Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: Inhibition of ERK1/2 does not prevent palmitate inhibition of MafA expression and has no effect on PDX-1 and C/EBPβ expression. MIN6 cells were cultured for 24 h with 2 mmol/l or 11 mmol/l glucose, ± 0.5 mmol/l palmitate, in the presence or absence of U0126 (30 μmol/l). MafA ( A ), PDX-1 ( B ), and C/EBPβ ( C ) mRNA levels were measured by RT-PCR and normalized to β-actin mRNA. Results are expressed as fold increase of the ratio of gene/β-actin mRNA over control value and are means ± SE of three replicate experiments. ** P < 0.01, *** P < 0.001 vs. vehicle. Vehicle, □; U0126, ■.

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Inhibition, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: Overexpression of PAS kinase prevents the inhibitory effects of palmitate on insulin gene expression. A : Representative hPASK and p85 immunoblots of control, Adv-Luc-infected, or Adv-hPASK wild-type infected MIN6 cells cultured for 24 h in 2 or 11 mmol/l glucose ± 0.5 mmol/l palmitate. B : Insulin pre-mRNA levels. Results are expressed as fold increase of the ratio of mPre-INS2/β-actin mRNA over control value and are mean ± SE of seven replicate experiments. * P < 0.05, ** P < 0.01. C : Representative hPASK and p85 immunoblots of control, Adv-Luc-infected, Adv-hPASK wild-type or Adv-hPASK kinase-dead infected rat islets cultured for 24 h in the presence of 16.7 mmol/l glucose + 0.5 mmol/l palmitate. D : Pre-INS2, MafA, PDX-1, and C/EBPβ mRNA levels were measured by RT-PCR and normalized to cyclophillin mRNA. Results are expressed as fold increase of the ratio of rGene/rCyclophillin mRNA over control value (Adv-Luc) and are means ± SE of three replicate experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. Cont, control; KD, kinase dead; Luc, luciferase; WT, wild type.

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Over Expression, Expressing, Western Blot, Infection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Luciferase

Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: PDX-1 protein expression in MIN6 cells overexpressing hPASK wild-type or kinase-dead. MIN6 cells were infected with adenoviruses expressing Luc ( A-C ), wild-type hPASK ( D-F ), or kinase-dead hPASK ( G-I ), and cultured for 24 h with 2 mmol/l glucose ( A , D , and G ), 11 mmol/l glucose ( B , E , and H ), or 11 mmol/l glucose + palmitate ( C , F , and I ). Cells were immunostained for PDX-1 and examined under a fluorescence microscope. Images are representative of three replicate experiments. KD, kinase dead; Luc, luciferase; WT, wild type. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Expressing, Infection, Cell Culture, Fluorescence, Microscopy, Luciferase