Relationship of plasma extracellular-superoxide dismutase level withinsulin resistance in type 2 diabetic patients

T Adachi, M Inoue1, H Hara, E Maehata2 and S Suzuki1

Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu 502-8585, Japan1Division of Endocrinology and Metabolism, Department of Internal Medicine, Showa University Fujigaoka Hospital, Yokohama, Kanagawa 227-8501, Japan2Central Clinical Laboratory, Mitsui Memorial Hospital, Tokyo 101-8643, Japan

(Requests for offprints should be addressed to T Adachi; Email: adachi@gifu-pu.ac.jp)

Abstract

Extracellular-superoxide dismutase (EC-SOD) is a se-cretory glycoprotein located in blood vessel walls at highlevels and may be important in the antioxidant capabilityof vascular walls. The aim of this study was to assess plasmalevels of EC-SOD and to evaluate the relationship of theEC-SOD level with insulin resistance in type 2 diabeticpatients. We determined plasma EC-SOD in 122 patientsand found for the first time that the EC-SOD level wasstrongly and positively related to adiponectin (r=0503,P

a proposed mediator of insulin resistance, overexpressed inadipose tissue and skeletal muscle of obese and type 2diabetic subjects (Iwata et al. 2001).

The present study was undertaken to clarify the re-lationship between the plasma EC-SOD level and indicesof insulin-resistance, and the change in the EC-SOD levelin insulin-resistant patients by administration of pioglita-zone, a thiazolidinedione (TZD) insulin-sensitizing drug.

Materials and methods

Study population

One hundred and twenty-two diabetic patients (82 men,average age 59988 years, and 40 women, average age584122 years) who showed a normal plasma creatininelevel of below 11 mg/dl and did not receive insulin orpioglitazone treatment were eligible for this study on therelationship between the plasma EC-SOD level andcharacteristics of insulin resistance. The study on thechange in the plasma EC-SOD level by pioglitazonetreatment included 19 diabetic patients (10 men and ninewomen). They showed higher diabetes/insulin resistance-parameters: for example, fasting plasma glucose (FPG) of888279 mM; hemoglobin A1c (HbA1c) of 8512%and homeostasis model assessment-insulin resistance index(HOMA-R) of 6787. The subjects were administered15 mg (female patients) or 30 mg (male patients) ofpioglitazone once daily. All the study subjects providedwritten informed consent according to the institutionalguidelines.

Laboratory measurements

Fasting blood samples were obtained. The EC-SODconcentrations in the plasma were determined by ELISA

described in our previous reports (Adachi et al. 1994)with minor modifications. The plasma adiponectinconcentration was assayed with an ELISA kit (OtsukaPharmaceutical, Tokyo, Japan). The plasma TNF-concentration was assayed with a Quantikine HSimmunoassay kit (R & D Systems, Minneapolis, MN,USA). Other characteristics were measured by standardclinical laboratory methods.

Statistical analysis

Data were analyzed with the t-test (Students t-test orWelchs t-test) and the Wilcoxon signed-rank test. Astepwise multiple-regression analysis was performed toassess the influence of variables on EC-SOD concen-trations. Variables were deleted from the regression analy-sis if the F value was not significant (,40) at any step ofthe calculations. A P value less than 005 was consideredsignificant.

Results

Plasma EC-SOD level in diabetic patients

We assayed the plasma EC-SOD level in 122 diabeticpatients (averageS.D. of EC-SOD level: 895275 ng/ml) and found the level was higher (P,0001) in females(1044332 ng/ml) than in males (819208 ng/ml).The plasma adiponectin level was lower in males than infemales, consistent with a previous report (Nishizawa et al.2002), whereas there were no significant gender differ-ences in FPG, HbA1c, immunoreactive insulin (IRI),body-mass index (BMI) and HOMA-R, as shown inTable 1. The plasma EC-SOD levels were significantlyand inversely related to FPG (r=0209, P=0022),

Table 1 Clinical characteristics of the study population

(A) Plasma concentration (B) Relation vs EC-SOD

Male Female Male Female Total

r F r F r F

EC-SOD (ng/ml) 819208 1044332**FPG (mM) 8220 8020 0072 0390 641 0209 459HbAlc (%) 7011 7113 0073 0210 0099 IRI (mU/ml) 8466 10274 0019 0445 763 0133 BMI (kg/m) 23322 24746 0174 0370 573 0187 HOMA-R 3131 3627 0072 0506 1095 0190 Adiponectin (g/ml) 6532 9151** 0380 133 0490 955 0503 3327TC (mg/dl) 1967307 2107363* 0117 0163 0048 HDL-C (mg/dl) 518105 549126 0020 0152 0107 LDL-C (mg/dl) 1242276 1316340 0135 0250 0129 TG (mg/dl) 1117544 1233666 0118 0148 0026

(A) Data are presented as meanS.D. Significant differences between males and females (*P

BMI (r=0187, P=0040) and HOMA-R (r=0190,P=0039), whereas they were strongly and positivelyrelated to adiponectin (r=0503, P

EC-SOD is the major SOD isozyme in extracellularfluids but is distributed mainly in the blood vessel wall(Strlin et al. 1995). In the vasculature, EC-SOD forms anequilibrium between the plasma phase and endothelial cellsurface (Karlsson & Marklund 1988), and the change inthe plasma EC-SOD level might reflect the changes in theexpression of EC-SOD and/or EC-SOD-tissue binding.In this study, parameters associated with diabetes/insulinresistance showed more significant relationships to EC-SOD in female subjects than in male subjects (Table 1). Ithas been reported that cardiovascular risk factors, includingmale sex, high BMI and smoking, decreased the plasmaEC-SOD level in an unselected middle-aged population(Marklund et al. 1997). The plasma levels of EC-SODmay be modulated by lifestyle and show a complexcovariation with many of the conventional cardiovascularrisk factors. From these observations, it is speculated thatthe cardiovascular risk factors obscure the relationshipbetween EC-SOD and parameters associated with insulinresistance, especially in male subjects.

The major observation indicated in Table 1 is thatplasma EC-SOD levels in diabetic patients were stronglypositively correlated with adiponectin, whereas they weresignificantly inversely related to parameters associated withinsulin resistance. Moreover, stepwise multiple-regressionanalyses showed that adiponectin especially highly affectsEC-SOD level. The plasma level of adiponectin has beenreported to relate inversely to BMI and HOMA-R (Hottaet al. 2000). The plasma EC-SOD level was also inverselyrelated to the above parameters, suggesting that factorsrelated to the pathogenesis of insulin resistance play animportant role in the regulation of the plasma EC-SOD.Increases in the plasma levels of EC-SOD and adiponectinby the pioglitazone treatment indicated in Fig. 2 wereconsistent with the above observations. It has beenreported that administration of pioglitazone normalized theoverexpression of TNF- and decreased the plasmaTNF- level in experimental animals (Hofmann et al.1994, Murase et al. 1998). We also observed that thepioglitazone treatment significantly decreased the plasmaTNF- level in diabetic patients, as shown in Fig. 2C. Themechanism by which this occurs is unclear; however,TNF- is known to suppress EC-SOD expression incultured cells (Marklund 1992, Strlin & Marklund 2000).From these results, it is speculated that the decline in theTNF- level by the pioglitazone treatment results in anincrease in EC-SOD expression in the vascular system.

In insulin-resistant patients, the decline in the activitiesof antioxidative enzymes (Cavarape et al. 2001) and theincrease in superoxide production (Yamagishi et al. 2001)may lead to the progression of atherosclerosis via theenhancement of LDL oxidation (Heinecke et al. 1986). Ithas been reported that exogenous addition of EC-SODand overexpression of EC-SOD prevented endothelialcell-mediated oxidative modification of LDL (Laukkanenet al. 2000, Takatsu et al. 2001). Moreover, mice lacking

EC-SOD were reported to be more prone to developdiabetes than wild-type controls (Sentman et al. 1999).The plasma TNF- level was reported to be higher inpatients with insulin resistance (Nilsson et al. 1998,Mishima et al. 2001) and correlated well with the degreeof atherosclerosis, as shown by factors such as LDL-cholesterol concentration (Skoog et al. 2002). In view of allthese findings, it appears that both a decrease in EC-SODexpression and a decrease in the capability of the endo-thelial cell surface to possess EC-SOD in the diabetes/insulin-resistant state probably lead to a decrease in thecapability of EC-SOD to protect the endothelial cellfunction, and may increase atherosclerotic development ininsulin-resistant patients. Increasing the EC-SOD expres-sion might be one approach to ameliorate the pathogenesisof atherosclerosis in these patients.

Acknowledgements

This work was supported in part by a Grant-in-Aidfor Scientific Research from the Japan Society for thePromotion of Science (to T A) and a grant for specificresearch from Gifu Pharmaceutical University (to T A)

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Received 26 December 2003Accepted 11 February 2004

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