Identification of Sphingolipid Metabolism Perturbations in Endothelial Cells Induced by the Acid Sphingomyelinase Inhibitor Imipramine Using Stable Isotope Labeling and Targeted Lipidomics
Authors: Berdyshev Evgeny , Justice Matthew J. , Bronova Irina , Schweitzer Kelly S. , Petrache Irina 
Published: November 2023

Imipramine is a tricyclic antidepressant that inhibits acid sphingomyelinase (ASM). ASM‐driven ceramide generation has been implicated in a variety of cell responses including lung cell apoptosis, autophagy, and the release of endothelial microparticles in response to cigarette smoking. However, others and we have identified that levels of other sphingolipid metabolites, in addition to ceramide, are significantly altered following pharmacological or molecular ASM inhibition. These static quantitative assessments suggest a dynamic crosstalk among various biochemical pathways of the sphingolipid metabolism and implicate sphingolipid metabolites other than ceramide as putative mediators of respective cellular functions. We set out to explore how ASM inhibition affects sphingolipid metabolism in human pulmonary artery endothelial cells (HPAEC).Imipramine (50 μM; 30 min) treatment induced autophagy in HPAEC, as detected by increased LC3B‐II and microscopic evidence of autophagosome formation. A snapshot analysis of sphingolipids by liquid chromatography tandem mass spectrometry (LC‐MS/MS) either in total cell lysates or in individual subcellular fractions revealed modest upregulation of ceramides and virtually no change in sphingomyelin content induced by imipramine treatment from 30 min to 16h. However, imipramine drastically reduced cellular and lysosome fraction levels of sphingosine. To further define the contribution of catabolic and de novo (biosynthetic) sphingolipid pathways induced by imipramine, HPAECs were labeled with U‐[13C,15N]‐serine for 5h or 8h followed by imipramine treatment (50 μM; for the final 2h or 3h of labeling, respectively). The analysis of the de novo‐formed (labeled) vs. non‐labeled sphingolipids revealed that the majority of ceramides that were increased in response to imipramine were formed from pre‐existing sphingolipids, with only a modest contribution of the short‐chain (N‐16:0) but not long‐chain (N‐24:1/24:0) ceramides formed through the de novo pathway. Imipramine drastically reduced non‐labeled and labeled (de novo‐formed) sphingosine and sphingosine‐1‐phosphate but did not affect the levels and the rate of label incorporation into dihydrosphingosine, dihydrosphingosine‐1‐phosphate, and glucosyl/galactosylceramides. The analysis of relative proportion of the label incorporation into individual ceramide molecular species suggests that in HPAECs, at normal growth conditions, N‐16:0‐ceramide is formed largely through the salvage pathway while ceramides with long‐chain fatty acids (N‐24:1/24:0) are preferentially formed through the de novo pathway.In summary, our study suggests a particular role for the loss of sphingosine rather than the changes in ceramides in imipramine induction of autophagy in HPAECs. In addition, the pulse‐labeling experiment largely excludes a role for the de novo‐formed sphingolipids in imipramine‐induced autophagic response.Support or Funding InformationThis study was supported by RO1HL077328 (IP); R21DA029249 (IP); F31HL126459 (MJ)