of neurotransmitter release. 40 However, the effects of CB1Rs on insulin release from b cells have not been firmly established, and the available data are occasionally inconsistent. We found that synthetic and endogenous ligands of CB1Rs reduced cAMP accumulation and insulin secretion in b cells and the CB1R inverse agonist prevented such effects. Moreover, Ex-4-and forskolinmediated intracellular accumulation of cAMP was reduced by CB1R agonism in CB1R-transfected CHO-GLP-1R cells, but not in vectortransfected cells that intrinsically lack CB1Rs, further eliminating the possibility of any non-specific effects of CB1R ligands. This is consistent with recent reports that show decreased intracellular cAMP levels in isolated islets 16 and MIN6 cells 28 due to ACEA. Taken together, we conclude that ECs influence insulin secretion in an autocrine manner by acting as a brake on AC activity.
Moreover, knockout of IR abolished the ability of CB1R to regulate the expression of insulin, GCK and GLUT2, indicating that CB1Rs regulate their expression in IR signalling-dependent manner. All of these factors combined are likely to be responsible for improved insulin secretion by CB1R antagonism. The molecular basis of the defective insulin secretion involves defects in the signal transduction pathways, especially such as insulin receptor signalling pathway, that are activated by increased glucose in pancreatic b-cells. [41][42][43] b-cell dysfunction due to defective insulin receptor signalling leads to alterations in mechanisms that are responsible for the production and secretion of insulin, and this alteration involves a reduction of GLUT2 and GCK expression. 44 bIRKO mice showed defective GSIS, progressive glucose intolerance, as well as reduction in GCK and GLUT2 gene expression in the islets of both non-diabetic and diabetic bIRKO mice. 44 In addition, islets from diabetic Zucker rats showed impaired GSIS associated with a reduction in the expressions of GLUT2 and GCK, 7 and mice with a b-cell-specific deletion of glucose-sensing machinery revealed severe hyperglycaemia and F I G U R E 4 Effects of CB1R on gene expression depends on insulin receptor (IR). A, Western blot analysis of IR b-subunit (IRb) and GAPDH in bIRWT and bIRKO cells. B, Quantitative real-time PCR analysis of CB1R expression in bIRWT and bIRKO cells. Data were normalized to 18S ribosomal RNA levels. C, Quantitative realtime PCR analysis of CB1R, insulin, GCK and GLUT2 expression in bIRWT and bIRKO cells transfected with control (siCtrl) or CB1R (siCB1R) siRNA. D, Schematic unifying the regulation of b-cell function by ECs and CB1Rs. Data are shown as the mean AE SEM from three independent experiments. *P < .05; **P < .01 infant death due to impaired GSIS. However, re-expression of GLUT2 in b cells rescued the mice from infant death and restored normal GSIS, 8,9 showing that altered glucose-sensing machinery of b cells leads to changes in GSIS. Taken together, it is possible to hypothesize that blockade of CB1R increases insulin secretion and improves insulin responsiveness, at least in part, by up-regulation of insulin, GCK and GLUT2 gene expressions.
EC levels in circulating blood as well as the pancreas are reported to be elevated in diabetes and obesity, 10,12,45,46 but it is highly unlikely that islet-derived ECs contribute to blood levels as they degrade within the islets. However, it is possible that increased EC tone (due to increased EC synthesis, receptor expression or activity) affects the well-described incretin and glucose unresponsiveness of b cells in type 2 diabetes. Additionally, it was recently found that AEA impaired insulin-stimulated AKT phosphorylation and decreased glucose uptake in skeletal muscle cells, 47 while CB1R antagonism enhanced insulin responsiveness in skeletal muscle. 48 Therefore, CB1R antagonists that have poor brain penetrance (to lessen CNS side effects) might be useful as therapeutic agents in type 2 diabetes where they would be expected to improve b-cell function, improve glucose uptake into muscle and prevent or reduce hepatic steatosis.