Articles in PresS. Am J Physiol Lung Cell Mol Physiol (March 6, 2015). doi:10.1152/ajplung.00335.2014
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TITLE:
Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells
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AUTHORS: Robyn G. Lottes1, Danforth A. Newton1, Demetri D. Spyropoulos2 and John E. Baatz1
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Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425
Department of Pathology & Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425
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Key Words: Alveolar type II cells, lactate oxidation, mitochondrial function, metabolism, hypoxia
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Running Title:
Lactate Oxidation in ATII Cells
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Corresponding Author: John E. Baatz, PhD
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Department of Pediatrics
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Medical University of South Carolina
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C.P. Darby Children’s Research Institute
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MSC 917
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173 Ashley Avenue
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Charleston, SC 29425
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Email: [email protected]
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Copyright © 2015 by the American Physiological Society.
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ABSTRACT
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Due to the many energy-demanding functions they perform and their physical location in
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the lung, alveolar epithelial type II (ATII) cells have a rapid cellular metabolism and the potential
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to influence substrate availability and bioenergetics both locally in the lung and throughout the
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body. A thorough understanding of ATII cell metabolic function in the healthy lung is necessary
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for determining how metabolic changes may contribute to pulmonary disease pathogenesis;
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however, lung metabolism is poorly understood at the cellular level. Here, we examine lactate
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utilization by primary ATII cells and the ATII model cell line, MLE-15, and link lactate
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consumption directly to mitochondrial ATP generation. ATII cells cultured in lactate undergo
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mitochondrial respiration at near-maximal levels, twice the rates of those grown in glucose, and
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oxygen consumption under these conditions is directly linked to mitochondrial ATP generation.
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When both lactate and glucose are available as metabolic substrate, the presence of lactate
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alters glucose metabolism in ATII to favor reduced glycolytic function in a dose-dependent
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manner, suggesting that lactate is utilized in addition to glucose when both substrates are
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available. Lactate use by ATII mitochondria is dependent on monocarboxylate transporter
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(MCT)-mediated import, and ATII cells express MCT1, the isoform that mediates lactate import
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by cells in other lactate-consuming tissues. The balance of lactate production and consumption
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may play an important role in the maintenance of healthy lung homeostasis, whereas disruption
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of lactate consumption by factors that impair mitochondrial metabolism, such as hypoxia, may
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contribute to lactic acid build-up in disease.
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INTRODUCTION From a metabolic perspective, the lung is a unique physiological environment. The cells
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that compose the alveolar epithelium form the barrier between external air and the pulmonary
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vasculature in the best-oxygenated environment in the body. While terminally-differentiated
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alveolar epithelial type I (ATI) cells form the passive surface across which gas exchange occurs,
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alveolar epithelial type II (ATII) cells perform a variety of energetically-costly functions including
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pulmonary surfactant production (16), fluid transport and homeostasis (27), immune functions
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(6, 23), and progenitor roles for self-renewal and transdifferentiation to repopulate ATI cells (5,
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19). Additionally, the lung is the only organ apart from the heart itself that receives the entire
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cardiac output upon every passage through the body. Metabolic activity in the cells that
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compose the alveolar epithelium could potentially influence substrate availability, energy
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production, and redox balance locally and throughout the whole body (20, 32). While the lung
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was thought to depend primarily on glucose for energy production, a number of landmark
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studies of whole lung metabolism demonstrated that other substrates are taken up from
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pulmonary circulation and oxidized. In particular, lactate was shown to be rapidly oxidized by
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lung tissue. Despite an early flurry of investigation regarding whole lung lactate metabolism, and
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despite growing interest in the contribution of lactate metabolism to diseases including cancer
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and fibrosis, the normal balance of lactate generation and consumption in the lung remains to
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be elucidated at the cellular level.
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Lactic acid is primarily produced as the end-product of anaerobic glycolytic metabolism
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and is generally considered a metabolic waste product that is exported from tissue and recycled
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in the liver to regenerate glucose. However, in reality, a comparatively low percentage of lactate
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is metabolized via gluconeogenesis, with the majority being used as substrate to fuel
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mitochondrial respiration in distinct cell types elsewhere in the body (4). For example, lactate in
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circulation is utilized as an important substrate by cardiac tissue, where it is preferentially
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utilized to fuel respiration (9). Additionally, highly oxidative cells in skeletal muscle consume
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lactate produced by neighboring glycolytic cells to fuel their own mitochondrial metabolism (3).
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In working muscle, the myocytes that compose fast-twitch glycolytic fibers undergo rapid
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anaerobic glycolysis and excrete lactate into the extracellular space (18). Neighboring cells of
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the highly oxidative slow-twitch fibers import the extracellular lactate for use as mitochondrial
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substrate. Though there has been debate regarding a similar cell-cell lactate shuttle in the brain,
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recent investigation supports an analogous connection between astrocytes and neurons
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wherein the latter consumes lactate produced by the former (2, 13). In this manner, glycolysis
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and oxidative metabolic processes are linked between distinct cell types in the same tissue or
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distant tissues (4).
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Lactate metabolism in the lung has received little attention in modern investigation,
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though several landmark studies of whole-organ metabolism previously indicated that lactate
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oxidation occurs in the lung. Wolfe, et al. used adult isolated perfused rate lung to demonstrate
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rapid oxidization of lactate by whole lung tissue, even when the perfusate contained glucose at
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a concentration several times that of lactate (34). Under these conditions, CO2 derived from
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lactate exceeded that from glucose. Later studies by Fox and colleagues using both perfused
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whole lung and isolated alveolar epithelial cells from fetal rat lung demonstrated rates of CO2
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generation from lactate that were approximately 20 times the rate of glucose oxidation and the
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highest rate for any substrate examined (11, 12). In these studies, glucose and lactate showed
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reciprocal inhibition, indicating similar pathways of metabolism. The observation that labeled
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lactate in pulmonary circulation is incorporated into lung lipids in both adult (31) and perinatal
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lung (28) further implicates ATII cells as a site of lactate consumption in the lung, as ATII cells
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synthesize the majority of lipid components in pulmonary surfactant. Despite this, lactate
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consumption has not been directly linked to cellular energetics in ATII cells.
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Cell consumption of lactate for ATP generation is achieved through conversion of lactate
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to pyruvate by lactate dehydrogenase (LDH) enzyme activity. Pyruvate is shuttled into the
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mitochondria and oxidized through reactions of the TCA cycle, and the reducing equivalents
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generated serve as electron donors for oxidative phosphorylation. Lactate transport both into
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and out of the cell is mediated by monocarboxylate transporter (MCT) proteins (17). Although
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MCT isoforms 1-4 all transport lactate and pyruvate bi-directionally, different isoforms more
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heavily favor import versus export. Specifically, the lower-affinity MCT4 is associated mainly
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with lactate efflux from glycolytic cells (8, 26), while the higher-affinity isoform MCT1 is
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associated with import into oxidative lactate-consuming cells. Both MCT1 and MCT4 have been
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found in the developing lung (15), and MCT1, 2, and 4 are present in adult whole lung samples
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(21). However, the specific cell types in the mature lung expressing each MCT isoform have not
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been determined, and the contribution of MCT-mediated transport to overall control of cell
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metabolism in the lung is unknown.
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We recently reported that ATII cells have a highly oxidative metabolic phenotype and are
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heavily dependent on mitochondrial function for energy production (24). This metabolic
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phenotype is characteristic of cells capable of lactate consumption in other tissues (3). This led
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us to hypothesize that ATII cells import lactate and utilize it as substrate for mitochondrial
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energy production. Here, we show that ATII cells can utilize lactate for oxidative ATP
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production. Culture in lactate alone induces high levels of oxygen consumption, resulting in a
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shift to an extremely oxidative, minimally glycolytic phenotype. Also, we provide evidence that
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the presence of lactate impacts glucose metabolism when both substrates are available. This
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metabolic strategy is dependent on the function of LDH and MCT proteins, and for the first time,
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we define ATII cells as a cell population in the mature lung that expresses MCT1. Finally, we
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address the impact of hypoxia on lactate-fueled mitochondrial respiration, inspired by the
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observation that pulmonary hypoxia develops in a number of lung diseases which have been
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recently associated with lactate build-up in patient lung tissue. Despite the apparent role of
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lactate in pulmonary disease pathogenesis, a thorough understanding of lactic acid production,
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consumption, and balance in the healthy lung is lacking. This work therefore represents a critical
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step toward understanding from a metabolic point of view the normal ATII processes that may
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be disrupted in, and contribute to, disease pathogenesis.
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METHODS Reagents. Culture media and supplements were obtained from Life Technologies
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(Grand Island, NY) except for fetal bovine serum (FBS) from Atlanta Biologicals (Lawrenceville,
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GA). Extracellular flux assay medium and consumables were purchased from Seahorse
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Bioscience (North Billerica, MA), and general chemicals, including D-glucose and sodium L-
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lactate, from Sigma-Aldrich (St. Louis, MO).
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Cell Culture. Cultures of the mouse lung epithelial 15 were maintained in a humidified
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incubator under ambient air (21% O2) and 5% CO2 at 37°C in HITES medium (Glucose-free,
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pyruvate-free RPMI; 2% FBS; 2 mM Glutamax; 10 µg/mL insulin; 10 µg/mL transferrin; 0.5
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µg/mL sodium selenite; supplemented with 5.5 mM glucose). Cells were plated in HITES
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medium and allowed to attach to culture dishes. After 1-2 hours of incubation, culture medium
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was replaced with HITES supplemented with 5.5 mM glucose, 5.5 mM lactate, or indicated
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concentrations. Hypoxia exposures were performed by incubation in a hypoxic chamber
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(Biospherix, Redfield, NY) with 1.5% O2, 5% CO2 at 37°C for 20 hours.
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Isolation and Culture of Primary ATII Cells. All procedures were conducted under
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approved Institutional Animal Care and Use Committee protocols. ATII cells were isolated from
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C57B/6 mouse lungs as described previously (24). Cells were cultured on Matrigel (BD
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Biosciences, San Jose, CA)-coated plates in glucose-free, pyruvate-free RPMI medium (Life
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Technologies) with 5% FBS and Small Airway Epithelial Cell supplement (Lonza, Walkersville,
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MD), formulated with indicated concentrations of lactate and/or glucose.
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Extracellular Flux Analysis. Cellular O2 consumption and H+ generation were
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assessed using a Seahorse Bioscience XF24 or XF96 instrument, as previously described (24).
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Cells were plated in 24-well assay plates at 6.5x104 cells/well or 96-well assay plates at 1.7x104
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cells/well and assayed in unbuffered XF Base Medium (Seahorse Bioscience) formulated with
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glucose or lactate and HITES media supplements (as above) except for FBS, which was
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excluded in MLE-15 cell assay media, but provided at 5% in primary cell media to maintain
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primary cell phenotype and viability. Glutamax supplement was excluded for indicated oxygen
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consumption experiments. Data were transformed using “Level (Direct) AKOS” algorithm (14)
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using the Seahorse XF24 or XF96 software package. PPR was calculated from measured
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ECAR with compensation for media buffering capacity. Media buffering capacities were
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calculated empirically for each batch. Primary cell assay media buffering capacity was
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calculated at 2.9*10-3 M. For MLE-15 assay medias, values ranged from 1.7*10-3 to 2.3*10-3 M,
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and correction was done individually for each experiment according to the value calculated on
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the day of assay. Values were normalized to well protein content.
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In-assay injections of carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP),
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oligomycin A, or an antimycin A/rotenone mixture were performed to assess spare respiratory
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capacity, coupling of oxygen consumption to ATP production, and non-mitochondrial respiration,
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respectively. α-cyano-4-hydroxycinnamate (CHC) was injected into assay wells to examine the
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effect of MCT inhibition. Inhibition of LDH was obtained by addition of 20 mM sodium oxamate
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to media 1 hour prior to assay.
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RNA & Protein Analyses. RNA harvest, cDNA generation, and qPCR assays were
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performed as previously described (24). Ct values for genes of interest were normalized to β-
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actin expression. Fold-change values for target genes were calculated using ΔΔCt analysis and
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values for biological replicates were averaged.
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Protocols used for cell lysis for protein assays, protein quantitation, electrophoresis and
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Western blot analysis were performed according to Lottes, et al (24). Mouse skeletal muscle
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tissue (provided by Dr. Kyu-Ho Lee, Medical University of South Carolina) was processed using
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a Dounce homogenizer prior to lysis. Blots were probed with goat anti-MCT1 (Santa Cruz
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Biotechnology, Inc. Dallas, TX) and rabbit anti-B Actin (Sigma). Secondary antibodies (LI-COR,
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Lincoln, NE) were used to visualize bands on a LI-COR Odyssey imager, with densitometry and
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normalization achieved using LI-COR Image StudioTM.
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ATP Concentration. ATP content of cultured cells was measured using the CellTitre Glo Luminescent assay (Promega, Madison, WI) following manufacturer’s instructions. Cell Count & EdU Incorporation. MLE-15 were plated onto 12-well plates at 5x104
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cells/well and cultured in HITES medium formulated with glucose, lactate, or combined
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substrates. At each interval, 3 individual wells were trypsinized and exposed to the vital dye
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trypan blue. Cells excluding trypan blue were counted using a hemocytometer. Relative rates of
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DNA synthesis were determined using EdU nucleotide analog incorporation as previously
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described (30). Each sample well was stained subsequently with ToPRO3 nuclear stain for
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normalization.
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Statistics. Significant difference was assessed using Student’s t-Test or ANOVA with
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Tukey post-hoc analysis. p values < 0.05 were considered significant. All error bars represent ±
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standard deviation. Statistical details for each experiment are also provided in figure legends.
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RESULTS Lactate is a substrate for oxidative ATP production in ATII cells. Metabolic flux
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analysis was performed using cells cultured in medium containing either lactate or glucose as
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metabolic substrate. MLE-15 cells cultured in lactate had oxygen consumption rates (OCR, a
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measure of mitochondrial activity) approximately twice those observed for cells metabolizing
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glucose (Figure 1). Alternatively, cells in lactate-formulated medium displayed minimal
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extracellular proton production rates (PPR, a measure of glycolysis) compared to those in
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glucose. Together, OCR and PPR values demonstrate a shift into a highly oxidative metabolism
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in the presence of lactate and absence of glucose. Similar results were also obtained with
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primary mouse ATII cells cultured in lactate versus those in glucose (Fig. 1).
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Numerous processes, including mitochondrial production of ATP and non-mitochondrial
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oxidation, contribute to total cellular OCR and can be measured by injection of various inhibitors
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during the flux assay. Following basal measurements, inhibition of ATP synthase via oligomycin
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injection resulted in a decrease in OCR, indicative of respiration coupled to ATP production. In
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glucose- and lactate-cultured cells, approximately 50% and 65% of basal oxygen consumption,
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respectively, is dedicated to mitochondrial ATP production (Table 1). This indicates a similar
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degree of coupling of O2 consumption to mitochondrial ATP generation by percentage of total
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O2 consumed, though in terms of OCR per µg protein, the amount of oxygen consumed to fuel
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ATP production is greater in the lactate-cultured cells due to their high basal rates. Similarly,
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non-mitochondrial oxygen consumption accounted for a similar proportion of total oxygen
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consumption in glucose- and lactate-grown cells (28% of each respective mean basal value).
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Given the greatly increased OCR in cells cultured in lactate compared to those in
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glucose, we sought to determine if this was the result of a simple increase in mitochondrial
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activity (use of spare reserve) or an actual increase in functional respiratory capacity of cells
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grown in lactate. Exposing cells to the mitochondrial uncoupling agent FCCP to force maximal
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electron transfer/OCR demonstrated that ATII cells grown in glucose, but not lactate, maintained
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a robust mitochondrial reserve above basal levels, indicating that the greater OCR in lactate is
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due to increased usage of existing mitochondrial spare respiratory capacity (Figure 2).
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Lactate must first be converted to pyruvate before it can be utilized as substrate for
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mitochondrial metabolism. This is achieved by the reverse activity of LDH, which oxidizes
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lactate to generate pyruvate. Thus, inhibition of LDH would be expected to reduce the ability of
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cells to respire using lactate as a metabolic substrate. Exposure to the competitive LDH inhibitor
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oxamate prior to assay resulted in decreased OCR in lactate-cultured cells, confirming that
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lactate is consumed by ATII via conversion to pyruvate (Figure 3). In contrast, OCR was
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increased in cells cultured in glucose and exposed to LDH inhibitor.
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Glutamine, an amino acid commonly supplemented in culture medium, can also be
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utilized as substrate for mitochondrial respiration via TCA cycle reactions. To ensure that the
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above results were not due to glutamine consumption, OCR was measured in cells assayed in
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XF base medium with and without the addition of glutamine-alanine dipeptide supplement. No
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significant difference was found between the conditions, suggesting that glutamine oxidation
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contributes minimally to total flux in ATII in these experiments (data not shown).
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Finally, to determine the impact of lactate availability on cellular metabolism when
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glucose is available, OCR and PPR were measured for MLE-15 cultured in media formulated
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with 5.5 mM glucose and increasing concentrations of lactate. At 2.75 mM lactate and above,
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the presence of lactate decreased PPR in a dose-dependent manner (Figure 4). There was no
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significant effect of media lactate on OCR, regardless of lactate concentration.
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Hypoxia suppresses lactate metabolism. Exposure of MLE-15 cells to 1.5% O2 for 20 hours prior to assay resulted in a similar decrease in OCR to approximately 50% of normoxic
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values for cells cultured in either glucose or lactate (Figure 5A). This indicates that mitochondrial
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metabolism in general is suppressed by hypoxia to a similar degree regardless of substrate.
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Similar results were obtained using primary ATII cells cultured in lactate versus glucose and
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subjected to hypoxia (Figure 5B). Thus, hypoxic exposure leads to decreased use of both
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lactate and glucose for mitochondrial metabolism in ATII.
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Lactate oxidation by ATII cells is dependent on MCT transport function. α-cyano-4-
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hydroxycinnamate (CHC) inhibits MCT protein isoforms at the cytosolic membrane, leading to
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reduced lactate transport into and out of the cell. Addition of CHC to medium during flux assays
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resulted in decreased PPR by MLE-15 cells regardless of substrate, with PPR of lactate-grown
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cells decreased to essentially zero (Figure 6A). However, CHC treatment lead to a decrease of
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OCR by approximately 50% for cells cultured in lactate, while in glucose-grown MLE-15, OCR
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was unaffected by CHC (Figure 6B).
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ATII cells express the lactate transporter MCT1. The MCT1 transporter mediates
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lactate import in lactate-consuming skeletal muscle, and in some cell types, is hypoxia-
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inducible. MCT1 mRNA expression was measured in lysates from MLE-15 and isolated primary
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ATII cells, and protein expression was assessed in MLE-15 lysates compared to mouse skeletal
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muscle tissue as positive control. Both MLE-15 and primary ATII cells express MCT1 mRNA.
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We evaluated the impact of hypoxic exposure on expression of MCT1 expression, and found no
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significant difference in mRNA expression in response to hypoxia in either MLE-15 or primary
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ATII cells (Figure 7A). Expression of MCT1 protein was confirmed in MLE-15 (Figure 7B, inset),
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and cultures exposed to hypoxia showed no significant difference in MCT1 protein level
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compared to normoxic cultures (Figure 7B). Altogether, these data indicate that the expression
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of MCT1 is not hypoxia-responsive in ATII cells.
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Lactate metabolism alone is sufficient to maintain bioenergetic homeostasis but
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not cell growth. ATP concentrations of MLE-15 cells cultured in lactate, both those cultured
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under normoxic and hypoxic conditions, did not differ significantly from glucose-grown controls
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(Figure 8A). Thus, lactate consumption provides sufficient substrate for mitochondrial respiration
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to maintain ATP homeostasis. However, MLE-15 cells cultured in lactate alone were not able to
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maintain cell growth. In medium containing only lactate, MLE-15 cell DNA synthesis (based on
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EdU nucleotide analog incorporation) occurred at approximately half the rate of cells cultured in
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glucose (Figure 8B). Culture in medium containing both substrates did not result in rates
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significantly different from culture in glucose alone over the 20 hour growth period. These
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findings were reiterated in cell counts over 5 days of culture. Compared to cultures maintained
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in glucose, those in lactate alone showed very little change in cell number over the growth
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period of 5 days (Figure 8C). The glucose-only and combined substrate conditions resulted in
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similar increases in cell number over time, confirming that the presence of 5.5 mM lactate did
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not itself inhibit growth. While the combined substrate condition showed higher mean cell count
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at day 5, this difference was not quite statistically (p < 0.05) significant until day 5, when the
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combined substrate condition showed a trend toward higher cell numbers than glucose alone,
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though this did not reach significance.
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DISCUSSION A series of landmark studies utilizing the isolated perfused whole lung experimental
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model demonstrated that lactate oxidation occurs in the lung tissue and suggested that lactate
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serves as an important precursor for both pulmonary cell energy production and lipid synthesis
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(7). Despite significant insight into pulmonary metabolism gained by whole-organ studies,
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investigators stressed that the model is limited in that it can provide no information about the
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function of specific cell types (7), and therefore the importance of lactate to lung bioenergetics at
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the cell-specific level were previously unknown. Furthermore, while oxidation of lactate to CO2
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was observed in these studies, the actual contribution of lactate to cellular oxygen-consumption
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or ATP production was not demonstrated or quantified. Here, we have examined the utilization
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of lactate by isolated primary and model ATII cells for oxidative energy production, and
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demonstrated that ATII cells consume lactate for use as substrate for rapid mitochondrial ATP
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generation. Using MLE-15 cells as a model for ATII metabolism, we additionally demonstrate
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that the availability of lactate regulates glucose metabolism. Also, we show that mature ATII
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cells specifically express the MCT1 isoform of the monocarboxylate transporter, often
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associated with lactate import, and MCT-mediated transport governs both lactic acid import and
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export in these cells. Overall, this work further demonstrates the metabolic adaptability of ATII
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cells to changing extracellular conditions and provides the first detailed assessments of
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mitochondrial metabolism in cells consuming lactate.
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Substrate availability created a dramatic shift in metabolic phenotype as ATII cells
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cultured in medium containing lactate in the absence of glucose adopted a highly-oxidative
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metabolism, consuming oxygen at rates approximately double that of cells cultured in glucose
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and performing minimal glycolysis as indicated by very low acid generation. Also, cells cultured
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in lactate maintained ATP homeostasis, even when exposed to hypoxia, despite the loss of
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glycolytic function. The need to compensate for the loss of glycolysis-derived ATP likely
308
contributes to the rapid rates of O2 consumption observed, since lactate is not metabolized
309
through the glycolytic pathway. Although inhibition of cell replication and division in lactate
310
culture would be expected to limit some of the major energy-demanding functions, it remains
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possible that increased ATP turnover due to other processes such as surfactant production or
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ion transport could also contribute to increased respiration.
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Addition of FCCP to flux assay medium was unable to stimulate increased O2
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consumption by cells cultured in lactate, indicating that respiration in lactate-fed cells is
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performed essentially at maximal mitochondrial capacity. Because cells in lactate alone are
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unresponsive to FCCP, and because OCR of lactate-cultured MLE-15 was similar to FCCP-
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stimulated glucose-fed cells, we conclude that the increased OCR during lactate-fueled
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respiration was not the result of enhanced mitochondrial capacity. For glucose-and lactate-
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cultured cells, ~50% and 65% of total basal O2 consumption, respectively, was coupled to ATP
320
generation. The difference between lactate- and glucose-cultured cells is subtle and not
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statistically significant. Though the proportion of oxygen consumption dedicated to energy
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production is similar between the conditions in terms of percentage, this translates in absolute
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quantity to a much greater amount of oxygen consumed for ATP generation in the lactate
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condition, with approximately twice the amount of O2 dedicated to ATP generation in lactate-
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cultured cells versus those in glucose. Although the ATP-coupled value for lactate-cultured cells
326
was not significantly higher than that of glucose, this results in an apparently lower percentage
327
of oxygen consumption that is generally attributed to “remainder” processes other than ATP
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production and non-mitochondrial respiration (e.g., mitochondrial membrane proton leak).
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This work also demonstrates that the availability of lactate modulates ATII cell glucose
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metabolism, as the addition of increasing concentrations of lactate to glucose-formulated
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medium decreased extracellular acidification, demonstrating decreased lactic acid generation.
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This finding supports the conclusion that elevated extracellular lactate concentration leads to
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increased use of lactate in place of glucose to fuel mitochondrial respiration, resulting in
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decreased glucose use and the observed decrease in glycolytic acid generation. This supports,
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at the cellular level, previous findings of whole lung lactate consumption from Fischer and Dodia
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using the isolated perfused rat lung model, wherein the addition of lactate to perfusate
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suppressed both glucose utilization and lactic acid output (10). Combined with multiple studies
338
showing that lactate oxidation occurs in the pulmonary tissue (7, 31, 34), this provided strong
339
evidence that lactate competes with glucose as mitochondrial substrate in the lung, and our
340
investigation supports and extends these findings by identifying ATII cells as a specific subset of
341
pulmonary cells that readily utilize lactate. Further study will be necessary to confirm the extent
342
to which lactate is taken up and utilized by ATII cells when glucose is also present; however,
343
this work establishes that ATII cell glucose metabolism is responsive to extracellular lactate.
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This may have considerable implications for ATII cell function in disease, as plasma lactate can
345
exceed 20 mM in extreme cases of hypoxia or hypotension (25), and pulmonary tissue
346
concentrations increase more than 2-fold from normal levels in IPF patient parenchymal tissue
347
(22) and possibly in cystic fibrosis, based on elevated lactate measured in patient sputum
348
samples (1).
349
Lactate must be converted to pyruvate before it can be oxidized via TCA cycle reactions,
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and we demonstrated that LDH activity is necessary for ATII cell lactate respiration. The marked
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decrease in OCR resulting from LDH inhibition, with the support of observations that withholding
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glutamine from media did not appreciably change OCR, confirms that lactate is utilized as fuel
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for rapid respiration. In these experiments, significant cellular OCR remained following exposure
354
to oxamate. This was not unexpected due to oxamate being a competitive inhibitor of LDH.
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Furthermore, a significant proportion of OCR (approximately 50-60%) is not linked to ATP
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production in ATII cells, and is, therefore, not expected to be impacted by reduced LDH
357
turnover. The opposite effect was observed for glucose-fed MLE-15 cultures, as oxamate
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exposure resulted in modestly increased OCR. This is likely to be an effect of reduced pyruvate-
359
to-lactate conversion, which would also be inhibited by oxamate, leading to enhanced pyruvate
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available to the mitochondria.
361
Our findings also indicate that utilization of lactate is suppressed by hypoxia due to
362
decreased mitochondrial substrate demand. The ~50% decrease in lactate-fueled OCR in
363
hypoxia-exposed cells compared to normoxic controls is similar to the degree of decrease
364
measured in glucose-cultured cells, reported here and in previous publication (24). The clinical
365
significance of this finding rests in the relationship of pulmonary hypoxia with several lung
366
diseases, including IPF. Based on our findings, ATII cells under normoxic conditions can readily
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remove lactate from the extracellular milieu, facilitated by co-transport with extracellular protons.
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We propose that this process acts as a lactate and H+ sink in the healthy lung, simultaneously
369
controlling lung tissue pH by preventing lactic acid build-up and providing rapidly-respiring ATII
370
with oxidizable substrate for ATP generation and surfactant lipid production. Under hypoxic
371
conditions we observed significant reduction in mitochondrial respiration by ATII cells cultured in
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lactate, indicating that decreased mitochondrial metabolism limits the ability of ATII cells to
373
consume lactate and therefore to regulate lactate and pH balance in the tissue. Previous clinical
374
investigations have observed lactate build-up and/or release from the lungs of patients suffering
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from diseases associated with development of pulmonary hypoxia. We suggest that this could
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be due in part to reduced capacity for lactate uptake and consumption by ATII cells.
377
The finding that lactate alone is insufficient to support MLE-15 cell growth at the same
378
rate as glucose is not surprising, because multiple glycolytic intermediates are crucial for DNA
379
synthesis. These critical intermediates will not be generated by cells metabolizing lactate alone.
380
We likewise demonstrate that it is the absence of glucose, and not the presence of lactate, that
381
limits cell growth, as the combined substrate condition did not lead to reduced rate of growth or
382
DNA synthesis. Over time, the presence of lactate may enhance growth, as culture in combined
383
substrate lead to apparently higher mean cell count and DNA incorporation, though these
384
differences did not reach significance.
385
We report that MCT1, previously associated with import of lactate into cells capable of
386
lactate oxidization, is also expressed by ATII cells. MCT1 and MCT4 proteins are both found in
387
lysates of whole lung tissue, but cell-specific expression and the overall role of MCTs in lung cell
388
metabolism was previously undetermined (21). This reports shows that MCTs are important for
389
both lactate import and export by ATII cells, as inhibition altered extracellular acidification and/or
390
mitochondrial function, depending on the available substrate. Specific MCT1 expression by ATII
391
cells supports the finding that they consume extracellular lactate, and the importance of MCT to
392
lactate oxidization is apparent given that MCT inhibition suppressed oxygen consumption in
393
cells cultured in lactate.
394
While MCT4 contains functional hypoxia-response elements and is a hypoxia-inducible
395
HIF1 target, the inducibility of MCT1 appears to differ by cell type. In adipocytes, hypoxic
396
exposure induces MCT1 expression (29), while in vitro studies using multiple cell lines showed
397
a similar expression pattern to that observed here, with no change in hypoxia (33). In our hands,
398
MCT1 was not induced by 20 hour hypoxic exposure. Given that there was no measured
399
change in either mRNA or protein under hypoxia, it is unlikely that the decreased respiration in
400
lactate-cultured cells exposed to hypoxia is controlled at the level of substrate transport.
401
The capacity of ATII cells to consume lactate from the extracellular space has important
402
implications for whole lung homeostasis. Based on our findings, ATII cells may take up lactate
403
produced by neighboring cells in a relationship analogous to white-glycolytic and red-oxidative
404
fibers in skeletal muscle. Metabolic flux has not been measured for other pulmonary cell types
405
like ATI cells and pulmonary fibroblasts; however, in fibrotic lung disease, activated
406
myofibroblasts generate high levels of lactic acid (22) and rough comparison of ATII flux
407
measurements to those for normal human dermal fibroblasts indicate that ATII cells likely have
408
higher mitochondrial metabolism (35). Based on perfused lung studies, ATII cells may also
409
utilize lactate delivered in pulmonary circulation. Multiple investigators have demonstrated lactic
410
acid uptake from pulmonary circulation (20), oxidization (7, 31, 34), and incorporation into lung
411
lipids (28, 31). In this manner, local metabolic cooperation between lung cell phenotypes may
412
form a key component of normal alveolar tissue homeostasis, providing substrate for ATII cell
413
energy and lipid production while also preventing lactic acid build-up.
414
Likewise, dysregulation of local metabolic cooperation between cell types may lead to
415
conditions associated with certain diseases. Recent investigation into pro-fibrotic processes that
416
contribute to myofibroblast activation in idiopathic pulmonary fibrosis (IPF) have highlighted the
417
importance of lactate balance in the lung. Kottman and colleagues determined that not only is
418
lactate elevated in the lungs of IPF patients, but lactic acid directly contributes to tissue fibrosis
419
(22). By lowering extracellular pH, lactic acid build-up activates latent Transforming Growth
420
Factor Beta (TGFβ). TGFβ is a cytokine responsible for initiating conversion of fibroblasts into
421
the myofibroblast phenotype, and the study determined that increased lactic acid enhances
422
myofibroblast conversion and matrix deposition. Increased lactate generation was associated
423
with increased tissue expression of LDH5, the isoform most strongly favoring pyruvate-to-lactate
424
conversion (and not the reverse). Enhanced expression was localized roughly to the epithelium
425
near fibrotic foci, though the cells responsible have yet to be conclusively determined. While it is
426
now clear that lactic acid build-up is involved in IPF pathogenesis, the cells and molecular
427
processes responsible for lactic acid build-up in diseased tissue are unknown. Based on these
428
findings, it is possible that altered ATII mitochondrial metabolism leading to reduced ability to act
429
as a sink for lactate and H+ is a contributing factor.
430 431
While this study examined only the impact of hypoxia on lactate metabolism, any condition that suppresses respiration would be expected to limit lactate consumption. Genetic
432
aberrations, damage by reactive oxygen species, and other challenges besides oxygen
433
deprivation can affect mitochondrial function and, therefore, lactate consumption. Future study
434
will be aimed at addressing the role of ATII cell lactate metabolism in epithelial dysfunction,
435
tissue acidification, and pathogenesis of diseases including IPF.
436 437
DISCLOSURES:
438
No conflicts of interest, financial or otherwise, are declared by the authors.
439 440
AUTHOR CONTRIBUTIONS:
441 442
Conception and design: JEB, RGL, DAN, DDS; Analysis and interpretation: RGL, JEB, DAN; Drafting the manuscript for important intellectual content: RGL, JEB, DAN
443 444 445
446
FIGURE LEGENDS
447
Figure 1: Culture in lactate shifts ATII cells into a highly oxidative metabolic state. OCR
448
and PPR were measured for primary ATII cells (circle markers) and MLE-15 cells (diamond
449
markers) cultured in either 5.5 mM glucose (closed) or 5.5 mM lactate (open). For MLE-15, four
450
individual experiments were performed, and in each, samples were assayed minimally in
451
triplicate per condition. For primary cultures, 6 single-well experiments were performed for each
452
condition. For each cell type, * indicates significant difference (p
1 2
TITLE:
Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells
3 4 5
AUTHORS: Robyn G. Lottes1, Danforth A. Newton1, Demetri D. Spyropoulos2 and John E. Baatz1
6 7
1
8 9
2
Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425
Department of Pathology & Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425
10 11 12 13
Key Words: Alveolar type II cells, lactate oxidation, mitochondrial function, metabolism, hypoxia
14 15
Running Title:
Lactate Oxidation in ATII Cells
16 17 18 19
Corresponding Author: John E. Baatz, PhD
20
Department of Pediatrics
21
Medical University of South Carolina
22
C.P. Darby Children’s Research Institute
23
MSC 917
24
173 Ashley Avenue
25
Charleston, SC 29425
26 27
Email: [email protected]
28
Copyright © 2015 by the American Physiological Society.
29
ABSTRACT
30
Due to the many energy-demanding functions they perform and their physical location in
31
the lung, alveolar epithelial type II (ATII) cells have a rapid cellular metabolism and the potential
32
to influence substrate availability and bioenergetics both locally in the lung and throughout the
33
body. A thorough understanding of ATII cell metabolic function in the healthy lung is necessary
34
for determining how metabolic changes may contribute to pulmonary disease pathogenesis;
35
however, lung metabolism is poorly understood at the cellular level. Here, we examine lactate
36
utilization by primary ATII cells and the ATII model cell line, MLE-15, and link lactate
37
consumption directly to mitochondrial ATP generation. ATII cells cultured in lactate undergo
38
mitochondrial respiration at near-maximal levels, twice the rates of those grown in glucose, and
39
oxygen consumption under these conditions is directly linked to mitochondrial ATP generation.
40
When both lactate and glucose are available as metabolic substrate, the presence of lactate
41
alters glucose metabolism in ATII to favor reduced glycolytic function in a dose-dependent
42
manner, suggesting that lactate is utilized in addition to glucose when both substrates are
43
available. Lactate use by ATII mitochondria is dependent on monocarboxylate transporter
44
(MCT)-mediated import, and ATII cells express MCT1, the isoform that mediates lactate import
45
by cells in other lactate-consuming tissues. The balance of lactate production and consumption
46
may play an important role in the maintenance of healthy lung homeostasis, whereas disruption
47
of lactate consumption by factors that impair mitochondrial metabolism, such as hypoxia, may
48
contribute to lactic acid build-up in disease.
49
50 51
INTRODUCTION From a metabolic perspective, the lung is a unique physiological environment. The cells
52
that compose the alveolar epithelium form the barrier between external air and the pulmonary
53
vasculature in the best-oxygenated environment in the body. While terminally-differentiated
54
alveolar epithelial type I (ATI) cells form the passive surface across which gas exchange occurs,
55
alveolar epithelial type II (ATII) cells perform a variety of energetically-costly functions including
56
pulmonary surfactant production (16), fluid transport and homeostasis (27), immune functions
57
(6, 23), and progenitor roles for self-renewal and transdifferentiation to repopulate ATI cells (5,
58
19). Additionally, the lung is the only organ apart from the heart itself that receives the entire
59
cardiac output upon every passage through the body. Metabolic activity in the cells that
60
compose the alveolar epithelium could potentially influence substrate availability, energy
61
production, and redox balance locally and throughout the whole body (20, 32). While the lung
62
was thought to depend primarily on glucose for energy production, a number of landmark
63
studies of whole lung metabolism demonstrated that other substrates are taken up from
64
pulmonary circulation and oxidized. In particular, lactate was shown to be rapidly oxidized by
65
lung tissue. Despite an early flurry of investigation regarding whole lung lactate metabolism, and
66
despite growing interest in the contribution of lactate metabolism to diseases including cancer
67
and fibrosis, the normal balance of lactate generation and consumption in the lung remains to
68
be elucidated at the cellular level.
69
Lactic acid is primarily produced as the end-product of anaerobic glycolytic metabolism
70
and is generally considered a metabolic waste product that is exported from tissue and recycled
71
in the liver to regenerate glucose. However, in reality, a comparatively low percentage of lactate
72
is metabolized via gluconeogenesis, with the majority being used as substrate to fuel
73
mitochondrial respiration in distinct cell types elsewhere in the body (4). For example, lactate in
74
circulation is utilized as an important substrate by cardiac tissue, where it is preferentially
75
utilized to fuel respiration (9). Additionally, highly oxidative cells in skeletal muscle consume
76
lactate produced by neighboring glycolytic cells to fuel their own mitochondrial metabolism (3).
77
In working muscle, the myocytes that compose fast-twitch glycolytic fibers undergo rapid
78
anaerobic glycolysis and excrete lactate into the extracellular space (18). Neighboring cells of
79
the highly oxidative slow-twitch fibers import the extracellular lactate for use as mitochondrial
80
substrate. Though there has been debate regarding a similar cell-cell lactate shuttle in the brain,
81
recent investigation supports an analogous connection between astrocytes and neurons
82
wherein the latter consumes lactate produced by the former (2, 13). In this manner, glycolysis
83
and oxidative metabolic processes are linked between distinct cell types in the same tissue or
84
distant tissues (4).
85
Lactate metabolism in the lung has received little attention in modern investigation,
86
though several landmark studies of whole-organ metabolism previously indicated that lactate
87
oxidation occurs in the lung. Wolfe, et al. used adult isolated perfused rate lung to demonstrate
88
rapid oxidization of lactate by whole lung tissue, even when the perfusate contained glucose at
89
a concentration several times that of lactate (34). Under these conditions, CO2 derived from
90
lactate exceeded that from glucose. Later studies by Fox and colleagues using both perfused
91
whole lung and isolated alveolar epithelial cells from fetal rat lung demonstrated rates of CO2
92
generation from lactate that were approximately 20 times the rate of glucose oxidation and the
93
highest rate for any substrate examined (11, 12). In these studies, glucose and lactate showed
94
reciprocal inhibition, indicating similar pathways of metabolism. The observation that labeled
95
lactate in pulmonary circulation is incorporated into lung lipids in both adult (31) and perinatal
96
lung (28) further implicates ATII cells as a site of lactate consumption in the lung, as ATII cells
97
synthesize the majority of lipid components in pulmonary surfactant. Despite this, lactate
98
consumption has not been directly linked to cellular energetics in ATII cells.
99
Cell consumption of lactate for ATP generation is achieved through conversion of lactate
100
to pyruvate by lactate dehydrogenase (LDH) enzyme activity. Pyruvate is shuttled into the
101
mitochondria and oxidized through reactions of the TCA cycle, and the reducing equivalents
102
generated serve as electron donors for oxidative phosphorylation. Lactate transport both into
103
and out of the cell is mediated by monocarboxylate transporter (MCT) proteins (17). Although
104
MCT isoforms 1-4 all transport lactate and pyruvate bi-directionally, different isoforms more
105
heavily favor import versus export. Specifically, the lower-affinity MCT4 is associated mainly
106
with lactate efflux from glycolytic cells (8, 26), while the higher-affinity isoform MCT1 is
107
associated with import into oxidative lactate-consuming cells. Both MCT1 and MCT4 have been
108
found in the developing lung (15), and MCT1, 2, and 4 are present in adult whole lung samples
109
(21). However, the specific cell types in the mature lung expressing each MCT isoform have not
110
been determined, and the contribution of MCT-mediated transport to overall control of cell
111
metabolism in the lung is unknown.
112
We recently reported that ATII cells have a highly oxidative metabolic phenotype and are
113
heavily dependent on mitochondrial function for energy production (24). This metabolic
114
phenotype is characteristic of cells capable of lactate consumption in other tissues (3). This led
115
us to hypothesize that ATII cells import lactate and utilize it as substrate for mitochondrial
116
energy production. Here, we show that ATII cells can utilize lactate for oxidative ATP
117
production. Culture in lactate alone induces high levels of oxygen consumption, resulting in a
118
shift to an extremely oxidative, minimally glycolytic phenotype. Also, we provide evidence that
119
the presence of lactate impacts glucose metabolism when both substrates are available. This
120
metabolic strategy is dependent on the function of LDH and MCT proteins, and for the first time,
121
we define ATII cells as a cell population in the mature lung that expresses MCT1. Finally, we
122
address the impact of hypoxia on lactate-fueled mitochondrial respiration, inspired by the
123
observation that pulmonary hypoxia develops in a number of lung diseases which have been
124
recently associated with lactate build-up in patient lung tissue. Despite the apparent role of
125
lactate in pulmonary disease pathogenesis, a thorough understanding of lactic acid production,
126
consumption, and balance in the healthy lung is lacking. This work therefore represents a critical
127
step toward understanding from a metabolic point of view the normal ATII processes that may
128
be disrupted in, and contribute to, disease pathogenesis.
129
130 131
METHODS Reagents. Culture media and supplements were obtained from Life Technologies
132
(Grand Island, NY) except for fetal bovine serum (FBS) from Atlanta Biologicals (Lawrenceville,
133
GA). Extracellular flux assay medium and consumables were purchased from Seahorse
134
Bioscience (North Billerica, MA), and general chemicals, including D-glucose and sodium L-
135
lactate, from Sigma-Aldrich (St. Louis, MO).
136
Cell Culture. Cultures of the mouse lung epithelial 15 were maintained in a humidified
137
incubator under ambient air (21% O2) and 5% CO2 at 37°C in HITES medium (Glucose-free,
138
pyruvate-free RPMI; 2% FBS; 2 mM Glutamax; 10 µg/mL insulin; 10 µg/mL transferrin; 0.5
139
µg/mL sodium selenite; supplemented with 5.5 mM glucose). Cells were plated in HITES
140
medium and allowed to attach to culture dishes. After 1-2 hours of incubation, culture medium
141
was replaced with HITES supplemented with 5.5 mM glucose, 5.5 mM lactate, or indicated
142
concentrations. Hypoxia exposures were performed by incubation in a hypoxic chamber
143
(Biospherix, Redfield, NY) with 1.5% O2, 5% CO2 at 37°C for 20 hours.
144
Isolation and Culture of Primary ATII Cells. All procedures were conducted under
145
approved Institutional Animal Care and Use Committee protocols. ATII cells were isolated from
146
C57B/6 mouse lungs as described previously (24). Cells were cultured on Matrigel (BD
147
Biosciences, San Jose, CA)-coated plates in glucose-free, pyruvate-free RPMI medium (Life
148
Technologies) with 5% FBS and Small Airway Epithelial Cell supplement (Lonza, Walkersville,
149
MD), formulated with indicated concentrations of lactate and/or glucose.
150
Extracellular Flux Analysis. Cellular O2 consumption and H+ generation were
151
assessed using a Seahorse Bioscience XF24 or XF96 instrument, as previously described (24).
152
Cells were plated in 24-well assay plates at 6.5x104 cells/well or 96-well assay plates at 1.7x104
153
cells/well and assayed in unbuffered XF Base Medium (Seahorse Bioscience) formulated with
154
glucose or lactate and HITES media supplements (as above) except for FBS, which was
155
excluded in MLE-15 cell assay media, but provided at 5% in primary cell media to maintain
156
primary cell phenotype and viability. Glutamax supplement was excluded for indicated oxygen
157
consumption experiments. Data were transformed using “Level (Direct) AKOS” algorithm (14)
158
using the Seahorse XF24 or XF96 software package. PPR was calculated from measured
159
ECAR with compensation for media buffering capacity. Media buffering capacities were
160
calculated empirically for each batch. Primary cell assay media buffering capacity was
161
calculated at 2.9*10-3 M. For MLE-15 assay medias, values ranged from 1.7*10-3 to 2.3*10-3 M,
162
and correction was done individually for each experiment according to the value calculated on
163
the day of assay. Values were normalized to well protein content.
164
In-assay injections of carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP),
165
oligomycin A, or an antimycin A/rotenone mixture were performed to assess spare respiratory
166
capacity, coupling of oxygen consumption to ATP production, and non-mitochondrial respiration,
167
respectively. α-cyano-4-hydroxycinnamate (CHC) was injected into assay wells to examine the
168
effect of MCT inhibition. Inhibition of LDH was obtained by addition of 20 mM sodium oxamate
169
to media 1 hour prior to assay.
170
RNA & Protein Analyses. RNA harvest, cDNA generation, and qPCR assays were
171
performed as previously described (24). Ct values for genes of interest were normalized to β-
172
actin expression. Fold-change values for target genes were calculated using ΔΔCt analysis and
173
values for biological replicates were averaged.
174
Protocols used for cell lysis for protein assays, protein quantitation, electrophoresis and
175
Western blot analysis were performed according to Lottes, et al (24). Mouse skeletal muscle
176
tissue (provided by Dr. Kyu-Ho Lee, Medical University of South Carolina) was processed using
177
a Dounce homogenizer prior to lysis. Blots were probed with goat anti-MCT1 (Santa Cruz
178
Biotechnology, Inc. Dallas, TX) and rabbit anti-B Actin (Sigma). Secondary antibodies (LI-COR,
179
Lincoln, NE) were used to visualize bands on a LI-COR Odyssey imager, with densitometry and
180
normalization achieved using LI-COR Image StudioTM.
181 182 183
ATP Concentration. ATP content of cultured cells was measured using the CellTitre Glo Luminescent assay (Promega, Madison, WI) following manufacturer’s instructions. Cell Count & EdU Incorporation. MLE-15 were plated onto 12-well plates at 5x104
184
cells/well and cultured in HITES medium formulated with glucose, lactate, or combined
185
substrates. At each interval, 3 individual wells were trypsinized and exposed to the vital dye
186
trypan blue. Cells excluding trypan blue were counted using a hemocytometer. Relative rates of
187
DNA synthesis were determined using EdU nucleotide analog incorporation as previously
188
described (30). Each sample well was stained subsequently with ToPRO3 nuclear stain for
189
normalization.
190
Statistics. Significant difference was assessed using Student’s t-Test or ANOVA with
191
Tukey post-hoc analysis. p values < 0.05 were considered significant. All error bars represent ±
192
standard deviation. Statistical details for each experiment are also provided in figure legends.
193
194 195
RESULTS Lactate is a substrate for oxidative ATP production in ATII cells. Metabolic flux
196
analysis was performed using cells cultured in medium containing either lactate or glucose as
197
metabolic substrate. MLE-15 cells cultured in lactate had oxygen consumption rates (OCR, a
198
measure of mitochondrial activity) approximately twice those observed for cells metabolizing
199
glucose (Figure 1). Alternatively, cells in lactate-formulated medium displayed minimal
200
extracellular proton production rates (PPR, a measure of glycolysis) compared to those in
201
glucose. Together, OCR and PPR values demonstrate a shift into a highly oxidative metabolism
202
in the presence of lactate and absence of glucose. Similar results were also obtained with
203
primary mouse ATII cells cultured in lactate versus those in glucose (Fig. 1).
204
Numerous processes, including mitochondrial production of ATP and non-mitochondrial
205
oxidation, contribute to total cellular OCR and can be measured by injection of various inhibitors
206
during the flux assay. Following basal measurements, inhibition of ATP synthase via oligomycin
207
injection resulted in a decrease in OCR, indicative of respiration coupled to ATP production. In
208
glucose- and lactate-cultured cells, approximately 50% and 65% of basal oxygen consumption,
209
respectively, is dedicated to mitochondrial ATP production (Table 1). This indicates a similar
210
degree of coupling of O2 consumption to mitochondrial ATP generation by percentage of total
211
O2 consumed, though in terms of OCR per µg protein, the amount of oxygen consumed to fuel
212
ATP production is greater in the lactate-cultured cells due to their high basal rates. Similarly,
213
non-mitochondrial oxygen consumption accounted for a similar proportion of total oxygen
214
consumption in glucose- and lactate-grown cells (28% of each respective mean basal value).
215
Given the greatly increased OCR in cells cultured in lactate compared to those in
216
glucose, we sought to determine if this was the result of a simple increase in mitochondrial
217
activity (use of spare reserve) or an actual increase in functional respiratory capacity of cells
218
grown in lactate. Exposing cells to the mitochondrial uncoupling agent FCCP to force maximal
219
electron transfer/OCR demonstrated that ATII cells grown in glucose, but not lactate, maintained
220
a robust mitochondrial reserve above basal levels, indicating that the greater OCR in lactate is
221
due to increased usage of existing mitochondrial spare respiratory capacity (Figure 2).
222
Lactate must first be converted to pyruvate before it can be utilized as substrate for
223
mitochondrial metabolism. This is achieved by the reverse activity of LDH, which oxidizes
224
lactate to generate pyruvate. Thus, inhibition of LDH would be expected to reduce the ability of
225
cells to respire using lactate as a metabolic substrate. Exposure to the competitive LDH inhibitor
226
oxamate prior to assay resulted in decreased OCR in lactate-cultured cells, confirming that
227
lactate is consumed by ATII via conversion to pyruvate (Figure 3). In contrast, OCR was
228
increased in cells cultured in glucose and exposed to LDH inhibitor.
229
Glutamine, an amino acid commonly supplemented in culture medium, can also be
230
utilized as substrate for mitochondrial respiration via TCA cycle reactions. To ensure that the
231
above results were not due to glutamine consumption, OCR was measured in cells assayed in
232
XF base medium with and without the addition of glutamine-alanine dipeptide supplement. No
233
significant difference was found between the conditions, suggesting that glutamine oxidation
234
contributes minimally to total flux in ATII in these experiments (data not shown).
235
Finally, to determine the impact of lactate availability on cellular metabolism when
236
glucose is available, OCR and PPR were measured for MLE-15 cultured in media formulated
237
with 5.5 mM glucose and increasing concentrations of lactate. At 2.75 mM lactate and above,
238
the presence of lactate decreased PPR in a dose-dependent manner (Figure 4). There was no
239
significant effect of media lactate on OCR, regardless of lactate concentration.
240 241
Hypoxia suppresses lactate metabolism. Exposure of MLE-15 cells to 1.5% O2 for 20 hours prior to assay resulted in a similar decrease in OCR to approximately 50% of normoxic
242
values for cells cultured in either glucose or lactate (Figure 5A). This indicates that mitochondrial
243
metabolism in general is suppressed by hypoxia to a similar degree regardless of substrate.
244
Similar results were obtained using primary ATII cells cultured in lactate versus glucose and
245
subjected to hypoxia (Figure 5B). Thus, hypoxic exposure leads to decreased use of both
246
lactate and glucose for mitochondrial metabolism in ATII.
247
Lactate oxidation by ATII cells is dependent on MCT transport function. α-cyano-4-
248
hydroxycinnamate (CHC) inhibits MCT protein isoforms at the cytosolic membrane, leading to
249
reduced lactate transport into and out of the cell. Addition of CHC to medium during flux assays
250
resulted in decreased PPR by MLE-15 cells regardless of substrate, with PPR of lactate-grown
251
cells decreased to essentially zero (Figure 6A). However, CHC treatment lead to a decrease of
252
OCR by approximately 50% for cells cultured in lactate, while in glucose-grown MLE-15, OCR
253
was unaffected by CHC (Figure 6B).
254
ATII cells express the lactate transporter MCT1. The MCT1 transporter mediates
255
lactate import in lactate-consuming skeletal muscle, and in some cell types, is hypoxia-
256
inducible. MCT1 mRNA expression was measured in lysates from MLE-15 and isolated primary
257
ATII cells, and protein expression was assessed in MLE-15 lysates compared to mouse skeletal
258
muscle tissue as positive control. Both MLE-15 and primary ATII cells express MCT1 mRNA.
259
We evaluated the impact of hypoxic exposure on expression of MCT1 expression, and found no
260
significant difference in mRNA expression in response to hypoxia in either MLE-15 or primary
261
ATII cells (Figure 7A). Expression of MCT1 protein was confirmed in MLE-15 (Figure 7B, inset),
262
and cultures exposed to hypoxia showed no significant difference in MCT1 protein level
263
compared to normoxic cultures (Figure 7B). Altogether, these data indicate that the expression
264
of MCT1 is not hypoxia-responsive in ATII cells.
265
Lactate metabolism alone is sufficient to maintain bioenergetic homeostasis but
266
not cell growth. ATP concentrations of MLE-15 cells cultured in lactate, both those cultured
267
under normoxic and hypoxic conditions, did not differ significantly from glucose-grown controls
268
(Figure 8A). Thus, lactate consumption provides sufficient substrate for mitochondrial respiration
269
to maintain ATP homeostasis. However, MLE-15 cells cultured in lactate alone were not able to
270
maintain cell growth. In medium containing only lactate, MLE-15 cell DNA synthesis (based on
271
EdU nucleotide analog incorporation) occurred at approximately half the rate of cells cultured in
272
glucose (Figure 8B). Culture in medium containing both substrates did not result in rates
273
significantly different from culture in glucose alone over the 20 hour growth period. These
274
findings were reiterated in cell counts over 5 days of culture. Compared to cultures maintained
275
in glucose, those in lactate alone showed very little change in cell number over the growth
276
period of 5 days (Figure 8C). The glucose-only and combined substrate conditions resulted in
277
similar increases in cell number over time, confirming that the presence of 5.5 mM lactate did
278
not itself inhibit growth. While the combined substrate condition showed higher mean cell count
279
at day 5, this difference was not quite statistically (p < 0.05) significant until day 5, when the
280
combined substrate condition showed a trend toward higher cell numbers than glucose alone,
281
though this did not reach significance.
282
283 284
DISCUSSION A series of landmark studies utilizing the isolated perfused whole lung experimental
285
model demonstrated that lactate oxidation occurs in the lung tissue and suggested that lactate
286
serves as an important precursor for both pulmonary cell energy production and lipid synthesis
287
(7). Despite significant insight into pulmonary metabolism gained by whole-organ studies,
288
investigators stressed that the model is limited in that it can provide no information about the
289
function of specific cell types (7), and therefore the importance of lactate to lung bioenergetics at
290
the cell-specific level were previously unknown. Furthermore, while oxidation of lactate to CO2
291
was observed in these studies, the actual contribution of lactate to cellular oxygen-consumption
292
or ATP production was not demonstrated or quantified. Here, we have examined the utilization
293
of lactate by isolated primary and model ATII cells for oxidative energy production, and
294
demonstrated that ATII cells consume lactate for use as substrate for rapid mitochondrial ATP
295
generation. Using MLE-15 cells as a model for ATII metabolism, we additionally demonstrate
296
that the availability of lactate regulates glucose metabolism. Also, we show that mature ATII
297
cells specifically express the MCT1 isoform of the monocarboxylate transporter, often
298
associated with lactate import, and MCT-mediated transport governs both lactic acid import and
299
export in these cells. Overall, this work further demonstrates the metabolic adaptability of ATII
300
cells to changing extracellular conditions and provides the first detailed assessments of
301
mitochondrial metabolism in cells consuming lactate.
302
Substrate availability created a dramatic shift in metabolic phenotype as ATII cells
303
cultured in medium containing lactate in the absence of glucose adopted a highly-oxidative
304
metabolism, consuming oxygen at rates approximately double that of cells cultured in glucose
305
and performing minimal glycolysis as indicated by very low acid generation. Also, cells cultured
306
in lactate maintained ATP homeostasis, even when exposed to hypoxia, despite the loss of
307
glycolytic function. The need to compensate for the loss of glycolysis-derived ATP likely
308
contributes to the rapid rates of O2 consumption observed, since lactate is not metabolized
309
through the glycolytic pathway. Although inhibition of cell replication and division in lactate
310
culture would be expected to limit some of the major energy-demanding functions, it remains
311
possible that increased ATP turnover due to other processes such as surfactant production or
312
ion transport could also contribute to increased respiration.
313
Addition of FCCP to flux assay medium was unable to stimulate increased O2
314
consumption by cells cultured in lactate, indicating that respiration in lactate-fed cells is
315
performed essentially at maximal mitochondrial capacity. Because cells in lactate alone are
316
unresponsive to FCCP, and because OCR of lactate-cultured MLE-15 was similar to FCCP-
317
stimulated glucose-fed cells, we conclude that the increased OCR during lactate-fueled
318
respiration was not the result of enhanced mitochondrial capacity. For glucose-and lactate-
319
cultured cells, ~50% and 65% of total basal O2 consumption, respectively, was coupled to ATP
320
generation. The difference between lactate- and glucose-cultured cells is subtle and not
321
statistically significant. Though the proportion of oxygen consumption dedicated to energy
322
production is similar between the conditions in terms of percentage, this translates in absolute
323
quantity to a much greater amount of oxygen consumed for ATP generation in the lactate
324
condition, with approximately twice the amount of O2 dedicated to ATP generation in lactate-
325
cultured cells versus those in glucose. Although the ATP-coupled value for lactate-cultured cells
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was not significantly higher than that of glucose, this results in an apparently lower percentage
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of oxygen consumption that is generally attributed to “remainder” processes other than ATP
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production and non-mitochondrial respiration (e.g., mitochondrial membrane proton leak).
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This work also demonstrates that the availability of lactate modulates ATII cell glucose
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metabolism, as the addition of increasing concentrations of lactate to glucose-formulated
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medium decreased extracellular acidification, demonstrating decreased lactic acid generation.
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This finding supports the conclusion that elevated extracellular lactate concentration leads to
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increased use of lactate in place of glucose to fuel mitochondrial respiration, resulting in
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decreased glucose use and the observed decrease in glycolytic acid generation. This supports,
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at the cellular level, previous findings of whole lung lactate consumption from Fischer and Dodia
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using the isolated perfused rat lung model, wherein the addition of lactate to perfusate
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suppressed both glucose utilization and lactic acid output (10). Combined with multiple studies
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showing that lactate oxidation occurs in the pulmonary tissue (7, 31, 34), this provided strong
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evidence that lactate competes with glucose as mitochondrial substrate in the lung, and our
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investigation supports and extends these findings by identifying ATII cells as a specific subset of
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pulmonary cells that readily utilize lactate. Further study will be necessary to confirm the extent
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to which lactate is taken up and utilized by ATII cells when glucose is also present; however,
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this work establishes that ATII cell glucose metabolism is responsive to extracellular lactate.
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This may have considerable implications for ATII cell function in disease, as plasma lactate can
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exceed 20 mM in extreme cases of hypoxia or hypotension (25), and pulmonary tissue
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concentrations increase more than 2-fold from normal levels in IPF patient parenchymal tissue
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(22) and possibly in cystic fibrosis, based on elevated lactate measured in patient sputum
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samples (1).
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Lactate must be converted to pyruvate before it can be oxidized via TCA cycle reactions,
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and we demonstrated that LDH activity is necessary for ATII cell lactate respiration. The marked
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decrease in OCR resulting from LDH inhibition, with the support of observations that withholding
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glutamine from media did not appreciably change OCR, confirms that lactate is utilized as fuel
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for rapid respiration. In these experiments, significant cellular OCR remained following exposure
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to oxamate. This was not unexpected due to oxamate being a competitive inhibitor of LDH.
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Furthermore, a significant proportion of OCR (approximately 50-60%) is not linked to ATP
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production in ATII cells, and is, therefore, not expected to be impacted by reduced LDH
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turnover. The opposite effect was observed for glucose-fed MLE-15 cultures, as oxamate
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exposure resulted in modestly increased OCR. This is likely to be an effect of reduced pyruvate-
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to-lactate conversion, which would also be inhibited by oxamate, leading to enhanced pyruvate
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available to the mitochondria.
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Our findings also indicate that utilization of lactate is suppressed by hypoxia due to
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decreased mitochondrial substrate demand. The ~50% decrease in lactate-fueled OCR in
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hypoxia-exposed cells compared to normoxic controls is similar to the degree of decrease
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measured in glucose-cultured cells, reported here and in previous publication (24). The clinical
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significance of this finding rests in the relationship of pulmonary hypoxia with several lung
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diseases, including IPF. Based on our findings, ATII cells under normoxic conditions can readily
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remove lactate from the extracellular milieu, facilitated by co-transport with extracellular protons.
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We propose that this process acts as a lactate and H+ sink in the healthy lung, simultaneously
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controlling lung tissue pH by preventing lactic acid build-up and providing rapidly-respiring ATII
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with oxidizable substrate for ATP generation and surfactant lipid production. Under hypoxic
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conditions we observed significant reduction in mitochondrial respiration by ATII cells cultured in
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lactate, indicating that decreased mitochondrial metabolism limits the ability of ATII cells to
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consume lactate and therefore to regulate lactate and pH balance in the tissue. Previous clinical
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investigations have observed lactate build-up and/or release from the lungs of patients suffering
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from diseases associated with development of pulmonary hypoxia. We suggest that this could
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be due in part to reduced capacity for lactate uptake and consumption by ATII cells.
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The finding that lactate alone is insufficient to support MLE-15 cell growth at the same
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rate as glucose is not surprising, because multiple glycolytic intermediates are crucial for DNA
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synthesis. These critical intermediates will not be generated by cells metabolizing lactate alone.
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We likewise demonstrate that it is the absence of glucose, and not the presence of lactate, that
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limits cell growth, as the combined substrate condition did not lead to reduced rate of growth or
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DNA synthesis. Over time, the presence of lactate may enhance growth, as culture in combined
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substrate lead to apparently higher mean cell count and DNA incorporation, though these
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differences did not reach significance.
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We report that MCT1, previously associated with import of lactate into cells capable of
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lactate oxidization, is also expressed by ATII cells. MCT1 and MCT4 proteins are both found in
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lysates of whole lung tissue, but cell-specific expression and the overall role of MCTs in lung cell
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metabolism was previously undetermined (21). This reports shows that MCTs are important for
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both lactate import and export by ATII cells, as inhibition altered extracellular acidification and/or
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mitochondrial function, depending on the available substrate. Specific MCT1 expression by ATII
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cells supports the finding that they consume extracellular lactate, and the importance of MCT to
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lactate oxidization is apparent given that MCT inhibition suppressed oxygen consumption in
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cells cultured in lactate.
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While MCT4 contains functional hypoxia-response elements and is a hypoxia-inducible
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HIF1 target, the inducibility of MCT1 appears to differ by cell type. In adipocytes, hypoxic
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exposure induces MCT1 expression (29), while in vitro studies using multiple cell lines showed
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a similar expression pattern to that observed here, with no change in hypoxia (33). In our hands,
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MCT1 was not induced by 20 hour hypoxic exposure. Given that there was no measured
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change in either mRNA or protein under hypoxia, it is unlikely that the decreased respiration in
400
lactate-cultured cells exposed to hypoxia is controlled at the level of substrate transport.
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The capacity of ATII cells to consume lactate from the extracellular space has important
402
implications for whole lung homeostasis. Based on our findings, ATII cells may take up lactate
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produced by neighboring cells in a relationship analogous to white-glycolytic and red-oxidative
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fibers in skeletal muscle. Metabolic flux has not been measured for other pulmonary cell types
405
like ATI cells and pulmonary fibroblasts; however, in fibrotic lung disease, activated
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myofibroblasts generate high levels of lactic acid (22) and rough comparison of ATII flux
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measurements to those for normal human dermal fibroblasts indicate that ATII cells likely have
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higher mitochondrial metabolism (35). Based on perfused lung studies, ATII cells may also
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utilize lactate delivered in pulmonary circulation. Multiple investigators have demonstrated lactic
410
acid uptake from pulmonary circulation (20), oxidization (7, 31, 34), and incorporation into lung
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lipids (28, 31). In this manner, local metabolic cooperation between lung cell phenotypes may
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form a key component of normal alveolar tissue homeostasis, providing substrate for ATII cell
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energy and lipid production while also preventing lactic acid build-up.
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Likewise, dysregulation of local metabolic cooperation between cell types may lead to
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conditions associated with certain diseases. Recent investigation into pro-fibrotic processes that
416
contribute to myofibroblast activation in idiopathic pulmonary fibrosis (IPF) have highlighted the
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importance of lactate balance in the lung. Kottman and colleagues determined that not only is
418
lactate elevated in the lungs of IPF patients, but lactic acid directly contributes to tissue fibrosis
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(22). By lowering extracellular pH, lactic acid build-up activates latent Transforming Growth
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Factor Beta (TGFβ). TGFβ is a cytokine responsible for initiating conversion of fibroblasts into
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the myofibroblast phenotype, and the study determined that increased lactic acid enhances
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myofibroblast conversion and matrix deposition. Increased lactate generation was associated
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with increased tissue expression of LDH5, the isoform most strongly favoring pyruvate-to-lactate
424
conversion (and not the reverse). Enhanced expression was localized roughly to the epithelium
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near fibrotic foci, though the cells responsible have yet to be conclusively determined. While it is
426
now clear that lactic acid build-up is involved in IPF pathogenesis, the cells and molecular
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processes responsible for lactic acid build-up in diseased tissue are unknown. Based on these
428
findings, it is possible that altered ATII mitochondrial metabolism leading to reduced ability to act
429
as a sink for lactate and H+ is a contributing factor.
430 431
While this study examined only the impact of hypoxia on lactate metabolism, any condition that suppresses respiration would be expected to limit lactate consumption. Genetic
432
aberrations, damage by reactive oxygen species, and other challenges besides oxygen
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deprivation can affect mitochondrial function and, therefore, lactate consumption. Future study
434
will be aimed at addressing the role of ATII cell lactate metabolism in epithelial dysfunction,
435
tissue acidification, and pathogenesis of diseases including IPF.
436 437
DISCLOSURES:
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No conflicts of interest, financial or otherwise, are declared by the authors.
439 440
AUTHOR CONTRIBUTIONS:
441 442
Conception and design: JEB, RGL, DAN, DDS; Analysis and interpretation: RGL, JEB, DAN; Drafting the manuscript for important intellectual content: RGL, JEB, DAN
443 444 445
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FIGURE LEGENDS
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Figure 1: Culture in lactate shifts ATII cells into a highly oxidative metabolic state. OCR
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and PPR were measured for primary ATII cells (circle markers) and MLE-15 cells (diamond
449
markers) cultured in either 5.5 mM glucose (closed) or 5.5 mM lactate (open). For MLE-15, four
450
individual experiments were performed, and in each, samples were assayed minimally in
451
triplicate per condition. For primary cultures, 6 single-well experiments were performed for each
452
condition. For each cell type, * indicates significant difference (p
