Accumulation Of Sphingolipids Research Articles

Urothelial Urinary Bladder Cancer Is Characterized by Stage-Dependent Aberrations in Metabolism of Bioactive Sphingolipids

Although dysregulated sphingolipid metabolism was observed in many malignant tumors, bladder cancer has not yet been examined in this regard. This study aims to investigate the metabolism of bioactive sphingolipids across different stages of urothelial urinary bladder cancer (UBC). Forty-eight patients with UBC were included in this study. The neoplasms were classified as either non-muscle-invasive (NMIBC, n = 24) or muscle-invasive (MIBC, n = 24). Samples of the healthy bladder tissue were taken from the patients who underwent radical cystectomy. The content of sphingolipids was measured using an HPLC method, and the mRNA expression of sphingolipid transporters and metabolizing enzymes was evaluated using RT-PCR. Compared to the healthy bladder tissue, the UBC, regardless of the stage, showed an elevated expression of SphK1, Spns2, and ABCC1. The changes in the level of bioactive sphingolipids were strongly stage-dependent. MIBC showed accumulation of sphingosine-1-phosphate (S1P) and ceramide, whereas the content of these sphingolipids in the NMIBC tumor was not different from that of healthy tissue. Moreover, MIBC, compared to NMIBC, was characterized by higher levels of sphingosine and dihydroceramide. We conclude that profound alterations in sphingolipid metabolism develop upon UBC transition from non-muscle-invasive to muscle-invasive. They include the accumulation of S1P, resulting from the increased availability of sphingosine generated from ceramide, which also builds up due to a further activation of its de novo synthesis. We hypothesize that the dysregulation of S1P metabolism leading to the accumulation of this tumor-promoting sphingolipid contributes to the progression of UBC.

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification

Oxidised phosphatidylcholine induces sarcolemmal ceramide accumulation and insulin resistance in skeletal muscle.

Intracellular ceramide accumulation in specific cellular compartments is a potential mechanism explaining muscle insulin resistance in the pathogenesis of type 2 diabetes. Muscle sarcolemmal ceramide accumulation negatively impacts insulin sensitivity in humans, but the mechanism explaining this localised accumulation is unknown. Previous reports revealed that circulating oxidised LDL is elevated in serum of individuals with obesity and type 2 diabetes. Oxidised phosphatidylcholine, which is present in oxidised LDL, has previously been linked to ceramide pathway activation, and could contribute to localised ceramide accumulation in skeletal muscle. We hypothesised that oxidised phosphatidylcholine inversely correlates with insulin sensitivity in serum, and induces sarcolemmal ceramide accumulation and decreases insulin sensitivity in muscle. We used LC-MS/MS to quantify specific oxidised phosphatidylcholine species in serum from a cross-sectional study of 58 well-characterised individuals spanning the physiological range of insulin sensitivity. We also performed in vitro experiments in rat L6 myotubes interrogating the role of specific oxidised phosphatidylcholine species in promoting sarcolemmal ceramide accumulation, inflammation and insulin resistance in skeletal muscle cells. Human serum oxidised phosphatidylcholine levels are elevated in individuals with obesity and type 2 diabetes, inversely correlated with insulin sensitivity, and positively correlated with sarcolemmal C18:0 ceramide levels in skeletal muscle. Specific oxidised phosphatidylcholine species, particularly 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), increase total ceramide and dihydroceramide and decrease total sphingomyelin in the sarcolemma of L6 myotubes by de novo ceramide synthesis and sphingomyelinase activation. POVPC also increases inflammatory signalling and causes insulin resistance in L6 myotubes. These data suggest that circulating oxidised phosphatidylcholine species promote ceramide accumulation and decrease insulin sensitivity in muscle, help explain localised sphingolipid accumulation and muscle inflammatory response, and highlight oxidised phosphatidylcholine species as potential targets to combat insulin resistance.

In Silico Modeling of Fabry Disease Pathophysiology for the Identification of Early Cellular Damage Biomarker Candidates.

Fabry disease (FD) is an X-linked lysosomal disease whose ultimate consequences are the accumulation of sphingolipids and subsequent inflammatory events, mainly at the endothelial level. The outcomes include different nervous system manifestations as well as multiple organ damage. Despite the availability of known biomarkers, early detection of FD remains a medical need. This study aimed to develop an in silico model based on machine learning to identify candidate vascular and nervous system proteins for early FD damage detection at the cellular level. A combined systems biology and machine learning approach was carried out considering molecular characteristics of FD to create a computational model of vascular and nervous system disease. A data science strategy was applied to identify risk classifiers by using 10 K-fold cross-validation. Further biological and clinical criteria were used to prioritize the most promising candidates, resulting in the identification of 36 biomarker candidates with classifier abilities, which are easily measurable in body fluids. Among them, we propose four candidates, CAMK2A, ILK, LMNA, and KHSRP, which have high classification capabilities according to our models (cross-validated accuracy ≥ 90%) and are related to the vascular and nervous systems. These biomarkers show promise as high-risk cellular and tissue damage indicators that are potentially applicable in clinical settings, although in vivo validation is still needed.

Abstract C033: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes including macropinocytosis. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development. In our study, we aimed to characterize PIKfyve, a lipid kinase integral to lysosomal functioning, as a novel and targetable vulnerability in PDAC. We first confirmed that PIKfyve inhibition disrupted autophagic flux and disrupted lysosomal function. Next, to understand the metabolic effects of PIKfyve inhibition in PDAC cells, we employed a metabolism-focused CRISPR screen. Through this, we identified that PIKfyve inhibition increases PDAC cells’ dependency on de novo fatty acid synthesis through genes such as FASN and ACACA. Accordingly, PIKfyve inhibition triggered an increase in SREBP1-driven transcriptional and metabolic signatures favoring de novo fatty acid synthesis through increasing expression of genes such as FASN and ACACA. Further, employing targeted lipidomics, we determined that PIKfyve inhibition triggered an SREBP1-dependent accumulation of long-chain sphingolipids and that knockout of serine palmitoyl transferase subunits SPTLC1 and SPTLC2 sensitized PDAC cells to PIKfyve inhibition. Taken together, PIKfyve inhibition disrupts PDAC lipid homeostasis such that PDAC cells upregulate de novo synthesis of sphingolipids as a compensatory mechanism, suggesting that co-targeting de novo fatty acid synthesis and PIKfyve would result in synergistic effects. Given the importance of KRAS-MAPK signaling in controlling PDAC metabolic homeostasis, we hypothesized that the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Utilizing chromatin immunoprecipitation sequencing, we determined that KRAS or MEK inhibition reduces MYC binding to FASN and ACACA promoters. In line with this observation, KRAS or MEK perturbation decreased FASN and ACACA transcript and protein levels, suggesting that KRAS or MEK inhibition may sensitize PDAC cells to PIKfyve inhibition. Indeed, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC. Citation Format: Caleb Cheng, Jasmine P Wisniewski, Sydney Peters, Jing Hu, Rahul Mannan, Bailey Jackson, Rüya Pakkan, Pietro Morlacchi, Brian Magnuson, Tongchen He, Rupam Bhattacharyya, Yuanyuan Qiao, Costas A Lyssiotis, Arul M Chinnaiyan. Targeting PIKfyve-driven lipid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C033.

No differences in native T1 of the renal cortex between Fabry disease patients and healthy subjects in cardiac dedicated native T1 maps

BackgroundFabry disease (FD) is an X-linked inherited lysosomal storage disease that is caused by deficient activity of the enzyme alpha-galactosidase A. Cardiovascular magnetic resonance (CMR) imaging can detect cardiac sphingolipid accumulation using native T1 mapping. The kidneys are often visible in cardiac CMR native T1 maps, however it is currently unknown if the maps can be used to detect sphingolipid accumulation in the kidneys of FD patients. Therefore, the aim of this study was to evaluate if cardiac dedicated native T1 maps can be used to detect sphingolipid accumulation in the kidneys. MethodsFD patients (n=18, 41 ± 10 years, 44% male) and healthy subjects (n=38, 41 ± 16 years, 47% male) were retrospectively enrolled. Native T1 maps were acquired at 1.5T (MAGNETOM Aera) using MOLLI research sequences. Native T1 values were measured by manually delineating regions of interest (ROI) in the renal cortex, renal medulla, heart, spleen, blood, and liver. Endo- and epicardial borders were delineated in the myocardium and averaged across all slices. Blood ROIs were placed in the left-ventricular blood pool in the midventricular slice. ResultsThere were no differences in native T1 between the FD patients and the healthy subjects in the renal cortex (1034±88 ms vs 1056±59 ms, p=0.29), blood (1614±111 ms vs 1576 ± 100 ms, p=0.22), spleen (1143±45 ms vs 1132±70 ms, p=0.54) or liver (568±49 ms vs 557±47 ms, p=0.41). Native T1 was lower in the hearts of the FD patients compared to healthy subjects (951±79 vs 1006±38, p<0.01), and higher in the renal medulla (1635±144 vs 1514±81, p<0.01). The results were similar when stratified for sex. ConclusionCompared to healthy subjects, patients with FD and cardiac involvement had no differences in native T1 of the renal cortex. FD patients had higher native T1 in the renal medulla, which is not totally explained by differences in blood native T1 but may reflect a hyperfiltration state in the development of renal failure. The findings suggest that sphingolipid accumulation in the renal cortex in FD patients could not be detected with cardiac dedicated research native T1 maps.

Inflammation, Oxidative Stress, and Endothelial Dysfunction in the Pathogenesis of Vascular Damage: Unraveling Novel Cardiovascular Risk Factors in Fabry Disease.

Anderson-Fabry disease (AFD), a genetic disorder caused by mutations in the α-galactosidase-A (GLA) gene, disrupts lysosomal function, leading to vascular complications. The accumulation of globotriaosylceramide (Gb3) in arterial walls triggers upregulation of adhesion molecules, decreases endothelial nitric oxide synthesis, and induces reactive oxygen species production. This cascade results in fibrotic thickening, endothelial dysfunction, hypercontractility, vasospasm, and a pro-thrombotic phenotype. AFD patients display increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating heightened cardiovascular risk. Nailfold capillaroscopy (NFC) shows promise in diagnosing and monitoring microcirculatory disorders in AFD, though it remains underexplored. Morphological evidence of AFD as a storage disorder can be demonstrated through electron microscopy and immunodetection of Gb3. Secondary pathophysiological disturbances at cellular, tissue, and organ levels contribute to the clinical manifestations, with prominent lysosomal inclusions observed in vascular, cardiac, renal, and neuronal cells. Chronic accumulation of Gb3 represents a state of ongoing toxicity, leading to increased cell turnover, particularly in vascular endothelial cells. AFD-related vascular pathology includes increased renin-angiotensin system activation, endothelial dysfunction, and smooth muscle cell proliferation, resulting in IMT increase. Furthermore, microvascular alterations, such as atypical capillaries observed through NFC, suggest early microvascular involvement. This review aims to unravel the complex interplay between inflammation, oxidative stress, and endothelial dysfunction in AFD, highlighting the potential connections between metabolic disturbances, oxidative stress, inflammation, and fibrosis in vascular and cardiac complications. By exploring novel cardiovascular risk factors and potential diagnostic tools, we can advance our understanding of these mechanisms, which extend beyond sphingolipid accumulation to include other significant contributors to disease pathogenesis. This comprehensive approach can pave the way for innovative therapeutic strategies and improved patient outcomes.

Neuropathy and pain in Fabry disease

Fabry disease (FD) is a multiorgan lysosomal storage disorder caused by mutations in the alfa-galactosidase A (GLA ) gene. Pathogenic GLA mutations lead to impaired or even lost enzyme activity, which causes the accumulation of sphingolipids, e.g., globotriaosylceramide, in cells and tissues. The majority of FD patients experience triggerable pain, mainly acral and burning, which often begins in early childhood. While small fiber pathology is assumed to be the basis of FD pain, the underlying molecular mechanisms are not well understood. This review summarizes the clinical characteristics of neuropathy and neuropathic pain in FD, presents current treatment options, and gives an overview of the latest findings from experimental and human model systems on the pathomechanisms contributing to small fiber pathology and FD-associated pain.

Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

#1575 Cardio-renal syndrome in patients with Fabry disease on enzyme replacement therapy

Abstract Background and Aims Fabry disease (FD) is a rare X-linked lysosomal storage disorder in which mutations of the GLA gene cause a decreased or absent activity of the alpha-galactosidase A (α-Gal A) and intracellular accumulation of globotriaosylceramide and other sphingolipids [1]. FD causes a variety of symptoms, including heart and kidneys damage - cardiorenal syndrome (CRS) type 5. In patients with FD, CRS is known to increase the risk of cardiovascular events and death [2]. The aim of our study was to assess renal function and outcomes in patients with FD and CRS receiving enzyme replacement therapy (ERT). Method We performed a retrospective analysis of the medical records of 10 patients (pts) from 7 unrelated families with established diagnosis of FD and cardiac and renal involvement. Pts #1 and #3 are siblings, pt # 10 is their mother; pt #9 is mother of pt #8 (Table 1). The diagnosis was confirmed by DNA diagnostics in all plus levels of α-Gal A enzymatic activity, globotriaosylsphingosine concentrations in some patients. All the patients underwent ERT. Seven out of 10 patients received blockers of the renin-angiotensin-aldosterone system, 3 - did not receive due to contraindications. Results All 10 patients, including 3 women, had left ventricular hypertrophy - left ventricular wall thickness (LVWT) ≥ 1.2 cm. (Table 1). Average LVWT was 1.850 ± 0.097 cm. Two patients had atrial fibrillation. None of the patients had proteinuria 1 g/24 h or higher. In all еGFR was below 60 ml/min/1,73 m2: 4 patients had CKD-G3a, 4 – G3b, 1 – G4 and 1 – G5. Average еGFR was 41,111 ± 8.069 ml/min/1,73 m2 excluding hemodialysis (HD) patient. There were 4 unfavorable outcomes in our group: death occurred in 3 patients from cardiac pathology (congestive heart failure), one patient reached CKD-G5 at 25 years of age and started HD treatment. Patient on HD, brother of deceased pt #1, began receiving ERT after the start of renal replacement therapy. The death of pt #1, who had a very high Lyso-Gb3 level - 118.36 ng/ml (normal 0.05-3.0 ng/ml), appears to be related to the late diagnosis and delayed start of ERT. Conclusion Patients with FD and CRS are likely to have a high risk of cardiovascular complications, loss of kidney function and death, especially with a late start of enzyme replacement and cardio-renoprotective therapy. Early diagnosis of FD and timely initiation of treatment are important in preventing the serious complications and death.

Effect of an eight-week high-intensity interval training programme on circulating sphingolipid levels in middle-aged adults at elevated cardiometabolic risk (SphingoFIT)-Protocol for a randomised controlled exercise trial.

Evidence indicates that sphingolipid accumulation drives complex molecular alterations promoting cardiometabolic diseases. Clinically, it was shown that sphingolipids predict cardiometabolic risk independently of and beyond traditional biomarkers such as low-density lipoprotein cholesterol. To date, little is known about therapeutic modalities to lower sphingolipid levels. Exercise, a powerful means to prevent and treat cardiometabolic diseases, is a promising modality to mitigate sphingolipid levels in a cost-effective, safe, and patient-empowering manner. This randomised controlled trial will explore whether and to what extent an 8-week fitness-enhancing training programme can lower serum sphingolipid levels of middle-aged adults at elevated cardiometabolic risk (n = 98, 50% females). The exercise intervention will consist of supervised high-intensity interval training (three sessions weekly), while the control group will receive physical activity counselling based on current guidelines. Blood will be sampled early in the morning in a fasted state before and after the 8-week programme. Participants will be provided with individualised, pre-packaged meals for the two days preceding blood sampling to minimise potential confounding. An 'omic-scale sphingolipid profiling, using high-coverage reversed-phase liquid chromatography coupled to tandem mass spectrometry, will be applied to capture the circulating sphingolipidome. Maximal cardiopulmonary exercise tests will be performed before and after the 8-week programme to assess patient fitness changes. Cholesterol, triglycerides, glycated haemoglobin, the homeostatic model assessment for insulin resistance, static retinal vessel analysis, flow-mediated dilatation, and strain analysis of the heart cavities will also be assessed pre- and post-intervention. This study shall inform whether and to what extent exercise can be used as an evidence-based treatment to lower circulating sphingolipid levels. The trial was registered on www.clinicaltrials.gov (NCT06024291) on August 28, 2023.

Lysosomal storage diseases. Sphingolipidoses – leukodystrophy

Lysosomal storage diseases. Sphingolipidoses – leukodystrophy

Lipid Toxicity in the Cardiovascular-Kidney-Metabolic Syndrome (CKMS).

Recent studies of Cardiovascular-Kidney-Metabolic Syndrome (CKMS) indicate that elevated concentrations of derivatives of phospholipids (ceramide, sphingosine), oxidized LDL, and lipoproteins (a, b) are toxic to kidney and heart function. Energy production for renal proximal tubule resorption of critical fuels and electrolytes is required for homeostasis. Cardiac energy for ventricular contraction/relaxation is preferentially supplied by long chain fatty acids. Metabolism of long chain fatty acids is accomplished within the cardiomyocyte cytoplasm and mitochondria by means of the glycolytic, tricarboxylic acid, and electron transport cycles. Toxic lipids and excessive lipid concentrations may inhibit cardiac function. Cardiac contraction requires calcium movement from the sarcoplasmic reticulum from a high to a low concentration at relatively low energy cost. Cardiac relaxation involves calcium return to the sarcoplasmic reticulum from a lower to a higher concentration and requires more energy consumption. Diastolic cardiac dysfunction occurs when cardiomyocyte energy conversion is inadequate. Diastolic dysfunction from diminished ATP availability occurs in the presence of inadequate blood pressure, glycemia, or lipid control and may lead to heart failure. Similar disruption of renal proximal tubular resorption of fuels/electrolytes has been found to be associated with phospholipid (sphingolipid) accumulation. Elevated concentrations of tissue oxidized low-density lipoprotein cholesterols are associated with loss of filtration efficiency at the level of the renal glomerular podocyte. Macroscopically excessive deposits of epicardial and intra-nephric adipose are associated with vascular pathology, fibrosis, and inhibition of essential functions in both heart and kidney. Chronic triglyceride accumulation is associated with fibrosis of the liver, cardiac and renal structures. Successful liver, kidney, or cardiac allograft of these vital organs does not eliminate the risk of lipid toxicity. Lipid lowering therapy may assist in protecting vital organ function before and after allograft transplantation.

Endocrinological and metabolic profile of Gaucher disease patients treated with enzyme replacement therapy.

Gaucher disease (GD) is a lysosomal storage disease caused by glucocerebrosidase (GCase) enzyme deficiency. Gaucher cells transformed from the macrophages by progressive sphingolipid accumulation and infiltrate bone marrow, spleen, liver, and other organs. The accumulation of substrate causes inflammation, compromised cellular homeostasis, and disturbed autophagy. It has been hypothesized that this proinflammatory state of GD leads cytokines and chemokines release. As a result of inflammatory process, the cellular dysfunction caused by disruption of cellular signaling, organelle dysfunction, or autoimmune antibodies may affect endocrine profile of GD patients such as hormone levels, lipid profile, and bone mineral density status. A total of 13 patients confirmed to have GD, 12 non-neuronopathic type and one subacute neuronopathic type, were enrolled in our study. The median treatment duration in the enzyme therapy was 13.33 years (9-26 years). At least one endocrinological abnormality was detected in blood tests of nine patients. Hyperinsulinism was the most common finding although fasting blood glucose levels HgbA1c levels were normal in all patients. Two patients had osteopenia, and osteoporosis was detected in two patients. Low HDL levels were detected in six patients, but HDL levels below 23 mg/dL associated with disease severity have been detected in two patients who have not receiving enzyme replacement therapy. None of patients had thyroidal dysfunction. This study had revealed endocrinological abnormalities in GD patients that have not led any severe morbidity in our patients. However, thyroid hormone abnormalities, insulin resistance, or lipid profile abnormalities may cause unpredictable comorbidities. Endocrinological assessment in GD patients in routine follow-up may prevent possible clinical manifestation in long term as well as can define efficacy of ERT on endocrine abnormalities.

Abstract 2087: Targeting lipid metabolism in pancreatic cancer

Abstract Background PIKfyve is a lipid kinase that serves as the sole source of cellular PI(3,5)P2 and PI5P, critical phosphatidylinositols for autophagy and lysosome function. Pancreatic ductal adenocarcinoma (PDAC) is known to upregulate and depend on lysosomal functions such as autophagy to exist in its harsh, nutrient-disrupted microenvironment. Thus, we aimed to establish PIKfyve as a therapeutic target in PDAC. Using a PIKfyve-knockout genetically engineered mouse model of pancreatic cancer (KC and KPC), we determined that PIKfyve was necessary for PDAC development. Given these results, we hypothesized that PIKfyve was critical for maintaining metabolic homeostasis in PDAC and that inhibiting PIKfyve would be an efficacious therapeutic strategy for PDAC. Methods and results We first validated the known role of PIKfyve in regulating autophagy using CRISPRi mediated knockdown, PIKfyve inhibitors Apilimod and ESK981, and a PIKfyve degrader, which all demonstrated that PIKfyve perturbation disrupted autophagic flux and suppressed PDAC cell growth. Little is known about the metabolic impacts of PIKfyve inhibition in PDAC beyond disruption of autophagy. Thus, we paired RNA-seq and a metabolic CRISPR screen to discover that PIKfyve inhibition obligated PDAC cells to upregulate de novo lipid synthesis. Specifically, PDAC cells upregulated fatty acid synthesis genes ACACA and FASN upon PIKfyve inhibition. Additionally, these genes were synthetically critical in the context of PIKfyve inhibition. We further performed metabolomics and lipidomics which revealed accumulation of sphingolipids upon PIKfyve inhibition. Given this, we concluded that PIKfyve regulates lipid homeostasis in PDAC. To further establish PIKfyve as a target in PDAC, we investigated the role of PIKfyve upon inhibition of KRAS, which drives PDAC through MAPK pathway signaling. First, KRAS/MAPK inhibition diminished expression of FASN and ACACA, genes synthetically essential upon PIKfyve inhibition. Additionally, KRAS/MAPK inhibition upregulated autophagy which was attenuated upon concurrent PIKfyve inhibition. Given this metabolic conflict between KRAS/MAPK and PIKfyve, we assessed the effects of dual inhibiting KRAS/MAPK and PIKfyve in a syngeneic orthotopic model. Encouragingly, combination therapy of trametinib (MEK inhibitor) and ESK981 eliminated tumor burden in most of mice, while single treatments only had modest effects. Further, we established a patient-derived xenograft model and showed that while the treatment with individual compounds trametinib, MRTX-1133 (KRASG12D inhibitor), and ESK981 all suppressed tumor growth, the combination of ESK981 and trametinib or ESK981 and MRTX-1133 caused substantial tumor regression in nearly all mice. Taken together, targeting lipid metabolism through inhibition of PIKfyve is a promising therapeutic strategy for PDAC, particularly in combination with the numerous KRAS/MAPK inhibitors in clinical development. Citation Format: Caleb Cheng, Jasmine P. Wisniewski, Ahmet Korkaya, Bailey Jackson, Rahul Mannan, Somnath Mahapatra, Nicholas Rossiter, Rupam Bhattacharyya, Pietro Morlacchi, Sydney Peters, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Targeting lipid metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2087.

Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models.

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

GT-11 impairs insulin signaling through modulation of sphingolipid metabolism in C2C12 myotubes

AimsSphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. Main methodsWe investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). Key findingsTreatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SignificanceOur findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.

Exploring the efficacy and safety of Ambroxol in Gaucher disease: an overview of clinical studies.

Gaucher disease (GD) is mainly caused by glucocerebrosidase (GCase) enzyme deficiency due to genetic variations in the GBA1 gene leading to the toxic accumulation of sphingolipids in various organs, which causes symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, and neurological manifestations. GD is clinically classified into the non-neuronopathic type 1, and the acute and chronic neuronopathic forms, types 2 and 3, respectively. In addition to the current approved GD medications, the repurposing of Ambroxol (ABX) has emerged as a prospective enzyme enhancement therapy option showing its potential to enhance mutated GCase activity and reduce glucosylceramide accumulation in GD-affected tissues of different GBA1 genotypes. The variability in response to ABX varies across different variants, highlighting the diversity in patients' therapeutic outcomes. Its oral availability and safety profile make it an attractive option, particularly for patients with neurological manifestations. Clinical trials are essential to explore further ABX's potential as a therapeutic medication for GD to encourage pharmaceutical companies' investment in its development. This review highlights the potential of ABX as a pharmacological chaperone therapy for GD and stresses the importance of addressing response variability in clinical studies to improve the management of this rare and complex disorder.

Cloning human Benchwarmer gene (BNCH) harboring E164K in vector pcDNA3.1 by site-directed mutagenesis method

Benchwarmer (BNCH) gene encodes an orphan transmembrane transporter belonging to the Major Facilitator Superfamily (MFS), facilitating the transport of ions, amino acids, simple sugars and recently lysolipids. The loss of BNCH function caused lethality in several animal models with neurodegeneration and senescence. At the cellular level, dysregulation of BNCH leads to adverse phenotypes of lysosome and also autophagy (i.e. dyshomeostasis, accumulation of carbohydrates and sphingolipids, and enlarged lysosome). However, the molecular function and ligand of BNCH protein remain to be unrevealed. This study aims to create a radical substitution change in human BNCH coding gene to knock out the protein functions. More specifically, lysine (K) was used to replace the glutamic acid residue 164 (E164K) which is conserved in many animals (fly, zebrafish, mouse and human) and this E164K mutation recapitulated BNCH mutant phenotype. In conclusion, BNCH harboring E164K (BNCH*) was successfully produced by site-directed mutagenesis and cloned into pcDNA.3.1 vector. The construct was transformed into E. coli OmniMAX and that provides a valuable cell assay to search for the molecular ligand of BNCH.

Identification of a Novel Acid Sphingomyelinase Activity Associated with Recombinant Human Acid Ceramidase.

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels.