Introduction

Obesity has become a disease reaching pandemic proportions and is a leading cause of increased morbidity and mortality rates worldwide. In particular, obesity is associated with type 2 diabetes mellitus (T2DM), cardiovascular disease, and certain forms of cancer (1). Adipose tissue—specifically visceral-adipose tissue—has been postulated to be a major factor contributing to insulin resistance, which is associated with both obesity and T2DM (2,3,4). In addition to its function as energy-storage depot, adipose tissue is also a highly metabolically active endocrine organ secreting a wide array of cytokines and thereby modulating glucose and lipid metabolism (5). Adipose tissue–derived retinol-binding protein 4 (RBP-4) was recently reported to be associated with insulin resistance and several other components of the metabolic syndrome (MetS) in subjects with obesity, impaired glucose tolerance, or T2DM (6). In obesity and T2DM, a major factor contributing to the impaired insulin-stimulated glucose transport in adipocytes is the downregulation of glucose transporter 4 (GLUT-4) (7). RBP-4 expression is upregulated in adipose tissue of adipose-GLUT-4 knockout mice and elevation of serum RBP-4 causes systemic insulin resistance, whereas reduction of serum RBP-4 levels improves insulin action (8). Thus, elevated serum RBP-4 levels might play a causative role in the development of systemic insulin resistance through decreased GLUT-4 expression in adipocytes. A recent study reported that serum RBP-4 levels were independently associated with visceral fat suggesting that visceral obesity is an independent predictor of serum RBP-4 levels (9). In addition, RBP-4 mRNA correlated inversely with GLUT-4 mRNA in visceral fat indicating that visceral fat may be a major source of RBP-4 in insulin-resistant states (10).

As pronounced weight loss after bariatric surgery results in considerable improvements in glucose and lipid metabolism (11), we investigated the effects of bariatric surgery on changes in visceral fat, parameters of the MetS, and their association with serum RBP-4 concentrations in a prospective study.

Methods and Procedures

Subjects

A total of 25 women and 11 men with a BMI >35 kg/m² and at least one comorbidity or a BMI >40 kg/m² participated voluntarily in this prospective study. The patients were recruited consecutively since the year 2002. Exclusion criteria included secondary causes of obesity, pregnancy, lipid lowering, or antipsychotic medication. Two patients were taking oral contraceptive medication, two patients (one without MetS, one with persisting MetS) were taking antihypertensive drugs, and three female subjects (one without MetS, one with persisting MetS, and one in who MetS resolved during weight loss) were receiving thyroid hormone replacement therapy. Drug doses were not modified in the course of the study. Diabetic patients were also excluded due to the insulinotropic nature of many antidiabetic drugs, which would interfere with the used homeostasis model assessment (HOMA) index (12). At study entry 19 patients met the criteria for a diagnosis of the MetS according to International Diabetes Federation (IDF) definition (13). The study subjects were examined within a 2-month period before the bariatric procedure and 2 years after the surgery. Patients were evaluated for either Swedish adjustable gastric banding or gastric bypass surgery by an algorithm based on esophageal body motility (14). For all the patients this was the first bariatric surgery, none of them were reoperated. The surgical procedures were performed at the Department of Surgery, Medical University Innsbruck as previously described (15).

Informed consent was obtained from all subjects. All procedures were performed in accordance with institutional guidelines of the Clinical Division of General Internal Medicine at the Medical University Innsbruck and the local ethical committee approved the study.

Body composition and ultrasound measurements

Body composition (lean mass, fat mass) was determined by impedance analysis using InBody 3.0 Body Composition Analyzer from Biospace Europe (Dietzenbach, Germany) with an integrated scale. Patients' height was measured to the nearest 0.1 cm and BMI was calculated as body weight in kilograms divided by height in meters squared using the software Lookin Body Version 1, Body Composition Analysis Data Management System.

Subcutaneous- (SFD) and visceral-fat diameter (VFD) were determined as described by Pontiroli et al. (16). A 3.0 MHz curved array transducer was placed along the xypho-umbilical line next to the umbilicus and visceral and subcutaneous fat were measured after smooth expiration. VFD was measured from the internal surface of the Musculus rectus abdominis to the near wall of the aorta. SFD was measured at the same position as the distance between the external surface of the muscle and the skin. The thickness of the muscle and skin were excluded. Measurements were performed in triplicates. All measurements were taken in the morning in the fasted state.

Analyses

Blood was drawn after an overnight fast from the antecubital vein into EDTA tubes (1.6 mg/ml). Plasma was separated from erythrocytes by centrifugation at 3,000 rpm for 10 min at 4 °C immediately after collection. Plasma samples were stored at –80 °C until assayed.

Plasma triglycerides, total cholesterol, and high-density lipoprotein cholesterol (HDL-C) were quantified using a commercially available enzymatic kit (Roche Diagnostic Systems, Basel, Switzerland). Low-density lipoprotein cholesterol was calculated using the Friedewald formula (17). Plasma glucose was measured by the hexokinase method on a Cobas MIRA analyzer. Plasma insulin was determined by a micro particle enzyme immunoassay from Abbott (Wiesbaden, Germany). Leptin was measured using an enzyme-linked immunosorbent assay kit (R&D Systems, Wiesbaden, Germany). C-reactive protein concentration was determined using the C-reactive protein (Latex) ultrasensitive assay (Roche Diagnostic Systems, Basel, Switzerland). Total-adiponectin levels were determined using a radioimmunoassay (Linco Research, St Charles, MO). Serum RBP-4 was measured using an enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH). Transferrin was measured using an immunoturbidimetric assay on a Modular analyzer (Roche Diagnostic Systems, Basel, Switzerland). The HOMA of insulin resistance (HOMA-IR) as an alternative method to assess insulin resistance based on known relationships between fasting glucose and serum insulin concentrations was calculated by the following formula: fasting serum insulin concentration (IU/ml) blood glucose concentration (mmol/l)/22.5 (ref. 18).

The MetS was diagnosed according to the IDF definition: central obesity (defined as waist circumference 94 cm for Caucasian men and 80 cm for Caucasian women) plus any two of the following parameters: triglyceride level 150 mg/dl or specific treatment for this lipid abnormality, HDL-C 50 mg/dl in females and HDL-C 40 mg/dl in males or specific treatment for this lipid abnormality, systolic blood pressure 130 mm Hg or diastolic blood pressure 85 mm Hg or treatment of previously diagnosed hypertension, raised fasting plasma glucose 100 mg/dl or previously diagnosed T2DM.

Statistical analyses

Data are expressed as mean s.d. The Shapiro–Wilk test was used to determine normal distribution of the data. As not all data were normally distributed, the Wilcoxon test for paired samples was used to determine significant changes before and after bariatric surgery. Mann–Whitney U-test was used for comparisons between groups. With regard to changes of RBP-4 levels between three groups defined by the absence, presence, or resolution of MetS during the study period, one-way ANOVA with Bonferroni correction for multiple comparisons was applied. To assess correlations between data, the Spearman rho-correlation coefficient was calculated. In addition, regression analyses were performed where applicable. Binary logistic regression was used to assess the significance of covariate-adjusted relations between percent changes in parameters and the diagnosis of MetS at follow-up. A two sided P value 0.05 was considered statistically significant. All analyses were performed using SPSS 13 for Windows (SPSS, Chicago, IL).

Results

Baseline characteristics and anthropometric measurements of the study population before and after surgery are given in Table 1. With regard to gender and the type of bariatric procedure performed (29 Swedish adjustable gastric banding, 7 gastric bypass), no significant differences of baseline, follow-up or changes in RBP-4 levels were observed.

Table 1 - Anthropometric and biochemical characteristics of the study population before and after surgery.

Full table (62K)

At study entry VFD was associated with weight (r = 0.348, P = 0.038), waist-to-hip ratio (WHR) (r = 0.834, P < 0.001), diastolic blood pressure (r = 0.382, P = 0.041), HOMA-IR (r = 0.430, P = 0.012), HDL-C (r = –0.580, P < 0.001), and leptin (r = –0.531, P = 0.001).

At follow-up, VFD was correlated with BMI (r = 0.493, P = 0.003), fat mass (r = 0.401, P = 0.017), WHR (r = 0.684, P < 0.001), HOMA-IR (r = 0.696, P < 0.001), HDL-C (r = 0.480, P = 0.004), adiponectin (r = 0.476, P = 0.008), and RBP-4 (r = 0.358, P = 0.035). RBP-4 was associated with waist (r = 0.370, P = 0.031), WHR (r = 0.583, P < 0.001), and HOMA-IR (r = 0.354, P = 0.043).

Two years after bariatric surgery mean weight loss amounted to 26.68 15.10 kg, corresponding to a BMI decrease of 9.07 4.93 kg/m². Loss in fat mass accounted for 76% of total weight loss. During the 2 years follow-up, VFD decreased by 60.6% and RBP-4 serum levels by 16.6% (Table 2). RBP-4 reduction was proportional to initial RBP-4 concentration (r = 0.716, P < 0.001). With regard to changes in parameters over time, RBP-4 was associated with WHR (r = 0.415, P = 0.016) and VFD (r = 0.424, P = 0.010) (Table 3). By stepwise multiple regression analysis, baseline HOMA-IR was the best predictor for changes in RBP-4 levels (r² = 0.217, P = 0.008). There were no gender-specific differences in changes of parameters.

Table 2 - Abdominal-fat distribution, adipocytokines, and inflammatory markers before and after surgery.

Full table (46K)

Table 3 - Cross-correlation matrix for changes in parameters of the metabolic syndrome, anthropometric measures, and adipocytokines.

Full table (28K)

According to IDF definition, the MetS was present in 19 patients at baseline (10 males, 9 females) and 9 patients at follow-up (7 males, 2 females). Sex, age, and total-fat mass–adjusted logistic regression analysis revealed that VFD and RBP-4 were significant predictors for the diagnosis of MetS at follow-up, when changes in BMI, WHR, VFD, HOMA-IR, leptin, adiponectin, and RBP-4 were used as independent variables. To investigate further the contribution of MetS to differences in RBP-4 concentrations, the subgroup of patients with MetS before surgery was compared with the subgroup of patients without MetS (Table 2). There were no significant baseline differences between the two groups, except for lean mass (P = 0.001), VFD (P = 0.023), and the parameters included in the IDF definition of the MetS.

In the group free of MetS, the fall in RBP-4 levels (–28.1% vs. –6.3%, P = 0.019) and decrease in VFD (–66.9% vs. –55.0%, P = 0.038) were significantly greater compared to the MetS group. In the MetS group, variation in RBP-4 levels was correlated with changes in WHR (r = 0.531, P = 0.023) and VFD (r = 0.505, P = 0.027), whereas no significant correlations were observed in the non-MetS group.

In the cohort of patients, who still had MetS at follow-up, RBP-4 levels did not significantly change (+2% 27.7, P = 0.441), in contrast to those patients in who MetS resolved after surgery (–13.7% 18.8, P = 0.037) (Figure 1).

Figure 1.

Retinol-binding protein 4 (RBP-4) reduction after bariatric surgery, split by diagnosis of metabolic syndrome. A, subgroup of patients who were free of metabolic syndrome at study entry (n = 17); B, subgroup of patients with metabolic syndrome at study entry, which resolved during weight loss (n = 10); C, subgroup of patients with metabolic syndrome at study entry, which persisted during weight loss (n = 9). Symbols indicate mean, error bars represent 95% confidence interval. n.s., not significant. *One-way ANOVA, Bonferroni adjustment applied.

Full figure and legend (8K)

Discussion

In this prospective study all subjects underwent bariatric surgery, which can serve as a model system of weight loss resulting in amelioration of most obesity-associated risk factors (19). During the 2 years after surgical intervention, patients lost an average of 51% of their excessive BMI. In addition, significant improvements in markers of insulin resistance and inflammation as well as a shift toward a more favorable lipid profile were observed. These observations are well in accordance with previously published data on bariatric surgery–induced changes in body composition, glucose, and lipid metabolism (1120,21,22).

Because of its unique anatomy and metabolic activity, the intra-abdominal visceral-fat deposition appears to play a key role in the development of insulin resistance (23,24,25). Therefore, we performed ultrasound measurements of the VFD and SFD to determine abdominal-fat distribution. The initial ratio of VFD to SFD was halved by the end of our study indicating a preferential mobilization of visceral-adipose tissue after bariatric surgery, which is commonly reported during long-term weight reduction (1626,27,28).

The peripheral hormone leptin, which adapts to changes in nutritional status (29), decreased significantly and was strongly correlated with loss of body weight, fat mass, SFD and improvements of insulin levels and HOMA-IR. This is in line with previous findings indicating that subcutaneous-adipose tissue is the most important source of circulating leptin (30,31,32). Conversely, weight loss after surgery was accompanied by a marked increase in adiponectin levels, which may contribute to the beneficial effect of weight loss on insulin resistance due to its insulin-sensitizing effect (33).

In insulin-resistant adipose specific GLUT-4 knockout mice, RBP-4 mRNA expression is increased in adipose tissue (8), but not in the liver, which is regarded as the major source of RBP-4 under physiologic conditions (34). Although subcutaneous-fat mass is greater than visceral-fat mass (35), we observed no correlation between RBP-4 levels and SFD. In contrast, reduction in VFD was associated with diminishing RBP-4 levels during pronounced weight loss. Consequently, visceral fat might contribute more to variation in RBP-4 levels in obese subjects than subcutaneous fat or the liver. This may be due to RBP-4 expression being higher in visceral-adipose tissue than in subcutaneous fat (10). Despite a notable number of reports describing a relationship of RBP-4 plasma levels and obesity or components of the MetS, these findings could not be confirmed by other studies (36,37,38). One possible explanation might be differences in age of the subjects investigated, as recent research indicates an influence of age on RBP-4 plasma levels (39,40). Gómez-Ambrosi et al. observed a decrease in RBP-4 levels only after gastric bypass surgery with concomitant pronounced reductions in total-fat mass, postulating that RBP-4 might be considered as a dynamic marker of negative energy balance (41). Decreasing RBP-4 levels being associated with malnutrition has also been shown in previous investigations (42). However, in our study, data from intermediate routine follow-ups showed that all but four patients were weight stable (<5% variation) within 3 months before follow-up. In addition, measurements of transferrin, which can also serve as a marker for malnutrition (42), were performed. Despite a significant decrease, baseline and follow-up values of all subjects were within the normal range and analysis revealed no association between RBP-4 and transferrin levels with regard to baseline, follow-up, or longitudinal measurements. These data on weight stability and the results of a marker for the metabolic status argue against the decrease of RBP-4 levels being due to a catabolic metabolic status in our patients.

Despite a longer observation period and greater weight loss in our study cohort, the decrease in RBP-4 serum levels was similar to that observed by another research group (43). However, patients free of MetS at baseline had a significant greater decrease of RBP-4 concentrations and reduction in VFD after surgery in comparison to the group with MetS. RBP-4 concentrations of some patients did not decrease after surgery despite significant reductions in BMI, fat mass, HOMA-IR, and VFD, and remarkably, MetS was still diagnosed in these patients at the end of the study. There was no single metabolic parameter in particular that was associated with the variations in RBP-4 levels during weight loss. Therefore, we hypothesize that RBP-4 could serve as a marker for identifying metabolically impaired patients, who are still at particular risk for the development of T2DM and cardiovascular disease after substantial weight loss, in contrast to metabolically healthy but obese individuals, who are protected or more resistant to the development of obesity-related metabolic disturbances (44). However, further studies in obese patients undergoing bariatric surgery are needed to investigate if the relationship between RBP-4 and the MetS is clinically meaningful as the extent to which RBP-4 contributes to the pathogenesis of insulin resistance and the MetS is not without controversy. Various other factors, i.e., free fatty acids released into the portal system by visceral fat, are likely to be involved in the development of these obesity-related complications. In addition, reports on RBP-4 contributing to systemic insulin resistance are ambiguous as several studies could not confirm the association between RBP-4 and insulin resistance as demonstrated by Graham et al. (6,36). Results from a recent study suggest that over-secretion of RBP-4 may negatively affect -cell function explaining the association between increased RBP-4 levels and T2DM (37). There is also some evidence that genetic variations in RBP-4 affect measures of insulin resistance, although the effect of increased fat mass on serum RBP-4 levels may outweigh the effect of genetic variants (45).

To our knowledge, the present results are the first to document the relationship between RBP-4 serum levels, visceral fat, and the MetS during weight loss in a prospective study. We demonstrate a marked decrease in RBP-4 levels after bariatric surgery, which correlates with a reduction in visceral-fat mass. Furthermore, the extent of changes in RBP-4 levels differs according to the severity of the MetS. These findings are compatible with the suggestion that RBP-4 may provide a mechanistic link between visceral-adipose tissue accumulation and the increased risk for metabolic complications.

Disclosure

The authors declared no conflict of interest.

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Acknowledgments

We thank Karin Salzmann for expert technical assistance.