Perilipin discerns chronic from acute hepatocellular steatosis

Research Article

Perilipin discerns chronic from acute hepatocellular steatosis Lena Maria Pawella1, Merita Hashani1, Eva Eiteneuer1, Marcus Renner1, Ralf Bartenschlager2, Peter Schirmacher1, Beate Katharina Straub1,⇑ 1

Department of General Pathology, Institute of Pathology, Im Neuenheimer Feld 224, D-69120 Heidelberg, Germany; 2Department for Infectious Diseases, Molecular Virology, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany

Background & Aims: Hepatocellular steatosis is the most frequent liver disease in the western world and may develop further to steatohepatitis, liver cirrhosis and hepatocellular carcinoma. We have previously shown that lipid droplet (LD)-associated proteins of the perilipin/PAT-family are differentially expressed in hepatocyte steatosis and that perilipin is expressed de novo. The aim of this study was to determine the conditions for the temporal regulation of de novo synthesis of perilipin in vitro and in vivo. Methods: Immunohistochemical PAT-analysis was performed with over 120 liver biopsies of different etiology and duration of steatosis. Steatosis was induced in cultured hepatocytic cells with combinations of lipids, steatogenic substances and DMSO for up to 40 days under conditions of stable down-regulation of adipophilin and/or TIP47. Results: Whereas perilipin and adipophilin were expressed in human chronic liver disease irrespective of the underlying etiology, in acute/microvesicular steatosis TIP47, and MLDP were recruited from the cytoplasm to LDs, adipophilin was strongly increased, but perilipin was virtually absent. In long-term steatosis models in vitro, TIP47, MLDP, adipophilin, and finally perilipin were gradually induced. Perilipin and associated formation of LDs were intricately regulated on the transcriptional (PPARs, C/EBPs, SREBP), post-transcriptional, and post-translational level (TAGamount, LD-fusion, phosphorylation-dependent lipolysis). In long-term steatosis models under stable down-regulation of adipophilin and/or TIP47, MLDP substituted for TIP47, and perilipin for adipophilin.

Keywords: PAT-protein family; Lipid droplet; Steatohepatitis; Adipophilin; MLDP; TIP47. Received 3 March 2013; received in revised form 6 November 2013; accepted 11 November 2013; available online 19 November 2013 ⇑ Corresponding author. Address: Institute of Pathology, University Clinic Heidelberg, Im Neuenheimer Feld 224, D-69120 Heidelberg, Germany. Tel.: +49 6221 56 38933; fax: +49 6221 56 5251. E-mail address: [email protected] (B.K. Straub). Abbreviations: ADRP, adipose differentiation-related protein; AFLD, alcoholic fatty liver disease; ASH, alcoholic steatohepatitis; CE, cholesterol-ester; C/EBP, CCAAT/ enhancer-binding protein; DMSO, dimethylsulfoxide; HFD, high fat diet; HSL, hormone-sensitive lipase; IRI, ischemia-reperfusion injury; LD, lipid droplet; LTX, liver transplantation; MLDP, myocardial lipid droplet protein; NAFLD, nonalcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PAT, perilipin/ adipophilin/TIP47-protein-family; PPAR, peroxisome proliferator activatingreceptor; RT, room temperature; TAG, triacylglyceride; TIP47, tail-interacting protein 47 kDa.

Conclusions: LD-maturation in hepatocytes in vivo and in vitro involves sequential expression of TIP47, MLDP, adipophilin and finally perilipin. Thus, perilipin might be used for the differential diagnosis of chronic vs. acute steatosis. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Hepatocellular steatosis is the most frequent liver pathology in western countries and the consequence of various nutritional factors and associated diseases such as alcoholic fatty liver disease (AFLD), obesity, and type II diabetes (non-alcoholic fatty liver disease/NAFLD), chronic hepatitis C (HCV), genetic disorders like lipodystrophy or Wilson’s disease, as well as medication with, e.g., tamoxifen or corticosteroids, but also hypoxic injury, parenteral nutrition, and starvation. AFLD and NAFLD may progress to steatohepatitis (ASH, NASH), liver cirrhosis and even hepatocellular carcinoma (HCC) [1]. Histologically, hepatocellular steatosis is defined by the accumulation of lipid droplets (LDs) in hepatocytes concomitant with an increase of triacylglyceride (TAG) content to more than 5% of liver weight. Intracellular LDs generally consist of a TAG- and/or cholesterol ester-(CE) rich core, which is surrounded by a phospholipid monolayer and associated amphiphilic proteins conferring a LDspecific protein composition dependent on the origin and metabolic status of a cell. LDs are metabolically active, highly dynamic organelles as they represent the regulatory site for neutral lipid hydrolysis and storage [2]. The perilipin/PAT-protein family (concerning nomenclature see [3]), including perilipin (perilipin1) [4], adipophilin (perilipin2, ADRP) [5], TIP47 (perilipin3) [6], S3–12 (perilipin4) [7], and MLDP (perilipin5, OXPAT [3,8]) play the most important role in the biogenesis, stabilization, and degradation of LDs. The exchangeable PAT-proteins (ePATs) TIP47 and MLDP are stable in the cytosol and recruited to LDs under certain metabolic conditions. In contrast, perilipin and adipophilin, the two constitutively expressed PATs (cPATs), only exist bound to LDs; otherwise they are prone to degradation. Adipophilin and TIP47 are almost ubiquitously expressed, whereas perilipin is specific for adipocytes, sebaceous gland epithelial cells, steroidogenic cells, steatotic hepatocytes, and derived tumors [9]. In adipocytes, perilipin promotes lipolysis via hormone-sensitive lipase that is

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Research Article regulated by catecholamines and protein kinase A in a phosphorylation-specific manner [10]. Perilipin-deficient mice are resistant to obesity [11,12], mice with absence of adipophilin show a hepatic TAG-reduction by more than 50% and a higher metabolic rate compared to control mice [13,14]. Recently, two heterozygous PLIN1-frameshift mutations (loss-of-function) were described in patients with partial lipodystrophy, severe dyslipidemia, insulin-resistant diabetes, and hepatic steatosis [15]. We have previously shown that the PAT-proteins perilipin, adipophilin, and TIP47 are differentially expressed in hepatocyte steatogenesis and tumorigenesis in situ [16,17]. Perilipin was unexpectedly negative in some, though moderately steatotic human liver specimens, in cultured cells [18], and fatty livers of morbidly obese mice [19], but found positive in a mouse model of chronic, but not acute ethanol ingestion [20]. Therefore, the circumstances of perilipin induction remain unclear. Thus, the aim of this study was to determine the conditions for the temporal regulation of de novo synthesis of perilipin both in cell culture and in vivo.

Materials and methods Tissues and cultured cells Cryopreserved and formalin-fixed, paraffin-embedded human liver specimens were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany; Supplementary Table 1). Written informed consent was obtained from each patient in accordance with the regulations of the tissue bank and the approval of the ethics committee of the University of Heidelberg (No. 206/2005). Human cell lines were cultured and seeded as listed in Supplementary Table 2. Substances and their working concentrations are listed in Supplementary Table 3.

DNA isolation, bisulfite conversion, and pyrosequencing DNA was isolated using Gentra Puregene Kit (Qiagen, Hilden, Germany). 400 ng DNA per sample were sodium bisulfite-converted using EpiTect Bisulfite Conversion Kit (Qiagen). Bisulfite pyrosequencing was performed on PyroMark Q24 (Qiagen) according to standard protocols, templates were amplified using PyroMark PCR Kit (Qiagen) and primer pairs designed with PyroMark Assay Design SW 2.0 (Qiagen; for sequences see Supplementary Table 5) and data evaluated with Pyro Q-CpG 1.0.9 (Biotage). siRNA- and lentiviral transfection of HuH7 cells siRNA-treatment was performed as described [18]. For lentiviral transfection, pAPM vector (originated from pALPS) carrying a microRNA-based shRNA cassettes targeting adipophilin, TIP47, or non-targeting shRNAs were used (see Supplementary Table 5). For double knockdown, the puromycin resistance gene was replaced by a hygromycin B resistance gene from pAHM NT in the XbaI/NotI restriction enzyme sites. For transfection, 62 ll of 2 M CaCl2 and 500 ll 2 HBS solution was added to 4 ml fresh DMEM; 6.4 lg pAPM, 6.4 lg psPAX.2, and 2.1 lg pMD2.G (3:3:1 ratio) was diluted in DMEM, and added to HEK-293T cells (1.2  106 cells/6-cm dish). After 1 d, medium was changed and 4  104 HuH7 target cells were seeded into 6-well plates for 1 d. Then, the supernatant of HEK-293T cells was harvested, and 1 ml of filtrated viral supernatant was added to HuH7 cells for 6 h, then replaced with viral supernatant in DMEM for 1 d (1:1 ratio). After repetition of the procedure the following day, fresh DMEM with 20% FBS was applied for 1 d. Selection was carried out using 1.2 lg puromycin/ml DMEM or 400 lM hygromycin B for 10 or 20 days, respectively. After selection, 0.3 lg of puromycin and 100 lM hygromycin B were constantly applied on selected cells.

Results Perilipin is expressed in chronic steatosis irrespective of the cause

Antibodies and reagents Primary and secondary antibodies as well as fluorescent dyes are listed in Supplementary Table 4.

Immunofluorescence microscopy and immunohistochemistry Immunofluorescence microscopy of cultured cells and frozen tissues, and immunohistochemistry of formalin-fixed, paraffin-embedded tissue was performed as published [16,17]. Epifluorescence was done with an Olympus IX81 photomicroscope (Hamburg, Germany). Confocal laser scanning microscopy was performed with a Nikon AR1 microscope (Duesseldorf, Germany).

Gel electrophoresis and immunoblotting Cultured cells were harvested in cell lysis buffer (Cell Signaling/New England Biolabs, Frankfurt, Germany), and equivalent aliquots of whole protein lysates were determined by Bradford assay. SDS-PAGE, protein transfer onto polyvinylidene difluoride membranes and immunoblot analyses were undertaken as described [16,17]. Densitometric evaluation of 3 independent experiments was performed by normalization to actin as loading control and to starting value.

Total RNA isolation, cDNA synthesis, and real-time PCR Total cellular RNA was extracted from cultured cells using the NucleoSpin RNA II kit according to the manufacturers’ protocol (Macherey-Nagel, Düren, Germany). For semiquantitative real-time PCR analysis, 1 lg total RNA was reversely transcribed and cDNA was amplified using Poly-dT primers and Multi Cycler PTC (Biozym, Oldendorf, Germany). A cycling program using TaqMan SYBRÒ Green-Master Mix (ABImed, Langenfeld, Germany) was applied for real-time PCR analysis (ABI Prism7300, Applied Biosystems, Darmstadt, Germany): 2 min 50 °C, 15 min

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95 °C, 40 times repeat 15 s 95 °C and 1 min 60 °C. Triplicate reactions were performed. Beta-2-microglobulin was used as endogenous calibrator (for primer sequences see Supplementary Table 5).

Previously, we had noted that PAT-protein-expression correlated with the degree of steatosis but no obvious differences in PATexpression were observed between etiologies such as AFLD/ASH and NAFLD/NASH, and chronic hepatitis C [16], yet, some cases of marked microvesicular steatosis showed strikingly low amounts or even total absence of perilipin. Therefore, we extended the immunohistochemical PAT-analysis to other known causes of hepatocellular steatosis. In about 30 different liver specimens, perilipin was detected in steatohepatitis (NASH), and in chronic hepatitis C as expected, as well as in long-standing drug-induced steatosis, e.g., after cortisone- or tetracyclin-therapy, Wilson’s disease, and even genetic diseases in newborn children such as in glycogenosis and mitochondriopathy, but not in normal livers (Fig. 1, Table 1A). Thereby, perilipin was detected in livers with chronic steatosis irrespective of etiology, gender, and age. Perilipin is absent in acute-onset steatosis Interestingly, we noted that in ballooned cells in NASH (Fig. 1) and in liver specimens with acute injury, like in liver infarcts, ischemia-reperfusion injury (IRI), HCV reinfection, or parenteral nutrition (PN) in liver transplants, as well as in acute druginduced steatosis, adipophilin was markedly induced and the ePATs TIP47 and MLDP were often recruited from the cytoplasm to small LDs, whereas perilipin was reduced or absent. To analyse time-dependent PAT-expression in human samples with especially acute steatosis, we chose liver biopsies of patients who

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JOURNAL OF HEPATOLOGY Adipophilin

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Fig. 1. Perilipin is expressed in hepatocellular steatosis irrespective of the cause. Immunohistochemical analysis of perilipin, adipophilin, TIP47, and MLDP in liver specimens with NASH (for ballooned cells see arrows), cortisone-induced hepatocellular steatosis, chronic hepatitis C, Wilson’s disease and mitochondrial disorders (MCP). For control, immunohistochemical PAT-staining in normal human liver is shown with negativity for perilipin, cytoplasmic MLDP and TIP47 staining and adipophilin and TIP47 staining restricted to LDs of hepatic stellate cells (arrows).

had undergone liver transplantation (LTX) and where control liver biopsies at defined time points were available to study the dynamics of PAT-induction by drugs and toxins, hypoxia (IRI, ischemia), PN, and HCV-reinfection. We investigated nearly 90 liver biopsies and resection specimens of 24 patients (Supplementary Table 1B). In non-steatogenic donor livers, only marginal amounts of PAT-proteins were detectable. Yet, only few days

after LTX, due to IRI, drug therapy, PN, necrosis or HCV-reinfection, microvesicular LD-accumulation was noted with positivity for adipophilin, TIP47, and MLDP, but virtually not perilipin (Fig. 2, Supplementary Table 1B). Only after several weeks, increasing perilipin reactivity was noted in LDs of increasing size (Fig. 2A). In the majority of the studied LTX-patients the donor liver showed mild pre-existing macrovesicular steatosis with

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Fig. 2. Perilipin is virtually absent from liver biopsies with acute steatosis. Immunohistochemical analysis for perilipin, adipophilin, TIP47, and MLDP in liver transplant specimens with drug-induced steatosis. (A) A 4 month old child received a liver specimen (LTX) with nearly no steatosis (arrows: perilipin and adipophilin-positive LDs). In follow-up liver biopsies (after 2 weeks with mild IRI and PN; and after 10 months following cortisone-therapy), mild microvesicular steatosis is seen with positivity for adipophilin, TIP47, and MLDP, but virtually no perilipin, and only moderate increase for perilipin after 10 months. (B) Liver biopsy of another transplanted child is shown, with therapy-induced steatosis (TX) positive for adipophilin, TIP47, and MLDP, but again virtually no perilipin. In the row below, higher magnification is shown for better visualization of the small LDs in acute microvesicular steatosis.

positivity of perilipin and adipophilin (Supplementary Table 1B). Yet, in early liver biopsies post LTX, though an increase of microvesicular steatosis was noted, perilipin was markedly diminished together with a break-down of large pericentral LDs. Simultaneously, high expression of TIP47, adipophilin, and MLDP appeared. Interestingly, in cases with strong liver damage due to massive necrosis or liver biopsies with incipient liver failure, strong TIP47, MLDP, and adipophilin immunoreactivity at minute LDs was noted (Supplementary Table 1B, cases 4, 11, 636

15, 16, 19, 20, 22–24). In summary, in all cases of acute steatosis/liver damage studied, TIP47, MLDP, and adipophilin were strongly increased in small-size LDs corresponding to microvesicular steatosis, but perilipin was generally absent or diminished (for an estimation of PAT-expression at different-sized LDs in micro- and macrovesicular steatosis see Supplementary Fig. 1). As primary human hepatocytes often express perilipin [16] and dedifferentiate rapidly in culture, cells of the lines HepG2, PLC, Hep3B, and HuH7 were incubated with oleate in

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JOURNAL OF HEPATOLOGY combinations with cholesterol, LDL, ethanol, and ciglitazone for up to 10 days. Adipophilin, was up-regulated as measured by immunoblot and immunofluorescence microscopy, whereas TIP47 and MLDP levels were often unchanged but appeared redirected towards LDs. Under conditions of hypoxia, induced by DMOG or by incubation in a hypoxia chamber, HIF-1a was detectable after a few hours and adipophilin was increased 1 to 2 days after treatment, whereas perilipin could not be detected. Sequential PAT-expression preceding perilipin induction in long-term cell culture models To test for perilipin expression in a chronic steatosis setting in vitro, we subjected HuH7 cells to long-term treatments for up to 40 days. With combinations of oleate, and different compounds known to induce adipogenesis (PAM/preadipocyte-adipocyte differentiation medium) with dimethylsulfoxide (DMSO), a factor known to induce differentiation and cell growth arrest [21], we succeeded to induce perilipin in chronic steatosis in vitro. Fastest and strongest perilipin induction was achieved with a combination of PAM-containing medium with DMSO (Fig. 3B), whereas control media, DMSO or oleate alone were less effective at inducing perilipin (Fig. 3A). PAT-protein expression followed a hierarchy: a protein peak in TIP47 was detected first, followed by MLDP, adipophilin, and finally perilipin (Fig. 3C) with declining TIP47, MLDP, and adipophilin levels as soon as perilipin levels were raising. Perilipin always localized around BODIPYpositive LDs of all sizes and consistently colocalized with adipophilin, predominantly in non-proliferating cells (Fig. 3D). Interestingly, whereas all hepatoma-derived cells showed perilipin induction in long-term culture, Caki-2, HEK-293, MCF-7, Hela, HCT 116, LN229, and HaCaT cells contained only very low amounts of perilipin protein even under PAM-DMSO-treatment, suggesting a cell-type-specific role of perilipin in LD-maturation (data not shown). In summary, these results show that perilipin is de novo expressed in cultures of hepatic cells under conditions mimicking chronic steatosis and follows the expression of TIP47, MLDP, and adipophilin. Regulation of PAT-expression in chronic steatosis In the steatosis models, perilipin expression was largely dependent on cell proliferation and the differentiation status of the cells as well as on the duration of the treatment (Fig. 3). Therefore, to determine transcriptional, post-transcriptional and post-translational modulators of perilipin expression, we analysed transcription factors known to play an important role in hepatic lipid metabolism (Fig. 3B). Expression of PPARa (peroxisome proliferator-activating receptor a), a transcription factor regulating fatty acid oxidation, and PPARc, the master regulator of adipogenesis, increased during PAM and PAM-DMSO treatments. Antibodies against C/EBPa (CCAAT/enhancer-binding protein a) revealed a time-dependent decrease of the active isoform (43 kDa), whereas the inhibiting isoform of C/EBPa (30 kDa) was not detected. The active isoforms of C/EBPb (LAP⁄/ LAP (liver enriched activating protein)) was up-regulated with and without DMSO (for reported molecular weights see [22,23]). The inhibiting isoform LIP as well as SREBP1 was only moderately up-regulated (Fig. 3B). HepG2 cells treated with oleate for up to 40 days showed the same concomitant

up-regulation of PPARa and c, SREBP1, LAP⁄, and LAP, yet C/EBPa was up-regulated in parallel with perilipin (data not shown). To dissect the role of PPARs in perilipin induction, we used known PPARa and PPARc agonists (PPARa: fenofibrate, PPARc: troglitazone) and antagonists (PPARa: GW6471; PPARc: GW9662) to treat HuH7 cells. PPARc-agonist troglitazone induced perilipin expression faster, but also fenofibrate- and control-treated cells showed perilipin induction (Supplementary Fig. 2A and B). In HuH7 cells after perilipin induction for 21 days in PAM-DMSO containing medium and subsequent treatment with PPAR-antagonists, perilipin amount was markedly reduced by inhibition of PPARa, pointing to the fundamental role of PPARa in hepatic steatogenesis and perilipin induction (Supplementary Fig. 2C and D). As PAT-expression was also prominent in control treated (0.12% DMSO), long-term cultured HuH7 cells, lipid load and the known stabilizing effect of LDs on PAT-expression appeared as an important post-translational cofactor besides the already mentioned transcriptional mechanisms. Concerning post-transcriptional mechanisms, only perilipin splice variant A was detected in immunoblot and PCR; no significant amounts of perilipin B or C were detected. Treatment with the demethylizing agent 5-Aza-2-deoxycytidine together with PAM or PAM-DMSO only slightly increased perilipin expression. Yet, analyses of the methylation status of HuH7 cells before and after perilipin induction showed maximal methylation irrespective of the perilipin expression status. Also in 9 liver biopsies of chronic and acute steatosis, no clear correlation between methylation and perilipin expression status could be noted. In conclusion, PAT-expression during LD-maturation is regulated especially at the transcriptional, as well as posttranslational level. Perilipin plays an important role in hormone-dependent lipolysis in liver To further the understanding of the role of perilipin in hepatocyte steatogenesis, we treated HuH7 cells with isoproterenol and forskolin to induce lipolysis. As expected, glycerol release upon isoproterenol and forskolin treatment increased significantly and both the number of LDs and their size gradually declined. To study the role of perilipin in hormone-dependent lipolysis, HuH7 cells were treated for 21 days with PAM-DMSO-containing medium before lipolysis was induced. Perilipin was rapidly phosphorylated, hormone-sensitive lipase HSL translocated to LDs and glycerol release was significantly increased. Whereas all PAT mRNA-levels dropped significantly, adipophilin and TIP47 protein levels remained unaltered (Supplementary Fig. 3); in addition perilipin and MLDP levels were slightly reduced. Also in human liver specimens in situ, we could demonstrate perilipin phosphorylation by using immunofluorescence microscopy, immunoblot as well as in ESI-MS of immunoprecipitated perilipin. This result suggests that perilipin plays an important role in hormone dependent lipolysis in hepatocytes. Sequential PAT-protein expression during LD-maturation To further analyse the extent by which certain PAT-proteins contribute to LD-maturation, HuH7 cells stably expressing lentiviral shRNAs targeting TIP47 (HuH7_shT), adipophilin (HuH7_shA), or both (HuH7_shT/A) were subjected to induction of steatosis

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JOURNAL OF HEPATOLOGY (Fig. 4A). The viability of shRNA-treated cells was not significantly affected; yet, the capacity to store TAGs was greatly reduced in cells with reduced adipophilin, but unaffected in those with reduced TIP47 levels (Fig. 4B). In PAM-DMSO-treated HuH7_shA and shT/A-cells, perilipin was upregulated faster (Fig. 4C) and coated only few large LDs and the overall TAGamount was low (Fig. 4D). In contrast, TAG levels in HuH7_shTcells increased along steatosis induction and increasing MLDP, adipophilin, and perilipin levels. In HuH7 cells with siRNAs targeting adipophilin and TIP47, frequent LD-fusions were detected concomitant with an increase of LD-size. In summary, these findings indicate that perilipin may replace adipophilin, and MLDP may function alternatively to TIP47. Moreover, TIP47, MLDP, adipophilin, and perilipin are sequentially expressed during hepatocellular LD maturation (Fig. 4E).

Discussion In this study, we demonstrate that perilipin marks chronic steatosis in vivo and in vitro and thereby represents a final stage of LD-maturation, which sequentially involves the ePATs TIP47 and MLDP, as well as the cPAT adipophilin. Immunohistochemical analysis of PAT-proteins may therefore be of help for the histopathological definition of liver diseases involving steatosis. Whereas TIP47- and MLDP-expression are markers for acute/ microvesicular steatosis, perilipin was localized at differently sized LDs in chronic/macrovesicular steatosis, and adipophilin was ubiquitously expressed both in acute and chronic steatosis (cf. Supplementary Fig. 1). Fuji and coauthors have already shown that adipophilin marks ballooned cells in steatohepatitis [24]; besides adipophilin, we could identify TIP47 and MLDP at minuscule LDs in ballooned cells, suggestive of acute hepatocellular damage during ballooning, however, perilipin was not detected. Additionally, we and others have proposed adipophilin as a general marker for LD-accumulation in liver and other organs [5,25]. Our results show that the sequential PAT-expression, as observed in acute and chronic steatosis, shows close analogy to LD maturation during adipogenesis, where most of the perilipin/PAT-family proteins have first been described. In mouse 3T3-L1 adipocytes, within minutes after oleate loading, TIP47 and S3–12 coat small LDs and are sequentially replaced by adipophilin and perilipin at LDs of increasing size [26,27]. We found that perilipin was induced in different long-term steatosis cell culture models using combinations of different agents including oleate, DMSO, PPARa and PPARc agonists as well as hormones such as thyroxine, insulin, and cortisone. In leptin-deficient, heavily obese mice of up to 1 year of age, perilipin was not observed in significant amounts [16], indicating the indispensable role of additional cofactors besides pure lipid loading in the induction of perilipin in the liver. Recently, Orlicky and coauthors succeeded in inducing hepatic perilipin expression in a

mouse model of chronic ethanol and high fat diet (HFD) consumption over a period of 6 weeks, whereas short-term consumption failed to induce perilipin in situ [20]. In human liver specimens, however, ethanol was not the only factor inducing perilipin, as in proven cases of NAFLD/NASH, and in other diverse etiologies, such as in genetic diseases in newborns, perilipin was highly expressed. Whereas in adipogenesis, perilipin is known to be regulated by PPARc [28], the mode of regulation of the plin1 gene in human hepatocytes was so far not well defined. Our experiments point to a complex role of transcriptional, post-transcriptional and post-translational mechanisms during LD maturation in human liver. Experiments with PPARa and PPARc agonists and antagonists suggest that in the liver perilipin is not only induced via PPARc, but also at least indirectly by PPARa via its prosteatotic role by induction of LDs coated by adipophilin and TIP47 [16]. Again, this may be in analogy to plurivacuolar adipose tissue; in this case, PPARa targets adipophilin preceding perilipin expression in univacuolar adipocytes. Therefore, we hypothesize that a concerted regulation of PPARa and -c is necessary for LD maturation (e.g., see [29]). Interestingly, in liver, in addition to perilipin, other PPARc-target genes are found, such as the lipid droplet-associated cell death–inducing DNA fragmentation factor 45-like effector (CIDE) proteins CIDEc [30,31]. Targeted deletion of PPARa or PPARc in hepatocytes protected mice against HFDinduced hepatic steatosis [32,33], however, so far, perilipin has not been investigated with this respect. In our cell culture models, we noticed a general increase of PPARa and -c paralleling perilipin induction, yet also control-treated cells showed slightly increased perilipin levels, pointing to additional mechanisms. In addition to PPARs, we detected a decrease of C/EBPa levels and a concomitant induction of C/EBPb [LAP⁄/LAP] in agreement with the described insulin and dexamethasone action on C/EBPs [34]. C/EBPs are known regulators of terminal differentiation and proliferation in hepatocytes and adipocytes (reviewed in [23,35]) and ablation of C/EBPb in Leprdb/db mice prevents liver steatosis [36]. Interestingly, in our setting, perilipin expression was considerably increased upon administration of DMSO, a factor known to maintain and induce hepatic differentiation [37], arrest cell growth [21], and affect the cellular epigenetic profile [38]. In accordance with that, perilipin was predominantly found in highly confluent, non-proliferating, differentiated cells. So far, no evident influence of the methylation status on perilipin expression in hepatocyte steatosis could be noted, yet, a certain influence of methylation by indirect action mechanisms may take place. Besides, post-transcriptional and -translational regulation of PAT-expression plays an important role via alternative splicing, phosphorylation-dependent lipolysis and proteasomal degradation if no lipids are present in cells [39]. Rising TAG-amounts stabilize newly expressed adipophilin and perilipin at LDs, as already described for fatty livers of dystrophic mice [19] and Y-1 adrenal cortical cells [39]. Additionally, PAT-proteins and their splice variants may also function separately concerning

Fig. 3. Sequential PAT-expression preceding perilipin induction in long-term steatosis models. (A) Immunoblot analysis of perilipin (Peri), adipophilin (AP), TIP47, and MLDP in whole cell lysates of HuH7 cells treated 3 to 30 days with either albumin-coupled oleate (OA), DMSO, DMSO, and oleate (DMSO-OA), or control medium (Control). (B) Immunoblot analysis of LD-associated proteins and transcription factors of adipo- and lipogenesis (PPARa, PPARc, C/EBPa, and C/EBPb (LAP/LAP/LIP) and SREBP1) in whole cell lysates of HuH7 cells following PAM or PAM-DMSO treatment for up to 40 days. Actin served as loading control. (C) Densitometric evaluation of immunoblot analyses of 3 independent PAM-DMSO treatments in HuH7 cells. (D) Immunofluorescence microscopy of perilipin (red) with BODIPY (BP), adipophilin (AP), Ki67, ZO-1 (each green) and DAPI (blue) in HuH7 cells 0 and 10 d after treatment with DMSO and oleate. Note ZO-1-positive bile canalicular structures after 10 d of treatment. Top row: confocal laser scanning microscopy; bottom row: epifluorescence. Bar: 25 lm.

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JOURNAL OF HEPATOLOGY the TAG- or CE-rich LDs they coat [40]. Whereas phosphorylation-dependent lipolysis has been demonstrated for perilipin and MLDP, the exact role of PAT-proteins for lipolysis in liver and in particular of TIP47 and adipophilin remains to be clarified. In our siRNA and lentiviral assays, adipophilin and TIP47 did not significantly rescue each other as already described [41]. Yet, the ePAT MLDP rescued TIP47 and the cPAT perilipin rescued adipophilin suggesting partly overlapping functions of these PAT-proteins during LD-maturation. Even under conditions of down-regulation of adipophilin, perilipin was only present at LDs showing at least minimal signals for adipophilin, providing evidence that adipophilin precedes perilipin incorporation. Down-regulation of TIP47 affected TAG-amounts only marginally, possibly due to rescue functions of MLDP, adipophilin, and perilipin (for controversial results in mice see [42]). Down-regulation of adipophilin however reduced TAG-amounts to basal values despite steatogenic treatment and simultaneous up-regulation of perilipin. Thereby, adipophilin is the major determinant of TAG-amount in hepatocytes and the best suited therapeutic target for the reduction of hepatic steatosis (for mice see [13,14]). The role of perilipin in regard to human fatty liver disease and its progression has not been finally clarified, but even a beneficial role seems possible. Whereas adipophilin is present in virtually every steatotic liver disease, in cirrhosis and HCC, and together with TIP47 and MLDP increased in acute liver damage such as in necrosis and liver failure, perilipin is only present in chronic, not acute liver disease and decreases during carcinogenesis [17]. Perilipin deficiency in humans has been shown to result in partial lipodystrophy, severe dyslipidemia, insulin-resistant diabetes, and liver steatosis [15]. In this line, PPARc-action on perilipin may also explain favourable effects of administration of PPARc-agonists for patients with NASH [43], while apparently contradictorily, PPARc has a prosteatotic role in fatty liver disease [32]. Our in situ and in vivo data therefore demonstrate that steatogenesis is a common reaction pattern to hepatocellular damage with a characteristic temporal evolution, in which perilipins/ PAT-proteins play a primordial role. With prolonged liver damage, nearly irrespectively of the underlying etiology, TIP47 and MLDP-positive microvesicular steatosis evolves into adipophilin and perilipin-positive macrovesicular steatosis. In chronic lipid overload due to exogenous and/or endogenous factors, the formation of large adipophilin and especially perilipin-positive LDs may enable hepatocytes to efficiently and stably store larger amounts of TAGs/CE for a longer time period and to minimize the LD surface thereby preventing lipotoxicity. The differential PAT-protein coverage of LDs in hepatocytes may thereby help explain the different clinical significance of micro- and macrovesicular steatosis and opens up possibilities to therapeutically address steatotic liver diseases.

Financial support The study was funded by grants of the Deutsche Forschungsgemeinschaft to BKS (STR-1160/1-1), RB (SFB/TRR77, project A1) and PS (SFB/TRR77, project B5). MH held a stipend of the Erasmus Basileus-Program, BKS was stipend of the OlympiaMorata program of the Medical Faculty of Heidelberg University.

Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgement The authors thank Elisabeth Specht-Delius, Zlata Antoni and Benedek Gyoengyoesi for technical assistance, Gualtiero Alvisi for help with the establishment of the lentiviral transfection, and Esther Herpel and Judith Lehman-Koch from the Tissue Bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) for providing us with human tissues.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.11. 007.

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Fig. 4. Effects of stable down-regulation of adipophilin and TIP47 on PAT-expression, TAG-levels, and LD-morphology in long-term steatosis models. (A) Immunoblot analysis of perilipin (Peri), adipophilin (AP), TIP47, MLDP and actin (loading control) in whole cell lysates of HuH7 cells with stable lentiviral down-regulation of TIP47 (shT), adipophilin (shA, AP), both (shT/A) and control transfection (control shRNA; cont). (B) Relative TAG-concentrations in HuH7_shT, HuH7_shA, HuH7_shT/A, and HuH7_cont during PAM-DMSO treatment. (C) Relative perilipin and MLDP mRNA levels in transfected HuH7 cells during PAM-DMSO treatment. (D) Confocal laser scanning microscopy of HuH7 cells after 10 days of PAM-DMSO incubation show increased staining for perilipin (red) at fewer, but larger LDs (BP, green) in HuH7_shA and HuH7_shT/A cells (DAPI, blue). Bar: 25 lm. (E) Proposed model for PAT-protein composition during LD-maturation in hepatocytes.

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