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3. Early activation of hepatic stellate cells induces rapid initiation of retinyl ester breakdown while maintaining lecithin: Retinol acyltransferase (LRAT) activity

Author

Haaker, Maya W, Goossens, Vera, Hoogland, Nina A N, van Doorne, Hidde, Wang, Ziqiong, Jansen, Jeroen W A, Kaloyanova, Dora V, van de Lest, Chris H A, Houweling, Martin, Vaandrager, A Bas, Helms, J Bernd, Haaker, Maya W, Goossens, Vera, Hoogland, Nina A N, van Doorne, Hidde, Wang, Ziqiong, Jansen, Jeroen W A, Kaloyanova, Dora V, van de Lest, Chris H A, Houweling, Martin, Vaandrager, A Bas, and Helms, J Bernd

Abstract

Lechthin:retinol acyltrasnferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release.

Published
2024

5. Inhibition of polyploidization in Pten-deficient livers reduces steatosis

Author

Moreno, Eva, Matondo, Augustine B, Bongiovanni, Laura, van de Lest, Chris H A, Molenaar, Martijn R, Toussaint, Mathilda J M, van Essen, Saskia C, Houweling, Martin, Helms, J Bernd, Westendorp, Bart, de Bruin, Alain, Cell Biology, Metabolism and Cancer, Pathobiologie, CS_Locomotion, Veterinaire biochemie, Equine Musculoskeletal Biology, Dep Biomolecular Health Sciences, Cell Biology, Metabolism and Cancer, Pathobiologie, CS_Locomotion, Veterinaire biochemie, Equine Musculoskeletal Biology, and Dep Biomolecular Health Sciences

Subjects
EXPRESSION, PTEN, GENES, Hepatology, Liver Neoplasms, Peroxisome Proliferator-Activated Receptors, PTEN Phosphohydrolase, non-alcoholic fatty liver disease, FATTY LIVER, hepatocellular carcinoma, polyploidization, Lipids, HEPATOCYTE PLOIDY, atypical E2Fs, Mice, Phosphatidylinositol 3-Kinases, FUSION, Liver, conditional knockout mice, Hepatocytes, steatosis, Animals, E2F8, Proto-Oncogene Proteins c-akt, SUPPRESSION
Abstract

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

Published
2022

6. Lipidomic profiling of rat hepatic stellate cells during activation reveals a two-stage process accompanied by increased levels of lysosomal lipids

Author

Molenaar, Martijn R, Haaker, Maya W, Vaandrager, A Bas, Houweling, Martin, Helms, J Bernd, Molenaar, Martijn R, Haaker, Maya W, Vaandrager, A Bas, Houweling, Martin, and Helms, J Bernd

Abstract

Hepatic stellate cells (HSCs) are liver-resident cells best known for their role in vitamin A storage under physiological conditions. Upon liver injury, HSCs activate into myofibroblast-like cells, a key process in the onset of liver fibrosis. Lipids play an important role during HSC activation. Here, we provide a comprehensive characterization of the lipidomes of primary rat HSCs during 17 days of activation in vitro. For lipidomic data interpretation, we expanded our previously described Lipid Ontology (LION) and associated web application (LION/Web) with the LION-PCA heatmap module, which generates heatmaps of the most typical LION-signatures in lipidomic datasets. Furthermore, we used LION to perform pathway analysis to determine the significant metabolic conversions in lipid pathways. Together, we identify two distinct stages of HSC activation. In the first stage, we observe a decrease of saturated phosphatidylcholine, sphingomyelin, and phosphatidic acid and an increase in phosphatidylserine and polyunsaturated bis(monoacylglycero)phosphate (BMP), a lipid class typically localized at endosomes and lysosomes. In the second activation stage, BMPs, hexosylceramides, and ether-linked phosphatidylcholines are elevated, resembling a lysosomal lipid storage disease profile. The presence of isomeric structures of BMP in HSCs was confirmed ex vivo in MS-imaging datasets of steatosed liver sections. Finally, treatment with pharmaceuticals targeting the lysosomal integrity led to cell death in primary HSCs but not in HeLa cells. In summary, our combined data suggest that lysosomes play a critical role during a two-stage activation process of HSCs.

Published
2023

7. Lipidomic profiling of rat hepatic stellate cells during activation reveals a two-stage process accompanied by increased levels of lysosomal lipids

Author

Veterinaire biochemie, Cell Biology, Metabolism & Cancer - Metabolomics, Molenaar, Martijn R, Haaker, Maya W, Vaandrager, A Bas, Houweling, Martin, Helms, J Bernd, Veterinaire biochemie, Cell Biology, Metabolism & Cancer - Metabolomics, Molenaar, Martijn R, Haaker, Maya W, Vaandrager, A Bas, Houweling, Martin, and Helms, J Bernd

Published
2023

11. Inhibition of polyploidization in Pten-deficient livers reduces steatosis

Author

Cell Biology, Metabolism and Cancer, Pathobiologie, CS_Locomotion, Veterinaire biochemie, Equine Musculoskeletal Biology, Dep Biomolecular Health Sciences, Moreno, Eva, Matondo, Augustine B, Bongiovanni, Laura, van de Lest, Chris H A, Molenaar, Martijn R, Toussaint, Mathilda J M, van Essen, Saskia C, Houweling, Martin, Helms, J Bernd, Westendorp, Bart, de Bruin, Alain, Cell Biology, Metabolism and Cancer, Pathobiologie, CS_Locomotion, Veterinaire biochemie, Equine Musculoskeletal Biology, Dep Biomolecular Health Sciences, Moreno, Eva, Matondo, Augustine B, Bongiovanni, Laura, van de Lest, Chris H A, Molenaar, Martijn R, Toussaint, Mathilda J M, van Essen, Saskia C, Houweling, Martin, Helms, J Bernd, Westendorp, Bart, and de Bruin, Alain

Published
2022

12. Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Author

Molenaar, Martijn R, Yadav, Kamlesh K, Toulmay, Alexandre, Wassenaar, Tsjerk A, Mari, Muriel C, Caillon, Lucie, Chorlay, Aymeric, Lukmantara, Ivan E, Haaker, Maya W, Wubbolts, Richard W, Houweling, Martin, Vaandrager, Arie Bas, Prieur, Xavier, Reggiori, Fulvio, Choudhary, Vineet, Yang, Hongyuan, Schneiter, Roger, Thiam, Abdou Rachid, Prinz, William A, Helms, J Bernd, dB&C FR-RMSC RMSC, Dep Scheikunde, Sub Biochemistry of Membranes begr1-6-12, Veterinaire biochemie, Celbiologie, IOV CCB, dB&C I&I, dB&C FR-RMSC FR, dB&C FR-RMSC RMSC, Dep Scheikunde, Sub Biochemistry of Membranes begr1-6-12, Veterinaire biochemie, Celbiologie, IOV CCB, dB&C I&I, dB&C FR-RMSC FR, Molecular Dynamics, Center for Liver, Digestive and Metabolic Diseases (CLDM), Microbes in Health and Disease (MHD), HAL-SU, Gestionnaire, Utrecht University [Utrecht], National Institutes of Health [Bethesda] (NIH), University Medical Center Groningen [Groningen] (UMCG), Microfluidique, Émulsion et Biologie, Laboratoire de physique de l'ENS - ENS Paris (LPENS), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Département de Physique de l'ENS-PSL, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Département de Physique de l'ENS-PSL, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), University of New South Wales [Sydney] (UNSW), Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), University of Fribourg, Laboratoire de physique de l'ENS - ENS Paris (LPENS (UMR_8023)), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-École normale supérieure - Paris (ENS Paris), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris)

Subjects
Male, Retinyl Esters, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Lecithin, Biochemistry, Seipin, Mice, 0302 clinical medicine, GTP-Binding Protein gamma Subunits, Lipid droplet, LECITHIN, Lipid bilayer, Cells, Cultured, chemistry.chemical_classification, 0303 health sciences, JUNCTIONS, 3. Good health, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, lipids (amino acids, peptides, and proteins), STORAGE, endocrine system, food.ingredient, PROTEINS, BIOGENESIS, ENDOPLASMIC-RETICULUM, Biophysics, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Mice, Transgenic, HEPATIC STELLATE CELLS, Biology, complex mixtures, 03 medical and health sciences, Cricetulus, food, COARSE-GRAINED MODEL, Report, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, Triglycerides, 030304 developmental biology, Organelles, FATTY-ACID, Endoplasmic reticulum, technology, industry, and agriculture, Fatty acid, Lipid Droplets, Cell Biology, Yeast, eye diseases, Mice, Inbred C57BL, REVERSE, chemistry, 030217 neurology & neurosurgery, Biogenesis
Abstract

Molenaar et al. show that seipin does not play a role in the biogenesis of lipid droplets composed mostly or only of the neutral lipid retinyl ester, even in cells that produce triacylglycerols., Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.

Published
2021

13. Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Author

dB&C FR-RMSC RMSC, Dep Scheikunde, Sub Biochemistry of Membranes begr1-6-12, Veterinaire biochemie, Celbiologie, IOV CCB, dB&C I&I, dB&C FR-RMSC FR, Molenaar, Martijn R, Yadav, Kamlesh K, Toulmay, Alexandre, Wassenaar, Tsjerk A, Mari, Muriel C, Caillon, Lucie, Chorlay, Aymeric, Lukmantara, Ivan E, Haaker, Maya W, Wubbolts, Richard W, Houweling, Martin, Vaandrager, Arie Bas, Prieur, Xavier, Reggiori, Fulvio, Choudhary, Vineet, Yang, Hongyuan, Schneiter, Roger, Thiam, Abdou Rachid, Prinz, William A, Helms, J Bernd, dB&C FR-RMSC RMSC, Dep Scheikunde, Sub Biochemistry of Membranes begr1-6-12, Veterinaire biochemie, Celbiologie, IOV CCB, dB&C I&I, dB&C FR-RMSC FR, Molenaar, Martijn R, Yadav, Kamlesh K, Toulmay, Alexandre, Wassenaar, Tsjerk A, Mari, Muriel C, Caillon, Lucie, Chorlay, Aymeric, Lukmantara, Ivan E, Haaker, Maya W, Wubbolts, Richard W, Houweling, Martin, Vaandrager, Arie Bas, Prieur, Xavier, Reggiori, Fulvio, Choudhary, Vineet, Yang, Hongyuan, Schneiter, Roger, Thiam, Abdou Rachid, Prinz, William A, and Helms, J Bernd

Published
2021

14. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system

Author

Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, Helms, J Bernd, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, and Helms, J Bernd

Published
2020

15. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system

Author

LS Veterinaire biochemie, dB&C FR-RMSC RMSC, LS Interne geneeskunde, dCSCA RMSC-1, Onderzoek, dCSCA AVR, Sub Biochemie Algemeen, Biochemisch laboratorium, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, Helms, J Bernd, LS Veterinaire biochemie, dB&C FR-RMSC RMSC, LS Interne geneeskunde, dCSCA RMSC-1, Onderzoek, dCSCA AVR, Sub Biochemie Algemeen, Biochemisch laboratorium, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Haaker, Maya W, Kruitwagen, Hedwig S, Vaandrager, Arie B, Houweling, Martin, Penning, Louis C, Molenaar, Martijn R, van Wolferen, Monique E, Oosterhoff, Loes A, Spee, Bart, and Helms, J Bernd

Published
2020

19. Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Author

Ajat, Mokrish, Molenaar, Martijn, Brouwers, Jos F H M, Vaandrager, Arie B., Houweling, Martin, Helms, J. Bernd, Sub Biochemie Algemeen, LS Veterinaire biochemie, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Sub Biochemie Algemeen, LS Veterinaire biochemie, Sub MS-faciliteit, Dep Biochemie en Celbiologie, and dB&C FR-RMSC FR

Subjects
0301 basic medicine, food.ingredient, Lecithin, Cell Line, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, food, Tandem Mass Spectrometry, Hepatic stellate cells, Lipid droplet, Retinyl palmitate, Lipidomics, Animals, Humans, Diacylglycerol O-Acyltransferase, Retinyl esters, Molecular Biology, DGAT1, Chemistry, Liver Diseases, Wild type, Retinol, Esters, Cell Biology, Lipid droplets, Lipids, 030104 developmental biology, Biochemistry, Acyltransferase, LRAT, Hepatic stellate cell, lipids (amino acids, peptides, and proteins), Acyltransferases, 030217 neurology & neurosurgery
Abstract

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC???MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT???/??? HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT???/??? HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ?? 9.0% small (??? 700 nm) lipid droplets in LRAT???/??? HSCs and 25.6 ?? 1.4% large (1400???2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (??? 700 nm) lipid droplets strongly increased both in wild type and in LRAT???/??? HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.

Published
2017

20. Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead

Author

Afd Chemical Biology and Drug Discovery, dB&C FR-RMSC RMSC, LS Veterinaire biochemie, Sub MS-faciliteit, LS Biochemie van parasieten, Dep Biochemie en Celbiologie, Bexkens, Michiel L, Mebius, Mirjam M, Houweling, Martin, Brouwers, Jos F, Tielens, Aloysius G M, van Hellemond, Jaap J, Afd Chemical Biology and Drug Discovery, dB&C FR-RMSC RMSC, LS Veterinaire biochemie, Sub MS-faciliteit, LS Biochemie van parasieten, Dep Biochemie en Celbiologie, Bexkens, Michiel L, Mebius, Mirjam M, Houweling, Martin, Brouwers, Jos F, Tielens, Aloysius G M, and van Hellemond, Jaap J

Published
2019

22. Lecithin:Retinol Acyl Transferase (LRAT) induces the formation of lipid droplets

Author

Molenaar, Martijn R., primary, Wassenaar, Tsjerk A., additional, Yadav, Kamlesh K., additional, Toulmay, Alexandre, additional, Mari, Muriel C., additional, Caillon, Lucie, additional, Chorlay, Aymeric, additional, Haaker, Maya W., additional, Wubbolts, Richard W., additional, Houweling, Martin, additional, Vaandrager, A. Bas, additional, Reggiori, Fulvio, additional, Thiam, Abdou Rachid, additional, Prinz, William A., additional, and Helms, J. Bernd, additional

Published
2019
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23. Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice

Author

Massafra, Vittoria, Milona, Alexandra, Vos, Harmjan R, Ramos, Rúben J J, Gerrits, Johan, Willemsen, Ellen C L, Ramos Pittol, José M, Ijssennagger, Noortje, Houweling, Martin, Prinsen, Hubertus C M T, Verhoeven-Duif, Nanda M, Burgering, Boudewijn M T, van Mil, Saskia W C, dB&C FR-RMSC FR, and dB&C FR-RMSC FR

Subjects
Male, 0301 basic medicine, Glutamine Synthetase, Proteome, Cytoplasmic and Nuclear, Glutamine, Wistar, Gene Expression, Receptors, Cytoplasmic and Nuclear, Inbred C57BL, Mice, chemistry.chemical_compound, INT-747, Chenodeoxycholic acid, Receptors, Urea, Mice, Knockout, Bile acid, Gastroenterology, Obeticholic acid, Liver Proteome, Liver, Biochemistry, Urea cycle, Metabolome, Dietary Proteins, medicine.medical_specialty, medicine.drug_class, Knockout, Cps1, Biology, Chenodeoxycholic Acid, Bile Acids and Salts, 03 medical and health sciences, Ammonia, Internal medicine, medicine, Journal Article, Animals, Rats, Wistar, Liver X receptor, Hepatology, Catabolism, Rats, Mice, Inbred C57BL, 030104 developmental biology, Endocrinology, chemistry, Hepatocytes, Farnesoid X receptor
Abstract

Background & Aims The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. Methods To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15 NH 4 Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. Results In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. Conclusions In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.

Published
2017

24. Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Author

Tuohetahuntila, Maidina, Molenaar, Martijn R., Spee, Bart, Brouwers, Jos F., Wubbolts, Richard, Houweling, Martin, Yan, Cong, Du, Hong, VanderVen, Brian C, Vaandrager, Arie B., Helms, J. Bernd, Sub Biochemie Algemeen, Onderzoek, dB&C FR-RMSC FR, LS Veterinaire biochemie, Sub MS-faciliteit, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C I&I, Sub Biochemie Algemeen, Onderzoek, dB&C FR-RMSC FR, LS Veterinaire biochemie, Sub MS-faciliteit, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Dep Biochemie en Celbiologie, dCSCA RMSC-1, and dB&C I&I

Subjects
0301 basic medicine, Male, medicine.drug_class, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Mice, Structure-Activity Relationship, 0302 clinical medicine, Lysosome, Lipid droplet, medicine, Hepatic Stellate Cells, Animals, Retinoid, Lipase, Enzyme Inhibitors, Rats, Wistar, Molecular Biology, Mice, Knockout, biology, Cell Biology, Lipid Droplets, Sterol Esterase, Hepatic stellate cell activation, Lipids, In vitro, Cell biology, Rats, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, chemistry, 030220 oncology & carcinogenesis, biology.protein, Hepatic stellate cell, Cholesteryl ester, Female, Lysosomes
Abstract

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously pro-vided evidence for the presence of two distinct LD pools, a pre-existing and a dynamic LD pool. Here we investigate the mech-anisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neu-tral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro. The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, tar-geted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa ؊/؊ mice. Lalistat partially inhibited the induction of activation marker ␣-smooth muscle actin (␣-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

Published
2017

25. Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

Author

Haaker, Maya W., Kruitwagen, Hedwig S., Vaandrager, Arie B., Houweling, Martin, Penning, Louis C., Molenaar, Martijn R., Wolferen, Monique E., Oosterhoff, Loes A., Spee, Bart, and Helms, J. Bernd

Subjects
ACYLTRANSFERASES, LIPIDOSES, FREE fatty acids, ADENOSINE monophosphate, CATS, LIVER
Abstract

Background: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. Objective: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. Animals: Liver organoids obtained from 6 cats. Methods: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. Results: Both T863 (a diacylglycerol O‐acyltransferase 1 [DGAT1] inhibitor) and 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P <.0001) and 46% (P =.0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. Conclusions and Clinical Importance: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid‐lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species‐specific system and provide the basis for further clinical testing of drugs to treat steatosis. [ABSTRACT FROM AUTHOR]

Published
2020
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27. Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Author

Sub Biochemie Algemeen, Onderzoek, dB&C FR-RMSC FR, LS Veterinaire biochemie, Sub MS-faciliteit, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C I&I, Tuohetahuntila, Maidina, Molenaar, Martijn R., Spee, Bart, Brouwers, Jos F., Wubbolts, Richard, Houweling, Martin, Yan, Cong, Du, Hong, VanderVen, Brian C, Vaandrager, Arie B., Helms, J. Bernd, Sub Biochemie Algemeen, Onderzoek, dB&C FR-RMSC FR, LS Veterinaire biochemie, Sub MS-faciliteit, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C I&I, Tuohetahuntila, Maidina, Molenaar, Martijn R., Spee, Bart, Brouwers, Jos F., Wubbolts, Richard, Houweling, Martin, Yan, Cong, Du, Hong, VanderVen, Brian C, Vaandrager, Arie B., and Helms, J. Bernd

Published
2017

28. Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice

Author

dB&C FR-RMSC FR, Massafra, Vittoria, Milona, Alexandra, Vos, Harmjan R, Ramos, Rúben J J, Gerrits, Johan, Willemsen, Ellen C L, Ramos Pittol, José M, Ijssennagger, Noortje, Houweling, Martin, Prinsen, Hubertus C M T, Verhoeven-Duif, Nanda M, Burgering, Boudewijn M T, van Mil, Saskia W C, dB&C FR-RMSC FR, Massafra, Vittoria, Milona, Alexandra, Vos, Harmjan R, Ramos, Rúben J J, Gerrits, Johan, Willemsen, Ellen C L, Ramos Pittol, José M, Ijssennagger, Noortje, Houweling, Martin, Prinsen, Hubertus C M T, Verhoeven-Duif, Nanda M, Burgering, Boudewijn M T, and van Mil, Saskia W C

Published
2017

29. Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Author

Sub Biochemie Algemeen, LS Veterinaire biochemie, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Ajat, Mokrish, Molenaar, Martijn, Brouwers, Jos F H M, Vaandrager, Arie B., Houweling, Martin, Helms, J. Bernd, Sub Biochemie Algemeen, LS Veterinaire biochemie, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dB&C FR-RMSC FR, Ajat, Mokrish, Molenaar, Martijn, Brouwers, Jos F H M, Vaandrager, Arie B., Houweling, Martin, and Helms, J. Bernd

Published
2017

30. Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Author

Md. Ajat, Mohd Mokrish, Molenaar, Martijn, Brouwers, Jos F. H. M., Vaandrager, Arie B., Houweling, Martin, Helms, J. Bernd, Md. Ajat, Mohd Mokrish, Molenaar, Martijn, Brouwers, Jos F. H. M., Vaandrager, Arie B., Houweling, Martin, and Helms, J. Bernd

Abstract

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.

Published
2017

31. Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice

Author

CMM, CMM Groep Van Mil, Child Health, CMM Groep Burgering, Cancer, Genetica Sectie Metabole Diagnostiek, Genetica, Massafra, Vittoria, Milona, Alexandra, Vos, Harmjan R., Ramos, Rúben J J, Gerrits, Johan, Willemsen, Ellen C.L., Ramos Pittol, José M., Ijssennagger, Noortje, Houweling, Martin, Prinsen, Hubertus C.M.T., Verhoeven-Duif, Nanda M., Burgering, Boudewijn M.T., van Mil, Saskia W.C., CMM, CMM Groep Van Mil, Child Health, CMM Groep Burgering, Cancer, Genetica Sectie Metabole Diagnostiek, Genetica, Massafra, Vittoria, Milona, Alexandra, Vos, Harmjan R., Ramos, Rúben J J, Gerrits, Johan, Willemsen, Ellen C.L., Ramos Pittol, José M., Ijssennagger, Noortje, Houweling, Martin, Prinsen, Hubertus C.M.T., Verhoeven-Duif, Nanda M., Burgering, Boudewijn M.T., and van Mil, Saskia W.C.

Published
2017

32. ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells[S]

Author

Maidina, Tuohetahuntila, Molenaar, Martijn R, Spee, Bart, Brouwers, Jos F, Houweling, Martin, Vaandrager, Arie B, Helms, J Bernd, LS Virologie, LS Veterinaire biochemie, Sub Biochemie Algemeen, Onderzoek, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C FR-RMSC FR, LS Virologie, LS Veterinaire biochemie, Sub Biochemie Algemeen, Onderzoek, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dCSCA RMSC-1, and dB&C FR-RMSC FR

Subjects
0301 basic medicine, Lipolysis, QD415-436, Biology, Biochemistry, vitamin A, 03 medical and health sciences, Mice, 0302 clinical medicine, Endocrinology, Lipid droplet, Lipidomics, Hepatic Stellate Cells, Animals, Diacylglycerol O-Acyltransferase, Enzyme Inhibitors, Research Articles, Triglycerides, chemistry.chemical_classification, Mice, Knockout, Lipogenesis, Phenylurea Compounds, hemic and immune systems, Cell Biology, Lipase, Lipid Droplets, heavy isotope labeling, lipolysis and fatty acid metabolism, In vitro, Cell biology, Rats, 030104 developmental biology, Enzyme, chemistry, 030220 oncology & carcinogenesis, Adipose triglyceride lipase, Hepatic stellate cell, Fatty Acids, Unsaturated, lipidomics, Cholesterol Esters
Abstract

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.

Published
2016

33. ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells

Author

LS Virologie, LS Veterinaire biochemie, Sub Biochemie Algemeen, Onderzoek, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C FR-RMSC FR, Maidina, Tuohetahuntila, Molenaar, Martijn R, Spee, Bart, Brouwers, Jos F, Houweling, Martin, Vaandrager, Arie B, Helms, J Bernd, LS Virologie, LS Veterinaire biochemie, Sub Biochemie Algemeen, Onderzoek, Sub MS-faciliteit, Dep Biochemie en Celbiologie, dCSCA RMSC-1, dB&C FR-RMSC FR, Maidina, Tuohetahuntila, Molenaar, Martijn R, Spee, Bart, Brouwers, Jos F, Houweling, Martin, Vaandrager, Arie B, and Helms, J Bernd

Published
2016

35. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells

Author

Tuohetahuntila, Maidina, Spee, Bart, Kruitwagen, Hedwig S, Wubbolts, Richard, Brouwers, Jos F, van de Lest, Chris H, Molenaar, Martijn R, Houweling, Martin, Helms, J Bernd, Vaandrager, Arie B, Tuohetahuntila, Maidina, Spee, Bart, Kruitwagen, Hedwig S, Wubbolts, Richard, Brouwers, Jos F, van de Lest, Chris H, Molenaar, Martijn R, Houweling, Martin, Helms, J Bernd, and Vaandrager, Arie B

Abstract

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.

Published
2015

36. Effects of a single glucocorticoid injection on propylene glycol-treated cows with clinical ketosis

Author

Applied Veterinary Research, Infection & Immunity, Fertility & Reproduction, Regenerative Medicine, Stem Cells & Cancer, dFAH AVR, dB&C FR-RMSC FR, dB&C I&I, Sub Junior Docenten, LS Veterinaire biochemie, Sub Immunologie, LS Biochemie van parasieten, LS Evidence Based Vet Medicine, Sub GZ Herkauwer, van der Drift, Saskia G A, Houweling, Martin, Bouman, Marina, Koets, Ad P, Tielens, Aloysius G M, Nielen, Mirjam, Jorritsma, Ruurd, Applied Veterinary Research, Infection & Immunity, Fertility & Reproduction, Regenerative Medicine, Stem Cells & Cancer, dFAH AVR, dB&C FR-RMSC FR, dB&C I&I, Sub Junior Docenten, LS Veterinaire biochemie, Sub Immunologie, LS Biochemie van parasieten, LS Evidence Based Vet Medicine, Sub GZ Herkauwer, van der Drift, Saskia G A, Houweling, Martin, Bouman, Marina, Koets, Ad P, Tielens, Aloysius G M, Nielen, Mirjam, and Jorritsma, Ruurd

Published
2015

38. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells

Author

Fertility & Reproduction, Regenerative Medicine, Stem Cells & Cancer, dB&C FR-RMSC FR, dB&C I&I, Infection & Immunity, Onderzoek, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, LS Veterinaire biochemie, Sub MS-faciliteit, LS Equine Muscoskeletal Biology, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Algemeen B&C, Tuohetahuntila, Maidina, Spee, Bart, Kruitwagen, Hedwig S, Wubbolts, Richard, Brouwers, Jos F, van de Lest, Chris H, Molenaar, Martijn R, Houweling, Martin, Helms, J Bernd, Vaandrager, Arie B, Fertility & Reproduction, Regenerative Medicine, Stem Cells & Cancer, dB&C FR-RMSC FR, dB&C I&I, Infection & Immunity, Onderzoek, LS Celbiologie-Algemeen, Sub Center for Cell Imaging, LS Veterinaire biochemie, Sub MS-faciliteit, LS Equine Muscoskeletal Biology, Sub Biochemie Algemeen, Dep Biochemie en Celbiologie, LS Algemeen B&C, Tuohetahuntila, Maidina, Spee, Bart, Kruitwagen, Hedwig S, Wubbolts, Richard, Brouwers, Jos F, van de Lest, Chris H, Molenaar, Martijn R, Houweling, Martin, Helms, J Bernd, and Vaandrager, Arie B

Published
2015

41. Ketosis in dairy cows: etiologic factors, monitoring, treatment

Author

Advances in Veterinary Medicine, Dep Gezondheidszorg Landbouwhuisdieren, Stegeman, Arjan, Tielens, Lodewijk, Jorritsma, Ruurd, Schonewille, Thomas, Houweling, Martin, van der Drift, S.G.A., Advances in Veterinary Medicine, Dep Gezondheidszorg Landbouwhuisdieren, Stegeman, Arjan, Tielens, Lodewijk, Jorritsma, Ruurd, Schonewille, Thomas, Houweling, Martin, and van der Drift, S.G.A.

Published
2013

42. Bifunktionalisierte Fettsäuren zur Visualisierung und Identifizierung von Protein-Lipid-Interaktionen in lebenden Zellen

Author

Haberkant, Per, primary, Raijmakers, Reinout, additional, Wildwater, Marjolein, additional, Sachsenheimer, Timo, additional, Brügger, Britta, additional, Maeda, Kenji, additional, Houweling, Martin, additional, Gavin, Anne-Claude, additional, Schultz, Carsten, additional, van Meer, Gerrit, additional, Heck, Albert J. R., additional, and Holthuis, Joost C. M., additional

Published
2013
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43. In Vivo Profiling and Visualization of Cellular Protein-Lipid Interactions Using Bifunctional Fatty Acids

Author

Haberkant, Per, primary, Raijmakers, Reinout, additional, Wildwater, Marjolein, additional, Sachsenheimer, Timo, additional, Brügger, Britta, additional, Maeda, Kenji, additional, Houweling, Martin, additional, Gavin, Anne-Claude, additional, Schultz, Carsten, additional, van Meer, Gerrit, additional, Heck, Albert J. R., additional, and Holthuis, Joost C. M., additional

Published
2013
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44. Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Author

Smolders, Lucas A, primary, Meij, Björn P, additional, Onis, David, additional, Riemers, Frank M, additional, Bergknut, Niklas, additional, Wubbolts, Richard, additional, Grinwis, Guy CM, additional, Houweling, Martin, additional, Groot Koerkamp, Marian JA, additional, van Leenen, Dik, additional, Holstege, Frank CP, additional, Hazewinkel, Herman AW, additional, Creemers, Laura B, additional, Penning, Louis C, additional, and Tryfonidou, Marianna A, additional

Published
2013
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