Results (
Thai) 2:
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To test this hypothesis, 12 patients with type 2 diabetes were examined twice, once during a euglycemic (5.0 mmol/l) clamp experiment and another time during a hyperglycemic (10.5 mmol/l) clamp. The experiments were performed in a single-blind fashion with the order of conditions balanced across patients. On both clamp conditions, insulin was infused at a constant rate of 2.5 mU/kg per min for 125 min. Simultaneously, a glucose solution was infused at a variable rate to achieve target glycemic levels. Twelve patients with type 2 diabetes were studied (5 men and 7 women). Patients' characteristics were as follows (means ± SE [range]): age 55.3 ± 2.9 years (39-69), BMI 30.1 ± 1.8kg/m^sup 2^ (21.5-40.7), known diabetes duration 11 ± 2 years (3-25), HbA^sub 1c^ 9.0 ± 0.5% (7.0-14.2, upper limit of the reference range 6.7). For treatment of diabetes, 10 patients received insulin at a dose of 52 ± 14 units/day (16-168), with 6 of these patients following a multiple injection regimen. Metformin was taken by eight and sulfonylureas by two of the patients. The subjects were instructed not to take these oral hypoglycemic agents in the morning before the experiments. Each patient gave written informed consent, and the study was approved by the local ethics committee.
Each patient was tested twice, once during a euglycemic clamp and another time during a hyperglycemic clamp. The study was performed in a single-blind cross-over design with the order of conditions balanced across patients. The time interval between the experimental sessions was at least 1 week. The patients were not informed that the main outcome variable of the study was the amount of ingested food, since this information could have influenced eating behavior. Instead, they were told that the study aimed to assess the influence of blood glucose concentration on several metabolic and endocrine parameters. For the same reason, we refrained from assessing feelings of hunger and satiety by ratings to avoid subjects paying undue attention to their eating behavior.
On the days of the experiments, subjects reported to the medical research unit at 6:30 A.M. Eating as well as the ingestion of caloric drinks was allowed until 10:00 A.M. on the preceding day but not thereafter. The patients who were on insulin therapy received their regular dose of long-acting but not short-acting insulin subcutaneously immediately after reporting to the laboratory. The experiments took place in a sound-attenuated room with the patients sitting with his/her trunk in an almost upright position (~60°) and his/her legs in a horizontal position on a bed. A cannula was inserted into a vein on the back of the hand, which was placed in a heated box (50-55°C) to obtain arterialized venous blood. A second cannula was inserted into an antecubital vein of the contralateral arm. Both cannulae were connected to long thin tubes, which enabled blood sampling and adjustment of the rate of glucose infusion from an adjacent room without awareness of the patient. With this experimental setting we were able to keep the patients completely unaware of their respective blood glucose levels throughout the clamps.
After a 30-min baseline period starting at 7:30 A.M., subjects received a bolus injection of regular human insulin (Avenus, Bad Soden, Germany) at a dose of 0.02 units/kg body wt followed by a constant rate infusion of insulin (2.5 mU . kg body wt^sup -1^ . min^sup -1^) during the next 125 min. Arterialized blood was drawn at 5-min intervals to measure glucose concentration (Glucose Analyser; Beckman Coulter, Munich, Germany). A 20% glucose solution was simultaneously infused at a variable rate to control plasma glucose levels. The target glycemic level was 5.0 mmol/l during the euglycemic clamp and 10.5 mmol/l during the hyperglycemic clamp. Blood samples for determination of serum or plasma levels of insulin, C-peptide, leptin, and ghrelin were collected every 30 min throughout the experiments.
Ninety minutes after the beginning of the clamp (9:30 A.M.), a typical German breakfast buffet of a total of 1,633.6 kcal was offered (Table 1). The patients were instructed to eat as much as they wanted during the next 30 min (i.e., until the end of the clamp) and told they did not have to worry about their blood glucose level since it would be controlled by insulin infusion for the next 3 h. After the clamp, blood glucose was monitored for at least 2.5 h until stable levels between 4.5 and 11.0 mg/dl were achieved.
Assays
Serum insulin, C-peptide, amylin, and leptin concentrations were assessed using enzyme-linked immunosorbent assays (insulin: Dako Cytomation, Cambridgeshire, U.K.; interassay coefficient of variation [CV] 7.5%, intra-assay CV 6.7%; C-peptide: Dako Cytomation; interassay CV 5.2%, intra-assay CV 4.7%; amylin: Linco Research, St. Charles, MO; interassay CV 15.4%, intra-assay CV 8.2%; leptin: Diagnostic Systems Laboratories, Sinsheim, Germany; interassay CV 4.4%, intra-assay CV 3.8%). For determination of total plasma GLP-1 as well as total plasma ghrelin levels, blood was collected in prefilled tubes containing EDTA (1 mg/ml) and aprotinin (500 units/ml) and than assessed using commercial radioimmunoassay (Linco Research; GLP-1: interassay CV 22%, intra-assay CV 23%; ghrelin: interassay CV 14.7%, intra-assay CV 10%).
View Image - Figure 1-Mean ± SE blood glucose concentrations during the baseline period (-30 to 0 min) and the euglycemic ([black circle]) and hyperglycemic ([white circle]) clamp (0-125 min) performed in 12 patients with . Gray area indicates the time period when the patients were allowed to eat.
Statistical analysis
Data are reported as means ± SE. Statistical analyses were generally based on repeated-measures ANOVA including factors for the condition (hyperglycemia versus euglycemia) and the multiple measurements during the clamps. For subsequent pairwise comparisons, Student's t test was used. To determine whether differences in food intake between the euglycemic and hyperglycemic condition were correlated with other patient variables, Pearson's correlation coefficients were calculated. A P value <0.05 was considered significant.During the final 30 min of the clamps, the patients were allowed to eat as much as they liked from a standard breakfast buffet.
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