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