Sigma protease inhibitor

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.

At each of the five time points during the OGTT, additional blood samples will be collected for measurement of satiety hormones including insulin. 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) [72 (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 Milliplex MAP Human Gut Hormone Panel Kit (Millipore, St Charles, MO). Surrogate markers of insulin resistance, HOMA and QUICKI, will be quantified as per our previous work [99 (link)].

At each time point, approximately 5 mL of venous blood was collected. Samples were aliquoted into a 2 mL into a K2EDTA plasma vacutainer, 2 mL into a serum vacutainer, and 1 mL into an Eppendorf tube containing EDTA, diprotinin-A (0.034 g/L blood; MP Biomedicals), sigma protease inhibitor (1 g/L blood; Sigma Aldrich), and Roche Pefabloc® SC (1 g/L of blood, Sigma Aldrich) to preserve analytes. Tubes were centrifuged for 15 min at 4°C. Supernatant was removed from each sample and transferred into a new labeled Eppendorf tube for storage at −80°C until analysis. Blood samples collected in Study A were assessed by a custom Human Metabolic Array [C-peptide, glucagon, gastric inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1 active), insulin and peptide YY (total)] (Milliplex Multiplex, Eve Technologies, University of Calgary). A glucose oxidase assay kit used plasma samples to measure plasma glucose concentrations at all time points (Sigma Aldrich). Serum paracetamol concentrations, determined using a paracetamol assay kit (Cambridge Life Science), were used to track gastric emptying. Several studies have demonstrated paracetamol concentrations to be a valid measure of gastric emptying (10 (link), 11 (link)).

The animals were anesthetized using isoflurane and denied access to food overnight for a 12 h fast; 1 mL of blood was collected from the portal vein in a chilled tube 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). Plasma was collected after centrifugation and stored in −80 °C until insulin, peptide tyrosine tyrosine (PYY) and glucagon-like peptide 1 (GLP-1) were measured using a Rat Metabolic Multiplex Array (MRDMET) (Millipore, St. Charles, MO, USA) (Eve Technologies, Calgary, AB, Canada). The animals were henceforth euthanized via decapitation and heart, liver, kidney, cecum, colon and male testes were weighed and stored in −80 °C until analysis. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was used to estimate insulin resistance using the following formula [80 (link)]:

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)).

Pancreatic cells, stomach and colon from mice were lysed by adding 5x diluted lysis buffer (50 mM Tris-base, 0.25 M NaCl, 10 mM EDTA, 0.5% NP40, 1% TritonX-100, 4 mM NaF, pH 7.5). The final lysates were centrifuged at 15,000 g, 4°C for 15 min. Protein concentration was estimated using Coomassie protein assay (Thermo Scientific). Mouse stomach and colon were used as positive control. All solutions contained 1× Sigma protease inhibitor (Sigma S8820). Protein samples were loaded on 10% polyacrylamide precast gels (Invitrogen), separated by electrophoresis, and blotted to PVDF membranes (Invitrogen). Membranes were blocked with 5% skim milk solution in TBS-Tween (0.1%) buffer for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against gastric HKα1(1:1000 rabbit monoclonal, Abcam, EPR12251 or polyclonal Calbiochem 119101), HKβ (1:1000 mouse monoclonal 2G11, Sigma, A274), non-gastric HKα2 (1:1000 rabbit polyclonal, Sigma, HPA039526) or antibody (C384-M79) [28 (link)], kindly donated by J. J. H. H. M. De Pont and H. G. P. Swarts, and β-Actin (1:1000 mouse monoclonal C4, Santa Cruz, Sc-47778). Blots were then incubated with appropriate HRP-conjugated antibodies (DAKO or Santa Cruz), developed with EZ-ECL (Biological Industries) and visualized on Fusion FX (Vilber Lourmat) and band intensity was calculated using Bio1D software.

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.