Vella A. et al. Appendix 3

Effects of Dipeptidyl Peptidase 4 Inhibition on Gastrointestinal Function, Meal Appearance and Glucose Metabolism in Type 2 Diabetes

Subjects

Subjects were eligible to participate in the study if they fulfilled the following criteria: -

·  Age 35 to 65 years with type 2 diabetes

·  HbA1c < 9%.

·  Body mass index > 19 or < kg/m2, or a total weight < 130kg.

·  Stable weight and did not engage in regular vigorous exercise

·  No history of a) proliferative retinopathy; b) significant nephropathy, (i.e., plasma creatinine > 1.4 mg/dl in women and 1.5 mg/dl in men, and/or proteinuria); c) symptomatic autonomic neuropathy; d) clinically significant atherosclerotic vascular disease (e.g., history of MI or angina); e) a known systemic illness.

·  Negative Bowel Disease Questionnaire (1).

·  Participants able to complete a 3 week wash-out of current anti-diabetic medications during this time they will self-monitor their blood sugars. Discontinued if fasting glucose > 250mg/dL or random glucose > 300mg/dL.

·  Patients on diuretics or cyclic hormone therapy must be on a stable dose (at least 3 months prior to screening) and the maintenance dose may not be adjusted during the study.

·  Not taking medication known to alter gastric emptying.

Fourteen subjects with type 2 diabetes gave written informed consent to participate in the study. All subjects were instructed to follow a weight maintenance diet (55% carbohydrate, 30% fat, and 15% protein) during the two phases of the study. All agents used for the treatment of diabetes were discontinued three weeks before the study.

Volunteer Characteristics
Age (years) / 53.1 + 2.0
Gender (M/F) / 5 / 9
BMI (Kg/M2) / 33.9 + 1.5
HbA1c (%) / 6.1 + 0.2
Treatment prior to study / 4 diet alone
7 Metformin monotherapy
3 Metformin + Oral hypoglycemic agent

Table 1: Volunteer Characteristics


Analytical techniques

Plasma samples were placed on ice, centrifuged at 4°C, separated, and stored at -20°C until assayed. Glucose concentrations were measured using a glucose oxidase method (Yellow Springs Instrument, Yellow Springs, OH). Insulin was measured using a chemiluminescence assay with reagents obtained from Beckman (Access Assay; Beckman, Chaska, MN). Glucagon and C-peptide were measured by RIA using reagents supplied by Linco Research (St. Louis, MO). Samples tubes utilized for measurement of GLP-1 had 100 mM of DPP-4 inhibitor (Linco Research, St. Louis, MO) added. Active GLP-1 concentrations were measured using an N-terminal immunoassay supplied by Linco Research (St. Louis, MO). Body composition was measured using dual-energy X-ray absorptiometry (DPX scanner; Lunar, Madison, WI). Plasma [6,6-2H2] glucose and [1-13C] glucose enrichments were measured using gas chromatographic mass spectrometry (Thermoquest, San Jose,CA) to simultaneously monitor the C-1 and C-2 and C-3 to C-6 fragments, as described by Beylot et al. (2) and [6-3H] glucose specific activity by liquid scintillation counting following deproteinization and passage over anion and cation exchange columns.

Calculation of glucose appearance and disappearance rates

The systemic rates of meal appearance (Rameal), endogenous glucose production (EGP) and glucose disappearance (Rd) were calculated using Steele’s two compartment model (3) as previously described (4) In brief, Rameal was calculated by multiplying rate of appearance of [1-13C] glucose (obtained form the infusion rate of [6-3H] glucose and the clamped plasma ratio of [6-3H] glucose and [1-13C] glucose) by the meal enrichment (i.e. the ratio of total glucose to tracer in the meal). EGP was calculated from the infusion rate of [6,62H2] glucose and the clamped plasma ratio of [6,62H2] glucose to endogenous glucose concentration. Glucose disappearance was calculated by subtracting the change in glucose mass from the overall rate of glucose appearance (i.e. Rameal+EGP). Values from –30 to 0 minutes were averaged and considered as basal. Area above basal and area under the curve was calculated using the trapezoidal rule.

Figure 1: Effect of Vildagliptin on gastric emptying T1/2 (min). Black horizontal bars represent mean values for the placebo and vildagliptin group. Each individual’s T1/2 in the presence or absence of vildagliptin is represented as (¡) linked by a solid line.

1. Talley NJ, Phillips SF, Melton J, 3rd, Wiltgen C, Zinsmeister AR: A patient questionnaire to identify bowel disease. Ann Intern Med 111:671-674, 1989

2. Beylot M, Previs SF, David F, Brunengraber H: Determination of the 13C-labeling pattern of glucose by gas chromatography-mass spectrometry. Anal Biochem 212:526-531, 1993

3. Steele R, Bjerknes C, Rathgeb I, Altszuler N: Glucose uptake and production during the oral glucose tolerance test. Diabetes 17:415-421, 1968

4. Basu R, Di Camillo B, Toffolo G, Basu A, Shah P, Vella A, Rizza R, Cobelli C: Use of a novel triple-tracer approach to assess postprandial glucose metabolism. Am J Physiol Endocrinol Metab 284:E55-69, 2003