Plasma lipidomic analysis shows a disease progression signature in mdx mice

Duchenne muscular dystrophy (DMD) is a rare genetic disorder affecting paediatric patients. The disease course is characterized by loss of muscle mass, which is rapidly substituted by fibrotic and adipose tissue. Clinical and preclinical models have clarified the processes leading to muscle damage and myofiber degeneration. Analysis of the fat component is however emerging as more evidence shows how muscle fat fraction is associated with patient performance and prognosis. In this article we aimed to study whether alterations exist in the composition of lipids in plasma samples obtained from mouse models. Analysis of plasma samples was performed in 4 mouse models of DMD and wild-type mice by LC–MS. Longitudinal samplings of individual mice covering an observational period of 7 months were obtained to cover the different phases of the disease. We report clear elevation of glycerolipids and glycerophospholipids families in dystrophic mice compared to healthy mice. Triacylglycerols were the strongest contributors to the signatures in mice. Annotation of individual lipids confirmed the elevation of lipids belonging to these families as strongest discriminants between healthy and dystrophic mice. A few sphingolipids (such as ganglioside GM2, sphingomyelin and ceramide), sterol lipids (such as cholesteryl oleate and cholesteryl arachidonate) and a fatty acyl (stearic acid) were also found to be affected in dystrophic mice. Analysis of serum and plasma samples show how several lipids are affected in dystrophic mice affected by muscular dystrophy. This study sets the basis to further investigations to understand how the lipid signature relates to the disease biology and muscle performance.

Abstract 425: Brown Fat Activation Enhances the Lipid-lowering and Anti-atherogenic Effect of Statin Treatment

Introduction: Brown adipose tissue (BAT) produces heat by burning triglyceride (TG)-derived fatty acids. As a result, BAT accelerates the formation and hepatic clearance of cholesterol-enriched lipoprotein remnants via the apoE-LDL receptor (LDLR) pathway, thereby alleviating hypercholesterolemia. Since statins upregulate hepatic LDLR expression, the aim of this study was to investigate whether BAT activation and statin treatment cooperate in lowering hyperlipidemia and atherosclerosis. Methods and Results: APOE*3-Leiden.CETP mice were fed a Western-type diet and treated with the selective β3-AR agonist CL316243 that activates BAT (CL, 20 μg/day; s.c.), atorvastatin (statin, 0.0036% w/w, in diet) or both. BAT activation alone and combined with statin treatment increased energy expenditure (+15%) as a result of increased fat oxidation (+50%). Furthermore, BAT activation decreased the lipid droplet content within BAT (-50%) and induced browning of subcutaneous white adipose tissue. BAT activation alone and combined with statin treatment also lowered plasma TG levels (-60%). In addition, plasma cholesterol was lowered by both BAT activation (-29%) and statin (-31%), but even further by the combination (-44%). The uptake of TG-derived fatty acids from glycerol tri[3H]oleate and [14C]cholesteryl oleate-labeled VLDL-mimicking particles into BAT was enhanced by BAT activation (+234%) and on top of statin (+220%), but not by statin alone. The hepatic uptake of the cholesterol-enriched remnants tended to be increased by BAT activation (+18%) or statin (+22%), and was markedly increased by the combination of BAT activation and statin treatment (+70%). Hepatic cholesterol levels were reduced by statin alone and the combination (-41%). Importantly, atherosclerosis development was decreased by BAT activation (-42%) and statin (-50%) alone and further decreased by the combination (-70%). Conclusions: BAT activation enhances the lipid-lowering and anti-atherogenic effect of statin treatment. We postulate that combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular diseases.

Abstract 330: Ezetimibe Prevents Atherogenesis Through Increased Catabolism and Fecal Excretion of LDL-cholesterol and Reduced Atherosclerotic Plaque Inflammation in Apolipoprotein E Knock-out Mice Fed a Paigen Diet

Background: Intestinal cholesterol absorption inhibitor ezetimibe (EZE) added to a statin therapy has demonstrated benefits in the IMPROVE-IT trial by further reducing LDL-cholesterol levels than statin therapy alone. We investigated the mechanisms by which EZE could contribute to cardiovascular events reduction in apolipoprotein E knock-out (apoE ko) mice. Methods: ApoE ko mice were fed a Paigen diet without (control) or with EZE (7mg/kg/day) for 6 weeks. To evaluate the effects of EZE on LDL-cholesterol metabolism and excretion, a first set of mice was injected intravenously with 3 H-cholesteryl oleate labeled human LDL. A second set of mice was used for in vivo SPECT/CT imaging of 99m Tc-cAbVCAM1-5, a single domain antibody directed against the Vascular Cell Adhesion Molecule-1 (VCAM-1), which was used as a marker of inflamed atherosclerotic plaques. The same mice were sacrificed for autoradiography and histology of aortic atherosclerotic plaques. Results: Compared with control, EZE treatment for 6 weeks induced a significant 41% and 65% reduction in plasma total cholesterol levels and atherosclerotic plaque area, respectively. After injection of 3 H-cholesteryl oleate labeled human LDL, mice treated with EZE showed a 173% higher LDL-cholesteryl ester catabolism (p<0.001 vs. control). At time 96 hours after radiolabeled LDL injection, 3 H-tracer hepatic recovery was reduced by 61% with EZE (p<0.001). Meanwhile, LDL-derived 3 H-tracer excretion in the feces was increased by 107% in the fecal cholesterol fraction (p<0.001). Similar trends were observed for hepatic cholesterol levels and fecal cholesterol mass excretion, with a 75% reduction and 99% increase with EZE, respectively (both p<0.001). After intravenous injection of 99m Tc-cAbVCAM1-5, mice treated with EZE also showed a significant 52% reduction in aortic uptake, which was confirmed by significant reduction in tracer uptake in ex vivo biodistribution and autoradiography analysis. Conclusion: EZE promotes anti-atherosclerotic effects through increased LDL-cholesterol catabolism and LDL-derived cholesterol fecal excretion, and reduced inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding EZE to a statin therapy.