Feedback Regulation of HMG‐CoA Reductase in Livers of Mice

Abstract

IntroductionHMG‐CoA reductase is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG‐CoA to mevalonate, a rate‐limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One of these mechanisms involves sterol‐induced ubiquitination of HMGCR, which marks the enzyme for degradation from ER membranes that is augmented by nonsterol isoprenoids. In this study, we examine the contribution of this sterol‐accelerated ubiquitination/degradation to overall regulation of HMGCR in the livers of mice.MethodsForty mice, including 20 wild‐type (WT) and 20 knock‐in (Ki) mice that express 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 of six proteins, and qRTPCR of 26 genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis.ResultsNormalization of mRNA levels to protein levels indicates that HMGCR Ki mouse livers contain a higher level of HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP‐2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP‐1 and its target genes increased, likely due to SREBP‐1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation.ConclusionThe increase in HMGCR protein relative to mRNA suggests that significant posttranscriptional 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 SREBP‐2 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.Support or Funding InformationNIH grants HL020948, GM112409, Ruth L. Kirschstein National Research Service Award (NRSA) Short‐Term Institutional Research Training Grant (T‐35)