Diabetes Alters the Occupancy of the Hepatic 3-Hydroxy-3-methylglutaryl-CoA Reductase Promoter

William R Lagor,Eric D De Groh,Gene C Ness

doi:10.1074/jbc.m504346200

William R Lagor, Eric D De Groh + Show 1 more

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https://doi.org/10.1074/jbc.m504346200

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Abstract

Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) protein and mRNA are substantially decreased in diabetic animals and rapidly restored by the administration of insulin. To begin to examine the underlying molecular mechanisms, measurements of transcription by nuclear run-on assays and an investigation of occupancy of the promoter were performed. The rate of transcription was substantially reduced in the diabetic rats and fully restored within 2 h after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The cAMP-response element was heavily protected in all conditions, including diabetes, feeding of dietary cholesterol, or statin treatment. Striking enhancements in footprints from diabetic animals were visible at -142 and at -161 (in the sterol-response element). Protections at a newly identified NF-Y site at -70/-71 were observed in normal animals and not in diabetics. This NF-Y site was found to be required for efficient HMGR transcription in luciferase assays. CREB-1 was able to bind the HMGR cAMP-response element in vitro and the promoter in vivo. This evidence supports an essential role for cAMP-response element-binding protein in transcription of hepatic HMGR and identifies at least two sites where in vivo occupancy is regulated by insulin.

Highlights

Hepatic HMG-CoA reductase is responsible for the majority of the regulatable cholesterol synthesis in the body
Nuclear run-on assays were performed to determine whether insulin acts to increase transcription of the HMG-CoA reductase gene
HMG-CoA reductase transcription was greatly diminished in the diabetic animals (Fig. 1A)

Summary

Introduction

Hepatic HMG-CoA reductase is responsible for the majority of the regulatable cholesterol synthesis in the body. The hamster HMG-CoA reductase gene requires about 300 bp of sequence upstream of the transcription start site for high level expression [8] This proximal promoter was found to contain sequences sufficient for sterol regulation in cultured cells [9] and shares about 90% sequence identity with the rat promoter (GenBankTM accession number S78687 [10]). Because of the problems inherent in a cell culture model, especially for a gene that is sterol-sensitive, we decided to perform in vivo footprinting in rat liver. This approach allows for a complete unbiased survey of the HMGR promoter. In this report we show that diabetes alters the occupancy of the HMG-CoA reductase promoter in live animals

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