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Introduction

Diabetes is a serious global health issue and has become one of the most common epidemics worldwide. The International Diabetes Federation estimated that ~7% of the world's population aged between 25 and 79 years of age had diabetes in 2010; by 2030 the figure will rise to 8% (1). Non-insulin dependent diabetes mellitus is a leading cause of morbidity worldwide and contributes significantly to premature mortality (2). Early-onset type 2 diabetes (EOD) has emerged as an increasing public health problem, particularly in developing areas such as Asia, which are undergoing rapid socio-economic changes (3,4). A recent national investigation completed in China showed that the prevalence of diabetes and pre-diabetes in individuals aged 18–39 years was ~45% (5).

It is established insulin resistance and reduced β-cell glucose sensitivity are present in patients with type 2 diabetes (6). Previously, the concept that insulin resistance was the primary genetic component of type 2 diabetes became widely believed (7); however, previous studies have indicated that the first-phase insulin secretion is the earliest detectable defect in β-cell function, and impaired β-cell function precedes insulin resistance in the pathogenesis of type 2 diabetes (8,9). Impaired β-cell function is regarded as a key factor in the progression from glucose intolerance to overt type 2 diabetes, and it could be the result of the loss of β-cell mass and/or functional defects (10). Moreover, the relative β-cell volume was decreased from ~20 to 50% in autopsy specimens of patients with type 2 diabetes (11). These results suggested that diabetes is associated with impaired β-cell function; however, the development of β-cell hypofunction is poorly understood with regards to type 2 diabetes.

In the present study, we evaluated the function and deterioration of the islet β-cell in patients with type 2 diabetes by using a 3-year follow-up and compared the difference between late-onset diabetes (LOD) and EOD with the use of contrastive analysis.

Materials and methods

Sample

Patients with type 2 diabetes were recruited from the Department of Endocrinology of Huashan Hospital affiliated to Fudan University (Shanghai, China) between 2003 and 2009. A total of 48 patients (36 men and 12 women) with EOD (age at diagnosis, <40 years) and 55 patients (27 men and 28 women) with LOD (age at diagnosis, ≥40 years) were selected for the present study. Plasma glucose concentrations were measured using an Abbott Bichromatic Analyzer (Abbott Laboratories, Lake Bluff, IL, USA), and diabetes was diagnosed according to the World Health Organization plasma glucose criteria (fasting plasma glucose, ≥126 mg/dl; or 2-h plasma glucose, ≥200 mg/dl (12). In addition, anti-insular cellular antibody (anti-ICA; cat. no. 02068), anti-glutamate decarboxylase antibody (anti-GAD; cat. no. 020107) and anti-insulin antibody (anti-IAA; cat. no. 02114A) all are negative. Patients were excluded if they had type 1 diabetes as defined by acute presentation with diabetic ketoacidosis, heavy ketonuria or continuous requirement of insulin within half a year of diagnosis, or unknown diabetes type. Disease course was no more than 5 years in all subjects and all patients received oral blood glucose lowering drugs based on alimentary control.

Ethical approval was obtained from Huashan Hospital Clinical Research Ethics Committee. All patients provided written informed consent for data analysis and research purpose at the time of assessment.

Clinical studies

Participants underwent routine physical examinations including the following measurements: Height, weight, waist circumference, hip circumference and resting blood pressure. Height was measured in meters (without shoes), and weight in kilograms (without heavy clothing). Body mass index (BMI) was calculated in kg/m2. Waist circumference was measured using a tape measure while subjects were standing, at the midpoint between the bottom of rip cage and the top of lateral border of iliac crests during minimal respiration. Hip circumference was measured around the maximal circumference of the buttocks, at approximately the level of the pubic symphysis. Waist-hip ratio (WHR) was determined by dividing the mean waist circumference by the mean hip circumference. Systolic blood pressure (SBP) was measured with the subject in the supine position following a 5-min rest.

After an overnight fast, venous blood was sampled for measurement of plasma glucose, glycated hemoglobin A1c (HbA1c), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), anti-ICA, anti-GAD and anti-IAA. All kits were purchased from Youbo Biotechnoglgy Co., Ltd. (Shanghai, China).

Islet β-cell secretory activity was measured by means of glucagon stimulation test (Express Technology Co., Ltd., Beijing, China). C-peptide (CPR0) levels were measured at fasting state. Glucagon stimulation test was performed by intravenous loading of 1 mg glucagon. The acute response to glucagon was measured by the C-peptide levels 6 min after glucagon challenge (CPR6).

BMI, HbA1c, CPR0 and CPR6 were measured at the beginning of the experiment, and thereafter, these indexes were examined once every year.

Biochemical assays and laboratory analyses

Blood glucose and lipid levels were analyzed using an automatic biochemical analyzer (AU5800; Beckman Coulter, Inc., Brea, CA, USA) according to the manufacturer's instructions. HbA1c was determined using high-performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan). Blood glucose was measured by a glucose oxidase method using a kit (DiaSys Diagnostic Systems GmbH, Holzheim, Germany). HDL-C was measured by a direct method of immunosuppression using a kit (DiaSys Diagnostic Systems GmbH). LDL-C was measured by a direct measuring method using the a kit (Sekisui Chemical, Co., Ltd., Osaka, Japan). TG and TC measurements were performed using an enzymic kit (Beckman Coulter, Inc.). C-peptide was assayed using a radio-immuno method (Shangsha Yike Biotech Co., Ltd., Changsha, China).

Statistical analysis

Measurement data and non-normally distributed data are expressed as the mean ± standard deviation and interquartile range, respectively. All data were analyzed by use of Kolmogorov-Smirnov normality test and if the data were not normally distributed, a log transformation was performed on these data. χ2 test and two sided t-tests were used for comparison of enumeration and measurement data, respectively. The correlation between islet β-cell function and various indexes were analyzed using Pearson correlation analysis. All statistical analyses were performed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA).

Results

Baseline general characteristics in the EOD and LOD groups

At the baseline, subjects with EOD had lower levels of SBP, DBP, BMI, CPR0, CPR6 and greater levels of HbA1c and TG compared to subjects with LOD (all P<0.01) (Table I). Compared with the baseline, CPR0, CPR6 and TG were decreased in the EOD and LOD groups at 12, 24 and 36 months (Table II).

Table I. Baseline general characteristics of study participants.

Table I.

Baseline general characteristics of study participants.

Characteristic	EOD	LOD
Gender (male/female)	36/12	27/28
Age (years)	35.44±5.11	59.96±8.50a
SBP (mmHg)	118.35±15.44	136.18±21.21a
DBP (mmHg)	78.33±9.65	84.47±10.42a
BMI (kg/m2)	23.31±2.93	25.17±2.99a
FBS (mmol/l)	9.63±3.26	10.09±1.58
HbA1c (%)	10.88±2.32	9.83±1.46a
CPR0 (nmol/l)b	1.1–2.10	1.98–3.46a
CPR6 (nmol/l)b	2.00–4.20	5.29–7.48a
TC (mmol/l)	4.53±0.99	5.15±1.15
TG (mmol/l)b	1.31–3.57	1.00–2.30a
HDL (mmol/l)b	0.96–1.21	1.00–1.24
LDL (mmol/l)b	1.83–3.46	2.22–3.71

a P<0.01, vs. LOD group.

b Indicated that the data were not normally distributed, and these data were expressed as interquartile range (25th and 75th percentiles). EOD, early-onset diabetes; LOD, late-onset diabetes; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index; FBS, fasting blood sugar; HbA1c, glycated hemoglobin A1c; CPR, C-peptide; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Table II. Clinical data of study participants during the 3-year follow-up.

Table II.

Clinical data of study participants during the 3-year follow-up.

	EOD				LOD
Characteristic	Baseline	12 month	24 month	36 month	Baseline	12 month	24 month	36 month
BMI (kg/m2)	23.31±2.93	23.72±2.70	23.81±2.12	23.86±2.63	25.17±2.99	25.11±2.91	25.36±2.98	26.03±3.24
HbA1c (%)	10.88±2.32	9.01±2.09	8.49±1.67	8.39±1.67	9.83±1.46	8.93±1.32	8.38±1.37	8.29±1.31a
CPR0 (nmol/l)	1.72±0.76	1.68±0.69	1.67±078	1.45±0.64	2.89±1.19	2.40±0.95	2.18±0.78	2.16±0.86a
CPR6 (nmol/l)	2.96±1.67	2.84±1.30	2.71±0.93	2.44±0.84	6.66±2.43	5.61±1.30	4.85±1.22	5.20±1.56a

a P<0.01 vs. baseline. EOD, early-onset diabetes; LOD, late-onset diabetes; BMI, body mass index; HbA1c, glycated hemoglobin A1c; CPR, C-peptide.

There was a decreasing trend in the EOD group (Figs. 1–3). HbA1c, CPR0, CPR6 levels were 10.88±2.32, 1.72±0.76 and 2.96±1.67 nmol/l, respectively, when the subjects were recruited, and these were decreased to 8.39±1.67, 1.45±0.64 and 2.44±0.84 nmol/l after 3 years. Compared with the baseline, significant differences were detected in these indexes at 36 months (P<0.01). Furthermore, a decreasing trend was similarly observed in the LOD group (Figs. 1–3). At the baseline, HbA1c, CPR0 and CPR6 levels were 9.83±1.46, 2.89±1.19 and 6.66±2.43 nmol/l, respectively, which decreased to 8.29±1.31, 2.16±0.86 and 5.20±1.56 nmol/l after 3 years. Thus, these indexes showed significant changes during the 3-year follow-up (P<0.01).

Figure 1. Changes in HbA1c in the 3-year follow-up. EOD, early-onset diabetes; LOD late-onset diabetes; HbA1c, glycated hemoglobin A1c.

Figure 3. Changes in CPR6 in the 3-year follow-up. EOD, early-onset diabetes; LOD late-onset diabetes; CPR6, C-peptide 6 min after glucagon challenge.

Comparison of β-cell function failure in the EOD and LOD groups

An >50% reduction in CPR0 or CPR6 was set as the standard of β-cell function failure. A total of 14 subjects (29.17%) in the EOD group and 6 subjects (10.91%) in the LOD group were up to the >50% reduction standard 3 years later, and there was significant difference between the two groups (χ2=5.861, P<0.05) (Fig. 4).

Figure 4. Ratio of β-cell function failure in EOD and LOD groups 3 years later. EOD, early-onset diabetes; LOD late-onset diabetes.

Effect of WHR and BMI on CPR0 and CPR6

Pearson correlation analysis indicated a positive correlation between CPR0 and WHR (P<0.01), HbA1c (P<0.05) in the EOD group after a 3-year follow-up. Furthermore, there was a positive correlation between CPR0 and BMI (P<0.05) in the LOD group.

Discussion

Type 2 diabetes (T2DM) is a complex population, which possesses the characteristics of heterogeneity and disuniformity (13). Previous studies have suggested that following with the progressive aggravation of insulin resistance, the islet β-cell function declines gradually during the pathogenic course of type 2 diabetes (14). However, numerous studies using a variety of techniques have demonstrated that individuals with impaired glucose tolerance or overt type 2 diabetes are characterized by β-cell dysfunction independently of obesity and insulin resistance (15,16). Using a glucose tolerance test, Elbein et al have discovered that first-degree relatives of type 2 diabetic individuals with normal glucose tolerance already possess the characteristic signs of β-cell dysfunction (17). Cnop et al have also reported that in first-degree relatives of type 2 diabetic individuals, the decline in glucose tolerance over time is strongly associated with the loss of β-cell function (18). Thus, early interventions to slow the decline in β-cell function should be considered in high-risk individuals.

Insulin secretion includes two states, the basal (postabsorptive) and stimulated (postprandial) states (19). The postabsorptive state predominates during the interprandial phases and plays a key effect during the period of overnight fast; the postprandial state regulates glucose metabolism as carbohydrate is abundant and must be get rid of (20). Under normal physiological status, postprandial insulin secretion is biphasic and divided into two phases, first-phase and second-phase insulin release (20). Early-phase insulin secretion contains the first phase and part of the second-phase of insulin secretion. Shen et al (21) have found that early-phase insulin secretion is involved in preventing hypernomic postprandial blood glucose and reducing plasma glucose fluctuations.

As the glucagon stimulation test is widely used to evaluate endogenous insulin secretion and shows good reproducibility in the clinic, it is frequently employed to assess β-cell function (22,23). The first measurable sign of β-cell dysfunction that can be observed in type 1 and type 2 diabetes is a defect in the first phase of insulin secretion (24). At 6 min after glucagon stimulation insulin is rapidly secreted, reaching an initial peak value, and this early phase of insulin release lasts no more than 10 min (25). Furthermore, as the 6-min C-peptide level response to 1 mg glucagon is significantly correlated with the area under the curve of C-peptide levels, it is used to assess the first phase of insulin secretion and the residual pancreatic β-cell function (26).

In the present study, glucagon stimulation test (0 and 6 min C-peptide: CPR0 and CPR6) was used to evaluated the β-cell function once a year during the 3-year follow-up in patients with type 2 diabetes. The results suggested that the subjects with EOD had lower BMI, SBP, DBP, CPR0, CPR6 and greater HbA1c and TG compared to subjects with LOD. In the EOD patients, the islet β-cell function declined and could not secrete sufficient insulin, so the plasma glucose was not well controlled and the HbA1c level increased. Hyperglycemia and blood lipid disorder play important roles in the course of islet β-cell deterioration (27). Glucotoxicity and lipotoxicity induced the progressive loss of β-cell function (28).

Currently, there are a number of hypotheses regarding the possible mechanism underlying impaired β-cell function (29). First, chronic hyperglycemia has been shown to inhibit β-cell response, which has been demonstrated both in vitro and in vivo (30). Exposure of β-cells to sustained levels of hyperglycemia may deplete the insulin secretory granules from the β-cell, leaving less insulin available for release in response to further hyperglycemia (31). Lowering of glucose levels allows more complete granulation of β-cells and thus improved acute insulin responses (32). In addition, it has been suggested that increased glucose levels activate the hexosamine pathway and contribute to excess generation of reactive oxygen species, resulting in the inhibition of insulin gene transcription and insulin secretion (33).

Another potential cause of β-cell dysfunction is lipotoxicity, accumulated fatty acids and their metabolic products may have a negative effect on the conversion of proinsulin to insulin, resulting in insulin deficiency (34). Additionally, Zhou and Grill has found that continuous sustained exposure to free fatty acids reduces insulin release in response to glucose by inhibiting glucose oxidation in isolated rat islets (35). It has been shown that the accumulated free fatty acids generated by the hydrolysis of TGs in the islets can decrease an elevation in nitric oxide production, inducing β-cell apoptosis (36). Previous epidemiological studies have demonstrated that triglyceride level is an independent determinant of cardiovascular risk, particularly in association with coronary heart disease (37), and reducing the triglyceride level may restrain atherosclerosis progression. The incidence rate of cardiovascular risk in diabetes mellitus is >3 times compared with the healthy population (38), and the lipid disorder displays a significant effect on the occurrence of macrovascular complications in diabetes mellitus (39).

Compared with the baseline, a decline in islet β-cell function was observed in the EOD and LOD groups over a 3-year follow-up, most notably in the EOD group. An >50% reduction in CPR0 or CPR6 was as the threshold β-cell function failure, and the ratio of β-cell function failure in the EOD group was higher compared with the LOD group. These results suggested that islet β-cell function deteriorated more rapidly in patients with EOD compared with LOD. A correlation analysis indicated that during the β-cell function decline, CPR0 showed a positive association with BMI in the EOD group and with WHR and HbA1c in the LOD group. These results suggest that progressive insulin resistance may accelerate β-cell function failure. Furthermore, the loss of insulin sensitivity contributed to adaptive increase in insulin secretion.

The UK prospective diabetes study group (40) has reported that islet β-cell function is reduced by 50% at the time of diagnosis, and a decreasing rate of 5% every year is observed in patients with type 2 diabetes. Along with the increasing age of the normal glucose tolerance population, the islet β-cell function decreases at a rate of 0.5% every year (41). van Haeften et al (42) has reported that the adaptivity of β-cell function to insulin sensitivity already reaches the maximal degree and the β-cell function may decline in patients with abnormal glucose tolerance. During the chronic progression from normal glucose tolerance to overt diabetes, β-cell function failure may be a more important factor than insulin resistance (6).

Previous investigations have shown that lowering the plasma glucose to near normal by intensive insulin therapy may partially recover the β-cell function and insulin secretion (43). Thus, this schema is consistent with therapeutic interventions aimed at preserving β-cell function/insulin secretion and at reducing the burden of insulin resistance to prevent the cardiovascular disease in the patients with EOD.

References