Metabolic heterogeneity of idiopathic pulmonary fibrosis: a metabolomic study

Abstract

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown cause characterised by progressive fibrotic formation in lung tissue. We hypothesise that disrupted metabolic pathways in IPF contribute to disease pathogenesis.

Methods: Metabolomics of human IPF was performed using mass spectroscopy (IPF lung=8; donor lung=8). Gene expression of key metabolic enzymes was measured using microarrays. Of the 108 metabolites whose levels were found altered, 48 were significantly increased, whereas 60 were significantly decreased in IPF samples compared with normal controls.

Results: Specific metabolic pathways mediating the IPF remodelling were found with a downregulated sphingolipid metabolic pathway but an upregulated arginine pathway in IPF. In addition, disrupted glycolysis, mitochondrial beta-oxidation and tricarboxylic acid cycle, altered bile acid, haem and glutamate/aspartate metabolism were found in IPF samples compared with control.

Conclusions: Our results show alterations in metabolic pathways for energy consumption during lung structural remodelling, which may contribute to IPF pathogenesis. We believe that this is the first report of simultaneously and systemically measuring changes of metabolites involving nine metabolic pathways in human severe IPF lungs. The measurement of the metabolites may serve in the future diagnosis and prognosis of IPF.

Keywords: interstitial fibrosis; lung transplantation.

Figure 1

Downregulated sphingolipid metabolism in idiopathic pulmonary fibrosis (IPF) lungs. (A) Data for metabolic intermediates in the normal lung (NL) are shown in green boxes and data for IPF are represented in purple. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalised to protein concentration (n=8 for each box). Samples of patients with IPF exhibited lower levels of sphinganine, sphingosine and sphingomyelin. (B) The classical sphingolipid is shown. (C) Analysis of gene expression for key enzymes of the sphingolipids metabolism; data for control lung are shown in open blue boxes and data for IPF lung are represented in open green boxes (y-axis label of metabolic changes is shown as counts×10⁶). The genes encoding sphingosine 1-phosphate (S1P) metabolic pathway showed downregulated sphingomyelinase (SMPD1 and SMPD4) as well as downregulated delta(4)-desaturase, sphingolipid 1 (DEGS1), suggesting a reduced ceramide production, and downregulated ACER3 (alkaline ceramidase 3) indicates reduced sphingosine and S1P production. Genes encoding DEGS1 (p=1.25e−10), ACER3 (p=2.08e−7), SMPD1 (p=2e−6) and SMPD4 (p=6.67e−14) were significantly changed in IPF lungs compared with normal (y-axis label for gene expression encoding enzymes is shown as fold changes of relative RNA levels of enzymes).

Figure 2

Arginine metabolism is increased in the idiopathic pulmonary fibrosis (IPF) lung. (A) The classical arginine metabolic pathway. (B) In all graphs, the metabolic data for control lung (NL) are shown in green boxes, and data for IPF lung are represented in purple boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalised to protein concentration (n=8 for each box; y-axis label of metabolic changes is shown as counts×10⁶). We found significantly increased polyamines putrescine and spermidine as well as increased 4-hydroxyproline and creatine (p<0.05, increase highlighted in red open box and decrease in green open box). Increased polyamine levels indicate increased cell proliferation, and creatine may provide some of the energy required. Increased 4-hydroxyproline is a marker of fibrosis. Decreased aspartate and fumarate may indicate that arginine metabolism is shuttling intermediates away from the tricarboxylic acid cycle. 5-MTA, 5′-methylthioadenosine; ECM, extracellular matrix; OAT,Ornithine aminotransferase.

Figure 3

Glycolysis is downregulated in idiopathic pulmonary fibrosis (IPF) lungs, with possible shuttling of intermediates to the sorbitol and pentose phosphate pathways. (A) Analysis of gene expression for key enzymes of glycolysis. Gene-encoding enzymes, including the genes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), phosphofructokinase (PFK), phosphoglycerate mutase 1 (PGAM1), PGAM4, glucose-6-phosphatase catalytic subunit 3 (G6PC3) and SLC2A4RG, were significantly downregulated in IPF compared with normal suggesting a decreased rate of glycolysis (p<0.05). (B) The classical glycolysis/pentose/energy pathways are shown. (C) Data for metabolic intermediates in the normal lung (NL) are shown in green boxes, and data for IPF are represented in purple boxes. Levels of glycolytic intermediates fructose 1,6-bisphosphate (fructose 1,6-BP) and phosphoenolpyruate were significantly decreased in IPF, suggesting decreased glycolysis. Fructose levels were significantly increased, whereas sorbitol levels showed an upwards trend in IPF, possibly indicating shuttling of glycolytic intermediates towards the sorbitol pathway. The y-axis label of metabolic changes is shown as counts×10⁶ and the y-axis label for gene expression encoding enzymes as fold changes of relative RNA levels of enzymes. DHAP, dihydroxyacetone phosphate.

Figure 4

Mitochondrial transport. In all graphs, the metabolic data for normal lung (NL) are shown in green boxes, and the data for idiopathic pulmonary fibrosis (IPF) lung are represented in purple boxes. Significant increase of fatty acids palmitoleate, caproate and myristate and decrease of acylcarnitines and carnitine shuttle (palmitoylcarnitine, hexanoylcarnitine and octanoylcarnitine) were found in IPF lung compared with normal (p<0.05, increase highlighted in red open box and decrease in green open box). The y-axis label of metabolic changes is shown as counts×10⁶.

Figure 5

Tricarboxylic acid (TCA) cycle. We found overall decreased TCA cycle metabolites and enzymes in idiopathic pulmonary fibrosis (IPF) lungs. In all graphs, the genes encoding metabolic enzymes for control lung (NL) are shown in blue open boxes and IPF lung in green open boxes, whereas metabolic data for control lung are shown in green boxes and IPF lung are represented in purple boxes. Significant increases in the enzyme succinyl coenzyme A synthetase (SUCLA2) and the metabolite cis-aconitate decreases in isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), fumarate, succinate dehydrogenase (SDH) and citrate synthase (CS) and accumulation of cis-aconitate are found in IPF compared with control (p<0.05). The y-axis label for gene expression encoding enzymes is shown as fold changes of relative RNA levels of enzymes.

Figure 6

Haem metabolism. In all graphs, the genes encoding metabolic enzymes for normal lung (NL) are shown in blue open boxes and data for IPF lung are represented in green open boxes, whereas metabolic data for control lung are shown in green boxes. Significantly decreased haem and biliverdin but increased bilirubin levels were found in IPF compared with control (p<0.05). Significantly increased gene expression of uridine 5′-diphospho-glucuronosyltransferase 1 (UGT1A1) was found in IPF lung (p<0.05). The y-axis label of metabolic changes is shown as counts×10⁶ and the y-axis label for gene expression encoding enzymes as fold changes of relative RNA levels of enzymes. ROS, reactive oxygen species.