Glycogen Synthase Kinase 3α Deficiency Attenuates Atherosclerosis and Hepatic Steatosis in High Fat Diet–Fed Low Density Lipoprotein Receptor–Deficient Mice
2014; Elsevier BV; Volume: 184; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2014.07.028
ISSN1525-2191
AutoresNicole Banko, Cameron S. McAlpine, Daniel E. Venegas-Pino, Preeya Raja, Yuanyuan Shi, Mohammad I. Khan, Geoff H. Werstuck,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoStudies have implicated signaling through glycogen synthase kinase (GSK) 3α/β in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr−/−;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat–diet fed Ldlr−/− mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr−/−;Gsk3a+/− mice, 80% smaller in Ldlr−/−;Gsk3a−/− mice; P < 0.05), compared with Ldlr−/−;Gsk3a+/+ controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1–derived macrophages that were pretreated with the GSK3α/β inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways. Studies have implicated signaling through glycogen synthase kinase (GSK) 3α/β in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr−/−;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat–diet fed Ldlr−/− mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr−/−;Gsk3a+/− mice, 80% smaller in Ldlr−/−;Gsk3a−/− mice; P < 0.05), compared with Ldlr−/−;Gsk3a+/+ controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1–derived macrophages that were pretreated with the GSK3α/β inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways. Atherosclerosis is a disease of the medium and large arteries that is characterized by inflammation and the accumulation of lipids in the arterial wall. Cardiovascular risk factors, including hyperglycemia, dyslipidemia, obesity, hypertension, and cigarette smoke, promote the accelerated development of atherosclerotic lesions.1Libby P. Ridker P.M. Hansson G.K. Progress and challenges in translating the biology of atherosclerosis.Nature. 2011; 473: 317-325Crossref PubMed Scopus (2645) Google Scholar Atherosclerosis typically originates in regions of the artery that contain bifurcations, branches, or inner curvatures, which tend to disrupt linear blood flow.2Schwartz C.J. Mitchell J.R. Observations on localization of arterial plaques.Circ Res. 1962; 11: 63-73PubMed Google Scholar Damaged endothelial cells express cell surface proteins, such as vascular cell adhesion molecule-1 and P-selectin, which mediate the attachment and infiltration of monocytes.3Cybulsky M.I. Iiyama K. Li H. Zhu S. Chen M. Iiyama M. Davis V. Gutierrez-Ramos J.C. Connelly P.W. Milstone D.S. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.J Clin Invest. 2001; 107: 1255-1262Crossref PubMed Scopus (970) Google Scholar Intimal monocytes differentiate into macrophages that endocytose low-density lipoprotein (LDL) and modified LDL particles that have entered the intima. This leads to the development of lipid-engorged macrophages, called foam cells, which form fatty streaks in the artery wall. Macrophage foam cells amplify the inflammatory response to vascular injury by secreting growth factors and cytokines.3Cybulsky M.I. Iiyama K. Li H. Zhu S. Chen M. Iiyama M. Davis V. Gutierrez-Ramos J.C. Connelly P.W. Milstone D.S. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.J Clin Invest. 2001; 107: 1255-1262Crossref PubMed Scopus (970) Google Scholar Advanced lesions are characterized by the cytokine-induced migration of vascular smooth muscle cells from the tunica media into the intima. Foam cells ultimately undergo apoptosis, leading to the production of a growing acellular region of the lesion, known as the necrotic core. Necrosis is believed to be a key feature of unstable plaques that are prone to rupture.4Seimon T. Tabas I. Mechanisms and consequences of macrophage apoptosis in atherosclerosis.J Lipid Res. 2009; 50: S382-S387Crossref PubMed Scopus (297) Google Scholar Lesion rupture triggers thrombosis, which can occlude the artery and cause myocardial infarction or stroke. Although great advances have been made in our understanding of the cellular processes involved in disease progression, the underlying molecular mechanisms that link these risk factors to pro-atherogenic processes are not clearly understood. During the past decade, it has become evident that the development and progression of atherosclerosis is associated with impaired protein processing in the endoplasmic reticulum (ER) of vascular cells, a condition known as ER stress.5Feng B. Yao P.M. Li Y. Devlin C.M. Zhang D. Harding H.P. Sweeney M. Rong J.X. Kuriakose G. Fisher E.A. Marks A.R. Ron D. Tabas I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages.Nat Cell Biol. 2003; 5: 781-792Crossref PubMed Scopus (712) Google Scholar, 6Zhou J. Lhoták S. Hilditch B.A. Austin R.C. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice.Circulation. 2005; 111: 1814-1821Crossref PubMed Scopus (251) Google Scholar Cardiovascular risk factors, including hyperglycemia, dyslipidemia, obesity, and cigarette smoke, have been associated with elevated levels of ER stress and/or activation of the unfolded protein response (UPR) to ER stress.7Werstuck G.H. Khan M.I. Femia G. Kim A.J. Tedesco V. Trigatti B. Shi Y. Glucosamine-induced endoplasmic reticulum stress is associated with accelerated atherosclerosis in a hyperglycemic mouse model.Diabetes. 2006; 55: 93-101Crossref PubMed Scopus (113) Google Scholar, 8Li Y. Schwabe R.F. DeVries-Seimon T. Yao P.M. Gerbod-Giannone M.C. Tall A.R. Davis R.J. Flavell R. Brenner D.A. Tabas I. Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis.J Biol Chem. 2005; 280: 21763-21772Crossref PubMed Scopus (360) Google Scholar, 9Özcan U. Cao Q. Yilmaz E. Lee A.-H. Iwakoshi N.N. Özdelen E. Tuncman G. Görgun C. Glimcher L.H. Hotamisligil G.S. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science. 2004; 306: 457-461Crossref PubMed Scopus (2972) Google Scholar, 10Somborac-Bacura A. van der Toorn M. Franciosi L. Slebos D.J. Zanic-Grubisic T. Bischoff R. van Oosterhout A.J. Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells.Exp Physiol. 2013; 98: 316-325Crossref PubMed Scopus (66) Google Scholar Elevated levels of UPR proteins, including glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein homologous protein (CHOP), are detected in atherosclerosis-prone regions of the endothelium before the detection of intimal macrophages/foam cells.11Khan M.I. Pichna B. Shi Y. Bowes A.J. Werstuck G.H. Endoplasmic reticulum stress plays a causal role in the development of atherosclerosis in a hyperglycemic mouse model.Antioxid Redox Signal. 2009; 11: 2289-2298Crossref PubMed Scopus (55) Google Scholar, 12Civelek M. Manduchi E. Riley R.J. Stoeckert Jr., C.J. Davies P.F. Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis.Circ Res. 2009; 105: 453-461Crossref PubMed Scopus (172) Google Scholar ER stress and UPR activation are also observed in macrophages/foam cells at all stages of atherosclerotic lesion development. ER stress–inducing agents promote the expression of inflammatory cytokines, the accumulation of lipids, and the initiation of macrophage/foam cell apoptosis.5Feng B. Yao P.M. Li Y. Devlin C.M. Zhang D. Harding H.P. Sweeney M. Rong J.X. Kuriakose G. Fisher E.A. Marks A.R. Ron D. Tabas I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages.Nat Cell Biol. 2003; 5: 781-792Crossref PubMed Scopus (712) Google Scholar, 6Zhou J. Lhoták S. Hilditch B.A. Austin R.C. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice.Circulation. 2005; 111: 1814-1821Crossref PubMed Scopus (251) Google Scholar, 8Li Y. Schwabe R.F. DeVries-Seimon T. Yao P.M. Gerbod-Giannone M.C. Tall A.R. Davis R.J. Flavell R. Brenner D.A. Tabas I. Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis.J Biol Chem. 2005; 280: 21763-21772Crossref PubMed Scopus (360) Google Scholar Little is known about the molecular mechanisms that may link ER stress and/or UPR induction to the development of atherosclerosis. We have previously identified a potential role for glycogen synthase kinase (GSK)-3α and/or β in downstream ER stress signaling.13Kim A.J. Shi Y.Y. Austin R.C. Werstuck G.H. Valproate protects cells from endoplasmic reticulum stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase 3.J Cell Sci. 2005; 118: 89-99Crossref PubMed Scopus (234) Google Scholar Conditions of ER stress can enhance GSK3α/β activity,14Song L. De Sarno P. Jope R.S. Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation.J Biol Chem. 2002; 277: 44701-44708Crossref PubMed Scopus (252) Google Scholar and dietary supplementation with valproate, a small molecule with GSK3α/β inhibitory properties, can attenuate accelerated atherogenesis in hyperglycemic, hyperhomocysteinemic, and high-fat–diet (HFD) fed Apoe−/− mice.15Werstuck G.H. Kim A.J. Brenstrum T. Ohnmacht S.A. Panna E. Capretta A. Examining the correlations between GSK-3 inhibitory properties and anticonvulsant efficacy of valproate and valproate-derived compounds.Bioorg Med Chem Lett. 2004; 14: 5465-5467Crossref PubMed Scopus (46) Google Scholar, 16Bowes A.J. Khan M.I. Shi Y.Y. Robertson L. Werstuck G.H. Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.Am J Pathol. 2009; 174: 330-342Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar However, the interpretation of these studies is limited by the lack of specificity of valproate as a GSK3α/β inhibitor, and the inability to distinguish between the respective roles of the α and β forms of GSK3. The kinase domains of GSK3α/β are 98% homologous, and there is a broad overlap in substrate specificity17Woodgett J.R. Molecular cloning and expression of glycogen synthase kinase-3/factor A.EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1149) Google Scholar; however, GSK3α and β are not functionally equivalent, and it is now clear that they have unique and distinct functions, in addition to some overlapping, redundant functions.18Soutar M.P. Kim W.Y. Williamson R. Peggie M. Hastie C.J. McLauchlan H. Snider W.D. Gordon-Weeks P.R. Sutherland C. Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain.J Neurochem. 2010; 115: 974-983Crossref PubMed Scopus (94) Google Scholar, 19Markou T. Cullingford T.E. Giraldo A. Weiss S.C. Alsafi A. Fuller S.J. Clerk A. Sugden P.H. Glycogen synthase kinases 3alpha and 3beta in cardiac myocytes: regulation and consequences of their inhibition.Cell Signal. 2008; 20: 206-218Crossref PubMed Scopus (48) Google Scholar Although GSK3β knockout mice are not viable,20Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation.Nature. 2000; 406: 86-90Crossref PubMed Scopus (1219) Google Scholar GSK3α knockout mice exist with little or no overt phenotype.21MacAulay K. Doble B.W. Patel S. Hansotia T. Sinclair E.M. Drucker D.J. Nagy A. Woodgett J.R. Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism.Cell Metab. 2007; 6: 329-337Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar Herein, we crossed Gsk3α−/− mice with low-density lipoprotein receptor–deficient (Ldlr−/−) mice to directly and specifically examine the effects of GSK3α deficiency on HFD-induced atherosclerosis. We monitor effects on ER stress/UPR activation, hepatic lipids, and metabolic parameters, including plasma lipids and body weight. Finally, we examine the effects of GSK3α deficiency on cytokine expression in isolated peritoneal macrophages. All animal experiments were conducted with approval of the McMaster University (Hamilton, ON, Canada) Animal Research Ethics Board. Ldlr−/− mice (B6.129S7-Ldlrtm1Her/J) were obtained from Jackson Labs (Bar Harbor, ME), and Gsk3α−/− mice were a generous gift from Drs. Bradley Doble (McMaster University) and James Woodgett (University of Toronto).21MacAulay K. Doble B.W. Patel S. Hansotia T. Sinclair E.M. Drucker D.J. Nagy A. Woodgett J.R. Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism.Cell Metab. 2007; 6: 329-337Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 22Patel S. Macaulay K. Woodgett J.R. Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α) in glucose metabolism: effect of strain variation.PLoS One. 2011; 6: e15845Crossref PubMed Scopus (31) Google Scholar Both strains were established in a C57Bl6 genetic background. Ldlr−/−;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− littermates were identified by PCR genotyping (Supplemental Figure S1) and used in the described experiments. Five-week-old mice of each genotype were randomly placed on a defined chow diet (model TD92078; Harlan Teklad, Madison, WI) or a HFD containing 21% milk fat and 0.2% cholesterol (42% kcal from fat) (model TD97363; Harlan Teklad). Subsets of mice were given chow or HFD containing 625 mg/kg sodium valproate.23McAlpine C. Bowes A.J. Khan M.I. Shi Y.Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3β activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler Thromb Vasc Biol. 2012; 32: 82-91Crossref PubMed Scopus (45) Google Scholar All mice had unlimited access to both food and water during the study. Body weights were monitored, and fasted and nonfasted blood glucose and lipids were quantified as previously described.7Werstuck G.H. Khan M.I. Femia G. Kim A.J. Tedesco V. Trigatti B. Shi Y. Glucosamine-induced endoplasmic reticulum stress is associated with accelerated atherosclerosis in a hyperglycemic mouse model.Diabetes. 2006; 55: 93-101Crossref PubMed Scopus (113) Google Scholar Lipid levels of nonfasted mice were measured using a colorimetric diagnostic kit for both total cholesterol and total triglycerides (Thermo Scientific, Middletown, VA). Plasma was fractionated using fast-performance liquid chromatography (FPLC) with the FRAC-950 FPLC (GE Life Sciences, Piscataway, NJ), and cholesterol concentration was measured using the infinity cholesterol reagent (Thermo Scientific). Plasma IL-10 was quantified by ELISA (eBioscience Inc., San Diego, CA). All mice were anesthetized (isoflurane) and harvested at 15 weeks of age. The vasculature was flushed with 0.9% saline and perfusion fixed with 10% neutral buffer formalin. Hearts, aortas, and liver were collected for further analysis. Serial sections (4 μm thick) of the aortic root were stained with hematoxylin and eosin (H&E). Images were collected using a Leitz LABORLUX S microscope (Leica Microsystems, Inc., Concord, ON, Canada) connected to a DP71 camera (Olympus Imaging, Center Valley, PA), and lesion area was quantified using ImageJ software version 1.43m (NIH, Bethesda, MD).24Venegas-Pino D.E. Banko N. Khan M.I. Shi Y. Werstuck G.H. Quantitative analysis and characterization of atherosclerotic lesions in mice.J Vis Exp. 2013; 82: 50933PubMed Google Scholar Serial sections were immunostained using the Vectastain avidin-biotin complex system and primary antibodies against GRP78/94 (anti-KDEL; SPA-827; Enzo Life Sciences, Inc., Farmingdale, NY) or CHOP (anti-GADD153; sc-575; Santa Cruz Biotechnology, Santa Cruz, CA), and visualized with the appropriate biotinylated secondary antibody and Nova Red.25Hager L. Li L. Pun H. Liu L. Hossain M.A. Maguire G.F. Naples M. Baker C. Magomedova L. Tam J. Adeli K. Cummins C.L. Connelly P.W. Ng D.S. Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice.J Biol Chem. 2012; 287: 20755-20768Crossref PubMed Scopus (46) Google Scholar Non-specific staining was controlled for using a similar section and preimmune IgG. Neutral lipid accumulation was determined by Oil Red O staining of sections of frozen liver and aortic sinus. Staining with Sudan IV was used to examine atherosclerosis in en face–prepared aortas. All staining was quantified using ImageJ software and normalized to cross-sectional area. Total cholesterol and triglycerides were determined in liver lysates using colorimetric diagnostic kits (Thermo Scientific). Total protein lysates were prepared from frozen mouse liver solubilized in 4× SDS-PAGE sample buffer [0.5 mol/L Tris-HCl (pH 6.8), glycerol, and 10% SDS] and quantified by Bradford assay. Samples were diluted in SDS-PAGE loading buffer to a protein concentration of 4 μg/μL. Total protein liver lysates were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were stained with antibodies against GSK3α (number 07-389; Cell Signaling, Beverly, MA), GSK3β (number 610202; BD Transduction Laboratories, Mississauga, ON, Canada), or β-actin (number A3854; Sigma-Aldrich, Oakville, ON, Canada) as the loading control. Membranes were incubated for 1 hour with the appropriate horseradish peroxidase secondary antibody (Invitrogen Life Technologies, Inc., Burlington, ON, Canada) and developed using Immobilon Western chemiluminescent horseradish peroxidase substrate (EMD Millipore, Etobicoke, ON, Canada). Total protein lysates were prepared from livers or isolated macrophages from Ldlr−/−;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− mice. GSK3 activity was measured by monitoring the incorporation of 32P onto phospho-glycogen synthase peptide-2 (Upstate, Billerica, MA), as previously described.15Werstuck G.H. Kim A.J. Brenstrum T. Ohnmacht S.A. Panna E. Capretta A. Examining the correlations between GSK-3 inhibitory properties and anticonvulsant efficacy of valproate and valproate-derived compounds.Bioorg Med Chem Lett. 2004; 14: 5465-5467Crossref PubMed Scopus (46) Google Scholar Kinase activity was determined in total liver extracts and on immunoprecipitated GSK3α and GSK3β. Neutral lipid accumulation was determined by Oil Red O staining of sections of frozen liver and aorta. Cryosections (8 μm thick) were collected on precoated glass slides and fixed in 37% formaldehyde for 10 minutes. Sections were stained with a 0.5% w/v stock solution of Oil Red O dissolved in 100% isopropanol and diluted in double-distilled water to a 60% working solution. Nuclei were stained with Meyer's hematoxylin solution (MHS32; Sigma-Aldrich), and slides were mounted in Crystal-mount media. Lipid staining was quantified using ImageJ software and normalized to cross-sectional area. Thioglycolate-elicited peritoneal macrophages were isolated from HFD-fed 15-week-old Ldlr−/;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− mice and cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies) containing 10% fetal bovine serum. Thp-1 monocytes were differentiated into macrophages in 100 nmol/L phorbol myristate acetate (Sigma-Aldrich). Cells were treated with 2.5 to 4 μg/mL tunicamycin, 5 mmol/L glucosamine, or 600 μmol/L palmitic acid for 18 hours. Transcripts encoding ER stress markers and inflammatory cytokines were quantified by RT-PCR using primers described in Table 1.26Sage A.T. Walter L.A. Shi Y.Y. Khan M.I. Kaneto H. Capretta A. Werstuck G.H. Hexosamine biosynthesis pathway (HBP) flux promotes endoplasmic reticulum (ER) stress, lipid accumulation, and inflammatory gene expression in hepatic cells.Am J Physiol Endocrinol Metab. 2010; 298: E499-E511Crossref PubMed Scopus (62) Google ScholarTable 1PCR Primer SequencesGene nameForwardReverseMouse DNA LDLR5′-CCATATGCATCCCCAGTCTT-3′5′-AATCCATCTTGTTCAATGGCCCGATC-3′ Gsk3a5′-CCCCCACCAAGTGATTTCACTGCTA-3′5′-AACATGAAATCCGGGCTCCAACTCTAT-3′5′-AACATGAAATCCGGGCTCCAACTCTAT-3′Mouse RNA Il1b5′-CTGCTTCCAAACCTTTGACC-3′5′-AGCTTCTCCACAGCCACAAT-3′ Il65′-ATCCAGTTGCCTTCTTGGGACTGA-3′5′-TAAGCCTCCGACTTGTGAAGTGGT-3′ Il105′-CATGGGTCTTGGGAAGAGAA-3′5′-AACTGGCCACAGTTTTCAGG-3′ Tnf/TNF-α5′-AGCCCCCAGTCTGTATCCTT-3′5′-GAGGCAACCTGACCACTCTC-3′ Hspa5/GRP785′-ACCTGGGTGGGGAAGACTTT-3′5′-TCTTCAAATTTGGCCCGAGT-3′ Ddit3/CHOP5′-TATCTCATCCCCAGGAAACG-3′5′-CTGCTCCTTCTCCTTCATGC-3′ Actb/β-actin5′-GGCACCACACCTTCTACAATG-3′5′-TCTTCAAATTTGGCCCGAGT-3′Human RNA IL1B5′-GTCAGCTCTCTCCTTTCA-3′5′-AATGTGGCCGTGGTTTCT-3′ IL65′-CCACACAGACAGCCAACTCAC-3′5′-AGGTTGTTTTCTGCCAGTGC-3′ IL85′-CTGTGGAGTTTTGGCTGTTTT-3′5′-CTCCCAGTCTTGTCATTG-3′ IL105′-GCCTAACATGCTTCGAGATC-3′5′-TGATGTCTGGGTCTTGGTTC-3′ HSPA5/GRP785′-CGAGGAGGAGGACAAGAAGG-3′5′-CACCTTGAACGGCAAGAACT-3′ DDIT3/CHOP5′-TGCCTTTCTCTCGGACACT-3′5′-TGTGACCTCTGCTGGTTCTG-3′ ACTB/β-actin5′-ACCGAGCGCGGCTACAG-3′5′-CTTAATGTGACGCACGATTTCC-3′ Open table in a new tab Total RNA was extracted from peritoneal macrophages isolated from Ldlr−/−;Gsk3a+/+, Ldlr−/−;Gsk3a+/−, and Ldlr−/−;Gsk3a−/− mice using the RNeasy Mini Kit (catalog number 74,104; Qiagen, Inc., Toronto, ON, Canada). RNA concentration/purity was determined by measuring the absorbance at 260/280 nm. RT-PCR was performed using 2 μg of total RNA, which was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Invitrogen Life Technologies) following the manufacturer's guidelines. Amplification of PCR product was performed using the Mastercycler gradient thermocycler (Eppendorf, Mississauga, ON, Canada). RT-PCR was conducted using a GeneAmp 7300 sequence detection system (Applied Biosystems, Foster City, CA) and SYBR GreenER qPCR SuperMix (Invitrogen Life Technologies). All PCRs were performed in biological and technical triplicates to obtain gene expression profiles using primers for the selected inflammatory cytokines (Table 1). Results from RT-PCR were analyzed using Data Assist software version 3.0 (Invitrogen Life Technologies). All in vitro experiments are representative of at least three independent experiments. The numbers of mice in each experimental group are indicated. All data are expressed as means ± SD. An unpaired Student's t-test or one-way analysis of variance test was used, as appropriate, to determine statistical significance. P < 0.05 was considered statistically significant. We have previously shown that dietary supplementation with valproate (625 mg valproate/kg body weight), a compound with GSK3α/β inhibitory properties, can attenuate accelerated atherogenesis in hyperglycemic, hyperhomocysteinemic, and HFD-fed Apoe−/− mice.16Bowes A.J. Khan M.I. Shi Y.Y. Robertson L. Werstuck G.H. Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.Am J Pathol. 2009; 174: 330-342Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 23McAlpine C. Bowes A.J. Khan M.I. Shi Y.Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3β activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler Thromb Vasc Biol. 2012; 32: 82-91Crossref PubMed Scopus (45) Google Scholar To examine the anti-atherogenic potential of valproate in a different mouse strain, we supplemented the diets (standard chow or HFD) of 5-week-old female Ldlr−/− mice with 625 mg valproate/kg body weight. At 15 weeks of age, all mice were sacrificed and analyzed. Valproate supplementation had no significant effect on body weight, nonfasting blood glucose, or plasma lipid levels relative to mice fed nonsupplemented diets (Table 2). No significant atherosclerosis was detected at the aortic sinus or ascending aorta of chow-fed Ldlr−/− mice, in the presence or absence of valproate supplementation. As expected, the HFD-fed mice had significantly increased liver weight and plasma lipid levels relative to chow-fed mice; however, total body weight was not significantly different (Table 2). Mice fed a HFD developed large advanced atherosclerotic lesions in the aortic sinus and ascending aorta by 15 weeks of age (Figure 1). Mice fed a valproate-supplemented HFD had significantly smaller lesions (5.7 ± 3.8 mm3 versus 10.0 ± 3.2 mm3; P < 0.05) containing smaller necrotic cores (1.5 ± 1.1 mm3 versus 3.6 ± 1.5 mm3; P < 0.05) compared with mice fed a nonsupplemented HFD. These results indicate that valproate supplementation can attenuate atherogenesis in Ldlr−/− mice.Table 2Metabolic Parameters of Valproate-Supplemented Ldlr−/− MiceParametersChow dietHFDWithout valproateWith valproateWithout valproateWith valproateBody weight (g)20.20 ± 0.1021.60 ± 0.3021.40 ± 0.4621.96 ± 0.16Liver weight (g)0.84 ± 0.100.83 ± 0.031.32 ± 0.09∗P < 0.05 relative to age-matched chow-fed mice.1.10 ± 0.06∗P < 0.05 relative to age-matched chow-fed mice.Fat pad weight (g)0.35 ± 0.060.29 ± 0.030.52 ± 0.11∗P < 0.05 relative to age-matched chow-fed mice.0.47 ± 0.10∗P < 0.05 relative to age-matched chow-fed mice.NFBG (mmol/L)10.00 ± 1.708.40 ± 1.207.30 ± 2.207.90 ± 0.40Cholesterol (mmol/L)6.32 ± 0.674.42 ± 0.5115.87 ± 1.11∗P < 0.05 relative to age-matched chow-fed mice.17.67 ± 4.47∗P < 0.05 relative to age-matched chow-fed mice.Triglyceride (mmol/L)0.02 ± 0.250.01 ± 0.010.58 ± 0.22∗P < 0.05 relative to age-matched chow-fed mice.0.89 ± 0.28∗P < 0.05 relative to age-matched chow-fed mice.Lesion volume (mm3)0.11 ± 0.100.10 ± 0.0910.0 ± 3.2∗P < 0.05 relative to age-matched chow-fed mice.5.7 ± 3.8∗P < 0.05 relative to age-matched chow-fed mice.†P < 0.05 relative to age-matched HFD (valproate)-fed mice.Necrotic volume (mm3)003.6 ± 1.5∗P < 0.05 relative to age-matched chow-fed mice.1.5 ± 1.1∗P < 0.05 relative to age-matched chow-fed mice.†P < 0.05 relative to age-matched HFD (valproate)-fed mice.n = 5 to 10 per group.HFD, high fat diet; NFBG, nonfasting blood glucose.∗ P < 0.05 relative to age-matched chow-fed mice.† P < 0.05 relative to age-matched HFD (valproate)-fed mice. Open table in a new tab n = 5 to 10 per group. HFD, high fat diet; NFBG, nonfasting blood glucose. Evidence from our previous in vitro and in vivo investigations has suggested that valproate attenuates atherogenesis by inhibiting GSK3α and/or β.16Bowes A.J. Khan M.I. Shi Y.Y. Robertson L. Werstuck G.H. Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.Am J Pathol. 2009; 174: 330-342Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 23McAlpine C. Bowes A.J. Khan M.I. Shi Y.Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3β activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler Thromb Vasc Biol. 2012; 32: 82-91Crossref PubMed Scopus (45) Google Scholar However, valproate is a non-specific drug that can affect many other metabolic pathways in addition to GSK3α/β.27Williams R.S. Cheng L. Mudge A.W. Harwood A.J. A common mechanism of action for three mood-stabilizing drugs.Nature. 2002; 417: 292-295Crossref PubMed Scopus (547) Google Scholar To specifically examine the potential role of GSK3α in the progression and development of atherosclerosis, we crossed GSK3α-deficient mice with atherosclerosis-prone Ldlr−/− mice to generate Ldlr−/−;Gsk3a+/− and Ldlr−/−;Gsk3a−/− genotypes (Supplemental Figure S1). Ldlr−/−;Gsk3a+/− and Ldlr−/−;Gsk3a−/− mice were born at expected mendelian frequencies and do not display any overt developmental phenotype. Total hepatic GSK3α/β activity was assessed using an established kinase assay and found to be reduced by approximately 20% in GSK3α-deficient mice (Supplemental Figure S2). GSK3α-specific activity was reduced 50% and 100% in Gsk3a+/− and Gsk3a−/− mice, respectively. In isolated peritoneal macrophages, GSK3β activity was not significantly affected by GSK3α deficiency. GSK3α deficiency did not detectibl
Referência(s)