The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. subsequently treated as indicated. Protein extraction and Western blot analysis of intracellular proteins in HRPTECs. Total cellular extracts from HRPTECs were prepared, and Western blot was carried out using a denaturing 8% Novex Tris-glycine gels (Invitrogen, Carlsbad, CA) or 10% NuPage Bis-Tris SDS-PAGE gels under reducing conditions, as described previously (24). Membranes were washed and reprobed with an antibody against -actinin (Sigma) to control for small variations in protein loading and transfer. Images were acquired using the Adobe Photoshop program (Adobe Systems, San Jose, CA) and processed using Multi Guage (Fuji Film, Tokyo, Japan) for densitometric analysis. Signal intensities in control lanes were arbitrarily assigned a value of 1 1.00. Oxygen consumption measurements. Cells were incubated under normoxic (21% O2) or hypoxic (1% O2) conditions in medium containing 5.5 mmol/L (low) or 25 mmol/L (high) glucose and then resuspended in normoxic medium. Average oxygen consumption rates in HRPTECs treated with reagents in normoxia or hypoxia for 4 h were measured in a sealed chamber using a Clark-type electrode. Measurement of cell ATP. HRPTECs were incubated with reagents under normoxic or hypoxic conditions for 4 h. ATP production was monitored by glucose-6-phosphate formation. Briefly, cells were extracted with perchloric acid (6%) and centrifuged (8,000for 10 min). Subsequently, the extract was neutralized with K2CO3 (5 mol/L) neutralized to pH 7. NADP+ (0.5 mmol/L) and glucose 6-phosphate dehydrogenase (0.25 units) were then added and ATP production was monitored from the NADPH content by spectrophotometry at 340 nm. Cell proteins were determined in parallel dishes for the normalization of the ATP values. Imaging of reactive oxygen species. The oxidative fluorescent dihydroethidium (DHE) (Sigma) was used to evaluate the intracellular production of superoxide (O2?) (25). In brief, after incubation overnight, cells with or without 1 mmol/L metformin or 1 mmol/L AICAR under normoxic and hypoxic conditions were washed with serum-free and phenol-redCfree DMEM and loaded with 5 mol/L DHE. After incubation for 10 min in the Rabbit Polyclonal to BAZ2A dark, the cells were washed with PBS and were subjected to fluorescence microscopy. NADPH content. NADPH content was determined using a NADP/NADPH Quantification kit (BioVision, Mountain View, CA) and the protocol supplied by the manufacturer. Immunocytochemistry. HRPTECs were cultured on four-chamber glass slides (BD Ambrisentan (BSF 208075) Biosciences) to reach 80% confluence. After exposure to 1 mmol/L metformin or 1 mmol/L AICAR for 4 h under normoxic or hypoxic conditions, the cells were fixed with 100% ethanol for 10 min and were incubated with an antiCHIF-1 antibody (1:100; BD Ambrisentan (BSF 208075) Biosciences) at 4C overnight. Then, cells were rinsed in PBS and subsequently incubated with Alexa Fluor 594 donkey anti-mouse secondary antibody (Invitrogen) at 1:200 dilution overnight at 4C. Finally, slides were analyzed by cofocal laserCscanning microscopy. Detection of cellular hypoxia. Cellular hypoxia was detected by adding pimonidazole hydrochloride (200 mmol/L hypoxyprobe-1; Hydroxyprobe, Burlington, MA), which binds to cells or tissues with pO2 levels 10 mmHg, to HRPTECs that were treated with 1 mmol/L metformin or 1 mmol/L AICAR and exposed to hypoxia (1% O2) for 4 h. To detect hypoxic conditions in each group of rats, pimonidazole (60 mg/kg) was injected intraperitoneally 1 h before they were killed. Staining was performed according to the manufacturers instructions. Animals. Male ZDF/Gmi-rats and their heterozygous (ZDF/Gmi-+/ 0.05 were considered statistically significant. RESULTS Metformin inhibits hypoxia-induced HIF-1 protein accumulation. We investigated the impact of metformin on hypoxia-induced HIF-1 expression. HRPTECs faintly expressed HIF-1 protein under normoxic condition (Fig. 1 0.01) and a significant ~85% attenuation in hypoxia-induced HIF-1 protein accumulation in HRPTECs treated with 1 mmol/L metformin, compared with hypoxia-treated controls ( 0.01) (Fig. 1andD 0.01 vs. control under normoxic conditions; ** 0.01 vs. control under hypoxic Ambrisentan (BSF 208075) conditions. 0.01 vs. control under hypoxic conditions; $$ 0.01 vs. 0.01 mmol/L metformin-treated cells under hypoxic conditions. and target genes. HRPTECs were treated with or.