Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes (original) (raw)

Study subjects. Nine subjects with type 2 diabetes (six men and three women, age 48 ± 4, weight 108 ± 7 kg, body surface area 2.2 ± 0.1 m2, body mass index 37 ± 3 kg/m2) were studied before and after 2 weeks of treatment with aspirin. All the patients were initially screened to rule out any other systemic disease and any biochemical evidence of abnormal renal or hepatic functions. Patients with a history of alcohol abuse, symptomatic coronary heart disease, stroke, current use of insulin for glycemic control, significant hepatic enzyme elevation (more than twice the upper limit of normal), serum creatinine greater than 1.5 mg/dl, history of ketoacidosis or current metabolic acidosis, history of gastric ulcer, dyspepsia, upper or lower gastrointestinal bleed, history of allergy to aspirin, or bleeding diathesis, or currently on oral anticoagulants, were excluded from the study. Women who were pregnant, lactating, or of childbearing potential and not using a barrier or hormonal method of contraception were also excluded from the study. The subjects were asked to stop their antidiabetic medications 1–2 weeks before the study period. Two subjects were diet-controlled and taking no antidiabetic medications, whereas others were on either a sulfonylurea and/or metformin. Given the relatively short biological half-lives of sulfonylurea and metformin compared with thiazolidinediones, it is likely that there were no residual effects of these compounds on any of the base-line parameters. Furthermore any residual effects of these agents would lead to an underestimation of the effect of high-dose aspirin on glucose metabolism. For 3 days prior to each inpatient study, the subjects were given a diet containing 35 kcal/kg body weight, consisting of 60% carbohydrate, 20% fat, and 20% protein, prepared by the Yale/New Haven Hospital General Clinical Research Center (GCRC) metabolic kitchen, and asked to abstain from alcohol, caffeine, and exercise, except for normal daily activities. The same diet was continued during their stay in the GCRC. The protocol was reviewed and approved by the Yale University Human Investigation Committee. Informed written consent was obtained from each subject.

Study design. Subjects were screened at the outpatient facilities of the GCRC, including physical examination, urine analysis, and a check of routine chemistries, fasting lipid panel, cell blood count, prothrombin time/partial thromboplastin time, serum glutamic oxaloacetic transaminase, alkaline phosphatase, and β human chorionic gonadotrophin for females. Stools were checked for blood by hemoccult cards. After the washout period, each subject was admitted to the GCRC at 2100 hours 1 day prior to the day of the study. Each subject was started on 200 μg misoprostol four times per day, to prevent aspirin-induced gastric ulcers, 3 days before the first inpatient visit (5 days before the first base-line clamp study), and the misoprostol was continued for the duration of the study. After their admission, subjects underwent a 24-hour urine collection for assessing urine creatinine and nitrogen excretion.

Mixed-meal tolerance test. After an overnight fast, a mixed-meal tolerance test was performed. At 0730 hours, a catheter was placed in the antecubital vein for blood collection. After obtaining fasting blood samples for plasma glucose, insulin, fatty acids, and C-peptide concentrations, the subjects ingested a liquid meal consisting of 33% of their total caloric requirement (240 kcal/8 ounces, 55% carbohydrate) at 0800 hours. Blood samples were then obtained periodically every half hour for the next 5 hours for plasma concentrations of glucose, insulin, C-peptide, and fatty acids.

Hyperinsulinemic-euglycemic clamp study. A hyperinsulinemic-euglycemic clamp (20) was performed in the morning following an overnight fast. Two indwelling catheters were placed at 0400 hours: one for administration of insulin, glucose, and K+ was inserted into an antecubital vein, and a second for blood drawing was placed in a retrograde direction into a hand vein. The hand was kept warm in a hot box at 70°C to “arterialize” the blood samples. Basal samples were drawn for determination of plasma glucose, insulin, C-peptide, fatty acids, glucagon, and lactate.

A primed (181 mg/m2) continuous infusion (1.81 mg/m2/min) of [6,6-2H2]glucose was infused for 4 hours to assess rates of glucose production. During the last 40 minutes of this infusion, blood samples were drawn for the determination of enrichment of plasma [6,6-2H2]glucose as well as plasma glucose and hormone concentrations. At 0800 hours, a two-step priming dose of insulin was administered for 10 minutes (480 mU/m2/min for 5 minutes, followed by 240 mU/m2/min for 5 minutes), followed immediately by a constant insulin infusion rate of 120 mU/m2/min for a total of 5 hours as previously described (20). The plasma glucose concentration was monitored every 5 minutes and allowed to decrease to 100 mg/dl, then maintained between 90 and 100 mg/dl throughout the study period by administration of glucose (20 g of dextrose per 100 ml enriched to approximately 2.5% with [6,6-2H2]glucose). During the last 40 minutes of the fifth hour of the insulin infusion, blood samples were obtained every 10 minutes for measurements of plasma glucose, fatty acid, insulin, and [6,6-2H2]glucose enrichment. To avoid insulin-induced hypokalemia, potassium was administered at a rate of 6 milli-equivalent/h. Indirect calorimetry was performed to determine whole-body glucose oxidation before and during the last 30 minutes of the hyperinsulin-emic clamp (21).

Treatment period. Upon completion of the base-line study, subjects were started on enteric-coated aspirin (6.2 g/d) divided in five equal doses (at 0700, 1100, 1500, 1900, and 2300 hours) for 2 weeks. Blood salicylate and electrolyte concentrations were monitored every 1–2 days, and the aspirin dose was adjusted to keep salicylate concentrations between 25 and 35 mg/dl. One week after the initiation of aspirin, patients were readmitted for mixed-meal tolerance testing. In addition, the subjects were seen as outpatients every other day during the treatment period to measure safety parameters (adverse event query, vital signs, stool guaiac) and to monitor compliance by blood salicylate level.

At the end of 2 weeks of study, subjects were readmitted to GCRC and underwent the same procedures as during the base-line period, with a mixed-meal tolerance test on the first day and then hyperinsulinemic-euglycemic clamp on the second day. At the completion of the study, the subjects were instructed to restart their antidiabetic medications.

Substrate and hormone measurements. Plasma glucose was measured at the bedside with a Beckman Instruments Inc. Glucose Analyzer II (Fullerton, California, USA). Plasma insulin and glucagons were determined by RIA (Linco Research, St. Charles, Missouri, USA), with an interassay coefficient of variation of 10.8% and an intra-assay coefficient of variation of 9.9% for insulin, and an interassay coefficient of variation of 7.4% and an intra-assay coefficient of variation of 6.9% for glucagon. Plasma C-peptide was also measured by RIA (Linco Research), with an interassay coefficient of variation of 10% and an intra-assay coefficient of variation of 7.9%. Plasma fatty acid concentrations were determined using a microfluorometric method (22), and plasma lactate concentrations were measured using a lactate dehydrogenase method (23). Urine nitrogen was done at the Mayo Medical Laboratories (Rochester, Minnesota, USA). [6,6-2H2]glucose enrichment was measured by gas chromatography–mass spectrometry analysis (24).

Calculations. Rate of endogenous glucose production (EGP) (mg/kg/min) = isotope infusion rate × [(enrichmentinfusate / enrichmentplasma) – 1].

Glucose disposal rate (Rd) (mg/kg/min) = endogenous glucose production during the clamp (cEGP) + glucose infusion rate (GIR). GIR is the mean rate of infusion of exogenous glucose from minutes 200–300 of the clamp period (mg/kg/min). cEGP was calculated as follows: cEGD = GIR × [(enrichmentinfusate /enrichmentplasma) – 1].

Insulin clearance rate (ml/min) = insulin infusion rate (pmol/min) / plasma insulin concentration (pmol/ml).

Statistical analysis. Data are expressed as mean ± SE. Paired t test was used to assess the differences between measurements before and after treatment. The area under the curve was used to compare different substrate and hormone profiles during the mixed-meal tolerance test and the clamp period before and after treatment.