Impaired glucose-induced glucagon suppression after partial ...

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The clinical diagnoses chronic pancreatitis, pancreatic carcinoma, pancreatic adenoma or ampullary cancer were confirmed by an independent pathologist in all cases. All post- operative experiments were conducted after full clinical recovery of the patients. The mean time interval between the first oral glucose challenge and surgery was 8.6 ± 7.6 days, and the second test was carried out 23.9 ± 27 days after surgery. Experimental procedures The experiments were performed as described (18) in the morning after an overnight fast with subjects in a supine position throughout the experiments. Both ear lobes were made hyperemic using Finalgon � (Nonivamid 4 mg/g, Nicoboxil 25 mg/g). The experiments were started by the ingestion of the oral glucose load (75 g glucose in 300 ml) over 5 min, and capillary and venous blood samples were drawn at t = -5, 0, 15, 30, 60, 90, 120, 150, 180, 210, and 240. Capillary blood samples (approximately 100 μl) were added to NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. Venous blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol � ; 20000 KIU/ml, 200 μl per 10 ml blood; Bayer AG, Leverkusen, Germany) and kept on ice. After centrifugation at 4 °C, plasma for hormone analyses was kept frozen at -28 °C. Measurements Glucose was measured as described (18, 19) using a glucose oxidase method with a Glucose Analyser 2 (Beckman Instruments, Munich, Germany). Insulin was measured as described (18) using an insulin microparticle enzyme immunoassay (MEIA), IMx Insulin, Abbott Laboratories, Wiesbaden, Germany. Cross- reactivity with proinsulin was < 0.005%. The intra-assay coefficient of variation was 4 %. C-peptide was measured as described (18) using an enzyme-linked immunoabsobent 6
assay (ELISA) from DAKOP Ltd., Cambrigshire, UK. Intra-assay coefficient of variation was 3.3 to 5.7 %, inter-assay variation was 4.6 to 5.7 %. Human insulin and C-peptide were used as standards. IR-glucagon was measured by a radioimmunoassay using antibody no. 4305 in ethanol- extracted plasma, as described (20). The detection limit was < 1 pmol/l. Intra-assay coefficient of variation was below 6.7 %. This assay specifically reacts with the free unmodified C-terminus of the glucagon molecule. Therefore, it is theoretically possible that other circulating forms glucagon than the 3500 dalton glucagon (especially proglucagon 1-61) were detected as well. However, the quantitative contribution of these forms to the overall glucagon immunoreactivity appears to be rather negligible (21). Calculations: The Matsuda index of insulin sensitivity and HOMA insulin resistance were calculated as described (22, 23). The maximum glucose-induced suppression of glucagon levels was calculated by expressing the nadir glucagon levels between t = 15 min and t = 240 min in relation to the respective baseline levels. Statistical Analysis Subject characteristics are reported as mean ± SD, results are presented as mean ± SEM. Time course evaluations were carried out by paired or unpaired analysis of variance (ANOVA), as appropriate, using Statistica version 5.0 (Statsoft Europe, Hamburg, Germany). All other parameters were compared by two-way ANOVA or Student’s t-test, as appropriate. A p-value < 0.05 was taken to indicate significant differences. Regression analyses were carried out using GraphPad Prism 4. 7
Page 1 and 2: J Clin Endocrin Metab. First publis
Page 3 and 4: Introduction: In health, glucose ho
Page 5: Materials and methods Study design
Page 9 and 10: However, after pancreatic head rese
Page 11 and 12: cell secretion (34, 35). Consistent
Page 13 and 14: A number of previous studies in rat
Page 15 and 16: References: 1. Meier JJ, Butler PC
Page 17 and 18: 36. Robertson RP, Sutherland DE, Se
Page 19 and 20: Figure legends: Figure 1: Plasma co

Impaired glucose-induced glucagon suppression after partial ...

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