Abstract 457: Feedback Regulation of 3-Hydroxy-3-methylglutaryl-coenzyme a Reductase in Livers of Mice

Abstract

Introduction: HMG-CoA reductase is a membrane protein of the endoplasmic reticulum that catalyzes reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, which exert feedback control on HMGCR through multiple mechanisms. These mechanisms ensure constant synthesis of essential nonsterol isoprenoids, while avoiding toxic cholesterol accumulation. One mechanism involves sterol-induced ubiquitination of HMGCR, marking the enzyme for degradation from ER membranes, a process augmented by nonsterol isoprenoids. We examine the contribution of sterol-accelerated ubiquitination/degradation to overall regulation of HMGCR in livers of mice. Methods: Forty mice, including 20 wild-type (WT) and 20 knock-in (Ki) mice expressing ubiquitination-resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1, 0.3, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis, and qRT-PCR of genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis. Results: Normalization of mRNA to protein levels indicates that HMGCR Ki livers contain a more HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP1 and its target genes increased, likely due to SREBP1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation. Conclusion: The increase in HMGCR protein relative to mRNA suggests that significant post-transcriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3, 1%), a greater degree of transcriptional control from sterol-mediated inhibition of SREBP2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.