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Role of endogenous CCK in the inhibition of gastric emptying by peptone and Intralipid in rats
Regulatory Peptides 88 (2000) 47–53 www.elsevier.com / locate / regpep
Role of endogenous CCK in the inhibition of gastric emptying by peptone and Intralipid in rats Wesley O. White, Gary J. Schwartz, Timothy H. Moran* Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618 720 Rutland Avenue Baltimore, MD 21205, USA Received 6 September 1999; received in revised form 13 December 1999; accepted 13 December 1999
Abstract To assess the role of endogenous cholecystokinin in the control of gastric emptying of peptone solutions and Intralipid suspensions, we examined the ability of a dose range of the CCK-A antagonist, devazepide to accelerate the gastric emptying of various caloric concentrations of peptone and Intralipid in rats. In the absence of devazepide, both peptone and Intralipid emptying slowed with increasing concentration. Devazepide’s effect on peptone gastric emptying diminished with increasing peptone concentration. The threshold dose for accelerating the emptying of 0.2 kcal / ml peptone was lower than the threshold dose for affecting 0.5 kcal / ml peptone and devazepide had no effect on the gastric emptying of 1.0 kcal / ml peptone. In contrast, devazepide affected Intralipid gastric emptying at all three Intralipid concentrations and the threshold dose decreased with increasing Intralipid concentration. However, the magnitude of the effect of devazepide on peptone or Intralipid gastric emptying was partial and did not increase as a function of concentration. These data demonstrate a role for endogenous CCK in the emptying of peptone and Intralipid but suggest that endogenous CCK does not account for the increased slowing of gastric emptying evident with increased caloric concentration 2000 Elsevier Science B.V. All rights reserved. Keywords: Endogenous cholecystokinin; Control of gastric emptying; Intralipid suspensions
1. Introduction Exogenous administration of the brain–gut peptide cholecystokinin (CCK) inhibits gastric emptying in a variety of species [1–4]. A physiological role for CCK in the control of gastric emptying was originally suggested by experiments demonstrating that the dose of exogenous CCK required for inhibition of gastric emptying was comparable to doses required for stimulation of pancreatic secretion [1]. Additionally, the doses of exogenous CCK that inhibited gastric emptying were associated with plasma concentrations that were within the physiological range
*Corresponding author. Tel.: 1 1-410-955-2344; fax: 1 1-410-6140013. E-mail address: [email protected] (T.H. Moran)
[5]. The availability of potent and specific CCK antagonists has allowed a more direct assessment of the role of endogenous CCK in the controls of nutrient gastric emptying. Administration of CCK-A specific receptor antagonists such as devazapide or loxiglumide has been shown to accelerate nutrient gastric emptying in a variety of species and in a range of experimental settings [6–9]. We have demonstrated roles for endogenous CCK in the control of liquid gastric emptying of fats, proteins, and carbohydrates in rhesus monkeys [9,10]. In these experiments, devazapide administration accelerated the gastric emptying of Intralipid and peptone test meals in a dose related fashion. Similarly, devazapide accelerated the gastric emptying of 5% glucose and 10% maltose in a dose related fashion while not affecting the slowed emptying of hypertonic carbohydrate or sodium chloride solutions. Devazapide administration has also been shown to acceler-
0167-0115 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00119-6
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W.O. White et al. / Regulatory Peptides 88 (2000) 47 – 53
ate the gastric emptying of maltose, intralipid and peptone in the rat [8,11–13]. Increasing nutrient concentration has been demonstrated to result in slower rates of liquid gastric emptying in a variety of species [14–16]. Although a role for CCK in the mediation of nutrient gastric emptying has been demonstrated, whether CCK mediates the increased inhibition on gastric emptying that accompanies increased nutrient concentration has not been routinely assessed. Thus, in the rat, the ability of devazapide to affect maltose or peptone gastric emptying has only been assessed at single concentrations of each nutrient [11,13]. While Holzer and colleagues have examined the ability of devazapide to affect the gastric emptying of two concentrations of Intralipid, a fat suspension, they employed the phenol-red technique for measuring gastric emptying [12]. Phenol-red stays with the water rather than the lipid phase and Friedman and colleagues have demonstrated that the use of this technique does not adequately assess emptying of the lipid phase [17]. In the present experiments, we have examined the role of endogenous CCK in the control of liquid gastric emptying of various concentrations of peptone solutions and Intralipid emulsions in the rat. Specifically, these experiments examine the ability of a dose range of the specific CCK-A antagonist devazapide to accelerate the gastric emptying of the 5 ml intragastric loads of peptone and intralipid at various concentrations. These experiments are aimed at determining whether endogenous CCK plays a role in the increased inhibition of gastric emptying associated with increased caloric concentration.
2. Materials and methods The subjects were male Sprague-Dawley rats (Charles River), weighing 250–300 g at the start of the study. Animals were housed in individual hanging wire-mesh cages and maintained at 228C on a 12–12 h light–dark cycle with lights on at 0600 h. The rats had free access to water. Shortly after arriving in the laboratory, the animals were placed on a restricted feeding schedule with pelleted chow (Purina Rat Chow) only available for 5 h per day beginning at approximately 1200 h. Gastric emptying sessions took place in the mornings after rats had been food deprived for approximately 16 h. Rats were adapted to the gastric emptying procedure before experimental manipulations were performed. A polyethylene feeding tube attached to a syringe was inserted through the mouth and advanced down the esophagus, until the end the tube was in the stomach. 5.0 ml of phenol-red-containing saline solution was infused over a five s interval, and the tube was then removed. Five mm later the tube was reinserted, and solution remaining in the stomach was withdrawn (‘initial recovery’). The stomach was rinsed with 5.0 ml of saline. This procedure was
repeated once daily for each rat until the volume of the original saline solution recovered after 5 mm was stable (1 week). On experimental days, 30 mm prior to the gastric emptying tests, each rat’s stomach was prewashed with 5.0 ml of warmed saline solution infused over 5 s. The stomach contents were immediately recovered. The prewash frequently contained small amounts of hair and cage debris. Prewashes were repeated until contents removed from the stomach were clear. In the initial experiment, we measured the emptying of different concentrations of peptone (n 5 6) and Intralipid (n 5 8) across time. Peptone meals containing 0.05, 0.125 and 0.25 g / ml (300, 750, and 1500 mOsm, respectively) were prepared by adding peptone (Sigma type II from meat) to distilled water containing 0.0625 mg / ml phenolred. These peptone meals contained 0.2, 0.5 and 1.0 kcal / ml respectively. Intralipid meals were prepared by diluting 10% or 20% Intralipid emulsions (10.0 g soybean oil, 1.2 g phospholipids from powdered egg yolk, 2.25 g glycerin, and 110 kcal per 100 ml; Kabi Pharmacia) with distilled water. Intralipid meals containing 0.2, 0.5 and 1.0 kcal / ml were used. All solutions were administered at a pH of 7.0 and at 378C. The infusion volume remained constant at 5.0 ml. At intervals of 5, 10, 20 or 40 mm after the meals were infused, remaining gastric contents were withdrawn. For peptone meals, the stomach was then washed with a 5 ml volume of saline. Meal volumes remaining in the initial withdrawal and withdrawn wash volume were determined by dye dilution spectrophotometry using a modification of the Hunt & Stubbs procedure [15]. For Intralipid meals, initial recovery was followed by two 5 ml warmed saline washes. The gastric contents initially recovered and the two washes were combined, and the amount of fat present was determined using a modification of the Dole procedure [17,18]. Each recovered gastric sample was combined with 5.0 ml distilled water, 7.5 ml heptane, and 12.5 ml Dole’s mixture (40 parts isopropyl alcohol, 10 parts heptane, 1 part 1 N H2504). Samples were shaken vigorously for 10 s and vortexed for 30 s. After phase separation (fifteen min) a 5.0 ml sample of the upper, fat-containing heptane layer was transferred to an aluminum dish. After overnight evaporation, the amount of fat in the dish was weighed, and the total fat in the original 9.95 ml heptane phase was calculated, that is, the amount of fat in the dish was multiplied by 1.99. Fat extraction was also performed on uninfused 5.0 ml samples of the various fat concentrations. The amount of fat contained in these samples represented the highest amount of fat that could be recovered at the end of a gastric emptying interval, and the samples served as standards against which the amount of fat recovered from infused solutions could be compared. Data were analyzed by repeated measures ANOVA for the factors of concentration and emptying time. Effects at single concentrations or time points were assessed with
W.O. White et al. / Regulatory Peptides 88 (2000) 47 – 53
analyses of simple effects and individual differences among conditions were assessed by planned t comparisons. To facilitate comparisons between peptone and Intralipid, data were expressed as a percent of the initial protein or fat load that remained at the time the stomach was emptied. In the second experiment, we assessed the effect of pretreatment with the specific CCKA receptor antagonist devazepide on peptone, intralipid and hyperosmotic saline gastric emptying. In these experiments, we used a single emptying timepoint-10 mm, and examined the effect of a dose range of devazepide 0–1000 mg / kg. Devazepide was dissolved in 1:1 dimethyl sulfoxide:distilled water. Devazepide doses of 10, 32, 100, 320, and 1000 mg / kg in a volume of 1.0 ml / kg were injected intraperitoneally 30 mm prior to the gastric emptying tests. Control vehicle injections consisted of 1.0 ml / kg 1:1 dimethyl sulfoxide:distilled water. We assessed the effects of the entire range of devazepide doses on the gastric emptying of Intralipid at concentrations of 0.5, 1.0 or 2.0 kcal / ml and peptone in concentrations of 0.2, 0.5 and 1.0 kcal / ml. We also assessed the effects of a single devazepide dose (320 mg / kg) on the 10 mm gastric emptying of 2.25% hyperosmotic saline (750 mOsm). This NaCl concentration has the same osmolarity as the 0.5 kcal / ml peptone solution. One group of eight rats was used the 0.2 and 0.5 kcal / ml protein and the hyperosmolar saline conditions. A second group of eight rats was used in the 1.0 kcal / ml protein condition and all the Intralipid conditions. Within these groups, all conditions involved within-subjects manipulations, with eight rats serving in each condition. All rats in a group received each treatment or treatment combination, with treatments randomized and balanced across each group. Each nutrient and concentration was run during a block of sessions. The dosage of devazepide administered was randomized across animals within a block of sessions. At weekly intervals during testing, we assessed the 10 min emptying of physiological saline with phenol red to assure a stable baseline against which the effects of nutrients and devazepide could be assessed. Again, data were analyzed by repeated measures ANOVA. For comparisons of the magnitude of effects of devazepide on peptone and Intralipid emptying, data were expressed as a percent of the initial protein or fat load remaining.
3. Results Ten min gastric emptying of physiological saline did not change over the six control sessions occurring before between and after each set of experimental conditions, F (5, 35) 5 0.565, P 5 0.73. The fact that we could repeatedly recover a consistent average volume of saline following a 10 min emptying period indicated that the test day differences in gastric emptying were due to the particular
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Fig. 1. Peptone gastric emptying as a function of time. Data (mean6S.E.) are expressed as a % of the initial 5 ml infused volume.
solutions infused and were not due to carryover effects that altered gastric emptying dynamics. Fig. 1 shows the mean6S.E. of the percent of peptone remaining at each time point for the 3 peptone concentrations. There were significant effects of both peptone concentration, F (2,10) 5 180.05, P , 0.001 and emptying time, F (3,15) 5 228.44, P , 0.001. Analyses of simple effects demonstrated that a greater % of initial volume remained at each time point for increasing peptone concentrations (P , 0.001 for all time points) and, for any peptone concentration, % of initial volume remaining decreased with time (P , 0.001 for all concentrations). There was also a significant concentration by time interaction F (6,30) 5 11.688, P , 0.001, such that the rate of emptying slowed as concentration increased. For Intralipid emptying, the mean (6S.E.) number of g of fat extracted from our 5.0 ml Intralipid standard emulsions containing 0.2, 0.5, or 1.0 kcal / ml were 0.08260.001 5 g, 0.20760.0019 g, and 0.45560.0034 g respectively. These values were in good agreement with the expected fat contents of 0.086 g, 0.216 g, and 0.434 g of fat. Fig. 2 shows the mean percentage of fat remaining, relative to the mean standard value, at various times points following infusion for the 3 concentrations. For each concentration, the amount of fat remaining in the stomach at 5 min was significantly less than that in the standard solutions. ANOVA demonstrated significant effects for both Intralipid concentration, F(2,14) 5 25.976, P , 0.001 and emptying time F(3,21) 5 22.406, P , 0.001. Less fat emptied from more concentrated Intralipid suspensions than from less concentrated suspensions (P , 0.001 at all time points). For each concentration, the differences among the time points demonstrated different patterns. At the 1.0 kcal / ml Intralipid concentration the % fat remaining at the 5, 10 and 20 mm timepoints did not differ. Only at the 40
50
W.O. White et al. / Regulatory Peptides 88 (2000) 47 – 53
Fig. 2. Intralipid gastric emptying as a function of time. Data (mean6S.E.) are expressed as a % of the initial fat load remaining in the stomach.
mm timepoint was the % fat remaining reduced relative to the amount remaining at 5 mm (P , 0.01). For the 0.5 kcal / ml concentration, the amount of fat remaining did not differ at the 5 and 10 mm timepoints. The amount of fat remaining was reduced at both 20 and 40 mm (p , 0.05). At the 0.2 kcal / ml concentration, the amount of fat at the 10, 20 and 40 mm timepoints were all significantly reduced compared to the amount remaining at the 5 mm timepoint (P , 0.05). Fig. 3 shows how a range of devazepide doses affected the gastric emptying of 0.2, 0.5, and 1.0 kcal / ml peptone solutions. Overall ANOVA indicated that as peptone concentration increased, a greater portion of the infused peptone remained in the stomach at the 10 mm emptying time point, F(2,14) 5 267.587, P , 0.001, and as dose of devazepide increased, the slowed gastric emptying of peptone was partially reversed in that a smaller portion of the infused peptone remained in the stomach, F(5,35) 5 6.18, P , 0.001. In all cases, the effect of devazepide was partial in that the percentage remaining never reached the level found for the 10 min gastric emptying of a 5 ml physiological saline load. The ability of devazepide to partially reverse the slowed emptying of protein depended on the concentration of the peptone solution. Planned t comparisons demonstrated that the emptying of the 0.2 kcal / ml solution was partially reversed by 100, 320, and 1000 mg / kg devazepide, and the emptying of the 0.5 kcal / ml solution was partially reversed by 100 and 1000 m / kg doses. The emptying of the 1.0 kcal / ml solution was not affected by any dose of devazepide. Fig. 4 shows the degree to which different doses of devazepide reversed the slowed gastric emptying of fat suspensions containing 0.5, 1.0 and 2.0 kcal / ml. ANOVA indicated that as Intralipid concentration increased, the
Fig. 3. Effect of a dose range of the CCK A antagonist devazepide on the 10 min gastric emptying of the three peptone concentrations. Data (mean6S.E.) are expressed as a % of the initial 5 ml infused volume. Data for % of initial 5 ml load physiological saline are included for comparison. * indicates significant effect of devazepide as compared to vehicle (0) dose.
proportion of fat remaining in the stomach at the end of the 10 mm emptying period increased, F(2,14) 5 703.79, P , 0.001, and that as devazepide dosage increased, the amount of fat remaining decreased, F(5,35) 5 6.33, P , 0.001. There was also a significant interaction between Intralipid concentration and devazepide dose, such that the effect of devazepide on gastric emptying depended upon the Intralipid concentration, F(10,70) 5 2.94, P , 0.005. As Intralipid concentration increased the dosage necessary to significantly reverse slowed emptying decreased. Specifically, the lowest dose of devazepide necessary to partially reverse slowed emptying of the 0.5, 1.0, and 2.0 kcal / ml solutions was, respectively, 320, 100, and 10 micrograms / kg. Although there was a decrease in the threshold devazepide dose with increasing Intralipid concentration, the magnitude of the devazepide reversal did not increase.
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Fig. 5. Effect of devazepide (320 mg / kg) on the slowed gastric emptying of hypertonic saline. * indicates significant difference from % of 0.9% NaCl remaining.
Fig. 4. Effect of a dose range of the CCK A antagonist devazepide of the 10 min gastric emptying of three Intralipid concentrations. Data (mean6S.E.) are expressed as a % of the initial fat load remaining in the stomach. Data for % of initial 5 ml load physiological saline are included for comparison. * indicates significant effect of devazepide as compared to vehicle (0) dose.
The devazepide sensitive component was 24.3%, 26.1% and 14.1% for the 0.5, 1.0 and 2.0 kcal / ml Intralipid concentrations F(2,14) 5 0.949, P . 0.41. Fig. 5 shows the effect of a single dose of devazepide (320 mg / kg) on the gastric emptying of 2.25% NaCl. This saline concentration emptied significantly more slowly that physiological saline (P , 0.01) and devazepide had no effect on this slowed emptying (P . 0.25).
4. Discussion These data demonstrate a number of phenomena. First, increasing concentrations of peptone and lipid empty progressively more slowly from the stomach. Second, the CCKA antagonist devazepide had a partial effect on the gastric emptying of peptone and Intralipid and no effect on
the gastric emptying of hypertonic saline. Third, although there were nutrient specific changes in the threshold devazepide dose for affecting emptying with increasing concentration, the magnitude of the effect of devazepide did not increase with increasing nutrient concentration. These results suggest that although endogenous CCK plays a role in the control of gastric emptying of proteins and fats in rats, CCK does not mediate the increased slowing with increased concentration. The results were somewhat different for peptone and Intralipid. Under baseline conditions, peptone emptying slowed as peptone concentration increased and the effect of concentration was apparent even at the 5 min time point. This pattern of results for the time course of gastric emptying of peptone meals is similar to what has been previously reported. Similar to peptone, Intralipid emptied in a concentration dependent manner. In contrast, the concentration dependent aspect of Intralipid emptying was not fully evident at earlier time points. More concentrated Intralipid suspensions appear to empty more rapidly at the outset of the emptying period and then there is a period in which little or no emptying occurs. These data suggest that the feedback from intestinal fat may be slower than with peptone. This delayed feedback allows greater emptying to occur at the outset but this greater intestinal fat load results in a more sustained inhibition before a constant rate of emptying over time is established. In these experiments we used different methods for assessing the gastric protein and fat contents. The proportion of infused protein remaining in the stomach was measured by standard dye dilution techniques [15]. This method was appropriate for the protein solution since the dye and protein were both in solution. The fat content of
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the gastric contents was directly assessed by a modification of the Dole procedure [18]. By extracting the fat from the gastric contents we were able to directly assess the lipid phase of gastric contents. This provided a direct measure of the fat remaining in the stomach. Friedman and colleagues [17] have demonstrated that dye dilution does not adequately assess fat gastric emptying since lipid suspensions separate into fat and water soluble components. Devazepide’s effect on peptone gastric emptying diminished with increasing peptone concentration. The threshold dose for affecting the emptying of 0.2 kcal / ml peptone was lower than the threshold dose for affecting 0.5 kcal / ml peptone and devazepide had no effect on the gastric emptying of 1.0 kcal / ml peptone. The diminishing effect of devazepide may have been the result of the increasing osmotic load of the more concentrated peptone solutions. The results with 2.25% NaCl demonstrate that high osmolarity in the absence of nutrient content slows gastric emptying and this effect is CCK independent since a high concentration of devazepide had no effect on the gastric emptying of the 2.25% NaCl load. The absence of a demonstrable CCK mediated component of the slow gastric emptying of 1.0 kcal / ml peptone implies that the osmotic concentration of this solution may be the main determinant of its gastric emptying rate. Green et al. [8] have previously demonstrated the ability of devazepide to affect the gastric emptying of a 4.5% peptone test meal. In that case the effect of devazepide was dose related with a significant reversal at a dose of 100 mg / kg and maximal reversal at a dose of 1 mg / kg. While a direct comparison to saline emptying was not made, it appears, as in the present study, that the CCK antagonist induced reversal of peptone slowed gastric emptying was only partial. Similarly, we have previously demonstrated that devazepide results in a partial reversal of slowed emptying of 4.5% peptone in rhesus monkey. Thus, the present data are consistent with the view that the slow gastric emptying of peptone only partially reflects the actions of endogenous CCK. CCK’s actions in peptone emptying are most evident at low concentrations. The increased slowing with increased peptone concentration does not depend on a CCK action. In contrast to the results with peptone, devazepide affected Intralipid gastric emptying at all three Intralipid concentrations and the threshold dose for affecting Intralipid gastric emptying decreased with increasing Intralipid concentration. Despite this greater potency, however, the magnitude of the effect of devazepide on Intralipid gastric emptying was partial and did not increase as a function of Intralipid concentration. These results are in apparent contrast with our prior data on the ability of devazepide to completely reverse the slowed gastric emptying of 0.5 kcal / ml Intralipid in the rhesus monkey [9]. However, in those experiments we were using the phenol dye dilution technique and we were not directly measuring the amount of fat in the gastric contents. As demonstrated
by Friedman et al. [17], this technique likely underestimated the volume of fat remaining in the stomach. Holzer et al. [12] have demonstrated that intraintestinal Intralipid inhibits the gastric emptying of a saline test meal in a dose related fashion. One ml intraduodenal infusions of 5 and 10% Intralipid inhibited the 5 mm emptying of saline test meals from 93% to 69 and 20% respectively. Devazepide pretreatment had no effect on the inhibition produced by 5% Intralipid but reversed the inhibition produced by 10% to the level produced by 5% Intralipid. Thus, these data showed a role for endogenous CCK in the intestinal feedback of gastric inhibition produced by intestinal lipids and demonstrated that a portion of this inhibition was CCK independent in that it was not blocked by devazepide pretreatment. Our data are consistent with the idea that there are CCK dependent and CCK independent components to lipid induced inhibitions of gastric emptying. The data from the present experiments demonstrate that while endogenous CCK plays a role in the inhibition of gastric emptying produced by peptone and Intralipid, endogenous CCK does not account for increased inhibition of emptying due to increased nutrient concentration. With peptone, it is likely that mechanisms related to the osmolarity of the solutions play a role in this concentration dependent phenomena. How the increased inhibition of gastric emptying with increased lipid concentration is mediated remains to be demonstrated.
Acknowledgements This work was supported by DK19302.
References [1] Debas HT, Farooq O, Grossman MI. inhibition of gasteric emptying is a phsyological action of cholecystokinin. Gastroenterology 1975;68:211–7. [2] Moran TH, McHugh PR. Cholecystokinin suppresses food intake by inhibiting gastric emptying. Amer J Physiol 1982;242:R491–7. [3] Conover KL, Collins SM, Weingarten HP. A comparison of CCK induced changes in gastric emptying and feeding in the rat. Am J Physiol 1988;255:R21–6. [4] Yamagishi T, Debas HT. Cholecystokinin inhibits gastric emptying by acting on both proximal stomach and pylorus. Am J Physiol 1978;234:E375–8. [5] Kleibeuker JH, Beekhuis H, Jansen JBMJ, Piers DA, Lamars CBHW. Cholecystokinin is a physiological hormonal mediator of fat-induced inhibition of gastric emptying in man. Europ J Clin Invest 1988;18:173–7. [6] Scarpignat OC, Varga G, Corradi C. Effect of cCK and its antagonists on gastric emptying. J Physiol 1993;87:291–300. [7] Shillabeer G, Davison JS. Proglumide, a cholecystokinin antagonist, increases gastric emptying in rats. Am J Physiol 1987;252:R353–60. [8] Green T, Dimaline R, Peikin S, Dockray GJ. Action of the cholecystokinin antagonist L364,718 on gastric emptying in the rat. Am J Physiol 1988;255:G685–9.
W.O. White et al. / Regulatory Peptides 88 (2000) 47 – 53 [9] Moran TH, Ameglio PJ, Schwartz GJ, Peyton HJ, McHugh PR. Endogenous cholecystokinin in the control of gastric emptying of liquid nutrient loads in rhesus monkeys. Am J Physiol 1993;242:R371–5. [10] Moran TH, Field DG, Knipp S, Carrigan TS, Schwartz GJ. Endogenous CCK in the control of gastric emptying of glucose and maltose. Peptides 1997;18:547–50. [11] Forster ER, Dockray GJ. The role of cholecystokinin in inhibition of gastric emptying by peptone in the rat. Exper Physiol 1992;77:693– 700. ¨ [12] Holzer HH, Turkelson CM, Solomon TE, Raybould HE. Intestinal lipid inhibits gastric emptying via CCK and vagal capsaicin-sensitive afferent pathway in rats. Am J Physiol 1994;267:G625–9. [13] Raybould HE, Lloyd KCK. Integration of postprandial function in the proximal gastrointestinal tract. Annals NY Acad Sci 1994;713:144–56.
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[14] McHugh PR, Moran TH. Calories and gastric emptying; a regulatory capacity with implications for feeding. Am J Physiol 1979;236:R254–60. [15] Hunt JN, Stubbs DF. The volume and energy content of meals as determinants of gastric emptying. J Physiol London 1975;245:209– 25. [16] Schwartz GJ, Berkow G, McHugh PR, Moran TH. Gastric branch vagotomy blocks nutrient and cholecystokinin induced suppression of gastric emptying. Am J Physiol 1993;264:R630–7. [17] Friedman MI, Ramirez I, Tordoff MG. Gastric emptying of ingested fat emulsion in rats: implications for studies of fat-induced satiety. Am J Physiol 1996;270:R688–92. [18] Dole VP. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J Clin Invest 1956;35:150–4.
Role of endogenous CCK in the inhibition of gastric emptying by peptone and Intralipid in rats Wesley O. White, Gary J. Schwartz, Timothy H. Moran* Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618 720 Rutland Avenue Baltimore, MD 21205, USA Received 6 September 1999; received in revised form 13 December 1999; accepted 13 December 1999
Abstract To assess the role of endogenous cholecystokinin in the control of gastric emptying of peptone solutions and Intralipid suspensions, we examined the ability of a dose range of the CCK-A antagonist, devazepide to accelerate the gastric emptying of various caloric concentrations of peptone and Intralipid in rats. In the absence of devazepide, both peptone and Intralipid emptying slowed with increasing concentration. Devazepide’s effect on peptone gastric emptying diminished with increasing peptone concentration. The threshold dose for accelerating the emptying of 0.2 kcal / ml peptone was lower than the threshold dose for affecting 0.5 kcal / ml peptone and devazepide had no effect on the gastric emptying of 1.0 kcal / ml peptone. In contrast, devazepide affected Intralipid gastric emptying at all three Intralipid concentrations and the threshold dose decreased with increasing Intralipid concentration. However, the magnitude of the effect of devazepide on peptone or Intralipid gastric emptying was partial and did not increase as a function of concentration. These data demonstrate a role for endogenous CCK in the emptying of peptone and Intralipid but suggest that endogenous CCK does not account for the increased slowing of gastric emptying evident with increased caloric concentration 2000 Elsevier Science B.V. All rights reserved. Keywords: Endogenous cholecystokinin; Control of gastric emptying; Intralipid suspensions
1. Introduction Exogenous administration of the brain–gut peptide cholecystokinin (CCK) inhibits gastric emptying in a variety of species [1–4]. A physiological role for CCK in the control of gastric emptying was originally suggested by experiments demonstrating that the dose of exogenous CCK required for inhibition of gastric emptying was comparable to doses required for stimulation of pancreatic secretion [1]. Additionally, the doses of exogenous CCK that inhibited gastric emptying were associated with plasma concentrations that were within the physiological range
*Corresponding author. Tel.: 1 1-410-955-2344; fax: 1 1-410-6140013. E-mail address: [email protected] (T.H. Moran)
[5]. The availability of potent and specific CCK antagonists has allowed a more direct assessment of the role of endogenous CCK in the controls of nutrient gastric emptying. Administration of CCK-A specific receptor antagonists such as devazapide or loxiglumide has been shown to accelerate nutrient gastric emptying in a variety of species and in a range of experimental settings [6–9]. We have demonstrated roles for endogenous CCK in the control of liquid gastric emptying of fats, proteins, and carbohydrates in rhesus monkeys [9,10]. In these experiments, devazapide administration accelerated the gastric emptying of Intralipid and peptone test meals in a dose related fashion. Similarly, devazapide accelerated the gastric emptying of 5% glucose and 10% maltose in a dose related fashion while not affecting the slowed emptying of hypertonic carbohydrate or sodium chloride solutions. Devazapide administration has also been shown to acceler-
0167-0115 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00119-6
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ate the gastric emptying of maltose, intralipid and peptone in the rat [8,11–13]. Increasing nutrient concentration has been demonstrated to result in slower rates of liquid gastric emptying in a variety of species [14–16]. Although a role for CCK in the mediation of nutrient gastric emptying has been demonstrated, whether CCK mediates the increased inhibition on gastric emptying that accompanies increased nutrient concentration has not been routinely assessed. Thus, in the rat, the ability of devazapide to affect maltose or peptone gastric emptying has only been assessed at single concentrations of each nutrient [11,13]. While Holzer and colleagues have examined the ability of devazapide to affect the gastric emptying of two concentrations of Intralipid, a fat suspension, they employed the phenol-red technique for measuring gastric emptying [12]. Phenol-red stays with the water rather than the lipid phase and Friedman and colleagues have demonstrated that the use of this technique does not adequately assess emptying of the lipid phase [17]. In the present experiments, we have examined the role of endogenous CCK in the control of liquid gastric emptying of various concentrations of peptone solutions and Intralipid emulsions in the rat. Specifically, these experiments examine the ability of a dose range of the specific CCK-A antagonist devazapide to accelerate the gastric emptying of the 5 ml intragastric loads of peptone and intralipid at various concentrations. These experiments are aimed at determining whether endogenous CCK plays a role in the increased inhibition of gastric emptying associated with increased caloric concentration.
2. Materials and methods The subjects were male Sprague-Dawley rats (Charles River), weighing 250–300 g at the start of the study. Animals were housed in individual hanging wire-mesh cages and maintained at 228C on a 12–12 h light–dark cycle with lights on at 0600 h. The rats had free access to water. Shortly after arriving in the laboratory, the animals were placed on a restricted feeding schedule with pelleted chow (Purina Rat Chow) only available for 5 h per day beginning at approximately 1200 h. Gastric emptying sessions took place in the mornings after rats had been food deprived for approximately 16 h. Rats were adapted to the gastric emptying procedure before experimental manipulations were performed. A polyethylene feeding tube attached to a syringe was inserted through the mouth and advanced down the esophagus, until the end the tube was in the stomach. 5.0 ml of phenol-red-containing saline solution was infused over a five s interval, and the tube was then removed. Five mm later the tube was reinserted, and solution remaining in the stomach was withdrawn (‘initial recovery’). The stomach was rinsed with 5.0 ml of saline. This procedure was
repeated once daily for each rat until the volume of the original saline solution recovered after 5 mm was stable (1 week). On experimental days, 30 mm prior to the gastric emptying tests, each rat’s stomach was prewashed with 5.0 ml of warmed saline solution infused over 5 s. The stomach contents were immediately recovered. The prewash frequently contained small amounts of hair and cage debris. Prewashes were repeated until contents removed from the stomach were clear. In the initial experiment, we measured the emptying of different concentrations of peptone (n 5 6) and Intralipid (n 5 8) across time. Peptone meals containing 0.05, 0.125 and 0.25 g / ml (300, 750, and 1500 mOsm, respectively) were prepared by adding peptone (Sigma type II from meat) to distilled water containing 0.0625 mg / ml phenolred. These peptone meals contained 0.2, 0.5 and 1.0 kcal / ml respectively. Intralipid meals were prepared by diluting 10% or 20% Intralipid emulsions (10.0 g soybean oil, 1.2 g phospholipids from powdered egg yolk, 2.25 g glycerin, and 110 kcal per 100 ml; Kabi Pharmacia) with distilled water. Intralipid meals containing 0.2, 0.5 and 1.0 kcal / ml were used. All solutions were administered at a pH of 7.0 and at 378C. The infusion volume remained constant at 5.0 ml. At intervals of 5, 10, 20 or 40 mm after the meals were infused, remaining gastric contents were withdrawn. For peptone meals, the stomach was then washed with a 5 ml volume of saline. Meal volumes remaining in the initial withdrawal and withdrawn wash volume were determined by dye dilution spectrophotometry using a modification of the Hunt & Stubbs procedure [15]. For Intralipid meals, initial recovery was followed by two 5 ml warmed saline washes. The gastric contents initially recovered and the two washes were combined, and the amount of fat present was determined using a modification of the Dole procedure [17,18]. Each recovered gastric sample was combined with 5.0 ml distilled water, 7.5 ml heptane, and 12.5 ml Dole’s mixture (40 parts isopropyl alcohol, 10 parts heptane, 1 part 1 N H2504). Samples were shaken vigorously for 10 s and vortexed for 30 s. After phase separation (fifteen min) a 5.0 ml sample of the upper, fat-containing heptane layer was transferred to an aluminum dish. After overnight evaporation, the amount of fat in the dish was weighed, and the total fat in the original 9.95 ml heptane phase was calculated, that is, the amount of fat in the dish was multiplied by 1.99. Fat extraction was also performed on uninfused 5.0 ml samples of the various fat concentrations. The amount of fat contained in these samples represented the highest amount of fat that could be recovered at the end of a gastric emptying interval, and the samples served as standards against which the amount of fat recovered from infused solutions could be compared. Data were analyzed by repeated measures ANOVA for the factors of concentration and emptying time. Effects at single concentrations or time points were assessed with
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analyses of simple effects and individual differences among conditions were assessed by planned t comparisons. To facilitate comparisons between peptone and Intralipid, data were expressed as a percent of the initial protein or fat load that remained at the time the stomach was emptied. In the second experiment, we assessed the effect of pretreatment with the specific CCKA receptor antagonist devazepide on peptone, intralipid and hyperosmotic saline gastric emptying. In these experiments, we used a single emptying timepoint-10 mm, and examined the effect of a dose range of devazepide 0–1000 mg / kg. Devazepide was dissolved in 1:1 dimethyl sulfoxide:distilled water. Devazepide doses of 10, 32, 100, 320, and 1000 mg / kg in a volume of 1.0 ml / kg were injected intraperitoneally 30 mm prior to the gastric emptying tests. Control vehicle injections consisted of 1.0 ml / kg 1:1 dimethyl sulfoxide:distilled water. We assessed the effects of the entire range of devazepide doses on the gastric emptying of Intralipid at concentrations of 0.5, 1.0 or 2.0 kcal / ml and peptone in concentrations of 0.2, 0.5 and 1.0 kcal / ml. We also assessed the effects of a single devazepide dose (320 mg / kg) on the 10 mm gastric emptying of 2.25% hyperosmotic saline (750 mOsm). This NaCl concentration has the same osmolarity as the 0.5 kcal / ml peptone solution. One group of eight rats was used the 0.2 and 0.5 kcal / ml protein and the hyperosmolar saline conditions. A second group of eight rats was used in the 1.0 kcal / ml protein condition and all the Intralipid conditions. Within these groups, all conditions involved within-subjects manipulations, with eight rats serving in each condition. All rats in a group received each treatment or treatment combination, with treatments randomized and balanced across each group. Each nutrient and concentration was run during a block of sessions. The dosage of devazepide administered was randomized across animals within a block of sessions. At weekly intervals during testing, we assessed the 10 min emptying of physiological saline with phenol red to assure a stable baseline against which the effects of nutrients and devazepide could be assessed. Again, data were analyzed by repeated measures ANOVA. For comparisons of the magnitude of effects of devazepide on peptone and Intralipid emptying, data were expressed as a percent of the initial protein or fat load remaining.
3. Results Ten min gastric emptying of physiological saline did not change over the six control sessions occurring before between and after each set of experimental conditions, F (5, 35) 5 0.565, P 5 0.73. The fact that we could repeatedly recover a consistent average volume of saline following a 10 min emptying period indicated that the test day differences in gastric emptying were due to the particular
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Fig. 1. Peptone gastric emptying as a function of time. Data (mean6S.E.) are expressed as a % of the initial 5 ml infused volume.
solutions infused and were not due to carryover effects that altered gastric emptying dynamics. Fig. 1 shows the mean6S.E. of the percent of peptone remaining at each time point for the 3 peptone concentrations. There were significant effects of both peptone concentration, F (2,10) 5 180.05, P , 0.001 and emptying time, F (3,15) 5 228.44, P , 0.001. Analyses of simple effects demonstrated that a greater % of initial volume remained at each time point for increasing peptone concentrations (P , 0.001 for all time points) and, for any peptone concentration, % of initial volume remaining decreased with time (P , 0.001 for all concentrations). There was also a significant concentration by time interaction F (6,30) 5 11.688, P , 0.001, such that the rate of emptying slowed as concentration increased. For Intralipid emptying, the mean (6S.E.) number of g of fat extracted from our 5.0 ml Intralipid standard emulsions containing 0.2, 0.5, or 1.0 kcal / ml were 0.08260.001 5 g, 0.20760.0019 g, and 0.45560.0034 g respectively. These values were in good agreement with the expected fat contents of 0.086 g, 0.216 g, and 0.434 g of fat. Fig. 2 shows the mean percentage of fat remaining, relative to the mean standard value, at various times points following infusion for the 3 concentrations. For each concentration, the amount of fat remaining in the stomach at 5 min was significantly less than that in the standard solutions. ANOVA demonstrated significant effects for both Intralipid concentration, F(2,14) 5 25.976, P , 0.001 and emptying time F(3,21) 5 22.406, P , 0.001. Less fat emptied from more concentrated Intralipid suspensions than from less concentrated suspensions (P , 0.001 at all time points). For each concentration, the differences among the time points demonstrated different patterns. At the 1.0 kcal / ml Intralipid concentration the % fat remaining at the 5, 10 and 20 mm timepoints did not differ. Only at the 40
50
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Fig. 2. Intralipid gastric emptying as a function of time. Data (mean6S.E.) are expressed as a % of the initial fat load remaining in the stomach.
mm timepoint was the % fat remaining reduced relative to the amount remaining at 5 mm (P , 0.01). For the 0.5 kcal / ml concentration, the amount of fat remaining did not differ at the 5 and 10 mm timepoints. The amount of fat remaining was reduced at both 20 and 40 mm (p , 0.05). At the 0.2 kcal / ml concentration, the amount of fat at the 10, 20 and 40 mm timepoints were all significantly reduced compared to the amount remaining at the 5 mm timepoint (P , 0.05). Fig. 3 shows how a range of devazepide doses affected the gastric emptying of 0.2, 0.5, and 1.0 kcal / ml peptone solutions. Overall ANOVA indicated that as peptone concentration increased, a greater portion of the infused peptone remained in the stomach at the 10 mm emptying time point, F(2,14) 5 267.587, P , 0.001, and as dose of devazepide increased, the slowed gastric emptying of peptone was partially reversed in that a smaller portion of the infused peptone remained in the stomach, F(5,35) 5 6.18, P , 0.001. In all cases, the effect of devazepide was partial in that the percentage remaining never reached the level found for the 10 min gastric emptying of a 5 ml physiological saline load. The ability of devazepide to partially reverse the slowed emptying of protein depended on the concentration of the peptone solution. Planned t comparisons demonstrated that the emptying of the 0.2 kcal / ml solution was partially reversed by 100, 320, and 1000 mg / kg devazepide, and the emptying of the 0.5 kcal / ml solution was partially reversed by 100 and 1000 m / kg doses. The emptying of the 1.0 kcal / ml solution was not affected by any dose of devazepide. Fig. 4 shows the degree to which different doses of devazepide reversed the slowed gastric emptying of fat suspensions containing 0.5, 1.0 and 2.0 kcal / ml. ANOVA indicated that as Intralipid concentration increased, the
Fig. 3. Effect of a dose range of the CCK A antagonist devazepide on the 10 min gastric emptying of the three peptone concentrations. Data (mean6S.E.) are expressed as a % of the initial 5 ml infused volume. Data for % of initial 5 ml load physiological saline are included for comparison. * indicates significant effect of devazepide as compared to vehicle (0) dose.
proportion of fat remaining in the stomach at the end of the 10 mm emptying period increased, F(2,14) 5 703.79, P , 0.001, and that as devazepide dosage increased, the amount of fat remaining decreased, F(5,35) 5 6.33, P , 0.001. There was also a significant interaction between Intralipid concentration and devazepide dose, such that the effect of devazepide on gastric emptying depended upon the Intralipid concentration, F(10,70) 5 2.94, P , 0.005. As Intralipid concentration increased the dosage necessary to significantly reverse slowed emptying decreased. Specifically, the lowest dose of devazepide necessary to partially reverse slowed emptying of the 0.5, 1.0, and 2.0 kcal / ml solutions was, respectively, 320, 100, and 10 micrograms / kg. Although there was a decrease in the threshold devazepide dose with increasing Intralipid concentration, the magnitude of the devazepide reversal did not increase.
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Fig. 5. Effect of devazepide (320 mg / kg) on the slowed gastric emptying of hypertonic saline. * indicates significant difference from % of 0.9% NaCl remaining.
Fig. 4. Effect of a dose range of the CCK A antagonist devazepide of the 10 min gastric emptying of three Intralipid concentrations. Data (mean6S.E.) are expressed as a % of the initial fat load remaining in the stomach. Data for % of initial 5 ml load physiological saline are included for comparison. * indicates significant effect of devazepide as compared to vehicle (0) dose.
The devazepide sensitive component was 24.3%, 26.1% and 14.1% for the 0.5, 1.0 and 2.0 kcal / ml Intralipid concentrations F(2,14) 5 0.949, P . 0.41. Fig. 5 shows the effect of a single dose of devazepide (320 mg / kg) on the gastric emptying of 2.25% NaCl. This saline concentration emptied significantly more slowly that physiological saline (P , 0.01) and devazepide had no effect on this slowed emptying (P . 0.25).
4. Discussion These data demonstrate a number of phenomena. First, increasing concentrations of peptone and lipid empty progressively more slowly from the stomach. Second, the CCKA antagonist devazepide had a partial effect on the gastric emptying of peptone and Intralipid and no effect on
the gastric emptying of hypertonic saline. Third, although there were nutrient specific changes in the threshold devazepide dose for affecting emptying with increasing concentration, the magnitude of the effect of devazepide did not increase with increasing nutrient concentration. These results suggest that although endogenous CCK plays a role in the control of gastric emptying of proteins and fats in rats, CCK does not mediate the increased slowing with increased concentration. The results were somewhat different for peptone and Intralipid. Under baseline conditions, peptone emptying slowed as peptone concentration increased and the effect of concentration was apparent even at the 5 min time point. This pattern of results for the time course of gastric emptying of peptone meals is similar to what has been previously reported. Similar to peptone, Intralipid emptied in a concentration dependent manner. In contrast, the concentration dependent aspect of Intralipid emptying was not fully evident at earlier time points. More concentrated Intralipid suspensions appear to empty more rapidly at the outset of the emptying period and then there is a period in which little or no emptying occurs. These data suggest that the feedback from intestinal fat may be slower than with peptone. This delayed feedback allows greater emptying to occur at the outset but this greater intestinal fat load results in a more sustained inhibition before a constant rate of emptying over time is established. In these experiments we used different methods for assessing the gastric protein and fat contents. The proportion of infused protein remaining in the stomach was measured by standard dye dilution techniques [15]. This method was appropriate for the protein solution since the dye and protein were both in solution. The fat content of
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the gastric contents was directly assessed by a modification of the Dole procedure [18]. By extracting the fat from the gastric contents we were able to directly assess the lipid phase of gastric contents. This provided a direct measure of the fat remaining in the stomach. Friedman and colleagues [17] have demonstrated that dye dilution does not adequately assess fat gastric emptying since lipid suspensions separate into fat and water soluble components. Devazepide’s effect on peptone gastric emptying diminished with increasing peptone concentration. The threshold dose for affecting the emptying of 0.2 kcal / ml peptone was lower than the threshold dose for affecting 0.5 kcal / ml peptone and devazepide had no effect on the gastric emptying of 1.0 kcal / ml peptone. The diminishing effect of devazepide may have been the result of the increasing osmotic load of the more concentrated peptone solutions. The results with 2.25% NaCl demonstrate that high osmolarity in the absence of nutrient content slows gastric emptying and this effect is CCK independent since a high concentration of devazepide had no effect on the gastric emptying of the 2.25% NaCl load. The absence of a demonstrable CCK mediated component of the slow gastric emptying of 1.0 kcal / ml peptone implies that the osmotic concentration of this solution may be the main determinant of its gastric emptying rate. Green et al. [8] have previously demonstrated the ability of devazepide to affect the gastric emptying of a 4.5% peptone test meal. In that case the effect of devazepide was dose related with a significant reversal at a dose of 100 mg / kg and maximal reversal at a dose of 1 mg / kg. While a direct comparison to saline emptying was not made, it appears, as in the present study, that the CCK antagonist induced reversal of peptone slowed gastric emptying was only partial. Similarly, we have previously demonstrated that devazepide results in a partial reversal of slowed emptying of 4.5% peptone in rhesus monkey. Thus, the present data are consistent with the view that the slow gastric emptying of peptone only partially reflects the actions of endogenous CCK. CCK’s actions in peptone emptying are most evident at low concentrations. The increased slowing with increased peptone concentration does not depend on a CCK action. In contrast to the results with peptone, devazepide affected Intralipid gastric emptying at all three Intralipid concentrations and the threshold dose for affecting Intralipid gastric emptying decreased with increasing Intralipid concentration. Despite this greater potency, however, the magnitude of the effect of devazepide on Intralipid gastric emptying was partial and did not increase as a function of Intralipid concentration. These results are in apparent contrast with our prior data on the ability of devazepide to completely reverse the slowed gastric emptying of 0.5 kcal / ml Intralipid in the rhesus monkey [9]. However, in those experiments we were using the phenol dye dilution technique and we were not directly measuring the amount of fat in the gastric contents. As demonstrated
by Friedman et al. [17], this technique likely underestimated the volume of fat remaining in the stomach. Holzer et al. [12] have demonstrated that intraintestinal Intralipid inhibits the gastric emptying of a saline test meal in a dose related fashion. One ml intraduodenal infusions of 5 and 10% Intralipid inhibited the 5 mm emptying of saline test meals from 93% to 69 and 20% respectively. Devazepide pretreatment had no effect on the inhibition produced by 5% Intralipid but reversed the inhibition produced by 10% to the level produced by 5% Intralipid. Thus, these data showed a role for endogenous CCK in the intestinal feedback of gastric inhibition produced by intestinal lipids and demonstrated that a portion of this inhibition was CCK independent in that it was not blocked by devazepide pretreatment. Our data are consistent with the idea that there are CCK dependent and CCK independent components to lipid induced inhibitions of gastric emptying. The data from the present experiments demonstrate that while endogenous CCK plays a role in the inhibition of gastric emptying produced by peptone and Intralipid, endogenous CCK does not account for increased inhibition of emptying due to increased nutrient concentration. With peptone, it is likely that mechanisms related to the osmolarity of the solutions play a role in this concentration dependent phenomena. How the increased inhibition of gastric emptying with increased lipid concentration is mediated remains to be demonstrated.
Acknowledgements This work was supported by DK19302.
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