Journal: Iranian Journal of Basic Medical Sciences

Article Title: Considering the protective effect of exendin-4 against oxidative stress in spiral ganglion neurons

doi: 10.22038/IJBMS.2023.69190.15076

Figure Lengend Snippet: Exendin-4 has antagonistic effect on kanamycin-induced SGNs damage

Article Snippet: Exendin-4 (exenatide) was purchased from MedChemExpress (New Jersey, USA), and the cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Techniques:

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Considering the protective effect of exendin-4 against oxidative stress in spiral ganglion neurons

doi: 10.22038/IJBMS.2023.69190.15076

Figure Lengend Snippet: Kanamycin-induced ANF damage was reduced by exendin-4

Article Snippet: Exendin-4 (exenatide) was purchased from MedChemExpress (New Jersey, USA), and the cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Techniques:

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Considering the protective effect of exendin-4 against oxidative stress in spiral ganglion neurons

doi: 10.22038/IJBMS.2023.69190.15076

Figure Lengend Snippet: Exendin-4 (100 nM) treatment could effectively antagonize the oxidative damage of SGNs induced by kanamycin (1 mM)

Article Snippet: Exendin-4 (exenatide) was purchased from MedChemExpress (New Jersey, USA), and the cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Techniques:

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Considering the protective effect of exendin-4 against oxidative stress in spiral ganglion neurons

doi: 10.22038/IJBMS.2023.69190.15076

Figure Lengend Snippet: Effect of exendin-4 on the protein expression of Nrf2 and HO-1

Article Snippet: Exendin-4 (exenatide) was purchased from MedChemExpress (New Jersey, USA), and the cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Techniques: Expressing

Journal: Plant biotechnology journal

Article Title: Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

doi: 10.1111/pbi.12008

Figure Lengend Snippet: Evaluation of transgene integration. (a) Schematic representation of the flanking sequence probe (0.81 kb) and expected products of the untransformed and transplastomic tobacco chloroplast genome when digested with HindIII (H). Southern blot probe was generated using BamHI (B) and BglII (B′). Primers used for transgene amplification are represented by arrows. Amino acid sequence indicates the hinge, furin cleavage site, and EX4 (bold letter). Asterisk represents resistant amino acid against DPP-IV. C-terminal 9-amino acid is represented by dotted line. (b) The amplification of genomic DNA fragment with 3P/3M and 5P/2M primer sets to check transgene integration. M, DNA size marker; WT, untransformed; #5, 6, and 9, transplastomic lines; Ve, CTB-EX4 containing pLD vector. (c) Southern blot analysis of CTB-EX4 transplastomic lines showing homoplasmy.

Article Snippet: The buffer was then removed and cells were incubated in Krebs-Ringer bicarbonate buffer with the various concentrations of purified CTB-EX4, commercial EX4 (California Peptide Research) and glucose (10 mM) for 45 min at 37°C.

Techniques: Sequencing, Southern Blot, Generated, Amplification, Marker, Plasmid Preparation

Journal: Plant biotechnology journal

Article Title: Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

doi: 10.1111/pbi.12008

Figure Lengend Snippet: Quantification and functional evaluation of CTB-EX4. Western blot analysis of total leaf homogenate (H, 5 μg) and soluble (S, 5 μg) protein probed with anti-CTB (a) or anti-EX4 (b) antibodies. Lane 1, 6.25; 2, 12.5; 3, 25 ng of purified cholera toxin B subunit; +, with DTT; −, without DTT; #6 and 9, CTB-EX4 transplastomic lines; WT, untransformed. (c) Percentage of CTB-EX4 in the total leaf protein from mature leaves at different harvesting time. (d) GM1 ELISA assay for evaluation of CTB-EX4 pentamer assembly. CTB, positive control (10 ng); F, fresh leaf; L, lyophilized CTB-EX4 transplastomic plant extracts (5 μg of total leaf protein), respectively; WT, untransformed total leaf protein (5 μg), BSA, negative control. Data shown are means ± SD of three independent experiments.

Article Snippet: The buffer was then removed and cells were incubated in Krebs-Ringer bicarbonate buffer with the various concentrations of purified CTB-EX4, commercial EX4 (California Peptide Research) and glucose (10 mM) for 45 min at 37°C.

Techniques: Functional Assay, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

Journal: Plant biotechnology journal

Article Title: Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

doi: 10.1111/pbi.12008

Figure Lengend Snippet: Lyophilization and characterization. (a) Amount of CTB-EX4 protein in fresh (F) and lyophilized (L) leaves. (b) Western blot analysis of fresh and lyophilized leaves expressing CTB-EX4. Equal quantity (50 mg) of fresh and lyophilized material was extracted in same volume (300 μl) of extraction buffer. Samples were loaded in a serial dilution as indicated. Western blot analysis with anti-CTB (c) and anti-EX4 (d) polyclonal antibody to evaluate long-term stability of lyophilized CTB-EX4 after storage at room temperature for 10 months. F, fresh leaf; L, 10-month old lyophilized leaf. Total leaf protein (5 μg) was loaded in each lane. CTB, Positive or negative control (20 ng). Samples were incubated for 10 min with DTT (100 mM) or boiled for 2 minutes. (e) Western blot analysis to evaluate stability of PA in lyophilized lettuce leaves after storage at room temperature for 2 (1), 4 (2), and 6 (3) months. PA, standard (100 ng), WT, untransformed lettuce. Total soluble protein (3 μg) was loaded in each lane. (f) Antigen stability after 3 months of storage, and lyophilization for different durations: (1) 24, (2) 48 and (3) 72 hrs. Fold increase of total protein (g) and specific antigen (h) after lyophilization. PA, lettuce transplastomic plant expressing PA; CTB-Pins, lettuce transplastomic plant expressing CTB-Proinsulin; white bar, fresh material; grey bar, lyophilized material. (i) Western blot analysis of fresh (F) and lyophilized (L) leaves expressing PA. Total soluble protein (10 μl) was loaded after equal quantity (50 mg) was extracted in same volume (300 μl) of extraction buffer. (j) Microbial burden of leaves expressing PA. 1, fresh leaf; 2, lyophilized leaf; 3, commercially available freeze-dried alfalfa capsules. (k) Simplified diagram of capsulation of lyophilized transplastomic leaf material. Data shown are means ± SD of three independent experiments.

Article Snippet: The buffer was then removed and cells were incubated in Krebs-Ringer bicarbonate buffer with the various concentrations of purified CTB-EX4, commercial EX4 (California Peptide Research) and glucose (10 mM) for 45 min at 37°C.

Techniques: Western Blot, Expressing, Serial Dilution, Negative Control, Incubation

Journal: Plant biotechnology journal

Article Title: Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

doi: 10.1111/pbi.12008

Figure Lengend Snippet: Purification of CTB-EX4 and pancreatic cell line assay. Western blot (a) and silver staining (b) of purified CTB-EX4. 1–3, CTB standard proteins of 12.5, 25, and 37.5 ng; 4, wild type total leaf protein (5 μg); 5, soluble fraction of CTB-EX4 before purification (5 μg); 6, soluble fraction of CTB-EX4 after purification (5 μg); 7–9, washed fractions; 10–12, elution fractions. (c, d) Furin cleavage assay of purified CTB-EX4. CTB, standard (25 ng); −F, without furin; +F, with furin; M, protein size marker; M′, ultra-low range protein size marker; arrow head, cleaved CTB (12.7 kDa); arrow, cleaved EX4 (4.2 kDa); dots and numbers, locations of monomer and oligomers of CTB-EX4. Proteins were resolved on 12% (c) and 16% (d) of Tricine-SDS-PAGE. (e) Mouse pancreatic cell line assay. Commercial EX4 (1X =5 nM) and partially purified CTB-EX4 (1X = 32 nM, concentration based on the molecular weight of the CTB) were added to beta-TC6 cells. The graph was normalized to PBS value which was used as a negative control. Insulin secretion was compared at three different concentrations in each group, with duplicate samples, using 88 wells of insulin detection kit. Data shown are means ± SD (n=6).

Article Snippet: The buffer was then removed and cells were incubated in Krebs-Ringer bicarbonate buffer with the various concentrations of purified CTB-EX4, commercial EX4 (California Peptide Research) and glucose (10 mM) for 45 min at 37°C.

Techniques: Purification, Western Blot, Silver Staining, Cleavage Assay, Marker, SDS Page, Concentration Assay, Molecular Weight, Negative Control

Journal: Plant biotechnology journal

Article Title: Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

doi: 10.1111/pbi.12008

Figure Lengend Snippet: Evaluation of functionality of CTB-EX4 in mice after oral gavage or injection. Glucose (2g/kg body weight) was injected intraperitoneally at t=60 (arrows). (a) Mice were orally gavaged with lyophilized untransformed (WT) and CTB-EX4 plant leaf materials (EX4) at t=0. One outlier was removed from control group because of no glucose spike at t=90. (b) Mice were given i.p. injection of PBS (200 μl) and commercial EX4 resuspended in PBS (0.01 μg in 200 μl) at t=0. Glucose measurements were made 2 or 3 times for each mouse, for a total of 288 evaluations of blood glucose levels in mouse sera. Single factor ANOVA was performed to test significant difference between groups statistically. *P < 0.05, **P < 0.01.

Article Snippet: The buffer was then removed and cells were incubated in Krebs-Ringer bicarbonate buffer with the various concentrations of purified CTB-EX4, commercial EX4 (California Peptide Research) and glucose (10 mM) for 45 min at 37°C.

Techniques: Injection

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A Monolayers were stimulated with GLP or Exendin-4 for 5 min, 15 min, 30 min. (10 nM) as described in MATERIALS AND METHODS. Bars show % decrease in bioluminescence compared with unstimulated (no ligand) monolayer (means ± SE; *p < 0.05 compared with unstimulated monolayer). The experiment was repeated three times and compared with untreated controls and pre-immune serum treated controls. B: Cell fractionation of HuH7 monolayers was performed as described in MATERIALS AND METHODS. Samples from membrane (M), cytoplasm(C) and nuclear (N) fractions were subjected to Western blot analysis using anti-GLP-1R antibody (1:500). Blots were also probed for Na+-K+-ATPase, Lamin A/C and β-actin to confirm equal protein loading. The results are representative of 2 independent experiments. C: Confocal imaging of GLP-1R was performed on filter grown monolayers of HuH-7 cells. Cells were stained with rabbit polyclonal antibody against GLP-1R (1:200) followed by Alexa Fluor secondary antibody. Rhodamine (blue arrow) was used to stain the cytoskeleton. In 1) GLP-1R (yellow arrow) is seen localized to the membrane and in 2) upon agonist stimulation GLP-1R is decreased from the membrane.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Cell Fractionation, Western Blot, Imaging, Staining

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B: Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; *p < 0.05 compared with untreated steatotic cells). The experiment was repeated three times in triplicate and compared with FFA exposed and non Exendin4 treated controls. C.: HepG2 cells were grown in either control media or methionine-choline deficient (MCD) media. Cells were then treated with Exendin-4 for 24 h. Following treatment, intracellular lipids (polar and neutral) were stained with Nile Red (NR) (0.5μg/ml). Flow cytometry was performed as described in Materials and Methods. 3T3L1 cells were served as a positive control. The figure is representative of three independent experiments.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Staining, Flow Cytometry, Positive Control

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH-7 cells were treated with GLP/Exendin-4 (10nM) following the time course indicated: 5, 15, 30, 60, 90 and 120 m and Western blot was performed. β-actin was used as loading control. A: Phosphorylation of PDK was induced. B–C: AKT phosphorylation was also increased in a time dependent manner as was the phosphorylation of PKC-ζ. All data presented are representative of the mean ± SE of at least 3 experiments *p<0.05 vs. basal or untreated.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. (B and C, respectively). These studies represent multiple independent experiments. (*p<.05 vs. control).

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Transfection, Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: In our previous work we demonstrated that GLP-1 or Exendin-4 increased cAMP production. Here we propose that the GLP-1 action shares key downstream components of the insulin signaling pathway, including PKCζ, which has been shown to be a key factor in NAFLD.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: