Vaspin

5 mL whole blood samples is obtained from all of the normal subjects before their medical check-up (more precisely, it was after overnight fast), in tubes containing EDTA for adipokines and biochemical investigations. Blood samples were centrifuged at 1000g for 10 min. Plasma specimens were then frozen and stored at −80 °C until analysis. Human total adiponectin (R&D Systems, Minneapolis, USA) and vaspin (Adipogen, Seoul, South Korea) plasma levels were measured with ELISAs which had been reported previously [26 (link)]. Fasting insulin concentrations were measured with a commercially available ELISA immunoassay kit (ALPCO Diagnostics, Salem, NH, USA). White blood cell count, high-sensitive C-reactive protein (hsCRP), fasting plasma glucose (FPG), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and lipid profiles including total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), uric acid (UA) and creatinine (Cr) were measured by colorimetric enzymatic assay systems (Roche MODULAR P-800, Swiss Confederation).

Human aortic endothelial cells (HAECs) were obtained from Lonza Inc. (CC-2535, Walkersville, MD, USA) and maintained in endothelial basal medium (CC-3162, Lonza) supplemented with various growth factors that are required for the growth of endothelial cells and 2% fetal bovine serum at 37°C in a humidified incubator supplemented with 5% CO₂.
THP-1 cells, a human monocytic cell line (ATCC® TIB-202™, Rockville, MD, USA), were grown in RPMI-1640 medium containing 10% fetal bovine serum. In all experiments, cells were used at six or fewer passages.
TNFα (210-TA-020, R&D Systems, Inc. Minneapolis, MN, USA) was used as the representative proinflammatory cytokine and phosphate buffered saline was used as the vehicle. Cells were transferred to medium containing 1% fetal bovine serum and incubated in media containing 10 ng/mL TNFα for the indicated amount of time. Vaspin was obtained from Adipogen Inc. (AG-40A0064, Incheon, Korea) and phosphate buffered saline was used as the vehicle. Cells were transferred to medium containing 1% fetal bovine serum and incubated in media containing various concentrations of Vaspin for the indicated amount of time before treatment with TNFα. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (123040, Calbiochem, Darmstadt, Germany), an activator of AMPK, was used as a positive control.

Blood samples were centrifuged at 1000 g for 10 minutes. Plasma specimens were then frozen and stored at −80°C until analysis. Human total Adiponectin (R&D Systems, Minneapolis, American) and Vaspin (Adipogen, Seoul, South Korea) plasma levels were measured with commercially available ELISAs as indicated before [9 (link)]. While high-sensitive C-reactive protein (hsCRP), fibrinogen (FIB), creatinine (Cr), blood urea nitrogen (BUN), uric acid (UA), fasting blood glucose (FBG), 2-hour postprandial glucose (PG2h), hemoglobin A1c (HbA1c), and lipid profiles, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (ApoA), and apolipoprotein B (ApoB) were measured by colorimetric enzymatic assay systems (Roche MODULAR P-800, Swiss Confederation).