Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.

Center for Research on Diabetes Metabolism and Nutrition 3rd Faculty of Medicine Charles University Prague Prague Czech Republic

Center for Research on Diabetes Metabolism and Nutrition 3rd Faculty of Medicine Charles University Prague Prague Czech Republic Centre of Toxicology and Health Safety The National Institute of Public Health Prague Czech Republic 2nd Internal Medicine Department University Hospital Kralovske Vinohrady Prague Czech Republic

Department of Biochemistry Cell and Molecular Biology Division of Cell and Molecular Biology 3rd Faculty of Medicine Charles University Prague Prague Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Laboratory for Metabolism and Bioenergetics 3rd Faculty of Medicine Charles University Prague Prague Czech Republic

Laboratory of Molecular Structure Characterization Institute of Microbiology of the CAS v v i Prague Czech Republic

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$a Pavlikova, Nela $u Department of Biochemistry, Cell and Molecular Biology-Division of Cell and Molecular Biology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

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