Cytosolic glucose concentration reflects the balance between glucose entry across the

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (R) in fibroblasts. Adrenaline increased cytosolic glucose concentration purchase EPZ-5676 in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls ( 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose. (6) constructed a fluorescence resonance energy transfer (FRET)-based glucose nanosensor, specifically expressed in cytosol. Nanosensor consists of a bacterial periplasmic glucose/galactose-binding protein, flanked with two green fluorescent protein variants, cyan (CFP) and yellow fluorescent protein (YFP). This construct undergoes a decrease in YFP/CFP ratio upon substrate binding and has been used successfully to estimate changes in cytosolic free glucose concentration in COS-7, CHO, HEK293, C2C12, and other cell types (5, 6). Determination of intracellular concentration of free glucose is usually of particular desire for insulin-sensitive cells (muscle mass and excess fat) to study the glucose handling under different conditions. 3T3-L1 fibroblasts are derived from mouse embryonic tissue and differentiate into adipocytes DH5 and purified using a commercially available protocol, the PureYield plasmid miniprep system (Promega). Plasmid was launched into 3T3-L1 cells by using the Cell Collection Nucleofector kit L (for Nucleofector II, Amaxa). Briefly, after trypsinization and centrifugation of the cell suspension, the pellet (1 106 to 5 106 cells) was resuspended in 100 l Rabbit Polyclonal to GPRIN3 of supplemented Nucleofector Answer L and mixed with 3C5 g of plasmid DNA. After nucleofection and centrifugation of the cell suspension, the cell pellet was resuspended in cell culture medium. Cells were seeded onto poly-l-lysine-coated coverslips and incubated in a humidified atmosphere at 37 C and 5% CO2. Fluorescence Microscopy purchase EPZ-5676 Imaging was performed 24C48 h after nucleofection using a fluorescence microscope Zeiss Axiovert 135 (Zeiss, Oberkochen, Germany) equipped with a Till photonics system purchase EPZ-5676 and a CCD video camera. Dual emission intensity ratios were recorded using monochromator Polychrome IV (Till Photonics, Gr?feling, Germany) with 436 nm/10 nm excitation and two emission filters (465 nm for CFP and 535 nm for YFP) and neutral density filter (optical density = 0.6). YFP and CFP images were acquired simultaneously using a Dual View image splitter (Optical Insights, Tucson, AZ). For analysis, background light intensity was subtracted from the individual YFP and CFP emission. Images of fluorescent 3T3-L1 cells were acquired through a water immersion objective C-Apochromat (63, numerical aperture (NA) = 1.2) at intervals of 10 or 20 s. Exposure time was 700 ms. Determination of Changes in YFP/CFP Ratio and Statistical Analysis FRET was decided as the YFP/CFP emission intensity ratio. Ratio changes (R) were calculated as purchase EPZ-5676 the difference between average ratios during superfusion with glucose-free medium and superfusion with glucose medium. Intracellular glucose concentrations at respective extracellular glucose were calculated using Equations 1 and 2. The minimum R (Rmin) at the complete absence of glucose was measured between two superfusions with glucose-free medium, and the maximum R (Rmax), at saturation with glucose, was measured after permeabilization with -escin (100 purchase EPZ-5676 m) at 10 mm extracellular glucose. where [Glc]extra represents extracellular glucose concentration, S[Glc]extra is the normalized value of R at certain extracellular glucose concentrations, and is the glucose binding affinity of FLIPglu-600 and was decided to be 0.589 mm (6). Unless stated otherwise, statistics are in the.