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

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.

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…A recent study explored the effect of IH in OSA on insulin resistance and the results from the in vitro model, which utilized gas-permeable dishes and OSA pathophysiology, were in accord with the animal model and patient cohort observations, including changes in NF-κB modulation (65). Moreover, adipocytes grown on the same type of cultureware and that were subjected to clinically-relevant IH exhibited an accumulation of triglycerides, which correlates with the observed link between obesity and OSA in patients (42). Conversely, culturing adipocytes in suboptimal settings without pericellular pO 2 monitoring has led to conflicting results on whether hypoxia increases HIF expression (87,121,122).…”

“…Namely, there is a lack of consensus in the usage of terms and units when it comes to measurements of pericellular O 2 levels. Many researchers describe the pericellular oxygen availability as concentration given in percent (18,19,(42)(43)(44)(45)(46)(47)(48). However, this is not accurate from the physical point of view as these studies are actually referring to the volume fraction of oxygen in the ambient air that corresponds to the actual molar concentration of oxygen in an aqueous solution-the medium.…”

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

“…We used an IPGphor focusing unit (GE Healthcare, Uppsala, Sweden) for the isoelectric focusing of samples loaded on 7 cm pH 4–7 Immobiline DryStrips (GE Healthcare, Uppsala, Sweden). We rehydrated each strip for 48 hours, as described previously [ 24 ]. After rehydration, pH 4–7 strips were focused with a limited current of 50 μA/strip at 20°C using the following conditions: gradient 0→150 V for 2 h, 150 V for 1 h, gradient 150→300 V for 1 h, 300 V for 2 h, gradient 300→1200 V for 3 h, 1200 V for 1 h, gradient 1200→3500 V for 5 h, and 3500 V for 5.5 h.…”

Markers of Acute Toxicity in Pancreatic Beta-Cells Exposed to Lethal Doses of the Organochlorine Pollutant DDT Determined by a Proteomic Approach

“…We used an IPGphor focusing unit (GE Healthcare, Uppsala, Sweden) for isoelectric focusing of samples loaded on 7 cm pH 4-7 Immobiline DryStrips (GE Healthcare, Uppsala, Sweden). We rehydrated each strip for 48 hours, as described previously (20). After rehydration, pH 4-7 strips were focused with a limited current of 50 μA/strip at 20 °C using the following conditions: gradient 0→150 V for 2 h, 150 V for 1 h, gradient 150→300 V for 1 h, 300 V for 2 h, gradient 300→1200 V for 3 h, 1200 V for 1 h, gradient 1200→3500 V for 5 h, and 3500 V for 5.5 h.…”