The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware

<div><p>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. <i>In vitro</i> 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.</p></div

Triglyceride accumulation under exposure to 1%, 4% and 20% O₂.

<p>3T3-L1 cells were cultured and differentiated under desired O₂ concentrations for 14 days. Lipid accumulation was evaluated using Oil Red O staining. (A) Data are expressed as fold-change relative to a control condition (20% O₂), (B) Data are normalized to total protein content. * p < 0.05 for comparison with 20% O₂.</p

A graphical interpretation of the identified proteins in the reversed TCA cycle.

<p>Tricarboxylic acid cycle is depicted with marked proteins upregulated by exposure to 1% O₂ and 4% O. Glutamine enters the reversed TCA cycle as α-ketoglutarate and is subsequently processed to citrate and acetyl-CoA. <i>Enzymes in metabolic pathways are shown as diamonds accompanied by enzyme nomenclature numbers</i>, <i>oval symbols represent substrates or products of metabolic reactions</i>. <i>Colored diamonds represent enzymes identified in proteomic analysis</i>. <i>1</i>.<i>3</i>.<i>5</i>.<i>1 –Succinate dehydrogenase</i>, <i>4</i>.<i>2</i>.<i>1</i>.<i>3 –Aconitate hydratase</i>, <i>1</i>.<i>1</i>.<i>1</i>.<i>41 –Isocitrate dehydrogenase</i>.<i>"</i></p

Proteins upregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).

<p>Proteins upregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).</p

Proteins downregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).

<p>Proteins downregulated in 1% O₂ and 4% O₂ compared to control conditions (20% O₂).</p