Abstract
The intestinal porcine epithelial cell line IPEC-J2, cultured under the air–liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium–cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.
Highlights
Cell culture models use either the isolation of primary cells or cell lines
The aim of the present study was to analyse what effects the air–liquid interface (ALI) culture of IPEC-J2 has on metabolism pathway showed 53 genes in the carbohydrate metabolism, 75 genes of the lipid metabolism and 15 genes of the amino acid metabolism with significant variations in expression (Figure 1b)
QPCR analyses resulted in a downregulation of MCT1 in the ALI cultures
Summary
Cell culture models use either the isolation of primary cells or cell lines. Primary cells are non-transformed and not tumour derived. Within many various continuous cell lines IPEC-1 and IPEC-J2 provide an exceptional option, as they are both originally isolates from new born piglets and are non-transformed, and not tumour derived.[1] Especially IPEC-J2 cells show morphological and functional similarity to porcine enterocytes. This cell line represents a wellestablished model for simulation of the human intestinal barrier.[2,3]
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