Demonstration of Glycated Insulin in Human Diabetic Plasma and Decreased Biological Activity Assessed by Euglycemic-Hyperinsulinemic Clamp Technique in Humans

• Published in: Diabetes (New York, N.Y.) Vol. 52; no. 2; pp. 492 - 498
• Main Authors: HUNTER, Steven J, BOYD, Alison C, SHERIDAN, Brian, BARNETT, Christopher R, MCNULTY, Helene, BELL, Patrick M, FLATT, Peter R, O'HARTE, Finbarr P. M, MCKILLOP, Aine M, WIGGAM, M. Ivan, MOONEY, Mark H, MCCLUSKEY, Jane T, LINDSAY, John R, ENNIS, Cieran N, GAMBLE, Raymond
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01.02.2003
• Subjects: Animals; Binding, Competitive; Biological activity; Biological and medical sciences; Blood Glucose - metabolism; CHO Cells; Chromatography, High Pressure Liquid; Complications and side effects; Cricetinae; Diabetes; Diabetes Mellitus, Type 2 - blood; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Genetic aspects; Glucose; Glucose Clamp Technique; Glycated Hemoglobin A - analysis; Glycosylation; Health aspects; Humans; Hyperglycemia; Hyperinsulinism - blood; Hyperlipidemia; Insulin - administration & dosage; Insulin - analogs & derivatives; Insulin - blood; Insulin - isolation & purification; Insulin - metabolism; Insulin - pharmacology; Insulin resistance; Male; Mass spectrometry; Medical sciences; Peptides; Physiology; Plasma; Scientific imaging; Spectrometry, Mass, Electrospray Ionization; Type 2 diabetes; Endocrinopathy; Human; Pancreatic hormone; Hypoglycemic agent; Diabetes mellitus; Insulin; Biological activity; Blood plasma
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/diabetes.52.2.492

Demonstration of Glycated Insulin in Human Diabetic Plasma and Decreased Biological Activity Assessed by Euglycemic-Hyperinsulinemic Clamp Technique in Humans Steven J. Hunter 1 , Alison C. Boyd 2 , Finbarr P.M. O’Harte 2 , Aine M. McKillop 2 , M. Ivan Wiggam 1 , Mark H. Mooney 2 , Jane T. McCluskey 2 , John R. Lindsay 1 , Cieran N. Ennis 1 , Raymond Gamble 1 , Brian Sheridan 1 , Christopher R. Barnett 2 , Helene McNulty 2 , Patrick M. Bell 1 and Peter R. Flatt 2 1 Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, U.K 2 School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, Northern Ireland, U.K Abstract The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA 1c 8.1 ± 0.2%, plasma glucose 8.7 ± 1.3 mmol/l [means ± SE]) revealed two major insulin-like peaks with retention times of 14–16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H) 3+ species at 1,991.1 m/z, representing monoglycated insulin with an intact M r of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin ( M r 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 ± 2.3 pmol/l, corresponding to ∼9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe 1 -glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 μg · kg −1 · min −1 , followed by 2 h at 83.0 μg · kg −1 · min −1 ; corresponding to 0.4 and 2.0 mU · kg −1 · min −1 ). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin ( P < 0.01) and ∼70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes. Footnotes Address correspondence and reprint requests to Professor P.R. Flatt, School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, N. Ireland BT52 1SA, U.K. E-mail: pr.flatt{at}ulster.ac.uk . Received for publication 9 August 2002 and accepted in revised form 14 November 2002. EGP, endogenous glucose production; ESI-MS, electrospray ionization mass spectrometry; HPLC, high-pressure liquid chromatography; RIA, radioimmunoassay; RP, reverse phase; TFA, trifluoroacetic acid. DIABETES