Inhibition of Dipeptidyl Peptidase-4 Augments Insulin Secretion in Response to Exogenously Administered Glucagon-Like Peptide-1, Glucose-Dep
http://www.100md.com
内分泌学杂志 2005年第4期
Department of Medicine (B.A.), Lund University, SE-221 84 Lund, Sweden; and Novartis Institutes for Biomedical Research (T.E.H.), Cambridge, Massachusetts 02139
Address all correspondence and requests for reprints to: Dr. Bo Ahrén, Department of Medicine, Lund University, B11 BMC, SE-221 84 Lund, Sweden. E-mail: bo.ahren@med.lu.se.
Abstract
Inhibition of dipeptidyl peptidase-4 (DPP-4) is currently being explored as a new approach to the treatment of type 2 diabetes. This concept has emerged from the powerful and rapid action of the enzyme to inactivate glucagon-like peptide-1 (GLP-1). However, other bioactive peptides with potential influence of islet function are also substrates of DPP-4. Whether this inactivation may add to the beneficial effects of DPP-4 inhibition is not known. In this study, we explored whether DPP-4 inhibition by valine-pyrrolidide (val-pyr; 100 μmol/kg administered through gastric gavage at t = –30 min) affects the insulin and glucose responses to iv glucose (1 g/kg) together with GLP-1 (10 nmol/kg), glucose-dependent insulinotropic polypeptide (GIP; 10 nmol/kg), pituitary adenylate cyclase-activating polypeptide 38 (PACAP38; 1.3 nmol/kg), or gastrin-releasing peptide (GRP; 20 nmol/kg) given at t = 0 in anesthetized C57BL/6J mice. It was found that the acute (1–5 min) insulin response to GLP-1 was augmented by val-pyr by 80% (4.2 ± 0.4 vs. 7.6 ± 0.8 nmol/liter), that to GIP by 40% (2.7 ± 0.3 vs. 3.8 ± 0.4 nmol/liter), that to PACAP38 by 75% (4.6 ± 0.5 vs. 8.1 ± 0.6 nmol/liter), and that to GRP by 25% (1.8 ± 0.2 vs. 2.3 ± 0.3 nmol/liter; all P < 0.05 or less). This was associated with enhanced glucose elimination rate after GLP-1 [glucose elimination constant (KG) 2.1 ± 0.2 vs. 3.1 ± 0.3%/min] and PACAP38 (2.1 ± 0.3 vs. 3.2 ± 0.3%/min; both P < 0.01), but not after GIP or GRP. The augmented insulin response to GRP by val-pyr was prevented by the GLP-1 receptor antagonist, exendin3 (9-39), raising the possibility that GRP effects may occur secondary to stimulation of GLP-1 secretion. We conclude that DPP-4 inhibition augments the insulin response not only to GLP-1 but also to GIP, PACAP38, and GRP.
Introduction
THE ENZYME DIPEPTIDYL peptidase-4 (DPP-4) is a cell surface serine peptidase, which is distributed throughout the endothelial lining of the vasculature, and which also is circulating in a soluble form (1). The enzyme inactivates a number of biologically active peptides containing Xaa-Ala or Xaa-Pro aminoterminal sequences by removing their N-terminal dipeptide residues. Glucagon-like peptide-1 (GLP-1) is one of the substrates of DPP-4, being inactivated by removal of the N-terminal histidine-alanine dipeptide moiety (2). Because GLP-1 has been shown to exert antidiabetic actions (3), prevention of its inactivation by inhibition of DPP-4 is currently being explored as a new treatment for type 2 diabetes (4, 5). DPP-4 inhibition has thereby been demonstrated to be antidiabetic both in animal models of diabetes (6, 7, 8, 9) and in patients with type 2 diabetes (10, 11). This is consistent with findings that animals with genetic deletion of DPP-4 activity exhibit improved glucose tolerance (12, 13). Due to recognition of their aminoterminal sequences, several other peptides are, however, also potential substrates of DPP-4, such as glucose-dependent insulinotropic polypeptide (GIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and gastrin-releasing peptide (GRP) (1, 2, 14, 15, 16). Like GLP-1, these peptides may be involved in the regulation of glucose homeostasis and insulin secretion because all of them are potent insulinotropic peptides and augment glucose-stimulated insulin secretion (17, 18, 19, 20). They have, however, different relations to islet function. Thus, GLP-1 and GIP are incretins released from the gut in association with meal ingestion and, therefore, of importance for the postprandial insulin response (3, 20, 21), whereas PACAP and GRP are neuropeptides being signals of pancreatic nerves and, therefore, involved in the neural regulation of islet function (17). Prevention of inactivation of these peptides may contribute to the antidiabetic ability of DPP-4 inhibitors, provided that DPP-4 inhibition increases the active component of the peptides to such an extent that their insulinotropic actions or other antidiabetic effects are augmented. In the present study, therefore, mice were given the DPP-4 inhibitor, valine-pyrrolidide (val-pyr), followed by iv administration of these peptides to explore whether their actions are perturbed. Val-pyr has previously been shown to inhibit DPP-4 activity to such an extent that it augments GLP-1 levels in pigs and mice and results in enhanced insulinotropic activity of exogenously added GLP-1 (8, 18).
Subjects and Methods
Animals
Female C57BL/6J mice, weighing 21–24 g, obtained from Taconic A/S (Ry, Denmark), were used. The animals were kept in a 12-h light schedule (lights on at 0600 h) and given a standard pellet diet (fat, 11.4%; carbohydrate, 62.8%; and protein, 25.8% on an energy base, total energy 12.6 kJ/g) and tap water ad libitum. The Lund University Ethical Committee (Lund, Sweden) approved the study.
In vivo experiments
The studies were performed in late morning after removal of food from the cages 4 h earlier. The animals were anesthetized with an ip injection of midazolam (0.2 mg/mouse, Dormicum, Hoffman-La Roche, Basel, Switzerland) and a combination of fluanisone (0.4 mg/mouse) and fentanyl (0.02 mg/mouse, Hypnorm, Janssen, Beerse, Belgium). Thirty minutes later, val-pyr was given through a gastric tube, with an outer diameter of 1.2 mm (100 μmol/kg; a kind gift from Novartis Institutes for Biomedical Research, Cambridge, MA). After another 30 min, a blood sample was taken from the retrobulbar, intraorbital, capillary plexus in heparinized tubes, whereafter D-glucose (British Drug Houses, Poole, UK; 1 g/kg) was rapidly injected iv alone or together with synthetic GLP-1 (Peninsula Europe Laboratories, 10 nmol/kg), synthetic porcine GIP (Sigma, St. Louis, MO; 10 nmol/kg), synthetic ovine PACAP38 (Peninsula Europe Laboratories; 1.3 nmol/kg), synthetic porcine GRP (Sigma; 20 nmol/kg), synthetic GRP14-27 (Sigma; 20 nmol/kg), or GRP together with synthetic exendin3 (9-39) (Peninsula Europe Laboratories; 30 nmol/kg). Controls were given saline. The doses selected for the peptides have previously been shown to be maximal in augmenting glucose-stimulated insulin secretion after iv administration in mice (19, 22, 23, 24). The volume load was 10 μl/g body weight. At specific time points after the iv injection, blood samples, consisting of 75 μl blood each, were collected. A total of seven samples were taken during each experiment, at t = 0, 1, 5, 10, 20, 30, and 50 min. The removal of this amount of blood has previously been shown not to alter baseline glucose levels in mice (19). Blood was kept in heparinized tubes; then, after immediate centrifugation, plasma was separated and stored at –20 C until analysis was carried out.
Assays
Insulin was determined radioimmunochemically with the use of a guinea pig antirat insulin antibody, 125I-labeled human insulin as tracer, and rat insulin as standard (Linco Research, St. Charles, MO). Free and bound radioactivity was separated by use of an anti-IgG (goat antiguinea pig) antibody (Linco Research). The sensitivity of the assay was 12 pmol/liter, and the coefficient of variation was less than 3% within assays and less than 5% between assays. Plasma glucose concentrations were determined with the glucose oxidase technique.
Calculations and statistics
Data and results are reported as means ± SEM. The acute insulin response to iv glucose (AIR) with or without administration of GLP-1, GIP, PACAP38, or GRP was calculated as the mean of suprabasal 1- and 5-min values. The glucose elimination was quantified as the glucose elimination constant (KG), the reduction in circulating glucose between min 1 and 20 after iv administration after logarithmic transformation of the individual plasma glucose values, and expressed as percent elimination of glucose per minute. Statistical comparisons were performed with Student’s t test.
Results
Figure 1 shows that the iv administration of glucose (1 g/kg) rapidly increased the circulating glucose and insulin levels, which both peaked at 1 min. Administration of val-pyr through a gavage 30 min before the iv glucose administration did not affect the glucose elimination rate or the insulin response to glucose.
FIG. 1. Plasma levels of insulin and glucose (inset) immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) in mice pretreated 30 min before the iv administration with administration of val-pyr (100 μmol/kg) or saline through a gastric gavage. Means ± SEM are shown. There were eight animals in each group.
Figure 2 shows the insulin and glucose responses to iv administration of glucose plus each of the peptides GLP-1, GIP, PACAP38, or GRP with or without preadministration with val-pyr. All these peptides augmented the insulin response to glucose. When val-pyr was administered 30 min before the peptides, the increase in insulin levels induced by the respective peptides was augmented. This altered insulin response to the peptides by val-pyr was associated with augmented glucose elimination after GLP-1 and PACAP38 but not after GIP and GRP. Table 1 shows the calculated AIR and KG for the individual experimental groups. The insulin response to GLP-1 was augmented by 80%, that to GIP by 40%, that to PACAP38 by 75%, and that to GRP by 25%. This was associated with enhanced glucose elimination rate after GLP-1 and PACAP38 (both P < 0.01), but not after GIP or GRP.
FIG. 2. Plasma levels of insulin and (inset) glucose immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) plus GLP-1 (10 nmol/kg), PACAP38 (1.3 nmol/kg), GIP (10 nmol/kg), or GRP (20 nmol/kg) in mice pretreated 30 min before the iv administration with administration of val-pyr (100 μmol/kg) or saline through a gastric gavage. Means ± SEM are shown. There were eight animals in each group in the experiments with GLP-1 and PACAP38 and 12 animals in each group in the experiments with GIP and GRP. Dotted lines show the mean insulin and glucose curves after iv administration of glucose alone (from Fig. 1).
TABLE 1. The AIR (i.e. the mean suprabasal 1 and 5 min insulin) and the KG (i.e. the fractional glucose elimination per minute during min 1–20) after iv administration of glucose (1 g/kg) alone or together with GLP-1 (10 nmol/kg), GIP (10 nmol/kg), PACAP38 (1.3 nmol/kg), or GRP (20 nmol/kg) preceded by administration through gastric gavage of val-pyr (100 μmol/kg) or saline (control) 30 min before the iv injection
The augmentation by val-pyr of GRP-induced insulin secretion was examined in more detail. First, it was examined whether a C-terminal peptide form of GRP (GRP14-27) augments glucose-stimulated insulin secretion in mice. Figure 3 shows that both GRP and GRP(14-27) augmented the insulin response to glucose. The AIR was 0.40 ± 0.04 nmol/liter after administration of glucose alone, and this was increased to 1.3 ± 0.10 nmol/liter by glucose + GRP (P < 0.001) and to 1.1 ± 0.080 by glucose + GRP(14–27) (P = 0.007). There was no significant difference between the effects of GRP vs. GRP(14–27). Second, it was examined whether a GLP-1 receptor antagonist, exendin3 (9-39), is able to affect the augmentation by val-pyr of GRP-induced insulin secretion. It was found that AIR after administration of GRP and glucose was 1.2 ± 0.1 nmol/liter (n = 6). This was augmented to 2.1 ± 0.2 nmol/liter (n = 6; P = 0.012) by val-pyr. However, when exendin3 (9-39) was administered together with GRP, val-pyr could no longer augment the insulin response. Thus, when exendin3 (9-39) and GRP were given with glucose, the AIR was 0.90 ± 0.10 nmol/liter, and when val-pyr was added, AIR was 0.92 ± 0.11 nmol/liter (n = 6 in both groups, not significant).
FIG. 3. Plasma levels of insulin and glucose (inset) in mice immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) together with GRP (20 nmol/kg) or GRP14-27 (20 nmol/kg). Means ± SEM are shown. There were eight animals in each group.
Discussion
DPP-4 is a ubiquitously distributed glycoprotein enzyme, which is involved in the metabolism of circulating peptide fragments, and inactivates a number of biologically active peptides through removal of the two N-terminal amino acids of the peptides by peptidase activity, provided that the amino acid in position number 2 is either proline or alanine (1). Because DPP-4 inactivates the main incretin hormone, GLP-1, the enzyme is thought to limit the potentiation of insulin secretion by this hormone. This is consistent with results that DPP-4 inhibition by val-pyr augments the insulin response to exogenous administration of GLP-1 (18). This has been the background for the development of DPP-4 inhibition in the treatment of type 2 diabetes because such inhibition augments incretin activity (4, 5). Indeed, both under experimental conditions and in subjects with type 2 diabetes, DPP-4 inhibition has been proven to exert antidiabetic effects (6, 7, 8, 9, 10, 11). However, because DPP-4 inactivates a number of biologically active peptides (1), the antidiabetic action of DPP-4 inhibition on GLP-1 activity might be further augmented by broader effects, including prevention of inactivation of non-GLP-1 peptides.
The present study shows that DPP-4 inhibition by val-pyr augments the insulin response not only to GLP-1 but also to PACAP38, GIP and GRP. Because all these peptides have been shown to be substrates for DPP-4 (1, 2, 14, 15, 16), it may be assumed that the augmentation of the insulin responses to these peptides is due to prevention of their rapid inactivation by the inhibition of DPP-4. This may be the case for GLP-1, GIP, and PACAP38 (2, 14, 16); therefore, the results of augmented insulin response to these peptides by val-pyr are expected. In contrast, it should be emphasized that although GRP has been shown to be a substrate for DPP-4 in vitro, it is not known whether DPP-4 degrades GRP in vivo. Furthermore, it has been demonstrated that the insulin-releasing property of GRP resides in the C-terminal end of the peptide (25); therefore, even though DPP-4 removes the two N-terminal amino acids of GRP in vivo, it is not clear how this would augment insulin secretion. Therefore, we examined the augmentation by val-pyr of GRP-induced insulin secretion in more detail. First, we showed that the C-terminal form of GRP, GRP(14-27), had similar insulin-releasing property as GRP. This shows that the N-terminal end of GRP is not required for its insulin-releasing action. We also showed that exendin3 (9-39), which is a GLP-1 receptor antagonist (26), prevented the augmentation by val-pyr of GRP-induced insulin secretion. These results suggest that insulin secretion after GRP is at least partially dependent on GLP-1. Based on this observation, we suggest that GRP causes a release of GLP-1, which in turn contributes to the insulin response to GRP. This is consistent with a previous report that GLP-1 levels are reduced in mice with genetic deletion of the GRP receptors (27). Hence, although val-pyr augments GRP-induced insulin secretion, this is most likely dependent on prevention of inactivation of GLP-1, rather than on prevention of inactivation of GRP. Therefore, our results may also indicate that GRP is not a substrate for DPP-4 under in vivo conditions, although this needs to be examined in more detail.
Val-pyr is not specific in inhibiting DPP-4 because at a minimum the related protease DPP-8, although an intracellular enzyme, also is inhibited by the compound. However, previous studies in DPP-4-deficient mice have shown that the effects of this compound on insulin secretion and glucose homeostasis are due to its inhibition of DPP-4 (12). It has also been shown that val-pyr does not affect glucose-stimulated insulin secretion from isolated islets (8), showing that a direct ?-cell effect of the compound is less likely to explain its effects. This latter view is consistent with the present finding that the insulin response to iv glucose was not affected by val-pyr. Hence, the augmenting effect by val-pyr on insulin secretion is most likely a consequence of an augmentation of the insulin response to the respective peptides due to the increase in plasma concentrations of the active peptides caused by the prevention of their degradation by DPP-4. However, whether prevention of inactivation of these peptides contributes to the antidiabetic action of DPP-4 inhibitors remains to be established.
The augmented insulin responses to GLP-1 and PACAP38 by val-pyr resulted in increased glucose elimination, whereas this was not observed after GIP and GRP, despite augmented insulin response also after these peptides. This pattern is consistent with the relative magnitude of augmentation of insulin secretion by val-pyr for the four peptides, being higher for GLP-1 and PACAP38 and lower for GIP and GRP. At first sight, it may be surprising that glucose elimination was not augmented also after the augmented insulin responses to GIP and GRP after val-pyr, but it should be emphasized that in mice, insulin-independent mechanisms contribute to a large extent (70%) to glucose elimination (28). Therefore, the smaller changes in insulin response observed for GIP and GRP may not have been sufficient to augment glucose elimination in these mice.
A recent study showed that DPP-4 inhibition by val-pyr failed to augment insulin secretion and glucose tolerance after oral glucose in mice subjected to double knockout of the GLP-1 receptor and GIP receptor (29). This would suggest that the antidiabetic action of DPP-4 inhibition in these animals is entirely explained by prevention of inactivation of the two main incretin hormones, GLP-1 and GIP. However, although this may be the case after acute administration of glucose through gastric gavage, other mechanisms may still contribute to long-term effects of DPP-4 inhibition, including those that might occur in the intermeal period. In particular, the present findings that the insulin response to the neuropeptide, PACAP38, is augmented by DPP-4 inhibition suggest that enhancement of neurally induced islet effects may be an additional mechanism underlying antidiabetic actions of DPP-4 inhibitors. Neural mechanisms may operate during the cephalic phase of insulin secretion during meal ingestion and additionally as mechanisms of the compensatory increase in insulin secretion and islet mass during insulin resistance (17). Hence, beneficial effects of DPP-4 inhibition may go beyond augmentation of insulin responses during an oral glucose load, as has been shown in long-term studies (30), and such effects may not be explained solely by prevention of incretin hormone inactivation. Therefore, these additional substrates of DPP-4 should be further evaluated for their potential to contribute to the insulin secretion seen in response to DPP-4 inhibitor treatment. It should be emphasized, however, that our conclusion is valid in these model experiments in mice and does not imply that such an action contributes to the beneficial antidiabetic effect of DPP-4 in clinical trials (10, 11). This is because it is not yet known whether these bioactive peptides are involved in the insulin response to meal ingestion in humans.
In conclusion, we have shown in this study that DPP-4 inhibition by val-pyr augments the insulin responses to iv administration of the incretin hormones, GLP-1 and GIP, and the neuropeptides, PACAP38 and GRP, in model experiments in mice. These results show that prevention of the inactivation of these four peptides independently augments islet function. This suggests that prevention of inactivation of several biologically active peptides may underlie the antidiabetic action of DPP-4 inhibitors. This suggests that biologically active peptides other than GLP-1 and GIP may participate in the antidiabetic actions of DPP-4 inhibitors.
Acknowledgments
We are grateful to Lilian Bengtsson and Lena Kvist for expert technical assistance.
References
Mentlein R 1999 Dipeptidyl-peptidase IV (CD26): role in the inactivation of regulatory peptides. Regul Pept 85:9–24
Deacon CF, Johnsen AH, Holst JJ 1995 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 80:952–957
Ahrén B 1998 Glucagon-like peptide 1 (GLP-1): a gut hormone of potential interest in the treatment of diabetes. Bioessays 20:642–651
Drucker DJ 2003 Therapeutic potential of dipeptidyl peptidase IV inhibitors for the treatment of type 2 diabetes. Exp Opin Investig Drugs 12:87–100
Holst JJ, Deacon CF 1998 Inhibition of activity of dipeptidyl peptidase IV as a treatment for type 2 diabetes. Diabetes 47:1663–1670
Pederson RA, White HA, Schlenzig D, Pauly RP, McIntosh CHS, Dermuth HU 1998 Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidylpeptidase IV inhibitor isoleucine thiazolidide. Diabetes 47:1253–1258
Balkan B, Kwasnik L, Miserendino R, Holst JJ, Li X 1999 Inhibition of dipeptidyl peptidase IV with NVP DPP728 increases plasma GLP-1 (7-36 amide) concentrations and improves oral glucose tolerance in obese Zucker rats. Diabetologia 42:1324–1331
Ahrén B, Holst JJ, M?rtensson H, Balkan B 2000 Improved glucose tolerance and insulin secretion by inhibition of dipeptidyl peptidase IV in mice. Eur J Pharmacol 404:239–245
Kvist Reimer M, Holst JJ, Ahrén B 2002 Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice. Eur J Endocrinol 146:717–727
Ahrén B, Landin-Olsson M, Jansson PA, Svensson M, Holmes D, Schweizer A 2004 Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels and reduced glucagon levels in type 2 diabetes. J Clin Endocrinol Metab 89:2078–2084
Ahrén B, Simonsson E, Larsson H, Landin-Olsson M, Torgeirsson H, Jansson PA, Sandqvist M, B?venholm P, Efendic S, Eriksson JW, Dickinson S, Holmes D 2002 Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4 week study period in type 2 diabetes. Diabetes Care 25:869–875
Marguet D, Baggio L, Kobayashi T, Bernard AM, Pierres M, Nielsen PF, Ribel U, Watanabe T, Drucker DJ, Wagtmann N 2000 Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26. Proc Natl Acad Sci USA 97:6874–6879
Nagakura T, Yasuda N, Yamazaki K, Ikuta H, Yoshikawa S, Asano O, Tanaka I 2001 Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats. Biochem Biophys Res Commun 284:501–506
Lambeir AM, Durinx C, Proost P, van Damme J, Scharpé S, De Meester I 2001 Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion. FEBS Lett 507:327–330
Pauly RP, Rosche F, Wermann M, McIntosh CH, Pederson RA, Demuth HU 1996 Investigation of glucose-dependent insulinotropic polypeptide (1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry: a novel kinetic approach. J Biol Chem 271:23222–23229
Zhu L, Tamvakopoulos C, Xie D, Dragovic J, Shen X, Fenyk-Melody JE, Schmidt K, Bagchi A, Griffin PR, Thornberry NA, Sinha Roy R 2003 The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-1(1–38). J Biol Chem 278:22418–22423
Ahrén B 2000 Autonomic regulation of islet hormone secretion. Implications for health and disease. Diabetologia 43:393–410
Deacon CF, Hughes TE, Holst JJ 1998 Dipeptidyl peptidase IV inhibition potentiates the insulinotropic effect of glucagon-like peptide 1 in the anesthetized pig. Diabetes 47:764–769
Filipsson K, Pacini G, Scheurink AJW, Ahrén B 1998 PACAP stimulates insulin secretion but inhibits insulin sensitivity in mice. Am J Physiol 274:E834–E842
Meier JJ, Nauck MA, Schmidt WE, Gallwitz B 2002 Gastric inhibitory polypeptide: the neglected incretin revisited. Regul Pept 107:1–13
Vilsb?ll T, Holst JJ 2004 Incretins, insulin secretion and type 2 diabetes mellitus. Diabetologia 47:357–366
Ahrén B, Hedner P, Lundquist I 1983 Interaction of gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK-8) with basal and stimulated insulin secretion in mice. Acta Endocrinol 102:96–102
Ahrén B, Pacini G 1999 Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice. Am J Physiol 277:E996–E1004
Pettersson M, Ahrén B 1987 Gastrin releasing peptide (GRP): Effects on basal and stimulated insulin and glucagon secretion in the mouse. Peptides 8:55–60
Kawai K, Mukai H, Yuzawa K, Suzuki S, Kuzuya N, Fuji K, Munekata E, Yamashita K 1990 Effects of neuromedin B and GRP-10 on gastrin and insulin release from cultured tumor cells of a malignant gastrinoma. Endocrinol Jpn 37:857–865
Ahrén B, Pacini G 1999 Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice. Am J Physiol 277:E996–E1004
Persson K, Gingerich RL, Nayak S, Wada K, Wada E, Ahrén B 2000 Reduced GLP-1 and insulin responses and glucose intolerance after gastric glucose in GRP receptor-deleted mice. Am J Physiol 279:E956–E962
Pacini G, Thomaseth K, Ahrén B 2001 Contribution to glucose intolerance of insulin-independent vs. insulin-dependent mechanisms in mice. Am J Physiol 281:E693–E703
Hansotia T, Baggio LLK, Delmeire D, Hinke SA, Yamada Y, Tsukiyama K, Seino Y, Holst. JJ, Schuit F, Drucker DJ 2004 Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing glucoregulatory actions of DPP-IV inhibitors. Diabetes 53:1326–1335
Pospisilik JA, Stafford SG, Demuth HU, Brownsey R, Parkhouse W, Finegood DT, McIntosh CH, Pederson RA 2002 Long-term treatment with the DPP-4 inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hypertinsulinemia, and ?-cell glucose responsiveness in VDF (fa/fa) Zucker rats. Diabetes 512:943–950(Bo Ahrén and Thomas E. Hu)
Address all correspondence and requests for reprints to: Dr. Bo Ahrén, Department of Medicine, Lund University, B11 BMC, SE-221 84 Lund, Sweden. E-mail: bo.ahren@med.lu.se.
Abstract
Inhibition of dipeptidyl peptidase-4 (DPP-4) is currently being explored as a new approach to the treatment of type 2 diabetes. This concept has emerged from the powerful and rapid action of the enzyme to inactivate glucagon-like peptide-1 (GLP-1). However, other bioactive peptides with potential influence of islet function are also substrates of DPP-4. Whether this inactivation may add to the beneficial effects of DPP-4 inhibition is not known. In this study, we explored whether DPP-4 inhibition by valine-pyrrolidide (val-pyr; 100 μmol/kg administered through gastric gavage at t = –30 min) affects the insulin and glucose responses to iv glucose (1 g/kg) together with GLP-1 (10 nmol/kg), glucose-dependent insulinotropic polypeptide (GIP; 10 nmol/kg), pituitary adenylate cyclase-activating polypeptide 38 (PACAP38; 1.3 nmol/kg), or gastrin-releasing peptide (GRP; 20 nmol/kg) given at t = 0 in anesthetized C57BL/6J mice. It was found that the acute (1–5 min) insulin response to GLP-1 was augmented by val-pyr by 80% (4.2 ± 0.4 vs. 7.6 ± 0.8 nmol/liter), that to GIP by 40% (2.7 ± 0.3 vs. 3.8 ± 0.4 nmol/liter), that to PACAP38 by 75% (4.6 ± 0.5 vs. 8.1 ± 0.6 nmol/liter), and that to GRP by 25% (1.8 ± 0.2 vs. 2.3 ± 0.3 nmol/liter; all P < 0.05 or less). This was associated with enhanced glucose elimination rate after GLP-1 [glucose elimination constant (KG) 2.1 ± 0.2 vs. 3.1 ± 0.3%/min] and PACAP38 (2.1 ± 0.3 vs. 3.2 ± 0.3%/min; both P < 0.01), but not after GIP or GRP. The augmented insulin response to GRP by val-pyr was prevented by the GLP-1 receptor antagonist, exendin3 (9-39), raising the possibility that GRP effects may occur secondary to stimulation of GLP-1 secretion. We conclude that DPP-4 inhibition augments the insulin response not only to GLP-1 but also to GIP, PACAP38, and GRP.
Introduction
THE ENZYME DIPEPTIDYL peptidase-4 (DPP-4) is a cell surface serine peptidase, which is distributed throughout the endothelial lining of the vasculature, and which also is circulating in a soluble form (1). The enzyme inactivates a number of biologically active peptides containing Xaa-Ala or Xaa-Pro aminoterminal sequences by removing their N-terminal dipeptide residues. Glucagon-like peptide-1 (GLP-1) is one of the substrates of DPP-4, being inactivated by removal of the N-terminal histidine-alanine dipeptide moiety (2). Because GLP-1 has been shown to exert antidiabetic actions (3), prevention of its inactivation by inhibition of DPP-4 is currently being explored as a new treatment for type 2 diabetes (4, 5). DPP-4 inhibition has thereby been demonstrated to be antidiabetic both in animal models of diabetes (6, 7, 8, 9) and in patients with type 2 diabetes (10, 11). This is consistent with findings that animals with genetic deletion of DPP-4 activity exhibit improved glucose tolerance (12, 13). Due to recognition of their aminoterminal sequences, several other peptides are, however, also potential substrates of DPP-4, such as glucose-dependent insulinotropic polypeptide (GIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and gastrin-releasing peptide (GRP) (1, 2, 14, 15, 16). Like GLP-1, these peptides may be involved in the regulation of glucose homeostasis and insulin secretion because all of them are potent insulinotropic peptides and augment glucose-stimulated insulin secretion (17, 18, 19, 20). They have, however, different relations to islet function. Thus, GLP-1 and GIP are incretins released from the gut in association with meal ingestion and, therefore, of importance for the postprandial insulin response (3, 20, 21), whereas PACAP and GRP are neuropeptides being signals of pancreatic nerves and, therefore, involved in the neural regulation of islet function (17). Prevention of inactivation of these peptides may contribute to the antidiabetic ability of DPP-4 inhibitors, provided that DPP-4 inhibition increases the active component of the peptides to such an extent that their insulinotropic actions or other antidiabetic effects are augmented. In the present study, therefore, mice were given the DPP-4 inhibitor, valine-pyrrolidide (val-pyr), followed by iv administration of these peptides to explore whether their actions are perturbed. Val-pyr has previously been shown to inhibit DPP-4 activity to such an extent that it augments GLP-1 levels in pigs and mice and results in enhanced insulinotropic activity of exogenously added GLP-1 (8, 18).
Subjects and Methods
Animals
Female C57BL/6J mice, weighing 21–24 g, obtained from Taconic A/S (Ry, Denmark), were used. The animals were kept in a 12-h light schedule (lights on at 0600 h) and given a standard pellet diet (fat, 11.4%; carbohydrate, 62.8%; and protein, 25.8% on an energy base, total energy 12.6 kJ/g) and tap water ad libitum. The Lund University Ethical Committee (Lund, Sweden) approved the study.
In vivo experiments
The studies were performed in late morning after removal of food from the cages 4 h earlier. The animals were anesthetized with an ip injection of midazolam (0.2 mg/mouse, Dormicum, Hoffman-La Roche, Basel, Switzerland) and a combination of fluanisone (0.4 mg/mouse) and fentanyl (0.02 mg/mouse, Hypnorm, Janssen, Beerse, Belgium). Thirty minutes later, val-pyr was given through a gastric tube, with an outer diameter of 1.2 mm (100 μmol/kg; a kind gift from Novartis Institutes for Biomedical Research, Cambridge, MA). After another 30 min, a blood sample was taken from the retrobulbar, intraorbital, capillary plexus in heparinized tubes, whereafter D-glucose (British Drug Houses, Poole, UK; 1 g/kg) was rapidly injected iv alone or together with synthetic GLP-1 (Peninsula Europe Laboratories, 10 nmol/kg), synthetic porcine GIP (Sigma, St. Louis, MO; 10 nmol/kg), synthetic ovine PACAP38 (Peninsula Europe Laboratories; 1.3 nmol/kg), synthetic porcine GRP (Sigma; 20 nmol/kg), synthetic GRP14-27 (Sigma; 20 nmol/kg), or GRP together with synthetic exendin3 (9-39) (Peninsula Europe Laboratories; 30 nmol/kg). Controls were given saline. The doses selected for the peptides have previously been shown to be maximal in augmenting glucose-stimulated insulin secretion after iv administration in mice (19, 22, 23, 24). The volume load was 10 μl/g body weight. At specific time points after the iv injection, blood samples, consisting of 75 μl blood each, were collected. A total of seven samples were taken during each experiment, at t = 0, 1, 5, 10, 20, 30, and 50 min. The removal of this amount of blood has previously been shown not to alter baseline glucose levels in mice (19). Blood was kept in heparinized tubes; then, after immediate centrifugation, plasma was separated and stored at –20 C until analysis was carried out.
Assays
Insulin was determined radioimmunochemically with the use of a guinea pig antirat insulin antibody, 125I-labeled human insulin as tracer, and rat insulin as standard (Linco Research, St. Charles, MO). Free and bound radioactivity was separated by use of an anti-IgG (goat antiguinea pig) antibody (Linco Research). The sensitivity of the assay was 12 pmol/liter, and the coefficient of variation was less than 3% within assays and less than 5% between assays. Plasma glucose concentrations were determined with the glucose oxidase technique.
Calculations and statistics
Data and results are reported as means ± SEM. The acute insulin response to iv glucose (AIR) with or without administration of GLP-1, GIP, PACAP38, or GRP was calculated as the mean of suprabasal 1- and 5-min values. The glucose elimination was quantified as the glucose elimination constant (KG), the reduction in circulating glucose between min 1 and 20 after iv administration after logarithmic transformation of the individual plasma glucose values, and expressed as percent elimination of glucose per minute. Statistical comparisons were performed with Student’s t test.
Results
Figure 1 shows that the iv administration of glucose (1 g/kg) rapidly increased the circulating glucose and insulin levels, which both peaked at 1 min. Administration of val-pyr through a gavage 30 min before the iv glucose administration did not affect the glucose elimination rate or the insulin response to glucose.
FIG. 1. Plasma levels of insulin and glucose (inset) immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) in mice pretreated 30 min before the iv administration with administration of val-pyr (100 μmol/kg) or saline through a gastric gavage. Means ± SEM are shown. There were eight animals in each group.
Figure 2 shows the insulin and glucose responses to iv administration of glucose plus each of the peptides GLP-1, GIP, PACAP38, or GRP with or without preadministration with val-pyr. All these peptides augmented the insulin response to glucose. When val-pyr was administered 30 min before the peptides, the increase in insulin levels induced by the respective peptides was augmented. This altered insulin response to the peptides by val-pyr was associated with augmented glucose elimination after GLP-1 and PACAP38 but not after GIP and GRP. Table 1 shows the calculated AIR and KG for the individual experimental groups. The insulin response to GLP-1 was augmented by 80%, that to GIP by 40%, that to PACAP38 by 75%, and that to GRP by 25%. This was associated with enhanced glucose elimination rate after GLP-1 and PACAP38 (both P < 0.01), but not after GIP or GRP.
FIG. 2. Plasma levels of insulin and (inset) glucose immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) plus GLP-1 (10 nmol/kg), PACAP38 (1.3 nmol/kg), GIP (10 nmol/kg), or GRP (20 nmol/kg) in mice pretreated 30 min before the iv administration with administration of val-pyr (100 μmol/kg) or saline through a gastric gavage. Means ± SEM are shown. There were eight animals in each group in the experiments with GLP-1 and PACAP38 and 12 animals in each group in the experiments with GIP and GRP. Dotted lines show the mean insulin and glucose curves after iv administration of glucose alone (from Fig. 1).
TABLE 1. The AIR (i.e. the mean suprabasal 1 and 5 min insulin) and the KG (i.e. the fractional glucose elimination per minute during min 1–20) after iv administration of glucose (1 g/kg) alone or together with GLP-1 (10 nmol/kg), GIP (10 nmol/kg), PACAP38 (1.3 nmol/kg), or GRP (20 nmol/kg) preceded by administration through gastric gavage of val-pyr (100 μmol/kg) or saline (control) 30 min before the iv injection
The augmentation by val-pyr of GRP-induced insulin secretion was examined in more detail. First, it was examined whether a C-terminal peptide form of GRP (GRP14-27) augments glucose-stimulated insulin secretion in mice. Figure 3 shows that both GRP and GRP(14-27) augmented the insulin response to glucose. The AIR was 0.40 ± 0.04 nmol/liter after administration of glucose alone, and this was increased to 1.3 ± 0.10 nmol/liter by glucose + GRP (P < 0.001) and to 1.1 ± 0.080 by glucose + GRP(14–27) (P = 0.007). There was no significant difference between the effects of GRP vs. GRP(14–27). Second, it was examined whether a GLP-1 receptor antagonist, exendin3 (9-39), is able to affect the augmentation by val-pyr of GRP-induced insulin secretion. It was found that AIR after administration of GRP and glucose was 1.2 ± 0.1 nmol/liter (n = 6). This was augmented to 2.1 ± 0.2 nmol/liter (n = 6; P = 0.012) by val-pyr. However, when exendin3 (9-39) was administered together with GRP, val-pyr could no longer augment the insulin response. Thus, when exendin3 (9-39) and GRP were given with glucose, the AIR was 0.90 ± 0.10 nmol/liter, and when val-pyr was added, AIR was 0.92 ± 0.11 nmol/liter (n = 6 in both groups, not significant).
FIG. 3. Plasma levels of insulin and glucose (inset) in mice immediately before and at 1, 5, 10, 20, 30, and 50 min after the iv administration of glucose (1 g/kg) together with GRP (20 nmol/kg) or GRP14-27 (20 nmol/kg). Means ± SEM are shown. There were eight animals in each group.
Discussion
DPP-4 is a ubiquitously distributed glycoprotein enzyme, which is involved in the metabolism of circulating peptide fragments, and inactivates a number of biologically active peptides through removal of the two N-terminal amino acids of the peptides by peptidase activity, provided that the amino acid in position number 2 is either proline or alanine (1). Because DPP-4 inactivates the main incretin hormone, GLP-1, the enzyme is thought to limit the potentiation of insulin secretion by this hormone. This is consistent with results that DPP-4 inhibition by val-pyr augments the insulin response to exogenous administration of GLP-1 (18). This has been the background for the development of DPP-4 inhibition in the treatment of type 2 diabetes because such inhibition augments incretin activity (4, 5). Indeed, both under experimental conditions and in subjects with type 2 diabetes, DPP-4 inhibition has been proven to exert antidiabetic effects (6, 7, 8, 9, 10, 11). However, because DPP-4 inactivates a number of biologically active peptides (1), the antidiabetic action of DPP-4 inhibition on GLP-1 activity might be further augmented by broader effects, including prevention of inactivation of non-GLP-1 peptides.
The present study shows that DPP-4 inhibition by val-pyr augments the insulin response not only to GLP-1 but also to PACAP38, GIP and GRP. Because all these peptides have been shown to be substrates for DPP-4 (1, 2, 14, 15, 16), it may be assumed that the augmentation of the insulin responses to these peptides is due to prevention of their rapid inactivation by the inhibition of DPP-4. This may be the case for GLP-1, GIP, and PACAP38 (2, 14, 16); therefore, the results of augmented insulin response to these peptides by val-pyr are expected. In contrast, it should be emphasized that although GRP has been shown to be a substrate for DPP-4 in vitro, it is not known whether DPP-4 degrades GRP in vivo. Furthermore, it has been demonstrated that the insulin-releasing property of GRP resides in the C-terminal end of the peptide (25); therefore, even though DPP-4 removes the two N-terminal amino acids of GRP in vivo, it is not clear how this would augment insulin secretion. Therefore, we examined the augmentation by val-pyr of GRP-induced insulin secretion in more detail. First, we showed that the C-terminal form of GRP, GRP(14-27), had similar insulin-releasing property as GRP. This shows that the N-terminal end of GRP is not required for its insulin-releasing action. We also showed that exendin3 (9-39), which is a GLP-1 receptor antagonist (26), prevented the augmentation by val-pyr of GRP-induced insulin secretion. These results suggest that insulin secretion after GRP is at least partially dependent on GLP-1. Based on this observation, we suggest that GRP causes a release of GLP-1, which in turn contributes to the insulin response to GRP. This is consistent with a previous report that GLP-1 levels are reduced in mice with genetic deletion of the GRP receptors (27). Hence, although val-pyr augments GRP-induced insulin secretion, this is most likely dependent on prevention of inactivation of GLP-1, rather than on prevention of inactivation of GRP. Therefore, our results may also indicate that GRP is not a substrate for DPP-4 under in vivo conditions, although this needs to be examined in more detail.
Val-pyr is not specific in inhibiting DPP-4 because at a minimum the related protease DPP-8, although an intracellular enzyme, also is inhibited by the compound. However, previous studies in DPP-4-deficient mice have shown that the effects of this compound on insulin secretion and glucose homeostasis are due to its inhibition of DPP-4 (12). It has also been shown that val-pyr does not affect glucose-stimulated insulin secretion from isolated islets (8), showing that a direct ?-cell effect of the compound is less likely to explain its effects. This latter view is consistent with the present finding that the insulin response to iv glucose was not affected by val-pyr. Hence, the augmenting effect by val-pyr on insulin secretion is most likely a consequence of an augmentation of the insulin response to the respective peptides due to the increase in plasma concentrations of the active peptides caused by the prevention of their degradation by DPP-4. However, whether prevention of inactivation of these peptides contributes to the antidiabetic action of DPP-4 inhibitors remains to be established.
The augmented insulin responses to GLP-1 and PACAP38 by val-pyr resulted in increased glucose elimination, whereas this was not observed after GIP and GRP, despite augmented insulin response also after these peptides. This pattern is consistent with the relative magnitude of augmentation of insulin secretion by val-pyr for the four peptides, being higher for GLP-1 and PACAP38 and lower for GIP and GRP. At first sight, it may be surprising that glucose elimination was not augmented also after the augmented insulin responses to GIP and GRP after val-pyr, but it should be emphasized that in mice, insulin-independent mechanisms contribute to a large extent (70%) to glucose elimination (28). Therefore, the smaller changes in insulin response observed for GIP and GRP may not have been sufficient to augment glucose elimination in these mice.
A recent study showed that DPP-4 inhibition by val-pyr failed to augment insulin secretion and glucose tolerance after oral glucose in mice subjected to double knockout of the GLP-1 receptor and GIP receptor (29). This would suggest that the antidiabetic action of DPP-4 inhibition in these animals is entirely explained by prevention of inactivation of the two main incretin hormones, GLP-1 and GIP. However, although this may be the case after acute administration of glucose through gastric gavage, other mechanisms may still contribute to long-term effects of DPP-4 inhibition, including those that might occur in the intermeal period. In particular, the present findings that the insulin response to the neuropeptide, PACAP38, is augmented by DPP-4 inhibition suggest that enhancement of neurally induced islet effects may be an additional mechanism underlying antidiabetic actions of DPP-4 inhibitors. Neural mechanisms may operate during the cephalic phase of insulin secretion during meal ingestion and additionally as mechanisms of the compensatory increase in insulin secretion and islet mass during insulin resistance (17). Hence, beneficial effects of DPP-4 inhibition may go beyond augmentation of insulin responses during an oral glucose load, as has been shown in long-term studies (30), and such effects may not be explained solely by prevention of incretin hormone inactivation. Therefore, these additional substrates of DPP-4 should be further evaluated for their potential to contribute to the insulin secretion seen in response to DPP-4 inhibitor treatment. It should be emphasized, however, that our conclusion is valid in these model experiments in mice and does not imply that such an action contributes to the beneficial antidiabetic effect of DPP-4 in clinical trials (10, 11). This is because it is not yet known whether these bioactive peptides are involved in the insulin response to meal ingestion in humans.
In conclusion, we have shown in this study that DPP-4 inhibition by val-pyr augments the insulin responses to iv administration of the incretin hormones, GLP-1 and GIP, and the neuropeptides, PACAP38 and GRP, in model experiments in mice. These results show that prevention of the inactivation of these four peptides independently augments islet function. This suggests that prevention of inactivation of several biologically active peptides may underlie the antidiabetic action of DPP-4 inhibitors. This suggests that biologically active peptides other than GLP-1 and GIP may participate in the antidiabetic actions of DPP-4 inhibitors.
Acknowledgments
We are grateful to Lilian Bengtsson and Lena Kvist for expert technical assistance.
References
Mentlein R 1999 Dipeptidyl-peptidase IV (CD26): role in the inactivation of regulatory peptides. Regul Pept 85:9–24
Deacon CF, Johnsen AH, Holst JJ 1995 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 80:952–957
Ahrén B 1998 Glucagon-like peptide 1 (GLP-1): a gut hormone of potential interest in the treatment of diabetes. Bioessays 20:642–651
Drucker DJ 2003 Therapeutic potential of dipeptidyl peptidase IV inhibitors for the treatment of type 2 diabetes. Exp Opin Investig Drugs 12:87–100
Holst JJ, Deacon CF 1998 Inhibition of activity of dipeptidyl peptidase IV as a treatment for type 2 diabetes. Diabetes 47:1663–1670
Pederson RA, White HA, Schlenzig D, Pauly RP, McIntosh CHS, Dermuth HU 1998 Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidylpeptidase IV inhibitor isoleucine thiazolidide. Diabetes 47:1253–1258
Balkan B, Kwasnik L, Miserendino R, Holst JJ, Li X 1999 Inhibition of dipeptidyl peptidase IV with NVP DPP728 increases plasma GLP-1 (7-36 amide) concentrations and improves oral glucose tolerance in obese Zucker rats. Diabetologia 42:1324–1331
Ahrén B, Holst JJ, M?rtensson H, Balkan B 2000 Improved glucose tolerance and insulin secretion by inhibition of dipeptidyl peptidase IV in mice. Eur J Pharmacol 404:239–245
Kvist Reimer M, Holst JJ, Ahrén B 2002 Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice. Eur J Endocrinol 146:717–727
Ahrén B, Landin-Olsson M, Jansson PA, Svensson M, Holmes D, Schweizer A 2004 Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels and reduced glucagon levels in type 2 diabetes. J Clin Endocrinol Metab 89:2078–2084
Ahrén B, Simonsson E, Larsson H, Landin-Olsson M, Torgeirsson H, Jansson PA, Sandqvist M, B?venholm P, Efendic S, Eriksson JW, Dickinson S, Holmes D 2002 Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4 week study period in type 2 diabetes. Diabetes Care 25:869–875
Marguet D, Baggio L, Kobayashi T, Bernard AM, Pierres M, Nielsen PF, Ribel U, Watanabe T, Drucker DJ, Wagtmann N 2000 Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26. Proc Natl Acad Sci USA 97:6874–6879
Nagakura T, Yasuda N, Yamazaki K, Ikuta H, Yoshikawa S, Asano O, Tanaka I 2001 Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats. Biochem Biophys Res Commun 284:501–506
Lambeir AM, Durinx C, Proost P, van Damme J, Scharpé S, De Meester I 2001 Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion. FEBS Lett 507:327–330
Pauly RP, Rosche F, Wermann M, McIntosh CH, Pederson RA, Demuth HU 1996 Investigation of glucose-dependent insulinotropic polypeptide (1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry: a novel kinetic approach. J Biol Chem 271:23222–23229
Zhu L, Tamvakopoulos C, Xie D, Dragovic J, Shen X, Fenyk-Melody JE, Schmidt K, Bagchi A, Griffin PR, Thornberry NA, Sinha Roy R 2003 The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-1(1–38). J Biol Chem 278:22418–22423
Ahrén B 2000 Autonomic regulation of islet hormone secretion. Implications for health and disease. Diabetologia 43:393–410
Deacon CF, Hughes TE, Holst JJ 1998 Dipeptidyl peptidase IV inhibition potentiates the insulinotropic effect of glucagon-like peptide 1 in the anesthetized pig. Diabetes 47:764–769
Filipsson K, Pacini G, Scheurink AJW, Ahrén B 1998 PACAP stimulates insulin secretion but inhibits insulin sensitivity in mice. Am J Physiol 274:E834–E842
Meier JJ, Nauck MA, Schmidt WE, Gallwitz B 2002 Gastric inhibitory polypeptide: the neglected incretin revisited. Regul Pept 107:1–13
Vilsb?ll T, Holst JJ 2004 Incretins, insulin secretion and type 2 diabetes mellitus. Diabetologia 47:357–366
Ahrén B, Hedner P, Lundquist I 1983 Interaction of gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK-8) with basal and stimulated insulin secretion in mice. Acta Endocrinol 102:96–102
Ahrén B, Pacini G 1999 Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice. Am J Physiol 277:E996–E1004
Pettersson M, Ahrén B 1987 Gastrin releasing peptide (GRP): Effects on basal and stimulated insulin and glucagon secretion in the mouse. Peptides 8:55–60
Kawai K, Mukai H, Yuzawa K, Suzuki S, Kuzuya N, Fuji K, Munekata E, Yamashita K 1990 Effects of neuromedin B and GRP-10 on gastrin and insulin release from cultured tumor cells of a malignant gastrinoma. Endocrinol Jpn 37:857–865
Ahrén B, Pacini G 1999 Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice. Am J Physiol 277:E996–E1004
Persson K, Gingerich RL, Nayak S, Wada K, Wada E, Ahrén B 2000 Reduced GLP-1 and insulin responses and glucose intolerance after gastric glucose in GRP receptor-deleted mice. Am J Physiol 279:E956–E962
Pacini G, Thomaseth K, Ahrén B 2001 Contribution to glucose intolerance of insulin-independent vs. insulin-dependent mechanisms in mice. Am J Physiol 281:E693–E703
Hansotia T, Baggio LLK, Delmeire D, Hinke SA, Yamada Y, Tsukiyama K, Seino Y, Holst. JJ, Schuit F, Drucker DJ 2004 Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing glucoregulatory actions of DPP-IV inhibitors. Diabetes 53:1326–1335
Pospisilik JA, Stafford SG, Demuth HU, Brownsey R, Parkhouse W, Finegood DT, McIntosh CH, Pederson RA 2002 Long-term treatment with the DPP-4 inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hypertinsulinemia, and ?-cell glucose responsiveness in VDF (fa/fa) Zucker rats. Diabetes 512:943–950(Bo Ahrén and Thomas E. Hu)