Mouse apolipoprotein A-IV gene: nucleotide sequence and induction by a high-lipid diet.

Apolipoprotein A-IV (apo A-IV) functions in conjunction with other apolipoproteins to form lipoprotein particles which are involved in lipid homeostasis. In this report we present the nucleotide sequence of the mouse apo A-IV gene and demonstrate its induction in the liver by chronically high dietary lipid. The apo A-IV gene consists of three exons and two introns. The introns separate evolutionarily conserved and functional polypeptide domains. Intron 1 divides most of the apo A-IV signal peptide from the amino terminus of the mature plasma protein. The second intron separates a highly evolutionarily conserved, variant amphipathic peptide repeat from the remainder of the mature apo A-IV protein. The 5' flanking region has several interesting features. The apo A-IV gene has variant TATA and CAT box sequences, TTTAAA and CCAACG, respectively. There are five G-rich direct repeats of 10 nucleotides and a short inverted repeat in the 5' flanking region. We speculate that these sequence elements in the 5' flanking region may be involved in the regulation of apo A-IV gene expression. We also show that chronically high dietary lipid induces liver apo A-IV levels 10-fold in C57BL/6 mice, a strain susceptible to atherosclerotic lesions, while we observed no induction in nonsusceptible BALB/c and C3H mice.     

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 microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.     

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Gastrointestinal satiety signals IV. Apolipoprotein A-IV

The focus of this article is to review evidence that apolipoprotein A-IV (apo A-IV) acts as a satiety factor. Additionally, information regarding the general involvement of apo A-IV in the regulation of food intake and body weight is stated. Apo A-IV is a glycoprotein synthesized by the human intestine. In rodents, both the small intestine and liver secrete apo A-IV, but the small intestine is the major organ responsible for circulating apo A-IV. There is now solid evidence that the hypothalamus, especially the arcuate nucleus, is another active site of apo A-IV expression. Intestinal apo A-IV synthesis is markedly stimulated by fat absorption and does not appear to be mediated by the uptake or reesterification of fatty acids to form triglycerides. Rather, the local formation of chylomicrons acts as a signal for the induction of intestinal apo A-IV synthesis. Intestinal apo A-IV synthesis is also enhanced by a factor from the ileum, probably peptide tyrosine-tyrosine (PYY). The inhibition of food intake by apo A-IV is mediated centrally. The stimulation of intestinal synthesis and secretion of apo A-IV by lipid absorption are rapid; thus apo A-IV likely plays a role in the short-term regulation of food intake. Other evidence suggests that apo A-IV may also be involved in the long-term regulation of food intake and body weight, as it is regulated by both leptin and insulin. Chronic ingestion of a high-fat diet blunts the intestinal as well as the hypothalamic apo A-IV response to lipid feeding. It also suppresses apo A-IV gene expression in the hypothalamus. Whereas it is tempting to speculate that apo A-IV may play a role in diet-induced obesity, we believe the confirmation of such a proposal awaits further experimental evidence.     

Apo A-IV: an update on regulation and physiologic functions

Apolipoprotein (apo) A-IV, first identified 28 years ago as a plasma lipoprotein moiety, is now known to participate in the regulation of various metabolic pathways. It is synthesized primarily in the enterocytes of the small intestine during fat absorption. After entry into the bloodstream, the 46-kDa glycoprotein apo A-IV appears associated with chylomicrons, high-density lipoproteins, and in the lipoprotein-free fraction. It has a role in lipid absorption, transport and metabolism, and may act as a post-prandial satiety signal, an anti-oxidant and a major factor in the prevention of atherosclerosis. After summarizing and discussing these functions for reader's comprehension, the current review focuses on the regulation of apo A-IV by nutrients, biliary components, drugs, hormones and gastrointestinal peptides. The understanding of the involved mechanisms that underline apo A-IV regulation may in the long run allow us to switch on its gene, which may confer multiple beneficial effects, including the protection from atherosclerosis.     

Nonsynonymous polymorphic sites in the apolipoprotein (apo) A-IV gene are associated with changes in the concentration of apo B- and apo A-I-containing lipoproteins in a normal population.

The aims of this study were to detect polymorphic sites in the apolipoprotein (apo) A-IV gene, to establish their frequencies, to determine potential haplotypes, and to investigate the role of these polymorphisms in lipid metabolism. A sequencing study of four individuals led to the identification of two synonymous mutations (codons 9 and 54) and three nonsynonymous mutations (Val-8----Met, Gln360----His, and Thr347----Ser) and of a VNTR polymorphism within a series of three or four CTGT repeats in the noncoding region of exon 3. Frequencies of these polymorphisms were determined in 291 students by using naturally occurring (BstEII for the synonymous mutation in codon 54, HinfI for Thr347----Ser, and Fnu4HI for Gln360----His) or artificially introduced restriction-enzyme cutting sites (BstEII for the synonymous mutation in codon 9 and MamI for Val-8----Met), subsequent to PCR amplification. The four-base deletion/insertion polymorphism and its localization cis or trans to the mutations in codons 347 and 360 were studied by direct sequencing of PCR-amplified DNA from 87 students. Frequencies of the rarer alleles were .007 for apo A-IV-8:Met, .04 for the synonymous mutation in codon 9, .14 for the synonymous mutation in codon 54, .16 for apo A-IV347:Ser, .07 for apo A-IV360:His, and .39 for the four-base of insertion. Apo A-IV360:His in all cases was cis-localized to the (CTGT)3 repeat and apo A-IV347:Thr; and apo A-IV347:Ser was cis-localized to the (CTGT)4 repeat and apo A-IV360:Gln. Four haplotypes formed from these three polymorphic sites were thus found. The apo A-IV347:Ser allele was associated both with significantly lower plasma apo B concentrations in both sexes and with significantly lower LDL-cholesterol concentrations in men. Heterozygous carriers of apo A-IV360:His exhibited significantly higher concentrations of LDL-cholesterol and lower Lp(a) concentrations, compared with apo A-IV360:Gln homozygotes. We could not confirm the previously reported association of apo A-IV360:His with elevated HDL-cholesterol concentrations. In the population, the Val-8----Met polymorphism was not associated with significantly different lipid concentrations, but in a family study the Met-8 allele was associated with lower HDL-cholesterol and higher LDL-cholesterol concentrations. In conclusion, our results indicate an important role of the apo A-IV gene locus in the metabolism of apo B and, to a lesser extent, apo A-I containing lipoproteins.     

Human apolipoprotein A-IV polymorphism: frequency and effect on lipid and lipoprotein levels

Summary Human apolipoprotein (apo) A-IV is genetically polymorphic, the apo A-IV polymorphism being controlled by two common alleles, A-IV¹ and A-IV². We have developed a method for typing the apo A-IV polymorphism by Western blotting using polyclonal rabbit antiapo A-IV as the first and gold-labeled antirabbit IgG as the second antibody. Apolipoprotein phenotypes were determined in plasma samples from 473 tiroleans. The frequencies of the apo A-IV alleles in this sample were f(A-IV¹)=0.919, f(A-IV²)=0.077, and f(A-IV³)=0.004. Although average triglyceride levels were lower in apo A-IV 2-1 heterozygotes, average total serum cholesterol and triglyceride levels were not significantly different among apo A-IV types. High density lipoprotein (HDL) cholesterol was significantly increased in individuals with the A-IV 2-1 phenotype. We estimate that genetic variation at the apo A-IV gene locus accounts for 11% of the total variability in HDL-cholesterol levels in Tiroleans. The effects of the apo A-IV polymorphism described here are consistant with, and may serve to enrich, our limited knowledge of the role of apo A-IV in lipid metabolism.     

Apolipoprotein A-IV stimulates duodenal vagal afferent activity to inhibit gastric motility via a CCK1 pathway

Apolipoprotein A-IV (apo A-IV), a peptide expressed by enterocytes in the mammalian small intestine and released in response to long-chain triglyceride absorption, may be involved in the regulation of gastric acid secretion and gastric motility. The specific aim of the present study was to determine the pathway involved in mediating inhibition of gastric motility produced by apo A-IV. Gastric motility was measured manometrically in response to injections of either recombinant purified apo A-IV (200 microg) or apo A-I, the structurally similar intestinal apolipoprotein not regulated by triglyceride absorption, close to the upper gastrointestinal tract in urethane-anesthetized rats. Injection of apo A-IV significantly inhibited gastric motility compared with apo A-I or vehicle injections. The response to exogenous apo A-IV injections was significantly reduced by 77 and 55%, respectively, in rats treated with the CCK(1) receptor blocker devazepide or after functional vagal deafferentation by perineural capsaicin treatment. In electrophysiological experiments, isolated proximal duodenal vagal afferent fibers were recorded in vitro in response to close-arterial injection of vehicle, apo A-IV (200 microg), or CCK (10 pmol). Apo A-IV stimulated the discharge of duodenal vagal afferent fibers, significantly increasing the discharge in 4/7 CCK-responsive units, and the response was abolished by CCK(1) receptor blockade with devazepide. These data suggest that apo A-IV released from the intestinal mucosa during lipid absorption stimulates the release of endogenous CCK that activates CCK(1) receptors on vagal afferent nerve terminals initiating feedback inhibition of gastric motility.     

Obesity induced by a high-fat diet downregulates apolipoprotein A-IV gene expression in rat hypothalamus

Apolipoprotein A-IV (apo A-IV) is an anorectic protein produced in the intestine and brain that has been proposed as a satiety signal. To determine whether diet-induced obesity alters apo A-IV gene expression in the intestine and hypothalamus, rats were fed a high-fat (HF), low-fat (LF), or standard chow (CHOW) diet for 2, 4, 6, 8, or 10 wk. Rats fed the HF diet had significantly greater body weights than rats given the LF and CHOW diets. Intestinal and plasma apo A-IV levels were comparable across dietary groups and time. LF and CHOW rats had comparable hypothalamic apo A-IV mRNA across the course of the experiment. However, HF rats had a slow and progressive diminution in hypothalamic apo A-IV mRNA over time that became significantly lower than that of LF or CHOW rats by 10 wk. Intragastric infusion of lipid emulsion to animals that were fasted overnight significantly stimulated hypothalamic apo A-IV mRNA in LF and CHOW rats but had no effect in HF rats. These results demonstrate that chronic consumption of a HF diet significantly reduces apo A-IV mRNA levels and the response of apo A-IV gene expression to dietary lipids in the hypothalamus. This raises the possibility that dysregulation of hypothalamic apo A-IV could contribute to diet-induced obesity.     

Diurnal changes in intestinal apolipoprotein A-IV and its relation to food intake and corticosterone in rats

To further investigate the role of intestinal aplipoprotein A-IV (apo A-IV) in the management of daily food intake, we examined the diurnal patterns in apo A-IV gene and protein expression in freely feeding (FF) and food-restricted (FR; food provided 4 h daily for 4 wk) rats that were killed at 3-h intervals throughout the 24-h diurnal cycle. In FF rats, the intestinal apo A-IV mRNA and protein levels showed a circadian rhythm concomitant with the feeding pattern. The daily pattern of fluctuation of apo A-IV, however, was altered in FR rats, which had a marked increase in intestinal apo A-IV levels during the 4-h feeding period of light phase. In both FF and FR rats, increased plasma corticosterone (Cort) levels temporally coincided with the increasing phase of intestinal apo A-IV mRNA and protein expression. Depletion of Cort by adrenalectomy abolished the diurnal rhythm by decreasing the apo A-IV expression during the dark period but did not change the feeding rhythm. Exposure of adrenalectomized rats to consistent Cort level (50-mg continuous release Cort pellet) resulted in fixed apo A-IV levels throughout the day. These results indicate that intestinal apo A-IV exhibits a diurnal rhythm, which can be regulated by endogenous Cort independently of the light-dark cue. The fact that intestinal apo A-IV levels were consistent with the food intake during the normal diurnal cycle as well as during the cycle of 4-h feeding each day suggests that intestinal apo A-IV is involved in the regulation of daily food intake.     

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.