HIF1A Reduces Acute Lung Injury by Optimizing Carbohydrate Metabolism in the Alveolar Epithelium
Figure 10
Consequences of alveolar-epithelial Hif1a deletion on mitochondrial function during ALI.
(A–G) Hif1af/f SurfactantCre+ mice or littermate controls (SurfactantCre+) matched in age, weight, and gender were exposed to ventilator-induced lung injury (VILI; pressure-controlled mechanical ventilation at an inspiratory pressure of 45 mbar with an inspired oxygen concentration of 100%, exposure time 120 min). 13C glucose was administered i.p. 30 min prior to the experimental procedure. Determination of 13C carbohydrates during VILI was performed using liquid chromatography–tandem mass spectrometry (LC-MS). (A) 13C malate. (B) Tricarboxylic acid (TCA) cycle flux rates were determined by measuring the ratio of 13C2 glutamate to total creatine (n = 4 for all experiments). (C–F) Hif1af/f SurfactantCre+ mice or age-, gender-, and weight-matched littermate controls (SurfactantCre+) were exposed to VILI (see above). (C) After 3 h of VILI exposure, mitochondrial fractions were obtained from lung tissue and analyzed for Complex IV activity using ELISA. Activity is given as OD (optical density) change over time (mean ± s.d., n = 3). (D) Frozen lung tissue was lysed and mitochondrial proteins resolved by SDS-PAGE. Resultant Western blots were probed with anti-COX4-2 antibody. A representative blot of three is shown. (E) Pulmonary ATP content in Hif1af/f SurfactantCre+ mice or littermate controls (SurfactantCre+) were exposed to VILI (inspired oxygen concentration 100%, exposure time 120 min, pressure-controlled ventilation with an inspiratory pressure of 45 mbar). (F) Hydrogen peroxide lung tissue levels in Hif1af/f SurfactantCre+ mice or littermate controls (SurfactantCre+) exposed to VILI. Data are expressed as the mean fluorescence levels from three independent experiments normalized by protein concentration. Error bars represent s.d. (n = 3). (G and H) BL6C57 mice were treated with Rotenone—an inhibitor of mitochondrial respiration—or vehicle control 1 h prior to the beginning of the experimental procedure. IL-6 levels in lung tissue homogenates (G) or Albumin levels in BAL (H) were evaluated using a mouse enzyme-linked immunosorbent assay (ELISA) following exposure to VILI (see above). Results are presented as mean ± s.d. (n = 6).