Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.
ABSTRACT: Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein?coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10-12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in ?-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.
Project description:The cannabinoid 1 receptor (CB1) regulates insulin sensitivity and glucose metabolism in peripheral tissues. CB1 is expressed on pancreatic beta (β)-cells where its functions have not been fully described. We generated a β-cell-specific CB1-knockout (β-CB1-/-) mouse to study the long-term consequences of CB1 ablation on β-cell function in adult mice. β-CB1-/- mice had increased basal- and stimulated-insulin secretion and intra-islet cAMP levels, resulting in primary hyperinsulinemia, as well as increased β-cell viability, proliferation, and islet area. Hyperinsulinemia led to insulin resistance, which was aggravated by a high fat/high glucose diet and weight gain, although β-cells maintained their insulin secretory capacity in response to glucose. Strikingly, islets from β-CB1-/- mice were protected from diet-induced inflammation. Mechanistically we show that this is a consequence of curtailment of oxidative stress and reduced activation of Nlrp3 inflammasome in β-cells. Our data demonstrate CB1 to be a negative regulator of β-cells and a mediator of islet inflammation under conditions of metabolic stress. Overall design: Gene expression profiles from islets from wildtype (β-CB1+/+) (N=4) and a β-cell-specific CB1-knockout (β-CB1-/-) mice (N=3) were examined using gene expression microarray analysis.
Project description:AIMS:Endocannabinoids are lipid mediators involved in the regulation of glucose homeostasis. They interact with the canonical cannabinoid receptors CB1 and CB2, and it is now apparent that some cannabinoid receptor ligands are also agonists at GPR55. Thus, CB1 antagonists such as SR141716A, also known as rimonabant, and AM251 act as GPR55 agonists in some cell types. The complex pharmacological properties of cannabinoids make it difficult to fully identify the relative importance of CB1 and GPR55 in the functional effects of SR141716A, and AM251. Here, we determine whether SR141716A and AM251 regulation of mouse and human islet function is through their action as GPR55 agonists. METHODS:Islets isolated from Gpr55+/+ and Gpr55-/- mice and human donors were incubated in the absence or presence of 10 µM SR141716A or AM251, concentrations that are known to activate GPR55. Insulin secretion, cAMP, IP1, apoptosis and ?-cell proliferation were quantified by standard techniques. RESULTS:Our results provide the first evidence that SR141716A and AM251 are not GPR55 agonists in islets, as their effects are maintained in islets isolated from Gpr55-/- mice. Their signalling through Gq-coupled cascades to induce insulin secretion and human ?-cell proliferation, and protect against apoptosis in vitro, indicate that they have direct beneficial effects on islet function. CONCLUSION:These observations may be useful in directing development of peripherally restricted novel therapeutics that are structurally related to SR141716A and AM251, and which potentiate glucose-induced insulin secretion and stimulate ?-cell proliferation.
Project description:Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse ?-cell lines, human islets and CB1R-null (CB1R-/- ) mice, we have now investigated the role of CB1Rs in modulating ?-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 ?-cell lines and human islets. In addition, silencing CB1R in mouse ? cells resulted in an increased expression of pro-insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in ? cells lacking insulin receptor. Furthermore, CB1R-/- mice had increased pro-insulin, GCK and GLUT2 expression in ? cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve ?-cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to ?-cell function in type 2 diabetes.
Project description:The endocannabinoid system has gained attention as an important modulator of activity in the central nervous system. Initial studies focused on cannabinoid receptor 1 (CB1), which is widely expressed in the brain, but recent work also implicates cannabinoid receptor 2 (CB2) in modulating neuronal activity. Both receptors are capable of reducing neuronal activity, generating interest in cannabinoid receptor agonists as potential anticonvulsants. CB1 (Cnr1) and CB2 (Cnr2) single-knockout mice have been generated, with the former showing heightened seizure sensitivity, but not overt seizures. Given overlapping and complementary functions of CB1 and CB2 receptors, we queried whether double-knockout mice would show an exacerbated neurological phenotype. Strikingly, 30% of double-knockout mice exhibited provoked behavioral seizures, and 80% were found to be epileptic following 24/7 video-electroencephalographic monitoring. Single-knockout animals did not exhibit seizures. These findings highlight the importance of the endocannabinoid system for maintaining network stability.
Project description:There is increasing evidence to suggest that cannabis can ameliorate muscle-spasticity in multiple sclerosis, as was objectively shown in experimental autoimmune encephalomyelitis models. The purpose of this study was to investigate further the involvement of CB1 and CB2)cannabinoid receptors in the control of experimental spasticity.Spasticity was induced in wildtype and CB1-deficient mice following the development of relapsing, experimental autoimmune encephalomyelitis. Spastic-hindlimb stiffness was measured by the resistance to flexion against a strain gauge following the administration of CB1 and CB2 agonists.As previously suggested, some CB2-selective agonists (RWJ400065) could inhibit spasticity. Importantly, however, the anti-spastic activity of RWJ400065 and the therapeutic effect of non-selective CB1/CB2 agonists (R(+)WIN55,212-2 and CP55, 940) was lost in spastic, CB1-deficit mice.The CB1 receptor controls spasticity and cross-reactivity to this receptor appears to account for the therapeutic action of some CB2 agonists. As cannabinoid-induced psychoactivity is also mediated by the CB1 receptor, it will be difficult to truly dissociate the therapeutic effects from the well-known, adverse effects of cannabinoids when using cannabis as a medicine. The lack of knowledge on the true diversity of the cannabinoid system coupled with the lack of total specificity of current cannabinoid reagents makes interpretation of in vivo results difficult, if using a purely pharmacological approach. Gene knockout technology provides an important tool in target validation and indicates that the CB1 receptor is the main cannabinoid target for an anti-spastic effect.
Project description:Cannabinoids exert antiproliferative properties in a variety of malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). In our study, we quantitatively evaluated the immunoreactivity for cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptors as well as for the endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGLL). Furthermore, quantitative real-time RT-PCR for CB1, CB2, FAAH and MGLL in normal pancreas and pancreatic cancer tissues was performed. Levels of endocannabinoids were determined by liquid chromatography/mass spectrometry. Immunoreactivity scores and QRT-PCR expression levels were correlated with the clinico-pathological (TNM, survival, pain) status of the patients. Evaluation of endocannabinoid levels revealed that these remained unchanged in PDAC compared to the normal pancreas. Patients with high CB1 receptor levels in enlarged nerves in PDAC had a lower combined pain score (intensity, frequency, duration; p = 0.012). There was a significant relationship between low CB1 receptor immunoreactivity or mRNA expression levels (p = 0.0011 and p = 0.026, respectively), or high FAAH and MGLL cancer cell immunoreactivity (p = 0.036 and p = 0.017, respectively) and longer survival of PDAC patients. These results are underlined by a significant correlation of high pain scores and increased survival (p = 0.0343). CB2 receptor immunoreactivity, CB2 receptor, FAAH and MGLL mRNA expression levels did not correlate with survival. Therefore, changes in the levels of endocannabinoid metabolizing enzymes and cannabinoid receptors on pancreatic cancer cells may affect prognosis and pain status of PDAC patients.
Project description:Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that ? cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of ? cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and ?/? cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ?-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.
Project description:Mixed cannabinoid receptor 1 and 2 (CB1 and CB2) agonists such as ?(9)-tetrahydrocannabinol (?(9)-THC) can produce tolerance, physical withdrawal, and unwanted CB1-mediated central nervous system side effects. Whether repeated systemic administration of a CB2-preferring agonist engages CB1 receptors or produces CB1-mediated side effects is unknown.We evaluated antiallodynic efficacy, possible tolerance, and cannabimimetic side effects of repeated dosing with a CB2-preferring agonist AM1710 in a model of chemotherapy-induced neuropathy produced by paclitaxel using CB1 knockout (CB1KO), CB2 knockout (CB2KO), and wild-type (WT) mice. Comparisons were made with the prototypic classic cannabinoid ?(9)-THC. We also explored the site and possible mechanism of action of AM1710.Paclitaxel-induced mechanical and cold allodynia developed to an equivalent degree in CB1KO, CB2KO, and WT mice. Both AM1710 and ?(9)-THC suppressed established paclitaxel-induced allodynia in WT mice. In contrast to ?(9)-THC, chronic administration of AM1710 did not engage CB1 activity or produce antinociceptive tolerance, CB1-mediated cannabinoid withdrawal, hypothermia, or motor dysfunction. Antiallodynic efficacy of systemic administration of AM1710 was absent in CB2KO mice and WT mice receiving the CB2 antagonist AM630, administered either systemically or intrathecally. Intrathecal administration of AM1710 also attenuated paclitaxel-induced allodynia in WT mice, but not CB2KO mice, implicating a possible role for spinal CB2 receptors in AM1710 antiallodynic efficacy. Finally, both acute and chronic administration of AM1710 decreased messenger RNA levels of tumor necrosis factor-? and monocyte chemoattractant protein 1 in lumbar spinal cord of paclitaxel-treated WT mice.Our results highlight the potential of prolonged use of CB2 agonists for managing chemotherapy-induced allodynia with a favorable therapeutic ratio marked by sustained efficacy and absence of tolerance, physical withdrawal, or CB1-mediated side effects.
Project description:Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.
Project description:Glucokinase-mediated glucose signaling induces insulin secretion, proliferation, and apoptosis in pancreatic ?-cells. However, the precise molecular mechanisms underlying these processes are not clearly understood. Here, we demonstrated that glucokinase activation using a glucokinase activator (GKA) significantly upregulated the expression of Fibulin-5 (Fbln5), a matricellular protein involved in matrix-cell signaling, in isolated mouse islets. The islet Fbln5 expression was induced by ambient glucose in a time- and dose-dependent manner and further enhanced by high-fat diet or the deletion of insulin receptor substrate 2 (IRS-2), whereas the GKA-induced increase in Fbln5 expression was diminished in Irs-2-deficient islets. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase inhibitor, KATP channel opener, Ca2+ channel blocker and calcineurin inhibitor, while it was augmented by harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1?A inhibitor. Although deletion of Fbln5 in mice had no significant effects on the glucose tolerance or ?-cell functions, adenovirus-mediated Fbln5 overexpression increased glucose-stimulated insulin secretion in INS-1 rat insulinoma cells. Since the islet Fbln5 expression is regulated through a glucokinase/KATP channel/calcineurin/nuclear factor of activated T cells (NFAT) pathway crucial for the maintenance of ?-cell functions, further investigation of Fbln5 functions in the islets is warranted.