Hepatocyte nuclear factor-4 mediates apolipoprotein A-IV transcriptional regulation by fatty acid in newborn swine enterocytes.

Hepatocyte nuclear factor-4alpha (HNF-4alpha) regulates transcription of several genes involved in lipid metabolism, including that of apolipoprotein (apo) A-IV, which is tightly regulated by lipid absorption and enhances enterocyte chylomicron secretion. Studies were performed to define the role of HNF-4alpha in the regulation of apo A-IV gene transcription by dietary fatty acid in neonatal swine small intestine. HNF-4alpha mRNA was expressed in liver > intestine > kidney in suckling, weanling, and weaned pigs. Jejunal HNF-4alpha mRNA and protein and apo A-IV and swine microsomal triglyceride transfer protein (MTP) large subunit mRNA expression were induced in parallel in 2-day-old swine by a 24-h high-fat intraduodenal infusion. In IPEC-1 cells, incubation with oleic acid (OA) resulted in coordinate induction of both HNF-4alpha, apo A-IV, and MTP mRNA, similar to that observed in vivo. When HNF-4alpha expression was driven by doxycycline by using the TET-On system in the absence of OA to observe the effect of HNF-4alpha directly on apo A-IV and MTP mRNA levels in the absence of other factors that might be concomitantly induced by fatty acid absorption, apo A-IV and MTP expression were increased. In luciferase reporter gene assays in IPEC-1 cells using apo A-IV/C-III intergenic region constructs, TET-On-regulated HNF-4alpha expression without OA increased luciferase activity, and incubation with OA did not further increase activity. These data suggest that acute induction of the apo A-IV and MTP genes by dietary lipid in newborn intestine occurs, at least in part, via ligand-independent transactivation by HNF-4alpha that is itself induced by a lipid-mediated mechanism.

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

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.