Δ9-Tetrahydrocannabinol Treatment Modifies Insulin Secretion in Pancreatic Islets from Prediabetic Mice Under Hypercaloric Diet.

Background: The endocannabinoid system over-activation is associated with type-2 diabetes mellitus onset, involving physiological, metabolic, and genetic alterations in pancreatic islets. The use of Δ9-Tetrahydrocannabinol (THC) as treatment is still controversial since its effects and mechanisms on insulin secretion are unclear. The aim of this study was to evaluate the effects of THC treatment in pancreatic islets from prediabetic mice. Methods: Prediabetes was induced in mice by hypercaloric diet, and then treated with THC for 3 weeks. Blood glucose and body weight were determined, after behavior tests. Histological changes were evaluated in whole pancreas; in isolated islets we analyzed the effect of THC exposure in glucose-stimulated insulin secretion (GSIS), gene expression, intracellular cyclic adenosine monophosphate (cAMP), and cytosolic calcium changes. Results: THC treatment in prediabetic mice enhanced anxiety and antidepressive behavior without changes in food ingestion, decreased oral-glucose tolerance test, plasma insulin and weight, with small alterations on pancreatic histology. In isolated islets from healthy mice THC increased GSIS, cAMP, and CB1 receptor (CB1r) expression, meanwhile calcium release was diminished. Small changes were observed in islets from prediabetic mice. Conclusions: THC treatment improves some clinical parameters in prediabetic mice, however, in isolated islets, modifies GSIS, intracellular calcium and gene expression, suggesting specific effects related to diabetes evolution.

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

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.

Novel CB1 receptor antagonist BAR-1 modifies pancreatic islet function and clinical parameters in prediabetic and diabetic mice.

BACKGROUDS: Cannabinoid receptor antagonists have been suggested as a novel treatment for obesity and diabetes. We have developed a synthetic cannabinoid receptor antagonist denominated BAR-1. As the function and integrity of a ß-cell cellular structure are important keys for diabetes onset, we evaluated the effects of pharmacological administration of BAR-1 on prediabetic and diabetic rodents. METHODS: CD-1 mice fed a hypercaloric diet or treated with streptozotocin were treated with 10 mg/kg BAR-1 for 2, 4 or 8 weeks. Body weight, oral glucose tolerance test, HbA1c, triglycerides and insulin in serum were measured. In isolated islets, we evaluated stimulated secretion and mRNA expression, and relative area of islets in fixed pancreases. Docking analysis of BAR-1 was complemented. RESULTS: BAR-1 treatment slowed down weight gain in prediabetic mice. Fasting glucose-insulin relation also decreased in BAR-1-treated mice and glucose-stimulated insulin secretion was increased in isolated islets, without effects in oral test. Diabetic mice treated with BAR-1 showed a reduced glucose and a partial recovery of islet integrity. Gene expression of insulin and glucagon showed biphasic behaviour, increasing after 4 weeks of BAR-1 administration; however, after 8 weeks, mRNA abundance decreased significantly. Administration of BAR-1 also prevents changes in endocannabinoid element expression observed in prediabetic mice. No changes were detected in other parameters studied, including the histological structure. A preliminary in-silico study suggests a close interaction with CB1 receptor. CONCLUSIONS: BAR-1 induces improvement of islet function, isolated from both prediabetic and diabetic mice. Effects of BAR-1 suggest a possible interaction with other cannabinoid receptors.

Prolonged activation of human islet cannabinoid receptors in vitro induces adaptation but not dysfunction.

BACKGROUND: Although in vivo studies have implicated endocannabinoids in metabolic dysfunction, little is known about direct, chronic activation of the endocannabinoid system (ECS) in human islets. Therefore, this study investigated the effects of prolonged exposure to cannabinoid agonists on human islet gene expression and function. METHODS: Human islets were maintained for 2 and 5 days in the absence or presence of CB1r (ACEA) or CB2r (JWH015) agonists. Gene expression was quantified by RT-PCR, hormone levels by radioimmunoassay and apoptosis by caspase activities. RESULTS: Human islets express an ECS, with mRNAs encoding the biosynthetic and degrading enzymes NAPE-PLD, FAAH and MAGL being considerably more abundant than DAGLα, an enzyme involved in 2-AG synthesis, or CB1 and CB2 receptor mRNAs. Prolonged activation of CB1r and CB2r altered expression of mRNAs encoding ECS components, but did not have major effects on islet hormone secretion. JWH015 enhanced insulin and glucagon content at 2 days, but had no effect after 5 days. Treatment with ACEA or JWH015 for up to 5 days did not have marked effects on islet viability, as assessed by morphology and caspase activities. CONCLUSIONS: Maintenance of human islets for up to 5 days in the presence of CB1 and CB2 receptor agonists causes modifications in ECS element gene expression, but does not have any major impact on islet function or viability. GENERAL SIGNIFICANCE: These data suggest that the metabolic dysfunction associated with over-activation of the ECS in obesity and diabetes in humans is unlikely to be secondary to impaired islet function.

Chronic activation of cannabinoid receptors in vitro does not compromise mouse islet function.

We have demonstrated previously that mouse and human islets express ECS (endocannabinoid system) elements, and that short-term activation of islet cannabinoid CB1r and CB2r (cannabinoid type 1 and 2 receptors respectively) stimulates insulin secretion in vitro. There is evidence that the ECS is overactive in Type 2 diabetes, impairing glucose homoeostasis, but little is known about whether it is implicated in islet dysfunction. Therefore the aim of the present study was to investigate the effect of chronic exposure of isolated mouse islets to cannabinoid receptor agonists on islet gene expression and function. Quantitative RT-PCR (reverse transcription-PCR) indicated that mRNAs encoding synthesis [NAPE-PLD (N-acyl-phosphatidyl ethanolamide-hydrolysing phospholipase D)] and degradation [FAAH (fatty acid amide hydrolase)] of the endocannabinoid AEA (anandamide) were the most abundant ECS elements in mouse islets, with much lower levels of CB1r, CB2r, DAGL (diacylglycerol lipase) and MAGL (monoacylglycerol lipase) mRNAs. Maintenance of islets for up to 7 days in the presence of the CB1r agonist ACEA [N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eiscosatetraenamide] or the CB2r agonist JWH015 [(2-methyl-1propyl-1H-indol3-yl)-1-napthalenylmethanone] did not compromise islet viability, as assessed by islet morphology and caspase activities, but there were some changes in mRNAs encoding ECS components. Neither glucose-stimulated insulin secretion nor acute insulin secretory responses to ACEA or JWH015 at 16 mM glucose were substantially modified by a 48 h or 7 day pre-exposure to these cannabinoid receptor agonists, but the stimulation of secretion at 3 mM glucose by 100 nM ACEA was significantly reduced after prolonged treatment with ACEA. Despite JWH015-induced reductions in islet glucagon content at 48 h and 7 days, there were no reductions in arginine-induced glucagon secretion from islets pre-exposed to JWH015 or ACEA. These data indicate that treatment of islets with agonists of CB1r and CB2r for up to 7 days does not have any major impact on islet function, suggesting that the impairments in glucose homoeostasis observed following overactivation of the ECS should be sought in relation to insulin resistance rather than ß-cell dysfunction.

Expression and function of monoacylglycerol lipase in mouse ß-cells and human islets of Langerhans.

Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB2 cannabinoid receptors regulates insulin secretion from mouse and human ß-cells. The current study aimed to investigate the expression and function, in mouse and human ß-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). We found that MGL mRNA is expressed by MIN6 ß-cells, mouse islets, human islets and enriched human islet ß-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some ß- and -α-cells. Blockade of MGL activity with the pharmacological inhibitor URB602 led to increased [Ca(2+)](i )and enhanced insulin secretion from MIN6 ß-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous 2-AG in islets that is amplified when its degradation is blocked.

CB1 cannabinoid receptor expression is regulated by glucose and feeding in rat pancreatic islets.

Endocannabinoid system is involved in food intake and energy balance. Beside the hypothalamus, pancreatic islet also expresses CB1 cannabinoid receptor, however little is known about its physiological role and regulation. Since gene expression of many specific proteins of the islet depends on the concentration of glucose, we studied CB1 receptor expression in response to fasting and feeding. Whole pancreas or islets were isolated from food-deprived adult Wistar rats, with or without a previous 1.5 g/kg glucose oral-intake. CB1, insulin and glucagon expressions were analyzed by confocal immunofluorescence and PCR. In vitro, rat islets were cultured at different glucose concentrations, in the presence of anandamide, or with Rimonabant analog BAR-1. CB1, insulin, glucagon, glucokinase, and PDX-1 expression were determined by real-time RT-PCR, and insulin secretion and islet content by ELISA. CB1 expression in pancreatic islets is upregulated during food restriction, and decreases in response to glucose intake or feeding. In cultured islets, 16 mmol/l glucose, BAR-1, and anandamide at low glucose reduced CB1 mRNA. Insulin, glucagon, glucokinase and PDX-1 expression increased in islets treated with anandamide at low glucose, while BAR-1 modified PDX-1 and glucagon mRNA at high glucose. Basal insulin secretion and insulin content in islets increased with anandamide, but not the glucose-stimulated response. Our results suggest that the endocannabinoid system has an important role in gene expression on islets and its close relationship with glucose response.

Macrophage migration inhibitory factor is a therapeutic target in treatment of non-insulin-dependent diabetes mellitus.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in the pathogenesis of a variety of autoimmune inflammatory diseases. Here, we investigated the role of MIF in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) using MIF(-/-) mice and a mouse model of streptozotocin (STZ)-induced NIDDM. Following single injection of STZ, MIF(+/+) BALB/c mice showed a significant increase in blood glucose levels, developed polyuria, and succumbed to disease. In contrast, no such increase in blood glucose was observed in MIF(-/-) BALB/c mice treated with STZ. These mice produced significantly less inflammatory cytokines and resistin as compared with MIF(+/+) mice and failed to develop clinical disease. Finally, oral administration of a small-molecule MIF antagonist, CPSI-1306, to outbred ICR mice following induction of NIDDM significantly lowered blood glucose levels in the majority of animals, which was also associated with a significant reduction in the levels of the proinflammatory cytokines IL-6 and TNF-alpha in the sera. Taken together, these results demonstrate that MIF is involved in the pathogenesis of NIDDM and is a therapeutic target to treat this disease.

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression.

Efecto de la biotina sobre la expresión genética y el metabolismo / Effect of biotin upon gene expression and metabolism

During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.

En décadas recientes, diversas investigaciones han demostrado que las vitaminas afectan la expresión genética. Los casos mejor estudiados son los de las vitaminas A y D. Existe menos información para las vitaminas hidrosolubles sobre su efecto en la expresión de los genes, sin embargo, se sabe que éstas también los modifican. La biotina es una vitamina hidrosoluble que actúa como grupo prostético de las carboxilasas. Además de su función como cofactor de enzimas, participa en el desarrollo embrionario, en la proliferación celular, en funciones inmunológicas y en el metabolismo. Ha habido un notable avance en la identificación de genes cuya expresión está regulada por la biotina. Asimismo, se han investigado los mecanismos moleculares a través de los cuales la biotina efectúa estas acciones. Estos estudios brindan nuevas claves para entender el papel de la biotina en la expresión genética, en el metabolismo, y en otras funciones biológicas de esta vitamina.

[Effect of biotin upon gene expression and metabolism]. / Efecto de la biotina sobre la expresión genética y el metabolismo.

During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.