Glibenclamide

ABSTRACT

Progressive deterioration of β-cell function is proposed as a disease-related factor of sulphonylurea (SU) failure in type 2 diabetes. If it gradually worsens over time then disease duration may mirror the progressive β-cell deterioration. The aim of the present study is to assess whether or not disease duration is influential in remodelling the secretion pattern of insulin-like molecules and in glucose control of SU-treated type 2 diabetes. A research model is used to investigate proinsulin secreting capacity over time, using two groups of patients: i) disease duration

KEY WORDS: Diabetes mellitus. Insulin. Proinsulin. Sulfonylurea compounds.

Introduction

In the human pancreatic islet, processing of proinsulin to insulin occurs predominantly in secretory vesicles, which provide a large intracellular store of insulin for rapid release when the β-cells receive the appropriate stimulation.1 In vitro experiments in the 1980s showed that decreased secretory response or reversible impairment of insulin secretion occurs when pancreatic islets experience prolonged exposure to a stimulatory concentration of glucose or sulphonylurea (SU) compounds.2-5 This phenomenon was described as 'desensitisation of insulin secretion' and is thought to be an important step in the manifestation of type 2 diabetes in those patients who fall into the SU failure (SUf) group.5-8

In SU-treated diabetes it is believed that the pancreatic islets are gradually over-stimulated as a result of long-term exposure to a full dose of sulphonylurea, and this leads to the depletion of releasable insulin in β-cells.9-11 In previous work, the authors found that type 2 diabetics showed severe insulin deficiency and that those with SUf were less able to control blood glucose when responding to a glucose challenge.12 The mechanisms underlying such pathological changes remain unclear, however, but it was proposed that desensitisation to stimuli is an evolving process that worsens over time, and that disease duration might mirror progressive β-cells deterioration.5

This study aims to discover whether or not disease duration influences glucose control and effects a remodelling of the secretory pattern of insulin-like molecules in SU-treated type 2 diabetes.

Materials and methods

A total of 124 patients with type 2 diabetes (diagnosed according to WHO 1999 guidelines) volunteered for the study. All were subjected to clinical or biochemical screen for evidence of cardiac, hepatic, kidney or thyroid abnormalities. Patients with no complications and not on insulin treatment (only full-dose SU treatment) were recruited for a glibenclamide challenge test13 to assess response to SU treatment.

Finally, 99 patients completed the study, as 13 were excluded on evidences of other disease, seven failed to attend for oral glucose tolerance test (OGTT) and five were excluded because they missed certain timed laboratory tests.

Glibenclamide challenge test

Glibenclamide (7.5 mg) was taken orally after a fasting glucose test, followed by a series of blood glucose tests at 60 min, 120 min and 180 min. Glucose decrease rate (%) was calculated using the formula (Glu^sub 0min^-Glu^sub (x)min^,)/Glu^sub 0min^ x 100%, where Glu^sub (x)min^ denotes glucose level at a certain time. A patient with a glucose decrease rate greater than 25% at any time point was classified as SUr, while the remainder were classified as SUf.

Study group details

Details of the group that comprised patients with disease duration

Details of the group that comprised patients with disease duration ≥ 5 years were as follows: n= 37 (male: 22; female: 15); mean age: 60.2 (range: 39-83); SUr: 17, SUf: 20

Blood sample collection

All patients took a last dose of oral hypoglycaemic agent the day before the study. After overnight fast, all subjects had a standard 75-g OGTT. Five blood samples were collected at O min, 30 min, 60 min, 120 min and 180 min for glucose, TPI, IPI and SI analysis. Sera were separated within 30 min of collection. Glucose and SI were measured immediately after separation, while samples for TPI and IPI were stored at -20°C for later analysis.

Sample analysis

Glucose samples were analysed on Beckman CX5 autoanalyser using a glucose oxidase method. SI samples were analysed on an Access chemiluminescent immunoassay system (Beckman Instruments, Cheska, USA). Analytical sensitivity (95% confidence) was 0.03 miu/L, with a reportable range of 0.03-300 miu/L. Total imprecision was

TPI and IPI samples were analysed on a Bio-Rad autoimmunoassay system using enzyme-linked immunosorbent assay (ELISA) kits (Dako, Cambridgeshire, UK). Assay parameters were provided by the manufacture (detection limit - TPI: 0.07 pmol/L, IPI: 0.13 pmol/L). Inter- and intra-assay coefficients of variation were

Data analysis

A general linear model multivariate procedure (SPSS software) was used for data calculation and statistical analysis. Serum glucose, SI, TPI and IPI levels at each time point were analysed as dependent variables, while disease duration and SU status were fixed factors. Patient age, sex and body-mass index (BMI) were selected as covariates in the analytical model.

As this study had an unequal n design, estimated marginal means were reported and used for pairwise comparison. All dependent variables at each time point were compared respectively between two main groups (

Results

Results of tests of between-subject effects (related to age, sex and BMI) are shown in Table 1. Estimated marginal mean and mean difference for all variables and for pairwise comparison outcomes (P) are listed in Table 2 (SUr vs. SUf) and Table 3 (

Discussion

In vitro study has found consistently that prolonged exposure to SU desensitises pancreatic β-cells and reduces insulin secretion.3,9,11 In a previous in vivo study, the authors of the present study have demonstrated that OGTT in both SUr and SUf groups shows no marked difference in insulin related molecules level and a similar level of insulin deficiency.12

In the majority of SU-dependent patients, increasing disease duration means longer exposure to SU agents, and, according to the 'desensitisation' theory, this results in progressive insensitivity to SU agents. Consequently, disease duration could be an important factor in glucose control.

In the present study, it was expected results would demonstrate consistency with in vitro study which shows that patients exposed to SU agents ≥ 5 years would have lower SI secretory activity. Surprisingly, however, there was no evidence of time-related changes in SI level but a significantly lower proinsulin level at all time points. Furthermore, this group of patients was less able to reduce glucose level in response to a glucose load. Data were analysed for Homa IR index but no marked differences were found between groups. These finding indicated that IR was not a major causation in disease duration related glucose controlling ability, neither proinsulin secreting capacity. Results also suggest that the two groups of patients showed marked differences in proinsulin conversion, but that insensitivity to glucose stimulation occurs differently.

In responding to a glucose load, SU-treated patients in the present in vivo study showed time-related changes in proinsulin but not insulin levels; a finding that is inconsistent with other in vitro studies.2-5 For years, studies into the cause of elevated proinsulin level in type 2 diabetes have concentrated on the exocytosis pathway,1 and one hypothesis implicates dysfunction of proinsulin conversion machinery. However, if dysfunction of the converting machinery is responsible for elevation of proinsulin during OGTT, a decreased insulin level should reflect this. However, the results of the present study do not support this theory.

In a normal pancreatic islet, proinsulin is transferred in an energy-dependent manner from the rough endoplasmic reticulum to the Golgi apparatus for further processing.1 Most (96%) proinsulin is cleaved into insulin and C-peptide, with the remainder comprising of intermediates and non-processed proinsulin. Approximately 98% is finally secreted into the circulation by exocytosis, while the remaining 2% proinsulin is secreted directly into the circulation from the Golgi apparatus through an unregulated constitutive secreting pathway.1

Proinsulin bioactivity reduces blood glucose and its effect is longer lasting than that of insulin.15 Results of the present study support this, as patients who had higher proinsulin levels appeared to enjoy better glucose control, and unraveling of mechanisms behind the proinsulin-secreting pathway may prove in furthering knowledge of glucose control.

The present study investigated proinsulin-secreting capacity at various time points in two groups of patients selected to reflect early and late disease (i.e., disease duration of

This work was partly funded by an Institute of Biomedical Science Overseas Research Grant. The research project was approved by the local ethics committee and carried out at the Guang Zhou Red Cross Hospital, Guang Zhou, 510220, P. R. China.

References

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15 Karam HJ, Salber PR, Forsham PH. Pancreatic hormones and diabetes mellitus. In: Greenspan FS, ed. Basic and clinical endocrinology. Appleton & Lange, 1991: 592-650.

Y. N. CHEN, S. Y. CHEN*, L. J. ZENG, J. M. RAN* and M. Y. WU

The Central Laboratory and * Department of Endocrinology, Guang Zhou Red Cross Hospital, Guang Zhou, 510220, P. R. China

Accepted: 7 December 2004

Correspondence to: Dr Yi Ni Chen

Email: stcyn@zsu.edu.cn

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