Endothelial Lipase Is Involved in Cold-Induced High-Density Lipoprotein Turnover and Reverse Cholesterol Transport in Mice

Abstract

The physiologic activation of thermogenic brown and white adipose tissues (BAT/WAT) by cold exposure triggers heat production by adaptive thermogenesis, a process known to ameliorate hyperlipidemia and protect from atherosclerosis. Mechanistically, it has been shown that thermogenic activation increases lipoprotein lipase (LPL)-dependent hydrolysis of triglyceride-rich lipoproteins (TRL) and accelerates the generation of cholesterol-enriched remnants and high-density lipoprotein (HDL), which promotes cholesterol flux from the periphery to the liver. HDL is also subjected to hydrolysis by endothelial lipase (EL) (encoded by LIPG). Genome-wide association studies have identified various variants of EL that are associated with altered HDL cholesterol levels. However, a potential role of EL in BAT-mediated HDL metabolism has not been investigated so far. In the present study, we show that in mice, cold-stimulated activation of thermogenic adipocytes induced expression of Lipg in BAT and inguinal WAT but that loss of Lipg did not affect gene expression of thermogenic markers. Furthermore, in both wild type (WT) and Lipg-deficient mice, activation of thermogenesis resulted in a decline of HDL cholesterol levels. However, cold-induced remodeling of the HDL lipid composition was different between WT and Lipg-deficient mice. Notably, radioactive tracer studies with double-labeled HDL indicated that cold-induced hepatic HDL cholesterol clearance was lower in Lipg-deficient mice. Moreover, this reduced clearance was associated with impaired macrophage-to-feces cholesterol transport. Overall, these data indicate that EL is a determinant of HDL lipid composition, cholesterol flux, and HDL turnover in conditions of high thermogenic activity.

Keywords: HDL; brown adipose tissue; cholesterol; endothelial lipase; lipidomics.

Figure 1

Loss of endothelial lipase (EL) does not compromise thermogenic response. Wild type (WT) mice were housed for 7 days either in a thermoneutral (30°C) or cold (6°C) environment and gene expression of Lipg was measured in iBAT (A) and ingWAT (B). WT and Lipg^−/− mice were housed for 7 days either in a thermoneutral (30°C) or cold (6°C) environment and gene expression of indicated genes was measured in iBAT (C), ingWAT (D) and liver (E). iBAT, interscapular brown adipose tissue (BAT); ingWAT, inguinal white adipose tissue (WAT). Each bar represents the mean of four to six mice per group ± SEM. Statistics were performed using two-way ANOVA and p-values lower than 0.05 were considered statistically significant; different letters indicate significant differences between groups.

Figure 2

Loss of EL increases high-density lipoprotein (HDL) cholesterol and impairs HDL lipid remodeling. WT and Lipg^−/− mice were housed for 7 days either in a thermoneutral (30°C) or cold (6°C) environment and plasma samples were taken after a 4 h fasting period. Triglyceride (A,B) and cholesterol (C,D) levels were measured in total plasma (A,C) and fast-performance liquid chromatography (FPLC) fractions (B,D); TRL, triglyceride-rich lipoprotein; LDL, low density lipoprotein; IDL, intermediate-size lipoprotein; HDL, high density lipoprotein. For HDL lipidomic analysis, individual FPLCs were run for each mouse (n = 4/group) and per mouse FPLC fractions 20–22 were pooled as HDL. Alterations in HDL lipid levels are shown for indicated lipid classes in comparison to WT 30°C, PC, phosphatidyl-cholines; PE, phosphatidyl- ethanolamines; PI, phosphatidyl-inositols; Lyso-PC, lysophosphatidyl-cholines; CE, cholesterol ester; TG, triglycerides. (E). Each bar/dot represents the mean of three mice per group ± SEM (A–D). Statistics were performed using two-way ANOVA and p-values lower than 0.05 were considered statistically significant. Data from (A,B) were subjected to logarithmic transformation before two-way ANOVA.

Figure 3

Loss of EL dampens cold-induced HDL turnover. WT and Lipg^−/− mice were housed for 7 days either in a thermoneutral (30°C) or cold (6°C) environment before they received an injection of ¹²⁵I-tyramine cellobiose (TC) and ³H-cholesteryl oleoyl ether (CEt) radiolabeled HDL (A). At indicated time points ³H-CEt activity in plasma was analyzed (B). At indicated time points ¹²⁵I-TC activity in plasma was analyzed. Five hours after injection of radiolabeled HDL fractional catabolic rate (FCR) was determined in plasma for ³H-CEt (C) and ¹²⁵I-TC (D) and in organs for ³H-CEt (E) and ¹²⁵I-TC (F). iBAT, interscapular BAT; ingWAT, inguinal WAT; epiWAT, epididymal WAT. Each bar represents the mean of five to six mice per group ± SEM. Statistics were performed using two-way ANOVA and p-values lower than 0.05 were considered statistically significant and are indicated as follows for (A,B): *WT30 vs. KO 30; #WT6 vs. KO 6; $WT30 vs. WT 6; and for (C–F): different letters indicate significant differences between groups.

Figure 4

EL-deficient mice present diminished reverse cholesterol transport (RCT) independent of housing temperature. Plasma FPLC profile of western-type diet-fed WT and Lipg^−/− mice after 7 days of housing at 6 or 30°C (A). Five days prior to injection of ex vivo ³H-radiolabeled macrophages, WT and Lipg^−/− mice were housed in a thermoneutral (30°C) or cold (6°C) environment. Then, the mice remained on indicated housing temperature and macrophages were allowed 48 h of circulation before mice were sacrificed (B). Clearance from plasma is determined by ³H-cholesterol radioactivity in plasma (C) and liver uptake by measuring ³H-cholesterol radioactivity in liver (D). Excretion of ³H-cholesterol was measured by collecting feces during 48 h of macrophage circulation and determining ³H-cholesterol activity (E). Each bar represents mean of five to seven mice per group ± SEM. Statistics were performed using two-way ANOVA, and p-values lower than 0.05 were considered statistically significant (different letters indicate significant differences between groups).