ITT was performed in mice fasted for 6 h and then injected intraperitoneally (i.p.) with human insulin (Humulin, Novo Nordisk) at 0.75 U/kg. Blood glucose levels were measured by One-touch Ultra glucose meter before and at 15, 30, 60 and 120 min after injection. Skeletal muscle insulin signaling was performed on mice either fasted for 6 h (9:00 am-3:00 pm) or overnight 16 h (16 h, 5:00 pm to 9:00 am) and i.p. injected with human insulin (Humulin, Novo Nordisk) at 1.0 U/kg. Exactly 15 min later, tissues were excised and immediately snap frozen in liquid nitrogen. Protein extracts from tissues were analyzed by western blot.
Humulin
Manufactured by Novo Nordisk
Sourced in Denmark
Humulin is a laboratory-produced insulin that functions as a replacement for the natural insulin produced by the human body. It is designed to provide a regulated supply of insulin to maintain stable blood sugar levels.
Automatically generated - may contain errors
Lab products found in correlation
Cholera toxin, by Fujifilm (2 mentions)
Triiodothyronine, by MP Biomedicals (2 mentions)
Penicillin, by Fujifilm (2 mentions)
Streptomycin, by Fujifilm (2 mentions)
Amphotericin b, by Bristol-Myers Squibb (2 mentions)
Minispec lf90ii, by Bruker (2 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Inveon multimodality preclinical scanner, by Siemens (1 mentions)
Ham s f 12 nutrient mixture, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fasting insulin, by Mercodia (1 mentions)
C peptide, by Crystal Chem (1 mentions)
7 protocols using humulin
1
Insulin Signaling Pathway in Mice
2
Optimized Culture Conditions for Keratinocyte Growth
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KCM was prepared according to previously described methods [10] (link). Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher, MA, USA) and DMEM with Ham's F-12 medium (Gibco) were mixed at a 1:1 ratio. The medium was supplemented with 5% autologous human serum, 0.3 μM saxizon (Takeda yakuhin kogyo, Osaka, Japan), 140.0 mU/mL humulin (Novo Nordisk, Bagsvaerd, Denmark), 2.0 nM triiodothyronine (MP Biomedicals, CA, USA), 0.2 μM epidermal growth factor (Higeta-Shoyu, Tokyo, Japan), 1.0 nM cholera toxin (Wako Pure Chemicals, Osaka, Japan), 100 U/mL penicillin, 69 μM streptomycin (Wako Pure Chemicals), and 0.4 μg/mL of amphotericin B (Bristol-Myers Squibb, NY, USA).
3
Insulin-Regulated Glucose Uptake in Mice
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Small-animal PET/CT was performed on a Siemens INVEON multimodality preclinical scanner. Mice (Dlk1+/+ n = 15, Dlk1−/− n = 13) were fasted overnight prior to PET/CT scanning at baseline (day 0) and one day later (day 1). Mice were subcutaneously injected with 75 μl saline or 0.75 U/(Kg body weight) short acting insulin (Humulin, NOVO Nordisk, DK) respectively, 15 min. Before injection of 18F-Fluoro-deoxy-glucose (FDG). Mice were anesthetized (2% isoflurane), and a bolus injection of FDG (day 0 95.5 ± 9.1 MBq; day 1: 91.6 ± 13.4 MBq) was administered via a tail vein catheter. Prior to the PET scan a two-bed CT scan was performed for attenuation correction of the PET data and anatomic orientation. CT and PET images were co-registered using a transformation matrix and CT-based attenuation correction was applied to the PET data. The PET data were reconstructed using the Siemens INVEON pre-clinical software and data analysis of the PET/CT fused images was performed with INVEON software version 4.2 (IRW, Siemens). FDG uptake in each volume of interest (VOI) was reported as standardized uptake values (SUV). VOIs of the skeletal limb hind muscles were normalized using brain uptake as a reference tissue that metabolizes glucose independent of insulin.
4
Metabolic Phenotyping of Mouse Models
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Tissue samples were fixed, processed, and stained with hematoxylin and eosin (H&E) and visualized under microscopy. Fat and lean body masses were measured by NMR-based Bruker minispec LF90II (Bruker company, German). Insulin tolerance tests were performed in mice fasted for 6 h and then injected intraperitoneally (i.p.) with human insulin (Humulin; Novo Nordisk) at 0.75 U/kg for mice on normal chow diet, and 2.5 U/kg for mice on HFD. Glucose tolerance tests were performed in 6 h fasted chow-fed mice injected by i.p. or 16 h fasted HFD-fed mice administered by gastric gavage with glucose (1.5 g/kg body weight). Blood glucose levels were measured by One-touch Ultra glucose meter before and at 15, 30, 60, and 120 min after administration.
5
Metabolic Assessment in Mice
6
Keratinocyte Culture Medium for Cell Sheets
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We used the keratinocyte culture medium (KCM) for cell culture and manufacture of cell sheets. KCM comprised a basal mixture of three parts Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, MA, USA) and one part Ham's F-12 nutrient mixture (Gibco), containing 5% autologous human serum of each patient, insulin (140.0 mU/mL, Humulin, Novo Nordisk, Bagsvaerd, Denmark), triiodothyronine (2.0 nM, MP Biomedicals, CA, USA), saxizon (0.3 μM, Takeda Yakuhin Kogyo, Osaka, Japan), epidermal growth factor (0.2 μM, Higeta-Shoyu, Tokyo, Japan), cholera toxin (1.0 nM, Wako Pure Chemicals, Osaka, Japan), penicillin (100 U/mL, Wako Pure Chemicals), streptomycin (69 μM, Wako Pure Chemicals), and amphotericin B (0.4 μg/mL, Bristol-Myers Squibb, NY, USA).
7
Glucose Homeostasis Assessment in Mice
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Oral glucose tolerance tests (OGTTs) were performed after at least a 3-day resting period following the MR session (described below). Six-hour fasted mice (food removed at ~07:30 h) received a glucose load of 1.5 g/kg by oral gavage, and blood glucose was monitored from the tail tip with a glucometer (Breeze, Bayer, Zürich, Switzerland) for 2 h. The area under the curve (AUC) in the glucose versus time plot was calculated to assess glucose clearance from the blood. An insulin tolerance test (ITT) was then performed after a minimum 3-day resting period by intraperitoneal injection of insulin (0.75 IU/kg, Humulin, Novo Nordisk, Bagsvaerd, Denmark, diluted in 0.9% NaCl). Blood glucose was monitored from the tail tip for 3 h and the area above the curve (AAC) was calculated for the glucose versus time plots. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure fasting insulin (Mercodia, Uppsala, Sweden) and C-peptide (Crystal Chem, Downers Grove, IL, USA) levels in the plasma. Insulin sensitivity was estimated with the quantitative insulin sensitivity check index (QUICKI) as the inverse of the log 10 sum of fasting insulin (μIU/mL) and fasting glucose (mg/dL).
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