Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Apolipoprotein A-IV interacts synergistically with melanocortins to reduce food intake.

Apolipoprotein (apo) A-IV is an anorexigenic gastrointestinal peptide that is also synthesized in the hypothalamus. The goal of these experiments was to determine whether apo A-IV interacts with the central melanocortin (MC) system in the control of feeding. The third ventricular (i3vt) administration of a subthreshold dose of apo A-IV (0.5 microg) potentiated i3vt MC-induced (metallothionein-II, 0.03 nmol) suppression of 30-min feeding in Long-Evans rats. A subthreshold dose of the MC antagonist (SHU9119, 0.1 nmol, i3vt) completely attenuated the anorectic effect of i3vt apo A-IV (1.5 microg). The i3vt apo A-IV significantly elevated the expression of c-Fos in neurons of the paraventricular nucleus of the hypothalamus, but not in the arcuate nucleus or median eminence. In addition, c-Fos expression was not colocalized with proopiomelanocortin-positive neurons. These data support a synergistic interaction between apo A-IV and melanocortins that reduces food intake by acting downstream of the arcuate.

The role of apolipoprotein AIV on the control of food intake.

Apolipoprotein AIV (apo AIV) is a protein synthesized by the human intestine. The synthesis and secretion of apo AIV are stimulated by fat absorption. In 1992, Fujimoto et al. [1] first demonstrated that apo AIV is a satiety signal secreted by the small intestine following the ingestion of a lipid meal. This initial observation was followed by a number of studies supporting apo AIV's role as a satiety signal. This review article discusses the regulation of synthesis of apo AIV in the small intestine as well as the hypothalamus. In addition, the evidence that apo AIV is a satiety factor and its role of apo AIV in diet induced obesity will be discussed. We hope this review will serve as a catalyst to promote apo AIV research in the future. With most of the required reagents available, e.g., the apo AIV knockout and transgenic animals and apo AIV antibodies, the next few years should bring considerable new information on the function of apo AIV.

Ingested fat and satiety.

Apolipoprotein A-IV (apo A-IV) is secreted by the intestine associated with chylomicron. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. The stimulation of apo A-IV synthesis in the jejunum and ileum is attenuated by intravenous leptin infusion. Intestinal apo A-IV synthesis is also stimulated by a factor from the ileum, probably peptide tyrosine-tyrosine (PYY), which has been demonstrated to affect satiety. Apo A-IV has been proposed to physiologically control food intake, and this inhibitory effect is centrally mediated. Recently, apo A-IV was demonstrated in the hypothalamus. The 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 normally limited by endogenous apo A-IV. Central administration of neuropeptide Y (NPY) significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner. The stimulation of intestinal synthesis and secretion of apo A-IV by lipid absorption are rapid; thus, apo A-IV is capable of short-term regulation of food intake. Evidence also suggests apo A-IV's involvement in long-term regulation of food intake and bodyweight. The chronic ingestion of high fat blunts the intestinal apo A-IV response to lipid feeding and may therefore explain why chronic intake of high fat predisposes animals and humans to obesity.

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.

Intestinal satiety protein apolipoprotein AIV is synthesized and regulated in rat hypothalamus.

Apolipoprotein AIV (apo AIV) is a satiety protein secreted by the small intestine. We demonstrate for the first time that apo AIV protein and apo AIV mRNA are present in rat hypothalamus, a site intimately involved in the integration of signals for regulation of food intake and energy metabolism. We further characterized the regulation of hypothalamic apo AIV mRNA levels. Food-deprived animals showed a pronounced decrease in gene expression of apo AIV in the hypothalamus, with a concomitant decrease in the jejunum. Refeeding fasted rats with standard laboratory chow for 4 h evokes a significant increase of apo AIV mRNA in jejunum but not in hypothalamus. However, lipid refeeding to the fasted animals restored apo AIV mRNA levels both in hypothalamus and jejunum. Intracerebroventricular administration of apo AIV antiserum not only stimulated feeding, but also decreased apo AIV mRNA level in the hypothalamus. These data further confirm the central role of apo AIV in the regulation of food intake.

Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.

Studies of the structure and function of apolipoprotein A-I (apoA-I) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoA-I from other plasma apolipoproteins. To reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoA-I. The recombinant baculovirus containing full-length human apoA-I cDNA, when introduced into Spodoptera frugiperda, directs the synthesis of preproapoA-I, which is subsequently secreted into the growth medium as proapoA-I, indicating correct processing of the signal peptide during secretion. To prevent the extensive degradation of secreted proapoA-I, leupeptin and pepstatin A were added to the serum free cell culture medium. The protein was simply purified by filtration of the medium, which contained up to 80 mg/l proapoA-I, followed by chromatography on phenyl-sepharose CL-4B. The resultant proapoA-I was found to bind lipid and to activate lecithin:cholesterol acyltransferase as effectively as apoA-I from human plasma. The advantage of this expression system is the ease of purification of intact, biologically active apoA-I in high yield.