The Roles of Leptin and Adiponectin
2005; Elsevier BV; Volume: 166; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)62476-5
ISSN1525-2191
AutoresXiaokun Ding, Neeraj K. Saxena, Songbai Lin, Amin Xu, Shanthi Srinivasan, Frank A. Anania,
Tópico(s)Liver physiology and pathology
ResumoAlthough leptin is a key adipokine promoting liver fibrosis, adiponectin may prevent liver injury. To determine the role of these adipokines in liver fibrosis and to understand their expression in vivo, fa/fa rats and their lean littermates were subjected to bile duct ligation (BDL). Histomorphometry for collagen and α-smooth muscle actin (α-SMA) revealed that lean rats, but not fa/fa littermates, had significant fibrosis with abundant hepatic stellate cell (HSC) activation. The lean-BDL rats had significantly higher leptin concentrations in the hepatic vein than lean sham-operated, fa/fa BDL, or fa/fa sham-operated rats. Co-localization of leptin and α-SMA in activated HSCs was observed by immunohistochemistry. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis confirmed the presence of leptin and α-SMA in activated, but not quiescent, HSCs, whereas only quiescent HSCs synthesized adiponectin mRNA and protein. Adiponectin overexpression in activated HSCs reduced proliferation, augmented apoptosis, and reduced expression of α-SMA and proliferating cell nuclear antigen. Adiponectin receptors (AdipoR1 and AdipoR2) were detected in both activated and quiescent HSCs, but only activated HSCs produced significant apoptosis after treatment with either globular or full-length adiponectin. Adiponectin may act to reverse HSC activation, maintain HSC quiescence, or significantly, may have important therapeutic implications in liver fibrosis. Although leptin is a key adipokine promoting liver fibrosis, adiponectin may prevent liver injury. To determine the role of these adipokines in liver fibrosis and to understand their expression in vivo, fa/fa rats and their lean littermates were subjected to bile duct ligation (BDL). Histomorphometry for collagen and α-smooth muscle actin (α-SMA) revealed that lean rats, but not fa/fa littermates, had significant fibrosis with abundant hepatic stellate cell (HSC) activation. The lean-BDL rats had significantly higher leptin concentrations in the hepatic vein than lean sham-operated, fa/fa BDL, or fa/fa sham-operated rats. Co-localization of leptin and α-SMA in activated HSCs was observed by immunohistochemistry. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis confirmed the presence of leptin and α-SMA in activated, but not quiescent, HSCs, whereas only quiescent HSCs synthesized adiponectin mRNA and protein. Adiponectin overexpression in activated HSCs reduced proliferation, augmented apoptosis, and reduced expression of α-SMA and proliferating cell nuclear antigen. Adiponectin receptors (AdipoR1 and AdipoR2) were detected in both activated and quiescent HSCs, but only activated HSCs produced significant apoptosis after treatment with either globular or full-length adiponectin. Adiponectin may act to reverse HSC activation, maintain HSC quiescence, or significantly, may have important therapeutic implications in liver fibrosis. Leptin, a 16-kd hormone, has an array of biological effects. 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We also demonstrate, for the first time, that adiponectin, an adipokine thought to be exclusively secreted by adipocytes,29Wang Y Xu A Knight C Xu LY Cooper GJS Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin.J Biol Chem. 2002; 77: 19521-19529Crossref Scopus (317) Google Scholar may be critical to either maintaining the hepatic stellate cell (HSC) quiescent phenotype or reverse hepatic fibrosis by induction of activated HSC apoptosis. Taken together, our data provide one mechanism to account for the recent data in vivo demonstrating the exquisite sensitivity to carbon tetrachloride by adiponectin knockout mice.22Pittas AG Joseph NA Greenberg AS Adipocytokines and insulin resistance.J Clin Endocrinol Metab. 2004; 89: 447-452Crossref PubMed Scopus (403) Google Scholar We also demonstrate that AdipoR1 and AdipoR2 are detected in both quiescent and activated HSCs by mRNA and protein and that activated HSCs have the potential to respond to both adiponectin isoforms because we demonstrate that fundamental biological properties of the activated HSC can be altered by adiponectin exposure. Super Frost Plus slides, hematoxylin stain, and mounting media are from Fisher Scientific (Pittsburgh, PA). Anti-smooth muscle α-actin antibodies (Oncogene Research Products, San Diego, CA); anti-leptin antibodies (R&D Systems, Minneapolis, MN); anti-proliferating cell nuclear antigen (PCNA), anti-β-actin (Sigma Chemical Co., St. Louis, MO); anti-adiponectin antibody (a gift from Dr. Aimin Xu, University of Hong Kong, Hong Kong, China); anti-FLAG (Sigma); normal donkey serum, biotin-SP-conjugated AffiniPure donkey anti-rabbit IgG, biotin-SP-conjugated AffiniPure donkey anti-mouse IgG, peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA); TSA Reagent Plus Cyanine3 system, TSA Reagent Plus FITCH system (Perkin Elmer Life Sciences, Boston MA); avidin-biotin blocking kit, Elite kit, biotinylated anti-mouse or anti-rabbit IgG (H+L), Nova Red substrate (Vector Laboratories, Burlingame, CA). Diaminobenzidine tetrahydrochloride, nickel sulfate hexahydrate, sodium acetate, hydrogen peroxide (30%), methanol, paraformaldehyde, and Sirius Red stain (Sigma). Rat leptin endocrine immunoassay was purchased as a service from Linco Research, St. Charles, MO. Dulbecco's modified Eagle's medium, trypsin-ethylenediamine tetraacetic acid, penicillin-streptomycin were all purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from (HyClone, Logan, UT). Antibodies to caspase 3, cleaved caspase 3, and mitogen-activated protein kinase (MAPK) were all purchased from Cell Signaling (Beverly, MA); antibodies to adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) were purchased from Alpha Diagnostic (San Antonio, TX). Globular adiponectin (gAd) was purchased from Peprotech (Rocky Hill, NJ); full-length adiponectin (fAd) was purchased from Biovender (Candler, NC). Matrigel was purchased from BD Biosciences (Bedford, MA). Four-week-old fa/fa rats and their lean littermates were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed for 4 additional weeks in a temperature-controlled environment (20 to 22°C) with a 12:12 hour light:dark cycle, and fed ad libitum with Purina Laboratory Chow (Ralston Purina, St. Louis, MO) and water. The bile duct ligation (BDL) protocol was approved by the Institutional Animal Care and Use Committee of Emory University. Rats were assigned to one of four groups (n = 10): BDL (lean, or LLB), BDL (fa/fa, or FFB) (n = 10), sham-operated (lean, LLN), and sham-operated (fa/fa, or FFN). BDL or sham operation was performed as described elsewhere.30Kurosawa H Que FG Roberts LR Fesmier PJ Gores GJ Hepatocytes in the bile duct-ligated rat express Bcl-2.Am J Physiol. 1997; 272: G1587-G1593PubMed Google Scholar Twenty-one days after surgery, we anesthetized rats, established intravenous access via the right atrium, and also established portal venous access with a 16-gauge intravenous catheter. To calculate concentration of leptin for each rat, 1 ml of whole blood was collected from the portal vein and 1 ml hepatic vein blood was collected from the right atrium after ligation of the vena cava. Total hepatectomy was performed and the animal was exsanguinated. Livers harvested were weighed and a portion was either snap-frozen in liquid nitrogen or placed in formalin fixative and paraffin-embedded for staining and immunohistochemical analysis. Serum from whole blood was used to measure alkaline phosphatase, total bilirubin, alanine aminotransferase, and aspartate aminotransferase. Serum chemistry was performed with a serial multichannel analyzer in the Department of Veterinary Resources at Emory University. Plasma was collected using ethylenediamine tetraacetic acid as an anticoagulant. Controls, supplied by the manufacturer, were run simultaneously with samples. All assays were run three times in duplicate with standards from Linco Research (St. Charles, MO). The manufacturer also assisted in these determinations. Picrosirius red staining was used to detect collagen fibrils as described elsewhere.31Janqueira L Bignolas G Bretani R Picrosirius staining plus polarization microscopy: a specific method for collagen detection in tissue sections.Histochem J. 1979; 11: 447-455Crossref PubMed Scopus (2002) Google Scholar Paraffin-embedded tissues were sectioned at 6 μm. Deparaffinized sections were placed in 95% ethanol and rinsed thoroughly in double- distilled water. Primary incubation with anti-α-SMA (1:800) was performed for 36 to 48 hours at 4°C in a closed humidity chamber. Slides were viewed with a Nikon E1000M microscope (Melville, NY), and photographed with a Cool Snap color digital camera (Roper Scientific GmbH, Germany). Quantitative analysis of collagen in Picrosirius-stained or α-SMA-stained liver sections was performed by morphometric analysis. All images were quantified using Image-ProPlus version 4.5, a commercially available software package from Media Cybernetics (Silver Spring, MD). Imaging tissue sections was automated using an Image-Pro Plus macro calibrated for each microscope objective. All sections were examined by the same person in which 10 random areas of interest were examined per liver section which was identified by computer-generated field identification. The operator who performed the collagen quantitation analyses was blinded to the identification of the individual slides examined. At least 15 different liver sections were examined for five individual animals for each treatment group. The tissue section on the slide was automatically scanned using a preselected threshold such that captured bright-field images were digitized into a series of picture elements (pixels). Areas of interest were selected from a specific section on each slide with the position of the tissue section recorded as counted from the slide label. Similar sections were then analyzed on each subsequent slide and the images saved for analysis. Background images were stored and subtracted from each capture area of interest. Data for both collagen and α-SMA were expressed as the mean percentage of total hepatic area in the tissue sections: the total area stained was divided by the total area of the slide (μm2) and multiplied by 100 to give percentage of area stained. This calculation resulted in the determination of the percent area staining positively for collagen fibers providing a quantitative value on a continuous scale. Identical image analysis was performed on 6-μm sections in which staining with anti-α-SMA-antibody was used. Paraffin-embedded tissues were sectioned at 6 μm and deparaffinized. Deparaffinized sections were placed in 95% ethanol and rinsed thoroughly in double- distilled water. To detect leptin, slides were boiled in 10 mmol/L sodium citrate (pH 6.0) for 5 minutes and then allowed to cool for an additional 20 minutes. Three percent solution of H2O2 prepared in KPBS (potassium-phosphate buffered saline; Sigma) for slide incubation for 10 minutes was used for subsequent washes. Slides were incubated with avidin reagent for 15 minutes and subsequently washed, followed by incubation with biotin for 15 minutes, and washed again. Primary incubation with anti-α-SMA (1:800) was for 36 to 48 hours at 4°C in a closed humidity chamber. Biotinylated secondary antibodies (1:600) were used for 1 hour at room temperature, and slides were incubated with either diaminobenzidine tetrahydrochloride (DAB) alone or nickel sulfate-DAB (NiDAB) for 20 minutes. Slides were monitored for color precipitate and anti-leptin (1:200) to stain tissues as described previously for anti-α-SMA. In place of DAB or NiDAB substrate, Nova Red substrate was used to detect leptin. Slides were subsequently counterstained in hematoxylin, decolorized in acid, and prepared for examination. Slides were viewed with a Nikon E1000M microscope, and photographed with a Cool Snap color digital camera (Roper Scientific). IPLab (Scanalytics, Fairfax, VA) was used for image analysis. Total RNA from either whole liver or HSCs from BDL-injured rats was obtained using Trizol reagent (Invitrogen). The cDNA template was prepared using oligo-dT and random hexamer primers and Maloney murine leukemia virus reverse transcriptase as previously described in detail.32Rasmussen RP Quantification on the LightCycler.in: Meuer S Witwer CT Nakagawata K Rapid Cycle Real Time PCR. Springer, New York2000: 21-34Google Scholar After the reverse transcription reaction, the cDNA template was amplified by polymerase chain reaction with Taq polymerase (Invitrogen). The following genes as listed in Table 1 were subjected to real-time RT-PCR analysis and their abundance was normalized against 18srRNA. All nucleotide sequences designed and outlined in Table 1 spanned two introns to prevent genomic DNA amplification.Table 1Genes Used in Real-Time RT-PCR AnalysisAccession no.GeneSequence 5′ → 3′NM013076Leptin (F)TTGTCACCAGGATCAATGACATTTLeptin (R)GACAAACTCAGAATGGGGTGAAGNM021578TGF-β1 (F)TGCCCTCTACAACCAACACATGF-β1 (R)GTTGGACAACTGCTCCACCTX06801α-SMA (F)CCGAGATCTCACCGACTACCα-SMA (R)TCCAGAGCGACATAGCACAGXM213440α1(1) collagen (F)GAGTGAGGCCACGCATGAα1(1) collagen (R)AGCCGGAGGTCCACAAAGXM66209α2(1) collagen (F)TGATACCTCCGCTGGTGACCα2(1) collagen (R)TAGGCACGACGTTACTGCAAAY033885Adiponectin (F)AATCCTGCCCAGTCATGAAGAdiponectin (R)TCTCCAGGAGTGCCATCTCTBC061838Adiponectin receptor 1 (F)GGACTTGGCTTGAGTGGTGTAdiponectin receptor 1 (R)AGGAATCCGAGCAGCATAAAXM232323Adiponectin receptor 2 (F)GGCAGATAGGCTGGCTAATGAdiponectin receptor 2 (R)CACCAGCAACCACAAAGATGF, forward primer; R, reverse primer. Open table in a new tab F, forward primer; R, reverse primer. For distinguishing the specific PCR product from nonspecific products and primer-dimers, melting curve analyses were used.33Rire KM Rasmussen RP Wittwer CT Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.Anal Biochem. 1997; 245: 154-160Crossref PubMed Scopus (1293) Google Scholar, 34Wilhelm J Pingoud A Real-time polymerase chain reaction.Chembiochem. 2003; 4: 1120-1128Crossref PubMed Scopus (291) Google Scholar Because different DNA products melt at different temperatures, it was possible to distinguish genuine products from primer dimers or nonspecific products. Indeed, great care was taken to use PCR primers, which yielded a single product, an empirical process often requiring the design of several primer pairs. All PCR products were verified by sequencing dye termination technology; products were separated on 1% agarose gels, stained with ethidium bromide, and photographed using ultraviolet illumination. For quantification, we used real-time RT-PCR (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany) using SYBR green as the fluorophore (Molecular Probes, Eugene, OR). Quiescent stellate cells were isolated as described in detail elsewhere.35Friedman SL Roll FJ Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan.Anal Biochem. 1987; 161: 207-218Crossref PubMed Scopus (315) Google Scholar Sprague-Dawley rats were purchased from (Charles River, Boston, MA). All rats received humane care, and the Institutional Animal Care and Use Committee of Emory University approved the HSC isolation protocol. In brief, in situ perfusion of the liver with 20 mg/dl of Pronase (Boehringer Mannheim, Indianapolis, IN) followed by collagenase (Cresecent Chemical, Hauppauge, NY), dispersed cell suspensions were layered on a discontinuous density gradient of 8.2% and 15.6% Accudenz (Accurate Chemical and Scientific, Westbury, NY). The resulting upper layer consisted of more than 95% HSCs. Cells were placed in modified medium 199 OR containing 20% FBS (Flow Laboratories, Naperville, IL). The purity of cells was assessed by immunolocalization of α-SMA in the monolayer as well as by intrinsic autofluorescence. The viability of all cells was verified by phase-contrast microscopy as well as the ability to exclude propidium iodide. Cell viability of cultures used for experiments was greater than 95%. Subconfluent activated cells in culture (75%) 7 to 10 days after isolation were washed twice with phosphate-buffered saline (PBS) and serum-starved for 16 hours with 0.1% FBS and 1% penicillin-streptomycin in Dulbecco's modified Eagle's medium. Culture-activated HSCs and quiescent HSCs, harvested immediately after liver perfusion, were washed in PBS and resuspended in ice-cold RIPA buffer (10 μmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail, 10 μl/per ml; Sigma) for 30 minutes on ice. HSC lysates were centrifuged at 14,000 rpm for 30 minutes at 4°C. The supernatant was harvested and protein concentrations were determined using Bradford reagent (Sigma).36Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (219734) Google Scholar Proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 μg/lane) and transblotted to nitrocellulose membranes.37Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (208226) Google Scholar Membranes were stained with Ponceau S (0.1%) to verify equal loading and transfer of proteins. After blocking with 1% bovine serum albumin in TBS-Tween 20 (20 mmol/L Tris-Cl (pH 7.5), 137 mmol/L NaCl, 0.05% Tween-20), membranes were incubated for 3 hours at room temperature with primary antibodies. Specific antibody binding was detected with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA). Equal protein loading was controlled by immunoblot of β-actin (dilution, 1:2000). To verify activation of HSCs with respect to quiescent phenotype, immunoblot for α-SMA (dilution, 1:2000) was also performed. To detect AdipoR1 and AdipoR2, immunoblot analysis was performed exactly as described above except that 100 μg/lane of protein was loaded on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred as previously described.37La
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