NPH insulin (Novo Nordisk A/S) was used for daily treatment in diabetic rats. For in vivo experiments, human insulin (Novo Nordisk A/S), and a long‐acting glucagon‐analogue (Novo Nordisk A/S) were used. Both were formulated in 5 mmol/L phosphate, 140 mmol/L NaCl, 70 ppm polysorbate‐20, pH 7.4. For in vitro experiments human insulin (Novo Nordisk internal reference solution) and native glucagon (Novo Nordisk A/S) dissolved in 20% H2O and 80% DMSO were used.
Human insulin
Manufactured by Novo Nordisk
Sourced in Denmark, United States, China
Human insulin is a lab-produced form of the natural insulin hormone found in the human body. It is used to regulate blood sugar levels in individuals with diabetes. The core function of human insulin is to facilitate the uptake and utilization of glucose by cells, thereby maintaining normal blood sugar levels.
Automatically generated - may contain errors
Lab products found in correlation
Rat mouse insulin elisa kit, by Merck Group (2 mentions)
Nph insulin, by Novo Nordisk (2 mentions)
2 deoxy d 1 14c glucose, by PerkinElmer (2 mentions)
Glucometer elite, by Bayer (2 mentions)
Accu check blood glucose meter, by Roche (2 mentions)
Insulin elisa kit, by Cloud-Clone (2 mentions)
D glucose, by Merck Group (2 mentions)
Glucagon, by Novo Nordisk (2 mentions)
3 3h glucose, by PerkinElmer (2 mentions)
3 3h glucose, by Novo Nordisk (2 mentions)
Cinnamaldehyde, by Merck Group (2 mentions)
Eugenol, by Merck Group (2 mentions)
Glucose, by Fujifilm (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Control sirna, by RiboBio (1 mentions)
Mimics control, by RiboBio (1 mentions)
Mir 155 inhibitor, by RiboBio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Inhibitor control, by RiboBio (1 mentions)
Hexokinase method, by Thermo Fisher Scientific (1 mentions)
59 protocols using human insulin
1
Insulin and Glucagon Formulations for Diabetes Research
2
Glucose and Insulin Tolerance Tests
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Before and after feeding NC or HFD, the glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. For the GTT, mice were starved for 16 h and were injected with 1.5 g kg−1 (bodyweight) glucose (Wako Pure Chemical Industries, Osaka, Japan) intraperitoneally. For the ITT, mice were injected with 0.75 U kg−1 (bodyweight) human insulin (Novo Nordisk, Bagsvaerd, Denmark) intraperitoneally without starvation. Tail vein blood was collected at 0, 15, 30, 60, and 120 min after administration to measure blood glucose levels with ONE TOUCH Ultra (Johnson & Johnson Services, New Jersey, USA).
3
Modulating Insulin Signaling Pathways
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An siRNA targeting mouse PDK4 (siG140711153320), an siRNA targeting mouse C/EBPβ (siG09723100032), an siRNA targeting mouse HDAC4 (siB1271190307) and a control siRNA were purchased from RiboBio Co., Ltd (Guangzhou, China). Western-blot and qRT-PCR analysis showed successful reduction of C/EBPβ, HDAC4 and PDK4 expression in cells transfected with siC/EBPβ, siHDAC4 and siPDK4, respectively (Fig 7E and S8 Fig ). Hepa1-6 cells were transfected with 50nM siRNA-PDK4, siRNA-C/EBPβ, siRNA-HDAC4 or scrambled control siRNAs using Lipofectamine 2000 (Invitrogen) for 48-72h according to the manufacturer's instructions. The effect of PDK4, C/EBPβ or HDAC4 knockdown on insulin-stimulated AKT phopshorylation was examined in hepa1-6 cells. After stimulation with 50IU/L human insulin (Novo Nordisk) for 15 min at 37°C (S6 Fig ), the medium was removed and the cells were immediately lysed with ice-cold lysis buffer for western-blot.
4
Glucose and Insulin Tolerance Tests
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For glucose tolerance test (GTT), mice were fasted overnight for 12h, and then injected intraperitoneally with glucose (2g/kg body weight). For insulin tolerance test (ITT), mice were fasted overnight for 6h, and then injected intraperitoneally with human insulin (Novo Nordisk) at a dose of 0.75IU/kg body weight. Blood glucose levels were measured from mouse tail vein with an automatic glucometer (One Touch Lifescan, Johnson & Johnson, USA) before glucose and insulin injection and at the indicated time after injection. Serum insulin levels were measured by enzyme-linked immunosorbent assay according to vendor’s instructions (Millipore Rat/Mouse Insulin ELISA Kit, EZRMI-13K).
5
Modulating miR-155 Expression and Insulin Signaling
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The mouse or human miR-155 mimics, mimics control, miR-155 inhibitor and inhibitor control were purchased from RiboBio Co., Ltd (Guangzhou, China). miRNAs were transiently transfected into cells (including hepa1-6, 7402 and C2C12 cells) at a working concentration of 100nM using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s procedure. All RNA oligonucleotides treatments proceeded for 48-72h. The effect of in vitro overexpression of miR-155 by using miRNA mimics and the repression of endogenous miR-155 expression by a miR-155 inhibitor on insulin-stimulated AKT phopshorylation was examined in hepa1-6 cells. After stimulation with 50IU/L human insulin (Novo Nordisk) for 15 min at 37°C (S6 Fig ), the medium was removed and the cells were immediately lysed with ice-cold lysis buffer.
6
Glucose and Insulin Tolerance Tests
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GTT was performed by an intraperitoneal injection of d -glucose (2 g/kg body weight, i.p.) after a 16 h overnight fast. Blood glucose levels were measured at 0, 30, 60, and 90 min after injection and the area under the curve (AUC) for blood glucose was calculated. For ITT, mice were fasted for 6 h and then injected with human insulin (Novo-Nordisk, Bagsværd, Denmark) at 0.75 U/kg body weight. Blood glucose levels were measured at 0, 15, 30, 60, and 90 min. PTT was performed to estimate gluconeogenesis. Mice were starved for 16 h and then injected intraperitoneally with pyruvate (2 g/kg body weight, i.p.) dissolved in saline. Blood glucose levels were measured in the tail blood. Blood glucose levels were measured using the hexokinase Method (Thermo Fisher Scientific, Lafayette, CO, USA).
7
Formulation and Characterization of Insulin-Loaded PLGA-Chitosan Nanoparticles
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A solution was prepared by dissolving 100 mg of PLGA copolymer (PLGA 50/50) and 50 mg of TPGS in 5 ml of acetone. The solution underwent sonication for 60 s using a Sonicator S-4000 probe sonicator (Misonix) (100 W, 22.5 kHz, 50% amplitude) while consistently stirring at 4 °C. A volume of 2 ml of human insulin (100 IU/ml, Novo Nordisk) was added to the solution with continuous stirring, and the solution was subjected to sonication for another 60 s. The solution was kept in an ice bath, forming a water-in-oil (W/O) emulsion, then it underwent sonication again for 180 s, using an equivalent volume of a 0.1% w/v chitosan solution prepared by dissolving chitosan in 1% v/v acetic acid. The sonication process was carried out under continuous stirring at a speed of 500 r.p.m. for 1 h. Following the completion of the coating reaction, the mixture underwent a purification process involving three cycles of centrifugation; this was done to separate the NP pellet from the supernatant, which contained the leftover chitosan solution (Figure 2 ).
8
Adipose Tissue Explant and Cell Culture Protocol
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Human omental adipose tissues (AT) were obtained from 10 subjects with normal weight (BMI: 21.3 ± 2.0 kg/m2) and 8 subjects with simple obesity (BMI: 31.6 ± 7.4 kg/m2) undergoing laparoscopic cholecystectomy (see Table S2 for more characteristics). Mouse epidydimal AT were from 14-wk-old C57BL/6 mice fed with a regular diet or a high-fat diet (60% fat, Research Diets, Inc., New Brunswick, USA) for 10 weeks. For explant culture, AT were finely minced and 40 pieces per well were incubated in 5 ml MEM supplemented with 0.5% BSA, 50 μM ascorbic acid (Sigma–Aldrich, Shanghai, China), and 1% Penicillin/Streptomycin for 24 h.13 Lactate dehydrogenase (LDH) release test (kit from Beyotime Institute of Biotechnology) was performed to evaluate the viability of explants during 24 h culture (Fig. S4A ).14 (link) For adipocytes and SVC culture, minced AT was digested and separated into adipocytes and SVC fractions as described before.15 (link) Isolated cells were then cultured in 5 ml MEM with 10% ELV-depleted FBS and 1% Penicillin/Streptomycin for 24 h. In some experiments, cultured human omental AT explants were treated with or without human insulin (Novo Nordisk, Copenhagen, Denmark) for 24 h. The culture medium (20–30 mL) was collected for ELV isolation as described above.
9
Metabolic Profiling of PDZ-RhoGEF Knockout Mice
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Fasting glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on age matched (8 month-old) wild type and PDZ-RhoGEF KO mice. For GTT, PDZ-RhoGEF wild type mice (n = 8) and KO mice (n = 8) were starved for 16 hr before intraperitoneal (IP) challenge with 1 g D-glucose/kg of body weight and their blood glucose monitored from the tail vein using an automated glucose monitor (Actusoft, Roche). ITT was performed following a three hour fast (n = 8). Human insulin (1 U/kg of body weight, Novo Nordisk) was given to the animals by intraperitoneal injection and the blood glucose level was measured every 15 min up to one hour as described above. Serum was collected from wild type (n = 6) and PDZ-RhoGEF KO mice (n = 8) after 16 hr of starvation for determination of fasting glucose, insulin, triglycerides, and glycerol. Glucose levels were measured by the automated glucose monitor (Actusoft, Roche). Insulin levels were determined by Ultrasensitive insulin ELISA kit (Crystal Chem Inc., Downers Grove, IL) in triplicates. The levels of fasting triglycerides were determined by the serum triglyceride determination kit (Sigma-Aldrich, St. Louis, MO).
10
Reagents and Compounds for Cell Culture
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Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 Medium, DMEM/F-12, Fetal Bovine Serum (FBS), and Penicillin-Streptomycin mixture were obtained from Milipore Sigma (Merck KGaA, Darmstadt, Germany). Human Insulin was purchased from NovoNordisk (Bagsværd, Denmark); Thiazolyl Blue Tetrazolium Bromide (MTT; cat. no. M5655), 5-Fluorouracil (5-FU; cat.no F6627), Verapamil hydrochloride (cat. no. V4629), 2-Acetoxybenzoic acid (Aspirin; cat. no. A2093), Losartan potassium (cat. no. 61188), Chloroquine diphosphate salt (cat. no. C6628), Cimetidine (cat. no. C4522), Itraconazole (cat. no. I6657), 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (Tacrine hydrochloride; cat. no. A79922) and Isonicotinic acid hydrazide (Isoniazid; cat. no. I3377) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Pravastatin (cat. no. 0010342) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).
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