Fig 1: Acute METH exposure upregulates enzymes of proline synthetic pathway from glutamate. (A) Schematic depicting the metabolic link between glutamate and proline. Synthesis of proline begins with the conversion of glutamine to glutamate by the enzyme glutaminase (GLS). Glutamate is then converted by P5C synthase (P5CS) to glutamic-?-semialdehyde that spontaneously cycles to ?1-pyrroline-5-carboxylate (P5C). P5C is subsequently catalyzed to l-proline by P5C-reductase (PYCR). Conversely, catabolism of proline to glutamate is catalyzed in two consecutive steps by proline oxidase (POX) and P5C dehydrogenase (P5CDH). Glutamate is finally converted to glutamine by glutamine synthase (GS). (B–D) Effect of acute METH treatment on proline catabolic enzymes. SH-SY5Y cells were treated with increasing concentrations of METH for 24 h. Post-treatment cells were harvested and lysed. Cell lysates were subjected to immunoblot analyses. (B) Representative immunoblot of enzymes involved in catabolism of proline to glutamate (n = 3) (Fig. S1). (C,D) Densitometry analysis of POX and P5CDH, respectively normalized to ß-actin. (E–H) Effect of acute METH treatment on proline metabolic enzymes. (E) Representative immunoblot of enzymes, P5CS, PYCR1 and PYCR2, which are essential for proline biosynthesis from glutamate (n = 3). (F–H) Densitometry analysis of P5CS, PYCR1, and PYCR2, respectively normalized to ß-actin. Data presented in panels (C,D,F–H) are mean values of three independent experiments with error bars representing SEM. *p < 0.05, **p < 0.005 represent statistical comparison of untreated vs METH-treated cells.