Apolipoprotein AIV (apo AIV) is a satiety protein secreted by the small intestine. We demonstrate for the first time that apo AIV protein and apo AIV mRNA are present in rat hypothalamus, a site intimately involved in the integration of signals for regulation of food intake and energy metabolism. We further characterized the regulation of hypothalamic apo AIV mRNA levels. Food-deprived animals showed a pronounced decrease in gene expression of apo AIV in the hypothalamus, with a concomitant decrease in the jejunum. Refeeding fasted rats with standard laboratory chow for 4 h evokes a significant increase of apo AIV mRNA in jejunum but not in hypothalamus. However, lipid refeeding to the fasted animals restored apo AIV mRNA levels both in hypothalamus and jejunum. Intracerebroventricular administration of apo AIV antiserum not only stimulated feeding, but also decreased apo AIV mRNA level in the hypothalamus. These data further confirm the central role of apo AIV in the regulation of food intake.
- satiety factor
- competitive reverse transcription polymerase chain reaction
- food intake
- central nervous system
apolipoprotein AIV (apo AIV) is a glycoprotein secreted only by the small intestine in humans (12). In rodents, both the small intestine and the liver secrete apo AIV; however, the small intestine is the major organ responsible for the circulating apo AIV (1). apo AIV is secreted in association with chylomicrons (CM) and displaced rapidly from the CM by other apolipoproteins during CM metabolism in the circulation (18, 28). It is well established that the output of apo AIV into intestinal lymph increases markedly as a result of lipid feeding (12, 15). This effect is associated with increased synthesis of apo AIV by the small intestine. Hayashi et al. (14) demonstrated that it is the formation and secretion of CM during lipid absorption, not the uptake and reesterification of monoglycerides and fatty acids to form triglycerides, that stimulate intestinal apo AIV synthesis and secretion.
The pattern of apo AIV secretion resembles that of several gut peptides that facilitate digestion and absorption and also reduce meal size, such as cholecystokinin (27, 3), the bombesin family of peptides (11), and glucagon-like peptide-1 (25). Our previous studies clearly demonstrated that apo AIV is a circulating signal released in response to fat feeding and that it mediates the anorectic effect of a lipid meal (6). A subsequent study in rats provided evidence that apo AIV is present in the cerebrospinal fluid (CSF) and that CSF levels increase markedly during fat absorption (8). Furthermore, the intracerebroventricular infusion of near-physiological doses of exogenous apo AIV dose dependently inhibited food intake. Ad libitum-fed rats normally do not consume food during the light period. Interestingly, ad libitum-fed rats all had food intake during the light cycle after the intracerebroventricular infusion of apo AIV antiserum (7). These observations collectively suggest that apo AIV controls food intake by acting within the central nervous system (CNS). Unanswered questions that remain, however, are 1) how does apo AIV, a protein with a molecular mass of 44 kDa, gain access to CNS sites and 2) does the brain synthesize apo AIV? With the use of RNA dot-blot analysis of whole brain RNA, one report suggests that the brain probably does not make apo AIV (4). However, this conclusion may be incorrect in view of the fact that whole brain RNA was used and the fact that RNA dot-blot analysis is not as sensitive as some other methodologies currently available to detect mRNA. The present study was designed to determine whether apo AIV is synthesized in the hypothalamus and, if so, whether it is regulated by physiological signals involved in homeostasis.
Male Sprague-Dawley rats (280–320 g) were housed in a temperature- and humidity-controlled room, illuminated from 0600 to 1800 (12:12-h light-dark cycle). They were divided into four groups. In the control group, rats had free access to Teklad laboratory standard chow (Madison, WI). Fasting rats were food-deprived for 28 h (starting at 2300). In the other two groups, rats were first fasted for 24 h (food deprivation also starting at 2300), then refed with standard laboratory chow or fed 5 ml of Intralipid (Abbott Laboratories, North Chicago, IL) by gavage. Water was available ad libitum in all groups of rats. Animals were killed early in the dark (0300) because we found previously that food intake is greatest at that time (10, 19) and because serum apo AIV peaks at the same time (9). All procedures involving the animals were performed in accordance with the institutional guidelines of the Animal Care Committee at the University of Cincinnati.
Rats receiving intracerebroventricular administration of apo AIV antibodies (or control solution) were anesthetized with 86 mg/kg ketamine and 1.4 mg/kg acepromazine. A 21-gauge cannula (Plastics One, Roanoke, VA) was placed stereotaxically into the third ventricle using a previously described method (21, 23). Cannula placement was verified 1 wk after surgery by injection (1 μl icv) of angiotension II (10 ng/μl). Animals not consuming at least 7 ml water 30 min postinjection were excluded.
Intracerebroventricular infusion of apo AIV antibody.
Two weeks after cannula placement, animals were subdivided into two groups of equal body weight. Goat anti-rat apo AIV serum (28 μg of protein in 5 μl of sterile saline) was injected into the third ventricle at a rate of 1 μl/min, starting at 1100. This time period was chosen because it is about midway through the light period, and rats would not normally be expected to eat at this time (19). The specificity of the apo AIV antiserum for apo AIV has been described before (14). As a control, normal goat serum (28 μg of protein in 5 μl of saline) was injected into the third ventricle at the same rate. Feeding and drinking behavior were observed for 60 min, after which the rats were killed by decapitation and the hypothalamus was removed for measuring the apo AIV mRNA level by competitive RT-PCR analysis.
PCR primers and RT-PCR conditions.
Reverse transcription was done using total RNA extracted by Tri-Reagent (Molecular Research Center, Cincinnati, OH) from the rats. First-strand cDNA and PCR amplification were performed in a single tube that contained a Ready-To-Go-PCR bead (Amersham Pharmacia Biotech, Piscataway, NJ). Each reaction (50 μl final volume) contained 10 mM Tris · HCl (pH 9.0), 60 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, Moloney Murine Leukemia Virus (M-MuLV)-RT, 2.0 units of Taq DNA polymerase, ribonuclease inhibitor (porcine), and stabilizers. Each reaction also contained 10 pM each of following primers: P-1: f5′-CTTTGCCAACGAGCTAAAGG-3′ and P-2: r5′-GCTGCTTGTTCAGGTCTTCC-3′. The reaction was placed in an MJ PTC-100 thermocycler at 42°C for 30 min. At the end of the reverse transcription reaction, the thermocycler was programmed to increase temperature to 95°C for 5 min to inactivate the RT and to completely denature the template. The thermocycler was then programmed for 29 cycles consisting of denaturation at 92°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s. The amplification was terminated by a 5-min final extension step at 72°C. PCR products were separated on a 1.5% agarose gel, stained with Gelstar (FMC, Rockland, ME), and photographed under ultraviolet (UV) illumination to verify the size of the RT-PCR products (343 bp for rat apo AIV).
Competitor design and the establishment of competitive RT-PCR.
A competitor of apo AIV mRNA was constructed according to the supplier's instruction (Ambion, Austin, TX). Primer P-3 (f5′-GCGTAATACGACTCACTATAGGGAGAGGAGACTAACCCCCCGTGCCAACG-3′) includes a T7 promoter primer, the P-1 sequence in its middle, and a downstream target specific sequence. P-4 primer (r5′-CTTATCCCCCAG-GGGCTCCA-3′) synthesis from the target cDNA at a site ∼50 nucleotides downstream of the P-2 binding site, which assures that the competitor is reverse transcribed as efficiently as the endogenous target (Fig. 1). Besides, 2′ modified nucleotides (CTP and UTP) incorporated during transcription confer nuclease resistance to the RNA competitor, making the competitor molecules stable in the presence of most nucleases.
For in vitro transcription, the above PCR product (389 bp) containing the cDNA specific for the apo AIV competitor was transcribed by T7 RNA polymerase, purified by 5% acrylamide-8 M urea gel, and quantified according to the manufacturer's instruction (Ambion, Austin, TX). The cRNA was stored at −20°C and used as an internal standard.
A series of concentrations of internal standard cRNA were added to each tube containing 200 ng of total RNA. The conditions for competitive RT-PCR and primers (P-1 and P-2 described above) are the same as the one described above for RT-PCR. After 29 cycles, the products were resolved on a 1.5% agarose-Gelstar gel. The fluorescence of each band was measured. Data are presented as the log ratio of density of the amplified cRNA internal standard (306 bp) to the density of target apo AIV amplification product (343 bp) and plotted against the log of known amounts of internal standard cRNA added to the test sample to generate a competitive PCR linear regression curve. The absolute amount of target apo AIV mRNA was calculated from the curve as the point of equivalency (p.o.e., see arrow, Fig. 3), at which the ratio of internal standard to target RNA was equal to 1.
The following control experiments were performed to ensure the specificity of the RT-PCR. First, the in vitro transcribed RNAs (target and internal standard) were amplified without RT to ensure that the amplification products obtained derived only from the RNAs and not from remaining DNA sequences caused by incomplete digestion of the templates. Second, a no-template negative-control reaction was carried out to control for contamination of the PCR components. In both cases, no PCR product was detectable by GelStar staining (data not shown).
Western blot analysis.
Antiserum against rat apo AIV was raised from goat as described previously (14). Samples of hypothalamus were homogenized at 4°C in a buffer containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, and 1 mM EDTA, then centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant (containing 40 μg protein) was separated by 12% polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and blotted with polyclonal antibody against rat apo AIV (1:3,000 dilution). The immune complexes were revealed by using an enhanced chemiluminescence detection system (Amersham Life Science, Princeton, NJ).
Northern blot hybridization.
Total RNA was isolated from rat jejunum using Tri-Reagent (Molecular Research Center) according to the manufacturer's suggested protocol. Ten micrograms of total RNA were fractionated on denaturing 1.2% agarose-formaldehyde electrophoresis gel, transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech), and UV cross-linked. The membranes were respectively hybridized with32P-labeled apo AIV cDNA probe synthesized from random prime labeling. The blots were stripped and reprobed with18S by a similar protocol. The levels of expression of a specific mRNA were quantified using a PhosphorImager (Molecular Dynamics) and normalized to 18S signal.
Results are presented as means ± SE. Hypothalamic apo AIV mRNA concentrations are expressed in ficomoles per microgram of total RNA. Data were analyzed by one-way ANOVA followed by Tukey's test. AP value <0.05 between group mean values was considered statistically significant.
Apo AIV mRNA and protein were found in the hypothalamus.
We were interested in determining whether apo AIV protein is present in the hypothalamus. As shown in Fig.2 B, Western blotting demonstrated the presence of a single immunoreactive band of 44 kDa, corresponding to the molecular mass of rat apo AIV both in hypothalamus and jejunum in the rat. Although the Western blot data demonstrate the presence of apo AIV in the hypothalamus, the next question is does the hypothalamus have the capability of synthesizing apo AIV?
Oligonucleotides deduced from the cloned rat apo AIV gene (2) were used for screening total RNA extracted from rat hypothalamus. A single apo AIV-PCR product of the expected size of 343 bp was found both in hypothalamus and jejunum of rats (Fig.2 A). After insertion into plasmid and subcloning, the nucleotide sequence of the amplified product was found to be identical to the mRNA sequence of the apo AIV gene (data not shown) (2). Thus these data support the idea that the hypothalamus synthesizes apo AIV because it has both the apo AIV mRNA and apo AIV protein present in the hypothalamus.
Hypothalamic and jejunal mRNA levels were changed in different feeding states.
To determine whether hypothalamic apo AIV mRNA changes in response to fasting, hypothalamic apo AIV mRNA was measured by the sensitive and quantitative competitive RT-PCR method and jejunal apo AIV mRNA was determined by Northern blot hybridization. Figure3 illustrates representative gel and linear regression curves for the amount of individual apo AIV transcript in the hypothalamus. The point of equality (arrow) represents the absolute amount of mRNA (fmol/μg total RNA) for apo AIV.
Apo AIV mRNA content in ad libitum-fed rat hypothalamus was 227.7 ± 25.7 fmol/μg total RNA (mean ± SE,n = 6; Fig. 4). Fasting reduced the hypothalamic apo AIV mRNA content to 80.5 ± 11.4 fmol/μg RNA, a value that was significantly lower than that measured in ad libitum-fed animals (P < 0.01, Fig.4). Fasting also reduced jejunal apo AIV mRNA level (P< 0.01, Fig. 5). Several important conclusions can be inferred from these data. First, apo AIV is synthesized in an area of the brain important in the regulation of energy homeostasis. Second, fasting reduces apo AIV gene expression in the hypothalamus. Finally, apo AIV gene expression changes in parallel in the hypothalamus and the jejunum.
Next, we were interested in determining whether refeeding after a 24-h fast has any effect on the apo AIV mRNA levels in the hypothalamus. Animals were fasted for 24 h and then allowed to refeed either pelleted chow or lipids for 4 h. Refeeding animals with pelleted chow resulted in a hypothalamic apo AIV content of 99.9 ± 16.8 fmol/μg RNA, a value that was not significantly different from that observed in fasting animals (Fig. 4). In contrast, the level of apo AIV mRNA in the hypothalamus of rats that had been refed with lipid was restored to levels observed in ad libitum-fed animals (217.9 ± 39.7 fmol/μg RNA, Fig. 4). Unlike hypothalamic apo AIV mRNA levels, jejunal apo AIV mRNA levels were restored to ad libitum-fed levels after refeeding of either chow or lipid (Fig. 5).
Hypothalamic apo AIV levels were decreased in animals treated with apo AIV antiserum.
Table 1 and Fig.6 show the influences of intracerebroventricular injection of anti-rat apo AIV antibody on the feeding and drinking behavior and apo AIV gene expression. Third-ventricular administration of goat anti-rat apo AIV serum elicited feeding in ad libitum-fed rats, which is consistent with our previous report (Table 1) (7). When we studied the hypothalamic apo AIV levels after antibody treatment, we were amazed to find that hypothalamic apo AIV levels in the apo AIV antiserum-treated animals were significantly lower (P < 0.05) than those observed in the animals treated with normal goat serum (Fig. 6). Periprandial drinking behavior accompanied this feeding response. The administration of normal goat serum into the third ventricle had no effect on feeding or drinking. The administration of neither normal goat nor goat anti-rat apo AIV caused any observable physiological reaction such as sedation or ataxia.
These experiments provide the first direct evidence that apo AIV protein as well as its mRNA are present in rat hypothalamus. These results therefore refute a previous report that rat brain does not synthesize apo AIV (4). In that report, total brain RNA was extracted and analyzed by RNA dot blotting, a method that is not sensitive enough to detect apo AIV mRNA especially when it is situated in a localized area of the brain. By limiting the assay to hypothalamic total RNA and by using competitive RT-PCR (an extremely sensitive method), we were able to detect mRNA in the rat hypothalamus.
There are several reports that exogenous apo AIV acts as a satiety hormone, and both intravenous and third-cerebroventricular apo AIV decrease food intake in fasted and ad libitum-fed rats (7,8). The hypothalamus is an obvious site for apo AIV to inhibit food intake because it is intimately involved in the regulation of energy homeostasis (20, 26, 29). In fact, the present study clearly demonstrates that both the apo AIV protein as well as its mRNA are found in the hypothalamus, an area of the brain that has receptors for other important metabolic signals that circulate in the blood, such as leptin and insulin. It is tempting to speculate that apo AIV may interact with these and other signals that contribute to the regulation of body adiposity because of several key neuropeptides that are also involved with metabolism. These neuropeptides are synthesized in the hypothalamus, including neuropeptide Y (5), proopiomelanocortin (13, 16), agouti-related protein (22), and cocaine-amphetamine-related transcript (17).
The present results demonstrate that hypothalamic apo AIV mRNA levels decrease during fasting and are totally restored by lipid refeeding. Chow refeeding, on the other hand, had little or no effect. These data suggest that hypothalamic apo AIV mRNA may be regulated by the amount of fat intake in the small intestine. Fukagawa et al. (9) reported that serum apo AIV concentrations are significantly higher in free-feeding than in food-deprived rats. The observation that both hypothalamic and serum apo AIV levels are markedly downregulated by fasting and upregulated by fat (re)feeding is potentially very important and suggests a physiological role of apo AIV in the regulation of food intake.
To further explore the role of hypothalamic apo AIV in the regulation of food intake, an experiment was conducted during the light phase when most ad libitum-fed rats do not eat. When anti-rat apo AIV antibodies were injected into the third ventricle of these rats, feeding was elicited in six of seven rats, which is consistent with our previous report (7). Interestingly, this manipulation also significantly decreased the apo AIV mRNA level in the hypothalamus. Exactly how the apo AIV antiserum treatment resulted in a marked decrease in the apo AIV mRNA level in the hypothalamus is unclear at the moment. There are potentially a couple of mechanisms to explain why this may occur.
First, the apo AIV level in the third ventricle (reduced by antibody treatment) may regulate hypothalamic apo AIV mRNA levels. As far as we know, this regulation of hypothalamic peptides by the peptide concentration in the third ventricle is unique and certainly warrants future investigation. A second possible mechanism is that apo AIV antibodies may penetrate into adjacent central nervous system tissue after injection and immunoneutralize endogenous peptides. The time required for antiserum to act varies markedly and is probably related to the time required for uptake from the ventricle and diffusion to the site of neutralization of endogenous peptide. This time was less than 1 h in the case of apo AIV antiserum, which suggests that the site of action was near the ventricle. The finding that rats administered with antibody actually initiated feeding at a time when they would normally not eat suggests that central apo AIV might function to delay meal onset. If so, this would distinguish it from other so-called satiety signals such as CCK, which limit meal size but have no effect on meal initiation (24).
In conclusion, the present experiments indicate that apo AIV is synthesized in the hypothalamus in addition to the intestine. Although the physiological role and the mechanism of how hypothalamic apo AIV is regulated are far from clear, the data thus far are suggestive of its role in feeding and energy homeostasis. The observation that apo AIV is synthesized in the hypothalamus further suggests that this protein may interact with other signals involved in the regulation of energy homeostasis such as leptin, insulin, and NPY.
This work was supported by research grants from National Institute of Diabetes and Digestive and Kidney Diseases (DK-54504, DK-56910, and DK-56863).
Address for reprint requests and other correspondence: P. Tso, Dept. of Pathology and Laboratory Medicine, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267 (E-mail:).
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- Copyright © 2001 the American Physiological Society