Air-liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air-liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1 alpha as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1 alpha content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

Automated summary

Improved oxygen availability in ALI cultures of small intestinal enterocytes leads to morphological, metabolic, and functional changes. A microarray analysis identified 830 significantly regulated genes in SMC and ALI cultures, with alterations in 31 pathways, mainly metabolic pathways, carbon metabolism, glycolysis, and HIF signaling. HIF-1 alpha showed increased mRNA levels and reduced nuclear content in ALI. Enhanced mRNA levels of oxidative phosphorylation genes were confirmed by western blot analysis, while COX subunit 5B protein was decreased. ALI cultures exhibited higher cytochrome C oxidase activity and decreased glucose levels with reduced lactate production. Energy metabolism analysis showed a shift towards oxidative phosphorylation and suppressed glycolysis in ALI.