Enzyme linked immunosorbent assay

Samples were collected in the fasting state. Triglycerides and LDL‐C were measured on fresh serum samples using traditional enzymatic methods. Non‐HDL‐C was calculated as total cholesterol minus HDL‐C. All other measurements of apolipoproteins and lipoprotein particle subclasses (triglyceride‐rich lipoprotein particles [TRLP], LDLP and high‐density lipoprotein particles [HDLP]) were performed on available frozen plasma EDTA or serum samples from the modified intent‐to‐treat population (mITT) population. Lipoprotein particle concentration and average lipoprotein particle size were measured by nuclear magnetic resonance (NMR) spectroscopy at baseline and 26 weeks (LabCorp, Burlington, NC, USA). The lipoprotein insulin resistance (LPIR) score, a weighted combination of six lipoprotein subclass measures (large TRLP, large HDLP, small LDLP and mean sizes of TRLP, LDLP and HDLP), was also calculated.²⁷ Preheparin serum LPL mass was measured by enzyme‐linked immunosorbent assay (ALPCO, Salem, NH, USA). ApoA‐I, apoB (Roche, Indianapolis, IN, USA) and apoC‐III (Kamiya, Seattle, WA, USA) were measured by immunoturbidimetry at baseline and at 4, 12 and 26 weeks.

The protocols used in this study were reviewed and approved by the Animal Experimentation and Ethics Committee of the Catholic University of Daegu (Gyeongsan, South Korea). A total of 103 C57/BL/6 male mice were used in this study, with 7–10 mice in each group. Blood and abdominal SAT samples were obtained from mice sacrificed at various time points after feeding a normal diet (ND) or a HFD from the age of 6 weeks until the age of 16, 26, 36, 47, or 77 weeks (Jeong et al. 2014 (link); Kim et al. 2014 (link)). In addition, blood and tissue samples were collected from obese mice with increased insulin sensitivity by thiazolidinedione (TZD) treatment. Blood samples collected by cardiac puncture from sacrificed mice were centrifuged at 2400 × g for 15 min at 4°C, and the plasma obtained was additionally centrifuged at 12,500 × g for 15 min. Non-fasting plasma concentrations of glucose and insulin were measured by glucometer and enzyme-linked immunosorbent assay (ALPCO Diagnostic, Salem, NH), respectively (Jeong et al. 2014 (link); Kim et al. 2014 (link)).

Thirty minutes before anesthesia (isoflurane induction: 4.0% in 1:1 O₂/air mix; maintenance: 2.0% in 1:1 O₂/air mix), rats received an injection of analgesia (buprenorphine; 0.1 mg/kg subcutaneously). Bilateral flank incisions were performed and the kidneys were approached retroperitoneally. The renal arteries and veins were stripped from the adventitia. All visible renal nerve bundles were cut under a dissection microscope (25X) and the vessels were coated with a solution of 10% phenol in ethanol (24) (link). In the sham group, the same surgical procedure was performed, but renal nerves remained intact and no phenol solution was used. Analgesic (carprofen; 4.0 mg/kg subcutaneously) was used directly after surgery, and 24 h and 48 h after the procedure. The animals recovered from the surgery within 1 week. The efficacy of this procedure was evaluated by measuring renal tissue norepinephrine content, assessed by enzyme-linked immunosorbent assay (Alpco Diagnostics, Salem, New Hampshire, No 17-NORHU-E01-RES), and normalized to protein concentration.