Regulation of the human apolipoprotein AIV gene expression in transgenic mice

The apolipoprotein (Apo) AI-CIII-AIV gene cluster has a complex pattern of gene expression that is modulated by both gene- and cluster-specific cis-acting elements. In particular the regulation of Apo AIV expression has been previously studied in vivo and in vitro including several transgenic mouse lines but a complete, consistent picture of the tissue-specific controls is still missing. We have analysed the role of the Apo AIV 3' flanking sequences in the regulation of gene expression using both in vitro and in vivo systems including three lines of transgenic mice. The transgene consisted of a human fragment containing 7 kb of the 5' flanking region, the Apo AIV gene itself and 6 kb of the 3' flanking region (-7+6 Apo AIV). Accurate analysis of the Apo AIV mRNA levels using quantitative PCR and Northern blots showed that the 7+6 kb Apo AIV fragment confers liver-specific regulation in that the human Apo AIV transgene is expressed at approximately the same level as the endogenous mouse Apo AIV gene. In contrast, the intestinal regulation of the transgene did not follow, the pattern observed with the endogenous gene although it produced a much higher intestinal expression following the accepted human pattern. Therefore, this animal model provides an excellent substrate to design therapeutic protocols for those metabolic derangements that may benefit from variations in Apo AIV levels and its anti-atherogenic effect.     

Regulation of intestinal and hypothalamic apolipoprotein A-IV

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.     

Regulation of apolipoprotein A1 synthesis in avian muscles

Until recently, liver and intestinal mucosa were believed to be the sole sites of synthesis of apolipoprotein A1 (Apo-A1), the major protein component of serum high density lipoprotein particles. We recently showed (Shackelford, J.E., and Lebherz, H.G. (1983) J. Biol. Chem. 258, 7175-7180) that chick breast muscle also synthesizes and secretes Apo-A1 but does so at high rates only around the time of hatching. In the present work, we investigate the regulation of synthesis of Apo-A1 in chicken muscles. 1) The primary translation product encoded for by muscle Apo-A1 mRNA is about 2600 daltons larger than the mature serum protein which is consistent with the idea that, like its mammalian liver counterpart, chick muscle Apo-A1 mRNA codes for an NH2-terminal extension (prepro segment) which is 24 amino acids long. 2) The developmentally regulated rise and fall in muscle Apo-A1 synthesis which occurs around the time of hatching is associated with a large accumulation followed by depletion of Apo-A1 mRNA molecules during this period. 3) Reinitiation of Apo-A1 synthesis to high levels in mature breast muscle occurred in vivo following surgical denervation and in vitro by maintaining breast muscle explants for several days in defined culture media. 4) Cardiac, but not smooth, muscles also synthesize and secrete Apo-A1 at high levels around the time of hatching. These and other observations are discussed in terms of possible regulatory "signals" which may control Apo-A1 synthesis in avian muscles.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.     

Regulation of apolipoprotein E synthesis in rat ovarian granulosa cells

Apoprotein E (apo-E) is a surface component of several classes of plasma lipoproteins. It functions as a ligand for receptor-mediated uptake of lipoproteins. Granulosa cells from ovaries of diethylstilbestrol-stimulated hypophysectomized immature rats cultured in serum-free medium with [35S]methionine secretes a 34-kDa protein which reacts with a monospecific anti-rat apo-E antibody and represents 0.2% of total secreted protein. Protease mapping confirms that this protein is apoprotein E. The secreted apoprotein E may be complexed with lipid since it floats in the ultracentrifuge at density less than 1.21 micrograms/ml. Freshly isolated granulosa cells contain receptors for follicle stimulating hormone (FSH) but not for human chorionic gonadotropin (hCG) or prolactin. Apoprotein E secretion is stimulated 2-fold by FSH, but hCG and prolactin have no effect. When granulosa cells develop hCG and prolactin receptors after 48 h of culture with FSH, apoprotein E secretion is not stimulated by addition of FSH, hCG, or prolactin although steroidogenesis is induced. The addition of 10(-7) M androgen plus FSH stimulates a marked increase in progestin synthesis over FSH alone, but androgen has little added effect on apoprotein E secretion. Cholera toxin (1.25 micrograms/ml) and dibutyryl cAMP (5 mg/ml), both of which increase intracellular cAMP, stimulate apo-E secretion 9-fold and 12-fold, respectively. The dibutyryl cAMP effect is dependent on both dose (greater than or equal to 0.5 mg/ml required) and time (onset at 24 h, maximum at 48 h, and back to near baseline at 96 h). Isobutylmethylxanthine, a phosphodiesterase inhibitor, augments FSH-stimulated apoprotein E synthesis 2.5-fold, supporting a role for cAMP in mediating the FSH effect. This is the first demonstration of the hormonal regulation of apoprotein E synthesis in an extrahepatic tissue.     

Supermolecular organization of apolipoprotein E in solution]

The aggregation behaviour and stability in solution of apolipoprotein E (apoE) isolated from human blood plasma very low density lipoproteins were investigated. The equilibrium denaturation of fluorescein-labeled apoE by guanidine-hydrochloride determined by anisotropy and overall intensity of fluorescence, shift of the emission spectrum maximum and gel-chromatographic behaviour was characterized by reversibility, biphasity, apoE concentration dependence and the existence of native structure of the apoE monomer. The contribution of the long-living component to the kinetic dependence of fluorescence anisotropy in the presence of the 6 M denaturant increased with an increase in apoE concentration. The data obtained fit into the following scheme: oligomer (upon aging of the preparation) in equilibrium tetramer in equilibrium native monomer in equilibrium denaturated monomer. The presence in the tetrameric structure of apoE of two domains is postulated; one of those is formed by lipid-binding fragments during aggregation of individual molecules of apoE. Monoclonal antibody 3D12F11 (subclass IgG1) showed a high affinity for the apoE (Kd = 3.5 +/- 0.5 nM) without any effect on the apoprotein binding to heparin-Sepharose and apoE-induced destruction of dipalmitoylphosphatidylcholine liposomes. It is concluded that the 3D12F11 epitope is localized outside heparin- and lipid-binding sites of the apoprotein molecule.