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The relationship between HbA1c, insulin resistance and changes of insulin secretion in Indonesian Type 2 diabetic subjects.

INTRODUCTION

Diabetes Mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The vast majority of cases of diabetes fall into two categories namely Type 1 diabetes (Type 1 DM) and the more prevalent Type 2 diabetes (Type 2 DM). Other categories include specific types of diabetes relate to genetic defects, the exocrine pancreas disease, endocrinopathies, drug induced and Gestational diabetes (American Diabetes Association, 2013). Type 2 DM is caused by a combination of genetic factors related to impaired insulin secretion and insulin resistance and environmental factors such as obesity, overeating, lack of exercise and stress, as well as aging. The main pathophysiological feature of type 2 DM are impaired insulin secretion and increased insulin resistance (Kaku, 2010).

The main cause of hyperglycemia in Type 2 DM are impaired of insulin secretion and increased insulin resistance (Okita, et al, 2013). Insulin resistance is defined as a state of decreasing ability of insulin to stimulate the uptake and metabolism of glucose in target cells at physiological concentration. Insulin resistance usually precedes the development of type 2 DM. The quantification of insulin resistance condition can be performed by evaluating the peripheral insulin sensitivity using mathematical formula, such as Homeostasis model assessment (HOMA-IR) for [beta]-cell function and QUICKI (Quantitative Insulin sensitivity check index) of insulin sensitivity (Ascaso et al, 2003; Hsieh et al, 2006; Radikova, 2003).

Recent studies have reported an increase in intact proinsulin levels in insulin-resistance individuals. Two major hypotheses have been expounded to explain the hyperproinsulinemia in type 2 diabetes: the increased release of proinsulin might result from an intrinsic defect in proinsulin processing, leading to an increased release of immature insulin precursors and thus contributing to the impairment in [beta]-cell function in type 2 DM. Alternatively, hyperproinsulinemia may be caused by an increased secretory demand on the [beta]-cells, leading to depletion of the "readily releaseable" insulin granule from the "reserve pool", which is thought to contain greater amounts of immature insulin precursor (Breuer et al., 2010). However, the molecular patophysiology of insulin resistance, insulin secretion profile and HbA1c as one of the diabetic criteria in development of Indonesian Type 2 diabetes patients still unclear.

HbA1c measurement currently has been recommended for diagnosis of diabetes by American Diabetes Association (ADA) (American Diabetes Association 2013). HbA1c, or glycosylated hemoglobin, is formed through the non-enzymatic binding of circulating glucose to hemoglobin (glycation). Higher levels of glucose in the blood contribute to more binding and consequent higher levels of glycosylated hemoglobin. Because glycation occurs over the entire 90-120 day life span of the red blood cells, so HbA1c can be interpreted as an average of the blood glucose present over 3-4 months. HbA1c is a more comprehensive measure of total glycemic exposure than FPG due to the representation of blood glucose in the postprandial state in addition to the fasting state, so HbA1c has been recommended for diagnosis of diabetes (Gomero et al., 2008).

Subjects And Methods:

The study was approved by institutional Human ethics committee (Medical Faculty, Brawijaya University) and informed consent was obtained from all participants.

A. Subjects:

This study included 67 subjects having general check-up in Central Laboratory of Dr. Saiful Anwar Hospital for a period of six month. The inclusion criterion of them were males and females with type 2 diabetes mellitus with or without complications or any co-morbid condition like hypertension, coronary artery disease, etc, and also non diabetic healthy individual as the controls. All the subjects, including the controls, were fully informed about the study and their voluntary informed consents were taken. The subjects were categorized into three groups which include: healthy controls (n = 21), prediabetes (n = 10), and diabetes (n = 36).

B. Methods

Clinical identification of normal or healthy control, prediabetes, and diabetes was done according to American Diabetes Association criteria. The diagnostic criteria of diabetes was assessed according to American Diabetes Association (ADA) i.e. subjects with a fasting plasma glucose > 126 mg/dL and/or 2 hour plasma glucose level > 200 mg/dL and/or HbA1c > 6.5% were considered to have diabetes; subjects with a fasting plasma glucose 100 to 125 mg/dL (IFG) or 2 hour plasma glucose level 140 to 199 mg/dL (IGT) or HbA1c 5.7 to 6.4% were considered to have increased risk for diabetes (prediabetes); subjects with a fasting plasma glucose < 110 mg/dL or 2 hour plasma glucose level < 140 mg/dL or HbA1c < 5.6% were regarded as a having normal glucose tolerance (NGT) (American Diabetes Association 2013).

Fasting venous blood was collected from all of subject. It was centrifuged (at 1500 g for 15 minutes). The separated plasma was used to assay the HbA1c. HbA1c was measured with ion-exchange high-performance liquid chromatography using an automated analyzer (BioRad D10). The separated serum was divided into four aliquot. One was designed for immediate assay of glucose and lipid profile which included Triglyceride (TG), total cholesterol (CHOL), high density lipoprotein (HDL), lowdense lipoprotein (LDL). The other aliquots were stored at -20[degrees]C for subsequent assay for insulin and proinsulin.

The assay of sample analysis was carried out by using different reagent kits as per procedure which was defined by manufacturer. The immediate assay of sample analysis was measured on a fully automated analyzer. The fasting plasma glucose was measured by the hexokinase method. The serum triglyceride was measured by the enzymatic method (GPO-POD method, End Point). For determination of total cholesterol, an enzymatic (CHOD-POD) colorimetric method was used. The direct measurement for HDL and LDL were done by using enzymatic methods.

Insulin and total proinsulin concentration were measured by Sandwich enzyme immunoassay method. Insulin concentration was measured using kit from Ucsn, China. Total proinsulin concentration was measured using kit from Novateinbio, Cambridge. This assay measures total human proinsulin and no cross-reactivity with insulin and C-peptide. Homeostasis model assessment of insulin resistance (HOMA-IR) was used for the direct measurement of insulin resistance and was calculated as follows:

HOMA-IR = [fasting insulin ([micro]U/mL) x fasting glucose (mg/dL)]/405 (Osman et al. 2012; Shirai 2004)

The quantitative insulin sensitivity check index (QUICKI) was calculated from fasting plasma glucose ([G.sub.o] in mg/dL) and insulin ([I.sub.o] in [micro]IU/mL) concentrations, as follows: QUICKI = 1/(log [I.sub.o] + log [G.sub.o]) (Radikova 2003)

The results were analyzed statistically using SPSS version 16.0 statistical software. The results were expressed as mean [+ or -] SD if the variables were continuous, and as percentage, if categorical. Multivariate analysis of variance was used for differences in continuous variables. Multiple regression was applied for correlation studies. All statistical tests were two-side and a P < 0.05 was considered to be significant.

Results:

The incidence of diabetes increases worldwide and become a global problem. It would be the leading cause of morbidity and mortality in the future. The characteristics of the study subjects have been shown in Table 1. The fasting glucose concentration in individual with Normal Glucose Tolerance (n = 10) (74.81 [+ or -] 7.07 mg/dL) and patient with prediabetes (n = 12) (109.83 [+ or -] 5.83 mg/dL) were significantly different (P < 0.01) with type 2 DM patients (n = 26) (191.91 [+ or -] 83.56 mg/dL). Our study showed increasing fasting plasma glucose, HbA1c, Total Cholesterol, total proinsulin, and insulin resistance (HOMA-IR) of [beta]-cell function. Beside that, our study presented the decreasing of fasting insulin and QUICKI index of insulin sensitivity.

Beside blood glucose level, HbA1c measurement currently has been recommended for diagnosis of diabetes by American Diabetes Association (ADA)(American Diabetes Association 2013). Our study indicated that the level of HbA1c progressively increased and had correlation with increasing level of proinsulin, decreasing level of fasting insulin, increasing of insulin resistance index (HOMA-IR), and proinsulin:insulin ratio, as shown in Fig 1. Beside that, our study showed the different profile of fasting proinsulin, insulin, total insulin + proinsulin level, and also the insulin resistance condition in Normal Glucose Tolerance (NGT), prediabetes, and type 2 diabetes patients, as shown in Fig 2. The level of fasting proinsulin in prediabetes is the same as in diabetic patients, but it is significantly higher normal glucose tolerance (NGT) as the control. The progressively decreasing of fasting insulin concentration was showed among NGT, prediabetic and diabetic patients, but total level of fasting insulin + proinsulin in diabetic patients was significantly lower than in NGT and prediabetic patients. The insulin resistance condition of diabetic patients was significantly higher than in NGT and prediabetic patients. Our study also showed the statistically different profile of Fasting Plasma Glucose (FPG) and HbA1c concentration in NGT, prediabetes and diabetic patients, as shown in Fig 3. Fasting Plasma Glucose (FPG) concentration in diabetic patients was significantly higher than in NGT and prediabetes, but HbA1c level was increasing progressively among NGT, prediabetes, and diabetic patients.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion:

Insulin is a peptide hormone secreted by [beta]-cell of the pancreatic islet of Langerhans for maintaining normal blood glucose level by facilitating cellular glucose uptake. Insulin synthesized in the [beta]-cell of the pancreatic islet of Langerhans as it precursor, proinsulin. Proinsulin is synthesized in the ribosome of the rough endoplasmic reticulum. Secretory vesicles transfer proinsulin from the RER to the Golgi apparatus. Most proinsulin was processed while it passes Golgi apparatus to secretory granules and secreted into blood circulation as insulin. However, some proinsulin remains without processing and present in secretory granules and secreted into blood circulation with insulin (Wilcox 2005).

In health, about 80-90% of total insulin concentrations are derived from mature, biologically fully active, insulin. While in healthy, non diabetic subjects, these precursors constitute only a minor fraction of the total amount of secreted insulin, the release of proinsulin is significantly increase in patient with type 2 diabetes as well as in prediabetes (Breuer et al. 2010). In our study, 90.53% of total insulin concentration was circulated in blood circulation as insulin and the rest was in proinsulin form. In prediabetes and type 2 diabetes subjects, the release of proinsulin increased to 22.66% and 24.31% of total insulin concentration. There was no significantly different between increasing proinsulin and insulin concentration in prediabetes and type 2 diabetes subjects. So, we also tend to support the concept of a primary defect in proinsulin processing in pathogenesis of type 2 diabetes.

Our study showed the insulin resistance condition of normal was the same as in prediabetes subjects, but significantly different with in diabetes subjects. Nevertheless, the proinsulin level of prediabetes and diabetes subjects was higher than normal. We indicate proinsulin activates receptors that also bind insulin and IGF-1, but have different metabolic activity. Most studies indicate a very low metabolic activity of proinsulin and low affinity for insulin receptor (IR) or IGF-1 receptor. Proinsulin has been regarded as a weak-affinity ligand for the insulin receptor with a low metabolic potency. Proinsulin and insulin bind to and activate the two Insulin Receptor (IR) isoforms, but the degradation responses of IR are different. The degradation response of IR-proinsulin is longer than IR-insulin binding. This delayed degradation responses possibly related to the low binding affinity of proinsulin to the IR. It may result in longer ligand-receptor interactions and effects the number of glucose uptake into the cell (Malaguarnera et al. 2012)(Prager and Schernthaner 1982)

The other function of insulin prevents uncontrolled hydrolysis of triglyceride and limits gluconeogenesis, thereby maintaining normal fasting blood glucose level. An increasing of serum glucose will induce pancreatic [beta]-cell to increase insulin secretion for maintaining the homeostasis of normal blood glucose. The condition was known as compensatory hyperinsulinemia condition (Wilcox 2005). In our study, we indicate prediabetes subjects as the compensatory hyperglycemia condition. On this condition, we found the increasing level of intact proinsulin, decreasing level of insulin, but total concentration of insulin + proinsulin and insulin resistance condition were not different from normal. It means that the by [beta]-cell of the pancreatic islet of Langerhans worked hard to secrete the insulin, unfortunately it failed to proceed the intact proinsulin well, so the secretory granule of the [beta]-cell secreted the intact proinsulin in higher level to the plasma. An elevated serum level of proinsulin predicts the development of Type 2 DM and indicates an advance state of [beta]-cell exhaustion in type 2 DM (Wilcox 2005).

Decreasing insulin level can stimulate hydrolysis triglyceride, so it will increase free fatty acid (FFA) level. Increasing FFA concentration reduce the inhibiting effect of insulin upon the gluconeogenesis and glycogenolysis, hereby the blood glucose level increased. Increasing blood FFA concentration and cellular triglyceride mainly in the liver, muscle and pancreatic Langerhans specifies the lipotoxicity of diabetogenic effect. Chronic exposure to abnormally high level of glucose over many years (chronic hyperglycemia), glucotoxicity, and lipotoxicity can lead to toxic effects on [beta]-cell. So, chronic hyperglycemia can induce insulin secretion defect and worsen insulin resistance. (Okita et al. 2013)(Robertson et al. 2004)(Defronzo 2004)(Kaku 2010). In our study, decreasing total proinsulin + insulin level indicated insulin secretion defect and support the glucotoxicity and lipotoxicity phenomenon in type 2 DM (Dimitrova and Georgiev 2007).

In our study, HbA1c has strong correlation with duration of Diabetes (r = 0.867, P value < 0.001), FPG (r = 0.967, P value < 0.001), 2h-postprandial Plasma Glucose (r = 0.979, P value < 0.001), Insulin Resistance or HOMA index of p-cell function (r = 0.939, P value < 0.001) and QUICKI index of insulin sensitivity (r = 0.901, P value < 0.001). We indicates that increasing blood glucose levels contribute to more binding of glucose to hemoglobin (glycation reaction) and consequent higher level of glycosylated hemoglobin (HbA1c). Some research also support that glycation occurs over the entire 90-120 day life span of the red blood cells, so HbA1c can be interpreted as an average of the blood glucose present over 3-4 months. HbA1c is a more comprehensive measure of total glycemic exposure than FPG due to the representation of blood glucose in the postprandial state in addition to the fasting state (Gomero et al. 2008)(Liang et al. 2010)(Ibrahim et al. 2010).

Conclusion:

Increasing blood glucose level in type 2 DM contributed to HbA1c, correlated with change of insulin secretion profile, by increasing proinsulin level and decreasing total insulin, and related with worsen insulin resistance.

ARTICLE INFO

Article history:

Received 2 April 2014

Received in revised form 13 May 2014

Accepted 28 May 2014

Available online 27 June 2014

ACKNOWLEDGEMENT

Author thanks to "Directorate General of Higher Education" through Penelitian Unggulan Perguruan Tinggi (Pemula) second batch 2013 for sponsor and financial support.

REFERENCE

American Diabetes Association, 2013. "Standards of Medical Care in Diabetes 2013." Diabates Care 36:S11-S66.

Ascaso, F., Juan et al., 2003. "Diagnosing Insulin Resistance by Simple Quantitative Methods in Subjects With Normal Glucose Metabolism." Diabetes Care, 26(12): 3320-25.

Breuer, G.K., Thomas et al., 2010. "Proinsulin levels in patients with pancreatic diabetes are associated with functional changes in insulin secretion rather than pancreatic b -cell area." European Journal of Endocrinology, 163: 551-58.

Defronzo, A., Ralph, 2004. "Pathogenesis of type 2 diabetes mellitus." The Medical Clinics of North Amerca, 88: 787-835.

Dimitrova, S. and I. Penchev Georgiev, 2007. "RELATIVE CONTRIBUTION OF DECREASED INSULIN SENSITIVITY TO DETERIORATION OF GLUCOSE HOMEOSTASIS." Bulgarian Journal of Veterinary Medicine, 10(4): 205-22.

Gomero, Ada, Thomas McDade, Sharon Williams and Stacy Tesller Lindau, 2008. "Dried Blood Spot Measurement of Glycosylated Hemoglobin (HbA1c) in Wave 1 of the National Social Life, Health and Aging Project (NSHAP)." NORC and The University of Chicago.

Hsieh, Chang-hsun et al., 2006. "Insulin resistance & secretion in subjects with normal fasting plasma glucose." Indian J Med Res., 124(November): 527-34.

Ibrahim, Hasni et al., 2010. "The use of HbA1C in the diagnosis of diabetes mellitus type 2 in high risk subjects." Int J Diabetes & Metab, 18:25-28.

Kaku, Kohei, 2010. "Pathophysiology of Type 2 Diabetes and Its Treatment Policy." Journal of the Japan Medical Association, 53(1): 41-46.

Liang, Chi-chau, Kun-wu Tsan, Shih-ming Ma, Shan-fan Chow, and Chin-chu Wu, 2010. "The Relationship between Fasting Glucose and HbA1c Among Customers of Health Examination Services." Formos J Endocrin Metab 1(3): 1-5.

Malaguarnera, Roberta et al., 2012. "Proinsulin Binds with High Affinity the Insulin Receptor Isoform A and Predominantly Activates the Mitogenic Pathway." Endocrinology, 153(5): 2152-63.

Okita, Kohei et al., 2013. "Homeostasis model assessment of insulin resistance for evaluating insulin sensitivity in patients with type 2 diabetes on insulin therapy." Endocrine Journal, 60(3): 283-90.

Osman, Mona M., Abeer I. Abd. El-mageed, Eman El-hadidi, Rania S. Shahin and Nanees A. Adel A. Mageed, 2012. "Clinical Utility of Serum Chemerin as a Novel Marker of Metabolic Syndrome and Type 2 Diabetes." Life Science Journal, 9(2):1098-1108.

Prager, Rudolf and Guntram Schernthaner, 1982. "Receptor Binding Properties of Human Insulin (Recombinan DNA) and HUman Proinsulin and Their Interaction at the Receptor Site." Diabetes Care 5(Suppl 2): 104-6.

Radikova, Zofia, 2003. "ASSESSMENT OF INSULIN SENSITIVITY/RESISTANCE IN EPIDEMIOLOGICAL STUDIES." Endocrine Regulation 37: 189-94.

Robertson, R., Paul, Jamie Harmon, Phuong Oanh T. Tran, and Vincent Poitout, 2004. "Beta Cell Glucose Toxicity, Lipotoxicity, and Oxidative Stress in Type 2 Diabetes." Diabetes, 53(Supplement 1): S119-24.

Shirai, K., 2004. "Obesity as the core of the metabolic syndrome and the management of coronary heart disease. Title." Curr. Med. Res. Opin, 20(3): 295-304.

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(1,2) Srihardyastutie, A, (3) D.W.Soeatmadji, (2) Fatchiyah, and (2) Aulanni'am

(1) Biology Doctoral Program, Faculty of Science, Brawijaya University

(2) Faculty of Science Brawijaya University

(3) Medical Faculty, Brawijaya University

Corresponding Author: Aulanni'am, Faculty of Science Brawijaya University E-mail: aulanibiochems@gmail.com
Table 1: Characteristics of different study groups.

                                                NGT

Fasting Plasma Glucose (mg/dL)          74.81 [+ or -] 7.07
HbA1c (%)                               4.58 [+ or -] 0.41
Total Cholesterol (mg/dL)              195.24 [+ or -] 39.32
Triglyceride (mg/dL)                   120.86 [+ or -] 71.65
HDL (mg/dL)                            54.43 [+ or -] 15.99
LDL (mg/dL)                            132.14 [+ or -] 38.93
Total proinsulin (ng/mL)               42.79 [+ or -] 11.78
Fasting Insulin (ng/mL)                409.06 [+ or -] 19.04
Insulin Resistance Index (HOMA--IR)   1360.40 [+ or -] 146.02
Insulin Sensitivity (QUICKI)           0.2219 [+ or -] 0.004

                                            Prediabetes

Fasting Plasma Glucose (mg/dL)         109.83 [+ or -] 5.83
HbA1c (%)                               6.08 [+ or -] 0.47
Total Cholesterol (mg/dL)              192.92 [+ or -] 53.27
Triglyceride (mg/dL)                   113.25 [+ or -] 48.97
HDL (mg/dL)                            47.00 [+ or -] 13.36
LDL (mg/dL)                            126.25 [+ or -] 48.06
Total proinsulin (ng/mL)               100.19 [+ or -] 45.37
Fasting Insulin (ng/mL)                341.85 [+ or -] 22.06
Insulin Resistance Index (HOMA--IR)   1667.70 [+ or -] 125.50
Insulin Sensitivity (QUICKI)           0.2187 [+ or -] 0.002

                                             Type 2 DM

Fasting Plasma Glucose (mg/dL)         191.91 [+ or -] 83.56
HbA1c (%)                                9.58 [+ or -] 2.82
Total Cholesterol (mg/dL)              207.74 [+ or -] 57.94
Triglyceride (mg/dL)                   188.56 [+ or -] 98.77
HDL (mg/dL)                             45.56 [+ or -] 13.34
LDL (mg/dL)                            142.24 [+ or -] 50.20
Total proinsulin (ng/mL)               102.60 [+ or -] 33.69
Fasting Insulin (ng/mL)                319.44 [+ or -] 30.66
Insulin Resistance Index (HOMA--IR)   2705.10 [+ or -] 1126.71
Insulin Sensitivity (QUICKI)           0.2105 [+ or -] 0.007
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Author:Srihardyastutie, A.; Soeatmadji, D.W.; Fatchiyah; Aulanni'am
Publication:Advances in Natural and Applied Sciences
Article Type:Clinical report
Date:Jul 1, 2014
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