Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of resveratrol for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of resveratrol for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of resveratrol. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of curcumin for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of curcumin for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of curcumin. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of Zyflamend® for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of Zyflamend® for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of Zyflamend®. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: Cells were treated with vehicle or the indicated concentration of LY294002 for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 2 hours. Western blot analysis was used to determine levels of Phospho-Akt and Akt (A) or Phospho-p65 and p65 (C). B, Cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of LY294002 for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. D, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of LY294002. Aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01. E-H, Cells were treated with vehicle or the indicated concentration of resveratrol (E), curcumin (F), EGCG (G) or Zyflamend® (H) for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 2 hours. Western blot analysis was used to determine levels of Phospho-Akt and Akt.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Concentration Assay, Western Blot, Transfection, Luciferase, Activity Assay, Incubation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A and B, Western blot analysis was used to determine levels of Phospho-Akt and Akt (A) and Phospho-p65 and p65 (B) in the mammary glands of four mice in each of the indicated treatment groups; C, 5 μg of nuclear protein was incubated with a 32P-labeled oligonucleotide containing NF-κB binding sites. Binding of nuclear protein from mammary glands is shown for each of the indicated treatment groups. D, Paracrine interactions between macrophages and other cell types, e.g., preadipocytes can explain the elevated levels of aromatase in the mammary glands of obese mice. In obesity, lipolysis is increased resulting in increased concentrations of free fatty acids. Saturated fatty acids stimulate the PI3K/Akt pathway leading to activation of NF-κB in macrophages and increased production of proinflammatory mediators (TNF-α, IL-1β and PGE2). Each of these proinflammatory mediators can act in a paracrine manner to induce aromatase. Polyphenols block the activation of the PI3K/Akt/NF-κB pathway in macrophages and thereby suppress the induction of proinflammatory mediators and aromatase.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Western Blot, Incubation, Labeling, Binding Assay, Activation Assay, Blocking Assay

Journal: BMJ Open Diabetes Research & Care

Article Title: Statins impair glucose uptake in human cells

doi: 10.1136/bmjdrc-2014-000017

Figure Lengend Snippet: Morphology of cells used throughout the experiments. (A) Human visceral preadipocytes were differentiated for 10 days according to the manufacturer's protocol. Next, the cells were stained with Oil Red O and visualized under phase contrast microscope. Magnification ×250. (B) Human skeletal muscle cells (Lonza) were differentiated for 5 days in Dulbecco's modified Eagle's medium (DMEM) medium + 2% horse serum. The cells were fixed in ice-cold methanol and stained with DAPI and anti-desmin-Alexa Fluor488 antibody. Fluorescence microscopy, magnification ×200. DAPI (left panel) and antidesmin stain (right panel). (C) Human skeletal muscle myoblasts (Lonza) were differentiated for 5 days in DMEM medium + 2% horse serum. The cells were fixed in ice-cold methanol and stained with DAPI and anti-desmin-Alexa Fluor488 antibody. Fluorescence microscopy, magnification ×200. DAPI (left panel) and antidesmin stain (right panel).

Article Snippet: Poietics Human Visceral Preadipocytes, Clonetics Human Primary Hepatocytes (NHEPS), human skeletal muscle cells (SkMc), and human skeletal muscle myoblasts (HSMM) were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer’s protocol.

Techniques: Staining, Microscopy, Modification, Fluorescence

Journal: BMJ Open Diabetes Research & Care

Article Title: Statins impair glucose uptake in human cells

doi: 10.1136/bmjdrc-2014-000017

Figure Lengend Snippet: Lovastatin decreases glucose uptake in non-malignant cell lines. (A) Differentiated human visceral preadipocytes, normal human skeletal muscle cells (SkMc), differentiated human skeletal muscle myoblasts (HSMM) or differentiated human hepatocellular carcinoma cells (HepG2/C3A) were incubated for 48 h with 10 µM lovastatin. Next, the cells were washed with phosphate-buffered saline (PBS) and incubated with either 300 µM 6-( N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG) in PBS or with 1.5 µL of [1,2-3H]-deoxy-D-glucose (2-DOG; 8.0 mCi/mL radionuclide concentration) for 30 min at 37°C. After three-time wash in cold PBS, the cells were analyzed in flow cytometry (6-NBDG) or in scintillation counter (2-DOG). The figure presents mean fluorescence intensity (MFI) or mean counts per minute (cpm)±SD; *p<0.05 vs controls in Student t test. (B) Normal human SkMc or differentiated human HepG2/C3A were incubated for 48 h with 5 µM (L5) or 20 µM (L20) lovastatin. Next, culture media were diluted and incubated with the reaction buffer composed of 10 mM Amplex Red, 10 U/mL horseradish peroxidase, and 100 U/mL glucose oxidase. The samples were light-protected and incubated at room temperature for 30 min. Next, absorbance at 560 nm was measured with spectrophotometer. Glucose values were calculated according to the glucose standard curve and normalized to the protein content measured with Bio-Rad Protein Assay; *p<0.05 vs controls in Student t test. (C) Normal human SkMc or differentiated human HepG2/C3A were incubated for 48 h with 10 µM atorvastatin (Ator), 10 µM fluvastatin (Flu), 10 µM simvastatin (Sim) and 1 µM cerivastatin (Cer). Next, the cells were washed with PBS and incubated with 300 µM 6-NBDG in PBS for 30 min at 37°C. After three-time wash in cold PBS, the cells were trypsinized and analyzed by flow cytometry. The figure presents MFI±SD; *p<0.05 vs controls in one-way analysis of variance and Tukey's post hoc test.

Article Snippet: Poietics Human Visceral Preadipocytes, Clonetics Human Primary Hepatocytes (NHEPS), human skeletal muscle cells (SkMc), and human skeletal muscle myoblasts (HSMM) were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer’s protocol.

Techniques: Incubation, Concentration Assay, Flow Cytometry, Fluorescence, Spectrophotometry

Journal: BMJ Open Diabetes Research & Care

Article Title: Statins impair glucose uptake in human cells

doi: 10.1136/bmjdrc-2014-000017

Figure Lengend Snippet: Decreased glucose uptake results from inhibition of mevalonate pathway. (A) Differentiated human visceral preadipocytes were incubated for 30 min with 10 mg/mL methyl-β-cyclodextrin (MβCD). Next, the cells were washed with phosphate-buffered saline (PBS) and incubated with 300 µM 6-( N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG) in PBS for 30 min at 37°C. After three-time wash in cold PBS, the cells were trypsinized and analyzed by flow cytometry. The figure presents mean fluorescence intensity (MFI)±SD; *p<0.05 vs controls in Student t test. (B) Differentiated human visceral preadipocytes or differentiated human skeletal muscle myoblasts (HSMM) were incubated for 48 h with 10 µM lovastatin (L). For the last 30 min of incubation, 0.2 mg/mL of water-soluble cholesterol–MβCD (chol) was added. Next, the cells were washed with PBS and incubated with 300 µM 6-NBDG in PBS for 30 min at 37°C. After a three-time wash in cold PBS, the cells were trypsinized and analyzed by flow cytometry. The figure presents MFI±SD; *p<0.05 in one-way analysis of variance and Tukey's post hoc test. (C) Differentiated human visceral preadipocytes, normal human skeletal muscle cells (SkMc), differentiated HSMM or differentiated human hepatocellular carcinoma cells (HepG2/C3A) were incubated for 48 h with 10 µM L and 200 mM mevalonic acid (M). Next, the cells were washed with PBS and incubated with 300 µM 6-NBDG in PBS for 30 min at 37°C. After three-time wash in cold PBS, the cells were trypsinized and analyzed by flow cytometry. The figure presents MFI±SD; *p<0.05 in one-way analysis of variance and Tukey's post hoc test.

Article Snippet: Poietics Human Visceral Preadipocytes, Clonetics Human Primary Hepatocytes (NHEPS), human skeletal muscle cells (SkMc), and human skeletal muscle myoblasts (HSMM) were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer’s protocol.

Techniques: Inhibition, Incubation, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs

doi: 10.3892/ijmm.2016.2729

Figure Lengend Snippet: Metformin suppresses the maturation of human visceral preadipocytes with no suppression of cell growth. (A) Metformin suppresses lipid droplet accumulation in human visceral preadipocytes. Oil Red O staining of HPrAD-vis cells was performed after 2 weeks of culture with or without metformin. The stained area was reduced in metformin-treated cells compared to non-treated cells, ×200. (B) The relative stained area of metformin-treated cells and non-treated cells. Cells were cultured for 1 or 2 weeks with or without metformin. The stained areas per ×200 field were measured using Image J. (C) Adiponectin secretion from HPrAD-vis cells is decreased following treatment with metformin. The cells were cultured for 1 or 2 weeks with or without metformin (n=3). The adiponectin concentration in the culture media was determined by ELISA with the specific antibody Acrp30. (D) Metformin did not suppress the growth of fatty cells. Cells were incubated with or without metformin for 1 week. Cell proliferation was measured using a WST-8 assay (n=6). (E) Genes involved in the differentiation and maturation of preadipocytes are downregulated by metformin. Cells were incubated for 1 week with or without metformin. The relative expression of PPARγ and C/EBPα in metformin-treated cells compared to non-treated cells was determined using RT-qPCR with relative quantification (n=3). β-actin was used as an internal control gene. (F) Effect of metformin on gene expression was confirmed by western blot analysis. The cells were incubated for 1 week with or without metformin. The protein expression level of C/EBPα decreased after treatment. HPrAD-vis, human visceral preadipocytes; ELISA, enzyme-linked immunosorbent assay; PPARγ, peroxisome proliferator-activated receptor γ; C/EBPα, CCAAT-enhancer-binding protein α.

Article Snippet: PoieticsTM human visceral preadipocytes (HPrAD-vis) were purchased from Lonza (Walkersville, MD, USA).

Techniques: Staining, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Control, Gene Expression, Western Blot, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs

doi: 10.3892/ijmm.2016.2729

Figure Lengend Snippet: Hierarchical clustering of miRNAs from HPrAD-vis cells. Hierarchical clustering was performed for miRNA expression profiles of control HPrAD-vis cells and cells cultured with 5 mM metformin for 1 (left-side panel) or 2 weeks (left-side panel). The samples are arranged in columns and miRNAs in rows. The miRNA clustering tree is shown on the left, and the sample clustering tree is shown at the top of each heat map. The heat maps show the relative expression intensity for each miRNA in which the base-2 logarithm of the intensity is median-centered for each row. The color-coding is indicated as a horizontal bar at the bottom left. The six miRNAs with an asterisk are common between data from 1 week incubation with metformin (P<0.05) and 2 weeks incubation with the reagent (P<0.005) regardless of fold-change (n=5). miRNAs, microRNAs; HPrAD-vis, human visceral preadipocytes.

Article Snippet: PoieticsTM human visceral preadipocytes (HPrAD-vis) were purchased from Lonza (Walkersville, MD, USA).

Techniques: Expressing, Control, Cell Culture, Incubation