Serum Retinol-binding Protein 4 Levels in Patients with Diabetic Retinopathy

The Journal of International Medical Research 2010; 38: 95 99 Serum Retinol-binding Protein 4 Levels in Patients with Diabetic Retinopathy Z-Z LI 1, X-Z LU 2, J-B LIU 1 AND L CHEN 1 1 Department of Endocrinology, Qilu Hospital, Shandong University, Jinan, China; 2 Department of Ophthalmology, Shandong Shierming Eye Hospital, Jinan, China Retinol-binding protein 4 (RBP4) has been reported to be involved in the development of insulin resistance and diabetes. This study was designed to investigate serum levels of RBP4 in patients with type 2 diabetes with and without diabetic retinopathy. Based on ophthalmological examination, 92 patients with type 2 diabetes were divided into three subgroups: those without diabetic retinopathy (NDR; n = 40); those with simple diabetic retinopathy (SDR; n = 37); and those with proliferative diabetic retinopathy (PDR; n = 15). The serum RBP4 level was significantly elevated in individuals with PDR compared with those with NDR or SDR. There was a significant positive correlation between serum RBP4 level and the urine albumin excretion rate (r = 0.219). This study showed that RBP4 may be involved in the process of diabetic retinopathy and may be a novel biomarker for its diagnosis and treatment in diabetic patients. KEY WORDS: TYPE 2 DIABETES MELLITUS; INSULIN RESISTANCE; DIABETIC RETINOPATHY; PROLIFERATIVE DIABETIC RETINOPATHY; RETINOL-BINDING PROTEIN 4 Introduction Diabetic retinopathy is a common microvascular complication of diabetes and a leading cause of adult vision loss and blindness. 1 Although progress has been made in the last decade in exploring its pathogenesis, the underlying molecular mechanisms of diabetic retinopathy remain unclear. Polyol accumulation, free radical damage and non-enzymatic glycation of proteins are believed to play a possible role in the pathogenic process. 2 Diabetic retinopathy is now recognized as a low-grade chronic inflammatory condition. 3 There are several inflammatory markers that may be involved in the pathogenesis of diabetic retinopathy. Retinol-binding protein 4 (RBP4), an adipokine, was predicted to be involved in the incidence and development of insulin resistance and diabetes. 4,5 Studies have shown that circulating RBP4 levels are higher in patients with diabetes than those without diabetes, 6,7 and that RBP4 levels correlate not only with indices of obesity and insulin resistance but also with inflammatory factors. 8 The hypothesis has been proposed that RBP4 is associated with the inflammatory process of diabetic retinopathy in type 2 diabetes and that RBP4 concentrations would be higher in diabetic individuals with retinopathy than in those without. The present study was designed to evaluate the serum RBP4 level in type 2 diabetic patients with or without diabetic retinopathy, and to evaluate the relationship between retinopathy stages and metabolic indices. 95

Patients and methods PATIENTS AND STUDY DESIGN In this observational study, patients with type 2 diabetes who were attending Qilu Hospital of Shandong University, Jinan, China, between February and June 2009 for treatment of their diabetes were sequentially enrolled. All patients underwent a complete ophthalmic examination, including ocular fundus and retinal fluorescein angiography performed by an experienced ophthalmologist (X-Z. L.). Based on the examination reports, the patients were divided into the following three groups: those with no diabetic retinopathy (NDR); those with simple diabetic retinopathy (SDR); and those with proliferative diabetic retinopathy (PDR). Patients with serious acute and chronic complications of diabetes, liver, kidney or heart failure or other severe diseases were excluded and no patients were included who were taking any medication that might have affected RBP4 levels. All patients underwent a complete physical examination, including blood pressure measurement and a routine biochemical analysis of their blood. The body mass index, waist and hip circumferences, and waist-to-hip ratio were calculated. The study protocol was approved by the Clinical Research Ethics Committee of Qilu Hospital, Shandong University, and written informed consent was obtained from all the patients. BIOCHEMICAL ASSAYS Blood was collected from each patient after an overnight fast. Serum was obtained by centrifugation and kept at 80 C for subsequent assays. Fasting blood glucose (FPG), 2-h blood glucose (PBG), total cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined by automatic biochemical analysis. Glycosylated haemoglobin (Hb A1c ) was detected by cation exchange column chromatography. Serum RBP4 concentrations were measured by enzyme immunoassay (EIA) using a RBP4 (human) EIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA) according to the manufacturer s instruction. The urinary albumin excretion rate (UAER) was analysed using immunoturbidimetry with an Array 360 automatic analyser (Beckman Coulter, Fullerton, CA, USA) and the mean was calculated from three consecutive 24-h urine samples. One set of blood samples was taken during this period. STATISTICAL ANALYSIS All analyses were carried out using the SPSS statistical package, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows. The mean ± SD was calculated for continuous variables. Each variable was examined for normal distribution and significantly skewed variables were log transformed. Comparisons of data among the three groups were performed using one-way analysis of variance. Comparisons between categorical variables were analysed using Pearson χ 2 -test. Correlations between variables were analysed by Pearson correlation analysis. All reported P-values were two-tailed and P < 0.05 was considered to be statistically significant. Results A total of 92 patients (46 men, 46 women) aged 33 82 years who had a history of type 2 diabetes of 1 25 years were enrolled in the study. Their demographic, physical and biochemical data after stratification into the three groups are shown in Table 1. Duration of diabetes was significantly longer in PDR 96

TABLE 1: Demographic, physical and biochemical characteristics of the 92 Chinese patients with type 2 diabetes stratified according to eye examination status: no diabetic retinopathy (NDR), simple diabetic retinopathy (SDR) and proliferative diabetic retinopathy (PDR) Characteristic NDR SDR PDR Male/female, n 21/19 17/20 8/7 Age (years) 56.20 ± 11.25 59.24 ± 11.30 62.53 ± 10.56 Duration of diabetes (years) 7.79 ± 5.72 10.52 ± 6.54 12.27 ± 5.76 a Body mass index (kg/m 2 ) 24.13 ± 2.67 24.13 ± 3.32 25.82 ± 3.40 Waist/hip ratio 0.92 ± 0.08 0.90 ± 0.07 0.93 ± 0.07 SBP (mmhg) 134 ± 18 142 ± 18 146 ± 31 DBP (mmhg) 77 ± 11 78 ± 13 77 ± 9 FBG (mmol/l) 8.82 ± 2.84 8.02 ± 2.55 8.62 ± 1.71 PBG (mmol/l) 11.12 ± 3.01 10.28 ± 3.02 10.99 ± 2.17 Hb A1c (%) 8.84 ± 2.18 8.29 ± 1.98 8.61 ± 1.33 TC (mmol/l) 5.76 ± 1.30 5.87 ± 1.14 5.90 ± 1.10 TG (mmol/l) 1.94 ± 1.13 1.63 ± 0.94 2.50 ± 1.50 b HDL-C (mmol/l) 1.35 ± 0.35 1.43 ± 0.39 1.18 ± 0.27 LDL-C (mmol/l) 3.39 ± 1.03 3.73 ± 1.31 3.84 ± 1.15 UAER (mg/day) 29.62 ± 38.86 52.56 ± 68.23 114.20 ± 73.89 c,d RBP4 (mg/l) 17.63 ± 5.05 16.89 ± 6.75 25.43 ± 5.70 c,d Data show mean ± SD unless stated otherwise. Comparisons of data among the three groups were performed using one-way analysis of variance and comparisons between categorical variables were analysed using Pearson s χ 2 -test: a P < 0.05 vs NDR; b P < 0.05 vs SDR; c P < 0.01 vs NDR; d P < 0.01 vs SDR. SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; PBG, 2-h blood glucose; Hb A1c, glycosylated haemoglobin; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; UAER, urine albumin excretion rate; RBP4, retinol-binding protein 4. patients than in NDR patients (P < 0.05). Levels of FBG, PBG and Hb A1c were similar and at a high level in all groups, which demonstrated poor metabolic control. Levels of TG were significantly higher in PDR patients than in SDR patients (P < 0.05). Levels of TC, HDL-C and LDL-C were similar in all groups. The UAER and serum RBP4 level were significantly higher in PDR patients than in either NDR or SDR patients (all P < 0.01). Correlation analyses showed a significant positive correlation between serum RBP4 levels and UAER (r = 0.219, P < 0.05). Age and HDL-C levels were also correlated with serum RBP4 (r = 0.428, P < 0.01; and r = 0.254, P < 0.05, respectively). Discussion The serum concentration of RBP4 was shown to be positively correlated with UAER in the present study, and a significantly higher level of RBP4 was observed in patients with PDR compared with patients with NDR or SDR, suggesting that RBP4 might be involved in the pathogenesis of diabetic retinopathy. Serum concentrations of RBP4 have been reported to be elevated in insulin-resistant individuals with obesity, impaired glucose tolerance and type 2 diabetes, and also in lean normoglycaemic subjects with a strong family history of type 2 diabetes. 4,5 Furthermore, serum RBP4 has been shown to be associated with variables related to adiposity, 9 insulin resistance 5 and metabolic syndrome. 10 It has 97

been suggested that RBP4 interferes with insulin signalling in skeletal muscle and liver, which results in insulin resistance and hyperglycaemia. 4,11 In the literature, however, there are some conflicting data related to RBP4 levels in diabetes. 12,13 These reported differences may be due to differences in methodology or research populations. Takebayashi et al. 14 reported a positive correlation of RBP4 levels with urinary albumin excretion, which is consistent with the current study. Raila et al. 15 reported that plasma RBP4 was increased in type 2 diabetic patients with microalbuminuria as compared with a normoalbuminuric group. These results imply that excretion or degradation of RBP4, together with other adipocytokines, may have been impaired from the early stages of diabetic nephropathy. Diabetic retinopathy and diabetic nephropathy are both diabetic microangiopathic conditions and have similar mechanisms of pathogenesis, including oxidative stress, the polyol pathway and inflammation, 16 factors which may also influence RBP4 levels. Further studies evaluating RBP4 as a biomarker of systemic insulin resistance and type 2 diabetes should, therefore, take renal function into consideration. Diabetic retinopathy is recognized as a lowgrade chronic inflammatory disease. 3 Chronic subclinical inflammation may underlie the vascular pathology of diabetic retinopathy. It is known, for example, that retinal expression of inflammatory factors is correlated with diabetic blood retinal barrier breakdown and ischaemia-related neovascularization. 17 As RBP4 levels are correlated with inflammatory factors, 8 it may be predicted that BRP4 could be involved in the inflammatory process of diabetic retinopathy. Although further studies are needed, we suggest that RBP4, together with numerous other factors, including adipokines and inflammatory factors, might be involved in the progression of eye lesions in patients with diabetes. Additionally, measuring RBP4 levels may be a novel method for evaluating the severity of diabetic retinopathy in patients with type 2 diabetes and RBP4 could become a helpful biomarker for the diagnosis and treatment of patients with diabetic retinopathy. Acknowledgements We thank the Key Laboratory of Cardiovascular Remodelling, the Function Research Centre for technical support and we are grateful to the Cryomedicine Laboratory, Qilu Hospital of Shandong University for their valuable cooperation. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 13 September 2009 Accepted subject to revision 23 September 2009 Revised accepted 20 December 2009 Copyright 2010 Field House Publishing LLP References 1 Aiello LP, Gardner TW, King GL, et al: Diabetic retinopathy. Diabetes Care 1998; 21: 143 156. 2 Yokoi M, Yamagishi SI, Takeuchi M, et al: Elevations of AGE and vascular endothelial growth factor with decreased total antioxidant status in the vitreous fluid of diabetic patients with retinopathy. Br J Ophthalmol 2005; 89: 673 675. 3 Adamis AP: Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol 2002; 86: 363 365. 4 Yang Q, Graham TE, Mody N, et al: Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 2005; 436: 356 362. 5 Graham TE, Yang Q, Bluher M, et al: Retinolbinding protein 4 and insulin resistance in 98

lean, obese, and diabetic subjects. N Engl J Med 2006; 354: 2552 2563. 6 Takebayashi K, Suetsugu M, Wakabayashi S, et al: Retinol binding protein-4 levels and clinical features of type 2 diabetes patients. J Clin Endocrinol Metab 2007; 92: 2712 2719. 7 Jia W, Wu H, Bao Y, et al: Association of serum retinol-binding protein 4 and visceral adiposity in Chinese subjects with and without type 2 diabetes. J Clin Endocrinol Metab 2007; 92: 3224 3229. 8 Balagopal P, Graham TE, Kahn BB, et al: Reduction of elevated serum retinol binding protein in obese children by lifestyle intervention: association with subclinical inflammation. J Clin Endocrinol Metab 2007; 92: 1971 1974. 9 Jia W, Wu H, Bao Y, et al: Association of serum retinol binding protein 4 and visceral adiposity in Chinese subjects with and without type 2 diabetes. J Clin Endocrinol Metab 2007; 92: 3224 3229. 10 Qi Q, Yu Z, Ye X, et al: Elevated retinol-binding protein 4 levels are associated with metabolic syndrome in Chinese people. J Clin Endocrinol Metab 2007; 92: 4827 4834. 11 Muoio DM, Newgard CB: Metabolism: A is for adipokine. Nature 2005; 436: 337 338. 12 Broch M, Vendrell J, Ricart W, et al: Circulating retinol-binding protein-4, insulin sensitivity, insulin secretion, and insulin disposition index in obese and nonobese subjects. Diabetes Care 2007; 30: 1802 1806. 13 von Eynatten M, Lepper PM, Liu D, et al: Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease. Diabetologia 2007; 50: 1930 1937. 14 Takebayashi K, Suetsugu M, Wakabayashi S, et al: Retinol binding protein-4 levels and clinical features of type 2 diabetes patients. J Clin Endocrinol Metab 2007; 92: 2712 2719. 15 Raila J, Henze A, Spranger J, et al: Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients. Kidney Int 2007; 72: 505 511. 16 Kalani M: The importance of endothelin-1 for microvascular dysfunction in diabetes. Vasc Health Risk Manag 2008; 4: 1061 1068. 17 Qaum T, Xu Q, Joussen AM, et al: VEGFinitiated blood retinal barrier breakdown in early diabetes. Invest Ophthalmol Vis Sci 2002; 42: 2408 2413. Author s address for correspondence Dr Li Chen Department of Endocrinology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, Jinan 250012, China. E-mail: qilul@yahoo.cn 99