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Identifier 000421762
Title Study of the molecular mechanisms involved in the pathogenesis of the metabolic syndrome (MetS) with systems biology approaches
Alternative Title Μελέτη των μοριακών μηχανισμών που εμπλέκονται στην παθογένεση του μεταβολικού συνδρόμου (ΜΣ) με μεθόδους βιολογίας συστημάτων
Author Νάσιας, Δημήτρης
Thesis advisor Καρδάσης, Δημήτριος
Abstract Metabolic syndrome (MetS) is a multifactorial disorder characterized by the presence of several comorbidities that predispose to cardiovascular disease and diabetes. Long-term exposure to excess dietary fat is a predominant factor that leads to obesity and MetS. However, the presently available animal models to study the molecular mechanisms underlying the MetS pathology have significant limitations. The purpose of the present study was first to identify global gene expression changes in metabolic tissues and circulating miRNAs in a humanized mouse model of diet-induced MetS, the apoE3L.CETP mice. Secondly, we aimed to assess the impact of hepatic FOXO1 ablation in apoE3L.CETP mice during the development of MetS. Male apoE3L.CETP mice received a high (HFD) or a low (LFD) fat diet for different time periods and the progression of MetS pathology was monitored. In a parallel study that was performed in collaboration with the group of Dr Thomas Lutz in Zurich, a separate group of mice was divided into responders (R) or non-responders (NRs) and received the same HFD for 16 weeks. The hepatic transcriptome was analyzed by Affymetrix expression arrays. We found that mice receiving the HFD progressively developed manifestations of MetS and displayed an increasing number of differentially expressed transcripts at 4, 8 and 12 weeks compared to LFD. Significantly changed genes were functionally annotated to metabolic diseases including insulin resistance, hepatic steatosis, metabolic syndrome and disorders of glucose and lipid metabolism. Pathway analysis revealed the downregulation of genes in cholesterol and fatty acid biosynthesis whereas genes of glycolysis or gluconeogenesis were not changed. A group of genes related to lipid droplet formation was upregulated in the HFD group and this was in line with the development of hepatic steatosis in these mice. In the serum of the apoE3L.CETP mice we identified 3 miRNAs that were up-regulated in the HFD group but not in the LFD group. We found that responder mice have a distinct gene signature that differentiates them from mice that do not respond well to the diet. The comparison of two diet intervention studies (HFD vs LFD and R vs NR) revealed a limited number of common differentially expressed genes but the expression of the common genes were affected in a similar way in both studies. As a following step, we sought to identify transcriptomic changes in the liver that are related to bariatric interventions in the context of MetS. Improvement of comorbidities in the MetS and sustained weight loss can be achieved by the Roux-en-Y gastric bypass (RYGB) surgical operation. For this purpose, we collaborated with the same group in Zurich in order to obtain liver samples and perform transcriptomics. Male apoE3L.CETP mice were fed a HFD diet for 6 weeks and underwent RYGB or Sham surgery. Sham groups were either fed ad libitum or were body weight-matched to the RYGB mice, to discriminate and identify surgical effects from body weight loss associated effects. RYGB-operated mice achieved a significant weight loss. In addition, RYGB surgery ameliorated the glucose metabolism and lipid metabolism, and altered hepatic transcriptome according to the global transcriptomic analysis in the livers of RYGB, Sham AL and BWm groups of mice. The comparison of the RYGB mice to BWm or Sham AL mice revealed 1137 (656↑, 481↓) and 411 s (300↑, 206↓) differentially expressed transcripts respectively. Ingenuity Pathway Analysis (IPA) indicated that the observed gene expression changes in the three different comparison groups were mainly associated with the lipid metabolism such as “concentration of lipid, fatty acid metabolism, concentration of cholesterol, and synthesis of lipid” at the level of biological functions. In addition, we examined the transcriptomic alterations that occur in the gonadal white adipose tissue (gWAT) of apoE3L.CETP mice in discrete stages of the development of MetS since WAT is a major determinant of metabolic health and disease. Adipose RNA was analyzed on Affymetrix Mouse Gene 2.0 ST arrays followed by bioinformatical analysis. The microarray analysis revealed that the transcriptomic profile of WAT was more robustly altered compared with the hepatic transcriptome in apoE3L.CETP mice. Indeed, the statistical analysis of transcriptomic data under specific criteria (p-value < 0.05 and |log2FC(fold change) |≥1.5) yielded a total of 481, 946 and 1377 genes that were found to be differentially expressed between the HFD and the LFD groups of mice at 4, 8 and 12 weeks, respectively. Functional analysis allowed identifying sets of genes significantly enriched with immune and inflammatory responses, especially at 8 and 12w. Moreover, a large number of genes related to extracellular matrix (ECM) were found to be differentially expressed, suggesting tissue remodeling during the progression of MetS. The downregulation of metabolic pathways including Glycolysis, Cholesterol Biosynthesis and Fatty Acid Biosynthesis, could be associated with a modulation of nutrients metabolism upon HFD feeding. Finally, genes with an established role in the regulation of adipose metabolism and maintenance or with a systemic effect in whole-body homeostasis were differentially expressed during MetS progression, including the leptin (lep), the glucose transporter, Slc2a4 (Solute carrier family 2, member 4), the Insulin receptor substrate 1 (Irs1) and the Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A). To elucidate the effect of FOXO1 silencing in the liver of apoE3L.CETP mice during the MetS progression, adult male mice were intravenously injected with the AAV-sc-RNA as control group or with the AAV-sh-Foxo1. All mice were fed a chow diet for 15 weeks post-injection before switching to a HFD for 3 months. Animals were monitored for their body weight, lipid profile, glucose levels and glucose tolerance before and after the diet intervention. We identified a reduction of FOXO1 at both mRNA and protein levels in certain sh-FOXO1 mice (5 out of 11 mice). Moreover, fluorescent microscopy revealed FOXO1 localization in the nuclei of the control group, whereas FOXO1 was barely detectable in the AAV-shFOXO1 group. The silencing of FOXO1 in the liver of apoE3L.CETP mice induced alterations only in some clinical circulating parameters. More precisely, the clinical characterization of mice revealed that there were no significant differences in glucose and lipid measurements at baseline and additionally histological alterations were similar to both groups of mice, related to HFD feeding. Importantly, there were significant systemic improvements concerning the glucose tolerance and the insulin sensitivity. Furthermore, RNA sequencing analysis revealed a significant response of hepatic transcriptome and of the skeletal muscles transcriptome to a less extent. Regarding hepatic transcriptomic changes, genes related to FOXO signaling pathway (mmu04068) and also to Insulin resistance (mmu04931) were down-regulated such as the insulin receptor (Insr, p-0.019758593), up-stream of FOXO1 signaling cascade. Gluconeogenesis may be reduced due to significant ablation of FOXO1 and relevant pathways that could regulate the glucose production and uptake when the HFD increases fasting blood glucose in both groups during the MetS progression. These data claim that FOXO1 modulation could be a therapeutic approach to counterfeit key-components of the MetS. In summary, the present transcriptomic analysis in the livers and the sera of apoE3L.CETP mice receiving a HFD or a LFD and in the livers of apoE3L.CETP mice subjected to RYGB surgery, combined with extensive bioinformatics analysis, revealed characteristic hepatic gene signatures, indicative of the molecular changes that occur at the different stages of the pathogenesis of the MetS. This approach was further completed by the transcriptomic signatures identified in the WAT of apoE3L.CETP mice and the clinical alterations that were observed after the hepatic silencing of FOXO1 in the context of MetS progression. Collectively, the patterns of gene expression and serum miRNAs identified in the present study provide a detailed insight to MetS pathology and could be exploited for diagnostic or therapeutic purposes.
Language English
Subject FOXO1
Gene signatures
White adipose tissue
Γονιδιακές υπογραφές
Κυκλοφορούντα miRNAs
Λευκός λιπώδης ιστός
Issue date 2019-03-27
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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