Rapid Publication Physiological Role of Cholecystokinin in Meal-Induced Insulin Secretion in Conscious Rats Studies With L 364718, A Specific Inhibitor of CCK-Receptor Binding LUCIANO ROSSETTI, GERALD I. SHULMAN, AND WALTER S. ZAWALICH

culties associated with measuring low levels and multiple SUMMARY It has been suggested that the gut hormone forms of CCK, we employed a potent and highly specific cholecystokinin (CCK), by modulating insulin output antagonist of CCK binding to its membrane receptor to block from pancreatic p-cells, plays an important role in the its in vivo actions (8). The results support the concept that enteroinsular axis. To investigate this hypothesis, CCK is an important in vivo incretin factor. eight rats were studied on two different occasions: after injection of L 364718, a specific antagonist of CCK MATERIALS AND METHODS binding to its membrane receptor, and after vehicle injection. In both studies a mixture of casein (11%) and ANIMALS glucose (9%) was infused through a chronic indwelling Male Sprague-Dawley rats (Charles River, Wilmington, MA), intraduodenal catheter to evoke CCK secretion. Plasma weighing 280-330 g were used. They were maintained on was analyzed for insulin, glucose, glucagon, and tyrosine many times during the procedure. Prior standard Ralston-Purina rat chow (Si Louis, MO) and were administration of the CCK antagonist significantly housed in an environmentally controlled room with a 12-h attenuated the increase in plasma insulin and light/dark cycle. One week before the first study an internal glucagon after casein infusion. These results support jugular catheter, extending to the right atrium, and an in- the concept that cholecystokinin plays an important traduodenal catheter were implanted. The catheters were physiologic role in the in vivo regulation of filled with heparin-povidone solution, sealed, and tunneled postprandial plasma insulin and glucagon subcutaneously around the side of the neck to the back of concentrations after protein ingestion. Diabetes the head. The catheters were externalized through an inci- 36:1212-15, 1987 sion in the back of the neck. Only animals that were active and eating normally within 36 h after the surgery were used. All rats were fasted for 12 h before each study. nsulin-mediated disposition of ingested nutrients is EXPERIMENTAL PROTOCOL thought to be facilitated by the release of various gut Throughout the study the rats were allowed to move freely factors (1,2). In this capacity these compounds, termed within the confines of a large cage, and the connecting tub- incretins (2), facilitate the release of insulin from the pan- I ing was suspended overhead by means of a pulley system. creatic 3-cell. Gastric inhibitory polypeptide (GIP) is a major The venous catheter was used for blood withdrawal and factor involved in this enteroinsular axis (2-6). Recent stud- infusion of the L 364718, and the intraduodenal catheter was ies, however, support the involvement of other factors as well used for infusion of a 9% glucose/11% casein solution (pH (7). Our studies were conducted to determine the influence 7; Sigma, St. Louis, MO). The total volume of blood withdrawn of cholecystokinin (CCK) on the plasma insulin response was <2.5 ml/study; to avoid volume depletion and anemia, after intraduodenal nutrient infusion. Because of the diffi- 1.5 ml of blood (diluted in 1.5 ml of heparinized saline) ob- tained by heart puncture from a littermate of the test animal was transfused through the venous catheter. Each rat was From the Yale University Schools of Medicine and Nursing, New Haven, Con- studied twice with a 5- to 7-day interim. Four animals under- necticut. Address correspondence and reprint requests to Luciano Rossetti, MD, 2071 went study 1 first, and four underwent study 2 first. LMP Building, Yale-New Haven Hospital, 333 Cedar Street, New Haven, CT Study 1. At 0900 h the heparin-povidone solution was as- 06510. Received for publication 24 April 1986 and accepted in revised form 28 July pirated from the jugular catheter, and a slow infusion (15 |xl/ 1987. min) of isotonic saline was initiated to preserve the catheter

1212 DIABETES, VOL. 36, OCTOBER 1987 patency. Plasma samples for the determination of glucose - 300 and insulin concentrations were obtained at 15-min intervals from -60 to 0 min. Plasma samples for glucagon and ty- rosine concentrations were obtained at -30 min. At -30 Q. and 0 min, 1 mg/kg body wt i.v. of the CCK antagonist — 250 L 364718 (Merck, Sharp, and Dohme, West Point, PA) was O administered in 0.05% methylcellulose vehicle. At 0 min a * + primed (2.27 ml/kg) continuous (0.16 ml/kg) intraduodenal o * + infusion of a 9% glucose/11% casein solution was started 200 and maintained throughout the 60 min study. Plasma sam- ples for glucose and insulin determinations were obtained o at 3-min intervals for the first 21 min of the infusion and at 10-min intervals thereafter. Plasma samples for tyrosine and 150 to glucagon concentrations were obtained at 30 and 60 min. Study 2. The experimental protocol was identical to study 1 except that at -30 and 0 min, a 0.05% methylcellulose ve- hicle injection was administered alone. In control studies, 100 i -IOA 71Q + four rats underwent the same experimental protocol de- L364,718 3 + 60MIN scribed in studies 1 and 2, but the intraduodenal infusion 30 MIN was represented by glucose alone. FIG. 2. Plasma glucagon concentrations after casein infusion. Plasma glucagon levels in response to intraduodenal infusion of 11% casein/ ANALYTICAL PROCEDURES 9% glucose mixture (pH 7) after vehicle (open columns) or L 364718 infusion (hatched columns). Values are means ± SE of 4 Plasma glucose was measured by the glucose oxidase measurements. *P < .01 vs. vehicle-infused rats. fP < .01 vs. basal. method (glucose analyzer, Beckman, Palo Alto, CA) and plasma insulin by radioimmunoassay with rat insulin (lot 615- D63-12-3, Lilly, Indianapolis, IN) as standard (9). The insulin samples from any paired study (1 and 2) were processed + L364.718 in the same assay. The intra-assay C.V. was <9% and the X I interassay C.V. was <20%. Plasma tyrosine concentrations were determined by an automated ion-exchange chromato- graphic technique (Dionex D-500, Sunnyvale, CA) with an L-6 column and lithium citrate buffer. Plasma glucagon was determined with the COOH-terminal 30,000-Mr antibody of Faloona and Unger (10).

CALCULATIONS Plasma insulin responses during the casein-glucose infusion represent the mean increment in plasma insulin concentra- tion above baseline during the 0- to 60-min interval. The basal insulin concentration is derived from the mean of four 18 determinations from -60 to 0 min. All data are presented as means ± SE. Statistical comparison between study 1 and e study 2 have been performed by paired Student's t test. 14 RESULTS z 10 Body weight, plasma glucose, and insulin concentra- _J tions. At the time of the study, body weight (302 ± 6 vs. to 304 ± 7 g), fasting plasma glucose (118 ±2 vs. 116 ± z 6 2 mg/dl), and plasma insulin concentration (2.42 ± 0.10 vs. 2.40 ± 0.11 ng/ml) were similar in the rats injected with the vehicle or the CCK antagonist, respectively. Plasma glucose and insulin responses to casein/glu- 0 10 20 30 40 50 60 cose intraduodenal infusion. After the intraduodenal in- TIME (mini fusion of 11 % casein/9% glucose in the control group, insulin secretion was stimulated, with a peak response at 15 min of FIG. 1. Plasma glucose and insulin after casein infusion. 4: plasma 20.0 ± 3.5 ng/ml and a mean value of 10.6 ± 1.5 ng/ml over glucose responses to intraduodenal infusion of 11% casein/ 9% glucose solution (pH 7) after vehicle (O) or L 364718 (A) the last 40 min of the infusion (Fig. 1). The administration of administration. L 364718 suspended in methylcellulose was given as CCK inhibitor L 364718 at -30 and 0 min before the intra- intravenous bolus at -30 and 0 min. Values are means ± SE (n = 8). duodenal infusion significantly reduced the plasma insulin B: plasma insulin responses to intraduodenal infusion of 11% casein/ 9% glucose solution (pH 7) after vehicle (O) or L 364718 (A) response; the peak value averaged 9.5 ± 1.6 ng/ml administration. Values are means ± SE (n = 8). (P < .01) at 15 min, with a mean value of 5.8 ± 0.8 ng/ml

DIABETES, VOL. 36, OCTOBER 1987 1213 (P< .01) over the last 40 min of the infusion. The mean proximate the values obtained in study 1 (229 ± 49 ng/ml). incremental insulin area (0-60 min) in the L 364718-treated Consistent with the finding that protein, but not glucose, is group was also significantly reduced, i.e., 229 ± 49 vs. an effective stimulant of CCK secretion (16), the casein- 512 ± 69 ng/ml (P < .01). The plasma glucose incremental induced increment in insulin output above that noted to glu- area (0-60 min) was 1916 ± 290 mg/dl in the vehicle-in- cose alone was virtually abolished by L 364718. Because jected rats and 2454 ± 237 mg/dl in the CCK-antagonist- the effect of casein on insulin release is probably mediated injected rats. After a primed (2.20 mg/kg body wt) con- by its positive impact on CCK output from duodenal cells tinuous (15 mg • kg~1 • min~1) infusion of glucose alone, (16), the physiologic role of CCK in postprandial elevations both the plasma glucose incremental areas (0-60 min; in insulin levels due to protein ingestion in rats is empha- 3673 ± 378 vs. 3241 ± 391 mg/dl) and the plasma insulin sized. incremental areas (0-60 min; 258 ± 71 vs. 273 ± 63 ng/ml) The small difference in the glucose profile after the admin- were similar in the vehicle-injected group and in the istration of equal amounts of casein/glucose solution in the L 364718-injected groups. two experimental protocols is clearly disproportionate to the Plasma glucagon and tyrosine concentrations. The basal relevant impairment in the insulin response and deserves plasma glucagon concentration was 139 ± 4 and 148 ± 12 comment. A marked degree of hyperglycemia after pg/ml in the control and L 364718-treated rats, respectively L 364718, an effect due to diminished circulating levels of (Fig. 2). In both experimental protocols, a significant increase insulin, might have been anticipated. This was, however, not in plasma glucagon concentration was observed at 30 and observed. Because CCK is also known to increase glucagon 60 min after the casein infusion. However, the magnitude of output from the a-cells (17), the contribution of glucagon to this increment was significantly reduced in the rats treated this situation was considered. Our results demonstrate that with L 364718 (190 ± 17 vs. 254 ± 3 pg/ml at 30 min and casein administration increases glucagon output from a- 211 ± 8 vs. 244 ± 7 pg/ml at 60 min, P < .01) in the treated cells and that this effect is reduced by the CCK antagonist. and nontreated animals, respectively. Plasma tyrosine con- The hyperglycemic effect of hypoinsulinemia in response to centrations were similar in the two protocols at 0 min (65 ± 2 L 364718 administration may be largely counteracted by the vs. 62 ± 5 |xM) and 30 min (128 ± 5 vs. 110 ± 8 (iM); a hypoglycemic effect of hypoglucagonemia. barely significant difference was detected at 60 min Because tyrosine is the most representative amino acid in (215 ± 19 vs. 165 ± 5 |xM, P < .05). casein, we monitored protein absorption by measuring the levels of tyrosine in plasma. No significant differences were detected at 0 or 30 min from the beginning of the intra- DISCUSSION duodenal infusion of casein and glucose. The reduction of CCK is a potent glucose-dependent stimulus for insulin se- tyrosine concentrations in the L 364718-treated rats at 60 cretion in the perfused rat pancreas and in isolated rat islets min confirms the inhibitory action of the blocker on exocrine (11-13). Our findings suggest that it plays a similar role in pancreas as well. However, it is unlikety that fluctuations in vivo. In the experiments reported here, a newly developed amino acid levels contributed significantly to the differences and highly specific antagonist of CCK, the benzodiazepine in insulin and glucagon responses. The fact that insulin and derivative L 364718, was employed. Our previous results glucagon levels were significantly reduced when amino acid with this compound in isolated perifused islets have dem- levels were similar in both groups argues against a major onstrated that at low doses (1 nM), it reduces CCK-8S-in- role for amino acids in observed differences in pancreatic duced insulin output (in the presence of 7 mM glucose) (14); hormone secretion. however, even at excessive levels (1 JJLM) it has no adverse impact on glucose-, glyceraldehyde-, or a-ketoisocaproate- ACKNOWLEDGMENTS induced insulin release. Previous studies with other CCK- We thank V. Diaz for performing the insulin radioimmunoas- sensitive tissues have demonstrated a similar remarkable say, R. Jacob for the tyrosine measurements, and Dr. Rosa degree of inhibitory selectivity (8). Hendler for the glucagon radioimmunoassay. We are also By studying chronically catheterized rates in randomized indebted to Dr. Ralph DeFronzo for helpful comments during order on two occasions, we have been able to detect dra- the performance of the study and critical review of the man- matic effects of this inhibitor on insulin release. The rise in uscript. plasma insulin concentrations, in response to a primed con- This work was supported in part by NIH Grants AM-34381 tinuous intraduodenal infusion of casein (11%) and glucose and AM-01248 and American Heart Association Grant 84- (9%) mixture, was significantly reduced by prior exposure 709. LR. is the recipient of a fellowship from the Juvenile of the B-cells to L 364718. Incremental areas were less than Diabetes Foundation (386266). G.I.S. is the recipient of a half of the level reached in the same rats in the absence of grant from the Juvenile Diabetes Foundation. the antagonist (229 ± 49 vs. 512 ± 69 ng/ml). Note that the insulin concentrations observed here in the presence of the REFERENCES 1. Unger RH, Eisentraut AH: Entero-insular axis. Arch Intern Med 123:261- inhibitor approximate the levels we have previously observed 66, 1969 after intraduodenal infusion of glucose alone (15). In control 2. Cruetzfeldt W: The incretin concept today. Diabetologia 16:75-85, 1979 3. Dupre J, Ross SA, Watson D, Brown JD: Stimulation of insulin secretion studies in which glucose alone was infused intraduodenally by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab 37:826- in two instances, in the presence or absence of the specific 28,1973 CCK antagonist L 364718, the incremental insulin areas were 4. 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