ted microscope with a proprietary XYZ stage KU55933 manufacturer enclosed in a controlled environment chamber with differential interference contrast transmitted light and a solid state module for fluorescence, Lipid Assays Adherent adipocytes in a 48-well plate were washed with PBS and 400 ml of PBS and 12 ml of AdipoRed reagent added. Plates were read on a Synergy 2 Multi-Mode Microplate Reader with fluorescent reading capabilities controlled by BioTek’s Gen5TM Reader Control and Data Analysis Software. AdipoRed uptake Hypoxia and Adipocyte Lipid Metabolism and a Nikon Coolsnap ES2 HQ camera. Z-stack images were transferred to Imaris, a multidimensional analysis program for analysis of fluorescence intensity data. Surfaces were created based on A488 emission to track cells and extract fluorescent intensity. The intensity sum was divided by the number of cells in the viewing field, and resulting average intensity per cell was reported. HBS Increases during Human Adipocyte Differentiation Given the observed increase in GFAT transcript levels with differentiation, we next studied HBS over the course of adipocyte differentiation. When compared to undifferentiated SVF, mature differentiated human visceral adipocytes demonstrated increased expression on Western blot analysis of adipocyte protein lysates with antibody specific for OGlcNAc, the glycosylation moiety that is covalently linked to multiple cellular proteins when HBS is activated, indicative of increased HBS over the course of adipocyte differentiation. PCR RNA was prepared from cells using an RNeasy lipid kit and treated with DNase. Equal amounts of input RNA were used for all reactions. RNA 
was reverse-transcribed using random hexamer primers. QRTPCR was performed using SYBR Green reagent and transcript-specific primers on an ABI7900 thermocycler. GAPDH and actin were used as endogenous controls and provided similar results in all cases. Fold changes relative to actin are reported. The 2ddCT relative quantification method was used to calculate fold difference in transcript levels between samples; efficiencies of amplification for all primer pairs were verified to be equivalent over a range of template concentrations. Hypoxia Inhibits HBS in Human Adipocytes Given that hypoxia is implicated in adipose tissue dysfunction in obesity, we next studied the role of hypoxia in regulating HBS. Hypoxia inhibited HBS in mature human visceral adipocytes based on Western blot analysis of adipocyte protein lysates and immunofluorescence staining of mature adipocytes with antibody specific for OGlNAc.Hypoxia inhibits lipogenesis over the course of differentiation in human visceral adipocytes based on AdipoRed staining, with no effect on cell viability, based on viability assays using propidium iodide nuclear staining, Hoechst dye staining, XTT metabolism, and trypan blue exclusion. Similar results with respect to lipid accumulation and viability were observed in subcutaneous adipocytes. Given the correlation of HBS 18194435 with lipogenesis during differentiation, we next asked whether HBS was required for lipogenesis during adipocyte differentiation. Inhibition of HBS with the small molecule inhibitor azaserine attenuated lipogenesis independent of hypoxia, consistent with a positive 12176911 effect of HBS on adipocyte lipogenesis. Similar results were observed in subcutaneous adipocytes. Addition of glucosamine, a substrate for and promoter of HBS, slightly increased adipocyte lipogenesis in hypoxic conditions,, also consistent w
