Apolipoprotein A-IV expression is acutely up-regulated 7-fold by a high-fat diet in newborn swine, suggesting a role in lipid absorption. Using an adeno-associated viral (AAV) expression vector, we have developed a stably transfected newborn swine enterocyte cell line that over expresses apo A-IV and have demonstrated an associated 5-fold increase in basolateral secretion of newly synthesized triacylglycerol and cholesteryl ester, as well as a 2-fold increase in phospholipid secretion, predominantly in the chylomicron/VLDL density range. These results suggest a role for apo A-IV in facilitating enterocyte lipoprotein secretion. Further studies are proposed using an AAV vector expression system, tetracycline regulatable gene expression technology and subcellular fractionation techniques to test the main hypothesis that apo A-IV enhances lipid transport in newborn swine enterocytes by allowing the assembly and secretion of larger lipoprotein particles through the normal chylomicron pathway by providing additional particle surface stability. Experiments will be performed to test the following specific hypotheses: a) the enhancement of lipid secretion is due to increased size of secreted lipoproteins, and the particles have a normal complement of other apolipoproteins; b) absorbed fatty acid type (chain length or saturation) has an effect on the lipid secretory enhancement by apo A-IV overexpression; c) Apo A-IV overexpression enhances the secretion of absorbed cholesterol; d) the enhancement of lipid secretion follows a dose-response with the amount of apo A-IV expressed and available for lipoprotein packaging; e) the a-helical regions of the apo A-IV protein are responsible for the enhancement of lipid secretion; and f) overexpressed apo A-IV follows the same secretory pathway as triacylglycerol-rich lipoproteins. Studies of the transcriptional regulation of apo A-IV gene expression in newborn swine small intestine have identified a possible transcriptional repressor that binds to ACCTTC in the proximal promoter during conditions of low fat absorption and becomes unbound during high fat absorption. Luciferase reporter transfection studies confirmed the activation of the apo A-IV promoter by fatty acid in this region with additional participation by HNF-4 in cotransfection experiments. Further studies are proposed to test the main hypothesis that dietary lipid activates apo A-IV gene expression in newborn swine small intestine by stimulating the transactivation of specific lipid-responsive regions of the apo A-IV/C-III intergenic region. The following specific hypotheses will be tested: a) the protein that binds to the ACCTTC motif in the proximal apo A-IV promoter is a unique transcription factor and functions as a repressor of apo A-IV transcription in the absence of lipid absorption; b) lipid-responsive elements in the swine apo A-IV/C-III intergenic region participate in the transcriptional activation of the apo A-IV gene by lipid; c) The nuclear hormone receptor HNF-4 is important in fatty acid-mediated regulation of the apo A-IV gene. The results of these studies will provide information to guide the optimal provision of dietary lipid to the neonate.
| Yao, Ying; Lu, Song; Huang, Yue et al. (2011) Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 300:G357-63 |
| Lu, Song; Yao, Ying; Wang, Heng et al. (2003) Regulation of apo A-IV transcription by lipid in newborn swine is associated with a promoter DNA-binding protein. Am J Physiol Gastrointest Liver Physiol 284:G248-54 |
| Wang, H; Hunter, F; Black, D D (1998) Effect of feeding diets of varying fatty acid composition on apolipoprotein expression in newborn swine. Am J Physiol 275:G645-51 |
| Black, D D; Wang, H; Hunter, F et al. (1996) Intestinal expression of apolipoprotein A-IV and C-III is coordinately regulated by dietary lipid in newborn swine. Biochem Biophys Res Commun 221:619-24 |
| Wang, H; Zhan, R; Hunter, F et al. (1996) Effect of acute feeding of diets of varying fatty acid composition on intestinal apolipoprotein expression in the newborn swine. Pediatr Res 39:1078-84 |
