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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.

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. K E Y W O R D SAICAR, diacylglycerol O-acyltransferase 1 inhibitor, hepatic organoids, perilipin 2, steatosis Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside; AMPK, adenosine monophosphate-activated protein kinase; AMPKK, AMPK kinase; CYP3A132, cytochrome 3A132; DAG, diacylglycerol; DGAT, diacylglycerol O-acyltransferase; FFA, free fatty acid; HMBS, hydroxymethylbilane synthase; HPRT-1, hypoxanthine phosphoribosyltransferase 1; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; NAFLD, non-alcoholic fatty liver disease; PLIN2, perilipin 2; RPS5, ribosomal protein S5; TAG, triacylglycerol; TTR, transthyretin; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta; ZMP, 5-amino-4-imidazolecarboxamide ribose-monophosphate.Maya W. Haaker and Hedwig S. Kruitwagen contributed equally to this work.

2Lipid droplets are unique and nearly ubiquitous organelles that store neutral lipids in a 3 hydrophobic core, surrounded by a monolayer of phospholipids. The primary neutral 4 lipids are triacylglycerols and steryl esters. It is not known whether other classes of 5 neutral lipids can form lipid droplets by themselves. Here we show that production of 6 retinyl esters by lecithin:retinol acyl transferase (LRAT) in yeast cells, incapable of 7 producing triacylglycerols and steryl esters, causes the formation of lipid droplets. By 8 electron microscopy, these lipid droplets are morphologically indistinguishable from 9 those in wild-type cells. In silico and in vitro experiments confirmed the propensity of 10 retinyl esters to segregate from membranes and to form lipid droplets. The hydrophobic 11 N-terminus of LRAT displays preferential interactions with retinyl esters in membranes 12 and promotes the formation of large retinyl ester-containing lipid droplets in mammalian 13 cells. Our combined data indicate that the molecular design of LRAT is optimally suited 14 to allow the formation of characteristic large lipid droplets in retinyl ester-storing cells. 15 16 Keywords 17 18 LRAT; vitamin A; retinol; retinyl ester; retinoids; lipid droplets; lipid droplet size; 19 nucleation; lipid:protein interaction; hepatic stellate cells; liver 20 3 61 the autophagic pathway in HSC activation (Hernandez-Gea and Friedman, 2011; Thoen et 62 al., 2011). 63 64 Surprisingly, inhibition of DGAT1 does not affect the dynamics of the preexisting LDs 65 nor does it affect the synthesis of retinyl esters in isolated primary HSCs (Ajat et al., 2017; 66 4 Tuohetahuntila et al., 2016). However, HSCs contain a specialized enzyme called 67 lecithin:retinol acyltransferase (LRAT) that catalyzes a trans-esterification reaction 68 between the sn-1 position of phosphatidylcholine (PC) and all-trans-retinol to form 69all-trans-retinyl ester (Fig. 1A,B) (Golczak et al., 2012; Ruiz and Bok, 2010). As LRAT is 70 the main contributor to retinyl ester storage in the liver (Liu and Gudas, 2005; O'Byrne et 71 al., 2005), we investigated the possibility that LRAT-mediated retinyl ester synthesis 72 drives the generation of the relatively large, retinyl ester-containing LDs in quiescent 73 HSCs. 74 75 5 Results 76 77 LRAT expression generates UV-positive lipid droplets 78 Primary and quiescent HSCs spontaneously transdifferentiate into activated HSCs 79 (myofibroblasts) ex vivo upon isolation and subsequent culture, resulting in LD 80 disappearance. Quiescent and activated HSCs can be identified based on their high 81 expression of desmin, whereas these two HSC populations can be distinguished from 82 each other by an increased alpha smooth muscle actin (α-SMA) expression in activated 83HSCs (Blaner et al., 2009; Friedman, 2008) (Fig. 1C,D). In addition, LRAT expression 84

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