GTTs were performed at 7 months of age (n=5-6/sex/genotype) [24 (link),27 (link),32 (link),33 (link)]. Mice were fasted for 12 hours before commencement of the experiment. Each mouse received an intraperitoneal (IP) injection of 10% glucose at a dose of 1 g/kg body weight. Blood glucose was measured before the glucose injection and at 15, 30, 45, 60, and 90 minutes after injection. ITTs were performed at 7.25 months of age in a fed state (n= 5-6). Recombinant human insulin (Humulin-R; Eli Lilly & Co, Indianapolis, Indiana) was prepared by diluting Humulin-R (100 U/ml) to 0.075 U/mL in sterile saline (0.9%). Each mouse received an IP injection of the 0.075 U/ml insulin solution at a dose of 0.75 U/kg body weight. Blood glucose measurements were performed before the insulin injection and at 15, 30, 45, and 60 minutes after injection.
Humulin r
Manufactured by Eli Lilly
Sourced in United States, Japan, Canada, Brazil, Denmark, France
Humulin R is a laboratory product manufactured by Eli Lilly. It is a human insulin solution used for research and development purposes.
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Lab products found in correlation
D glucose, by Merck Group (17 mentions)
Accu chek, by Roche (11 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (11 mentions)
Onetouch ultra glucometer, by LifeScan (8 mentions)
Contour glucometer, by Bayer (7 mentions)
Accu chek performa, by Roche (6 mentions)
Accu chek glucometer, by Roche (5 mentions)
3 3h glucose, by PerkinElmer (5 mentions)
G5767, by Merck Group (5 mentions)
Onetouch ultra, by LifeScan (4 mentions)
Comprehensive lab animal monitoring system, by Columbus Instruments (4 mentions)
Pyruvate, by Merck Group (4 mentions)
Sodium pyruvate, by Merck Group (4 mentions)
Onetouch glucometer, by LifeScan (4 mentions)
G8270, by Merck Group (3 mentions)
Accu check performa, by Roche (3 mentions)
Onetouch ultra test strip, by LifeScan (3 mentions)
Nova max glucometer, by Nova Biomedical (3 mentions)
Mouse insulin elisa kit, by Mercodia (3 mentions)
Alphatrak 2, by Zoetis (3 mentions)
427 protocols using humulin r
1
Glucose and Insulin Tolerance Tests
2
Insulin Effect on Liver and Muscle
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The mice were killed in a CO2-saturated atmosphere, immediately followed by decapitation. We extracted liver and muscle samples from the mice 5 min after an intraperitoneal bolus of 1 U kg-1 insulin (Humulin®R, Eli Lilly, São Paulo, Brazil) or saline solution (0.9% NaCl wt/vol). The liver and muscle samples were snap frozen in liquid nitrogen and stored for subsequent protein. After, these tissues were homogenized using a lysate buffer (10 mmol L-1 EDTA, 100 mmol L-1 Tris base, 100 mmol L-1 sodium pyrophosphate, 100 mmol L-1 sodium fluoride, 10 mmol L-1 sodium orthovanadate, 2 mmol L-1 PMSF, 1% Triton X-100 and 1 μg mL-1 aprotinin). Pancreatic islets were isolated from the mice immediately after the exercise, as previously described [36 (link)].
3
Insulin and Glucose Tolerance Tests
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Blood glucose levels were monitored at time points as indicated. For the insulin tolerance test (ITT), mice fasted for 6 h, and insulin (0.75 units/kg; Humulin R, Eli Lilly, Indianapolis, IN, USA) was administered i.p. The i.p. glucose tolerance test (IPGTT) was performed at the end of the study; mice fasted for 16 h, and glucose was then injected i.p. (1 g/kg body weight). The AUC of glucose was calculated during the course of the tests.
4
Glucose and Insulin Tolerance Tests in Mice
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For the glucose tolerance test, mice at 15~16 weeks of age were fasted for 14 hours (8:00 pm to 10:00 am) or 7 hours (9:00 am to 4:00 pm), followed by the intraperitoneal injection of glucose at a dose of 1 g/kg body weight. For the insulin tolerance test, mice were fasted for 14 hours and injected with human insulin (Humulin R, Eli Lilly) at a dose of 1 unit/kg body weight. Blood glucose was determined at the indicated time-points by a glucometer (ACCU-CHEK, Aviva; Roche). Plasma insulin levels were determined using the Mouse Insulin ELISA kit (Shibayagi) according to the manufacturer’s instructions.
5
Glucose and Insulin Tolerance Tests
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Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed as previously described,18 (link) using 1 g/kg of D-glucose (Sigma-Aldrich) and 0.75 U/kg insulin (Humulin R; Eli Lilly and Company, Indianapolis, IN, USA) by intraperitoneal injection after an 8-h fasting. Mice were weighed, and the blood glucose of each mouse was measured to get the value for 0 time point. Blood glucose was measured at 15, 30, 60, 90, and 120 minutes after the glucose and insulin injection. Blood was drawn from a tail vein, and blood glucose levels were measured using Auto-Check (Diatech Korea Co., Seoul, Korea).
6
Insulin Tolerance Test in Mice
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ITT was performed in accordance with a recommendation from the Mouse Metabolic Phenotyping Center (MMPC) Consortium [12] (link). After 6-h-fasted blood glucose levels were measured, insulin (Humulin R, Eli Lilly Japan, Tokyo) was injected intraperitoneally at 0.5 U/kg of body weight. Blood glucose levels were measured at 15, 30, 60, and 90 min after the injection as described earlier.
7
Insulin Sensitivity Assessment in Mice
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Animals were weighed on a weekly basis systematically at 9:00-10:00 during the entire protocol. They were five-hour fasted prior blood glucose determination (One Touch Ultra, LifeScan Inc., Wayne, PA, United States). Additionally, to assess insulin sensitivity, all mice underwent an insulin tolerance test (ITT) by the end of the 18th week. Mice were short-fasted for 5 h, basal blood glucose was determined and shortly after this, 100 μl saline solution containing a standardized dose of 0.025 IU of human-recombinant short-acting insulin (Humulin R, Lilly, Indianapolis, IN, United States) was intraperitoneally administered in every animal. Blood glucose measurement was repeated thereafter at 30 min and 60 min. An additional solution of dextrose in sterile water was ready to use in case an animal might be at risk of death by the hypoglycemic effect of short-acting insulin. Once the protocol was finished, all animals were given free access to food and water. No animal losses occurred during the ITT.
8
Insulin-Induced Glucose Monitoring in Mice
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Following an overnight fast, mice received an injection of insulin (0.75 units/kg body wt i.p.; Humulin R; Eli Lilly, Indianapolis, IN). Blood glucose was monitored (OneTouch Ultra; LifeScan) before and at serial time points after insulin administration.
9
Hyperinsulinemic Euglycemic Clamp Procedure
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Hyperinsulinemic euglycemic clamp experiments were carried out in all patients at baseline. Clamp experiments were performed as previously described [7] . After a 12-h overnight fast, catheters were inserted into the antecubital vein of both arms for infusion and in the dorsal vein for blood sample collection. A heated box was used on the blood-taking arm to obtain arterialized venous blood. Insulin (Humulin R, Eli Lilly, USA) was administered intravenously at a rate of 40 mU/kg/min for 150 min. Blood samples were drawn using an intravenous catheter in a heated vein, and glucose concentrations were measured at 5-min intervals. Dextrose 20% was infused at variable rates to maintain a glucose level of 5.0 mmol/L. The time taken to reach euglycemia was calculated from the beginning of the experiment to euglycemia. Glucose disposal rate (GDR, mg/kg/min) was measured during steady-state intervals.
10
Glucose Metabolism in Tsc1 and S6k1 Mice
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At six weeks of age cohorts of control, Tsc1-/-, S6k1-/- and Tsc1-/-;S6k1-/- mice were placed on either normal chow diet (NCD) (PicoLab Rodent Diet 20, 5053, LabDiet) composed of 25% protein, 13% fat, and 62% carbohydrate, or a high fat diet (HFD), (AIN-76A, 58R2, TestDiet) composed of 15% protein, 25.7% carbohydrate, 59.3% fat for eight weeks. Mice were weighed weekly.
Glucose Tolerance Test (GTT) was performed in mice fasted for sixteen hours after an intraperitoneal (IP) injection of 10% glucose (1 mg/g body weight). Blood glucose was measured at 0, 15, 30, 60, and 120 minutes in blood obtained via tail nicking using the OneTouch glucometer (LifeScan, Inc., Milpitas, CA). Similarly, insulin sensitivity testing was performed after a 4-hour fast and 0.5 mU/g IP injection of regular insulin (Humulin R, Eli Lilly) followed by glucose measurements at 0, 30, 60, 90, and 120 minutes.
Glucose Tolerance Test (GTT) was performed in mice fasted for sixteen hours after an intraperitoneal (IP) injection of 10% glucose (1 mg/g body weight). Blood glucose was measured at 0, 15, 30, 60, and 120 minutes in blood obtained via tail nicking using the OneTouch glucometer (LifeScan, Inc., Milpitas, CA). Similarly, insulin sensitivity testing was performed after a 4-hour fast and 0.5 mU/g IP injection of regular insulin (Humulin R, Eli Lilly) followed by glucose measurements at 0, 30, 60, 90, and 120 minutes.
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