We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2 ؊/؊ mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2 ؊/؊ mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2 ؊/؊ mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7-to 9-day-old PEX2 ؊/؊ mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2 ؊/؊ mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2 ؊/؊ mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2 ؊/؊ mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2 ؊/؊ mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisomedeficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunoXuorescence and cell permeabilization techniques. Our present Wndings clearly show and conWrm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal -oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.

Regulation of hepatic cholesterol biosynthesis, lipogenesis, and insulin signaling intersect at the transcriptional level by control of SREBP and Insig genes. We previously demonstrated that peroxisome-deficient PEX2 ؊/؊ mice activate SREBP-2 pathways but are unable to maintain normal cholesterol homeostasis. In this study, we demonstrate that oral bile acid treatment normalized hepatic and plasma cholesterol levels and hepatic cholesterol synthesis in early postnatal PEX2 mutants, but SREBP-2 and its target gene expressions remained increased. SREBP-2 pathway induction was also observed in neonatal and longer surviving PEX2 mutants, where hepatic cholesterol levels were normal. Abnormal expression patterns for SREBP-1c and Insig-2a, and novel regulation of Insig-2b, further demonstrate that peroxisome deficiency widely affects the regulation of related metabolic pathways. We have provided the first demonstration that peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, especially the integrated stress response mediated by PERK and ATF4 signaling. Our studies suggest a mechanism whereby ER stress leads to dysregulation of the endogenous sterol response mechanism and concordantly activates oxidative stress pathways. Several metabolic derangements in peroxisome-deficient PEX2 ؊/؊ liver are likely to trigger ER stress, including perturbed flux of mevalonate metabolites, altered bile acid homeostasis, changes in fatty acid levels and composition, and oxidative stress.