Chinese Medical Journal 2012;125(10):1686-1689
Cigarette smoking increases levels of retinol-binding protein-4 in healthy men with normal glucose tolerance

Correspondence to:LI Ming,Department of Endocrinology, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China (Tel: 86-10-65295073. Fax:.
smoking; adipocytokines; retinol-binding protein-4; insulin resistance
Background  Smoking is related with insulin resistance and type 2 diabetes mellitus. Retinol-binding protein-4 is a new adipocytokine associated with insulin resistance. We investigated the serum levels of a series of adipocytokines including retinol-binding protein-4 in smokers and non-smokers to explore the possible roles of adipocytokines on smoking induced insulin resistance.
Methods  A total of 136 healthy male subjects (92 smokers and 44 non-smokers) with normal glucose tolerance were enrolled in the study. Adipocytokines including retinol-binding protein-4, visfatin, leptin, resistin, adiponectin were measured for the comparison between the two groups. Serum lipid profile, glucose, true insulin and proinsulin levels were measured as well in both groups. Food intake spectrum was also investigated.
Results  Both groups had similar profile of food consumption; visfatin, leptin, resistin and adiponectin, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase, as well as blood pressure and body mass index, were similar in both groups. Triglycerides, retinol-binding protein-4 and homeostatic model assessment index for insulin resistance were higher in smoker group ((2.58±2.53) vs. (1.60±0.94) mmol/L, (26.05±8.50) vs. (21.83±8.40) µg/ml, and 2.25±2.08 vs. 1.58±1.15, respectively).
Conclusion  Smoking may have effect on insulin sensitivity, which is correlated with retinol-binding protein-4.
Multiple clinical trials and research trials have revealed that smoking is correlated with cardiovascular disease and hyperlipidemia.1 In a study including 21 068 US male physicians aged 40 to 84 years, an association between cigarette smoking and the incidence of type 2 diabetes mellitus was documented. Smokers had a dose-dependent increased risk of developing type 2 diabetes mellitus.2
Adipose tissue is an endocrine organ secreting biologically active factors called adipocytokines that are thought to contribute to the development of insulin resistance, type 2 diabetes, and cardiovascular disease (CVD). Adiponectin is secreted exclusively by adipocytes in adipose tissue, and low levels in individuals had consistently predicted the presence of insulin resistance, type 2 diabetes and CVD risk. Leptin, resistin and visfatin are the other adipocytokines implicated in the pathogenesis of insulin resistance.3 Retinol binding protein 4 (RBP-4) is a recently identified adipocytokine that has caused a controversial scientific discussion on whether and how it links to adiposity, insulin resistance, and type 2 diabetes.4
Insulin resistance is one of the characteristics of diabetes and can be regulated by smoking. It was reported that smoking affected insulin sensitivity, which may be mediated by series of hormones, such as adipocytokines. Many studies have reported on hypoadiponectinemia and hyperleptinemia in smokers.5,6 However, the underlying mechanisms of this relationship are not yet fully understood. We aimed to investigate the profile of adipocytokines in smokers and non- smokers and to explore how insulin sensitivity is affected by smoking.
The healthy male subjects from outpatient clinic were collected during 2005 to 2006 in Beijing Chaoyang Hospital (Jingxi Branch). To exclude the influence of sex hormones, female was not included in the current study. Subjects with liver or kidney diseases were also excluded.
Study design
All subjects were screened with 75 g oral glucose tolerance test (OGTT). The subjects with normal glucose tolerance (NGT) were divided into two groups according to their smoking history as 92 smokers and 44 non-smokers. Being a smoker was defined as smoking cigarettes for at least 2 years, with more than 5 cigarettes per day. The average smoking history is 4.6 years. Only a “never-smoker” was accepted as a non-smoker. The age of non-smokers was (46.4±9.8) years; the age of smokers was (43.0±7.3) years. NGT is designated as following: fasting glucose <5.6 mmol/L and 2-hour blood glucose <7.8 mmol/L. Written informed consent for the procedures was obtained from all subjects.
Biochemical measurements
Regular food was taken 3 days before the test was taken, and the amount of carbohydrate was no less than 200 g, and calcium blocker was paused to avoid the effect on insulin secretion. High fat food was avoided in the previous supper, after fasting for 810 hours, blood was taken in the next morning, fasting blood glucose, fasting true insulin (FTI), fasting proinsulin (FPI), lipid such as triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c); liver function such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and kidney function were tested. Another set of blood samples were aliquoted and stored at –80°C until analysis for leptin, RBP-4, resistin and adiponectin. After bolus of 75 g of glucose, 2-hour blood glucose, true insulin and proinsulin were tested. Height, weight, waist circumference, hip circumference, and average of 3 measurements of blood pressure (both systolic blood pressure (SBP) and diastolic blood pressure (DBP)) were recorded. Life style and food intake spectrum were also investigated. Information on current cigarette smoking and alcohol use were assessed with standardized questionnaires. Insulin sensitivity was estimated using the homeostatic model assessment (HOMA) index (ie, HOMA-IR=FTI×FBG/22.5), body mass index (BMI) was calculated as weight (kg) divided by the square of height (m2). Glucose measurement was based on glucose oxidase method. Measurements of true insulin, proinsulin, and adipocytokines were based on enzyme-linked immunosorbent assay (ELISA) and performed in a single laboratory at the Department of Endocrinology in Peking Union Medical College Hospital.
Statistical analysis
Data were presented as means ± standard deviation (SD) for numeric variable. Continuous variables were compared between groups using student t test; categorical variables were compared using the chi squared (χ2) test. Linear regression analysis was used to determine correlation coefficients between various parameters. Prior to regression analysis, data were tested for normality of distribution by the Shapiro-Wilk test and consequently HOMA-IR, leptin, true insulin, proinsulin were Ln-transformed to obtain normal distributions. Partial correlation coefficient was also obtained by controlling for age, BMI and fasting glucose. All tests were two tailed and significance was determined as P <0.05. All calculations were performed using a standard statistical package (SPSS 11.5, SPSS Inc., USA).
Clinical and biomedical features of participants
A total of 136 male participants with NGT were included in this study, including 44 non-smokers and 92 smokers. As shown in Table 1, both groups have similar profile of food consumption and alcohol intake. The age of non-smoker is significantly higher than smoker ((46.4±9.8) years vs. (43.0±7.3) years, P <0.05). No significant difference was observed between the two groups in height, weight, waist circumference, hip circumference, BMI, SBP, and DBP. However, there is a significant increase in TG in smoking group ((2.58±2.53) vs. (1.60±0.94) mmol/L, P <0.01), and a slight decrease in HDL-c ((1.18±0.29) vs. (1.29±0.28) mmol/L, P=0.057). There is no difference was observed in TC and LDL-c.

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Table 1. Clinical and biochemical characteristics in healthy participants with or without smoking
Smoking on insulin sensitivity and insulin secretion
As shown in Table 2, after adjusted for age, BMI and FBG, smoking group has slightly higher levels of fasting (Ln) true insulin (2.03±0.67 vs. 1.76±0.61, P=0.024) and HOMA-IR (2.25±2.08 vs. 1.58±1.15, P=0.025). Therefore, after excluding subjects with impaired glucose tolerance and other abnormalities, we had shown that smoking actually worsens insulin resistance.

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Table 2. Effect of smoking on insulin resistance in healthy participants with smoking or without smoking
Smoking on serum leptin, visfatin, RBP-4, resistin and adiponectin
No significant difference was observed between smoker and non-smoker group in serum leptin, visfatin, resistin and adiponectin. In contrast, smoker group had a higher RBP-4 levels than non-smoker group ((26.05±8.50) µg/ml vs. (21.83±8.40) µg/ml, P <0.05), as shown in Table 3.

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Table 3. Levels of adipocytokines in healthy participants with smoking or without smoking
Correlations between RBP-4 and metabolic parameters
The serum RBP-4 level was positively correlated with TC, TG, LDL-c, AST, ALT, Ln2hTI and Ln2hPI (P <0.01 or P <0.05), as shown in Table 4.

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Table 4. Correlations between RBP-4 and metabolic parameters
Smoking has been viewed as one of the causes of death and disability worldwide; it is highly related with the development of series of abnormalities including cardiovascular disease and cancer. The effect of smoking on lipid metabolism has been revealed by many other studies. There are smoking related lipid metabolism disorders including increased TC, LDL-c, TG and decreased HDL-c.7,8 Cigarette smoking and its cessation are reported to alter lipid metabolism. It is well established that smoking stimulates lipolysis in vivo. The lipolytic effect of smoking has been attributed to the nicotine component being mediated via release of catecholamines.9 The results of our study show that smoking group has a high level of TG, but no significant differences were observed in TC, LDL-c and HDL-c levels.
It was reported that smoking is related with reduced insulin sensitivity.10 In this study, after excluding subjects with impaired glucose tolerance and other abnormalities, we also show that smoking actually worsens insulin resistance. Smoking affects lipid metabolism and insulin sensitivity, possibly through insulin signaling pathway. Among many mechanism raised by the researchers, it has been thought that adipocytokines such as adiponectin and leptin play a role in such a process. Circulating adiponectin levels are lower in smokers,5,8,9 smoking cessation is associated with increased plasma adiponectin levels,11 and in obese smokers and obese non-smokers, the leptin concentrations are significantly higher than in non-obese non-smokers.6 Circulating adiponectin levels are positively correlated with insulin sensitivity evaluated by using different insulin sensitivity techniques,12 and circulating leptin levels are negatively correlated with insulin sensitivity.13 But, we found that no significant difference was observed between smoker and non-smoker in serum leptin, visfatin, resistin and adiponectin; it may be explained by screening criteria and length of smoking, which can be explained by criteria of subject selection. Nevertheless, we found that smoker had a higher RBP-4 level than non-smoker.
Previous studies reported that plasma RBP-4 levels were elevated in human subjects with impaired glucose tolerance and type 2 diabetes,4 and age-associated difference in RBP-4 plasma levels was observed in woman but not in men.14 In this study, the age of non-smoker is slightly older than smoker. Since only male participants with NGT were included and even further adjustment for age was performed, we still found that smokers had elevated RBP-4 levels and insulin resistance. Moreover, the RBP-4 levels were also found to be related to various clinical parameters known to be associated with insulin resistance, such as serum lipids, true insulin and proinsulin. Thus we postulate that RBP-4 may play an important role in smoking related insulin resistance. Furthermore, smoking may directly mediated insulin sensitivity through RBP-4. RBP-4 is produced by adipocyte and reported to regulate insulin action pathway and play a role in development of insulin resistance in many tissue including adipocyte and muscles.15 RBP-4 increased hepatic gluconeogenesis by enhancing the expression of phosphoenolpyruvate carboxykinase in the liver and attenuated insulin signaling in skeletal muscle. The level of glucose transporter-4 (GLUT4) protein in human adipocytes correlated positively with the rate of glucose disposal and inversely with the serum RBP-4 level.4 Therefore, whether the role of RBP-4 on insulin resistance is primary or secondary to GLUT-4 still remains unclear; however, these results suggest that RBP-4 might serve as a novel link between cigarette smoking and insulin resistance.
In conclusion, association of tobacco smoking with insulin sensitivity and adipocytokines in male subjects with NGT was evaluated. Smoking is found to induce insulin resistance and RBP-4 is one of the key adipocytokines which may mediate this effect. Further studies with larger sample sizes in different populations are needed to confirm our findings, and the detailed underlying mechanism should be explored.
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