Mitochondrial dysfunction contributes to the increased vulnerabilities of adiponectin knockout mice...

STEATOHEPATITIS/METABOLIC LIVER DISEASE

Mitochondrial Dysfunction Contributes to theIncreased Vulnerabilities of Adiponectin Knockout

Mice to Liver InjuryMingyan Zhou,1,2,3 Aimin Xu,1,3,4 Paul K. H. Tam,5 Karen S. L. Lam,3,4 Lawrence Chan,7 Ruby L. C. Hoo,3,4 Jing Liu,1,2,3

Kim H. M. Chow,1,2,3 and Yu Wang1,2,6

Adiponectin is an adipocyte-derived hormone with a wide range of beneficial effects onobesity-related medical complications. Numerous epidemiological investigations in diverseethnic groups have identified a lower adiponectin level as an independent risk factor fornonalcoholic fatty liver diseases and liver dysfunctions. Animal studies have demonstratedthat replenishment of adiponectin protects against various forms of hepatic injuries, sug-gesting it to be a potential drug candidate for the treatment of liver diseases. This study wasdesigned to investigate the cellular and molecular mechanisms underlying the hepatopro-tective effects of adiponectin. Our results demonstrated that in adiponectin knockout(ADN-KO) mice, there was a preexisting condition of hepatic steatosis and mitochondrialdysfunction that might contribute to the increased vulnerabilities of these mice to secondaryliver injuries induced by obesity and other conditions. Adenovirus-mediated replenishmentof adiponectin depleted lipid accumulation, restored the oxidative activities of mitochon-drial respiratory chain (MRC) complexes, and prevented the accumulation of lipid peroxi-dation products in ADN-KO mice but had no obvious effects on mitochondrial biogenesis.The gene and protein levels of uncoupling protein 2 (UCP2), a mitochondrial membranetransporter, were decreased in ADN-KO mice and could be significantly up-regulated byadiponectin treatment. Moreover, the effects of adiponectin on mitochondrial activities andon protection against endotoxin-induced liver injuries were significantly attenuated inUCP2 knockout mice. Conclusion: These results suggest that the hepatoprotective propertiesof adiponectin are mediated at least in part by an enhancement of the activities of MRCcomplexes through a mechanism involving UCP2. (HEPATOLOGY 2008;48:1087-1096.)

Abbreviations: ADN, adiponectin; ADN-KO, adiponectin knockout; ALT, alanine aminotransferase; DB, 12-week-old leptin receptor�/�; DKO, leptin receptor�/�/adiponectin�/� double knockout; HF, high fat; LPS, lipopolysaccharide; Luci, luciferase; MDA, malondialdehyde; MRC, mitochondrial respiratory chain; mRNA,messenger RNA; mtDNA, mitochondrial DNA; NADPH, nicotinamide adenine dinucleotide phosphate; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholicsteatohepatitis; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; SSBP1, single-stranded DNA binding protein 1; TBARS,thiobarbituric acid reactive substance; TEM, transmission electron microscopy; TNF�, tumor necrosis factor alpha; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling; UCP2, uncoupling protein 2; UCP2-KO, uncoupling protein 2 knockout.

From the 1Department of Pharmacology, 2Genome Research Center, 3Research Center of Heart, Brain, Hormone, and Healthy Aging, 4Department of Medicine, and5Department of Surgery, Li Ka Shing Faculty of Medicine, and 6Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis,University of Hong Kong, Hong Kong, China; and 7Division of Diabetes, Endocrinology, and Metabolism, Departments of Medicine and Molecular and Cellular Biology,Baylor College of Medicine, Houston, TX.

Received January 6, 2008; accepted May 21, 2008.Supported by grants from Seeding Funds for Basic Research of the University of Hong Kong (Y.W.), by Hong Kong Research Grant Council grants HKU 778007 (Y.W.)

and HKU 7645/06M (A.X.), by the U.S. National Institutes of Health grant HL-51586 (L.C.), and by the Area of Excellent Scheme (AoE/P-10-01) established underthe University Grants Committee of the Hong Kong Special Administrative Region.

Address reprint requests to: Yu Wang, Department of Pharmacology and Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discoveryand Synthesis, University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China. E-mail: [email protected]; fax: 852 28170859.

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.22444Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

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Obesity and its related metabolic syndrome arenow reaching an epidemic level worldwide.Nonalcoholic fatty liver disease (NAFLD) is be-

ing increasingly recognized as a hepatic manifestation ofthe metabolic syndrome, which is closely associated withthe development of insulin resistance, type 2 diabetes, andcardiovascular diseases.1,2 NAFLD and nonalcoholic ste-atohepatitis (NASH) predispose people to the progressivedevelopment of hepatic fibrosis, cirrhosis, and end-stageliver cancers. A growing body of evidence from animalmodels suggests a “two-hit” hypothesis for the develop-ment of NAFLD.3-5 In this theory, the first hit is theoccurrence of a fatty liver (steatosis), and it is followed bya second event leading to the development of NASH. Thesecondary liver injuries can be induced by endotoxin ex-posure, alcohol consumption, drug compounds, and virusinfections, for example. To date, there have been very feweffective drugs available for the treatment of NAFLD andNASH.

Adiponectin is an adipokine abundantly producedfrom adipocytes.6 This adipokine has recently attractedgreat attention because of its antidiabetic, anti-inflamma-tory, and anti-atherogenic activity. A previous study fromour group provided the first evidence demonstrating thatadiponectin possesses potent protective effects against al-coholic fatty liver disease, NAFLD, and steatohepatitis.7

In both ethanol-fed and ob/ob obese mice, chronic treat-ment with recombinant adiponectin markedly attenuatedhepatomegaly and steatosis and significantly decreasedhepatic inflammation and serum alanine aminotransfer-ase (ALT) levels. Consistent with our data, studies from anumber of independent groups have demonstrated thehepatoprotective effects of adiponectin in different ani-mal models with various forms of liver injuries, includingthose induced by carbon tetrachloride, lipopolysaccharide(LPS)/D-galactosamine, pharmacological compounds,bile duct ligations, and a methionine-deficient diet.8-13

These animal-based findings have been further corrobo-rated by clinical observations showing an inverse associa-tion between serum levels of adiponectin and liverdysfunctions.7,14-18 Plasma adiponectin levels are signifi-cantly lower in patients with NAFLD in comparison withsex-matched and age-matched healthy controls. More-over, NASH patients with lower levels of adiponectinshow higher grades of inflammation, and this suggeststhat adiponectin deficiency is an important risk factor forthe development of fatty livers, steatohepatitis, and otherforms of liver injuries. Therefore, adiponectin and its ago-nists represent emerging therapeutic agents for the treat-ment and/or prevention of liver dysfunctions.

Although adiponectin deficiency has been shown topredispose mice to various liver injuries,9,10 the underly-

ing mechanisms remain largely unknown. Here we haveexamined the liver functions of adiponectin knockout(ADN-KO) mice and their wild-type littermates underboth normal and obese conditions. Our results show apreexisting condition of steatosis accompanied by mito-chondrial defects in the liver tissues of ADN-KO mice.Adiponectin treatment improves liver functions by in-creasing mitochondrial respiratory chain (MRC) activi-ties and reducing hepatic lipid accumulation in thesemice. Furthermore, we also provide evidence suggestingthat uncoupling protein 2 (UCP2), a mitochondrialmembrane carrier protein, is critically important in me-diating the hepatoprotective effects of adiponectin.

Materials and Methods

Animal Studies. ADN-KO mice with a C57BL/6Jbackground and leptin receptor�/�/adiponectin�/� dou-ble knockout (DKO) mice were generated as we describedrecently.19 Uncoupling protein 2 knockout (UCP2-KO)mice with a C57BL/6J background were purchased fromthe Jackson Laboratory (Bar Harbor, ME). Animals wereprovided with standard chow (LabDiet 5053, LabDiet,Purina Mills, Richmond, IN) unless otherwise specified.To establish a dietary obese mouse model, 4-week oldmale C57BL/6J and ADN-KO mice were fed a 19.33kJ/g high-fat diet (D12451, Research Diet, New Bruns-wick, NJ) that was 49.85% fat, 20% protein, and 30.15%carbohydrate for 8 weeks. All animals were kept under12-hour light-dark cycles at 22 to 24°C. For adiponectintreatment, the recombinant adenovirus expressing adi-ponectin or luciferase was tail-vein–injected into mice 2weeks prior to tissue collection.19,20 Note that the amountof the injected adenovirus (108 pfu) caused no toxicity inmice as judged by their body weight gains, food and waterintake, liver functions, and other behavioral variables.The increased expression levels of adiponectin were con-firmed by both enzyme-linked immunosorbent assay andWestern blotting analysis. For the induction of acute liverinjury, 200 �g of LPS was intraperitoneally injected intoadiponectin or UCP2-KO mice, and the liver tissue wascollected at different time points as indicated. All theanimal experimental procedures were approved by theCommittee on the Use of Live Animals for Teaching andResearch of the University of Hong Kong and were car-ried out in accordance with the Guide for the Care and Useof Laboratory Animals.

Isolation of Mitochondria from Liver Tissues andMeasurement of MRC Activities. Mice were sacrificedunder deep anesthesia, and the liver tissues were immedi-ately collected for hepatic mitochondria isolation with theprocedures that we described previously.21 The resulting

1088 ZHOU ET AL. HEPATOLOGY, October 2008

mitochondrial pellet was resuspended in a 50 mM trishy-droxymethylaminomethane solution (pH 8.0) to a pro-tein concentration of 5 to 8 mg/mL and kept at �80°Cfor subsequent measurements of MRC activities. Briefly,complex I activity was determined by the measurement ofthe colorimetric changes during the oxidation of NADHat the wavelength of 340 nm. The specific activities werecalculated by the subtraction of values obtained in thepresence of the complex I–specific inhibitor rotenone.The dual complex activities of complexes II and III weremeasured by the monitoring of the colorimetric changesduring the reduction of cytochrome C at the wavelengthof 550 nm in the presence or absence of the specific in-hibitor antimycin A. The activity of complex IV was an-alyzed by the measurement of the oxidation of reducedcytochrome C with a commercially available cytochromeC oxidase assay kit (Sigma, St Louis, MO). The activity ofcomplex V was evaluated with a coupled enzymatic (pyru-vate kinase and lactate dehydrogenase) assay for measur-ing the hydrolytic activities of ATPase. The specificactivity was calculated by subtraction of the readings inthe presence of oligomycin.

Electron Microscopy Analysis of Mitochondrial Ul-trastructures and Histological Staining for the Eval-uation of Hepatic Steatosis and Inflammation.Transmission electron microscopy (TEM) was used toevaluate the ultrastructures of liver mitochondria. Briefly,�1 mm3 of liver tissues from the same anatomical loca-tions was fixed in 2.5% glutaraldehyde in 100 mM so-dium cocodylate, treated with 1% osmium tetroxide in a0.1 M NaCo buffer, washed, dehydrated, and embeddedin Epon 812 resin. Random thin sections (100 nm) werecut and stained with aqueous uranyl acetate and Reyn-old’s lead acetate. The images were examined with a Phil-ips EM208S transmission electron microscope (Philips,Eindhoven, The Netherlands). To evaluate hepatic ste-atosis, liver tissues were embedded in Tissue-Teck O.C.T.compound (Sakura Finetek USA, Inc., Torrance, CA),and 8-�m sections were prepared with a Leica 2800EFrigocut microtome cryostat. The air-dried frozen sec-tions were used for Red Oil O and hematoxylin-eosinstaining.22

Thiobarbituric Acid Reactive Substance (TBARS)and Lucigenin Assays. The concentrations of the lipidperoxidation product malondialdehyde (MDA) in serum,liver lysates, or mitochondrial suspensions were deter-mined with a commercial TBARS assay kit (CaymanChemical, Ann Arbor, MI). The results were calculatedagainst the total protein contents. The activity of nicotin-amide adenine dinucleotide phosphate (NADPH) oxi-dase was assessed with the lucigenin assay as described.23

Quantitative Reverse-Transcription PolymeraseChain Reaction (RT-PCR). Total RNA was extractedfrom the liver tissues with the TRIzol reagent (InvitrogenCorp., Carlsbad, CA) and transcribed into complemen-tary DNA with the ImProm-II reverse transcription sys-tem (Promega, Madison, WI). Mitochondrial andnuclear genomic DNA was obtained by proteinase K di-gestion and phenol-chloroform extraction. Fifty nano-grams of complementary DNA or 4 ng of genomic DNAwas used for quantification of the gene expression or mi-tochondrial DNA (mtDNA) copy number, respectively.Quantitative RT-PCR was performed with GreenERqPCR Supermix with specific primers (Invitrogen). Thereactions were carried out on a 7000 Sequence DetectionSystem (Applied Biosystems, Foster City, CA). For geneexpression, quantification was achieved with Ct valuesnormalized against glyceraldehyde 3-phosphate dehydro-genase as an internal control. For mtDNA copy numberdetermination, a T cell receptor was used as an internalcontrol. The primer sequences are listed in Supplemen-tary Table 1.

Western Blotting Analysis. Fifty micrograms of mi-tochondrial proteins was separated by 12% sodium dode-cyl sulfate–polyacrylamide gel electrophoresis, transferredonto a polyvinylidene difluoride membrane, and probedwith sheep antiserum against UCP2 (Santa Cruz, CA) ata dilution of 1:1000. After incubation with a horseradishperoxidase–conjugated rabbit anti-sheep immunoglobu-lin G antibody, the proteins were visualized with en-hanced chemiluminescence reagents (GE Healthcare,Uppsala, Sweden). The specificity of this antibody wasvalidated by immunoprecipitation and mass spectrometryanalysis, which confirmed the purified protein as UCP2(data not shown).

Terminal Deoxynucleotidyl Transferase-MediatedDeoxyuridine Triphosphate Nick-End Labeling(TUNEL) Assay. The liver tissues were obtained 6 hoursafter LPS injection. Apoptosis was assessed by TUNELassay with an in situ cell death detection kit (Roche, Indi-anapolis, IN). The liver sections were viewed at 488-nmexcitation/512-nm emission under a fluorescence micro-scope (Leica Microsystems, Bensheim, Germany).TUNEL-positive cells were manually counted in 20 ran-dom fields (�400) per sample to determine the averagenumber of positive-staining apoptotic cells. DNase Itreatment of additional sections was performed, and theyserved as positive controls.

Statistical Analysis. All the results were derived fromat least three independent experiments. Values are ex-pressed as the mean � standard error. The statistical cal-culations were performed with the Statistical Package forthe Social Sciences version 11.5 software package (SPSS,

HEPATOLOGY, Vol. 48, No. 4, 2008 ZHOU ET AL. 1089

Inc., Chicago, IL). Differences between groups were de-termined by the Student t test. Comparisons with P �0.05 were considered statistically significant.

Results

Mice Without Adiponectin Exhibit Increased Vul-nerabilities to Obesity-Induced Liver Injuries and aPreexisting Condition of Hepatic Steatosis. Althoughmany epidemiological studies have observed a close asso-ciation between low serum levels of adiponectin and in-creased risks of NAFLD and NASH,7,14-18 there is nolaboratory evidence showing that adiponectin deficiencycan exacerbate obesity-induced NAFLD and NASH inanimal models. To this end, we established two types ofobese models with an adiponectin-null background (micewhose obesity was induced by a high-fat diet and micewho were genetically obese because of the lack of func-tional leptin receptor�/�), and we evaluated the develop-ment of fatty liver diseases in these animals. Our resultsdemonstrated that in both dietary obese mice and genet-ically obese mice, adiponectin deficiency resulted in sig-nificantly increased hepatomegaly (Fig. 1A), exacerbatedhepatic steatosis (Fig. 1B), and a more severe phenotypeof liver injury, as reflected by the elevated levels of tumornecrosis factor alpha (TNF�) and the lipid peroxidationproduct MDA (Fig. 1C). In addition, ALT, a well-estab-lished marker of liver injury, was also significantly ele-vated in both obese models with adiponectin deficiency(data not shown). These results, along with several previ-ous studies,7-11,13 suggest that adiponectin deficiency is acommon risk factor that increases the vulnerability of theliver to various acute and chronic injuries.

We also analyzed the liver tissues of ADN-KO miceand their wild-type littermates under basal conditions. Toour surprise, we found that there was a preexisting condi-tion of hepatic steatosis in the liver tissues of 3-week-oldADN-KO mice even with the standard chow feeding(Fig. 2A). The increased lipid accumulation was also de-tected in the liver tissues of 13-week-old ADN-KO mice.On the other hand, TNF� levels were not altered in3-week-old ADN-KO mice but were significantly in-creased in 13-week-old ADN-KO mice in comparisonwith those of wild-type mice (Fig. 2B). These data suggestthat the lack of adiponectin might cause a “first hit” con-dition of hepatic steatosis, which predisposes the mice tothe secondary liver injuries and inflammation induced byobesity, chemicals, and endotoxins, for example.

Adiponectin Deficiency Is Associated with Abnor-mal Mitochondrial Morphologies and DecreasedMRC Activities. Mitochondrial dysfunction plays a cen-tral role in various forms of hepatic steatosis and liver

injuries.24-28 Using TEM, we then compared the mito-chondrial ultrastructures in the liver tissues of ADN-KOmice with those of their wild-type littermates. As shownin Fig. 3, this analysis demonstrated a profound morpho-logical change of hepatic mitochondria in both 3- and13-week-old ADN-KO mice. First, the electron densitiesof the mitochondrial matrix were kept well in wild-typeC57 mice but were lost distinctly in ADN-KO mice. Sec-ond, the average sizes of mitochondria in ADN-KO micewere about 1.5- to 2-fold larger than those of the wild-type controls. Moreover, mitochondrial swelling and me-gamitochondria were present in ADN-KO mice. Some of

Fig. 1. Adiponectin deficiency exacerbates liver injury in both high fat(HF)–fed obese mice and genetically obese mice. The two types of obesemodels were established as described in the Materials and Methodssection. The liver tissues were collected from 12-week-old C57(C57�HF) and ADN-KO (ADN-KO�HF) mice that had been fed an HFdiet for 8 weeks or from 12-week-old leptin receptor�/� (DB) and DKOmice, and they were evaluated for (A) hepatomegaly, (B) hepatic ste-atosis, and (C) liver inflammatory injuries as described in the text. Dataare presented as fold changes against the corresponding control groups.*P � 0.05 and **P � 0.01 versus the C57�HF group; #P � 0.05versus the DB group (n � 6-8).


the mitochondria in ADN-KO mice showed ruptures atthe outer membrane, which were possibly due to a mito-chondrial permeability transition. To further evaluatewhether the mitochondrial functions were altered inADN-KO mice, we purified the mitochondria from theliver tissues and measured the activities of each individual

MRC complex. Our results demonstrated that the activ-ities of complex I, complex II�III, complex IV, and com-plex V were significantly decreased in both 3- and 13-week-old ADN-KO mice in comparison with those of theage-matched and strain-matched C57 controls (Fig. 4).Moreover, in both dietary obese mice and geneticallyobese mice, targeted disruption of the adiponectin genealso led to a significant reduction in the MRC activities incomparison with those of littermate controls with normaladiponectin levels (Supplementary Fig. 1). Taken to-gether, these data suggest that the mitochondrial abnor-malities might contribute to the increased vulnerabilitiesof ADN-KO mice to various liver injuries.

Adiponectin Treatment Enhances MRC Activitiesbut Has No Obvious Effects on Mitochondrial Bio-genesis. We next investigated whether replenishment ofadiponectin can restore the MRC activities and reduce thelipid accumulation in the liver tissues of ADN-KO mice.To this end, the recombinant adenoviruses encodingmouse adiponectin or luciferase (as a control) were ad-ministered to ADN-KO mice through tail-vein injection.Consistent with our previous findings,20 serum levels ofadiponectin started to rise within 2 days after adenoviralinjection, reached peak levels at day 8, and remainedwithin a physiological concentration (approximately 5�g/mL) at 2 weeks after treatment (Supplementary Fig.2A). A nonheating and nonreducing sodium dodecyl sul-

Fig. 2. Hepatic steatosis preexists in the liver tissues of ADN-KO mice.Liver sections derived from 3- or 13-week-old C57 and ADN-KO micewere subjected to (A) Red Oil O staining for evaluating the fatty liverstatus and (B) quantitative RT-PCR analysis for measuring TNF� mRNAlevels. Data are shown as fold changes against the age-matched C57control mice. *P � 0.05 versus C57 mice (n � 6).

Fig. 3. Representative electron photomicrographs demonstrating thealtered mitochondrial ultrastructures in the liver tissues of 3- and 13-week-old ADN-KO mice. Note that there are profound mitochondrialmorphological changes, including mitochondrial swelling, mega mito-chondria, and mitochondrial outer membrane ruptures, in the livers ofADN-KO mice. Scale bars represent 1 �m.

Fig. 4. MRC activities are significantly reduced in the liver tissues ofADN-KO mice. The activities of hepatic MRC complexes I, II�III, IV, andV of both 3- and 13-week-old ADN-KO mice were measured as describedin the Materials and Methods section and calculated against the reactiontime and the total amount of proteins. *P � 0.05 compared to C57 mice(n � 4-7).


fate–polyacrylamide gel electrophoresis analysis showedthat circulating adiponectin in ADN-KO mice infectedwith the adenovirus formed all three quaternary struc-tures, including trimer, hexamer, and high-molecular-weight oligomeric complexes (Supplementary Fig. 2B);this pattern was similar to that of endogenous adiponectinin wild-type mice.29 Furthermore, over 60% of the adi-ponectin expressed in the liver and circulated in serumwas the high-molecular-weight oligomer, which has beenproposed to be the major active form possessing hepato-protective functions.6 This evidence suggests that the ad-enovirus-mediated expression system could deliveradiponectin with proper biological activities. As shown inFig. 5A,B, adenovirus-mediated adiponectin replenish-ment in 13-week-old ADN-KO mice significantly de-creased the lipid accumulation and enhanced the MRCactivities in liver tissues. Similar results have also beenobserved in 3-week-old ADN-KO mice (SupplementaryFig. 3). Moreover, the elevated mitochondrial MDA con-tents in the liver tissues of 13-week-old ADN-KO micewere significantly reduced by adiponectin treatment (Fig.5C). On the other hand, the activities of NADPH oxi-dase, a major source of cytoplasmic reactive oxygen spe-cies (ROS) production, showed no obvious changes inADN-KO mice before and after adiponectin treatment.These results suggest that the hepatoprotective propertiesof adiponectin might be at least partly attributable to itsstimulatory effects on mitochondrial activities and theoxidative phosphorylations of the MRC complexes.

The components of oxidative respiratory chain com-plexes are encoded by the coordination of the nuclear andmitochondrial genomes. To investigate whether the de-creased MRC activities in ADN-KO mice are due to im-paired mitochondrial biogenesis, we evaluated themtDNA copy number and the messenger RNA (mRNA)levels of several mitochondrial genome-encoded genes.This analysis demonstrated that in both 3- and 13-week-old ADN-KO mice, the mRNA levels of several MRCcomplexes genes, such as NADH dehydrogenase subunit1, cytochrome B, cytochrome C oxidase subunit 1, andATP synthase subunit 6, were significantly decreased by�30% in comparison with those of the wild-type mice.The mtDNA copy number was decreased by �35% in13-week-old ADN-KO mice but not in 3-week-oldADN-KO mice. However, adiponectin treatment had noobvious effects on mtDNA or mRNA expressions of mi-tochondria-encoded genes, mitochondria transcriptionfactor A, peroxisome proliferator-activated receptor-� co-activator-1�, DNA polymerase �, and mitochondrialRNA polymerase (data not shown), and this suggests thatadiponectin-mediated improvement of mitochondrial

functions might not be related to the de novo mitochon-drial biogenesis.

Expression of UCP2 Is Decreased in ADN-KO Miceand Can Be Induced by Adiponectin Treatment.UCP2 is a mitochondrial inner membrane carrier proteinthat has been suggested to regulate the proton leak, un-coupling, and mitochondrial respirations.30 A growingbody of evidence suggests that UCP2 possesses hepato-protective and anti-inflammatory activities by enhancing

Fig. 5. Adiponectin treatment reduces lipid accumulation and im-proves MRC activities in the liver tissues of ADN-KO mice. Thirteen-week-old ADN-KO mice were treated with 108 pfu of a recombinant adenovirusencoding luciferase (ADN-KO�Luci) or an adenovirus encoding adi-ponectin (ADN-KO�ADN). The liver tissues were collected after 2 weeksand subjected to (A) Red Oil O staining and (B) mitochondria isolationfor MRC activity measurements. Note that the MRC complex activities ofADN-KO�Luci mice were not different from those of the ADN-KO mice inFig. 5. (C) The MDA contents and NADPH oxidase activity were measuredwith the liver lysates derived from C57 and ADN-KO mice treated withoutor with the recombinant adenoviruses according to the proceduresdescribed in the Materials and Methods section. *P � 0.05 and **P �0.01 versus ADN-KO�Luci; #P � 0.05 versus C57 mice (n � 5-10).


the mitochondrial functions, reducing mitochondrialROS levels, and inhibiting the production of proinflam-matory cytokines.31-33 Using a genome-wide microarrayanalysis, we found that the mRNA levels of UCP2 weresignificantly decreased in high-fat diet–fed ADN-KOmice and DKO mice in comparison with age-matchedand strain-matched control animals (data not shown). Wethen evaluated the expressions of UCP2 in ADN-KOmice by comparing them with those of wild-type controls.Interestingly, our results showed that in mitochondriapurified from liver tissues of both 3- and 13-week-oldADN-KO mice, the protein levels of UCP2 were signifi-cantly decreased in comparison with those of wild-typemice (Fig. 6A). Moreover, a lower level of UCP2 expres-sion was also detected in 16- and 18-day embryos ofADN-KO mice (data not shown), and this indicated thatthe reduced UCP2 might contribute to the preexistingconditions of hepatic steatosis and mitochondrial dys-function in ADN-KO mice. On the other hand, replen-ishment of adiponectin dramatically increased both thegene and protein levels of UCP2 (Fig. 6A,B).

The Stimulatory Effects of Adiponectin on MRCActivities Are Abolished in UCP2-KO Mice. To inves-

tigate the role of UCP2 in mediating the hepatic actionsof adiponectin, we measured the MRC activities inUCP2-KO mice treated with or without adiponectin(Fig. 7). In comparison with the wild-type controls, theactivities of the MRC complexes II�III, IV, and V werenot changed in UCP2-deficient mice, whereas the activityof MRC complex I was decreased by �40%. In contrastto its effects in C57 and ADN-KO mice, adiponectintreatment failed to raise the activities of the MRC com-plexes I, II�III, IV, and V in UCP2-KO mice, and thissuggested an obligatory role of this mitochondrial carrierprotein in mediating the stimulatory activities of adi-ponectin on mitochondrial functions.

We also evaluated the role of UCP2 in mediating thehepatoprotective properties of adiponectin in LPS-in-duced acute liver injury. As shown in Fig. 8, the liversections of both ADN-KO and UCP2-KO mice exposedto LPS showed extensive areas of ballooned, hypereosino-philic hepatocytes, necrosis, histological features of apo-

Fig. 7. UCP2 deficiency attenuates the stimulatory effects of adi-ponectin on the MRC activities. The activities of mitochondrial MRCcomplexes (I, II�III, IV, and V) were measured and compared among theliver samples derived from 5-week-old C57, ADN-KO, and UCP2-KO micethat had been treated with or without a recombinant adenovirus encodingadiponectin for 2 weeks through tail-vein injections. Data were calculatedas fold changes against the values of C57 mice. #P � 0.05 versus C57mice; *P � 0.05 and **P � 0.01 versus ADN-KO mice (n � 4-6).

Fig. 6. The reduced expression of UCP2 in the liver tissue of ADN-KOmice can be restored by adenovirus-mediated replenishment of recom-binant adiponectin. (A) Mitochondrial proteins (50 �g) were analyzed byWestern blotting using antibodies against UCP2 and single-stranded DNAbinding protein 1 (SSBP1) as the loading control. (B) QuantitativeRT-PCR was performed for measuring the mRNA levels of UCP2 in livertissues of C57 and ADN-KO mice treated without or with a recombinantadenovirus encoding luciferase (ADN-KO�Luci) or adiponectin (ADN-KO�ADN). Data are presented as fold changes against the C57 controlgroups. *P � 0.05 versus C57 mice; #P � 0.05 versus ADN-KO�Luci(n � 4-6).


ptosis (that is, condensed nuclear chromatin with reducedcytoplasmic volume), and a large number of TUNEL-positive hepatocytes. Adenovirus-mediated adiponectintreatment prevented LPS-induced massive apoptosis ofhepatocytes and decreased LPS-induced elevation ofTNF� and ALT levels in ADN-KO mice but not inUCP2-KO mice. These data suggest that up-regulation ofUCP2 by adiponectin might play a critical role in medi-ating its protective effects against liver injuries.

DiscussionAlthough the hepatoprotective properties of adipo-

nectin have been suggested by many clinical and animalstudies,7,12,14-18 the detailed cellular and molecular mech-anisms remain elusive. Here we have found that the livertissues of ADN-KO mice show aberrant mitochondrialultrastructures and decreased MRC activities in compar-ison with those of the C57 control mice (Figs. 3 and 4).Notably, the mitochondrial dysfunction can be detectedin the liver tissues of 3-week-old ADN-KO mice (Fig. 3),and this suggests that it is an early event that predisposesADN-KO mice to NAFLD, NASH, and other forms ofliver injuries (Fig. 1). Using adenovirus-mediated overex-pression approaches, we have found that adiponectin

treatment almost completely depletes the lipid accumula-tion in ADN-KO mice and restores the activities of com-plexes I, II�III, IV, and V in both 3- and 13-week-oldADN-KO mice (Fig. 5 and Supplementary Fig. 3). Onthe other hand, it has little effect on mtDNA copy num-bers and the expression levels of genes involved in mito-chondrial biogenesis, and this suggests that thestimulatory effects of adiponectin on mitochondrial func-tions might not involve de novo mitochondrial biogenesis.

A growing body of evidence suggests that mitochon-drial dysfunctions might represent a central mechanismlinking obesity with its related metabolic complications.34

Fatty liver disease is an important component of the met-abolic syndrome associated with obesity and is causallyinvolved in the development of insulin resistance and type2 diabetes.35 In patients with NASH, the hepatic mito-chondria exhibit ultrastructural lesions and decreased ac-tivity of the respiratory chain complexes.27,36 Under thiscondition, the decreased activity of the respiratory chainresults in an accumulation of ROS that oxidize fat depos-its to form lipid peroxidation products, which in turncause steatohepatitis, necrosis, inflammation, and fibro-sis. The increased mitochondrial ROS formation in ste-atohepatitis could directly damage mtDNA and therespiratory chain polypeptides and induce nuclear factorkappa B activation and hepatic TNF� production.37 Ox-idative phosphorylation reactions mediated by the MRCcomplexes are directly involved in regulating the intracel-lular ROS levels and preventing the hepatic accumulationof lipids and lipid peroxidation products. In addition,mitochondria are the major sites for fat oxidation. Mito-chondrial dysfunction can increase hepatic lipid accumu-lation by promoting lipogenesis in the liver.28 In thisstudy, we found increased lipid accumulation inADN-KO mice even when the animals were fed standardchow (Fig. 2). This preexisting hepatic steatotic conditionmight be the direct result of dysregulated mitochondrialfunctions in these mice. This conclusion is supported byour observation that adiponectin-mediated reduction ofhepatic lipid accumulation is associated with the restora-tion of the impaired MRC activities and the reduction ofthe elevated mitochondrial MDA levels in ADN-KOmice (Fig. 5). Therefore, stimulation of mitochondrialfunctions might represent an important mechanism bywhich adiponectin exerts its multiple beneficial effects onvarious obesity-related pathologies.38-40

Impaired mitochondrial oxidative phosphorylation isthe major culprit for the increased ROS production andliver injury associated with steatohepatitis. It is knownthat UCP2 possesses antioxidant activities through inhi-bition of ROS production from mitochondria.30,41 UCP2can also inhibit the production of proinflammatory cyto-

Fig. 8. The protective effects of adiponectin against LPS-induced liverinjury are attenuated in UCP2-deficient mice. Recombinant adenovirusesencoding luciferase (Luci) or adiponectin (ADN) were tail-vein–injectedinto ADN-KO and UCP2-KO mice. One week later, the mice were treatedwith LPS as described in the Materials and Methods section. (A) The thinsections of the liver tissues collected 6 hours after LPS treatment weresubjected to histological evaluation and TUNEL staining. The specificity ofthe TUNEL assay was tested with samples for which the terminal trans-ferase was not added on the slides (data not shown). (B) Serum levelsof ALT were determined with commercial reagents from Sigma-Aldrich.(C) TNF� mRNA levels in liver tissues were quantified by real-timeRT-PCR. *P � 0.05 versus all other groups (n � 4).


kines in both macrophage and Kupffer cells, the majorsources of liver inflammation.31 Here we have providedevidence supporting a key role of UCP2 in mediating thebeneficial effects of adiponectin on mitochondrial func-tions. The protein and mRNA levels of UCP2 are de-creased in the liver tissues of ADN-KO mice and can besignificantly up-regulated by adiponectin treatment (Fig.6). Furthermore, the effects of adiponectin on MRC ac-tivities and endotoxin-induced liver injuries are dramati-cally attenuated in UCP2-deficient mice, and thissuggests that increased UCP2 expression might be neededfor adiponectin to elicit its hepatoprotective functions.Although this is the first report on the regulation of UCP2expression by adiponectin in the liver, a number of studieshave shown that low adiponectin levels are associated withreduced UCP2 expression in adipose tissue. For example,abnormal down-regulation of adiponectin and UCP2 ex-pression has been reported in adipose tissue from first-degree relatives of patients with type 2 diabetes.42,43

Lipodystrophy in antiretroviral-treated human immuno-deficiency virus patients is associated with systemic insu-lin resistance, mitochondrial impairment, UCP2 down-regulation, and reduced adiponectin levels in adiposetissue.44 Increased insulin sensitivity in 11�-hydroxys-teroid dehydrogenase type-1–deficient mice and Crebbpheterozygous mice is associated with increased adiponec-tin and UCP2 mRNA levels.45 In agouti yellow (Ay/a)obese mice, adiponectin treatment increases the expres-sion of UCP1 in brown adipose tissue, UCP2 in whiteadipose tissue, and UCP3 in skeletal muscle.46 Chevillotteand colleagues47 showed that UCP2 controls adiponectingene expression by regulating ROS production. Takentogether, these results suggest the existence of a reciprocalrelationship between uncoupling proteins and adiponec-tin in various tissues. However, the detailed signalingmechanisms underlying adiponectin-stimulated UCP2expression remain poorly understood and warrant furtherinvestigation.

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