Abstract
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.
Highlights
Cultured 3T3-L1 cells are widely used as a model in adipocyte research due to their ability to differentiate and accumulate triglycerides in lipid droplets, and due to their structural and metabolic characteristics that mimick primary adipocytes [1]
Adipocytes cultured and differentiated under 4% O2 accumulated 4.2 ± 0.8 resp. 2.2 ± 0.2 times more lipids than cells exposed to 20% O2 or 1% O2, Fig 1A
When compared to cells cultured under 20% and 1% O2, growing adipocytes under 4% O2 improved cell viability by 220 ± 20% and 230 ± 40%, respectively, (p < 0.05)
Summary
Cultured 3T3-L1 cells are widely used as a model in adipocyte research due to their ability to differentiate and accumulate triglycerides in lipid droplets, and due to their structural and metabolic characteristics that mimick primary adipocytes [1]. Pericellular oxygen levels are strongly affected by cell metabolic activity and cell number (confluency), both of which continuously change during a typical culture and/ or experimental procedures. These changes in oxygen consumption rate modify the pericellular O2 levels, and reaching and maintaining a stable pericellular O2 concentration represents a major challenge for in vitro models
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