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:

Journal:

Article Title: Effects on Rotavirus Cell Binding and Infection of Monomeric and Polymeric Peptides Containing ?2?1 and ?x?2 Integrin Ligand Sequences

doi: 10.1128/JVI.78.21.11786-11797.2004

Figure Lengend Snippet: MA104 cell treatment with high concentrations of DGEA does not induce cell surface expression of α2β1, β2, and αvβ3 integrins (A), but the loss of DGEA-mediated inhibition of SA11 infectivity is inhibited by genistein (B). (A) Flow cytometric histograms. Cells were treated with 2.0 mM DGEA, control peptide GHRP, or mock treated, and their surface integrin expression levels were determined by flow cytometry after staining with monoclonal antibodies AK7 (α2I), P4H9-A11 (β2), and LM609 (αvβ3). Cells were also stained with isotype control monoclonal antibody MOPC21 at the same concentration as each test monoclonal antibody (control). Mock-treated and peptide-treated cells stained with MOPC21 gave identical histograms (control). In panel B, cells were mock treated or treated with 10 μM genistein prior to incubation with peptide DGEA or GHRP at concentrations ranging from 0.015 to 1.0 mM, followed by assay of SA11 rotavirus infectivity, as described in Materials and Methods. The infectivity titer of SA11 in cells treated with peptide with and without genistein is expressed as a percentage of the titer obtained in the absence of peptide or genistein.

Article Snippet: Peptides GFOGER, Gly-Phe-Hyp-Gly-Ala-Arg (GFOGAR), Asp-Gly-Ala-Ala (DGAA), and Gly-His-Arg-Pro (GHRP) that were ≥90% pure by high-performance liquid chromatography were purchased from Auspep, Victoria, Australia, and dissolved in Dulbecco's modified Eagle's medium at pH 7.5, as described previously ( 14 ).

Techniques: Expressing, Inhibition, Infection, Control, Flow Cytometry, Staining, Bioprocessing, Concentration Assay, Incubation