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Hepatocyte-Specific Deletion of Lysosomal Acid Lipase Leads to Cholesteryl Ester but Not Triglyceride Or Retinyl Ester Accumulation

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Hepatocyte-Specific Deletion of Lysosomal Acid Lipase Leads to Cholesteryl Ester but Not Triglyceride Or Retinyl Ester Accumulation JBC Papers in Press. Published on April 25, 2019 as Manuscript RA118.007201 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.007201 Hepatocyte-specific deletion of lysosomal acid lipase leads to cholesteryl ester but not triglyceride or retinyl ester accumulation Laura Pajed1, Carina Wagner1, Ulrike Taschler1, Renate Schreiber1, Stephanie Kolleritsch1, Nermeen Fawzy1, Isabella Pototschnig1, Gabriele Schoiswohl1, Lisa-Maria Pusch1, Beatrix I. Wieser2, Paul Vesely2, Gerald Hoefler2,4, Thomas O. Eichmann1,3, Robert Zimmermann1,4, and Achim Lass1,4* From the 1Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, 8010 Graz, Austria; 2Diagnostic & Research Center for Molecular BioMedicine, Institute of Pathology, Medical University of Graz, Graz, Austria; 3Center for Explorative Lipidomics, BioTechMed-Graz, Graz, Austria; 4BioTechMed-Graz, Graz, Austria Downloaded from Running title: Lysosomal acid lipase in neutral lipid metabolism *Corresponding author: Achim Lass, Institute of Molecular Biosciences, University of Graz, Heinrichstraße 31/II, 8010 Graz, Austria, Phone: +43 316 380 1900; Fax: +43 316 380 9016; http://www.jbc.org/ E-mail: [email protected] Keywords: lysosomal acid lipase, neutral lipid ester metabolism, cholesterol, triglyceride, vitamin A, hepatocyte, liver, hyperlipidemia, metabolic disorder, lipid metabolism at Bibliothek der Karl-Franzens-Universität Graz on April 29, 2019 ABSTRACT livers from hep-LAL-ko mice indicates that LAL Lysosomal acid lipase (LAL) hydrolyzes is limiting for CE turnover, but not for TG and RE cholesteryl (CE) and retinyl (RE) esters and turnovers. Furthermore, in vitro hydrolase activity triglyceride (TG). Mice globally lacking LAL assays revealed the existence of non-LAL acid accumulate CE most prominently in the liver. The hydrolytic activities for TG and RE. The severity of the CE accumulation phenotype corresponding acid lipase(s) catalyzing these progresses with age and is accompanied by reactions remains to be identified. hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic In humans, mutations in the LIPA gene RE levels are even decreased. To dissect the (encoding lysosomal acid lipase, LAL) are functional role of LAL for neutral lipid ester causative for hepatic cholesteryl ester (CE) and mobilization in the liver, we generated mice triglyceride (TG) accumulation, associated with specifically lacking LAL in hepatocytes (hep- plasma hyperlipidemia (e.g. elevated total LAL-ko). On a standard chow diet, hep-LAL-ko cholesterol and TG levels), hepatomegaly, and the mice exhibited increased hepatic CE accumulation, development of liver disease (e.g. elevated serum but unaltered TG and RE levels. Feeding the hep- transaminases, jaundice, steatosis, fibrosis, and LAL-ko mice a vitamin A excess/high-fat diet cirrhosis) (1, 2). If residual LAL activity of 1 – (VitA/HFD) further increased hepatic cholesterol 12% is present in affected individuals, the levels, but hepatic TG and RE levels in these mice symptoms are less severe and patients are typically were lower than in control mice. Performing in diagnosed during early childhood with CE storage vitro activity assays with lysosomal enriched disease (CESD) (3, 4). If residual activity is below fractions from liver of mice globally lacking LAL, 1%, the clinical symptoms are very pronounced, we detected residual acid hydrolytic activities and affected individuals do not survive beyond the against TG and RE. Interestingly, this non-LAL age of one year (1). This severe form of LAL acid TG hydrolytic activity was elevated in deficiency is termed Wolman disease (5). lysosomal enriched fractions from livers of hep- In mice and similar to humans, genetic LAL-ko mice upon VitA/HFD feeding. In disruption of the Lipa gene (LAL-knockout; -ko) conclusion, the neutral lipid ester phenotype in causes CE and TG accumulation in the liver and Lysosomal acid lipase in neutral lipid metabolism animals develop hepatomegaly (6). In contrast to compared to littermates. Furthermore, lysosomal humans however, the phenotype of LAL-ko mice enriched fractions of liver from hep-LAL-ko mice resembles more the clinical symptoms of CESD on VitA/HFD contained increased acid TG patients and mice live up to 7 - 8 months of age hydrolase activities, which were not inhibited by (7). LAL-ko mice show increased hepatic the presence of the LAL specific inhibitor Lalistat expression of genes involved in fatty acid (FA) and 2, suggesting the presence of so far cholesterol (CHOL) biosynthesis (8). These uncharacterized acid lipase(s). changes in hepatic lipid homeostasis are likely a compensatory mechanism for the lysosomal lipid/ Results CHOL entrapment, further aggravating the lipid Generation of mice lacking LAL specifically in accumulation phenotype with age (8). Irrespective hepatocytes of the age and disease progression of the animals, Lipatm1a mice, carrying the targeted mutation the hepatic CE content is always manifold 1a allele, which harbors reporter and selection Downloaded from increased (e.g. 14.7- and 42.5-fold in 1.5 and 8 genes and the floxed Lipa exon 4, were crossed months old mice, respectively (7)). Remarkably with FLP expressing mice to remove the reporter and in contrast to the pronounced CE accumulation and resistance genes (Fig. 1A). Offspring carrying phenotype, younger LAL-ko mice show little (7) the floxed Lipa allele were crossed with mice http://www.jbc.org/ or no hepatic TG accumulation (9), while at a more expressing Cre recombinase under the control of advanced age (8 months) hepatic TG content is the albumin promoter, which resulted in mice several fold increased (e.g. 9.7-fold (7)). lacking LAL expression specifically in hepatocytes Furthermore, it is puzzling that LAL-ko mice (hep-LAL-ko, Fig. 1A). To confirm the exhibit decreased hepatic RE content, although hepatocyte-specific LAL knock-out, we isolated at Bibliothek der Karl-Franzens-Universität Graz on April 29, 2019 recombinant LAL has been shown to hydrolyze primary hepatocytes and non-parenchymal cells retinyl palmitate (RP), and liver homogenates of (NPCs). Purities of the cell preparations were LAL-ko mice show 90% lower acid RE hydrolase confirmed by high mRNA expression levels of the activity (9, 10). These age-dependent and hepatocyte markers glucose 6-phosphatase diverging changes in hepatic neutral lipid ester (G6Pase) and glycerol kinase (GK) in hepatocytes content (i.e. of CE vs TG and RE) of LAL-ko mice and high mRNA expression of the endothelial cell raise the question whether LAL is limiting for CE marker CD31 as well as macrophage marker F4/80 hydrolysis and changes in hepatic TG and RE in NPCs (Fig. 1B). Analysis of Lipa mRNA levels contents are consequences of deregulated CHOL indicated >96% decreased Lipa expression levels homeostasis. The answer can hardly be deduced in total liver and hepatocytes, while the levels in from the LAL-ko mouse model because of its NPC fraction were virtually unchanged, consistent severe phenotype, which progresses with age and with a hepatocyte-specific deletion of Lipa (Fig. involves many tissues and organs, inducing a very 1C). complex pathology. To limit defective LAL to the Next, we compared lipolytic activities of liver, we generated mice lacking LAL specifically isolated WT and hep-LAL-ko hepatocytes by in hepatocytes (hep-LAL-ko). These mice were performing in vitro activity assays at acidic pH expected to exhibit a rather mild hepatic neutral (pH 4.5) using cholesteryl oleate, triolein, and RP lipid phenotype. Furthermore, the phenotype as substrates. We observed that irrespective of the should be alleviated by the uptake of circulating substrates, acid hydrolytic activities in lysates of LAL, derived from other liver cell types and other hepatocytes from hep-LAL-ko mice were at least tissues, similar as observed in LAL-ko mice with 65% lower compared to WT littermates (Fig. 1D). tissue specific transgenic expression of LAL (11). Furthermore, addition of Lalistat 2, a specific LAL As expected, we observed a moderate CE inhibitor (12), decreased hydrolytic activities of accumulation in liver of hep-LAL-ko mice, which WT lysates to levels similar to that of hep-LAL-ko aggravated upon vitamin A excess/high fat diet lysates, while addition of Orlistat, a more general (VitA/HFD) feeding. Interestingly, under standard serine hydrolase inhibitor (13), further reduced chow diet, hepatic TG and RE contents of these acid hydrolytic activities of both WT and hep- mice remained unaltered. Paradoxically however, LAL-ko lysates (Fig. 1D). Interestingly, addition liver of hep-LAL-ko mice fed a VitA/HFD of Lalistat 2 to lysates prepared from hepatocytes exhibited lower hepatic TG and RE contents as of hep-LAL-ko mice decreased TG and RE 2 Lysosomal acid lipase in neutral lipid metabolism hydrolase activities by 30 and 50%, respectively. VitA/CHOL/olive oil gavage. Two hours after Together, results indicate that hepatocyte-specific lipid bolus administration, plasma RP levels were deletion of LAL leads to a pronounced reduction similarly increased in hep-LAL-ko mice and WT of acid neutral lipid ester hydrolase activities. The littermates and were back to basal levels after 4 presence of Lalistat 2 inhibitable activities in and 8 hours (Fig. 3A). The similar decrease in lysates of hep-LAL-ko hepatocytes indicate the plasma RP levels 4 hours after lipid bolus presence of LAL protein, which may derive
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