SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Dynamic changes in the mouse hepatic lipidome following warm ischemia reperfusion injury.

Liver failure secondary to metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most common cause for liver transplantation in many parts of the world. Moreover, the prevalence of MASLD not only increases the demand for liver transplantation, but also limits the supply of suitable donor organs because steatosis predisposes grafts to ischemia-reperfusion injury (IRI). There are currently no pharmacological interventions to limit hepatic IRI because the mechanisms by which steatosis leads to increased injury are unclear. To identify potential novel mediators of IRI, we used liquid chromatography and mass spectrometry to assess temporal changes in the hepatic lipidome in steatotic and non-steatotic livers after warm IRI in mice. Our untargeted analyses revealed distinct differences between the steatotic and non-steatotic response to IRI and highlighted dynamic changes in lipid composition with marked changes in glycerophospholipids. These findings enhance our knowledge of the lipidomic changes that occur following IRI and provide a foundation for future mechanistic studies. A better understanding of the mechanisms underlying such changes will lead to novel therapeutic strategies to combat IRI.

Farnesoid X receptor prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease.

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.

Endoplasmic reticulum stress and mitochondrial dysfunction during aging: Role of sphingolipids.

The endoplasmic reticulum (ER) plays a key role in the regulation of protein folding, lipid synthesis, calcium homeostasis, and serves as a primary site of sphingolipid biosynthesis. ER stress (ER dysfunction) participates in the development of mitochondrial dysfunction during aging. Mitochondria are in close contact with the ER through shared mitochondria associated membranes (MAM). Alteration of sphingolipids contributes to mitochondria-driven cell injury. Cardiolipin is a phospholipid that is critical to maintain enzyme activity in the electron transport chain. The aim of the current study was to characterize the changes in sphingolipids and cardiolipin in ER, MAM, and mitochondria during the progression of aging in young (3 mo.), middle (18 mo.), and aged (24 mo.) C57Bl/6 mouse hearts. ER stress increased in hearts from 18 mo. mice and mice exhibited mitochondrial dysfunction by 24 mo. Hearts were pooled to isolate ER, MAM, and subsarcolemmal mitochondria (SSM). LC-MS/MS quantification of lipid content showed that aging increased ceramide content in ER and MAM. In addition, the contents of sphingomyelin and monohexosylceramides are also increased in the ER from aged mice. Aging increased the total cardiolipin content in the ER. Aging did not alter the total cardiolipin content in mitochondria or MAM yet altered the composition of cardiolipin with aging in line with increased oxidative stress compared to young mice. These results indicate that alteration of sphingolipids can contribute to the ER stress and mitochondrial dysfunction that occurs during aging.

Circulating sphingolipids in heart failure.

Lack of significant advancements in early detection and treatment of heart failure have precipitated the need for discovery of novel biomarkers and therapeutic targets. Over the past decade, circulating sphingolipids have elicited promising results as biomarkers that premonish adverse cardiac events. Additionally, compelling evidence directly ties sphingolipids to these events in patients with incident heart failure. This review aims to summarize the current literature on circulating sphingolipids in both human cohorts and animal models of heart failure. The goal is to provide direction and focus for future mechanistic studies in heart failure, as well as pave the way for the development of new sphingolipid biomarkers.

Exhaled breath condensate identifies metabolic dysregulation in patients with radiation-induced lung injury.

Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatography/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyruvate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle deficiency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the development of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.

Sphingosine kinase 1 mediates sexual dimorphism in fibrosis in a mouse model of NASH.

OBJECTIVE: Men with non-alcoholic fatty liver disease (NAFLD) are more likely to progress to non-alcoholic steatohepatitis (NASH) and liver fibrosis than women. However, the underlying molecular mechanisms of this dimorphism is unclear. We have previously shown that mice with global deletion of SphK1, the enzyme that produces the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), were protected from development of NASH. The aim of this study was to elucidate the role of hepatocyte-specific SphK1 in development of NASH and to compare its contribution to hepatosteatosis in male and female mice. METHODS: We assessed mouse livers in early-stage fibrosis induced by high fat feeding, using single harmonic generation microscopy, LC-MS/MS analysis of hydroxyproline levels, and expression of fibrosis markers. We identified an antifibrotic intercellular signaling mechanism by culturing primary mouse hepatocytes alongside, and in co-culture with, LX2 hepatic stellate cells. RESULTS: We generated hepatocyte-specific SphK1 knockout mice (SphK1-hKO). Unlike the global knockout, SphK1-hKO male mice were not protected from diet-induced steatosis, inflammation, or fibrogenesis. In contrast, female SphK1-hKO mice were protected from inflammation. Surprisingly, however, in these female mice, there was a ∼10-fold increase in the fibrosis markers Col1α1 and 2-3 fold induction of alpha smooth muscle actin and the pro-fibrotic chemokine CCL5. Because increased fibrosis in female SphK1-hKO mice occurred despite an attenuated inflammatory response, we investigated the crosstalk between hepatocytes and hepatic stellate cells, central players in fibrosis. We found that estrogen stimulated release of S1P from female hepatocytes preventing TGFß-induced expression of Col1α1 in HSCs via S1PR3. CONCLUSIONS: The results revealed a novel pathway of estrogen-mediated cross-talk between hepatocytes and HSCs that may contribute to sex differences in NAFLD through an anti-fibrogenic function of the S1P/S1PR3 axis. This pathway is susceptible to pharmacologic manipulation, which may lead to novel therapeutic strategies.

Sphingolipids in Adipose: Kin or Foe?

Obesity research has shifted in recent years to address not only the total amount of adipose tissue present in an individual but also to include adipose tissue functions such as endocrine function and thermogenesis. Data suggest that sphingolipids are critical regulators of metabolic homeostasis, and that disruption of their levels is associated with metabolic disease. Abundant data from mouse models has revealed both beneficial and deleterious roles for sphingolipids in adipose function, and numerous human studies have shown that obesity alters circulating sphingolipid profiles. Sphingolipids comprise a large family of interrelated metabolites, and pinpointing specific functions for specific lipids will be required to fully exploit the therapeutic potential of targeting sphingolipids to treat obesity and related disorders.

Analysis of the Sphingolipidome in NAFLD.

The relationship between sphingolipid levels and NAFLD pathology has been recognized for some time. Numerous studies using pharmacological and genetic approaches in vitro and in animal models of NAFLD have demonstrated that modifications to sphingolipid metabolism can attenuate various facets of NAFLD pathology. However, a more precise understanding of the role of sphingolipids and NAFLD pathology is essential to creating therapeutics that target this pathway. This chapter touches on the scale and variety of sphingolipid metabolites at play in NAFLD, which vary widely in their chemical structures and biological functions. With advances in liquid chromatography and tandem mass spectrometry approaches, each of thousands of individual sphingolipid species and sphingolipid metabolites can be identified and precisely quantified. These approaches are beginning to reveal specific sub-classes and species of sphingolipids that change in NAFLD, and as such, enzymes that generate them can be identified and potentially serve as therapeutic targets. Advances in lipidomics technology have been, and will continue to be, critical to these gains in our understanding of NAFLD.

MYCN upregulates the transsulfuration pathway to suppress the ferroptotic vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis is an iron-dependent, oxidative form of cell death that is countered mainly by glutathione peroxidase 4 (GPX4) and the production of glutathione (GSH), which is formed from cysteine. The identification of the cancers that may benefit from pharmacological ferroptotic induction is just emerging. We recently demonstrated that inducing ferroptosis genetically or pharmacologically in MYCN-amplified neuroblastoma (NB) is a novel and effective way to kill these cells. MYCN increases iron metabolism and subsequent hydroxyl radicals through increased expression of the transferrin receptor 1 (TfR1) and low levels of the ferroportin receptor. To counter increased hydroxyl radicals, MYCN binds to the promoter of SLC3A2 (solute carrier family 3 member 2). SLC3A2 is a subunit of system Xc-, which is the cysteine-glutamate antiporter that exports glutamate and imports cystine. Cystine is converted to cysteine intracellularly. Here, we investigated other ways MYCN may increase cysteine levels. By performing metabolomics in a syngeneic NB cell line either expressing MYCN or GFP, we demonstrate that the transsulfuration pathway is activated by MYCN. Furthermore, we demonstrate that MYCN-amplified NB cell lines and tumors have higher levels of cystathionine beta-synthase (CBS), the rate-limiting enzyme in transsulfuration, which leads to higher levels of the thioether cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine). In addition, MYCN-amplified NB tumors have high levels of methylthioadenosine phosphorylase (MTAP), an enzyme that helps salvage methionine following polyamine metabolism. MYCN directly binds to the promoter of MTAP. We propose that MYCN orchestrates both enhanced cystine uptake and enhanced activity of the transsulfuration pathway to counteract increased reactive oxygen species (ROS) from iron-induced Fenton reactions, ultimately contributing to a ferroptosis vulnerability in MYCN-amplified neuroblastoma.

Systemic Elevation of n-3 Polyunsaturated Fatty Acids (n-3-PUFA) Is Associated with Protection against Visual, Motor, and Emotional Deficits in Mice following Closed-Head Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) causes neuroinflammation and neurodegeneration leading to various pathological complications such as motor and sensory (visual) deficits, cognitive impairment, and depression. N-3 polyunsaturated fatty acid (n-3 PUFA) containing lipids are known to be anti-inflammatory, whereas the sphingolipid, ceramide (Cer), is an inducer of neuroinflammation and degeneration. Using Fat1+-transgenic mice that contain elevated levels of systemic n-3 PUFA, we tested whether they are resistant to mild TBI-mediated sensory-motor and emotional deficits by subjecting Fat1-transgenic mice and their WT littermates to focal cranial air blast (50 psi) or sham blast (0 psi, control). We observed that visual function in WT mice was reduced significantly following TBI but not in Fat1+-blast animals. We also found Fat1+-blast mice were resistant to the decline in motor functions, depression, and fear-producing effects of blast, as well as the reduction in the area of oculomotor nucleus and increase in activated microglia in the optic tract in brain sections seen following blast in WT mice. Lipid and gene expression analyses confirmed an elevated level of the n-3 PUFA eicosapentaenoic acid (EPA) in the plasma and brain, blocking of TBI-mediated increase of Cer in the brain, and decrease in TBI-mediated induction of Cer biosynthetic and inflammatory gene expression in the brain of the Fat1+ mice. Our results demonstrate that suppression of ceramide biosynthesis and inflammatory factors in Fat1+-transgenic mice is associated with significant protection against the visual, motor, and emotional deficits caused by mild TBI. This study suggests that n-3 PUFA (especially, EPA) has a promising therapeutic role in preventing neurodegeneration after TBI.

Sphingolipids in metabolic disease: The good, the bad, and the unknown.

The bioactive sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) are a recent addition to the lipids accumulated in obesity and have emerged as important molecular players in metabolic diseases. Here we summarize evidence that dysregulation of sphingolipid metabolism correlates with pathogenesis of metabolic diseases in humans. This review discusses the current understanding of how ceramide regulates signaling and metabolic pathways to exacerbate metabolic diseases and the Janus faces for its further metabolite S1P, the kinases that produce it, and the multifaceted and at times opposing actions of S1P receptors in various tissues. Gaps and limitations in current knowledge are highlighted together with the need to further decipher the full array of their actions in tissue dysfunction underlying metabolic pathologies, pointing out prospects to move this young field of research toward the development of effective therapeutics.

Sphingolipid Mediators of Myocardial Pathology.

Cardiomyopathy is the leading cause of mortality worldwide. While the causes of cardiomyopathy continue to be elucidated, current evidence suggests that aberrant bioactive lipid signaling plays a crucial role as a component of cardiac pathophysiology. Sphingolipids have been implicated in the pathophysiology of cardiovascular disease, as they regulate numerous cellular processes that occur in primary and secondary cardiomyopathies. Experimental evidence gathered over the last few decades from both in vitro and in vivo model systems indicates that inhibitors of sphingolipid synthesis attenuate a variety of cardiomyopathic symptoms. In this review, we focus on various cardiomyopathies in which sphingolipids have been implicated and the potential therapeutic benefits that could be gained by targeting sphingolipid metabolism.

Depletion of adipocyte sphingosine kinase 1 leads to cell hypertrophy, impaired lipolysis, and nonalcoholic fatty liver disease.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Sphingosine kinase 1 is required for myristate-induced TNFα expression in intestinal epithelial cells.

Saturated fatty acids (SFA) have been known to trigger inflammatory signaling in metabolic tissues; however, the effects of specific SFAs in the intestinal epithelium have not been well studied. Several previous studies have implicated disruptions in sphingolipid metabolism by oversupply of SFAs in inflammatory process. Also, our previous studies have implicated sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) as having key roles in the regulation of inflammatory processes in the intestinal epithelium. Therefore, to define the role for specific SFAs in inflammatory responses in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, significantly induced the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and it was SK1-dependent. Interestingly, myristate-induced TNFα expression was not suppressed by inhibition of S1P receptors (S1PRs), hinting at a potential novel intracellular target of S1P. Additionally, myristate regulated the expression of TNFα via JNK activation in an SK1-dependent manner, suggesting a novel S1PR-independent target as a mediator between SK1 and JNK in response to myristate. Lastly, a myristate-enriched milk fat-based diet (MFBD) increased expression of TNFα in colon tissues and elevated the S1P to sphingosine ratio, demonstrating the potential of myristate-involved pathobiologies in intestinal tissues. Taken together our studies suggest that myristate regulates the expression of TNFα in the intestinal epithelium via regulation of SK1 and JNK.

FTY720/fingolimod decreases hepatic steatosis and expression of fatty acid synthase in diet-induced nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD), a leading cause of liver dysfunction, is a metabolic disease that begins with steatosis. Sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate (S1P), have recently received attention for their potential roles in insulin resistance and hepatic steatosis. FTY720/fingolimod, a prodrug for the treatment of multiple sclerosis, is phosphorylated in vivo to its active phosphorylated form by sphingosine kinase 2 and has been shown to interfere with the actions of S1P and to inhibit ceramide biosynthesis. Therefore, in this study we investigated the effects of FTY720 in a diet-induced animal model of NAFLD (DIAMOND) that recapitulates the hallmarks of the human disease. The oral administration of FTY720 to these mice fed a high-fat diet and sugar water improved glucose tolerance and reduced steatosis. In addition to decreasing liver triglycerides, FTY720 also reduced hepatic sphingolipid levels, including ceramides, monohexosylceramides, and sphingomyelins, particularly the C16:0 and C24:1 species, as well as S1P and dihydro-S1P. FTY720 administration decreased diet-induced fatty acid synthase (FASN) expression in DIAMOND mice without affecting other key enzymes in lipogenesis. FTY720 had no effect on the expression of SREBP-1c, which transcriptionally activates FASN. However, in agreement with the notion that the active phosphorylated form of FTY720 is an inhibitor of histone deacetylases, FTY720-P accumulated in the liver, and histone H3K9 acetylation was markedly increased in these mice. Hence, FTY720 might be useful for attenuating FASN expression and triglyceride accumulation associated with steatosis.

Sphingolipids in adipose tissue: What's tipping the scale?

Adipose tissue lies at the heart of obesity, mediating its many effects upon the rest of the body, with its unique capacity to expand and regenerate, throughout the lifespan of the organism. Adipose is appreciated as an endocrine organ, with its myriad adipokines that elicit both physiological and pathological outcomes. Sphingolipids, bioactive signaling molecules, affect many aspects of obesity and the metabolic syndrome. While sphingolipids are appreciated in the context of these diseases in other tissues, there are many discoveries yet to be uncovered in the adipose tissue. This review focuses on the effects of sphingolipids on various aspects of adipose function and dysfunction. The processes of adipogenesis, metabolism and thermogenesis, in addition to inflammation and insulin resistance are intimately linked to sphingolipids as discussed below.