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.     

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.     

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.     

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.     

Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitis

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the "shotgun" quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ～50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.     

Differential effects of gastric bypass and banding on circulating gut hormone and leptin levels

Objective: To quantify plasma concentrations of hormones that regulate energy homeostasis in order to establish possible mechanisms for greater weight loss after Roux‐en‐Y gastric bypass (RYGBP) compared with gastric banding (BND). Research Methods and Procedures: Four groups of women were studied: lean (n = 8; mean BMI, 21.6 kg/m²); BND (n = 9; BMI, 35.8; 25% weight loss), RYGBP (n = 9; BMI, 34.2; 36% weight loss), and controls matched for BMI to the surgical groups (n = 11; BMI, 34.4). Results: Fasting total peptide YY (PYY) and PYY_(3–36) immunoreactivity were similar among all groups, but the postprandial response in the RYGBP group was exaggerated, such that 30 minutes after the meal, total and PYY_(3–36) levels were 2‐ to 4‐fold greater compared with all other groups. Maximal postprandial suppression of total ghrelin was blunted in the BND group (13%) compared with RYGBP (27%). Postprandial suppression of octanoylated ghrelin was also less in BND (29%) compared with RYGBP (56%). Fasting insulin was lower in RYGBP (6.6 μU/mL) compared with BND (10.0 μU/mL). Compared with lean controls, leptin concentrations were significantly higher in BND but not in RYGBP. There was a greater increase in post‐meal satiety in the RYGBP group compared with BND and overweight controls. Discussion: The differences between RYGBP and BND subjects in postprandial concentrations of PYY and ghrelin would be expected to promote increased satiety and earlier meal termination in RYGBP and may aid in greater weight loss. The differences in insulin and leptin concentrations associated with these procedures may also reflect differences in insulin sensitivity and energy partitioning.     

GLP-1 reduces intestinal lymph flow, triglyceride absorption, and apolipoprotein production in rats

Glucagon-like peptide 1 (GLP-1) is a gastrointestinal hormone secreted in response to meal ingestion by enteroendocrine L cells located predominantly in the lower small intestine and large intestine. GLP-1 inhibits the secretion and motility of the upper gut and has been suggested to play a role in the "ileal brake." In this study, we investigated the effect of recombinant GLP-1-(7-36) amide (rGLP-1) on lipid absorption in the small intestine in intestinal lymph duct-cannulated rats. In addition, the effects of rGLP-1 on intestinal production of apolipoprotein (apo) B and apo A-IV, two apolipoproteins closely related to lipid absorption, were evaluated. rGLP-1 was infused through the jugular vein, and lipids were infused simultaneously through a duodenal cannula. Our results showed that infusion of rGLP-1 at 20 pmol.kg(-1).min(-1) caused a dramatic and prompt decrease in lymph flow from 2.22 +/- 0.15 (SE) ml/h at baseline (n = 6) to 1.24 +/- 0.06 ml/h at 2 h (P < 0.001). In contrast, a significant increase in lymph flow was observed in the saline (control) group: 2.19 +/- 0.20 and 3.48 +/- 0.09 ml/h at baseline and at 6 h of lipid infusion, respectively (P < 0.001). rGLP-1 also inhibited intestinal triolein absorption (P < 0.05) and lymphatic apo B and apo A-IV output (P < 0.05) but did not affect cholesterol absorption. In conclusion, rGLP-1 dramatically decreases intestinal lymph flow and reduces triglyceride absorption and apo B and apo A-IV production. These findings suggest a novel role for GLP-1 in lipid absorption.     

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

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was 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/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Induction of liver apolipoprotein A-IV mRNA in porphyric mice.

We have isolated cDNA clones for mRNAs that are induced by porphyria from a mouse liver library. Of the three inducible clones isolated, we have identified one as being apolipoprotein A-IV (apo A-IV) by its extensive homology with a rat apolipoprotein A-IV cDNA sequence. The level of liver apo A-IV mRNA increases rapidly in response to either of two porphyrogenic drugs. When the ferrochelatase-inhibited drug, 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) is used, a 6 and 28 fold induction of liver apo A-IV mRNA is observed in male and female mice, respectively. If the heme-destroying porphyrogenic drug, allylisopropylacetamide (AIA) is the inducing agent, liver apo A-IV mRNA levels increase 2-3 fold in both males and females. The level of apo A-IV mRNA reaches a maximum within 6-10 hr. after drug administration. Intestine apo A-IV mRNA levels do not change during either of these drug-induced porphyrias. RNA from acute-phase responsive liver or liver from mice treated with bilirubin, porphobilinogen, or protoporphyrin IX show no increase in apo A-IV mRNA. These results indicate that apo A-IV induction is tied to a disruption in porphyrin-heme biosynthesis but is not directly affected by several heme intermediates nor by the major heme degradation product, bilirubin.     

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.     

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.     

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.     

Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.     

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.     

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 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.