The study approved by the local Institutional Review Board was conducted at the Vanderbilt University Medical Center. Participants were enrolled among subjects status post GB surgery (with a least 12 months follow up) undergoing an elective abdominal surgical procedure (e.g. cholecystectomy, abdominal wall hernia repair, and/or abdominoplasty), subjects with class II and III obesity undergoing GB surgery, and non-obese (BMI: 20–29) control subjects requiring an elective abdominal surgery.
Obese and normal weight controls were selected to match age distribution of the post-GB group. The study employed the following exclusion criteria: diagnosis of neoplastic disease, inflammatory bowel disease, acute cholecystitis, and pregnancy.
During the surgical procedure, immediately after entering the abdominal cavity, two <0.5-cm3
biopsies of abdominal subcutaneous and intra-abdominal omental fat tissue were obtained. The tissue samples were washed in PBS solution and then soaked in RNAlater preservative solution (Qiagen, Courtaboeuf, France) and stored at −80°C until analysis. All the samples were stored in the same −80°C freezer and then analyzed together. Total RNA was extracted from the adipose tissue biopsies using the RNeasy total RNA Mini kit (Qiagen). The integrity of total RNA was checked by electrophoresis through an agarose gel. Adiponectin gene expression was studied by quantitative, real-time RT-PCR using the specific protocol for the iCycler iQ Detection System (BioRad, Hercules, CA, USA) with SYBR green fluorophore. Reactions were performed in a total volume of 20μL—including 10μL 2x SYBR Green PCR Master Mix (Applied Biosystems), 5μL of each primer at 5μM concentration, and 1μL of the previously reverse-transcribed cDNA template. The PCR primer sequence for adiponectin and GAPDH was chosen based on the sequences available in GenBank (www.ncbi.nlm.nih.gov/Genbank
). Melting curves were used to determine the specificity of the gene products, which was subsequently confirmed by running the PCR products on agarose gels. The threshold cycle (CT) value for each reaction, reflecting the amount of PCR needed to identify a target gene was calculated. Specimens were run in duplicate and the CT values averaged. GAPDH was used as internal control housekeeping gene to normalize the PCRs for the amount of RNA added to the reverse transcription reactions. The data was analyzed using the 2−ΔΔCT
] where ΔΔCT = (CTadiponectin
that allows to determine the fold change in the target gene in the omentum compared to subcutaneous fat from the same subject.
A fasting blood sample was also obtained from each subjects immediately before the induction of general anesthesia and immediately spun in a refrigerated (4°C) centrifuge at 3,000 rpm for 10 minutes. The extracted plasma was used for glucose and insulin level determinations. Plasma glucose concentration was determined by the glucose oxidase method. Immunoreactive insulin was determined in plasma with a double-antibody system. Insulin resistance was assessed by the homeostatic model assessment (HOMA): [fasting plasma glucose (mmol/l) × fasting insulin (μU/ml)/22.5]. The extracted plasma was used for adiponectin determinations by high-sensitivity enzymatic assay (Millipore, Inc.).
Cryopreserved human subcutaneous adipose stem cells (hASCs) purchased from Zen-Bio, (Zen-Bio Inc., RTP, NC) were maintained and differentiated according to the supplier’s specifications. Differentiated adipocytes maintained in AM-1 medium (Zen- Bio Inc., RTP, NC) were exposed for 24 hours to 5, 10 and 100 ng/ml of des-(1–3)-IGF-I (des-IGF-I) (Diagnostic System Laboratories), then harvested by Trizol reagent (Invitrogen Corp., Carlsbad, CA).
Comparisons of continuous variables between the groups were completed by unpaired Student’s t tests. Categorical variables were compared using the chi-square test or Fisher’s exact test. Time course experiments were analyzed by ANOVA. The SPSS statistical software program (version 17.0, SPSS, Chicago, USA) was used for all analyses. All tests were 2-tail. P values of less than 0.05 were considered to indicate statistical significance.