Overexpression of Apolipoprotein A-IV Enhances Lipid Secretion in IPEC-1 Cells by Increasing Chylomicron Size

Song Lu,Ying Yao,Xiangying Cheng,Sonya Mitchell,Shuangying Leng,Songmei Meng,James W Gallagher,Gregory S Shelness,Gabriel S Morris,James Mahan,Sharon Frase,Charles M Mansbach,Richard B Weinberg,Dennis D Black

doi:10.1074/jbc.m502501200

Abstract

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Highlights

Apolipoprotein A-IV2 is a lipid-binding protein that is expressed predominantly in the mammalian small intestine [1,2,3,4]
We previously demonstrated that when cultured with oleic acid, high level constitutive overexpression and secretion of Apolipoprotein A-IV (apoA-IV) enhanced radiolabeled basolateral triacylglycerol and cholesteryl ester secretion by 4.9-fold, and phospholipid by 2-fold, mainly as lipoproteins in the chylomicron and very low density lipoprotein (VLDL) density range [21]
Doxycycline-regulatable Swine ApoA-I mRNA Expression and Basolateral Secretion of ApoA-I Protein and Labeled Triacylglycerol in IPEC-1 Cells—To determine whether the enhancement of basolateral labeled triacylglycerol secretion was specific for apoA-IV, IPEC-1 cells were transfected with a regulatable porcine apoA-I construct

Summary

Introduction

Apolipoprotein A-IV (apoA-IV)2 is a lipid-binding protein that is expressed predominantly in the mammalian small intestine [1,2,3,4]. RT-PCR analysis of the mRNA from IPEC-1 cells expressing doxycycline-regulatable human apoA-IV and mutants demonstrated a progressive increase in mRNA levels as measured by real-time PCR with increasing medium doxycycline concentration.

Results

Conclusion