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Division of Endocrinology and Metabolism (K.A., Z.R., J.M., F.G.S.T., E.T., C.K., D.E.K.), Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; the Department of Information Engineering (C.D.M., C.C.), University of Padova, 35122 Padova, Italy; Department of Medical Physiology (J.J.H., C.F.D.), The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark; Novartis Pharmaceuticals Corporation (Y.H.), Cambridge, Massachusetts 02139; and Novartis Pharmaceuticals Corporation (M.L.-S., D.S., J.E.F.), East Hanover, New Jersey 07936
Address all correspondence and requests for reprints to: James E. Foley, Ph.D., Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, New Jersey 07936. E-mail: james.foley{at}novartis.com.
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Abstract |
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Research Design and Methods: Participants with T2DM (n = 16) who had a baseline hemoglobin A1c of 7.1 ± 0.2% completed a crossover study with 6 wk of treatment with vildagliptin and 6 wk with placebo. At the completion of each arm, participants had a study of postprandial metabolism and a two-step glucose clamp performed at 20 and 80 mU/min·m2 insulin infusions.
Results: Vildagliptin increased postprandial glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide by 3- and 2-fold, respectively, reduced fasting plasma glucose and postprandial plasma glucose by 1.3 ± 0.3 mmol/liter and 1.6 ± 0.3 mmol/liter (both P <0.01), and improved glucose responsiveness of insulin secretion by 50% (P < 0.01). Vildagliptin lowered postprandial glucagon by 16% (P <0.01). Examined by glucose clamp, insulin sensitivity and glucose clearance improved after vildagliptin (P < 0.01).
Conclusions: Vildagliptin improves islet function in T2DM and improves glucose metabolism in peripheral tissues.
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Introduction |
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Pharmacological inhibition of the enzyme dipeptidyl peptidase 4 (DPP-4), which prolongs incretin action, has emerged as a novel treatment for type 2 diabetes mellitus (T2DM) (10, 11, 12, 13). DPP-4 inhibitors, such as vildagliptin and sitagliptin, improve glucose responsiveness of insulin secretion in T2DM (10, 11, 12, 13). DPP-4 inhibition also reduces postprandial glucagon in T2DM (11, 14, 15), which also contributes to improved glucose homeostasis (16, 17). These effects are direct (14, 15) and regarded as the main mechanisms by which DPP-4 inhibition lowers glucose. The increases in intact GLP-1 attained with DPP-4 inhibition do not appear to be sufficient to induce weight loss (18). Although a small early delay in gastric emptying has been observed after a single dose of vildagliptin (19), vildagliptin does not slow gastric emptying in a clinically meaningful manner after multiple dosing in patients with T2DM (20). Vildagliptin has been associated with attenuated insulin resistance during meals (12, 21). It is not known, however, whether DPP-4 inhibition improves peripheral glucose utilization.
The current study was undertaken to evaluate the effects of vildagliptin treatment on peripheral glucose utilization in T2DM, using a double-blinded, placebo-controlled, crossover study in patients with T2DM. A two-step hyperinsulinemic glucose clamp was performed to assess glucose utilization.
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Subjects and Methods |
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This was a double-blinded, placebo-controlled, randomized-order, crossover study (Fig. 1
). Vildagliptin (50 mg twice daily) or placebo was given 30 min before breakfast and dinner for 6 wk, and participants were then crossed over to the alternate treatment for 6 wk. To be eligible, participants were required to have T2DM, a hemoglobin A1c (HbA1c) of no more than 8.0%, and a fasting plasma glucose (FPG) of no more than 11 mmol/liter and be treated by diet and exercise alone or with metformin. Those treated with a sulfonylurea, repaglinide, or nateglinide were eligible provided these agents were withdrawn and replaced by metformin monotherapy, individually adjusted (standard clinical practice) during a 4- to 6-wk period to meet the criterion of FPG of no more than 11 mmol/liter before randomization. Metformin was maintained at a stable dose during the trial. Written informed consent was obtained, and the research protocol was approved by the University of Pittsburgh Institutional Review Board.
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Eighteen participants were randomized. One withdrew consent, another was excluded because of noncompliance with study medications, and 16 completed the study. The clinical characteristics were nine men and seven women with a mean age 56 ± 2 yr, a mean body mass index of 31.4 ± 1.0 kg/m2, and mean HbA1c at randomization of 7.1 ± 0.2%. Five were drug-naive at randomization, and 11 were on metformin with a mean daily dose of 1.7 g.
Metabolic evaluations
At completion of each 6-wk block of vildagliptin or placebo, participants were admitted to the University of Pittsburgh General Clinical Research Center for a 2-d metabolic evaluation. On the evening of admission, participants received study medication before dinner and fasted overnight. In the morning, a mixed macronutrient meal (7 kcal/kg; 50% carbohydrate, 35% fat, and 15% protein) was given along with study medication 30 min before the meal. One gram of [1-13C]glucose was added to the meal to trace ingested glucose. Blood samples were collected at –30, –15, 0, 10, 20, 30, 60, 90, 120, 150, 180, 240, and 300 min for measurements of plasma glucose, insulin, and C-peptide and at selected time points for measurement of [1-13C]glucose plasma enrichment, intact GLP-1, intact GIP, glucagon, and DPP-4 activity.
On the following day, withholding that morning’s dose of study medication, a two-step insulin infusion-glucose clamp was performed, as shown in Fig. 2. A primed (1.1 mmol/m2), continuous infusion of [6,6-d2]glucose (0.014 mmol/min·m2) was started 3 h before insulin infusions. A 60-min infusion of [U-13C]palmitate (11 nmol/min·kg) was given to measure palmitate rate of appearance (Ra) and utilization (Rd) during fasting conditions and resumed for a 60-min infusion during the final hour of each of the two steps of insulin infusion. During the first step of the clamp, insulin was infused at 20 mU/min·m2 for 3 h (LO), and plasma glucose was maintained at an isoglycemic level (i.e. maintaining the FPG of the study morning). This LO step was done to evaluate the effect of vildagliptin at a low physiological level of insulin and without altering ambient FPG. The LO step was then followed by an infusion at 80 mU/min·m2 (HI) for 2 h. During HI, plasma glucose was maintained at euglycemia (5.0 mmol/liter) to study the effect of vildagliptin at an upper physiological insulin level. Plasma glucose was maintained by an adjustable infusion of 20% dextrose, labeled with [6,6-d2]glucose, to maintain stable plasma enrichment. A heated hand was used for blood sampling. Systemic indirect calorimetry, using an open-circuit spirometry metabolic monitor system (Delta Trac, Anaheim, CA), was performed for 30-min intervals during fasting and at steady-state conditions during LO and HI. To estimate protein oxidation, an overnight urine collection was obtained for nitrogen determination.
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Plasma glucose was measured by the glucose oxidase method (YSI Glucose Analyzer, Yellow Springs, OH). Insulin, C-peptide, and GLP-1 were measured by commercially available ELISA kits, and glucagon was measured by RIA using a commercially available kit (Linco Research Inc., St. Charles, MO). Intact GIP was measured using a RIA (antiserum 98171, specific for the intact N terminus of GIP) (22). DPP-4 activity (mU/ml·min) was measured using a fluorometric method, which is based on the ability of DPP-4 to degrade H-GLy-Pro-7-amino-4-methyl-coumarin to fluorescent 7-amino-4-methyl-coumarin. Free fatty acids (FFA) and [U-13C]palmitate were extracted (23), separated, derivatized (24), and measured on a gas chromatograph (Agilent, GC-6890; Santa Clara, CA) equipped with a flame ionization detector. 13C enrichment of plasma glucose and [6,6-d2]glucose enrichment of plasma glucose were measured in glucose pentaacetate derivatives prepared by addition of 2:1 acetic anhydride-pyridine using, respectively, gas chromatography-combustion-isotope ratio mass spectrometry and gas chromatography-mass spectrometry.
Calculations
Rates of insulin secretion were calculated as previously described by using a model for C-peptide secretion and parameters of 
static (an index of insulin secretion in response to a given glucose concentration) and dynamic (an index of insulin secretion in response to a change in glucose concentration) were determined (25). The systemic concentration of ingested glucose was calculated using the ratio of plasma [1-13C]glucose enrichment to the meal [1-13C]glucose enrichment multiplied by postprandial plasma glucose (PPG). Rates of glucose appearance and utilization (Ra and Rd) were calculated using non-steady-state equations (26), and glucose metabolic clearance rate (MCR glucose) was determined as the quotient of glucose Rd and plasma glucose. Rates of glucose and fat oxidation were calculated using the equations developed by Frayn (27).
Statistical analysis
Data are presented as mean ± SEM. A paired t test was used to compare treatments (vildagliptin vs. placebo), and two-way repeated-measurement ANOVA was used to examine for time and treatment effects. A P value of <0.05 was considered significant. Statistical evaluations were performed using Sigma Stat 3.0 (Handel Scientific, San Rafael, CA).
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Results |
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Vildagliptin strongly suppressed plasma activity for DPP-4 as measured at 2 h (0.2 ± 0.04 vs. 9.5 ± 0.5 mU/ml·min for placebo; P < 0.01) and 12 h after drug administration (1.4 ± 0.3 vs. 9.2 ± 0.4 mU/ml·min; P < 0.01). Fasting plasma glucose was lower at the end of 6 wk treatment with vildagliptin (7.0 ± 0.2 vs. 8.2 ± 0.3 mmol/liter; P < 0.01). Neither fasting insulin (47 ± 9 vs. 50 ± 8 pmol/liter, vildagliptin vs. placebo) nor glucagon (91 ± 6 vs. 96 ± 5 ng/liter, vildagliptin vs. placebo) differed across treatment arms. Fasting intact GIP was similar across arms (11 ± 2 vs. 9 ± 1 pmol/liter, vildagliptin vs. placebo). Fasting intact GLP-1 was 3-fold higher during vildagliptin treatment (10 ± 2 vs. 3 ± 0.2 pmol/liter; P < 0.01).
Effects of vildagliptin on PPG
Vildagliptin reduced PPG by 1.6 ± 0.3 mmol/liter (P < 0.01). Plasma enrichment for [1-13C]glucose (that had been used to label ingested glucose) was highly similar across treatment arms. These data were used to estimate systemic concentrations of ingested and endogenous glucose. Plasma concentrations of endogenous glucose were significantly reduced with vildagliptin (P < 0.01), as shown in Fig. 3, whereas those for ingested glucose were similar across treatments (data not shown). Intact GLP-1 rose and remained 3-fold higher during vildagliptin treatment during postprandial conditions, and intact GIP was 2-fold higher (P < 0.01 for both) (Fig. 4). Postprandial glucagon was significantly reduced (P < 0.01). The reduction in glucagon correlated with that for endogenous plasma glucose (r = 0.55; P < 0.05). Postprandial insulin and C-peptide were nearly identical across treatments. Physiological modeling of PPG, insulin, and C-peptide revealed a 50% increase in static with vildagliptin (69 ± 7 vs. 46 ± 5 10–9/min; P < 0.01). This parameter reflects the response of insulin secretion to ambient plasma glucose. There was not a significant treatment difference for dynamic (597 ± 81 vs. 471 ± 92 10–9), a parameter indicative of the responsiveness of insulin secretion to changes in plasma glucose.
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On the day after meal studies, a two-step (LO and HI) insulin infusion was carried out (Fig. 2
). Study medication was withheld that morning. Fasting rates of glucose appearance (glucose Ra) were similar across treatments (1.76 ± 0.05 vs. 1.80 ± 0.05 mg/kg·min, vildagliptin vs. placebo), but glucose flux normalized to FPG (MCR glucose) was significantly higher with vildagliptin (1.45 ± 0.07 vs. 1.30 ± 0.07 ml/kg·min; P < 0.01; Fig. 5). The improvement in fasting MCR obtained with vildagliptin correlated with improvement in PPG (r = –0.73; P < 0.01). Fasting palmitate flux was lower with vildagliptin (120 ± 8 vs. 145 ± 11 µmol/min; P < 0.01).
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). Suppression of endogenous glucose production (EGP) was equivalent across arms (–1.48 ± 0.11 vs. –1.48 ± 0.12 mg/kg·min). During the HI insulin infusion (80 mU/m2·min), euglycemia was achieved and was matched across arms (5.3 ± 0.1 vs. 5.2 ± 0.1 mmol/liter, vildagliptin vs. placebo), and plasma insulin was matched across treatment arms. Plasma glucagon was slightly but significantly lower with vildagliptin (P < 0.01). Plasma FFA, suppression of EGP, and suppression of palmitate flux were similar across arms. Insulin-stimulated systemic glucose utilization was significantly higher under vildagliptin treatment (6.05 ± 0.49 vs. 5.35 ± 0.51 mg/kg·min; P < 0.05).
Systemic indirect calorimetry was performed during fasting, LO, and HI conditions. Under fasting conditions, energy expenditure, respiratory quotient, and rates of glucose and fat oxidation were closely similar across treatments. This similarity persisted during LO and HI, although glucose oxidation was slightly greater with vildagliptin (P = 0.08).
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Discussion |
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- and β-cell function, which are key mechanisms of action for improving glucose homeostasis in T2DM (16, 17). A more novel finding from the current study is that vildagliptin improves peripheral glucose utilization. Under steady-state euglycemic conditions, vildagliptin treatment increased insulin-stimulated glucose utilization. Also, during fasting conditions as well as during a modest level of insulin stimulation conducted under conditions maintaining FPG, vildagliptin treatment was associated with a significant increase in glucose clearance. The most likely explanation for at least some of the improved insulin-stimulated glucose utilization is that improved glucose control alleviates glucose toxicity (28). However, mean HbA1c at randomization was 7.1%, indicative of good baseline glucose control, so that participants with relatively mild severity of hyperglycemia were investigated, and therefore, alleviation of glucose toxicity may not be the full explanation for the increased insulin sensitivity that was observed. Previous research on the effects of glucose toxicity on insulin resistance noted effects in response to much larger reductions in hyperglycemia (29). Another possible component is reduced lipotoxicity. Stored triglycerides in muscle and liver may be reduced because of the modest but significant reduction in fasting FFA flux. This hypothesis requires further study.
In the current study, the reduction in postprandial glucagon achieved with vildagliptin treatment correlated with lower postprandial concentrations of endogenous glucose, which indicates that amelioration of -cell dysfunction is one of the important mechanisms by which DPP-4 inhibition improves glucose homeostasis in T2DM (16, 17). A slight and nonsignificant reduction in fasting glucagon was also observed and might therefore have contributed to an uncertain extent to the effects of vildagliptin on glucose clearance. However, in HI insulin infusion studies, hepatic glucose production was fully suppressed, so it is unlikely that there was a contribution of treatment effects on glucagon to the increased glucose utilization associated with vildagliptin treatment.
Another possible component of the improved peripheral glucose utilization is a direct effect of either GLP-1 or GIP on glucose uptake. The current study cannot distinguish whether this effect is direct or secondary to the improved metabolic state associated with vildagliptin treatment. A single-dose study, where islet function is taken into account, is required to address this question.
Metformin and thiazolidinediones (TZD) are two classes of diabetic medications that are generally regarded as insulin sensitizers. A recent metaanalysis (30) found that metformin improves hepatic insulin sensitivity by 18% and increases fasting MCR by 18% but does not improve insulin-stimulated MCR. TZD agents (considered as a class) improved hepatic insulin sensitivity EGP by 19% and increased fasting MCR by 15% and insulin-stimulated MCR by 34% (30). Therefore, the 15% improvement in glucose MCR (during both fasting and insulin-stimulated conditions) that was associated with vildagliptin is intermediate between the improvements induced by metformin and a TZD. It was recently reported that metformin increases total GLP-1 in patients with T2DM and, when used in combination with DPP-4 inhibition, raises intact GLP-1 to higher levels than that attained with DPP-4 inhibition alone (31). In the current study, the effects of vildagliptin to increase glucose MCR was similar in those on metformin and vildagliptin and those on vildagliptin monotherapy.
Other findings of the current study continue to bolster the body of data that DPP-4 inhibition improves glucose-dependent insulin secretion in patients with T2DM (10, 11, 12, 13, 14). Postprandial insulin concentrations were highly similar across treatment arms, and insulin secretion was nearly identical, although at lower glucose concentrations with vildagliptin. Compartmental analysis of the dynamics of insulin secretion revealed that vildagliptin induced a 50% increase in static, which is the parameter denoting the response of insulin secretion to ambient glucose (25). There was not a statistically significant increase in dynamic, which is the component of insulin secretion that is responsive to change in glucose concentrations (25).
A recent study has examined the effect of vildagliptin on gastric emptying in patients with T2DM (20), which was ascertained by nuclear imaging and found to not be altered by DPP-4 inhibition. Also Vella et al. (20) labeled the meal with stable isotopes and ascertained that entry of ingested glucose into the peripheral pool was not altered by vildagliptin, a process that involves not only rates of gastric emptying but also first-pass splanchnic uptake of ingested glucose. In the current study, we have made similar observations based on labeling of the meal with [1-13C]glucose. Together, these data indicate that splanchnic tissues are unlikely to be the site of increased glucose utilization.
In summary, using a random-order crossover design with double-blinded administration of vildagliptin and placebo, we observed that DPP-4 inhibition improves glucose responsiveness of insulin secretion, increases postprandial suppression of glucagon, and augments peripheral glucose utilization. This investigation, although fully confirming earlier findings of the effects of DPP-4 inhibition on islet function in T2DM, has also yielded the novel finding that DPP-4 inhibition is associated with increased glucose clearance. The mechanisms responsible for the peripheral effects of vildagliptin were not elucidated by the current study and warrant further investigation.
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Acknowledgments |
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Footnotes |
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This trial (NCT 00351546) is registered with ClinicalTrials.gov.
Disclosure Summary: K.A., Z.R., J.M., F.G.S.T., E.T., C.K., C.D.M., and C.C. have nothing to disclose. J.J.H. has previously consulted for and received research support from Novartis Pharma. C.F.D. received research support from Novartis Pharma. Y.H., M.L.-S., D.S., and J.E.F. are employed by and have an equity interest in Novartis Pharma. D.E.K. consulted for and received grant support from Novartis Pharma and is now an employee of Merck.
First Published Online November 27, 2007
1 K.A. and Z.R. contributed equally to this work. 
Abbreviations: DPP-4, Dipeptidyl peptidase 4; EGP, endogenous glucose production; FFA, free fatty acid; FPG, fasting plasma glucose; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; HbAA1c, hemoglobin A1c; MCR, metabolic clearance rate; PPG, postprandial plasma glucose; T2DM, type 2 diabetes mellitus; TZD, thiazolidinediones.
Received June 20, 2007.
Accepted November 19, 2007.
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) are shown for plasma glucose (A), plasma enrichment of [1-13C]glucose (B), and estimations of the plasma concentration of endogenous glucose (C) and plasma glucagon (D). *, P < 0.05, by two-way repeated-measurements ANOVA.
