1 Department of Physiology, University of British Columbia, Vancouver, Canada
2 Probiodrug GmbH, Halle (Saale), Germany
3 Department of Biochemistry, University of British Columbia, Vancouver, Canada
4 School of Kinesiology, Simon Fraser University, Burnaby, Canada
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ABSTRACT |
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INTRODUCTION |
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Also known as the incretins, GIP and GLP-1 make up the endocrine component of the entero-insular (gut-pancreas) axisa concept describing the neural, endocrine, and substrate signaling pathways between the small intestine and the islets of Langerhans (9). Together, the incretins are responsible for >50% of nutrient-stimulated insulin release. In addition, the incretins share a number of non-insulin-mediated effects that contribute to effective glucose homeostasis. GIP and GLP-1 have both been shown to inhibit gastric motility and secretion (10,11), promote ß-cell glucose competence (12), and stimulate insulin gene transcription and biosynthesis (13,14). In addition, GIP has been reported to play a role in the regulation of fat metabolism (15), and GLP-1 has been shown to stimulate ß-cell differentiation and growth (16), as well as to restore islet-cell glucose responsiveness (17).
We have previously shown that acute administration of the specific DP IV inhibitor P32/98 (isoleucyl-thiazolidine) in Zucker rats enhances insulin secretion and glucose tolerance (18), improvements that were much more profound in the diabetic, fatty animals than in their lean littermates (19). Balkan et al. (20) confirmed these findings using the DP IV inhibitor NVP-DPP728 and went on to provide evidence for the previously postulated stabilization of, and rise in, plasma active GLP-1736 (GLP-1a) after inhibitor treatment. However, despite its efficacy, the use of DP IV inhibitors on an acute scale is unlikely to exploit the longer term incretin actions involving altered intracellular protein function and gene expression. It was therefore hypothesized that chronic DP IV inhibitor treatment of diabetic animals, in addition to improving glucose tolerance, would enhance ß-cell glucose responsiveness, replication, and turnover, and thus result in sustained improvements in ß-cell function.
In the present study, two groups of Vancouver diabetic fatty (VDF) rats were treated for 3 months with the DP IV inhibitor P32/98. VDF rats are a substrain of the fatty (fa/fa) Zucker rat, which display abnormalities characteristic of type 2 diabetes, including mild hyperglycemia, hyperinsulinemia, glucose intolerance, hyperlipidemia, impaired insulin secretion, and peripheral and hepatic insulin resistance (21). Parameters including body weight, food and water intake, and oral glucose tolerance were regularly examined to track the progression of the disease and study the possible therapeutic effects of the inhibitor. At the end of the treatment period, ex vivo fat and muscle insulin sensitivity were assessed, and pancreas perfusion was performed to measure ß-cell glucose responsiveness. The results provided the first demonstration that long-term DP IV inhibitor treatment causes progressive and sustained improvements in glucose tolerance, insulin sensitivity, and ß-cell glucose responsiveness.
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RESEARCH DESIGN AND METHODS |
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Animals.
We randomly assigned six pairs of male fatty (fa/fa) VDF Zucker rat littermates to a control or treatment (P32/98) group at 440 g body wt (age 11 ± 0.5 weeks). Animals were housed on a 12-h light/dark cycle (lights on at 0600) and allowed access to standard rat diet and water ad libitum. 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 # A99-006.
Protocol for daily monitoring and drug administration.
The treatment group received P32/98 (10 mg/kg) by oral gavage twice daily (0800 and 1700) for 100 days, and the control animals received concurrent doses of vehicle consisting of a 1% cellulose solution. Every 2 days, body weight, morning and evening blood glucose, and food and water intake were assessed. Blood samples were acquired from the tail, and glucose was measured using a SureStep analyzer (Lifescan Canada, Burnaby, Canada). Food and water intake were measured by subtraction.
Protocol for monthly assessment of glucose tolerance.
Every 4 weeks from the start of the experiment, an oral glucose tolerance test (OGTT; 1 g/kg) was performed after an 18-h fast and complete drug washout (12 circulating half-lives for P32/93). No 0800 dose was administered in this case. Blood samples (250 µl) were collected from the tail using heparinized capillary tubes, centrifuged, and stored at -20°C. In the case of the 12-week OGTT, blood was collected directly into tubes containing the DP IV inhibitor P32/98 (final concentration 10 µmol/l) for analysis of active GLP-1 (EGLP-35K; Linco Research, St. Charles, MO). Plasma insulin was measured by radioimmunoassay using a guinea pig anti-insulin antibody (GP-01), as previously described (23), and blood glucose was measured as described above. Plasma DP IV activity was determined using a colorimetric assay measuring the liberation of p-nitroanilide (A405 nm) from the DP IV substrate H-gly-pro-pNA (Sigma; Parkville, Ontario, Canada). It is important to note that the assay involves a 20-fold sample dilution and therefore underestimates the actual degree of inhibition occurring in the undiluted sample when using rapidly reversible inhibitors such as P32/98.
Estimations of insulin sensitivity made from OGTT data were performed using the composite insulin sensitivity index proposed by Matsuda and DeFronzo (24). Calculation of the index was made according to the following equation:
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Protocol for 24-h glucose, insulin, and DP IV profile.
To determine the effects of DP IV inhibition over a 24-h period, blood glucose, insulin, and DP IV activity levels were measured as described above, every 3 h for 24 h, 6 weeks into the study. Drug dosing was continued at the appropriate times during the profile.
Skeletal muscle insulin sensitivity.
Uptake of 14C-labeled glucose in soleus muscle strips was measured as an indicator of skeletal muscle insulin sensitivity. In brief, after an overnight fast and 18 h after the last dose of P32/98, the animals were anesthetized with pentobarbital sodium (Somnotol; 50 mg/kg). The soleus muscles of both hindlimbs were exposed and isolated. After freeing the muscle by severing the proximal and distal tendons, strips of
2535 mg were pulled from the muscle (the two outer thirds of each muscle were used). After being weighed, the strips were fixed onto stainless steel clips at their resting length and allowed to stabilize for 30 min in a Krebs-Ringer bicarbonate buffer supplemented with 3 mmol/l pyruvate, continuously gassed with 95% O2:5% CO2 and held at 37°C in a shaking water bath. These conditions were maintained for the duration of the experiment, unless otherwise stated.
To assess glucose uptake in response to insulin, muscle strips underwent two preincubations (30 and 60 min) followed by a 30-min test incubation. Both the second preincubation and the test incubation contained 0 or 800 µU/ml insulin. The test incubation was performed in media supplemented with [3H]inulin (0.1 µCi/ml) as a measure of extracellular space and the nonmetabolizable glucose analogue [14C]-3-O-methylglucose (0.05 µCi/ml) for measurement of glucose uptake. After incubation, each strip was blotted dry and digested with proteinase K (0.25 µg/ml), and the radioactivity of the muscle digests was measured with a liquid scintillation-counting dual-isotopic program.
Adipose tissue insulin sensitivity.
To estimate insulin sensitivity in adipose tissue, glycogen synthase (GS) and acetyl-CoA carboxylase (ACC) levels were measured, as previously described (25,26). In brief, 3-cm3 samples of ependymal adipose tissue were obtained from anesthetized animals and subjected to 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. Then 2-ml aliquots of the adipocyte suspension containing 0, 100, 250, 800, and 1,500 µU/ml insulin were incubated for 30 min and immediately flash frozen on liquid nitrogen and stored at -70°C. Before ACC and GS assessment, stored samples were thawed, homogenized in buffer (pH 7.2) containing 20 mmol/l MOPS, 250 mmol/l sucrose, 2 mmol/l EDTA, 2 mmol/l EGTA, 2.5 mmol/l benzamidine, and centrifuged (15 min at 15,000g).
For measurement of ACC activity, 50-µl aliquots of supernatant, preincubated in the presence or absence of 20 mmol/l citrate, were added to 450 µl of [14C]HCO3 containing assay buffer (pH 7.4; 50 mmol/l HEPES, 10 mmol/l MgSO4, 5 mmol/l EDTA, 5.9 mmol/l ATP, 7.8 mmol/l glutathione, 2 mg/ml BSA, 15 mmol/l KHCO3, 150 µmol/l acetyl CoA). After 3 min, the reaction was arrested by the addition of 200 µl of 5 mol/l HCl. Samples were dried for 6 h, resuspended in 400 µl of distilled water, combined with 3 ml scintillation cocktail, and counted on a Beckman LS 6001C ß-counter.
GS activity was measured using a modification of a filter paper method (26): 25 µl of the cell extracts, prepared as indicated above, were added to assay buffer (pH 7.0; 75 mmol/l MOPS, 75 mmol/l NaF, 10 mg/ml glycogen, 2 mmol/l UDP-[14C]glucose) held at 30°C in the presence or absence of 15 mmol/l glucose-6-phosphate. Each reaction was stopped by spotting 50 µl of the reaction mixture onto Whatmann 3MM filter paper and immersing the paper in 66% ethanol. After three ethanol washes, the samples were air dried and the [14C] activity (UDP-[14C]glucose incorporation into glycogen) was determined.
Protocol for pancreas perfusion.
After excision of soleus and ependymal adipose tissue samples, the pancreas was isolated and perfused with a low-to-high glucose (4.4 to 8.8 mmol/l) perfusion protocol, as previously described (27). After exposure through a mid-line incision on the ventral aspect, the pancreas was isolated, all minor vessels were ligated, and a glucose perfusate was introduced through the celiac artery. Perfusion effluent was collected at 1-min intervals via the portal vein, with a perfusion rate of 4 ml/min. Samples were stored at -20°C until analysis.
Immunohistochemistry and ß-cell mass determination.
Pancreata were removed from anesthetized animals (50 mg/kg sodium pentobarbital) and placed directly into fixative for 48 h (44% formaldehyde, 47% distilled H2O, 9% glacial acetic acid). After being embedded in paraffin, 5-µm tissue sections were cut, mounted onto slides, and dried ready for staining. To assess ß-cell area, sections were stained first with a guinea pig anti-insulin primary antibody and then with peroxidase-conjugated goat anti-guinea pig secondary. Slides were developed using diaminobenzidine and counterstained with hematoxylin. Analyses were performed using Northern Eclipse Software (Empix Imaging, Mississauga, Ontario, Canada), as previously described (28).
Statistical Analysis.
Students t test and ANOVA were used, where appropriate, to test statistical significance of the data (P < 0.05). Analyses were performed using Prism 3.0 data analysis software (GraphPad Software, San Diego, CA).
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RESULTS |
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DISCUSSION |
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Daily monitoring revealed a 12.5% decrease in body weight gain (4% reduction in final body weight) in the treated animals compared to untreated controls (Fig. 1A). Although not statistically significant, mean food intake in the treated animals averaged 0.4 g · day-1 · rat-1 (41 g/rat over the course of the study) less than those in the control group. The cumulative 41 g/rat nonsignificant difference in food intake over the course of the experiment might partially account for the decreased weight gain in the treated animals. These findings rule out neither the possibility that the gastric inhibitory actions of the incretins nor the reported satiety effects of GLP-1 played a role in the decrease in weight gain.
Monitored every 2 days, morning and evening blood glucose values showed no significant response to the inhibitor treatment, a likely reflection of two points. First, the blood-sampling times (0800 and 1700) corresponded to postabsorptive and early feeding states, respectively, with blood glucose values in the ranges of 4.55.5 and 6.08.0 mmol/l, respectively. In light of the hypothesized, glucose-dependent mechanism of action of the treatment, large decreases in glucose values would not be anticipated at these glycemic levels. Second, both morning and evening blood samples were collected immediately before drug dosing, at times of minimum DP IV inhibition, when the potential for any acute therapeutic effects of the treatment were at a minimum. Both points are supported by the 24-h profile shown in Fig. 2.
The unaltered postabsorptive blood glucose values notwithstanding, DP IV inhibitor treatment effectively reduced both prandial blood glucose and blood glucose responses to an OGTT (Figs. 2 and 3). During the 24-h profile, the control animals exhibited a 105% rise in plasma insulin in response to a 5.2-mmol/l increase in blood glucose, whereas the treated animals displayed a 160% insulin response to a much smaller glucose excursion (3.0 mmol/l). Although these differences were likely attributable, at least in part, to an acute increase in circulating incretin levels induced by P32/98, the pronounced early-phase insulin peak exhibited during the OGTT was not (the OGTT took place after complete drug washout). The latter data suggest not only an increased insulin sensitivity, but also an enhanced ß-cell glucose responsiveness, in treated animals. Ultimately, an increase in ß-cell glucose responsiveness was clearly demonstrated through pancreas perfusion. After exposure to an elevated (8.8 mmol/l) glucose perfusate, pancreata from the control animals showed an absence of first-phase insulin release, whereas those from the treated group exhibited an immediate 3.2-fold insulin response (Fig. 5). The absence of an early-phase insulin release seen in the control group is characteristic of the VDF rat and is a hallmark of type 2 diabetes (21). Considering the lack of altered ß-cell area or islet morphology, these data suggest that long-term treatment with P32/98 causes an improvement in the ability of the existing ß-cell population to sense and respond to increases in glucose concentration. These findings are consistent with the reported effects of GLP-1 on ß-cell differentiation, as well as numerous reports showing the glucose-sensitizing effects of GIP and GLP-1 in both islets and immortalized ß-cell models (30,31).
Elevated fasting blood glucose in the face of hyperinsulinemia and poor clearance of an oral glucose load are consistent with the hepatic and muscle insulin resistance, respectively, described in the fa/fa Zucker rat. Findings in the present study showed that DP IV inhibitor treatment at least partially corrected both of these metabolic deviations, suggesting improvements in both sites of insulin resistance. An increased glucose-to-insulin ratio evident during the postabsorptive state of the 24-h profile (Fig. 2), as well as fasting values of the 12-week OGTT (Figs. 3 and 4), were consistent with a decrease in insulin resistance in the treated animals. The latter increase in insulin sensitivity was shown to be significant at both 4 and 12 weeks using the composite insulin sensitivity index of Matsuda and DeFronzo (24). This mathematical analysis was previously validated (with high correlation) against the hyperinsulinemic-euglycemic clamp technique in 153 subjects with varying degrees of insulin resistance. The relative insulin sensitivity of the treated animals improved with each successive OGTT, ultimately reaching a relative index score 1.56 ± 0.26 times that of the control animals. The results of the 24-h glucose/insulin/DP IV profile and the OGTT were corroborated by direct measurements of glucose uptake in soleus muscle strips, which clearly demonstrated improved glucose uptake in both the nonstimulated and the insulin-stimulated states (Fig. 6). Though somewhat controversial, both GIP and GLP-1 (and exendin-4) have been reported to increase muscle insulin sensitivity through the stimulation of glycogen synthesis and glucose uptake (3235). In addition, a number of whole animal studies using GLP-1 or related GLP-1 receptor agonists have observed similar improvements in glucose tolerance and insulin sensitivity. Young et al. (36) showed that long-term administration of the GLP-1 agonist exendin-4 causes glucose-lowering and insulin-sensitizing effects in a number of diabetic animal models, including the fa/fa Zucker rat. Also, a number of subchronic infusion studies have revealed improvements in glycemic control, glucose tolerance, and insulin sensitivity (3739). Our findings are consistent with the results of these previous investigations. However, the indirect contributions of a long-term improvement in glycemia or long-term enhancement of a number of other DP IV substrates (in particular, the insulin secretagogues vasoactive intestinal peptide, pituitary adenylyl cyclase-activating peptide, gastrin-releasing peptide, and neuropeptide Y) over the course of the treatment cannot be ruled out as a causative factor for the metabolic improvements observed. Further experiments will be required to address this issue and that of the relative effect of DP IV inhibitor therapy on each of the major insulin-sensitive tissues.
An important facet shared by the OGTT, the muscle glucose uptake and the pancreas perfusion protocols, was that cessation of drug treatment occurred 18 h before these experimental procedures. Any divergence between groups, therefore, reflected long-term, lasting changes in metabolic state, rather than an acute effect of the drug. Drug washout was confirmed by DP IV activity measurements. It is interesting that an increase in DP IV activity was observed in the treated animals over the course of the study, which was likely a compensatory response to chronic inhibition of DP IV activity in the treated group (>90% of the 24-h cycle). The significance of this finding is not fully understood, as the circulating, soluble form of DP IV measured in the present study represents only 510% of the entire DP IV pool (2). Therefore, further investigation into the effects of DP IV inhibitor therapy on other sources of DP IV activity in the circulation (particularly lymphocytic and endothelial DP IV) is warranted. It is likely that the compensatory change in circulating DP IV levels could be avoided by once-daily treatment and/or a lower inhibitor dosage. Therapeutic dosages required to improve glucose tolerance on an acute scale in humans (0.2 mg/kg) are 100-fold lower than those used in the present study (40,41).
Several therapeutic strategies exploiting the antidiabetic effects of GIP and GLP-1 are currently being pursued. The success of DP IV inhibition is owed in part to a mechanism of action that impacts only a single physiological regulatory system. DP IV inhibition slows the rate of incretin inactivation, but leaves the nutrient-dependent mechanism of incretin release intact and the glucose dependence of their anti-diabetic actions unaltered. The present study showed for the first time that long-term DP IV inhibitor therapy leads to enhanced peripheral insulin sensitivity and ß-cell glucose responsiveness, improvements that culminate in markedly improved glucose tolerance well beyond the clearance time of the drug. Further, the findings of this study exemplify the importance of the noninsulinotropic effects of GIP and GLP-1 in the regulation of ß-cell function. In conclusion, the data presented here establish the potential utility of DP IV inhibitors in the treatment of diabetes.
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ACKNOWLEDGMENTS |
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We would like to thank Cuilan Nian, Narinder Dhatt, and Susan Collins for their excellent technical support.
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FOOTNOTES |
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Received for publication 28 August 2001 and accepted in revised form 9 January 2002.
ACC, acetyl-CoA carboxylase; DP IV, dipeptidyl peptidase IV; GIP, glucose-dependent insulinotropic polypeptide-(142); GLP-1, glucagon-like peptide 1-(7-36)amide; GLP-1a, active GLP-1736; GS, glycogen synthase; OGTT, oral glucose tolerance test.
H.-U.D. is the Chief Executive Officer and Chief Scientific Officer of and a shareholder in Probiodrug GmbH, a pharmaceutical company in the process of developing a DP IV inhibitor treatment for diabetes and its complications. R.A.P. and C.H.S.M. are both members of a scientific advisory panel to Probiodrug and receive consulting fees for their participation. R.A.P. and C.H.S.M. also receive grant/research support from Probiodrug to support studies on the drug candidate P32/98 and its utility in treating diabetes and its complications.
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REFERENCES |
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