Regulation of apolipoprotein A-IV gene expression in rat liver: Effects of polyunsaturated fatty acids, obesity and insulin resistance

Apolipoprotein A-IV (apoA-IV) is a protein component of triglyceride-rich lipoproteins, high-density lipoproteins and of the lipoprotein-free plasma fraction. ApoA-IV confers protection against atherosclerosis by participating in reverse cholesterol transport from peripheral tissues to the liver, and may function as a satiety factor. Polymorphisms in the complex containing the apoA-I, C-III and A-IV genes are associated with the response of plasma lipid levels to diet, with hyperlipoproteinemia and with coronary artery disease. In diabetic patients apoA- IV is increased and correlated to the atherosclerotic complications in large vessels and to the degree of metabolic control. ApoA-IV is subject to nutritional and hormonal regulation. In earlier work we found that apoA-IV gene transcription in rat liver is suppressed by dietary omega-3 polyunsaturated fatty acids (PUFA) contained in a lipid lowering fish oil diet. In genetically obese insulin resistant rats (Zucker fatty rats) the expression of apoA-IV was increased and the apoA-IV gene was insensitive to suppression by dietary PUFA. Therefore this model seems suitable for studying the effects of PUFA, obesity and insulin resistance on the apolipoprotein genes. The objective of this proposal are-: 1) To gain insight into the regulation of the apoA-IV gene by PUFA. To determine whether PUFA reduce hepatic apoA-IV gene transcription by a direct effect on hepatocytes, primary rat hepatocyte cultures will be incubated with PUFA and the abundance, of the primary apoA-IV transcript will be measured. To locate PUFA response elements, apoA-IV-Iuciferase reporter gene constructs will be transiently transfected into primary hepatocyte cultures incubated with PUFA and subjected to deletion mapping. DNA-protein interactions and the trans factors binding at PUFA response regions will be studied. 2) To characterize the post-transcriptional mechanisms causing overexpression of apoA-IV in the liver of obese rats. The abundance and the kinetics of the nuclear apoA-IV mRNA precursors will be studied by reverse transcription -PCR and ribonuclease protection assays. 3) To study the regulation of apoA-IV gene expression by insulin. Primary rat hepatocyte cultures will be incubated with insulin/glucose. To analyze transcriptional effects the abundance of the primary apoA-IV transcript will be determined, and transfection experiments will be carried out. Additionally post-transcriptional effects of insulin will be studied. 4) To determine whether insulin resistance is responsible for the overexpression and lacking regulation of ApoA-IV by PUFA in obese rats. For this purpose insulin resistance will be alleviated in Zucker fatty rats by treatment with an insulin sensitizing thiazolidine drug (rosiglitazone). It will be determined whether the improvement in insulin sensitivity can correct the post-transcriptional overexpression of apoA-IV and restore the sensitivity of apoA-IV to

Apolipoprotein A-IV (apoA-IV) is a lipid transporting plasma protein, which protects against atherosclerosis. ApoA-IV is regulated by dietary fatty acids and by hormones. Recently a closely related gene, designated as apoA- V was discovered in the vicinity of apoA-IV. ApoA-V lowers plasma triglycerides and its variation contributes to lipid disorders in man. Our aims were to characterize the regulation of the apoA-IV and apoA-V genes in rat liver by polyunsaturated fatty acids (PUFA) insulin, insulin resistance and obesity. We found that dietary PUFA contained in a fish oil diet lower plasma trigycerides and suppress the activity of the apoA-IV gene in rat liver within hours. This effect could not be reproduced in tissue culture suggesting that it may require extra cellular metabolism of fatty acids or signals from other cells. The expression of the apoA-V gene did not change. Thus, apoA-V does not appear to contribute to the lipid lowering by dietary PUFA. To study the effects of resistance to insulin, a common abnormality in obesity, insulin resistance was alleviated in genetically obese Zucker fatty rats by rosiglitazone, a drug used in the treatment of human diabetes. Rosiglitazone normalized the increased expression of apoA-IV in these rats. The expression of apoA-V in obese rats did not differ from lean animals. Rosiglitazone lowered plasma triglycerides in Zucker fatty rats while liver apoA-V mRNA increased. These findings sugges that apoA-V may not be involved in the elevation of triglycerides seen in Zucker fatty rats, but may contribute to the lipid lowering effect of rosiglitazone. This may be of interest for the development of triglyceride lowering substances. Furthermore, we extended our studies on apoA-IV to human obesity. Our aim was to determine whether the increase in plasma apoA-IV described in obese patients correlates with the degree of overweight and to analyze the effect of weight reduction on plasma apoA-IV. In a group of obese adolescents we found that the plasma apoA-IV concentration decreased almost two fold during 3 weeks of weight reduction. Moreover, the distribution of apoA- IV in plasma changed. The mechanisms underlying this dramatic decrease remain to be clarified. ApoA-IV levels before and after weight reduction and the changes in plasma apoA-IV were not related to the degree of overweight or weight loss.