Colorimetric assay

At each time point, blood was collected in a vacutainer treated with EDTA (BD Vacutainer^®) and placed on ice immediately for later glucose analysis via the glucose oxidase method using a colorimetric assay (Cayman Chemical Company, Ann Arbor, MI, USA). In addition, EDTA tubes were treated with diprotinin-A (0.034 g/L blood; MP Biomedicals), sigma protease inhibitor (1 g/L blood; Sigma-Aldrich, St. Louis, MO, USA) and Roche Pefabloc (1 g/L of blood) for analysis of gut and metabolic hormones. After blood collection, samples were centrifuged and the supernatant was collected and frozen at −80 °C for later determination. Metabolic-related hormones were simultaneously quantified by a multiplex assay (Millipore, St. Charles, MO, USA) according to the manufacturer’s protocol. Analytes included C-Peptide, ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) (active), glucagon, insulin, leptin and pancreatic polypeptide YY (PYY) (total). The assay sensitivities of these markers ranged from 0.6–87 pg/mL.

For the lipogenesis assay, mature adipocytes from iWAT and 3T3-L1 adipocytes were isolated to measure lipid accumulation by a colorimetric assay (Cayman Chemical Company, MI, USA)^{51 (link)}. Approximately 75 μL of Lipid Droplets Assay Fixative (Cayman Chemical Company, MI, USA) was added to each well and incubated for 15 min. Wells were washed with a wash solution twice for 5 min each and left to dry completely by placing the plate under a blowing hood. Dye extraction solution was added, the wells were gently mixed for 20 min and the degree of lipogenesis was quantified from lipid droplets in cells by obtaining the absorbance at a single fixed wavelength of 490 nm with a microplate reader. For the lipolysis assay, isolated adipocytes from iWAT were incubated in phenol red-free DMEM supplemented with 2% fatty acid-free bovine serum albumin (BSA) for the indicated time at 37 °C in the presence or absence of 10 μm isoproterenol. The glycerol content in the medium was measured colorimetrically at 540 nm according to the instructions of the Lipolysis colorimetric assay Kit (Sigma-Aldrich, USA) against a set of glycerol standards. The cells were then washed with ice-cold PBS and lysed in 1% Triton X-100 buffer, and the protein concentration was determined and used to normalise glycerol release. All experiments were carried out in triplicate.

Lipid peroxidation was assessed using thiobarbituric acid-reactive substances (TBARS) as indicators, following Mihara and Uchiyama [70 (link)]. Advanced oxidation protein products (AOPP) were determined spectrophotometrically according to Barsotti et al. [71 (link)], and nitric oxide species (NOx) were monitored by a colorimetric assay (Cayman Chemical, Ann Arbor, MI, USA) as described by Castillo et al. [72 (link)]. A detailed description of all these methods for measuring oxidant activity in the brain can be found in Coimbra-Costa et al. [28 (link)].

Hepatic lipid content was measured from liver homogenates as described previously^{54 (link)}. Briefly, frozen liver samples (~50 mg) were homogenized in 1.5 ml of 40 mM Tris-HCl buffer (pH 7.4) on ice and centrifuged at 12,000 rpm for 15 minutes at 4 °C. Cholesterol levels were measured using the Infinity Cholesterol Reagent (Thermo Scientific, Middletown, VA) and triglyceride levels were measured by a colorimetric assay (Cayman Chemicals, Ann Arbor, MI). Protein concentration was determined using the BCA assay kit (Thermo Scientific, Middletown, VA).