Systemic injection of MK-801, a noncompetitive antagonist of N-methyl-d-aspartate (NMDA) receptor ion channels, increases meal size and delays satiation. We examined whether MK-801 increases food intake by directly interfering with actions of cholecystokinin (CCK). Prior administration of MK-801 (100 μg/kg ip) reversed the inhibitory effects of CCK-8 (2 and 4 μg/kg ip) on real feeding of both liquid and solid foods. MK-801 alone did not alter 30-min sham intake of 15% sucrose compared with intake after saline. Furthermore, while CCK-8 (2 or 4 μg/kg ip) reduced sham intake, this reduction was not attenuated by MK-801 pretreatment. To ascertain whether MK-801 attenuation of CCK-induced reduction of real feeding was associated with attenuated inhibition of gastric emptying, we tested the effect of MK-801 pretreatment on CCK-induced inhibition of gastric emptying of 5-ml saline loads. Ten-minute gastric emptying was accelerated after MK-801 (3.9 ± 0.2 ml) compared with saline vehicle (2.72 ± 0.2 ml). CCK-8 (0.5 μg/kg ip) reduced 10-min emptying to 1.36 ± 0.3 ml. Pretreatment with MK-801 did not significantly attenuate CCK-8-induced reduction of gastric emptying (0.9 ± 0.4 ml). This series of experiments demonstrates that blockade of NMDA ion channels reverses inhibition of real feeding by CCK. However, neither inhibition of sham feeding nor inhibition of gastric emptying by CCK is attenuated by MK-801. Therefore, increased food intake after NMDA receptor blockade is not caused by a direct interference with CCK-induced satiation. Rather, increased real feeding, either in the presence or absence of CCK, depends on blockade of NMDA receptor participation in other postoral feedback signals such as gastric sensation or gastric tone.
- sham feeding
- gastric emptying
meal size and meal termination are controlled by interplay between positive and negative feedback signals. Satiation signals, such as gastric distension, CCK, and the presence of nutrients in the small intestine, limit the intake of food. Reduction of food intake by exogenous CCK depends on activation of small unmyelinated vagal sensory fibers (12, 15). However, the neurotransmitter(s) released in the brain when these fibers are activated remains unknown. Several lines of evidence suggest that glutamate may be a vagal sensory neurotransmitter (16, 21, 26). A number of laboratories, including our own, have reported that MK-801 (dizolcipine), a noncompetitive antagonist of N-methyl-d-aspartate (NMDA) receptor ion channels, increases feeding (1, 3-7, 13, 20-23, 26) as well as drinking (7, 24). MK-801 increases meal size initiated by an overnight food deprivation or the presentation of a highly palatable food (3) and has also been shown to increase intake of chow when administered just before dark onset in ad libitum-fed rats (7). We have also shown that direct injection of MK-801 into the dorsal vagal complex increases the size of deprivation-induced meals (21).
Previous results suggest that blockade of NMDA receptors attenuates reduction of food intake by exogenous CCK (1). In the work reported here, we have compared the effect of NMDA receptor blockade on CCK-induced reduction of real feeding and sham feeding to determine whether increased food intake after NMDA receptor blockade is the direct result of interference with CCK-mediated feedback signals. We also examined the effect of NMDA receptor blockade on inhibition of gastric emptying by CCK. We found that while NMDA receptor blockade reverses inhibition of real feeding by CCK, it fails to reverse inhibition of sham feeding or gastric emptying by CCK. These results suggest that MK-801 increases feeding by interfering with postoral satiation signals that are modulated by CCK.
Adult (350-400 g) male Sprague-Dawley rats (Simonsen Laboratories, CA) were individually housed in hanging wire bottom cages in a temperature-controlled vivarium with ad libitum access to standard pelleted rodent chow and water except during experiments or overnight fasts as indicated below. The rats were maintained in a 12:12-h light-dark cycle (lights on at 0700) and were habituated to laboratory conditions for at least 5-7 days before surgery or the initiation of experiments.
MK-801 (dizolcipine, RBI, Natick, MA) and CCK octapeptide sulfate (CCK-8; American Peptide, Sunnyvale, CA) were dissolved in physiological sterile saline and were administered intraperitoneally in a volume of 1.0 ml/kg body wt.
Gastric cannulation. Rats used in gastric-emptying studies were implanted with stainless steel gastric cannulas, according to a modification of the procedure previously described by Yox and Ritter (25). Briefly, the animals were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL), and the flanged end of a stainless steel gastric cannula (13 mm long, 6 mm ID, 8 mm OD) was inserted through the ventral wall of the nonglandular portion of the stomach near the greater curvature. The cannula was secured with a purse-string suture, a piece of Marlex mesh was placed around it, and the nonflanged end of the cannula was externalized through an incision in the left paramedian abdominal wall. The cannula was kept closed with a stainless steel screw, except during experiments. A minimum of 2 wk was allowed for recovery from surgery.
Experiment 1: Effects of MK-801 on CCK-Induced Inhibition of Solid and Liquid Food Intake
Solid food. Food-deprived (17 h) rats (n = 10) were injected with one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, or CCK/MK-801. A preweighed amount of standard rodent chow was presented to the rats, and food intake was recorded at 0.5, 1, 2, 4, and 6 h postinjection, taking into account spillage that was collected in a tray placed below the cage. MK-801 (100 μg/kg ip) was administered 10 min before CCK-8 (2 or 4 μg/kg ip) injection and 30 min before food presentation. CCK was administered 20 min before food. Each drug treatment was separated by a minimum of 48 h and was bracketed by a NaCl/NaCl condition. Two separate studies were performed. In the first study, the dose of CCK used was 2 μg/kg, whereas in the second study, a 4 μg/kg dose of CCK was administered. For each experiment, a minimum of two repetitions of each drug combination were conducted. Thus each experimental datum represents the mean from at least two tests separated by a saline-only baseline test.
Liquid food. A separate group of rats (n = 8) was trained to drink a 15% sucrose solution (wt/vol) from a calibrated 25-ml glass burette. This group received the identical drug treatment combinations as described in experiment 1; however, only the 2 μg/kg CCK dose was tested. Sucrose intake was measured every 5 min for 30 min in the place of measuring chow intake.
Experiment 2: Effects of MK-801 on CCK-Induced Inhibition of Gastric Emptying
Gastric emptying after CCK. Rats (n = 9) were fitted with chronic gastric cannulas as described under Surgical Procedures. The rats were deprived of food, but not water, overnight for 17 h before the start of gastric-emptying measurements. At the beginning of each gastric-emptying experiment, each rat was removed from its home cage, the gastric cannula was opened, and the stomach was gently washed with warm (37°C) tap water. A drainage tube was attached to the open cannula, and the rat was placed in a Plexiglas gastric emptying cage, which has been described previously (25). The drainage tube exited through a longitudinal slot in the wire-mesh floor of the cage and rested in a graduated cylinder. After connection of the drainage tube and while the rats rested in the Plexiglas cages, the stomach was flushed twice with warm 0.9% NaCl, via the drainage tube, by means of a syringe attached to the drainage tube. The second saline wash contained phenol red (60 mg/l) to saturate gastric mucosal binding of phenol red and minimize loss of the dye due to adsorption during subsequent emptying measurements (8). Phenol red recovery after this procedure was 95-97% of the infused loads. After the final dye-containing wash, ≥30 min was allowed to drain any remaining wash solution from the stomach. After this draining period, MK-801 (100 μg/kg) or NaCl was administered 15 min before a 5 ml 0.9% NaCl load. CCK (0.5 μg/kg) was given 10 min after MK-801 administration. The dose of CCK employed in this experiment was chosen based on previous studies examining the effects of CCK on gastric emptying (17). Rats received one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, or CCK/MK-801. Five minutes after CCK or NaCl administration, 5 ml of warm 0.9% NaCl, containing 0.006% phenol red, were instilled into the stomach via the drainage tube, and the tube was clamped. At the end of a 10-min emptying period, the clamp was removed, the volume remaining in the stomach was withdrawn, and the stomach was washed twice with saline and allowed to drain for another 30 min into a collection flask. Collected volume was measured, and the gastric contents were centrifuged at 10,000 rpm for 5 min to remove any particulate matter. A 1-ml sample from the centrifuged gastric contents was buffered with 24 ml of 0.014 M Na3PO4·12H2O, and the spectrophotometric absorbance of each buffered sample was compared with that of a 1-ml buffered sample from the originally instilled phenol red solution to determine the volume of the original test load remaining in the stomach at the end of the 10-min emptying period. Each drug treatment was preceded and followed by gastric-emptying measurements after control injections of 0.9% NaCl. A minimum of two tests was conducted for each drug treatment, and all injections or infusions were separated by ≥48 h.
Experiment 3: Effects of MK-801 on CCK-Induced Suppression of Sham Feeding
A separate group of rats (n = 7) equipped with chronic gastric cannulas was trained to sham feed 15% sucrose solution. After overnight food deprivation, rats were removed from their cages, the stainless steel screw was removed from their gastric cannulas, and their stomachs were gently lavaged with warm tap water (37°C). After a drainage tube had been attached to their open cannula, the rats were placed in Plexiglas sham-feeding boxes. The rats then received one of the following drug combinations: NaCl/NaCl, NaCl/MK-801, CCK/NaCl, and CCK/MK-801. MK-801 (100 μg/kg) or NaCl was administered 15 min before CCK (2 or 4 μg/kg) or NaCl. Calibrated glass burettes filled with 15% sucrose solution were presented immediately after CCK or NaCl injection, and intake was recorded every 5 min for the ensuing 30 min. Two separate studies were performed. In the first study, the dose of CCK used was 2 μg/kg, whereas in the second study, a 4 μg/kg dose of CCK was administered. For each experiment, a minimum of two tests were conducted for each drug combination. All drug administrations were separated by a period of 48 h during which no experimental manipulations occurred. Each drug combination was bracketed by a vehicle-only baseline combination.
The cumulative food intake was analyzed with two-way repeated-measures ANOVA with treatment and time as main factors using Sigma Stat software. The gastric-emptying data were analyzed using one-way repeated measure with treatment as the main factor. When a significant effect of treatment or an interaction was detected, Bonferroni tests were used in the post hoc analyses to isolate the effect. Intakes for all rats are expressed as means ± SE in milliliters for liquid food and in grams for solid food.
Effects of MK-801 on CCK-Induced Inhibition of Solid and Liquid Food Intake
Solid foods. Two-way repeated-measures ANOVA demonstrated significant main effects of drug combination [F(3,199) = 13.6, P < 0.001 for 2 μg/kg dose of CCK and F(3,199) = 27.5, P < 0.001 for 4 μg/kg dose of CCK] and time [F(4,199) = 677.2, P < 0.001 for 2 μg/kg dose of CCK and F(4,199) = 525.7, P < 0.001 for 4 μg/kg dose of CCK], as well as an interaction between drug combination and time [F(12,199) = 4.1, P < 0.001 for 2 μg/kg dose of CCK and F(12,199) = 9.2, P < 0.001 for 4 μg/kg dose of CCK]. When rats were tested on solid chow food, prior treatment with MK-801 increased overall food intake significantly compared with saline treatment (P < 0.001). The increase in intake was evident at 60 min postinjection and continued to be significantly high throughout the duration of the experiment and up to 6 h postinjection. As expected, both doses of CCK-8 used significantly decreased food intake compared with saline (CCK 2 μg/kg, P = 0.038; CCK 4 μg/kg, P < 0.001). There was a significant difference in the magnitude of food suppression produced by 2 μg/kg dose of CCK compared with 4 μg/kg dose of CCK [F(1,99) = 10.4, P = 0.01]. While the lowest dose of CCK produced a 32% suppression of intake at 30 min and lasted up to 60 min postinjection, the higher dose of CCK produced a 69.8% reduction of intake in the first 30 min, and this effect lasted up to 4 h (%suppression, 69.8, 44.0, 29.8, and 23.9 for 0.5, 1, 2, and 4 h, respectively). When rats received MK-801 treatment before CCK injection at either dose used, MK-801 completely reversed CCK-induced suppression of solid food intake (P < 0.001 for both 2 and 4 μg/kg dose of CCK). This reversal was complete beginning at 30 min, and the subsequent intake was similar to the level of MK/NaCl condition (Figs. 1 and 2).
Liquid foods. Repeated-measures ANOVA demonstrated a significant main effect of drug combination [F(3,191) = 14.4, P < 0.001] and time [F(5,191) = 254.6, P < 0.001]. Also, a significant interaction between drug combination and time was found [F(15,191) = 13.4, P < 0.001]. Administration of 2 μg/kg dose of CCK produced a significant suppression of sucrose intake beginning at 10 min and lasting throughout the 30-min period whereas prior administration of MK-801 significantly attenuated CCK-induced inhibition of intake at 20, 25, and 30 min. For example, systemic administration of MK-801 alone (100 μg/kg) significantly increased 30-min sucrose intake [MK/NaCl, 15.2 ± 0.1 vs. NaCl/NaCl, 10.7 ± 0.1 ml; P < 0.01]. Injection of CCK (2 μg/kg) decreased sucrose intake significantly [NaCl/CCK, 6.2 ± 0.3 vs. NaCl/NaCl, 10.7 ± 0.1 ml; P < 0.001]. Coadministration of MK-801 and CCK-8 significantly attenuated suppression of sucrose intake by CCK [NaCl/CCK, 6.2 ± 0.3 vs. MK/CCK, 12.5 ± 0.1 ml; P < 0.001] (Fig. 3).
Effects of MK-801 on CCK-Induced Inhibition of Gastric Emptying
One-way repeated-measures ANOVA showed a significant effect of treatment on 10-min gastric emptying [F(3,84) = 13.8, P < 0.001]. Specifically, administration of MK-801 significantly increased 10-min gastric emptying of saline solution (ml emptied: 3.88 ± 0.24) compared with saline treatment (ml emptied: 2.7 ± 0.18, P = 0.009). CCK (0.5 μg/kg) significantly suppressed gastric emptying of saline (ml emptied: 1.36 ± 0.28, P = 0.047) compared with control. Pretreatment with MK-801 had no effect on CCK-induced suppression of gastric emptying (ml emptied: 0.88 ± 0.4, P = 1.0) (Fig. 4).
Effects of MK-801 on CCK-Induced Suppression of Sham Feeding
Repeated-measures ANOVA demonstrated significant main effects of drug combination [F(3,287) = 33.1, P < 0.001 for 2.0 μg/kg dose of CCK and F(3,287) = 5.4, P = 0.003 for 4.0 μg/kg dose of CCK] and time [F(5,287) = 268.0, P < 0.001 for 2.0 μg/kg dose of CCK and F(5,287) = 205.8, P < 0.001 for 4.0 μg/kg dose of CCK], as well as an interaction between drug combination and time [F(15,287) = 11.1, P < 0.001 for 2.0 μg/kg dose of CCK and F(15,287) = 1.9, P = 0.022 for 4.0 μg/kg dose of CCK]. Treatment of rats with MK-801 alone did not cause any changes in sham feeding of 15% sucrose in either study (P = 0.36 and P = 0.6). Both doses of CCK-8 used significantly decreased sham intake compared with saline (2 μg/kg: 42% suppression, P < 0.001; 4 μg/kg: 53.2% suppression, P < 0.001). This suppression of intake produced by either dose of CCK became significant at 10 min and lasted throughout the experiment. When rats received MK-801 treatment before CCK, MK-801 had no effect on CCK-induced suppression of sham feeding regardless of the dose of CCK used (P < 0.05) (Figs. 5 and 6). The pattern of sham intake produced after administration of NaCl/CCK combination was almost identical with that obtained after MK/CCK combination during the 30-min period.
We and others previously have demonstrated that systemic (3-5, 7, 14, 20) or hindbrain (21, 26) administration of MK-801, a noncompetitive NMDA receptor ion channel antagonist, increases meal size. We have shown that this action of MK-801 is mediated by receptors in the dorsal vagal complex (21, 22). These results suggest that NMDA-type glutamatergic synapses may participate in control of food intake by vagal feedback processes. Indeed, results from our lab (4) and those of Berthoud et al. (2) indicated that capsaicin-induced vagal damage attenuates increased intake by MK-801. Therefore, it is possible that MK-801 increases meal size by attenuating specific vagal afferent satiation signals.
Previously, Bednar et al. (1) reported that systemic MK-801 attenuated the reduction of liquid diet in intraoral feeding tests. This result raised the possibility that MK-801 directly antagonized vagally mediated CCK-satiation signals. In our study reported here, we have further probed this hypothesis. We found that MK-801 completely reversed CCK-induced reduction of real feeding. MK-801-induced reversal of CCK's effect occurred when either liquid or solid food was offered. The feeding pattern after NMDA blockade in response to CCK administration differs somewhat between the solid vs. liquid food tested as well as between the doses of CCK employed. When rats were tested on either solid or liquid food, MK-801 produced a complete reversal of CCK-induced suppression of sucrose intake beginning at 20 min (liquid food) or 30 min (solid food) and lasted throughout the experiment, or in the case of solid food, until the feeding-suppressive effects of CCK wore off. Also, the pattern of results in the real feeding tests using the higher dose of CCK (4.0 μg/kg) differed from those found with the lower dose of CCK (2.0 μg/kg) where intakes after MK-801 were virtually the same with or without CCK. As noted, 4.0 μg/kg dose compared with 2.0 μg/kg dose of CCK produced a higher suppression of solid food intake and lasted until the end of the experiment. Therefore, it might be that MK-801 was unable to enhance intake at the later time points (4 and 6 h) due to the lasting opposing effects of a higher dose of CCK (4.0 μg/kg). In contrast to its effects on CCK-induced suppression of real feeding, MK-801 failed to attenuate reduction of sham feeding by CCK. This result suggests that MK-801 does not directly interfere with CCK-induced satiation.
It could be argued that the sham feeding preparation results in rates of intake that are already maximal and therefore cannot be increased by MK-801. In addition, it is possible that MK-801 evokes competing behaviors or motor impairments that prevent increased sham feeding. Indeed, we previously reported that MK-801 does not increase sham intake of 15% sucrose. However, the likelihood that rats are not able to increase their intake seems an improbable explanation for these results. First, MK-801 alone did not reduce the rate of sham feeding in this experiment, indicating that even when rats were eating at a very high rate MK-801 did not significantly reduce intake by impairing motor performance. Second, when CCK reduced sham feeding, the rate of intake was well below the maxima that occurred in the presence of MK-801. This fact is also consistent with the notion that increased intake was not prevented by a performance ceiling. Finally, we previously demonstrated that lesions of the area postrema, which do not substantially damage the nucleus of the solitary tract, dramatically increase liquid diet intake (22). However, these lesions do not prevent increased intake in the presence of MK-801. Taken together, these data indicate that failure of MK-801 to reverse CCK-induced reduction of sham feeding clearly indicates that NMDA-type ion channels are not required participants in the satiation effect of CCK during sham feeding.
CCK inhibits gastric emptying (9-11, 17) by increasing pyloric contraction and reducing intragastric pressure. Previously, we demonstrated that MK-801 produces small but significant increases in the rate of gastric emptying (5). Therefore, it is conceivable that MK-801 reverses CCK-induced inhibition of real feeding by reversing or attenuating CCK's effects on gastric emptying. However, we found that while MK-801 alone increased the rate of gastric emptying, it did not attenuate CCK-induced inhibition of gastric emptying. In fact, if anything, MK-801 slightly enhanced CCK-induced inhibition of gastric emptying. Therefore, MK-801's ability to reverse CCK's reduction of real feeding is not dependent on normalization of gastric emptying.
Our results indicate that blockade of NMDA-type ion channels does not directly reverse reduction of food intake by CCK in the absence of other postoral feedback signals. Rather, our data suggest that MK-801 might interfere with a postoral satiation signal that is modulated by CCK. Entry of nutrients into the small intestine activates vagal afferent fibers that participate in meal termination [see Ritter et al. (15) for review]. It seems plausible that MK-801 might interfere with transmission of satiation signals generated by intestinal nutrients. However, in previously published work (6), we demonstrated that MK-801 does not directly attenuate reduction of food intake by intestinal infusion by several nutrients. These results, in conjunction with the observation that MK-801 does not increase sham feeding, suggest that the satiation signal with which MK-801 interferes arises postorally and preintestinally. In other words, the available data suggest that NMDA-type glutamatergic mechanism(s) participate in gastric satiation signals.
Other investigators have demonstrated that CCK enhances vagal afferent discharge in response to gastric stimulation (for review, see Ref. 19). Furthermore, gastric loads enhance CCK's ability to reduce food intake (18). Therefore, it is plausible that MK-801 alters gastric afferent responses to CCK and thereby reverses CCK's effect on real feeding. This speculation is intriguing because, if true, it would mean that reduction of food intake by CCK during real feeding depends on neuroanatomically and neurochemically distinct afferent processes from CCK's demonstrated mediation of postgastric satiation signals. Further investigation will be needed to determine the role of NMDA ion channels in the control of food intake. Nevertheless, the current results indicate that CCK-modulated gastric afferent signals are a potential substrate through which NMDA-type ion channels participate in the control of meal size.
In conclusion, we have shown that pretreatment with MK-801 results in attenuation of CCK-induced inhibition of intake of both liquid and solid foods but does not attenuate inhibition of sham feeding by CCK, suggesting that MK-801 does not directly antagonize CCK-satiation signals. Taken together with our previous work, these findings suggest that MK-801-induced attenuation of CCK-induced inhibition of real feeding as well as MK-801-induced increases in meal size may involve direct or indirect interference with gastric sensation or gastric tone.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52849 and National Institute of Neurological Disorders and Stroke Grant NS-20561.
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