Peptide YY stimulates the expression of apolipoprotein A-IV gene in Caco-2 intestinal cells

The effect of peptide YY, a gastrointestinal hormone, on the expression of the apolipoprotein A-IV gene in the intestinal epithelial cell line Caco-2 was examined by semiquantitative RT-PCR followed by Southern hybridization with an inner oligonucleotide probe. Apolipoprotein A-IV mRNA levels were increased in response to peptide YY in a dose- and time-dependent fashion. Western blotting revealed that the exogenous peptide YY increased the intracellular concentration of apolipoprotein A-IV. In contrast, apolipoprotein A-I, B, and C-III mRNA did not respond to peptide YY. Differentiated Caco-2 cells expressed Y1- but not Y2- and Y5-receptor subtype mRNA. The present results suggest that peptide YY modulates apolipoprotein A-IV gene expression, likely via the Y1-receptor subtype in intestinal epithelial cells.     

The effects of probucol on lipoprotein metabolism in the rat

The effects of probucol on liver and intestinal apolipoprotein, LDL-receptor and hepatic lipase gene expression, as well as plasma lipid and apolipoprotein levels and liver lipase activity were evaluated in male rats. Administration of probucol decreased plasma triacylglycerols, without affecting plasma cholesterol. Plasma apo E and apo B concentrations increased after probucol. Since liver and intestinal apo B and apo E mRNA levels remained unchanged, this increase could be attributed to a delayed clearance by the LDL-receptor, whose mRNA levels dropped by 50% in the liver. For the HDL-apolipoproteins, only liver apo A-IV mRNA levels decreased after probucol, which was reflected by a fall of plasma apo A-IV. Neither hepatic lipase activity nor mRNA levels were significantly influenced by probucol.     

Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor alpha

We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated pathways, using a PPARalpha-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPARalpha ligand Wy14,643. In contrast, no effect was detected in the PPARalpha-null mice. Testing of eight main ginsenosides on PPARalpha reporter gene expression indicated that Rf was responsible for the effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPARalpha-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPARalpha.     

Adaptation of intestinal production of apolipoprotein A-IV during chronic feeding of lipid

We examined the effect of daily fat supplementation on intestinal gene expression and protein synthesis and plasma levels of apolipoprotein A-IV (apo A-IV). Rats were fasted overnight and then given intragastric bolus infusion of either saline or fat emulsion after 0, 1, 2, 4, 8, or 16 days of similar daily feedings. Four hours after the final saline or fat infusion, plasma and jejunal mucosa were harvested; plasma levels of apo A-IV, triglycerides, and leptin were measured, as well as mucosal apo A-IV mRNA levels and biosynthesis of apo A-IV protein. In response to fat, plasma apo A-IV showed an initial 40% increase compared with saline-injected control rats; with continued daily fat feeding, the plasma A-IV response showed rapid and progressive diminution such that by 4 days, plasma A-IV was not different between fat- and saline-fed groups. Jejunal mucosal apo A-IV synthesis and mRNA levels also showed time-dependent refractoriness to fat feeding. However, the kinetics of this effect were considerably slower than in the case of plasma, requiring 16 days for completion. There was no correlation between plasma leptin or triglyceride levels and intestinal apo A-IV synthesis or plasma apo A-IV. These results indicate rapid, fat-induced, posttranslational adapation of plasma apo A-IV levels and a slower, but similarly complete pretranslational adaptation of intestinal apo A-IV production, which are independent of plasma levels of leptin.     

Human apolipoprotein A-I forms small high density lipoprotein particles in rats in vivo.

The effects of injection of purified human or rat apolipoprotein (apo) A-I (1.7 mg/100 g body weight) on the size and composition of rat high density lipoprotein (HDL) particles have been investigated. The injection of human apo A-I results in the formation (over a period of 3 to 6 h) of a population of smaller HDL particles resembling human HDL3. This population of smaller particles contains human apo A-I and rat apo A-IV but lacks rat apo A-I and rat apo E. Small HDL3-like particles are not detected in rat plasma following the injection of rat apo A-I. Associated with the injection of either human or rat apo A-I is a gradual increase of plasma cholesterol levels of 20 to 50% (over 24 h) and the appearance of larger HDL particles. The results suggest that the smaller HDL particles in human plasma compared to rat plasma are not simply due to the action of lipid modifying enzymes or lipid transfer proteins but a specific property of human apo A-I.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

Gene and gene-product variation in the apolipoprotein A-I/C-III/A-IV cluster in the Dogrib Indians of the Northwest Territories.

The Dogrib, an Amerindian tribe residing in the Northwest Territories of Canada, were typed for DNA and protein polymorphism at the apolipoprotein A-I/C-III/A-IV gene cluster. Variation was seen at three previously described RFLPs detected with the enzymes SstI, PstI, and XmnI, though frequencies of these polymorphisms differ significantly from those reported in other populations. They exhibit no variation at two previously reported PvuII sites. No variation was seen in the APO A-I or APO A-IV gene products, with the Dogrib showing the most common isoelectric-focusing/immunoblot patterns of other world populations. Haplotype frequencies computed from inferred haplotypes and by maximum likelihood estimation did not differ significantly. The extent of nonrandom association of these sites is highly significant (P less than .00001), though pairwise analysis shows significance between the SstI and XmnI sites only. Levels of fasting triglyceride and fasting total cholesterol were determined for each individual. Analysis of covariance shows that fasting triglyceride levels in women vary significantly with the XmnI genotype. These results suggest that genetic variation at the APO A-I/C-III/A-IV gene cluster may be a useful tool for the study of quantitative lipoprotein variation in the Dogrib.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans. Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency

The extent to which lipid and apolipoprotein (apo) concentrations in tissue fluids are determined by those in plasma in normal humans is not known, as all studies to date have been performed on small numbers of subjects, often with dyslipidemia or lymphedema. Therefore, we quantified lipids, apolipoproteins, high density lipoprotein (HDL) lipids, and non-HDL lipids in prenodal leg lymph from 37 fasted ambulant healthy men. Lymph contained almost no triglycerides, but had higher concentrations of free glycerol than plasma. Unesterified cholesterol (UC), cholesteryl ester (CE), phosphatidylcholine (PC), and sphingomyelin (SPM) concentrations in whole lymph were not significantly correlated with those in plasma. HDL lipids, but not non-HDL lipids, were directly related to those in plasma. Lymph HDLs were enriched in UC. However, as the HDL cholesterol/non-HDL cholesterol ratio in lymph exceeded that in plasma, whole lymph nevertheless had a lower UC/CE ratio than plasma. Lymph also had a significantly higher SPM/PC ratio. The lymph/plasma (L/P) ratios of apolipoproteins were as follows: A-IV > A-I and A-II > C-III and E > B. Comparison with the L/P ratios of seven nonlipoprotein proteins suggested that apoA-IV was predominantly lipid free. Concentrations of apolipoproteins A-II, A-IV, C-III, and E in lymph, but not of apolipoproteins A-I or B, were positively correlated with those in plasma. The L/P ratios of apolipoproteins B, C-III, and E in two subjects with lipoprotein lipase (LPL) deficiency, and of apolipoproteins A-I and A-IV in a subject with lecithin:cholesterol acyltransferase (LCAT) deficiency, were low relative to those in normal subjects. Thus, the concentrations of lipids, apolipoproteins, and lipoproteins in human tissue fluid are determined only in part by their concentrations in plasma. Other factors, including the actions of LPL and LCAT, are at least as important.     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Vitamin A is linked to the expression of the AI-CIII-AIV gene cluster in familial combined hyperlipidemia

There is growing evidence of the capacity of vitamin A to regulate the expression of the genetic region that encodes apolipoproteins (apo) A-I, C-III, and A-IV. This region in turn has been proposed to modulate the expression of hyperlipidemia in the commonest genetic form of dyslipidemia, familial combined hyperlipidemia (FCHL). The hypothesis tested here was whether vitamin A (retinol), by controlling the expression of the AI-CIII-AIV gene cluster, plays a role in modulating the hyperlipidemic phenotype in FCHL. We approached the subject by studying three genetic variants of this region: a C1100-T transition in exon 3 of the apoC-III gene, a G3206-T transversion in exon 4 of the apoC-III gene, and a G-75-A substitution in the promoter region of the apoA-I gene. The association between plasma vitamin A concentrations and differences in the plasma concentrations of apolipoproteins A-I and C-III based on the different genotypes was assessed in 48 FCHL patients and 74 of their normolipidemic relatives. The results indicated that the subjects carrying genetic variants associated with increased concentrations of apoA-I and C-III (C1100-T and G-75-A) also presented increased plasma concentrations of vitamin A. This was only observed among the FCHL patients, which suggested that certain characteristics of these patients contributed to this association. The G3206-T was not associated with changes in either apolipoprotein concentrations or in vitamin A.In summary, we report a relationship between genetically determined elevations of proteins of the AI-CIII-AIV gene cluster and vitamin A in FCHL patients. More studies will be needed to confirm that vitamin A plays a role in FCHL which might also be important for its potential application to therapeutical approaches.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0-500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.     

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.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.