New adipokines vaspin and omentin. Circulating levels and gene expression in adipose tissue from morbidly obese women

The study was approved by the institutional review board. All participants gave written informed consent for participation in medical research. We analyzed the circulating levels of vaspin and omentin in 71 Spanish women of European descent: 31 lean (BMI < 25 Kg/m2) and 40 morbidly obese (BMI > 40). We also analyzed vaspin and omentin gene expression in paired samples of subcutaneous and visceral adipose tissue from 46 patients: 6 lean (BMI < 25 Kg/m2) and 40 morbidly obese (BM I > 40). Adipose tissue samples were obtained from morbidly obese women and from control women who underwent bariatric surgery by laparoscopic gastric by-pass and elective surgery, respectively. Subcutaneous adipose tissue biopsies were taken from the right hypocondrion region and visceral adipose tissue biopsies were taken from the greater epiploon region. For each type of surgery, samples were obtained by the same specialist. Morbidly obese women and controls were age matched. The weight of all subjects was stable for at least three months before surgery. Those patients who had an acute illness, acute or chronic inflammatory or infective diseases, or an end-stage malignant disease, were excluded from. Liver and renal diseases were specifically excluded by biochemical work-up. Control or morbidly obese patients diagnosed as type 2 diabetes mellitus or receiving hypolipemiant treatment were also excluded from the study.

BMI was calculated as weight divided by height squared (kg/m²) (according to the criteria of the World Health Organization [21]. Waist circumference (WC) was measured at the height of the iliac crest.

Morbidly obese women were further subclassified according to the presence or absence of the metabolic syndrome (MS). The MS and metabolic risks are defined according to the US National Cholesterol Education Program Adult Treatment Panel III guidelines and modified as recommended in the latest American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement [22] by adopting a lower cutoff for fasting glucose (5.6 mmol/L). The MS was defined as having 3 of the following metabolic risk factors: (1) central obesity (waist circumference 88 cm in women), (2) hypertriglyceridemia (fasting triglycerides 1.69 mmol/L (150 mg/dL)), (3) low HDL cholesterol (fasting HDL <1.29 mmol/L (50 mg/dl) in women), (4) glucose intolerance (fasting glucose 5.6 mmol/L (100 mg/dL)), and (5) hypertension (sitting blood pressure 130/85 mm Hg obtained as a mean of two readings taken after resting for at least 10 minutes or on regular antihypertensive medications).

Basal, fasting blood samples were taken after an overnight fast to determine glucose, insulin, and standard laboratory parameters. Plasma and serum samples were stored at -80°C until analytical measurements were performed, except for glucose, which was determined immediately after blood was drawn.

Fasting plasma glucose and lipid profile (triglycerides, total cholesterol, and high-density lipoprotein cholesterol) were measured using the usual enzymatic methods in an ADVIA Centaur auto analyzer. Low-density lipoprotein cholesterol was calculated as the difference between total cholesterol, HDL, and triglyceride content/5 if it was <400 mg/dL. Plasma insulin concentration was measured by commercial chemiluminescence assay for ADVIA Centaur (Siemens Medical Solutions S.L., Barcelona, Spain) according to the manufacturer's instructions. The homeostasis model assessment of insulin resistance (HOMA2-IR) was completed using the HOMA Calculator version 2.2.2 (http://​www.​dtu.​ox.​ac.​uk accessed May 2010). Glycosylated hemoglobin (HbA1c) was measured by a chromatographic method (Glico Hb Quick Column Procedure, Helena Laboratories, Beaumont, TX).

Circulating levels of TNF-RI, TNF-RII (Biosource Europe S.A., Nivelles, Belgium), IL 6 (Quantikine, R&D Systems, Minneapolis, USA), adiponectin, HMW adiponectin (Linco Research, Inc., St. Charles, USA), resistin (Biovendor, Modrice, Czech Republic), leptin (Biovendor, Modrice, Czech Republic), RBP4, lipocalin 2 (Biovendor, Modrice, Czech Republic), serum vaspin (Adipogen, Seoul, South Korea) and plasma omentin (Apotech, Axxora, Nottingham, UK) were measured in duplicate using enzyme-linked immunosorbent assays (ELISA) following the manufacturer's instructions. TNF-RI assay sensitivity was 50 pg/mL and the inter-assay and intra-assay coefficients of variation were less than 5.7 and 1.7%, respectively. TNF-RII assay sensitivity was 0.1 ng/mL and inter-assay and intra-assay coefficients of variation were less than 3.2 and 3.3%, respectively. IL 6 assay sensitivity was 0.039 pg/mL and inter-assay and intra-assay coefficients of variation were less than 9.6 and 6.9%, respectively. Adiponectin assay sensitivity was 0.78 ng/mL and inter-assay and intra-assay coefficients of variation were less than 8.4 and 7.4%, respectively. HMW adiponectin assay sensitivity was 0.5 ng/mL and inter-assay and intra-assay coefficients of variation were less than 3.8 and 2.6%, respectively. Resistin assay sensitivity was 33 pg/mL and inter-assay and intra-assay coefficients of variation were less than 6.9 and 3.4%, respectively. Leptin assay sensitivity was 0.2 ng/mL and inter-assay and intra-assay coefficients of variation were less than 7.6 and 4.4%, respectively. RBP4 assay sensitivity was 0.02 g/L and inter-assay and intra-assay coefficients of variation were less than 1.1 and 2.2%, respectively. LCN 2 assay sensitivity was 0.01 ng/mL and inter-assay and intra-assay coefficients of variation were less than 5.6 and 4.4%, respectively. Vaspin assay sensitivity was 12 pg/mL and inter-assay and intra-assay coefficients of variation were <6% and <4%. Omentin assay sensitivity was 0.4 ng/mL and inter-assay and intra-assay coefficients of variation were less than 6 and 2.7%, respectively.

Total RNA was isolated from adipose tissues with the RNeasy mini kit (Qiagen) according to the manufacturer's protocol and digested with DNase I (RNase-Free DNase set, Qiagen). RNA quality was evaluated by measuring the 260/280 nm absorbance ratio (≥1.8) and by electrophoresis. First-strand cDNA was synthesized using an equal amount of total RNA with the High Capacity RNA-to-cDNA Kit (Applied Biosystems). Real-time quantitative PCR was performed in a final volume of 20 μL, which contained 10 ng of reverse-transcribed cDNA, 10 μL of 2X Taq Man Fast Universal PCR Master Mix (Applied Biosystems) and 1 μL Taq Man Assay predesigned by Applied Biosystems^® for the detection of vaspin, omentin, and GAPDH, used as a housekeeping gene. All reactions were performed in triplicate and were carried out in 96-well plates by using the 7900HT Fast Real-Time PCR systems (Applied Biosystems).

All the values reported are expressed as mean ± SEM (standard error of the mean) and were analyzed using the statistical package SPSS/PC+ for Windows (v.15.0 Chicago, Illinois, USA). Differences between groups were calculated using either Student's t test or the One-way ANOVA analysis. The strength of association between variables was calculated using Pearson's method for parametric variables and the Spearman Rho correlation test for non-parametric contrasts. Multiple linear regression analysis with backward variable selection was performed to identify independent predictors of HOMA2-IR. The validity of the regression model and its assumptions was assessed with the plot of residuals vs. predicted values. Data were normally distributed. Logistic regression analysis was performed to identify independent predictors of the metabolic syndrome. Vaspin and omentin circulating levels were age and BMI adjusted in some analyses. P values < 0.05 were considered to be statistically significant.

The baseline patient characteristics given in Table 1 show the mean and SEM of the variables of interest. Patients were separated into control subjects (BMI < 25 kg/m²), and morbid subjects (BMI > 40 kg/m²). The two groups were well matched for age.

Biochemical analyses indicated that obese women had significantly higher levels of glucose, insulin, HOMA2-IR and HbA1c than the control group. Blood pressure was also increased in the morbidly obese women. The lipidemic profile differed significantly between groups. Obese patients showed higher triglyceride levels and lower HDL cholesterol.

Mean ± SD serum vaspin was 0.87 ± 0.96 μg/liter in the morbidly obese women and not significantly different from the values of the control group (1.66 ± 2.09 μg/liter) (Figure 1).

Serum vaspin concentrations did not correlate with metabolic variables (Table 2) and inversely correlated with levels of LCN2, leptin and IL 6 (Table 3).

We investigated the relationship between vaspin circulating levels and the presence of the MS but we found no correlation (Table 4). As expected, the MS correlated with the BMI, WC, HOMA2-IR, fasting glucose, blood pressure, HDL cholesterol and triglycerides.

Vaspin mRNA expression was significantly higher in morbidly obese women than in controls, in both SAT and VAT (Figure 2). We found no differences between visceral and subcutaneous vaspin expression in the adipose tissues of the obese group (p = 0.192) and the control group (p = 0.934) (Figure 2).

SAT and VAT vaspin mRNA expression did not correlate with obesity, glucose metabolism, insulin resistance parameters, blood pressure, other adipokines studied or inflammatory parameters (data not shown).

Mean ± SD plasma omentin was 1.97 ± 2.15 ng/mL in the morbidly obese group and significantly different from that of the control group (5.27 ± 5.33 ng/mL) (Figure 1).

Plasma omentin levels inversely correlated with fasting glucose and HOMA2-IR (Table 2) and did not correlate with the adipokines or cytokines studied (Table 3). When we investigated the relationship between omentin circulating levels and the presence of the MS, we found that omentin correlated negatively with the MS (Table 4). After adjustment for age and BMI, we subclassified the subjects in three tertiles in accordance with their omentin levels: tertile 1 (>4.33 ng/ml); tertile 2 (4.33-2.30 ng/ml); tertile 3 (<2.30 ng/ml). In a logistic regression analysis, the lowest omentin levels (tertile 2 and 3) were associated with the presence of MS while the highest tertile (tertile 1) was not (Table 5).

Omentin mRNA expression in SAT was similar in morbidly obese women and controls. However, omentin expression in VAT was significantly lower in morbidly obese women than in controls (Figure 2). When comparing both adipose tissues, we found that omentin expression was higher in VAT than in SAT in both the obese group (p = 0.009) and the control group (p = 0.034) (Figure 2).

SAT and VAT omentin mRNA expression did not correlate with obesity, glucose metabolism or insulin resistance parameters. Neither was there any correlation with lipid metabolism, blood pressure or the other adipokines and cytokines studied (data not shown).