TELEMOLD project: oximetry and exercise telemonitoring to improve long-term oxygen therapy.

BACKGROUND: Standard assessment of long-term oxygen therapy (LTOT) prescription involves hospital-based clinical tests. However, there is some evidence suggesting that oxygen demand during daily activities may not be correctly estimated by such tests, when compared with continuous ambulatory oximetry. The authors describe the results of a study aiming to evaluate the clinical relevance of a home telemonitoring system in LTOT optimization. SUBJECTS AND METHODS: Thirty-five chronic respiratory failure patients were monitored in real time with an oximeter sensor and an accelerometer. Signals were sent via Bluetooth(®) (Bluetooth SIG, Kirkland, WA) to a mobile phone and then via 3G or general packet radio service to a server. Continuous and secure access to data was established through an Internet site. RESULTS: Each patient was monitored an average of 7.6 ± 4.5 days (total, 83 ± 67 h). Valid records were on average 65 ± 24%. Records of rest, activity, and sleep time per patient were, on average, 28 ± 21%, 7 ± 6%, and 59 ± 25%, respectively. Significant desaturation during rest, activity, and sleep was found in 2, 26, and 9 patients, respectively. Patients' ratings of the user-friendliness of the equipments, assessed by questionnaire, were fairly good (76% reported it as easy/very easy). CONCLUSIONS: Our study suggests that a telemonitoring system combining oximetry and physical activity evaluation might contribute to a more adequate oxygen prescription, mainly during daily activities.

The Relationship Between Circulating Endogenous Cannabinoids and the Effects of Smoked Cannabis.

Background: The endogenous cannabinoid system (ECS), including the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG), plays an integral role in psychophysiological functions. Although frequent cannabis use is associated with adaptations in the ECS, the impact of acute smoked cannabis administration on circulating eCBs, and the relationship between cannabis effects and circulating eCBs are poorly understood. Methods: This study measured the plasma levels of AEA, 2-AG, and Δ-9-tetrahydrocannabinol (THC), subjective drug-effects ratings, and cardiovascular measures at baseline and 15-180 min after cannabis users (n=26) smoked 70% of a cannabis cigarette (5.6% THC). Results: Cannabis administration increased the ratings of intoxication, heart rate, and plasma THC levels relative to baseline. Although cannabis administration did not affect eCB levels relative to baseline, there was a significant positive correlation between baseline AEA levels and peak ratings of "High" and "Good Drug Effect." Further, baseline 2-AG levels negatively correlated with frequency of cannabis use (mean days/week) and with baseline THC metabolite levels. Conclusions: In a subset of heavy cannabis smokers: (1) more frequent cannabis use was associated with lower baseline 2-AG, and (2) those with lower AEA got less intoxicated after smoking cannabis. These findings contribute to a sparse literature on the interaction between endo- and phyto-cannabinoids. Future studies in participants with varied cannabis use patterns are needed to clarify the association between circulating eCBs and the abuse-related effects of cannabis, and to test whether baseline eCBs predict the intoxicating effects of cannabis and are a potential biomarker of cannabis tolerance.

Pharmacological activation of kainate receptors drives endocannabinoid mobilization.

Activation of both presynaptic metabotropic cannabinoid type 1 receptors (CB(1)s) and ionotropic kainate receptors (KARs) can efficiently modulate GABA release at many synapses of the CNS. The inhibitory effect of kainic acid (KA) has been ascribed to metabotropic actions, and KAR-induced release of secondary neuromodulatory agents may partly mediate these actions. Here, we investigated the involvement of the endocannabinoid system in the modulation of GABAergic synaptic transmission by pharmacological activation of KARs with KA in CA1 pyramidal neurons of the mouse hippocampus. We show that the depression of GABAergic synaptic transmission induced by KA (3 µm) is strongly inhibited by the simultaneous blockade of CB(1) and GABA(B) receptors with SR141716A (5 µm) and CGP55845 (5 µm), respectively. KA induces a calcium-dependent mobilization of the endocannabinoid anandamide (AEA) by activation of GluK2-containing KARs in postsynaptic pyramidal neurons. Consistently, the effect of KA is prolonged by the inhibitor of AEA degradation URB597 (1 µm) in a CB(1)-dependent manner, but it is not altered by blockade of degradation or synthesis of the other main endocannabinoid 2-arachidonoylglycerol (2AG). Hence, our work reveals that the pharmacological activation of KARs leads to the stimulation of secondary metabotropic signaling systems. In addition, these data further underline the profound mechanistic differences between exogenous and endogenous activation of KARs in the hippocampus.

Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes.

The tissue concentrations of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F442A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), alpha-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as 2-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated 2-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom 2 (sn-2), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased 2-AG and anandamide levels and the amounts of AA esterified on both the sn-2 and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.

Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.

Inhibitory effect of the anorexic compound oleoylethanolamide on gastric emptying in control and overweight mice.

Gastric emptying regulates food intake. Oleoylethanolamide (OEA), an endogenous acylethanolamide chemically related to the endocannabinoid anandamide, inhibits food intake, but its effect on gastric emptying is unknown. Here, we investigated the effect and the role of OEA on gastric emptying in mice fed either a standard (STD) or a high-fat diet (HFD) for 14 weeks. Gastric emptying was reduced by OEA, but not by its saturated analog, palmitoylethanolamide. The effect of OEA was unaffected by rimonabant (cannabinoid CB1 receptor antagonist), SR144528 (cannabinoid CB2 receptor antagonist), 5'-iodoresiniferatoxin (transient receptor potential vanilloid type 1 antagonist), or MK886 (peroxisome proliferator-activated receptor-alpha) antagonist. Compared to STD mice, HFD mice showed delayed gastric emptying and higher levels of gastric OEA. HFD-induced increase in OEA levels was accompanied by increased expression of the OEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-selective phospholipase D and decreased expression of the OEA-degrading enzyme fatty acid amide hydrolase. These results might suggest that elevation of gastric OEA could possibly contribute to the delayed gastric emptying observed in HFD-fed animals. HFD regulates OEA levels in the stomach through an increase of its biosynthesis and a decrease of its enzymatic degradation. The inhibitory effect of OEA on gastric emptying here observed might underlie part of the anorexic effects of this compound previously reported.

Endocannabinoid control of food intake and energy balance.

Marijuana and its major psychotropic component, Delta(9)-tetrahydrocannabinol, stimulate appetite and increase body weight in wasting syndromes, suggesting that the CB(1) cannabinoid receptor and its endogenous ligands, the endocannabinoids, are involved in controlling energy balance. The endocannabinoid system controls food intake via both central and peripheral mechanisms, and it may also stimulate lipogenesis and fat accumulation. Here we discuss the multifaceted regulation of energy homeostasis by endocannabinoids, together with its applications to the treatment of eating disorders and metabolic syndromes.

Regulation of hypothalamic endocannabinoid levels by neuropeptides and hormones involved in food intake and metabolism: insulin and melanocortins.

Endocannabinoids are paracrine/autocrine lipid mediators with several biological functions. One of these, i.e. the capability to stimulate food intake via cannabinoid CB(1) receptors, has been particularly studied, thus leading to the development of the first CB(1) receptor blocker, rimonabant, as a therapeutic tool against obesity and related metabolic disorders. Hypothalamic endocannabinoids stimulate appetite by regulating the expression and release of anorexic and orexigenic neuropeptides via CB(1) receptors. In turn, the tone of the latter receptors is regulated by hormones, including leptin, glucocorticoids and possibly ghrelin and neuropeptide Y, by modulating the biosynthesis of the endocannabinoids in various areas of the hypothalamus. CB(1) receptor stimulation is also known to increase blood glucose during an oral glucose tolerance test in rats. Here we investigated in the rat if insulin, which is known to exert fundamental actions at the level of the mediobasal hypothalamus (MBH), and the melanocortin system, namely alpha-melanocyte stimulating hormone (alpha-MSH) and melanocortin receptor-4 (MCR-4), also regulate hypothalamic endocannabinoid levels, measured by isotope-dilution liquid chromatography coupled to mass spectrometry. No effect on anandamide and 2-arachidonoylglycerol levels was observed after 2h infusion of insulin in the MBH, i.e. under conditions in which the hormone reduces blood glucose, nor with intra-cerebroventricular injection of alpha-MSH, under conditions in which the neuropeptide reduces food intake. Conversely, blockade of MCR-4 receptors with HS014 produced a late (6h after systemic administration) stimulatory effect on endocannabinoid levels as opposed to a rapid and prolonged stimulation of food-intake (observable 2 and 6h after administration). These data suggest that inhibition of endocannabinoid levels does not mediate the effect of insulin on hepatic glucose production nor the food intake-inhibitory effect of alpha-MSH, although stimulation of endocannabinoid levels might underlie part of the late stimulatory effects of MCR-4 blockade on food intake.

Dysregulation of peripheral endocannabinoid levels in hyperglycemia and obesity: Effect of high fat diets.

Increasing evidence indicates that endocannabinoid (EC) signalling is dysregulated during hyperglycemia and obesity, particularly at the level of anandamide (AEA) and/or 2-arachidonoylglycerol (2-AG) concentrations in tissues involved in the control of energy intake and processing, such as the liver, white adipose tissue and pancreas. Here we review this previous evidence and provide new data on the possible dysregulation of EC levels in organs with endocrine function (adrenal glands and thyroid), involved in energy expenditure (brown adipose tissue and skeletal muscle), or affected by the consequences of metabolic disorders (heart and kidney), obtained from mice fed for 3, 8 and 14 weeks with two different high fat diets (HFDs), with different fatty acid compositions and impact on fasting glucose levels. Statistically significant elevations (in the skeletal muscle, heart and kidney) or reductions (in the thyroid) of the levels of either AEA or 2-AG, or both, were found. Depending on the diet, these changes preceded or accompanied the development of overt obesity and/or hyperglycemia. In the adrenal gland, first a reduction and then an elevation of EC levels were observed. In the brown fat, a very early elevation of both AEA and 2-AG normalized levels was observed with one of the diets, whereas delayed decreases were explained by an increase of the amount of fat tissue weight induced by the HFDs. The potential implications of these and previous findings in the general framework of the proposed roles of the EC system in the control of metabolic, endocrine and cardiovascular and renal functions are discussed.

Endocannabinoids and the control of energy balance.

Two receptors have been cloned to date for the psychotropic compound Delta(9)-tetrahydrocannabinol, and termed cannabinoid CB(1) and CB(2) receptors. Their endogenous ligands, the endocannabinoids, have also been identified. CB(1) receptors and endocannabinoids are present in brain structures controlling energy intake and in peripheral cells (hepatocytes, adipocytes, pancreatic islet cells) regulating energy homeostasis. CB(2) receptors are more abundant in lymphocytes and macrophages, and participate in immune and inflammatory reactions. Metabolic hormones and peptides regulate the levels of the endocannabinoids and, hence, the activity of cannabinoid receptors in several tissues in a seemingly coordinated way. The endocannabinoids, particularly after stress and brief food deprivation, act in turn as local modulators of the expression and action of neurotransmitters, hormones and adipokines involved in metabolic control. Endocannabinoid overactivity seems to accompany metabolic and eating disorders and to contribute to the development of abdominal obesity, dyslipidemia and hyperglycemia. Accordingly, clinical trials have shown that CB(1) receptor antagonists are efficacious at reducing not only food intake, but also abdominal adiposity and its metabolic sequelae.

Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia.

CONTEXT: Cannabinoid CB(1) receptor blockade decreases weight and hyperinsulinemia in obese animals and humans in a way greatly independent from food intake. OBJECTIVE: The objective of this study was to investigate the regulation and function of the endocannabinoid system in adipocytes and pancreatic beta-cells. DESIGN, SETTING, AND PATIENTS: Mouse 3T3-F442A adipocytes and rat insulinoma RIN-m5F beta-cells, pancreas and fat from mice with diet-induced obesity, visceral and sc fat from patients with body mass index equal to or greater than 30 kg/m(2), and serum from normoglycemic and type 2 diabetes patients were studied. MAIN OUTCOME MEASURE: Endocannabinoid enzyme and adipocyte protein expression, and endocannabinoid and insulin levels were measured. RESULTS: Endocannabinoids are present in adipocytes with levels peaking before differentiation, and in RIN-m5F beta-cells, where they are under the negative control of insulin. Chronic treatment of adipocytes with insulin is accompanied by permanently elevated endocannabinoid signaling, whereas culturing of RIN-m5F beta-cells in high glucose transforms insulin down-regulation of endocannabinoid levels into up-regulation. Epididymal fat and pancreas from mice with diet-induced obesity contain higher endocannabinoid levels than lean mice. Patients with obesity or hyperglycemia caused by type 2 diabetes exhibit higher concentrations of endocannabinoids in visceral fat or serum, respectively, than the corresponding controls. CB(1) receptor stimulation increases lipid droplets and decreases adiponectin expression in adipocytes, and it increases intracellular calcium and insulin release in RIN-m5F beta-cells kept in high glucose. CONCLUSIONS: Peripheral endocannabinoid overactivity might explain why CB(1) blockers cause weight-loss independent reduction of lipogenesis, of hypoadiponectinemia, and of hyperinsulinemia in obese animals and humans.

Evolutionary origins of the endocannabinoid system.

Endocannabinoid system evolution was estimated by searching for functional orthologs in the genomes of twelve phylogenetically diverse organisms: Homo sapiens, Mus musculus, Takifugu rubripes, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, Plasmodium falciparum, Tetrahymena thermophila, Archaeoglobus fulgidus, and Mycobacterium tuberculosis. Sequences similar to human endocannabinoid exon sequences were derived from filtered BLAST searches, and subjected to phylogenetic testing with ClustalX and tree building programs. Monophyletic clades that agreed with broader phylogenetic evidence (i.e., gene trees displaying topographical congruence with species trees) were considered orthologs. The capacity of orthologs to function as endocannabinoid proteins was predicted with pattern profilers (Pfam, Prosite, TMHMM, and pSORT), and by examining queried sequences for amino acid motifs known to serve critical roles in endocannabinoid protein function (obtained from a database of site-directed mutagenesis studies). This novel transfer of functional information onto gene trees enabled us to better predict the functional origins of the endocannabinoid system. Within this limited number of twelve organisms, the endocannabinoid genes exhibited heterogeneous evolutionary trajectories, with functional orthologs limited to mammals (TRPV1 and GPR55), or vertebrates (CB2 and DAGLbeta), or chordates (MAGL and COX2), or animals (DAGLalpha and CB1-like receptors), or opisthokonta (animals and fungi, NAPE-PLD), or eukaryotes (FAAH). Our methods identified fewer orthologs than did automated annotation systems, such as HomoloGene. Phylogenetic profiles, nonorthologous gene displacement, functional convergence, and coevolution are discussed.

Presynaptic facilitation of glutamatergic synapses to dopaminergic neurons of the rat substantia nigra by endogenous stimulation of vanilloid receptors.

Growing evidence regarding the function of vanilloid receptor-1 (VR1) in the brain suggests potential central roles of this receptor, previously described to occur primarily in peripheral sensory neurons. In the present study, we used electrophysiological and biochemical techniques to investigate the function and the endogenous stimulation of VR1 in dopaminergic neurons of the substantia nigra pars compacta (SNc). The VR1 agonist capsaicin increased the frequency of both TTX-sensitive and -insensitive spontaneous EPSCs (sEPSCs) without affecting their amplitude, suggesting a presynaptic site of action. In contrast, no effect was detected with regard to GABAergic transmission. No increase in sEPSC frequency was observed in the presence of cadmium chloride, while the voltage-dependent calcium channel antagonist omega-conotoxin MVIIC did not prevent capsaicin action. The VR1 antagonists capsazepine and iodoresiniferatoxin (IRTX) blocked the effects of capsaicin. Importantly, IRTX per se reduced sEPSC frequency, suggesting a tonic activity of VR1. The endogenous VR1 agonist anandamide (AEA) produced an IRTX-sensitive increase in the frequency of sEPSCs on dopaminergic neurons that was more pronounced when protein kinase A had been activated. Furthermore, mass spectrometric analyses and binding experiments revealed high levels of endogenous AEA and specific binding of AEA to VR1 receptors in the SNc. These data suggest a tonic facilitation of glutamate release exerted by VR1 in the SNc through a stimulation of VR1 by endovanilloids, including anandamide. The increase in sEPSC frequency by VR1 onto midbrain dopaminergic neurons suggests the involvement of these receptors in motor and cognitive functions involving the dopaminergic system.

Mitochondrial CB1 receptors regulate neuronal energy metabolism.

The mammalian brain is one of the organs with the highest energy demands, and mitochondria are key determinants of its functions. Here we show that the type-1 cannabinoid receptor (CB(1)) is present at the membranes of mouse neuronal mitochondria (mtCB(1)), where it directly controls cellular respiration and energy production. Through activation of mtCB(1) receptors, exogenous cannabinoids and in situ endocannabinoids decreased cyclic AMP concentration, protein kinase A activity, complex I enzymatic activity and respiration in neuronal mitochondria. In addition, intracellular CB(1) receptors and mitochondrial mechanisms contributed to endocannabinoid-dependent depolarization-induced suppression of inhibition in the hippocampus. Thus, mtCB(1) receptors directly modulate neuronal energy metabolism, revealing a new mechanism of action of G protein-coupled receptor signaling in the brain.

Home telemonitoring of severe chronic respiratory insufficient and asthmatic patients.

For 9 months we evaluated a portable device to transfer patient-clinician data by Internet: oximetry, ECG, clinical questionnaires and messages from the doctor. Fifty-one patients with severe chronic respiratory insufficiency (CRI) were followed at the hospital Pulido Valente and Espírito Santo and 21 asthmatics (A) were followed at the latter hospital. The use and acceptance of this device was evaluated through questionnaires soliciting patients' and health professionals' opinions. Patients with CRI followed in Lisbon were also asked about hospital admissions and quality of life compared with a nine month period before the monitoring programme. CRI patients found learning to use the system more difficult; the majority (80%) reported problems with the equipment, qualified as rare/occasional in 62% of the cases. For 31 CRI patients followed in Lisbon, the use of the system was classified as correct in 12 patients, incorrect in 7 and reasonable in 12 patients. The first group had a reducded number and duration of hospital admissions and also improved quality of life. With this remote monitoring system 80% of CRI patients reported they were more/much more supported and 33 patients (75%) would use this system in the future. 81% of asthmatic patients would also like to maintain this type of monitoring. The service was considered useful by the researchers. We concluded that home telemonitoring was a positive contribution to the management of chronic patients and raised awareness of it should be considered in the future.

Regulation and possible role of endocannabinoids and related mediators in hypercholesterolemic mice with atherosclerosis.

In this study we analysed the possible modulation of endocannabinoids and related molecules during atherosclerosis development in mice. Wild-type and apolipoprotein E knockout (ApoE(-/-)) mice were fed either normal chow or high-cholesterol diet for 8-12 weeks, and tissue endocannabinoid levels were measured by liquid chromatography-mass spectrometry. We found increased levels of 2-AG in aortas and visceral adipose tissue (VAT) of ApoE(-/-) mice fed on high-cholesterol diet for 12 weeks as compared to ApoE(-/-) mice fed on normal chow or wild-type mice fed on cholesterol. No significant difference in 2-AG levels was observed after 8 weeks of diet, and no changes in anandamide levels were found in any group. The levels of the anandamide-related mediators with anti-inflammatory or anti-lipogenic properties, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), decreased or increased only in VAT or in both tissues, respectively. Endocannabinoid- and OEA/PEA-degrading enzymes were expressed by macrophages within atherosclerotic lesions. In vitro, 2-AG and OEA-induced monocyte migration at 0.3-1microM, which corresponds to the levels observed in aortas. PEA 1microM also induced monocyte migration but counteracted the effect of 2-AG, whereas OEA enhanced it. Enhanced 2-AG levels in advanced atherosclerotic lesions may trigger the inflammatory process by recruiting more inflammatory cells and inducing extracellular matrix degradation via CB(2) receptors, and this possibility was supported in vitro but not in vivo by experiments with the CB(2) antagonist, SR144528.

Anti-inflammatory effect of palmitoylethanolamide on human adipocytes.

Obesity leads to the appearance of an inflammatory process, which can be initiated even with a moderate weight gain. Palmitoylethanolamide (PEA) is an endogenous lipid, secreted by human adipocytes, that possesses numerous anti-inflammatory properties. The main purpose of this study was to investigate the anti-inflammatory effect of PEA on human adipocytes, as well as in a murine model. The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-treated human subcutaneous adipocytes in primary culture and CF-1 mice was investigated by enzyme-linked immunosorbent assay. The effects of PEA on adipocyte TNF-alpha secretion were explored as well as some suspected PEA anti-inflammatory pathways: nuclear factor-kappaB (NF-kappaB) pathway, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) gene expression, and TNF-alpha-converting enzyme (TACE) activity. The effects of PEA on the TNF-alpha serum concentration in intraperitoneally LPS-treated mice were also studied. We demonstrate that the LPS induced secretion of TNF-alpha by human adipocytes is inhibited by PEA. This action is neither linked to a reduction in TNF-alpha gene transcription nor to the inhibition of TACE activity. Moreover, PPAR-alpha is not implicated in this anti-inflammatory activity. Lastly, PEA exhibits a wide-reaching anti-inflammatory action as the molecule is able to completely inhibit the strong increase in TNF-alpha levels in the serum of mice treated with high doses of LPS. In view of its virtual lack of toxicity, PEA might become a potentially interesting candidate molecule in the prevention of obesity-associated insulin resistance.

Role of insulin as a negative regulator of plasma endocannabinoid levels in obese and nonobese subjects.

OBJECTIVE: Endocannabinoids (ECs) control metabolism via cannabinoid receptors type 1 (CB1). Their plasma levels are elevated in overweight type 2 diabetes (T2D) and in obese patients, and decrease postprandially in normoweight individuals. We investigated in two different cohorts of nonobese or obese volunteers whether oral glucose in glucose tolerance tests (OGTT) or acute insulin infusion during euglycemic hyperinsulinemic clamp affect plasma EC levels. DESIGN AND METHODS: OGTT was performed in ten obese hyperinsulinemic patients (body mass index (BMI)=35.8 kg/m2, fasting insulin=14.83 mU/l), and ten normoweight normoinsulinemic volunteers (BMI=21.9 kg/m2, fasting insulin=7.2 mU/l). Insulin clamp was performed in 19 mostly nonobese men (BMI=25.8 kg/m2) with varying degrees of liver fat and plasma triglycerides (TGs), with (n=7) or without T2D. Plasma levels of ECs (anandamide and 2-arachidonoylglycerol (2-AG)) were measured by liquid chromatography-mass spectrometry, before and 60 and 180 min after OGTT, and before and 240 and 480 min after insulin or saline infusion. RESULTS: Oral glucose load decreased anandamide plasma levels to an extent inversely correlated with BMI, waist circumference, subcutaneous fat, fasting insulin and total glucose, and insulin areas under the curve during the OGTT, and nonsignificantly in obese volunteers. Insulin infusion decreased anandamide levels to an extent that weakly, but significantly, correlated negatively with TGs, liver fat and fasting insulin, and positively with high density lipoprotein cholesterol. OGTT decreased 2-AG levels to a lower extent and in a way weakly inversely correlated with fasting insulin. CONCLUSIONS: We suggest that insulin reduces EC levels in a way inversely related to anthropometric and metabolic predictors of insulin resistance and dyslipidemia.

Endocannabinoid dysregulation in the pancreas and adipose tissue of mice fed with a high-fat diet.

OBJECTIVE: In mice, endocannabinoids (ECs) modulate insulin release from pancreatic beta-cells and adipokine expression in adipocytes through cannabinoid receptors. Their pancreatic and adipose tissue levels are elevated during hyperglycemia and obesity, but the mechanisms underlying these alterations are not understood. METHODS AND PROCEDURES: We assessed in mice fed for up to 14 weeks with a standard or high-fat diet (HFD): (i) the expression of cannabinoid receptors and EC biosynthesizing enzymes (N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) and DAGLalpha) and degrading enzymes (fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) in pancreatic and adipose tissue sections by immunohistochemical staining; (ii) the amounts, measured by liquid chromatography-mass spectrometry, of the ECs, 2-AG, and anandamide (AEA). RESULTS: Although CB(1) receptors and biosynthetic enzymes were found mostly in alpha-cells, degrading enzymes were identified in beta-cells. Following HFD, staining for biosynthetic enzymes in beta-cells and lower staining for FAAH were observed together with an increase of EC pancreatic levels. While we observed no diet-induced change in the intensity of the staining of EC metabolic enzymes in the mesenteric visceral fat, a decrease in EC concentrations was accompanied by lower and higher staining of biosynthesizing enzymes and FAAH, respectively, in the subcutaneous fat. No change in cannabinoid receptor staining was observed following HFD in any of the analyzed tissues. DISCUSSION: We provide unprecedented information on the distribution of EC metabolic enzymes in the pancreas and adipose organ, where their aberrant expression during hyperglycemia and obesity contribute to dysregulated EC levels.

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.