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Laboratory of Food Biochemistry, Research Group of Food Science, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589 Japan
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • apolipoprotein A-IV • small intestine • neural blockers • dietary lipid • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although
the precise function of apo A-IV is not known, it has been shown to
modulate lipoprotein metabolism (Chen and Albers 1985
,
Dvorin et al. 1986
, Stein et al. 1986
,
Steinmetz and Utermann 1985
, Steinmetz et al. 1990
), food intake (Fujimoto et al. 1992
and
Fujimoto et al. 1993
) and gastric functions
(Okumura et al. 1994
, and Okumura et al. 1996
). For this reason, information on the expression of
apo A-IV in the small intestine should lead to a better
understanding of the regulation of lipoprotein metabolism, feeding
behavior and gastric functions.
Although a number of reports have demonstrated that the expression,
synthesis and lymphatic output of intestinal apo A-IV were
stimulated by dietary fat (Apfelbaum et al. 1987
,
Gordon et al. 1982
, Hayashi et al. 1990
,
Kalogeris et al. 1996
), the mechanism underlying this
phenomenon is not entirely clear. As dietary lipid stimulates the
release of several gastrointestinal peptide hormones which in turn
regulate feeding behavior and gastrointestinal functions (Aponte et al. 1985
, Jin et al. 1993
), it is possible
that some humoral factors released in response to dietary lipid are
involved in the stimulation of intestinal apo A-IV by lipid. In
fact, Kalogeris et al. (1998)
recently reported that the
synthesis and lymphatic output of apo A-IV protein in rat jejunum
were increased by intravenous infusion of peptide YY (PYY), a
gastrointestinal hormone whose release from enteroendocrine cells in
the distal bowel was stimulated by dietary lipid (Aponte et al. 1985
, Aponte et al. 1989
, Pappas et al. 1985
). This increase in apo A-IV synthesis was not
accompanied by any corresponding changes in mRNA levels
(Kalogeris et al. 1998
). In contrast, we observed that
apo A-IV mRNA levels increased in response to exogenous PYY in a
dose- and time-dependent fashion in differentiated Caco-2
intestinal cells (Sonoyama et al. 2000
). To date, no
experimental evidence has sufficiently explained the above inconsistent
observations. Nevertheless, PYY has been proposed to act as a humoral
factor mediating lipid stimulation of intestinal apo A-IV
synthesis.
Furthermore, experimental evidence suggests the existence of receptors
on intestinal afferent nerves that are capable of responding to
nutrient stimulation (Paintal 1973
). Thus, neural
factors may be other factors that mediate the upregulation of
intestinal apo A-IV synthesis via dietary lipid. To test this
hypothesis, the present study investigated the effect of neural
blockers on the expression of apo A-IV mRNA in the intestine of
unrestrained conscious rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In all experiments, male Wistar rats (Japan SLC, Hamamatsu, Japan),
which were 7-wk-old at the start of the experiments, were housed in
individual cages in a temperature-controlled (23 ± 2°C)
room with a dark period from 1900 to 500 h. The rats were
acclimatized for 3–5 d with free access to water and to a purified
diet consisting of 250 g/kg (24% of total energy) of casein, 650 g/kg
(64% of total energy) of sucrose, 50 g/kg of (12% of total energy)
corn oil, 40 g/kg of mineral mixture, and a 10 g/kg of vitamin mixture
(Sonoyama et al. 1995
). This diet is used as a standard
rat diet in our laboratory because we have found that it yields a
maximal growth rate.
In expt. 1, the effect of intravenous administration of hexamethonium, a ganglionic blocker, on apo mRNA levels in the intestine was investigated. A total of 12 rats was anesthetized by an intraperitoneal injection of Nembutal (sodium pentobarbital 50 mg/kg body weight; Abbott Laboratories, North Chicago, IL). A silicone tube (No. 00; 0.5 mm i.d., 1.0 mm o.d.; Dow Corning Co., Kanagawa, Japan) was inserted into the right cervical vein, and the distal end of the cannula was then exteriorized at the back of the neck and filled with saline containing 1 x 106 IU/L of heparin. After being allowed to recover for 2 d, food-deprived and unrestrained rats were then intravenously infused for 8 h with either hexamethonium bromide [10 mg/(kg · h), 1 mL/h, (Wako Pure Chemical Industries, Osaka, Japan)] or a vehicle (0.15 mol/L NaCl, 1 mL/h) (n = 6 per group). At the end of the infusion period, rats were anesthetized by an intraperitoneal injection of Nembutal. Following laparotomy, rats were killed by bleeding from abdominal aorta, and blood was collected for determination of plasma apo A-IV and apo A-I concentrations. Two 10-cm sections of the intestine were excised, one at 2 cm distal to the ligament of Treitz as the jejunal segment, and the other just proximal to the ileocecal valve as the ileal segment. The luminal contents were then washed with 10 mL of ice-cold saline. The mucosa was scraped with a glass slide and immediately plunged into liquid nitrogen. It was then stored at -80°C for RNA isolation.
In expt. 2, the effect of intravenous hexamethonium on the intestinal apo mRNA was examined in bile-drained rats. In a total of 12 rats, a cannula was inserted into the right cervical vein as described in expt. 1. After being allowed to recover for 2 d, the rats were deprived of food for 18 h and then anesthetized by an intraperitoneal injection of Nembutal. Rats were laparotomized and subjected to bile drainage. To achieve this, the tip of a polyethylene catheter (SP10; 0.28 mm i.d., 0.61 mm o.d.; Natsume Seisakusyo, Tokyo, Japan) was inserted into the common bile-pancreatic duct at a point 5 mm proximal to the ampulla of Vater and connected to a silicone tube (No. 00). The distal end of the cannula was then externalized at the back of the neck, and the bile was drained completely. On the next day of surgery, food-deprived and unrestrained rats were intravenously infused for 8 h with either hexamethonium bromide or a vehicle (n = 6 per group). Following infusion, the intestinal mucosa was isolated and stored as described in expt. 1.
In expt. 3, the effect of propranolol and atropine which are ß-adrenergic and cholinergic blockers, respectively, on the intestinal apolipoprotein mRNA was investigated. In a total of 24 rats, a cannula was inserted into the right cervical vein as described in expt. 1. After being allowed to recover for 2 d, food-deprived and unrestrained rats were intravenously infused for 8 h with either hexamethonium bromide [10 mg/(kg · h), 1 mL/h], propranolol hydrochloride [2 mg/(kg · h), 1 mL/h, Wako Pure Chemical Industries], atropine sulfate monohydrate [0.5 mg/(kg · h), 1 mL/h, Wako], or a vehicle (n = 6 per group). Following infusion, the intestinal mucosa was isolated and stored as described in expt. 1.
In expt. 4, whether stimulation of apo A-IV mRNA expression by
intestinal lipids was affected by an intravenous infusion of
hexamethonium was investigated. In a total of 18 rats, a cannula was
inserted into the right cervical vein as described in expt. 1. In
addition, following laparotomy a silicone tube (No. 00) was inserted
through the fistula into the ileum at a point 10 cm proximal to the
ileocecal valve, and the distal end of the cannula was exteriorized at
the back of the neck. After being allowed to recover for 2 d,
food-deprived and unrestrained rats were intravenously infused for
8 h with either hexamethonium bromide [10 mg/(kg · h), 1
mL/h] or a vehicle under (n = 6 and 12 per
hexamethonium- and vehicle-infused groups, respectively). Of these
18 rats, 6 rats from both the hexamethonium- and vehicle-infused
groups were simulataneously enteraly infused with a lipid emulsion
composed of 20 mmol/L of monoolein, 40 mmol/L oleic acid, 2.21 mmol/L
of phosphatidylcholine and 16.15 mmol/L of sodium taurocholate. The
composition of the lipid emulsion was identical to that reported by
Kalogeris et al. (1996)
. The remaining six rats in the
vehicle-infused group were simultaneously enteraly infused with
glucose-saline solution composed of 145 mmol/L of NaCl, 0.4 mmol/L
of KCl and 0.28 mol/L of glucose. Following infusion, the intestinal
mucosa was isolated and stored as described in expt. 1.
This study was approved by the Hokkaido University Animal Use Committee, and animals were maintained in accordance with the guidelines for the care and use of laboratory animals of Hokkaido University.
Isolation and analysis of RNA
Total RNA was isolated from the intestinal mucosa using Isogen (Nippon
Gene, Tokyo, Japan) according to the manufacturer’s protocol. Samples
of total RNA (10 µg/lane) were electrophoresed on denaturing 2.2
mol/L of formaldehyde, a 1% agarose gel, and then transferred to a
nylon membrane (Biodyne Plus, Pall, NY). Blots were hybridized with a
digoxigenin-labeled apo A-IV probe of a 54-base oligonucleotide
as previously described (Sonoyama et al. 1995
).
Prehybridization, hybridization and detection were all carried out with
a DIG luminescence detection kit (Boehringer Mannheim, Mannheim,
Germany). The hybridization was performed at 42°C overnight, and
posthybridization washing was performed twice with 0.1 x SSC,
0.1% SDS at 65°C for 15 min. Following detection, each filter was
then sequentially rehybridized with a digoxigenin-labeled apo
A-I probe of a 54-base oligonucleotide (Sonoyama et al. 1995
). The bands were developed on X-ray film and then
quantitated using NIH Image.
Immunoblotting for plasma apo A-IV and apo A-I quantitation
In expt. 1, the immunoblot analysis of plasma was performed for the
semiquantitaion of apo A-IV and apo A-I. Whole plasma were
subjected to 12% SDS-PAGE under reducing conditions
(Laemmli 1970
). Electrophoresed proteins were
electrophoretically transferred to nitrocellulose membrane (Hybond C
extra; Amersham International plc., Amersham, United Kingdom) and
immunostained with the rabbit antihuman apo A-IV IgG (Alpha
Biomedical Laboratories, Bellevue, WA) or the rabbit anti-rat apo
A-I serum (a gift from Dr. Fumihiko Horio, Nagoya University,
Nagoya, Japan) as previously described (Sonoyama et al. 1995
). The relative quantities of apo A-IV and apo A-I
were estimated by using NIH Image.
Statistical analysis
All results were expressed as means ± SEM. Student’s t test was applied to compare the mean values of two groups (expts. 1 and 2). ANOVA and the Tukey-Kramer HSD was applied to compare the mean values among four and three groups in expts. 3 and 4, respectively. All statistical calculations were carried out using JMP computer software (SAS Institute, NC). Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).In contrast, the ileal apo A-IV mRNA levels in the
hexamethonium-infused rats were significantly lower than those in
the vehicle-infused rats. Apo A-I, mRNA levels did not differ
between the groups in either jejunum or ileum. Similar results were
observed in the fed rats (data not shown). Relative concentrations of
plasma apo A-IV and apo A-I did not differ between the two
groups (Fig. 2
).
|
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). In the jejunum, however, the apo A-IV mRNA levels did not differ
between the groups (data not shown). There was no significant
difference in the apo A-I mRNA levels between the two groups in
both jejunum and ileum (data not shown).
|
), propranolol had no effect. Jejunal apo A-IV mRNA levels were
unaffected by these neural blockers, and apo A-I mRNA levels in
both jejunum and ileum also did not differ among the groups (data not
shown).
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). In addition, no significant difference was observed between
hexamethonium- and vehicle-infused rats receiving the ileal lipid
(Fig. 5)
. In the jejunum, no significant differences were observed in
the apo A-IV mRNA levels among the groups (data not shown). Apo
A-I mRNA levels were not affected by any of the treatments in
either the jejunum or ileum (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, despite the lower levels of apo A-IV mRNA in the ileum, the plasma concentrations of apo A-IV in hexamethonium-infused rats were comparable to those in the vehicle-infused rats. This may be due to translational and/or posttranslational regulation of intestinal apo A-IV synthesis and secretion. In addition, the infusion period of hexamethonium may have been too short to detect the decrease in plasma apo A-IV. Furthermore, since the apo A-IV expression is lower in the ileum than in the jejunum, the lower expression of apo A-IV in the ileum of rats infused with hexamethonium may not have contributed greatly to changes in plasma concentrations of apo A-IV.
We previously reported that bile diversion into the colon for 7 d
diminished ileal apo A-IV mRNA levels in rats (Sonoyama et al. 1994
). In addition, we showed that apo A-IV mRNA levels
increased in the residual ileum following massive small bowel resection
in rats, and that the increase was abolished by bile diversion into the
colon (Sonoyama et al. 1996
). Furthermore, we
demonstrated that the bile-pancreatic diversion into the ileum
increased ileal apo A-IV mRNA levels in rats (Sonoyama et al. 1997
). These observations suggest that a biliary component
stimulates the apo A-IV gene expression in the ileum. In the
present study, however, the ileal apo A-IV mRNA levels in
bile-drained rats (expt. 2) were comparable to those in normal rats
(expt. 1). This may be due to the relatively shorter period of
bile-drainage in expt. 2. As the ganglionic blocker hexamethonium
inhibits pancreaticobiliary secretion and intestinal motility, it may
diminish apo A-IV mRNA through a decreased supply of biliary
components to the ileum. In the present study, however, the lower
levels of ileal apo A-IV mRNA in rats infused with hexamethonium
were observed even in bile-drained rats. Thus, the lower expression
of the ileal apo A-IV gene by hexamethonium is not due to any
changes in bile availability in the ileum. In addition, the lower
expression of the ileal apo A-IV mRNA in rats infused with
hexamethonium may be secondary to changes in motility of the intestine,
mediated either through altered luminal pressure, stretch response
within the mucosa, or some other causes. In the present study, however,
we did not determine the motility of the intestine. Therefore further
investigations will be required to clarify the relationship between
intestinal motility and expression of apo A-IV gene.
In the present study, concomitant infusion of a ganglionic blocker did
not suppress the lipid-stimulated expression of apo A-IV mRNA
in the ileum. Thus, neural factors likely are associated with basal but
not lipid-stimulated expression of apo A-IV gene in the ileum.
Kalogeris et al. (1996)
demonstrated that ileal infusion
of lipid emulsion stimulated the apo A-IV synthesis in not only the
ileum but also the proximal jejunum, suggesting the presence of a
signal elicited from the ileum. In addition, Kalogeris et al. (1998)
reported that the synthesis and lymphatic output of apo
A-IV protein in rat jejunum were increased by an intravenous
infusion of PYY, a gastrointestinal peptide hormone whose release from
enteroendocrine cells in the distal bowel was stimulated by dietary
lipid (Aponte et al. 1985
, Aponte et al. 1989
, Pappas 1985
). Furthermore, we observed
that apo A-IV mRNA levels increased in response to exogenous PYY in
a dose- and time-dependent fashion in differentiated Caco-2
intestinal cells (Sonoyama et al. 2000
). These
observations suggest that PYY is a possible ileal-derived humoral
factor stimulating the expression, synthesis and output of apo A-IV
in the intestine. Since experimental evidence suggests that PYY release
from the distal intestine is regulated by neural factors
(Fu-Cheng et al. 1997
), it is of interest to investigate
whether there are some interactions between the humoral and neural
factors in the regulation of the apo A-IV expression in the
intestine.
In the present study, we mentioned only two classical
neurotransmittions, i.e., adrenergic and cholinergic neurons, in regard
to the neural regulation of the apo A-IV gene expression in the
intestine. However other neurotransmitters including dopamine,
5-hydroxytryptamine, histamine, adenosine and adenine nucleotide, and
also nitric oxide have been proposed as regulators of cellular function
in the gastrointestinal tract (Burks 1994
). In addition,
the present study did not reveal whether the neural regulation of
intestinal apo A-IV gene is mediated by the central or enteric
nervous system. Therefore, further studies will be necessary to
elucidate the precise mechanism for the neural regulation of the apo
A-IV expression in the small intestine.
In conclusion, we propose that basal but not lipid-stimulated expression of apo A-IV gene in the small intestine is at least in part regulated by cholinergic neurons in a site-specific manner.
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; PYY, peptide YY.
Manuscript received June 25, 1999. Initial review completed August 9, 1999. Revision accepted November 17, 1999.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.
Apfelbaum T. F., Davidson N. O., Glickman R. M. Apolipoprotein A-IV synthesis in rat intestine: regulation by dietary triglyceride. Am. J. Physiol. 1987;252:G662-G666
2.
Aponte G. W., Fink A. S., Meyer J. H., Tatemoto K., Taylor I. L. Regional distribution and release of peptide YY with fatty acids of different chain length. Am. J. Physiol. 1985;249:G745-G750
3. Aponte G. W., Park K., Hess R., Garcia R., Taylor I. L. Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat. FASEB J 1989;3:1949-1955[Abstract]
4. Burks T. F. Neurotransmission and neurotransmitters. Hohnson L. R. eds. Physiology of the Gastrointestinal Tract Third Edition, Chapt. 4 1994:211-242 Raven Press New York
5. Chen C. H., Albers J. J. Activation of lecithin: cholesterol acyltransferase by apolipoproteins E-2, E-3, and A-IV isolated from human plasma. Biochim. Biophys. Acta 1985;836:279-285[Medline]
6.
Dvorin E., Gordon N. L., Benson D. M., Gotto A. M. J. Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes. J. Biol. Chem. 1986;261:15714-15728
7.
Elshourbagy N. A., Boguski M. S., Liao W. S. L., Jefferson L. S., Gordon J. I., Taylor J. M. Expression of rat apolipoprotein A-IV and A-I genes: mRNA induction during development and in response to glucocorticoids and insulin. Proc. Natl. Acad. Sci. U.S.A. 1985;82:8242-8246
8. Fu-Cheng X., Anini Y., Chariot J., Castex N., Galmiche J.-P., Roze C. Mechanisms of peptide YY release induced by an intraduodenal meal in rats: neural regulation by proximal gut. Eur. J. Physiol. 1997;433:571-579[Medline]
9.
Fujimoto K., Cardelli J. A., Tso P. Increased apolipoprotein A-IV in rat mesenteric lymph after lipid meal acts as a physiological signal for satiation. Am. J. Physiol. 1992;262:G1002-G1006
10. Fujimoto K., Fukagawa K., Sakata T., Tso P. Suppression of food intake by apolipoprotein A-IV is mediated through the central nervous system in rats. J. Clin. Invest. 1993;91:1830-1833
11. Gordon J. I., Smith D. P., Alpers D. H., Strauss A. W. Cloning of a complementary deoxy-ribonucleic acid encoding a portion of rat intestinal preapolipoprotein A-IV messenger ribonucleic acid. Biochemistry 1982;21:5424-5431[Medline]
12. Hayashi H., Nutting D. F., Fujimoto K., Cardelli J. A., Black D., Tso P. Transport of lipid and apolipoprotein A-I and A-IV in intestinal lymph of the rat. J. Lipid Res. 1990;31:1613-1625[Abstract]
13. Jin H., Cai L., Lee K., Chang T.-M., Li P., Wagner D., Chey W. Y. A physiological role of peptide YY on exocrine pancreatic secretion in rats. Gastroenterology 1993;105:208-215[Medline]
14.
Kalogeris T. J., Tsuchiya T., Fukagawa K., Wolf R., Tso P. Apolipoprotein A-IV synthesis in proximal jejunum is stimulated by ileal lipid infusion. Am. J. Physiol. 1996;270:G277-G286
15.
Kalogeris T. J., Qin X., Chey W. Y., Tso P. PYY stimulates synthesis and secretion of intestinal apolipoprotein AIV without affecting mRNA expression. Am. J. Physiol. 1998;275:G668-G674
16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685[Medline]
17. Okumura T., Fukagawa K., Tso P., Taylor I. L., Pappas T. N. Intracisternal injection of apolipoprotein A-IV inhibits gastric secretion in pylorus-ligated conscious rats. Gastroenterology 1994;107:1861-1864[Medline]
18.
Okumura T., Fukagawa K., Tso P., Taylor I. L., Pappas T. N. Apolipoprotein A-IV acts in the brain to inhibit gastric emptying in the rat. Am. J. Physiol. 1996;270:G49-G53
19.
Paintal A. S. Vagal sensory receptors and their reflex effects. Physiol. Rev. 1973;53:159-227
20.
Pappas T. N., Debas H. T., Goto Y., Taylor I. L. Peptide YY inhibits meal-stimulated pancreatic and gastric secretion. Am. J. Physiol. 1985;248:G118-G123
21. Sonoyama K., Fujiwara R., Aoyama Y. Stimulating effect of ileal pancreaticobiliary secretion on ileal apolipoprotein A-IV mRNA expression in fasted rats. Biosci. Biotech. Biochem. 1997;61:887-889[Medline]
22. Sonoyama K., Nishikawa H., Kiriyama S., Niki R. Bile diversion lowers apolipoprotein A-I and A-IV mRNA levels in rat ileum. J. Nutr. Sci. Vitaminol. 1994;40:343-352
23. Sonoyama K., Nishikawa H., Kiriyama S., Niki R. Apolipoprotein mRNA in liver and intestine of rats is affected by dietary beet fiber or cholestyramine. J. Nutr. 1995;125:343-352
24. Sonoyama K., Nakamura Y., Kiriyama S., Niki R. Upregulation of apolipoprotein A-IV gene expression in residual ileum after massive small bowel resection requires the biliary secretion in rats. Proc. Soc. Exp. Biol. Med. 1996;211:273-280[Abstract]
25.
Sonoyama K., Suzuki K., Kasai T. Peptide YY stimulates the expression of apolipoprotein A-IV gene in Caco-2 intestinal cells. Proc. Soc. Exp. Biol. Med. 2000;223:270-275
26. Stein O., Stein Y., Lefevre M., Roheim P. S. The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine. Biochim. Biophys. Acta 1986;878:7-13[Medline]
27.
Steinmetz A., Utermann G. Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV. J. Biol. Chem. 1985;260:2258-2264
28.
Steinmetz A., Barbaras R., Ghalim N., Clavel V., Fruchart J. C., Ailhaud G. Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells. J. Biol. Chem. 1990;265:7859-7863
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