Pefabloc

Following overnight feed removal, a fasted blood samples was collected from a tail nick and glucose concentrations measured immediately using a One Touch Blood Glucose Meter (BD Biosciences). Immediately following the fasted blood sample, a 2 g/kg glucose load was administered via oral gavage, and additional blood glucose measurements made at 15, 30, 60, 90, and 120 min. At the fasting time point, a whole blood sample was collected into tubes containing diprotin-A (0.034 g/L blood: MP Biomedicals, Irvine, CA, USA), Sigma protease inhibitor (1 g/L blood: Sigma Aldrich, Oakville, ON, Canada), and Roche Pefabloc (1 g/L of blood: Roche, Mississauga, ON, Canada) and allowed to clot before centrifugation. Serum concentrations of active GLP-1, total PYY, total GIP, active ghrelin, leptin, and insulin were measured with the Milliplex Rat Gut Hormone Panel kit (Millipore, Billerica, MA, USA). The sensitivity of the kit is as follows (minimum detectable concentration in picograms per milliliter in brackets): GLP-1 (28), GIP (1), ghrelin (2), insulin (28), leptin (27), and PYY (16). The intra-assay variation is <7%, and inter-assay variation is <24%. Insulin resistance was calculated using HOMA-IR with the formula: fasting insulin (μU/ml) × fasting glucose (mmol/l) divided by 22.5. The composite insulin sensitivity index (CISI) was calculated as previously described (17 (link)).

One day prior to testing, rats (n = 6) were weighed and randomly assigned to 1 of 4 test groups: (1) Dextrose; (2) Ensure®; (3) Mixed meal; or (4) Saline. Following overnight feed deprivation with access to water, rats were given an oral gavage of the test solutions standardized to provide 2 g CHO/kg body weight or an equivalent volume of saline. Blood was sampled via tail nick at 0, 15, 30, 60, 90, and 120 min post-gavage and immediately analyzed for glucose using a blood glucose meter (OneTouch Glucose Meter, Lifescan Inc., Milpitas, CA). At the same time, blood was collected in a chilled tube containing diprotinin-A (0.068 mg/ml blood; MP Biomedicals, Irvine, CA), Sigma protease inhibitor (1 mg/ml blood; Sigma Aldrich, Oakville, ON, Canada) and Roche Pefabloc (1 mg/ml of blood; Roche, Mississauga, ON, Canada). Plasma was stored at −80°C until analysis for satiety hormones.

Five days prior to sacrifice, rats were fasted overnight for 12 h, and blood glucose was measured from a tail nick sample using a One Touch Ultra 2 glucose meter (Lifescan, Burnaby, Canada). Rats were gavaged with a 2 g/kg dose of glucose, and additional blood glucose measurements were made at 15, 30, 60, 90, and 120 min post gavage. At the 0-, 15-, 60-, and 120-min time points, additional blood was collected from the tail into chilled tubes containing diprotinin-A (0.034 mg/mL blood (MP Biomedicals, Irvine, CA, USA), Sigma protease inhibitor (1 mg/mL blood; Sigma Aldrich, Oakville, ON, Canada), and Roche Pefabloc (1 mg/mL of blood; Roche, Mississauga, ON, Canada). Samples were centrifuged, and the serum was stored at −80 °C until analysis for insulin.

At each of the five time points during the OGTT, additional blood samples will be collected for measurement of satiety hormones. Blood will be drawn into a cooled EDTA vacutainer tube containing diprotinin-A (0.034 mg/ml blood; MP Biomedicals, Irvine, CA); Sigma protease inhibitor (1 mg/ml blood; Sigma Aldrich, Oakville, ON, Canada) and Roche Pefabloc (1 mg/ml of blood; Roche, Mississauga, ON, Canada) [13 (link)]. Blood will be centrifuged within 30 min and plasma analyzed for ghrelin (active), insulin, leptin, glucose-dependent insulinotropic polypeptide (GIP) (total), GLP-1 (active), and PYY (total) concentrations using a Human Gut Hormone Panel Milliplex Kit (Millipore, St Charles, MO).

EFV was purchased from Toronto Research Chemicals Inc., Ontario, Canada. The PK and Pefabloc (protease inhibitor) were purchased from Roche, Germany. All other reagents/chemicals were purchased from Sigma-Aldrich, USA. The PrP monoclonal antibody (mAb) 4H11 used in this study to detect PrP has been previously described [42 (link)]. The antibodies anti-Cyp46A1 (ab82814, Abcam, USA), anti-flotillin-1 (610,821, Mouse BD Transduction Laboratories) and anti-β-actin (Cell signaling) were used. Secondary antibody conjugated with peroxidase, goat anti-mouse HRP, was obtained from Jackson Immuno Research, USA.