Interaction of Glucagon-like Peptide-1(7-36)Amide and Gastric Inhibitory Polypeptide or Cholecystokinin on Insulin and Glucagon Secretion From the Isolated Perfused Rat Pancreas Seiji Suzuki,

Koichi Kawai,

Shinichi

Ohashi,

Yasuko

Watanabe,

and Kamejiro

Yamashita

The interaction of three incratin candidates, glucagon-like paptida-1(7-36)amida (t-GLP-I), gastric inhibitory polypaptida (GIP), and sulfated COOH-terminal octapaptida of cholacystokinin (CCK-6-S). on insulin and gfucagon release from the isolated perfused rat pancreas was studied. Under the parfusata condition of 8.3 mmol/L glucose, coinfusion of 0.t nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each paptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusatas, and no cooperative affect was obsarvad by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L CCKI-S also resulted in an augmented insulin release greater than that obtained by the same dose of each paptida alone. A similar cooperative affect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any paptida at concentrations of 0.1,0.3,1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfuslon of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release. These results suggest that (I) t-GLP-1 and GIP cooperatively affect insulin and glucagon release at the nearly physiologic postprandial plasma concantrations of both paptides, and (2) t-GLP-1 and CCK-8-S cooperatively affect insulin release in a wide range of concentrations, although the physiologic significance of this interaction should be slight, considering the physiologic postprandial plasma concentration of CCK-8-h Copyright 0 1992 by W.B. Saunders Company

M ANY

GASTROINTESTINAL peptides are released postprandially. Some of them positively modulate insulin secretion and are called incretins.’ Gastric inhibitory polypeptide (GIP) is accepted as a major physiological incretin; however, incretins other than GIP have been postulated.’ The carbo@(C)-terminal octapeptide of cholecystokinin with a sulfated tyrosine residue in position seven (CCK-8-S) is a candidate for incretin, although this is still controversial,2.3 and glucagon-like peptide-1(7-36)amide (tGLP-1) is a new candidate for incretin. The insulinotropic activity induced by physiologic doses of these hormones seems to be small, under physiologic environmental conditions, considering the results obtained by isolated pancreas perfusion ~tudies.“~‘~~ Therefore, it is expected that these hormones synergistically or additively affect pancreatic p cells. An in vivo study in mice demonstrated that the coinfusion of threshold doses of GIP and CCK-8 synergistically enhances basal insulin release and that maximal doses additively enhance it9 A recent rat pancreas perfusion study demonstrated that the coinfusion of t-GLP-1 and GIP additively enhanced glucose-induced insulin release.” These results suggest the necessity of further study, using different doses of these hormones and different experimental designs. Furthermore, glucagon release after meals is also important for postprandial nutrient homeostasis. According to the results of studies with isolated pancreas perfusion, GIP and CCK-8-S stimulate glucagon release,“.‘* and t-GLP-1 suppresses it.r3-” However, there has been no previous study discussing the interaction of these three peptides in regard to glucagon release. Therefore, the interactions of t-GLP-1 and GIP or t-GLP-1 and CCK-8-S on insulin and glucagon release were examined, using isolated perfused rat pancreases. MATERIALS

AND METHODS

Materials

Human t-GLP-1 was synthesized by the stepwise solid-phase method, using an automatic synthesizer (model 430A, Applied Metabolism, Vol41, No 4 (April), 1992: pp 359-363

Biosystems, Foster City, CA), and then purified by high-pressure liquid chromatography (HPLC). The purity of the peptide was monitored by analytical reverse-phase HPLC on a column of Nucleosit 5C18 (4.6 x 150 mm; Gaskurokogyo, Tokyo, Japan), under the isocratic conditions of 0.1% trifluoroacetic acid and 39% acetonitrile, and proved to be at least 98% pure.13 CCK-8-S and human GIP were purchased from Peptide Institute (Osaka, Japan). These peptides were dissolved in 0.9% NaCl solution containing 0.2% bovine serum albumin (BSA). When infusing two peptides, the peptides were dissolved in the solution together. Rat Pancreas Perfusion The pancreases were isolated from male Wistar rats, weighing 300 to 380 g, under pentobarbital anesthesia, after an overnight fast. The isolated rat pancreases were perfused according to the method of Grodsky et a1.16A Krebs-Ringer bicarbonate buffer solution containing 4% Dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 0.2% BSA, 5 mmol/L each of pyruvate, fumarate, and glutamate, and 8.3 mmol/L glucose was equilibrated with a 95% 0, to 5% CO, mixture at 37°C. The pancreas was perfused from the celiac artery, with a flow rate of 2 mL/min. After 25 minutes of equilibration, the venous effluent was collected in a chilled tube at l-minute intervals, via a cannula inserted into the portal vein. An initial 10 minutes was the basal period, and then the peptide solution was administered for 15 minutes through a side-arm syringe at a rate of 0.1 mL/min, as shown in Figs 1 and 2. RLA Insulin (IRI) and glucagon (IRG) in the effluent perfusate were determined by radioimmunoassay (RIA), according to the meth-

From the Department of Internal Medicine, Institute of Clinical Medicine, Universityof Tsukuba, Japan; and the Research Institute for Polymers and Textiles, Tsukuba, Japan. Supported by Grant-in-Aid for Scientific Research No. 01570624 born the Ministy of Education, Science, and Culture, Japan. Address reprint requests to Koichi Kawai, MD, PhD, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki-ken, 305, Japan. Copyright 0 1992 by W.B. Saunders Company 0026-0495/92/4104-0003$03.0010 359

360

SUZUKI ET AL

A

B

60-

I

0

25

1

30

0

10

25

30

T I M E (min)

Fig 1. (A) Effects of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP, separately, and their combination, on IRI release from the isolated perfused rat pancreas. t-GLP-1 (n = 4); -o-o-, _.__ 0 ____0 ___-, GIP (n = 5); -A-A-, t-GLP-1 + GIP (n = 5). (B) Effects of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L CCK-8-S. separately, and their combination, on IRI release from the isolated rat -O-O-, t-GLP-1 pancreas. (n = 4); --A--A--, CCK-8-S t-GLP-1 + (n = 4); -m-m-, CCK-8-S (n = 5). Results are the mean + SEM.

ods of Herbert et al” and Faloona and Unger” with E-7 antibody (kindly donated by Dr H. von Schenck), respectively.

Effects of Coinfusion of t-GLP-1 and GIP, or t-GLP-1 and CCK-S-S, on Insulin Release From the Isolated Perfused Rat Pancreas

StatisticalAnatjk

The coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP revealed a cooperative effect on insulin release (Table 2, Fig l), but the coinfusion of 0.3, 1, or 3 nmol/L of t-GLP-1 and GIP did not result in an insulin release greater than that obtained by the same dose of each peptide alone (Table 2). The coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L CCK-8-S stimulated a greater insulin release than that obtained by the infusion of 0.1 nmol/L CCK-8-S alone. A similar augmentation of insulin release was observed with coinfusion of 0.3 nmol/L, 1 nmol/L, and 3 nmol/L each of t-GLP-1 and CCK-8-S (Table 2).

The degree of stimulation or inhibition of hormone release by peptides was calculated as follows: (area under the curve during the infusion of peptides [shown in Figs 1 and 2]-(area under the curve during its preceding basal level) x 1.5. Student’s t test for unpaired data was used to calculate the significance of the changes (Tables 1 through 4). A level ofP < .05 was considered significant. All data herein are expressed as means f SEM. RESULTS Effects of Various Doses of t-GLP-1, GIP, and CCK-8-S on Insulin Release From the isolated Perjked Rat Pancreas

As shown in Fig 1, 0.1 nmol/L t-GLP-1, GIP, and CCK-8-S stimulated insulin release under the perfusate condition of 8.3 mmol/L glucose. Table 1 shows the results obtained by infusion of each peptide alone. The perfusion of the vehicle caused a small decrease in the IRI level (-105 ~fr40 uU/15 min). In the case of t-GLP-1 and GIP, 0.3 nmol/L of these peptides stimulated an insulin release greater than that stimulated by 0.1 nmol/L of these peptides. However, there were no significant increases in the insulin release stimulated by 1 nmol/L and 3 nmol/L t-GLP-1 or GIP compared with that stimulated by 0.3 nmol/L t-GLP-1 or GIP. A range of 0.1 nmol/L to 1 nmol/L CCK-8-S stimulated insulin release in a dosedependent manner, and there was no difference between the effects induced by 1 nmol/L and 3 nmol/L CCK-8-S. The degree of stimulation by 0.3 to 3 nmol/L CCK-8-S was significantly smaller than that by the same doses of t-GLP-1 or GIP.

Effects of Various Doses of t-GLP-1, GIP, and CCK-8-S on Glucagon Release From the Pe$used Isolated Rat Pancreas

Under the perfusate condition of 8.3 mmol/L glucose, each 0.3,1, and 3 nmol/L of t-GLP-1, GIP, or CCK-8-S did Table 1. Effects of Various Doses of t-GLP-1, GIP, and CCK-8-S on Insulin Release From the Isolated Perfused Rat Pancreas InsulinOutput (pU/15 min) Dose

t-GLP-1

GIP

CCK-8-S

0.1 nmol/L

293 f 135 (4)

128 f 31 (5)

149 + 21 (4)

0.3 nmol/L

636 f 134* (5)

639 2 114” (5)

244 ?k40 (5)

1 nmol/L

683 k 105’ (5)

668 -+ 89’ (5)

435 -t 82’ (4)

3 nmol/L

741 -t 94* (5)

718 f 95+ (4)

386 + 87* (5)

NOTE. Values are means f SEM of incremental areas between the basal levels and the response curves shown in Fig 1, and are calculated as described in Methods. The numbers in parentheses are the numbers of experiments. *P < .05 v 0.1 nmol/L t-GLP-1, GIP, or CCK-8-S.

361

INTERACTION OF INCRETINS

Table 2. Effects of Various Doses of t-GLP-1 + GIP and t-GLP-1 +

Table 3. Effects of Various Doses of t-GLP-1, GIP, and CCK-8-S on

CCK-8-S on Insulin Release From the Isolated Perfused Rat Pancreas

Glucagon Release From the Isolated Perfused Bat Pencreas

t-GLP-1 (nmol/L)

GIP (nmol/L)

InsulinOutput (pull5 min)

CCK-8-S (nmollL)

-

-

-105?40(5)

GlucagonOutput (ngI15 min) t-GLP-1

Dose

CCK-8-S

GIP -1.18

k 0.49 (4)

-0.37

f 0.89 (5)

0.3 nmol/L

-0.72

-c 0.90 (4)

1 nmol/L

-0.60

2 0.21 (5)

0.91 2 0.53 (5)

- 1.05 -t 0.72 (4)

3 nmol/L

- 1.29 + 0.32 (4)

1.00 + 1.31 (4)

0.23 t 0.65 (5)

0.3

0.1 0.3

-

629 f 156 (5)

1

1

-

759 2 117(5)

3

3

-

628 t 99 (5)

0.1

-

0.1

449 2 128t (5)

basal levels and the response curves shown in Fig 2, and are calculated

0.3

-

0.3

830 2 89t (5)

as described in Methods. The numbers in parentheses are the numbers

1

772 2 40t (5)

of experiments.

3

1,011 2 94t (5)

0.1

1 3

-

553 2 89* (5)

NOTE. Values are means + SEM of incremental areas between the basal levels and the response curves shown in Fig 1, and are calculated as described in Methods. The numbers in parentheses are the numbers of experiments.

lP < .Ol v 0.1 nmol/L GIP (Table 1). tP < .Ol v the same concentration of CCK-8-S (Table 1).

not affect the glucagon release significantly, compared with the control experiments (Fig 2 and Table 3. The result of the control experiment is shown in Table 4). Both 1 nmol/L and 3 nmol/L t-GLP-1 tended to reduce glucagon release, and 3 nmol/L GIP tended to stimulate it (Table 3). CCK-8-S did not cause any effects on glucagon release (Table 3, Fig 2). Effects of Coinfusion of t-GLP-1 and GIP, or t-GLP-I and CCK-S-S, on Glucagon Release From the Isolated Perjksed Rat Pancreas

The secretory responses to coinfusion of each 1 nmol/L of t-GLP-1 and GIP were significantly greater than the responses to 1 nmol/L t-GLP-1 or 1 nmol/L GIP, alone (Fig 2A). A prominent peaked glucagon release was ob-

NOTE. Values are means 2 SEM of incremental areas between the

served 4 minutes after the coinfusion. A similar profile was observed when each 0.3 nmol/L and 3 nmol/L of t-GLP-1 and GIP was coinfused. Conversely, the coinfusion of 1 nmol/L of t-GLP-1 and CCK-8-S did not cause any significant change in the glucagon secretion (Fig. 2B). Coinfusion of 0.3 nmol/L and 3 nmol/L of t-GLP-1 and CCK-8-S also did not cause any significant effects. A similar synergism of t-GLP-1 and GIP in the stimulation of glucagon release was observed under the perfusate condition of 2.8 mmol/L glucose. In this condition, 0.3 and 1 nmol/L of t-GLP-1 suppressed glucagon release, and the same doses of GIP stimulated it, significantly. The coinfusion of each 0.3 or 1 nmol/L of t-GLP-1 and GIP synergistically stimulated glucagon release (data not shown). DISCUSSION

It is generally accepted that gastrointestinal hormones play an important role in postprandial glucose homeostasis through the regulation of pancreatic hormone release. GIP is such a leading hormone, although its share in the potentiation of postprandial insulin release has been re-

A

B

500 h

Fig 2. (A) Effects of 1 nmol/L t-GLP-1 and 1 nmol/L GIP, separately, and and their tombhation, on IBG mlease from isolated perfused rat pancreas. -O-O-, t-GLP-1 (n = 5); --O--O--, GIP (n = 4); -A-A-, t-GLP-1 + GIP (n =5). (B) Effects of 1 nmol/L MLP-1 and 1 nmol/L cCK-8-S

I ~paratew#aod~~ onIBGreleaeefromleolatedperfused rat pancreas. -O-@-, t-GLP-1 (n=5); -A-A-,CCK4-S (n = 5); +-C,t-GLP-1 + CCK8-B(n=5).Besultsarethemean~ SEM.

5 400 (5) a V (J

300

LT 200

100

0

3

10

25

30

0

T I M E (min>

10

25

30

362

SUZUKI ET AL

Table 4. Effects of Various Doses of t-GLP-1 + GIP and t-GLP-1 + CCKB-S on Glucagon Release From the Isolated Perfused Rat Pancreas t-GLP-1 (nmollL1

GIP (nllWl/L)

CCK-ES (nmol/L)

GlucagonOutput (ng/15 min)

-

-

-

-1.27

r 0.65 (5)

0.1

0.1

-

-0.74

k 0.51 (5)

0.3

0.3

-

1.31 + 0.70* (5)

1

1

-

2.56 + 1.14* (5)

3

-

2.91 r 0.54* (5)

0.3

3 -

0.3

1

-

1

-1.12

f 0.84 (5)

3

-

3

-0.23

2 0.40 (5)

0.06 f 0.50 (5)

NOTE. Values are means 2 SEM of incremental areas between the basal levels and the response curves

shown

in Fig 2, and are calculated

as described in Methods. The numbers in parentheses are the numbers of experiments.

lP < .05 v t-GLP-1 or GIP alone.

ported as less than 50%.19 In this study, we have clearly demonstrated that insulin release was augmented by coinfusion of t-GLP-1 and GIP with each nearly physiologic postprandial plasma concentration.6,*$21 However, this cooperability was not observed with concentrations of each peptide of more than 0.3 nmol/L. The second messenger of GIP and t-GLP-1 in islet l3 cells has been reported to be cyclic adenosine monophosphate (cAMP).~*‘~ Therefore, GIP and t-GLP-1 probably cooperatively stimulate insulin release by an increase in intracellular cAh4P through the hormone receptors of each, and the stimulation of insulin release by these hormones should reach its plateau with 0.3 nmol/L. A recent study has demonstrated the cooperative effects of 0.5 nmol/L t-GLP-1 and 0.1 to 10 nmol/L GIP on insulin release from the isolated perfused rat pancreas,” although the experimental design and presentation of the data differ from ours. The physiologic postprandial GIP concentration is approximately 0.1 nmol/L,*l and that of t-GLP-1 has been reported at approximately 0.05 nmol/L.6*20 Therefore, the synergistic interaction of GIP and t-GLP-1 seems to be important in exerting the incretin effect of each. CCK-8-S and t-GLP-1 cooperatively stimulated the insulin release through a wide range of their concentrations. This might reflect a difference in the signal transduction mechanism in islet p-cells. The second messenger of CCK-

8-S has been reported to be a phosphoinositide.” A previous in vivo study in mice demonstrated that a bolus injection of threshold doses of GIP and CCK-8 markedly enhanced basal insulin release, and that maximal doses elicited a pure additive effectg In our study, only an additive effect was observed, even with the near threshold doses of both peptides. Fehmann et al*’ demonstrated that the insulinotropic action of 0.5 nmol/L t-GLP-1 was strongly potentiated by the addition of 20 to 100 pmol/L CCK-8. This result was considerably different from ours in its degree of potentiation. This may be because the experimental designs of the pancreas perfusion studies were different; they stimulated insulin release by increasing glucose concentrations of perfusate simultaneously with the addition of these peptides. The physiologic postprandial plasma CCK8-S level was nearly 5 pmol/L,Z~26~27 and its threshold dose for the stimulation of insulin release was reported to be 10 pmol/L in a rat pancreas perfusion study.” Considering these reports, the present results suggest that CCK-g-S is not an important incretin candidate, as was already suggested by other experimental evidence.j Previous pancreas perfusion studies demonstrated that GIP and CCK-8 stimulated glucagon release,” and t-GLP-1 suppressed it.13-” In this study, 0.3 to 3 nmol/L t-GLP-1, GIP, and CCK-8-S did not elicit any significant effects on glucagon release under 8.3 mmol/L glucose, because the effects of these hormones were not observed under high glucose concentrations of perfusate.11.29The combination of t-GLP-1 and GIP stimulated glucagon release unexpectedly with a delayed prominent peak, though the combination of t-GLP-1 and CCK-8-S did not elicit any effect on glucagon release. There has been no previous report concerning the effect of the combination of t-GLP-1 and GIP, or t-GLP-1 and CCK-8-S, on glucagon release. The mechanism of this synergism is not explainable as yet. If islet (Ycells possess specific receptors for both GIP and t-GLP-1, the second messengers in this case should be different, because GIP stimulates glucagon release, while t-GLP-1 inhibits it. However, when both hormones simultaneously bind to their own receptor, intracellular CAMP levels might be increased additively, which possibly provokes the stimulation of glucagon release. In summary, the present study indicates that t-GLP-1 and GIP secreted postprandially cooperatively stimulate insulin release and transiently stimulate glucagon release.

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INTERACTION

OF INCRETINS

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