Proteolytic Inhibition of CTSD in Hyperlipidemic Mice

Experiments were performed according to the Dutch regulation and approved by the Committee for Animal Welfare of Maastricht University. Ldlr^−/− mice on a C57BL/6 background were housed under standard conditions and given free access to food and water. Female 12 week-old Ldlr^−/− mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet (SAFE, Augy, France) for 3 weeks and were divided in three groups (n = 11 for each group). The HFC diet contained 21% milk butter, 0.2% cholesterol, 46% carbohydrates and 17% casein. To examine whether proteolytic inhibition of CTSD would decrease hepatic inflammation, Ldlr^−/− mice were injected intraperitoneally with pepstatin A (PepA; 50 µg/g body weight; P5318, Sigma, Zwijndrecht, the Netherlands), a proteolytic inhibitor of aspartyl proteases. Mice were injected with DMSO (8%) or PepA two times every week. An overview of all experimental groups is depicted in Supplementary Fig. S1. Collection of blood and tissue specimens, fluorescence-activated cell sorting (FACS), liver histology, plasma alanine transaminase levels (ALT), RNA isolation, cDNA synthesis and qPCR were determined as described previously^{4 (link), 15 , 41 , 42 (link)}.