Upon release into circulation, the potent insulin secretagogues glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are rapidly cleaved and inactivated by the enzyme dipeptidyl peptidase IV (DP IV). Long-term administration of specific DP IV inhibitors, so as to enhance circulating active GIP and GLP-1 levels, has been shown to improve glucose tolerance and β-cell glucose responsiveness and to reduce hyperinsulinemia in the Vancouver diabetic fatty (VDF) rat model of type 2 diabetes. Using the VDF model, the current study was undertaken to examine the effects of long-term DP IV inhibitor treatment on insulin sensitivity. Euglycemic-hyperinsulinemic clamps were performed on two sets of conscious VDF rats treated with or without the DP IV inhibitor P32/98 (20 mg · kg−1 · day−1 for 12 weeks). The protocol consisted of three sequential 90-min periods with insulin infusion rates of 0, 5, and 15 mU · kg−1 · min−1 and included a constant infusion of [ 3H]glucose for measure of hepatic and peripheral insulin sensitivity. Relative to untreated littermates, the treated animals showed a left shift in the sensitivity of hepatic glucose output to insulin (average reduction ∼6 μmol · kg−1 · min−1) and a marked gain in peripheral responsiveness to insulin, with glucose disposal rates increasing 105 and 216% in response to the two insulin steps (versus 2 and 46% in controls). These results provide the first demonstration of improved hepatic and peripheral insulin sensitivity after DP IV inhibitor therapy, and coupled with apparent improvements in β-cell function, they offer strong support for the utility of these compounds in the treatment of diabetes.

Glucose-dependent insulinotropic polypeptide (GIP)1-42 and glucagon-like peptide-1 (GLP-1)7-36amide make up the endocrine arm of the enteroinsular axis, a concept describing the pathways that relay the presence of luminal nutrients in the small intestine to the endocrine pancreas, eliciting insulin release (1). Recent studies have highlighted the importance of GIP and GLP-1 not only as potent insulin secretagogues, but also as enhancers of β-cell function and stimulators of β-cell growth, survival, and differentiation (27). Upon release into the circulation, GIP and GLP-1 are rapidly degraded by the ubiquitous serine protease dipeptidyl peptidase IV (DP IV; EC 3.4.14.5), resulting in a circulating half-life of ∼1−2 min for the parent compounds (811). The NH2-terminally truncated peptide products GIP3-42 and GLP-19-36amide have been shown in vitro to be inactive at the receptor level (antagonist and partial agonist, respectively) and noninsulinotropic in both β-cell models and the perfused rat pancreas (1214). DP IV-mediated NH2-terminal cleavage has since been established as the primary mechanism of incretin inactivation, with a number of research groups making significant contributions (811, 15). Identification of this physiological regulatory system has given rise to the development of specific DP IV inhibitors, a promising therapeutic paradigm involving protection of the full-length active forms of endogenously secreted GIP and GLP-1, and subsequent enhancement of their numerous antidiabetic effects (1620).

Recently, we showed in the Vancouver diabetic fatty (VDF) rat (fa/ fa, a model of type 2 diabetes) that long-term treatment with the DP IV inhibitor P32/98 produced lasting improvements in glucose tolerance, hyperglycemia, hyperinsulinemia, and β-cell glucose responsiveness (21). These findings were corroborated by two recent examinations of the long-term effects of DP IV inhibition in the ZDF rat (22) and type 2 diabetic humans (23). Oral glucose tolerance tests (OGTTs) as well as ex vivo soleus glucose uptake data from our previous study provided strong, yet indirect, evidence that insulin sensitivity was heightened in the DP IV inhibitor-treated animals (21). The following study was therefore undertaken to more accurately define the effects of DP IV inhibitor treatment on insulin sensitivity.

Toward these ends, a euglycemic-hyperinsulinemic clamp was performed on conscious VDF rats after 3 months of treatment with oral DP IV inhibitor (P32/98). The results provide the first demonstration that DP IV inhibitor therapy increases both hepatic and peripheral insulin sensitivity in the VDF model, providing strong support for the use of incretin-based therapies in the treatment of diabetes.

Materials.

The DP IV inhibitor P32/98 (di-[2S,3S] -2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate) was synthesized as previously described (24). Glucose for intravenous infusion was obtained from Abbott Laboratories (Montreal, PQ, Canada). All other chemicals were obtained from Sigma Canada (Toronto, ON, Canada) unless otherwise stated.

Animals.

Eleven pairs of male VDF (fa/fa) littermates were randomly assigned to a control or treatment (P32/98) group at 400 g body weight (10 ± 0.5 weeks of age). Animals were housed on a 12-h light/dark cycle (lights on at 6:00 a.m.) and allowed ad libitum access to standard rat food and water. The treatment group received P32/98 (10 mg/kg) by oral gavage twice a day (8:00 a.m. and 5:00 p.m.) for 12 weeks, whereas the control animals received concurrent doses of 1% cellulose vehicle. The techniques used in this study were in compliance with the guidelines of the Canadian Council on Animal Care and were approved by the University of British Columbia Council on Animal Care (certificate A99-0006).

Assessment of oral glucose tolerance.

After 11 weeks of treatment, an OGTT (1 g/kg) was performed after a 16-h fast and complete drug washout (∼11 circulating half-lives for P32/98). No dose of inhibitor was administered on the morning of the OGTT. Blood samples (250 μl) were collected from the tail vein using heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) and centrifuged, and the plasma was stored at −20°C. Plasma insulin was measured by radioimmunoassay using a guinea pig anti-insulin antibody (GP-01) as previously described (25). Whole blood glucose was measured using a hand-held glucose monitor (Lifescan Canada, Burnaby, BC, Canada).

Estimation of insulin sensitivity from OGTT blood glucose and plasma insulin profiles was performed using the composite insulin sensitivity index (CISI) proposed by Matsuda and DeFronzo (26). Calculation of the index was made according to the following equation:

\[\mathrm{CISI}{=}1,000/{[}(\mathrm{FBG}{\times}\mathrm{FPI}){\times}(\mathrm{MG}{\times}\mathrm{MI}){]}^{1/2}\]

where FBG is the fasting blood glucose concentration, FPI is the fasting plasma insulin concentration, MG is the mean glucose concentration, and MI is the mean insulin concentration (area under the curve [AUC]/120 min) over the course of the OGTT.

Euglycemic-hyperinsulinemic clamps.

After 12 weeks of treatment, animals were anesthetized with sodium pentobarbital (Somnotol; 36 mg/kg) and a midline incision was made on the ventral aspect of the neck. Chronic cannulas were then inserted into the left carotid artery (PE-50 cannula; Clay Adams) and the right jugular vein (dual PE-10 cannula encased in Silastic tubing; Dow Corning), brought around the neck subcutaneously, and passed through a small skin incision at the base of the neck. After ≥4 days of recovery with at least 2 days of consecutive weight gain, the animals underwent a euglycemic-hyperinsulinemic clamp. Treatment was discontinued the day of catheter implantation and re-initiated 2 days later; the final bolus of inhibitor was given at 5:00 p.m. the day before the clamp. The protocol consisted of three sequential 90-min periods: priming, which involved a bolus (2 μCi) followed by continuous infusion of d-[3-3H]glucose (0.03 μCi/min; Amersham), and two insulin infusion steps (HumulinR; Eli Lilly Canada, Montreal, PQ, Canada) of 5 and 15 mU · kg−1 · min−1, respectively (30 and 90 pmol · kg− 1 · min−1). Blood samples (30 μl) were taken every 5 min and centrifuged briefly, and the plasma was analyzed for glucose using the glucose oxidase method (Beckman Glucose Analyzer 2; Beckman Instruments, Palo Alto, CA). During the final 30 min of each period, three 150-μl blood samples were taken at 15-min intervals for determination of plasma tracer and insulin levels. Samples were deproteinated (BaOH2/ZnSO4) and evaporated to dryness (to remove [3H]H2O), and tracer levels were measured using a liquid scintillation counter after resuspension in distilled H2O.

Inhibition of lipolysis in adipocytes.

Samples of epididymal adipose tissue (∼3 cm3) were obtained under anesthesia 3 days after the clamp procedure. After a 16-min collagenase digestion (0.5 mg/ml), recovered adipocytes were washed three times and allowed to stabilize for 1 h in 37°C Krebs buffer repetitively gassed with 95% O2, 5% CO2. Aliquots of adipocyte suspension (2 ml) were then prestimulated for 10 min with 0, 0.3, 0.6, 1.5, or 4.75 nmol/l insulin, after which lipolysis was stimulated with a maximally stimulating dose of isoproterenol (10−7 mol final concentration). The reaction was allowed to proceed for 30 min at 37°C, after which the samples were boiled for 10 min and centrifuged at 4°C for 15 min (12,000g). The aqueous phase of the supernatant was recovered and stored at −70°C. Glycerol determinations were made using a colorimetric glycerol kit (Boehringer Mannheim).

Measurement of PEPCK activity and plasma glucagon levels.

PEPCK activity was measured as follows: liver samples (0.5 g) were homogenized in 1 ml homogenization buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 0.25 mol/l sucrose, and 50 mmol/l KCl, pH 7.2) and centrifuged for 30 min at 10,000g (4°C). After protein determination (BCAprot; Pierce, Rockville, MD), samples containing 100 μg protein were combined with reaction buffer (50 mmol/l Tris-HCl, 2 mmol/l MnCl2, 2.5 mmol/l phosphoenolpyruvate, 10 mmol/l NaHCO3, 5 units/ml malate dehydrogenase, and 0.15 mmol/l β-NADH) in a 96-well plate. The reaction was initiated with the addition of 0.4 mmol/l dGDP (final concentration) and followed at 340 nm on a microtiter plate reader. One unit of PEPCK activity corresponds to the conversion of 1 μmol β-NADH to NAD in 1 min. Plasma glucagon levels were measured using a COOH-terminally directed glucagon radioimmunoassay kit (Ab 1032K; Linco Research, St. Charles, MO).

Calculations and analysis.

Hepatic glucose output (HGO) and glucose disposal rate (GDR) were calculated according to the method of Steele (27). In summary, HGO was calculated by subtracting the glucose infusion rate (GIR) from the tracer-determined rate of glucose appearance into the plasma compartment. Similarly, at steady state, the GDR is equal to the sum of the rates of endogenous (HGO) and exogenous (GIR) glucose entry into the plasma compartment.

Data (means ± SE) were compared using Prism 3.02 data analysis software (GraphPad Software, La Jolla, CA) within groups using a paired t test and between groups using a Student’s t test (P < 0.05).

In the current study, two groups of 11 VDF rats were treated with the DP IV inhibitor P32/98 for 12 weeks, after which they underwent a euglycemic-hyperglycemic clamp. Fourteen animals (seven per group) were successfully clamped (the remainder failed due to loss of aortic cannula patency). The data presented below are compiled from these 14 animals.

Oral glucose tolerance.

One week before surgical preparation (week 11), an OGTT was performed on all animals. Animals were fasted, and inhibitor dosing was discontinued for 16 h, an interval sufficient to ensure complete drug washout. Plasma DP IV activity measurements made to confirm inhibitor washout showed a 41% increase in DP IV activity in the treated animals versus controls (33.3 ± 0.9 and 23.6 ± 0.6 mU/ml, respectively), a finding consistent with our previous study. Fasting plasma glucose levels in the treated animals were ∼2 mmol/l lower than in control littermates (6.5 ± 0.3 vs. 8.6 ± 0.4 mmol/l), despite comparable fasting insulin levels (1.71 ± 0.23 and 1.69 ± 0.19 nmol/l, respectively) (Fig. 1). The difference in fasting blood glucose increased to nearly 4 mmol/l over the 120-min course of the OGTT, with glucose levels peaking at 10.4 ± 0.4 mmol/l in the treated group compared with 14.2 ± 0.7 mmol/l in the control group. Significant insulin responses were elicited in both groups despite marked hyperinsulinemia; however, the early-phase insulin response in the treated animals measured 260% of that of the controls (Fig. 1). Calculation of AUC revealed no difference in total insulin secretion between the two groups (data not shown). Analysis of these data according to a composite insulin sensitivity index (ISI) revealed mean ISI scores (arbitrary units) of 0.93 ± 0.07 and 1.46 ± 0.13 (P < 0.05) for the control and treated animals, respectively, suggesting enhanced insulin sensitivity after DP IV inhibitor treatment (Fig. 1, inset).

Euglycemic-hyperinsulinemic clamp.

Baseline glucose, insulin, and DP IV activity (treated 35.6 ± 1.7, control 24.4 ± 1.0 mU/ml) values measured at the outset of the clamp were comparable to those observed during the OGTT ∼10 days earlier; the treated group exhibited reduced fasting plasma glucose concomitant with unchanged plasma insulin values (Fig. 2). Fasting plasma glucose levels appeared slightly elevated under clamp conditions, a phenomenon attributable to the difference in measurement technique, and in keeping with our own observations and with the literature on measurement of whole blood versus plasma glucose levels (28). Figure 2 clearly shows that steady-state insulin levels between groups were comparable, rising ∼0.7 nmol/l during the first insulin step and a further ∼2 nmol/l during the second. These values, although hyperinsulinemic, correspond closely to plasma values measured both during the OGTT (Fig. 1) and during normal feeding (21). P32/98-treated animals displayed an immediate requirement for exogenous glucose infusion (GIR) in response to 5 mU · kg−1 · min− 1 insulin; under the same conditions, control animals showed a reduced and significantly delayed (∼25 min) response to the same stimulus (Fig. 2). Elevation of the insulin infusion rate to 15 mU · kg−1 · min−1 elicited a further increase in GIR in both groups, with plateau levels reaching 39.2 ± 5.3 and 26.8 ± 4.5 μmol · kg−1 · min−1 in the treated and control groups, respectively.

HGO, calculated from plasma tracer levels, was significantly reduced in the treated animals compared with controls during each stage of the clamp (Fig. 3A and B). Basal HGO averaged 6.5 ± 0.9 μmol · kg−1 · min−1 less in the treated than in the control group (12.7 ± 0.9 μmol · kg−1 · min−1). A difference of similar magnitude was found at each insulin infusion level (Fig. 3A and B). Calculation of GDR revealed little or no response to insulin in the control animals, consistent with previous clamp studies of obese Zucker rats (29). GDR in the treated group, however, showed a return of insulin responsiveness, with steady-state levels 105 and 216% above basal during each of the two insulin steps, respectively (Fig. 4). Basal GDR in the treated animals was reduced relative to controls (Fig. 4).

PEPCK and glucagon determinations.

To further investigate the changes in hepatic insulin sensitivity, liver PEPCK levels and fasting plasma glucagon levels were measured. Measurement of PEPCK activity in liver samples obtained at termination showed no significant difference between the treated and untreated animals, suggesting a non- PEPCK-dependent pathway for the left shift in insulin sensitivity. PEPCK activity averaged 25.9 ± 2.9 and 31.5 ± 4.3 mU/mg tissue in the treated and control groups, respectively (Fig. 3C). Values for the treated animals were comparable to those obtained in control Wistar rats (24.7 ± 0.2 mU/ mg). No difference was detected in fasting plasma glucagon levels either with time or as a result of the treatment (Fig. 3D).

Inhibition of lipolysis in adipocytes.

Using glycerol release as an indicator, inhibition of isoproterenol-stimulated lipolysis by insulin was examined in isolated epididymal adipocytes (Fig. 5). Concentration response of inhibition by insulin showed a left shift in adipocytes isolated from treated animals compared with controls; estimated half-maximal concentrations (EC50 values) were 0.29 ± 0.01 and 1.11 ± 0.01 nmol/l, respectively (P < 0.01).

The protection of full-length GIP1-42 and GLP-17-36 amide (incretins) in the circulation using DP IV inhibitors represents a significant advancement in the search for new and effective alternative treatments for diabetes. Since the early 1990s, numerous studies have revealed a pleiotropy of antidiabetic effects triggered by interaction of the incretins with their respective cell-surface receptors. Among them are stimulation of β-cell glucose competence, proliferation, differentiation, growth, and cell survival; inhibition of glucagon secretion; and several reports indicating stimulation of glucose uptake in muscle cells (27, 30,31). Recently, we showed that long-term DP IV inhibitor therapy caused marked improvements in glucose tolerance, hyperinsulinemia, and β-cell function in the VDF rat, findings that highlight the potential utility of these compounds in diabetes therapy. In the same study, OGTT data and an in vitro determination of insulin-stimulated glucose uptake in isolated muscle provided indirect evidence for a treatment-induced improvement in insulin sensitivity. The present study was carried out to characterize the effects of DP IV inhibitor therapy on insulin sensitivity in the same model, using a euglycemic-hyperinsulinemic clamp. The data collected constitute the first conclusive demonstration that DP IV inhibitor therapy improves both hepatic and peripheral insulin sensitivity.

Extensive literature exists on the obese Zucker rat, including numerous studies examining insulin resistance by means of euglycemic-hyperinsulinemic clamp (32). In one such study, Terrettaz et al. (29) tested HGO and GDR responses in obese Zucker rats over a wide range of insulin concentrations, allowing a comprehensive evaluation of both responsiveness (the efficacy or magnitude of the response to insulin) and sensitivity (the potency or EC50 of the response to insulin) to the hormone. The authors concluded that the obese animals demonstrate marked hepatic insulin resistance (characterized by a fully responsive yet right-shifted HGO and severely impaired glucose disposal (characterized by a total lack of responsiveness over a wide range of insulin concentrations) compared with lean littermates (29). As in these early reports, the control VDF rats in the current study showed a responsive yet right-shifted hepatic glucose response to insulin and an extremely blunted peripheral response (Figs. 3 and 4). It is apparent that long-term treatment with the DP IV inhibitor P32/98 partially reversed both of these functional pathologies, causing a left shift in the HGO response to insulin (Fig. 3) and a partial restoration of peripheral insulin-stimulated glucose uptake (Fig. 4). The primary physiological consequence of these improvements appears to be a reduction in fasting plasma glucose and a marked enhancement of glucose tolerance. Similar changes in HGO and GDR have been shown for a number of classes of oral antidiabetic therapies in obese Zucker rats (3335).

The finding that P32/98 treatment improves hepatic insulin sensitivity supports our previous work showing reduced hyperinsulinemia in the basal fed state and reduced fasting plasma glucose in the same model (21). Similar reductions in insulinemia and fasting plasma glucose have been shown in a number of studies using long-term GLP-1 (or GLP-1 mimetic) treatment (3638). In keeping with the previous discussion of HGO sensitivity to insulin, the defining feature of P32/98 treatment on hepatic insulin sensitivity appeared to be a left shift in insulin responsiveness. Because fasting insulin levels did not differ between groups, a reduction in basal HGO in the treated animals is implicit. And because basal (fasting) glucose output from the liver is primarily determined by the rates of gluconeogenesis and glycogenolysis (both of which are potently stimulated by glucagon), a potential underpinning for these results might have been a reduction in glucagon levels in the treated group. Examination of fasting (complete drug washout) glucagon levels, however, although measured using an antibody blind to NH2-terminal truncation, did not support such a hypothesis (Fig. 3D). Also, PEPCK activity was measured as an index of gluconeogenic enzyme expression, a group of enzymes that exhibit coordinate hormonal expression with respect to insulin and glucagon. Because PEPCK does not undergo short-term regulation via phosphorylation or allosteric effectors and because, physiologically, it is rate limiting for gluconeogenesis, PEPCK activity measurement provides an indicator of gluconeogenic potential. Although a nonsignificant (∼15%) decrease in PEPCK activity was demonstrated in the treated animals, the exact mechanisms responsible for the HGO sensitivity shift remain unclear and warrant further study (Fig. 3C).

Whereas treatment-induced alterations in HGO preferentially affected sensitivity to insulin rather than responsiveness, alterations in GDR appeared to comprise shifts in both responsiveness and sensitivity to insulin. Control animals displayed a basal GDR of 12.6 ± 1.1 μmol · kg−1 · min−1, nearly twofold that of the treated group (7.1 ± 1.2 μmol · kg−1 · min−1). Further, the P32/98-treated group displayed two- and threefold responses to 5 and 15 mU · kg− 1 · min−1 insulin, respectively (Fig. 4B), whereas the control animals showed their first sign of peripheral insulin responsiveness (46%) only during the latter infusion step. These data suggest a marked left shift in insulin-stimulated peripheral glucose uptake and are consistent with our previous demonstration of increased glucose uptake in soleus muscle (21) and with several reports of incretin-stimulated increases in glucose uptake (30,39). A number of studies using GLP-1 receptor agonists over the long term have suggested similar improvements in insulin sensitivity (38). It is unclear whether direct acute incretin effects on glucose disposal, as reported by some groups (40,41) and refuted by others (42), play a role.

On that note, although the literature on DP IV inhibition focuses primarily on a mechanism of action involving the enhancement of circulating active GIP and GLP-1, the role of other peptide substrates of DP IV in the improvements evidenced here, and previously, should not be discounted. Natural substrates of DP IV include all tested members of the glucagon/vasoactive intestinal polypeptide (VIP) superfamily of polypeptides (43), including glucagon (44,45), as well as a number of neuroendocrine and immune factors (46). Many of these peptides play significant roles in the regulation of energy metabolism and are likely to contribute toward the improvements resulting from DP IV inhibitor treatment. For instance, recent work by Huypens et al. (47) has highlighted the importance of the counterregulatory hormone glucagon in the maintenance of glucose competence of the β-cell and in proper insulin secretion. Considering that blood glucose is maintained by circulating glucagon levels for approximately two-thirds of the 24-h cycle, DP IV inhibitor− induced enhancement of NH2-terminally intact glucagon (active; glucagon1-29) is likely to contribute toward the reported improvements in β-cell function.

In addition to the examination of HGO and GDR in vivo, an in vitro examination of adipocyte insulin sensitivity was performed 3 days after the clamp. The VDF Zucker rat displays excessive fat accumulation and pronounced hyperlipidemia (including elevated free fatty acids [FFAs]), pathologies intimately associated with human type 2 diabetes. It has been suggested that >50% of the insulin resistance in diabetic patients is FFA-induced, with elevated plasma FFAs stimulating insulin secretion, peripheral glucose underutilization, and hepatic glucose overproduction (increased gluconeogenesis) (48, 49). Physiologically, these FFA effects likely serve to preserve glucose stores when supply is limited. However, in times of plenty (e.g., a typical Western diet), these effects become counterproductive, inhibiting the utilization of glucose (48, 49). The demonstration that insulin-induced inhibition of lipolysis is sensitized in adipocytes from DP IV inhibitor-treated animals suggests a potential mechanism of action for the improvements in hepatic and peripheral insulin sensitivity discussed above. A reduction in FFA release from adipocytes, secondary to a sensitized inhibition of lipolysis by insulin, might attenuate the glucose sparing effects of plasma FFAs, thereby reducing the severity of insulin resistance. These findings warrant further investigation into the effects of DP IV inhibition on lipid metabolism.

The success of DP IV inhibitors as a therapeutic strategy in the treatment of diabetes is owed in great part to the pleiotropic nature of its primary effectors, the incretins GIP and GLP-1. Previously believed to be mere enhancers of β-cell function, the incretins are being shown to possess numerous non-insulin-dependent functions, including stimulation of cell survival and modulation of peripheral energy disposal (liver and muscle) (50). The findings of this study corroborate such reports and further exemplify the importance of the non-insulinotropic effects of GIP and GLP-1 in the regulation of glucose homeostasis. In conclusion, the addition of improved hepatic and peripheral insulin sensitivity to the list of beneficial metabolic effects of long-term DP IV inhibitor therapy provides strong support for the use of these compounds in the treatment of diabetes.

This study was provided for by grants from the Canadian Institute for Health Research (CIHR) and the Department of Science and Technology of Sachsen Anhalt (9704/00116 to H.-U.D.). J.A.P. is grateful to the CIHR, Michael Smith Foundation for Health Research, and the Killam Trust Foundation for scholarship support.

Further, we would like to thank Diane Finegood, Brian Topp, Horatio Vinerean, and Christian Lehn-Brand for sharing their expertise and advice on the art of glucose clamping. Last but not least, many thanks are in order to Madeleine Speck and Shalea Piteau for their excellent technical support.

1.
Creutzfeldt W: The incretin concept today.
Diabetologia
16
:
75
–85,
1979
2.
Trumper A, Trumper K, Trusheim H, Arnold R, Goke B, Horsch D: Glucose-dependent insulinotropic polypeptide is a growth factor for beta (INS-1) cells by pleiotropic signaling.
Mol Endocrinol
15
:
1559
–1570,
2001
3.
Xu G, Stoffers DA, Habener JF, Bonner-Weir S: Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.
Diabetes
48
:
2270
–2276,
1999
4.
Stoffers DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, Habener JF, Egan JM: Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas.
Diabetes
49
:
741
–748,
2000
5.
Hui H, Wright C, Perfetti R: Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells.
Diabetes
50
:
785
–796,
2001
6.
Buteau J, Roduit R, Susini S, Prentki M: Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells.
Diabetologia
42
:
856
–864,
1999
7.
Ehses J, Casilla V, Doty T, Pospisilik J, Demuth H-U, Pederson R, McIntosh C: Glucose-dependent insulinotropic polypeptide (GIP) stimulates cell proliferation and promotes survival of β-(INS-1)-cells (Abstract).
Diabetes
51 (Suppl. 2)
:
A1385
,
2002
8.
Mentlein R, Gallwitz B, Schmidt WE: Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum.
Eur J Biochem
214
:
829
–835,
1993
9.
Kieffer TJ, McIntosh CH, Pederson RA: Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV.
Endocrinology
136
:
3585
–3596,
1995
10.
Hansen L, Deacon CF, Orskov C, Holst JJ: Glucagon-like peptide-1-(7-36)amide is transformed to glucagon-like peptide-1-(9–36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine.
Endocrinology
140
:
5356
–5363,
1999
11.
Deacon CF, Nauck MA, Meier J, Hucking K, Holst JJ: Degradation of endogenous and exogenous gastric inhibitory polypeptide in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide.
J Clin Endocrinol Metab
85
:
3575
–3581,
2000
12.
Brown JC, Dahl M, Kwauk S, McIntosh CH, Otte SC, Pederson RA: Actions of GIP.
Peptides
2
:
241
–245,
1981
13.
Knudsen LB, Pridal L: Glucagon-like peptide-1-(9–36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor.
Eur J Pharmacol
318
:
429
–435,
1996
14.
Hinke SA, Gelling RW, Pederson RA, Manhart S, Nian C, Demuth HU, McIntosh CH: Dipeptidyl peptidase IV-resistant [ D-Ala(2)]glucose-dependent insulinotropic polypeptide (GIP) improves glucose tolerance in normal and obese diabetic rats.
Diabetes
51
:
652
–661,
2002
15.
Pauly RP, Rosche F, Wermann M, McIntosh CH, Pederson RA, Demuth HU: 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,
1996
16.
Pauly R, Demuth H-U, Rosche F, Schmidt J, White H, McIntosh C, Pederson R: Inhibition of dipeptidyl peptidase IV (DP IV) in rat results in improved glucose tolerance (Abstract).
Regul Pept
64
:
148
,
1996
17.
Pederson RA, White HA, Schlenzig D, Pauly RP, McIntosh CH, Demuth HU: Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide.
Diabetes
47
:
1253
–1258,
1998
18.
Holst JJ, Deacon CF: Inhibition of the activity of dipeptidyl-peptidase IV as a treatment for type 2 diabetes.
Diabetes
47
:
1663
–1670,
1998
19.
Balkan B, Kwasnik L, Miserendino R, Holst JJ, Li X: 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,
1999
20.
Ahren B, Holst JJ, Martensson H, Balkan B: Improved glucose tolerance and insulin secretion by inhibition of dipeptidyl peptidase IV in mice.
Eur J Pharmacol
404
:
239
–245,
2000
21.
Pospisilik JA, Stafford SG, Demuth HU, Brownsey R, Parkhouse W, Finegood DT, McIntosh CH, Pederson RA: Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucose responsiveness in VDF (fa/fa) Zucker rats.
Diabetes
51
:
943
–950,
2002
22.
Sudre B, Broqua P, White R, Ashworth D, Evans D, Haigh R, Junien J-L, Aubert M: Chronic inhibition of circulating dipeptidyl peptidase IV by FE 999011 delays the occurrence of diabetes in male Zucker diabetic fatty rats.
Diabetes
51
:
1461
–1469,
2002
23.
Ahren B, Simonsson E, Larsson H, Landin-Olsson M, Torgeirsson H, Jansson P-A, Sandqvist M, Bavenholm P, Efendic S, Eriksson J, Dickinson S, Holmes D: Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4-week study period in type 2 diabetes.
Diabetes Care
25
:
869
–875,
2002
24.
Demuth HU: Recent developments in inhibiting cysteine and serine proteases.
J Enzyme Inhib
3
:
249
–278,
1990
25.
Jia X, Elliott R, Kwok YN, Pederson RA, McIntosh CH: Altered glucose dependence of glucagon-like peptide I(7-36)-induced insulin secretion from the Zucker (fa/fa) rat pancreas.
Diabetes
44
:
495
–500,
1995
26.
Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp.
Diabetes Care
22
:
1462
–1470,
1999
27.
Steele R: Influences of glucose loading and injected insulin on hepatic glucose output.
Ann N Y Acad Sci
82
:
420
–440,
1959
28.
Weitgasser R, Davalli AM, Weir GC: Measurement of glucose concentrations in rats: differences between glucose meter and plasma laboratory results.
Diabetologia
42
:
256
,
1999
29.
Terrettaz J, Assimacopoulos-Jeannet F, Jeanrenaud B: Severe hepatic and peripheral insulin resistance as evidenced by euglycemic clamps in genetically obese fa/fa rats.
Endocrinology
118
:
674
–678,
1986
30.
Yang H, Egan JM, Wang Y, Moyes CD, Roth J, Montrose MH, Montrose-Rafizadeh C: GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor.
Am J Physiol
275
:
C675
–C683,
1998
31.
Mizuno A, Kuwajima M, Ishida K, Noma Y, Murakami T, Tateishi K, Sato I, Shima K: Extrapancreatic action of truncated glucagon-like peptide-I in Otsuka Long-Evans Tokushima Fatty rats, an animal model for non-insulin-dependent diabetes mellitus.
Metabolism
46
:
745
–749,
1997
32.
McIntosh C, Pederson R: Noninsulin-dependent animal models of diabetes mellitus. In:
Experimental Models of Diabetes.
McNeill JH, Ed. Boca Raton, FL, CRC Press,
1999
, p.
337
–398
33.
Bowen L, Stein PP, Stevenson R, Shulman GI: The effect of CP 68722, a thiozolidinedione derivative, on insulin sensitivity in lean and obese Zucker rats.
Metabolism
40
:
1025
–1030,
1991
34.
Shibata T, Matsui K, Yonemori F, Wakitani K: JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models.
Br J Pharmacol
125
:
1744
–1750,
1998
35.
Hevener AL, Reichart D, Olefsky J: Exercise and thiazolidinedione therapy normalize insulin action in the obese Zucker fatty rat.
Diabetes
49
:
2154
–2159,
2000
36.
Larsen J, Jallad J, Damsbo P: One week continuous infusion of GLP-1 (7–37) improves glycemic control in NIDDM (Abstract).
Diabetes
45
:
233A
,
1996
37.
Rachman J, Barrow B, Levy J, Turner R: Near normalisation of diurnal glucose concentrations by continuous administrations of glucagon-like peptide-1 (GLP-1) in subjects with NIDDM.
Diabetologia
40
:
205
–211,
1997
38.
Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B, Denaro M: Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta).
Diabetes
48
:
1026
–1034,
1999
39.
Wang Y, Montrose-Rafizadeh C, Adams L, Raygada M, Nadiv O, Egan JM: GIP regulates glucose transporters, hexokinases, and glucose-induced insulin secretion in RIN 1046-38 cells.
Mol Cell Endocrinol
116
:
81
–87,
1996
40.
Sandhu H, Wiesenthal SR, MacDonald PE, McCall RH, Tchipashvili V, Rashid S, Satkunarajah M, Irwin DM, Shi ZQ, Brubaker PL, Wheeler MB, Vranic M, Efendic S, Giacca A: Glucagon-like peptide 1 increases insulin sensitivity in depancreatized dogs.
Diabetes
48
:
1045
–1053,
1999
41.
Meneilly GS, McIntosh CH, Pederson RA, Habener JF, Gingerich R, Egan JM, Finegood DT, Elahi D: Effect of glucagon-like peptide 1 on non-insulin-mediated glucose uptake in the elderly patient with diabetes.
Diabetes Care
24
:
1951
–1956,
2001
42.
Ahren B, Larsson H, Holst JJ: Effects of glucagon-like peptide-1 on islet function and insulin sensitivity in noninsulin-dependent diabetes mellitus.
J Clin Endocrinol Metab
82
:
473
–478,
1997
43.
Lambeir A-M, Durinx C, Proost P, Van Damme J, Scharpe S, De Meester I: Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in insulin secretion.
FEBS Lett
507
:
327
–330,
2001
44.
Pospisilik JA, Hinke SA, Pederson RA, Hoffmann T, Rosche F, Schlenzig D, Glund K, Heiser U, McIntosh CH, Demuth H: Metabolism of glucagon by dipeptidyl peptidase IV (CD26).
Regul Pept
96
:
133
–141,
2001
45.
Hinke SA, Pospisilik JA, Demuth HU, Mannhart S, Kuhn-Wache K, Hoffmann T, Nishimura E, Pederson RA, McIntosh CH: Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon: characterization of glucagon degradation products and DPIV-resistant analogs.
J Biol Chem
275
:
3827
–3834,
2000
46.
Mentlein R: Dipeptidyl-peptidase IV (CD26): role in the inactivation of regulatory peptides.
Regul Pept
85
:
9
–24,
1999
47.
Huypens P, Ling Z, Pipeleers D, Schuit F: Glucagon receptors on human islet cells contribute to glucose competence of insulin release.
Diabetologia
43
:
1012
–1019,
2000
48.
Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.
Diabetes
46
:
3
–10,
1997
49.
McGarry JD: Banting Lecture 2001: Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes.
Diabetes
51
:
7
–18,
2002
50.
Alcantara AI, Morales M, Delgado E, Lopez-Delgado MI, Clemente F, Luque MA, Malaisse WJ, Valverde I, Villanueva-Penacarrillo ML: Exendin-4 agonist and exendin(9–39)amide antagonist of the GLP-1(7-36)amide effects in liver and muscle.
Arch Biochem Biophys
341
:
1
–7,
1997

Address correspondence and reprint requests to Dr. R.A. Pederson, Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. E-mail: pederson@interchange.ubc.ca.

Received for publication 20 April 2002 and accepted in revised form 29 May 2002.

H.-U.D. is a chief executive officer, the chief scientific officer, and a shareholder in Probiodrug. C.H.S.M. and R.A.P. are members of a scientific advisory panel to Probiodrug and receive consulting fees for their participation; in addition, they receive grant/research support from Probiodrug.

AUC, area under the curve; CISI, composite insulin sensitivity index; DP IV, dipeptidyl peptidase IV; EC50, half-maximal concentration; FFA, free fatty acid; GDR, glucose disposal rate; GIP, glucose-dependent insulinotropic polypeptide; GIR, glucose infusion rate; ISI, insulin sensitivity index; GLP-1, glucagon-like peptide-1; HGO, hepatic glucose output; OGTT, oral glucose tolerance test.