Pyruvate metabolism dictates fibroblast sensitivity to GLS1 inhibition during fibrogenesis

Abstract

Fibrosis is a chronic disease characterized by excessive extracellular matrix (ECM) production which leads to destruction of normal tissue architecture and disruption of organ function. Fibroblasts are key effector cells of this process and respond to a host of pro-fibrotic stimuli, including notably the pleiotropic cytokine, TGF-{beta}1, which promotes fibroblast to myofibroblast differentiation. This is accompanied by the simultaneous rewiring of metabolic networks to meet the biosynthetic and bioenergetic needs of contractile and ECM-synthesizing cells, but the exact mechanisms involved remain poorly understood. In this study, we report that extracellular nutrient availability profoundly influences the TGF-{beta}1 transcriptome of primary human lung fibroblasts (pHLFs) and the "biosynthesis of amino acids" emerges as a top enriched transcriptional module influenced by TGF-{beta}1. We subsequently uncover a key role for pyruvate in influencing the pharmacological impact of glutaminase (GLS1) inhibition during TGF-{beta}1-induced fibrogenesis. In pyruvate replete conditions which mimic the physiological concentration of pyruvate in human blood, GLS1 inhibition is ineffective in blocking TGF-{beta}1-induced fibrogenesis, as pyruvate is able to be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1-inhibition is required to fully block TGF-{beta}1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor in order to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.