At 16 weeks of age, i.e., 6 weeks after commencing HFD and cold exposure interventions, the mice were fasted for 16 h and injected intraperitoneally with a 10% D-glucose solution at a dose of 1.0 g per kg body weight. Blood samples were collected from the tip of the tail at 0, 15, 30, 60 and 90 min after glucose administration, and blood glucose levels were measured using a glucometer (Accu Check II; Roche, Castle Hill, New South Wales, Australia). Two days later, the mice were fasted for 6 h from 8:00 a.m. and were injected intraperitoneally with insulin at a dose of 1.0 IU per kg body weight. Blood samples were obtained from the tail tip at 0, 15, 30 and 60 min after insulin injection for determination of glucose levels as stated above. Cold exposure was performed subsequently to the glucose and insulin tolerance tests on the same day.
Accucheck 2
Manufactured by Roche
Sourced in Australia
The AccuCheck II is a compact and user-friendly laboratory instrument designed for accurate measurement of various parameters. It features a high-resolution display and intuitive controls for easy operation. The AccuCheck II is intended to provide reliable data for research and analysis purposes.
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10 protocols using accucheck 2
1
Glucose and Insulin Tolerance Tests
2
Insulin and Glucose Tolerance Testing
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Insulin tolerance tests were performed on male mice at 15 weeks of age (unless otherwise stated) at 14:00–16:00 h. Briefly, the mice were fasted for 6 h and then intraperitoneally injected with insulin (0.5 IU/kg) (Novo Nordisk Pharmaceuticals, Baulkham Hills, NSW, Australia). Tail tip blood samples were then collected at 0, 15, 30, 45, 60, 75 and 90 min after insulin injection, and blood glucose levels were measured using a glucometer (AccuCheck II; Roche, New South Wales, Castle Hill, Australia). 7 days later (unless otherwise stated), mice underwent a glucose tolerance test, where they were fasted for 16–24 h before intraperitoneal injection between 13:00 and 15:00 h of a 10% d -glucose solution (1.0 g/kg) (Astra Zeneca, North Ryde, NSW, Australia). Blood samples were obtained from the tail tip at 0, 20 and 60 min after glucose injection and blood glucose levels were measured using a glucometer. Serum was also separated, immediately frozen and stored at −20 °C for later measurement of serum insulin using an ELISA kit from Linco Research (St Charles, Missouri, USA) or a RIA kit from Millipore (Millipore, Billerica, MA, USA).
3
Mouse glucose, insulin, and triglyceride analysis
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Blood glucose was measured using a glucometer (AccuCheck II, Roche, Australia) while insulin was determined by radioimmunoassay (Linco/Millpore, USA). At the end of the study, mice were killed by cervical dislocation and the relevant tissue samples were immediately freeze-clamped. Triglyceride levels in plasma and liver were determined by a Peridochrom triglyceride GPO-PAP kit (Roche Diagnostics, USA) [19] (link).
4
Glucose Tolerance Test in Mice
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Mice were fasted for 6 hours before receiving intraperitoneal injections of glucose (2 g/kg). Blood glucose levels were monitored at the 0, 15, 30, 45, 60 and 90 min following glucose injection using a Glucometer (Accu-check II, Roche Diagnostics, Australia). Plasma insulin levels were determined by a sensitive rodent insulin radioimmunoassay kit (Millipore, Missouri, USA).
5
Glucose Tolerance Test in Mice
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Mice were weighed weekly during feeding and daily during the CRS period using an electronic laboratory scale. Glucose tolerance was assessed prior to and following the 4‐week CRS period using a GTT. Mice were fasted for 4 h prior to GTTs. Blood was obtained via tail tipping and blood glucose determined using an Accu‐check II glucometer (Roche Diagnostic). A 20% (2 g kg−1) glucose bolus was administered via intraperitoneal injection and blood glucose levels recorded at 15, 30, 60, 120, 180 min post injection. Total area under the glucose curve (AUC) was calculated using all time‐points as a measure of glucose tolerance (GTT‐AUC).
6
Glucose and Insulin Tolerance Assays
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Mice were fasted for 6 h before intraperitoneal (i.p.) injection of a 10% d -glucose solution (1.0 g/kg body weight)38 (link),46 (link) at age indicated in the main text. Blood samples were obtained from the tail tip at the indicated times, and glucose levels were measured using a glucometer (AccuCheck II; Roche, New South Wales, Castle Hill, Australia). Serum from mice administrated with glucose was collected and stored for subsequent insulin assay using a Rat/Mouse insulin RIA kits (Linco Research, St Charles, MO, USA). Serum insulin levels at fed and fasted states of these mice were also determined using the same insulin RIA kits following the manufacturers’ instructions. ITT was carried out to determine insulin-induced hypoglycemia in mice at ages indicated in the main text. Briefly, the mice were injected with insulin (1 IU/kg body weight) intraperitoneally to induce hypoglycemia after being fasted for 6 h, then blood samples were collected from the tail tip at the indicated times, glucose levels were measured using a glucometer (Roche) as shown in the results.
7
Intraperitoneal Glucose and Insulin Tolerance Tests
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Intraperitoneal glucose tolerance test (GTT) (n= 10 in controls; n = 12 in butyrate group) and insulin tolerance test (ITT) (n = 5 per group) were performed in overnight fasted rats. Blood samples were obtained from the tail tip at 0, 15, 30, 45, 60, 90, 120 min for GTT. For ITT, blood samples were collected at 0, 15, 30, 45, 60 min. Glucose levels were measured using a glucometer (AccuCheck II; Roche, Castle Hill, NSW, Australia). The doses used during these tests were 1.5 g/kg body weight and 0.5 U/kg body weight for GTT and ITT, respectively.
8
Oral Glucose Tolerance and Metabolic Markers
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After overnight fasting, a mixed meal load (3.7 kcal, 2.5 mL/300 g body weight) was administered orally. The liquid mixed meal consisted of a commercial product (1 can: 300 kcal/200 mL: 12% carbohydrate, 24% protein, 22% fat, Mediwell Diabetic Meal; Maeil Dairies, Seoul, South Korea). Blood was drawn by eye bleed at 0, 30, 60, 90, and 120 min after the meal administration for measurement of glucose (AccuCheck II; Roche, Indianapolis, Indy, USA), and plasma samples were stored at −20°C, until further assays. GLP-1 (Alpco diagnostics), insulin and C-peptide (Mercodia) levels were measured using the corresponding ELISA kits. The insulin sensitivity (Homeostasis model assessment of insulin resistance (HOMA-IR)=fasting glucose×fasting insulin÷22.5) and β-cell function index (Homeostasis model assessment of β-cell function (HOMA-β)=20×fasting insulin (µU/mL)/(fasting glucose (mM/L) – 3.5)) were also calculated.
9
Metabolic Profiling of Mice
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Body weight and food intake were monitored daily throughout the experiment. Before the start of the study and at 5 weeks, following 5–7 h of food removal, tail vein blood was collected for glucose measurement with a glucometer (Accu-Check II; Roche, Castle Hill, Perth, Australia). Before the start of the study and at the end of the study, plasma was collected and stored at −80°C for subsequent biochemical testing. Mice were anesthetized with a ketamine/xylazine mixture (up to 100 mg/kg body weight ketamine and 20 mg/kg body weight xylazine) was administered via intraperitoneal injection. Mice fixation was then performed via transcardial perfusion with heparinized phosphate buffered saline (PBS; 10–20 mL/mouse) followed by 4% paraformaldehyde (PFA; 10–20 mL/mouse; #C007, ProSciTech). At the completion of the PFA perfusion, the right lobe of the liver was dissected and immersed in 4% PFA-filled glass scintillation vials for further analysis.
10
Glucose Tolerance Test in Rats
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Seven weeks after the NaB treatment (16 weeks after the beginning of the experiment), all rats were fasted for 12 h and then underwent a glucose tolerance test. Briefly, glucose was orally administered (1•5 g/kg body weight). Blood samples were collected sequentially from the tail vein, and glucose levels were tested at 15, 30, 45, 60, 90 and 120 min after gavage. Glucose levels were measured using a glucometer (AccuCheck II; Roche). To compare the time-course changes of the glucose concentration among groups, statistical analysis of AUC was performed using the GraphPad Prism software (GraphPad Software, Inc.).
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