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Expression and function of cannabinoid receptors in mouse islets Chen Li, James E. Bowe & Peter M. Jones Published online: 01 Sep 2010.
To cite this article: Chen Li, James E. Bowe & Peter M. Jones (2010) Expression and function of cannabinoid receptors in mouse islets , Islets, 2:5, 293-302, DOI: 10.4161/isl.2.5.12729 To link to this article: http://dx.doi.org/10.4161/isl.2.5.12729
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research paper
Research paper
Islets 2:5, 293-302; September/October 2010; ©2010 Landes Bioscience
Expression and function of cannabinoid receptors in mouse islets Chen Li, James E. Bowe, Peter M. Jones and Shanta J. Persaud* Diabetes Research Group; King’s College London; London, UK
Key words: mouse islets, insulin secretion, cannabinoids, cyclic AMP, calcium
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Abbreviations: ACEA, arachidonyl-2-chloroethylamide; AEA, anandamide; 2-AG, 2-arachidonylglycerol; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; cyclic AMP, cyclic adenosine 3', 5'-monophosphate; DAB, 3,3'-diaminobenzidine; ECL, enhanced chemiluminescence; FITC, fluorescein isothiocyanate; JWH015, (2-methyl-1-propyl-1H-indol-3-yl)-1naphthalenylmethanone
The endocannabinoid system plays a key role in energy homeostasis, with agonists and antagonists of CB1 receptors acting centrally to stimulate and inhibit food intake, respectively. In addition to their established effects on the central nervous system, cannabinoid receptor agonists also exert peripheral effects by modulating cellular cyclic AMP and calcium levels and there have been reports that they regulate β-cell function. However, the few reports to date on islet expression of cannabinoid receptors and effects of agonists on insulin secretion have failed to reach a consensus. We have therefore investigated cannabinoid receptor expression by mouse islet β- and α-cells and the effects of selective receptor agonists on cyclic AMP and calcium levels, and on dynamic insulin secretory responses. CB1 and CB2 mRNA and protein expression by islets was detected by RT-PCR and western blotting respectively, and cellular location of the receptors was identified by immunohistochemistry with insulin and glucagon antibody co-staining. Cyclic AMP generation was quantified by enzyme immunoassay and changes in calcium levels were measured by microfluorimetry of Fura-2-loaded mouse islet cells. Dynamic insulin secretion was quantified by radioimmunoassay after perifusion of isolated islets. We found that mouse islets expressed both CB1 and CB2 receptors, and they were localized to β-cells. Activation of mouse β-cell CB1 and CB2 receptors resulted in decreased cyclic AMP, increased calcium and potentiation of glucose-stimulated insulin secretion. Thus, activation of islet cannabinoid receptors by locally produced endocannabinoids such as 2-aminoglycerol may be another regulatory pathway by which islets stimulate insulin secretion to maintain glucose homeostasis.
Introduction The endocannabinoid system is composed of a family of locally produced, endogenous cannabimimetic phospholipid derivatives, the cannabinoid receptors and enzymes for endocannabinoid biosynthesis and degradation. Two subtypes of cannabinoid receptors, CB1 and CB2, have been cloned, both of which belong to the classic seven transmembrane G-protein-coupled receptor family.1-3 CB1 receptors are ubiquitously expressed, with high levels of expression in areas of the brain involved in regulation of appetite and satiety, body temperature, locomotion and mood.4-8 They have also been identified in peripheral tissues such as liver, skeletal muscle, gastrointestinal tract and adipose tissue.9-12 In contrast, CB2 receptors are reported to be localized mainly to immune cells, such as macrophages, B lymphocytes and NK cells, although they are also present in the brainstem,13 and other brain regions.14,15 In the CNS the endocannabinoid system is activated when endogenous cannabinoids are produced on demand by Ca 2+ -sensitive enzymes and inactivated by agonist
uptake from the extracellular space and intracellular enzymatic hydrolysis.8,16 The activation of both cannabinoid receptor subtypes causes downstream effects via heterotrimeric Gi/o-proteins, which are coupled to inhibition of adenylate cyclase.17,18 Cannabinoid receptor activation is reported to decrease intracellular Ca 2+ levels through closure of voltage-gated Ca 2+ channels in neuronal cells, 19 but there are also reports that cannabinoids can induce transient elevations in Ca 2+.20 The effects of cannabinoids on alterations in Ca 2+ levels in ATt20 anterior pituitary cells are thought to occur via CB1 receptors since overexpression of CB2 receptors did not affect Ca 2+ channel activity.21 The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) are most abundant in the key brain areas participating in the regulation of food intake and satiety,22-25 and CB1 receptors have been pharmacologically targeted to provide therapies for appetite suppression. Thus, rimonabant, a CB1 receptor antagonist, has been used clinically as an anti-obesity agent, and there is considerable interest in the regulatory role of
*Correspondence to: Shanta Persaud; Email:
[email protected] Submitted: 05/24/10; Revised: 06/11/10; Accepted: 06/12/10 Previously published online: www.landesbioscience.com/journals/islets/article/12729 DOI: 10.4161/islets.2.5.12729 www.landesbioscience.com Islets
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effects of CB1 and CB2 receptor activation on insulin secretion from mouse islets.
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Results
Figure 1. Cannabinoid receptor expression by mouse islets. (A) Amplicons of the correct sizes coding for CB1R (351 bp) and CB2R (506 bp) cannabinoid receptor subtypes were amplified from mouse islet cDNA. No products were amplified when non-reverse-transcribed RNA was used as a template. Results are representative of data obtained from 3 mouse islet preparations. (B) 20 µg of mouse islet and cerebral cortex (mouse brain) extracts were fractionated on 10% polyacrylamide gels and CB1 (64 kDa) and CB2 (35 kDa) receptors were identified by western blotting. Data are representative of 6 experiments using samples obtained from six mice.
CB1 receptors in metabolic balance. There is less information available on the functional roles of CB2 receptors, but several studies have indicated their importance in anti-inflammatory responses.26,27 Given the key roles of islet β-cells in maintaining fuel homeostasis and the established involvement of the central endocannabinoid system in energy balance regulation, the potential role of cannabinoids in regulating insulin secretion has attracted attention in recent years. It has been reported that the endocannabinoids 2-AG and anandamide are generated by RINm5F cells,28 but the currently available literature on islet cannabinoid receptor expression and function is confusing and often contradictory. Thus, there are reports of CB1 and/or CB2 receptor expression by islets,29-35 but there is no consensus on whether these receptors are expressed by β-cells or whether they are functionally coupled to regulation of insulin secretion. CB1 and CB2 receptors are reported to be expressed by rat islet β-cells,30 but the same study indicated that mouse β-cells expressed CB2 but not CB1 receptors, which is supported by another group.34 In direct contrast, another study has reported expression of CB1 but not CB2 receptors by mouse β-cells33 and there is also a report of CB1 receptors being localized to islet δ-cells, with no expression of CB2 receptors by the endocrine pancreas.35 Furthermore, measurements of downstream signalling following cannabinoid receptor activation with receptor-selective agonists has led to disparate findings, with reports of both stimulation28 and inhibition29,33 of intracellular Ca 2+ and insulin secretion. To clarify the role of cannabinoids in mouse endocrine pancreas we have therefore carried out a systematic investigation of cannabinoid receptor expression by mouse islet cells, coupling of cannabinoid receptors to cellular cyclic AMP and Ca 2+ levels and
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Expression of CB1 and CB2 cannabinoid receptors by mouse β-cells. PCR amplifications using primers for the mouse CB1 and CB2 receptor subtypes produced single products of 351 bp and 506 bp respectively from cDNAs prepared from whole mouse islets, but not from non reverse-transcribed RNA (Fig. 1A). Sequencing of amplicons indicated that there was >98% homology between predicted sequences and those of the amplified products. Consistent with the RT-PCR amplifications, western blotting of mouse islet proteins with CB1 and CB2 receptor antibodies demonstrated that 64 kDa (CB1R) and 35 kDa (CB2R) proteins were detected. Proteins of the same size were also detected in mouse cerebral cortex protein samples, which were used as positive controls (Fig. 1B). Since western blotting gives no indication of the localization of immunoreactive proteins in the mixed cell populations of whole islets, further investigation of the presence of CB1 and CB2 receptors in β-cells was carried out by immunohistochemical analysis of mouse pancreas sections. Figure 2A shows immunostaining of a single mouse islet using antibodies directed against CB1 receptors (FITC secondary antibody), insulin (Texas Red secondary antibody) and glucagon (Texas Red secondary antibody) demonstrating co-localization of CB1 receptors with insulin in β-cells. The merged images indicated some CB1 receptor-positive, insulin-negative (green) cells. Co-staining with glucagon antibodies suggested that these are not α-cells, but they may be the CB1 receptor-expressing δ-cells that were identified in another study.35 CB1 immunoreactivity was not detectable in the surrounding exocrine pancreas. The western blots (Fig. 1B) had suggested lower levels of CB2 than CB1 receptor immunoreactivity in protein-matched mouse islet samples, and it was difficult to detect CB2 receptors in mouse pancreas using fluorescence-tagged secondary antibodies. Immunohistochemistry was therefore carried out on consecutive mouse pancreas sections using a streptavidin-conjugated horseradish peroxidase/biotin-based amplification system with the chromagen diaminobenzidine (DAB). The cells in these sections do not overlay directly, but it can be seen from Figure 2B that CB2 receptors were expressed by islet endocrine cells, and the pattern of expression was consistent with β-cell, rather than α-cell, localization. Effect of cannabinoid receptor agonists on intracellular cyclic AMP levels in mouse islets. The pharmacological CB1 receptor-selective agonist ACEA, and a CB2 receptor-selective cannabinoid, JWH015, were used to determine whether activation of cannabinoid receptors affected β-cell cyclic AMP levels as it does in other cell types. It can be seen from Figure 3 that the adenylate cyclase activator forskolin (10 µM) stimulated cyclic AMP production by mouse islets (6,256 ± 250% of the 20 mM glucose response) and this robust increase in cyclic AMP was significantly reduced by both CB1 and CB2 receptor activation with 10 µM ACEA and JWH015 respectively. Noradrenaline,
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Figure 2. Immunohistochemical detection of cannabinoid receptor expression in mouse pancreas. (A) Mouse pancreas sections were immunoprobed with a rabbit anti-CB1 receptor antibody and the immunoreactivity was revealed using a FITC-conjugated donkey anti-rabbit secondary antibody (green). The sections were also probed with anti-insulin and anti-glucagon primary antibodies and Texas Red-conjugated secondary antibody (red). Fluorescent images were then merged to illustrate CB1 receptor co-localization to insulin-expressing β-cells (yellow), but not to glucagon-expressing α-cells. Results are representative of pancreas sections from six mice. (B) Mouse pancreas sections were immunoprobed with a rabbit anti-CB2 receptor antibody, which was recognised by a DAB-reactive horseradish peroxidase-conjugated secondary antibody (brown). Consecutive pancreas sections were stained with insulin and glucagon antibodies, which were also detected by DAB staining. The DAB-labeled sections were also counterstained with hematoxylin (blue). CB2 receptor staining is consistent with expression by β-cells. Results are representative of pancreas sections from six mice.
which inhibits cyclic AMP generation in β-cells through the activation of α2-adrenergic receptors, also significantly reduced the cyclic AMP increases in response to forskolin in mouse islets, to a greater extent than either ACEA or JWH015, but in all cases cyclic AMP levels were still significantly higher (p < 0.001) than in the presence of 20 mM glucose alone. Effect of cannabinoid receptor agonists on intracellular Ca 2+ levels in mouse islet cells. Figure 4 shows that activation of both CB1 and CB2 receptors resulted in reversible increases in [Ca 2+]i levels in Fura-2-loaded mouse islet cells without any loss of function after exposure to cannabinoid receptor agonists since the cells were then able to mount an appropriate elevation in [Ca 2+]i in response to the K ATP channel blocker, tolbutamide. Thus, the CB1 receptor-selective agonist ACEA elevated [Ca 2+]i when applied at 1 µM and 10 µM, but no increase in the amplitude of the [Ca 2+]i response was obtained with 10 µM ACEA (53 ± 12% and 37 ± 6% peak stimulation of 100 µM tolbutamide response at 1 µM and 10 µM ACEA). The stimulatory effects of 10 µM ACEA on [Ca 2+]i were completely abolished in the presence of a CB1 receptor-selective antagonist, AM251 (Fig. 4A). Activation of CB2 receptors with JWH015 elevated [Ca 2+]i, by 14 ± 1% and 43 ± 4% at 1 µM and 10 µM respectively (peak stimulation of maximal response to 100 µM tolbutamide; Fig. 4B), and exposure of islet cells to the endocannabinoid 2-AG, which activates both CB1 and CB2 receptors, also increased [Ca 2+]i in a concentration-dependent manner (33 ± 2% and 46 ± 1% of the tolbutamide response at 1 µM at 10 µM respectively). Antagonism of CB1 receptors by 10 µM AM251 reduced, but did not abolish,
the stimulatory effects of 10 µM 2-AG (54 ± 10% of peak stimulation to 10 µM 2-AG), consistent with blockade of CB1, but not CB2, receptors. Effect of cannabinoid receptor agonists on insulin secretion. Neither ACEA nor 2-AG stimulated insulin release from mouse islets at 2 mM glucose (Fig. 5A and B), but robust insulin secretory responses to 20 mM glucose were observed from the same islets, indicating that the lack of stimulation by ACEA and 2-AG was not due to impaired islet viability. Despite their lack of effect on insulin secretion at 2 mM glucose, both ACEA and 2-AG potentiated glucose-stimulated insulin secretion from mouse islets in parallel experiments, causing a gradual increase over the plateau phase response to 20 mM glucose (Fig. 6A and B). The elevated insulin output declined slowly towards the pre-stimulation glucose plateau after removal of the cannabinoid agonists. Exposure of mouse islets to 10 µM of the CB2 receptor agonist, JWH015, resulted in a small but significant increase in insulin secretion at 2 mM glucose (Fig. 5C) and a transient potentiation of glucosestimulated insulin release (Fig. 6C), with more rapid onset of insulin secretion than that seen with either ACEA or 2-AG. Discussion Cannabinoid receptors play important roles in maintaining energy balance,6,8,9,12,16,36-40 and most research has focused on their function in the brain. In recent years there has been interest in the roles that these receptors play in fuel homeostasis through regulation of insulin secretion, but to date there is no consensus
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Figure 3. Islet cannabinoid receptors are coupled to inhibition of adenylate cyclase. Groups of 5 isolated mouse islets were pre-incubated for 2 hours in a physiological buffer supplemented with 2 mM glucose prior to a 30 minute exposure at 37°C to the agents shown in the Figure. The black bar shows cyclic AMP levels in the presence of 20 mM glucose. The cyclic AMP concentration of islet extracts was quantified using an enzyme immunoassay. Data are presented as means ± SEM, n = 6–12, ##p < 0.001 versus 20 mM glucose; **p < 0.001 versus 20 mM glucose + 10 µM forskolin. Results shown here are representative of two separate experiments using islets isolated from four mice.
on the expression of cannabinoid receptors by pancreatic β-cells, nor on the functional effects of receptor activation. Our data demonstrate that mouse islets express mRNAs for CB1 and CB2 receptors, and we also identified CB1 and CB2 receptor proteins by western blotting. The smeared immunoreactive 64 kDa protein detected with the anti-CB1 receptor antibody most likely reflects size variation caused by the presence of glycosylated residues, which is consistent with earlier reports of consensus sequences for three potential glycosylation sites within the CB1 receptor extracellular N-terminus.18,41 Mouse cerebral cortex was used as a positive control in our studies because it expresses elements of the endocannabinoid system,1,13,18 and the mouse islet CB1 and CB2 receptor immunoreactive proteins migrated with those of the cerebral cortex samples. CB1 receptors are reported to be expressed by RINm5F,28 INS-1 and βTC6 insulin-secreting cells,35 suggesting a β-cell localization, and this was supported by the detection of CB1 receptor co-localization with insulin, but not glucagon, in mouse pancreas sections in the current studies. These observations are in contrast to other studies that observed co-expression of CB1 receptors only with glucagon in α-cells of dispersed mouse islets,29,30 although a more recent paper indicates that CB1 receptors are detectable in some β-cells in fixed pancreas.42 It has been reported that CB2 receptors, the other major cannabinoid receptor subtype, are not expressed by mouse pancreas,33,35 but other recent studies observed expression of CB2 receptors by both mouse αand β-cells.29,30,34,42 We were unable to reproducibly detect CB2
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receptors in mouse pancreas using fluorescence-labeled secondary antibodies, but DAB immunohistochemical staining was consistent with these receptors being expressed by mouse islet β-cells, adding to the reports of CB2 receptor expression outside of the immune system. It is perhaps surprising that both cannabinoid receptors were identified in β-cells in mouse pancreas, given that several other studies have suggested that one or both, receptors are not localized to β-cells.29,30,33-35 However, results obtained by immunohistochemistry depend, to a large extent, on the choice of antibodies and the experimental procedures employed. The experiments described in this paper were performed on fixed mouse pancreas sections under conditions where antibody dilutions were optimized in preliminary studies, negative controls were included and detection systems (fluorescence or chromagen) were selected to allow the most sensitive detection with minimal background immunoreactivity. Furthermore, the immunohistochemistry studies were complemented by western blotting with the same antibodies, which provided information that the immunoreactive proteins that were detectable were of the correct molecular weights. The established coupling of CB1 and CB2 receptors is via Gi/oproteins to inhibit cyclic AMP generation.18 Inhibitory effects of cannabinoid receptor activation on adenylate cyclase activity in β-cells have not been reported previously. Noradrenaline, which couples to β-cell α2 adrenergic receptors to inhibit adenylate cyclase activity,43 significantly inhibited mouse islet cyclic AMP production in response to the adenylate cyclase activator
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Figure 4. Islet cannabinoid receptors are coupled to increases in intracellular calcium. Fura-2-loaded mouse islet cells were exposed to the test agents shown by the grey bars, in a physiological buffer supplemented with 2 mM glucose. ACEA (A), JWH015 (B) and 2-AG (C) induced reversible increases in [Ca2+]i and the CB1 receptor antagonist AM251 abolished and decreased the stimulatory effects of ACEA and 2-AG, respectively. Changes in [Ca2+]i in individual cells are expressed as 340 nm/380 nm ratiometric data (means ± SEM n = 4–7), and each part is representative of three separate experiments. 100 µM tolbutamide (Tolb) was used at the end of each experiment as a positive control.
forskolin. In the same experiments activation of CB1 or CB2 receptors with ACEA and JWH015 respectively also inhibited forskolin-stimulated cyclic AMP generation, providing the first direct evidence that the cannabinoid receptors are functionally coupled to reduce adenylate cyclase activity in mouse islet cells. Cannabinoid receptors also inhibit Ca 2+ influx through a Gi/o-dependent pathway,18 and CB1 and CB2 receptor activation is reported to reduce the amplitude of mouse islet Ca 2+ oscillations.29,33 However, a CB1 receptoractivated Gq/11-coupled Ca 2+ -sensitive PLC pathway has been identified using a CB1 receptor agonist in transfected CB1 receptor-expressing HEK293 cells and cultured hippocampal neurons, which exerted a positive impact on [Ca 2+]i.44 Consistent with this stimulatory effect on Ca 2+, the endocannabinoid 2-AG was reported to cause a transient [Ca 2+]i increase in NG10815 neuronal cells via CB1 receptor activation20 and in human HL60 cells through CB2 receptor stimulation.45 Stimulatory effects on intracellular Ca 2+ levels have also been observed in RINm5F insulinoma cells in response to both CB1 and CB2 receptor agonists,31 and we observed similar elevations in [Ca 2+]i in the current study. Thus, pharmacological activation of CB1 and CB2 receptors with ACEA and JWH015 induced reversible elevations in Ca 2+ in dispersed islets cells, as did the endocannabinoid 2-AG. The partial inhibition by AM251 of 2-AG effects on [Ca 2+]i indicates that
stimulation by 2-AG was mediated in part by CB1 receptor activation and it is likely that the residual response to 2-AG obtained in the presence of AM251 was via CB2 receptor activation. One
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Figure 5. Effect of cannabinoid receptor agonists on basal insulin secretion from mouse islets. Groups of 80 isolated mouse islets were transferred to chambers and perifused with a physiological buffer containing 2 mM glucose and supplemented as shown. 10 µM JWH015 (C) induced a small, but significant increase (p < 0.01) in insulin secretion at 2 mM glucose, but 10 µM ACEA (A) and 2-AG (B) were without significant effect (p > 0.2). Data are presented as a percentage of insulin secretion at 2 mM glucose (means ± SEM, n = 3–4), and each part is representative of four separate experiments using islets obtained from 30 mice.
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possible reason for the discrepancies between our data and those previously reported using mouse islets29,33 is that we examined the effects of cannabinoids on basal Ca 2+ levels, where stimulation was observed, while the earlier studies showed inhibition of glucosestimulated elevations in Ca 2+. There have been reports that cannabinoid receptor activation either inhibits29,33 or stimulates28,32 insulin secretion, which is perhaps not surprising given our observations that both CB1 and CB2 receptor agonists can inhibit β-cell cyclic AMP production and stimulate increases in Ca 2+. All of the studies reported to date have determined the insulin secretagogue effects of cannabinoid receptor agonists in static incubation studies where insulin secreted from β-cells into the supernatant were determined over a fixed time-course (30 min-2 hours). As these static incubation protocols do not give any dynamic information about the insulin secretory responses, in the current studies we used a perifusion system to investigate the time-course and reversibility of cannabinoid receptor agonists on insulin secretion from mouse islets. These perifusion experiments allow identification of direct stimulatory effects of agonists on insulin secretion since potential inhibitory agents
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Figure 6. CB1 and CB2 receptor agonists potentiate glucose-stimulated insulin secretion from mouse islets. Groups of 80 isolated mouse islets were transferred to chambers and perifused with buffers supplemented with 2 mM glucose for the first 10 minutes and with 20 mM glucose thereafter. Twenty-minute exposure to 10 µM ACEA (A), 2-AG (B) and JWH015 (C) significantly (p < 0.01) potentiated glucose-induced insulin secretion. Data are presented as a percentage of insulin secretion at 2 mM glucose (means ± SEM, n = 4), and each part is representative of three separate experiments using islets obtained from 20 mice.
such as somatostatin and GABA, which might be released from islets in response to cannabinoid receptor activation, are rapidly removed. However, in the static incubation protocols performed in earlier studies there could be a build up of such agents which may feed back on β-cells to inhibit insulin secretion. Our data indicate that neither ACEA nor 2-AG had any effect on basal insulin output, while the CB2 receptor agonist JWH015 induced a small, reversible increase in insulin secretion at 2 mM glucose. An earlier study has also indicated that 2-AG does not affect basal insulin secretion,29 and this is the first report of initiation of insulin secretion following activation of CB2 receptors. Our observations of potentiation of glucose-stimulated insulin secretion by all three cannabinoids used are in contrast to reports of inhibitory effects of the endocannabinoids 2-AG and anandamide, and the CB1 receptor agonist ACEA, in static incubations of mouse islets.29,33 The profiles of stimulation following CB1 and CB2 receptor activation were different, with ACEA causing a slow, sustained increase in insulin output, while activation of CB2 receptors with JWH015 stimulated a more rapid, transient stimulatory response. The mixed cannabinoid agonist, 2-AG, most likely regulated insulin secretion primarily
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through activation of CB1 receptors, since the secretory profiles with 2-AG at 2 mM and 20 mM glucose were very similar to those of ACEA. The stimulatory effects of the pharmacological cannabinoids ACEA and JWH015, and those of the endocannabinoid 2-AG, on insulin secretion may be coupled to their capacity to elevate [Ca 2+]i, since they all caused rapid elevations in Ca 2+ levels in mouse islet cells. These stimulatory effects on Ca 2+ appear to be sufficient to counteract the potentially inhibitory effects of reduced cyclic AMP production, as we did not see cannabinoid-induced reductions in insulin secretion in any of the experimental protocols that we used. Thus, the net effect of cannabinoids on insulin secretion may reflect a balance between their stimulatory input to increase [Ca 2+]i and their inhibitory effect via Gi/o to decrease cyclic AMP levels, and in some earlier studies the negative input may have prevailed over the stimulatory one.29,33 The data presented in this paper have cast some light on the pathophysiological significance of the islet cannabinoid signalling system. Thus, under normal circumstances islets may synthesise 2-AG from locally generated diacylglycerol via diacylglycerol lipase enzymes that we and others have identified in islets.32,46 As shown here and earlier in RINm5F cells31 and human β-cells,32 2-AG can activate β-cell cannabinoid receptors to stimulate insulin secretion. However, it has been demonstrated that 2-AG is upregulated in the pancreas and epididymal fat of diet-induced obese mice, as it is in the visceral fat of obese humans.28 Therefore, under conditions of obesity it might be expected that elevated local and/or circulating 2-AG could lead to hyperstimulatation of insulin secretion and consequent exacerbation of the adipocyte hypertrophy and elevated lipid levels. The importance of peripheral cannabinoid receptors in this dysregulation of energy homeostasis in obesity is confirmed by observations that the CB1 receptor antagonist rimonabant reversed the diet-induced obese phenotype in mice,47 and also improved lipid profiles and reduced insulin resistance in clinical use, independently of reduced food intake.40 In addition, direct administration of rimonabant to islets in vitro inhibited basal insulin hypersecretion due to obesity and it also inhibited glucose-stimulated insulin secretion from islets isolated from lean rats,48 consistent with a stimulatory role for endogenous cannabinoids on insulin secretion. In summary, this study has shown that CB1 and CB2 receptor mRNAs and proteins are expressed by primary mouse β-cells and activation of either receptor leads to increased intracellular Ca 2+ levels and reductions in cyclic AMP. Perifusion experiments have revealed that CB1 and CB2 receptors are coupled to potentiation of glucose-stimulated insulin secretion from mouse islets, which may occur through the elevations in [Ca 2+]i. While the islet endocannabinoid system can contribute to glucose homeostasis under normal physiological circumstances, its overstimulation in obesity may lead to a vicious cycle, resulting in hyperinsulinemia and enhanced lipogenesis. Material and Methods Materials. Collagenase XI, Research Park Memorial Institute (RPMI) medium, penicillin/streptomycin, L-glutamine, Fura-2
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AM, noradrenaline, ethidium bromide and anti-glucagon antibody were purchased from Sigma-Aldrich (Dorset, UK). 2-AG, ACEA and JWH015 were from Tocris Biosciences (Bristol, UK). Fetal bovine serum (FBS) and 10% polyacrylamide BisTris gels were from Invitrogen (Paisley, UK). RNeasy mini kits and gel extraction kits were obtained from Qiagen (West Sussex, UK) and PCR primers were from Operon Biotech (Cologne, Germany). Antibodies against CB1 and CB2 receptors were from Autogen Bioclear (Wilts, UK), the anti-insulin antibody was from Dako UK Ltd., (Cambridgeshire, UK), horseradish peroxidase-linked goat anti-rabbit secondary antibody and Texas Redand FITC-conjugated secondary antibodies were from Thermo Scientific (Epsom, UK). ECL western Blotting Detection reagents, RainbowTM molecular weight markers and the cyclic AMP Biotrak Enzymeimmunoassay System were obtained from GE Healthcare (Buckinghamshire, UK). Standard PCR was carried out using a Px2 Thermal RT-PCR cycler (Thermo Scientific, Epsom, UK). ICR mice were purchased from Harlan (Oxfordshire, UK). Mouse islet isolation. Islets were isolated from male ICR mice by collagenase digestion of the exocrine pancreas, essentially as described49 and maintained in culture overnight in RPMI 1640 medium (11 mM glucose, 10% FBS) prior to use. Detection of mRNAs by RT-PCR. RNA was isolated from mouse islets and reverse-transcribed into cDNA as described previously,50 which was amplified for 35 cycles using synthetic oligonucleotide primers specific for mouse CB1 and CB2 receptors respectively (CB1 sense: 5'-CCT GGG CTG GAA CTG CAA-3'; antisense: 5'-CCG AAG ACG TCA TAC ACC ATG A-3'; 351 bp product), CB2 sense: 5'-GGA TGC CGG GAG ACA GAA GTG A-3'; antisense: 5'-CCC ATG AGC GGC AGG TAA GAA AT-3'; 506 bp product). The amplified PCR products were separated on 1.8% agarose gels, extracted using the Qiagen Gel Extraction kit according to the manufacturer’s protocol, and their identities were confirmed by sequencing using fluorescent di-deoxy termination methods (Molecular Biology Unit, King’s College London). Protein extraction and western blotting. Mouse islet and cerebral cortex extracts were fractionated by electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes. CB1 and CB2 receptor expression was identified by immunoprobing with rabbit polyclonal antibodies raised against amino acids 1–150 at the N-terminus of CB1 and amino acids 301–360 at the C-terminus of CB2 (1/1,000 dilutions of both CB receptor antibodies). After incubation with a 1 in 7,500 dilution of horseradish peroxidase-linked goat anti-rabbit secondary antibody blots were developed by ECL and standard curves were constructed of distances migrated by RainbowTM molecular weight markers. Immunohistochemistry. Mouse pancreases were fixed in 4% paraformaldehyde, wax embedded and sections were cut onto microscope slides. Dewaxed and rehydrated cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS and incubated for two hours at room temperature with guinea pig anti-insulin polyclonal antibody (1/50 dilution), mouse anti-glucagon monoclonal antibody (1/50 dilution) and rabbit CB1 or CB2 receptor
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antibodies (both at 1/200 dilution). Immunoreactive proteins were detected by incubation for two hours at room temperature with Texas Red- (insulin and glucagon) or FITC-(CB receptors) conjugated secondary antibodies (1/50 dilutions) followed by visualization under a Nikon TE2000 fluorescence microscope. Control protocols were performed by incubating pancreas sections in the presence of fluorophore-conjugated secondary antibodies in the absence of primary antibodies and no fluorescence was detected. Cyclic AMP quantification. Groups of five isolated mouse islets were incubated at 37°C for 30 minutes in 200 µl of physiological salt solution51 supplemented with agents of interest, then the islets were lysed and sonicated on ice. Cyclic AMP contents of cell lysates were determined using the cyclic AMP Biotrak Enzymeimmunoassay System, according to the manufacturer’s protocols. Single-cell calcium microfluorimetry. Approximately 300 isolated mouse islets were gently dissociated by incubation at 37°C with 0.02% w/v EDTA then cells were seeded onto 2.2 cm circular acid/ethanol washed sterile glass cover-slips. After maintenance overnight under standard tissue culture conditions cells were loaded for 40 minutes at 37°C with 5 µM of the Ca 2+ fluorophore Fura-2 AM and perifused with physiological References 1. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992; 258:1946-9. 2. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365:61-5. 3. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, et al. International union of pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002; 54:161-202. 4. Bisogno T, Berrendero F, Ambrosino G, Cebeira M, Ramos JA, Fernandez-Ruiz JJ, et al. Brain regional distribution of endocannabinoids: Implications for their biosynthesis and biological function. Biochem Biophys Res Commun 1999; 256:377-80. 5. Bellocchio L, Mancini G, Vicennati V, Pasquali R, Pagotto U. Cannabinoid receptors as therapeutic targets for obesity and metabolic diseases. Curr Opin Pharmacol 2006; 6:586-91. 6. Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R. The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev 2006; 27:73-100. 7. Bifulco M, Grimaldi C, Gazzerro P, Pisanti S, Santoro A. Rimonabant: Just an Antiobesity drug? Current evidence on its pleiotropic effects. Mol Pharmacol 2007; 71:1445-56. 8. Di Marzo V. The endocannabinoid system: Its general strategy of action, tools for its pharmacological manipulation and potential therapeutic exploitation. Pharmacol Res 2009; 60:77-84. 9. Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J, Radaeva S, Batkai S, et al. Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J Clin Invest 2005; 115:1298-305. 10. Liu YL, Connoley IP, Wilson CA, Stock MJ. Effects of the cannabinoid CB1 receptor antagonist SR141716 on oxygen consumption and soleus muscle glucose uptake in Lep(ob)/Lep(ob) mice. Int J Obes 2005; 29:183-7.
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We are grateful to Diabetes UK and the Henry Lester Trust for funding. James Bowe is a Diabetes Research and Wellness Foundation Research Fellow.
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Volume 2 Issue 5
