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• W2057142803 abstract "Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2AkitaLdlr−/−). Ins2AkitaLdlr−/− mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr−/− mice, 20-week-old Ins2AkitaLdlr−/− mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2AkitaLdlr−/− mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2AkitaLdlr−/− mice. Thus, the Ins2AkitaLdlr−/− mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis. Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2AkitaLdlr−/−). Ins2AkitaLdlr−/− mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr−/− mice, 20-week-old Ins2AkitaLdlr−/− mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2AkitaLdlr−/− mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2AkitaLdlr−/− mice. Thus, the Ins2AkitaLdlr−/− mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis. Accelerated atherosclerosis is the critical manifestation of macrovascular disease in both type 1 and 2 diabetics and the major etiology of morbidity and mortality in these individuals (1Calles-Escandon J. Cipolla M. Diabetes and endothelial dysfunction: a clinical perspective.Endocr. Rev. 2001; 22: 36-52Crossref PubMed Scopus (559) Google Scholar, 2Retnakaran R. Zinman B. Type 1 diabetes, hyperglycaemia, and the heart.Lancet. 2008; 371: 1790-1799Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In 2000, the global excess mortality attributable to diabetes mellitus was estimated at 2.9 million deaths (3Roglic G. Unwin N. Bennett P.H. Mathers C. Tuomilehto J. Nag S. Connolly V. King H. The burden of mortality attributable to diabetes: realistic estimates for the year 2000.Diabetes Care. 2005; 28: 2130-2135Crossref PubMed Scopus (664) Google Scholar), with 80% the result of major cardiovascular events (4Coccheri S. Approaches to prevention of cardiovascular complications and events in diabetes mellitus.Drugs. 2007; 67: 997-1026Crossref PubMed Scopus (110) Google Scholar). Clinical studies of diabetic cardiovascular disease have mainly focused on type 2 diabetes. However, despite its younger age of onset, type 1 diabetes is also associated with a significantly increased risk of cardiovascular diseases, with an age-adjusted risk greater than 10 times the general population, which even exceeds that of type 2 diabetes (2Retnakaran R. Zinman B. Type 1 diabetes, hyperglycaemia, and the heart.Lancet. 2008; 371: 1790-1799Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 5Libby P. Nathan D.M. Abraham K. Brunzell J.D. Fradkin J.E. Haffner S.M. Hsueh W. Rewers M. Roberts B.T. Savage P.J. et al.Report of the National Heart, Lung, and Blood Institute-National Institute of Diabetes and Digestive and Kidney Diseases Working Group on cardiovascular complications of type 1 diabetes mellitus.Circulation. 2005; 111: 3489-3493Crossref PubMed Scopus (228) Google Scholar–8Laing S.P. Swerdlow A.J. Slater S.D. Burden A.C. Morris A. Waugh N.R. Gatling W. Bingley P.J. Patterson C.C. Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes.Diabetologia. 2003; 46: 760-765Crossref PubMed Scopus (562) Google Scholar). The mechanism of type 1 diabetes-accelerated atherosclerosis is not well studied, and a major reason has been the lack of a good animal model (9Hsueh W. Abel E.D. Breslow J.L. Maeda N. Davis R.C. Fisher E.A. Dansky H. McClain D.A. McIndoe R. Wassef M.K. et al.Recipes for creating animal models of diabetic cardiovascular disease.Circ. Res. 2007; 100: 1415-1427Crossref PubMed Scopus (181) Google Scholar). The most commonly used animal model of type 1 diabetes is the streptozotocin (STZ)-treated mouse. In this model, injection of STZ ablates pancreatic β-cells. Deficiencies of this model include considerable mouse-to-mouse variation; age dependency of pancreatic β-cell destruction; toxicity to other cells and tissues, including DNA damage and induction of carcinogenesis; instability of the diabetes phenotype due to pancreatic islet β-cell regeneration; and the difficulty of inducing diabetes in females (10Bugger H. Boudina S. Hu X.X. Tuinei J. Zaha V.G. Theobald H.A. Yun U.J. McQueen A.P. Wayment B. Litwin S.E. et al.Type 1 diabetic akita mouse hearts are insulin sensitive but manifest structurally abnormal mitochondria that remain coupled despite increased uncoupling protein 3.Diabetes. 2008; 57: 2924-2932Crossref PubMed Scopus (146) Google Scholar–12Bolzan A.D. Bianchi M.S. Genotoxicity of streptozotocin.Mutat. Res. 2002; 512: 121-134Crossref PubMed Scopus (323) Google Scholar). The variability and instability of STZ-treated mouse model make it especially difficult to perform the long-term experiments required for atherosclerosis studies. The Akita mouse (Ins2Akita) carries a single copy of a dominant mutation in the Ins2 gene (Cys96Tyr). This mutation disrupts intramolecular disulfide bond formation causing improper folding of proinsulin. Proinsulin accumulates intracellularly and, by engorging the endoplasmic reticulum (ER) and triggering the ER stress response, leads to apoptosis of pancreatic β-cells (13Yoshinaga T. Nakatome K. Nozaki J. Naitoh M. Hoseki J. Kubota H. Nagata K. Koizumi A. Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface.Biol. Chem. 2005; 386: 1077-1085Crossref PubMed Scopus (28) Google Scholar). Despite the co-expression of a normal insulin gene allele, by 3 to 4 weeks of age, Ins2Akita mice exhibit hypoinsulinemia, hyperglycemia, polydipsia, and polyuria in the absence of obesity (14Wang J. Takeuchi T. Tanaka S. Kubo S.K. Kayo T. Lu D. Takata K. Koizumi A. Izumi T. A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse.J. Clin. Invest. 1999; 103: 27-37Crossref PubMed Scopus (469) Google Scholar–16Izumi T. Yokota-Hashimoto H. Zhao S. Wang J. Halban P.A. Takeuchi T. Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse.Diabetes. 2003; 52: 409-416Crossref PubMed Scopus (166) Google Scholar). On the C57BL/6J background on a chow diet, Ins2Akita mice have persistent hyperglycemia with fasting blood glucose levels of greater than 400 mg/dl (14Wang J. Takeuchi T. Tanaka S. Kubo S.K. Kayo T. Lu D. Takata K. Koizumi A. Izumi T. A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse.J. Clin. Invest. 1999; 103: 27-37Crossref PubMed Scopus (469) Google Scholar–16Izumi T. Yokota-Hashimoto H. Zhao S. Wang J. Halban P.A. Takeuchi T. Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse.Diabetes. 2003; 52: 409-416Crossref PubMed Scopus (166) Google Scholar). The Ins2Akita model has been used to study diabetic microvascular complications, such as retinopathy, neuropathy, and nephropathy (9Hsueh W. Abel E.D. Breslow J.L. Maeda N. Davis R.C. Fisher E.A. Dansky H. McClain D.A. McIndoe R. Wassef M.K. et al.Recipes for creating animal models of diabetic cardiovascular disease.Circ. Res. 2007; 100: 1415-1427Crossref PubMed Scopus (181) Google Scholar). However, macrovascular diabetic complications, such as atherosclerotic cardiovascular disease, have not been examined. In our initial studies we found Ins2Akita mice on the C57BL/6J background fed the 0.02% cholesterol AIN76a diet (low cholesterol, low fat) from weaning to 20 weeks of age averaged total cholesterol levels of ∼112 mg/dl and triglycerides of 52 mg/dl and had no signs of atherosclerotic lesions at the aortic root (data not shown). The latter was not surprising since mice are normally atherosclerosis-resistant and much higher lipid levels are required to foster lesion development (17Breslow J.L. Mouse models of atherosclerosis.Science. 1996; 272: 685-688Crossref PubMed Scopus (572) Google Scholar). Therefore, to enable studies of the effect of hyperglycemia on atherosclerosis, we bred the Ins2Akita trait onto the atherosclerosis-susceptible Ldlr−/− background and compared Ins2AkitaLdlr−/− to Ldlr−/− controls. We chose the Ldlr−/− background over the apoE−/− background because its plasma lipid profile more closely resembles that of most atherosclerosis-prone humans. We also chose the 0.02% cholesterol AIN76a diet to avoid the additional stresses of obesity and insulin resistance apart from hyperglycemia present in other models (18Mulvihill E.E. Assini J.M. Sutherland B.G. DiMattia A.S. Khami M. Koppes J.B. Sawyez C.G. Whitman S.C. Huff M.W. Naringenin decreases progression of atherosclerosis by improving dyslipidemia in high-fat-fed low-density lipoprotein receptor-null mice.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 742-748Crossref PubMed Scopus (123) Google Scholar). On the 0.02% cholesterol AIN76a diet at 20 weeks of age, Ins2AkitaLdlr−/− mice had higher levels of total, VLDL, and LDL cholesterol and triglycerides, as well as increased aortic root cross-sectional lesion areas. Liver gene expression revealed alteration in lipid homeostasis genes and increased expression of pro-inflammatory cytokine genes. Immunofluorescence staining showed that the expression levels of several pro-inflammatory cytokines were also increased in the atherosclerotic lesions and artery walls of Ins2AkitaLdlr−/− mice. These data suggest the Ins2AkitaLdlr−/− mouse is a promising model for mechanistic studies of accelerated macrovascular disease associated with type 1 diabetes. Ldlr−/− mice (B6.129S7-Ldlrtm1Her/J, stock no. 002207) and heterozygous Ins2Akita mice (C57BL/6-Ins2Akita/J, stock no. 003548) both on the C57BL/6J background were obtained from the Jackson Laboratory and subsequently crossed to generate Ldlr−/− and Ins2AkitaLdlr−/− mice. All animals were housed in the Rockefeller University Laboratory Animal Research Center under a protocol approved by the Institutional Animal Care and Use Committee in a specific pathogen-free environment in rooms with a light-dark cycle. Experimental mice were weaned and separated by gender at 28 days of age and fed a semisynthetic, modified AIN76 diet containing 0.02% cholesterol (Research Diets, New Brunswick, NJ) for 16 weeks until euthanasia at 20 weeks of age. Three days prior to sacrifice mice were fasted for 6 h following the dark (feeding) cycle with free access to water. After fasting, mice were injected intraperitoneally with 2 mg glucose/g body weight. Plasma glucose values were obtained from venous blood from a small tail clip at 0, 15, 30, 60, and 120 min using the Ascensia Elite XL (Bayer HealthCare, Tarrytown, NY) handheld blood glucometer. On the day of euthanasia, mice were fasted for 6 h following the dark (feeding) cycle. Immediately prior to euthanasia, the fasting plasma glucose was measured, and then mice were anesthetized by intraperitoneal injection with sodium pentobarbital (Henry Schein, Melville, NY). Mice were exsanguinated by left-ventricular puncture, and blood was collected into EDTA-containing syringes. Plasma was prepared by spinning at 16,000 g for 10 min. The circulation was flushed with PBS, and the heart was removed and stored frozen in Tissue-Tek OCT compound as we described before (19Teupser D. Persky A.D. Breslow J.L. Induction of atherosclerosis by low-fat, semisynthetic diets in LDL receptor-deficient C57BL/6J and FVB/NJ mice: comparison of lesions of the aortic root, brachiocephalic artery, and whole aorta (en face measurement).Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1907-1913Crossref PubMed Scopus (132) Google Scholar). Liver and other tissues were collected and stored in RNAlater solution (Life Technologies, Carlsbad, CA). Total cholesterol concentrations were determined enzymatically by a colorimetric method (Roche, Indianapolis, IN). Lipoproteins fractions were isolated by spinning 60 μl of plasma in a TL-100 ultracentrifuge (Beckman Coulter, Brea, CA) at its own density (1.006 g/ml) at 70,000 RPM for 3 h to harvest the supernatant and after adjusting the infranatant with solid KBr to a density of 1.063 g/ml, then spinning it for 70,000 RPM for 18 h to harvest the supernatant. The cholesterol content of each supernatant and the final infranatant were measured and taken to be VLDL (<1.006 g/ml), LDL (1.006 ≤ d ≤ 1.063 g/ml), and HDL (d >1.063 g/ml) cholesterol. Cholesterol concentrations in all three fractions were then determined enzymatically by a colorimetric method (Roche, Indianapolis, IN). Plasma triglyceride levels were determined enzymatically in the original plasma sample. Plasma insulin was measured using a rat/mouse insulin ELISA kit (EZRMI-13K, Linco Research, St. Charles, MO). To quantify atherosclerosis at the aortic root, OCT-embedded hearts were sectioned and stained with oil red O as described (19Teupser D. Persky A.D. Breslow J.L. Induction of atherosclerosis by low-fat, semisynthetic diets in LDL receptor-deficient C57BL/6J and FVB/NJ mice: comparison of lesions of the aortic root, brachiocephalic artery, and whole aorta (en face measurement).Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1907-1913Crossref PubMed Scopus (132) Google Scholar). The heart was oriented so that the three valves of the aortic root were in the same plane, and 12 μm sections were saved onto glass slides. Sections were stained with oil red O. The lesion area was quantified in every fourth section, and the average was reported for five measurements. Total RNA was isolated from the livers of male Ins2AkitaLdlr−/− and Ldlr−/− mice using the RNeasy mini kit (Qiagen USA, Valencia, CA) according to the manufacturer-supplied protocol. Quantitative real-time PCR (QPCR) was performed using gene-specific primers and the SYBR green PCR kit (Life Technologies, Carlsbad, CA) in an ABI 7900 system (Life Technologies) as described before (20Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). All samples were quantified using the comparative Ct method for relative quantification of gene expression, normalized to GAPDH (21Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124358) Google Scholar). The primer sets utilized in this study are shown in supplementary Table I. Total RNA samples were prepared from the livers of Ins2AkitaLdlr−/− and Ldlr−/− mice using the RNeasy mini kit (Qiagen USA, Valencia, CA). cRNA was prepared and hybridized to the MouseRef-8 v2.0 Illumina Genome-Wide Expression BeadChip Array (Illumina, San Diego, CA). The hybridized BeadChip was washed and labeled with streptavidin-Cy3 and scanned with Illumina BeadScan by the Rockefeller University Genomics Resource Center according to the manufacturer's protocol (Illumina). BeadStudio 3.2 software was used for background correction of the imported scanned image. Gene expression profiles were analyzed using the GeneSpring GX 10.0 software (Agilent Technologies, Santa Clara, CA). In addition to microarray analysis, gene set enrichment analysis (GSEA) was conducted using the GSEA desktop application (http://www.broad.mit.edu/GSEA) (Broad Institute, Cambridge, MA) (22Subramanian A. Kuehn H. Gould J. Tamayo P. Mesirov J.P. GSEA-P: a desktop application for gene set enrichment analysis.Bioinformatics. 2007; 23: 3251-3253Crossref PubMed Scopus (913) Google Scholar, 23Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc. Natl. Acad. Sci. USA. 2005; 102: 15545-15550Crossref PubMed Scopus (26834) Google Scholar). This application ranks the expression of members of a gene set in Ins2AkitaLdlr−/− versus Ldlr−/−. To determine whether a gene set is significantly enriched, GSEA identifies significant changes in gene sets by assigning each a calculated enrichment score (ES). For each gene set, ES was calculated by using weighted Kolmogorov-Smirnov statistics to measure the proximity of the gene set to the top of the Ins2AkitaLdlr−/−-effect ranked list. A highly positive ES indicated that the gene set or pathway was collectively upregulated by the Akita mutation intervention, while a highly negative ES indicated downregulation. To account for differences in numbers of genes in each set, the normalized ES (NES) was used to compare analysis results across gene sets. Gene set analysis was conducted using the canonical pathway (C2) and the transcription factor target (C3) databases from the molecular signature database (MSigDb) available at the Broad Institute, MIT, website. Positively or negatively enriched gene sets were considered significant with a P value ≤ 0.05 and a false discovery rate (FDR) ≤ 25%. On the basis of the difference in the expression of each gene between Ins2AkitaLdlr−/− and Ldlr−/− mice, global functions, networks, and canonical pathways were also analyzed by Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) (Ingenuity Systems, Redwood City, CA). IPA is web-based software designed to organize biological information in a way that allows one to gain a high-level overview of the general biology associated with microarray data (24Calvano S.E. Xiao W. Richards D.R. Felciano R.M. Baker H.V. Cho R.J. Chen R.O. Brownstein B.H. Cobb J.P. Tschoeke S.K. et al.A network-based analysis of systemic inflammation in humans.Nature. 2005; 437: 1032-1037Crossref PubMed Scopus (1219) Google Scholar–26Liu X. Lu R. Xia Y. Sun J. Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo.BMC Genomics. 2010; 11: 722Crossref PubMed Scopus (37) Google Scholar). In this study, the gene list from GeneSpring GX 10.0 was generated with gene identifiers, and the difference in gene expression was entered into IPA. The IPA Canonical Pathway Analysis tool was used to identify the signaling pathways associated with the database. The functional analysis identified the molecular and cellular function that was most significant to the data set as a whole and generated functional interpretation of microarray data (26Liu X. Lu R. Xia Y. Sun J. Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo.BMC Genomics. 2010; 11: 722Crossref PubMed Scopus (37) Google Scholar). Immunohistochemistry were performed on 12 μm sections of aortic roots freshly embedded in OCT. Sections were first fixed in 100% ice-cold acetone for 15 min and then washed with PBS for 20 min. Sections were permeabilized with PBS + 0.1% Triton ×100 (PBST) for 10 min. Nonspecific binding was reduced by incubating slides in 10% rabbit sera diluted in PBST for 20 min at room temperature. Sections were then incubated with rabbit antibodies against mouse Mcp1 (1:50; Abcam, Cambridge, MA), tumor necrosis factor (Tnf)α (1:100; Abcam,) or interleukin (Il)1β (1:50; Abcam) at 4°C for 12-15 h. Sections were rinsed with PBS and incubated with Alexa 594-labeled goat anti-rabbit secondary antibodies (1:500; Life Technologies). The nuclei were stained by mounting the slides with DAPI medium (Vector Laboratories, Burlingame, CA). Images were acquired with a Nikon fluorescence microscopy (Nikon, Melville, NY). All data are expressed as mean ± SD unless indicated otherwise. Statistically significant differences between two groups were analyzed by t-test for data normally distributed and by the Mann-Whitney test for data not normally distributed using Prism version 4.0 (GraphPad Prism Software, San Diego, CA). Grubbs's test was also performed to detect significant outliers (P < 0.05). At 20 weeks of age, Ins2AkitaLdlr−/− mice had significantly decreased body weight in males, but female body weight remained the same (supplementary Fig. IA). The Akita mutation also increased mortality of Ldlr−/− mice in both males and females (supplementary Fig. IB). Ins2AkitaLdlr−/− mice were severely hyperglycemic with fasting blood glucose levels of 423 ± 162 mg/dl in males and 392 ± 130 mg/dl in females, compared with Ldlr−/− mice with glucose levels of 153 ± 29 mg/dl in males and 129 ± 17 mg/dl in females (Fig. 1A). In addition, Ins2AkitaLdlr−/− mice had low fasting plasma insulin levels of 0.31 ± 0.05 ng/ml in males and 0.28 ± 0.07 ng/ml in females, compared with Ldlr−/− mice with insulin levels of 0.89 ± 0.32 ng/ml in males and 0.61 ± 0.22 ng/ml in females (Fig. 1B). Finally, Ins2AkitaLdlr−/− mice of both genders showed impaired glucose tolerance compared with Ldlr−/− mice as measured by the IPGTT (Fig. 1C, D). Also at 20 weeks of age, Ins2AkitaLdlr−/− mice had increased fasting cholesterol levels of 1,252 ± 536 mg/dl in males and 1,067 ± 347 mg/dl in females, compared with Ldlr−/− mice with cholesterol levels of 473 ± 124 mg/dl in males and 688 ± 173 mg/dl in females. Similarly, Ins2AkitaLdlr−/− mice had increased fasting triglyceride levels of 467 ± 239 mg/dl in males and 368 ± 181 mg/dl in females, compared with Ldlr−/− mice with triglyceride levels of 65 ± 26 mg/dl in males and 148 ± 64 mg/dl in females (Fig. 2A). Comparison of lipoprotein cholesterol levels between Ins2AkitaLdlr−/− and Ldlr−/− mice revealed VLDL cholesterol levels increased 7-fold in males and 1.8-fold in females; LDL cholesterol levels increased 2-fold in males but only 24% in females; and HDL cholesterol levels were unchanged in males but slightly and significantly increased in females (Fig. 2B). The Ins2AkitaLdlr−/− and Ldlr−/− mice were sacrificed at 20 weeks of age, and aortic root cross-sectional lesion areas were determined (Fig. 3). Compared with Ldlr−/− mice, Ins2AkitaLdlr−/− mice had increased cross-sectional lesion areas of 224% in males and 30% in females (P < 0.001and P < 0.05, respectively). To elucidate possible molecular mechanisms through which the Akita mutation might augment hyperlipidemia and atherosclerosis in Ldlr−/− mice, gene expression microarray studies were performed comparing livers from Ins2AkitaLdlr−/− and Ldlr−/− mice. Genes with more than 1.5-fold expression changes are listed in supplementary Table II. To confirm the results of the microarray analysis and to investigate other genes involved in lipid homeostasis that were not revealed by the microarray analysis, hepatic gene expression in Ins2AkitaLdlr−/− and Ldlr−/− mice were evaluated by QPCR (Fig. 4). Expression of several key genes involved in lipid homeostasis, including sterol-regulatory element binding protein (Srebp)1c, liver X receptor (Lxr)α, Abca1, Lpl, Cyp7b1, and Cyp27a1, were significantly altered in Ins2AkitaLdlr−/−. Furthermore, the expression of pro-inflammatory cytokine genes, including Il1α, Il1β, Il2, Tnfα, and Mcp1, were significantly increased in Ins2AkitaLdlr−/− mice (Fig. 5).Fig.5Akita mutation increases mRNA levels of pro-inflammatory cytokine genes in the livers of Ins2AkitaLdlr−/− mice. Total RNA was isolated from the livers of 20-week-old male Ldlr−/− and Ins2AkitaLdlr−/− mice. The expression levels of pro-inflammatory cytokine genes were measured by quantitative PCR (n = 5 per group, *P < 0.05 and ***P < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Microarray results were further analyzed by GSEA and IPA to identify gene sets and pathways affected by breeding the Ins2Akita mutation onto the Ldlr−/− background. GSEA is a computational method that determines whether an a priori defined set of genes shows statistically significance between two biological states (22Subramanian A. Kuehn H. Gould J. Tamayo P. Mesirov J.P. GSEA-P: a desktop application for gene set enrichment analysis.Bioinformatics. 2007; 23: 3251-3253Crossref PubMed Scopus (913) Google Scholar). Comparing Ins2AkitaLdlr−/− to Ldlr−/− mice, GSEA analysis identified seven downregulated and seven upregulated gene sets (Table 1). The downregulated gene sets in Ins2AkitaLdlr−/− mice include pathways involved in complement and coagulation cascades, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) interactions in vesicular transport, biosynthesis of steroids, ether lipid metabolism, 1 and 2 methylnaphthalene degradation, linoleic acid metabolism, and bile acid biosynthesis. The upregulated gene sets include pathways involved in carbon fixation, glutathione metabolism, oxidative phosphorylation, pentose phosphate pathway, metabolism of xenobiotics by cytochrome p450, citrate cycle, and hematopoietic cell lineage.TABLE 1GSEA Analysis of Altered Hepatic Gene Networks in Ins2 LDLR MiceRankGene Set (Pathway)Genes Within Gene SetNESPFDRNegatively Enriched Gene Sets1HSA04610_COMPLEMET_AND_COAGULATION CASCADESCfh, Serping1, Cfi, Plg, C4b, Serpina5, C3ar1, Tfpi, Cpb2, Serpind1, Proc, F2r, F12, Masp1, C1s, Masp2, C9, C6, C8a, F8, F7, F2,Cfd, C8b, Klkb1, F11−2.32<0.0010.0052HSA04130_SNARE_INTERACTIONS_IN_VESICULAR_TRANSPORTSnap23, Bnip1, Sec22b, Stx3, Gosr2, Stx18, Vamp2, Ykt6, Vamp5, Stx7, Bet1−2.14<0.0010.0033HSA00100_BIOSYNTHESIS_OF_STEROIDSDhcr7,Vkorc1, Mvd, Hsd17b7, Pmvk, Sqle, Idi1, Sc4mol,Nsdhl,Fdps, Ggcx, Ebp−2.13<0.0010.0034HSA00565_ETHER_LIPID_METABOLISMAgps, Enpp6, Pla2g10, Pla2g12a, Chpt1, Ppap2c, Pafah2, Ppap2a,Enpp2, Agpat4, Pla2g6, Agpat2, Ppap2b, Pla2g12b−2.040.0050.0045HSA00624_1_AND_2_METHYLNAPHTHALENE DEGRADATIONDhrs2, Nat6, Dhrs2, Acad9, Acad8, Dhrs1, Adh4−1.570.0270.1176HSA00591_LINOLEIC_ACID_METABOLISMPla2g10, Pla2g12a, Rdh11, Rdh14, Cyp1a2, Hsd3b7, Pla2g6, Pla2g12b−1.530.0200.1317HSA00120_BILE_ACID_BIOSYNTHESISAcaa2, Cyp7a1, Acad9, Baat, Aldh2, Rdh11, Rdh14, Srd5a2. Soat2, Cyp27a1, Hsdeb7, Srd5a1, Adh4−1.530.0100.115Positively Enriched Gene Sets1HSA00710_CARBON_FIXATIONMdh2, Tktl1, Fbp2, Aldoa, Tpi1, Got2, Aldob1.88<0.0010.0152HSA00480_GLUTATHIONE_METABOLISMGsta1, Idh2, Gpx3, Gsta2, Mgst3, Gpx4, Gstm3, Gstm1, Gstm2, Gpx7, Gstk1, Gstt21.740.0020.0603HSA00190_OXIDATIVE_PHOSPHORYLATIONAtp6v1b2, Sdhb, Ndufa5, Ndufv2, Ndufb4, Atp5a1, Ndufa7, Ndufs4, Ndufs8, Atp6v0b, Atp5 h, Uqcrc1, Cox10, Ppa1, Ndufb5, Ndufb7, Ndufv1, Cox7b, Ndufa10, Cox5b, Cox7a2, Cox6a1, Cox8a, Ndufb9, Cox4i1, Atp5e, Cox7c, Atp5j2, Ndub10, Ndufs6, Atp5g1, Ndufs7, Ndufb8, Cox6c, Ndufa21.70<0.0010.0744HSA00030_PENTOSE_PHOSPHATE_PATHWAYTktl1, Prps1, H6pd, Fbp2, Aldoa, Aldob, Pgls1.620.0080.1495HSA00980_METABOLISM_OF_XENOBIOTICS_BY_CYTOCHROME_P450Gsta1, Aldh3b1, Gsta2, Mgst3, Ephx1, Gstm3, Adh7, Gstm1, Gstm2, Aldh1a3, Gstk1, Gstt2, Cyp1b11.600.0070.1406HSA00020_CITRATE_CYCLEIdh2, Mdh2, Sdh8, Idh3b, Aco2, Ogdh1.600.0150.1207HSA04640_HEMATOPOIETIC_CELL_LINEAGEIl3ra, Cd8b, Epo, Cd44, Tnf, Cd36, Cd22, Ilr5a, Il7, Il7r, Il2ra, Gp1ba, Itgam, Itga2b, Cd34, Csf3r, Cd3e, Csf3, Cd5, Il1r2, Cd7, Epor, Gp1bb1.540.0070.215 Open table in a new tab In addition to GSEA, the microarray results were analyzed by IPA Canonical Pathway Analysis. The genes from microarray results were overlaid onto the global signaling pathways developed from information contained in the Ingenuity Pathways Knowledge Base (26Liu X. Lu R. Xia Y. Sun J. Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo.BMC Genomics. 2010; 11: 722Crossref PubMed Scopus (37) Google Scholar). The IPA revealed differential expression of genes in lipid and carbohydrate metabolism and cell death (Table 2). Both analyses indentified significantly changed gene sets or pathways related to lipid homeostasis in the" @default.
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• W2057142803 title "Hyperglycemic Ins2AkitaLdlr−/− mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease" @default.
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