Elena Baixeras

Presence of functional cannabinoid receptors in human endocrine pancreas

Aims/hypothesisWe examined the presence of functional cannabinoid receptors 1 and 2 (CB1, CB2) in isolated human islets, phenotyped the cells producing cannabinoid receptors and analysed the actions of selective cannabinoid receptor agonists on insulin, glucagon and somatostatin secretion in vitro. We also described the localisation on islet cells of: (1) the endocannabinoid-producing enzymes N-acyl-phosphatidyl ethanolamine-hydrolysing phospholipase D and diacylglycerol lipase; and (2) the endocannabinoid-degrading enzymes fatty acid amidohydrolase and monoacyl glycerol lipase.MethodsReal-time PCR, western blotting and immunocytochemistry were used to analyse the presence of endocannabinoid-related proteins and genes. Static secretion experiments were used to examine the effects of activating CB1 or CB2 on insulin, glucagon and somatostatin secretion and to measure changes in 2-arachidonoylglycerol (2-AG) levels within islets. Analyses were performed in isolated human islets and in paraffin-embedded sections of human pancreas.ResultsHuman islets of Langerhans expressed CB1 and CB2 (also known as CNR1 and CNR2) mRNA and CB1 and CB2 proteins, and also the machinery involved in synthesis and degradation of 2-AG (the most abundant endocannabinoid, levels of which were modulated by glucose). Immunofluorescence revealed that CB1 was densely located in glucagon-secreting alpha cells and less so in insulin-secreting beta cells. CB2 was densely present in somatostatin-secreting delta cells, but absent in alpha and beta cells. In vitro experiments revealed that CB1 stimulation enhanced insulin and glucagon secretion, while CB2 agonism lowered glucose-dependent insulin secretion, showing these cannabinoid receptors to be functional.Conclusions/interpretationTogether, these results suggest a role for endogenous endocannabinoid signalling in regulation of endocrine secretion in the human pancreas.

Regulation of brain anandamide by acute administration of ethanol

The endogenous cannabinoid acylethanolamide AEA (arachidonoylethanolamide; also known as anandamide) participates in the neuroadaptations associated with chronic ethanol exposure. However, no studies have described the acute actions of ethanol on AEA production and degradation. In the present study, we investigated the time course of the effects of the intraperitoneal administration of ethanol (4 g/kg of body mass) on the endogenous levels of AEA in central and peripheral tissues. Acute ethanol administration decreased AEA in the cerebellum, the hippocampus and the nucleus accumbens of the ventral striatum, as well as in plasma and adipose tissue. Parallel decreases of a second acylethanolamide, PEA (palmitoylethanolamide), were observed in the brain. Effects were observed 45-90 min after ethanol administration. In vivo studies revealed that AEA decreases were associated with a remarkable inhibition of the release of both anandamide and glutamate in the nucleus accumbens. There were no changes in the expression and enzymatic activity of the main enzyme that degrades AEA, the fatty acid amidohydrolase. Acute ethanol administration did not change either the activity of N-acyltransferase, the enzyme that catalyses the synthesis of the AEA precursor, or the expression of NAPE-PLD (N-acylphosphatidylethanolamine-hydrolysing phospholipase D), the enzyme that releases AEA from membrane phospholipid precursors. These results suggest that receptor-mediated release of acylethanolamide is inhibited by the acute administration of ethanol, and that this effect is not derived from increased fatty acid ethanolamide degradation.

Plasma profile of pro-inflammatory cytokines and chemokines in cocaine users under outpatient treatment: influence of cocaine symptom severity and psychiatric co-morbidity.

The treatment for cocaine use constitutes a clinical challenge because of the lack of appropriate therapies and the high rate of relapse. Recent evidence indicates that the immune system might be involved in the pathogenesis of cocaine addiction and its co‐morbid psychiatric disorders. This work examined the plasma pro‐inflammatory cytokine and chemokine profile in abstinent cocaine users (n = 82) who sought outpatient cocaine treatment and age/sex/body mass‐matched controls (n = 65). Participants were assessed with the diagnostic interview Psychiatric Research Interview for Substance and Mental Diseases according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM‐IV‐TR). Tumor necrosis factor‐alpha, chemokine (C‐C motif) ligand 2/monocyte chemotactic protein‐1 and chemokine (C‐X‐C motif) ligand 12 (CXCL12)/stromal cell‐derived factor‐1 (SDF‐1) were decreased in cocaine users, although all cytokines were identified as predictors of a lifetime pathological use of cocaine. Interleukin‐1 beta (IL‐1β), chemokine (C‐X3‐C motif) ligand 1 (CX3CL1)/fractalkine and CXCL12/SDF‐1 positively correlated with the cocaine symptom severity when using the DSM‐IV‐TR criteria for cocaine abuse/dependence. These cytokines allowed the categorization of the outpatients into subgroups according to severity, identifying a subgroup of severe cocaine users (9–11 criteria) with increased prevalence of co‐morbid psychiatric disorders [mood (54%), anxiety (32%), psychotic (30%) and personality (60%) disorders]. IL‐1β was observed to be increased in users with such psychiatric disorders relative to those users with no diagnosis. In addition to these clinical data, studies in mice demonstrated that plasma IL‐1β, CX3CL1 and CXCL12 were also affected after acute and chronic cocaine administration, providing a preclinical model for further research. In conclusion, cocaine exposure modifies the circulating levels of pro‐inflammatory mediators. Plasma cytokine/chemokine monitoring could improve the stratification of cocaine consumers seeking treatment and thus facilitate the application of appropriate interventions, including management of heightened risk of psychiatric co‐morbidity. Further research is necessary to elucidate the role of the immune system in the etiology of cocaine addiction.

Oleoylethanolamide enhances β-adrenergic-mediated thermogenesis and white-to-brown adipocyte phenotype in epididymal white adipose tissue in rat.

β-adrenergic receptor activation promotes brown adipose tissue (BAT) β-oxidation and thermogenesis by burning fatty acids during uncoupling respiration. Oleoylethanolamide (OEA) can inhibit feeding and stimulate lipolysis by activating peroxisome proliferator-activating receptor-α (PPARα) in white adipose tissue (WAT). Here we explore whether PPARα activation potentiates the effect of β3-adrenergic stimulation on energy balance mediated by the respective agonists OEA and CL316243. The effect of this pharmacological association on feeding, thermogenesis, β-oxidation, and lipid and cholesterol metabolism in epididymal (e)WAT was monitored. CL316243 (1 mg/kg) and OEA (5 mg/kg) co-administration over 6 days enhanced the reduction of both food intake and body weight gain, increased the energy expenditure and reduced the respiratory quotient (VCO2/VO2). This negative energy balance agreed with decreased fat mass and increased BAT weight and temperature, as well as with lowered plasma levels of triglycerides, cholesterol, nonessential fatty acids (NEFAs), and the adipokines leptin and TNF-α. Regarding eWAT, CL316243 and OEA treatment elevated levels of the thermogenic factors PPARα and UCP1, reduced p38-MAPK phosphorylation, and promoted brown-like features in the white adipocytes: the mitochondrial (Cox4i1, Cox4i2) and BAT (Fgf21, Prdm16) genes were overexpressed in eWAT. The enhancement of the fatty-acid β-oxidation factors Cpt1b and Acox1 in eWAT was accompanied by an upregulation of de novo lipogenesis and reduced expression of the unsaturated-fatty-acid-synthesis enzyme gene, Scd1. We propose that the combination of β-adrenergic and PPARα receptor agonists promotes therapeutic adipocyte remodelling in eWAT, and therefore has a potential clinical utility in the treatment of obesity.

Opposite clinical phenotypes of glucokinase disease: Description of a novel activating mutation and contiguous inactivating mutations in human glucokinase (GCK) gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Role of the satiety factor oleoylethanolamide in alcoholism

Oleoylethanolamide (OEA) is a satiety factor that controls motivational responses to dietary fat. Here we show that alcohol administration causes the release of OEA in rodents, which in turn reduces alcohol consumption by engaging peroxisome proliferator‐activated receptor‐alpha (PPAR‐α). This effect appears to rely on peripheral signaling mechanisms as alcohol self‐administration is unaltered by intracerebral PPAR‐α agonist administration, and the lesion of sensory afferent fibers (by capsaicin) abrogates the effect of systemically administered OEA on alcohol intake. Additionally, OEA is shown to block cue‐induced reinstatement of alcohol‐seeking behavior (an animal model of relapse) and reduce the severity of somatic withdrawal symptoms in alcohol‐dependent animals. Collectively, these findings demonstrate a homeostatic role for OEA signaling in the behavioral effects of alcohol exposure and highlight OEA as a novel therapeutic target for alcohol use disorders and alcoholism.

Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice.

ABSTRACT Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6−/−) mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis. Summary: The administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis in HFD-induced obesity.

IL-6 cooperates with peroxisome proliferator-activated receptor-α-ligands to induce liver fatty acid binding protein (LFABP) up-regulation

LFABP plays a critical role in the uptake and intracellular transport of fatty acids (FA) and other peroxisome proliferator‐activated receptor alpha (PPARα) ligands. PPARα activation by PPARα ligands bound to LFABP results in gene expression of FA oxidation enzymes and de novo LFABP. The cytokine IL‐6 is involved in regulating liver lipid oxidation.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle.

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB 1 -/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

ADIPONECTIN PROMOTER ACTIVATOR NP-1 REDUCES BODY WEIGHT AND HEPATIC STEATOSIS IN HIGH-FAT DIET-FED ANIMALS

Enhancement of adiponectin level has been shown to have beneficial effects, including antiobesity, antidiabetic, and hepatoprotective effects. This evidence supports the therapeutic utility of adiponectin in complicated obesity. The present study characterized the in vivo effects of sustained adiponectin release by NP-1, a new class of thiazol derivative that increases adiponectin levels. Acute administration of NP-1 reduced feeding, increased plasma adiponectin, and improved insulin sensitivity without inducing malaise, as revealed by conditioned taste aversion studies. Short-term (7 days) treatment with NP-1 also reduced feeding and body weight gain and increased phosphorylation of AMPK in muscle, a main intracellular effector of adiponectin. NP-1 was also evaluated in diet-induced obesity, and adult male Wistar rats were fed two different types of diet: a standard high-carbohydrate/low-fat diet (SD) and a high-fat diet (HFD). Once obesity was established, animals were treated daily with NP-1 (5 mg/kg) for 14 consecutive days. Chronic NP-1 induced body weight loss and reduction of food intake and resulted in both a marked decrease in liver steatosis and an improvement of biochemical indexes of liver damage in HFD-fed rats. However, a marked induction of tolerance in adiponectin gene transcription and release was observed after chronic NP-1 with respect to the acute actions of this drug. The present results support the role of adiponectin signaling in diet-induced obesity and set in place a potential use of compounds able to induce adiponectin release for the treatment of obesity and nonalcoholic fatty liver, with the limits imposed by the induction of pharmacological tolerance.