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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: Hepatology. 2008 Oct;48(4):1087–1096. doi: 10.1002/hep.22444

Mitochondria dysfunction contributes to the increased vulnerabilities of adiponectin knockout mice to liver injury

Mingyan Zhou 1,2,5, Aimin Xu 1,3,5, Paul KH Tam 4, Karen SL Lam 3,5, Lawrence Chan 7, Ruby LC Hoo 3,5, Jing Liu 1,2,5, Kim HM Chow 1,2,5, Yu Wang 1,2,6,*
PMCID: PMC2597507  NIHMSID: NIHMS65278  PMID: 18698578

Abstract

Adiponectin is an adipocyte-derived hormone with a wide range of beneficial effects on obesity-related medical complications. Numerous epidemiological investigations in diverse ethnic groups have identified lower adiponectin level as an independent risk factor for non-alcoholic fatty liver diseases and liver dysfunctions. Animal studies have demonstrated that replenishment of adiponectin protects against various forms of hepatic injuries, suggesting it to be a potential drug candidate for the treatment of liver diseases. This study was designed to investigate the cellular and molecular mechanisms underlying the hepato-protective effects of adiponectin. Our results demonstrated that in adiponectin knockout (ADN-KO) mice, there was a pre-existing condition of hepatic steatosis and mitochondria dysfunction, which might contribute to the increased vulnerabilities of these mice to secondary liver injuries induced by obesity and other conditions. Adenovirus-mediated replenishment of adiponectin depleted lipid accumulation, restored the oxidative activities of mitochondria respiratory chain (MRC) complexes and prevented the accumulation of lipid peroxidation products in ADN-KO mice, but had no obvious effects on mitochondria biogenesis. The gene and protein levels of uncoupling protein 2 (UCP2), a mitochondria membrane transporter, were decreased in ADN-KO mice and could be significantly up-regulated by adiponectin treatment. Moreover, the effects of adiponectin on mitochondria activities and on protection against endotoxin-induced liver injuries were significantly attenuated in UCP2 knockout mice. These results suggest that the hepatoprotective properties of adiponectin are mediated, at least in part, by enhancing the activities of MRC complexes through a mechanism involving UCP2.

Keywords: Adiponectin, metabolic syndrome, non-alcoholic fatty liver disease, mitochondria respiratory chain complex, uncoupling protein 2

Introduction

Obesity and its related metabolic syndrome are now reaching an epidemic level worldwide. Nonalcoholic fatty liver disease (NAFLD) is being increasingly recognized as a hepatic manifestation of the metabolic syndrome, which is closely associated with the development of insulin resistance, Type 2 Diabetes and cardiovascular diseases (1, 2). NAFLD and nonalcoholic steatohepatitis (NASH) predispose the progressive development of hepatic fibrosis, cirrhosis and end-stage liver cancers. A growing body of evidence from animal models suggests a “two-hit” hypothesis responsible for the development of NAFLD (3-5). With this theory, the first hit is the occurrence of fatty liver (steatosis), followed by a second event leading to the development of NASH. The secondary liver injuries can be induced by endotoxin exposure, alcohol consumption, drug compounds and virus infections etc. To date, there have been very few effective drugs available for the treatments of NAFLD and NASH.

Adiponectin is an adipokine abundantly produced from adipocytes (6). This adipokine has recently attracted great attention due to its anti-diabetic, anti-inflammatory and anti-atherogenic activitities. A previous study from our group has provided the first evidence demonstrating that adiponectin possesses potent protective effects against alcoholic and nonalcoholic fatty liver disease and steatohepatitis (7). In both ethanol-fed and ob/ob obese mice, chronic treatment with recombinant adiponectin markedly attenuated hepatomegaly and steatosis, and significantly decreased the hepatic inflammation and serum alanine aminotransferase (ALT) levels. Consistent with our data, studies from a number of independent groups have demonstrated the hepato-protective effects of adiponectin in different animal models with various forms of liver injuries, including those induced by carbon tetrachloride, lipopolysaccharide (LPS)/D-galactosamine (GalN), pharmacological compounds, bile duct ligations and methionine-deficient diet etc (8-13). These animal-based findings were further corroborated by clinical observations showing an inverse association between serum levels of adiponectin and liver dysfunctions (7, 14-18). Plasma adiponectin levels are significantly lower in patients with non-alcoholic fatty liver disease (NAFLD) compared to the sex and age matched healthy controls. Moreover, NASH patients with lower levels of adiponectin show higher grades of inflammation, suggesting that adiponectin deficiency is an important risk factor for the development of fatty liver, steatohepatatis and other forms of liver injuries. Therefore, adiponectin and its agonists represent emerging therapeutic agents for the treatment and/or prevention of liver dysfunctions.

Although adiponectin deficiency has been shown to predispose mice to various liver injuries (9, 10), the underlying mechanisms remain largely unknown. Here, we have examined the liver functions of adiponectin knockout mice (ADN-KO) and their wild-type littermates under both normal and obese conditions. Our results show a pre-existing condition of steatosis accompanied by mitochondrial defects in the liver tissues of ADN-KO mice. Adiponectin treatment improves liver functions through increasing mitochondrial respiratory chain (MRC) activities and reducing hepatic lipid accumulation in these mice. Furthermore, we have also provided evidence suggesting that uncoupling protein 2 (UCP2), a mitochondria membrane carrier protein, is critically important in mediating the hepato-protective effects of adiponectin.

Materials and Methods

Animal studies

Adiponectin knock-out (ADN-KO) mice in C57BL/6J background and the leptin receptor (Lepr)−/−/adiponectin−/− double knockout (DKO) mice were generated as we described recently (19). UCP2 knockout (UCP2-KO) mice in C57BL/6J background were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were provided with standard chow (LabDiet 5053; LabDiet, Purina Mills, Richmond, IN) unless specified. To establish a dietary obese mouse model, 4-week old male C57BL/6J and ADN-KO mice were fed with a high-fat diet (D12451, Research Diet, New Brunswick, NJ) containing 19.33 kJ/g from 49.85% fat, 20% protein and 30.15% carbohydrate for 8 weeks. All animals were kept under 12-hour light-dark cycles at 22-24 °C. For adiponectin treatment, the recombinant adenovirus expressing adiponectin or luciferase was tail vein injected into mice at two weeks prior to tissue collection (19, 20). Note that the amount of injected adenovirus [108 plaque-forming units (pfu)] caused no toxicity in mice as judged by their body weight gains, food and water intakes, liver functions, as well as other behavioral variables. The increased expression levels of adiponectin were confirmed by both ELISA and Western Blotting analysis. For the induction of acute liver injury, 200 μg of lipopolysaccharide (LPS) was intraperitoneally injected into adiponectin or UCP2 knockout mice and the liver tissue collected at different time points as indicated. All the animal experimental procedures were approved by the Committee on the Use of Live Animals for Teaching and Research, University of Hong Kong, and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals.

Isolation of mitochondria from liver tissues and measurement of MRC activities

Mice were sacrificed under deep anesthesia and the liver tissues were immediately collected for hepatic mitochondria isolation following the procedures as we described previously (21). The resulting mitochondrial pellet was resuspended in 50 mM Tris-solution (pH 8.0) to a protein concentration of 5-8 mg/ml, and kept at −80° C for subsequent measurement of MRC activities. Briefly, complex I activity was determined by measuring the colorimetric changes during the oxidation of NADH at 340 nm wavelength. The specific activities were calculated by subtraction of values obtained in the presence of the complex I specific inhibitor rotenone. The dual complex activities of the complexes II and III were measured by monitoring the colorimetric changes during the reduction of cytochrome C at 550 nm wavelength, in the presence or absence of a specific inhibitor antimycin A. The activity of the complex IV was analyzed by measuring the oxidation of reduced cytochrome C using a commercially available Cytochrome C Oxidase Assay Kit (Sigma, St Louis, MO). The activity of the complex V was evaluated by a coupled enzymatic (pyruvate kinase and lactate dehydrogenase) assay for measuring the hydrolytic activities of ATPase. The specific activity was calculated by subtracting the readings in the presence of oligomycin.

Electron-microscopic analysis of mitochondrial ultrastructures and histological staining for evaluation of hepatic steatosis and inflammation

Transmission electronic microscope (TEM) was used to evaluate the ultrastructures of liver mitochondria. Briefly, ∼1 mm3 liver tissues from the same anatomical locations were fixed in 2.5% glutaraldehyde in 100 mM sodium cocodylate, treated with 1% osmium tetroxide in 0.1 M NaCo buffer, washed, dehydrated, and embedded in Epon 812 resin. Random thin sections (100 nm) were cut and stained with aqueous uranyl acetate and Reynold's lead acetate. The images were examined with a Philips EM208S transmission electron microscope (Philips, Eindhoven, Netherlands). To evaluate hepatic steatosis, liver tissues were embedded in Tissue-Teck O.C.T. compound (Sakura Finetek USA Inc., Torrance, CA) and eight micrometer sections were prepared using a Leica 2800E Frigocut Microtome Cryostat. The air dried frozen sections were used for Red Oil O and hematoxylin-eosin staining (22).

TBARS (thiobarbituric acid reactive substances) and lucigenin assays

The concentrations of the lipid peroxidation product malondialdehyde (MDA) in serum, liver lysates, or mitochondrion suspensions were determined using a commercial TBARS assay kit (Cayman Chemical, Ann Arbor, MI). The results were calculated against the total protein contents. The activity of NADPH oxidase was assessed by the lucigenin assay as described (23).

Quantitative RT-PCR

Total RNA was extracted from the liver tissues using the TRIzol® reagent (Invitrogen Crop., Carlsbad, CA) and transcribed into cDNA using ImProm-II™ Reverse Transcription System (Promega. Madison, WI). Mitochondrial and nuclear genomic DNA was obtained by proteinase K digestion and phenol-chloroform extraction. 50 ng of cDNA or 4 ng of genomic DNA was used for quantification of gene expression or mitochondrial DNA (mtDNA) copy number respectively. Quantitative RT-PCR was performed using GreenER™ qPCR Supermix with specific primers (Invitrogen Crop., Carlsbad, CA). The reactions were carried out on a 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). For gene expression, quantification was achieved using Ct values normalized against GAPDH as internal control. For mitochondrial DNA copy number determination, T cell receptor was used as internal control. The primer sequences were listed in Supplementary Table 1.

Western Blotting analysis

Fifty micrograms of mitochondrial proteins were separated by 12% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and probed with sheep antiserum against UCP2 (Santa Cruz, CA) at a dilution of 1:1000. After incubation with a horse-radish-peroxidase-conjugated rabbit anti-sheep IgG antibody, the proteins were visualized with the enhanced chemiluminescence reagents (GE Healthcare, Uppsala, Sweden). The specificity of this antibody was validated by immunoprecipitation and mass spectrometry analysis, which confirmed the purified protein as UCP2 (data not shown).

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) Assay

The liver tissues were obtained at 6 hour after LPS injection. Apoptosis was assessed by TUNEL assay using an In situ Cell Death Detection kit (Roche, Indianapolis, IN). The liver sections were viewed at 488 nm excitation/512 nm emission under a fluorescence microscope (Leica Microsystems, Bensheim, Germany). TUNEL-positive cells were manually counted in 20 random fields (× 400) per sample to determine the average numbers of positive staining apoptotic cells. DNase I treatment of additional sections were performed and served as positive controls.

Statistical analysis

All the results were derived from at least three independent experiments. Values are expressed as mean ± SE. The statistical calculations were performed with the Statistical Package for the Social Sciences version 11.5 software package (SPSS, Inc., Chicago, IL). Differences between groups were determined by Student's t-test. Comparisons with p < 0.05 were considered as statistically significant.

Results

Mice without adiponectin exhibit increased vulnerabilities to obesity-induced liver injuries and a pre-existing condition of hepatic steatosis

Although many epidemiological studies have observed a close association between low serum levels of adiponectin and the increased risks of NAFLD and NASH (7, 14-18), there is no laboratory evidence showing that adiponectin deficiency can exacerbate obesity-induced NAFLD and NASH in animal models. To this end, we established two types of obese models in adiponectin-null background, including high fat diet-induced obese mice and genetic obese mice due to the lack of functional leptin receptor (Lepr)−/−, and evaluated the development of fatty liver diseases in these animals. Our results demonstrated that in both dietary and genetic obese mice, adiponectin deficiency resulted in a significantly increased hepatomegaly (Figure 1A), exacerbated hepatic steatosis (Figure 1B), and a more severe phenotype of liver injury, as reflected by the elevated levels of tumor necrosis factor alpha (TNFα) and the lipid peroxidation products malondialdehyde (MDA) (Figure 1C). In addition, alanine transaminase (ALT), a well-established marker of liver injury, was also significantly elevated in both obese models with adiponectin deficiency (data not shown). These results, along with several previous studies (7-11, 13), suggest that adiponectin deficiency is a common risk factor that increases the vulnerability of liver to various acute and chronic injuries.

Figure 1.

Figure 1

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Adiponectin deficiency exacerbates liver injury in both high fat (HF)-fed and genetic obese mice. The two types of obese models were established as described in the Methods. The liver tissues were collected from 12-wk old C57 (C57+HF) and ADN-KO mice (ADN-KO+HF), which had been fed with high fat diet for 8 weeks, or 12-wk old leptin receptor (Lepr)-/- (DB) and leptin receptor (Lepr)-/-/adiponectin-/- double knockout (DKO) mice, and evaluated for hepatomegaly (A), hepatic steatosis (B) and liver inflammatory injuries (C) as described in the text. Data are presented as fold changes against the corresponding control groups. *, p < 0.05 and **, p < 0.01 versus C57+HF group; #, p < 0.05 versus DB group (n=6-8).

We also analyzed the liver tissues of ADN-KO mice and their wild type littermates under basal conditions. To our surprise, we found that there was a preexisting condition of hepatic steatosis in the liver tissues of 3-wk old ADN-KO mice even under the standard chow feeding (Figure 2A). The increased lipid accumulation was also detected in the liver tissues of 13-wk old ADN-KO mice. On the other hand, TNFα levels were not altered in 3-wk old ADN-KO mice, but were significantly increased in 13-wk old ADN-KO mice comparing with those of wild type mice (Figure 2B). These data suggest that the lack of adiponectin might cause a “first hit” condition of hepatic steatosis, which predisposes the mice to the secondary liver injuries and inflammation induced by obesity, chemical and endotoxin etc.

Figure 2.

Figure 2

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Hepatic steatosis preexists in the liver tissues of ADN-KO mice. Liver sections derived from 3-wk or 13-wk old C57 and ADN-KO mice were subjected to Red Oil O staining for evaluating the fatty liver status (A), and quantitative RT-PCR analysis for measuring TNFα mRNA levels (B). Data are shown as the fold changes comparing to the age matched C57 control mice. *, p < 0.05 versus C57 mice (n=6).

Adiponectin deficiency is associated with abnormal mitochondrial morphologies and decreased MRC activities

Mitochondrial dysfunction plays a central role in various forms of hepatic steatosis and liver injuries (24-28). We then compared the mitochondrial ultra-structures in the liver tissues of ADN-KO mice with its wild type littermates using a transmission electronic microscope (TEM). As shown in Figure 3, this analysis demonstrated a profound morphological change of hepatic mitochondria in both 3-wk and 13-wk old ADN-KO mice. Firstly, the electron densities of the mitochondrial matrix were kept well in wild-type C57 mice but lost distinctly in ADN-KO mice. Secondly, the average sizes of mitochondria in ADN-KO mice were about 1.5-2 folds larger than those of the wild-type controls. Moreover, mitochondrial swelling and megamitochondria were present in ADN-KO mice. Some of the mitochondria in ADN-KO mice showed ruptures at the outer membrane, possibly due to mitochondrial permeability transition. To further evaluate whether the mitochondria functions are altered in ADN-KO mice, we purified the mitochondria from the liver tissues and measured the activities of each individual mitochondrial respiratory chain (MRC) complex. Our results demonstrated that the activities of complex I, complex II+III, complex IV and complex V were significantly decreased in both 3-wk and 13-wk old ADN-KO mice, comparing with those of the age-and strain-matched C57 controls (Figure 4). Moreover, in both dietary and genetic obese mice, targeted disruption of the adiponectin gene also led to a significant reduction in the MRC activities comparing with those littermate controls with normal adiponectin levels (Supplementary Figure 1). Taken together, these data suggest that the mitochondrial abnormalities might contribute to the increased vulnerabilities of ADN-KO mice to various liver injuries.

Figure 3.

Figure 3

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Representative electron photomicrographs demonstrating the altered mitochondrial ultra-structures in the liver tissues of 3- and 13-wk old ADN-KO mice. Note that there are profound mitochondrial morphological changes, including mitochondrial swelling, mega mitochondrion and mitochondrial outer membrane ruptures etc, in the livers of ADN-KO mice. Scale bars represent 1 μm.

Figure 4.

Figure 4

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MRC activities are significantly decreased in the liver tissues of ADN-KO mice. The activities of hepatic MRC complex I, II+III, IV and V of both 3- and 13-wk old ADN-KO mice were measured as described in Methods and calculated against the reaction time and the total amount of proteins. *, p < 0.05 compared to C57 mice (n=4-7).

Adiponectin treatment enhances MRC activities, but has no obvious effects on mitochondria biogenesis

We next investigated whether replenishment of adiponectin can restore the MRC activities and reduce the lipid accumulation in the liver tissues of ADN-KO mice. To this end, the recombinant adenoviruses encoding mouse adiponectin or luciferase (as control) were administered into ADN-KO mice through tail vein injection. Consistent with our previous findings (20), serum levels of adiponectin started to rise within two days after adenoviral injection, reached peak levels at day 8, and sustained within a physiological concentration (approximately 5 μg/ml) at two weeks after treatment (Supplementary Figure 2A). A non-heating and non-reducing SDS-PAGE analysis showed that circulating adiponectin in ADN-KO mice infected with the adenovirus formed all three quaternary structures, including trimer, hexamer and high molecular weight (HMW) oligomeric complexes (Supplementary Figure 2B), a pattern similar to that of endogenous adiponectin in wild type mice (29). Furthermore, over 60% of the adiponectin expressed in liver and circulated in serum were the HMW oligomers, which had been proposed to be the major active form possessing hepatoprotective functions (6). These evidence suggest that the adenovirus-mediated expression system could deliver adiponectin with proper biological activities. As shown in Figure 5A and 5B, adenovirus-mediated adiponectin replenishment in 13-wk old ADN-KO mice significantly decreased the lipid accumulation and enhanced the MRC activities in liver tissues. Similar results have also been observed in 3-wk old ADN-KO mice (Supplementary Figure 3). Moreover, the elevated mitochondria MDA contents in the liver tissues of 13-wk old ADN-KO mice were significantly reduced by adiponectin treatment (Figure 5C). On the other hand, the activities of NADPH oxidase, a major source of cytoplasmic reactive oxygen species (ROS) production, showed no obvious changes in ADN-KO mice before and after adiponectin treatment. These results suggest that the hepatoprotective properties of adiponectin might be at least partly attributed to its stimulatory effects on mitochondrial activities and the oxidative phosphorylations of the MRC complexes.

Figure 5.

Figure 5

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Adiponectin treatment reduces lipid accumulation and improves MRC activities in the liver tissues of ADN-KO mice. 13-wk old ADN-KO mice were treated with 108 plaque-forming units (pfu) of recombinant adenovirus encoding luciferase (ADN-KO+Luci) or adenovirus encoding adiponectin (ADN-KO+ADN). The liver tissues were collected after two weeks and subjected to Red Oil O staining (A), mitochondria isolation for MRC activities measurements (B). Note that the MRC complex activities of ADN-KO+Luci mice were not different from those of the ADN-KO mice in Figure 5. The MDA contents and NADPH oxidase activity were measured using the liver lysates derived from C57 and ADN-KO mice treated without or with the recombinant adenoviruses following the procedures described in the Methods (C). *, p < 0.05 and **, p < 0.01, versus ADN-KO+Luci; #, p < 0.05 vs C57 mice (n=5-10).

The components of oxidative respiratory chain complexes are encoded by the coordination of the nuclear and mitochondrial genomes. To investigate whether the decreased MRC activities in ADN-KO mice are due to an impaired mitochondrial biogenesis, we evaluated the mitochondria DNA copy number (mtDNA) and the mRNA levels of several mitochondrial genome-encoded genes. This analysis demonstrated that in both 3- and 13-wk old ADN-KO mice, the mRNA levels of several MRC complexes genes, such as ND1, CytB, COX1 and ATP6, were significantly decreased by ∼30% comparing with those of the wild-type mice. The mitochondria DNA copy number was decreased by ∼35% in 13-wk old ADN-KO mice, but not in 3-wk old ADN-KO mice. However, adiponectin treatment had no obvious effects on both mtDNA and the mRNA expressions of mitochondria-encoded genes, mitochondria transcription factor A (Tfam), peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), DNA polymerase γ and mitochondrial RNA polymerase (POLRMT) (data not shown), suggesting that adiponectin-mediated improvement of mitochondria functions might not be related to the de novo mitochondria biogenesis.

Expression of uncoupling protein 2 (UCP2) is decreased in ADN-KO mice and can be induced by adiponectin treatment

UCP2 is a mitochondrial inner membrane carrier protein that has been suggested to regulate the proton leak, uncoupling and mitochondrial respirations (30). A growing body of evidence suggests that UCP2 possesses hepato-protective and anti-inflammatory activities through enhancing the mitochondrial functions, decreasing mitochondrial ROS levels, as well as inhibiting the production of pro-inflammatory cytokines (31-33). Using a genome-wide microarray analysis, we have found that the mRNA levels of UCP2 were significantly decreased in high fat-fed ADN-KO mice and DKO mice, comparing with their age- and strain-matched control animals (data not shown). We then evaluated the expressions of UCP2 in ADN-KO mice by comparing with those of wild-type controls. Interestingly, our results showed that in mitochondria purified from liver tissues of both 3-wk and 13-wk ADN-KO mice, the protein levels of UCP2 were significantly decreased comparing with those of wild-type mice (Figure 6A). Moreover, a lower level of UCP2 expression was also detected at 16- and 18-day embryos of ADN-KO mice (data not shown), indicating that the decreased UCP2 might contribute to the preexisting conditions of hepatic steatosis and mitochondria dysfunction in ADN-KO mice. On the other hand, replenishment of adiponectin dramatically increased both the gene and protein levels of UCP2 (Figure 6, A and B).

Figure 6.

Figure 6

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The reduced expression of UCP2 in the liver tissue of ADN-KO mice can be restored by adenovirus-mediated replenishment of recombinant adiponectin. A, 50 μg of mitochondria proteins were analyzed by Western Blotting using antibodies against UCP2 and single-stranded DNA binding protein 1 (SSBP1, as the loading control). B, Quantitative RT-PCR was performed for measuring the mRNA levels of UCP2 in liver tissues of C57 and ADN-KO mice treated without or with recombinant adenovirus-encoding luciferase (ADN-KO+Luci) or adiponectin (ADN-KO+ADN). Data are presented as fold changes against the C57 control groups. *, p < 0.05 vs C57 mice; #, p < 0.05 vs ADN-KO+Luci (n=4-6).

The stimulatory effects of adiponectin on MRC activities are abolished in UCP2 knockout mice

To investigate the role of UCP2 in mediating the hepatic actions of adiponectin, we measured the MRC activities in UCP2-KO mice treated with or without adiponectin (Figure 7). Comparing with the wild-type controls, the activities of the MRC complex II+III, IV and V were not changed in UCP2 deficient mice, whereas the activity of MRC complex I was decreased by ∼40%. In contrast to its effects in C57 and ADN-KO mice, adiponectin treatment failed to raise the activities of the MRC complexes I, II+III, IV and V in UCP2-KO mice, suggesting an obligatory role of this mitochondria carrier protein in mediating the stimulatory activities of adiponectin on mitochondrial functions.

Figure 7.

Figure 7

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UCP2 deficiency attenuates the stimulatory effects of adiponectin on the MRC activities. The activities of mitochondrial MRC complexes (I, II+III, IV and V) were measured and compared among the liver samples derived from 5-wk old C57, ADN-KO and UCP2-KO mice, which had been treated with or without recombinant adenovirus encoding adiponectin for 2 weeks through tail vein injections. Data were calculated as fold changes against the values of C57 mice. #, p < 0.05 vs C57 mice; *, p < 0.05 and **, p < 0.01 vs ADN-KO mice (n=4-6).

We also evaluated the role of UCP2 in mediating the hepato-protective properties of adiponectin in LPS-induced acute liver injury. As shown in Figure 8, the liver sections of both ADN-KO and UCP2-KO mice exposed to LPS showed extensive areas of ballooned, hypereosinophilic hepatocytes, necrosis, histological features of apoptosis (i.e. condensed nuclear chromatin with reduced cytoplasmic volume) and a large number of TUNEL-positive hepatocytes. Adenovirus-mediated adiponectin treatment prevented LPS-induced massive apoptosis of hepatocytes and decreased LPS-induced elevation of TNFα and ALT levels in ADN-KO mice, but not in UCP2-KO mice. These data suggest that upregulation of UCP2 by adiponectin might play a critical role in mediating its protective effects against liver injuries.

Figure 8.

Figure 8

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The protective effects of adiponectin against LPS-induced liver injury are attenuated in UCP2-deficient mice. Recombinant adenovirus encoding luciferase (Luci) or adiponectin (ADN) were tail vein injected into ADN-KO and UCP2-KO mice. One week later, the mice were treated with LPS as described in the Methods section. The thin sections of the liver tissues collected at 6 hour after LPS treatment were subjected to histological evaluation and TUNEL staining (A). The specificity of the TUNEL assay was tested with samples for which the terminal transferase (TdT) was not added on the slides (data not shown). Serum levels of ALT were determined using commercial reagents from Sigma-Aldrich (B). TNFα mRNA levels in liver tissues were quantified by real-time RT-PCR (C). *, p < 0.05 versus all other groups (n=4).

Discussion

Although the hepato-protective properties of adiponectin have been suggested by many clinical and animal studies (7, 12, 14-18), the detailed cellular and molecular mechanisms remain elusive. Here, we have found that the liver tssues of ADN-KO mice show aberrant mitochondria ultrastructures and decreased MRC activities comparing with those of the C57 control mice (Figure 3 and 4). Notably, the mitochondria dysfunction can be detected in the liver tissues of 3-wk old ADN-KO mice (Figure 3), suggesting it to be an early event that predisposes ADN-KO mice to NAFLD, NASH and other forms of liver injuries (Figure 1). Using adenovirus-mediated overexpression approaches, we have found that adiponectin treatment almost completely depletes the lipid accumulation in ADN-KO mice and restores the activities of complex I, II+III, IV and V in both 3-wk and 13-wk old ADN-KO mice (Figure 5 and Supplementary Figure 3). On the other hand, it has little effects on mtDNA copy numbers and the expression levels of genes involved in mitochondria biogenesis, suggesting that the stimulatory effects of adiponectin on mitochondria functions might not involve de novo mitochondria biogenesis.

A growing body of evidence suggests that mitochondrial dysfunctions might represent a central mechanism linking obesity with its related metabolic complications (34). Fatty liver disease is an important component of the metabolic syndrome associated with obesity and is causally involved in the development of insulin resistance and Type 2 Diabetes (35). In patients with NASH, the hepatic mitochondria exhibit ultrastructural lesions and decreased activity of the respiratory chain complexes (27, 36). Under this condition, the decreased activity of the respiratory chain results in the accumulation of reactive oxygen species (ROS) that oxidize fat deposits to form lipid peroxidation products, which in turn causes steatohepatitis, necrosis, inflammation, and fibrosis. The increased mitochondrial ROS formation in steatohepatitis could directly damage mitochondria DNA (mtDNA) and the respiratory chain polypeptides, induce NF-κB activation and the hepatic TNFα production (37). Oxidative phosphorylation reactions mediated by the MRC complexes are directly involved in regulating the intracellular ROS levels and preventing the hepatic accumulation of lipids and lipid peroxidation products. In addition, mitochondria are the major sites for fat oxidation. Mitochondrial dysfunction can increase hepatic lipid accumulation through promoting lipogenesis in the liver (28). In this study, we have found that an increased lipid accumulation in ADN-KO mice even when the animals were fed with standard chow (Figure 2). This pre-existing hepatic steatotic condition might be the direct consequence of dysregulated mitochondria functions in these mice. This conclusion is supported by our observation that adiponectin-mediated reduction of hepatic lipid accumulation is associated with the restoration of the impaired MRC activities and the reduction of the elevated mitochondrial MDA levels in ADN-KO mice (Figure 5). Therefore, stimulation of mitochondrial functions might represent an important mechanism whereby adiponectin exert its multiple beneficial effects on various obesity-related pathologies (38-40).

Impaired mitochondrial oxidative phosphorylation is the major culprit for the increased ROS production and liver injury associated with steatohepatitis. It is known that UCP2 possesses anti-oxidant activities through inhibition of ROS production from mitochondria (30, 41). UCP2 can also inhibit the production of pro-inflammatory cytokines in both macrophage and Kupffer cells, the major sources of liver inflammation (31). Here, we have provided evidence supporting a key role of UCP2 in mediating the beneficial effects of adiponectin on mitochondria functions. The protein and mRNA levels of UCP2 are decreased in the liver tissues of adiponectin knockout mice and can be significantly up-regulated by adiponectin treatment (Figure 6). Furthermore, the effects of adiponectin on MRC activities and endotoxin-induced liver injuries are dramatically attenuated in UCP2 deficient mice, suggesting that increased UCP2 expression might be obligatory for adiponectin to elicit its hepato-protective functions. Although this is the first report on the regulation of UCP2 expression by adiponectin in liver, a number of studies have shown that low adiponectin levels are associated with reduced UCP2 expression in adipose tissue. For example, abnormal down-regulation of adiponectin and UCP2 expression has been reported in adipose tissue from first-degree relatives of Type 2 Diabetic patients (42, 43). Lipodystrophy in antiretroviral-treated HIV patients is associated with systemic insulin resistance, mitochondrial impairment, UCP2 down-regulation and reduced adiponectin levels in adipose tissue (44). Increased insulin sensitivity in 11β-hydroxysteroid dehydrogenase type-1 deficient mice and Crebbp heterozygous mice is associated with increased adiponectin and UCP2 mRNA levels (45). In agouti yellow (Ay/a) obese mice, adiponectin treatment increases the expression of UCP1 in brown adipose tissue, UCP2 in white adipose tissue and UCP3 in skeletal muscle (46). Chevillotte E and colleagues have shown that UCP2 controls adiponectin gene expression through regulating ROS production (47). Taken together, these results suggest the existence of a reciprocal relationship between uncoupling proteins and adiponectin in various tissues. However, the detailed signaling mechanisms underlying adiponectin-stimulated UCP2 expression remain poorly understood and warrant further investigation.

Supplementary Material

Fig 1
Fig 2
Fig 3
Supp data

Acknowledgments

Financial support: This project is supported by the grants from Seeding Funds for Basic Research of the University of Hong Kong (Y. Wang), Hong Kong Research Grant Council grants HKU 778007 (Y. Wang) and HKU 7645/06M (A. Xu), and the Area of Excellent Scheme (AoE/P-10-01) established under the University Grants Committee, HKSAR.

Abbreviations

NAFLD

non-alcoholic fatty liver disease

NASH

non-alcoholic steatohepatitis

ADN

adiponectin

ADN-KO

adiponectin knockout mice

DKO

leptin receptor (Lepr)−/−/adiponectin−/− double knockout mice

UCP2

uncoupling protein 2

MDA

malondialdehyde

MRC

mitochondrial respiratory chain

ROS

reactive oxygen species

TEM

transmission electronic microscope

PGC-1α

peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α

Tfam

mitochondrial transcription factor A

ND1

NADH dehydrogenase subunit 1

CytB

cytochrome B

COX1

cytochrome C oxidase subunit 1

ATP6

ATP synthase subunit 6

mtDNA

mitochondrial DNA

POLRMT

mitochondrial RNA polymerase

Luci

lucifearase

TNFα

tumor necrosis factor alpha

LPS

lipopolysaccharide

References

  • 1.Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology. 2006;43:S99–S112. doi: 10.1002/hep.20973. [DOI] [PubMed] [Google Scholar]
  • 2.Williams R. Global challenges in liver disease. Hepatology. 2006;44:521–526. doi: 10.1002/hep.21347. [DOI] [PubMed] [Google Scholar]
  • 3.Brunt EM. Nonalcoholic steatohepatitis. Semin Liver Dis. 2004;24:3–20. doi: 10.1055/s-2004-823098. [DOI] [PubMed] [Google Scholar]
  • 4.Charlton M. Noninvasive indices of fibrosis in NAFLD: starting to think about a three-hit (at least) phenomenon. Am J Gastroenterol. 2007;102:409–411. doi: 10.1111/j.1572-0241.2006.01039.x. [DOI] [PubMed] [Google Scholar]
  • 5.Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–845. doi: 10.1016/s0016-5085(98)70599-2. [DOI] [PubMed] [Google Scholar]
  • 6.Wang Y, Lam KS, Yau MH, Xu A. Post-translational modifications of adiponectin: mechanisms and functional implications. Biochem J. 2008;409:623–633. doi: 10.1042/BJ20071492. [DOI] [PubMed] [Google Scholar]
  • 7.Xu A, Wang Y, Keshaw H, Xu LY, Lam KS, Cooper GJ. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest. 2003;112:91–100. doi: 10.1172/JCI17797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ding X, Saxena NK, Lin S, Xu A, Srinivasan S, Anania FA. The roles of leptin and adiponectin: a novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology. Am J Pathol. 2005;166:1655–1669. doi: 10.1016/S0002-9440(10)62476-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kamada Y, Tamura S, Kiso S, Matsumoto H, Saji Y, Yoshida Y, Fukui K, et al. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology. 2003;125:1796–1807. doi: 10.1053/j.gastro.2003.08.029. [DOI] [PubMed] [Google Scholar]
  • 10.Matsumoto H, Tamura S, Kamada Y, Kiso S, Fukushima J, Wada A, Maeda N, et al. Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice. World J Gastroenterol. 2006;12:3352–3358. doi: 10.3748/wjg.v12.i21.3352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Thakur V, Pritchard MT, McMullen MR, Nagy LE. Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol. 2006;290:G998–1007. doi: 10.1152/ajpgi.00553.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ikejima K, Okumura K, Kon K, Takei Y, Sato N. Role of adipocytokines in hepatic fibrogenesis. J Gastroenterol Hepatol. 2007;22(Suppl 1):S87–92. doi: 10.1111/j.1440-1746.2007.04961.x. [DOI] [PubMed] [Google Scholar]
  • 13.Masaki T, Chiba S, Tatsukawa H, Yasuda T, Noguchi H, Seike M, Yoshimatsu H. Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice. Hepatology. 2004;40:177–184. doi: 10.1002/hep.20282. [DOI] [PubMed] [Google Scholar]
  • 14.Aygun C, Senturk O, Hulagu S, Uraz S, Celebi A, Konduk T, Mutlu B, et al. Serum levels of hepatoprotective peptide adiponectin in non-alcoholic fatty liver disease. Eur J Gastroenterol Hepatol. 2006;18:175–180. doi: 10.1097/00042737-200602000-00010. [DOI] [PubMed] [Google Scholar]
  • 15.Hui JM, Hodge A, Farrell GC, Kench JG, Kriketos A, George J. Beyond insulin resistance in NASH: TNF-alpha or adiponectin? Hepatology. 2004;40:46–54. doi: 10.1002/hep.20280. [DOI] [PubMed] [Google Scholar]
  • 16.Vuppalanchi R, Marri S, Kolwankar D, Considine RV, Chalasani N. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study. J Clin Gastroenterol. 2005;39:237–242. doi: 10.1097/01.mcg.0000152747.79773.2f. [DOI] [PubMed] [Google Scholar]
  • 17.Yoneda M, Iwasaki T, Fujita K, Kirikoshi H, Inamori M, Nozaki Y, Maeyama S, et al. Hypoadiponectinemia plays a crucial role in the development of nonalcoholic Fatty liver disease in patients with type 2 diabetes mellitus independent of visceral adipose tissue. Alcohol Clin Exp Res. 2007;31:S15–21. doi: 10.1111/j.1530-0277.2006.00281.x. [DOI] [PubMed] [Google Scholar]
  • 18.Pagano C, Soardo G, Esposito W, Fallo F, Basan L, Donnini D, Federspil G, et al. Plasma adiponectin is decreased in nonalcoholic fatty liver disease. Eur J Endocrinol. 2005;152:113–118. doi: 10.1530/eje.1.01821. [DOI] [PubMed] [Google Scholar]
  • 19.Hoo RL, Chow WS, Yau MH, Xu A, Tso AW, Tse HF, Fong CH, et al. Adiponectin mediates the suppressive effect of rosiglitazone on plasminogen activator inhibitor-1 production. Arterioscler Thromb Vasc Biol. 2007;27:2777–2782. doi: 10.1161/ATVBAHA.107.152462. [DOI] [PubMed] [Google Scholar]
  • 20.Wang Y, Lam KS, Chan L, Chan KW, Lam JB, Lam MC, Hoo RC, et al. Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J Biol Chem. 2006;281:16391–16400. doi: 10.1074/jbc.M513907200. [DOI] [PubMed] [Google Scholar]
  • 21.Wang Y, Lam KS, Lam JB, Lam MC, Leung PT, Zhou M, Xu A. Overexpression of angiopoietin-like protein 4 alters mitochondria activities and modulates methionine metabolic cycle in the liver tissues of db/db diabetic mice. Mol Endocrinol. 2007;21:972–986. doi: 10.1210/me.2006-0249. [DOI] [PubMed] [Google Scholar]
  • 22.Xu A, Lam MC, Chan KW, Wang Y, Zhang J, Hoo RL, Xu JY, et al. Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice. Proc Natl Acad Sci U S A. 2005;102:6086–6091. doi: 10.1073/pnas.0408452102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Block K, Gorin Y, Hoover P, Williams P, Chelmicki T, Clark RA, Yoneda T, et al. NAD(P)H oxidases regulate HIF-2alpha protein expression. J Biol Chem. 2007;282:8019–8026. doi: 10.1074/jbc.M611569200. [DOI] [PubMed] [Google Scholar]
  • 24.Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion. 2006;6:1–28. doi: 10.1016/j.mito.2005.10.004. [DOI] [PubMed] [Google Scholar]
  • 25.Berson A, De Beco V, Letteron P, Robin MA, Moreau C, El Kahwaji J, Verthier N, et al. Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology. 1998;114:764–774. doi: 10.1016/s0016-5085(98)70590-6. [DOI] [PubMed] [Google Scholar]
  • 26.Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest. 2004;114:147–152. doi: 10.1172/JCI22422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Perez-Carreras M, Del Hoyo P, Martin MA, Rubio JC, Martin A, Castellano G, Colina F, et al. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2003;38:999–1007. doi: 10.1053/jhep.2003.50398. [DOI] [PubMed] [Google Scholar]
  • 28.Pessayre D. Role of mitochondria in non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 2007;22(Suppl 1):S20–27. doi: 10.1111/j.1440-1746.2006.04640.x. [DOI] [PubMed] [Google Scholar]
  • 29.Xu A, Chan KW, Hoo RL, Wang Y, Tan KC, Zhang J, Chen B, et al. Testosterone selectively reduces the high molecular weight form of adiponectin by inhibiting its secretion from adipocytes. J Biol Chem. 2005;280:18073–18080. doi: 10.1074/jbc.M414231200. [DOI] [PubMed] [Google Scholar]
  • 30.Baffy G. Uncoupling protein-2 and non-alcoholic fatty liver disease. Front Biosci. 2005;10:2082–2096. doi: 10.2741/1683. [DOI] [PubMed] [Google Scholar]
  • 31.Bai Y, Onuma H, Bai X, Medvedev AV, Misukonis M, Weinberg JB, Cao W, et al. Persistent nuclear factor-kappa B activation in Ucp2-/- mice leads to enhanced nitric oxide and inflammatory cytokine production. J Biol Chem. 2005;280:19062–19069. doi: 10.1074/jbc.M500566200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Horimoto M, Fulop P, Derdak Z, Wands JR, Baffy G. Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice. Hepatology. 2004;39:386–392. doi: 10.1002/hep.20047. [DOI] [PubMed] [Google Scholar]
  • 33.Rousset S, Emre Y, Join-Lambert O, Hurtaud C, Ricquier D, Cassard-Doulcier AM. The uncoupling protein 2 modulates the cytokine balance in innate immunity. Cytokine. 2006;35:135–142. doi: 10.1016/j.cyto.2006.07.012. [DOI] [PubMed] [Google Scholar]
  • 34.Parish R, Petersen KF. Mitochondrial dysfunction and type 2 diabetes. Curr Diab Rep. 2005;5:177–183. doi: 10.1007/s11892-005-0006-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Machado M, Cortez-Pinto H. Non-alcoholic fatty liver disease and insulin resistance. Eur J Gastroenterol Hepatol. 2005;17:823–826. doi: 10.1097/00042737-200508000-00008. [DOI] [PubMed] [Google Scholar]
  • 36.Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, Luketic VA, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology. 2001;120:1183–1192. doi: 10.1053/gast.2001.23256. [DOI] [PubMed] [Google Scholar]
  • 37.Fromenty B, Robin MA, Igoudjil A, Mansouri A, Pessayre D. The ins and outs of mitochondrial dysfunction in NASH. Diabetes Metab. 2004;30:121–138. doi: 10.1016/s1262-3636(07)70098-8. [DOI] [PubMed] [Google Scholar]
  • 38.Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med. 2001;7:941–946. doi: 10.1038/90984. [DOI] [PubMed] [Google Scholar]
  • 39.Berg AH, Combs TP, Du X, Brownlee M, Scherer PE. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med. 2001;7:947–953. doi: 10.1038/90992. [DOI] [PubMed] [Google Scholar]
  • 40.Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, et al. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci U S A. 2001;98:2005–2010. doi: 10.1073/pnas.041591798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L, et al. A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 1997;11:809–815. [PubMed] [Google Scholar]
  • 42.Lihn AS, Ostergard T, Nyholm B, Pedersen SB, Richelsen B, Schmitz O. Adiponectin expression in adipose tissue is reduced in first-degree relatives of type 2 diabetic patients. Am J Physiol Endocrinol Metab. 2003;284:E443–448. doi: 10.1152/ajpendo.00358.2002. [DOI] [PubMed] [Google Scholar]
  • 43.Pedersen SB, Nyholm B, Kristensen K, Nielsen MF, Schmitz O, Richelsen B. Increased adiposity and reduced adipose tissue mRNA expression of uncoupling protein-2 in first-degree relatives of type 2 diabetic patients: evidence for insulin stimulation of UCP-2 and UCP-3 gene expression in adipose tissue. Diabetes Obes Metab. 2005;7:98–105. doi: 10.1111/j.1463-1326.2005.00365.x. [DOI] [PubMed] [Google Scholar]
  • 44.Giralt M, Domingo P, Guallar JP, Rodriguez de la Concepcion ML, Alegre M, Domingo JC, Villarroya F. HIV-1 infection alters gene expression in adipose tissue, which contributes to HIV- 1/HAART-associated lipodystrophy. Antivir Ther. 2006;11:729–740. [PubMed] [Google Scholar]
  • 45.Morton NM, Paterson JM, Masuzaki H, Holmes MC, Staels B, Fievet C, Walker BR, et al. Novel adipose tissue-mediated resistance to diet-induced visceral obesity in 11 beta-hydroxysteroid dehydrogenase type 1-deficient mice. Diabetes. 2004;53:931–938. doi: 10.2337/diabetes.53.4.931. [DOI] [PubMed] [Google Scholar]
  • 46.Masaki T, Chiba S, Yasuda T, Tsubone T, Kakuma T, Shimomura I, Funahashi T, et al. Peripheral, but not central, administration of adiponectin reduces visceral adiposity and upregulates the expression of uncoupling protein in agouti yellow (Ay/a) obese mice. Diabetes. 2003;52:2266–2273. doi: 10.2337/diabetes.52.9.2266. [DOI] [PubMed] [Google Scholar]
  • 47.Chevillotte E, Giralt M, Miroux B, Ricquier D, Villarroya F. Uncoupling protein-2 controls adiponectin gene expression in adipose tissue through the modulation of reactive oxygen species production. Diabetes. 2007;56:1042–1050. doi: 10.2337/db06-1300. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Fig 1
NIHMS65278-supplement-Fig_1.tif (1.3MB, tif)
Fig 2
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Fig 3
NIHMS65278-supplement-Fig_3.tif (4.1MB, tif)
Supp data
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