Fig 2: De Novo Synthesis of Long-Chain Bases Is Essential for Nuclear Integrity in Yeast(A) Representative images of cells expressing Lcb1, Lcb2, or Tsc3 tagged with GFP. Scale bar, 10 μm.(B) Representative images of wild-type cells expressing Heh1-GFP treated with myriocin or rapamycin for 3 h. Scale bar, 10 μm.(C) Percentage of abnormal nuclei of wild-type cells treated with myriocin or rapamycin (n = 200).(D) Kinetics of the serine palmitoyltransferase (SPT) enzymatic activity in wild-type cells and tsc3Δ (see STAR Methods). Error bars represent SD (n = 3).(E) Fold change of the levels in LCBs upon tsc3Δ compared with wild-type cells, shown as mean ± SD (n = 3 biological replicates).(F) Representative live-cell images of tsc3Δ cells expressing Heh1-GFP alone or treated with PHS or ceramide for 3 h. Scale bar, 10 μm.(G) Percentage of nuclear abnormalities of tsc3Δ upon PHS or ceramide treatment (n = 200 cells).(H) Representative electron micrograph images of the nuclear envelope in wild-type yeast and cell harboring tsc3Δ. Scale bar, 500 nm; zoom scale bar, 100 nm.(I) Representative images of wild-type cells expressing Heh1-GFP treated with DHS, PHS, or ceramide for 3 h. Scale bar, 5 μm.(J) Percentage of abnormal nuclei in wild-type cells expressing Heh1-GFP treated with DHS, PHS, or ceramide (n = 200 cells).

Fig 3: Effects of short- and long-time incubation with PQQ on energy substrate metabolism in L6 myotubes challenged with palmitate. ATF2: activating transcription factor 2; ATGL: adipose triglyceride lipase; AMPK: 5’AMP-activated protein kinase; CD36: cluster of differentiation 36; CER: ceramides; CPT1: carnitine palmitoyltransferase 1; CREB: cAMP-response element binding protein; CS: citrate synthase; DAG: diacylglycerols; DGATs: diacylglycerol O-acyltransferases; FABPpm: plasma membrane-associated fatty acid binding protein; FATPs: fatty acid transport proteins; GLUT4: glucose transporter type 4; PGC-1α: peroxisome proliferator-activated receptor γ co-activator 1α; PQQ: pyrroloquinoline quinone; SIRT1: sirtuin 1; SPT1: serine palmitoyltransferase, long chain base subunit 1.

Fig 4: Scheme of the crosstalk of sphingolipid and eicosanoid metabolic pathways. The sphingolipid pathway is known to involve the SPT complex, which is negatively regulated by ORMDL family proteins. This pathway leads to the production of sphingosines and ceramides. The eicosanoid pathway involves PLA2 and 5-LO. When activated, PLA2 releases arachidonic acid (AA) from ER membranes, which is utilized by 5-LO and cyclooxygenases (COX) as a substrate for the production of precursors of leukotrienes and prostaglandins, respectively. The connection between eicosanoid and sphingolipid pathways is linked to the activity of their mediators. It has been shown that the nonvesicular C1P transfer regulates PLA2-dependent release of arachidonic acid (54) and that second messenger S1P induces expression of cyclooxygenase 2 (COX2) (55). Data in this study indicate that ORMDL3 interacts with 5-LO and that 5-LO interacts with SPT complex subunits, SPTLC1 and SPTLC2. The functional consequences of the leukotriene and sphingolipid pathway crosstalk are shown (in orange) and involve 1) inhibitory role of ORMDL3 on the activity of 5-LO and eicosanoid production; 2) activatory role of SPTLC1 on 5-LO activity; 3) inhibitory effect of 5-LO on ceramide levels. Moreover, reduced SPTLC1 levels are followed by decreased expression of SPTLC2 and ORMDL3 (dashed lines). Sphingolipid synthesis inhibitors, myriocin and fumonisin B1, which block the activity of SPT complex and ceramide synthase, respectively, affect the release of AA from membranes and subsequently the formation of LTB4 or PGD2. These data are in line with the regulatory roles of ceramides on the PLA2 activity (39, 54). Thus, the crosstalk between eicosanoid and sphingolipid pathways seems to be mediated via metabolic mediators (ceramides, S1P, and C1P) and physical interaction of ORMDL-SPT complex and 5-LO.

Fig 5: Changes in the metabolism of sphingolipids and eicosanoids in BMMCL with SPTLC1 or 5-LO knockdowns (KDs). A: Lysates from BMMCL transduced with empty vector (control), SPTLC1 shRNA (SPTLC1 KD), and 5-LO shRNA (5-LO KD) were assessed by immunoblotting with the indicated antibodies. B–E: Quantification of data as in A, normalized to expression in controls and HPRT load. Controls (n = 9), SPTLC1 KD (n = 8), and 5-LO KD (n = 8). F–L: LC-ESI-MS/MS analysis of sphingolipids in Ag-activated transduced BMMCL: control (n = 8), SPTLC1 KD (n = 8), and 5-LO KD (n = 9). F: The sum of total sphingosines, C18:1, C18:0, C20:1, and C20:0, is calculated. G–J, The values of distinct sphingosines C18:1 and C20:1 and sphinganines C18:0 and C20:0 are shown. K: The sum of total ceramide fatty acid chain molecular species, derived from C18:1 sphingosine. L: Non-2-hydroxylated ceramide molecular species derived from C18:1 sphingosine. M–P: UPLC MS/MS analysis of eicosanoids from supernatants of Ag-activated BMMCL: controls (n = 8; except TXB2 n = 7), SPTLC1 KD (n = 8; except TXB2 n = 7), and 5-LO KD (n = 7; except TXB2 n = 6). Quantitative data are mean ± SEM, calculated from n, which show numbers of biological replicates of independently transduced cells. P values were determined by one-way ANOVA with Bonferroni post hoc test except for LTB4, 6t-LTB4, and LTB5 data, which were compared by nonparametric Kruskal-Wallis test with Dunn's post hoc test.