Tacrolimus Ameliorates Metabolic Disturbance and Oxidative Stress Caused by Hepatitis C Virus Core Protein
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090102
ISSN1525-2191
AutoresKyoji Moriya, Hideyuki Miyoshi, Takeya Tsutsumi, Seiko Shinzawa, Hajime Fujie, Yoshizumi Shintani, Hiroshi Yotsuyanagi, Kohji Moriishi, Yoshiharu Matsuura, Tetsuro Suzuki, Tatsuo Miyamura, Kazuhiko Koike,
Tópico(s)Liver Disease and Transplantation
ResumoHepatic steatosis and insulin resistance are factors that aggravate the progression of liver disease caused by hepatitis C virus (HCV) infection. In the pathogenesis of liver disease and metabolic disorders in HCV infection, oxidative stress due to mitochondrial respiratory chain dysfunction plays a pivotal role. Tacrolimus (FK506) is supposed to protect mitochondrial respiratory function. We studied whether tacrolimus affects the development of HCV-associated liver disease using HCV core gene transgenic mice, which develop hepatic steatosis, insulin resistance, and hepatocellular carcinoma. Administration of tacrolimus to HCV core gene transgenic mice three times per week for 3 months led to a significant reduction in the amounts of lipid in the liver as well as in serum insulin. Tacrolimus treatment also ameliorated oxidative stress and DNA damage in the liver of the core gene transgenic mice. Tacrolimus administration reproduced these effects in a dose-dependent manner in HepG2 cells expressing the core protein. The intrahepatic level of tumor necrosis factor-α, which may be a key molecule for the pathogenesis in HCV infection, was significantly decreased in tacrolimus-treated core gene transgenic mice. Tacrolimus thus reversed the effect of the core protein in the pathogenesis of HCV-associated liver disease. These results may provide new therapeutic tools for chronic hepatitis C, in which oxidative stress and abnormalities in lipid and glucose metabolism contribute to liver pathogenesis. Hepatic steatosis and insulin resistance are factors that aggravate the progression of liver disease caused by hepatitis C virus (HCV) infection. In the pathogenesis of liver disease and metabolic disorders in HCV infection, oxidative stress due to mitochondrial respiratory chain dysfunction plays a pivotal role. Tacrolimus (FK506) is supposed to protect mitochondrial respiratory function. We studied whether tacrolimus affects the development of HCV-associated liver disease using HCV core gene transgenic mice, which develop hepatic steatosis, insulin resistance, and hepatocellular carcinoma. Administration of tacrolimus to HCV core gene transgenic mice three times per week for 3 months led to a significant reduction in the amounts of lipid in the liver as well as in serum insulin. Tacrolimus treatment also ameliorated oxidative stress and DNA damage in the liver of the core gene transgenic mice. Tacrolimus administration reproduced these effects in a dose-dependent manner in HepG2 cells expressing the core protein. The intrahepatic level of tumor necrosis factor-α, which may be a key molecule for the pathogenesis in HCV infection, was significantly decreased in tacrolimus-treated core gene transgenic mice. Tacrolimus thus reversed the effect of the core protein in the pathogenesis of HCV-associated liver disease. These results may provide new therapeutic tools for chronic hepatitis C, in which oxidative stress and abnormalities in lipid and glucose metabolism contribute to liver pathogenesis. Hepatitis C virus (HCV) is a major cause of liver disease; approximately 170 million people are chronically infected worldwide. Persistent HCV infection leads to the development of chronic hepatitis, cirrhosis, and, eventually, hepatocellular carcinoma (HCC), thereby being a serious problem from both medical and socioeconomic viewpoints.1Saito I Miyamura T Ohbayashi A Harada H Katayama T Kikuchi S Watanabe Y Koi S Onji M Ohta Y Choo Q Houghton M Kuo G Hepatitis C virus infection is associated with the development of hepatocellular carcinoma.Proc Natl Acad Sci USA. 1990; 87: 6547-6549Crossref PubMed Scopus (1077) Google Scholar, 2Simonetti RG Camma C Fiorello F Cottone M Rapicetta M Marino L Fiorentino G Craxì A Ciccaglione A Giuseppetti R Stroffolini T Pagliaro L Hepatitis C virus infection as a risk factor for hepatocellular carcinoma in patients with cirrhosis.Ann Intern Med. 1992; 116: 97-102Crossref PubMed Scopus (360) Google Scholar Recently, a growing amount of evidence showing that HCV infection induces alteration in lipid3Scheuer PJ Ashrafzadeh P Sherlock S Brown D Dusheiko GM The pathology of chronic hepatitis C.Hepatology. 1992; 15: 567-571Crossref PubMed Scopus (514) Google Scholar, 4Bach N Thung SN Schaffner F The histological features of chronic hepatitis C and autoimmune chronic hepatitis: a comparative analysis.Hepatology. 1992; 15: 572-577Crossref PubMed Scopus (470) Google Scholar, 5Barba G Harper F Harada T Kohara M Goulinet S Matsuura Y Eder G Schaff Z Chapman MJ Miyamura T Bréchot C Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.Proc Natl Acad Sci USA. 1997; 94: 1200-1205Crossref PubMed Scopus (572) Google Scholar, 6Moriya K Yotsuyanagi H Shintani Y Fujie H Ishibashi K Matsuura Y Miyamura T Koike K Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (576) Google Scholar, 7Lerat H Honda M Beard MR Loesch K Sun J Yang Y Okuda M Gosert R Xiao SY Weinman SA Lemon SM Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar and glucose metabolism has accumulated.8Caronia S Taylor K Pagliaro L Carr C Palazzo U Petrik J O'Rahilly S Shore S Tom BD Alexander GJ Further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis C virus infection.Hepatology. 1999; 30: 1059-1063Crossref PubMed Scopus (351) Google Scholar, 9Mehta SH Brancati FL Sulkowski MS Strathdee SA Szklo M Thomas DL Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States.Ann Intern Med. 2000; 133: 592-599Crossref PubMed Scopus (597) Google Scholar Augmentation of oxidative stress is also substantiated in HCV infection by a number of clinical and basic studies.10Choi J Ou JH Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G847-G851Crossref PubMed Scopus (257) Google Scholar, 11Koike K Miyoshi H Oxidative stress and hepatitis C viral infection.Hepatol Res. 2006; 34: 65-76Crossref PubMed Scopus (51) Google Scholar, 12Korenaga M Wang T Li Y Showalter LA Chan T Sun J Weinman SA Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.J Biol Chem. 2005; 280: 37481-37488Crossref PubMed Scopus (335) Google Scholar, 13Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Miyamura T Kimura S Koike K Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar We demonstrated previously that the core protein of HCV induces HCC in transgenic mice that have marked hepatic steatosis in the absence of inflammation.14Moriya K Fujie H Shintani Y Yotsuyanagi H Tsutsumi T Matsuura Y Kimura S Miyamura T Koike K The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1067Crossref PubMed Scopus (1097) Google Scholar In this animal model for HCV-associated HCC, there is augmentation of oxidative stress in the liver during the incubation period.10Choi J Ou JH Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G847-G851Crossref PubMed Scopus (257) Google Scholar Also noted is an accumulation of lipid droplets that are rich with carbon 18 monounsaturated fatty acids such as oleic and vaccenic acids, which is also observed in liver tissues of patients with chronic hepatitis C compared with those in patients with fatty liver due to simple obesity.15Moriya K Todoroki T Tsutsumi T Yotsuyanagi H Tsutsumi T Ishibashi K Takayama T Makuuchi M Watanabe K Miyamura T Kimura S Koike K Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C: analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar Recently, we have also shown, using the HCV transgenic mouse model, that the ability of insulin to lower plasma glucose levels is impaired in association with HCV infection,16Shintani Y Fujie H Miyoshi H Tsutsumi T Kimura S Moriya K Koike K Hepatitis C virus and diabetes: direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar which would be the basis for the frequent development of type 2 diabetes in patients with chronic hepatitis C.8Caronia S Taylor K Pagliaro L Carr C Palazzo U Petrik J O'Rahilly S Shore S Tom BD Alexander GJ Further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis C virus infection.Hepatology. 1999; 30: 1059-1063Crossref PubMed Scopus (351) Google Scholar, 9Mehta SH Brancati FL Sulkowski MS Strathdee SA Szklo M Thomas DL Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States.Ann Intern Med. 2000; 133: 592-599Crossref PubMed Scopus (597) Google Scholar Disturbances in lipid and glucose metabolism are notable features of HCV infection and may be profoundly involved in the pathogenesis of liver diseases. Although the mechanism underlying these phenomena is not yet well understood, the development of clues to correct these metabolic disturbances occurring in HCV infection, which have been recently connected to the poor prognosis of patients with chronic hepatitis C, is awaited. Moreover, a key role for oxidative stress in the pathogenesis of hepatitis C,11Koike K Miyoshi H Oxidative stress and hepatitis C viral infection.Hepatol Res. 2006; 34: 65-76Crossref PubMed Scopus (51) Google Scholar, 12Korenaga M Wang T Li Y Showalter LA Chan T Sun J Weinman SA Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.J Biol Chem. 2005; 280: 37481-37488Crossref PubMed Scopus (335) Google Scholar which may be closely associated with the aforementioned metabolic disorders, has been identified. The association of oxidative stress augmentation in HCV infection with mitochondrial respiratory dysfunction10Choi J Ou JH Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G847-G851Crossref PubMed Scopus (257) Google Scholar, 13Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Miyamura T Kimura S Koike K Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar, 17Piccoli C Scrima R Quarato G D'Aprile A Ripoli M Lecce L Boffoli D Moradpour D Capitanio N Hepatitis C virus protein expression causes calcium-mediated mitochondrial bioenergetic dysfunction and nitro-oxidative stress.Hepatology. 2007; 46: 58-65Crossref PubMed Scopus (106) Google Scholar suggests that one possibility to ameliorate such a condition is the use of agents that can protect the mitochondrial respiratory function. We have conducted information retrieval and screening for agents that can protect the mitochondrial respiratory function. Tacrolimus (FK506), which is widely used in organ transplantation, is one such agent with evidence showing protection of the mitochondrial respiratory function,18Cetinkale O Konukoğlu D Senel O Kemerli GD Yazar S Modulating the functions of neutrophils and lipid peroxidation by FK506 in a rat model of thermal injury.Burns. 1999; 25: 105-112Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19Kaibori M Inoue T Tu W Oda M Kwon AH Kamiyama Y Okumura T FK506, but not cyclosporin A, prevents mitochondrial dysfunction during hypoxia in rat hepatocytes.Life Sci. 2001; 69: 17-26Crossref PubMed Scopus (34) Google Scholar, 20Keswani SC Chander B Hasan C Griffin JW McArthur JC Hoke A FK506 is neuroprotective in a model of antiretroviral toxic neuropathy.Ann Neurol. 2003; 53: 57-64Crossref PubMed Scopus (73) Google Scholar, 21Kaymaz M Emmez H Bukan N Dursun A Kurt G Paçsaoğlu H Paçsaoğlu A Effectiveness of FK506 on lipid peroxidation in the spinal cord following experimental traumatic injury.Spinal Cord. 2005; 43: 22-26Crossref PubMed Scopus (18) Google Scholar although it shows no antiviral effect. We explored, using transgenic mouse and cultured cell models that express the HCV core protein, whether tacrolimus improves metabolic disturbances including lipid and glucose homeostases as well as oxidative stress augmentation through a possible involvement of mitochondrial function. The production of HCV core gene transgenic mice has been described previously.6Moriya K Yotsuyanagi H Shintani Y Fujie H Ishibashi K Matsuura Y Miyamura T Koike K Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (576) Google Scholar Mice were cared for according to institutional guidelines with the approval by the institutional review board of the animal care committee, fed an ordinary chow diet (Oriental Yeast Co., Ltd., Tokyo, Japan), and maintained in a specific pathogen-free state. Because there is a sex preference in the development of liver lesions in the transgenic mice, we used only male mice. At least five mice were used in each experiment, and the data were subjected to statistical analysis. HepG2 cell lines expressing the HCV core protein under the control of the CAG promoter (Hep39J, Hep396, and Hep397) or a control HepG2 line (Hepswx) carrying the empty vector were described previously.22Ruggieri A Murdolo M Harada T Miyamura T Rapicetta M Cell cycle perturbation in a human hepatoblastoma cell line constitutively expressing hepatitis C virus core protein.Arch Virol. 2004; 149: 61-74Crossref PubMed Scopus (17) Google Scholar, 23Aizaki H Harada T Otsuka M Seki N Matsuda M Li YW Kawakami H Matsuura Y Miyamura T Suzuki T Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame.Hepatology. 2002; 36: 1431-1438Crossref PubMed Scopus (27) Google Scholar Bulk HepG2 cells were also used as a control. Cholesterol esters and lipid standards were purchased from Sigma-Aldrich (St. Louis, MO), and glycogen and amyloglucosidase were obtained from Seikagaku Kogyo (Tokyo, Japan). Other chemicals were of analytical grade and were purchased from Wako Chemicals (Tokyo, Japan). Tacrolimus (FK506) was kindly provided by Astellas Pharma Inc. (Tokyo, Japan). Cyclosporine A (CyA) was purchased from Sigma-Aldrich. Tacrolimus (0.1 mg/kg b.wt., suspended in mannitol and hydroxychlorinated caster oil [HCO-60]), or vehicle only was administered to the core gene transgenic or control mice i.p., three times per week for 3 months beginning at 3 months of age. For in vitro experiments, tacrolimus was added to the culture medium at the final concentration of 0 nmol/L, 10 nmol/L, 100 nmol/L, or 1 μmol/L. CyA was also added to the culture medium at the same concentrations. Blood was drawn at different time points from the tail vein, and plasma glucose concentrations were measured using an automatic biochemical analyzer (DRI-CHEM 3000V, Fuji Film, Tokyo, Japan). The levels of serum insulin were determined by radioimmunoassay (Biotrak, Amersham Pharmacia Biotech, Piscataway, NJ) using rat insulin as a standard. For the determination of the fasting plasma glucose level, the mice were fasted for >16 hours before the study. An insulin tolerance test was performed as described previously.16Shintani Y Fujie H Miyoshi H Tsutsumi T Kimura S Moriya K Koike K Hepatitis C virus and diabetes: direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar Lipid extraction from the mouse liver tissues or cultured cells was performed as described previously.15Moriya K Todoroki T Tsutsumi T Yotsuyanagi H Tsutsumi T Ishibashi K Takayama T Makuuchi M Watanabe K Miyamura T Kimura S Koike K Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C: analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar, 24Todoroki T Imai K Matsumoto K Kano S Initial deactivation of Florisil adsorbent for column chromatographic separation of lipids.Analyst. 1983; 108: 1267-1269Crossref Google Scholar For the analysis of fatty acid compositions, the residue was methanolysed by the modified Morrison and Smith method with boron trifluoride as a catalyst.25Morrison WR Smith LM Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J Lipid Res. 1964; 5: 600-608Abstract Full Text PDF PubMed Google Scholar Fatty acid methyl esters were analyzed using a Shimadzu GC-7A gas chromatograph (Shimadzu Corp., Kyoto, Japan) equipped with a 30-m-long × 0.3-mm diameter support coated with ethylene glycol succinate.24Todoroki T Imai K Matsumoto K Kano S Initial deactivation of Florisil adsorbent for column chromatographic separation of lipids.Analyst. 1983; 108: 1267-1269Crossref Google Scholar Lipid peroxidation was estimated spectrophotometrically using thiobarbituric acid-reactive substances and is expressed in terms of malondialdehyde formed per milligram protein. Reduced glutathione and oxidized glutathione levels were measured as described previously.10Choi J Ou JH Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G847-G851Crossref PubMed Scopus (257) Google Scholar The total amount of glutathione was calculated by adding the amounts obtained for glutathione and oxidized glutathione. For the evaluation of DNA damage in cells, apurinic/apyrimidinic sites were determined using a DNA Damage Quantification Kit (Dojindo Molecular Technologies, Inc., Tokyo, Japan) following the manufacturer’s protocol. Cells were plated onto glass coverslips and examined for reactive oxygen species (ROS) production as a marker for oxidative stress. They were loaded for 2 hours with chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (Molecular Probes Inc., Eugene, OR) at a final concentration of 10 μmol/L.26Gelasco AK Raymond JR Indoxyl sulfate induces complex redox alterations in mesangial cells.Am J Physiol Renal Physiol. 2006; 290: F1551-F1558Crossref PubMed Scopus (98) Google Scholar Results were expressed as relative fluorescence intensity and normalized to the control cells. In some experiments, ROS was measured after the incubation with tacrolimus or CyA. For the determination of ketone body ratio (KBR), cells were cultured to confluence on a 3.5-cm dish, and the medium was replaced with 700 μl of fresh medium. For arterial KBR, the mice were fasted for >16 hours, followed by the drawing of arterial blood. After a 24-hour incubation, acetoacetate and β-hydroxybutyrate in the medium were measured by monitoring the production or consumption of NADH with a Ketorex kit (Sanwa Chemical, Nagoya, Japan).27Williamson DH Mellanby J Krebs HA Enzymic determination of d(−)-β-hydroxybutyric acid and acetoacetic acid in blood.Biochem J. 1962; 82: 90-96Crossref PubMed Scopus (1040) Google Scholar The KBR was calculated as the acetoacetate/β-hydroxybutyrate ratio. An Affymetrix GeneChip analysis cDNA array system (Mouse Genome 430A 2.0, Kurabo, Osaka, Japan) was used for the analysis. Two thousand species of mouse DNA fragments were spotted on the filter. Genes that were 1.5-fold increased or decreased in both of the two tacrolimus-treated mice compared with mice treated with vehicle were defined as up-regulated or down-regulated, respectively. RNA was prepared from mouse liver tissues using TRIzol LS (Invitrogen, Carlsbad, CA). The first-strand cDNAs were synthesized with a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Franklin Lakes, NJ). The fluorescent signal was measured with an ABI Prism 7000 system (Applied Biosystems, Tokyo, Japan). The genes encoding mouse tumor necrosis factor (TNF)-α, sterol regulatory element binding protein (SREBP)-1c, resistin, stearoyl-CoA desaturase (SCD)-1, and hypoxanthine phosphoribosyltransferase were amplified with the primer pairs 5′-GACAAGGTGGGCTACGGGCTTG-3′ and 5′-TCCCAAATGGGCTCCCTCT-3′, 5′-ACGGAGCCATGGATTGCACATTTG-3′ and 5′-TACATCTTTAAAGCAGCGGGTGCCGATGGT-3′, 5′-GAAGGCACAGCAGTCTTGA-3′ and 5′-GCGACCTGCAGCTTACAG-3′, 5′-TTCCCTCCTGCAAGCTCTAC-3′ and 5′-CGCAAGAAGGTGCTAACGAAC-3′, and 5′-CCAGCAAGCTTGCAACCTTAACCA-3′ and 5′-GTAATGATCAGTCAACGGGGGAC-3′, respectively. The sense and antisense primers were located in different exons to avoid false-positive amplification from contaminated genomic DNA. Each PCR product was confirmed as a single band of the correct size by agarose gel electrophoresis (data not shown). A plasmid encoding firefly luciferase under the control of the SREBP-1c promoter (pGL3-srebp-1cPro) and a control plasmid encoding Renilla luciferase (Promega, Madison, WI) were transfected into 293T cells. Tacrolimus was added at a final concentration of 100 nmol/L to the culture medium of 293T cells transfected with pGL3-srebp-1cPro with or without an expression plasmid of HCV core protein at 24 hours after transfection. Cells were harvested 24 hours after treatment. Luciferase activity was measured by using the dual-luciferase reporter assay system (Promega). Firefly luciferase activity was standardized with that of Renilla luciferase, and the results are expressed as the fold-increase in relative luciferase units. Data are presented as the mean ± SE. The significance of the difference in means was determined by a Mann-Whitney U test wherever appropriate. P < 0.05 was considered significant. The core gene transgenic mice exhibit insulin resistance in the absence of obesity from the age of 2 months.16Shintani Y Fujie H Miyoshi H Tsutsumi T Kimura S Moriya K Koike K Hepatitis C virus and diabetes: direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar In tacrolimus-treated mice, there was a slight, but not significant, reduction in body weight compared with control mice at the end of tacrolimus administration at 6 months of age (Figure 1A). Tacrolimus administration to the core gene transgenic mice restored the plasma glucose levels to within normal limit (Figure 1B) (P < 0.05), whereas it caused no significant reduction in the control mice. The plasma glucose levels in the vehicle-treated core gene transgenic mice were higher than those in the core gene transgenic mice reported previously,16Shintani Y Fujie H Miyoshi H Tsutsumi T Kimura S Moriya K Koike K Hepatitis C virus and diabetes: direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar probably owing to the older age of mice in the current study than in the previous one. The levels of serum insulin were also significantly reduced by the treatment with tacrolimus for 3 months in the core gene transgenic mice, whereas there was no significant change in the control mice (Figure 1C). The reduction in both plasma glucose and serum insulin levels indicates that the administration of tacrolimus restored the resistance to insulin action, which is attributed to the suppression of insulin action in the liver by the core protein.16Shintani Y Fujie H Miyoshi H Tsutsumi T Kimura S Moriya K Koike K Hepatitis C virus and diabetes: direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar Actually, an insulin tolerance test (1 U/kg b.wt.) demonstrated the improvement of insulin action in the tacrolimus-treated core gene transgenic mice (Figure 1D). We then studied whether tacrolimus administration affects lipid metabolism in the mice. The core gene transgenic mice developed a marked hepatic steatosis.6Moriya K Yotsuyanagi H Shintani Y Fujie H Ishibashi K Matsuura Y Miyamura T Koike K Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (576) Google Scholar, 14Moriya K Fujie H Shintani Y Yotsuyanagi H Tsutsumi T Matsuura Y Kimura S Miyamura T Koike K The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1067Crossref PubMed Scopus (1097) Google Scholar In addition, the composition of accumulated lipid was different from that in the fatty liver as a result of simple overnutrition: carbon 18 or 16 monounsaturated fatty acid levels were significantly increased.15Moriya K Todoroki T Tsutsumi T Yotsuyanagi H Tsutsumi T Ishibashi K Takayama T Makuuchi M Watanabe K Miyamura T Kimura S Koike K Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C: analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar As shown in Figure 1E, the tacrolimus treatment significantly reduced the lipid content in liver tissues compared with the vehicle treatment of the core gene transgenic mice (P < 0.05, n = 5 each), whereas there was no change in the control mice. The increased ratios of oleic to stearic acid [18:1 (n-9)/18:0] and palmitoleic to palmitic acid [16:1(n-9)/16:0] in the core gene transgenic mice returned to levels similar to those in control mice (Figure 1, F and G) (P < 0.05). Thus, the administration of tacrolimus for 3 months restored the abnormalities in lipid metabolism that were induced by the core protein of HCV. Histologically, tacrolimus significantly improved steatosis in the liver of core gene transgenic mice, in which micro- and macrovesicular lipid droplets were accumulated in hepatocytes, chiefly around the central veins of the liver (Figure 2A). There was no sign of inflammation in the liver with or without the tacrolimus treatment. To further prove the ameliorating effect of tacrolimus on lipid metabolism, we then performed experiments using HepG2 cells that express the core protein.22Ruggieri A Murdolo M Harada T Miyamura T Rapicetta M Cell cycle perturbation in a human hepatoblastoma cell line constitutively expressing hepatitis C virus core protein.Arch Virol. 2004; 149: 61-74Crossref PubMed Scopus (17) Google Scholar, 23Aizaki H Harada T Otsuka M Seki N Matsuda M Li YW Kawakami H Matsuura Y Miyamura T Suzuki T Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame.Hepatology. 2002; 36: 1431-1438Crossref PubMed Scopus (27) Google Scholar HepG2 cells, the lipid metabolism of which is somewhat different from that in normal hepatocytes,28Choi Y Park Y Pariza MW Ntambi JM Regulation of stearoyl-CoA desaturase activity by the trans-10,cis-12 isomer of conjugated linoleic acid in HepG2 cells.Biochem Biophys Res Commun. 2001; 284: 689-693Crossref PubMed Scopus (101) Google Scholar show a significant increase in the level of 5,8,11-eicosatrienoic acid [20:3 (n-9)], as a result of activations of the fatty acid enzymes, Δ9-, Δ6-, and Δ5-desaturases, by the core protein (H. Miyoshi and K. Koike, unpublished data). Incubation of the core-expressing HepG2 cells with tacrolimus at 100 nmol/L and 1 μmol/L for 48 hours significantly reduced the accumulation of 20:3(n-9), whereas CyA treatment increased the level of 20:3(n-9) in a dose-dependent manner in the core-expressing HepG2 cells (Figure 3, A and B). Neither tacrolimus nor CyA changed the 20:3(n-9) content in HepG2 cells that do not express the core protein. Because the usual dose of tacrolimus for liver transplantation naturally induces an immunosuppressed state in patients, we conducted a mouse study with a tacrolimus dose lower than that in the aforementioned study. In this low-dose experiment, tacrolimus at 0.02 mg/kg b.wt. (one-fifth of the previous one) was administered to mice for 1 month from the age of 3 months. Similar to the results with the dose of 0.1 mg/kg b.wt., there were significant decreases in the lipid content in the liver (9.5 ± 0.8 [0.02 mg/kg b.wt. tacrolimus] versus 18.7 ± 4.4 [vehicle only] mg/g liver; P < 0.05) and serum insulin concentration (96.6 ± 16.9 [0.02 mg/kg b.wt. tacrolimus] versus 1137.1 ± 88.0 [vehicle only] pmol/L; P < 0.05) in the core gene transgenic mice treated with tacrolimus. Histological changes are shown in Figure 2B. We next examined whether the 3-month administration of tacrolimus affects the redox state in the core gene transgenic mice. In the liver of the core gene transgenic mice, the ROS level was higher than that in the liver of control mice as determined by lipid peroxidation.10Choi J Ou JH Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G847-G851Crossref PubMed Scopus (257) Google Scholar Treatment with tacrolimus significantly reduced the level of thiobarbituric acid-reactive substances in the liver of the core gene transgenic mice (Figure 4A) (P < 0.05). As a result of oxidative stress overproduction, there was damage in the DNA of hepatocytes of the core gene transgenic mice from a young age.10Choi J Ou JH Mechanisms of liver injury. III. Oxidative st
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