Differential pattern of lipid droplet-associated proteins and de novo

Differential Pattern of Lipid Droplet–AssociatedProteins and De Novo Perilipin Expression in

Hepatocyte SteatogenesisBeate Katharina Straub,1,2 Pamela Stoeffel,1 Hans Heid,2 Ralf Zimbelmann,2 and Peter Schirmacher1

Fatty change (steatosis) is the most frequent liver pathology in western countries and iscaused by a broad range of disorders such as alcohol abuse and metabolic syndrome. Thesurface layer of lipid droplets (LDs) contains members of a protein family that share homol-ogous sequences and domains, the so-called PAT proteins, named after their constituents,perilipin, adipophilin, and TIP47. We characterized the LD-associated proteins in normaland diseased liver connected with LD accumulation. Adipophilin and TIP47 are expressed inLDs of vitamin A–storing hepatic stellate cells and additionally in LDs of steatotic hepato-cytes. Perilipin, which was thought to be characteristic for LDs of adipocytes and steroido-genic cells, becomes de novo expressed in hepatocytes of human steatotic liver. Perilipinsplice variant A was found in human steatotic hepatocytes by biochemical, molecular bio-logical, and immunohistochemical methods. Its association with LDs is different fromTIP47 and adipophilin, and depends on size and localization of the LDs, suggesting that thedifferent PAT proteins play specific roles during maturation of LDs. (HEPATOLOGY 2008;47:1936-1946.)

Fatty liver disease affects approximately 30% ofadults and 20% of children in the United Statesand its prevalence is still increasing in the highly

industrialized countries. Via steatohepatitis, fatty liverdisease progresses to cirrhosis in about 5%-10% of allcases over 10 years. Additionally, steatosis due to meta-bolic syndrome or alcohol abuse is recognized as an addi-tional risk factor for cirrhosis in hepatitis C virusinfection.1,2 To date, the diagnosis of fatty liver disease ismade histologically by the presence of microvesicular ormacrovesicular fat droplets. By definition, in steatosis,triglyceride content exceeds 5% of liver weight, but yetsmaller amounts may be clinically relevant, for example,

in cases of comorbidity. In order to monitor the process ofsteatosis, it is crucial to understand the mechanisms oflipid pathophysiology in human fatty liver disease.

Lipid droplets (LDs) are not only storage compart-ments for energy-rich fats, but also dynamic cell or-ganelles.3 Amphiphilic proteins of the PAT protein familywith its constituents perilipin, adipophilin (synonymouswith adipose differentiation–related protein, ADRP), andTIP47, play a major role in LD structure and formation(see reviews by Londos et al.,4 Martin et al.,5 and Wolinset al.6). Perilipin was reported to be exclusive for adipo-cytes and steroidogenic cells,7,8 whereas adipophilin9,10

and TIP4711 have been described as being expressednearly ubiquitously, that is, in the case of adipophilin, alsoin hepatocytes of steatotic livers.12,13 It is assumed thatPAT proteins play a crucial role in stabilization of LDsand in the control of lipolysis. Dependent on cat-echolamines, perilipin is phosphorylated by protein ki-nase A in adipocytes, changes its conformation, andpermits lipolysis by hormone-sensitive lipase.14 Further-more, overexpression of adipophilin or perilipin in cul-tured cells such as fibroblasts increases the storage oftriglycerides.6,15 With knockout mice, loss of perilipinresulted in a lean mouse with aberrant adipocyte lipolysis,enhanced leptin production, and resistance to diet-in-duced obesity.16,17 Mice lacking adipophilin were pro-tected against fatty liver disease, but showed unimpaired

Abbreviations: AP, adipophilin; ASH, alcoholic steatohepatitis; BSA, bovineserum albumin; BMI, body mass index; HBV, hepatitis B virus; HCC, hepatocel-lular carcinoma; HCV, hepatitis C virus; LD, lipid droplet; NASH, nonalcoholicsteatohepatitis; Peri, perilipin.

From the 1Department of General Pathology, Institute of Pathology, - Heidel-berg, Germany, and 2Division of Cell Biology, German Cancer Research Center, -Heidelberg, Germany

Received September 15, 2007; accepted January 28, 2008.Address reprint requests to: Beate K. Straub, M.D., Institute of Pathology, University

Clinic Heidelberg, Im Neuenheimer Feld 220/221, D-69120 Heidelberg, Germany.E-mail: [email protected]; fax: �49-6221-56-5251.

Copyright © 2008 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.22268Potential conflict of interest: Nothing to report.Supplementary material for this article can be found on the HEPATOLOGY Web

site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

1936

adipogenesis.12 Nevertheless, the pattern and role of dif-ferent PAT family proteins in hepatic steatogenesis hasnot been characterized.

To further the understanding of hepatocyte steatosis,we analyzed the presence and localization of LD-bindingproteins in normal and steatotic liver. Surprisingly, per-ilipin was found to be de novo expressed in steatotic hepa-tocytes. Differential and structure-associated localizationof PAT proteins in normal and steatotic hepatocytes sug-gest specific roles during maturation of LDs during he-patic steatosis.

Materials and Methods

Tissues and Cultured Cells. Bovine liver was from alocal slaughterhouse, and murine livers were from animalsof the central animal house of the German Cancer Re-search Center. Six-week-old obese mice (ob/ob) were pur-chased from Charles River Laboratories (Kisslegg,Germany). Cryopreserved and formalin-fixed, paraffin-embedded human tissue samples included adipose tissue,normal fetal and adult tissue, as well as steatotic liver

tissue and hepatocellular carcinoma (HCC) with promi-nent fatty change, and were provided by the tissue bank ofthe National Center for Tumor Diseases (NCT, Heidel-berg, Germany). For clinical details of the respective 81paraffin-embedded liver specimens subjected to immuno-histochemistry, see Tables 1 and 2. Independently, eightcryopreserved normal (3�) and steatotic (5�) liver spec-imens that were further subjected to immunoblot andimmunofluorescence microscopy included liver tissuefrom partial hepatectomies for liver metastases of colorec-tal cancer taken far away from the metastasis as well asdonor livers rejected due to steatosis; the paraffin-embed-ded tissue of one of these steatotic liver specimens wasincluded in group NAFLD (Table 2). For immunofluo-rescence microscopy, tissue was routinely snap-frozen inisopentane cooled with liquid nitrogen to about �130°C.For biochemical experiments, liver tissue was either useddirectly or frozen in liquid nitrogen and kept at �80°Cuntil needed. Cultured human epithelial cells were PLC,HepG2, Hep3B, and HuH718 (see Supplementary Table1). In some cases, PLC cells were fed with bovine serum

Table 1. Morphometric Analysis of Perilipin, Adipophilin, and TIP47 in Human Liver Specimens Grouped According toSteatosis

GroupSteatosis (Irrespective of

Underlying Etiology)Number of

CasesAge

[Years (Range)]Sex

[% Male]Perilipin Staining*

[Intensity: 0-3 (Range)]Adipophilin Staining†

[Intensity: 0-3 (Range)]TIP47 Staining†

[Intensity: 0-3 (Range)]

0 0-1% 15 48.4 (0-73)‡ 47 0.5 (0-1) 0.7 (0.5-1) 0.4 (0-1)1 2-5% 16 51.4 (33-64) 81.3 0.7 (0-2) 1.25 (1-2) 0.2 (0-1)2 6-19% 10 56.4 (40-72) 70 1.15 (0.5-2) 1.2 (0.5-2) 0.2 (0-1)3 20-39% 9 50.4 (39-71) 66.7 1.6 (0-2) 2.5 (1-3) 0.2 (0-0.5)4 40-59% 11 53.1 (23-83) 87 1.7 (0.5-2) 2.6 (2-3) 0.2 (0-1)5 60-79% 15 44.5 (18-63) 86.7 2.1 (1-3) 3.0 0.4 (0-1)6 80-100% 5 40 (19-54) 60 2.3 (2-3) 3.0 0.3 (0-0.5)

*Perilipin staining was accentuated in the perivenous zone. †Additional staining in hepatic stellate cells is observed in TIP47 and adipophilin immunohistochemicalreactions. ‡Normal liver specimens included one fetal human liver of approximately 14 weeks of gestation.

Table 2. Morphometric Analysis of Perilipin, Adipophilin, and TIP47 in Human Liver Specimens Grouped According toEtiology

Group Etiology/ DiagnosisNumber of

CasesAge

[Years (Range)]Sex

[% Male]Steatosis

[Volume % (Range)]

Perilipin Staining*[Intensity: 0-3

(Range)]

Adipophilin Staining†[Intensity: 0-3

(Range)]

TIP47 Staining†[Intensity: 0-3

(Range)]

1 Normal liver 9 49 (0-64)‡ 55.6 2.9 (0-5) 0.5 (0-1) 1.2 (0-2) 0.3 (0-1)2 Alcohol 16 51.3 (27-83) 75 35.8 (0-90)§ 1.2 (0-2) 1.9 (0.5-3) 0.2 (0-1)2a ASH 9 49.9 (27-83) 77.8 60 (30-90) 1.7 (0.5-3) 2.7 (2-3) 0.3 (0-1)2b cirrhosis 7 53 (44-64) 71.4 4.7 (0-15)§ 0.6 (0-2) 0.9 (0.5-1) 0.1 (0-1)3 Diabetes, super-

nutrition16 44 (18-71) 75 58.8 (15-90) 2.2 (0.5-3) 2.9 (2-3) 0.4 (0-1)

3a NAFLD 10 31.1 (18-61) 80 54.5 (15-75) 2.1 (0.5-3) 2.9 (2-3) 0.5 (0-1)3b NASH 6 52 (19-71) 66.7 65.8 (20-90) 2.3 (1-3) 2.8 (2-3) 0.2 (0-1)4 HBV 10 45.1 (30-67) 60 3.2 (1-5) 0.9 (0-2) 0.8 (0.5-1) 0.3 (0-1)5 HCV 18 50.5 (23-73) 66.6 21.0 (1-70) 1.1 (0.5-3) 1.8 (0.5-3) 0.2 (0-1)6 Mixed and others 12 55.8 (38-73) 83 33.2 (0-70) 1.4 (0.5-3) 1.9 (1-3) 0.1 (0-1)

*Perilipin staining was accentuated in the perivenous zone. †Additional staining in hepatic stellate cells is observed in TIP47 and adipophilin immunohistochemicalreactions. ‡Normal liver specimens included one fetal human liver of approximately 14 weeks of gestation. §This subgroup contained 5 cirrhotic liver specimens withminimal steatosis.

HEPATOLOGY, Vol. 47, No. 6, 2008 STRAUB ET AL. 1937

albumin (BSA)-coupled oleic acid (100 to 600 �M;Sigma, Taufkirchen, Germany) for 12-48 hours.

Antibodies and Reagents. Monoclonal antibodieswere: adipophilin (clone AP125), “pan”-perilipin (clonePeri 112.17), vimentin (3B4 and V9), desmin (clone D9;all from Progen Biotechnik, Heidelberg, Germany), aswell as actin (clone C4; MP Biomedicals, Solon, OH).The following polyclonal guinea pig antibodies from Pro-gen were used: adipophilin (GP40, GP41), “pan”-perili-pin (GP29), perilipin A (GP33), TIP47 (GP30, GP32),and cytokeratin 8/18 (GP11). Rabbit polyclonal antiseraincluded antibodies against desmin (Progen Biotechnik),human perilipin and mouse perilipin variants A and B(Novus Biologicals, Littleton, CO), and human perilipinvariant A (Sigma-Aldrich, St. Louis, MO ). Secondaryantibodies used were Alexa 488–coupled anti-mouse, an-ti-rabbit, and anti-guinea pig IgG antibodies (MoBiTec,Gottingen, Germany) as well as the respective Cy3 andCy5 coupled anti-mouse, anti-rabbit, and anti-guinea pigIgG antibodies (Dianova, Hamburg, Germany). Nile red(Sigma-Aldrich) was solubilized in ethanol at 1 mg/mLand then diluted 1:500 to 1:5,000 in phosphate-bufferedsaline (PBS) for immunofluorescence staining of LDs.

Immunofluorescence Microscopy. Tissue cryosec-tions of approximately 5 �m thickness were air-dried for1 hour and fixed in 4% paraformaldehyde for 20 minutesat room temperature. Sections were then incubated withthe primary antibodies diluted in PBS for 30 minutes, andthen washed twice with PBS for 5-10 minutes each. In-cubation with secondary antibodies was for 30 minuteswith two subsequent washes in PBS for 5-10 minutes.Sections then were rinsed in distilled water and mounteddirectly in Fluoromount G (Biozol Diagnostica, Eching,Germany). If staining for lipids was not necessary, tissuecryosections or cultured cells were fixed in methanol andacetone.19 Cultured cells were grown on glass coverslips toabout 70% confluency, washed repeatedly in PBS con-taining 2 mM MgCl2 at 37°C, and fixed with 2% para-formaldehyde in PBS for 10 minutes. Fixed cells werewashed repeatedly with PBS for 30 minutes, permeabil-ized in PBS with 0.05% Tween 20 for 10 minutes, andprocessed as described.19 Epifluorescence was with a ZeissAxiophot photomicroscope (Zeiss, Jena, Germany). Con-focal laser-scanning immunofluorescence microscopy waswith a Zeiss LSM 510 microscope.

Immunohistochemistry of Formalin-Fixed, Paraf-fin-Embedded Tissue. Formalin-fixed, paraffin-embed-ded human tissue sections of approximately 2 �m wereincubated in an oven at 60°C for 4 hours. After rehydra-tion in graded ethanol, antigen retrieval was performed in0.1 M Tris-HCl containing 5% urea, pH 9.5 in a steamerat 100°C for 15 minutes. Sections were preincubated with

3% H2O2 for 30 minutes and blocked with the serum ofthe species in which the secondary antibody was raised for30 minutes, and then with avidin and biotin for 10 min-utes each. Sections then were incubated with the primaryantibody for 1 hour or overnight. After subsequent washesin PBS for 20 minutes, sections were incubated with thesecondary antibody for 30 minutes, washed with PBS,and incubated with the avidin-biotin complex for 10 min-utes. After washing with PBS, sections were incubatedwith the peroxidase complex for 2-10 minutes. Sectionswere mounted with Eukitt (Riedel de Haen, Seelze, Ger-many).

Morphometrical and Statistical Analysis of Immu-nohistochemical Reactions. Hepatic steatosis was mea-sured with the point counting technique20 according tothe principles of Delesse (area density � volume density):therefore, representative areas of diastase/periodic acidSchiff stained liver specimens were selected, and the num-ber of LDs was counted microscopically in a high-powerfield (magnification: 400�) using a transparent grid with100 points. The number of LDs located on points wascounted, and thereby the percentage of steatosis was re-ceived. Three independent measurements were taken,and the arithmetic mean was calculated. Liver specimensstained with antibodies against perilipin, adipophilin, andTIP47 were graded according to the conventional immu-nohistochemical score IRS,21 that is, intensity was gradedfrom 0 (negativity) to 3 (strong positivity), and the num-ber of positive cells was graded from 0 (no stained cells; 1:1%-10% positive cells; 2: 11%-49% positive cells; 3:50%-79% positive cells) to 4 (80%-100% positive cells).Statistical analysis was performed with SPSS version 15.0for Windows (SPSS Inc., Chicago, IL). Two-sided signif-icance level was � � 0.05. The Mann-Whitney U test wasused to compare patient subgroups for quantitative vari-ables. Spearman correlations among continuous variableswere computed.

Gel Electrophoresis and Immunoblotting. SDS-polyacrylamide gel electrophoresis was performed as de-scribed.19,22 Samples were dissolved in SDS-containingsample buffer (250 mM Tris-HCl, pH 6.8, 20% SDS,25% glycerol, 125 mM dithiothreitol) and benzonase (1:1,000; Merck, Darmstadt, Germany) was added to de-grade DNA. Immunoblotting was with polyvinylidenefluoride membranes (Millipore, Bedford, MA ) followingstandard procedures. Unspecific binding sites of polyvi-nylidene fluoride membranes were blocked with 10%milk powder in Tris-buffered saline Tween 20 (TBST; 10mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20)for 2 hours. For antibodies directed against adipophilin,membranes were blocked in 0.5% BSA in TBST. Blotswere incubated with primary antibodies in TBST for 1

1938 STRAUB ET AL. HEPATOLOGY, June 2008

hour, followed by three washes in TBST for 10 minuteseach. Horseradish peroxidase–conjugated antibodies rec-ognizing rabbit, mouse or guinea pig IgG (diluted1:5,000 in TBST) were applied for 30 minutes, followedby 30-minute washes in TBST, and a 1-minute incuba-tion with enhanced chemiluminescence solution (ECL;Amersham Biosciences, Freiburg, Germany). Band inten-sity of X-ray films from immunoblots was quantified us-ing a high-resolution image scanning system (Epsonperfection 4870 photo; Meerbusch, Germany) and theevaluation software AIDA version 2.11 (Raytest,Straubenhardt, Germany). Signal intensity minus back-ground was calculated and normalized against actin. Themedian for the values of the normal human tissue wasgenerated and set as standard. Three measurements weremade and the standard deviation was calculated.

RNA Isolation, cDNA Synthesis, and PolymeraseChain Reaction Amplification. Human cells of thelines PLC/PRF-5, HepG2, Hep3B, and HuH7 were re-covered from petri dishes by scraping. RNA isolation wasperformed with the RNeasy Mini Kit according to themanufacturers’ protocol, including the on-column de-oxyribonuclease step (Qiagen, Hilden, Germany). TotalRNAs from the following human adult normal tissueswere purchased from BioCat (Heidelberg, Germany):adipose tissue (R1234003A-50-BC), liver tissue(R1234149-50-BC), adrenal gland (R1234004-50-BC),and testis (R1234260-50-BC). BioCat also supplied RNAfrom primary human hepatocytes of patients with a bodymass index (BMI) under 25 (RNA-L50-1-ZB), between 25and 30 (RNA-L50-2-ZB), and over 30 (RNA-L50-3-ZB).Copy DNAs (cDNAs) were synthesized from 10.0 �g totalRNA by AMV Reverse Transcriptase using random N6primer (Roche, Penzberg, Germany). Three �g portions ofthe obtained cDNA product were used for amplificationwith KOD Hot Start DNA Polymerase (Toyobo, Osaka,Japan) or Taq polymerase (Roche). Amplification primersfor adipophilin, TIP47, and perilipin were as follows:adipophilin (whole gene: forward: 5�-TTTA-GATCTTCCATGGCATCCGTTGCAGTT-3�; reverse:5�-TTTGTCGACTTAATGAGTTTTATGCTC-3�;ADRP-1082R: 5�-GTACACCTTGGATGTTGG-3�;ADRP-565R: 5�-GACTGTGTTAATGCTGCC-3�;ADRP-774F: 5�-AGCAGGCTCTCAGCAGGG-3�;ADRP-358F: 5�-GCCTATTCTGAATCAGCC-3�),TIP47 (whole gene: forward primer: 5�-TTTGGATC-CATGGCTGCCGACGGGGCA-3�; reverse primer:5�-TTTGGTACCCTACTTCTTCTCCTCCGG-3�;TIP-1022R: 5�-GGACTCGACCTGCTCTGG-3�;TIP-553R: 5�-ACGGACTTTGTCTTGTCC-3�; TIP-742F: 5�-AGGAACAGAGCTACTTCG-3�; TIP312F:5�-CAGATTGCATCAGCCAGC-3�), perilipin (whole

gene: forward primer (Peri f): 5�-TTTAGATCTTC-CATGGCAGTCAACAAAGGC-3�; reverse primer: 5�-TTTGTCGACTCAGCTCTTCTTGCGCAG-3’). Forperilipin splice variants, the following primer pairs wereused (see also Supplementary Fig. 1): (perilipin A: Pan-Peri F: 5�-GCAGCATTGAGAAGGTGG-3�; PanPeriR: 5�-CCATCAGCGACAGCCTGG-3�; perilipin B:PeriB R: 5�-GCAGCCCACACAGTGACC-3�; PeriB F:5�-CATGGCTCTGGCCTGAGG-3�; perilipin C:PeriC R: 5�-CAAAGCAGGGTCAGTGCC-3�; PeriC F:5�-ACAGAAGGGGTGAGAAGC-3�; perilipin D:PeriD R: 5�-CTGCATGGCCACTGAGGC-3�; PeriDF: 5�-CTGCCATTCGGAGGCTCG-3�). Polymerasechain reaction (PCR) products were separated by electro-phoresis on 1% agarose gels and stained with ethidiumbromide. The pBluescribe vector (Stratagene, La Jolla,CA) digested with Hinf I (Roche) was used as the molec-ular weight marker.

Cloning and Purification of Recombinant Proteins.The DNA of human perilipin (ACC-No NM 002666),adipophilin (ACC-No NM 001122), and TIP47(ACC-No AF 057140) were amplified from the corre-sponding full-length cDNAs using PCR and subse-quently processed according to standard protocols fromQiagen using the prokaryotic expression vector pQE 30,the E. coli strain M15 (pREP4), and Ni-NTA agarosematrix columns.

Results

LD-Binding Proteins in Normal Liver. In normalmouse, bovine, and human livers, LDs of hepatic stellatecells were positive for adipophilin and TIP47, and in cowalso for perilipin (Figs. 1A, 2A). Adipophilin, TIP47, andperilipin colocalized with the vitamin A autofluorescenceof LDs of hepatic stellate cells, as well as with markerproteins such as the intermediate filaments vimentin anddesmin (Fig. 1B,C). The few and small hepatocytic LDsin normal liver were faintly positive with antibodiesagainst TIP47 and adipophilin, and negative with severalpolyclonal antibodies against perilipin (Fig. 1C). Theconnective tissue of the portal tracts was not stained withany of the antibodies against PAT proteins used. In fetalhuman liver tissue (14 weeks of gestation), only a faintreactivity of antibodies against TIP47, adipophilin, andperilipin was detectable in LDs of some elongated sinu-soidal cells, most likely hepatic stellate cells, whereas hepa-tocytes were negative and the same was observed for adulthuman liver specimens without steatosis (Fig. 2A, ar-rows). In HCC cell lines PLC, HepG2, Hep3B, andHuH7, the few small existing LDs were surrounded by


TIP47 and adipophilin, whereas perilipin was faintlypresent in the cytoplasm (data not shown).

PAT Protein Expression in Hepatocellular Steato-sis. We next investigated the relation of LD-associatedproteins to hepatocellular lipid accumulation. In repre-sentative normal and steatotic liver specimens, includingthose with alcoholic as well as nonalcoholic steatosis (n �81, LD content ranging from 0%-90%) antibodiesagainst TIP47, adipophilin, and perilipin reacted well onparaffin-embedded tissues, despite the fact that process-

ing removed the lipid content of LDs (Fig. 2). All caseswere positive for adipophilin and TIP47, and the stainingintensity for adipophilin, but not TIP47, positively cor-related with the amount of LDs (Fig. 2A, Table 1, level ofsignificance for adipophilin: P � 0.01). Antibodiesagainst perilipin reacted positively at the LD-cytoplasminterface in all steatotic liver tissues analyzed (Fig. 2),whereas nonsteatotic hepatocytes were negative. Perilipinstaining also was positively correlated with the degree ofsteatosis (Table 1, level of significance P � 0.01). No

Fig. 1. Immunolocalization of PAT proteins in normal liver tissue. (A) Immunohistochemistry of TIP47, adipophilin, and perilipin in bovine liver.Arrows depict positive staining in the sinusoidal space. (B) Confocal laser scanning fluorescence microscopy of adipophilin (AP, green), and perilipin(Peri, green) together with desmin (Desm, red) and vimentin (Vim, red), specific for mesenchymal liver cells, demonstrates their localization to theLDs of hepatic stellate cells in bovine liver. The respective differential interfering contrast images (DIC) are shown on the right side. (C) No stainingfor perilipin (green) is observed in cytokeratin 8/18 positive hepatocytes (CK 8/18, blue) of bovine liver (stellate cells; vimentin, red). Bars: 20 �m.


statistically significant differences were found in the in-tensity of perilipin, adipophilin, and TIP47 when samplesof different underlying etiology, but with similar amountof steatosis were compared (Table 2). When comparingthe intensity of perilipin and adipophilin expression withsteatosis versus steatohepatitis, no significant correlationwas found. In liver specimens with steatohepatitis, espe-cially with cirrhosis, the amount of LDs in sinusoidal cellsstained by antibodies against TIP47 or adipophilin wasdiminished. Besides liver specimens with steatosis,TIP47, adipophilin, and perilipin were also presentaround LDs in HCC samples with prominent fattychange. Using cryopreserved normal and steatotic liverspecimens, the PAT proteins TIP47, adipophilin, andperilipin colocalized with the lipid-binding fluorophorenile red in hepatocytes of steatotic liver (Fig. 3A). Inter-estingly, TIP47 was associated with a minor subset of verysmall-size hepatocellular LDs (Fig. 3A, arrows), whereasadipophilin and perilipin stained larger size LDs. Perilipin

was accentuated near the terminal hepatic venules in astark portocentral gradient, and staining was thereforeobserved in about 60% of light microscopically visiblelipid droplets, whereas adipophilin almost evenly stainedall visible lipid droplets in perivenous and periportal hepa-tocytes (Figs. 2B, 3B). Upon immunohistologic analysis,a much higher number of small LDs in hepatocytes weredetected when compared to light microscopical evalua-tion of routine stained sections. To confirm the hepato-cellular expression of perilipin, which had not beendescribed in hepatocytes before, we perfomed triple con-focal immunofluorescence microscopy. In steatotic liver,perilipin was localized around LDs in cytokeratin 8/18–positive hepatocytes and not in vimentin-positive mesen-chymal cells of the liver (Fig. 3B).

Furthermore, using variant-specific reverse transcriptasePCR assay, all perilipin splice variants (A-D; deduced fromLu et al.23) were detected in human liver tissue and in pri-mary hepatocytes (Supplementary Fig. 2, lanes 2-5) accord-

Fig. 2. Immunohistochemistry for TIP47, adipophilin, and perilipin in normal and steatotic human liver. (A) Immunohistochemistry for PAT-family proteinsin normal human liver, adult steatotic human liver with moderate predominantly microvesicular fatty change, and with severe micro- and macrovesicular fattychange show positive reactions for TIP47, adipophilin (AP), and perilipin (Peri). TIP47 and adipophilin staining surround LDs of hepatic stellate cells in normalhuman liver (arrows), whereas hepatocytes show positivity around the few small lipid droplets. In adult human liver with micro- or macrovesicular steatosis,TIP47, adipophilin, and perilipin surround LDs of hepatocytes. The corresponding diastase/periodic acid Schiff-stained tissue sections (D-PAS) are depictedon the right side. Magnification: 400�. (B) Distribution of TIP47, adipophilin and perilipin staining in human steatotic liver. Perilipin staining is accentuatedperivenously, whereas adipophilin is found nearly ubiquitously, and TIP47 is localized predominantly in hepatic stellate cells and around very small sized LDsin hepatocytes. Abbreviations: CV: central vein, PF: portal field. Magnification: 100�.


Fig. 3. Immunofluorescence microscopy of LDs and PAT proteins in steatotic human liver. (A) Confocal laser scanning images of a human adultliver specimen with pronounced fatty degeneration, stained with antibodies against the intermediate filament protein cytokeratin 8/18 (CK8/18,green) as well as the PAT family members together with the lipophilic dye nile red. TIP47 (green), adipophilin (AP, green), and perilipin (Peri, green)surround multiple nile red positive LDs in hepatocytes (cf. merge picture; for the staining of TIP47 around small size LDs cf. arrows). (B) Perilipin (Peri,red) is localized in cytokeratin 8/18 positive steatotic hepatocytes (CK 8/18, green), and not in vimentin-positive hepatic stellate cells (Vim, blue)of fatty liver disease. Note the prominent portal-to-central perilipin-gradient. Abbreviations: CV: central vein, PF: portal field. DIC images are shownon the right side. Bars: 20 �m.


ing to their calculated molecular size (Supplementary Fig. 1),while in PLC, HepG2, Hep3B and HuH7 cells, perilipinmessenger RNA (mRNA) could not be detected (data notshown). The mRNAs of adipophilin and TIP47 werepresent in normal human liver and primary hepatocytes(Supplementary Fig. 2) as well as in the HCC cell lines.These data demonstrate that perilipin together with TIP47and adipophilin is present in situ in LDs of human steatotichepatocytes of fatty liver disease and that PAT proteins showdifferential hepatocellular expression in regard to hepatic mi-croarchitecture and size of hepatic LDs.

PAT Proteins Localize to Different LD Subpopu-lations. The differential localization pattern of PAT pro-teins in human steatotic liver tissue (Figs. 2 and 3) suggestedthat PAT proteins may localize to different sized fat droplets.In order to further investigate this question, we performeddouble and triple immunofluorescence microscopy of hu-man liver specimens (Fig. 4). TIP47 was present in smallersize LDs compared to other LD-binding proteins (see doublelocalization with anti-perilipin antibodies, Fig. 4). Adipophi-lin was concentrated mainly around larger size LDs, inperivenular hepatocytes mostly in colocalization with perili-pin. In the periportal parenchyma, however, adipophilin and

perilipin were differentially localized (Fig. 4). A rabbit poly-clonal antibody specific for perilipin A showed for most LDsthe same staining pattern in hepatocytes as pan-perilipin an-tibodies, but in some singular hepatocytes, only pan-perili-pin antibodies reacted with the LD interface, suggesting thata perilipin variant apart from variant A must be present. Thisdifferential staining pattern of pan-perilipin and perilipin Aantibodies was further confirmed by double label immuno-fluorescence microscopy (Fig. 4). Thus, PAT proteins andeven perilipin splice variants show differential localization tofat droplets in regard to size and zonal distribution.

Dynamic Pattern of Hepatocellular PAT ProteinExpression During Steatogenesis. Association of PATproteins with LDs and de novo expression during steato-genesis suggested to us that hepatocellular PAT proteinexpression may be quantitatively coupled to steatogenesis(see also morphometric analysis, Table 1). In order toconfirm the presence of PAT proteins in liver and to fur-ther test this hypothesis, whole-cell lysates from adult hu-man liver with different degrees of steatosis, humanprimary hepatocytes, and HCC -derived cell cultureswere semiquantitatively analyzed using immunoblots(Fig. 5A). TIP47 was detectable as a protein band of about

Fig. 4. Differential expression of PAT proteins in human steatotic liver. Confocal laser scanning microscopy of adult human liver with steatosisshows the differential patterns of PAT proteins in regard to size and acinar distribution of LDs. TIP47 (green) compared to Perilipin (Peri, red) localizesto LDs of hepatic stellate cells rather than hepatocytes. Adipophilin (AP, green) and perilipin (Peri, red) shows colocalization as well as differentiallocalization. Double staining of antibodies against pan-perilipins (panPeri, green) with antibodies against perilipin A (Peri A, red) reveals that perilipinvariants other than perilipin variant A must be present (cf. arrow). The respective DIC and phase contrast images are given on the right side. Bars:20 �m.


50 kDa in PLC, HepG2, Hep3B, and HuH7 cells andafter long exposition of respective X-ray films also in pri-mary human hepatocytes as well as in normal and stea-totic human liver. Polyclonal antibodies against humanadipophilin showed a protein band of about 53 kDa inlysates of normal and steatotic liver specimens, and livercancer cells (Fig. 5A). Perilipin was detected as a majorband in the molecular range of 65 kDa as well as twobands of around 50 and between 30 and 40 kDa in mu-rine and human adipose tissue; these protein bands prob-ably represent products of perilipin splice variants. In

human fatty liver, also a band of 65 kDa was recognizedby all employed anti-perilipin antibodies including perili-pin splice product A antibodies (Fig. 5A,B) and minorquantities of 50 and 30 kDa perilipin splice productsrecognized by pan-perilipin antibodies. The specificity ofall antibodies used was confirmed by immunoblots withprotein lysates of recombinant TIP47, adipophilin, andperilipin (Supplementary Fig. 3). Using representativenormal and steatotic liver specimens as well as adiposetissue, TIP47 and adipophilin were not present in humanadipose tissue, but were present in all liver specimens an-

Fig. 5. Immunoblot analysis of PAT proteins in normal and steatotic liver. (A) The following cells and tissues were probed with antibodies against TIP47,adipophilin (AP), perilipin (Peri), and perilipin variant A (Peri A) as well as with actin as loading control: cultured cells of the lines PLC (lane 1), HepG2 (lane2), Hep3B (lane 3), HuH7 (lane 4), primary human hepatocytes (phH, lane 5), microscopically normal human liver tissue (hLiver; lane 6), human liver tissuewith severe steatosis (hfLiver; lane 7), and human adipose tissue (hAdipo; lane 8). Whereas TIP47 and adipophilin are present in cultured cells and in normalliver, perilipin (variant A) is only present in primary human hepatocytes which showed light-microscopically visible lipid droplets, in human fatty liver andadipose tissue. (B) Immunoblot of human fatty liver tissue reacted with antibodies specific for perilipin (Peri). (C) Human liver specimens with different degreesof steatosis (1-2%: hLiver1-3, 10%-20%: hLiver4-5, 20%-40%: hLiver6-7, 60%: hLiver8; lanes 1-8), and of human adipose tissue (hAdipo; lane 9) wereprobed with antiboides against adipophilin (AP), perilipin A (Peri A) as well as with antibodies against actin as loading control. Molecular weight markersare indicated on the right margin. (D) Densitometric quantification of protein bands reactive with antibodies to adipophilin and perilipin A when normalizedto actin. Adipophilin is upregulated up to 9-fold in hLiver8 when compared to normal liver (hLiver1-3), perilipin A even up to 22-fold. RQ: relative quantities.


alyzed. Perilipin A was detected with different antibodiesin all steatotic livers as well as in human adipose tissue(Fig. 5C). Pan-perilipin antibodies reacted additionally witha band in the range of 50 kDa, representing perilipin B or aprotein modification; this band was found irrespective of thedegree of steatosis. TIP47 was not significantly correlatedwith the degree of steatosis, whereas amounts of adipophilinand perilipin A positively correlated with the amount ofhepatocytic fat (Fig. 5C). Using densitometric analysis, adi-pophilin was up-regulated up to nine-fold in human fattyliver when compared to microscopically normal humanliver, and perilipin A up to 22-fold (Fig. 5D).

Next, we tested this association of PAT proteins withsteatogenesis in a dynamic model. Therefore, PLC-cells werefed with BSA-bound oleic acid for 2 days by slight variationof the method described for 3T3-L1 cells.24 Multiple smalland large size LDs developed; their borders were stained byantibodies against adipophilin and TIP47, but not perilipin(Supplementary Fig. 4A). In immunoblot analyses, theamount of adipophilin and TIP47 was increased in PLC cellsfed with oleic acid in comparison to controls. In addition, weanalyzed a mouse model of obesity, the ob/ob mouse.25 Byimmunofluorescence microscopy and immunoblot analysesof steatotic liver specimens of 12-week-old and 24-week-oldmice, adipophilin and TIP47 was detected, but not perilipin(Supplementary Fig. 4B), again suggesting that expression ofadipophilin and TIP47 precedes perilipin in hepatocyte ste-atogenesis.

DiscussionIn this study, we show for the first time the differential

expression and subcellular localization of PAT proteins inhuman hepatocellular steatogenesis. Although adipophi-lin is generally present in hepatocytes of different species,perilipin is not significantly expressed in normal hepato-cytes or cultured cells derived therefrom, but becomes denovo expressed in steatotic hepatocytes. For adipophilin, afunctional correlation with liver steatogenesis has alreadybeen demonstrated10; human fatty liver and livers of micefed a high-fat diet showed an up-regulation of adipophi-lin.13 Adipophilin knock-out mice do not develop fattylivers, but show no difference in peripheral adipogenesisin comparison to control mice.12 The existence of perili-pin surrounding LDs in hepatocytes of human fatty liverwas surprising, because perilipin has been reported to berestricted to adipocytes and certain steroidogenic cells,7,8

and to be absent in normal and steatotic livers of variousspecies,12,13,26 although a “perilipin-like protein” in hu-man nonalcoholic fatty liver disease has recently been re-ported.27 In mouse and rat tissues, different variants of theperilipin protein, produced by alternative splicing, existwith cell-specific and tissue-specific regulation and ex-

pression patterns.23,28,29 Perilipins A and B appeared to bespecific for adipocytes, whereas perilipins C and D werefound in steroidogenic cells.23 In human fatty liver, wefound mRNAs for all perilipin splice variants. We con-firmed by immunohistochemistry and immunoblot anal-ysis that at least perilipin A is present, a splice variantreported as adipocyte-specific. In addition, the localiza-tion of perilipin A was different from the pattern seenwith pan-perilipin antibodies, which suggests the pres-ence and differential expression (and thus function) ofother perilipin variants. Our results demonstrate that adi-pophilin, TIP47, and perilipin are preferentially associ-ated with different sizes of LDs in steatotic hepatocytesand with different hepatocyte populations along the por-tocentral axis. Wolins et al.6,24 demonstrated that, inoleate-treated murine 3T3-L1 cells, TIP47 coats nascentLDs during rapid fat storage, and that adipophilin andperilipin are involved in sustained fat storage. Data fromPAT gene knockout mice indicate that TIP47 may func-tionally substitute for adipophilin,30 perilipin may substi-tute for adipophilin,31 and adipophilin also partlysubstitutes for perilipin in reverse.16 Therefore, concern-ing fatty liver disease, up-regulation of adipophilin13 andperilipin suggests that both proteins are important for thematuration and maintenance of hepatocellular LDs.

The already available antibodies against PAT proteinsmay thus allow the monitoring of, for example, the pro-cess of steatosis in fatty liver disease or the correlation ofthe presence of different LD-binding proteins to differ-ences in pathogenesis even in paraffin-embedded tissuesobtained under routine conditions. Their usefulness indifferent relevant, for example, dynamic (prognosis) diag-nostic questions, such as determination of remissive stageof steatosis, will have to be further analyzed. In this study,the expression of PAT proteins was most notably corre-lated with the proportion of LDs in liver steatosis of dif-ferent underlying etiology, but apparently irrespective ofthe etiology. Because perilipin appears to be important forhuman hepatocellular steatogenesis and is not present innonsteatogenic hepatocytes, it may represent a potentialtarget for the suppression of hepatic steatosis.

Acknowledgment: We thank Werner W. Franke forcontinuous advice, interest in the study, and generoussupport. We also thank Thomas Keenan for critically re-viewing the manuscript, Jens Schumacher for densito-metric quantification, and Kai Breuhahn for statisticalanalysis. We acknowledge the technical assistance ofMichaela Hergt and Elisabeth Specht-Delius. Thanks goalso to Thomas Longerich, Andras Kiss, and KatalinBorka for providing cryopreserved and paraffin-embed-ded human tissue samples. This work was funded in partby a grant from the Ministry of Science, Research, and the


Arts of Baden-Wurttemberg (Az: 23-7532.22-23-12/1)to B.K.S. B.K.S. is a member of the postdoctoral programof the Medical Faculty of Heidelberg University.

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Differential pattern of lipid droplet-associated proteins and de novo