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Central and peripheral mechanisms modulate food intake and energy balance in mammals and the precise role of the type 1 cannabinoid receptor (CB1) in these processes is still being explored. Using the zebrafish, Danio rerio, we show that rimonabant, a CB1-specific antagonist with an EC(50) of 5.15 × 10(-8) m, decreases embryonic yolk sac reserve use. We reveal a developmental overlap between CART genes and CB1 expression in the hypothalamus and medulla oblongata, two brain structures that play crucial roles in appetite regulation in mammals. We show that morpholino knockdown of CB1 or fasting decreases cocaine- and amphetamine-related transcript (CART)-3 expression. Strikingly, this down-regulation occurs only in regions coexpressing CB1 and CART3, reinforcing the link between CB1, CART, and appetite regulation. We show that rimonabant treatment impairs the fasting-induced down-regulation of CART expression in specific brain regions, whereas vehicle alone-treated embryos do not display this rescue of CART expression. Our data reveal that CB1 lies upstream of CART and signals the appetite through the down-regulation of CART expression. Thus, our results establish the zebrafish as a promising system to study appetite regulation.

Cannabinoid receptor CB1 is expressed abundantly in the brain and presumably in the peripheral tissues responsible for energy metabolism. It is unclear if the antiobesity effects of rimonabant, a CB1 antagonist, are mediated through the central or the peripheral CB1 receptors. To address this question, we generated transgenic mice with central nervous system (CNS)-specific knockdown (KD) of CB1, by expressing an artificial microRNA (AMIR) under the control of the neuronal Thy1.2 promoter. In the mutant mice, CB1 expression was reduced in the brain and spinal cord, whereas no change was observed in the superior cervical ganglia (SCG), sympathetic trunk, enteric nervous system, and pancreatic ganglia. In contrast to the neuronal tissues, CB1 was undetectable in the brown adipose tissue (BAT) or the liver. Consistent with the selective loss of central CB1, agonist-induced hypothermia was attenuated in the mutant mice, but the agonist-induced delay of gastrointestinal transit (GIT), a primarily peripheral nervous system-mediated effect, was not. Compared to wild-type (WT) littermates, the mutant mice displayed reduced body weight (BW), adiposity, and feeding efficiency, and when fed a high-fat diet (HFD), showed decreased plasma insulin, leptin, cholesterol, and triglyceride levels, and elevated adiponectin levels. Furthermore, the therapeutic effects of rimonabant on food intake (FI), BW, and serum parameters were markedly reduced and correlated with the degree of CB1 KD. Thus, KD of CB1 in the CNS recapitulates the metabolic phenotype of CB1 knockout (KO) mice and diminishes rimonabant's efficacy, indicating that blockade of central CB1 is required for rimonabant's antiobesity actions.

The increase in adiponectin levels in obese patients with untreated dyslipidemia and its mRNA expression in adipose tissue of obese animals are one of the most interesting consequences of rimonabant treatment. Thus, part of rimonabant's metabolic effects could be related to an enhancement of adiponectin secretion and its consequence on the modulation of insulin action, as well as energy homeostasis. The present study investigated the effects of rimonabant in adiponectin knockout mice (Ad(-/-)) exposed to diet-induced obesity conditions. Six-week-old Ad(-/-) male mice and their wild-type littermate controls (Ad(+/+)) were fed a high-fat diet for 7 mo. During the last month, animals were administered daily either with vehicle or rimonabant by mouth (10 mg/kg). High-fat feeding induced weight gain by about 130% in both wild-type and Ad(-/-) mice. Obesity was associated with hyperinsulinemia and insulin resistance. Treatment with rimonabant led to a significant and similar decrease in body weight in both Ad(+/+) and Ad(-/-) mice compared with vehicle-treated animals. In addition, rimonabant significantly improved insulin sensitivity in Ad(+/+) mice compared with Ad(+/+) vehicle-treated mice by decreasing hepatic glucose production and increasing glucose utilization index in both visceral and subcutaneous adipose tissue. In contrast, rimonabant failed to improve insulin sensitivity in Ad(-/-) mice, despite the loss in body weight. Rimonabant's effect on body weight appeared independent of the adiponectin pathway, whereas adiponectin seems required to mediate rimonabant-induced improvement of insulin sensitivity in rodents.

Background: The visceral (VAT) and subcutaneous (SCAT) adipose tissues play different roles in physiology and obesity. The molecular mechanisms underlying their expansion in obesity and following body weight reduction are poorly defined., Methodology: C57Bl/6 mice fed a high fat diet (HFD) for 6 months developed low, medium, or high body weight as compared to normal chow fed mice. Mice from each groups were then treated with the cannabinoid receptor 1 antagonist rimonabant or vehicle for 24 days to normalize their body weight. Transcriptomic data for visceral and subcutaneous adipose tissues from each group of mice were obtained and analyzed to identify: i) genes regulated by HFD irrespective of body weight, ii) genes whose expression correlated with body weight, iii) the biological processes activated in each tissue using gene set enrichment analysis (GSEA), iv) the transcriptional programs affected by rimonabant., Principal Findings: In VAT, "metabolic" genes encoding enzymes for lipid and steroid biosynthesis and glucose catabolism were down-regulated irrespective of body weight whereas "structure" genes controlling cell architecture and tissue remodeling had expression levels correlated with body weight. In SCAT, the identified "metabolic" and "structure" genes were mostly different from those identified in VAT and were regulated irrespective of body weight. GSEA indicated active adipogenesis in both tissues but a more prominent involvement of tissue stroma in VAT than in SCAT. Rimonabant treatment normalized most gene expression but further reduced oxidative phosphorylation gene expression in SCAT but not in VAT., Conclusion: VAT and SCAT show strikingly different gene expression programs in response to high fat diet and rimonabant treatment. Our results may lead to identification of therapeutic targets acting on specific fat depots to control obesity.

(1) We investigated the effect of the cannabinoid CB1 receptor antagonist, SR 141716, on indomethacin-induced small intestine inflammation and Escherichia coli lipopolysaccharide (LPS)-induced plasma TNF-alpha (TNF) release in comparison to the cannabinoid CB2 receptor antagonist, SR 144528, in rodents. (2) In rats, indomethacin induced significant ulcer formation in the small intestine; this was accompanied by an increase in tissue TNF levels and myeloperoxidase (MPO) activity. SR 141716 prevented the ulcers and the rise in TNF levels (ID50 3.3, 0.4 mg kg-1, respectively) and MPO activity. SR 144528 prevented intestinal ulcers only. (3) The effect of SR 141716 against indomethacin-induced ulcers and increase of plasma TNF levels after LPS was also studied in wild-type and CB1 receptor knockout mice. Indomethacin induced intestinal ulcers in mice, but not tissue TNF production and MPO activity. SR 141716 reduced the ulcers to a similar extent in wild-type and CB1 receptor knockout mice. In rats and wild-type mice, but not in CB1 receptor knockout mice, SR 141716 inhibited the LPS-induced increase in plasma TNF levels. (4) These findings provide evidence that the indomethacin model of intestinal lesions differs in rat and mouse and support the existence of several mechanisms for the antiulcer activity of SR141716, the most important involving the inhibition of TNF production. The potent anti-inflammatory activity of SR141716 in rodents indicated its potential therapeutic interest in chronic immune-inflammatory diseases.

1. This study was undertaken in cultured vascular smooth muscle cells to characterize the angiotensin II (AII) AT1 receptor subtype involved in DNA synthesis because (i) the AII receptor involved in vascular proliferation has previously been characterized in vitro in rat aortic cells and identified as an AT1 subtype and (ii) molecular cloning and biochemical studies have provided evidence for the existence of different AT1 receptor subtypes. 2. In cultured rat aortic vascular smooth muscle (VSMC), exposure to AII (0.1 to 100 nM) resulted in a concentration-dependent increase in [3H]-thymidine incorporation with an EC50 of 1.41 +/- 0.51 nM. Maximal stimulation was observed in the presence of 100 nM AII and corresponded to 271 +/- 40% of basal [3H]-thymidine incorporation. 3. To characterize the AII AT1 receptor subtype involved in this effect, cells were exposed to AII (3 nM) in the absence or presence of increasing concentrations of various AII receptor antagonists. The stimulatory effect of AII (3 nM) on [3H]-thymidine incorporation in VSMC was antagonized by the non-selective AT1/AT2 receptor antagonist, [Sar1, Ile8]-AII (IC50 = 5.6 nM), by the AT1A/AT1B receptor antagonist, losartan (IC50 = 10.5 nM) and the AT1 receptor antagonist, L-158809 (IC50 = 0.20 nM). The selective AT2 receptor ligand, CGP 42112A, antagonized AII-induced [3H]-thymidine incorporation with an IC50 of 6.3 +/- 1.3 microM while the AT2/AT1B receptor antagonist, PD 123319, was found to be almost inactive (IC50 > 10 microM). 4. Under the same experimental conditions, angiotensin III (AIII) was found to be at least 50 times less potent than All with an apparent EC50 of 81.6 +/- 7.7 nM. At the highest concentration tested (10 microM),the effect of AIII corresponded to 327 +/- 61% of basal [3H]-thymidine incorporation.5. These results confirm that All can stimulate DNA synthesis in VSMC through an AT, receptor.Furthermore, the pharmacological characterization of this AT1 receptor is compatible with the ATlA receptor subtype recently described on cultured mesangial cells since (i) the ATIA/ATIB receptor antagonist losartan is active at nanomolar concentrations, (ii) micromolar concentrations of the AT2/AT1B receptor antagonist PD 123319 are ineffective at antagonizing the AII-induced [3H]-thymidine incorporation and (iii) All is at least 50 times more potent than AIII in stimulating DNA synthesis.

1 Rabbit hypothalamic slices prelabelled with [3H]-noradrenaline and superfused with Krebs solution were stimulated electrically at a frequency of 5 Hz. Exposure to verapamil (0.1 to 10 microM) significantly increased, in a concentration-dependent manner, the electrically-evoked overflow of tritium, without affecting the spontaneous outflow of radioactivity. 2 Exposure to diltiazem in concentrations up to 100 microM had no effect on the electrically evoked release of [3H]-noradrenaline, but increased the basal outflow of radioactivity at 10 and 100 microM. 3 The preferential alpha 2-adrenoceptor antagonist, yohimbine (0.1 microM) significantly antagonized the inhibitory effect of clonidine or adrenaline on [3H]-noradrenaline overflow elicited by electrical stimulation. Verapamil (3 microM) also antagonized this inhibitory effect of the alpha 2-adrenoceptor agonists on [3H]-noradrenaline release. In contrast to these results, exposure to diltiazem (10 microM) was ineffective in blocking the action of the alpha 2-adrenoceptor agonist. 4 These results suggest that the two Ca2+-antagonists verapamil and diltiazem differ in their ability to affect central noradrenergic neurotransmission. While verapamil is a relatively potent alpha 2-adrenoceptor antagonist, diltiazem is devoid of presynaptic alpha 2-adrenoceptor antagonist properties.