The lipid droplet-associated protein perilipin 3 facilitates hepatitis C virus-driven hepatic steatosis
2016; Elsevier BV; Volume: 58; Issue: 2 Linguagem: Inglês
10.1194/jlr.m073734
ISSN1539-7262
AutoresDaniel Ferguson, Jun Zhang, Matthew A. Davis, Robert N. Helsley, Lise‐Lotte Vedin, Richard Lee, Rosanne M. Crooke, Mark J. Graham, Daniela Allende, Paolo Parini, J. Mark Brown,
Tópico(s)Hepatitis C virus research
ResumoHepatitis C virus (HCV) is an enveloped RNA virus responsible for 170 million cases of viral hepatitis worldwide. Over 50% of chronically infected HCV patients develop hepatic steatosis, and steatosis can be induced by expression of HCV core protein (core) alone. Additionally, core must associate with cytoplasmic lipid droplets (LDs) for steatosis development and viral particle assembly. Due to the importance of the LD as a key component of hepatic lipid storage and as a platform for HCV particle assembly, it seems this dynamic subcellular organelle is a gatekeeper in the pathogenesis of viral hepatitis. Here, we hypothesized that core requires the host LD scaffold protein, perilipin (PLIN)3, to induce hepatic steatosis. To test our hypothesis in vivo, we have studied core-induced hepatic steatosis in the absence or presence of antisense oligonucleotide-mediated knockdown of PLIN3. PLIN3 knockdown blunted HCV core-induced steatosis in transgenic mice fed either chow or a moderate fat diet. Collectively, our studies demonstrate that the LD scaffold protein, PLIN3, is essential for HCV core-induced hepatic steatosis and provide new insights into the pathogenesis of HCV. Hepatitis C virus (HCV) is an enveloped RNA virus responsible for 170 million cases of viral hepatitis worldwide. Over 50% of chronically infected HCV patients develop hepatic steatosis, and steatosis can be induced by expression of HCV core protein (core) alone. Additionally, core must associate with cytoplasmic lipid droplets (LDs) for steatosis development and viral particle assembly. Due to the importance of the LD as a key component of hepatic lipid storage and as a platform for HCV particle assembly, it seems this dynamic subcellular organelle is a gatekeeper in the pathogenesis of viral hepatitis. Here, we hypothesized that core requires the host LD scaffold protein, perilipin (PLIN)3, to induce hepatic steatosis. To test our hypothesis in vivo, we have studied core-induced hepatic steatosis in the absence or presence of antisense oligonucleotide-mediated knockdown of PLIN3. PLIN3 knockdown blunted HCV core-induced steatosis in transgenic mice fed either chow or a moderate fat diet. Collectively, our studies demonstrate that the LD scaffold protein, PLIN3, is essential for HCV core-induced hepatic steatosis and provide new insights into the pathogenesis of HCV. The hepatitis C virus (HCV) affects 3% of the world's population and is a leading cause of end stage liver failure, presenting a considerable global healthcare burden (1.Mohd Hanafiah K. Groeger J. Flaxman A.D. Wiersma S.T. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence.Hepatology. 2013; 57: 1333-1342Crossref PubMed Scopus (1965) Google Scholar). Liver tissue is the primary target for HCV infections, where the virus elegantly co-opts the hepatic lipid metabolic processes to promote viral assembly (2.Syed G.H. Amako Y. Siddiqui A. Hepatitis C virus hijacks host lipid metabolism.Trends Endocrinol. Metab. 2010; 21: 33-40Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3.Herker E. Ott M. Unique ties between hepatitis C virus replication and intracellular lipids.Trends Endocrinol. Metab. 2011; 22: 241-248Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Steatosis is a hallmark of HCV infection, occurring in 50–70% of chronically infected individuals (4.Lonardo A. Adinolfi L.E. Restivo L. Ballestri S. Romagnoli D. Baldelli E. Nascimbeni F. Loria P. Pathogenesis and significance of hepatitis C virus steatosis: an update on survival strategy of a successful pathogen.World J. Gastroenterol. 2014; 20: 7089-7103Crossref PubMed Scopus (73) Google Scholar). Importantly, there is an inverse correlation between steatosis and response to antiviral treatment and hepatic steatosis increases the risk for steatohepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) (4.Lonardo A. Adinolfi L.E. Restivo L. Ballestri S. Romagnoli D. Baldelli E. Nascimbeni F. Loria P. Pathogenesis and significance of hepatitis C virus steatosis: an update on survival strategy of a successful pathogen.World J. Gastroenterol. 2014; 20: 7089-7103Crossref PubMed Scopus (73) Google Scholar). Hepatic steatosis is characterized by a pathological accumulation of liver lipids resulting in increases in the size and number of lipid droplets (LDs). Although it is well-appreciated that HCV infection promotes hepatic steatosis, the exact mechanism is incompletely understood. The HCV core protein (core) is a viral structural protein that serves to form a capsid around the viral RNA genome and has been shown to induce steatosis in various models (5.Moriya 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 (580) Google Scholar, 6.Roingeard P. Hourioux C. Hepatitis C virus core protein, lipid droplets and steatosis.J. Viral Hepat. 2008; 15: 157-164Crossref PubMed Scopus (57) Google Scholar). Core is the first viral protein translated and undergoes two proteolytic processing events at the endoplasmic reticulum membrane (7.Pène V. Hernandez C. Vauloup-Fellous C. Garaud-Aunis J. Rosenberg A.R. Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment.J. Viral Hepat. 2009; 16: 705-715Crossref PubMed Scopus (36) Google Scholar). Once core undergoes the second cleavage by signal peptide peptidase, it is able to associate with host cytoplasmic LDs via a C-terminal binding domain composed of two amphipathic helices separated by a hydrophobic loop (8.Boulant S. Structural determinants that target the hepatitis C virus core protein to lipid droplets.J. Biol. Chem. 2006; 281: 22236-22247Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 9.McLauchlan J. Lemberg M.K. Hope G. Martoglio B. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets.EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (394) Google Scholar). The processing events that allow core to associate with LDs have been shown to be essential for steatosis formation (10.Harris C. Herker E. Farese R.V. Ott M. Hepatitis C virus core protein decreases lipid droplet turnover: a mechanism for core-induced steatosis.J. Biol. Chem. 2011; 286: 42615-42625Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Once core is associated with the LD, newly synthesized viral RNA is trafficked, by HCV nonstructural protein NS5A, to core where core forms a protein capsule (nucleocapsid) around the HCV genome (11.Bartenschlager R. Penin F. Lohmann V. André P. Assembly of infectious hepatitis C virus particles.Trends Microbiol. 2011; 19: 95-103Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Importantly, nucleocapsid formation by core occurs at the LD, which serves as the primary site of HCV viral particle assembly. The importance of the core-LD interaction is highlighted by independent studies demonstrating that LDs are required for HCV production and that disruption of core from the LD inhibits virus production (12.Boulant S. Targett-Adams P. McLauchlan J. Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.J. Gen. Virol. 2007; 88: 2204-2213Crossref PubMed Scopus (209) Google Scholar, 13.Miyanari Y. The lipid droplet is an important organelle for hepatitis C virus production.Nat. Cell Biol. 2007; 9: 1089-1097Crossref PubMed Scopus (976) Google Scholar). There is now unequivocal evidence that the ability of core to interact with host LDs is critical for both viral particle assembly as well as associated hepatic steatosis (10.Harris C. Herker E. Farese R.V. Ott M. Hepatitis C virus core protein decreases lipid droplet turnover: a mechanism for core-induced steatosis.J. Biol. Chem. 2011; 286: 42615-42625Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12.Boulant S. Targett-Adams P. McLauchlan J. Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.J. Gen. Virol. 2007; 88: 2204-2213Crossref PubMed Scopus (209) Google Scholar, 13.Miyanari Y. The lipid droplet is an important organelle for hepatitis C virus production.Nat. Cell Biol. 2007; 9: 1089-1097Crossref PubMed Scopus (976) Google Scholar). Host liver LDs, which are essential for HCV particle production, are dynamic organelles storing neutral lipids within a hydrophobic core. The neutral lipid core, composed mainly of TGs and cholesterol esters, is surrounded by a phospholipid monolayer that is decorated by a unique proteome (14.Yang L. Ding Y. Chen Y. Zhang S. Huo C. Wang Y. Yu J. Zhang P. Na H. Zhang H. et al.The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans.J. Lipid Res. 2012; 53: 1245-1253Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). LDs expand and contract based on cellular metabolic demand. When fatty acids are in excess, they can be incorporated into TGs by enzymes, such as diacylglycerol acyl transferase (DGAT), which catalyze the terminal step in TG synthesis (15.Yen C-L. E. Stone S.J. Koliwad S. Harris C. Farese R.V. DGAT enzymes and triacylglycerol biosynthesis.J. Lipid Res. 2008; 49: 2283-2301Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). When the cellular energy state is diminished, lipolytic enzymes can hydrolyze TGs from within the LD to release free fatty acids for β-oxidation. One of the primary lipolytic enzymes for TG hydrolysis is adipose TG lipase (ATGL), along with its cofactor, comparative gene identification 58 (CGI-58), which cooperate to release fatty acids from the hydrophobic TG-rich LD core (16.Zimmermann R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1513) Google Scholar, 17.Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 18.Schweiger M. Schreiber R. Haemmerle G. Lass A. Fledelius C. Jacobsen P. Tornqvist H. Zechner R. Zimmermann R. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism.J. Biol. Chem. 2006; 281: 40236-40241Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). Importantly, enzymes such as the ATGL-CGI-58 complex must gain access to the neutral lipid core of the LD in order to release fatty acids. Notably, certain LD proteins, specifically the perilipin (PLIN), adipocyte differentiation-related protein (ADRP), and tail-interacting protein at 47 kDa (TIP47) (PAT) family proteins, can act as scaffolding proteins and help regulate LD size by serving as gatekeepers that regulate access to the neutral lipid core and allow for the assembly of lipogenic/lipolytic enzymes (19.Bickel P.E. Tansey J.T. Welte M.A. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.Biochim. Biophys. Acta. 2009; 1791: 419-440Crossref PubMed Scopus (521) Google Scholar). In liver, the two main LD-associated scaffolding proteins are PLIN3, also known as TIP47, and PLIN2, also known as ADRP (19.Bickel P.E. Tansey J.T. Welte M.A. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.Biochim. Biophys. Acta. 2009; 1791: 419-440Crossref PubMed Scopus (521) Google Scholar, 20.Bell M. Wang H. Chen H. McLenithan J.C. Gong D-W. Yang R-Z. Yu D. Fried S.K. Quon M.J. Londos C. et al.Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance.Diabetes. 2008; 57: 2037-2045Crossref PubMed Scopus (150) Google Scholar). PLIN3 is a highly exchangeable protein involved in multiple processes, including lipid storage, lipid mobilization, and LD biogenesis (21.Skinner J.R. Shew T.M. Schwartz D.M. Tzekov A. Lepus C.M. Abumrad N.A. Wolins N.E. Diacylglycerol enrichment of endoplasmic reticulum or lipid droplets recruits perilipin 3/TIP47 during lipid storage and mobilization.J. Biol. Chem. 2009; 284: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22.Bulankina A.V. Deggerich A. Wenzel D. Mutenda K. Wittmann J.G. Rudolph M.G. Burger K.N.J. Höning S. TIP47 functions in the biogenesis of lipid droplets.J. Cell Biol. 2009; 185: 641-655Crossref PubMed Scopus (191) Google Scholar). PLIN2 is a nonexchangeable protein that is degraded if displaced from the LD (23.Masuda Y. Itabe H. Odaki M. Hama K. Fujimoto Y. Mori M. Sasabe N. Aoki J. Arai H. Takano T. ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells.J. Lipid Res. 2006; 47: 87-98Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Expression levels of PLIN2 and/or PLIN3 on the LD surface can vary in response to changing cellular metabolic needs to alter TG storage levels in the cell (24.Listenberger L.L. Ostermeyer-Fay A.G. Goldberg E.B. Brown W.J. Brown D.A. Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover.J. Lipid Res. 2007; 48: 2751-2761Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 25.Goodman J.M. Demonstrated and inferred metabolism associated with cytosolic lipid droplets.J. Lipid Res. 2009; 50: 2148-2156Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 26.Carr R.M. Patel R.T. Rao V. Dhir R. Graham M.J. Crooke R.M. Ahima R.S. Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2012; 302: R996-R1003Crossref PubMed Scopus (53) Google Scholar, 27.Chang B.H.J. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol. Cell. Biol. 2006; 26: 1063-1076Crossref PubMed Scopus (256) Google Scholar) by regulating accessibility of enzymes involved in lipid synthesis or lipolysis. Interestingly, previous evidence has shown that expression of core alters the normal composition of LD scaffolding proteins. Core expression in hepatoma cells resulted in increased PLIN3 expression and decreased PLIN2 expression (28.Sato S. Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.J. Biochem. 2006; 139: 921-930Crossref PubMed Scopus (142) Google Scholar). Furthermore, core expression has been shown to cause a redistribution of LDs from the cell periphery to the perinuclear region, which is associated with the displacement of PLIN2 from redistributed LDs (29.Boulant S. Douglas M.W. Moody L. Budkowska A. Targett-Adams P. McLauchlan J. Hepatitis C virus core protein induces lipid droplet redistribution in a microtubule- and dynein-dependent manner.Traffic. 2008; 9: 1268-1282Crossref PubMed Scopus (178) Google Scholar). The observation that core expression alters important LD scaffolding protein expression and changes the LD proteome may highlight a mechanism involved in core-induced steatosis, which is the focus of the current study. Previous studies have shown that core's association with the LD is an indispensable part of HCV replication and persistence, making it an attractive target for discovery of new targets to inhibit HCV infections (12.Boulant S. Targett-Adams P. McLauchlan J. Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.J. Gen. Virol. 2007; 88: 2204-2213Crossref PubMed Scopus (209) Google Scholar, 13.Miyanari Y. The lipid droplet is an important organelle for hepatitis C virus production.Nat. Cell Biol. 2007; 9: 1089-1097Crossref PubMed Scopus (976) Google Scholar, 30.Filipe A. McLauchlan J. Hepatitis C virus and lipid droplets: finding a niche.Trends Mol. Med. 2015; 21: 34-42Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Although the core-LD association is a critical part of the HCV life cycle, it is still unclear as to how the viral protein interacts with the LD resulting in a pathological accumulation of lipids in the liver. In order to understand the mechanisms involved in HCV-mediated pathogenesis, we have focused our studies on core-induced hepatic steatosis in vivo, given that hepatoma cell models have dramatically altered lipid metabolic processes when compared with primary hepatocytes. Core serves as a critical component in the viral life cycle of HCV and by defining how core interacts with the LD, we should be able to identify innovative targets to disrupt core's association with the LD and impair HCV infections. Given that core expression has been shown to increase expression of the LD scaffolding protein, PLIN3 (28.Sato S. Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.J. Biochem. 2006; 139: 921-930Crossref PubMed Scopus (142) Google Scholar), we hypothesized that PLIN3 is an important component for core to induce steatosis. To test our hypothesis, we have generated a hepatocyte-specific HCV core transgenic mouse model and studied core-induced steatosis with normal or diminished levels of PLIN3. Although HCV genotype 3a is most closely related to hepatic steatosis risk in people, we chose to use genotype 1b to allow comparisons to the vast majority of cell biology work with HCV core. The full-length coding sequence of HCV core (genotype 1b) was subcloned from the pCAGC191 vector (31.Suzuki R. Tamura K. Li J. Ishii K. Matsuura Y. Miyamura T. Suzuki T. Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus.Virology. 2001; 280: 301-309Crossref PubMed Scopus (54) Google Scholar) into the MluI- and ClaI-digested pLiv11 vector (32.Cheng D. MacArthur P.S. Rong S. Parks J.S. Shelness G.S. Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.J. Lipid Res. 2010; 51: 849-855Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). The pLiv11 vector contains the human apoE promoter along with a 3′-hepatic control region, which selectively drives transgene expression in hepatocytes (32.Cheng D. MacArthur P.S. Rong S. Parks J.S. Shelness G.S. Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.J. Lipid Res. 2010; 51: 849-855Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 33.Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice.J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar). The pLiv11-HCVcore transgenic cassette was separated from the vector backbone by digestion with restriction enzymes NotI (5′) and SpeI (3′). This linearized vector was then microinjected into fertilized embryos of B6D2 F1J mice. PCR was used to confirm the presence of the HCV core gene, apoE promoter, and the hepatic control region (data not shown) in founder mice. Genotyping was performed by PCR analysis on genomic DNA isolated from ear snips, as previously described (34.Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (302) Google Scholar), using primers specific for the core construct as follows: primer 1, 5′-GAG CAC AAA TCC TAA ACC CCA AAG-3′, primer 2, 5′-GAT GGT CAA ACA GGA CAG CAG AG-3′. From this effort, we established three independent founder strains with low (HCVcoreTg29), medium (HCVcoreTg15), or high (HCVcoreTg3) core protein expression. At the age of 6–8 weeks, subgroups of mice were fed ad libitum with either chow or a moderate fat diet (MFD) containing 20% of energy as lard and added cholesterol, 0.1% (w/w), for a total of 6 or 8 weeks. Mice treated with antisense oligonucleotide (ASO) received 50 mg/kg of either PLIN3 ASO or control (nontargeting) ASO via intraperitoneal injection once a week (total dose of 50 mg/kg per week) for 6–8 weeks. Second-generation ASOs were synthesized by Ionis Pharmaceuticals (Carlsbad, CA) and formulated in PBS (35.Crooke R.M. Graham M.J. Lemonidis K.M. Whipple C.P. Koo S. Perera R.J. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis.J. Lipid Res. 2005; 46: 872-884Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The PLIN3 ASO, Ionis 409003 (5′-CACAGTGTTGTCTAGGGCCT-3′), is a second-generation oligonucleotide that incorporates several chemical modifications to improve potency, duration of action, and tolerability. All of the internucleotide phosphates are chemically modified with a phosphorothioate substitution, in which one of the nonbridging oxygen atoms is substituted with sulfur. Additionally the compound incorporates five 2′-O-(2-methoxyethyl)-modified ribonucleosides at the 3′ and 5′ ends with ten 2′-O-deoxyribonucleosides in between to support RNaseH-1-mediated target mRNA degradation. These modifications improve the binding affinity for target mRNA as well as stability against exonuclease-mediated degradation. A control oligonucleotide, Ionis 141923 (5′-CCTTCCCTGAAGGTTCCTCC-3′), contains the same chemical modifications, with no complementarity to known genes, including LD proteins (26.Carr R.M. Patel R.T. Rao V. Dhir R. Graham M.J. Crooke R.M. Ahima R.S. Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2012; 302: R996-R1003Crossref PubMed Scopus (53) Google Scholar). For liver X receptor (LXR) agonist and fasting studies, 6-week-old C57BL/6 mice were fed ad libitum with chow for a period of 6 weeks while receiving treatment with either control or PLIN3 ASOs. For studies with LXR agonist, T0901317 was suspended in a vehicle containing 1.0% carboxymethylcellulose and 0.1% Tween 80. For a period of seven days mice were gavaged once daily with either vehicle or 25 mg/kg T0901317, as previously described (36.Warrier M. Shih D.M. Burrows A.C. Ferguson D. Gromovsky A.D. Brown A.L. Marshall S. McDaniel A. Schugar R.C. Wang Z. et al.The TMAO-generating enzyme flavin monooxygenase 3 is a central regulator of cholesterol balance..Cell Rep. January 14, 2015; doi:10.1016/j.celrep.2014.12.036Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37.Temel R.E. Sawyer J.K. Yu L. Lord C. Degirolamo C. McDaniel A. Marshall S. Wang N. Shah R. Rudel L.L. et al.Biliary sterol secretion is not required for macrophage reverse.Cell Metab. 2010; 12: 96-102Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). For fasting studies, subgroups of mice were fasted for 18 h prior to necropsy. All mice used in the studies were housed in a pathogen-free barrier facility at Wake Forest University School of Medicine or the Cleveland Clinic with the approval of the American Association for Accreditation of Laboratory Animal Care. The Institutional Animal Care and Use Committee from Wake Forest University or the Cleveland Clinic approved all protocols before execution of the studies. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore), and detected after incubation with the indicated antibodies using LiCor Odyssey Infrared Imaging system. The antibodies used include: anti-protein disulfide isomerase rabbit polyclonal (Cell Signaling #2446), anti- GAPDH rabbit monoclonal (Cell Signaling #5174), anti-α tubulin mouse monoclonal (Cell Signaling #3873), and anti-PLIN3 rabbit polyclonal (Proteintech #10694-1-AP). The monoclonal HCV core antigen antibody (C7-50) was obtained from Thermo Scientific. Densitometry was determined using Image Studio version 4.0.21. Tissue RNA extraction and quantitative (q)PCR were conducted as previously described (38.Brown J.M. Bell T.A. Alger H.M. Sawyer J.K. Smith T.L. Kelley K. Shah R. Wilson M.D. Davis M.A. Lee R.G. et al.Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal neutral sterol loss.J. Biol. Chem. 2008; 283: 10522-10534Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39.Temel R.E. Lee R.G. Kelley K.L. Davis M.A. Shah R. Sawyer J.K. Wilson M.D. Rudel L.L. Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2.J. Lipid Res. 2005; 46: 2423-2431Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Cyclophilin A was used for an invariant control and expression levels were calculated based on the ΔΔCT method. qPCR was conducted using the Applied Biosystems 7500 real-time PCR system. All qPCR primers are available upon request. Portions of livers were fixed in 10% buffered formalin and processed for hematoxylin and eosin (H&E) staining by the Clinical Pathology Laboratory in the Department of Pathology at Wake Forest University School of Medicine or the Imaging Core in the Lerner Research Institute at the Cleveland Clinic Foundation. Liver lipid extracts were made and TG, total cholesterol, free cholesterol, and cholesterol ester were measured using detergent-solubilized enzymatic assays as previously described (34.Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (302) Google Scholar, 38.Brown J.M. Bell T.A. Alger H.M. Sawyer J.K. Smith T.L. Kelley K. Shah R. Wilson M.D. Davis M.A. Lee R.G. et al.Targeted depletion of hepatic ACAT2-driven cholesterol esterification reveals a non-biliary route for fecal neutral sterol loss.J. Biol. Chem. 2008; 283: 10522-10534Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 40.Carr T.P. Andresen C.J. Rudel L.L. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (483) Google Scholar). Plasma TG levels were quantified enzymatically (L-Type TG M; Wako Diagnostics, Richmond, VA). Plasma lipoproteins were fractionated by size from 2.5 μl of individual plasma samples using a Superose 6 PC 3.2/30 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) followed by on-line determination of TGs and cholesterol, as previously described (41.Parini P. Johansson L. Broijersen A. Angelin B. Rudling M. Lipoprotein profiles in plasma and interstitial fluid analyzed with an automated gel-filtration system.Eur. J. Clin. Invest. 2006; 36: 98-104Crossref PubMed Scopus (105) Google Scholar). The lipid concentrations of the different lipoprotein fractions were calculated after integration of individual chromatograms. Hepatic LDs were isolated by sucrose gradient centrifugation essentially as described in (42.Harris L-A.L.S. Shew T.M. Skinner J.R. Wolins N.E. A single centrifugation method for isolating fat droplets from cells and tissues.J. Lipid Res. 2012; 53: 1021-1025Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Approximately 100 mg of tissue was minced with a razor blade on a cold surface. Minced tissue was transferred to a Potter-Elvehjem homogenizer, and then 200 μl of 60% sucrose was added to the tissue sample and incubated on ice for 10 min. Next, 800 μl of lysis buffer was added and mixed, and then incubated on ice for 10 min. Samples were homogenized with five strokes of a Teflon® pestle and transferred to a 2 ml centrifuge tube. Lysis buffer (600 μl) was carefully layered on top of homogenate and centrifuged for 2 h at 20,000 g at 4°C. The tube was then frozen at −80°C and cut at the 1,000 μl mark. The bottom piece of the centrifuge tube contained the non-LD fraction, which was allowed to thaw before being transferred to a new tube. The LD fraction was collected by cutting an ∼4–6 mm piece from the top of the ice cylinder and placing it in a new 2 ml tube. To increase the purity of the LD fraction, this process was repeated once more. Briefly, 200 μl of 60% sucrose was added to the LD fraction. Next, 800 μl of lysis buffer was added and mixed followed by careful layering with 600 μl of lysis buffer and then centrifugation for 2 h at 20,000 g at 4°C. After freezing at −80°C, the tube was cut and the LD fraction was collected by cutting an ∼4–6 mm piece from the top of the ice cylinder and placing it in a new tube. Protein analysis was performed using the modified Lowry assay, as previously described (43.Temel R.E. Parks J.S. Williams D.L. Enhancement of scavenger receptor class B type I-mediated selective cholesteryl ester uptake from apoA-I-/- high density lipoprotein (HDL) by apolipoprotein A-I requires HDL reorganization by lecithin cholesterol acyltransferase.J. Biol. Chem. 2003; 278: 4792-4799Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Lipids in the LD fraction were extracted according to the Folch method (44.Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipides from an
Referência(s)