Humulin u 100

Passage 3 TPCs were plated at 5 × 10³ cells/cm² in 6-well cell culture plates and maintained in complete DMEM until they reached 80-90%% confluence. Complete DMEM was then substituted with osteogenic medium (complete DMEM supplemented with 10 mM β-glyceraldehyde-3-phosphate, 50 μg/mL ascorbic acid, 100 nM dexamethasone) or osteogenic medium with 0.07 or 0.7 nM insulin (Humulin® U-100, Lilly, USA). The medium was replaced every 48-72 hours [15 (link), 16 (link)]. The cultures were maintained for 14 days. In addition, TPC osteogenic cultures (with 0, 0.07, and 0.7 nM insulin) were also maintained with or without 100 nM picropodophyllin (PPP; Selleckchem, # S766802), a small molecule inhibitor of IGF-I receptor. Control osteogenic cultures were cultured (+/- insulin) with an equal amount of DMSO.

Passage 3 TPCs were plated at 3 × 10³ cells/cm² in basal medium in 96-well plates. After 1-day culture, insulin (at 0 nM, 0.007 nM, 0.07 nM, and 0.7 nM concentrations; Humulin™ U-100, Lilly, USA) treatments were added and incubated for 3 days. Three replicate wells for each treatment group were used to measure the cell numbers via a mitochondrial metabolic assay (Cell Titer MTT 96 aqueous one solution cell proliferation assay, Promega) which was used in accordance with the manufacturer's instructions. In brief, 20 μL of the assay reagent containing tetrazolium was added into each well of the 96-well plate containing 100 μL of fresh media and incubated at 37°C for 2.5 hours. Absorbance was measured at 490 nm in a microplate reader (Tecan™ Infinite 200 PRO plate reader) to detect concentrations of the metabolic product, formazan. The mean value was calculated from replicate wells to provide a single data point. These optical density values from plated TPCs from each horse were reported.

Glucose (GTT), insulin (ITT) and pyruvate (PTT) tolerance tests were performed as described previously [37 (link)] after the mice were challenged with HFD and treated with carvedilol for 4 weeks. Body weight were 48.98 ± 1.22 g for HFD-fed + vehicle treatment cohort (n = 6) and 47.62 ± 0.91 g for HFD-fed + carvedilol treatment cohort (n = 6), P = 0.392. For GTT and PTT, mice were fasted overnight for 16 hours with water ad libitum provided. After measurement of fasted glucose levels, mice were intraperitoneally injected with a glucose solution (1.5 g/kg body weight) or sodium pyruvate solution (2 g/kg body weight) in normal saline. For ITT, mice were fasted for 2 hours and provided with water ad libitum. After measurement of basal glucose levels, 0.75 U/kg body weight of regular insulin (Humulin® U-100, Eli Lilly and Co., IN, USA) in normal saline was administered intraperitoneally. Blood samples were taken from a tail nick at 0, 15, 30, 60, 90, 120, and 150 minutes after injection and glucose levels were measured. The area under the curve (AUC) was determined to quantify the glucose, pyruvate and insulin tolerance.

Figure 1 shows the full-length proinsulin and pro-IAPP sequences, including the IAPP insertion sites within proinsulin (left) and cleavage site for the IAPP peptide (right) (Figure 1A). Schematics of all probe constructs are shown in detail in Figure 1B.
Linear HIP peptides (Figure 1B Top) were synthesized at >80% purity (Peptide 2.0, Chantilly, VA, USA) and resuspended at 2 mg/mL in 2XPBS. Cyclic C47/IAPP HIP peptides were synthesized at >98% purity (Peptide 2.0, Chantilly, VA, USA), and peptides were reconstituted at 2 mg/mL in PBS.
Production of recombinant three-dimensional HIPs (Figure 1B) was outsourced (Creative BioMart, Shirley, NY, USA). Double-stranded DNAs encoding three-dimensional (3D) HIPs were generated using complementary overlapping PCR primers (Integrated DNA Technologies, Coralville, IA, USA), with unique restriction enzyme sites at the 5′ and 3′ ends for directional cloning into pcDNA3.1.
DNA was subcloned into vectors for bacterial expression and purification (>90% purity). PI HIP recombinant proteins were denatured and refolded according to a proprietary protocol, and resuspended in a buffer (20 mM Tris-HCL (pH 8.0), 150 mM NaCl) at 0.5 mg/mL. Recombinant human insulin (Humulin U-100, 100 units/10 mL) was purchased from Eli Lilly, (Indianapolis, IN, USA). HPLC-purified (>99%) human proinsulin was purchased from AmideBio (Louisville, CO, USA).

An insulin tolerance test (ITT) was conducted to investigate the role of IES on insulin sensitivity. Regular insulin (Humulin U-100; Lilly, Indianapolis, IN, USA) in a saline solution (0.5U/kg) was intraperitoneally injected following a 14 h fast. Blood was collected from the tail vein to measure blood glucose level at baseline and 30, 60, and 120 min after insulin injection. The 12 GK rats with one pair of chronic IES electrodes were studied in two ITT sessions on separate days with IES using the optimized parameters from Experiment 1.1 and Sham-IES (same experimental setup but 0 mA output), respectively.