Granutest

After a 12-h overnight fast, participants in study groups 1 and 4 underwent a standard 75 g OGTT. Serum insulin and plasma glucose was measured at 0, 30 and 120 minutes. Serum insulin levels (excluding des-31,32 and intact proinsulin) were measured using the AutoDELFIA insulin kit (Perkin-Elmer, Wallac, Turku, Finland). Plasma glucose was analyzed using a glucose oxidase method (Granutest; Merck, Darmstadt, Germany) [26 (link)].

After a 12-hour overnight fast, participants were subjected to an IVGTT in which glucose min (0.3 g/kg body weight of 50% glucose) was injected in the contralateral antecubital vein over a period of 1 minute. At 20 min, a bolus of 3 mg tolbutamide/kg body weight (Orinase, Upjohn, Kalamazoo, MI, USA) was injected over 5 seconds to elicit a secondary pancreatic beta-cell response. Venous blood samples were drawn in triplicate at 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, and 180 minutes for analysis of plasma concentration of glucose and serum insulin. The plasma glucose concentration was analyzed by a glucose oxidase method (Granutest, Merck, Darmstadt, Germany). Serum insulin was determined by ELISA excluding des-31,32 and intact proinsulin [27 (link)].

Variants in GCK were identified using sequencing. Samples were collected from a population-based cohort of 6,058 individuals both with and without diabetes [50 (link)], 2,930 patients with newly-diagnosed diabetes [51 (link)], patients diagnosed with GCK-MODY [53 (link)] and from a population of 1,146 Danish children [52 (link)]. Individuals were included if they carried one missense GCK variant according to transcript NM_000162 and if a measure of fasting plasma glucose was available. Measures of fasting plasma glucose were examined using a glucose oxidase method (Granutest; Merck, Darmstadt, Germany) in the population based cohort and in samples from patients with known GCK-MODY [50 (link), 53 (link)], an enzymatic hexokinase method (Gluco-quant Glucose/HK, Roche Diagnostics) in newly diagnosed diabetes patients [51 (link)], and using a Dimension Vista® 1500 Analyzer (Siemens, Erlangen, Germany) in children [52 (link)]. Samples were excluded if fasting plasma glucose level exceeded 9 mM.