Mice underwent hyperinsulinemic-euglycemic clamps with deuterated glucose to determine whole-body and hepatic insulin sensitivity as described previously [18] (link), [19] (link). In brief, a silicon catheter (Silastic laboratory tubing, Dow Corning, Midland, MI) was placed into the right jugular vein under Isoflurane (CP Pharma, Burgdorf, Germany) anesthesia. Mice were allowed to recover for 4–5 days and fasted for 6 h on the day of the experiment (03:00–09:00a.m.). To assess basal whole-body glucose disposal, D-[6,6-2H2]glucose (98% enriched; Cambridge Isotope Laboratories, Andover, MA, USA) was infused at a rate of 4 μmol/kg/min for 120 min. The hyperinsulinemic-euglycemic clamp was performed with a primed (40 mU/kg)-continuous infusion (4 mU/kg/min; Huminsulin, Lilly, Giessen, Germany) for 180 min. Euglycemia was maintained by periodically adjusting a variable 20% glucose infusion. D-[6,6-2H2]glucose was co-infused together with insulin solution (0.4 μmol/kg/min) and variable glucose infusion to obtain stable tracer concentrations during varying glucose infusion rates. Blood samples were taken at 10-min intervals during the last 30 min of basal, and hyperinsulinemic-euglycemic clamps.
Huminsulin
Manufactured by Eli Lilly
Sourced in Germany, India
Huminsulin is a laboratory equipment product manufactured by Eli Lilly. It is designed for the measurement and analysis of insulin levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Skyscan 1178, by Bruker (2 mentions)
Phenomaster, by TSE Systems (2 mentions)
Accu chek, by Roche (2 mentions)
Elisa kit, by Crystal Chem (2 mentions)
Accutrend plus system, by Roche (2 mentions)
Isoflurane, by CP-Pharma (1 mentions)
D 6 6 2h2 glucose, by Cambridge Isotopes (1 mentions)
Silastic laboratory tubing, by Dow (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Peg 2k, by Merck Group (1 mentions)
Up100h, by Hielscher (1 mentions)
Glucometer, by Roche (1 mentions)
Accu check go, by Roche (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
8 protocols using huminsulin
1
Hyperinsulinemic-Euglycemic Clamp in Mice
2
Insulin and GAD Incubation Assay
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Human whole blood samples (1000 µL) were incubated with 50 µL of 1000 IU/mL human insulin (50 IU Huminsulin; Eli Lilly) or 50 µL of 10 µg/mL GAD (Sigma-Aldrich). All tubes were gently mixed prior to incubation.
All blood samples were incubated for 14 h at 37°C (98.6°F). Once every two hours during the incubation period, the samples were carefully mixed to re-suspend the cells, thereby ensuring for adequate insulin exposure by redistribution of insulin throughout the samples. After overnight incubation, a 50-µL aliquot was obtained from each tube of whole blood and analyzed by flow cytometry.
All blood samples were incubated for 14 h at 37°C (98.6°F). Once every two hours during the incubation period, the samples were carefully mixed to re-suspend the cells, thereby ensuring for adequate insulin exposure by redistribution of insulin throughout the samples. After overnight incubation, a 50-µL aliquot was obtained from each tube of whole blood and analyzed by flow cytometry.
3
Hyperinsulinemic-Euglycemic Clamp in Mice
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A silicon catheter was placed into the right-side jugular vein under anesthesia. Mice recovered for 4–5 days and were fasted for 6 h (3:00–9:00 a.m. ). The clamp was performed with a primed (40 mU/kg) continuous infusion (4 mU/kg/min) (Huminsulin; Lilly, Giessen, Germany) for 180 min (8 (link)). Whole-body insulin sensitivity is expressed as glucose infusion rates (mg/kg/min).
4
PLGA-TPGS Nanoparticles for Insulin Delivery
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About 100 mg of PLGA copolymer (PLG4:PLGA 80/20) and 50 mg of TPGS (Sigma, Bangalore, India) were dissolved in 5 mL of acetone (Fischer Scientific). The solution was sonicated for 60 seconds using a probe sonicator (Hielscher sonicator, ultrasonic processor UP 100H, Berlin, Germany [100 W, 30 kHz, 0.8 cycle, 80% amplitude]), and it was stirred continuously at 4°C. To this, 2 mL of Huminsulin (100 IU/mL, Eli-Lilly, Haryana, India) was added while stirring the solution and it was further sonicated for 60 seconds. The solution was kept in an ice bath and it formed a water-in-oil (W/O) emulsion. It was again sonicated for 180 seconds with 10 mL of aqueous PEG 2000 (PEG 2K; 1% w/v, Sigma, India) solution to form a double emulsion (W/O/W), and it was stirred continuously for 6 hours to remove the organic solvent. Then, it was centrifuged (Remi high-speed centrifuge C-24, Remi House, Mumbai, India) at 12,000 rpm (4°C) for 15 minutes and the ISTPPLG4 NPs were formed (Figure 1 ). The residue was freeze-dried (Christ Alpha 2–4 LSC freeze dryer, Martin Christ Gefriertocknungsanlagen GmbH, Osterode am Harz, Germany) for 12 hours, and the pellet obtained was stored at 4°C for further studies. The same protocol was also followed for the synthesis of copolymer PLG6 (PLGA 70/30).
5
Insulin Sensitivity Assessment in Animal Models
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The FBG of fasted animals was measured by glucometer (ACCU-CHECK® GO, Hoffmann-La Roche Ltd). Whole body insulin sensitivity was assessed by Oral Glucose Tolerance Test (OGTT)28 (link) at different time points (0, 15, 30, 60, 90, 120 min). Homeostasis model assessment- index of insulin resistance was calculated as:
Insulin tolerance test (ITT) was performed according to Lehnen et al., 2010. Briefly regular insulin (Huminsulin Eli Lilly & company India Pvt Ltd, India) (0.75 U/kg) was injected subcutaneously to animals and blood samples were collected from tail vein at 0 (just before insulin injection), 30, 60, 120 min (after insulin the injection) for glucose estimation using glucometer (Hoffmann-La Roche Ltd, India).
Insulin tolerance test (ITT) was performed according to Lehnen et al., 2010. Briefly regular insulin (Huminsulin Eli Lilly & company India Pvt Ltd, India) (0.75 U/kg) was injected subcutaneously to animals and blood samples were collected from tail vein at 0 (just before insulin injection), 30, 60, 120 min (after insulin the injection) for glucose estimation using glucometer (Hoffmann-La Roche Ltd, India).
6
Metabolic Profiling of High-Fat Diet Mice
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Blood glucose, cholesterol and triglycerides were measured in blood samples of overnight fasted mice with a glucose meter device (Accu-Chek, Roche) and the Accutrend Plus system (Roche). Fasting insulin concentrations were measured with an ELISA Kit (Crystal Chem). For insulin tolerance tests (ITT), mice were fasted for 6 h and then injected i.p. with insulin (1–1.5 U/Kg, Huminsulin, Lilly). Glucose concentrations were measured at specific time points up to 120 min after insulin injection.
For metabolic cage analysis55 (link), mice fed a HFD for 8 weeks were individually housed in metabolic chambers (PhenoMaster, TSE Systems) with free access to food and water, maintaining a 12 h : 12 h dark–light cycle. Mice were acclimatized in metabolic chambers for 24 h before initiation of data collection. The volume of oxygen consumption (VO2) and carbon dioxide production (VCO2) were determined every 20 min for a period of 3 days. Respiratory exchange ratio (RER) was defined as VCO2/VO2 and energy expenditure (EE) was determined by using the formula 3.941×VO2 + 1.106×VCO2; food uptake was also determined. Statistics were performed using ANCOVA analysis. Determination of lean and fat mouse mass was performed by using computed tomography (CT, Skyscan 1178; Bruker)56 .
For metabolic cage analysis55 (link), mice fed a HFD for 8 weeks were individually housed in metabolic chambers (PhenoMaster, TSE Systems) with free access to food and water, maintaining a 12 h : 12 h dark–light cycle. Mice were acclimatized in metabolic chambers for 24 h before initiation of data collection. The volume of oxygen consumption (VO2) and carbon dioxide production (VCO2) were determined every 20 min for a period of 3 days. Respiratory exchange ratio (RER) was defined as VCO2/VO2 and energy expenditure (EE) was determined by using the formula 3.941×VO2 + 1.106×VCO2; food uptake was also determined. Statistics were performed using ANCOVA analysis. Determination of lean and fat mouse mass was performed by using computed tomography (CT, Skyscan 1178; Bruker)56 .
7
Metabolic Profiling of Mice Fed High-Fat Diet
8
Assessing T-cell Activation in Blood Samples
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Lymphocytes were stimulated with whole blood by overnight incubation, and flow cytometry and quantification of CD69 on CD4+ and CD8+ T-lymphocytes were performed by FastImmune assay (BD Biosciences, Heidelberg, Germany), as previously described by Arneth (5 (link), 6 (link)). Each blood sample was divided into 4 1-mL aliquots (Tubes 1, 2, 3 and 4). Tube 1 was treated with 50 µL of 0.2 g/mL concanavalin A (ConA; Sigma-Aldrich), Tube 2 was not treated, Tube 3 was treated with 50 IU/mL human insulin (Huminsulin; Eli Lilly) and Tube 4 was treated with human GAD (Sigma-Aldrich). In addition, the 12 samples from the healthy volunteers were treated using a BioTrek Transfection Kit (Stratagene) and subsequently incubated.
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