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Effects of nateglinide on the secretion of glycated insulin and glucose tolerance in type 2 diabetes
Diabetes Research and Clinical Practice 61 (2003) 167 /173 www.elsevier.com/locate/diabres
Effects of nateglinide on the secretion of glycated insulin and glucose tolerance in type 2 diabetes J.R. Lindsay a, A.M. McKillop b, M.H. Mooney b, F.P.M. O’Harte b, P.R. Flatt b, P.M. Bell a,* a
Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast BT12 6BA, UK b School of Biomedical Sciences, University of Ulster, Coleraine, UK Received 1 November 2002; received in revised form 9 April 2003; accepted 17 April 2003
Abstract Aims: Glycation of insulin has been demonstrated within pancreatic b-cells and the resulting impaired bioactivity may contribute to insulin resistance in diabetes. We used a novel radioimmunoassay to evaluate the effect of nateglinide on plasma concentrations of glycated insulin and glucose tolerance in type 2 diabetes. Methods: Ten patients (5 M/5 F, age 57.89/1.9 years, HbA1c 7.69/0.5%, fasting plasma glucose 9.49/1.2 mmol/l, creatinine 81.69/4.5 mmol/l) received oral nateglinide 120 mg or placebo, 10 min prior to 75 g oral glucose in a random, single blind, crossover design, 1 week apart. Blood samples were taken for glycated insulin, glucose, insulin and C-peptide over 225 min. Results: Plasma glucose and glycated insulin responses were reduced by 9% (P/0.005) and 38% (P/0.047), respectively, following nateglinide compared with placebo. Corresponding AUC measures for insulin and C-peptide were enhanced by 36% (P/0.005) and 25% (P/0.007) by nateglinide. Conclusions: Glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, reduced glycated insulin release may contribute to the antihyperglycaemic action of nateglinide. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Nateglinide; Glycated insulin; Glucose tolerance; Type 2 diabetes
1. Introduction
* Corresponding author. Tel.: /44-28-90-894794; fax: /4428-90-263131. E-mail address: [email protected] (P.M. Bell).
There has been considerable debate about the relative importance of insulin resistance and b-cell secretory defects in the pathophysiology of type 2 diabetes [1,2]. Whilst there is little doubt that both mechanisms are of significance, several recent clinical studies have focused on defective first-
0168-8227/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(03)00107-4
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J.R. Lindsay et al. / Diabetes Research and Clinical Practice 61 (2003) 167 /173
phase insulin release and consequent postprandial hyperglycaemia. This has received particular attention given emerging evidence of a link between postprandial hyperglycaemia and cardiovascular mortality [3]. The normal physiology of b-cell secretion consists of an early first-phase insulin response that occurs immediately after exposure of b-cells to glucose and is complete by 10 min [4]. A subsequent second-phase insulin response occurs within 10 /20 min of b-cell exposure to glucose and can continue for several hours. Patients with diabetes display a typical delayed first phase insulin response and several new antihyperglycaemic agents are now available to help restore first phase insulin secretion [5]. Repaglinide, a benzoic acid derivative of meglitinide was the first meglitinide analogue to become available [6]. NategliD-phenylalanine derivative, has nide, a subsequently become available in the UK and is licensed for combination therapy with metformin [7]. Nateglinide stimulates a rapid, transient secretion of insulin from pancreatic b-cells that is dependent on ambient glucose concentrations, which may be more physiological [8,9]. The drug acts at a distinct binding site on the sulphonylurea receptor, which involves the inhibition of b-cell membrane K -ATP channels, triggering an influx of extracellular calcium and the secretion of insulin [10]. Following oral administration, peak plasma concentrations of nateglinide are observed within 1 h, with an elimination half-life of 1.8 h [11]. Previous in vitro and in vivo studies by our group support the concept that insulin is glycated under conditions of hyperglycaemia within the pancreatic b-cells where it is stored in secretory granules prior to co-secretion with insulin [12 /15]. We have also shown that secretion of glycated insulin from b-cells in vitro is readily stimulated by glucose, amino acids and other insulin secretogogues [15] and that glycated insulin exhibits impaired biological activity in animals and healthy human subjects [16 /18]. The aim of the current study was to use a newly developed radioimmunoassay [13,14,19,20] to evaluate the effects of nateglinide on plasma concentrations of glycated
insulin and associated metabolic responses in a group of patients with type 2 diabetes.
2. Materials and methods 2.1. Subjects Serum insulin and plasma glycated insulin profiles and associated parameters were determined following oral nateglinide 120 mg or placebo in conjunction with a 75 g oral glucose tolerance test in ten subjects with type 2 diabetes. All were outpatients, being treated by diet therapy (n /6) alone, metformin (n/3) or gliclazide (n / 1), with normal renal function (serum creatinine B/115 mmol/l). All drugs were stopped 1 week prior to the study period. The patients were admitted at 08:00 h to the Endocrinology and Diabetes Centre, Royal Victoria Hospital. An intravenous cannula was inserted into the antecubital vein, which was kept patent by means of heparinised saline. The subjects were given oral nateglinide 120 mg or placebo at time t //10 min followed by a 75 g oral glucose load at t /0 min in a randomised crossover design, 1 week apart. Venous blood samples were taken over 225 min, at t//15, /10, /5, 0, 5, 10, 15, 20, 30, 40, 60, 90, 120, 150, 180 and 210 min. Plasma glucose, insulin, C-peptide and glycated insulin were determined at each time-point. This study was approved by the ethics committee of The Queen’s University of Belfast and carried out following informed consent from all subjects. 2.2. Biochemical analyses Development of a specific radioimmunoassay for glycated insulin has been described in detail elsewhere [13,14]. In brief, an N-terminally glycated synthetic insulin peptide, closely related to the amino-terminal sequence of the insulin B-chain (Phe-Val-Asn-Gln-His-Leu-Tyr-Lys), was linked to ovalbumin using glutaraldehyde and used to raise specific antibodies in guinea pigs. This peptide comprised the naturally occurring 1 /6 sequence of insulin B-chain with a Tyr and Lys at positions 7 and 8, respectively. For radiometric
J.R. Lindsay et al. / Diabetes Research and Clinical Practice 61 (2003) 167 /173
determination of circulating glycated insulin, the insulin peptide was glycated under hyperglycaemic reducing conditions and iodinated using the solid phase iodogen method [21], generating a highly specific mono-iodinated I125-tyrosylated glycated peptide tracer. Antiserum G3/B/vi was used to establish a dextran-coated charcoal radioimmunoassay with a glycated insulin standard curve in the presence of insulin free serum. Assay sensitivity was 9 pmol/l with an intra-assay coefficient of variance of 1.8%. The glycated insulin antibody cross-reacted 52% with glycated proinsulin, however cross-reaction with non-glycated insulin, proinsulin and other pancreatic hormones was negligible [13,14]. Serum insulin was determined using the Abbott IMX insulin microparticulate enzyme immunoassay (MEIA; Abbott Laboratories Ltd, Berkshire, UK), which has a sensitivity of 6 pmol/l and an intra-assay coefficient of variance of 4%. Crossreactivity with proinsulin was B/0.005% with no detectable reaction with C-peptide. Cross-reactivity with glycated insulin was concentration-dependent, representing about 50%. C-peptide was measured using a commercial kit (Dako Diagnostics Ltd, Ely, Cambridgeshire, UK). Glucose concentrations were measured in plasma using the glucose oxidase method [22]. HbA1c was measured in whole blood by ion-exchange HPLC using the Menari HA-8140 kit (BIOMEN Ltd, Berkshire, UK). Serum creatinine was determined using the Johnston and Johnston Vitros 950 analyser (Orthoclinical Diagnostics, Buckinghamshire, UK) using a multilayered dry slide aminohydrolase technique [23].
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3. Results Study participants comprised ten diabetic subjects (5 M/5 F), with a mean age of 57.89/1.9 years, duration of diabetes 1.09/0.5 years and body mass index of 34.29/2.4 kg/m2. HbA1c and serum creatinine were 7.69/0.5% and 81.69/4.5 mmol/l, respectively. Fasting plasma glucose (9.69/ 1.4; 9.19/1.0 mmol/l), insulin (99.89/17.7; 108.99/ 19.8 pmol/l), C-peptide (3.39/0.4; 3.59/0.5 mg/l), and glycated insulin concentrations (4.49/1.9; 4.89/2.2 pmol/l) were similar for both nateglinide and placebo study mornings. Maximum glucose concentrations following nateglinide and placebo were 17.29/1.7 and 18.29/1.7 mmol/l, attained at t /60 and 90 min, respectively
2.3. Statistical analysis All experimental data are expressed as mean9/ S.E.M. Significant differences between groups of data were assessed using Wilcoxon Signed Rank test. Statistical significance was assumed if P B/ 0.05. The derived variables area under the curve (AUC) were calculated using the trapezoidal rule for glucose, insulin, C-peptide and glycated insulin between t //15 /210 min.
Fig. 1. Circulating glucose and insulin concentrations for diabetic subjects (n/10) who received oral nateglinide or placebo (t //10 min) prior to 75 g oral glucose load (t /0 min). Values are mean9/S.E.M. *, P B/0.05; **, P B/0.01.
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(Fig. 1). The glucose profiles were similar until t/ 60 min and then they diverged until the end of the sampling at which point the maximum mean difference was 2.9 mmol/l (P /0.005). AUC measures for glucose following nateglinide and placebo were 29869/326 and 32709/308 mmol min/l, respectively (P /0.005). Insulin responses were higher following nateglinide at each individual time point from t/15 until t/180 min. The maximum insulin response was 637.69/163.9 for nateglinide and 414.39/100.9 pmol/l for placebo (P /0.03) at t /90 min (Fig. 1). AUC for insulin was higher following nateglinide compared with placebo (106 3389/28 897 vs. 68 6299/16 545 pmol min/l; P /0.005). C-peptide responses mirrored insulin at all time points until t/210 with maximum concentrations following nateglinide of 11.89/1.4 and placebo of 8.49/1.0 mg/l (P /0.007), both detected at t/120 min, respectively (Fig. 2). C-peptide responses was
Fig. 2. Circulating C-peptide and glycated insulin concentrations for diabetic subjects (n/10) who received oral nateglinide or placebo (t //10 min) prior to 75 g oral glucose load (t/0 min). Values are mean9/S.E.M. *, P B/0.05; **, P B/0.01.
also higher following nateglinide than placebo at t/210 with a mean difference of 1.25 mg/l. Corresponding AUC measures for C-peptide were 20759/212 and 15579/176 mg min/l (P / 0.007). Glycated insulin responses to nateglinide and placebo showed a similar pattern of release with early peaks followed by a late plateau until the end of each sampling period (Fig. 2). Peak glycated insulin concentrations following nateglinide were 12.79/4.9, 15 min from drug ingestion and following placebo were 20.99/6.6 pmol/l, 5 min from drug ingestion (P /0.2). AUC measures for glycated insulin following nateglinide or placebo, respectively, were 10989/406 and 17649/507 pmol min/l (P /0.047).
4. Discussion Disturbances of insulin secretion and insulin action are well described features of type 2 diabetes, and a growing body of evidence supports a role for glucose toxicity as a contributor to the progressive impairment of b-cell function and insulin sensitivity [24]. Recent evidence also supports the concept that glycation of insulin in pancreatic b-cells under conditions of hyperglycaemia impairs hormone bioactivity and thereby contributes to insulin resistance [16 /18]. Using a novel radioimmunoassay, we have demonstrated elevated concentrations of glycated insulin in good and moderately controlled patients with type 2 diabetes compared with age- and sex-matched healthy controls [19]. In a subsequent study, we demonstrated that plasma concentrations of glycated insulin exhibited diurnal variation [20]. The observed temporal pattern to meal induced insulin secretion supports the view that insulin and glycated insulin are both secreted from pancreatic b-cells in vivo [20]. Consistent with previous publications, the present studies demonstrate that nateglinide was a highly effective insulin secretagogue and antihyperglycaemic agent when given prior to an oral glucose load [25]. Early insulinotropic effects were demonstrable within 25 min of administration with maximum insulinotropic and antihypergly-
J.R. Lindsay et al. / Diabetes Research and Clinical Practice 61 (2003) 167 /173
caemic effects between 70 and 220 min from administration. Integrated insulin and C-peptide responses were greater and the glycaemic excursion less with nateglinide compared with placebo and glucose alone. Nateglinide was given at a dose of 120 mg, the maximum effective dose in a previous study of patients with type 2 diabetes [25]. The present data show that secretion of glycated insulin is rapid following nateglinide given along with glucose as well as when glucose given with placebo. This is broadly consistent with the effects of nutrients and hormones tested in vitro and in vivo in animal models of diabetes [22,23]. The finding of a very early secretory peak of glycated and native insulin may partly reflect a neurally mediated secretory response. It is well known that parasympathetic, sympathetic and sensory nerves richly innervate the pancreatic islets and that cephalic insulin secretion in humans is observed within 3/4 min of a stimulus, triggered by olfactory, visual, gustatory and oropharyngeal mechanical receptors [26,27]. Release of glycated insulin may reflect a prominent early marker of the cephalic phase, due to preferential release of mature granules particularly rich in glycated insulin. We have also shown that secretion of glycated insulin occurs in a later phase, which may be analogous to the physiological insulin response. Whilst the first phase insulin response predominantly reflects immediate release of preformed hormone, the subsequent later phase is dependent on recruitment of reserve insulin stores and partially upon protein synthesis within the b-cell [4]. Recent biochemical and electrophysiological studies have suggested that secretory granules exist in different pools defined by the time course of their release from the b-cell [28]. Most granules belong to the reserve pool and need to be chemically modified, or even physically translocated, to become immediately available for exocytosis. The latter subset of granules is referred to as the readily releasable pool and requires one or more ATP-dependent reactions for mobilisation [29]. As previous studies [14,15] favour co-secretion of both glycated and native insulin from insulin secretory granules, it would seem possible that the observed early phase release may reflect
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release of glycated insulin from a rich peripheral primed granule pool that is about to or has docked with the cell membrane prior to exocytosis. Further secretion of glycated insulin from the bcell probably results from mobilised granule stores with a different proportion in glycated form. Alternatively, the later stages of secretion of glycated insulin could reflect release of newly formed glycated insulin. Although the time scale involved is rather short evidence from b-cell lines in vitro indicate that intracellular glycation of insulin is relatively rapid. This reflects the highly efficient mechanism for glucose transport, metabolism and production of the powerful glycating agent, glucose-6-phosphate at the inner leaflet of the endoplasmic reticulum where proinsulin is synthesised [12,15]. In principle, acute effects of sulphonylureas or repaglinide on the releasable pool of insulin may also lead to reduced plasma concentrations of glycated insulin. Clearly the effects observed during this study may not be unique to nateglinide. In conclusion, the current study demonstrates that secretion of glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, this action may contribute to the antihyperglycaemic action of nateglinide. Whether a reduction in plasma glycated insulin might have clinical significant effects in type 2 diabetes remains to be evaluated.
Acknowledgements These studies were supported in part by the Research and Development Office of the Department of Health and Personal Social Services (Northern Ireland), The Welcome Trust, and University of Ulster Research Strategy Funding.
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[2] R.E. Pratley, C. Weyer, The role of impaired early insulin secretion in the pathogenesis of type II diabetes mellitus, Diabetologia 44 (2001) 929 /945. [3] The DECODE study group, European Diabetes Epidemiology Group, Glucose tolerance and mortality: comparison of WHO and American diabetes association diagnostic criteria, Lancet 354 (1999) 617 /621. [4] M.A. Pfeifer, J.B. Halter, D.J. Porte, Insulin secretion in diabetes mellitus, Am. J. Med. 70 (1981) 579 /588. [5] R.E. Pratley, J.E. Foley, B.E. Dunning, Rapid acting insulinotropic agents: restoration of early insulin secretion as a physiologic approach to improve glucose control, Curr. Pharm. Des. 7 (2001) 1375 /1397. [6] W.J. Malaisse, Repaglinide, a new oral antidiabetic agent: a review of recent preclinical studies, Eur. J. Clin. Invest. 29 (Suppl. 2) (1999) 21 /29. [7] E.S. Horton, C. Clinkingbeard, M. Gatlin, J. Foley, S. Mallows, S. Shen, Nateglinide alone and in combination with metformin improves glycemic control by reducing mealtime glucose levels in type 2 diabetes, Diabetes Care 23 (2000) 1660 /1665. [8] Y. Sato, M. Nishikawa, H. Shinkai, E. Sukegawa, Possibility of ideal blood glucose control by a new oral hypoglycemic agent, N -[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166), and its stimulatory effect on insulin secretion in animals, Diabetes Res. Clin. Pract. 12 (1991) 53 /59. [9] T. Ikenoue, M. Akiyoshi, S. Fujitani, K. Okazaki, N. Kondo, T. Maki, Hypoglycaemic and insulinotropic effects of a novel oral antidiabetic agent, (/)-N -(trans-4isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166), Br. J. Pharmacol. 120 (1997) 137 /145. [10] M. Akiyoshi, M. Kakei, M. Nakazaki, H. Tanaka, A new hypoglycemic agent, A-4166, inhibits ATP-sensitive potassium channels in rat pancreatic beta-cells, Am. J. Physiol. 268 (1995) E185 /E193. [11] A.H. Karara, B.E. Dunning, J.F. McLeod, The effect of food on the oral bioavailability and the pharmacodynamic actions of the insulinotropic agent nateglinide in healthy subjects, J. Clin. Pharmacol. 39 (1999) 172 /179. [12] Y.H. Abdel-Wahab, F.P. O’Harte, H. Ratcliff, N.H. McClenaghan, C.R. Barnett, P.R. Flatt, Glycation of insulin in the islets of Langerhans of normal and diabetic animals, Diabetes 45 (1996) 1489 /1496. [13] A.M. McKillop, J.T. McCluskey, A.C. Boyd, M.H. Mooney, P.R. Flatt, F.P. O’Harte, Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues, J. Endocrinol. 167 (2000) 153 /163. [14] A.M. McKillop, M.H. Mooney, P. Harriott, P.R. Flatt, F.P. O’Harte, Evaluation of glycated insulin in diabetic animals using immunocytochemistry and radioimmunoassay, Biochem. Biophys. Res. Commun. 286 (2001) 524 / 528. [15] Y.H. Abdel-Wahab, F.P. O’Harte, C.R. Barnett, P.R. Flatt, Characterization of insulin glycation in insulin-
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Effects of nateglinide on the secretion of glycated insulin and glucose tolerance in type 2 diabetes J.R. Lindsay a, A.M. McKillop b, M.H. Mooney b, F.P.M. O’Harte b, P.R. Flatt b, P.M. Bell a,* a
Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast BT12 6BA, UK b School of Biomedical Sciences, University of Ulster, Coleraine, UK Received 1 November 2002; received in revised form 9 April 2003; accepted 17 April 2003
Abstract Aims: Glycation of insulin has been demonstrated within pancreatic b-cells and the resulting impaired bioactivity may contribute to insulin resistance in diabetes. We used a novel radioimmunoassay to evaluate the effect of nateglinide on plasma concentrations of glycated insulin and glucose tolerance in type 2 diabetes. Methods: Ten patients (5 M/5 F, age 57.89/1.9 years, HbA1c 7.69/0.5%, fasting plasma glucose 9.49/1.2 mmol/l, creatinine 81.69/4.5 mmol/l) received oral nateglinide 120 mg or placebo, 10 min prior to 75 g oral glucose in a random, single blind, crossover design, 1 week apart. Blood samples were taken for glycated insulin, glucose, insulin and C-peptide over 225 min. Results: Plasma glucose and glycated insulin responses were reduced by 9% (P/0.005) and 38% (P/0.047), respectively, following nateglinide compared with placebo. Corresponding AUC measures for insulin and C-peptide were enhanced by 36% (P/0.005) and 25% (P/0.007) by nateglinide. Conclusions: Glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, reduced glycated insulin release may contribute to the antihyperglycaemic action of nateglinide. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Nateglinide; Glycated insulin; Glucose tolerance; Type 2 diabetes
1. Introduction
* Corresponding author. Tel.: /44-28-90-894794; fax: /4428-90-263131. E-mail address: [email protected] (P.M. Bell).
There has been considerable debate about the relative importance of insulin resistance and b-cell secretory defects in the pathophysiology of type 2 diabetes [1,2]. Whilst there is little doubt that both mechanisms are of significance, several recent clinical studies have focused on defective first-
0168-8227/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(03)00107-4
168
J.R. Lindsay et al. / Diabetes Research and Clinical Practice 61 (2003) 167 /173
phase insulin release and consequent postprandial hyperglycaemia. This has received particular attention given emerging evidence of a link between postprandial hyperglycaemia and cardiovascular mortality [3]. The normal physiology of b-cell secretion consists of an early first-phase insulin response that occurs immediately after exposure of b-cells to glucose and is complete by 10 min [4]. A subsequent second-phase insulin response occurs within 10 /20 min of b-cell exposure to glucose and can continue for several hours. Patients with diabetes display a typical delayed first phase insulin response and several new antihyperglycaemic agents are now available to help restore first phase insulin secretion [5]. Repaglinide, a benzoic acid derivative of meglitinide was the first meglitinide analogue to become available [6]. NategliD-phenylalanine derivative, has nide, a subsequently become available in the UK and is licensed for combination therapy with metformin [7]. Nateglinide stimulates a rapid, transient secretion of insulin from pancreatic b-cells that is dependent on ambient glucose concentrations, which may be more physiological [8,9]. The drug acts at a distinct binding site on the sulphonylurea receptor, which involves the inhibition of b-cell membrane K -ATP channels, triggering an influx of extracellular calcium and the secretion of insulin [10]. Following oral administration, peak plasma concentrations of nateglinide are observed within 1 h, with an elimination half-life of 1.8 h [11]. Previous in vitro and in vivo studies by our group support the concept that insulin is glycated under conditions of hyperglycaemia within the pancreatic b-cells where it is stored in secretory granules prior to co-secretion with insulin [12 /15]. We have also shown that secretion of glycated insulin from b-cells in vitro is readily stimulated by glucose, amino acids and other insulin secretogogues [15] and that glycated insulin exhibits impaired biological activity in animals and healthy human subjects [16 /18]. The aim of the current study was to use a newly developed radioimmunoassay [13,14,19,20] to evaluate the effects of nateglinide on plasma concentrations of glycated
insulin and associated metabolic responses in a group of patients with type 2 diabetes.
2. Materials and methods 2.1. Subjects Serum insulin and plasma glycated insulin profiles and associated parameters were determined following oral nateglinide 120 mg or placebo in conjunction with a 75 g oral glucose tolerance test in ten subjects with type 2 diabetes. All were outpatients, being treated by diet therapy (n /6) alone, metformin (n/3) or gliclazide (n / 1), with normal renal function (serum creatinine B/115 mmol/l). All drugs were stopped 1 week prior to the study period. The patients were admitted at 08:00 h to the Endocrinology and Diabetes Centre, Royal Victoria Hospital. An intravenous cannula was inserted into the antecubital vein, which was kept patent by means of heparinised saline. The subjects were given oral nateglinide 120 mg or placebo at time t //10 min followed by a 75 g oral glucose load at t /0 min in a randomised crossover design, 1 week apart. Venous blood samples were taken over 225 min, at t//15, /10, /5, 0, 5, 10, 15, 20, 30, 40, 60, 90, 120, 150, 180 and 210 min. Plasma glucose, insulin, C-peptide and glycated insulin were determined at each time-point. This study was approved by the ethics committee of The Queen’s University of Belfast and carried out following informed consent from all subjects. 2.2. Biochemical analyses Development of a specific radioimmunoassay for glycated insulin has been described in detail elsewhere [13,14]. In brief, an N-terminally glycated synthetic insulin peptide, closely related to the amino-terminal sequence of the insulin B-chain (Phe-Val-Asn-Gln-His-Leu-Tyr-Lys), was linked to ovalbumin using glutaraldehyde and used to raise specific antibodies in guinea pigs. This peptide comprised the naturally occurring 1 /6 sequence of insulin B-chain with a Tyr and Lys at positions 7 and 8, respectively. For radiometric
J.R. Lindsay et al. / Diabetes Research and Clinical Practice 61 (2003) 167 /173
determination of circulating glycated insulin, the insulin peptide was glycated under hyperglycaemic reducing conditions and iodinated using the solid phase iodogen method [21], generating a highly specific mono-iodinated I125-tyrosylated glycated peptide tracer. Antiserum G3/B/vi was used to establish a dextran-coated charcoal radioimmunoassay with a glycated insulin standard curve in the presence of insulin free serum. Assay sensitivity was 9 pmol/l with an intra-assay coefficient of variance of 1.8%. The glycated insulin antibody cross-reacted 52% with glycated proinsulin, however cross-reaction with non-glycated insulin, proinsulin and other pancreatic hormones was negligible [13,14]. Serum insulin was determined using the Abbott IMX insulin microparticulate enzyme immunoassay (MEIA; Abbott Laboratories Ltd, Berkshire, UK), which has a sensitivity of 6 pmol/l and an intra-assay coefficient of variance of 4%. Crossreactivity with proinsulin was B/0.005% with no detectable reaction with C-peptide. Cross-reactivity with glycated insulin was concentration-dependent, representing about 50%. C-peptide was measured using a commercial kit (Dako Diagnostics Ltd, Ely, Cambridgeshire, UK). Glucose concentrations were measured in plasma using the glucose oxidase method [22]. HbA1c was measured in whole blood by ion-exchange HPLC using the Menari HA-8140 kit (BIOMEN Ltd, Berkshire, UK). Serum creatinine was determined using the Johnston and Johnston Vitros 950 analyser (Orthoclinical Diagnostics, Buckinghamshire, UK) using a multilayered dry slide aminohydrolase technique [23].
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3. Results Study participants comprised ten diabetic subjects (5 M/5 F), with a mean age of 57.89/1.9 years, duration of diabetes 1.09/0.5 years and body mass index of 34.29/2.4 kg/m2. HbA1c and serum creatinine were 7.69/0.5% and 81.69/4.5 mmol/l, respectively. Fasting plasma glucose (9.69/ 1.4; 9.19/1.0 mmol/l), insulin (99.89/17.7; 108.99/ 19.8 pmol/l), C-peptide (3.39/0.4; 3.59/0.5 mg/l), and glycated insulin concentrations (4.49/1.9; 4.89/2.2 pmol/l) were similar for both nateglinide and placebo study mornings. Maximum glucose concentrations following nateglinide and placebo were 17.29/1.7 and 18.29/1.7 mmol/l, attained at t /60 and 90 min, respectively
2.3. Statistical analysis All experimental data are expressed as mean9/ S.E.M. Significant differences between groups of data were assessed using Wilcoxon Signed Rank test. Statistical significance was assumed if P B/ 0.05. The derived variables area under the curve (AUC) were calculated using the trapezoidal rule for glucose, insulin, C-peptide and glycated insulin between t //15 /210 min.
Fig. 1. Circulating glucose and insulin concentrations for diabetic subjects (n/10) who received oral nateglinide or placebo (t //10 min) prior to 75 g oral glucose load (t /0 min). Values are mean9/S.E.M. *, P B/0.05; **, P B/0.01.
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(Fig. 1). The glucose profiles were similar until t/ 60 min and then they diverged until the end of the sampling at which point the maximum mean difference was 2.9 mmol/l (P /0.005). AUC measures for glucose following nateglinide and placebo were 29869/326 and 32709/308 mmol min/l, respectively (P /0.005). Insulin responses were higher following nateglinide at each individual time point from t/15 until t/180 min. The maximum insulin response was 637.69/163.9 for nateglinide and 414.39/100.9 pmol/l for placebo (P /0.03) at t /90 min (Fig. 1). AUC for insulin was higher following nateglinide compared with placebo (106 3389/28 897 vs. 68 6299/16 545 pmol min/l; P /0.005). C-peptide responses mirrored insulin at all time points until t/210 with maximum concentrations following nateglinide of 11.89/1.4 and placebo of 8.49/1.0 mg/l (P /0.007), both detected at t/120 min, respectively (Fig. 2). C-peptide responses was
Fig. 2. Circulating C-peptide and glycated insulin concentrations for diabetic subjects (n/10) who received oral nateglinide or placebo (t //10 min) prior to 75 g oral glucose load (t/0 min). Values are mean9/S.E.M. *, P B/0.05; **, P B/0.01.
also higher following nateglinide than placebo at t/210 with a mean difference of 1.25 mg/l. Corresponding AUC measures for C-peptide were 20759/212 and 15579/176 mg min/l (P / 0.007). Glycated insulin responses to nateglinide and placebo showed a similar pattern of release with early peaks followed by a late plateau until the end of each sampling period (Fig. 2). Peak glycated insulin concentrations following nateglinide were 12.79/4.9, 15 min from drug ingestion and following placebo were 20.99/6.6 pmol/l, 5 min from drug ingestion (P /0.2). AUC measures for glycated insulin following nateglinide or placebo, respectively, were 10989/406 and 17649/507 pmol min/l (P /0.047).
4. Discussion Disturbances of insulin secretion and insulin action are well described features of type 2 diabetes, and a growing body of evidence supports a role for glucose toxicity as a contributor to the progressive impairment of b-cell function and insulin sensitivity [24]. Recent evidence also supports the concept that glycation of insulin in pancreatic b-cells under conditions of hyperglycaemia impairs hormone bioactivity and thereby contributes to insulin resistance [16 /18]. Using a novel radioimmunoassay, we have demonstrated elevated concentrations of glycated insulin in good and moderately controlled patients with type 2 diabetes compared with age- and sex-matched healthy controls [19]. In a subsequent study, we demonstrated that plasma concentrations of glycated insulin exhibited diurnal variation [20]. The observed temporal pattern to meal induced insulin secretion supports the view that insulin and glycated insulin are both secreted from pancreatic b-cells in vivo [20]. Consistent with previous publications, the present studies demonstrate that nateglinide was a highly effective insulin secretagogue and antihyperglycaemic agent when given prior to an oral glucose load [25]. Early insulinotropic effects were demonstrable within 25 min of administration with maximum insulinotropic and antihypergly-
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caemic effects between 70 and 220 min from administration. Integrated insulin and C-peptide responses were greater and the glycaemic excursion less with nateglinide compared with placebo and glucose alone. Nateglinide was given at a dose of 120 mg, the maximum effective dose in a previous study of patients with type 2 diabetes [25]. The present data show that secretion of glycated insulin is rapid following nateglinide given along with glucose as well as when glucose given with placebo. This is broadly consistent with the effects of nutrients and hormones tested in vitro and in vivo in animal models of diabetes [22,23]. The finding of a very early secretory peak of glycated and native insulin may partly reflect a neurally mediated secretory response. It is well known that parasympathetic, sympathetic and sensory nerves richly innervate the pancreatic islets and that cephalic insulin secretion in humans is observed within 3/4 min of a stimulus, triggered by olfactory, visual, gustatory and oropharyngeal mechanical receptors [26,27]. Release of glycated insulin may reflect a prominent early marker of the cephalic phase, due to preferential release of mature granules particularly rich in glycated insulin. We have also shown that secretion of glycated insulin occurs in a later phase, which may be analogous to the physiological insulin response. Whilst the first phase insulin response predominantly reflects immediate release of preformed hormone, the subsequent later phase is dependent on recruitment of reserve insulin stores and partially upon protein synthesis within the b-cell [4]. Recent biochemical and electrophysiological studies have suggested that secretory granules exist in different pools defined by the time course of their release from the b-cell [28]. Most granules belong to the reserve pool and need to be chemically modified, or even physically translocated, to become immediately available for exocytosis. The latter subset of granules is referred to as the readily releasable pool and requires one or more ATP-dependent reactions for mobilisation [29]. As previous studies [14,15] favour co-secretion of both glycated and native insulin from insulin secretory granules, it would seem possible that the observed early phase release may reflect
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release of glycated insulin from a rich peripheral primed granule pool that is about to or has docked with the cell membrane prior to exocytosis. Further secretion of glycated insulin from the bcell probably results from mobilised granule stores with a different proportion in glycated form. Alternatively, the later stages of secretion of glycated insulin could reflect release of newly formed glycated insulin. Although the time scale involved is rather short evidence from b-cell lines in vitro indicate that intracellular glycation of insulin is relatively rapid. This reflects the highly efficient mechanism for glucose transport, metabolism and production of the powerful glycating agent, glucose-6-phosphate at the inner leaflet of the endoplasmic reticulum where proinsulin is synthesised [12,15]. In principle, acute effects of sulphonylureas or repaglinide on the releasable pool of insulin may also lead to reduced plasma concentrations of glycated insulin. Clearly the effects observed during this study may not be unique to nateglinide. In conclusion, the current study demonstrates that secretion of glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, this action may contribute to the antihyperglycaemic action of nateglinide. Whether a reduction in plasma glycated insulin might have clinical significant effects in type 2 diabetes remains to be evaluated.
Acknowledgements These studies were supported in part by the Research and Development Office of the Department of Health and Personal Social Services (Northern Ireland), The Welcome Trust, and University of Ulster Research Strategy Funding.
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