Three animal experiments were included in this study. First, inbred male mice of the heterozygous E3L strain were kept under specific-pathogen-free conditions (TNO, Leiden, The Netherlands; n=40). E3L mouse is the wildtype C57BL/6J mouse with a knocked-in mutated human apolipoprotein E3 gene (APOE3). This mutation caused reduced protein binding capacity to the low-density lipoprotein receptor (LDLr) leading to slower cholesterol clearance [10]. Animals were housed in Makrolon cages at a relative humidity of 50-60% and temperature at around 21°C under a 12:12 light/dark cycle with the light on at 7 am. Food and acidified tap water were available ad libitum. All mice were fed with standard lab chow (Ssniff-R, Uden, The Netherlands) until 12 weeks of age. Ten mice were sacrificed at 12 weeks of age to serve as a baseline reference group. Following that, the remaining 30 mice received 24% w/w lard and sucrose-containing energy-dense high-fat diet (HFD; #D12451, Research Diets, New Brunswick, NJ) between 12-20 weeks of age to induce obesity as illustrated in Figure 1. Diet and macronutrient composition are described in detail elsewhere [11]. Bodyweight and caloric intake were monitored regularly. Tail blood was sampled after 4 hours of fasting at 12 weeks (before HFD intervention) and 16 and 20 weeks for lipidomic analyses. At 20 weeks, the remaining mice were sacrificed and the subcutaneous, epidydimal, and mesenteric adipose tissue depots were isolated.

Schematic overview of the ApoE3L mice experiment

In the second study, the findings from the E3L experiment were subsequently validated in 12-week-old Ldlr-/-.Leiden and C57BL/6J mice. The Ldlr-/-.Leiden mouse is the wildtype C57BL/6J with a knocked-out LDLr gene to mimic humans with LDLR-defective mutations. These two types of mice are 94% genetically similar and the wildtype C57BL/6J is the most common strain used to study obesity. To induce obesity, the same HFD was fed to the Ldlr-/-.Leiden mice and wildtype C57BL/6J8 for 21 and 24 weeks, respectively.

Finally, to evaluate whether the identified candidate biomarkers were apparent at earlier ages, another lipidomic analysis of plasma of E3L mice collected at the age of 4 weeks was performed. In this study, animals were supplemented with arachidonic acid/docosahexaenoic acid (ARA/DHA) from 4-12 weeks of age. Lipid concentrations reflecting desaturase activities were analysed against 4-12 weeks weight gain between mice treated with ARA/DHA or placebo (n=15 each) [12].

All animal care and experimental procedures were approved by the Ethical Committee on Animal Care and Experimentation (Zeist, the Netherlands; approval reference numbers DEC-3117, -3076, -3260 and -3277) and were performed in compliance with ARRIVE guidelines and the European Community specifications regarding the use of laboratory animals.

During the HFD feeding period, mice were weighed regularly to record delta weight gain as the primary obesity endpoint. Additional obesity endpoints included final absolute body weight after completing HFD, overall adiposity (i.e. the total mass of all adipose tissue depots), and absolute mass of specific adipose tissue depots. Histological analysis of subcutaneous, epidydimal, and mesenteric adipose tissue depots allowed determination of the average adipocyte cell size, a determinant of white adipose tissue (WAT) quality [13].

Lipids were analysed as described elsewhere [14] with electrospray and atmospheric pressure chemical ionization (APCI) ultra-high performance liquid chromatography/high-resolution mass spectrometry (ESI-UHPLC-HRMS; Thermo Accela and LTQ OrbitrapTM). The method is explained in more detail in the supplementary materials.

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