Catalyst chemistry analyzer

GLUT4 levels at the surface of mononuclear cells were assessed using a commercially available ELISA (USCN Life Science Inc., United States) according to manufacturer’s instruction and absorbance was read at 450 nm. To ensure GLUT4 levels were indicative of surface amounts, levels were compared with samples that were sonicated. Sonicated samples had 2-3 fold higher GLUT4 levels compared with unsonicated samples (Schnurr et al., 2013 ). A BCA Protein Assay (Pierce, Theroms Scientific, United States) was used for protein adjustment in regard with GLUT4 and absorbance was read at 562 nm. Insulin levels were measured using an ELISA (Porcine/Canine; ALPCO, Salem NH), following the protocol by the manufacturer and taking absorbance readings at 450nm. All absorbance readings were done using Synergy HT multi-mode microplate reader (BioTek, United States). Plasma Glucose analysis was performed by the North Pole Veterinary Clinic (North Pole, AK) using the in-house diagnostic Catalyst® Chemistry Analyzer (IDEXX, United States). HOMA-IR was calculated for each individual with the linear approximation formula, which divides the product of insulin (in μU/mL) and glucose (in mmol/L) concentrations by 22.5 (Matthews et al., 1985 (link)). All samples were assayed in duplicates as an internal control.

The protocol for the assessment of GLUT4 protein concentrations translocated to the surface of mononuclear cells has been described elsewhere [25 (link)]. We controlled for the measurement of GLUT4 translocated to the cell membrane, and not total GLUT4, in comparing sonicated to unsonicated samples. We could detect the same trend as we have seen in an earlier study, namely that sonicated samples had 3 fold higher GLUT4 levels compared to unsonicated samples (data not shown) [27 ]. Protein content was determined using the BCA Protein Assay (Pierce, Thermo Scientific, United States). Insulin levels were measured using an ELISA (Porcine/Canine; ALPCO, Salem NH), following the protocol by the manufacturer. All absorbance readings were done using Synergy HT multi-mode microplate reader (BioTek, United States). Plasma Glucose analysis was performed by the North Pole Veterinary Clinic (North Pole, AK) using the in-house diagnostic Catalyst® Chemistry Analyzer (IDEXX, United States). All samples were assayed in duplicates as an internal control.