Enzyme-linked immunosorbent assay for circulating human resistin: resistin concentrations in normal subjects and patients with type 2 diabetes

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Abstract

Background: Resistin is a recently identified adipocyte-secreted hormone in rodents, and has been proposed to serve as a link between obesity and insulin resistance. The aim of this study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for human resistin and evaluate serum resistin concentrations in normal subjects and patients with type 2 diabetes. Methods: Using ELISA developed by two polyclonal antibodies, resistin concentrations were measured in 90 patients with type 2 diabetes and compared to 74 healthy control subjects. Results: This ELISA has high specificity and sensitivity over the concentration of range 0.5–100 ng/ml with good percentage recovery (97.1±4.7%) and reproducibility (within-day assay, CV=4.8–8.6%; between-day assay, CV=5.6–9.7%). The mean concentration of resistin in sera from type 2 diabetic patients was significantly higher than that in normal subjects (mean±S.E.: 20.8±0.7 vs. 14.9±0.5 ng/ml, p<0.001). A moderate positive correlation was observed between serum resistin levels and body mass indices in both normal subjects (r=0.412, p<0.0003) and patients with type 2 diabetes (r=0.395, p<0.0001). Conclusions: Our ELISA will be useful to confirm the physiological and pathophysiological role of resistin in humans.

Introduction

Type 2 diabetes is characterized by insulin resistance in insulin target tissues and impaired insulin secretion from pancreatic β cells. Obesity is an important risk factor for the development of insulin resistance [1]. Adipocytes are known to secrete a variety of mediators, including tumor necrosis factor α, leptin, free fatty acids and adiponectin that can influence the ability of the body to respond to insulin and metabolize glucose [1], [2], [3], [4], [5].

Resistin belongs to a family of cysteine-rich C-terminal domain proteins known as resistin-like molecules that are also termed FIZZs [6], [7]. Steppan et al. [7] demonstrated that serum resistin levels are significantly increased in both genetic (ob/ob and db/db) and high fat diet-induced obese mice, and that administration of recombinant resistin impaired glucose tolerance and insulin action in normal mice. Their study also showed that immunoneutralization of circulating resistin in obese mice reduced hyperglycemia and insulin resistance, and that resistin expression in mice and 3T3-L1 adipocytes was down-regulated by rosiglitazone, an insulin-sensitizing drug that is an agonist of the peroxisome proliferator-activated receptor-γ. Based on these observations, it was proposed that resistin may be an important link between obesity and insulin resistance in type 2 diabetes. However, recent human studies have cast doubt on the relevance of the rodent data to human obesity or insulin resistance and studies of resistin gene expressions have provided inconsistent results [8], [9], [10], [11], [12]. In addition, there is only limited information on the levels of resistin in human serum. It remains unclear whether resistin is a major factor in the development of insulin resistance-associated obesity. Therefore, a specific and sensitive method to quantitate circulating serum resistin concentrations is required for physiological and clinical studies. In the present study, we established an enzyme-linked immunosorbent assay (ELISA) system for human resistin and measured serum resistin levels in normal subjects and patients with type 2 diabetes.

Section snippets

Human subjects

Serum samples were taken from 90 patients with type 2 diabetes who fulfilled the World Health Organization criteria for diabetes mellitus [13] (43 males and 47 females; mean age±S.E. 53.8±0.8 years; mean body mass index (BMI) 23.4±0.9 kg/m2) and 74 normal subjects (36 males and 38 females; mean age 53.4±0.7 years; mean BMI 23.2±0.9 kg/m2). Patients with type 2 diabetes who were receiving insulin, thiazolidinediones or metformin therapy were excluded from this study. The clinical study protocol

Analytical evaluation

We analysed resistin levels in the adipose tissue, mononuclear cell preparation and serum samples by Western blot analysis (Fig. 1). Molecular weights of the protein bands reactive with the anti-recombinant human resistin IgG were calibrated with standard proteins. Anti-resistin IgG reacted strongly with the recombinant human resistin (9.75 kDa: lane 1). Notably, immunoreactive resistin was also detectable in the adipose tissue, mononuclear cell preparations and serum samples as observed by the

Discussion

Steppan et al. [7] reported that resistin is a signaling molecule that is induced during adipogenesis and secreted by adipocytes, and is a candidate of the adipocyte-derived factor that contributes to insulin resistance in vivo. It has not been proved yet whether association of resistin with obesity in diabetes mellitus of type 2 holds true in humans. Immunoreactive resistin is detectable in serum from normal mice and rats [7]. A simple and sensitive assay system for human resistin is required

Acknowledgements

This work was supported in part by the Kobe Pharmaceutical University Collaboration Fund and Science Research Promotion Fund of the Japan Private School Promotion Foundation.

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