- Email: [email protected]
Glucagon-like peptide-1 analogue LY315902: Effect on intestinal motility and release of insulin and somatostatin
Regulatory Peptides 106 (2002) 89 – 95 www.elsevier.com/locate/regpep
Glucagon-like peptide-1 analogue LY315902: Effect on intestinal motility and release of insulin and somatostatin Erik Na¨slund a,*, Staffan Skogar b, Suad Efendic c, Per M. Hellstro¨m b a
Division of Surgery, Karolinska Institutet Danderyd Hospital, SE-182 88 Danderyd, Stockholm, Sweden Department of Gastroenterology and Hepatology, Karolinska Hospital, SE-171 76 Stockholm, Sweden c Department of Endocrinology, Karolinska Hospital, SE-171 76 Stockholm, Sweden
b
Received 12 November 2001; received in revised form 7 January 2002; accepted 4 March 2002
Abstract LY315902 is an analogue of GLP-1 that yields a reduced clearance and longer half-life. The aim of the study is to assess the effect of LY315902 on fasting gastrointestinal motility, somatostatin and insulin release. Sprague – Dawley rats were fitted with three bipolar electrodes, 15, 25 and 35 cm distal to the pylorus. The effect of LY315902 and GLP-1 on migrating myoelectric complex (MMC) cycle length, duration and propagating velocity of activity fronts was studied for 60 min in conscious animals. The effect of LY315902 and GLP-1 on fasting small bowel motility was dose-dependent and treatment with exendin (9 – 39)amide, a GLP-1 receptor antagonist, together with LY315902 and GLP-1 completely antagonised the inhibitory effect of LY315902 and GLP-1 on fasting small bowel motility. Pretreatment with the nitric oxide (NO) synthase inhibitor NN-nitro-L-arginine (L-NNA) partly blocked the action of both LY315902 and GLP-1. Plasma insulin concentrations were not different from controls during infusion of LY315902 or GLP-1, while somatostatin concentrations were significantly higher during LY315902 and GLP-1 compared to saline. LY315902 has a longer duration of inhibitory action on the MMC than GLP-1, albeit similar effects on plasma insulin and somatostatin concentrations. The effect of LY315902 on motor control is mediated through the GLP-1 receptor and seems partly dependent on the L-arginine/NO pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Glucagon-like peptides; LY315902; Gastrointestinal motility; Migrating motor complex; Insulin; Somatostatin
1. Introduction GLP-1 is a peptide of 30 amino acids that is produced in the L-cells of the mucosa of the distal intestine and secreted after an intake of a mixed meal. It has about 50% sequence homology with glucagon and arises as the result of proteolytic cleavage of proglucagon in the gut [1,2]. The amino acid sequence of GLP-1 is highly conserved through evolution and all mammals seem to have identical GLP-1 sequence, indicating a physiologically important function of the peptide [3]. At physiological plasma levels, GLP-1 is insulinotropic and glucagonostatic, and hence, lowers blood glucose concentrations. It inhibits meal- and pentagastrin-induced gastric acid secretion [4], and increases insulin-induced glucose clearance [5]. Furthermore, GLP-1 delays gastric emptying
*
Corresponding author. Fax: +46-8-655-7766. E-mail address: [email protected] (E. Na¨slund).
of liquid [4] and solid meals [6], suggesting a role for GLP1 in the ‘‘ileal brake’’ mechanism [4]. There is evidence that the effect of GLP-1 on gastric secretion and motility is mediated via the vagus nerve in both animals and man [7– 9], and the blood glucose-lowering effect of GLP-1 is at least in part mediated through the peptide’s inhibitory effect on gastric emptying [10]. Peripheral administration of GLP1 in humans increases satiety and decreases food intake in normal weight [11,12], diabetic [13,14] and obese subjects [6,15]. LY315902 is an analogue of GLP-1 with three chemical modifications compared to GLP-1-(7 – 37)amide, which yields a reduced clearance and longer half-life. The modifications of LY315902 are the removal of the amino group at the N-terminal histidine residue, replacement of lysine with arginine at position 26 and the addition of an aliphatic octanoic acid moiety [16]. The primary aim of the present study was to compare the effect and mechanisms of actions of LY315902 and GLP-1 on fasting small bowel motility in the rat. Fasting motility is featured by the migrating myoelectric complex (MMC)
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 2 ) 0 0 0 5 4 - X
90
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
consisting of a repetitive cycle of motor events: phase I (quiescence), phase II (random spiking) and the characteristic phase III (regular spiking). This predictive pattern of motor events is easy to study and may act as a surrogate marker for how motility is effected by physiological regulation or pharmacological control. The secondary aim was to compare the effects of LY315902 and GLP-1 on plasma insulin and somatostatin release to the circulation.
In a first set of dose –response experiments, GLP-1 or LY315902 at doses of 5 – 100 pmol kg 1 min 1 was administered intravenously for 60 min and the effect on small bowel motility was recorded. Since a pharmacological motor response with quiescence was found first after the infusion of GLP-1 at 10 pmol kg 1 min 1 and LY315902
2. Materials and methods Fifty-eight male Sprague – Dawley rats (B&K, Sollentuna, Sweden) weighing 250– 300 g were used for the study. The local ethics committee for animal experimentation in northern Stockholm, Sweden approved the experimental protocol. 2.1. Preparation of rats for electromyography The rats were anaesthetised with pentobarbital (50 mg/kg intraperitoneally; Apoteksbolaget, Umea˚, Sweden) and, through a midline incision, three bipolar stainless steel electrodes (SS-5T, Clark Electromedical Instruments, Reading, UK) were implanted into the muscular wall of the small intestine 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus. All animals were supplied with a jugular vein catheter for the administration of drugs. The electrodes and catheter were tunnelled subcutaneously to exit at the back of the animal’s neck. In a separate group of animals (n=6), a subdiaphragmatic vagotomy and pyloroplasty were performed. After surgery, the animals were housed singly and allowed to recover for at least 7 days before experiments were undertaken. During recovery, the rats were trained to accept experimental conditions. Experiments were then carried out in conscious animals after an 18-h fasting period in wire-bottomed cages with free access to water. During the experiments, the rats were placed in Bollman cages. The electrodes were connected to an EEG preamplifier (7P5B) operating a Grass Polygraph 7B (Grass Instruments, Quincy, MA, USA). The time constant was set at 0.015 s and the low and high cut-off frequencies were set at 10 and 35 Hz, respectively. 2.2. Fasting small bowel motility studies All experiments started with a control recording of basal myoelectric activity with four activity fronts propagated over all three recording sites during a period of 60 min. The infusion of GLP-1 (Saxon Biochemicals, Hannover, Germany) or LY302915 (Eli Lilly, Stockholm, Sweden) was started immediately after the fifth activity front had passed the first electrode site using a microinjection pump (CMA 100, Carnegie Medicine, Stockholm, Sweden). The vehicle for all experiments was saline with 1% bovine serum albumin (Sigma, St. Louis, MO, USA).
Fig. 1. Median migrating motor complex (MMC) cycle length measured 15 (J1) (n), 25 (J2) (.) and 35 (J3) (E) cm distal to the pylorus during intravenous infusion of glucagon-like peptide-1 (GLP-1) (A) (n = 6) and the GLP-1 analogue LY315902 (B) (n = 6). Control data shown in each figure are values for all rats, for statistical comparison, data from the representative rat were used. p = 0.03 (*), Wilcoxon signed rank test for matched pairs.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
91
Fig. 2. Electromyographic recording from the rat jejunum at 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus showing the effect of glucagon-like peptide-1 (GLP-1) (upper panel) and its analogue LY315902 (lower panel) on the migrating myoelectric complex.
at 40 pmol kg 1 min 1, these doses were used for additional experiments. In a second set of experiments, an intravenous infusion of exendin (9– 39)amide (Peninsula Laboratories, Merseyside, UK) at a dose of 1000 pmol kg 1 min 1 was begun 15 min before the commencement of infusion of GLP-1 or LY315902 at a dose of 10 and 40 pmol kg 1 min 1, respectively. In a third set of experiments, the effect of guanethidine (Ismelin, CIBA-Geigy, Basle, Switzerland) administered as a intravenous bolus dose of 3 mg kg 1 was studied on the response to GLP-1 or LY315902 at 10 and 40 pmol kg 1 min 1, respectively. After a 1-h control period, GLP-1 or LY315902 was given intravenously for 60 min. Then, after normal propagated activity fronts of MMC were resumed, guanethidine was administered 10 min before the infusion of GLP-1 or LY325902 was started. In a fourth set of experiments, the effect of the nitric oxide (NO) synthase inhibitor NN-nitro-L-arginine (L-NNA) (Sigma) was studied on the response to GLP-1 or LY315902 at a dose of 10 and 40 pmol kg 1 min 1, respectively. After a 1-h control period, GLP-1 or LY315902 was given intravenously for 60 min. After propagated activity fronts were resumed, L-NNA at a dose of 1 mg kg 1 was given intravenously 10 min before the infusion with GLP-1 or LY315902 was repeated for 60 min. Subsequently, 300 mg
kg 1 of L-arginine (Sigma) was given 10 min before another period of GLP-1 or LY315902. In a fifth set of experiments, the effect of GLP-1 or LY315902 at 10 and 40 pmol kg 1 min 1, respectively, was studied in rats subjected to subdiaphragmatic vagotomy. After a 60-min control period, infusion with GLP-1 or LY315902 was given for 60 min. 2.3. Studies of plasma insulin and somatostatin concentrations In conscious animals, continuous infusions of saline (Natriumklorid 154 mmol/l, Baxter Medical, Kista, Sweden), GLP-1 or LY315902 at doses of 20 and 100 pmol kg 1 min 1, respectively, were carried out for 30 min. Thereafter, 5 ml of blood samples were obtained by heart puncture under phenobarbital sedation. Blood samples were collected using heparinized Vacutainer tubes (Becton Dickinson, Meylan Cedex, France), placed on ice and centrifuged for 3000 rpm at 4 jC for 10 min. The plasma was stored at 70 jC until the radioimmunoassay analysis is in one series. Immunoreactive insulin in rat plasma was measured by radioimmunoassay using antibodies against porcine insulin with the appropriate standard. The sensitivity of the assay was 1 pmol l 1 and the intra- and inter-assay coefficients of variation were 5% and 10%, respectively. The cross-reac-
Fig. 3. Electromyographic recordings of the migrating myoelectric complex (MMC) propagated through the rat jejunum at 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus. Effect of pretreatment with exendin (9 – 39)amide on the basal MMC pattern and inhibition of response to glucagon-like peptide-1 (GLP-1) and LY315902 (cf. Fig. 1).
92
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
tivity with proinsulin was 80 –85%, but there was no crossreactivity between C-peptide and insulin in the assay [17]. Immunoreactive somatostatin was analysed with radioimmunoassay as described previously. The detection limit was 2 pmol l 1 and the intra- and inter-assay coefficients of variation were 7% and 11%, respectively [18]. 2.4. Data and statistical analysis Data are shown as median (range) unless stated otherwise. The main characteristic feature of myoelectric activity of the small intestine in the fasted state, the activity front (phase III) of the migrating motor complex (MMC), was identified as a period of clearly distinguishable intense spiking activity with an amplitude at least twice that of the preceding baseline, propagating aborally through the whole recording segment and followed by a period of quiescence. The MMC cycle length, duration and propagation velocity of the activity fronts were calculated [19]. Also, in the fourth set of experiments, the time period for normal propagated activity fronts to recur after cessation of GLP-1 (10 pmol kg 1 min 1) or LY315902 (40 pmol kg 1 min 1) was recorded. When no activity front was observed during the 60-min infusion period, a value of 61 min was given for that experiment. When only one or two activity fronts were observed followed by a long period of quiescence, the MMC cycle length was calculated from the activity front preceding the start of the infusion to the phase III observed and then from the observed activity front during the infusion to the end of the infusion period +1 min. Data were compared using the Kruskal –Wallis test with the Mann – Whitney U-test as a post-test with correction for multiple comparisons or Wilcoxon rank sum test for matched pairs, whichever is appropriate. p<0.05 was considered statistically significant.
3. Results 3.1. Studies of the fasted motor pattern Under fasting control conditions, all rats exhibited a fasted motor pattern with recurring MMCs that were propagated through the intestinal segment under study (Fig. 1A and B).
3.2. Effects of GLP-1 and LY315902 on MMC Both LY315902 and GLP-1 increased the MMC cycle length in a dose-dependent manner (Figs. 1 and 2). However, GLP-1 at an infusion rate of 10 pmol kg 1 min 1 completely inhibited the MMC during the infusion period in all segments, while for LY315902, this first occurred at an infusion rate of 40 pmol kg 1 min 1. The characteristics of activity fronts in controls with a median (range) duration of 2.3 (2.3 – 6.3), 4.3 (3.2 –6.3) and 5 (2.4 –8.1) min for J1, J2 and J3, respectively, and propagation velocity of 1.5 (0.9 – 3.7) and 1.8 (1.1 – 2.5) cm min 1 for the segment J1 – J2 and J2 – J3, respectively, were not changed during infusion of LY315902 and until motor quiescence was induced. The general condition of the animals was not affected by infusion with LY315902 or GLP-1. The duration of quiescence until normal propagated activity fronts were resumed after the infusion of GLP-1 at a dose of 10 pmol kg 1 min 1 was significantly shorter than the period until the return of normal activity fronts after stopping the infusion of LY315902 at a dose of 40 pmol kg 1 min 1 (16.4 (2.7 – 20.5) and 152 (133 – 205) min, respectively, p<0.01). Pretreatment of the animals with exendin (9 – 39)amide completely antagonised the inhibitory effects of LY315902 and GLP-1 (Fig. 3). The control MMC cycle length was unchanged during the infusion of exendin (9 –39)amide alone or in combination with LY315902 or GLP-1. Similarly, the duration and propagation velocity of phase III of MMC were unchanged compared to control conditions (Table 1). After the pretreatment of rats with a bolus of guanethidine, both LY315902 and GLP-1 did not change the motor response compared to the infusion of LY315902 or GLP-1 (data not shown). Pretreatment of the animals with L-NNA intravenously partly blocked the action of both LY315902 and GLP-1. In three of the six animals, one or two propagated activity fronts were observed during L-NNA in combination with GLP-1 resulting in a median (range) cycle length of 46.8 (24.2 – 61.0), 43.7 (20.5 – 61.0) and 38.8 (15.5 – 61.0) min for J1, J2 and J3, respectively, compared to quiescence (61 min) during GLP-1 infusion alone ( p=0.07). The duration and propagation velocity of the activity fronts were not different compared to controls. After L-NNA, the total inhibitory effect of GLP-1 was reinstated by additional
Table 1 Effect of intravenous infusion of glucagon-like peptide-1 (GLP-1), the GLP-1 analogue LY315902 and the GLP-1 antagonist exendin (9 – 39)amide (Ex) on fasting duodenal motility in rats (n = 6) Characteristic
Control
Ex alone (1000 pmol kg min 1)
1
Cycle length (min) J1 18.1 (14.9 – 24.6) 19.2 (12.1 – 39.9) J2 19.3 (15.6 – 28.4) 22.2 (13.2 – 40.2) J3 20.5 (16.7 – 28.3) 23.0 (10.9 – 41.6)
GLP-1 alone (10 pmol kg min 1) 61 (61 – 61) 61 (61 – 61) 61 (61 – 61)
1
GLP-1 LY315902 alone LY315902 (10 pmol kg 1 min 1) + (40 pmol kg 1 (40 pmol/kg/min)+ 1 1 Ex (1000 pmol kg Ex (1000 pmol kg min ) min 1) 19.8 (12.7 – 27.8) 20.4 (13.9 – 27.3) 17.1 (14.5 – 29.0)
61 (61 – 61) 61 (61 – 61) 61 (61 – 61)
1
min 1)
20.3 (15.2 – 32.4) 27.4 (17.1 – 31.6) 28.3 (15.7 – 31.5)
Data shown as median (range). Not available (NA), when no activity fronts were present during 60-min infusion, an arbitrary number of 61 min was given. No effect was seen on the duration or propagating velocity of the activity fronts (data not shown). Kruskal – Wallis test, p=0.002 for cycle length.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
93
Table 2 Effect of intravenous infusion of glucagon-like peptide-1 (GLP-1) and the GLP-1 analogue LY315902 on plasma insulin and somatostatin concentrations in rats (n = 6) Saline Insulin (pmol l 1) Somatostatin (pmol l 1)
659 (173 – 952) 4.1 (2.7 – 7.4)
GLP-1 (20 pmol kg
1
min 1)
373 (242 – 1360) 9.8 (6.6 – 15.7)
GLP-1 (100 pmol kg
1
210 (97 – 1021) 24.3 (8.9 – 40.0)
min 1)
LY315902 (20 pmol kg
1
min 1)
255 (41 – 1021) 8.7 (6 – 41.6)
Median (range). Kruskal – Wallis test, p = 0.0007 for somatostatin, p = 0.06 (*) for GLP-1 100 vs. LY315902 100 pmol kg and Kruskal – Wallis test, p = 0.32 for insulin.
administration of L-arginine at a dose of 300 mg kg 1 before the administration of GLP-1. Similarly, in a separate set of rats, three of the six animals exhibited one or two propagated activity fronts during L-NNA in combination with LY315902 (40 pmol kg 1 min 1) resulting in a median (range) cycle length of 50.3 (20.9 – 61), 49.1 (16.8 – 61) and 47.4 (14.3 – 61) min for J1, J2 and J3, respectively, compared to the total quiescence (61 min) during LY315902 infusion alone ( p=0.07). The duration and propagation velocity of the activity fronts were not different from control. Again, after the L-NNA infusion, the full inhibitory effect of LY315902 was reinstated by an additional administration of L-arginine at a dose of 300 mg kg 1 before the administration of LY315902. In vagotomised animals, the action of LY315902 and GLP-1 was impaired. In three of the six animals, one or two propagated activity fronts were observed during GLP-1 infusion resulting in a median (range) MMC cycle length of 48.6 (24.9 –61), 48.8 (23.8 – 61) and 48.6 (19.9 –61) min in J1, J2 and J3, respectively. The duration and propagation velocity of the activity fronts were not different compared to control. During the infusion of LY315902, only one activity front was observed in a single animal. 3.3. Effect of GLP-1 and LY35902 on plasma insulin and somatostatin Plasma insulin concentrations were not different from controls during infusion of LY315902 or GLP-1 at any dose. Plasma somatostatin concentrations were higher during GLP-1 and LY315902 infusion compared to saline. In addition, increased somatostatin concentrations were seen with GLP-1 or LY315902 at high doses (Table 2).
4. Discussion This study demonstrates that the GLP-1 analogue LY315902 has a longer duration of inhibitory action on the MMC than GLP-1 and that a four times higher infusion dose is needed to achieve total inhibition of MMC with LY315902 compared to GLP-1. Since the motor response to GLP-1 as well as LY315902 was prevented by exendin (9– 39)amide, it is likely that the effect of LY315902 is mediated through the GLP-1 receptor, partly dependent on the L-arginine/NO pathway.
LY315902 (100 pmol kg
1
min 1)
386 (152 – 3112) 44.1 (21.9 – 61.3)* 1
min 1, Mann – Whitney U-test
The results of GLP-1 effects on fasting small bowel motility, plasma insulin and somatostatin concentrations are in accordance with that reported previously [20], yet our findings on LY315902 are novel, as this compound with regard to gastrointestinal motility has not been previously studied. GLP-1 has been suggested as treatment for noninsulin-dependent diabetes mellitus [21] and obesity [6]. However, the short half-life of GLP-1 has placed limitations on its therapeutic use [22]. In humans, the half-life of GLP-1 in plasma is short [22] and even shorter in rodents. The halflife of LY315902 has been reported to be 1.1 h after subcutaneous injection of 100 Ag kg 1 [16]. Our finding of a nine times longer period of absent activity fronts of the MMC after LY315902 compared to GLP-1 is in accordance with this longer half-life. The inhibitory response of GLP-1 and LY315902 on MMC was partly blocked by treatment with the NO synthase inhibitor L-NNA, an effect reverted by the addition of L-arginine in animals with a blocking effect of L-NNA. Thus, it seems likely that the effect of LY315902 as well as GLP-1 on MMC is partly dependent on the elaboration and release of NO in the enteric nervous system or smooth muscle. This is in accordance with a previous study from our group [20]. L-NNA at the dose administered in this experiment has previously been shown not to effect basal intestinal motility [23]. Yet, others have reported the inhibitory action of GLP-1 on gut motility not to be dependent on the L-arginine/NO pathway [24]. However, in this study by Giralt and Vergara, also conducted in rats, GLP-1 was given during a period of only 10 min, which is not likely to reveal an inhibitory action of GLP-1 by itself as activity fronts regularly occur with an interval of about 15 – 20 min. Therefore, prolonged infusion periods are needed in order to correctly judge whether a true inhibitory action on the MMC pattern prevails or not. Preferably, infusion times covering at least a period corresponding to three cycle lengths, that is 45 – 60 min, is recommendable. In addition, the inhibitory effect of GLP-1 and LY315902 on MMC was partly inhibited by vagotomy, suggesting that a central vagally mediated pathway for both GLP-1 and LY315902 [9] is in operation for the motility-inhibiting responses of the two agents. However, nervous sympathetic pathways have also been reported to mediate GLP-1 actions in the gastrointestinal tract [24]. The motility effect of GLP-1, as well as LY315902, seems to be a more sensitive target than the effect on plasma
94
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
insulin in the fasting state. Generally, GLP-1 is considered not to release insulin during euglycemic conditions and earlier studies have reported only a weak insulin-releasing effect in euglycemia [25]. As found earlier [20], we were able to support this in our present study and there was no difference between GLP-1 and LY315902 in terms of insulin response, which further supports our conclusion. In accordance with previous studies, both GLP-1 and LY315902 stimulated somatostatin release [20,26,27]. Determined from our present study, it is possible that the effect of LY315902 may be stronger than the one of GLP-1 in this respect. It has even been suggested that the inhibitory effect of GLP-1 on MMC may be mediated by somatostatin, as this peptide is known to inhibit gastrointestinal motility. However, the dose of somatostatin needed to achieve plasma concentrations that inhibit the MMC is far greater than the plasma concentrations of somatostatin seen even after high doses of GLP-1 and LY315902 (100 pmol kg 1 min 1) [20]. In addition, as the somatostatin induced inhibitory response on motility is not NO-dependent [20], this fact precludes the motility responses to either GLP-1 or LY315902 to be dependent on somatostatin. Thus, the effect of GLP-1 seems to be a direct one as indicated by our findings with exendin (9– 39)amide and independent of insulin [20]. In summary, this study demonstrates a longer duration of inhibitory action on the MMC of LY315902 than GLP-1, and that a four times higher infusion dose is needed to achieve total inhibition of MMC with LY315902 compared to GLP1. This effect seems to be mediated through the GLP-1 receptor, partly dependent on the L-arginine/NO pathway.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgements Eli Lilly, Stockholm, Sweden, kindly provided LY302915. This study was supported by grants from the Swedish Research Council, the Swedish Medical Society, Funds of the Karolinska Institutet, the Professor Nanna Svartz Fund, the Magnus Bergvall Fund, the Tore Nilsson Fund, the Ruth and Richard Juhlin Fund, Jeanssons foundation and the AMF-sjukfo¨rsa¨kring Jubile´e Foundation for Research in National Diseases.
[17]
[18]
[19]
[20]
References [21] [1] Holst JJ. Enteroglucagon. Annu Rev Physiol 1997;59:257 – 71. [2] Bell Gl, Santerre RF, Mullenbach GT. Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature 1983;302:716 – 8. [3] Ørskov C. Glucagon-like peptide-1, a new hormone of the enteroinsular axis. Diabetologica 1992;35:701 – 11. [4] Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Truncated GLP-1 (proglucagon 78 – 107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci 1993;38(4):665 – 73. [5] Ørskov C, Wettergren A, Holst JJ. Biological effects and metabolic rates of glucagonlike peptide-1 7 – 36 amide and glucagonlike pep-
[22]
[23]
[24]
tide-1 7 – 37 in healthy subjects are indistinguishable. Diabetes 1993; 42:658 – 61. Na¨slund E, Barkeling B, King N, Gutniak M, Blundell JE, Holst JJ, et al. Energy intake and appetite are suppressed by glucagon-like peptide-1 (GLP-1) in obese men. Int J Obes Relat Metab Disord 1999;23:304 – 11. Wettergren A, Wøjdemann M, Meisner S, Stadil F, Holst JJ. The inhibitory effect of glucagon-like peptide-1 (GLP-1) 7 – 36 amide on gastric acid secretion in humans depends on an intact vagal innervation. Gut 1997;40:597 – 601. Wettergren A, Wøjdemann M, Holst JJ. Glucagon-like peptide-1 inhibits gastropancreatic function by inhibiting central parasympathetic outflow. Am J Physiol 1998;275:G984 – 92. Imeryuz N, Yegen BC, Bozkurt A, Coskun T, Villanueva-Penacarrillo ML, Ulusoy NB. Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms. Am J Physiol 1997;273: G920 – 7. Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Ørskov C, Ritzel R, et al. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol 1997;273:E981 – 8. Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998;101:515 – 20. Gutzwiller JP, Go¨ke B, Drewe J, Hildebrand P, Ketterer S, Handschin D, et al. Glucagon-like peptide-1: a potent regulator of food intake in humans. Gut 1999;44:81 – 6. Gutzwiller JP, Drewe J, Go¨ke B, Schmidt H, Rohrer B, Lareida J, et al. Glucagon-like peptide-1 promotes satiety and reduces food intake in patients with diabetes mellitus type 2. Am J Physiol 1999;276: R1541 – 4. Toft-Nielsen M-B, Madsbad S, Holst JJ. Continuous subcutaneous infusion of glucagon-like peptide-1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care 1999;22: 1134 – 7. Na¨slund E, Gutniak M, Skogar S, Ro¨ssner S, Hellstro¨m PM. Glucagon-like peptide 1 increases the period of postprandial satiety and slows gastric emptying in obese men. Am J Clin Nutr 1998;68: 525 – 30. Chou JZ, Place GD, Waters DG, Kirkwood JA, Bowsher RR. A radioimmunoassay for LY315902, an analog of glucagon-like insulinotropic peptide, and its application in the study of canine pharmacokinetics. J Pharm Sci 1997;86:768 – 73. Grill V, Pigon J, Hartling SG, Binder C, Efendic S. Effects of dexamethosone on glucose-induced insulin and proinsulin release in low and high insulin responders. Metabolism 1990;39:251 – 8. Grill V, Gutniak M, Roovete A, Efendic S. A stimulating effect of glucose on somatostatin release is impaired on non-insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1984;59:293 – 7. Hellstro¨m PM, Bra¨nnstro¨m RO, Al-Saffar A. Computer program ‘‘MMC’’ to summarize characteristics of activity fronts of migrating myoelectric complex in rat small intestine. Surg Res Commun 1993; 14:51 – 63. Tolessa T, Gutniak M, Holst JJ, Efendic S, Hellstro¨m PM. Inhibitory effect of glucagon-like peptide-1 on small bowel motility. J Clin Invest 1998;102:764 – 74. Nauck MA, Holst JJ, Willms B. Glucagon-like peptide 1 and its potential in the treatment of non-insulin-dependent diabetes mellitus. Horm Metab Res 1997;29:411 – 6. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 1995;80:953 – 7. Hellstro¨m PM, Ljung T. Nitrergic inhibition of migrating myoelectric complex in the rat is mediated by vasoactive intestinal peptide. Neurogastroenterol Motil 1996;8:229 – 306. Giralt M, Vergara P. Sympathetic pathways mediate GLP-1 actions in the gastrointestinal tract of the rat. Regul Pept 1998;74:19 – 25.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95 [25] Gutniak MK, Juntti-Berggren L, Hellstro¨m PM, Guenifi A, Holst JJ, Efendic S. Glucagon-like peptide-1 enhances the insulinotropic effect of glibenclamide in NIDDM patients and in the perfused rat pancreas. Diabetes Care 1996;19:857 – 63. [26] Ørskov C, Holst JJ, Nielsen OV. Effect of truncated glucagon-like peptide-1 [proglucagon-(78 – 107) amide] on endocrine secretion from
95
pig pancreas, antrum, and nonantral stomach. Endocrinology 1988; 123:2009 – 13. [27] D’Alessio DA, Fujimoto WY, Ensinck JW. Effects of glucagonlike peptide I-(7 – 36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures. Diabetes 1989;38:1534 – 8.
Glucagon-like peptide-1 analogue LY315902: Effect on intestinal motility and release of insulin and somatostatin Erik Na¨slund a,*, Staffan Skogar b, Suad Efendic c, Per M. Hellstro¨m b a
Division of Surgery, Karolinska Institutet Danderyd Hospital, SE-182 88 Danderyd, Stockholm, Sweden Department of Gastroenterology and Hepatology, Karolinska Hospital, SE-171 76 Stockholm, Sweden c Department of Endocrinology, Karolinska Hospital, SE-171 76 Stockholm, Sweden
b
Received 12 November 2001; received in revised form 7 January 2002; accepted 4 March 2002
Abstract LY315902 is an analogue of GLP-1 that yields a reduced clearance and longer half-life. The aim of the study is to assess the effect of LY315902 on fasting gastrointestinal motility, somatostatin and insulin release. Sprague – Dawley rats were fitted with three bipolar electrodes, 15, 25 and 35 cm distal to the pylorus. The effect of LY315902 and GLP-1 on migrating myoelectric complex (MMC) cycle length, duration and propagating velocity of activity fronts was studied for 60 min in conscious animals. The effect of LY315902 and GLP-1 on fasting small bowel motility was dose-dependent and treatment with exendin (9 – 39)amide, a GLP-1 receptor antagonist, together with LY315902 and GLP-1 completely antagonised the inhibitory effect of LY315902 and GLP-1 on fasting small bowel motility. Pretreatment with the nitric oxide (NO) synthase inhibitor NN-nitro-L-arginine (L-NNA) partly blocked the action of both LY315902 and GLP-1. Plasma insulin concentrations were not different from controls during infusion of LY315902 or GLP-1, while somatostatin concentrations were significantly higher during LY315902 and GLP-1 compared to saline. LY315902 has a longer duration of inhibitory action on the MMC than GLP-1, albeit similar effects on plasma insulin and somatostatin concentrations. The effect of LY315902 on motor control is mediated through the GLP-1 receptor and seems partly dependent on the L-arginine/NO pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Glucagon-like peptides; LY315902; Gastrointestinal motility; Migrating motor complex; Insulin; Somatostatin
1. Introduction GLP-1 is a peptide of 30 amino acids that is produced in the L-cells of the mucosa of the distal intestine and secreted after an intake of a mixed meal. It has about 50% sequence homology with glucagon and arises as the result of proteolytic cleavage of proglucagon in the gut [1,2]. The amino acid sequence of GLP-1 is highly conserved through evolution and all mammals seem to have identical GLP-1 sequence, indicating a physiologically important function of the peptide [3]. At physiological plasma levels, GLP-1 is insulinotropic and glucagonostatic, and hence, lowers blood glucose concentrations. It inhibits meal- and pentagastrin-induced gastric acid secretion [4], and increases insulin-induced glucose clearance [5]. Furthermore, GLP-1 delays gastric emptying
*
Corresponding author. Fax: +46-8-655-7766. E-mail address: [email protected] (E. Na¨slund).
of liquid [4] and solid meals [6], suggesting a role for GLP1 in the ‘‘ileal brake’’ mechanism [4]. There is evidence that the effect of GLP-1 on gastric secretion and motility is mediated via the vagus nerve in both animals and man [7– 9], and the blood glucose-lowering effect of GLP-1 is at least in part mediated through the peptide’s inhibitory effect on gastric emptying [10]. Peripheral administration of GLP1 in humans increases satiety and decreases food intake in normal weight [11,12], diabetic [13,14] and obese subjects [6,15]. LY315902 is an analogue of GLP-1 with three chemical modifications compared to GLP-1-(7 – 37)amide, which yields a reduced clearance and longer half-life. The modifications of LY315902 are the removal of the amino group at the N-terminal histidine residue, replacement of lysine with arginine at position 26 and the addition of an aliphatic octanoic acid moiety [16]. The primary aim of the present study was to compare the effect and mechanisms of actions of LY315902 and GLP-1 on fasting small bowel motility in the rat. Fasting motility is featured by the migrating myoelectric complex (MMC)
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 2 ) 0 0 0 5 4 - X
90
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
consisting of a repetitive cycle of motor events: phase I (quiescence), phase II (random spiking) and the characteristic phase III (regular spiking). This predictive pattern of motor events is easy to study and may act as a surrogate marker for how motility is effected by physiological regulation or pharmacological control. The secondary aim was to compare the effects of LY315902 and GLP-1 on plasma insulin and somatostatin release to the circulation.
In a first set of dose –response experiments, GLP-1 or LY315902 at doses of 5 – 100 pmol kg 1 min 1 was administered intravenously for 60 min and the effect on small bowel motility was recorded. Since a pharmacological motor response with quiescence was found first after the infusion of GLP-1 at 10 pmol kg 1 min 1 and LY315902
2. Materials and methods Fifty-eight male Sprague – Dawley rats (B&K, Sollentuna, Sweden) weighing 250– 300 g were used for the study. The local ethics committee for animal experimentation in northern Stockholm, Sweden approved the experimental protocol. 2.1. Preparation of rats for electromyography The rats were anaesthetised with pentobarbital (50 mg/kg intraperitoneally; Apoteksbolaget, Umea˚, Sweden) and, through a midline incision, three bipolar stainless steel electrodes (SS-5T, Clark Electromedical Instruments, Reading, UK) were implanted into the muscular wall of the small intestine 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus. All animals were supplied with a jugular vein catheter for the administration of drugs. The electrodes and catheter were tunnelled subcutaneously to exit at the back of the animal’s neck. In a separate group of animals (n=6), a subdiaphragmatic vagotomy and pyloroplasty were performed. After surgery, the animals were housed singly and allowed to recover for at least 7 days before experiments were undertaken. During recovery, the rats were trained to accept experimental conditions. Experiments were then carried out in conscious animals after an 18-h fasting period in wire-bottomed cages with free access to water. During the experiments, the rats were placed in Bollman cages. The electrodes were connected to an EEG preamplifier (7P5B) operating a Grass Polygraph 7B (Grass Instruments, Quincy, MA, USA). The time constant was set at 0.015 s and the low and high cut-off frequencies were set at 10 and 35 Hz, respectively. 2.2. Fasting small bowel motility studies All experiments started with a control recording of basal myoelectric activity with four activity fronts propagated over all three recording sites during a period of 60 min. The infusion of GLP-1 (Saxon Biochemicals, Hannover, Germany) or LY302915 (Eli Lilly, Stockholm, Sweden) was started immediately after the fifth activity front had passed the first electrode site using a microinjection pump (CMA 100, Carnegie Medicine, Stockholm, Sweden). The vehicle for all experiments was saline with 1% bovine serum albumin (Sigma, St. Louis, MO, USA).
Fig. 1. Median migrating motor complex (MMC) cycle length measured 15 (J1) (n), 25 (J2) (.) and 35 (J3) (E) cm distal to the pylorus during intravenous infusion of glucagon-like peptide-1 (GLP-1) (A) (n = 6) and the GLP-1 analogue LY315902 (B) (n = 6). Control data shown in each figure are values for all rats, for statistical comparison, data from the representative rat were used. p = 0.03 (*), Wilcoxon signed rank test for matched pairs.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
91
Fig. 2. Electromyographic recording from the rat jejunum at 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus showing the effect of glucagon-like peptide-1 (GLP-1) (upper panel) and its analogue LY315902 (lower panel) on the migrating myoelectric complex.
at 40 pmol kg 1 min 1, these doses were used for additional experiments. In a second set of experiments, an intravenous infusion of exendin (9– 39)amide (Peninsula Laboratories, Merseyside, UK) at a dose of 1000 pmol kg 1 min 1 was begun 15 min before the commencement of infusion of GLP-1 or LY315902 at a dose of 10 and 40 pmol kg 1 min 1, respectively. In a third set of experiments, the effect of guanethidine (Ismelin, CIBA-Geigy, Basle, Switzerland) administered as a intravenous bolus dose of 3 mg kg 1 was studied on the response to GLP-1 or LY315902 at 10 and 40 pmol kg 1 min 1, respectively. After a 1-h control period, GLP-1 or LY315902 was given intravenously for 60 min. Then, after normal propagated activity fronts of MMC were resumed, guanethidine was administered 10 min before the infusion of GLP-1 or LY325902 was started. In a fourth set of experiments, the effect of the nitric oxide (NO) synthase inhibitor NN-nitro-L-arginine (L-NNA) (Sigma) was studied on the response to GLP-1 or LY315902 at a dose of 10 and 40 pmol kg 1 min 1, respectively. After a 1-h control period, GLP-1 or LY315902 was given intravenously for 60 min. After propagated activity fronts were resumed, L-NNA at a dose of 1 mg kg 1 was given intravenously 10 min before the infusion with GLP-1 or LY315902 was repeated for 60 min. Subsequently, 300 mg
kg 1 of L-arginine (Sigma) was given 10 min before another period of GLP-1 or LY315902. In a fifth set of experiments, the effect of GLP-1 or LY315902 at 10 and 40 pmol kg 1 min 1, respectively, was studied in rats subjected to subdiaphragmatic vagotomy. After a 60-min control period, infusion with GLP-1 or LY315902 was given for 60 min. 2.3. Studies of plasma insulin and somatostatin concentrations In conscious animals, continuous infusions of saline (Natriumklorid 154 mmol/l, Baxter Medical, Kista, Sweden), GLP-1 or LY315902 at doses of 20 and 100 pmol kg 1 min 1, respectively, were carried out for 30 min. Thereafter, 5 ml of blood samples were obtained by heart puncture under phenobarbital sedation. Blood samples were collected using heparinized Vacutainer tubes (Becton Dickinson, Meylan Cedex, France), placed on ice and centrifuged for 3000 rpm at 4 jC for 10 min. The plasma was stored at 70 jC until the radioimmunoassay analysis is in one series. Immunoreactive insulin in rat plasma was measured by radioimmunoassay using antibodies against porcine insulin with the appropriate standard. The sensitivity of the assay was 1 pmol l 1 and the intra- and inter-assay coefficients of variation were 5% and 10%, respectively. The cross-reac-
Fig. 3. Electromyographic recordings of the migrating myoelectric complex (MMC) propagated through the rat jejunum at 15 (J1), 25 (J2) and 35 (J3) cm distal to the pylorus. Effect of pretreatment with exendin (9 – 39)amide on the basal MMC pattern and inhibition of response to glucagon-like peptide-1 (GLP-1) and LY315902 (cf. Fig. 1).
92
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
tivity with proinsulin was 80 –85%, but there was no crossreactivity between C-peptide and insulin in the assay [17]. Immunoreactive somatostatin was analysed with radioimmunoassay as described previously. The detection limit was 2 pmol l 1 and the intra- and inter-assay coefficients of variation were 7% and 11%, respectively [18]. 2.4. Data and statistical analysis Data are shown as median (range) unless stated otherwise. The main characteristic feature of myoelectric activity of the small intestine in the fasted state, the activity front (phase III) of the migrating motor complex (MMC), was identified as a period of clearly distinguishable intense spiking activity with an amplitude at least twice that of the preceding baseline, propagating aborally through the whole recording segment and followed by a period of quiescence. The MMC cycle length, duration and propagation velocity of the activity fronts were calculated [19]. Also, in the fourth set of experiments, the time period for normal propagated activity fronts to recur after cessation of GLP-1 (10 pmol kg 1 min 1) or LY315902 (40 pmol kg 1 min 1) was recorded. When no activity front was observed during the 60-min infusion period, a value of 61 min was given for that experiment. When only one or two activity fronts were observed followed by a long period of quiescence, the MMC cycle length was calculated from the activity front preceding the start of the infusion to the phase III observed and then from the observed activity front during the infusion to the end of the infusion period +1 min. Data were compared using the Kruskal –Wallis test with the Mann – Whitney U-test as a post-test with correction for multiple comparisons or Wilcoxon rank sum test for matched pairs, whichever is appropriate. p<0.05 was considered statistically significant.
3. Results 3.1. Studies of the fasted motor pattern Under fasting control conditions, all rats exhibited a fasted motor pattern with recurring MMCs that were propagated through the intestinal segment under study (Fig. 1A and B).
3.2. Effects of GLP-1 and LY315902 on MMC Both LY315902 and GLP-1 increased the MMC cycle length in a dose-dependent manner (Figs. 1 and 2). However, GLP-1 at an infusion rate of 10 pmol kg 1 min 1 completely inhibited the MMC during the infusion period in all segments, while for LY315902, this first occurred at an infusion rate of 40 pmol kg 1 min 1. The characteristics of activity fronts in controls with a median (range) duration of 2.3 (2.3 – 6.3), 4.3 (3.2 –6.3) and 5 (2.4 –8.1) min for J1, J2 and J3, respectively, and propagation velocity of 1.5 (0.9 – 3.7) and 1.8 (1.1 – 2.5) cm min 1 for the segment J1 – J2 and J2 – J3, respectively, were not changed during infusion of LY315902 and until motor quiescence was induced. The general condition of the animals was not affected by infusion with LY315902 or GLP-1. The duration of quiescence until normal propagated activity fronts were resumed after the infusion of GLP-1 at a dose of 10 pmol kg 1 min 1 was significantly shorter than the period until the return of normal activity fronts after stopping the infusion of LY315902 at a dose of 40 pmol kg 1 min 1 (16.4 (2.7 – 20.5) and 152 (133 – 205) min, respectively, p<0.01). Pretreatment of the animals with exendin (9 – 39)amide completely antagonised the inhibitory effects of LY315902 and GLP-1 (Fig. 3). The control MMC cycle length was unchanged during the infusion of exendin (9 –39)amide alone or in combination with LY315902 or GLP-1. Similarly, the duration and propagation velocity of phase III of MMC were unchanged compared to control conditions (Table 1). After the pretreatment of rats with a bolus of guanethidine, both LY315902 and GLP-1 did not change the motor response compared to the infusion of LY315902 or GLP-1 (data not shown). Pretreatment of the animals with L-NNA intravenously partly blocked the action of both LY315902 and GLP-1. In three of the six animals, one or two propagated activity fronts were observed during L-NNA in combination with GLP-1 resulting in a median (range) cycle length of 46.8 (24.2 – 61.0), 43.7 (20.5 – 61.0) and 38.8 (15.5 – 61.0) min for J1, J2 and J3, respectively, compared to quiescence (61 min) during GLP-1 infusion alone ( p=0.07). The duration and propagation velocity of the activity fronts were not different compared to controls. After L-NNA, the total inhibitory effect of GLP-1 was reinstated by additional
Table 1 Effect of intravenous infusion of glucagon-like peptide-1 (GLP-1), the GLP-1 analogue LY315902 and the GLP-1 antagonist exendin (9 – 39)amide (Ex) on fasting duodenal motility in rats (n = 6) Characteristic
Control
Ex alone (1000 pmol kg min 1)
1
Cycle length (min) J1 18.1 (14.9 – 24.6) 19.2 (12.1 – 39.9) J2 19.3 (15.6 – 28.4) 22.2 (13.2 – 40.2) J3 20.5 (16.7 – 28.3) 23.0 (10.9 – 41.6)
GLP-1 alone (10 pmol kg min 1) 61 (61 – 61) 61 (61 – 61) 61 (61 – 61)
1
GLP-1 LY315902 alone LY315902 (10 pmol kg 1 min 1) + (40 pmol kg 1 (40 pmol/kg/min)+ 1 1 Ex (1000 pmol kg Ex (1000 pmol kg min ) min 1) 19.8 (12.7 – 27.8) 20.4 (13.9 – 27.3) 17.1 (14.5 – 29.0)
61 (61 – 61) 61 (61 – 61) 61 (61 – 61)
1
min 1)
20.3 (15.2 – 32.4) 27.4 (17.1 – 31.6) 28.3 (15.7 – 31.5)
Data shown as median (range). Not available (NA), when no activity fronts were present during 60-min infusion, an arbitrary number of 61 min was given. No effect was seen on the duration or propagating velocity of the activity fronts (data not shown). Kruskal – Wallis test, p=0.002 for cycle length.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
93
Table 2 Effect of intravenous infusion of glucagon-like peptide-1 (GLP-1) and the GLP-1 analogue LY315902 on plasma insulin and somatostatin concentrations in rats (n = 6) Saline Insulin (pmol l 1) Somatostatin (pmol l 1)
659 (173 – 952) 4.1 (2.7 – 7.4)
GLP-1 (20 pmol kg
1
min 1)
373 (242 – 1360) 9.8 (6.6 – 15.7)
GLP-1 (100 pmol kg
1
210 (97 – 1021) 24.3 (8.9 – 40.0)
min 1)
LY315902 (20 pmol kg
1
min 1)
255 (41 – 1021) 8.7 (6 – 41.6)
Median (range). Kruskal – Wallis test, p = 0.0007 for somatostatin, p = 0.06 (*) for GLP-1 100 vs. LY315902 100 pmol kg and Kruskal – Wallis test, p = 0.32 for insulin.
administration of L-arginine at a dose of 300 mg kg 1 before the administration of GLP-1. Similarly, in a separate set of rats, three of the six animals exhibited one or two propagated activity fronts during L-NNA in combination with LY315902 (40 pmol kg 1 min 1) resulting in a median (range) cycle length of 50.3 (20.9 – 61), 49.1 (16.8 – 61) and 47.4 (14.3 – 61) min for J1, J2 and J3, respectively, compared to the total quiescence (61 min) during LY315902 infusion alone ( p=0.07). The duration and propagation velocity of the activity fronts were not different from control. Again, after the L-NNA infusion, the full inhibitory effect of LY315902 was reinstated by an additional administration of L-arginine at a dose of 300 mg kg 1 before the administration of LY315902. In vagotomised animals, the action of LY315902 and GLP-1 was impaired. In three of the six animals, one or two propagated activity fronts were observed during GLP-1 infusion resulting in a median (range) MMC cycle length of 48.6 (24.9 –61), 48.8 (23.8 – 61) and 48.6 (19.9 –61) min in J1, J2 and J3, respectively. The duration and propagation velocity of the activity fronts were not different compared to control. During the infusion of LY315902, only one activity front was observed in a single animal. 3.3. Effect of GLP-1 and LY35902 on plasma insulin and somatostatin Plasma insulin concentrations were not different from controls during infusion of LY315902 or GLP-1 at any dose. Plasma somatostatin concentrations were higher during GLP-1 and LY315902 infusion compared to saline. In addition, increased somatostatin concentrations were seen with GLP-1 or LY315902 at high doses (Table 2).
4. Discussion This study demonstrates that the GLP-1 analogue LY315902 has a longer duration of inhibitory action on the MMC than GLP-1 and that a four times higher infusion dose is needed to achieve total inhibition of MMC with LY315902 compared to GLP-1. Since the motor response to GLP-1 as well as LY315902 was prevented by exendin (9– 39)amide, it is likely that the effect of LY315902 is mediated through the GLP-1 receptor, partly dependent on the L-arginine/NO pathway.
LY315902 (100 pmol kg
1
min 1)
386 (152 – 3112) 44.1 (21.9 – 61.3)* 1
min 1, Mann – Whitney U-test
The results of GLP-1 effects on fasting small bowel motility, plasma insulin and somatostatin concentrations are in accordance with that reported previously [20], yet our findings on LY315902 are novel, as this compound with regard to gastrointestinal motility has not been previously studied. GLP-1 has been suggested as treatment for noninsulin-dependent diabetes mellitus [21] and obesity [6]. However, the short half-life of GLP-1 has placed limitations on its therapeutic use [22]. In humans, the half-life of GLP-1 in plasma is short [22] and even shorter in rodents. The halflife of LY315902 has been reported to be 1.1 h after subcutaneous injection of 100 Ag kg 1 [16]. Our finding of a nine times longer period of absent activity fronts of the MMC after LY315902 compared to GLP-1 is in accordance with this longer half-life. The inhibitory response of GLP-1 and LY315902 on MMC was partly blocked by treatment with the NO synthase inhibitor L-NNA, an effect reverted by the addition of L-arginine in animals with a blocking effect of L-NNA. Thus, it seems likely that the effect of LY315902 as well as GLP-1 on MMC is partly dependent on the elaboration and release of NO in the enteric nervous system or smooth muscle. This is in accordance with a previous study from our group [20]. L-NNA at the dose administered in this experiment has previously been shown not to effect basal intestinal motility [23]. Yet, others have reported the inhibitory action of GLP-1 on gut motility not to be dependent on the L-arginine/NO pathway [24]. However, in this study by Giralt and Vergara, also conducted in rats, GLP-1 was given during a period of only 10 min, which is not likely to reveal an inhibitory action of GLP-1 by itself as activity fronts regularly occur with an interval of about 15 – 20 min. Therefore, prolonged infusion periods are needed in order to correctly judge whether a true inhibitory action on the MMC pattern prevails or not. Preferably, infusion times covering at least a period corresponding to three cycle lengths, that is 45 – 60 min, is recommendable. In addition, the inhibitory effect of GLP-1 and LY315902 on MMC was partly inhibited by vagotomy, suggesting that a central vagally mediated pathway for both GLP-1 and LY315902 [9] is in operation for the motility-inhibiting responses of the two agents. However, nervous sympathetic pathways have also been reported to mediate GLP-1 actions in the gastrointestinal tract [24]. The motility effect of GLP-1, as well as LY315902, seems to be a more sensitive target than the effect on plasma
94
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95
insulin in the fasting state. Generally, GLP-1 is considered not to release insulin during euglycemic conditions and earlier studies have reported only a weak insulin-releasing effect in euglycemia [25]. As found earlier [20], we were able to support this in our present study and there was no difference between GLP-1 and LY315902 in terms of insulin response, which further supports our conclusion. In accordance with previous studies, both GLP-1 and LY315902 stimulated somatostatin release [20,26,27]. Determined from our present study, it is possible that the effect of LY315902 may be stronger than the one of GLP-1 in this respect. It has even been suggested that the inhibitory effect of GLP-1 on MMC may be mediated by somatostatin, as this peptide is known to inhibit gastrointestinal motility. However, the dose of somatostatin needed to achieve plasma concentrations that inhibit the MMC is far greater than the plasma concentrations of somatostatin seen even after high doses of GLP-1 and LY315902 (100 pmol kg 1 min 1) [20]. In addition, as the somatostatin induced inhibitory response on motility is not NO-dependent [20], this fact precludes the motility responses to either GLP-1 or LY315902 to be dependent on somatostatin. Thus, the effect of GLP-1 seems to be a direct one as indicated by our findings with exendin (9– 39)amide and independent of insulin [20]. In summary, this study demonstrates a longer duration of inhibitory action on the MMC of LY315902 than GLP-1, and that a four times higher infusion dose is needed to achieve total inhibition of MMC with LY315902 compared to GLP1. This effect seems to be mediated through the GLP-1 receptor, partly dependent on the L-arginine/NO pathway.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgements Eli Lilly, Stockholm, Sweden, kindly provided LY302915. This study was supported by grants from the Swedish Research Council, the Swedish Medical Society, Funds of the Karolinska Institutet, the Professor Nanna Svartz Fund, the Magnus Bergvall Fund, the Tore Nilsson Fund, the Ruth and Richard Juhlin Fund, Jeanssons foundation and the AMF-sjukfo¨rsa¨kring Jubile´e Foundation for Research in National Diseases.
[17]
[18]
[19]
[20]
References [21] [1] Holst JJ. Enteroglucagon. Annu Rev Physiol 1997;59:257 – 71. [2] Bell Gl, Santerre RF, Mullenbach GT. Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature 1983;302:716 – 8. [3] Ørskov C. Glucagon-like peptide-1, a new hormone of the enteroinsular axis. Diabetologica 1992;35:701 – 11. [4] Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Truncated GLP-1 (proglucagon 78 – 107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci 1993;38(4):665 – 73. [5] Ørskov C, Wettergren A, Holst JJ. Biological effects and metabolic rates of glucagonlike peptide-1 7 – 36 amide and glucagonlike pep-
[22]
[23]
[24]
tide-1 7 – 37 in healthy subjects are indistinguishable. Diabetes 1993; 42:658 – 61. Na¨slund E, Barkeling B, King N, Gutniak M, Blundell JE, Holst JJ, et al. Energy intake and appetite are suppressed by glucagon-like peptide-1 (GLP-1) in obese men. Int J Obes Relat Metab Disord 1999;23:304 – 11. Wettergren A, Wøjdemann M, Meisner S, Stadil F, Holst JJ. The inhibitory effect of glucagon-like peptide-1 (GLP-1) 7 – 36 amide on gastric acid secretion in humans depends on an intact vagal innervation. Gut 1997;40:597 – 601. Wettergren A, Wøjdemann M, Holst JJ. Glucagon-like peptide-1 inhibits gastropancreatic function by inhibiting central parasympathetic outflow. Am J Physiol 1998;275:G984 – 92. Imeryuz N, Yegen BC, Bozkurt A, Coskun T, Villanueva-Penacarrillo ML, Ulusoy NB. Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms. Am J Physiol 1997;273: G920 – 7. Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Ørskov C, Ritzel R, et al. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol 1997;273:E981 – 8. Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998;101:515 – 20. Gutzwiller JP, Go¨ke B, Drewe J, Hildebrand P, Ketterer S, Handschin D, et al. Glucagon-like peptide-1: a potent regulator of food intake in humans. Gut 1999;44:81 – 6. Gutzwiller JP, Drewe J, Go¨ke B, Schmidt H, Rohrer B, Lareida J, et al. Glucagon-like peptide-1 promotes satiety and reduces food intake in patients with diabetes mellitus type 2. Am J Physiol 1999;276: R1541 – 4. Toft-Nielsen M-B, Madsbad S, Holst JJ. Continuous subcutaneous infusion of glucagon-like peptide-1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care 1999;22: 1134 – 7. Na¨slund E, Gutniak M, Skogar S, Ro¨ssner S, Hellstro¨m PM. Glucagon-like peptide 1 increases the period of postprandial satiety and slows gastric emptying in obese men. Am J Clin Nutr 1998;68: 525 – 30. Chou JZ, Place GD, Waters DG, Kirkwood JA, Bowsher RR. A radioimmunoassay for LY315902, an analog of glucagon-like insulinotropic peptide, and its application in the study of canine pharmacokinetics. J Pharm Sci 1997;86:768 – 73. Grill V, Pigon J, Hartling SG, Binder C, Efendic S. Effects of dexamethosone on glucose-induced insulin and proinsulin release in low and high insulin responders. Metabolism 1990;39:251 – 8. Grill V, Gutniak M, Roovete A, Efendic S. A stimulating effect of glucose on somatostatin release is impaired on non-insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1984;59:293 – 7. Hellstro¨m PM, Bra¨nnstro¨m RO, Al-Saffar A. Computer program ‘‘MMC’’ to summarize characteristics of activity fronts of migrating myoelectric complex in rat small intestine. Surg Res Commun 1993; 14:51 – 63. Tolessa T, Gutniak M, Holst JJ, Efendic S, Hellstro¨m PM. Inhibitory effect of glucagon-like peptide-1 on small bowel motility. J Clin Invest 1998;102:764 – 74. Nauck MA, Holst JJ, Willms B. Glucagon-like peptide 1 and its potential in the treatment of non-insulin-dependent diabetes mellitus. Horm Metab Res 1997;29:411 – 6. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 1995;80:953 – 7. Hellstro¨m PM, Ljung T. Nitrergic inhibition of migrating myoelectric complex in the rat is mediated by vasoactive intestinal peptide. Neurogastroenterol Motil 1996;8:229 – 306. Giralt M, Vergara P. Sympathetic pathways mediate GLP-1 actions in the gastrointestinal tract of the rat. Regul Pept 1998;74:19 – 25.
E. Na¨slund et al. / Regulatory Peptides 106 (2002) 89–95 [25] Gutniak MK, Juntti-Berggren L, Hellstro¨m PM, Guenifi A, Holst JJ, Efendic S. Glucagon-like peptide-1 enhances the insulinotropic effect of glibenclamide in NIDDM patients and in the perfused rat pancreas. Diabetes Care 1996;19:857 – 63. [26] Ørskov C, Holst JJ, Nielsen OV. Effect of truncated glucagon-like peptide-1 [proglucagon-(78 – 107) amide] on endocrine secretion from
95
pig pancreas, antrum, and nonantral stomach. Endocrinology 1988; 123:2009 – 13. [27] D’Alessio DA, Fujimoto WY, Ensinck JW. Effects of glucagonlike peptide I-(7 – 36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures. Diabetes 1989;38:1534 – 8.