Mihai Covasa

CCK and 5-HT act synergistically to suppress food intake through simultaneous activation of CCK-1 and 5-HT3 receptors

Cholecystokinin (CCK) and serotonin (5-HT) systems have been shown to cooperate interdependently in control of food intake. To assess mechanisms by which CCK and 5-HT systems interact in control of food intake we examined: (1) participation of CCK-1 and 5-HT3 receptors in 5-HT-induced suppression of sucrose intake; (2) the interaction between CCK and 5-HT in suppression of food intake; (3) the role of CCK-1 and 5-HT3 receptors in mediating this interaction. Intraperitoneal administration of 5-HT (0.25, 0.5 and 1.0 mg/kg) significantly reduced intake compared to control in a dose responsive fashion (r2=0.989). Suppression of food intake by 5-HT was significantly attenuated by prior treatment with the 5-HT3 receptor antagonist ondansetron at each 5-HT dose tested (P<0.05), while blockade of CCK-1 receptors by lorglumide had no effect on 5-HT-induced suppression of intake. Administration of CCK-8 (0.5 microg/kg) or 5-HT (0.5 mg/kg) alone significantly reduced sucrose intake by 22.9 and 22.2% respectively, compared to control (P<0.0001). Co-administration of CCK and 5-HT resulted in a synergistic suppression of intake leading to an overall 48.4% reduction in sucrose intake compared to saline (P<0.0001). Concomitant CCK-1 and 5-HT3 receptor blockade by lorglumide and ondansetron respectively, resulted in a complete reversal of the combined CCK and 5-HT-induced suppression of intake. Independent administration of lorglumide or ondansetron did not alter intake compared to control. These studies provide evidence that 5-HT causes suppression in food intake by acting at 5-HT3, not CCK-1 receptors. Furthermore, CCK and 5-HT interact to produce an enhanced suppression of food intake, an effect mediated through concomitant activation of CCK-1 and 5-HT3 receptors.

Dorsal hindbrain 5-HT3 receptors participate in control of meal size and mediate CCK-induced satiation

We have previously shown that systemic administration of ondansetron, a selective serotonin type-3 (5-HT3) receptor antagonist, attenuates cholecystokinin (CCK)-induced suppression of food intake. The exact location of 5-HT3 receptors mediating this action is not clear and may involve hindbrain 5-HT3 receptors. In this study, we first examined sucrose intake in response to direct injections of ondansetron into various sites of the dorsal hindbrain. Ondansetron (1.0 and 2.0 microg/100 nl) delivered into the medial nucleus of the solitary tract (NTS) significantly increased 15% sucrose intake (12.2 +/- 0.6 and 13.5 +/- 0.7 ml, respectively) compared to control (10.2 +/- 0.7 ml), while equivalent injections into ipsilateral adjacent sites such as the lateral NTS, dorsal medial nucleus of the vagus, and other areas of the dorsal hindbrain had no effect on sucrose intake. Second, we examined the effects of hindbrain 5-HT3 receptor blockade on suppression of intake by systemic CCK. Fourth ventricular (i.c.v.) administration of ondansetron (10.0 microg/3.0 microl) significantly attenuated suppression of intake by CCK (9.1 +/- 1.0 vs. 6.4 +/- 0.4 ml, respectively). Ondansetron alone had no effect on sucrose intake at any i.c.v. dose tested. In a separate group of rats, CCK administration suppressed 60-min intake significantly (8.9 +/- 0.8 ml) compared to control (12.4 +/- 0.4 ml). Administration of ondansetron into the medial NTS completely reversed suppression of intake by CCK (11.8 +/- 1.0 and 12.3 +/- 1.4 ml, for 0.5 microg and 1.0 microg/100 nl, respectively). These data demonstrate that 5-HT3 receptors located in the medial NTS participate in control of meal size and mediate CCK-induced suppression of food intake.

Altered dopamine D2 receptor function and binding in obese OLETF rat

A decrease in D2-like receptor (D2R) binding in the striatum has been reported in obese individuals and drug addicts. Although natural and drug rewards share neural substrates, it is not clear whether such effects also contribute to overeating on palatable meals as an antecedent of dietary obesity. Therefore, we investigated receptor density and the effect of the D2R agonist quinpirole (0.05, 0.5 mg/kg, S.C.) on locomotor activity and sucrose intake in a rat model of diet-induced obesity, the CCK-1 receptor-deficient Otsuka Long Evans Tokushima Fatty (OLETF) rat. Compared to age-matched lean controls (LETO), OLETF rats expressed significantly lower [125I]-iodosulpride binding in the accumbens shell (-16%, p<0.02). Whereas the high dose of quinpirole increased motor activity in both strains equally, the low dose reduced activity more in OLETF. Both doses significantly reduced sucrose intake in OLETF but not LETO rats. These findings demonstrate an altered D2R signaling in obese OLETF rats similar to drug-induced sensitization and suggest a link between this effect and avidity for sucrose in this model.

Intestinal infusions of oleate and glucose activate distinct enteric neurons in the rat.

Nutrients entering the small intestine trigger a variety of neural and endocrine reflexes that involve specific afferents, efferents and interneurons, many of which are located within the enteric nervous system (ENS). We hypothesized that intestinal nutrient stimuli might activate specific subpopulations of these neurons. To test this hypothesis, we utilized immunohistochemical detection of nuclear c-fos expression in the myenteric and submucosal plexuses of the rat small intestine following intraintestinal infusions of oleate or glucose. Additionally, we used dual label methods to detect both Fos-immunoreactivity and immunoreactivity for five phenotypic neuronal markers: neurokinin-1 receptor (NK-1R), neurofilament-M (NF-M), neuronal nitric oxide synthase (NOS), calbindin (Cal) and calretinin (Calr), to characterize neurons that were activated by intestinal infusion of oleate and glucose. We found that oleate and glucose activated myenteric neurons in the duodenum and jejunum, but not the ileum. Oleate and glucose infusions significantly increased the number of Fos-immunoreactive nuclei in the submucosal plexus of the duodenum and jejunum, however, only glucose increased Fos-immunoreactivity in the ileum. Oleate and glucose infusions were associated with a small increase in Fos-immunoreactivity in NOS-immunoreactive neurons in the myenteric plexus. In the submucosal plexus, the majority of neurons activated by intestinal infusion of oleate or glucose were immunoreactive to Cal and Calr. In the rat, many of these neurons have Dogiel Type II-like morphology, which is consistent with the possibility that these neurons function as mucosal afferents in reflexes activated by nutrient stimuli.

Gastric distension enhances CCK-induced Fos-like immunoreactivity in the dorsal hindbrain by activating 5-HT3 receptors

The combination of gastric distension and cholecystokinin (CCK) enhances both suppression of food intake and induction of c-Fos-like immunoreactivity (Fos-LI) in the dorsal vagal complex (DVC). Previously, we have shown that serotonin type-3 (5-HT3) receptor mediation of suppression of food intake by CCK requires gastric participation. Therefore, we hypothesized that 5-HT3 receptors mediate CCK-induced Fos-LI in the dorsal hindbrain through a mechanism that involves gastric distension. To test this hypothesis, we counted Fos-LI in the DVC of ondansetron (1 mg/kg; 5-HT3 receptor antagonist) and vehicle-treated rats following gastric balloon distension (5 ml), CCK (1 microg/kg) administration, or CCK combined with gastric distension. Ondansetron administration attenuated DVC Fos-LI by CCK administration. Likewise, ondansetron attenuated Fos-LI by gastric distension in the DVC, specifically within the nucleus of the solitary tract (NTS) and area postrema (AP) nuclei. The most pronounced attenuation of distension-induced Fos-LI by ondansetron occurred in the NTS, particularly in the medial and intermedial NTS. When combined, CCK and gastric distension enhanced Fos-LI in the DVC greater than each treatment alone. Furthermore, ondansetron administration attenuated the overall DVC enhanced Fos-LI induced by CCK + gastric distension, in particular at the NTS and AP nuclei. We found that, within the mid-to-caudal regions of the NTS and AP, 5-HT3 receptors most significantly mediate neuronal activation by CCK + distension. In conjunction with previous behavioral data, these results show that gastric distension enhances CCK-induced neuronal activation in the DVC by activating 5-HT3 receptors.

Intestinal nutrients elicit satiation through concomitant activation of CCK1 and 5-HT3 receptors

Previous studies demonstrate that cholecystokinin type-1 (CCK(1)) and serotonin type-3 (5-HT(3)) dependent pathways are independently involved in intestinal nutrient-induced meal termination. In the current study, we employed selective antagonists to investigate the relative contribution of CCK(1) and 5-HT(3) receptors in mediating the anorexia produced by duodenal infusion of Polycose or Intralipid in rats. Combined administration of 1 mg/kg ondansetron (Ond) and 1 mg/kg devazepide (Dev) reversed 132 mM Polycose-induced suppression to the level of control intake and significantly attenuated 263 mM Polycose-induced suppression greater than either antagonist alone. Similar results were observed when subthreshold doses of Ond (500 microg/kg) and Dev (5 microg/kg) were co-administered prior to 263 mM Polycose infusion. Suppression of intake resulting from 130 mM Intralipid was reversed to the level of control when Ond and Dev were co-administered at both independent effective doses (1 mg/kg each) and subthreshold doses (500 microg/kg and 5 microg/kg, respectively). Finally, combined administration of the antagonists increased sucrose intakes beyond intakes following control or treatment with either antagonist alone when rats were infused with saline. These data demonstrate that intestinal carbohydrates and lipids inhibit food intake through simultaneous CCK(1) and 5-HT(3) receptor activation and that these receptors appear to completely mediate the Intralipid-induced suppression of intake.

Cholecystokinin-induced satiety is mediated through interdependent cooperation of CCK-A and 5-HT3 receptors

Recent evidence supports a role for the serotonin-3 (5-HT3) receptors in the modulation of cholecystokinin (CCK)-induced satiation. Likewise, 5-HTs anorectic response has been linked to recruitment of peripheral CCK-A receptors. Evidence to date, however, does not elucidate whether there is a concomitant interaction between CCK-A and 5-HT3 receptors or whether each receptor functions independently in the negative feedback control of food intake elicited by CCK. In the present study, we used selective receptor antagonists to investigate the roles of CCK-A and 5-HT3 receptors in CCK-induced satiation. Intraperitoneal administration of CCK-8 reduced 30-min 15% sucrose intake in a dose-responsive manner. Prior treatment with ondansetron (1.0 mg/kg ip), a highly selective 5-HT3 receptor antagonist, attenuated CCK-induced suppression of food intake in a dose-responsive manner. Pretreatment with lorglumide (1.0 mg/kg ip), a selective CCK-A receptor antagonist, reversed CCK-induced inhibition of sucrose intake. Finally, simultaneous blockade of CCK-A and 5-HT3 receptors by lorglumide and ondansetron, as well as concomitant administration of the two antagonists with CCK, produced a significant synergistic increase in sucrose intake compared with intakes after administration of saline, CCK, or either antagonist alone. These findings support evidence that CCK-A and 5-HT3 receptors cooperate interdependently in control of short-term food intake. Most likely, this interconnection exists through a feed-forward parallel model arising from CCK-A and 5-HT3 receptors, where activation of one system engages the other to intensify the overall satiety signal.

NMDA NR2 receptors participate in CCK-induced reduction of food intake and hindbrain neuronal activation.

Previous work has shown that blockade of NMDAR by non-competitive (MK-801) and competitive (AP5) antagonists increase food intake by acting in the dorsal hindbrain. NMDAR are heteromeric complexes composed of NR1, NR2 and NR3 subunits. Competitive NR2B antagonists potently increase feeding when injected into the hindbrain. NR2 immunoreactivity is present in the hindbrain, vagal afferents and enteric neurons. NMDA receptors expressed on peripheral vagal afferent processes in the GI tract modulate responsiveness to GI stimuli. Therefore, it is possible that peripheral as well as central vagal NMDA receptors participate in control of food intake. To examine this possibility, we recorded intake of rodent chow, a palatable liquid food (15% sucrose), and non-nutrient (0.2% saccharin) solutions following intraperitoneal (i.p.) administration of D-CPPene, a competitive NMDA receptor antagonist that is selective for binding to the NR2B/A channel subunit. To assess participation of peripheral NMDA receptors in postoral satiation signals, we examined the ability of D-CPPene to attenuate reduction of feeding and hindbrain Fos expression following IP CCK administration. IP D-CPPene (2, 3 mg/kg) produced a significant increase in sucrose and chow intake but not saccharin. Pretreatment with D-CPPene (2 mg/kg) reversed CCK (2 microg/kg)-induced inhibition of sucrose intake, and attenuated CCK-induced Fos-Li in the dorsal hindbrain. These results confirm that antagonism of hindbrain NMDA receptors increases food intake. In addition our results suggest that NMDA receptors outside the hindbrain, perhaps in the periphery, participate in vagally mediated, CCK-induced reduction of food intake and NTS neuron activation.

Cholecystokinin1 receptors mediate the increase in Fos-like immunoreactivity in the rat myenteric plexus following intestinal oleate infusion

Intestinal infusion of nutrients, such as glucose and oleic acid, increase Fos-like immunoreactivity (Fos-LI) in both the enteric nervous system and neurons of the dorsal vagal complex (DVC) of the hindbrain. To test the hypothesis that increased Fos-LI in enteric neurons and the DVC, following intestinal nutrient infusions is mediated by cholecystokinin(1) receptors (CCK(1)), we counted enteric and DVC neurons that expressed Fos-LI following intestinal infusion of oleate or glucose, with and without pretreatment with the CCK(1) receptor antagonist, lorglumide. Both oleate and glucose infusions increased Fos-LI in the DVC. Oleate also increased Fos-LI in the myenteric and submucosal plexuses of the duodenum and the jejunum, but not the ileum, while glucose only increased Fos-LI in the submucosal plexus of the ileum. The CCK(1) receptor antagonist, lorglumide, abolished Fos-LI in the DVC following infusions of either oleate or glucose. In addition, lorglumide attenuated oleate-induced Fos-LI in the myenteric and submucosal plexuses of the duodenum and jejunum. However, lorglumide failed to attenuate glucose-induced Fos-LI in the submucosal plexus of the ileum. These data confirm previous reports indicating that CCK(1) receptors mediate increased DVC Fos-LI following intestinal infusion of oleate or glucose. CCK(1) receptors also contribute to increased Fos-LI in enteric neurons following intestinal oleate infusion. However, failure of lorglumide to attenuate the increase of Fos-LI in the ileal submucosal plexus following intestinal glucose suggests that some intestinal nutrients trigger Fos-LI induction via CCK(1) receptor-independent pathways.

Differential feeding behavior and neuronal responses to CCK in obesity-prone and -resistant rats.

Deficits in satiation signals are strongly suspected of accompanying obesity and contributing to its pathogenesis in both humans and rats. One such satiation signal is cholecystokinin (CCK), whose effects on food intake are diminished in animals adapted to a high fat diet. In this study, we tested the hypothesis that diet-induced obese prone (OP) rats exhibit altered feeding and vagal responses to systemic (IP) administration of CCK-8 compared to diet-induced obese resistant (OR) rats. We found that CCK (4.0 microg/kg) suppressed food intake significantly more in OP than OR rats. To determine whether enhanced suppression of feeding is accompanied by altered vagal sensory responsiveness, we examined dorsal hindbrain expression of Fos-like immunoreactivity (Fos-Li) following IP CCK injection in OP and OR rats. After 4.0 microg/kg CCK, there were significantly more Fos-positive nuclei in the NTS of OP compared to OR rats. Treatment with 8.0 microg/kg CCK resulted in no significant difference in food intake or in Fos-Li between OP and OR rats. Also, we found that OP rats were hyperphagic on a regular chow diet and gained more weight compared to OR rats. Finally OP rats had decreased relative fat pad mass compared to OR rats. Collectively, these results show that OP rats exhibit a different behavioral and vagal neuronal responses to CCK than OR rats.