Expression Of Lysosomal Acid Lipase Research Articles

Abstract 1056: Overexpression Of Lysosomal Acid Lipase

Background: Previous evidence suggests arterial smooth muscle cells (SMCs) comprise the majority of atherosclerotic lesion foam cells in human and mouse atheromas, have reduced expression of ABCA1 and lysosomal acid lipase (LAL) compared to macrophages, and retain cholesteryl esters in lysosomes rather than in the cytoplasm as in macrophage foam cells. Exogenous LAL can increase cholesterol efflux from SMC foam cells despite low ABCA1. Hypothesis: Overexpression of LAL increases cholesterol efflux from SMC foam cells by ABCA1-dependent and -independent mechanisms. Methods: Human vascular SMCs were transfected with 500 MOI AdV- LIPA or AdV- mCherry (adenovirus) control for 6 h and cholesterol loaded using 100 μg/ml aggregated LDL (agLDL) for 24 h. LAL activity was measured by specific enzymatic assay and location of stored cholesteryl esters (CE) was determined using LAMP-1 (lysosome marker) and BODIPY (neutral lipid) staining by confocal microscopy. Cholesterol efflux to 10 μg/ml apoliprotein A-I (ApoA-I) or 100 μg/ml HDL was determined following ABCA1 siRNA treatment or control, with cholesterol mass determined using Amplex Red assay. Student’s t test was used for statistical analysis of LAL activity, and Mann-Whitney U Test for analysis of colocalization of lipid pools. Results: LAL activity increased 1.67-fold 72 h after transfection with AdV- LIPA compared to SMCs transfected with AdV- mCherry (n=5, p = 0.001). Increased LAL expression in the absence of cholesterol acceptor resulted in a shift in CE storage from lysosomes to non-lysosomal compartments (Pearson correlation coefficient for BODIPY/LAMP-1 colocalization 0.27 in AdV- LIPA vs. 0.47 in AdV- mCherry SMCs, n=103, p < 0.00001). Preliminary cholesterol efflux studies indicate SMCs overexpressing LAL release cholesterol by both ABCA1-dependent and -independent mechanisms. Conclusion: Our study provides evidence that despite low ABCA1 and LAL expression, SMC foam cells mobilize lysosomal cholesterol for cellular trafficking and efflux following supplementation with LAL. Further studies will examine changes in gene expression and the mechanism of ABCA1-independent efflux following LAL supplementation in SMC foam cells.

Placental extract suppresses lipid droplet accumulation by autophagy during the differentiation of adipose-derived mesenchymal stromal/stem cells into mature adipocytes

ObjectivePlacental extract, which contains various bioactive compounds, has been used as traditional medicine. Many studies have demonstrated additional applications of placental extract and provided a scientific basis for the broad spectrum of its effects. We have previously reported that porcine placental extract (PPE) strongly suppresses adipogenesis in a 3T3-L1 preadipocyte cell line, inhibiting differentiation. This study aimed to examine the effect of PPE on the accumulation of lipid droplets (LD) in adipose-derived mesenchymal stromal/stem cells (ASC).ResultsThe study findings revealed that PPE decreased the size of LD during the differentiation of ASC into mature adipocytes. RT-qPCR analysis revealed that PPE increased the gene expression of lysosomal acid lipase A (Lipa), a lipolysis-related gene, in ASC-differentiated adipocytes. However, no differences were noted in the adipocyte differentiation markers (Pparg, Cebpa, and Adipoq), or the adipogenesis-related genes (Dgat1, Dgat2, Fasn, Soat1, and Soat2). In addition, PPE promoted autophagosome formation, which was partially co-localized with the LD, indicating that PPE accelerated the degradation of LD by inducing autophagy (termed lipophagy) during the differentiation of ASC into mature adipocytes. These results suggest that the use of PPE may be a potential novel treatment for regulating adipogenesis for the treatment of obesity.

LAL deficiency induced myeloid-derived suppressor cells as targets and biomarkers for lung cancer

BackgroundMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells in tumor microenvironment, which suppress antitumor immunity. Expansion of various MDSC subpopulations is closely associated with poor clinical outcomes in...

Abstract P6-10-14: Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC

Abstract Background: TNBCs have the highest mortality rate among all BC subtypes. There is thus an urgent and unmet need for effective targeted therapies in TNBC. Recently we, identified a novel agent ERX-41 that showed good efficacy in treating TNBC in preclinical mouse models, however, its molecular action remain unknown. In this study, we identified LIPA as novel molecular target of ERX-41. Methods: We have used CRISPR knockout pooled library and multiple TNBC models for identifying molecular target of ERX-41. Mechanistic studies were performed using LIPA mutants, RNA-seq, Turbo-ID mapping, Mass spectrometry, Immunoprecipitation, and Western blotting. The in vivo efficacy of ERX-41 was examined using four different patient-derived xenograft (PDX) models. We evaluated LIPA protein expression in TNBC using tissue microarray (TMA). Results: To identify the molecular target of ERX-41, we performed an unbiased CRISPR–Cas9 knockout (KO) screen in MDA-MB-231 cells and the results identified LIPA as a top hit. KO of LIPA alone (which encodes lysosomal acid lipase (LAL) abrogated cytotoxic response to ERX-41. Cellular thermal shift assays confirmed that ERX-41 binds to LAL. In silico modelling and mutational studies confirmed that ERX-41 interacts with LAL through residues in its LXXLL domain and that ERX-41 ability to induce ER stress and cell death in TNBC is independent of the lipase activity of LAL. Unbiased RNA-seq studies with and without ERX-41 in parental and LIPA KO SUM-159 cells revealed induction of genes involved in ER stress and UPR response by ERX-41 in parental SUM-159 cells but not in cells with LIPA KO. Ultrastructural studies using live-cell confocal microscopy show that LIPA KO abrogated ER morphological changes at 2 and 4 h after ERX-41 treatment. Further, subcellular localization studies showed LIPA localizes to endoplasmic reticulum (ER). Unbiased proteomic approaches (TurboID and DIA mass spec) identified a core set of proteins that were both LAL binders and affected by ERX-41 treatment. GO analyses of LAL binding proteins confirmed their involvement in protein folding. Tumor micro array (TMA) analyses confirmed that >80% of primary TNBC tumors had significant and detectable LAL protein expression in contrast, normal breast tissue had lower LAL expression. ERX-41 (10 mg/kg body weight) decreased growth of four distinct TNBC patient-derived xenografts (PDXs) in vivo. Conclusions: Our results identified a new molecular target (LAL) for ERX-41 and novel mechanism of action (disruption of protein folding and induction of ER stress) that may have utility in treating patients with TNBC. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Uday Pratap, Gangadhara R. Sareddy, Susan T. Weintraub, Ganesh V. Raj, Jung-Mo Ahn, Ratna K. Vadlamudi. Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-14.

Lysosomal acid lipase deficiency: A rare inherited dyslipidemia but potential ubiquitous factor in the development of atherosclerosis and fatty liver disease.

Lysosomal acid lipase (LAL), encoded by the gene LIPA, is the sole neutral lipid hydrolase in lysosomes, responsible for cleavage of cholesteryl esters and triglycerides into their component parts. Inherited forms of complete (Wolman Disease, WD) or partial LAL deficiency (cholesteryl ester storage disease, CESD) are fortunately rare. Recently, LAL has been identified as a cardiovascular risk gene in genome-wide association studies, though the directionality of risk conferred remains controversial. It has also been proposed that the low expression and activity of LAL in arterial smooth muscle cells (SMCs) that occurs inherently in nature is a likely determinant of the propensity of SMCs to form the majority of foam cells in atherosclerotic plaque. LAL also likely plays a potential role in fatty liver disease. This review highlights the nature of LAL gene mutations in WD and CESD, the association of LAL with prediction of cardiovascular risk from genome-wide association studies, the importance of relative LAL deficiency in SMC foam cells, and the need to further interrogate the pathophysiological impact and cell type-specific role of enhancing LAL activity as a novel treatment strategy to reduce the development and induce the regression of ischemic cardiovascular disease and fatty liver.

Lysosomal acid lipase, CSF1R, and PD-L1 determine functions of CD11c+ myeloid-derived suppressor cells.

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids. In the blood of LAL-deficient (Lal–/–) mice, increased CD11c+ cells were accompanied by upregulated programmed cell death ligand 1 (PD-L1) expression. Single-cell RNA sequencing of Lal–/– CD11c+ cells identified 2 distinctive clusters with a major metabolic shift toward glucose utilization and reactive oxygen species overproduction. Pharmacologically blocking pyruvate dehydrogenase in glycolysis not only reduced CD11c+ cells and their PD-L1 expression but also reversed their capabilities of T cell suppression and tumor growth stimulation. Colony-stimulating factor 1 receptor (CSF1R) played an essential role in controlling Lal–/– CD11c+ cell homeostasis and function and PD-L1 expression. Pharmacological inhibition of LAL activity increased CD11c, PD-L1, and CSF1R levels in both normal murine myeloid cells and human blood cells. Tumor-bearing mice and human patients with non–small cell lung cancer also showed CD11c+ cell expansion with PD-L1 and CSF1R upregulation and immunosuppression. There were positive correlations among CD11c, PD-L1, and CSF1R expression and negative correlations with LAL expression in patients with lung cancer or melanoma using The Cancer Genome Atlas database and patient samples. Therefore, CD11c+ cells switched their functions to immune suppression and tumor growth stimulation through CSF1R/PD-L1 upregulation and metabolic reprogramming.

Morroniside, a novel GATA3 binding molecule, inhibits hepatic stellate cells activation by enhancing lysosomal acid lipase expression

BackgroundLiver fibrosis can be easily developed into irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in liver fibrosis development. Morroniside, an iridoid glycoside isolated from Cornus officinalis Sieb. et Zucc., exerts hepatic protective effects. However, the mechanism of action underling the anti-liver fibrosis effects of morroniside have not been fully elucidated. PurposeTo explore whether Lipa served as a biomarker for liver fibrosis and investigate the anti-liver fibrosis effects of morroniside and the underlying action mechanism in liver fibrosis cell models. MethodsLAL expression was examined in the liver tissues of CCl4 and high-fat diet (HFD)-induced liver fibrosis animal models. α-smooth muscle actin (α-SMA) level, collagen and GATA family expressions were analyzed by Real-time PCR and Western blot. Putative transcription factor binding sites in the DNA sequences of Lipa was identified by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed to explore the protein-ligand interaction. ResultsWe found that the expression of hepatic LAL is lower in the liver fibrosis animal models than the control models. The reduced LAL expression is associated with HSCs activation, suggesting LAL is novel liver fibrosis biomarker. More importantly, our data showed that morroniside exerts anti-liver fibrosis effects in vitro. Mechanistic studies reveal that it binds to the hydrophobic sites of GATA3 and also reduces GATA3 expression, which increases LAL expression. ConclusionsThis study, for the first time, suggests LAL is a novel biomarker for liver fibrosis. Besides, morroniside exerts its anti-liver fibrosis effects by targeting GATA3 and LAL and hence inhibits HSC activation. These findings provide strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for liver injury prevention and treatment.

Lysosomal acid lipase promotes endothelial proliferation in cold-activated adipose tissue

ABSTRACT Oxidative tissues such as brown adipose tissue and muscle internalize large amounts of circulating lipids and glucose as energy source. Endothelial cells (ECs) provide a platform for regulated transport and processing of blood-borne nutrients. Next to this role, it has become recognized that intercellular crosstalk between ECs and underlying parenchymal cells is indispensable for maintenance of tissue homoeostasis. Here, we comment on our recent observation that capillary ECs in thermogenic adipose tissues take up and metabolize entire triglyceride-rich lipoprotein (TRL) particles in response to cold exposure. This process is dependent on CD36, lipoprotein lipase (LPL) and lysosomal acid lipase (LAL). Remarkably, loss of LAL specifically in endothelial cells results in impaired endothelial proliferation and diminished thermogenic adaptation. Mechanistically, cell culture experiments indicate that LAL-mediated TRL processing leads to the generation of reactive oxygen species, which in turn activate hypoxia-induced factor (HIF)-mediated proliferative responses. In the current manuscript, we provide in vivo evidence that LAL-deficiency impairs proliferation of endothelial cells in thermogenic adipose tissue. In addition, we show uptake of nanoparticle-labelled TRL and LAL expression in cardiac endothelial cells, suggesting a physiological function of endothelial lipoprotein processing not only in thermogenic adipose tissue but also in cardiac muscle.

Hepatic Lysosomal Acid Lipase Overexpression Alters Metabolism and Promotes Immune Infiltration on a Western Diet

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets (LD) in hepatocytes. If untreated, NAFLD can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, or hepatocellular carcinoma. The progression of NAFLD to NASH is associated with increases in cholesterol levels and decreased autophagy and lipophagy flux. Previous studies have shown that the expression of lysosomal acid lipase (LAL), which can hydrolyze both triglyceride and cholesteryl esters, inversely correlates with the severity of NAFLD and NASH. Based on this, we predicted that overexpressing LAL in the livers of mice fed a NASH inducing high fat diet containing high fructose, palmitate, and cholesterol (FPC) would prevent the development of NAFLD/NASH. Mice fed the FPC diet exhibited numerous markers of NASH including hepatomegaly, lipid accumulation, and inflammation. While LAL overexpression did not prevent hepatomegaly, alterations in LD size and number were observed. RNAseq on liver tissue revealed significant increases in mitochondrial and ribosomal mRNAs as a result of LAL overexpression on the FPC diet, and no changes in lipid biosynthetic pathways. Additionally, mRNAs related to immune cell infiltration and inflammation were significantly enriched in LAL overexpressing mice. Mass cytometry analysis also revealed increased hepatic immune cells in mice overexpressing LAL. Overall, hepatic overexpression of LAL drove immune cell infiltration and inflammation, and did not prevent the development of NAFLD suggesting that targeting LAL activation is not a viable route to treat NAFLD/NASH.

Lysosomal Acid Lipase, a Novel Biomarker for Hepatic Fibrosis

Background: Hepatic fibrosis (HF) can be easily developed irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in cholesterol ester metabolism and is considered a biomarker for liver cirrhosis. However, the association between LAL and HF, as well as the role of LAL in HF development are poorly understood. In this study, we aimed to determine whether LAL served a novel biomarker for HF development and reveal its underlying mechanism. Methods: The expression of LAL was surveyed in the liver tissues of CCl 4 and high-fat diet (HFD)-induced HF animals. Alpha smooth muscle actin level, collagen and Gata family expression was analyzed by Real-time PCR and Western blot. The identification of putative transcription factor binding sites in the DNA sequences of Lipa was located by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed using Desmond to explore the protein-ligand interaction. Findings: Our results showed that LAL expression was reduced in TGF-β1-induced HSC-T6 cells and liver tissues. Gata 3, a member in the Gata family that transcribes Lipa, was involved in the pathological process of HF. More importantly, morroniside, an active compound of Cornus officinalis Sieb. et Zucc., regulated the GATA3/Lipa axis, inhibited HSCs activation and enhanced extracellular matrix (ECM) degradation. These findings, for the first time, suggest a novel biomarker for HF, which is also a molecular target of morroniside for its anti-HF effects. Interpretation: This study provides strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for HF prevention and treatment. Funding: This work was supported by the Basic and Applied Basic Research Fund Project of Guangdong Province (NO.2019A1515110202), the China Postdoctoral Science Foundation (NO.2020M672604), The National Natural Science Foundation of China (82074019 and 81930114), Characteristic innovation projects of universities in Guangdong Province (2020KTSCX030), Guangdong Key Laboratory for Translational Cancer research of Chinese Medicine (2018B030322011), Key-Area Research and Development Program of Guangdong Province (No. 2020B1111100004). Research Grant Council of HKSAR (HKBU-22103017-ECS), Innovation & Technology Commission (PRP/015/19FX), National Natural Science Foundation of China (SCM-2016-NSFC-003) and Natural Science Foundation of Guangdong Province (2018A0303130122). Declaration of Interest: None to declare. Ethical Approval: All animals received humane care, and the experimental protocol was approved by the Committee of Laboratory Animals and was performed according to institutional guidelines.

Hepatic lysosomal acid lipase overexpression worsens hepatic inflammation in mice fed a Western diet

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets in hepatocytes. NAFLD development and progression is associated with an increase in hepatic cholesterol levels and decreased autophagy and lipophagy flux. Previous studies have shown that the expression of lysosomal acid lipase (LAL), encoded by the gene LIPA, which can hydrolyze both triglyceride and cholesteryl esters, is inversely correlated with the severity of NAFLD. In addition, ablation of LAL activity results in profound NAFLD. Based on this, we predicted that overexpressing LIPA in the livers of mice fed a Western diet would prevent the development of NAFLD. As expected, mice fed the Western diet exhibited numerous markers of NAFLD, including hepatomegaly, lipid accumulation, and inflammation. Unexpectedly, LAL overexpression did not attenuate steatosis and had only minor effects on neutral lipid composition. However, LAL overexpression exacerbated inflammatory gene expression and infiltration of immune cells in mice fed the Western diet. LAL overexpression also resulted in abnormal phagosome accumulation and lysosomal lipid accumulation depending upon the dietary treatment. Overall, we found that hepatic overexpression of LAL drove immune cell infiltration and inflammation and did not attenuate the development of NAFLD, suggesting that targeting LAL expression may not be a viable route to treat NAFLD in humans.

The Severity of the Clinical Expression of Lysosomal Acid Lipase in Patients with Cryptogenic Cirrhosis

Lysosomal acid lipase is an enzyme that intervenes in the last steps of lipid metabolism to hydrolyze cholesteryl esters. Patients with cryptogenic cirrhosis present a clear deficiency of lysosomal acid lipase with an unknown mechanism. Design: The present study has an analytical and retrospective design, of a sample of 55 patients with cryptogenic cirrhosis. The degree of association of lysosomal acid lipase was determined with the results of the enzymes alanine aminotransferase and alkaline phosphatase, as well as with the clinical manifestations of portal hypertension and splenic volume. The sensitivity and specificity of the test were determined for the diagnosis of the manifestations of portal hypertension. Results: The most frequent complication of portal hypertension was variceal hemorrhage with 40%, followed by ascites with 32.7%, and last, by hepatic encephalopathy, with 18.2%. Association by the x² test was without statistical significance with values of 0.177, 0.299, and 0.184 for encephalopathy, variceal hemorrhage, and ascites, respectively. The association of lysosomal acid lipase and splenic volume utilizing the Student t-test had a low degree of association and p >0.05. Through ROC curves, we obtained AUROC results close to 0.5. Conclusion: It is established that there is no correlation of the levels of activity of lysosomal acid lipase with the values of alanine aminotransferase and alkaline phosphatase, as well as with the presence or absence of clinical manifestations and by ultrasound of portal hypertension. Lysosomal acid lipase is not a good test for the diagnosis of the clinical manifestations of portal hypertension.

Lysosomal Acid Lipase Drives Adipocyte Cholesterol Homeostasis and Modulates Lipid Storage in Obesity, Independent of Autophagy.

Besides cytoplasmic lipase-dependent adipocyte fat mobilization, the metabolic role of lysosomal acid lipase (LAL), highly expressed in adipocytes, is unclear. We show that the isolated adipocyte fraction, but not the total undigested adipose tissue (ATs), from obese patients has decreased LAL expression compared with that from nonobese people. Lentiviral-mediated LAL knockdown in the 3T3L1 mouse cell line to mimic the obese adipocytes condition did not affect lysosome density or autophagic flux, but it did increase triglyceride storage and disrupt endoplasmic reticulum cholesterol, as indicated by activated SREBP. Conversely, mice with adipose-specific LAL overexpression (Adpn-rtTA x TetO-hLAL) gained less weight and body fat than did control mice fed a high-fat diet, resulting in ameliorated glucose tolerance. Blood cholesterol level in the former was lower than that of control mice, although triglyceridemia in the two groups of mice was similar. The adipose-specific LAL-overexpressing mouse phenotype depends on the housing temperature and develops only under mild hypothermic stress (e.g., room temperature) but not at thermoneutrality (30°C), demonstrating the prominent contribution of brown AT (BAT) thermogenesis. LAL overexpression increased levels of BAT free cholesterol, decreased SREBP targets, and induced the expression of genes involved in initial steps of mitochondrial steroidogenesis, suggesting conversion of lysosome-derived cholesterol to pregnenolone. In conclusion, our study demonstrates that adipose LAL drives tissue-cholesterol homeostasis and affects BAT metabolism, suggesting beneficial LAL activation in anti-obesity approaches aimed at reactivating thermogenic energy expenditure.

Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

The endocannabinoid (EC) system has been implicated in the pathogenesis of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). With the current study we aimed to verify the modulatory effect of endocannabinoid receptor 1 (CB1)-signaling on perilipin 2 (PLIN2)-mediated lipophagy. Here, we demonstrate that a global knockout of the cannabinoid receptor 1 gene (CB1−/−) reduced the expression of the lipid droplet binding protein PLIN2 in the livers of CB1−/− and hepatitis B surface protein (HBs)-transgenic mice, which spontaneously develop hepatic steatosis. In addition, the pharmacologic activation and antagonization of CB1 in cell culture also caused an induction or reduction of PLIN2, respectively. The decreased PLIN2 expression was associated with suppressed lipogenesis and triglyceride (TG) synthesis and enhanced autophagy as shown by increased colocalization of LC3B with lysosomal-associated membrane protein 1 (LAMP1) in HBs/CB1−/− mice. The induction of autophagy was further supported by the increased expression of LAMP1 in CB1−/− and HBs/CB1−/− mice. LAMP1 and PLIN2 were co-localized in HBs/CB1−/− indicating autophagy of cytoplasmic lipid droplets (LDs) i.e., lipophagy. Lipolysis of lipid droplets was additionally indicated by elevated expression of lysosomal acid lipase. In conclusion, these results suggest that loss of CB1 signaling leads to reduced PLIN2 abundance, which triggers lipophagy. Our new findings about the association between CB1 signaling and PLIN2 may stimulate translational studies analyzing new diagnostic and therapeutic options for NAFLD.

Lipid accumulation impairs lysosomal acid lipase activity in hepatocytes: Evidence in NAFLD patients and cell cultures

AimsIt has been hypothesized that the activity of lysosomal acid lipase (LAL), a key enzyme involved in lipid metabolism, is involved in the NAFLD phenotype. To clarify the role of LAL in NAFLD, we studied 164 consecutive patients with biopsy-proven NAFLD and fat-loaded HepG2 cells. MethodsLAL activity was measured (i) on dried blood spots (DBS) from NAFLD patients and dyslipidemic subjects without fatty liver and (ii) on liver biopsies from NAFLD patients. LAL activity and expression were evaluated in HepG2 cells cultured in the presence of free fatty acids (FAs), with or without a PPAR-alpha agonist. ResultsLAL activity was significantly reduced in patients with NAFLD compared to dyslipidemic subjects. LAL activity measured in liver biopsies from NAFLD patients was highly correlated to that measured on DBS and was independent of LAL expression in the liver. In a fully adjusted model, LAL activity on DBS was associated only with platelets and, when normalized by platelet count, it did not differ according to fibrosis stage. In vitro, FA loading of HepG2 fully replicated the impairment of LAL activity observed in NALFD patients. In these cells, the activation of PPAR-alpha receptors prevented and corrected FA-induced LAL impairment, by stimulating FA oxidation and LAL expression. ConclusionsLAL activity is reduced in NAFLD patients, independently from disease progression. In vitro, impaired LAL activity induced by FA loading was rescued by PPAR-alpha activation. These data suggest that the pharmacological modulation of LAL should be explored in the management of NAFLD patients.

Von Hippel-Lindau Protein Maintains Metabolic Balance to Regulate the Survival of Naive B Lymphocytes.

SummaryB lymphocytes undergo metabolic reprogramming upon activation to meet the bioenergetic demands for proliferation and differentiation. Yet, little is known if and how the fate of naive B cells is metabolically regulated. Here, we specifically delete von Hippel-Lindau protein (VHL) in B cells using CD19-Cre and demonstrate that metabolic balance is essential for naive B cell survival. Loss of VHL disturbs glycolytic and oxidative metabolic balance and causes severe reduction in mature B cells. Mechanistically, the metabolic imbalance in VHL-deficient B cells, arising from over-stabilization of hypoxia-inducible factor-1α (HIF-1α), triggers reductive glutamine metabolism leading to increased Fas palmitoylation and caspase-8-mediated apoptosis. Blockade of reductive glutamine metabolic flux by lactate supplementation and ATP citrate lyase inhibition restores the metabolic balance and rectifies the impaired survival of VHL-deficient B cells. Hence, we unravel that the VHL/HIF-1α pathway is required to maintain the metabolic balance of naive B cells and ensure their survival.

Abstract 612: Low Expression of Lysosomal Acid Lipase in Smooth Muscle Cells Relative to Macrophages Provides New Insights into Foam Cell Lipid Accumulation

Background and Hypothesis: Smooth muscle cells (SMCs) are the predominant cell type within the intima of human atherosclerosis-prone arteries and promote initial retention of atherogenic lipoproteins in the deep intima. We previously found that ≥50% of foam cells in intermediate coronary atheromas are of SMC origin and that intimal SMCs have reduced expression of the ATP-binding cassette transporter A1 (ABCA1). Previously we also found that ABCA1 expression is acutely dependent on the flux of cholesterol out of lysosomes and subsequent generation of oxysterols for promotion of gene transcription by the nuclear liver X receptor (LXR). Hypothesis: In the present studies we tested the hypothesis that SMCs have lysosomal dysfunction that contributes to foam cell formation. Methods and Results: Human monocyte-derived macrophages (HMMs) and arterial SMCs were treated with aggregated LDL (agLDL) to increase intracellular cholesterol. Unlike HMMs, agLDL treatment failed to upregulate ABCA1 expression in SMCs and did not significantly increase 27-hydroxycholesterol levels. Also in contrast to HMMs, SMCs did not downregulate new cholesterol synthesis and displayed minimal increases in cholesteryl ester formation with agLDL loading. This data suggested retention of lipids within lysosomal compartments. Indeed, confocal microscopy revealed retention of lipids identified by the neutral lipid dye BODIPY within lysosomal compartments stained with LAMP1 in SMCs, while HMMs showed mostly cytosolic lipid droplets [Pearson correlation of colocalization of +0.3197±0.0123 in SMCs and -0.0897±0.0143 in HMMs (p<0.0001, avg±SEM, n>100 cells)]. LIPA mRNA levels and LAL protein were markedly reduced in SMCs relative to HMMs, with LAL activity being 23.4-times higher in HMMs compared to SMCs (p<0.0001, n=4). Similarly, we found reduced LAL levels in mouse SMCs relative to macrophages. Incubation of SMCs with medium containing LAL reduced lysosomal lipid accumulation, resulting in decreased new cholesterol synthesis and increased cholesterol efflux. Conclusions: We find that arterial SMCs have a relative deficiency in LAL expression resulting in defects in downstream sterol regulatory events compared to macrophages. Our results provide a novel reason for SMC foam cell formation and a potential therapeutic target.

Lysosomal acid lipase promotes cholesterol ester metabolism and drives clear cell renal cell carcinoma progression.

Clear cell renal cell carcinoma (ccRCC) is characterized histologically by accumulation of cholesterol esters, cholesterol and other neutral lipids. Lysosomal acid lipase (LAL) is a critical enzyme involved in the cholesterol ester metabolism. Here, we sought to determine whether LAL could orchestrate metabolism of cholesterol esters in order to promote ccRCC progression. Quantitative reverse-transcription PCR and western blots were conducted to assess the expression of LAL in human ccRCC tissues. We analysed the relationship between LAL levels and patient survival using tissue microarrays. We used cell proliferation assays, colony formation assays, cell death assays, metabolic assays and xenograft tumour models to evaluate the biological function and underlying mechanisms. LAL was up-regulated in ccRCC tissue. Tissue microarray analysis revealed higher levels of LAL in advanced grades of ccRCC, and high LAL expression indicated lower patient survival. Suppressing LAL expression not only blocked the utilization of cholesterol esters but also impaired proliferation and cellular survival. Furthermore, immunohistochemistry staining showed that LAL expression was correlated with Akt phosphorylation. Suppressing LAL expression decreased the phosphorylation level of Akt and Src and reduced the level of 14,15-epoxyeicosatrienoic acids in ccRCC cells. Supplement of 14,15-epoxyeicosatrienoic acids rescued proliferation in vitro and in vivo. LAL promoted cell proliferation and survival via metabolism of epoxyeicosatrienoic acids and activation of the Src/Akt pathway.

Abstract 5421: Differential expression of lysosomal acid lipase (LAL) between benign and malignant tissues among human organs

Abstract Background: Lysosomal acid lipase (LAL), a lysosomal enzyme is able to degrade cholesteryl esters to free fatty acids and cholesterol. The information on the association of LAL with malignancies is limited. This study is to determine the difference in expression level of LAL between benign and malignant tissues among human organs. Method: Immunohistochemistry (IHC) for human LAL was performed on A tissue microarray (TMA) containing 360 benign and malignant tissues from 36 human organs. The expression level of LAL was represented by an IHC score calculated by multiplying area score (0-3) with intensity score (0-3), ranged from 0 to 9. Student’s t-test was used to compare the mean IHC score between benign and malignant tissues in same organs. Results: Eighteen out of 36 human organs have IHC stains on both benign and malignant tissues. As shown in Table 1, LAL expression level was higher in malignancy than in benign tissues in 9 organs. The difference in LAL expression level was significantly (or nearly lower in malignant tissues of adrenal gland, stomach, liver, testis and kidney. Conclusion: The expression level of LAL varies among human organs. As compared with elevation of LAL expression level, decreased expression level of LAL in malignancies could be of more significance. Table 1Difference in expression level of LALOrganmalignantBenignpM/BnMeanSEnMeanSEvalueRatioAdrenal Gland31.670.73560.950.02-1.36Stomach131.170.4583.130.820.035-2.7Liver131.350.3483.560.490.001-2.6Larynx80.250.0940.630.380.22-2.5Testis41.630.4743.750.750.054-2.3Uterus131.620.4272.570.970.302-1.6Kidney122.920.3134.50.870.051-1.5Prostate152.970.51441.22.389-1.3Urinary Bladder110.680.1750.70.30.9561Cervix41.630.4751.250.560.6361.3Colon162.340.3991.50.280.1451.6Esophagus41.630.992100.6951.6Ovary43.631.0741.940.930.2781.9Skin83.591.3251.850.720.3491.9Breast142.860.5861.380.550.1392.1Pancreas101.80.5590.830.140.1262.2Brain32.170.4450.80.560.144`2.7Lung81.970.7450.600.290.193.3 Citation Format: Mohamed EI Gassim, Jinghe Mao, Tangen Ma, Xinchun Zhou. Differential expression of lysosomal acid lipase (LAL) between benign and malignant tissues among human organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5421. doi:10.1158/1538-7445.AM2017-5421

The hepatic expression of lysosomal acid lipase (LAL) is reduced in NAFLD patients, and associated with features of genetically determined LAL deficiency and NAFLD activity score

The hepatic expression of lysosomal acid lipase (LAL) is reduced in NAFLD patients, and associated with features of genetically determined LAL deficiency and NAFLD activity score