Coordination of alanine synthesis and uptake contributes to fibroblast activation in pulmonary fibrosis

Pulmonary fibroblasts increase glutamine uptake and consumption to drive their activation and differentiation into myofibroblasts, accompanied by enhanced de novo synthesis of non-essential amino acids (NEAAs) via transaminases. However, the role of NEAAs in fibroblast activation remains unclear. We show that the pan-transaminase inhibitor AOA and GPT inhibitors (L-cycloserine and β-chloro-L-alanine) suppress TGFβ-induced a-SMA expression and collagen production in primary lung fibroblasts. The glutamine-glutamate-α-ketoglutarate axis contributes to the expression of a key enzyme GPT2, and GPT2 knockdown confirms its essential role in fibroblast activation. Metabolomic profiling revealed that these inhibitors and GPT2 knockdown reduced intracellular alanine levels. Supplementation with exogenous alanine reversed the inhibition of fibroblast activation. Stable isotope tracing confirmed that alanine serves as a carbon and nitrogen source for proline, particularly under glutamine-deprived conditions. Moreover, SLC38A2 was identified as the primary alanine transporter in activated fibroblasts, with its expression upregulated by TGFβ stimulation and extracellular alanine deprivation. Interestingly, SLC38A2 knockdown inhibited both alanine and glutamine uptake, thus reducing GPT2 expression and fibroblast activation. Collectively, we emphasize the crucial role of alanine biosynthesis and uptake during fibroblast activation, and targeting SLC38A2 may offer a potential therapeutic strategy for pulmonary fibrosis.