The function of the endocannabinoid system in the pancreatic islet and its implications on metabolic syndrome and diabetes

ABSTRACT

The following review focuses on the scientific studies related to the role of endocannabinoid system (ECS) in pancreatic islet physiology and dysfunction. Different natural or synthetic agonists and antagonists have been suggested as an alternative treatment for diabetes, obesity and metabolic syndrome. Therapeutic use of Cannabis led to the discovery and characterization of the ECS, a signaling complex involved in regulation of various physiological processes, including food intake and metabolism. After the development of different agonists and antagonists, evidence have demonstrated the presence and activity of cannabinoid receptors in several organs and tissues, including pancreatic islets. Insulin and glucagon expression, stimulated secretion, and the development of diabetes and other metabolic disorders have been associated with the activity and modulation of ECS in pancreatic islets. However, according to the animal model and experimental design, either endogenous or pharmacological ligands of cannabinoid receptors have guided to contradictory and paradoxical results that suggest a complex physiological interaction. In consensus, ECS activity modulates insulin and glucagon secretions according to glucose in media; over-stimulation of cannabinoid receptors affects islets negatively, leading to glucose intolerance, meanwhile the treatment with antagonists in diabetic models and humans suggests an improvement in islets function.

Graphical Abstract

KEYWORDS:

• Endocannabinoid system
• pancreatic islets
• diabetes
• insulin secretion
• cannabinoids

Overview of the endocannabinoid system components

The endocannabinoid system (ECS) is a lipid signaling complex involved in metabolism regulation and specific physiological processes. Today, ECS role in the development and treatment of some diseases, including metabolic syndrome and type-2 Diabetes Mellitus, is still under research.^1,2 The ECS was discovered after the use of tetrahydrocannabinol (THC) and the activation of a transmembrane G protein-coupled receptor (GPR) in the nervous system, renamed cannabinoid receptor 1 (CB1).^3,4 CB2 and GPR55 receptors were discovered by their interaction with endogenous ligands identified as endocannabinoids (EC): N-arachidonyl ethanolamine, also named anandamide (AEA), and 2-arachidonylglycerol (2-AG), both derived from phospholipids containing arachidonic acid (AAc).^5–7 Enzymes involved in biosynthesis and degradation of EC are present in many cells, anchored in the plasmatic membrane or close to: AEA production is regulated by N-acyl phosphatidylethanolamine phospholipase D (NAPE-D), and degradation by fatty acid-acyl hydrolase (FAAH), meanwhile 2-AG depends of diacylglycerol lipase (DAGL) for synthesis and monoacyl-glycerol lipase (MAGL) for metabolism.

Figure 1. Mechanisms of action of cannabinoid receptors proposed for pancreatic beta cell: CB1 and CB2 receptors decreases cAMP, and GPR55 is associated with intracellular calcium increase.

Schematic representation of a pancreatic beta-cell with cannabinoid receptors CB1, CB2 and GPR55, their signaling cascade with implications in insulin secretion.

Figure 2. Mechanisms of action of cannabinoid receptors proposed for pancreatic alpha cell. CB2 receptor probably is coupled to PLC and PKC activity, increasing glucagon secretion.

Suggested representation of a pancreatic alpha-cell with cannabinoid receptor CB2 signaling, involved in glucagon secretion.

Figure 3. Modulation of insulin secretion with natural agonists, synthetic agonists and antagonists, and genetic deletion of CB receptors: (+) stimulation and enhance, (-) inhibition, (???) unknown effect or incomplete description. AEA (Anandamide), ACPA (arachidonyl cyclopropylamine), ACEA (arachidonyl-2-chloroethylamine), abn-CBD (abnormal Cannabidiol), CBD (Cannabidiol) and THC (Tetrahydrocannabinol).

Schematic regulation of insulin secretion in pancreatic beta-cell through the effects of different natural agonists, synthetic agonists and antagonists on cannabinoid receptors, and their genetic deletion.

After the psychoactive effects of delta-9-tetrahydrocannabinol in brain, CB1 receptor functions were discovered in basal ganglia, hippocampus, hypothalamus, cerebellum, olfactory cortex, septum, amygdala, brainstem, nerve terminals, spinal cord nodes and periaqueductal gray matter. The effects are associated to food intake modulation, motor coordination and pain sensitivity.^8–12 CB1 receptor activity inhibits neurotransmitters release in the presynaptic terminals, such as glutamate, dopamine or GABA, mobilizing vesicles or regulating ion channels activity.^11,12 2-AG synthesis is provided by DAGL located at postsynaptic neurons, and its inactivation by MAGL activity is present in presynaptic neurons, establishing a retrograde mechanism of action.^13–15 In a similar way, synthesis and degradation of AEA by NAPE-PLD and FAAH are present in the plasmatic membrane of postsynaptic neurons, plus the high expression of CB1 receptor, suggests a local predominance of this system.^16,17 CB2 receptor is highly expressed in immune cells, and in lower proportion at neurons from cerebellum and brainstem, liver, pancreas, muscle and adipose tissue.¹⁸ It has been suggested the participation of a membrane transporter for ECs, not yet characterized, could facilitate release and uptake at both synapse membranes, and promote intracellular signaling mechanisms.^19,20

CB1 and CB2 receptors are expressed in several peripheral tissues, executing physiological and pharmacological responses according to specific interactions with other proteins. Mostly CB receptors, as homo or heterodimers, are coupled to Gi/o proteins, inhibit adenylate cyclase (AC), regulate the activity of Ca²⁺ and K⁺ ion channels, promote mitogen-activating protein kinase (MAPK) signaling with p42/44 MAPK, p38 MAPK and c-JUN N-terminal kinase, and regulate intracellular location of specific nuclear transcription factors.^21,22 Some cells present CB1 and CB2 receptors associated to Gs proteins, inducing changes in cAMP, intracellular Ca²⁺ streams, and increase phospholipases activity.²³

Orphan GPR55 receptor is expressed in some tissues involved in vascular physiology and nutrients metabolism. Some refer to GPR55 as CB3 receptor after specific binding to AEA, cannabidiol (CBD), abnormal cannabidiol (abn-CBD), virodhamine, palmitoylethanolamide and oleoylethanolamide.^24,25 GPR55 activity is coupled to Gα12/13 proteins, associated with RhoA, cdc42 and rac1 activation. Its interaction with Gαq proteins induces changes in intracellular Ca²⁺ and extracellular signal-regulated kinase ERK ½ phosphorylation.^26,27

ECS plays a key role in the energy metabolism and homeostasis of lipids and glucose through hypothalamic and mesolimbic pathways, and in adipocytes, liver and pancreatic islets as peripheral pathways.^28–32 At physiological concentrations, agonists and antagonists of CB receptors have shown specific short-effects on food ingestion, glucose and lipid metabolism; chronic or pharmacological exposure correlates to cellular dysfunction, ECS dysregulation, and the development of metabolic and eating disorders including abdominal obesity, dyslipidemia, metabolic syndrome and diabetes.^31,32 In this review we spotlight these effects in pancreatic islets.

The ECS function in pancreatic islets

The presence and activity of the ECS in pancreatic islets have been reported in different models: cell lines, primary culture of intact islets or dispersed cells; in histological slides of pancreas obtained from rats, mice, and humans, under healthy, obesity of diabetic conditions.^33–45 Gene expression and localization of cannabinoid receptors, synthesis/degradation enzymes, and the local ECs production have been determined by different approaches, including immunochemestry, immunoflorescence, conventional PCR and Real-Time PCR, leading to controversial and inconclusive results: the inherent characteristics of mice, rats and human islets are well documented under different ages and in diabetes development.^33–45 The use of antibodies from diverse sources recognizes different epitopes that contributes to the inexact location of each ECS element. Apparently, the expression of receptors and enzymes is associated to specific culture conditions, tissue extraction, and the health state of patients, donors, or animals. The shared conclusion leads to a dynamic ECS expression and activity in islets, close-related to the changes in glucose input, diet and the development of diabetes.^43,44

In beta-cell lines and islets isolated from mice and rat, CB1 and CB2 receptors activated with AEA from physiological (10–100 nM) to pharmacological (1 μM) concentrations, or with synthetic agonists, potentiate glucose-stimulated insulin secretion after increasing intracellular Ca²⁺ concentration, but reducing cyclic AMP production.^34,35,42 In opposition, there are evidences that AEA and 2-AG decrease insulin secretion with variable effects on intracellular calcium.^37,38,41 The conditions and origin of cell culture, ligands concentration, time of exposure, experimental approaches, even the source of reagents could be the reasons of these disparities, evoking opposite results; it is probable that some agonists change to inverse agonists, or under specific conditions CB receptors present different G-proteins coupling.^{18–20,46–49} Under physiological circumstances, ECS is associated to food ingestion and energy balance,^28–32 including insulin and glucagon secretions as modulators of glucose homeostasis and metabolism. The over-stimulation of CB receptors and the increasing presence of ECs in blood has been correlated with hyperinsulinemia, hyperglycemia and lipogenesis, suggesting negative effects on islets function.^33,45,50 Our hypothesis is ESC present a role as paracrine regulator: under fasting state, glucagon secretion is promoted among with a discrete basal insulin secretion. Since CB1 receptor and enzymes for synthesis and degradation of AEA are mainly expressed in islets, probably this endocannabinoid control basal insulin secretion in beta-cells. CB2 receptor and enzymes involved in 2-AG production are low expressed in islets, probably in correlation to the percent of alpha-cells, suggesting glucagon secretion could be regulated by CB1 and CB2 receptors.^51,52 Considering evidences of GPR55 activity involved in insulin secretion,^47,53–55 the ECS role in islets suggests an integral and paracrine regulation of metabolic homeostasis, therefore, a potential therapeutic option for diabetes. Solid evidences suggest LH-21 and abnormal cannabidiol as pharmacological strategy to improve beta-cell function in human and mouse islets, using different mechanism depend and independent of GPR55 interaction (Figure 1 and figure 2).⁵⁵

Islets exposed to endogenous CB receptors agonists

In beta-cell lines and dispersed islets from mice, short-term activation of CB1 receptor by AEA reduced glucose-induced insulin secretion, but cytosolic Ca²⁺ oscillations were inhibited only in 50% of the cells, suggesting that CB1r is not present in all islet cells.^37,38 2-AG exposure presented a similar effect, related to the permissive pulsatile insulin release.⁴⁰ In opposition, pancreatic islets from rat and beta-cell lines treated for 1 or 24 hours with pharmacological concentrations of AEA, increased basal and glucose-stimulated insulin secretion.^35,42 2-AG promotes cAMP production, elevation in intracellular Ca²⁺, thus induces insulin secretion and decreases basal glucagon secretion.^33,42–44 The explanation for these contrary observations is not clear, authors suggest different expression of ECS elements within islets, possibly a random distribution and activity, even between the same cell lineage.^43,51,52 AEA and 2-AG binding to CB receptors are well-described in neurons and other cell types, but there are not specific studies in pancreatic islets, suggesting a possible cross interaction and activation using pharmacological doses.^18,20 In obese and diabetic models, AEA and 2-AG production inside the islets is increased, probably induce an over-activation of the ECS, leading to interference in metabolic mechanisms involved in insulin secretion (decrease in cAMP, changes in gene expression, insulin resistance).^33,45,56 The mechanism of CB1r action suggested for beta-cells is the formation of heterotrimeric complex with insulin receptor and Giα protein, which results in inhibition of insulin signaling by autophosphorylation.⁵⁰

Islets treated with synthetic CB receptors agonists

The development of pharmacological analogs and agonists, first for CB1 receptor and then for CB2 receptor, has been a useful in the study of mechanisms of action, binding and biological effects.^{1,2,5,45,51,52,56–58} CB1 receptor agonist methanandamide increased intracellular Ca²⁺ oscillations in 31.3% of the beta-cells tested (10 out of 32), similarly to the secondary effect of AEA.³⁷ Selective agonists for CB1 receptor, arachidonyl cyclopropylamine (ACPA) and arachidonyl-2-chloroethylamine (ACEA), as well as JWH-015 for CB2 receptor, have been usually studied in isolated islets from animal models and humans.^{43,50–52,56–59} At short exposure, ACEA increases the release of intracellular Ca²⁺ by activating the phosphatidyl-inositol-triphosphate (IP3) receptor in the endoplasmic reticulum, however the effect on insulin secretion is not clear since some studies demonstrate an inhibition, while others propose a significant increase of basal secretion.^51,58 Isolated islets from mice and human chronically exposed to ACEA induced an adaptable ECS response, probably involving a process of saturation and self-regulation of receptors’ expression, meanwhile acute exposition decreased insulin secretion and apoptosis.^51,52,56 Acute and chronic effects of JWH-015 were most evident in alpha-cells, evoking increase in glucagon secretion, content, and gene expression.^51,52

Islets exposed to synthetic CB receptors antagonists

The synthetic compound SR141716A, also called rimonabant, is a highly selective antagonist for CB1 receptor, suggested for the treatment of overweight, obesity and diabetes, but suspended until today.^42,60,61 Clinical studies demonstrated rimonabant reduced significantly adipose mass on obese and diabetic patients, increasing adiponectin expression, lipolysis, and decreasing pro-inflammatory and other cardiovascular risk factors.^60–64 The study of rimonabant in obesity (RIO) showed several benefits in obese and non-obese patients: 20 mg/day treatment improved carbohydrate and lipid metabolisms in peripheral tissues, mainly skeletal muscle, adipose tissue and liver.⁶³ The effects on insulin secretion and sensitivity induced a significant reduction in glycated hemoglobin percent and LDL level.⁶³

In Zucker fatty rats and mice with diet-induced metabolic syndrome, chronic rimonabant treatment improved oral glucose tolerance and insulin resistance. Specific analysis of pancreatic islets correlated insulin expression, content and secretion with slowing beta-cell dysfunction: 60% reduction on islet-cell surface (in these models, the islet mass and area increases in correlation with dysfunction), and 50% less disorganized islets, with a normal distribution of cells.^60,64,65

However, rimonabant was discontinued after undesired side effects reported, including dizziness, psychiatric disorders, symptoms of depression, anxiety, and the development of suicidal intention.^66,67 In the last decades two different CB1 receptor antagonists have been synthetized, BAR-1 and Ibipinabant.^42,68–70 In isolated pancreatic islets from rats, BAR-1 increased insulin, glucokinase, PDX-1 and CB1r gene expression, and basal insulin secretion was modified, in opposition to AEA effect.⁴² BAR-1 treatment of prediabetic and diabetic mice reduced oral glucose tolerance without effect on HbA1c and triglycerides; glucose-stimulated insulin secretion was enhanced, but gene expression for insulin, glucagon, PDX-1 and some elements of ECS showed significant decrease.⁶⁸ Streptozocin-induced diabetic mice presented a discrete increase in islets mass and insulin-positive cells after BAR-1 exposure, suggesting a protective effect against apoptosis.⁶⁸ Ibipinabant is another rimonabant analog tested in animal models that improved glucose tolerance, insulin sensitivity, weight loss, triglycerides in blood, and induced a significant reduction in beta-cell lost.^69,70

In the last decade, AM251, AM281 and LY320135 were proposed as synthetic antagonists or reverse agonists for cannabinoid receptors. Isolated human and mouse islets short-term exposed to AM251 increased beta-cell proliferation after cytokines stimulation.^46,47,71 As a CB1 receptor antagonist AM251 exerts stimulatory effect on glucose-stimulated insulin secretion in isolated islets from mice and human, but as GPR55 agonist enhances GLP-1 receptor activity, and therefore, potentiates insulin secretion.

Genetic ablation of CB1 receptor in islets

Obese mice and rats with modified gene expression or knock-out for CB1 receptor have reduced food intake and body fat deposits, showed a sustained loss weight, reversible changes in several metabolites, and increased plasmatic levels of leptin, insulin and adiponectin.^{47,48,56,72,73} In mice with specific CB1 receptor -/- knock-out in pancreatic beta-cells, food intake was diminished while a significant increase in fasting insulin and insulin secretion during a glucose-lipid challenge was observed.⁷³ Isolated islets from these animals increased glucose-stimulated insulin secretion, preceded by elevated cAMP levels. Gene ablation also modified beta-cells viability, induced a discrete proliferation, and protection against diet-induced inflammation, supporting the interaction with insulin receptor and other signaling pathways.^{22,56,73–75} These results suggest CB1 receptor has a negative regulatory function in beta-cell as mediator of inflammation under metabolic stress condition. Pancreatic beta-cells and hepatocytes express a specific isoform of CB1 receptor; exposure to the reverse agonist JD-5037 increase insulin secretion associated to cAMP production, suggesting it as a potential therapeutic target for metabolic disorders.⁴⁸

Islets exposed to natural phytocannabinoids

The use of some Cannabis derivatives as alternative treatment for diabetic patients is still on debate. Chronic marijuana diabetic users usually present low fasting insulin levels, reduced HOMA-IR index, adequate waist circumference, and low prevalence of complications.^3,4,76,77 Diabetic rats treated chronically with 3 to 5 mg/day of THC presented improvement in glucose tolerance, total cholesterol, HDL and LDL, small decrease in body weight, increased total area of islets, and upregulation of gene expression for CB1 receptor, glucose transporter GLUT2, uncoupling protein UCP2, and protein kinase B.^49,78 These studies suggest THC treatment could improve hyperlipidemia, hyperglycemia, and islet dysfunction.^25,26 However, we assume that therapeutic effects of THC should be promoted as agonist of GPR55 receptor, after CB1 receptor presents oversaturation at nanomolar range. Unpublished data from our group suggest chronic exposure to THC increases insulin secretion in isolated islets, and improves oral glucose tolerance test in prediabetic mice, but decrease gene expression meanwhile islet structure remains unchanged (García-Luna and Vilches-Flores, 2023). To date, there are no direct evidences of the effect of THC on human islets.

Other receptors and signaling pathways related to ECS

CB1 receptor agonists AEA, N-arachidonoyl dopamine and N-arachidonylglycine can activate the transient potential receptor V1 (TRVP-1) or GPR18 receptor in pancreatic islets, elevate intracellular Ca²⁺ and potentiate insulin secretion, through coupling with the PI-PLC pathway and insulin receptor signaling.^38,58,74 CB1 receptor coupled to heterodimers of Gαi proteins interacts with insulin receptor through direct binding to the tyrosine-kinase domain, affecting the activation loop; consequently, phosphorylation of pro-apoptotic protein Bad is reduced, leading to apoptotic activity.^50,56 CB1 receptor blockade or reduced expression improves insulin signaling by increase in cAMP production, leading to improvements on hyperglycemia, cell survival, and islet cells proliferation.⁷² Other evidence demonstrate ECs regulate CB1 receptor interaction with focal adhesion kinase (FAK), mediating insulin exocytosis process by cytoskeleton rearrangement.^48,79 In addition, CB1 receptor can interact with the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway; in isolated islets, antagonists for CB1 and CB2 receptors decreased glucose-stimulated insulin secretion and mTOR activity, meanwhile in vivo they induced glucose intolerance and insulin resistance.⁷⁵

ECS expressed in adipose tissue, liver and pancreatic islet present a complex interaction and coordination between them to regulate glucose homeostasis. Treatment with CB2 receptor agonists JWH-015 and SER 601 induces changes in gene expression of the ESC elements, in liver, adipose tissue and pancreatic islets from mice; insulin secretion, glucose and lipid metabolisms, cell signaling, and immune responses in diabetic animal models are improved.^{31,33,40,41,45,51,52,68,80} The function of GPR55 as part of the ECS within pancreatic islet is still under study, pointing to different pharmacological strategies will be target in future. Synthetic agonists abbnormal cannabidiol (abn-CBD) and O-1602 improve the function and viability of beta-cells from humans and rats, potentiate glucose-stimulated insulin secretion through increase cAMP and intracellular Ca²⁺, reduce cytokine-induced apoptosis, cytotoxic effects, and increase AKT phosphorylation (Figure 3).^53–55

Implications of the ECS in the development of pathologies associated with the islet

Under physiological conditions the ECS in pancreatic islets participates integrally in the regulation of insulin secretion, according to food intake and in close coordination with other peripheral organs. We have exposed how CB receptors on islets, modulated by agonists and antagonists, are close-related to glucose metabolism, synthesis, secretion and action of insulin. ECs high production and the over-activation of cannabinoid receptors have important implications in white adipose tissue, hepatocytes and pancreatic islets, inducing deficiencies in hormonal secretion, hyperglycemia and metabolism imbalance.^{29–33,45,62} Impaired or over-activated ECS presents a direct correlation with weight gain, decreased insulin sensitivity, hyperinsulinemia, glucose intolerance and hyperlipidemia, contributing to the development of obesity and type-2 diabetes mellitus.^{35,39–42,52,60,64–66}

The chronic hyperactivity of CB receptors and the elevated EC production have been associated with significant alterations in insulin secretion, dysfunction of pancreatic islets and type-2 diabetes progression. It is not clear the therapeutic role of THC, cannabidiol and other agonists of the cannabinoid receptors in islets, but evidence suggest a complex interaction and adaptative response over chronic exposure. CB1 receptor antagonists are proposed as therapeutic alternative to delay islet cell dysfunction, reduce obesity and improve insulin sensitivity, decrease blood triglyceride levels and weight, through insulin- and glucagon-mediated actions.^49,61,63,77

Acknowledgments

This review and our research line is supported by PAPIIT-DGAPA UNAM, grant project IN225819.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the PAPIIT, DGAPA-UNAM [IN225819].