Biochimicn et Riophysicu Acra, 1165 (1992) 93-101 0 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00
93
BBALIP 54042
Regulation of intestinal apo A-IV mRNA abundance in rat pups during fasting and refeeding Masao Sato a, Katsumi Imaizumi a, Haruhiko Mori a and Michihiro Sugano b a Laboratory of Nutrition Chemistry and b Laboratory of Food Chemistry, Department of Food Science and Technology, School of Agriculture Kyushu U~i~~er$~~,Fukuok~ (Japan~
(Received 20 May 1992)
Key words: Apolipoprotein
A-IV, mRNA; intestinal apo A-IV; Serum triacylglycerol; Fat absorption; Fat secretion; (Rat pups)
The amount of intestinal apolipo~ro~~in (ape> A-IV mRNA was examined in rat pups during fasting and refeeding. When 14-day old pups were fasted for 15 h, apo A-IV mRNA levels in the whole intestine decreased to 20% of the prefasting level. Refeeding
casein and lactose, and the artificial milk composed of Intralipid, casein and lactose, caused an elevation of the apo A-IV mRNA after 3 h, without accompanying an elevation of serum triacylglycerols and apo A-IV (fat-independent elevation of apo A-IV mRNA). Refeeding Intralipid alone simultaneously elevated the apo A-IV mRNA, and serum triacylglycerols and apo A-IV after 3 h ~fat-dependent elevation of ape A-IV mRNA). Administration of physiologicaI saline during fasting partly suppressed the reduction of the apo A-IV mRNA ~40% of the prefasting level), and the dietary fat-independent elevation of the message disappeared. Refeeding dam’s milk to the pups, fasted without water administration, increased the apo A-IV mRNA after 3 and 15 h, although the elevation of serum triacylglycerols and apo A-IV occurred only after 15 h. Refeeding the milk increased the apo A-IV mRNA after 3 h and 15 h. Refeeding dam’s milk to the pups fasted with saline administration accelerated the fat-dependent elevation of the apo A-IV mRNA. Simultaneously refeeding Intralipid and Pluronic L-81, an inhibitor of lymphatic fat transport, delayed the elevation of the apo A-IV mRNA and serum triacylglycerols and apo A-IV. Transcription rates of the apo A-IV mRNA, de~e~iued by nuclear run/on assay, were similar before and after fasting and refeeding Intralipid. During fasting, administration of puromycin, as compared with actinomycin D, enhanced the disappearance rate of the apo A-IV message. Intestinal mRNA for apo B, but not for apo A-I and /3-actin, similarly changed to the apo A-IV message. Thus, it can be concluded that: (1) dietary fat-dependent and -independent factors are involved in the elevation of the intestinal apo A-IV message; (2) the elevation of the message is not mediated by lipid uptake in the enterocytes but rather stimulated by the events leading to secretion of chytomicrons; and, (3) dietary fat-dependent elevation of the message appears to be due to the stabilization of the message.
Introduction A~olipopro~ei~ (ape> A-IV was first discovered as a component of serum high-density lipoproteins in adult rats by Swaney et al. [l]. This apoprotein was subsequently identified in mesenteric lymph chylomicrons in rats [2,3], human chylus fluid [4] and rat renal lymph
[.5]. Apo A-IV is also distributed in lipoprotein-free fractions of. mesenteric lymph 161 and sera [7,8] of mammals. cDNA of apo A-IV was first constructed by Gordon et al. [9] from rat intestinal epithelial cells, and now primary sequences of apo A-IV of rat [lo], mouse [ 111 and man [121 have been deduced from cDNA
Correspondence to: K. Imaizumi, Laboratory of Nutrition Chemistry, Department of Food Science and Technology, School of Agriculture, Kyushu University, Fukuoka 812, Japan.
sequence analyses. The function of this apoprotein still remains unclear, although it may be involved in cholesterol transport [13], tria~l~lycerol clearance in the blood serum 1141 and uptake of high-density lipoproteins by the cells [15]. Secretion of apo A-IV into mesenteric lymph in rats increases when fat emulsions were infused and large chylomicrons isolated from the lymph contain considerable amounts of this apoprotein [Z]. Oral intake of fat emulsions in humans increases the serum level of apo A-IV [16]. These early results suggest that dietary fats are involved in the regulation of apo A-IV synthesis and transport in the intestine. Detailed molecular mechanisms, however, remain largely unsolved. Frost et al, 1171 showed that absorption and transport of dietary fats are more active in rat pups suckled with dam’s milk as compared to adult rats raised on commercially available rat chow. Levels of serum apo
94 A-IV [18] and intestinal apo A-IV mRNA [19] also dramatically change developmentally after birth: higher in the suckling period and lower after weanling. The present study was carried out to examine the regulation of the intestinal apo A-IV mRNA IeveI in rat pups during fasting and refeeding. The results show that factors dependent on and independent of dietary fats elevate the intestinal apo A-IV mRNA and that continuous absorption and transport of dietary fats may stabilize the message. Materials
quently, 0.5 ml of saline containing 10% glucose was administered at 8, 10, 12, 14, 16, 18, 20 and 22 h. The lipid emulsion was made of 7 ml Intralipid and 3 ml phosphate-buffered saline (pH 7.4). The Pluronic L-81 lipid emulsion was made of 7 ml Intralipid and 3 ml Pluronic L-81 solution (8.8 pmol egg phosphatidylcholine, 57 kmol sodium taurocholatc and 1.0 mg Pluronic L-81 in the phosphate-buffered saline). The pups were killed at 0, 3 and 6 h in the Pluronic L-XI phase and at 9, 12, 15 and 24 h in the saline administration phase.
and Methods
Animuls l4-day old Wistar rat pups with their dams (8-10 pups per dam) were obtained from Seiwa Experimental Animal Co., Fukuoka. Animals were maintained under controlled temperature (23 + 1°C) and light (light period 06:00-18:OO). All aspects of the experiment were approved by Kyushu University Animal Policy and Welfare Committee. The pups were fasted for 15 h without water (dehydrated pups), or administered with 0.75 ml of physiological saline by a stomach tube made of silicone for every 3 h (hydrated pups). Some pups were returned to their dams to feed their milk and others were refed one of the folIowing liquid diets with a stomach tube every 2 h: (1) commercially available Intralipid (10% w/v soybean oil, 1.2% W/Y egg lecithin and 2.5% w/v glycerol, Kabivitrum AB, Stockholm); (2) artificial milk [ZO] composed of 9.2% (w/v) casein and 3.0% (w/v) lactose which were soluhilized directly in ln~raiipid (pH 7.0); (319.2% (w/v) casein in water; and (4) 3.0% (w/v) lactose in water. The pups were killed by withdrawing aortic blood under diethyl ether anesthesia. The whole intestine from the duodenum to the ileum was immediateIy excised, flushed with ice-cold physiological saline to remove the intestinal contents, and stored in liquid nitrogen untif used. Treatment of pups with drugs Some pups were given intraperitoneally a 600 pg of puromycin (2 mg/ml saline) immediately after removal from their dams, and followed by injecting 80 pg puromycin twice every 2 h during fasting, according to the schedule used for adult rats [21]. This treatment for adult rats has been reported to minimally influence transport of dietary fats [22]. Actinomycin D (1 mg/kg body weight in saline/506 v/v ethanol) was also given intraperitoneally to the pups just before fasting 1221. Pups were also treated with Pluronic L-81 [23], a potent inhibitor of chylomicron formation, which was obtained from Dr. P. l-so, Louisiana State University, Shreveport, LA. After 15 h fasting, 0.5 ml of Pluronic L-81 lipid emulsion or lipid emuIsion alone was administered by the stomach tube at 0, 2, 4 and 6 h. Subse-
Electroimmunoassays of appolipoproteinA-IV The serum apo A-IV was measured by rocket immunoelectrop~oresis as described in Ref. 18. Concentration of apo A-IV in the serum was determined by comparing its rocket heights with those in a series of dilutions of pooled serum obtained from S-week-old male Sprague-Dawley rats, and expressed as an arbitrary unit. Lipid unalyses Serum triacylglycerols were measured by using an enzyme assay kit, Triglyceride G-Test (Wake Pure Chemicals, Osaka). Mucosal lipids in the pup’s whole intestine were extracted in chloroform/met~anol mixture (2 : 1, v/v) 1241. The lipids were quantitatively spotted on thin-layer plates (Silica-gel G, Merck) by Drumond microsyringe with the solvent system composed of hexane/ diethyl ether/ formic acid (80:20:2, v/v) [25]. A series concentration of triolein was simultaneously appIied to the plate as an internat standard. After the plate was sprayed with 3% (w/v) Cu acetate -7% (w/w> phosphate solution 1261 and heated at 150°C for 30 min, the triacylglycerol contents were determined by scanning densitometry (Desital Densitorol DMU-33C, Toyo Science industrial, Tokyo) at 650 nm. Isolation and quantitation of mRNA by slot blot hybridization ussay and Northern analysis Total RNA was isolated from the whole intestine by the guanidine thiocyanate procedure according to the method of Chirgwin et al. [27] and stored at -40°C. The RNA sample was denatured with glyoxsal at 50°C for 1 h as described by Thomas [28]. Constant amounts of RNA supplemented with yeast RNA to provide a final amount of 0.8 Fug of RNA per sample were blotted onto the nitrocellulose filter (Zeta Probe membrane, Bio-Rad Japan, Tokyo) prewetted with 20 X SSC (3 M NaCl, 1 M sodium citrate, (pH 7.0)) with a vacuum-manifold apparatus (Bio-Rad Japan). For Northern analysis, the denatured total RNA was electrophoresed in a 0.8% (w/v) Agarose gel prepared in 20 mM sodium phosphate buffer, pH 6.5 [281. After pre-hybridization with a hybridization solution (5 X
95
oJ,*, 0
( 025
05
,
, 075
10
Dilutton Factor
b
cl
I/
0---"-Y-
0
02
04
06
08
Intestinal Total RNA (vg)
Fig. 1. Slot blot hybridization assay for intestinal apo A-IV mRNA in rat pups. (A) Standard curve constructed with a series of dilution of the intestinal total RNA prepared from nonfasted pups. (B) Relationship between the densitometric area and three different amounts of total RNA prepared from fasted (open circle) and nonfasted (closedcircle)pups, respectively.
SSC, 50% w/v formamide, 20 mM sodium phosphate, pH 6.5, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrrolidone, 0.02% w/v bovine serum albumin, and 100 mg/ml denatured herring sperm DNA) for 5 h at 42”C, hybridization reactions were performed in the hybridization solution containing cDNA labeled with [5’“PI dCTP (1.48 MBq/mmoI, Amersham Japan, Tokyo) by maItiprime DNA labeling system according to the Amersham’s instruction for 18 h at 42°C. The cDNA clones for rat apo A-IV [lOI and apo B 1291 were obtained from Dr. J.I. Gordon (Washington Univ. School of Med., St Louis, MO) and Dr. A. Matsumoto (National Institute of Health and Nutrition), respectively. Thirty-oligomer to rat apo A-I cDNA was synthesized by Research Laboratory for Genetic Informations, Kyusyu University, Fukuoka. cDNA for @-actin was obtained from Wako Pure Chemicals. Blots were exposed to X-ray film (Fuji Film Industry, Tokyo), and autoradiograms of the filters were analyzed by quantitative scanning densitomet~ (Desital Desitorol DMU33C). Total RNA from one of the pups before fasting was used to construct a standard curve (Fig. 1). The levels of mRNA were expressed as an arbitrary unit. The levels of mRNA in one of the pups before fasting was assumed to be 100. When autoradiograms for three different amounts of RNA gave a linear densitometric area as shown in Fig. 1, the mean value was adopted as the abundance of the sample’s mRNA. A similar standard curve was constructed for the messages of apo A-I, apo B and @-actin.
Whole intestine of rat pups obtained immediately after killing, were washed with ice-cold 10 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl. Nuclei were isolated from the whole intestine, according to the method of Nawa et al. [30], and modified as follows. The tissue segment (1 g wet wt.> was suspended in 6 ml homogenizing buffer (10 mM Tris (pH 8.0)) containing 0.32 M sucrose, 3 mM CaCl,, 2 mM Mg acetate, 0.1 mM EDTA, 0.1% (w/v) Nonidet P-40, 1.0 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride, and homogenized in loose-fatting Potter-E~vehjem Teflon homogenizer (500 rpm and 5-6 strokes). The nucleus solution was layered on the dilution buffer (10 mM Tris-HCl, pH 8.0 containing 2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride), and centrifuged at 50,000 x g for 60 min at 2°C. The precipitated nuclei were suspended in storage buffer (50 mM Tris, pH 8.0, containing 45% glycerol, 5 mM MgCl,, and 0.1 mM EDTA), counted by hemocytometer, and stored in liquid nitrogen. RNA transcription was measured according to the method of Protko et al. [31]. Briefly, nuclei (8 X 10’) were incubated in 25 mM Tris (pH 8.0) containing 20% (w/v) glycerol, 2 mM MgCl,, 150 mM KCl, 1 mM dithiothreitol, 1 mM ATP (Wake Pure Chemicals), 0.5 mM CTP (Yamasa Shoyu, Tokyo), 0.5 mM GTP (Sigma Chemicals), 8 mM phosphocreatine (Nakalai Tesque), 40 mg of creatine kinase/ml, 50 units/ml of RN&n (Sigma) and [a-32 P]UTP (1.48 MBq/mmol; Amersham Japan). RNA synthesis was terminated after 30 min, and nuclei were treated with 20 mg/ml of DNAase I (BMY, Tokyo), 100 mg/ml of proteinase K (BMY) and 50 mg/ml of yeast tRNA (BMY), The RNA was extracted twice with phenol/chloroform (1: 1, v/v) and precipitated in ethanol. The newly transcribed RNA was hybridized to DNA immobilized onto nitrocellulose filters (Zeta Probe membrane, Bio-Rad). Plasmid DNA was linearized, heat-denatured, spotted on the filters and baked at 80°C. Prehybridization was carried out in 50% (v/v) formamide containing 5 x Denhardt’s, 5 x SSC, 50 mM sodium phosphate (pIi 7.41, 5 mM EDTA, 0.1% (w/v) SDS and 100 mg of tRNA/ml. Hybridization was carried out in the same solution with labeled RNA added for 2 days at 42°C. After hybridization, the filters were washed and treated with RNAases A and Tl. The sample filters were counted for 5 min in a liquid scintillation counter (Tri-carb 2250, Packard Japan, Tokyo). Statistics
Differences within groups were compared by analysis of variance, and group differences were compared by the independent two-tailed r-test. Differences were
96 considered significant when the probability of the difference occurring by chance was less than 5 in 100 (P < 0.05) for both t-test and analysis of variance. Results When 14-day-old pups were nursed normally by their dams, the levels of intestinal apo A-IV mRNA and the serum apo A-IV measured every 3 h for 24 h were essentially similar (data not shown), suggesting no significant fluctuations in a day. When these pups were fasted for 15 h without giving water, serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA determined by slot blot and Northern blot assay decreased (Fig. 2). Since the validity of the slot blot assay was supported by the Northern blot assay as shown in Fig. 2 (the lowest figure), the slot blot assay was used
0
3
6
9
Time After Refeedmg (hr)
Fig. 3. Effect of refeeding artificial milk, casein, lactose and Intralipid on serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA. Pups fasted for 15 h without administration of water were refed liquid diets every 2 h. Each point and bar show means& different from the values S.E. for 3 pups per group. ” Significantly after 15 h fasting at P < 0.05. h Significantly different from the values of the pups refed lntralipid at P < 0.05.
C k
OJ, 0
I
,
2
4
.
I
I
1
6
8
10
’
I
I
12
14
-
Time After Fasting (hr) Fig. 2. Effect of fasting on serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA. Pups were removed from their dams at 3 pm. and subsequently fasted for 15 h without administration of water. Northern blotting of apo A-IV mRNA in the intestine from the fed pups before fasting (A) and after 15 h fasting (B) is shown in the inlet of the lowest figure. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the prefasting values at P < 0.05.
for the subsequent experiments. The apo A-IV message reached the bottom level (20% of the prefasting level) after 8 h fasting. As shown in Fig. 3, administration of artificial milk to pups fasted without administration of water (dehydrated pups) elevated the apo A-IV mRNA after 3 h, although the serum triacylglycerols and apo A-IV did not change. Similar patterns were also observed when dehydrated pups were given casein or lactose solution. The administration of Intralipid alone to dehydrated pups, however, caused a simultaneous elevation of the message, and serum triacylglycerols and apo A-IV after 3 h. Fig. 4 showed that fasting caused a reduction in the intestinal apo B mRNA to 40% of the prefasting level. Refeeding the artificial milk elevated the apo B mRNA to 80% of the prefasting level after 6 h. The messages for apo A-I and p-actin, however, showed essentially no changes during fasting and refeeding the milk. Refeeding the saline to the pups fasted without administration of water (dehydrated), also enhanced the apo A-IV mRNA after 3 h (Fig. 5). When pups
97
a
E
oJr 0
1
n
n
3 6 9 Time After Refeeding (hr)
1
12
Fig. 4. Effect of refeeding artificial milk on intestinal messages for apo A-I, B, and p-actin. Experimental conditions are shown in the legend to Fig. 3. Each point and bar show means + S.E. for 3 pups per group. a Signifcantly different from the values after 15 h fasting at P < 0.05.
were administered the saline during fasting (hydrated), the reduction of the intestinal apo A-IV mRNA was significantly suppressed and the message decreased to 40% of the prefasting level. In the next experiment, pups that were hydrated or dehydrated during fasting were nursed again by their dams. Firstly, pups were not administered water for 15 h. As shown in Fig. 6, these pups soon appeared to be suckled, as intestinal triacylglycerols increased almost to the maximum level as early as 3 h after nursing. The stomach contents also increased after 3 h nursing and maintained constant weight throughout the nursing period (data not shown). Significant elevation of the serum triacylglycerols and apo A-IV was delayed until after 15 h nursing. These elevations were suppressed when pups were again removed from their dams after 12 h. Elevations of the intestinal apo A-IV mRNA were observed after 3 and 15 h. Secondly, pups were fasted 15 h with saline administration. The patterns of the serum triacylglycerols and apo A-IV, and the intestinal mRNA markedly differed from those pups dehydrated during fasting, although intestinal accumulation of the milk triacylglycerols similarly occurred (Fig. 7). The level of serum triacylglycerols and apo A-IV increased gradually during nursing. The apo A-IV mRNA increased after 6 h, but subsequently decreased again to the fasting level. The apo B mRNA showed a pattern similar to that of the apo A-IV message. The messages for apo A-I and p-actin did not markedly change during fasting and nursing. As shown in Fig. 8, when Intralipid was refed to the hydrated pups, the patterns of the elevation of the serum triacylglycerols and apo A-IV resembled those pups nursed with dams (Fig. 7). Absorption and subsequent secretion of the dietary fat occurred as early as 1.5 h after. The apo A-IV mRNA increased 6 h after, although as shown in Fig. 3, the message increased after 3 h when the dehydrated pups were refed Intralipid alone. Subsequently, the level of apo A-IV mRNA was maintained to more than prefasting level.
Again, apo B mRNA followed the pattern of the apo A-IV mRNA, and the messages for apo A-I and p-actin showed no change. To examine the effect of the inhibitor of fat secretion from the intestine on the levels of serum triacylglycerols and apo A-IV, and the intestinal apo A-IV mRNA, dehydrated pups were refed with Intralipid alone or the Intralipid supplemented with Pluronic L-81. Fig. 9 showed that refeeding Intralipid alone caused elevations of intestinal triacylglycerols and apo A-IV mRNA, and serum triacylglycerols and apo A-IV. Administration of the saline following Intralipid promptly decreased all the parameters. Refeeding the mixture of Intralipid and Pluronic L-81, as compared with Intralipid alone, caused a marked accumulation of the mucosal triacylglycerols. Maximum elevations of the serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA delayed and occurred after the saline administration. As shown in Fig. 10, Pluronic L-81 treatment suppressed the elevation of the intestinal apo B mRNA throughout the period, although refeeding Intralipid alone promptly increased the message. Table I shows the rate of apo A-IV mRNA transcription in the intestinal nuclei prepared from the pups suckled by dams (Fed), fasted for 15 h (Fast), and refed Intralipid for 6 h (Refed). There were no significant differences among the transcription rates for the apo A-IV mRNA. To examine the effect of inhibitors for protein and RNA syntheses on the disappearance rate of the intestinal apo A-IV mRNA during fasting, pups were treated with puromycin and actinomycin D. As shown in Fig. 11, a half-life of the apo A-IV mRNA reduction in the pups untreated with drugs was 3.7 h. The administration of actinomycin D, an inhibitor of RNA synthesis, accelerated the disappearance rate of the apo
0
3
6
9
12 15 18 2l
P
27
Time After Fasting (hr)
Fig. 5. Effect of hydration and dehydration during fasting on intestinal apo A-IV mRNA. Pups were separated into two groups: one group received physiological saline every 2 h for the initial 15 h (hydrated), and the other was not (dehydrated). Subsequently both groups were administered the saline every 3 h. Arrow indicates the start of the administration of the saline to the dehydrated pups. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the corresponding values of the dehydrated pups at P < 0.05. b Significantly different from the values after 15 h fasting at P < 0.05.
0
3
6
0
9
3
0
3
0
3
12 15 18 21 24 27
6
9
12
15
18
-
Time after Refeeding (hr)
6
9
12 15 18 21 24 27
6
9
12 15 18 21 24 27
Time after Refeeding (hr)
Fig. h. Effect of refeeding dam’s milk to dehydrated pups on the serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and ape A-IV mRNA. After fasting for 15 h without administration of water, the pups were returned to their dams. At after 12 h, returned to the dams, some pups were fasted again (black circle with dotted line). Pups were killed every 3 h. Each point and bar show means&SE. for 3 pups per group. ” Significantly different from the values after IS h fasting at P < 0.05.
A-IV mRNA during fasting, and puromycin, an inhibitor of protein synthesis, further accelerated the disappearance.
apo A-IV and tri~cylglycerols. These elevations dependent on dietary fat were maintained as long as the fat feeding continued. Thus, present study confirmed the early observation by Gordon et al. [9] who showed that intragastric administration of plant oil to adult rats, fasted overnight, increases the intestinal apo A-IV mRNA levels. There are very few studies on the regulation of apo A-IV mRNA Ievel affected by factors other than dietary fats. One report describes a higher
Discussion
Present study showed that refeeding Intralipid alone pups, fasted without water administration promptly increases the intestinal apo A-IV mRNA, and serum to
01,
,
,
0
3
6
9
12
,
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04
15
18
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9
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15
18
afx A-W
l
a
12
n aen
a a a
3
a
6
9
12
a
a
15
16
Time after Refeeding (hr.1
=
0
3
6
9
12
15
Time after Refeeding (hr
113
)
Fig. 7. Effect of refeeding dam’s milk to hydrated pups on the serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and messages for apo A-I. apo A-IV, apo B and p-actin. Pups were administered the saline every 2 h for 15 h fasting, then returned to their dams. Each point and har show mean + SE. for 3 pups per group. a Significantly different from the values after IS h fasting at P < 0.05.
99
0
3
6
9
12
15
Or 0
3
6
9
12
15 Time After Refeeding (hr)
Time After Refeeding (hr)
Fig. 8. Effect of refeeding Intralipid to hydrated pups on serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and the messages for apo A-I, A-IV, B, and p-actin. Pups were administered physiological saline every 2 h for 15 h fasting. At after 15 h fasting, the pups were refed with Intralipid alone every 2 h. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the values 15 h fasting at P < 0.05.
dent elevation of the apo A-IV mRNA disappeared when the pups were administered with saline during fasting, some endocrine and/or neural informations provoked by an expansion of the stomach after refeeding may be responsible for this phenomenon. Present study showed that refeeding artificial milk delays the elevation of serum triacylglycerols and apo A-IV, and the intestinal apo A-IV mRNA dependent on dietary fat. Dietary-fat-dependent elevation of the apo A-IV mRNA was also delayed in dehydrated pups when they were nursed again. Although nursing again
level of apo A-IV mRNA in the cultured rat hepatocytes when the medium was supplemented with insulin and dexamethazone alone, or both [19]. Present study showed that feeding fat-free diets such as saline, casein and lactose solution also increases the intestinal apo A-IV mRNA level. This experiment suggests that factor(s) other factor (s) than dietary fats, participate in the elevation of apo A-IV mRNA level in the intestine. This dietary-fat-independent elevation for the intestinal message was also observed for the apo B, but not for apo A-I and p-actin. Since the dietary fat indepen-
0
3
0
3
6
9
12
15
18
21
24
6
9
12
15
18
21
24
Time After Refeeding (hr)
Fig. 9. Effect of refeeding Pluronic L-81.Intralipid mRNA. After 15 h fasting without administration with Pluronic L-81 (Pluronic L-811, and the other
0
0
3
3
6
6
9
9
12
12
15
15
18
18
2-l
21
24
24
Time After Refeeding (hr)
emulsion on serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and apo A-IV of water pups were separated into two groups: one group refed with Intralipid supplemented Intralipid alone (Control). Arrow indicates the start of the saline-glucose administration. Each point shows mean of two pups.
l Pluronic L-81
r---
0
3
6
9
12
15
18
0
21 2S
Time After Refeeding (hr) Fig.
IO. Effect
intestinal
apo
of refeeding 6 mRNA.
Piuronic
Pups were
L-Wlntralipid treated
emulsion
as described
on
in the
legend to Fig. 9. Each point shows mean of two pups.
hydrated pups during fasting accelerated the elevation of the apo A-IV mRNA, the elevation did not last as compared with the pups refed Intralipid alone. Absorption process of the dietary fat was not delayed as these milk did not disturb the mucosal accumulation of triacylglycerols. Thus, milk component(s) other than fat may participate in the regulation of the level of the intestinal apo A-IV mRNA in pups refed dietary fat. To investigate the effect of secretory pathway of the intestinal tria~lglycero~s on the level of the mucosal apo A-IV mRNA, the pups were administered Pturonic L-81 which has been reported to inhibit the chylomicron transport in adult rat 2231. The present study showed that treating pups with Pluronic L-81 delayed both the elevations of serum triacylglycerols and the intestinal apo A-IV mRNA. Thus, the amount of intestinal apo A-IV mRNA dependent on dietary fat is closely related to the events involved in the packaging and secretion of chylomicrons rather than the absorptive process of the tuminal fats. In this regard, the present results agree with the results reported by ~ayashi et al. [32] who showed that an elevation of the translational activity for apo A-IV foIlows active lymphatic transport of dietary triacylglycerols. At present, two factors have been reported with regard to the prompt processing of dietary fats to lymphatic chylomicrons in adult rats: (1) availability of phospholipids synthesized endogenously and supplied exogenously [33], and (2) lymph flow [341. Some dietary components have been reported to influence secretion
TABLE
1
Sy~i~~~i~mte of’apo A-W ml&VA by ~zuclear nm /on assuy Pups with dams (Fed) tered lntralipid
were fasted for 15 h (Fasted),
for 6 h (Refed).
Values
are meansiS.E.
per group. Treatment
Rate of RNA (ppm)
Fed Fasted Refed
9.23 + 0.98 7.24 + 1.01 10.1
+0.30
synthesis
4
2
6
8
Time After Fasting (hr)
and adminisfor 5 rats
Fig. ii.
Effect
of transcription
pearance
of intestinal
separated
into three
and translation
apo A-IV
mRNA
groups as described
with actinomycin
D and puromycin,
At later
the amount
treated
period,
during
inhibitors fasting.
on disapPups were
in the text: pups treated
or without
of the apo A-IV
treatment mRNA
with the drugs was too small to be determined.
(Control). in the pups Each point
shows means for 3 pups per group.
of intestinal lipids to the lymph. Intraduodenal infusions of a fat-free mixture of dextrin and casein [35] or casein and sucrose with or without ethanol [36] all significantly increase intestinal lymph Iipids. On the contrary, lower levels of lymph lipid output were reported during sucrose and starch infusion period [40]. Dietary proteins and carbohydrates inhibit secretion of c~ytomicrons directly or indirectly through stimulation of some gastrointestinal hormones, which appear to inhibit bowel motility [38]. Concerning rat pups, the mechanisms on how dietary casein and/or lactose in the milk influence the utilization of exogenous phospholipid for chylomicron synthesis and secretion [33], and permeability of blood fluid to interstitial spaces of intestinal mucosa, which facilitates chylomicron output from the cells to lymph [34], are stilt unknown. To determine whether the dieta~-fat-dependent level of the intestinal apo A-IV mRNA is due to an enhanced transcriptional activity or post transcriptional events, nuclear run/on assay was carried out. The present results showed no remarkable differences in the transcription rate during the suckling period by dams, after 15 h fasting and after 6 h refeeding Intralipid. Thus, the dietary-fat-dependent level of the intestinal apo A-IV mRNA appeared to be regulated mainly during the post-transcriptional events. In addition, since puromycin as compared with actinomycin D further accelerated the disappearance rate of the message during fasting, dietary fat may modulate the synthesis of some protein(s) which participates in the stabilization of the intestinal message. In the present study, lymphatic secretion of the translation products of apo A-IV mRNA was determined indirectly by measuring the level of the serum apo A-IV, Since the serum level of apo A-IV was well correlated to the serum triacylglycerol level, the determination of the serum apo A-IV can partly reflect the translational activity of apo A-IV protein in the intestine, although Windmuellar and Wu have shown that
101 apo A-IV synthesized in intestine is also transported into portal system 1391. Fig. 7 showed that serum apo A-IV continues to increase after refeeding dam’s milk although the intestinal apo A-IV mRNA only transiently increased. Therefore, dietary fat and/or other milk component(s) may also be involved in the translational process of apo A-IV. In addition to apo A-IV message, apo B mRNA, but not messages for apo A-I and p-actin, in the intestine was also enhanced following Intralipid administration alone. Regulation of apo B mRNA in the pup’s intestine was apparently similar to that of apo A-IV mRNA, but not identical as the saline administration following the mixture of Pluronic L-81 and Intralipid elevated the apo A-IV mRNA, but not apo B mRNA. In addition, Davidson et al. did not find any significant changes in the synthetic rate of apo B in the adult rat intestinal segment [40] and its mRNA [41]. Also, when given high fat diet, the intestinal apo B synthesis in piglets appeared to be unaffected by the triacylglycerol absorption, even though apo A-IV synthesis increased [421. Thus, different factors derived from dietary fats appears to be involved in the transcriptional and posttranscriptional regulation of these apoprotein synthesis in the intestine. Acknowledgment
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (03303011). References
5 6 7 8 9 10
Swaney, J.B., Braithwaite, F. and Eder, A.A. (1977) Biochemistry 16, 271-278. Imaizumi, K., Fainaru, M. and Havel, R.J. (1978) J. Lipid Res. 19, 712-722. Green, P.H.R., Tall, A.R. and Glickman, R.M. (1978) J. Clin. Invest. 61, 528-534. Green, P.H.R., Glickman, R.M., Saudek, C.D., Blum, C.B. and Tall, A.R. (1979) J. Clin. Invest. 64, 233-242. Roheim, P.S., Edelstein, D. and Pinter, G.G. (1976) Proc. Nat]. Acad. SC. USA 73, 1757-1760. Wu, A.L. and Windmueller, H.G.(1978) J. Biol. Chem. 254, 316-7322. Weisgraber, K.H, Bersot, T.P. and Mahley, R.W. (1978) Biochem. Biophys. Res. Commun. 85, 287-292. Beisiegel, U. and Utermann, G. (1979) Eur. J. Biochem. 93, 601-608. Gordon, J.I., Smith, D.P., Alpers, D.H. and Strauss, A.W. (1982) Biochemistry 21, 5424-5431. Boguski, M.S., Elshourbagy, N., Taylor, J.M.N. and Gordon, J.I. (1984) Proc. Natl. Acad. Sci. USA, 81, 5021-5025.
S.M., Lusis, A.J., Leboeuf, R.C. and 11 Williams, S.C., Bruckheimer, Kinniburgh, A.J. (1986) Mol. Cell. Biol. 6, 3807-3814. N.A., Walker, D.W., Boguski, M.S., Gordon, J.I. 12 Elshourbagy, and Taylor, J.M. (1986) J. Biol. Chem. 261, 1998-2002. 13 Stein, O., Stein. Y., Lefevre, M. and Roheim, P.S. (1986) Biochim. Biophys. Acta 878, 7-13. 14 Goldberg, I.J., Scheraldi, C.A., Yacoub, L.K., Saxena, U. and Bisgaier, C.L. (1990) J. Biol. Chem. 265, 4266-4272. 15 Dvorin, E., Gorder, N.L., Benson, D.M. and Gotto A.M. (1986) J. Biol. Chem. 261, 15714-15718. 16 Green, P.H.R., Glickman, R.M., Riley, J.W. and Quinet, E. (1980) J. Clin. Invest. 65, 911-919. 17 Frost, S.C., Clark, W.A. and Wells, M.A. (1983) J. Lipid Res. 24, 899-903. 18 Imaizumi, K., Lu, Y.-F. and Sugano, M., (1987) Biochim. Biophys. Acta 917, 269-278. 19 Elshourbagy, N.A., Boguski, M.S., Liao, W.S.L., Jefferson, L.S., Gordon, J.I. and Taylor, J.M. (1985) Proc. Natl. Acad. Sci. USA 82, 8242-8246. 20 Keen, CL., Lonnerdal, B., Clegg, M. and Hurley, L.S. (1981) J. Nutr. 111, 226-230. 21 Sabesin, S.M. and Isselbacher, K.J. (196.51 Science 147, 1149-1151. 22 Vahouny, G.V., Ito, M., Blenchermann, E.M., Gallo, L.L. and Treadwell, C.R. (1977) J. Lipid Res. 18, 745-752. 23 Tso, P. and Gollamudi, S.R. (1984) Am. J. Physiol. 247, G32-G36. 24 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 266, 497-506. 25 Storry, J.E. and Tuckley, B. (1967) Lipids 2, 501-502. 26 Fewster, M.E., Burns, B.J. and Mead, J.F. (1969) J. Chromatogc 43, 120-126. 27 Chirgwin, J.M., Przybyla, A.E., McDonald, R.J. and Rutter, W.J. (1979) Biochemistry 24, 5294-5299. 28 Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA. 77, 5201-5205. 29 Matsumoto, A., Aburatani, H., Shibasaki, Y., Kodama, T., Takaku, F. and Itakura, H. (1987) Biochem. Biophys. Res. Commun. 142, 92-99. 30 Nawa, K., Nakamura, T., Kuwatori, A., Noda, C. and Ichihara, A. (19861 J. Biol. Chem. 261, 16883-16888. 31 Prostko, CR., Fritz, R.S. and Kletzien, R.F. (1989) Biochem. J. 258, 295-299. 32 Hayashi, H., Nutting, D.F., Fujimoto, K., Cardelli, J.A., Black, D. and Tso, P. (1990) J. Lipid Res. 31, 1613-1625. 33 Tso, P., Kendrick, M., Balint, J.A. and Simmonds, W.J. (1981) Gastroenterology 80, 60-65. 34 Tso, P., Pitts, V. and Granger, D.N. (1985) Am. J. Physiol. 249, G21 -G28. 35 Risser, T.R., Reaven, G.M. and Reaven, E.P. (1978) Am. J. Physiol. 234, E277-E281. 36 Boquillon, M. (1976) Lipids 11, 848-852. 37 Keim, M.L. and Marlett, J.A. (1980) J. Nutr. 110, 1354-1364. 38 Sernka, T. and Jacobson, E. (1978) in Gastrointestinal Physiology - The Essentials, pp. 58-77, Williams and Wilkins, Baltimore. 39 Windmueller, H.G. and Wu, A.-L. (1981) J. Biol. Chem. 256, 3012-3016. 40 Davidson, N.O., Kollmer, M.E. and Glickman, R.M. (1986) J. Lipid Res. 27, 30-39. 41 Davidson, N.O., Drewek, M.J., Gordon, J.I. and Elovson, J. (1988) J. Clin. Invest. 82, 300-308. 42 Black, D.D. and Davidson, N.O. (1989) J. Lipid Res. 30, 207-218.
93
BBALIP 54042
Regulation of intestinal apo A-IV mRNA abundance in rat pups during fasting and refeeding Masao Sato a, Katsumi Imaizumi a, Haruhiko Mori a and Michihiro Sugano b a Laboratory of Nutrition Chemistry and b Laboratory of Food Chemistry, Department of Food Science and Technology, School of Agriculture Kyushu U~i~~er$~~,Fukuok~ (Japan~
(Received 20 May 1992)
Key words: Apolipoprotein
A-IV, mRNA; intestinal apo A-IV; Serum triacylglycerol; Fat absorption; Fat secretion; (Rat pups)
The amount of intestinal apolipo~ro~~in (ape> A-IV mRNA was examined in rat pups during fasting and refeeding. When 14-day old pups were fasted for 15 h, apo A-IV mRNA levels in the whole intestine decreased to 20% of the prefasting level. Refeeding
casein and lactose, and the artificial milk composed of Intralipid, casein and lactose, caused an elevation of the apo A-IV mRNA after 3 h, without accompanying an elevation of serum triacylglycerols and apo A-IV (fat-independent elevation of apo A-IV mRNA). Refeeding Intralipid alone simultaneously elevated the apo A-IV mRNA, and serum triacylglycerols and apo A-IV after 3 h ~fat-dependent elevation of ape A-IV mRNA). Administration of physiologicaI saline during fasting partly suppressed the reduction of the apo A-IV mRNA ~40% of the prefasting level), and the dietary fat-independent elevation of the message disappeared. Refeeding dam’s milk to the pups, fasted without water administration, increased the apo A-IV mRNA after 3 and 15 h, although the elevation of serum triacylglycerols and apo A-IV occurred only after 15 h. Refeeding the milk increased the apo A-IV mRNA after 3 h and 15 h. Refeeding dam’s milk to the pups fasted with saline administration accelerated the fat-dependent elevation of the apo A-IV mRNA. Simultaneously refeeding Intralipid and Pluronic L-81, an inhibitor of lymphatic fat transport, delayed the elevation of the apo A-IV mRNA and serum triacylglycerols and apo A-IV. Transcription rates of the apo A-IV mRNA, de~e~iued by nuclear run/on assay, were similar before and after fasting and refeeding Intralipid. During fasting, administration of puromycin, as compared with actinomycin D, enhanced the disappearance rate of the apo A-IV message. Intestinal mRNA for apo B, but not for apo A-I and /3-actin, similarly changed to the apo A-IV message. Thus, it can be concluded that: (1) dietary fat-dependent and -independent factors are involved in the elevation of the intestinal apo A-IV message; (2) the elevation of the message is not mediated by lipid uptake in the enterocytes but rather stimulated by the events leading to secretion of chytomicrons; and, (3) dietary fat-dependent elevation of the message appears to be due to the stabilization of the message.
Introduction A~olipopro~ei~ (ape> A-IV was first discovered as a component of serum high-density lipoproteins in adult rats by Swaney et al. [l]. This apoprotein was subsequently identified in mesenteric lymph chylomicrons in rats [2,3], human chylus fluid [4] and rat renal lymph
[.5]. Apo A-IV is also distributed in lipoprotein-free fractions of. mesenteric lymph 161 and sera [7,8] of mammals. cDNA of apo A-IV was first constructed by Gordon et al. [9] from rat intestinal epithelial cells, and now primary sequences of apo A-IV of rat [lo], mouse [ 111 and man [121 have been deduced from cDNA
Correspondence to: K. Imaizumi, Laboratory of Nutrition Chemistry, Department of Food Science and Technology, School of Agriculture, Kyushu University, Fukuoka 812, Japan.
sequence analyses. The function of this apoprotein still remains unclear, although it may be involved in cholesterol transport [13], tria~l~lycerol clearance in the blood serum 1141 and uptake of high-density lipoproteins by the cells [15]. Secretion of apo A-IV into mesenteric lymph in rats increases when fat emulsions were infused and large chylomicrons isolated from the lymph contain considerable amounts of this apoprotein [Z]. Oral intake of fat emulsions in humans increases the serum level of apo A-IV [16]. These early results suggest that dietary fats are involved in the regulation of apo A-IV synthesis and transport in the intestine. Detailed molecular mechanisms, however, remain largely unsolved. Frost et al, 1171 showed that absorption and transport of dietary fats are more active in rat pups suckled with dam’s milk as compared to adult rats raised on commercially available rat chow. Levels of serum apo
94 A-IV [18] and intestinal apo A-IV mRNA [19] also dramatically change developmentally after birth: higher in the suckling period and lower after weanling. The present study was carried out to examine the regulation of the intestinal apo A-IV mRNA IeveI in rat pups during fasting and refeeding. The results show that factors dependent on and independent of dietary fats elevate the intestinal apo A-IV mRNA and that continuous absorption and transport of dietary fats may stabilize the message. Materials
quently, 0.5 ml of saline containing 10% glucose was administered at 8, 10, 12, 14, 16, 18, 20 and 22 h. The lipid emulsion was made of 7 ml Intralipid and 3 ml phosphate-buffered saline (pH 7.4). The Pluronic L-81 lipid emulsion was made of 7 ml Intralipid and 3 ml Pluronic L-81 solution (8.8 pmol egg phosphatidylcholine, 57 kmol sodium taurocholatc and 1.0 mg Pluronic L-81 in the phosphate-buffered saline). The pups were killed at 0, 3 and 6 h in the Pluronic L-XI phase and at 9, 12, 15 and 24 h in the saline administration phase.
and Methods
Animuls l4-day old Wistar rat pups with their dams (8-10 pups per dam) were obtained from Seiwa Experimental Animal Co., Fukuoka. Animals were maintained under controlled temperature (23 + 1°C) and light (light period 06:00-18:OO). All aspects of the experiment were approved by Kyushu University Animal Policy and Welfare Committee. The pups were fasted for 15 h without water (dehydrated pups), or administered with 0.75 ml of physiological saline by a stomach tube made of silicone for every 3 h (hydrated pups). Some pups were returned to their dams to feed their milk and others were refed one of the folIowing liquid diets with a stomach tube every 2 h: (1) commercially available Intralipid (10% w/v soybean oil, 1.2% W/Y egg lecithin and 2.5% w/v glycerol, Kabivitrum AB, Stockholm); (2) artificial milk [ZO] composed of 9.2% (w/v) casein and 3.0% (w/v) lactose which were soluhilized directly in ln~raiipid (pH 7.0); (319.2% (w/v) casein in water; and (4) 3.0% (w/v) lactose in water. The pups were killed by withdrawing aortic blood under diethyl ether anesthesia. The whole intestine from the duodenum to the ileum was immediateIy excised, flushed with ice-cold physiological saline to remove the intestinal contents, and stored in liquid nitrogen untif used. Treatment of pups with drugs Some pups were given intraperitoneally a 600 pg of puromycin (2 mg/ml saline) immediately after removal from their dams, and followed by injecting 80 pg puromycin twice every 2 h during fasting, according to the schedule used for adult rats [21]. This treatment for adult rats has been reported to minimally influence transport of dietary fats [22]. Actinomycin D (1 mg/kg body weight in saline/506 v/v ethanol) was also given intraperitoneally to the pups just before fasting 1221. Pups were also treated with Pluronic L-81 [23], a potent inhibitor of chylomicron formation, which was obtained from Dr. P. l-so, Louisiana State University, Shreveport, LA. After 15 h fasting, 0.5 ml of Pluronic L-81 lipid emulsion or lipid emuIsion alone was administered by the stomach tube at 0, 2, 4 and 6 h. Subse-
Electroimmunoassays of appolipoproteinA-IV The serum apo A-IV was measured by rocket immunoelectrop~oresis as described in Ref. 18. Concentration of apo A-IV in the serum was determined by comparing its rocket heights with those in a series of dilutions of pooled serum obtained from S-week-old male Sprague-Dawley rats, and expressed as an arbitrary unit. Lipid unalyses Serum triacylglycerols were measured by using an enzyme assay kit, Triglyceride G-Test (Wake Pure Chemicals, Osaka). Mucosal lipids in the pup’s whole intestine were extracted in chloroform/met~anol mixture (2 : 1, v/v) 1241. The lipids were quantitatively spotted on thin-layer plates (Silica-gel G, Merck) by Drumond microsyringe with the solvent system composed of hexane/ diethyl ether/ formic acid (80:20:2, v/v) [25]. A series concentration of triolein was simultaneously appIied to the plate as an internat standard. After the plate was sprayed with 3% (w/v) Cu acetate -7% (w/w> phosphate solution 1261 and heated at 150°C for 30 min, the triacylglycerol contents were determined by scanning densitometry (Desital Densitorol DMU-33C, Toyo Science industrial, Tokyo) at 650 nm. Isolation and quantitation of mRNA by slot blot hybridization ussay and Northern analysis Total RNA was isolated from the whole intestine by the guanidine thiocyanate procedure according to the method of Chirgwin et al. [27] and stored at -40°C. The RNA sample was denatured with glyoxsal at 50°C for 1 h as described by Thomas [28]. Constant amounts of RNA supplemented with yeast RNA to provide a final amount of 0.8 Fug of RNA per sample were blotted onto the nitrocellulose filter (Zeta Probe membrane, Bio-Rad Japan, Tokyo) prewetted with 20 X SSC (3 M NaCl, 1 M sodium citrate, (pH 7.0)) with a vacuum-manifold apparatus (Bio-Rad Japan). For Northern analysis, the denatured total RNA was electrophoresed in a 0.8% (w/v) Agarose gel prepared in 20 mM sodium phosphate buffer, pH 6.5 [281. After pre-hybridization with a hybridization solution (5 X
95
oJ,*, 0
( 025
05
,
, 075
10
Dilutton Factor
b
cl
I/
0---"-Y-
0
02
04
06
08
Intestinal Total RNA (vg)
Fig. 1. Slot blot hybridization assay for intestinal apo A-IV mRNA in rat pups. (A) Standard curve constructed with a series of dilution of the intestinal total RNA prepared from nonfasted pups. (B) Relationship between the densitometric area and three different amounts of total RNA prepared from fasted (open circle) and nonfasted (closedcircle)pups, respectively.
SSC, 50% w/v formamide, 20 mM sodium phosphate, pH 6.5, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrrolidone, 0.02% w/v bovine serum albumin, and 100 mg/ml denatured herring sperm DNA) for 5 h at 42”C, hybridization reactions were performed in the hybridization solution containing cDNA labeled with [5’“PI dCTP (1.48 MBq/mmoI, Amersham Japan, Tokyo) by maItiprime DNA labeling system according to the Amersham’s instruction for 18 h at 42°C. The cDNA clones for rat apo A-IV [lOI and apo B 1291 were obtained from Dr. J.I. Gordon (Washington Univ. School of Med., St Louis, MO) and Dr. A. Matsumoto (National Institute of Health and Nutrition), respectively. Thirty-oligomer to rat apo A-I cDNA was synthesized by Research Laboratory for Genetic Informations, Kyusyu University, Fukuoka. cDNA for @-actin was obtained from Wako Pure Chemicals. Blots were exposed to X-ray film (Fuji Film Industry, Tokyo), and autoradiograms of the filters were analyzed by quantitative scanning densitomet~ (Desital Desitorol DMU33C). Total RNA from one of the pups before fasting was used to construct a standard curve (Fig. 1). The levels of mRNA were expressed as an arbitrary unit. The levels of mRNA in one of the pups before fasting was assumed to be 100. When autoradiograms for three different amounts of RNA gave a linear densitometric area as shown in Fig. 1, the mean value was adopted as the abundance of the sample’s mRNA. A similar standard curve was constructed for the messages of apo A-I, apo B and @-actin.
Whole intestine of rat pups obtained immediately after killing, were washed with ice-cold 10 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl. Nuclei were isolated from the whole intestine, according to the method of Nawa et al. [30], and modified as follows. The tissue segment (1 g wet wt.> was suspended in 6 ml homogenizing buffer (10 mM Tris (pH 8.0)) containing 0.32 M sucrose, 3 mM CaCl,, 2 mM Mg acetate, 0.1 mM EDTA, 0.1% (w/v) Nonidet P-40, 1.0 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride, and homogenized in loose-fatting Potter-E~vehjem Teflon homogenizer (500 rpm and 5-6 strokes). The nucleus solution was layered on the dilution buffer (10 mM Tris-HCl, pH 8.0 containing 2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride), and centrifuged at 50,000 x g for 60 min at 2°C. The precipitated nuclei were suspended in storage buffer (50 mM Tris, pH 8.0, containing 45% glycerol, 5 mM MgCl,, and 0.1 mM EDTA), counted by hemocytometer, and stored in liquid nitrogen. RNA transcription was measured according to the method of Protko et al. [31]. Briefly, nuclei (8 X 10’) were incubated in 25 mM Tris (pH 8.0) containing 20% (w/v) glycerol, 2 mM MgCl,, 150 mM KCl, 1 mM dithiothreitol, 1 mM ATP (Wake Pure Chemicals), 0.5 mM CTP (Yamasa Shoyu, Tokyo), 0.5 mM GTP (Sigma Chemicals), 8 mM phosphocreatine (Nakalai Tesque), 40 mg of creatine kinase/ml, 50 units/ml of RN&n (Sigma) and [a-32 P]UTP (1.48 MBq/mmol; Amersham Japan). RNA synthesis was terminated after 30 min, and nuclei were treated with 20 mg/ml of DNAase I (BMY, Tokyo), 100 mg/ml of proteinase K (BMY) and 50 mg/ml of yeast tRNA (BMY), The RNA was extracted twice with phenol/chloroform (1: 1, v/v) and precipitated in ethanol. The newly transcribed RNA was hybridized to DNA immobilized onto nitrocellulose filters (Zeta Probe membrane, Bio-Rad). Plasmid DNA was linearized, heat-denatured, spotted on the filters and baked at 80°C. Prehybridization was carried out in 50% (v/v) formamide containing 5 x Denhardt’s, 5 x SSC, 50 mM sodium phosphate (pIi 7.41, 5 mM EDTA, 0.1% (w/v) SDS and 100 mg of tRNA/ml. Hybridization was carried out in the same solution with labeled RNA added for 2 days at 42°C. After hybridization, the filters were washed and treated with RNAases A and Tl. The sample filters were counted for 5 min in a liquid scintillation counter (Tri-carb 2250, Packard Japan, Tokyo). Statistics
Differences within groups were compared by analysis of variance, and group differences were compared by the independent two-tailed r-test. Differences were
96 considered significant when the probability of the difference occurring by chance was less than 5 in 100 (P < 0.05) for both t-test and analysis of variance. Results When 14-day-old pups were nursed normally by their dams, the levels of intestinal apo A-IV mRNA and the serum apo A-IV measured every 3 h for 24 h were essentially similar (data not shown), suggesting no significant fluctuations in a day. When these pups were fasted for 15 h without giving water, serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA determined by slot blot and Northern blot assay decreased (Fig. 2). Since the validity of the slot blot assay was supported by the Northern blot assay as shown in Fig. 2 (the lowest figure), the slot blot assay was used
0
3
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9
Time After Refeedmg (hr)
Fig. 3. Effect of refeeding artificial milk, casein, lactose and Intralipid on serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA. Pups fasted for 15 h without administration of water were refed liquid diets every 2 h. Each point and bar show means& different from the values S.E. for 3 pups per group. ” Significantly after 15 h fasting at P < 0.05. h Significantly different from the values of the pups refed lntralipid at P < 0.05.
C k
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I
,
2
4
.
I
I
1
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8
10
’
I
I
12
14
-
Time After Fasting (hr) Fig. 2. Effect of fasting on serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA. Pups were removed from their dams at 3 pm. and subsequently fasted for 15 h without administration of water. Northern blotting of apo A-IV mRNA in the intestine from the fed pups before fasting (A) and after 15 h fasting (B) is shown in the inlet of the lowest figure. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the prefasting values at P < 0.05.
for the subsequent experiments. The apo A-IV message reached the bottom level (20% of the prefasting level) after 8 h fasting. As shown in Fig. 3, administration of artificial milk to pups fasted without administration of water (dehydrated pups) elevated the apo A-IV mRNA after 3 h, although the serum triacylglycerols and apo A-IV did not change. Similar patterns were also observed when dehydrated pups were given casein or lactose solution. The administration of Intralipid alone to dehydrated pups, however, caused a simultaneous elevation of the message, and serum triacylglycerols and apo A-IV after 3 h. Fig. 4 showed that fasting caused a reduction in the intestinal apo B mRNA to 40% of the prefasting level. Refeeding the artificial milk elevated the apo B mRNA to 80% of the prefasting level after 6 h. The messages for apo A-I and p-actin, however, showed essentially no changes during fasting and refeeding the milk. Refeeding the saline to the pups fasted without administration of water (dehydrated), also enhanced the apo A-IV mRNA after 3 h (Fig. 5). When pups
97
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1
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12
Fig. 4. Effect of refeeding artificial milk on intestinal messages for apo A-I, B, and p-actin. Experimental conditions are shown in the legend to Fig. 3. Each point and bar show means + S.E. for 3 pups per group. a Signifcantly different from the values after 15 h fasting at P < 0.05.
were administered the saline during fasting (hydrated), the reduction of the intestinal apo A-IV mRNA was significantly suppressed and the message decreased to 40% of the prefasting level. In the next experiment, pups that were hydrated or dehydrated during fasting were nursed again by their dams. Firstly, pups were not administered water for 15 h. As shown in Fig. 6, these pups soon appeared to be suckled, as intestinal triacylglycerols increased almost to the maximum level as early as 3 h after nursing. The stomach contents also increased after 3 h nursing and maintained constant weight throughout the nursing period (data not shown). Significant elevation of the serum triacylglycerols and apo A-IV was delayed until after 15 h nursing. These elevations were suppressed when pups were again removed from their dams after 12 h. Elevations of the intestinal apo A-IV mRNA were observed after 3 and 15 h. Secondly, pups were fasted 15 h with saline administration. The patterns of the serum triacylglycerols and apo A-IV, and the intestinal mRNA markedly differed from those pups dehydrated during fasting, although intestinal accumulation of the milk triacylglycerols similarly occurred (Fig. 7). The level of serum triacylglycerols and apo A-IV increased gradually during nursing. The apo A-IV mRNA increased after 6 h, but subsequently decreased again to the fasting level. The apo B mRNA showed a pattern similar to that of the apo A-IV message. The messages for apo A-I and p-actin did not markedly change during fasting and nursing. As shown in Fig. 8, when Intralipid was refed to the hydrated pups, the patterns of the elevation of the serum triacylglycerols and apo A-IV resembled those pups nursed with dams (Fig. 7). Absorption and subsequent secretion of the dietary fat occurred as early as 1.5 h after. The apo A-IV mRNA increased 6 h after, although as shown in Fig. 3, the message increased after 3 h when the dehydrated pups were refed Intralipid alone. Subsequently, the level of apo A-IV mRNA was maintained to more than prefasting level.
Again, apo B mRNA followed the pattern of the apo A-IV mRNA, and the messages for apo A-I and p-actin showed no change. To examine the effect of the inhibitor of fat secretion from the intestine on the levels of serum triacylglycerols and apo A-IV, and the intestinal apo A-IV mRNA, dehydrated pups were refed with Intralipid alone or the Intralipid supplemented with Pluronic L-81. Fig. 9 showed that refeeding Intralipid alone caused elevations of intestinal triacylglycerols and apo A-IV mRNA, and serum triacylglycerols and apo A-IV. Administration of the saline following Intralipid promptly decreased all the parameters. Refeeding the mixture of Intralipid and Pluronic L-81, as compared with Intralipid alone, caused a marked accumulation of the mucosal triacylglycerols. Maximum elevations of the serum triacylglycerols and apo A-IV, and intestinal apo A-IV mRNA delayed and occurred after the saline administration. As shown in Fig. 10, Pluronic L-81 treatment suppressed the elevation of the intestinal apo B mRNA throughout the period, although refeeding Intralipid alone promptly increased the message. Table I shows the rate of apo A-IV mRNA transcription in the intestinal nuclei prepared from the pups suckled by dams (Fed), fasted for 15 h (Fast), and refed Intralipid for 6 h (Refed). There were no significant differences among the transcription rates for the apo A-IV mRNA. To examine the effect of inhibitors for protein and RNA syntheses on the disappearance rate of the intestinal apo A-IV mRNA during fasting, pups were treated with puromycin and actinomycin D. As shown in Fig. 11, a half-life of the apo A-IV mRNA reduction in the pups untreated with drugs was 3.7 h. The administration of actinomycin D, an inhibitor of RNA synthesis, accelerated the disappearance rate of the apo
0
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12 15 18 2l
P
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Time After Fasting (hr)
Fig. 5. Effect of hydration and dehydration during fasting on intestinal apo A-IV mRNA. Pups were separated into two groups: one group received physiological saline every 2 h for the initial 15 h (hydrated), and the other was not (dehydrated). Subsequently both groups were administered the saline every 3 h. Arrow indicates the start of the administration of the saline to the dehydrated pups. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the corresponding values of the dehydrated pups at P < 0.05. b Significantly different from the values after 15 h fasting at P < 0.05.
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3
6
0
9
3
0
3
0
3
12 15 18 21 24 27
6
9
12
15
18
-
Time after Refeeding (hr)
6
9
12 15 18 21 24 27
6
9
12 15 18 21 24 27
Time after Refeeding (hr)
Fig. h. Effect of refeeding dam’s milk to dehydrated pups on the serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and ape A-IV mRNA. After fasting for 15 h without administration of water, the pups were returned to their dams. At after 12 h, returned to the dams, some pups were fasted again (black circle with dotted line). Pups were killed every 3 h. Each point and bar show means&SE. for 3 pups per group. ” Significantly different from the values after IS h fasting at P < 0.05.
A-IV mRNA during fasting, and puromycin, an inhibitor of protein synthesis, further accelerated the disappearance.
apo A-IV and tri~cylglycerols. These elevations dependent on dietary fat were maintained as long as the fat feeding continued. Thus, present study confirmed the early observation by Gordon et al. [9] who showed that intragastric administration of plant oil to adult rats, fasted overnight, increases the intestinal apo A-IV mRNA levels. There are very few studies on the regulation of apo A-IV mRNA Ievel affected by factors other than dietary fats. One report describes a higher
Discussion
Present study showed that refeeding Intralipid alone pups, fasted without water administration promptly increases the intestinal apo A-IV mRNA, and serum to
01,
,
,
0
3
6
9
12
,
,
04
15
18
0
1
3
6
I
9
e, a0 ‘II lii3 Y.r 15 =x6 .m 0‘ C .c ,O T+m c,zz 5 $E 0~ 0
,
/
15
18
afx A-W
l
a
12
n aen
a a a
3
a
6
9
12
a
a
15
16
Time after Refeeding (hr.1
=
0
3
6
9
12
15
Time after Refeeding (hr
113
)
Fig. 7. Effect of refeeding dam’s milk to hydrated pups on the serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and messages for apo A-I. apo A-IV, apo B and p-actin. Pups were administered the saline every 2 h for 15 h fasting, then returned to their dams. Each point and har show mean + SE. for 3 pups per group. a Significantly different from the values after IS h fasting at P < 0.05.
99
0
3
6
9
12
15
Or 0
3
6
9
12
15 Time After Refeeding (hr)
Time After Refeeding (hr)
Fig. 8. Effect of refeeding Intralipid to hydrated pups on serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and the messages for apo A-I, A-IV, B, and p-actin. Pups were administered physiological saline every 2 h for 15 h fasting. At after 15 h fasting, the pups were refed with Intralipid alone every 2 h. Each point and bar show means k S.E. for 3 pups per group. a Significantly different from the values 15 h fasting at P < 0.05.
dent elevation of the apo A-IV mRNA disappeared when the pups were administered with saline during fasting, some endocrine and/or neural informations provoked by an expansion of the stomach after refeeding may be responsible for this phenomenon. Present study showed that refeeding artificial milk delays the elevation of serum triacylglycerols and apo A-IV, and the intestinal apo A-IV mRNA dependent on dietary fat. Dietary-fat-dependent elevation of the apo A-IV mRNA was also delayed in dehydrated pups when they were nursed again. Although nursing again
level of apo A-IV mRNA in the cultured rat hepatocytes when the medium was supplemented with insulin and dexamethazone alone, or both [19]. Present study showed that feeding fat-free diets such as saline, casein and lactose solution also increases the intestinal apo A-IV mRNA level. This experiment suggests that factor(s) other factor (s) than dietary fats, participate in the elevation of apo A-IV mRNA level in the intestine. This dietary-fat-independent elevation for the intestinal message was also observed for the apo B, but not for apo A-I and p-actin. Since the dietary fat indepen-
0
3
0
3
6
9
12
15
18
21
24
6
9
12
15
18
21
24
Time After Refeeding (hr)
Fig. 9. Effect of refeeding Pluronic L-81.Intralipid mRNA. After 15 h fasting without administration with Pluronic L-81 (Pluronic L-811, and the other
0
0
3
3
6
6
9
9
12
12
15
15
18
18
2-l
21
24
24
Time After Refeeding (hr)
emulsion on serum triacylglycerols and apo A-IV, and intestinal triacylglycerols and apo A-IV of water pups were separated into two groups: one group refed with Intralipid supplemented Intralipid alone (Control). Arrow indicates the start of the saline-glucose administration. Each point shows mean of two pups.
l Pluronic L-81
r---
0
3
6
9
12
15
18
0
21 2S
Time After Refeeding (hr) Fig.
IO. Effect
intestinal
apo
of refeeding 6 mRNA.
Piuronic
Pups were
L-Wlntralipid treated
emulsion
as described
on
in the
legend to Fig. 9. Each point shows mean of two pups.
hydrated pups during fasting accelerated the elevation of the apo A-IV mRNA, the elevation did not last as compared with the pups refed Intralipid alone. Absorption process of the dietary fat was not delayed as these milk did not disturb the mucosal accumulation of triacylglycerols. Thus, milk component(s) other than fat may participate in the regulation of the level of the intestinal apo A-IV mRNA in pups refed dietary fat. To investigate the effect of secretory pathway of the intestinal tria~lglycero~s on the level of the mucosal apo A-IV mRNA, the pups were administered Pturonic L-81 which has been reported to inhibit the chylomicron transport in adult rat 2231. The present study showed that treating pups with Pluronic L-81 delayed both the elevations of serum triacylglycerols and the intestinal apo A-IV mRNA. Thus, the amount of intestinal apo A-IV mRNA dependent on dietary fat is closely related to the events involved in the packaging and secretion of chylomicrons rather than the absorptive process of the tuminal fats. In this regard, the present results agree with the results reported by ~ayashi et al. [32] who showed that an elevation of the translational activity for apo A-IV foIlows active lymphatic transport of dietary triacylglycerols. At present, two factors have been reported with regard to the prompt processing of dietary fats to lymphatic chylomicrons in adult rats: (1) availability of phospholipids synthesized endogenously and supplied exogenously [33], and (2) lymph flow [341. Some dietary components have been reported to influence secretion
TABLE
1
Sy~i~~~i~mte of’apo A-W ml&VA by ~zuclear nm /on assuy Pups with dams (Fed) tered lntralipid
were fasted for 15 h (Fasted),
for 6 h (Refed).
Values
are meansiS.E.
per group. Treatment
Rate of RNA (ppm)
Fed Fasted Refed
9.23 + 0.98 7.24 + 1.01 10.1
+0.30
synthesis
4
2
6
8
Time After Fasting (hr)
and adminisfor 5 rats
Fig. ii.
Effect
of transcription
pearance
of intestinal
separated
into three
and translation
apo A-IV
mRNA
groups as described
with actinomycin
D and puromycin,
At later
the amount
treated
period,
during
inhibitors fasting.
on disapPups were
in the text: pups treated
or without
of the apo A-IV
treatment mRNA
with the drugs was too small to be determined.
(Control). in the pups Each point
shows means for 3 pups per group.
of intestinal lipids to the lymph. Intraduodenal infusions of a fat-free mixture of dextrin and casein [35] or casein and sucrose with or without ethanol [36] all significantly increase intestinal lymph Iipids. On the contrary, lower levels of lymph lipid output were reported during sucrose and starch infusion period [40]. Dietary proteins and carbohydrates inhibit secretion of c~ytomicrons directly or indirectly through stimulation of some gastrointestinal hormones, which appear to inhibit bowel motility [38]. Concerning rat pups, the mechanisms on how dietary casein and/or lactose in the milk influence the utilization of exogenous phospholipid for chylomicron synthesis and secretion [33], and permeability of blood fluid to interstitial spaces of intestinal mucosa, which facilitates chylomicron output from the cells to lymph [34], are stilt unknown. To determine whether the dieta~-fat-dependent level of the intestinal apo A-IV mRNA is due to an enhanced transcriptional activity or post transcriptional events, nuclear run/on assay was carried out. The present results showed no remarkable differences in the transcription rate during the suckling period by dams, after 15 h fasting and after 6 h refeeding Intralipid. Thus, the dietary-fat-dependent level of the intestinal apo A-IV mRNA appeared to be regulated mainly during the post-transcriptional events. In addition, since puromycin as compared with actinomycin D further accelerated the disappearance rate of the message during fasting, dietary fat may modulate the synthesis of some protein(s) which participates in the stabilization of the intestinal message. In the present study, lymphatic secretion of the translation products of apo A-IV mRNA was determined indirectly by measuring the level of the serum apo A-IV, Since the serum level of apo A-IV was well correlated to the serum triacylglycerol level, the determination of the serum apo A-IV can partly reflect the translational activity of apo A-IV protein in the intestine, although Windmuellar and Wu have shown that
101 apo A-IV synthesized in intestine is also transported into portal system 1391. Fig. 7 showed that serum apo A-IV continues to increase after refeeding dam’s milk although the intestinal apo A-IV mRNA only transiently increased. Therefore, dietary fat and/or other milk component(s) may also be involved in the translational process of apo A-IV. In addition to apo A-IV message, apo B mRNA, but not messages for apo A-I and p-actin, in the intestine was also enhanced following Intralipid administration alone. Regulation of apo B mRNA in the pup’s intestine was apparently similar to that of apo A-IV mRNA, but not identical as the saline administration following the mixture of Pluronic L-81 and Intralipid elevated the apo A-IV mRNA, but not apo B mRNA. In addition, Davidson et al. did not find any significant changes in the synthetic rate of apo B in the adult rat intestinal segment [40] and its mRNA [41]. Also, when given high fat diet, the intestinal apo B synthesis in piglets appeared to be unaffected by the triacylglycerol absorption, even though apo A-IV synthesis increased [421. Thus, different factors derived from dietary fats appears to be involved in the transcriptional and posttranscriptional regulation of these apoprotein synthesis in the intestine. Acknowledgment
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (03303011). References
5 6 7 8 9 10
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