Abstract
The liver is the central organ regulating cholesterol synthesis, storage, transport, and elimination. Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play roles in hepatic triacylglycerol metabolism and VLDL assembly. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and thus be responsible for the hydrolysis of HDL-derived CE; this could contribute to the final step in the reverse cholesterol transport (RCT) pathway, wherein cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. In this study, we interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d-deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would (1) reduce in vivo RCT flux and (2) provoke liver CE accumulation after a high-cholesterol diet challenge. We found that liver-specific Ces1d-deficient mice did not show any difference in the flux of in vivo HDL-to-feces RCT nor did it cause additional liver CE accumulation after high-fat, high-cholesterol Western-type diet feeding. These findings challenge the importance of Ces1d as a major hepatic CE hydrolase.
Highlights
Because carboxylesterase 1d (Ces1d) is localized in the endoplasmic reticulum (ER) lumen [22,23,24,25,26], lumenal contents were released by sonication and detergent incubation of microsomes prepared from pCI-neo and Ces1d cells to allow access of the substrate for lipase activity measurements. pCI-neo microsomal fractions did not show lipase activity, whereas microsomal fractions isolated from Ces1d cells exhibited robust lipase activity
To assess the contribution of liver Ces1d in the process of removal of HDL-derived cholesteryl ester (CE) from the body, in vivo reverse cholesterol transport (RCT) assay was performed by injecting HDL[3H]-cholesteryl oleate into liver-specific Ces1d-KO mice (LKO) mice and control Lox mice
Cholesterol levels in the plasma HDL fraction were measured, and the results showed that Lox and LKO mice had comparable HDL-Total cholesterol (TC), free cholesterol (FC), and CE levels (Fig. 1E); the decreased cholesterol level in the plasma represents decreased cholesterol associated with the fraction of apoB-containing lipoproteins
Summary
All animal procedures were conducted in compliance with protocols approved by the University of Alberta’s Animal Care and Use Committee and in accordance of the Canadian Council on Animal Care policies and regulations. Measurement of CE hydrolase activity in the microsomes of Ces1d-expressing McA cells. The supernatants were centrifuged at 425,866 g at 4◦C for 15 min, and the microsomal pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.0), sonicated to release lumenal contents (including Ces1d), and used for the CE hydrolase activity assay. Micelles containing 0.1 μmol cholesteryl[14C]oleate (specific activity 400,000 disintegrations per minute/μmol) were used as the substrate for the assay with 100 ug of microsomal fractions. 4◦C for 45 min and recovery of the clear infranatant under the fat cake, and 100 μg of the cytosolic protein containing HSL was assessed for CE hydrolase activity as described above. Lipase activity in the cell lysate and microsomal fraction was measured utilizing 4-methylumbelliferyl heptanoate as the substrate as described previously [29]. At 48 h after injection, the liver and gallbladder were collected after a 12 h fast
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