Cocaine-induced behavioral sensitization is associated with changes in the expression of endocannabinoid and glutamatergic signaling systems in the mouse prefrontal cortex.

BACKGROUND: Endocannabinoids modulate the glutamatergic excitatory transmission by acting as retrograde messengers. A growing body of studies has reported that both signaling systems in the mesocorticolimbic neural circuitry are involved in the neurobiological mechanisms underlying drug addiction. METHODS: We investigated whether the expression of both endocannabinoid and glutamatergic systems in the prefrontal cortex (PFC) were altered by an acute and/or repeated cocaine administration schedule that resulted in behavioral sensitization. We measured the protein and mRNA expression of the main endocannabinoid metabolic enzymes and the cannabinoid receptor type 1 (CB1). We also analyzed the mRNA expression of relevant components of the glutamate-signaling system, including glutamate-synthesizing enzymes, metabotropic receptors, and ionotropic receptors. RESULTS: Although acute cocaine (10 mg/kg) produced no significant changes in the endocannabinoid-related proteins, repeated cocaine administration (20 mg/kg daily) induced a pronounced increase in the CB1 receptor expression. In addition, acute cocaine administration (10 mg/kg) in cocaine-sensitized mice (referred to as cocaine priming) induced a selective increase in the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). These protein changes were accompanied by an overall decrease in the ratios of endocannabinoid synthesis/degradation, especially the N-acyl phosphatidylethanolamine phospholipase D/FAAH and diacylglycerol lipase alpha/MAGL ratios. Regarding mRNA expression, while acute cocaine administration produced a decrease in CB1 receptors and N-acyl phosphatidylethanolamine phospholipase D, repeated cocaine treatment enhanced CB1 receptor expression. Cocaine-sensitized mice that were administered priming injections of cocaine mainly displayed an increased FAAH expression. These endocannabinoid changes were associated with modifications in glutamatergic transmission-related genes. An overall decrease was observed in the mRNA expression of the glutamate-synthesizing gene kidney-type glutaminase (KGA), the metabotropic glutamate receptors (mGluR3 and GluR), and subunits of NMDA ionotropic receptors (NR1, NR2A, NR2B and NR2C) after acute cocaine administration, while mice repeatedly exposed to cocaine only displayed an increase in NR2C. However, in cocaine-sensitized mice primed with cocaine, this inhibition was reversed and a strong increase was detected in the mGluR5, NR2 subunits, and both GluR1 and GluR3. CONCLUSIONS: These findings indicate that cocaine sensitization is associated with an endocannabinoid downregulation and a hyperglutamatergic state in the PFC that, overall, contribute to an enhanced glutamatergic input into PFC-projecting areas.

Diet-dependent modulation of hippocampal expression of endocannabinoid signaling-related proteins in cannabinoid antagonist-treated obese rats.

Diet-induced obesity produces changes in endocannabinoid signaling (ECS), influencing the regulation of energy homeostasis. Recently, we demonstrated that, in high-fat-diet-fed rats, blockade of CB1 receptor by AM251 not only reduced body weight but also increased adult neurogenesis in the hippocampus, suggesting an influence of diet on hippocampal cannabinoid function. To further explore the role of hippocampal ECS in high-fat-diet-induced obesity, we investigated whether the immunohistochemical expression of the enzymes that produce (diacylglycerol lipase alpha and N-acyl phosphatidylethanolamine phospholipase D) and degrade (monoacylglycerol lipase and fatty acid amino hydrolase) endocannabinoids may be altered in the hippocampus of AM251 (3 mg/kg)-treated rats fed three different diets: standard diet (normal chow), high-carbohydrate diet (70% carbohydrate) and high-fat diet (60% fat). Results indicated that AM251 reduced caloric intake and body weight gain, and induced a modulation of the expression of ECS-related proteins in the hippocampus of animals exposed to hypercaloric diets. These effects were differentially restricted to either the 2-arachinodoyl glycerol or anandamide signaling pathways, in a diet-dependent manner. AM251-treated rats fed the high-carbohydrate diet showed a reduction of the diacylglycerol lipase alpha : monoacylglycerol lipase ratio, whereas AM251-treated rats fed the high-fat diet showed a decrease of the N-acyl phosphatidylethanolamine phospholipase D : fatty acid amino hydrolase ratio. These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a diet-specific manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets.

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

BACKGROUND: 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. AIMS: To study the ability of IL-6 to modulate the expression of the LFABP in hepatocytes. METHODS: HepG2 and mouse primary hepatocytes were used to test LFABP mRNA and protein expression after IL-6 and PPARα-ligand treatments. Mice lacking IL-6 and wild-type C57Bl/6 were subjected to a fasting/re-feeding cycle to monitor hepatic LFABP mRNA kinetics after food intake. RESULTS: In hepatocyte cultures, IL-6 treatment stimulated a LFABP mRNA sustained expression. Combined treatment of IL-6 plus PPARα ligands further enhanced LFABP gene and protein expression. In contrast, pretreatment with the PPARα-antagonist GW-6471 prevented the up-regulation of LFABP mRNA induced by IL-6 in the late phase of LFABP kinetics. Furthermore, the up-regulation of LFABP mRNA observed in the liver of wild-type mice 8 h after re-feeding was absent in mice lacking IL-6. CONCLUSIONS: IL-6 induces LFABP kinetics in hepatocytes and is partially dependent on PPARα. The maximum increase in LFABP expression occurs when the stimulation with IL-6 and PPARα-ligands takes place simultaneously. The in vivo results indicate a postprandial regulation of LFABP that correlates with the presence of IL-6. These effects may have important implications in the postprandial increase in FA uptake and intracellular trafficking in the liver.

Oleoylethanolamide dose-dependently attenuates cocaine-induced behaviours through a PPARα receptor-independent mechanism.

Oleoylethanolamide (OEA) is an acylethanolamide that acts as an agonist of nuclear peroxisome proliferator-activated receptor alpha (PPARα) to exert their biological functions, which include the regulation of appetite and metabolism. Increasing evidence also suggests that OEA may participate in the control of reward-related behaviours. However, direct experimental evidence for the role of the OEA-PPARα receptor interaction in drug-mediated behaviours, such as cocaine-induced behavioural phenotypes, is lacking. The present study explored the role of OEA and its receptor PPARα on the psychomotor and rewarding responsiveness to cocaine using behavioural tests indicative of core components of addiction. We found that acute administration of OEA (1, 5 or 20 mg/kg, i.p.) reduced spontaneous locomotor activity and attenuated psychomotor activation induced by cocaine (20 mg/kg) in C57Bl/6 mice. However, PPARα receptor knockout mice showed normal sensitization, although OEA was capable of reducing behavioural sensitization with fewer efficacies. Furthermore, conditioned place preference and reinstatement to cocaine were intact in these mice. Our results indicate that PPARα receptor does not play a critical, if any, role in mediating short- and long-term psychomotor and rewarding responsiveness to cocaine. However, further research is needed for the identification of the targets of OEA for its inhibitory action on cocaine-mediated responses.

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.

Expression of the cannabinoid system in muscle: effects of a high-fat diet and CB1 receptor blockade.

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) ß and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.

D-Pinitol from Ceratonia siliqua Is an Orally Active Natural Inositol That Reduces Pancreas Insulin Secretion and Increases Circulating Ghrelin Levels in Wistar Rats.

To characterize the metabolic actions of D-Pinitol, a dietary inositol, in male Wistar rats, we analyzed its oral pharmacokinetics and its effects on (a) the secretion of hormones regulating metabolism (insulin, glucagon, IGF-1, ghrelin, leptin and adiponectin), (b) insulin signaling in the liver and (c) the expression of glycolytic and neoglucogenesis enzymes. Oral D-Pinitol administration (100 or 500 mg/Kg) resulted in its rapid absorption and distribution to plasma and liver compartments. Its administration reduced insulinemia and HOMA-IR, while maintaining glycaemia thanks to increased glucagon activity. In the liver, D-Pinitol reduced the key glycolytic enzyme pyruvate kinase and decreased the phosphorylation of the enzymes AKT and GSK-3. These observations were associated with an increase in ghrelin concentrations, a known inhibitor of insulin secretion. The profile of D-Pinitol suggests its potential use as a pancreatic protector decreasing insulin secretion through ghrelin upregulation, while sustaining glycaemia through the liver-based mechanisms of glycolysis control.

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

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/ß), 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/ß, 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.

CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity.

De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in the expression of lipogenic enzymes.

Pharmacological administration of the isoflavone daidzein enhances cell proliferation and reduces high fat diet-induced apoptosis and gliosis in the rat hippocampus.

Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17ß-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.

Anti-obesity efficacy of LH-21, a cannabinoid CB(1) receptor antagonist with poor brain penetration, in diet-induced obese rats.

BACKGROUND AND PURPOSE: Peripheral blockade of cannabinoid CB(1) receptors has been proposed as a safe and effective therapy against obesity, putatively devoid of the adverse psychiatric side effects of centrally acting CB(1) receptor antagonists. In this study we analysed the effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist with poor brain penetration, in an animal model of diet-induced obesity. EXPERIMENTAL APPROACH: To induce obesity, male Wistar rats were fed a high-fat diet (HFD; 60 kcal% fat) whereas controls received a standard diet (SD; 10 kcal% fat). Following 10 weeks of feeding, animals received a daily i.p. injection of vehicle or 3 mg·kg(-1) LH-21 for 10 days. Plasma and liver samples were used for biochemical analyses whereas visceral fat-pad samples were analysed for lipid metabolism gene expression using real-time RT-PCR. In addition, the potential of LH-21 to interact with hepatic cytochrome P450 isoforms and cardiac human Ether-à-go-go Related Gene (hERG) channels was evaluated. KEY RESULTS: LH-21 reduced feeding and body weight gain in HFD-fed animals compared with the control group fed SD. In adipose tissue, this effect was associated with decreased gene expression of: (i) leptin; (ii) lipogenic enzymes, including SCD-1; (iii) CB(1) receptors; and (iv) both PPARα and PPARγ. Although there were no significant differences in plasma parameters between HFD- and SD-fed rats, LH-21 did not seem to induce hepatic, cardiac or renal toxicity. CONCLUSIONS AND IMPLICATIONS: These results support the hypothesis that treatment with the peripherally neutral acting CB(1) receptor antagonist, LH-21, may promote weight loss through modulation of visceral adipose tissue.

Elaidyl-sulfamide, an oleoylethanolamide-modelled PPARα agonist, reduces body weight gain and plasma cholesterol in rats.

We have modelled elaidyl-sulfamide (ES), a sulfamoyl analogue of oleoylethanolamide (OEA). ES is a lipid mediator of satiety that works through the peroxisome proliferator-activated receptor alpha (PPARα). We have characterised the pharmacological profile of ES (0.3-3 mg/kg body weight) by means of in silico molecular docking to the PPARα receptor, in vitro transcription through PPARα, and in vitro and in vivo administration to obese rats. ES interacts with the binding site of PPARα in a similar way as OEA does, is capable of activating PPARα and also reduces feeding in a dose-dependent manner when administered to food-deprived rats. When ES was given to obese male rats for 7 days, it reduced feeding and weight gain, lowered plasma cholesterol and reduced the plasmatic activity of transaminases, indicating a clear improvement of hepatic function. This pharmacological profile is associated with the modulation of both cholesterol and lipid metabolism regulatory genes, including the sterol response element-binding proteins SREBF1 and SREBF2, and their regulatory proteins INSIG1 and INSIG2, in liver and white adipose tissues. ES treatment induced the expression of thermogenic regulatory genes, including the uncoupling proteins UCP1, UCP2 and UCP3 in brown adipose tissue and UCP3 in white adipose tissue. However, its chronic administration resulted in hyperglycaemia and insulin resistance, which represent a constraint for its potential clinical development.