Time-Resolved and Tissue-Specific Systems Analysis of the Pathogenesis of Insulin Resistance

BACKGROUND: The sequence of events leading to the development of insulin resistance (IR) as well as the underlying pathophysiological mechanisms are incompletely understood. As reductionist approaches have been largely unsuccessful in providing an understanding of the pathogenesis of IR, there is a need for an integrative, time-resolved approach to elucidate the development of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Male ApoE3Leiden transgenic mice exhibiting a humanized lipid metabolism were fed a high-fat diet (HFD) for 0, 1, 6, 9, or 12 weeks. Development of IR was monitored in individual mice over time by performing glucose tolerance tests and measuring specific biomarkers in plasma, and hyperinsulinemic-euglycemic clamp analysis to assess IR in a tissue-specific manner. To elucidate the dynamics and tissue-specificity of metabolic and inflammatory processes key to IR development, a time-resolved systems analysis of gene expression and metabolite levels in liver, white adipose tissue (WAT), and muscle was performed. During HFD feeding, the mice became increasingly obese and showed a gradual increase in glucose intolerance. IR became first manifest in liver (week 6) and then in WAT (week 12), while skeletal muscle remained insulin-sensitive. Microarray analysis showed rapid upregulation of carbohydrate (only liver) and lipid metabolism genes (liver, WAT). Metabolomics revealed significant changes in the ratio of saturated to polyunsaturated fatty acids (liver, WAT, plasma) and in the concentrations of glucose, gluconeogenesis and Krebs cycle metabolites, and branched amino acids (liver). HFD evoked an early hepatic inflammatory response which then gradually declined to near baseline. By contrast, inflammation in WAT increased over time, reaching highest values in week 12. In skeletal muscle, carbohydrate metabolism, lipid metabolism, and inflammation was gradually suppressed with HFD. CONCLUSIONS/SIGNIFICANCE: HFD-induced IR is a time- and tissue-dependent process that starts in liver and proceeds in WAT. IR development is paralleled by tissue-specific gene expression changes, metabolic adjustments, changes in lipid composition, and inflammatory responses in liver and WAT involving p65-NFkB and SOCS3. The alterations in skeletal muscle are largely opposite to those in liver and WAT

Plasma and Liver Lipidomics Response to an Intervention of Rimonabant in ApoE*3Leiden.CETP Transgenic Mice

Background: Lipids are known to play crucial roles in the development of life-style related risk factors such as obesity, dyslipoproteinemia, hypertension and diabetes. The first selective cannabinoid-1 receptor blocker rimonabant, an anorectic anti-obesity drug, was frequently used in conjunction with diet and exercise for patients with a body mass index greater than 30 kg/m2 with associated risk factors such as type II diabetes and dyslipidaemia in the past. Less is known about the impact of this drug on the regulation of lipid metabolism in plasma and liver in the early stage of obesity. Methodology/Principal Findings: We designed a four-week parallel controlled intervention on apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE&z.ast;3Leiden.CETP) transgenic mice with mild overweight and hypercholesterolemia. A liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric approach was employed to investigate plasma and liver lipid responses to the rimonabant intervention. Rimonabant was found to induce a significant body weight loss (9.4%, p<0.05) and a significant plasma total cholesterol reduction (24%, p<0.05). Six plasma and three liver lipids in ApoE&z.ast;3Leiden.CETP transgenic mice were detected to most significantly respond to rimonabant treatment. Distinct lipid patterns between the mice were observed for both plasma and liver samples in rimonabant treatment vs. non-treated controls. This study successfully applied, for the first time, systems biology based lipidomics approaches to evaluate treatment effects of rimonabant in the early stage of obesity. Conclusion: The effects of rimonabant on lipid metabolism and body weight reduction in the early stage obesity were shown to be moderate in ApoE&z.ast;3Leiden.CETP mice on high-fat diet. © 2011 Hu et al

Lipidomics Reveals Multiple Pathway Effects of a Multi-Components Preparation on Lipid Biochemistry in ApoE*3Leiden.CETP Mice

Background: Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses. Methodology/Principal Findings: ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p <0.001), CETP levels (-31%,

Effects of propagermanium intervention on body weight and metabolic parameters.

<p>Mice were fed a high-fat diet (HFD) for 24 weeks or treated with the CCR2 inhibitor propagermanium started after 6 weeks (HFD+Pro_6w, early intervention) or 12 weeks of HFD (HFD+Pro_12w, late intervention). Chow-fed mice were included as a reference. (A) HFD feeding increased body weight compared with chow and was not affected by propagermanium intervention. (B) Fasting blood glucose levels over time. (C) HFD-induced increased fasting plasma insulin levels, which were reduced in HFD+Pro_6w. (D) C-peptide levels were increased at week 24 in HFD-fed mice and attenuated by early propagermanium intervention. (E) HFD-induced increases in HOMA-IR relative to chow were significantly reduced by early, but not late, propagermanium intervention. (F) Plasma adiponectin levels were increased by early propagermanium treatment in week 24. All data are mean±SEM, n = 5 (chow), and n = 13–15 for HFD and intervention groups. * <i>p<</i>0.05 (compared with chow) # <i>p<</i>0.05 compared with HFD.</p

Time course development of insulin resistance, adipose and liver inflammation.

<p>Mice were maintained on a high-fat diet (HFD) 0, 6, 12 and 24 weeks prior to clamp experiment to induce insulin resistance. (A) shows HFD-induced decrease in glucose infusion rate (GIR) under hyperinsulinemic conditions, indicating development of whole body insulin resistance. (B) HFD feeding resulted in a gradual decrease of tissue-specific uptake of glucose into white adipose tissue (WAT). (C) HFD feeding increased hepatic glucose production after 24 weeks of HFD, indicating development of hepatic insulin resistance. ‘Basal’ indicates prior to hyperinsulinemic clamp conditions and ‘clamp’ indicates hyperinsulinemic clamp conditions. (D) Representative microphotographs of HPS-stained WAT cross-sections, showing WAT inflammation (i.e. crown-like structures) already after 6 weeks of HFD feeding. (E) Representative microphotographs of HE-stained liver cross-sections, showing NASH development after 24 weeks of HFD feeding (arrow heads indicate clusters of inflammatory cells). (F) Quantitative analysis of crown-likes structures (CLS), demonstrating gradual development of WAT inflammation by HFD feeding. (G) Hepatic inflammation (number of inflammatory cell aggregates per 100x field) was induced after 24 weeks of HFD feeding. All data are mean±SEM, n = 7-11/group. * <i>p<</i>0.05 ** <i>p<</i>0.01 *** <i>p<</i>0.001 compared with t = 0. # <i>p<</i>0.05, ###<i>p<</i>0.01 compared to week 24.</p

Effects of propagermanium intervention on WAT inflammation markers.

<p>(A) HFD feeding increased CD68 expression (general macrophage marker), which was not affected by propagermanium. (B) HFD increased CD11c expression, suggesting increased number of ‘pro-inflammatory’ (M1) macrophages in WAT. (C) Arginase-1 expression (anti-inflammatory M2 macrophage marker) did not differ between the groups. Early intervention significantly lowered HFD-induced expression of (D) Mcp-1 and showed slightly lowered (E) Tnf-α expression levels in WAT. All data are mean±SEM.* <i>p<</i>0.05.</p

Effects of propagermanium intervention on NASH development.

<p>(A) HFD feeding induced pronounced macrovesicular steatosis, which was reduced in HFD+Pro_6w group. (B) Microvesicular steatosis was induced by HFD and was not affected by propagermanium treatment. (C) Quantification of the number of inflammatory cell aggregates per field in the liver (lobular inflammation), which was increased by HFD and tended to be reduced by early propagermanium treatment. (D) Lobular inflammation strongly correlates with the ratio of M1/M2 markers (CD11c/Arginase-1 gene expression). (E) M1/M2 ratio is decreased by propagermanium treatment, indicating a shift towards M2 phenotype. All data are mean±SEM. * <i>p<</i>0.05.</p

Unraveling the Transcriptional Dynamics of NASH Pathogenesis Affecting Atherosclerosis

The prevalence of non-alcoholic steatohepatitis (NASH) is rapidly increasing and associated with cardiovascular disease (CVD), the major cause of mortality in NASH patients. Although sharing common risk factors, the mechanisms by which NASH may directly contribute to the development to CVD remain poorly understood. The aim of this study is to gain insight into key molecular processes of NASH that drive atherosclerosis development. Thereto, a time-course study was performed in Ldlr&minus;/&minus;.Leiden mice fed a high-fat diet to induce NASH and atherosclerosis. The effects on NASH and atherosclerosis were assessed and transcriptome analysis was performed. Ldlr&minus;/&minus;.Leiden mice developed obesity, hyperlipidemia and insulin resistance, with steatosis and hepatic inflammation preceding atherosclerosis development. Transcriptome analysis revealed a time-dependent increase in pathways related to NASH and fibrosis followed by an increase in pro-atherogenic processes in the aorta. Gene regulatory network analysis identified specific liver regulators related to lipid metabolism (SC5D, LCAT and HMGCR), inflammation (IL1A) and fibrosis (PDGF, COL3A1), linked to a set of aorta target genes related to vascular inflammation (TNFA) and atherosclerosis signaling (CCL2 and FDFT1). The present study reveals pathogenic liver processes that precede atherosclerosis development and identifies hepatic key regulators driving the atherogenic pathways and regulators in the aorta