CONTROL OF GASTRIC EMPTYING IN THE PIG: INFLUENCE OF CHOLECYSTOKININ, SOMATOSTATIN AND PROKINETIC AGENTS

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1 Experimental Physiology (1995), 80, Printed in Great Britain CONTROL OF GASTRIC EMPTYING IN THE PIG: INFLUENCE OF CHOLECYSTOKININ, SOMATOSTATIN AND PROKINETIC AGENTS P. C. GREGORY*, M. McFADYEN AND D. V. RAYNER-r The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK (MANUSCRIPT RECEIVED 21 MAY 1994, ACCEPTED I SEPTEMBER 1994) SUMMARY The influence of systemic arterial infusions of cholecystokinin octapeptide (CCK8) and somatostatin, and injections of cisapride, metoclopramide and atropine on gastric emptying were studied in eight pigs. Gastric emptying of dry matter (DM) and liquids (Cr-EDTA as marker) was measured, in pigs fitted with a gastric cannula, by evacuation of gastnc contents either immediately after the pigs had finished feeding, or 3 h after being fed a meal containing 1200 g of a finely ground barley diet mixed with water. Gastric emptying of DM and liquids during the feeding period was not significantly altered by cisapride (0.15 and 0-3 mg kg-'-1), metoclopramide (0-2 mg kg-'), CCK8 (250 ng kg-' h-1) or somatostatin (1.8 and 4-5 jug kg-1 h-1); atropine (0.06 mg kg-') slowed emptying of DM (by %; P < 0 001) and of liquids (by %, P < 0.01). In contrast, the amount of DM emptied within 3 h of feeding was significantly reduced with CCK8 (250 ng kg-1 h-1; by %, P < 0.001) and with somatostatin (1 8 jug kg-1 h-1; by 10± 4%; P < 0001). There was no increase in emptying of DM or liquids with cisapride or metoclopramide; indeed, there was actually a reduction in liquid emptying (by %; P < 0 05) with cisapride (0 3 mg kg-'). Gastric emptying in the pig is therefore resistant both to stimulation by prokinetic agents, and to inhibition by some putative neurohormonal modulators at the doses used; gastric emptying during feeding is even more resistant to alteration than is emptying between meals. It is concluded that in the pig, neither CCK nor somatostatin are involved in regulation of gastric emptying during the feeding period, but that both may play a minor role in regulation of emptying between meals. The low sensitivity to CCK may be a factor in the rapid gastric emptying of the pig. INTRODUCTION It has recently been demonstrated (Gregory, McFadyen & Rayner, 1990) that the rate of gastric emptying of energy in pigs fed a finely ground diet mixed with water is considerably greater, calculated on the basis of metabolic body weight, than that of even a liquid meal in man (Brener, Hendrix & McHugh, 1983) or monkey (McHugh & Moran, 1979). A rapid rate of gastric emptying has been suggested to predispose towards obesity (Hunt, 1980) and indeed the pig is sometimes used as a model for obesity in man (Houpt, Houpt & Pond, 1979). Therefore, we considered it important to investigate possible factors responsible for this rapid gastric emptying in the pig. In man the prokinetic agents metoclopramide (Behar & Ramsby, 1978) and cisapride (Lazzaroni, Sangaletti & Porro, 1987) accelerate gastric emptying, even in subjects with a * Present address: Solvay Pharma Deutschland, Gastrointestinal Pharmacology, Hans-Bokciler Allee 20, D Hannover, Germany. J To whom reprint requests should be addressed.

2 160 P. C. GREGORY, M. McFADYEN AND D. V. RAYNER normal rate of emptying, suggesting that the normal rate of emptying is not the maximum rate, i.e. it is to some extent inhibited (Johnson, 1989). It needs to be determined if the same also holds true for the pig. The rate of gastric emptying of a meal is controlled according to its size and content via a complex interrelationship of myogenic, neural and hormonal mechanisms (Cooke, 1975). Two of the major gastrointestinal peptides concemed with regulation of gastrointestinal motility are cholecystokinin and somatostatin (Mukhopadhyay, Thor, Copeland, Johnson & Weisbrodt, 1977; Neri, Cuccurullo & Marzio, 1990). Both are released after feeding or in response to duodenal fat (Schusdziarra, Haris, Conlon, Anmura & Unger, 1978; Lilja, Wiener, Inoue, Fried, Greeley & Thompson, 1984); furthermore, duodenal infusion of fat induces satiety in the pig (Gregory & Rayner, 1987), which can be blocked by prior administration of a cholecystokinin (CCK) antagonist (Gregory, McFadyen & Rayner, 1989a), and duodenal infusion of fat also causes severe slowing of gastric emptying during the feeding period in the pig (Gregory, McFadyen & Rayner, 1989b). In man the rate of gastric emptying is slowed by infusions of low, apparently physiological, doses of CCK (Liddle, Morita, Conrad & Williams, 1986), while somatostatin has been variously reported to slow (Bloom, Ralphs, Besser, Hall, Coy, Kastin & Schally, 1975) or accelerate (Mogard, Maxwell, Wong, Reedy, Sytnik & Walsh, 1988) gastric emptying. The present study was designed to look at the influence of these various agents on gastric emptying in the pig. Their effects on emptying during the period of feeding and on emptying in the period between meals have been compared, since different patterns and rates of emptying have been observed in the pig under these two conditions (Gregory et al. 1990). Since none of these agents altered the rate of emptying during the feeding period, the influence of one further agent, atropine, was tested as a control. A brief report of some of these results has been published (Rayner & Gregory, 1989). METHODS Experiments were carried out on a total of eight female Large White x Landrace pigs. When they reached a body weight of kg they were fitted vith a gastric cannula (plastic; barrel diameter 25 mm) and with a polyvinyl chloride (PVC) catheter (1 mm diameter) inserted through the saphenous artery and positioned during X-ray screening by image intensifier to lie in the abdominal aorta. All surgery was performed with full aseptic precautions under halothane anaesthesia as previously described (Gregory & Rayner, 1987). The animals were first sedated with azaperone (4% w/v, 1 ml kg-' I.M., Stresnil; Janssen Pharmaceuticals) and were then anaesthetized with a halothane-oxygen mixture by face mask. Following introduction of an endotracheal tube, anaesthesia was maintained by varying the concentration of halothane (1-3 %) in a halothane-nitrous oxide-oxygen mixture (N20:02 approximately 60:40), using positive pressure ventilation if necessary. The animals were given at least 2 weeks to recover from surgery and were then housed in metabolism cages under continuous lighting. They were fed twice daily (09.00 and h) with NRS9, a barleybased diet (Gregory & Rayner, 1987) mixed with twice its weight of water. All experiments were carried out on the rate of gastric emptying of dry matter (DM) and liquids (using Cr-EDTA as a marker) of the morning meal, using the gastric evacuation technique described previously (Gregory, McFadyen & Rayner, 1989b). The stomach was washed free of any digesta remaining from the overnight meal1 h before starting the experiments. The animals were fed a meal of 1200 g NRS9 mixed with water containing Cr-EDTA (38 mg Cr I`), and the gastric contents were evacuated immediately after the pigs had finished the meal or 3 h after beginning to feed. Cisapride (0-15 and 0.3 mg kg-'; Janssen Pharmaceutica n.v., Beerse, Belgium), metoclopramide (0-2 mg kg-';

3 GASTRIC EMPTYING IN THE PIG Maxolon; SmithKline Beecham Pharmaceuticals, Welwyn Garden City, UK) and atropine (0-06 mg kg-1; atropine sulphate; Sigma Chemical Co., Poole, UK) were injected intra-arterially 20 min before feeding, while somatostatin (1 8 and 4.5 jug kg-' h-'; Sigma Chemical Co.) and sulphated cholecystokinin octapeptide (250 ng kg-' h-'; Sigma Chemical Co.) were infused from 10 min before feeding until the stomach was evacuated. Each gastrointestinal peptide or prokinetic agent was investigated separately and the results were compared with the control rate of emptying measured within the same week in the same animals without infusion, using Student's paired t test. 161 RESULTS Gastric emptying during the feeding period There were no significant changes in the rates of gastric emptying of either DM or liquids after injection of cisapride (0 15 or 0-3 mg kg-') or metoclopramide, or during infusion of CCK or somatostatin (Table 1), although there was a trend towards a decrease in the rate of DM emptying with CCK (seen in 8 of 10 experiments) and with somatostatin (4-5 jtg kg-' h-'; also seen in 8 of 10 experiments). Injection of atropine, however, caused severe reduction of emptying of DM (by 53 ± 6 %; P < 0.001) and of liquids (by %; P < 0-01), and was accompanied by a % increase in the time to eat the meal (P < 0.01). Gastric emptying between meals During the 3 h period after feeding (Table 2) significant reductions in the amount of DM emptied resulted from infusions of CCK (by %; P < 0.001) and of somatostatin (by %; P < 0.001); there were also significant reductions in the amount of liquid emptied with somatostatin infusion (by %; P < 0.001). Table 1. Gastric emptying during the feeding period in the pig Control With treatment f<ate of gastric emptying Rate of gastric emptying Treatment Meal time Dry matter Liquid Meal time Dry matter Liquid Peptide or agent Dose n (min) (g min-') (ml min-') (min) (g min-') (ml min-') Cholecystokinin 250ngkg-'h' ±0-9 68±06 44± ± Somatostatin 1.8,ugkg- 'h ±2-1 42±06 42±3 179±3-3 37± Somatostatin 45,ugkg- 'h ± ±2 170± 13 40± Cisapride 0 15 mgkg-' 4 120± ±5 129± Cisapride 0-3mgkg-' ± ± ± Metoclopramide 0-2 mg kg-' ± ± ± ± 6 Atropine 0-06mgkg-' 7 206± *** ** 20±2t* Results are means + S.E.M. of the rates of gastric emptying, measured using a gastric evacuation technique in 8 pigs. Cholecystokinin and somatostatin were infused from 10 min before feeding until the end of gastric evacuation. Atropine, cisapride and metoclopramide were injected slowly 20 min before feeding. The pigs were fed a meal of 1200 g NRS9 (1036 g DM) mixed with water containing Cr-EDTA. The meal time was measured as the time from the start of feeding until the time to mid-evacuation. n, number of observations. Statistics by Student's paired t test showed that there were no significant changes in gastric emptying during the feeding period with any of the treatments except with atropine. **P<0.01; ***p<0-001.

4 162 P. C. GREGORY, M. McFADYEN AND D. V. RAYNER Table 2. Gastric emptying over a per-iod of 3 h after feeding in the pig Control gastric emptying Gastric emptying with treatment Treatment Dry matter Liquid Dry matter Liquid Peptide or agent Dose n (g) (ml) (g) (ml) Cholecystokinin 250 ng kg-' h *** Somatostatin 1l8,ug kg-' h-' *** 1803 ± 31*** Cisapride 0.3 mg kg-' ± ± 130* Metoclopramide 0.2 mg kg-' ± Values are means + S.E.M. in 8 pigs of the amounts of dry matter and liquid in the meal emptied from the stomach 3 h after feeding a meal of 1200 g NRS9 (1036 g DM) mixed with water containing Cr-EDTA. Drugs were administered as described in Table 1. n, number of observations. Statistics were by Student's paired t test. * P < 0 05; *** P < Neither cisapride nor metoclopramide caused any tendency towards an increase in the rate of emptying of DM or liquids: indeed, with cisapride there was a significant decrease in liquid emptying (by %; P < 0.05). DISCUSSION The results of the present study show that the rate of gastric emptying in the pig is completely resistant to, or is only slightly affected by, a variety of agents at doses which cause significant stimulation or inhibition of gastric emptying in man. Their lack of action at doses which are effective in other species suggests that their potency may be different in the pig. However, studies at different concentrations will be needed for complete assessment. One possible cause of the lack of a stimulatory effect of cisapride or metoclopramide is the pig's very rapid gastric emptying (Gregory et al. 1990), since in man these prokinetic agents show their strongest effects in individuals with delayed gastric emptying (Behar & Ramsby, 1978; Johnson, 1989). However, these prokinetic agents failed to increase gastric emptying either during the feeding period, or over the 3 h period following the meal, even though the rate of emptying over this latter period is slower than during the feeding period (Gregory et al. 1990). This indicates that, although the rate of emptying cannot be measured at this time, it is nevertheless resistant to stimulation. The major findings of this study concern the effects of CCK. This peptide is believed to be involved in the physiological regulation of gastric emptying in dog (Debas, Farooq & Grossman, 1975) and man (Liddle et al. 1986); indeed loxiglumide, a CCK antagonist, increases the rate of gastric emptying in the latter (Meyer, Beglinger, Zach, Rovati, Setnikar & Stalder, 1988). However, the CCK-A antagonist MK-329 does not significantly alter the rate of gastric emptying during feeding in the pig (Rayner & Miller, 1993), suggesting that CCK may not be a physiological regulator of gastric emptying in this species. In the present study a continuous infusion of CCK8 (250 ng kg-' h-1) had no significant effect on gastric emptying of DM or liquids during the feeding period, and caused only a 14 % slowing of emptying of DM and no effect on emptying of liquids in the 3 h period following the meal. In contrast, this same rate of infusion inhibits emptying of a saline meal in the dog by 62 % (Debas et al. 1975) and in the monkey by 92 % (Moran & McHugh, 1982), while in man

5 GASTRIC EMPTYING IN THE PIG 163 infusion of CCK8 (30 ng kg-1 h-1) reduced emptying of a water meal by 63 % (Liddle et al. 1986), and infusion of CCK33 (the 33 amino acid cholecystokinin peptide) at 0.75 Ivy dog units kg-' h-' (equivalent to 30 ng kg-' h-1 CCK8; Moran & McHugh, 1982) reduces emptying of solids by 57 % (Kleibeuker, Beekhuis, Jansen, Piers & Lamers, 1988). It is evident, therefore, that CCK has a much smaller effect on gastric emptying of both solids and liquids in the pig than in man or dog. Furthermore, the rate of infusion used in the present study (250 ng kg-1 h-') was found in the dog to be at the top of the physiological range, i.e. it induces maximal gallbladder emptying (Debas et al. 1975). Since plasma concentrations of CCK in the pig increase after feeding to levels similar to those in man and dog (Lilja et al. 1984) and are in the same range (D. V. Rayner, unpublished observation) as reported in man (Liddle et al. 1986), it seems likely that CCK has, at the most, a very minor role in the physiological regulation of gastric emptying in the pig. Indeed, it is possible that the lack of a strong inhibitory effect of CCK could be a factor in the more rapid rate of emptying in the pig in comparison with man (Gregory et al. 1990). The reasons for the lack of sensitivity to CCK in the pig are not clear, but presumably it is ineffective both in increasing pyloric tone and in lowering fundic tone (Yamagashi & Debas, 1978), since pyloric tone and fundic tone are mainly involved in the control of gastric emptying of solids and liquids, respectively (Kelly, 1980). Interestingly, the satiety effect of CCK (McLaughlin & Baile, 1980) and exocrine pancreatic secretion in response to CCK (McLaughlin, Peikin & Baile, 1982) are less easily evoked in obese than in lean rats. This raises the possibility that a more generalized loss of CCK sensitivity could be an important factor in obesity. A further finding of the present study was that somatostatin (1.8 jug kg-' h-') moderately but significantly inhibited emptying of DM (by 19 %) and of liquids (by 9 %) over the 3 h period following the meal. Therefore, somatostatin could be involved in the control of gastric emptying in the pig. Nevertheless, the rate of infusion used was relatively high, around the top of, or even slightly above, the physiological range, suggesting that somatostatin is unlikely to be a major factor. Previous studies with somatostatin infusions in man have yielded controversial results. Infusion of 11 jug kg-' h-l' somatostatin inhibited emptying of an unnamed meal by 70 % (Bloom et al. 1975); infusion of 5 jtg kg-1 h-1 caused a transient stimulation (at 40 min) of emptying of a glucose meal (Johansson, Efendic, Wisen, Uvnas- Wallensten & Luft, 1978) but had no effect on emptying between min; in contrast, infusions of 0-16 and 0.32,ug kg-' h-' somatostatin increased emptying of peptone meals (Mogard et al. 1988). It is possible that the conflicting results were caused by the differences in infusion rates in the various studies, but recent results from our laboratory show that gastric (abomasal) emptying in sheep is strongly inhibited at both 0-2 and 0-9 sg kg-'h-1 (P. C. Gregory & S. Miller, unpublished observations). Further studies are evidently needed to clarify the influence of lower infusion rates in the pig. Although both CCK (250 ng kg-1 h-1) and somatostatin (1-8 jug kg-1 h-1) inhibited emptying following the meal, the same infusions had no significant effect on emptying during the feeding period. There was, in fact, a trend towards slowing of emptying of DM with CCK infusion, but another study (Rayner & Miller, 1990) has confirmed that the infusion of CCK (250 ng kg-1 h-') has no significant effect on emptying during the meal in the pig. A reduced sensitivity to inhibitory mediators may be at least partly responsible for the more rapid emptying seen during feeding compared with after feeding in the pig (Gregory et al. 1990); further observations are required.

6 164 P. C. GREGORY, M. McFADYEN AND D. V. RAYNER In conclusion, this study has shown that gastric emptying in the pig, unlike that in man, is resistant to stimulation by the prokinetic agents cisapride and metoclopramide. It is probable, therefore, that the rapidity of emptying seen in the pig does not simply reflect a higher maximal rate of emptying than man, but means that the normal rate of emptying is closer to maximal in the pig than in man. A possible reason for this is that gastric emptying in the pig is far less sensitive to the inhibitory effects of CCK than it is in man. It remains to be determined if the insensitivity of gastric emptying in the pig to inhibition is specific for CCK (and whether there is also a loss of sensitivity to other effects of CCK), or whether it is part of a more generalized insensitivity of gastric emptying to inhibitory regulators. Clarification of these points could perhaps eventually help towards a better understanding of factors involved in obesity. The authors thank Dr W. A. McKelvey and Mr C. A. Simpson for assistance with the surgical preparation of the animals and Mr D. Wood for veterinary care of the animals. REFERENCES BEHAR, J. & RAMSBY, G. (1978). Gastric emptying and antral motility in reflux esophagitis. Effect of oral metoclopramide. Gastroenterology 74, BLOOM, S. R., RALPHS, D. N., BESSER, G. M., HALL, R., Coy, D. H., KASTIN, A. J. & SCHALLY, A. V. (1975). Effect of somatostatin on motilin levels and gastric emptying. Gut 16, 834. BRENER, W., HENDRIX, T. R. & MCHUGH, P. R. (1983). Regulation of the gastric emptying of glucose. Gastroenterology 85, COOKE, A. R. (1975). Control of gastric emptying and motility. Gastroenterology 68, DEBAS, H. T., FAROOQ, 0. & GROSSMAN, M.I. (1975). Inhibition of gastric emptying is a physiological action of cholecystokinin. Gastroenterology 68, GREGORY, P. C., MCFADYEN, M. & RAYNER, D. V. (1989a). Duodenal infusion of fat, cholecystokinin secretion and satiety in the pig. Physiology and Behaviour 45, GREGORY, P. C., MCFADYEN, M. & RAYNER, D. V. (1989b). Control of gastric emptying in the pig: influence of duodenal infusions of glucose and emulsified fat. Quarterly Journal of Experimental Physiology 74, GREGORY, P. C., MCFADYEN, M. & RAYNER, D. V. (1990). Pattern of gastric emptying in the pig: relation to feeding. British Journal of Nutr ition 64, GREGORY, P. C. & RAYNER, D. V. (1987). The influence of gastrointestinal infusions of fats on regulation of food intake in pigs. Journal of Physiology 385, HouPT, K. A., HOUPT, T. R. & POND, W. G. (1979). The pig as a model for the study of obesity and control of food intake: A review. Yale Journal of Biology and Medicine 52, HUNT, J. N. (1980). A possible relation between the regulation of gastric emptying and food intake. American Journal of Physiology 239, GĪ 4. JOHANSSON, C., EFENDIC, S., WISEN,O., UVNAS-WALLENSTEN, K. & LUFT, R. (1978). Effects of shorttime somatostatin infusion on the gastric and intestinal propulsion in humans. Scandinavian Jour-nal of Gastroenterology 13, JOHNSON, A. G. (1989). The effects of cisapride on atroduodenal co-ordination and gastric emptying. Scandinavian Journal of Gastroenterology 24, suppl. 165, KELLY, K. A. (1980). Gastric emptying of liquids and solids: roles of proximal and distal stomach. American Journal of Physiology 239, G KLEIBEUKER, J. H., BEEKHUIS, H., JANSEN, J. B. M. J., PIERS, D. A. & LAMERS, C. B. H. W. (1988). Cholecystokinin is a physiological hormonal mediator of fat-induced inhibition of gastric emptying in man. European Journal of Clinical Investigation 18, LAZZARONI, M., SANGALETTI, 0. & PORRO, G. B. (1987). Effect of cisapride on gastric emptying and ileal transit time of balanced liquid meal in healthy subjects. Digestion 37,

7 GASTRIC EMPTYING IN THE PIG 165 LIDDLE, R. A., MORITA, E. T., CONRAD, C. K. & WILLIAMS, J. A. (1986). Regulation of gastric emptying in humans by cholecystokinin. Journal of Clinical Investigation 77, LILJA, P., WIENER, I., INOUE, K., FRIED, G. M., GREELEY, G. H. JR & THOMPSON, J. C. (1984). Release of cholecystokinin in response to food and intraduodenal fat in pigs, dogs and man. Surgery, Gynecology and Obstetrics 159, MCHUGH, P. R. & MORAN, T. H. (1979). Calories and gastric emptying: A regulatory capacity with implications for feeding. American Journal of Physiology 236, R MCLAUGHLIN, C. L. & BAILE, C. A. (1980). Decreased sensitivity of Zucker obese rats to the putative satiety agent cholecystokinin. Physiology and Behaviour 25, MCLAUGHLIN, C. L., PEIKIN, S. R. & BAILE, C. A. (1982). Decreased pancreatic exocrine response to cholecystokinin in Zucker obese rats. American Journal of Physiology 242, G MEYER, B., BEGLINGER, C., ZACH, D., ROVATI, L., SETNIKAR, I. & STALDER, G. A. (1988). Cholecystokinin (CCK) is a physiological regulator of gastric emptying in man. Gastroenterology 94, A301 (abstract). MOGARD, M. H., MAXWELL, V., WONG, H., REEDY, T. J., SYTNIK, B. & WALSH, J. H. (1988). Somatostatin may not be a hormonal messenger of fat-induced inhibition of gastric ftlnctions. Gastroenterology 94, MORAN, T. H. & MCHUGH, P. R. (1982). Cholecystokinin suppresses food intake by inhibiting gastric emptying. American Journal of Physiology 242, R MUKHOPADHYAY, A., THOR, P., COPELAND, E., JOHNSON, L. & WEISBRODT, N. (1977). Effect of cholecystokinin on myoelectric activity of the small bowel of the dog. American Journal of Physiology 232, E NERI, M., CUCCURULLO, F. & MARZIO, L. (1990). Effect of somatostatin on gallbladder volume and small intestinal motor activity in humans. Gastroenterology 98, RAYNER, D. V. & GREGORY, P. C. (1989). The role of gastrointestinal tract in the control of voluntary food intake. British Society of Animal Production, Occasional Publication No. 13, RAYNER, D. V. & MILLER, S. (1990). Cholecystokinin effects on wet and dry meals in pigs. Proceedings of the Nutrition Society 49, 222A. RAYNER, D. V. & MILLER, S. (1993). Voluntary intake and gastric emptying in pigs: effects of fat and a CCK inhibitor. Physiology and Behaviour 54, SCHUSDZIARRA, V., HARRIS, V., CONLON, J. M., ARIMURA, A. & UNGER, R. (1978). Pancreatic and gastric somatostatin release in response to intragastric and intraduodenal nutrients and HCl in the dog. Journal of Clinical Investigation 62, YAMAGASHI, T. & DEBAS, H. T. (1978). Cholecystokinin inhibits gastric emptying by acting on both proximal stomach and pylorus. American Journal of Physiology 234, E