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Glutamine Metabolism Is Required for Collagen Protein Synthesis in Lung Fibroblasts.

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is characterized by the transforming growth factor (TGF)-β-dependent differentiation of lung fibroblasts into myofibroblasts, leading to excessive deposition of extracellular matrix proteins, which distort lung architecture and function. Metabolic reprogramming in myofibroblasts is emerging as an important mechanism in the pathogenesis of IPF, and recent evidence suggests that glutamine metabolism is required in myofibroblasts, although the exact role of glutamine in myofibroblasts is unclear. In the present study, we demonstrate that glutamine and its conversion to glutamate by glutaminase are required for TGF-β-induced collagen protein production in lung fibroblasts. We found that metabolism of glutamate to α-ketoglutarate by glutamate dehydrogenase or the glutamate-pyruvate or glutamate-oxaloacetate transaminases is not required for collagen protein production. Instead, we discovered that the glutamate-consuming enzymes phosphoserine aminotransferase 1 (PSAT1) and aldehyde dehydrogenase 18A1 (ALDH18A1)/Δ¹-pyrroline-5-carboxylate synthetase (P5CS) are required for collagen protein production by lung fibroblasts. PSAT1 is required for de novo glycine production, whereas ALDH18A1/P5CS is required for de novo proline production. Consistent with this, we found that TGF-β treatment increased cellular concentrations of glycine and proline in lung fibroblasts. Our results suggest that glutamine metabolism is required to promote amino acid biosynthesis and not to provide intermediates such as α-ketoglutarate for oxidation in mitochondria. In support of this, we found that inhibition of glutaminolysis has no effect on cellular oxygen consumption and that knockdown of oxoglutarate dehydrogenase has no effect on the ability of fibroblasts to produce collagen protein. Our results suggest that amino acid biosynthesis pathways may represent novel therapeutic targets for treatment of fibrotic diseases, including IPF.

SUBMITTER: Hamanaka RB

PROVIDER: S-EPMC6827066 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aerobic glycolysis has been shown to contribute to the abnormal activation of lung fibroblasts with excessive collagen deposition in lipopolysaccharide (LPS)-induced pulmonary fibrosis. Targeting aerobic glycolysis in lung fibroblasts might therefore be considered as a promising therapeutic approach for LPS-induced pulmonary fibrosis. In the present study, the aim was to investigate whether metformin, a widely used agent for treating type 2 diabetes, could alleviate LPS-induced lung fibroblast collagen synthesis and its potential underlying mechanisms. Different concentrations of metformin were used to treat the human lung fibroblast MRC-5 cells after LPS challenge. Indicators of aerobic glycolysis in MRC-5 cells were detected by measuring glucose consumption and lactate levels in culture medium in addition to lactate dehydrogenase activity in cellular lysates. The glucose consumption, lactate levels and the lactate dehydrogenase activity were measured respectively using colorimetric/fluorometric and ELISA kits. The effects of metformin in AMP-activated protein kinase (AMPK) activation was assessed by mitochondrial complex I activity kits. Collagen I, α-smooth muscle actin (α-SMA) and collagen III were used as markers of collagen synthesis, which was measured using western blotting, whereas phosphorylated (p-) AMPK, AMPK, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and mTOR were detected by western blotting. Metformin significantly decreased mitochondrial complex I activity and upregulated the expression of p-AMPK/AMPK protein in a concentration-dependent manner. Furthermore, the aerobic glycolysis mediated by PFKFB3 and collagen synthesis in LPS-treated MRC-5 cells was gradually inhibited with increasing concentrations of metformin. However, this inhibitory role of metformin on PFKFB3-meditaed aerobic glycolysis and collagen synthesis was prevented by treatments with 3BDO and compound C, which are specific mTOR activator and AMPK inhibitor, respectively. Taken together, the findings from this study suggested that metformin may prevent PFKFB3-associated aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts via regulating the AMPK/mTOR pathway.

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Glutamine Metabolism Is Required for Collagen Protein Synthesis in Lung Fibroblasts.

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