Journal: Journal of Diabetes Investigation

Article Title: Genome‐wide scan for circulating vascular adhesion protein‐1 levels: MACROD 2 as a potential transcriptional regulator of adipogenesis

doi: 10.1111/jdi.12805

Figure Lengend Snippet: The effect of knockdown of MACRO domain containing 2 ( MACROD 2 ; si MACROD 2) on (a) MACROD 2 and (b) vascular adhesion protein‐1 ( VAP ‐1 ) gene expression in human primary preadipocytes ( n = 14). (c) VAP ‐1 concentration in culture medium ( n = 10). (d) Expression of adipogenic genes including FABP 4 , ADIPOQ , CEBPA , PPARG 2 and SREBP 1 ( n = 14) as compared with control (siControl). Data is expressed as mean ± standard error. * P < 0.05; ** P < 0.01; # P < 0.1. mRNA , messenger ribonucleic acid.

Article Snippet: Human visceral preadipocytes, preadipocyte growth supplement and preadipocyte differentiation supplement were purchased from ScienCell Research Laboratories (San Diego, California, USA).

Techniques: Knockdown, Gene Expression, Concentration Assay, Expressing, Control

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

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