Aging Influences the Metabolic and Inflammatory Phenotype in an Experimental Mouse Model of Acute Lung Injury

Abstract

Increased age is a risk factor for poor outcomes from respiratory failure and acute respiratory distress syndrome (ARDS). In this study, we sought to define age-related differences in lung inflammation, muscle injury, and metabolism after intratracheal lipopolysaccharide (IT-LPS) acute lung injury (ALI) in adult (6 months) and aged (18-20 months) male C57BL/6 mice. We also investigated age-related changes in muscle fatty acid oxidation (FAO) and the consequences of systemic FAO inhibition with the drug etomoxir. Aged mice had a distinct lung injury course characterized by prolonged alveolar neutrophilia and lack of response to therapeutic exercise. To assess the metabolic consequences of ALI, aged and adult mice underwent whole body metabolic phenotyping before and after IT-LPS. Aged mice had prolonged anorexia and decreased respiratory exchange ratio, indicating increased reliance on FAO. Etomoxir increased mortality in aged but not adult ALI mice, confirming the importance of FAO on survival from acute severe stress and suggesting that adult mice have increased resilience to FAO inhibition. Skeletal muscles from aged ALI mice had increased transcription of key fatty acid metabolizing enzymes, CPT-1b, LCAD, MCAD, FATP1 and UCP3. Additionally, aged mice had increased protein levels of CPT-1b at baseline and after lung injury. Surprisingly, CPT-1b in isolated skeletal muscle mitochondria had decreased activity in aged mice compared to adults. The distinct phenotype of aged ALI mice has similar characteristics to the adverse age-related outcomes of ARDS. This model may be useful to examine and augment immunologic and metabolic abnormalities unique to the critically ill aged population.

Keywords: Critical illness; Fats; Inflammation; Nutrition.

Figure 1.

Comparison of injury and resolution patterns in aged and adult mice. (A) Aged and adult mice underwent alveolar lavage under sham and ALI conditions. Adult mice had a significantly higher BAL cell count at peak inflammation but similar counts at all other time points (group size 4–10 per time point). (B) BAL protein levels were measured in aged and adult mice. Protein levels were similar at all time points (group size 3–6 per time point). (C) Aged mice had prolonged alveolar neutrophilia compared to adult mice. BAL neutrophil percentage was similar at peak inflammation but increased in aged mice at day 5 (group size 4–10 per time point). (D) IT-LPS results in robust systemic and alveolar TNFα production. Aged and adult mice have similar TNFα levels in both BAL and blood after lung injury (group size 4–6). (E, F) Aged mice exhibit resistance to exercise. Exercise reduces alveolar cell count (group size 8–11) and plasma G-CSF (group size 4–5) in adult but not aged mice after lung injury.

Figure 2.

Aged and adult mice have distinct metabolic phenotypes after ALI. (A) Aged and adult mice were metabolically phenotyped before and after lung injury (n = 6 per group). Aged mice had significantly reduced (p = .048) RER after ALI compared to younger animals. (B) Aged and adult mice had similar food intake prior injury. Aged mice had prolonged anorexia and consumed significantly less food per hour after lung injury (p = .0003). (C, D). Both groups had decreased energy expenditure after ALI. When adjusting for baseline variation, differences in energy expenditure did not achieve statistical significance over the entire course of lung injury (p = .051). However, significant differences existed over individual light/dark cycles. Aged mice had higher energy expenditure at baseline, and reduced energy expenditure on light cycles 2–4 and dark cycles 2–5.

Figure 3.

Comparison of fatty acid oxidation after ALI. (A–E) Transcription of key mediators of fatty acid oxidation (UCP3, CPT-1b, FATP1, MCAD, LCAD) was measured in gastrocnemius in aged and adult animals at baseline and after lung injury (group size 3–14 per time point). Transcription varied dynamically with IT-LPS. Aged mice consistently had higher transcription in early phase (day 1, 2, 3) ALI. (F) Oxidative stress was measured in skeletal muscle at baseline and after ALI by thiobarbituric acid reactive substance (TBARS) assay. No difference was seen at any time point (group size 5–7 per time point). (G) Mitochondrial CPT-1b protein expression was measured by western blot at baseline and day 10. Aged mice had increased CPT-1b expression at both baseline and day 10. Western blot was performed using 2 gels for electrophoresis. Same age baseline and day 10 injury were run on the same gel. (H) and (I) CPT-1b activity and malonyl inhibition were measured by radioassay at baseline and day 10 time points. Both aged and adult mice had increased CPT-1b activity at day 10 compared to baseline. Older mice had reduced activity when normalized for CPT-1 protein expression compared to younger animals. (J) Aged and adult mice were treated with either the CPT-1 inhibitor etomoxir or vehicle under control and ALI conditions. CPT-1 inhibition and ALI increased mortality in aged animals compared to vehicle or etomoxir alone. Aged ALI + vehicle control mice were sacrificed on day 10. All other conditions were sacrificed on day 7. All animals that survived to sacrifice showed signs of lung injury recovery.