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Endocrinology, doi:10.1210/en.2003-1554
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Endocrinology Vol. 145, No. 7 3232-3238
Copyright © 2004 by The Endocrine Society

Diurnal Rhythm of Apolipoprotein A-IV in Rat Hypothalamus and Its Relation to Food Intake and Corticosterone

Min Liu, Ling Shen, Yin Liu, Daisuke Tajima, Randall Sakai, Stephen C. Woods and Patrick Tso

Cincinnati Obesity Research Center (M.L., R.S., S.C.W., P.T.), Department of Pathology and Laboratory Medicine (M.L., L.S., Y.L., D.T., P.T.), Department of Psychiatry (R.S., S.C.W.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Min Liu, Ph.D., Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0507. E-mail: lium{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apolipoprotein A-IV (apo A-IV) is a satiety protein synthesized in the small intestine and hypothalamus. To further understand the roles of central apo A-IV in the management of daily food intake, we have examined the diurnal patterns of hypothalamic apo A-IV gene and protein expression in freely feeding and food-restricted (food provided 4 h daily between 1000 h and 1400 h) rats. In freely feeding rats, the hypothalamic apo A-IV mRNA and protein levels fluctuated, with high levels during the light phase, peaking at 0900 h (3 h after lights on), and low levels during the dark phase, with a nadir at 2100 h (3 h after lights off). The daily patterns of the fluctuation, however, were altered in food-restricted rats, which had a marked decrease in hypothalamic apo A-IV mRNA and protein levels during the 4 h-feeding period of the light phase. Although corticosterone (CORT) secretion temporally coincided with the decreasing phase of apo A-IV in the hypothalamus, depletion of CORT by adrenalectomy significantly decreased, rather than increased, hypothalamic apo A-IV mRNA and protein levels. These results indicate that the diurnal expression of hypothalamic apo A-IV is regulated by factors other than the circulating CORT, for example, the reduced food intake and body weight in adrenalectomized animals. The fact that hypothalamic apo A-IV level and food intake were inversely related during the normal diurnal cycle as well as in the period of restricted feeding suggests that hypothalamic apo A-IV is involved in the regulation of daily food intake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOLIPOPROTEIN A-IV (apo A-IV) is a component of intestinally synthesized and secreted triacylglycerol-rich lipoproteins (1). Intestinal apo A-IV synthesis is markedly stimulated by fat absorption and appears to be mediated by the formation of chylomicrons (2). Apo A-IV has been proposed as a physiological controller of food intake, and the inhibition of food intake by apo A-IV is mediated centrally (3, 4). Intracerebroventricular administration of apo A-IV significantly inhibits food intake in a dose-dependent manner without eliciting signs of toxicity (5, 6). Blocking the action of endogenous apo A-IV with its antibody increases meal size, implying that endogenous apo A-IV exerts an inhibitory tone on feeding (5, 7). Recently, apo A-IV has been demonstrated to be present in the hypothalamus, and the hypothalamic apo A-IV gene expression is reduced by food deprivation and restored by lipid refeeding (7). In view of these seemingly antagonistic effects elicited by apo A-IV, we speculated that, under physiological conditions, feeding behavior may reflect levels of apo A-IV in the hypothalamus.

In previous studies, we demonstrated that rat serum apo A-IV has a circadian rhythm (8). With ad libitum feeding, serum apo A-IV concentration fluctuates in parallel with the feeding pattern. It is unknown whether hypothalamic apo A-IV gene and protein expression also fluctuate across the 24-h light/dark cycle in association with spontaneous food intake. Although we have demonstrated that the administration of apo A-IV into the third-cerebroventricle resulted in the inhibition of food intake, the role of central endogenous apo A-IV in the management of daily food intake is still not well defined. Therefore, the first aim of the present series of experiments was to determine whether the gene and protein expression of apo A-IV in the hypothalamus exhibits a circadian rhythm in ad libitum-fed rats.

Animals maintained on a food-restricted (FR) regimen (food availability for 4 h during light phase) consume approximately 25–30% less food and maintain a stable body weight in contrast to rats maintained on a free-feeding (FF) regimen with progressing weight gain (9, 10). In the present study, a food restriction paradigm (11), which alters feeding behavior while leaving the light-dark cues unchanged, was used to achieve the second aim of these experiments, i.e. to determine whether there is a direct relationship between hypothalamic apo A-IV and feeding.

The role of adrenal glucocorticoids in the regulation of feeding and the development of obesity has been well demonstrated. The peak and nadir of glucocorticoid diurnal secretion over 24 h coincide with the initiation and termination, respectively, of the active feeding period and locomotor activities (12, 13). Central administration of corticosterone (CORT) or its analogs stimulates food intake and promotes obesity (14, 15). Most obese rodent models are hypercorticosteronemic (16, 17). However, at present there are no studies that have examined the effects of CORT on apo A-IV gene and protein expression in the hypothalamus. Thus, the third aim of the present study was to examine the relationship of the diurnal changes of hypothalamic apo A-IV gene and protein expression with circulating CORT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and procedure
Adult male Sprague Dawley rats (250–275 g, Harlan, Indianapolis, IN) were housed individually in a light- and temperature-controlled room (lights on 0600–1800, 21 C). Unless stated otherwise, food (pelleted chow, Teklad Rodent Chow, Harlan) and water were available ad libitum. Animals were allowed to acclimate to these housing conditions for 1 wk before experiments were started. All procedures were performed in accordance with institutional guidelines of the Institutional Animal Care & Use Committee at the University of Cincinnati.

Experimental protocol
Rats were divided randomly into two groups: one group was allowed to feed freely; and the other was FR, with food available from 1000–1400 h for 4 wk. Water was available ad libitum to all rats. Body weight, 24-h food intake for FF rats, and 4 h-food intake for FR rats were recorded daily. The animals were killed by decapitation at 3-h intervals (five to six FF rats/time point and four FR rats/time point) throughout a 24-h period. Utmost care was taken to cause minimal stress to the rats before decapitation, which was completed within 1 min of disturbing a cage. The brain was rapidly removed from each rat, and the entire hypothalamus was dissected according to the atlas of Paxinos and Watson (18) and immediately frozen in liquid nitrogen. The hypothalamus was stored at –80 C until mRNA and protein extraction. Trunk blood was collected and centrifuged, and plasma was stored at –80 C. Separate aliquots of plasma were taken for assay of apo A-IV measured by ELISA and CORT determined by RIA.

To determine the effect of CORT on the diurnal feeding rhythm and diurnal variation in hypothalamic apo A-IV gene and protein expression, another two groups of rats were used. One group was sham-operated (SHAM, six rats), and another was adrenalectomized (ADX, seven rats) by a dorsolateral approach to remove the adrenal glands bilaterally (19). Drinking water for all ADX rats was replaced with 0.9% saline. Body weight and food intake were recorded daily after surgery. All the animals were killed by decapitation at 2100 h, on d 8 after surgery. We chose this time point because the CORT level is relatively high, and hypothalamic apo A-IV gene and protein levels are dramatically changed at that time. Trunk blood was collected, and the hypothalamus was quickly removed as described above. Only ADX rats with plasma CORT levels less than 1 µg/dl at the time of death were regarded as indicative of the completeness of ADX and were included in the results.

Real-time PCR for apo A-IV mRNA
Hypothalamic apo A-IV mRNA levels were determined by quantitative real-time RT-PCR. Cyclophilin mRNA levels from each sample were used as internal controls to normalize the mRNA levels. The sequences of the primers for apo A-IV and cyclophilin were determined using primer design software (Integrated DNA Technologies, Coralville, IA), following the manufacturer’s guidelines. The specificity of the designed primers for their respective target genes was confirmed by nucleotide BLAST search on the NCBI database. There were no sequence homologies of probes or primers for any genes other than the target genes in the same species. The sequences of primers for apo A-IV and cyclophilin are listed in Table 1Go.


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  		TABLE 1. Primer sequences for real-time RT-PCR

 
Hypothalamic total RNA was isolated with Tri Reagent according to the manufacturer’s instructions (Molecular Research Center, Inc., Cincinnati, OH), and total RNA concentration was determined spectrophotometrically at 260 nm. To ensure uniform and reproducible reaction conditions, both reverse transcription and PCR reagents were prepared as master mixes and aliquoted into individual tubes before using them for each batch of reactions. Total RNA (100 ng) from hypothalamus was reverse-transcribed to first-strand cDNA using random hexamers and Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ). Reactions in which RNA was omitted served as negative controls. Real-time RT-PCR was performed in a 25-µl final reaction vol with an iCycler iQ Detection System using iQ SYBR Green Supermix (Bio-Rad Laboratories Inc., Hercules, CA) according to the manufacturer’s instruction. Real-time RT-PCR conditions were as follows: 95 C for 3 min for one cycle, followed by 40 cycles of 95 C for 30 sec and 58 C for 30 sec on an iCycler iQ real-time PCR detection system (Bio-Rad). The standard curves were generated from corresponding tissues of normal, untreated Sprague Dawley rats. No other products were amplified because melting curves showed only one peak in each sample. Threshold cycle readings for each of the unknown samples were then used, and the results were transferred and analyzed in Excel using the {Delta}{Delta}CT method (PerkinElmer Applied Biosystems, Foster City, CA) (20).

Western blot for apo A-IV protein
Hypothalamic A-IV protein level was assessed by semiquantitative Western blot analysis. Antiserum against apo A-IV was raised from rabbit as described previously (2). The tissues were homogenized, and the supernatant (containing 30 µg hypothalamic protein) was separated by 12% PAGE, transferred onto nitrocellulose sheets, and blotted with apo A-IV antibody (1:3000 dilution). The amount of immune complexes was quantitated using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The blots were stripped and reincubated with monoclonal antibody against actin (Roche Molecular Biochemicals GmbH, Mannheim, Germany, 1:3000 dilution). The reacted membranes were exposed to x-ray film (Kodak Scientific imaging film, Rochester, NY). Film density, measured as transmittance, was expressed as volume-adjusted optical density. The amount of apo A-IV protein was normalized to the respective individual density values reflecting actin protein levels and was expressed as a ratio.

Measurement of plasma CORT
Total plasma CORT was measured by RIA using rabbit antiserum raised against CORT (B3–163) obtained from Endocrine Sciences (Calabasas Hills, CA). Briefly, 20-µl duplicate samples of plasma were heated at 60 C for 2 h to denature binding protein and were incubated overnight with CORT antibody. [3H]corticosterone (NEN Life Science Products Life Sciences, Inc., Boston, MA) was used as a radioactive tracer. Free and bound CORT were separated by incubating with charcoal. CORT concentrations were calculated using an equation derived from a standard curve.

Statistical analysis
Daily body weight changes are presented as percent of initial body weight. Data were analyzed by one-way (the time-course data of apo A-IV mRNA and protein levels) or two-way (the time-course data of apo A-IV mRNA and protein levels between FF and FR groups) ANOVA, followed by Tukey’s multiple comparison. Results were expressed as the mean ± SEM, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of FF and FR on body weight
As depicted in Fig. 1Go, the body weight of the FF increased steadily during the 4 wk of experimental period (Fig. 1AGo). Food restriction (food provided 4 h daily from 1000–1400 h) initially resulted in loss of body weight during the first 3 d. Thereafter, rats slowly increased their food intake and regained their initial body weight by d 9 and then gained weight at a steady rate. At the end of the 4th wk, the body weight of FR rats was 12% lower than that of FF rats (Fig. 1AGo). The lower body weight gain in FR rats can be attributed to a lower average daily food consumption (P < 0.01, Fig. 1BGo). FR rats, for example, consumed 20.8 ± 0.8 g compared with 28.4 ± 0.7 g for FF rats on d 26.



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  		FIG. 1. A, Body weight changes relative to the initial value in FF and FR rats. Availability of food was 4 h (from 1000–1400 h) each day in FR rats. B, Daily food intake of FF (n = 8) and FR (n = 32) rats.

 
Diurnal expression of hypothalamic apo A-IV mRNA and protein and relative to food intake in FF rats
Every 3 h, interval food intake was monitored throughout the 24-h light/dark cycle. Under ad libitum feeding conditions, food intake in the light phase was low, varying from 0.65–1.83 g/3 h. A dramatic increase in food intake was observed after the onset of the dark cycle (1800 h), and the active food intake was maintained from 1800–0300 h. Subsequently, the amount of food ingestion declined until 0600 h (Fig. 2AGo). Food intake during the light period was 4.1 ± 0.5 g and during the dark period was 24.3 ± 1.2 g, with the difference being highly significant (P < 0.01). Thus, the food intake showed a strong nocturnal feeding pattern.



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  		FIG. 2. A, A 3-h interval food intake (g) under free feeding and drinking conditions in rats. Quantitation of diurnal changes in hypothalamic apo A-IV mRNA (B), measured by real-time PCR, was performed with iCycler iQ Detection Software. The apo A-IV mRNA values are presented as a percentage of apo A-IV mRNA level at 0600 h set at 100%. Apo A-IV protein levels (C) were measured by Western blot analysis in FF rats. Values are expressed as means ± SEM; n = 5–6. Dissimilar superscripts denote statistically significant differences (P < 0.05).

 
In FF rats, hypothalamic apo A-IV mRNA levels and food intake were inversely related across the diurnal cycle. As shown in Fig. 2BGo, apo A-IV mRNA level increased from 0600 h onward and was sustained until 1500 h, with peak levels at 0900 h. A decrease in apo A-IV mRNA level to a lower range occurred at 1800 h, during the dark phase, with a nadir at 2100 h (3 h after lights off). Interestingly, this low level was sustained throughout the dark phase (Fig. 2BGo). The average level of apo A-IV mRNA in the hypothalamus in the light cycle (from 0600–1800 h) was significantly greater than that in the dark cycle (1800–0600 h; P < 0.01). Statistical analysis indicated that the levels of apo A-IV mRNA at 0900 and 1200 h in the light cycle differed significantly from those at 2100, 2400, and 0300 h in the dark cycle (P < 0.05 for each comparison, Fig. 2BGo). The results from protein analysis measured by Western blot confirmed that the diurnal fluctuation of apo A-IV protein level in the hypothalamus is consistent with apo A-IV mRNA levels (Fig. 2CGo).

Alterations in daily fluctuations of hypothalamic apo A-IV mRNA and protein expression: effects of FR
The daily pattern of fluctuation in hypothalamic apo A-IV mRNA and protein levels was changed in FR rats when compared with the FF rats. As shown in Fig. 3AGo, FR rats had a marked decrease in hypothalamic apo A-IV mRNA level, which occurred during the 4-h feeding period of the light phase. When compared with the pattern of hypothalamic apo A-IV gene expression between the FF and the FR rats (Fig. 3BGo), we found that both groups of animals had similar apo A-IV mRNA levels at 0600 h, and then began to separate. That is why we used the apo A-IV mRNA level at 0600 h as a control during real-time PCR datum analysis. In contrast to the increase observed in the FF rats during the light phase, the apo A-IV mRNA level started to decrease in the FR rats, and the difference between the two groups at 0900 and 1200 h (before and the mid-point of the 4 h of feeding) was statistically significant (P < 0.05). After 4 h of feeding, the hypothalamic apo A-IV levels in FR rats started to increase and maintained relatively higher levels during the entire dark phase. Although there is a slight trough that occurred during the dark phase in the FR rats, the magnitude is markedly reduced compared with FF rats. The fluctuation of the apo A-IV protein level showed a pattern similar to that of the mRNA level (Fig. 3CGo).



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  		FIG. 3. Diurnal changes in hypothalamic apo A-IV mRNA, as measured by real-time RT-PCR (A); and protein (C) levels, as measured by Western blot analysis, in FR rats. B, Comparison of hypothalamic apo A-IV mRNA levels between the FF and FR rats. The apo A-IV mRNA values are shown as a percentage of apo A-IV mRNA levels at 0600 h set at 100%. Values are expressed as means ± SEM; n = 4. Dissimilar superscripts denote statistically significant differences (P < 0.05).

 
The fact that the decrease in hypothalamic apo A-IV level precedes that of the actual presentation of the food in FR rats suggests a food anticipatory response. To monitor detailed changes of apo A-IV mRNA and protein levels, we killed an additional 20 FR rats (five rats/h) from 0800–1100 h, spanning the period before and after food was present. Compared with the levels at 0800 h, apo A-IV mRNA and protein levels at 0900 h were slightly lower. A marked decrease was observed at 1000 h, a time point when the food was just presented (Fig. 4Go). These data further demonstrate the anticipatory response of apo A-IV in the hypothalamus to food.



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  		FIG. 4. The relative hypothalamic apo A-IV mRNA (A), expressed as a percentage of apo A-IV mRNA levels at 0800 h set at 100%, and protein (B) levels in FR rats. Values are expressed as means ± SEM; n = 5.

 
Daily fluctuations in plasma CORT levels in FF and FR rats
In FF rats, circulating CORT showed a well-defined diurnal rhythm (Fig. 5AGo). Plasma CORT levels were low in the morning (from 0600–1200 h), ranging from 0.1–1.3 µg/dl. Then, CORT levels exhibited an increase from trough levels to 4.0 ± 0.71 µg/dl 3 h before the dark phase and a further rise to a peak at the light/dark transition phase (1800 h; 18.5 ± 0.67 µg/dl). Subsequently, plasma CORT levels declined to 10.5 ± 1.57 µg/dl at 2100 h, and 3.4 ± 0.58 µg/dl 3 h before returning to the nadir at 0600 h. Food availability restricted to 1000–1400 h shifted the daily rise in plasma CORT levels. CORT levels rose at 0900 h, preceding the feeding phase, and returned to the basal range at 1500 h, 1 h after the feeding (Fig. 5BGo). Plasma CORT levels and apo A-IV gene and protein expression across the light-dark cycle were determined in the same animals. Note that a sharp rise in CORT preceded the decreasing phase of apo A-IV protein expression in the hypothalamus of both FF and FR rats. The nadir of apo A-IV protein expression lagged 3–4 h behind the peak of plasma CORT (Fig. 5Go, A and B).



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  		FIG. 5. Diurnal changes in plasma CORT and hypothalamic apo A-IV protein levels in FF (Fig. 5AGo) and FR (Fig. 5BGo) rats. Values are expressed as means ± SEM; n = 5–6 (for FF rats); n = 4 (for FR rats). Dissimilar superscripts denote statistically significant differences (P < 0.05).

 
Effect of ADX on the diurnal feeding and apo A-IV gene and protein expression in the hypothalamus
In SHAM rats, plasma CORT showed a normal level as observed in intact FF rats at 2100 h (9.4 ± 2.48 µg/dl vs.10.5 ± 1.57 µg/dl). CORT was undetectable in ADX rats. ADX resulted in a reduction in body weight (–11.5 ± 2.68 g from the presurgical body weight), whereas SHAM controls gained 17.3 ± 3.03 g from their presurgical body weight in 7 d (P < 0.05 vs. ADX rats; Fig. 6AGo). Daily food intake was measured for 7 d, starting from the day before surgery. ADX rats ate significantly less than SHAM controls (P < 0.01, Fig 6BGo). The general feeding rhythm, however, was not altered by ADX (Fig. 6CGo).



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  		FIG. 6. Effects of ADX on body weight and food intake. Dynamic changes in body weight gain (A) and daily food intake (B) in SHAM or ADX rats compared with their presurgery state. C, Food intake during the 12-h light and dark cycles and 24-h period. As noted, the feeding rhythm is unaffected by ADX; n = 6 for the SHAM group; n = 7 for the ADX group. *, P < 0.05; **, P < 0.01; ADX vs. SHAM.

 
An important question is: if CORT down-regulates apo A-IV in the hypothalamus, does the hypothalamic apo A-IV gene and protein expression increase in the rats when their adrenal glands are removed? To answer this question, the levels of apo A-IV mRNA and protein in the hypothalamus were measured in SHAM and ADX rats killed by decapitation at 2100 h, a time point with a nadir of apo A-IV level and with a relatively high level of CORT in FF rats (Fig. 5AGo). SHAM controls maintained levels of apo A-IV gene (Fig. 7AGo) and protein (Fig. 7BGo) expression in the hypothalamus similar to those observed in the unoperated FF rats. Quantitative analyses showed that apo A-IV gene and protein expression in the hypothalamus were down-regulated, rather than up-regulated, in ADX rats by approximately 44% (mRNA) and 38% (protein) of the levels found in SHAM controls (P < 0.05, Fig. 7Go, A and B).



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  		FIG. 7. Effect of ADX on the expression of apo A-IV gene and protein in the hypothalamus. Both apo A-IV mRNA (A) and protein (B) exhibited significant down-regulation at 2100 h in response to ADX. Values are expressed as means ± SEM; n = 6 for the SHAM group; n = 7 for the ADX group. *, P < 0.05, ADX vs. SHAM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated, for the first time, that apo A-IV mRNA and protein expression in the hypothalamus of ad libitum-fed rats varies with time of day. Apo A-IV mRNA and protein levels displayed a significant diurnal rhythm, with a marked peak at 0900 h (3 h after lights on) and a nadir at 2100 h (3 h after lights off), which were inversely related to the feeding pattern of the animals. These data indicate that the presence and consumption of food during the dark period reduce the nocturnal gene and protein expression of apo A-IV in the hypothalamus. However, it is not possible to determine whether the decrease in hypothalamic apo A-IV is caused by the circadian rhythm or the presence and consumption of food from the experiment using ad libitum-fed rats only.

To differentiate these two possibilities, we performed an additional experiment in which the feeding was moved to the light phase and the animals were allowed free access to food during 1000–1400 h of the light phase. The animals in the FR group initially lost weight, then they soon began to regain the lost body weight. As shown in Fig. 3Go, the circadian rhythm of the hypothalamic apo A-IV expression was modified significantly in the FR rats compared with the FF rats. Instead of showing a decrease in the hypothalamic apo A-IV level during the dark phase in the FF rats, the nadir was shifted to the light phase, and it occurred before the actual feeding took place. Thus, it would appear that the food and/or active feeding caused the hypothalamic apo A-IV expression to shift and decrease at a different time.

In the ad libitum-fed rats, it would seem that the initiation of feeding activity is preceded by a gradual decline in the hypothalamic apo A-IV gene and protein expression in the FF rats. The fact that hypothalamic apo A-IV mRNA and protein levels started to decrease before food was available in the FR rats further supports this concept. To monitor changes of apo A-IV mRNA and protein levels in detail, an additional five FF rats were killed every hour from 0800–1100 h. Compared with the apo A-IV gene and protein levels at 0800 h, apo A-IV at 0900 h was slightly lower. However, a marked decrease was observed at 1000 h before food was presented. All of these data demonstrate that the anticipation of food may be one of the factors that down-regulate apo A-IV mRNA and protein expression in the hypothalamus.

The circadian rhythm of hypothalamic apo A-IV is consistent with our current understanding of the role of brain apo A-IV in the regulation of food intake. Central administration of apo A-IV significantly inhibits food intake in a dose-dependent manner without apparent toxic effect (6, 21, 22). Blocking the action of endogenous apo A-IV with a specific neutralizing antibody increases food intake during a period when there is little or no food intake (middle part of light phase) (5, 7). If brain apo A-IV were a physiological regulator of fat intake, hypothalamic apo A-IV levels would be predicted to be reduced when animals were eating. The lower satiety response of apo A-IV thereby may contribute to animal active feeding. Our current observations lend support to the concept that apo A-IV’s physiological role may be as a regulator of daily food intake.

The mechanism by which hypothalamic apo A-IV mRNA and protein expression fluctuates diurnally is not clear. The decrease of apo A-IV before the onset of the dark cycle in FF rats suggests that the diurnal rhythm of apo A-IV levels is unlikely to be light-entrained. Analysis of diurnal events that precede the reducing phases of apo A-IV expression may predict the regulatory factors of diurnal expression of apo A-IV mRNA and protein. In a carefully conducted study described in 1979, Wilkinson et al. (22) reported that peak CORT levels occurred just before feeding, making CORT a potential candidate in regulating the hypothalamic apo A-IV. Therefore, we determined circulating CORT levels in the animals in the present studies. A single peak occurred just before the active feeding period (the dark cycle) in FF rats, and this surge in CORT was shifted with the time of food availability as observed in FR rats. Interestingly, plasma CORT levels in FR rats rose at the onset of feeding and decreased to a basal range at the end of 4 h of feeding, thereby affirming a tight relationship of the circulating CORT with the onset of feeding behavior (23).

Analysis of the temporal relationship between CORT and apo A-IV gene and protein expression demonstrated that the peak in circulating CORT and the nadir of hypothalamic apo A-IV mRNA and protein levels coincide in both FF and FR rats. The surge in plasma CORT preceded the decrease in apo A-IV, suggesting that CORT may act as a signal for reduced apo A-IV expression. This possibility, however, was not supported by the observation that ADX significantly decreased, rather than increased, apo A-IV mRNA and protein levels in the hypothalamus. This result is opposite to the above hypothesis, indicating that the diurnal fluctuation of apo A-IV gene and protein expression is not closely related to circulating CORT. It is more likely that the reduced food intake and/or body weight in ADX rats act as a causative factor of the fluctuation of apo A-IV. Further experiments will be needed to determine the involved mechanism(s) and how the food intake affects hypothalamic apo AIV gene and protein expression.

In conclusion, the present study clearly demonstrates that there is a diurnal rhythm in hypothalamic apo A-IV mRNA and protein levels in rats fed ad libitum. This diurnal rhythm of apo A-IV can be altered by food restriction. The present study also provides evidence that the diurnal rhythm of apo A-IV gene and protein expression is independent of circulating CORT. Other factor(s) that may affect hypothalamic apo A-IV gene and protein expression, and the precise mechanisms and how the hypothalamic apo A-IV regulates feeding behavior under physiological conditions, remain to be determined.


   Acknowledgments
 
The authors acknowledge the technical assistance of Dr. M-D Zhang. We also thank Dr. Ronald Jandacek for suggestions on the manuscript.


   Footnotes
 
This work was supported by Research Grants DK54890, DK17844, DK56863, DK 63907, HL62542, DK54504, DK56910, and DK53444 from the National Institutes of Health, and by the Procter & Gamble Company.

Abbreviations: ADX, Adrenalectomized; apo A-IV, apolipoprotein A-IV; CORT, corticosterone; FF, freely feeding; FR, food-restricted; SHAM, sham-operated.

Received November 17, 2003.

Accepted for publication March 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Apfelbaum TF, Davidson NO, Glickman RM 1987 Apolipoprotein A-IV synthesis in rat intestine: regulation by dietary triglyceride. Am J Physiol 252:G662–G666
  2. Hayashi H, Nutting DF, Fujimoto K, Cardelli JA, Black D, Tso P 1990 Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat. J Lipid Res 31:1613–1625[Abstract]
  3. Liu M, Shen L, Tso P 1999 The role of enterostatin and apolipoprotein AIV on the control of food intake. Neuropeptides 33:425–433[CrossRef][Medline]
  4. Tso P, Liu M, Kalogeris TJ 1999 The role of apolipoprotein A-IV in food intake regulation. J Nutr 129:1503–1506[Abstract/Free Full Text]
  5. Fujimoto K, Fukagawa K, Sakata T, Tso P 1993 Suppression of food intake by apolipoprotein A-IV is mediated through the central nervous system in rats. J Clin Invest 91:1830–1833
  6. Liu M, Maiorano N, Shen L, Pearson K, Tajima D, Zhang DM, Woods SC, Seeley RJ, Davidson WS, Tso P 2003 Expression of biologically active rat apolipoprotein AIV in Escherichia coli. Physiol Behav 78:149–155[CrossRef][Medline]
  7. Liu M, Doi T, Shen L, Woods SC, Seeley RJ, Zheng S, Jackman A, Tso P 2001 Intestinal satiety protein apolipoprotein AIV is synthesized and regulated in rat hypothalamus. Am J Physiol Regul Integr Comp Physiol 280:R1382–R1387
  8. Fukagawa K, Gou HM, Wolf R, Tso P 1994 Circadian rhythm of serum and lymph apolipoprotein AIV in ad libitum-fed and fasted rats. Am J Physiol 267:R1385–R1390
  9. Kalra SP, Dube MG, Sahu A, Phelps CP, Kalra PS 1991 Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc Natl Acad Sci USA 88:10931–10935[Abstract/Free Full Text]
  10. Sahu A, White JD, Kalra PS, Kalra SP 1992 Hypothalamic neuropeptide Y gene expression in rats on scheduled feeding regimen. Brain Res Mol Brain Res 15:15–18[Medline]
  11. Krieger DT, Hauser H 1978 Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Proc Natl Acad Sci USA 75:1577–1581[Abstract/Free Full Text]
  12. Akana SF, Strack AM, Hanson ES, Dallman MF 1994 Regulation of activity in the hypothalamo-pituitary-adrenal axis is integral to a larger hypothalamic system that determines caloric flow. Endocrinology 135:1125–1134[Abstract]
  13. Akana SF, Strack AM, Hanson ES, Horsley CJ, Milligan ED, Bhatnagar S, Dallman MF 1999 Interactions among chronic cold, corticosterone and puberty on energy intake and deposition. Stress 3:131–146[Medline]
  14. Tempel DL, Leibowitz SF 1994 Adrenal steroid receptors: interactions with brain neuropeptide systems in relation to nutrient intake and metabolism. J Neuroendocrinol 6:479–501[CrossRef][Medline]
  15. Therrien M, Drouin J 1993 Molecular determinants for cell specificity and glucocorticoid repression of the proopiomelanocortin gene. Ann NY Acad Sci 680:663–671[Medline]
  16. Green PK, Wilkinson CW, Woods SC 1992 Intraventricular corticosterone increases the rate of body weight gain in underweight adrenalectomized rats. Endocrinology 130:269–275[Abstract/Free Full Text]
  17. Zakrzewska KE, Cusin I, Stricker-Krongrad A, Boss O, Ricquier D, Jeanrenaud B, Rohner-Jeanrenaud F 1999 Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat. Diabetes 48:365–370[Abstract]
  18. Paxinos G, Watson C 1982 The rat brain in stereotaxic coordinates. 4th ed. New York: Academic Press, Harcourt Brace & Company
  19. Chao HM, Sakai RR, Ma LY, McEwen BS 1998 Adrenal steroid regulation of neurotrophic factor expression in the rat hippocampus. Endocrinology 139:3112–3118[Abstract/Free Full Text]
  20. Erickson JC, Clegg KE, Palmiter RD 1996 Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 381:415–421[CrossRef][Medline]
  21. Fujimoto K, Cardelli JA, Tso P 1992 Increased apolipoprotein A-IV in rat mesenteric lymph after lipid meal acts as a physiological signal for satiation. Am J Physiol 262:G1002–G1006
  22. Fujimoto K, Machidori H, Iwakiri R, Yamamoto K, Fujisaki J, Sakata T, Tso P 1993 Effect of intravenous administration of apolipoprotein A-IV on patterns of feeding, drinking and ambulatory activity of rats. Brain Res 608:233–237[CrossRef][Medline]
  23. Dallman MF, Strack AM, Akana SF, Bradbury MJ, Hanson ES, Scribner KA, Smith M 1993 Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14:303–347[CrossRef][Medline]



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