title: Air-liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2.
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 cellsin 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. Cytochromecoxidase (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-1αprotein 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.
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Klasvogt
authors.affiliation: Leibniz Institute for Neurobiology , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Zuschratter
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Kröber
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Vorwerk
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Romina
authors.initials: R
authors.lastname: Wolter
authors.affiliation: Institute of Clinical Chemistry and Pathochemistry, Otto-von-Guericke University , Magdeburg 39120, Germany.
authors.forename: Berend
authors.initials: B
authors.lastname: Isermann
authors.affiliation: Leibniz Institute for Farm Animal Biology , Dummerstorf 18196, Germany.
authors.forename: Klaus
authors.identifier: 0000-0002-9523-6790
authors.initials: K
authors.lastname: Wimmers
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Hermann-Josef
authors.initials: HJ
authors.lastname: Rothkötter
authors.affiliation: Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Nossol