Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals

Leptin Augments Inflammatory and Profibrogenic Responses in the Murine Liver Induced by Hepatotoxic Chemicals KENICHI IKEJIMA,1 HAJIME HONDA,1 MUTSUKO YOSHIKAWA,1,2 MIYOKO HIROSE,1 TSUNEO KITAMURA,1 YOSHIYUKI TAKEI,1 AND NOBUHIRO SATO1,2

Lines of evidence suggested a possible link between leptin and hepatic fibrosis; however, whether leptin modulates the fibrogenesis in the liver remains unclear. The purpose of this study, therefore, was to evaluate the effect of leptin on inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. Male C57Bl/6 mice were given carbon tetrachloride (CCl4) (0.1 ␮L/g body wieght [BW], intraperitoneally [IP]) and/or recombinant murine leptin (1 ␮g/g BW, IP) simultaneously, and sacrificed up to 72 hours later. Further, some mice were given thioacetamide (TAA; 200 ␮g/g BW, IP) and leptin 3 times per week for 4 weeks to evaluate the effect of leptin on chronic fibrogenic responses. A simultaneous injection of leptin enhanced acute CCl4-induced necroinflammatory and subsequent fibrotic changes in the hepatic lobules. The steadystate messenger RNA (mRNA) levels of ␣1(I) procollagen and heat shock protein 47 (HSP47) in the liver were potentiated when leptin was injected together with CCl4. Expression of ␣ smooth muscle actin (␣-SMA) in the liver after CCl4 treatment was also augmented markedly in combination with leptin. Further, leptin increased transforming growth factor ␤1 (TGF-␤1) mRNA in the liver 24 hours after acute CCl4 about 4-fold higher than CCl4 alone. Moreover, leptin enhanced hepatic fibrosis and induction of ␣1(I) procollagen mRNA caused by chronic TAA administration. Collectively, these findings indicated that leptin augments both inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. It is postulated that the increase in systemic leptin levels enhances up-regulation of TGF-␤1, leading to activation of stellate cells, thereby augmenting the fibrogenic response in the liver. (HEPATOLOGY 2001;34:288-297.)

Abbreviations: Ob-R, leptin receptor; NASH, non-alcoholic steatohepatitis; TAA, thioacetamide; IP, intraperitoneal; BW, body weight; HE, hematoxylin-eosin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TNF-␣, tumor necrosis factor ␣; ELISA, enzyme-linked immunosorbent assay; ␣-SMA, ␣ smooth muscle actin; TGF-␤1, transforming growth factor ␤1; cDNA, complementary DNA; HSP47, heat shock protein 47; RT-PCR, reverse transcription-polymerase chain reaction; mRNA, messenger RNA; LPS, lipopolysaccharide; IL, interleukin; NAFLD, nonalcoholic fatty liver disease. From the 1Department of Gastroenterology and 2Division of Biochemistry, Central Laboratory for Medical Science, Juntendo University School of Medicine, Tokyo, Japan. Received January 16, 2001; accepted May 21, 2001. Supported in part by Uehara Memorial Foundation (Tokyo, Japan). Address reprint requests to: Nobuhiro Sato, M.D., Ph.D., Professor and Chairman, Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. E-mail: [email protected]; fax: (81) 3-3813-8862. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3402-0012$35.00/0 doi:10.1053/jhep.2001.26518

Leptin, an obese gene product, is a cytokine-type peptide hormone that is mainly produced by adipocytes and controls body weight.1 Indeed, a homozygous defect of this gene in the mouse (ob/ob mouse) presents an obese phenotype,2 and recombinant murine leptin has been shown to reduce food intake and body weight of this obese animal.3 Leptin receptors (Ob-R) have been shown in hypothalamic neurons, through which leptin is believed to regulate food intake and body weight.4 In fact, homozygous mutations of the leptin receptor gene have been identified both in mice (i.e., db/db mouse) and rats (Zucker [fa/fa] rat), which are also associated with obesity.5,6 There are several isoforms of Ob-R, which are splice variants with the same extracellular domain. The most ubiquitous form of Ob-R is the short form (Ob-Ra); however, this isoform has not convincingly been shown to be a signaling molecule. On the other hand, a long-form leptin receptor (Ob-Rb), which contains longer intracellular domain, is known to activate the Janus kinase-STAT-3 pathway, leading to transcriptional regulation of target genes.7,8 Ob-Rb is expressed not only in hypothalamic neurons but also in various peripheral tissues including lung, pancreatic beta islet, and kidney suggesting that leptin might exert diverse biological functions.9-11 Recently, it was reported that leptin is produced not only by adipose tissues but also by other tissues, including stomach.12 Interestingly, isolated hepatic stellate cells have been shown to produce leptin during the in vitro transactivation process.13 Further, it has been reported that serum leptin levels are increased in patients with alcohol-induced cirrhosis.14,15 These observations suggested a possible involvement of leptin in the fibrogenic process in the liver. Moreover, recent clinical studies have disclosed that obesity is a risk factor of the progression of chronic liver diseases including viral hepatitis,16 alcohol-induced liver disease,17 and nonalcoholic steatohepatitis (NASH).18 Although obese people show obvious hyperleptinemia,19 it is still unclear whether an increase in systemic leptin levels is involved in the progression of liver diseases. In the present study, therefore, we evaluated the effect of coadministration of leptin on inflammatory and profibrogenic responses in the liver after acute carbon tetrachloride (CCl4) administration. Further, the effect of leptin on chronic fibrogenesis in the liver was investigated in the second model of hepatic fibrosis induced by long-term administration of thioacetamide (TAA). MATERIALS AND METHODS Animal Experiments. Specific pathogen-free male C57Bl/6 mice 5 weeks after birth were purchased from Japan SLC Inc. (Hamamatsu, Japan). All animals received humane care and the experimental protocol was approved by the Committee of Laboratory Animals accord-

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ing to institutional guidelines. Mice were allowed free access of water and laboratory chow diet and housed for 1 week before experiments. Mice were then given an intraperitoneal (IP) injection of CCl4 (0.1 ␮L/g, 2% in olive oil; Wako Chemical Inc., Osaka, Japan) and/or recombinant murine leptin (1 ␮g/g body weight [BW]; R&D Systems Inc., Minneapolis, MN) simultaneously, and sacrificed by exsanguinations from inferior vena cava under light ether anesthesia 12, 24, and 72 hours later. According to the manufacturer’s note, the endotoxin levels in the recombinant leptin were below 0.1 ng/␮g as determined by the Limulus Amebocyte Lysate method. Some mice were sacrificed 12 hours after treatment with CCl4 and/or leptin to collect portal blood samples under aseptic conditions. Further, some mice were given repeated injections of TAA (200 ␮g/g, IP) and leptin (1 ␮g/g BW, IP) 3 times per week for 4 weeks to evaluate the extended time course of hepatic fibrogenesis. For histologic analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin, and hematoxylin-eosin (HE) and Azan staining were performed. Serum samples and liver tissues for RNA preparation were kept frozen at ⫺80°C until assayed. Measurement of Serum Transaminases. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured spectrophotometrically by a standard enzymatic method using a commercial kit (KAINOS Laboratories Inc., Tokyo, Japan). Measurement of Serum Tumor Necrosis Factor ␣. Serum tumor necrosis factor ␣ (TNF-␣) levels were determined by using an enzymelinked immunosorbent assay (ELISA) kit (R&D Systems Inc.) according to the manufacturer’s instruction. Measurement of Endotoxin Levels in the Portal Blood. The platelet-rich plasma were obtained from heparinized portal blood samples by centrifugation at 1,500 rpm for 10 minutes, and endotoxin levels were determined by a commercial kit based on the methods of Limulus Amebocyte Lysate test (Endospecy, Seikagaku Corp., Tokyo, Japan). Immunohistochemical Staining for ␣ Smooth Muscle Actin. To detect ␣ smooth muscle actin (␣-SMA) in the liver, formalin-fixed and paraffin-embedded tissue sections were deparaffinized, and immunohistochemical staining using a mouse monoclonal anti-SMA antibody (American Research Products, Inc., Belmont, MA) was performed. After rinsing the primary antibody, the sections were incubated with a biotinylated mouse anti-mouse IgG F (ab⬘) fragment, followed by incubation with the avidin biotin complex solution by using a VECTOR Mouse on Mouse Immunodetection kit (Vector Laboratories, Inc., Burlingame, CA). RNA Preparation. Total RNA was prepared from frozen livers by a guanidium/CsTFA centrifugation method using QuickPrep total RNA extraction kit (Amersham Pharmacia Biotech, Piscataway, NJ). The concentration of isolated RNA was determined from the optical density at 260 nm and its purity from the ratio of 260:280. The integrity of RNA was further verified by electorophoresis on formaldehyde-denaturing agarose gels. Plasmid Construction. To generate riboprobes for RNase protection assays, partial complementary DNA (cDNA) for mouse ␣1(I) procollagen, heat shock protein 47 (HSP47), and transforming growth factor ␤1 (TGF-␤1) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into pCRII-TOPO vector (Invitrogen Corp., Carlsbad, CA). Briefly, total liver RNA from CCl4treated mice (1 ␮g) were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Gibco/Life Technologies, Grand Island, NY) and an oligo dT12-18 primer (Invitrogen Corp., Carlsbad, CA) at 42°C for 1 hour. Obtained cDNA (1 ␮L) was amplified using Taq DNA polymerase (AmpliTaq Gold; PE Applied Biosystems, Foster City, CA). The primer sets used were as follows: for ␣1(I) procollagen forward, 5⬘- GAG CGG AGA GTA CTG GAT CG -3⬘; and reverse, 5⬘- TGC TGT AGG TGA AGC GAC TG -3⬘; yielding a product size of 419 bp (based on the sequence from Gene Bank accession No. U03419); for HSP47 forward, 5⬘- CTG CGA ACA CTC CAA GAT CA -3⬘; and reverse, 5⬘- CCA GAT GTT TCT GCA GGT CA -3⬘; yielding a 500-bp product size (Gene Bank accession No. X60676); for TGF-␤1 forward, 5⬘- TGA GTG GCT GTC TTT TGA CG -3⬘; and reverse, 5⬘- TGG TTG TAG AGG GCA AGG AC -3⬘;

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yielding a 456-bp product size (Gene Bank accession No. M13177). All primer sequences were selected in our laboratory based on the reported cDNA sequences, and generated using an oligo-synthesizer (custom oligonucleotides ordering system; Amersham Pharmacia, Tokyo, Japan). After a 10-minute denaturation period at 95°C, 35 cycles of 95°C for 45 seconds, 58°C for 45 seconds, and 72°C for 60 seconds followed by final extension at 72°C for 7 minutes were performed using GeneAmp PCR System 9700 (PE Applied Biosystems). The size and quantity of PCR products were verified by electrophoresis on 1.5% agarose gels followed by staining with ethidium bromide. Subsequently, PCR products were subcloned into pCRII-TOPO vector using TOPO TA Cloning kit (Invitrogen Corp.) according to the manufacturer’s instruction. The sequences of the inserts were verified by direct sequencing using ABI PRISM 310 genetic analyzer (PE Applied Biosystems) and the dye terminator cycle sequencing FS ready reaction kit, which were identical to the reported sequences for mouse ␣1(I) procollagen,20 HSP47,21 and TGF-␤1.22 RNase Protection Assay. The subcloned plasmids containing cDNA for mouse ␣1(I) procollagen, HSP47, and TGF-␤1 were linealized by restriction endonuclease digestion using Acc I, Bsu 36I, and Hind III, respectively, and served as a template of in vitro transcription. The radiolabeled riboprobe was then generated by using a MAXIscript in vitro transcription kit (Ambion Inc., Austin, TX) in the presence of ␣32P UTP (Amersham Pharmacia Biotech) and T7 RNA polymerase. The riboprobe for mouse cyclophilin and RNA ladder were also transcribed similarly with T7 RNA polymerase using pTRI-cyclophilinmouse antisense control template (Ambion Inc.) and RNA template set (Wako Chemical Inc.). RNase protection assay was performed using RPA III kit (Ambion Inc.) according to the manufacturer’s instruction. Briefly, 20 ␮g of total RNA was hybridized with the radiolabeled probes (1 ⫻ 105 cpm each) in the hybridization solution at 42°C overnight. The reaction was then incubated with RNase A/T1 at 37°C for 30 minutes and precipitated with RNase inactivation/ precipitation solution. The reaction was separated on a denaturing 5% polyacrylamide/urea gel and exposed to films at ⫺80°C. Subsequently, densitometrical analysis was performed using a Windows version of NIH Image software (Scion Image, version Beta 4.0.2, Scion Corp., Frederick, MD). Statistical Analysis. Data for serum transaminase levels and densitometrical analyses were expressed as means ⫾ SEM. Statistical differences between means were determined using analysis of variance (ANOVA) on ranks and a Student-Newman-Keuls post-hoc test. P ⬍ .05 was selected before the study to reflect significance. RESULTS Histopathologic Changes in the Liver After Coadministration of CCl4 and Leptin. To evaluate the effect of leptin in hepatic

inflammation and fibrogenesis, histologic changes in the liver after acute CCl4 administration with or without a simultaneous injection of recombinant murine leptin were examined. Figure 1 shows typical photomicrographs of acute CCl4-induced inflammatory and fibrotic changes in the liver by HE and Azan staining, respectively. As expected, a considerable degree of hepatocellular necrosis (Fig. 1C) and, after that, fibrotic change in the hepatic lobule (Fig. 1G) was observed 24 hours and 72 hours after a single CCl4 administration, respectively. In contrast, both inflammatory and fibrotic changes were much stronger when leptin was given together with CCl4 (Fig. 1D and 1H). Leptin per se had no effect on liver histology throughout the whole time course in this study (Fig. 1B and 1F). These findings indicated that leptin enhanced both hepatic necrosis and postnecrotic fibrosis in the liver after acute CCl4 treatment. Serum Transaminases After Coadministration of CCl4 and Leptin.

Serum transaminases were measured to evaluate the severity of liver injury after acute CCl4 treatment (Fig. 2). As expected, serum AST and ALT were elevated 24 hours after a single

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FIG. 1. Histopathologic changes in the liver after coadministration of CCl4 and leptin. (A-D) Necroinflammatory changes in the liver. Mice were given IP injections of CCl4 (0.1 ␮L/g BW) and/or recombinant murine leptin (1 ␮g/g BW) simultaneously, and liver samples were obtained 24 hours later. Representative photomicrographs of liver histology from controls (A), leptin treatment alone (B), CCl4 treatment alone (C), and simultaneous treatment of CCl4 and leptin (D) are shown (HE staining, original magnification ⫻200). (E-H) Fibrotic changes in the liver. Liver samples were obtained 72 hours after coinjection of CCl4 and/or leptin. Representative photomicrographs of liver histology from controls (E), leptin alone (F), CCl4 alone (G), and CCl4 plus leptin (H) are shown (Azan staining, original magnification ⫻100).

injection of CCl4, however, the levels were nearly 3 times higher when leptin was injected together with CCl4. Leptin per se had no effect on serum transaminases. These findings were consistent with the histologic findings stated above, confirming that leptin enhances liver injury after acute CCl4 treatment.

Serum TNF-␣ Levels After Coadministration of CCl4 and Leptin.

To elucidate the mechanism by which leptin exacerbates hepatic inflammation after acute CCl4 treatment, the changes of serum TNF-␣ levels after CCl4 and/or leptin administration were determined by ELISA (Fig. 3A). Serum TNF-␣ levels were elevated 12 hours after a single injection of CCl4 fol-

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FIG. 2. Serum transaminases after coadministration of CCl4 and leptin. Serum samples were collected 24 hours after injection of CCl4 and/or leptin, and serum AST and ALT levels were determined by a standard enzymatic method. Mean AST values (A) and ALT values (B) ⫾SEM in each group are plotted (n ⫽ 4, *P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and StudentNewman-Keuls test).

lowed by a gradual decrease to the basal levels. Simultaneous administration of leptin enhanced this CCl4-induced increase in serum TNF-␣ nearly 3-fold. In contrast, leptin alone had no effect on serum TNF-␣ levels over 24 hours. Because TNF-␣ is one of the key inflammatory cytokines in acute CCl4-induced liver injury, it is likely that augmented production of TNF-␣ is responsible for the exacerbation of inflammatory changes in the liver after coadministration of CCl4 and leptin. Effect of Leptin on Portal Endotoxin Levels After Acute CCl4 Treatment. Because gut-derived endotoxin has been shown to play

a pivotal role in the inflammatory responses during acute CCl4 intoxication, here we measured endotoxin levels in the portal blood after coadministration of CCl4 and leptin (Fig. 3B). Endotoxin levels in the portal blood were increased significantly 12 hours after CCl4 treatment alone; the value reached was 1.7-fold as high as control. The levels after coadministration of CCl4 and leptin were not significantly different as compared with CCl4 alone. Leptin per se had no effect on portal endotoxin levels.

Effect of Leptin on CCl4-Induced Increases in ␣1(I) Procollagen and HSP47 Messenger RNA in the Liver. Next, we evaluated the

steady state messenger RNA (mRNA) levels of ␣1(I) procollagen, a component of type I collagen, in the liver after acute CCl4 administration by RNase protection assay (Fig. 4). As expected, mRNA for ␣1(I) procollagen was barely detectable in the control liver; however, it was detected clearly in the

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 FIG. 3. Serum TNF-␣ levels and endotoxin levels in the portal blood after coadministration of CCl4 and leptin. (A) Serum TNF-␣ levels. Experimental design as in Fig. 1 except that serum samples were obtained 0, 12, and 24 hours after CCl4 and/or leptin. Serum TNF-␣ levels were determined by ELISA. Mean serum TNF-␣ levels ⫾ SEM in each group are plotted (n ⫽ 5, *P ⬍ .05 vs. controls; #P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and StudentNewman-Keuls test). (B) Plasma endotoxin levels in the portal blood. Portal blood samples were obtained 12 hours after CCl4 and/or leptin, and plasma endotoxin levels were measured by Limulus Amebocyte Lysate test. Mean ⫾ SEM in each group are plotted (n ⫽ 5, *P ⬍ .05 vs. controls by ANOVA on ranks and Student-Newman-Keuls test).

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control values. In contrast, leptin potentiated this CCl4-induced increase in HSP47 mRNA about 1.7-fold. Collectively, these results indicated that synthesis of collagen after acute CCl4 intoxication is up-regulated by coadministration of leptin, which were compatible with the histologic changes induced by the combination of CCl4 and leptin (Fig. 1E-H). Expression of ␣-SMA in the Liver After Coadministration of CCl4 and Leptin. Because activation of hepatic stellate cells is a key

event in the fibrogenesis in the liver, expression of ␣-SMA, a

FIG. 4. ␣1(I) procollagen mRNA in the liver after coadministration of CCl4 and leptin. Experimental design as in Fig. 2. Steady state mRNA levels for ␣1(I) procollagen in the liver 72 hours after CCl4 and/or leptin treatment were measured by using RNase protection assay as described detail in Methods. Representative photographs of protected bands simultaneously detected for ␣1(I) procollagen (363 bp) and cyclophilin (103 bp), a house keeping gene, from 4 individual experiments are shown (A). Yeast RNA was used as a negative control. The ratio of densitometrical results for ␣1(I) procollagen and cyclophilin (expressed as % controls) were plotted (B, mean ⫾ SEM, n ⫽ 4, *P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and Student-Newman-Keuls test).

liver 72 hours after a single injection of CCl4. Interestingly, ␣1(I) procollagen mRNA increased dramatically when leptin was given together with CCl4; the levels were about 3-fold higher than CCl4 alone (Fig. 4B). In addition, the steady state mRNA levels of HSP47, also known as collagen binding protein–1, which is a 47-kD glycoprotein that acts as a molecular chaperone during the processing and/or secretion of procollagen in the endoplasmic reticulum,23 were detected similarly using RNase protection assay (Fig. 5). In the control liver, HSP47 was detected in a quite low level, whereas it was clearly detected 72 hours after a single injection of CCl4, being nearly 6-fold higher than

FIG. 5. HSP47 mRNA in the liver after coadministration of CCl4 and leptin. Experimental design as in Fig. 4 except that a riboprobe for HSP47 was used. Representative photographs of protected bands simultaneously detected for HSP47 (408 bp) and cyclophilin (103 bp) from 4 individual experiments are shown (A). The ratio of densitometric results for HSP47 and cyclophilin (expressed as % controls) were plotted (B, mean ⫾ SEM, n ⫽ 4, *P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and Student-Newman-Keuls test).

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FIG. 6. Expression of ␣-SMA in the liver after coadministration of CCl4 and leptin. Experimental design as in Fig. 2. The expression and localization of ␣-SMA in the liver 72 hours after injection of CCl4 and/or leptin were detected by immunohistochemical staining using a monoclonal antibody for ␣-SMA as described detail in Methods. Representative photomicrographs from 4 individual experiments are shown (original magnification ⫻100). (A) control liver, (B) leptin alone, (C) CCl4 alone, (D) CCl4 and leptin.

typical marker of activation of hepatic stellate cells, was detected in the liver after acute CCl4 administration (Fig. 6). The liver sections 72 hours after an injection of CCl4 showed positive staining for ␣-SMA as expected (Fig. 6C). In the liver after simultaneous injections of CCl4 and leptin, however, expressed ␣-SMA stronger than CCl4 alone (Fig. 6D), indicating that extrinsic leptin augments the activation of hepatic stellate cells caused by acute CCl4.

TGF-␤1 mRNA in the Liver After Coadministration of CCl4 and Leptin. Further, to explore the mechanism by which leptin

enhances stellate cell activation, expression of TGF-␤1 mRNA in the liver was detected by RNase protection assay (Fig. 7). Steady-state mRNA levels for TGF-␤1 in the liver were increased about 2-fold 24 hours after single CCl4 injection. Leptin alone for 24 hours did not affect the expression of TGF-␤1 mRNA in the liver. In contrast, leptin increased TGF-␤1 mRNA in the liver 24 hours after CCl4 treatment about 4-fold higher than CCl4 alone. Effect of Leptin on TAA-Induced Hepatic Fibrosis. To evaluate whether leptin augments hepatic fibrogenesis in a chronic model, the effect of leptin on TAA-induced liver fibrosis was investigated. C57Bl/6 mice were given repeated injections of TAA (200 ␮g/g, IP) and/or recombinant murine leptin (1 ␮g/g, IP) 3 times per week for 4 weeks, and liver histology was assessed. Figure 8 shows representative photographs of picrosirius red staining in the liver. Chronic treatment of leptin alone had no effect on liver histology in terms of fibrosis (Fig. 8B); however, leptin markedly enhanced chronic TAA-induced hepatic fibrosis (Fig. 8C and D). Further, immunohistochemical analysis for ␣-SMA showed enhanced induction of ␣-SMA in the liver after a 4-week treatment with TAA and leptin (Fig. 9D) compared with TAA alone (Fig. 9C). Furthermore, steady-state mRNA levels of ␣1(I) procollagen were largely increased in the liver after 4-week treatment with TAA

and leptin, which were almost 2-fold higher than TAA treatment alone (Fig. 10). DISCUSSION Leptin Augments CCl4-Induced Acute Inflammatory Response in the Liver. CCl4 is a known hepatotoxin that induces a revers-

ible acute centrilobular liver necrosis followed by a phase of tissue repair and fibrogenesis. In this study, we showed that short-term leptin treatment enhances acute inflammatory responses and profibrogenic responses in the liver caused by a single injection of CCl4. Extrinsic leptin augmented CCl4induced hepatotoxicity, in terms of increased necrosis of parenchymal cells and transaminases levels (Figs. 1 and 2). The mechanisms of acute CCl4 hepatotoxicity involve immediate cleavage of CCl4 by cytochrome P450 2E1 (CYP2E1) in hepatocytes,24 which generate trichloromethyl radical, leading to lipid peroxidation and membrane damage.25 Subsequently, activated hepatic macrophages (Kupffer cells) produce toxic mediators (e.g., inflammatory cytokines, reactive oxygen intermediates, and eicosanoids), resulting in the injury of parenchymal cells.26 On the other hand, lines of evidence indicate that leptin modulates proinflammatory responses caused by endotoxin (lipopolysaccharide, LPS)27 or TNF-␣28; however, the profound role of leptin in proinflammatory responses has not been well established. Importantly, leptin has been shown to up-regulate phagocytosis and production of proinflammatory cytokines in macrophages caused by LPS, suggesting that exogenous leptin enhances inflammatory immune responses.29 In that study, both leptin-deficient ob/ob mice and Ob-R– deficient Zucker (fa/fa) rats produced less TNF-␣ and interleukin 6 (IL-6) after injection of LPS, and addition of leptin enhanced LPS-induced production of TNF-␣, IL-6, and IL-12 in isolated peritoneal macrophages in vitro. In the present study, we showed that serum TNF-␣

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bility that leptin potentiates the activation of Kupffer cells to endotoxin and/or toxic metabolites of CCl4, leading to augmented production of toxic mediators including TNF-␣, thereby exacerbating liver injury. Leptin Augments Profibrogenic Responses in the Liver Caused by Hepatotoxic Chemicals. The initial purpose of this study was to

FIG. 7. TGF-␤1 mRNA in the liver after coadministration of CCl4 and leptin. Experimental design as in Fig. 4, except that liver samples 24 hours after CCl4 and/or leptin treatment were analyzed for TGF-␤1 mRNA. Representative photographs of protected bands for TGF-␤1 (456 bp) and cyclophilin (103 bp) as a housekeeping gene from 4 individual experiments are shown (A). The ratio of densitometric results for TGF-␤1 and cyclophilin (expressed as % controls) were plotted (B, mean ⫾ SEM, n ⫽ 4, *P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and Student-Newman-Keuls test).

levels 12 hours after acute CCl4 administration were potentiated by a simultaneous injection of leptin (Fig. 3A). On the other hand, mild increases in portal endotoxin levels were observed both in CCl4 groups with and without leptin, the values being not significantly different (Fig. 3B), indicating that leptin does not affect the translocation of endotoxin from the gut. Taken together, these observations suggest the possi-

evaluate the effect of leptin on profibrogenic responses in the liver induced by hepatotoxic chemicals. As shown in Fig. 1, postinflammatory fibrotic changes in the hepatic lobules were increased when leptin was administered simultaneously. This observation was further confirmed by increased levels of ␣1(I) procollagen mRNA (Fig. 4), which reflect the synthesis of type I collagen in the liver. In addition, we showed that steady-state mRNA levels of HSP47 were increased by simultaneous injection of CCl4 and leptin (Fig. 5). HSP47 has been shown to play a pivotal role in the maturation of collagen fibrils23 and increases during hepatic fibrogenesis induced by various fibrogenic stimuli including CCl4 and bile duct ligation.30,31 Taken together, these findings indicate that leptin definitely enhanced production of extracellular matrix components in the liver induced by acute CCl4. Further, to elucidate the role of leptin on chronic profibrogenic responses in the liver, long-term TAA-induced hepatic fibrosis was used as a second model. Although CCl4 is one of the classical profibrogenic chemicals, it is difficult to distinguish inflammatory and profibrogenic responses, because CCl4 causes severe necroinflammatory changes in the liver. In contrast, TAA is much less inflammatory compared with CCl4, which is beneficial to investigate fibrogenesis in the liver. Coadministration of leptin enhanced TAA-induced fibrogenic responses in the liver, which were assessed by picrosirius red staining (Fig. 8), ␣-SMA immunohistochemistry (Fig. 9), and the steady-state mRNA levels of ␣1(I) procollagen (Fig. 10). These findings clearly indicated that leptin unequivocally augments chronic profibrogenic responses in the liver. TGF-␤1 is one of the key cytokines for tissue repair and fibrogenesis in the liver.32,33 The causative role of TGF-␤1 in stellate cell activation and hepatic fibrogenesis has been shown using transgenic mice,34,35 knockout mice, and adenoviral overexpression.36 In the present study, leptin enhanced the expression of ␣-SMA, a hallmark of stellate cell transactivation after the potentiation of TGF-␤1 mRNA in the liver after CCl4 administration (Figs. 6 and 7). These phenomena are, at least in part, a consequence of augmented proinflammatory responses caused by leptin and CCl4. On the other hand, sinusoidal endothelial cells express functional Ob-R, through which leptin increases TGF-␤1 mRNA in these cells.37 Collectively, these findings support the hypothesis that leptin most likely increases TGF-␤1 production, leading to the transacitvation of hepatic stellate cells thereby enhancing synthesis of extracelluar matrix components in the liver. It is concluded, therefore, that leptin is a profibrogenic cytokine in the liver. Clinical Relevance. As stated above, leptin augments both proinflammatory and profibrogenic responses induced by acute CCl4 through up-regulation of TGF-␤1. These findings may account for the relationship between obesity, steatosis, and progression of chronic liver diseases. Recently, obesity has been recognized as a risk factor of the development of chronic liver diseases caused by a variety of etiologies including chronic hepatitis C,16 alcohol,17 and nonalcoholic fatty liver disease (NAFLD).18 Because serum leptin levels are ele-

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FIG. 8. Hepatic fibrosis induced by long-term coadministration of TAA and leptin. Mice were given repeated IP injections of TAA (200 ␮g/g BW) and/or recombinant murine leptin (1 ␮g/g BW) 3 times per week for 4 weeks, and liver samples were obtained. Tissue sections were stained by picrosirius red staining as described in detail in Methods. Representative photomicrographs of controls (A), leptin treatment alone (B), TAA treatment alone (C), and simultaneous treatment of TAA and leptin (D) are shown (original magnification ⫻100).

vated in obese individuals,19 increased leptin in the blood presumably enhances profibrogenic responses in the liver in concert with the definitive chronic fibrogenic stimuli such as hepatitis C virus infection or alcohol. Further, our findings support the hypothesis that leptin is involved in the development of NASH. Although the pathogenesis of this new clinical

entity has not been elucidated yet, severe obesity appears to be a risk factor for NASH,18 where serum leptin levels are extremely high. Moreover, progression of liver fibrosis including cryptogenic cirrhosis is observed often in overweight individuals without any recognizable etiologies.38-40 Additional work will be required to elucidate the role of leptin in the

FIG. 9. Expression of ␣-SMA in the liver after long-term coadministration of TAA and leptin. Experimental design as in Fig. 8. The expression and localization of ␣-SMA in the liver after 4-week treatment with TAA and/or leptin were detected by immunohistochemical staining using a monoclonal antibody for ␣-SMA as described in detail in Methods. Representative photomicrographs from 4 individual experiments are shown (original magnification ⫻100). (A) Control liver, (B) leptin alone, (C) TAA alone, (D) TAA and leptin.

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FIG. 10. ␣1(I) procollagen mRNA in the liver after long-term coadministration of TAA and leptin. Experimental design as in Fig. 8. Steady-state mRNA levels for ␣1(I) procollagen in the liver after 4-week treatment with TAA and/or leptin were measured using RNase protection assay as in Fig. 4. Representative photographs of protected bands simultaneously detected for ␣1(I) procollagen (363 bp) and cyclophilin (103 bp), a housekeeping gene, from 4 individual experiments are shown (A). Yeast RNA was used as a negative control. The ratio of densitometric results for ␣1(I) procollagen and cyclophilin (expressed as % controls) were plotted (B, mean ⫾ SEM, n ⫽ 4, *P ⬍ .05 vs. CCl4 alone by ANOVA on ranks and Student-Newman-Keuls test).

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