Deficiency of Interleukin-1 Receptor Antagonist Deteriorates Fatty Liver and Cholesterol Metabolism in Hypercholesterolemic Mice
2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m412220200
ISSN1083-351X
AutoresKikuo Isoda, Shojiro Sawada, Makoto Ayaori, Taizo Matsuki, Reiko Horai, Yutaka Kagata, Kôji Miyazaki, Masatoshi Kusuhara, Mitsuyo Okazaki, Osamu Matsubara, Yoichiro Iwakura, Fumitaka Ohsuzu,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoAlthough the anti-inflammatory effect of interleukin-1 (IL-1) receptor antagonist (IL-1Ra) has been described, the contribution of this cytokine to cholesterol metabolism remains unclear. Our aim was to ascertain whether deficiency of IL-1Ra deteriorates cholesterol metabolism upon consumption of an atherogenic diet. IL-1Ra-deficient mice (IL-1Ra-/-) showed severe fatty liver and portal fibrosis containing many inflammatory cells following 20 weeks of an atherogenic diet when compared with wild type (WT) mice. Expectedly, the levels of total cholesterol in IL-1Ra-/- mice were significantly increased, and the start of lipid accumulation in liver was observed earlier when compared with WT mice. Real-time PCR analysis revealed that IL-1Ra-/- mice failed to induce mRNA expression of cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, with concurrent up-regulation of small heterodimer partner 1 mRNA expression. Indeed, IL-1Ra-/- mice showed markedly decreased bile acid excretion, which is elevated in WT mice to maintain cholesterol level under atherogenic diet feeding. Therefore, we conclude that the lack of IL-1Ra deteriorates cholesterol homeostasis under atherogenic diet-induced inflammation. Although the anti-inflammatory effect of interleukin-1 (IL-1) receptor antagonist (IL-1Ra) has been described, the contribution of this cytokine to cholesterol metabolism remains unclear. Our aim was to ascertain whether deficiency of IL-1Ra deteriorates cholesterol metabolism upon consumption of an atherogenic diet. IL-1Ra-deficient mice (IL-1Ra-/-) showed severe fatty liver and portal fibrosis containing many inflammatory cells following 20 weeks of an atherogenic diet when compared with wild type (WT) mice. Expectedly, the levels of total cholesterol in IL-1Ra-/- mice were significantly increased, and the start of lipid accumulation in liver was observed earlier when compared with WT mice. Real-time PCR analysis revealed that IL-1Ra-/- mice failed to induce mRNA expression of cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, with concurrent up-regulation of small heterodimer partner 1 mRNA expression. Indeed, IL-1Ra-/- mice showed markedly decreased bile acid excretion, which is elevated in WT mice to maintain cholesterol level under atherogenic diet feeding. Therefore, we conclude that the lack of IL-1Ra deteriorates cholesterol homeostasis under atherogenic diet-induced inflammation. When compared with other organs, the liver has one of the largest populations of macrophages, which are key components of the innate immune system. Resident hepatic macrophages, i.e. Kupffer cells, are derived from circulating monocytes that arise from bone marrow progenitors. Once localized within the liver, these cells differentiate to perform specialized functions, including phagocytosis. Kupffer cells also generate various products, including cytokines. These factors regulate not only the phenotypes of the Kupffer cells that produce them but also the phenotypes of neighboring cells, such as hepatocytes (1Laskin D.L. Weinberger B. Laskin J.D. J. Leukocyte Biol. 2001; 70: 163-170Crossref PubMed Google Scholar). Many recent studies suggest that several proinflammatory cytokines produced by activated Kupffer cells might be involved in the onset of liver disease, including alcoholic and nonalcoholic fatty liver disease (NAFLD) 1The abbreviations used are: NAFLD, nonalcoholic fatty liver disease; ACAT2, Acyl-CoA cholesterol acyltransferase 2; ALT, alanine aminotransferase; Ct, threshold cycle number; CYP7A1, cholesterol 7α-hydroxylase; CYP27A1, sterol 27-hydroxylase; IL-1, interleukin-1; IL-1Ra, IL-1 receptor antagonist; IL-1Ra-/-, IL-1Ra-deficient; LRH-1, liver receptor homolog-1; SHP, small heterodimer partner 1; SR-BI, scavenger receptor class B type I; WT, wild type; HPLC, high pressure liquid chromatography; LDL, low density lipoprotein; HDL, high density lipoprotein; VLDL, very low density lipoprotein; HMG, human menopausal gonadotropin; JNK, c-Jun N-terminal kinase. (2Diehl A.M. Immunol. Rev. 2000; 174: 160-171Crossref PubMed Scopus (268) Google Scholar, 3Tilg H. Diehl A.M. N. Engl. J. Med. 2000; 343: 1467-1476Crossref PubMed Scopus (846) Google Scholar, 4Fan J.G. Xu Z.J. Wang G.L. Ding X.D. Tian L.Y. Zheng X.Y. Zhonghua Gan. Zang. Bing. Za. Zhi. 2003; 11: 73-76PubMed Google Scholar). For instance, elevated circulating levels of tumor necrosis factor-α, IL-1β, and IL-6 have been observed in human patients (5McClain C.J. Barve S. Deaciuc I. Kugelmas M. Hill D. Semin. Liver Dis. 1999; 19: 205-219Crossref PubMed Scopus (334) Google Scholar) and animal models of both NAFLD (4Fan J.G. Xu Z.J. Wang G.L. Ding X.D. Tian L.Y. Zheng X.Y. Zhonghua Gan. Zang. Bing. Za. Zhi. 2003; 11: 73-76PubMed Google Scholar) and alcohol-induced liver injury (6Thurman R.G Bradford B.U. Iimuro Y. Frankenberg M.V. Knecht K.T. Connor H.D. Adachi Y. Wall C. Arteel G.E. Raleigh J.A. Forman D.T. Mason R.P. Front. Biosci. 1999; 4: e42-e46Crossref PubMed Google Scholar). In contrast to proinflammatory cytokines, anti-inflammatory cytokines are considered to have hepato-protective effects (5McClain C.J. Barve S. Deaciuc I. Kugelmas M. Hill D. Semin. Liver Dis. 1999; 19: 205-219Crossref PubMed Scopus (334) Google Scholar). IL-1 receptor antagonist (IL-1Ra) is one of the negative regulators to IL-1 signaling by binding and blocking the functional receptor (IL-1 receptor type-I) without activation (7Dinarello C.A. Int. Rev. Immunol. 1998; 16: 457-499Crossref PubMed Scopus (679) Google Scholar). IL-1Ra plays an anti-inflammatory role in acute and chronic inflammation (8Dinarello C.A. N. Engl. J. Med. 2000; 343: 732-734Crossref PubMed Scopus (320) Google Scholar). IL-1Ra is also produced by hepatocytes as well as macrophages/monocytes as an acute phase protein in vivo (9Gabay C. Gigley J. Sipe J. Arend W.P. Fantuzzi G. Eur. J. Immunol. 2001; 31: 490-499Crossref PubMed Scopus (51) Google Scholar). Furthermore, we recently reported that IL-1Ra-deficient (IL-1Ra-/-) mice showed decreased weight gain when consuming the same amount of food as wild-type mice and that body lipid accumulation remained impaired even when they were fed a high fat diet (10Matsuki T. Horai R. Sudo K. Iwakura Y. J. Exp. Med. 2003; 198: 877-888Crossref PubMed Scopus (178) Google Scholar). However, the function of IL-1Ra in NAFLD is still not yet well understood. Atherogenic diet has been widely used to study atherogenesis in animal models. Early atherogenic diets contained high concentrations of cholesterol (5%) and fat (30%) either supplemented with cholic acid (2%) (11Thompson J.S. J. Atheroscler. Res. 1969; 10: 113-122Abstract Full Text PDF PubMed Scopus (35) Google Scholar) or fed in combination with irradiation treatments (12Vesselinovitch D. Wissler R.W. J. Atheroscler. Res. 1968; 8: 497-523Abstract Full Text PDF PubMed Scopus (32) Google Scholar). These early diets produced high mortality and were subsequently modified to reduce the concentrations of cholesterol (1.25%), fat (15%), and cholate (0.5%) (13Paigen B. Morrow A. Brandon C. Mitchell D. Holmes P. Atherosclerosis. 1985; 57: 65-73Abstract Full Text PDF PubMed Scopus (491) Google Scholar). Although this modified atherogenic diet produces an atherogenic lipoprotein profile and fatty streak lesions (14Paigen B. Mitchell D. Reue K. Morrow A. Lusis A.J. Leboeuf R.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3763-3767Crossref PubMed Scopus (229) Google Scholar), it also induces inflammatory gene expression in the liver (15Liao F. Andalibi A. deBeer F.C. Fogelman A.M. Lusis A.J. J. Clin. Investig. 1993; 91: 2572-2579Crossref PubMed Scopus (254) Google Scholar). Furthermore, it has been reported that a fat-enriched diet induced NAFLD in animal models (4Fan J.G. Xu Z.J. Wang G.L. Ding X.D. Tian L.Y. Zheng X.Y. Zhonghua Gan. Zang. Bing. Za. Zhi. 2003; 11: 73-76PubMed Google Scholar). It is now well recognized that inflammation or cytokines increase serum lipid levels (16Hardardottir I. Grunfeld C. Feingold K.R. Curr. Opin. Lipidol. 1994; 5: 207-215Crossref PubMed Scopus (268) Google Scholar, 17Khovidhunkit W. Kim MS. Memon R.A. Shigenaga J.K. Moser A.H. Feingold K.R. Grunfeld C. J. Lipid Res. 2004; 45: 1169-1196Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). This increase in serum lipid levels can be considered part of the acute phase response that results in marked changes in the levels of a large number of circulating protein primarily due to alterations in the liver (18Macliewicz A. Kushner I. Baumann H. Acute Phase Proteins, Molecular Biology, Biochemistry and Clinical Applications. CRC Press, Inc., Boca Raton, FL1993Google Scholar). However, the role of IL-1Ra on lipid metabolism remains poorly understood. To address the question more directly whether deficiency of IL-1Ra promotes development of NAFLD and changes lipid metabolism, we employed IL-1Ra-/- mice we had recently generated (19Horai R. Asano M. Sudo K. Kanuka H. Suzuki M. Nishihara M. Takahashi M. Iwakura Y. J. Exp. Med. 1998; 187: 1463-1475Crossref PubMed Scopus (527) Google Scholar). The present study aimed to definitively test the hypothesis that deficiency of IL-1Ra promotes NAFLD and alters lipid metabolism employing IL-1Ra-/- and wild type (WT) mice on atherogenic diet. Animals and Experimental Procedure—The creation of IL-1Ra-/- mice used in this study has been described previously (19Horai R. Asano M. Sudo K. Kanuka H. Suzuki M. Nishihara M. Takahashi M. Iwakura Y. J. Exp. Med. 1998; 187: 1463-1475Crossref PubMed Scopus (527) Google Scholar). In these mutant mice, the genes for all four isoforms of the IL-1Ra were disrupted. These mice were backcrossed to C57BL/6J strain mice for eight generations. IL-1Ra genotyping was performed by a polymerase chain reaction analysis of tail DNA as described previously (19Horai R. Asano M. Sudo K. Kanuka H. Suzuki M. Nishihara M. Takahashi M. Iwakura Y. J. Exp. Med. 1998; 187: 1463-1475Crossref PubMed Scopus (527) Google Scholar). We used only male mice to rule out gender differences. IL-1Ra-/- mice and corresponding WT mice, derived by intercrossing WT littermates for knock-out, were studied at different times. Mice were maintained on a 12-h light/12-h dark cycle and fed a normal rodent diet containing 4.6% crude fat with less than 0.02% cholesterol (Clea Japan, Inc., Tokyo, Japan). Eight-week-old mice were switched to a high fat/cholesterol and cholate diet containing 15% total fat (8% cocoa butter), 1.25% cholesterol, and 0.5% sodium cholate (Clea Japan), hereafter referred to as the atherogenic diet. IL-1Ra-/- and WT mice were characterized immediately before and after consumption of the atherogenic diet for 1, 4, 8, and 20 weeks. The studies were carried out according to the protocols approved by the National Defense Medical College Board for Studies in Experimental Animals. Chemical Analysis of Serum and Tissue—On the day of analysis, food was removed from the cages in the morning, and the mice were fasted for 7 h. Blood was drawn from mice by cardiac puncture under light methoxyflurane anesthesia. Animals were sacrificed by cervical dislocation, and livers were immediately collected and weighed, and tissue samples were divided; some were fixed in 4% paraformaldehyde, and others were frozen in liquid nitrogen and stored at -80 °C. Blood was transferred into tubes, and serum was collected by centrifugation. Serum alanine aminotransferase (ALT) determination was performed using a commercially available assay kit according to the manufacturer's instructions (Sigma diagnostic kit; Sigma). Plasma analysis of albumin and bilirubin levels was performed using a Paramax RX automated analyzer (Dade International). The plasma total cholesterol, HDL cholesterol, and triglyceride levels were measured by enzymatic assays as described previously (20Hedrick C.C. Castellani L.W. Warden C.H. Puppione D.L. Lusis A.J. J. Biol. Chem. 1993; 268: 20676-20682Abstract Full Text PDF PubMed Google Scholar). Furthermore, plasma lipoproteins were analyzed by an on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides by HPLC at Skylight Biotech Inc. (Akita, Japan) according to the procedure as described by Usui et al. (21Usui S. Hara Y. Hosaki S. Okazaki M. J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar). 200 μl of 20× saline-diluted sera was injected into two tandem connected TSK gel LipopropakXL columns (300 × 7.8-mm; Tosoh), and cholesterol and triglyceride contents in lipoproteins separated by size were determined by using enzymatic reagents specially prepared by Kyowa Medex (Tokyo, Japan). Total cholesterol and triglyceride concentrations (in mg/dL) were calculated by comparison with total area under the chromatographic curves of a calibration standard of known concentration (22Okazaki M. Usui S. Hosaki S. Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. Second Ed. American Association of Clinical Chemistry Press, Washington, D. C.2000: 647-669Google Scholar). Hepatic lipids were extracted according to the methods of Folch et al. (23Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The extract was dissolved in 2-propanol and subsequently analyzed for total cholesterol, free cholesterol, and triglycerides using a commercially available reagent (TC kit, FC kit, and TG kit, Wako, Japan). Analysis of Fecal Bile Acids—Stools were collected from each animal over 72 h immediately prior to study, dried, weighed, and ground in a mechanical blender. Aliquots were taken for the measurement of total bile acid content by an enzymatic method as described (24Porter J.L. Fordtran J.S. Santa Ana C.A. Emmett M. Hagey L.R. Macdonald E.A. Hofmann A.F. J. Lab. Clin. Med. 2003; 141: 411-418Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The daily stool output (g/day/100 g of body weight) and fecal bile acid content (μmol/g of stool) were used to calculate the rate of bile acid excretion (μmol/day/100 g of body weight). Tissue Preparation and Histology—Livers were harvested, fixed overnight in 4% paraformaldehyde, embedded in OCT compounds (Tissue-Tek; Sakura Finetechnical Co., Tokyo, Japan), and sectioned (10-μm thickness). All samples were routinely stained with hematoxylin-eosin, Masson's trichrome, and oil red O. Analysis of Gene Expression by Real-time Quantitative PCR—Total RNA was isolated from each mouse liver using Tri Reagent (Sigma) according to the manufacturer's instructions. Purified RNA was reverse-transcribed according to the protocols supplied by the manufacturer. Quantitative gene expression analysis was performed on an ABI PRISM 7700 machine (Applied Biosystems) using SYBR Green technology. PCR primers (Table I) were designed using Primer Express 1.7 software with the manufacturer's default settings (Applied Biosystems) and validated for identical efficiencies (slope = -3.3 for a plot of Ct versus a log of ng of cDNA). In 96-well optical plates, 12.5 μl of SYBR Green master mix was added to 12.5 μl of cDNA (corresponding to 50 ng of total RNA input) and 300 nm forward and reverse primers in water. Plates were heated for 2 min at 50 °C and 10 min at 95 °C. Subsequently, 40 PCR cycles consisting of 15 s at 95 °C and 60 s at 60 °C were applied. At the end of the run, samples were heated to 95 °C with a ramp time of 20 min to construct dissociation curves to check that single PCR products were obtained. The absence of genomic DNA contamination in the RNA preparations was confirmed by using total RNA samples that had not been reverse-transcribed. 18S was used as the standard housekeeping gene. The ratio of target gene and 18S expression levels (relative gene expression numbers) was calculated by subtracting the threshold cycle number (Ct) of the target gene from the Ct of 18S and raising 2 to the power of this difference. Ct values are defined as the number of PCR cycles at which the fluorescent signal during the PCR reaches a fixed threshold. Target gene mRNA expression is thus expressed relative to 18S expression.Table IPCR primersForwardReverseACAT25′-GCCCCAGATTCTACAAGCAAGA-3′5′-TGTGGTAGATGGTTCGGAAATG-3′HMG-CoA reductase5′-TTTCTAGAGCGAGTGCATTAGCA-3′5′-GATTGCCATTCCACGAGCTATAT-3′18S rRNA5′-AGTCGGAGGTTCGAAGACGAT-3′5′-GCGGGTCATGGGAATAACG-3′CYP7A15′-GCTGAGAGCTTGAAGCACAAGA-3′5′-TTGAGATGCCCAGAGGATCAC-3′LXRα5′-GAGTTCTCCAGAGCCATGAATGA-3′5′-CATATGTGTGTTGCAGCCTCTCTA-3′SREBP25′-GTTCTGAGGAAGGCCATTGATT-3′5′-CCACATCACTGTCCACCAGACT-3′LDL receptor5′-CCAGTGTGACCGTGAACATGA-3′5′-TCCCCACTGTGACACTTGAACT-3′SHP5′-AGCTGGGTCCCAAGGAGTATG-3′5′-ACCAGGGCTCCAAGACTTCA-3′FXR5′-ATCTCCGCCGAACGAAGAA-3′5′-GGACCACGAAGATCAGATTGC-3′CYP27A15′-GCCTTGCACAAGGAAGTGACT-3′5′-CGCAGGGTCTCCTTAATCACA-3′LRH-15′-ACTGAGAAATTCGGACAGCTACTTC-3′5′-AGGTAGTCTTCTGCCTGCTTGCT-3′ABCA15′-CCCAGAGCAAAAAGCGACTC-3′5′-GGTCATCATCACTTTGGTCCTTG-3′SR-B15′-TTCAGGGCGTCCAGAA-3′5′-GATCTTGCTGAGTCCGTTCCA-3′IL-1Ra5′-CTTTACCTTCATCCGCTCTGAGA-3′5′-TCTAGTGTTGTGCAGAGGAACCA-3′IL-1 β5′-TGGTGTGTGACGTTCCCATT-3′5′-CAGCACGAGGCTTTTTTGTTG-3′CD685′-ATAGCCCAAGGAACAGAGGAAGA-3′5′-GGATGTAGGTGTCATCGTGAAG-3′TGF β5′-ACAAGGCTGCCCCGACTAC-3′5′-GTTGACTTTCTCCTGGTATGAGATAGC-3′ Open table in a new tab Statistical Analysis—The results are shown as the mean ± S.E. except the values of serum ALT, bilirubin, and albumin, which were determined using plasma pooled from groups of five mice. Two groups were compared using Student's t test or Student-Newman Keuls's test with the one-way analysis of variance. A value of p < 0.05 was regarded as a significant difference. Liver Size and Serum Transaminase—Analysis of livers of IL-1Ra-/- mice fed a standard laboratory rodent chow indicated that these mice appeared identical to those of WT mice as determined by morphological and histological studies (data not shown). We next tested the effect of an atherogenic diet (chow supplemented with 15% fat, 1.25% cholesterol, and 0.5% sodium cholate). In contrast to little changes in WT mice, there were dramatic morphological changes in the livers of IL-1Ra-/- mice fed the same diet (Fig. 1). Following 20 weeks of atherogenic diet, there was a prominent color and size change in the livers of IL-1Ra-/- mice versus WT mice (Fig. 1a). Four weeks after the start of the atherogenic diet, the liver weight to body weight ratios in the IL-1Ra-/- mice tended to become larger (but not significant) than those of WT (8.6 ± 0.9%, n = 5 versus 6.0 ± 0.6%, n = 5; p = NS; Fig. 1b). Following 8 weeks of the atherogenic diet, this parameter increased by 166% in IL-1Ra-/- mice when compared with WT mice (15.7 ± 2.6%, n = 5 versus 5.9 ± 0.5%, n = 5; p < 0.05). Following 20 weeks of the atherogenic diet, the parameter increased ∼3-fold when compared with before atherogenic diet feeding in IL-1Ra-/- mice; however, the atherogenic diet caused only a small increase in liver weight to body weight ratios in WT mice (17.6 ± 2.4%, n = 5 versus 6.7 ± 0.6%, n = 5; p < 0.01). In conclusion, continued atherogenic diet caused significantly greater enlargement of livers in IL-1Ra-/- mice than in WT mice. As markers of liver injury, serum ALT, bilirubin, and albumin were monitored throughout the study. Before atherogenic diet, the serum level of ALT in both IL-1Ra-/- and WT mice was 40 units/liters. Following 20 weeks of atherogenic diet, the serum levels of ALT and bilirubin increased by 93 and 875%, respectively, in IL-1Ra-/- mice when compared with WT mice (Fig. 1c). In contrast, the serum level of albumin decreased by 28% when compared with WT mice (Fig. 1c). We detected moderate ascites in all IL-1Ra-/- mice but not in WT mice 20 weeks after the start of atherogenic diet. These data suggest that the atherogenic diet induces a more severe liver injury in IL-1Ra-/- mice than in WT mice. Histopathological Changes—Histological examination of livers from both mice demonstrated a time-dependent increase of the number and size of intracellular vacuoles, characteristics of lipid deposits (Fig. 2). These changes, however, were prominent in IL-1Ra-/- but not WT livers. Furthermore, following 20 weeks of the atherogenic diet, the morphology of the IL-1Ra-/- livers had been substantially altered, including extensive portal fibrosis and collagen deposition in a pericellular distribution in the lobule (Fig. 2b). Moreover, both lobular and portal inflammation was detected in IL-1Ra-/- liver (Fig. 2b). Oil red O staining of livers from both types of mice revealed that the deposition of neutral lipid increased in a time-dependent manner (Fig. 3). However, the start of lipid deposition in IL-1Ra-/- livers occurred earlier when compared with the WT livers. Indeed, we could detect lipid deposits in IL-1Ra-/- liver after 1 week of atherogenic diet, with no deposits in WT liver. Furthermore, as expected from the gross morphology, neutral lipid accumulation was markedly more pronounced in the IL-1Ra-/- than the WT liver following 20 weeks of atherogenic diet. Alteration of Lipid Homeostasis in IL-1Ra-/- Mice—Following 4 weeks of the atherogenic diet, levels of total plasma cholesterol did not differ between IL-1Ra-/- and WT mice (224.9 ± 12.4 mg/dl versus 207.1 ± 20.5 mg/dl; n = 5: p = NS; Fig. 4a). However, after 8 weeks of the atherogenic diet, the levels of total cholesterol in IL-1Ra-/- mice increased by 198% when compared with WT mice (610 ± 224 mg/dl, versus 205 ± 12 mg/dl; n = 5; p < 0.05). After 20 weeks of atherogenic diet, moreover, the total cholesterol levels increased significantly in IL-1Ra-/- mice when compared with WT mice (942 ± 160 mg/dl versus 240 ± 13 mg/dl, n = 5; p < 0.01). High resolution HPLC analysis of plasma lipoproteins following 4 weeks of the atherogenic diet revealed no significant differences in the levels of chylomicron-, VLDL-, LDL-, and HDL-cholesterol between the IL-1Ra-/- and WT mice (Fig. 4, b and c). However, after 20 weeks of atherogenic diet, HPLC analysis revealed markedly increased cholesterol levels in the VLDL and LDL fractions in the IL-1Ra-/- mice when compared with the WT mice. The cholesterol levels in the VLDL and LDL fractions were 699 ± 126 mg/dl (n = 5, p < 0.01) and 192 ± 36 mg/dl (n = 5, p < 0.01), respectively in IL-1Ra-/- mice when compared with 136 ± 7 mg/dl (n = 5) and 50 ± 4 mg/dl (n = 5), respectively, in the WT mice. In contrast, levels of HDL-cholesterol were significantly lower in IL-1Ra-/- mice (31 ± 10 mg/dl, n = 5) when compared with those in WT mice (54 ± 3 mg/dl, n = 5; p < 0.05; Fig. 4, b and c). The hepatic cholesterol levels of both mice showed a time-dependent increase (Fig. 5). However, these changes were more prominent in the liver of IL-1Ra-/- than in WT mice, and the difference between these mice increased with prolonged diet. In agreement with the enlarged liver and distinct color change, hepatic cholesterol was significantly increased in IL-1Ra-/- mice after 20 weeks of the atherogenic diet regimen. No significant differences were observed for hepatic triglyceride levels between the IL-1Ra-/- and WT mice. The major component of hepatic cholesterol in both genotypes was cholesterol ester. Alteration of Bile Acids and Cholesterol Metabolism in IL-1Ra-/- Mice—The primary route of cholesterol elimination from the body is via bile, which is based on conversion of hepatic cholesterol to bile acids. Fecal bile acid excretion of WT mice increased with the duration of atherogenic diet; however, in IL-1Ra-/- mice, bile acid excretion was increasingly impaired under continued atherogenic diet (Fig. 6). Alteration of mRNA Expressions in IL-1Ra-/- Mice—Analyses of mRNA expression of genes involved in cytokine, monocyte activation, and the regulation of cholesterol, fatty acid, and bile acid metabolism were performed to identify pathways potentially responsible for the observed alterations in IL-1Ra-/- mice. Real-time PCR employed mRNA extracted from livers of both mice (Fig. 7). Prior to the start of the atherogenic diet (0 week), expression levels of cholesterol 7α-hydroxylase (CYP7A1) in IL-1Ra-/- mice were 30% lower than those in WT mice (p < 0.05; Fig. 7a). After 1 and 4 weeks of atherogenic diet, CYP7A1 expression decreased in both mice; however, transcript levels in IL-1Ra-/- mice were markedly lower (or absent). These results suggest that bile acid biosynthesis in IL-1Ra-/- mice might be attenuated as early as 1 week after the start of the atherogenic diet regimen. Although a previous study showed that lipopolysaccharide suppresses both CYP7A1 and sterol 27-hydroxylase (CYP27A1) (25Memon R.A. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Biol. Chem. 2001; 276: 30118-30126Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), expression levels of CYP27A1 in both IL-1Ra-/- and WT mice decreased after 1 week of the atherogenic diet, and there was no significant difference between these two groups (Fig. 7c). Although atherogenic diet increased small heterodimer partner 1 (SHP) mRNA in both mice, expression was 1.8-fold higher (p < 0.01) after 1 week and 3-fold higher (p < 0.001) after 4 weeks of the atherogenic diet in IL-1Ra-/- when compared with WT mice (Fig. 7b). Furthermore, expression of liver receptor homolog-1 (LRH-1) increased in both mice after 4 weeks of atherogenic diet; however, a 1.8-fold higher (p < 0.05) increase was observed in IL-1Ra-/- mice when compared with WT mice (Fig. 7c). Moreover, after 1 week of the atherogenic diet, mRNA expression of Acyl-CoA cholesterol acyltransferase 2 (ACAT2) was only marginally if at all increased in the IL-1Ra-/- mice, whereas a 2-fold increased level was observed after 4 weeks of the atherogenic diet (p < 0.05;Fig. 7a). In contrast, no differences were observed in hepatic expression of LDL receptor, HMG-CoA reductase, SREBP2, nuclear liver X-receptor α, and farnesoid X-receptor between IL-1Ra-/- and WT mice. Furthermore, hepatic expression of the HDL-cholesterol regulatory genes ATP binding cassette-A1 (ABCA1) and scavenger receptor class B type I (SR-BI) did not differ between IL-1Ra-/- and WT mice. IL-1Ra, as expected, was absent in IL-1Ra-/- mice, whereas the expression level of IL-1β in IL-1Ra-/- mice was significantly elevated even before atherogenic diet (p < 0.05) and more than 10-fold (p < 0.001) up-regulated following 4 weeks of the atherogenic diet (Fig. 7d). Moreover, 4 weeks of atherogenic diet increased transforming growth factor-β (4.5-fold, p < 0.01) and CD68 (7-fold, p < 0.01) in the IL-1Ra-/- mice when compared with WT mice. The present study demonstrates that IL-1Ra-/- mice develop severe NAFLD and portal fibrosis with many inflammatory cells following 20 weeks of the atherogenic diet when compared with WT mice. We also found that mRNA levels of IL-1β and transforming growth factor-β were significantly elevated in livers of IL-1Ra-/- mice. These findings are in accord with several previous studies implicating cytokine imbalances in murine models of NAFLD (4Fan J.G. Xu Z.J. Wang G.L. Ding X.D. Tian L.Y. Zheng X.Y. Zhonghua Gan. Zang. Bing. Za. Zhi. 2003; 11: 73-76PubMed Google Scholar) and fibrosis (26Baroni G.S. D'Ambrosio L. Curto P. Casini A. Mancini R. Jezequel A.M. Benedetti A. Hepatology. 1996; 23: 1189-1199Crossref PubMed Google Scholar, 27Boros D.L. Whitfield J.R. Infect. Immun. 1999; 67: 1187-1193Crossref PubMed Google Scholar). These findings suggest that cytokines, such as IL-1β, may play an important role in the pathogenesis of NAFLD and hepatic fibrosis. In our IL-1Ra-/- mice, excessive IL-1 signaling may induce inflammation in the liver and, thus, the IL-1 system might play a role in the development of NAFLD and fibrosis. The atherogenic diet we used in this study appears associated with these dramatic changes in the livers of IL-1Ra-/- mice, corroborating a recent study showing that high cholesterol levels induce expression of several genes involved in acute inflammation and that cholate induces expression of genes involved in extracellular matrix deposition in hepatic fibrosis (28Vergnes L. Phan J. Strauss M. Tafuri S. Reue K. J. Biol. Chem. 2003; 278: 42774-42784Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Expectedly, the levels of total cholesterol in IL-1Ra-/- mice were significantly higher, and the start of lipid deposition in livers of IL-1Ra-/- mice was observed earlier than in WT mice. These results suggest that deficiency of IL-1Ra yields abnormal lipid metabolism upon feeding on atherogenic diet. The primary route of cholesterol elimination from the body is via bile, based on both direct canalicular excretion of biliary cholesterol as well as conversion of hepatic cholesterol to bile acids (29Vlahcevic Z.R. Stravitz R.T. Heuman D.M. Hylemon P.B. Pandak W.M. Gastroenterology. 1997; 113: 1949-1957Abstract Full Text PDF PubMed Scopus (90) Google Scholar). In our study, WT but not IL-1Ra-/- mice demonstrated increased bile acid excretion to maintain physiological cholesterol levels, suggesting that impairment of bile acid excretion yields significant cholesterol accumulation in response to atherogenic diet in IL-1Ra-/- mice. In our study, a 2-fold increase in mRNA level of ACAT2 was observed in the IL-1Ra-/- mice when compared with WT mice after 4 weeks. ACAT2 is thought to function in intestinal cholesterol absorption and transport into chylomicrons as well as in providing cholesteryl esters for VLDL assembly in the liver. Indeed, ACAT2-deficient mice have reduced cholesterol absorption and are resistant to diet-induced hypercholesterolemia by a high fat, high cholesterol diet (30Buhman K.F. Accad M. Novak S. Choi R.S. Wong J.S. Homilton R.L. Turley S. Farese R.V. Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (297) Google Scholar). Thus, the increased mRNA level of ACAT2 in IL-1Ra-/- mice might contribut
Referência(s)