Coordinate Transcriptional Repression of Liver Fatty Acid-binding Protein and Microsomal Triglyceride Transfer Protein Blocks Hepatic Very Low Density Lipoprotein Secretion without Hepatosteatosis

Nathanael J Spann,Sohye Kang,Andrew C Li,Amelia Z Chen,Elizabeth P Newberry,Nicholas O Davidson,Simon T.Y Hui,Roger A Davis

doi:10.1074/jbc.m607148200

Abstract

Unlike the livers of humans and mice, and most hepatoma cells, which accumulate triglycerides when treated with microsomal triglyceride transfer protein (MTP) inhibitors, L35 rat hepatoma cells do not express MTP and cannot secrete very low density lipoprotein (VLDL), yet they do not accumulate triglyceride. In these studies we show that transcriptional co-repression of the two lipid transfer proteins, liver fatty acid-binding protein (L-FABP) and MTP, which cooperatively shunt fatty acids into de novo synthesized glycerolipids and the transfer of lipids into VLDL, respectively, act together to maintain hepatic lipid homeostasis. FAO rat hepatoma cells express L-FABP and MTP and demonstrate the ability to assemble and secrete VLDL. In contrast, L35 cells, derived as a single cell clone from FAO cells, do not express L-FABP or MTP nor do they assemble and secrete VLDL. We used these hepatoma cells to elucidate how a conserved DR1 promoter element present in the promoters of L-FABP and MTP affects transcription, expression, and VLDL production. In FAO cells, the DR1 elements of both L-FABP and MTP promoters are occupied by peroxisome proliferator-activated receptor alpha-retinoid X receptor alpha (RXRalpha), with which PGC-1beta activates transcription. In contrast, in L35 cells the DR1 elements of both L-FABP and MTP promoters are occupied by chicken ovalbumin upstream promoter transcription factor II, and transcription is diminished. The combined findings indicate that peroxisome proliferator-activated receptor alpha-RXRalpha and PGC-1beta coordinately up-regulate L-FABP and MTP expression, by competing with chicken ovalbumin upstream promoter transcription factor II for the DR1 sites in the proximal promoters of each gene. Additional studies show that ablation of L-FABP prevents hepatic steatosis caused by treating mice with an MTP inhibitor. Our findings show that reducing both L-FABP and MTP is an effective means to reduce VLDL secretion without causing hepatic steatosis.

Highlights

Reducing both L-FABP and MTP is an effective means to reduce VLDL secretion without causing hepatic steatosis
Hepatic production of apolipoprotein B-containing lipoproteins is the major pathway by which essential lipids and fatsoluble nutrients are transported to peripheral tissues for anabolic and energy requirements
Because the L-FABP promoter contains a similar DR1 element (Fig. 1A), we examined if its expression would vary in parallel with expression of MTP

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cells were cultured and transfected as described [37]. FAO cells were obtained as a gift from Franz Simon (University of Colorado). On the day of transfection, cells were transfected with 0.8 ␮g of promoter/luciferase reporter construct and with 6 ng of pRL-CMV plasmid as an internal control for normalization of L-FABP and MTP promoter activities. Reporter Gene Constructs and Expression Vectors—The wild type and mutant rat MTP reporter vectors (Ϫ135/ϩ66) were as described previously [37]. To generate the wild type rat L-FABP reporter vector (Ϫ141/ϩ66), genomic DNA was isolated and purified from FAO cells using the DNeasy tissue kit (Qiagen). For the in vitro mutagenesis, the wild type rat L-FABP (Ϫ141/ϩ66)luciferase reporter vector was used as the template along with two oligonucleotide primers (mutated bases underlined), each complementary to opposite strands of the vector as follows: forward, 5Ј-AAT CGA CAA TCA CTG TGC TAT GGC CTA TAT TT-3Ј; reverse, 5Ј-AAA TAT AGG CCA TAG CAC AGT GAT TGT CGA TT-3Ј.

Chromatin Immunoprecipitation

RESULTS

It should be noted that the vehicle

We also examined whether a

DISCUSSION