Alveolar type II cells maintain bioenergetic homeostasis in hypoxia through metabolic and molecular adaptation

Abstract

Lottes RG, Newton DA, Spyropoulos DD, Baatz JE. Alveolar type II cells maintain bioenergetic homeostasis in hypoxia through metabolic and molecular adaptation. Am J Physiol Lung Cell Mol Physiol 306: L947-L955, 2014. First published March 28, 2014 doi:10.1152/ajplung.00298.2013.-Although many lung diseases are associated with hypoxia, alveolar type II epithelial (ATII) cell impairment, and pulmonary surfactant dysfunction, the effects of O 2 limitation on metabolic pathways necessary to maintain cellular energy in ATII cells have not been studied extensively. This report presents results of targeted assays aimed at identifying specific metabolic processes that contribute to energy homeostasis using primary ATII cells and a model ATII cell line, mouse lung epithelial 15 (MLE-15), cultured in normoxic and hypoxic conditions. MLEs cultured in normoxia demonstrated a robust O 2 consumption rate (OCR) coupled to ATP generation and limited extracellular lactate production, indicating reliance on oxidative phosphorylation for ATP production. Pharmacological uncoupling of respiration increased OCR in normoxic cultures to 175% of basal levels, indicating significant spare respiratory capacity. However, when exposed to hypoxia for 20 h, basal O2 consumption fell to 60% of normoxic rates, and cells maintained only ϳ50% of normoxic spare respiratory capacity, indicating suppression of mitochondrial function, although intracellular ATP levels remained at near normoxic levels. Moreover, while hypoxic exposure stimulated glycogen synthesis and storage in MLE-15, glycolytic rate (as measured by lactate generation) was not significantly increased in the cells, despite enhanced expression of several enzymes related to glycolysis. These results were largely recapitulated in murine primary ATII, demonstrating MLE-15 suitability for modeling ATII metabolism. The ability of ATII cells to maintain ATP levels in hypoxia without enhancing glycolysis suggests that these cells are exceptionally efficient at conserving ATP to maintain bioenergetic homeostasis under O 2 limitation. mitochondrial function; metabolism THE ALVEOLAR EPITHELIUM FORMS the barrier between the pulmonary vasculature and the external milieu and serves as the surface across which O 2 and waste gases are exchanged. Because of their physical location, the cells that line alveoli in developed human lungs are normally exposed to an exceptionally well-oxygenated environment of ϳ13% O 2 in nondiseased lungs (i.e., a PO 2 of ϳ105 mmHg compared with a PO 2 of ϳ40 mmHg in peripheral blood) (33). However, decreases in alveolar O 2 tensions (pulmonary hypoxia) can result from a number of pathological conditions, including chronic obstructive pulmonary disease, lung cancers, and pulmonary hypertension and edema (34). In contrast, differentiation and development of the fetal lung distal epithelium normally occurs in low O 2 (1-5% O 2 ) (19), with hypoxia-related signaling in the pulmonary epithelium throughout gestation (8,9,28). This condition is critical for normal fetal lung ...

Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells

“…The ϳ50% decrease in lactate-fueled OCR in hypoxia-exposed cells compared with normoxic controls is similar to the degree of decrease measured in glucose-cultured cells, reported here and in a previous publication (24). The clinical significance of this finding rests in the relationship of pulmonary hypoxia with several lung diseases, including IPF.…”

“…All procedures were conducted under approved Institutional Animal Care and Use Committee protocols. ATII cells were isolated from C57B/6 mouse lungs as described previously (24). Cells were cultured on Matrigel (BD Biosciences, San Jose, CA)-coated plates in glucose-free, pyruvatefree RPMI medium (Life Technologies) with 5% FBS and Small Airway Epithelial Cell supplement (Lonza, Walkersville, MD), formulated with indicated concentrations of lactate and/or glucose.…”

“…Cellular O 2 consumption and H ϩ generation were assessed using a Seahorse Bioscience XF24 or XF96 instrument, as previously described (24). Cells were plated in 24-well assay plates at 6.5 ϫ 10 4 cells/well or 96-well assay plates at 1.7 ϫ 10 4 cells/well and assayed in unbuffered XF Base Medium (Seahorse Bioscience) formulated with glucose or lactate and HITES media supplements (as above) except for FBS, which was excluded in MLE-15 cell assay media, but provided at 5% in primary cell media to maintain primary cell phenotype and viability.…”

“…RNA harvest, cDNA generation, and qPCR assays were performed as previously described (24). Ct values for genes of interest were normalized to ␤-actin expression.…”

“…We recently reported that ATII cells have a highly oxidative metabolic phenotype and are heavily dependent on mitochondrial function for energy production (24). This metabolic phenotype is characteristic of cells capable of lactate consumption in other tissues (3).…”