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1 From the Department of Food and Nutrition, Hoseo University, Asan-Si, Korea (SP), and the Department of Internal Medicine, KonKuk University, Chungjoo-Si, Korea (SBC).
2 Supported by a grant from the G7 project of the Department of Public Health and Welfare in Korea. 3 Address reprint requests to S Park, Department of Food and Nutrition, Hoseo University, Sechul-Ri, Asan-Si, ChungNam-Do, 336-795, Korea. E-mail: smpark{at}office.hoseo.ac.kr.
ABSTRACT
Background: Most Koreans with type 2 diabetes are insulin deficient and insulin resistant. Continuous subcutaneous insulin infusion (CSII) provides a suitable amount of insulin to overcome insulin deficiency and achieve near-normal blood glucose concentrations. Our previous study showed, however, that CSII does not reduce oxidative stress even though it normalizes blood glucose concentrations.
Objective: The purpose of this study was to determine whether CSII plus -tocopherol supplementation for 2 mo would alter oxidative stress in Korean patients with type 2 diabetes.
Design: Ninety-eight subjects received CSII plus either 200 mg -tocopherol/d (n = 48) or a placebo (n = 50) for 2 mo. The general characteristics (age, duration of diabetes, body mass index, and blood glucose concentrations) of the 2 groups were not significantly different.
Results: Fasting and postprandial blood glucose concentrations of all subjects were normalized after CSII. Fasting plasma insulin concentrations did not differ significantly between the 2 groups after CSII. Lipid peroxide concentrations in plasma and red blood cells decreased and -tocopherol concentrations in plasma and red blood cells increased after -tocopherol supplementation. However, these changes were not affected significantly by CSII. Plasma vitamin C concentrations increased significantly after CSII plus -tocopherol supplementation. However, the activities of antioxidant enzymes in red blood cells did not change significantly after CSII plus -tocopherol supplementation.
Conclusion: -Tocopherol supplementation was beneficial in decreasing blood lipid peroxide concentrations without altering antioxidant enzyme activities in Korean patients with type 2 diabetes treated with CSII.
Key Words: Vitamin C • -tocopherol • lipid peroxide • glutathione peroxidase • superoxide dismutase • catalase • type 2 diabetes • glucose concentrations • insulin • Korea
INTRODUCTION
The prevalence of type 2 diabetes in the Korean population has steadily increased in recent years and currently >8% of the population are affected (1). The characteristics of Korean patients with type 2 diabetes differ from those of Western patients. For example, type 2 diabetes is associated predominantly with insulin deficiency in Koreans but with impaired insulin action on cellular glucose utilization characterized by insulin resistance or hyperinsulinemia in Western populations; additionally, Koreans have a smaller insulin secretory capacity (2–4). Blood glucose concentrations in Korean diabetic patients are less likely to be adequately controlled with oral hypoglycemic agents or diet and thus resemble concentrations in a subset of patients with type 2 diabetes, ie, patients with type 1.5 diabetes (3,4).
Korean patients with type 2 diabetes often need exogenous insulin to control their blood glucose concentrations. Continuous subcutaneous insulin infusion (CSII) with an insulin pump is suitable for mimicking physiologic insulin secretion from the pancreas (5). Intensive CSII can provide good glycemic control in patients with type 2 diabetes (6,7), but it may result in adverse effects such as weight gain, an increased number of hypoglycemic episodes, and hyperinsulinemia.
Few studies have been conducted on the relations, if any, between hyperinsulinemia and oxidative stress (8,9). Our previous study showed that after 2 wk of CSII therapy, lipid peroxide concentrations in plasma and red blood cells (RBCs) did not change significantly despite the maintenance of euglycemia in patients with type 2 diabetes (10). Thus, for CSII to effectively reduce oxidative stress in nonobese patients with type 2 diabetes, additional therapy is needed.
-Tocopherol acts as a free oxygen radical scavenger to reduce oxidative stress (11). However, it is unclear whether -tocopherol administration reduces oxidative stress in patients with type 2 diabetes; if it does, it may improve glucose disposal rates and pancreatic ß cell function and reduce the prevalence of diabetic complications (12,13). The purpose of this study was to determine whether CSII therapy alone or plus -tocopherol supplementation for 2 mo would decrease oxidative stress in Korean patients with type 2 diabetes.
SUBJECTS AND METHODS
Selection of subjects
Ninety-eight Korean patients with type 2 diabetes were selected for CSII therapy. Exclusion criteria were as follows: 1) proliferative retinopathy, 2) significant renal impairment (serum creatinine > 270 µmol/L), 3) documented coronary artery disease, 4) chronic liver disease, 5) diabetic foot ulceration and gangrene, 6) pulmonary infection, 7) heavy smoking and drinking, and 8) supplementation with multivitamins or traditional herbs in the previous 3 mo. All selected subjects started CSII to control their blood glucose concentrations after the baseline measurements were made. Regular insulin (Humulin R; Eli Lilly, Indianapolis) was the source of exogenous insulin. The study was approved by the Human Ethical Approval Committee of Konkuk University (ChungBuk-Do, Korea) and informed, written consent was obtained from each subject.
Experimental design
The 98 subjects selected were randomly assigned to 2 groups by rolling a die. One group (n = 48) received CSII plus 200 mg -tocopherol/d for 2 mo and the other group (n = 50) received CSII plus a digestive pill (Magenal F; Taepeungyang, Seoul, Korea; placebo group) for 2 mo. At the beginning of the study, all subjects were hospitalized for 2–4 wk in the Diabetes Center of KonKuk University for training on how to control their blood glucose concentrations with an insulin pump (Dana insulin pump; Sooil Development Co, Seoul, Korea). After being discharged from the hospital, patients from both groups continued to maintain good glycemic control at home. Follow-up examinations took place once every 2–3 wk at an outpatient clinic. Basal insulin rates and premeal boluses were adjusted to achieve euglycemia, as defined by preprandial and 2-h postprandial glucose concentrations of <5.5 and <7.8 mmol/L, respectively.
Because age, exercise, smoking, drinking, polyunsaturated fat intake, and dietary antioxidant vitamin intakes can affect oxidative status, the subjects were given specific rules to follow throughout the study to maintain their oxidative status. The subjects were instructed to maintain their smoking and drinking habits, to not take any multivitamin supplements or traditional herbs, and to consume 2 serving sizes of vegetable oil, 3 servings of fruit, and 5 servings of vegetables daily. In addition, it was recommended that the subjects engage in low-impact exercise for 30 min/d.
The subjects' food intakes were determined with a 24-h dietary recall method through a personal interview with a registered dietitian before and during the last week of the study. The nutrient content of the diet was derived with the use of a database (Computerized Analysis of Nutrients; The Korean Nutrition Society, Seoul, Korea) based on Korean Department of Agriculture sources. Compliance with the protocol was monitored with a telephone call once weekly.
Blood collection and biochemical analysis
After the subjects had fasted overnight, blood was collected in tubes containing EDTA before and after the administration of CSII plus -tocopherol or placebo for 2 mo. After centrifugation of blood at 3000 x g for 30 min at 4°C, the plasma supernatant fluid was separated and stored at -70°C until analyzed further. The white layer between the plasma and the RBCs was removed and the RBCs were washed with 0.9% NaCl 3 times. RBCs were suspended with saline in equal proportions and stored at -70°C.
Blood glucose concentrations were measured 7 times/d with a portable glucometer (Accutrend; Boehringer Mannheim, Mannheim, Germany) before and after meals and at bedtime. Plasma C-peptide and insulin concentrations were evaluated by radioimmunoassay (Linco Research, St Charles, MO) (14). Glycated hemoglobin (Hb A1c) was measured by HPLC (15).
Concentrations of the lipid peroxidation products of thiobarbituric acid–reactive substances, mainly malondialdehyde, were determined fluorometrically according to the modified method of Ohkawa et al (16) at excitation and emission wavelengths of 515 and 550 nm, respectively. 1,1,3,3-Tetraethoxypropane was used as the standard. The thiobarbituric acid–reactive substances assay is a common method for measuring lipid peroxidation, which can represent oxidative stress in several situations, including diabetes (17–19). Because plasma triacylglycerol concentrations decreased significantly after both treatments, the lipid peroxide concentrations in plasma were calculated as µmol/g triacylglycerol.
Plasma or RBC suspensions were deproteinized with ethanol containing RRR--tocopherol acetate as the internal standard. Tocopherols were extracted with hexane and evaporated to dryness under a stream of nitrogen. Concentrations of -tocopherol in plasma and RBCs were measured by HPLC (Waters, Milford, MA) with the use of a fluorescence detector with excitation and emission wavelengths of 205 and 340 nm, respectively (20). A C18 reversed-phase column was used for the stationary phase and methanol as the mobile phase; 2,2,5,7,8-pentamethyl-6-hydroxychroman was used as an internal standard. Concentrations of plasma and RBC -tocopherol were expressed as µmol/L and µmol/L packed RBCs, respectively. Plasma was treated with metaphosphoric acid to precipitate proteins, and vitamin C concentrations were measured from the supernatant fluid by reversed-phase HPLC with an ultraviolet detector. Vitamin C was quantitatively measured with the use of isoascorbic acid as an internal standard (21).
Activities of the antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD), and catalase were measured in both treatment groups. Glutathione peroxidase activity was measured with the use of a modified version of the method of Flohe and Gunzler (22) in which t-butyl hydroperoxide was used as the substrate. The results are expressed as nmol NADPH oxidized•min-1mg protein-1 in RBCs and in plasma. SOD activity was determined by monitoring the autooxidation of pyrogallol according to the method of Flohe and Otting (23). One unit of SOD activity is defined as the amount of the enzyme required to inhibit the rate of pyrogallol autooxidation by 50% and is expressed as mg protein in RBCs. Catalase activity in RBCs was determined with the use of the method described by Aebi (24). The decomposition rate of the substrate hydrogen peroxide by catalase was monitored at 240 nm in an ultraviolet-visible spectrophotometer (Pharmacia, Uppsala, Sweden) and is expressed as nmol H2O2 decomposed•min-1•mg protein-1.
Statistical analysis
Results are expressed as means ± SDs. The statistical analysis was performed with the use of SAS (25). Two-factor repeated-measures analyses of variance was conducted to determine whether there were treatment or time effects and time x treatment interactions. Unless otherwise stated, the time x treatment interactions were not significant. General characteristics such as age, duration of diabetes, body mass index, and blood glucose concentrations were compared between groups with the use of unpaired 2-sample Student's t tests if applicable. Pearson's correlation analysis was used to determine the relation between indicators of blood glucose control status and oxidative stress status. A P value < 0.05 was considered to be statistically significant.
RESULTS
General characteristics of subjects
Before the study began, there were no significant differences in clinical characteristics between the CSII + -tocopherol and placebo groups (Table 1). The average age of the subjects in both groups was 49.5 ± 9.8 y, and the average body mass index (BMI; in kg/m2) of both groups was within the normal range. Fasting and postprandial C-peptide concentrations indicated that all subjects had type 2 diabetes. Fifty percent of the subjects had diabetic complications. Thirty-three percent of the subjects smoked and 31% drank alcohol; these habits remained constant throughout the study.
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TABLE 1. . General characteristics of the subjects at baseline1
Daily nutrient intakes
The daily intakes of energy, carbohydrate, protein, fat, and vitamins C, A, and E in both groups before (baseline) and after CSII treatment are shown in Table 2. None of the intakes after treatment were significantly different from those at baseline. Daily energy intakes increased in both groups, but not significantly so. Two-factor repeated-measures analysis of variance showed no significant effects of time or treatment and no time x treatment interactions. Carbohydrate, protein, and fat intakes as a percentage of energy at baseline and after treatment showed a pattern typical of Korean patients with diabetes. All subjects consumed sufficient daily quantities of vitamin C, but intakes of vitamins A and E and of lipid-soluble vitamins were less than the recommended dietary allowances (26). Overall intakes of vitamins A and E were 55.0% and 69.5% of the recommended dietary allowances, respectively.
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TABLE 2. . Daily nutrient intakes of the subjects before and after 2 mo of treatment1
Changes in metabolic indexes
Measures of body weight and glucose, insulin, lipid, and Hb A1c concentrations in the blood are shown in Table 3. Fasting and postprandial blood glucose concentrations were not controlled in all subjects before CSII treatment. Both groups achieved euglycemic control with exogenous insulin after CSII. Good glycemic control after CSII was associated with a decrease in Hb A1c concentrations to normal ranges in all subjects. Fasting plasma insulin concentrations increased from baseline by 40.9 ± 4.6% in the CSII + -tocopherol group and by 49.0 ± 5.6% in the placebo group, but these increases were not significantly different between the 2 treatment groups.
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TABLE 3. . Body weight and metabolic variables before and after 2 mo of treatment1
Body weight increased by 3.6 ± 1.2 and 4.1 ± 1.4 kg in the CSII + -tocopherol and the placebo groups, respectively, after 2 mo of treatment. BMI increased to 23.7 ± 2.4 in the CSII + -tocopherol group and to 23.9 ± 2.8 in the placebo group (data not shown). The leaner a subject was at the beginning of the study, the more weight he or she gained; however, no significant treatment effects on body weight or BMI were observed.
Total, LDL-, and HDL-cholesterol and triacylglycerol concentrations in fasting plasma did not differ significantly between the 2 treatment groups at baseline. Concentrations of each of these indexes, except triacylglycerol, were close to the high end of the normal range; triacylglycerol concentrations were above normal ranges. Plasma total, LDL-, and HDL-cholesterol concentrations did not change significantly in the CSII + -tocopherol group after 2 mo of treatment. Plasma triacylglycerol concentrations decreased with CSII, but no additional effect of -tocopherol supplementation was observed.
Plasma and RBC lipid peroxide concentrations
Plasma and RBC lipid peroxide concentrations did not change significantly in the placebo group but decreased significantly in the CSII + -tocopherol group after 2 mo of treatment (Table 4). Correlations between fasting blood glucose and Hb A1c concentrations and plasma and RBC lipid peroxides indicated a significant association between blood glucose control and oxidative stress; however, fasting plasma insulin concentrations did not correlate with plasma and RBC lipid peroxide concentrations and thus showed no such association (Table 5).
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TABLE 4. . Plasma and red blood cell (RBC) lipid peroxides before and after 2 mo of treatment1
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TABLE 5. . Correlation coefficients among indicators of blood glucose control and oxidative stress status1
Antioxidants and antioxidant enzymes
Plasma and RBC -tocopherol and plasma vitamin C concentrations before and after treatment are shown in Table 6. At baseline, -tocopherol concentrations in plasma and RBC did not differ significantly between the 2 treatment groups. After 2 mo of treatment, plasma and RBC -tocopherol concentrations increased significantly from baseline in the CSII + -tocopherol group, but no significant time effect or time x treatment interaction on either index was observed. Significant time and treatment effects but no time x treatment interaction on plasma vitamin C concentrations were observed. Plasma vitamin C concentrations increased after treatment in both treatment groups, but more so in the CSII + -tocopherol group than in the placebo group. Plasma and RBC -tocopherol concentrations correlated negatively with blood glucose and Hb A1c concentrations, but they had no association with plasma insulin concentrations.
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TABLE 6. . Plasma and red blood cell (RBC) -tocopherol and plasma vitamin C concentrations before and after 2 mo of treatment1
The activities of plasma and RBC glutathione peroxidase and of SOD and catalase in RBCs were not significantly different between treatment groups either before or after the 2-mo treatment period. In addition, there was no time effect or time x treatment interaction for any of the 3 indexes (Table 7).
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TABLE 7. . Activities of glutathione peroxidase, superoxide dismutase, and catalase before and after 2 mo of treatment1
DISCUSSION
One possible reason oxidative stress did not improve with 2 wk of CSII in our previous study (10) is the duration of therapy. It is possible that 2 wk is insufficient for normalizing whole-body glucose metabolism. Another possible reason for the lack of improvement is the increase in plasma insulin concentrations, which may have resulted in an increase in the production of oxygen-derived free radicals (27,28). These 2 possible reasons may explain why oxidative stress was not reduced in the placebo group. The present study was designed to determine the effect of CSII and supplementation with -tocopherol on oxidative stress in Koreans with type 2 diabetes.
Min (3) reported that 77% of Korean patients with type 2 diabetes have characteristics similar to those of patients with so-called type 1.5 diabetes. In the present study, 88% of our subjects were nonobese [obesity was defined as a BMI >27 in males and >25 in females (29,30)]. Baseline plasma C-peptide and insulin concentrations of the subjects in the present study were lower than those reported by Min; therefore, most of our subjects likely had type 1.5 diabetes.
The pathophysiology of type 2 diabetes involves a combination of impaired insulin secretion, peripheral insulin resistance, and excessive hepatic glucose output (6). Because one of the characteristics of patients with type 2 diabetes is hyperinsulinemia, which is closely related to insulin resistance, intensive insulin therapy is not usually recommended. In some recent studies, intensive insulin therapy was undertaken in patients with type 2 diabetes even though CSII was shown previously to increase plasma insulin concentrations (5,31,32). The rationale behind CSII treatment for type 2 diabetes is based on the observation that hyperglycemia, rather than hyperinsulinemia, may impair insulin action and insulin secretion, creating a metabolic milieu that further promotes oxidative stress. These studies reported that CSII improves glycemic control in patients with type 2 diabetes and that the maintenance of euglycemia enhances insulin action and decreases future exogenous insulin requirements (5,31,32).
The present study showed that 2 mo of therapy with CSII alone (placebo treatment) did not reduce oxidative stress in the patients with type 2 diabetes. However, Berg et al (33) found that patients with type 1 diabetes maintained good glycemic control after 24 mo of CSII and that concentrations of Hb A1c and plasma lipid hydroperoxides decreased significantly from baseline. They suggested that hyperglycemia is an important factor in the generation of lipid hydroperoxides and reactive oxygen species in the circulation of type 1 diabetic patients. Sharma et al (34) reported a finding similar to ours. They reported decreased serum malondialdehyde concentrations and increased plasma vitamin E concentrations after glycemic control in patients with type 2 diabetes. However, serum malondialdehyde concentrations remained higher and plasma vitamin E concentrations remained lower in the patients with type 2 diabetes than in the healthy control subjects. Vitamin E supplementation further decreased serum malondialdehyde concentrations in the patients with type 2 diabetes.
Euglycemia promoted higher plasma ascorbic acid concentrations in our study. However, the degree of increase in plasma ascorbic acid concentrations seen in euglycemia was probably not sufficient to cause a significant decrease in oxidative stress or an increase in -tocopherol concentrations. It was previously reported that plasma and tissue ascorbic acid concentrations are lower in diabetic patients and experimental animal models of diabetes than in healthy individuals (35,36). The lower concentrations are explained by a decreased cellular uptake of ascorbic acid due to competition from glucose, increased urinary excretion of ascorbic acid, and defective enzymatic regeneration of ascorbic acid from dehydroascorbate due to reduced concentrations of glutathione in the diabetic patients (37). Low ascorbic acid concentrations in the plasma and tissue of patients with diabetes could also result in impaired recycling of -tocopherol.
In conclusion, oxidative stress persisted after glycemic control with CSII alone in the Korean patients with type 2 diabetes in the present study; however, daily supplementation with 200 mg -tocopherol plus CSII formed a remarkable defense against free radicals and lipid peroxides without any significant changes in antioxidant enzyme activities. This improvement in oxidative stress after -tocopherol supplementation may have been due to antioxidant action by scavenging free radicals. Thus, -tocopherol supplementation may be beneficial in improving the complications of diabetes associated with increased oxidative stress in patients with type 2 diabetes treated with CSII.
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