Hypoxia-inducible factor 2α overexpression in podocytes ameliorates lipid metabolism disorders in diabetic kidney disease by inhibiting S1P

Baicalin alleviates lipid metabolism disorders in diabetic kidney disease via targeting FKBP51.

Sphingosine 1-phosphate (S1P) is a potent vasculoprotective and neuroprotective signaling lipid, synthesized primarily by sphingosine kinase 2 (SK2) in the brain. We have reported pronounced loss of S1P and SK2 activity early in Alzheimer's disease (AD) pathogenesis, and an inverse correlation between hippocampal S1P levels and age in females, leading us to speculate that loss of S1P is a sensitizing influence for AD. Paradoxically, SK2 was reported to mediate amyloid β (Aβ) formation from amyloid precursor protein (APP) in vitro To determine whether loss of S1P sensitizes to Aβ-mediated neurodegeneration, we investigated whether SK2 deficiency worsens pathology and memory in male J20 (PDGFB-APPSwInd) mice. SK2 deficiency greatly reduced Aβ content in J20 mice, associated with significant improvements in epileptiform activity and cross-frequency coupling measured by hippocampal electroencephalography. However, several key measures of APPSwInd-dependent neurodegeneration were enhanced on the SK2-null background, despite reduced Aβ burden. These included hippocampal volume loss, oligodendrocyte attrition and myelin loss, and impaired performance in Y-maze and social novelty memory tests. Inhibition of the endosomal cholesterol exporter NPC1 greatly reduced sphingosine phosphorylation in glial cells, linking loss of SK2 activity and S1P in AD to perturbed endosomal lipid metabolism. Our findings establish SK2 as an important endogenous regulator of both APP processing to Aβ, and oligodendrocyte survival, in vivo These results urge greater consideration of the roles played by oligodendrocyte dysfunction and altered membrane lipid metabolic flux as drivers of neurodegeneration in AD.SIGNIFICANCE STATEMENT Genetic, neuropathological, and functional studies implicate both Aβ and altered lipid metabolism and/or signaling as key pathogenic drivers of Alzheimer's disease. In this study, we first demonstrate that the enzyme SK2, which generates the signaling lipid S1P, is required for Aβ formation from APP in vivo Second, we establish a new role for SK2 in the protection of oligodendrocytes and myelin. Loss of SK2 sensitizes to Aβ-mediated neurodegeneration by attenuating oligodendrocyte survival and promoting hippocampal atrophy, despite reduced Aβ burden. Our findings support a model in which Aβ-independent sensitizing influences such as loss of neuroprotective S1P are more important drivers of neurodegeneration than gross Aβ concentration or plaque density.

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

Background and PurposeDisordered lipid metabolism and disturbed mitochondrial bioenergetics play pivotal roles in the initiation and development of diabetic kidney disease (DKD). Berberine is a plant alkaloid, used in Chinese herbal medicine. It has multiple therapeutic actions on diabetes mellitus and its complications, including regulation of glucose and lipid metabolism, improvement of insulin sensitivity, and alleviation of oxidative damage. Here, we investigated the reno‐protective effects of berberine.Experimental ApproachWe used samples from DKD patients and experiments with models of DKD (db/db mice) and cultured podocytes, to characterize energy metabolism profiles using metabolomics. Molecular targets and mechanisms involved in the regulation of mitochondrial function and bioenergetics by berberine were investigated, along with its effects on metabolic alterations in DKD mice.Key ResultsMetabolomic analysis suggested altered mitochondrial fuel usage and generalized mitochondrial dysfunction in patients with DKD. In db/db mice, berberine treatment reversed the disordered metabolism, podocyte damage and glomerulosclerosis. Lipid accumulation, excessive generation of mitochondrial ROS, mitochondrial dysfunction, and deficient fatty acid oxidation in DKD mouse models and in cultured podocytes were suppressed by berberine. These protective effects of berberine were accompanied by activation of the peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) signalling pathway, which promoted mitochondrial energy homeostasis and fatty acid oxidation in podocytes.Conclusion and ImplicationsPGC‐1α‐mediated mitochondrial bioenergetics could play a key role in lipid disorder‐induced podocyte damage and development of DKD in mice. Restoration of PGC‐1α activity and the energy homeostasis by berberine might be a potential therapeutic strategy against DKD.

Background and Aims Sodium-glucose cotransporter 2 inhibitors (SGLT2i) and hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors have pleiotropic properties beyond blood glucose-lowering and erythropoietin-increasing effects. We studied the effects of SGLT2i and HIF-PH inhibitor on podocyte protection in diabetic kidney disease (DKD). Method We studied cultured human podocytes treated with high glucose, dapagliflozin or roxadustat. Podocyte-associated molecules expression and morphology changes were assessed. We then studied 5 DKD patients who were started on 3-month SGLT2i treatment and 5 patients who were not treated (control group). The histological patterns of podocyte-associated molecules were assessed in renal biopsies in DKD patients with and without SGLT2i treatment. Results Cultured podocytes exposed to hyperglycemia had reduced mRNA expression of nephrin, podocalyxin and synaptopodin, while treatment with dapagliflozin, roxadustat, or both restore their mRNA expression. Their intracellular protein levels were similarly reduced when treated with hyperglycemia; dapagliflozin, roxadustat, or both restore their intracellular levels. Under hyperglycemia, podocyte cell bodies were shrunken, and the expression of nephrin and podocin on the cell surface were reduced and became granular. Dapagliflozin, roxadustat, or their combination restored the distribution of nephrin and podocin in podocytes. Under hyperglycemia, podocalyxin at apical cell membrane appeared disorganized, and the expression of synaptopodin was reduced, especially at the cell processes, and the punctate appearance was disrupted. Treatment with dapagliflozin, but not roxadustat, partly restored the normal distribution of podocalyxin and synaptopodin. In human DKD, kidney biopsy showed disruption of nephrin, podocin, podocalyxin, and synaptopodin distribution. In the SGLT2i treated patient, their immunofluorescence staining pattern returned to a linear and continuous distribution. Conclusion SGLT2i and HIF-PH inhibitor have significant effects on restoring the podocyte morphology, cytoskeleton architecture, and intracellular levels of podocyte-associated molecule in a diabetic milieu.

Background: Sphingosine kinase 1 (SPHK1) is over-expressed in multiple cancers including breast and colon cancer. SPHK1 catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). S1P promotes tumorigenesis by inhibiting apoptosis and increasing cell proliferation and angiogenesis. Thus SPHK1 has been evaluated as a therapeutic target in cancer. The antidiabetic drug, metformin, has been reported to have protective effects in several cancers, including ovarian cancer. However, molecular mechanisms mediating the anti-tumor effects of metformin are not fully understood. In this study, we demonstrate that SPHK1 is a novel target of metformin and may predict metformin response in ovarian cancer. Methods: The S1P signaling cascade was profiled in several ovarian cancer cell lines. Four different ovarian cancer cell lines were treated with 1mM and 5mM metformin and the mRNA and protein levels of SPHK1 were measured by qRT-PCR and western blotting respectively. Next, the Tyk-nu, HeyA8, SNU119, Kuramochi cell lines were stably transfected to overexpression SPHK1 and metformin sensitivity was measured using MTT proliferation assays. Metabolomic analysis was performed on serum from ovarian cancer patients using metformin for diabetes and compared controls (IRB 13248A). Serum samples were analyzed using a Q-Exactive orbitrap mass spectrometer coupled to a Dionex ultimate UHPLC. Results: In all cell lines tested, metformin suppresses SPHK1 mRNA and protein levels. Analysis of the S1P signaling pathway showed that metformin sensitive cell lines (Tyknu and HeyA8) have high SPHK1 and low S1P lyase (SGPL1) expression, the opposite is true in metformin resistant cell lines (Kuramochi and SNU119). At the same time, S1P receptor 1 (S1PR1) is the main S1P receptor among five S1P receptors (S1PR1-S1PR5) in metformin sensitive cancer cells, however S1PR1 is not dominant in metformin resistant cancer cells. Overexpression of SPHK1 upregulates S1PR1 and increases metformin sensitivity in both metformin sensitive and resistant cell lines. The in vitro findings are augmented by preliminary metabolomic analysis of patient samples that revealed that ovarian cancer patients using metformin for diabetes have lower serum levels of S1P compared to controls. Conclusions: The findings of this study indicate that SPHK1 is a novel metformin target in ovarian cancer. Specifically, high SPHK1 expression sensitizes cells to metformin and S1P signaling profiles predicts metformin response. Based on these findings we propose that sphingolipid signaling be evaluated as a metformin-response signature in ongoing clinical trials of the drug as adjuvant treatment for ovarian cancer (NCT02122185). Citation Format: Tatsuyuki Chiyoda, Xiaojing Liu, Ernst Lengyel, Jason Locasale, Iris Romero. Sphingosine kinase 1 as a mediator and predictor of metformin's protective effect in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A68.

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Several studies describe a binding of p53 to HIF-1alpha in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1alpha. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1alpha plasmid constructs and/or p53 and HIF-1alpha reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1alpha-mediated transactivation of endogenous HIF-1alpha targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1alpha targets that were most dependent on HIF-1alpha function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1alpha-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1alpha target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1alpha target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1alpha targets, regardless of p53 activation, in primary astrocytes.

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Two new classes of drugs, sodium-glucose cotransporter-2 inhibitors (SGLT2is) and hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors (HIF-PHis), are likely to have a significant effect on the management of CKD. SGLT2is were approved by the Food and Drug Administration for the treatment of diabetes in 2013, and have now been shown to slow the progressive loss of kidney function in both diabetic and nondiabetic kidney disease (1⇓–3). SGLT2is demonstrate mortality benefit for patients with CKD, distinguishing them from renin-angiotensin-aldosterone system inhibitors (RAASis), which have otherwise been the mainstay of treatment for patients with diabetic or proteinuric CKD and which provide mortality benefit for a subset of patients with heart failure with reduced ejection fraction (4). Studies of SGLT2is in nondiabetic CKD have been extremely encouraging and we expect that these medications will soon become standard of care for diabetic or nondiabetic, and proteinuric or nonproteinuric, CKD. The potential benefits of SGLT2is have not been specifically explored in autosomal dominant polycystic kidney disease (ADPKD), because both major trials of SGLT2is in nondiabetic CKD (the ongoing EMPA-KIDNEY study [5] and the recently published DAPA-CKD study [1]) excluded patients with ADPKD. Separately, HIF-PHis are anticipated to have a major effect in the management of anemia of CKD. The current use of erythropoiesis-stimulating agents (ESAs) involves a complex balance between treating anemia and limiting potential adverse effects of cardiovascular events, hypertension, and loss of vascular access (6). HIF-PHis have beneficial effects on iron metabolism, hypertension, and inflammation (7). However, there are concerns that HIF activation could promote cyst growth (8) when used for treatment of patients with ADPKD. Here, we examine reasons why patients with ADPKD, who comprise approximately 5% of the ESKD population in the United States, warrant unique consideration regarding treatment with SGLT2is or HIF-PHis. We …

Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p< 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p<0.01 after 3 days). SK activity could be induced further in SKAP cells by the PKC activator PMA, although to a lesser extent than in vector alone cells, while the addition of MK-571 and FTC resulted in a marked increase in intracellular S1P in SKAP and vector alone cells, with a decrease in extracellular S1P levels. These experiments confirm the membrane localisation of SK1 in SKAP transfected cells. SKAP transfection did not affect sensitivity to imatinib or doxorubicin compared to vector alone. Conclusion: These data suggest that SKAP may act as a positive regulator of SK1 activity in cancer cells, an observation that has implications in carcinogenesis and chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256

Hyperglycemia induces all isoforms of transforming growth factor β (TGFβ), which in turn play key roles in inflammation and fibrosis that characterize diabetic nephropathy. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid, derived from sphingosine by the action of sphingosine kinase (SK). S1P mediates many biological processes, which mimic TGFβ signaling. To determine the role of SK1 and S1P in inducing fibrosis and inflammation, and the interaction with TGFβ-1, 2 and 3 signalling in diabetic nephropathy, human proximal tubular cells (HK2 cells) were exposed to normal (5mmol/L) or high (30mmol/L) glucose or TGFβ-1, -2, -3 ± an SK inhibitor (SKI-II) or SK1 siRNA. Control and diabetic wild type (WT) and SK1(-/-) mice were studied. Fibrotic and inflammatory markers, and relevant downstream signalling pathways were assessed. SK1 mRNA and protein expression was increased in HK2 cells exposed to high glucose or TGFβ1,-2,-3. All TGFβ isoforms induced fibronectin, collagen IV and macrophage chemoattractant protein 1 (MCP1), which were reversed by both SKI-II and SK1 siRNA. Exposure to S1P increased phospho-p44/42 expression, AP-1 binding and NFkB phosphorylation. WT diabetic mice exhibited increased renal cortical S1P, fibronectin, collagen IV and MCP1 mRNA and protein expression compared to SK1(-/-) diabetic mice. In summary, this study demonstrates that inhibiting the formation of S1P reduces tubulointerstitial renal inflammation and fibrosis in diabetic nephropathy.

The functional and structural changes in the proximal tubule play an important role in the occurrence and development of diabetic kidney disease (DKD). Diabetes-induced metabolic changes, including lipid metabolism reprogramming, are reported to lead to changes in the state of tubular epithelial cells (TECs), and among all the disturbances in metabolism, mitochondria serve as central regulators. Mitochondrial dysfunction, accompanied by increased production of mitochondrial reactive oxygen species (mtROS), is considered one of the primary factors causing diabetic tubular injury. Most studies have discussed how altered metabolic flux drives mitochondrial oxidative stress during DKD. In the present study, we focused on targeting mitochondrial damage as an upstream factor in metabolic abnormalities under diabetic conditions in TECs. Using SS31, a tetrapeptide that protects the mitochondrial cristae structure, we demonstrated that mitochondrial oxidative damage contributes to TEC injury and lipid peroxidation caused by lipid accumulation. Mitochondria protected using SS31 significantly reversed the decreased expression of key enzymes and regulators of fatty acid oxidation (FAO), but had no obvious effect on major glucose metabolic rate-limiting enzymes. Mitochondrial oxidative stress facilitated renal Sphingosine-1-phosphate (S1P) deposition and SS31 limited the elevated Acer1, S1pr1 and SPHK1 activity, and the decreased Spns2 expression. These data suggest a role of mitochondrial oxidative damage in unbalanced lipid metabolism, including lipid droplet (LD) formulation, lipid peroxidation, and impaired FAO and sphingolipid homeostasis in DKD. An in vitro study demonstrated that high glucose drove elevated expression of cytosolic phospholipase A2 (cPLA2), which, in turn, was responsible for the altered lipid metabolism, including LD generation and S1P accumulation, in HK-2 cells. A mitochondria-targeted antioxidant inhibited the activation of cPLA2f isoforms. Taken together, these findings identify mechanistic links between mitochondrial oxidative metabolism and reprogrammed lipid metabolism in diabetic TECs, and provide further evidence for the nephroprotective effects of SS31 via influencing metabolic pathways.

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.