1Centre de recherche de l'Hôpital Laval et Centre de recherche sur le métabolisme énergétique de l'Université Laval, Quebec, Canada; 2Laboratoire Neurogénétique et Stress, Institut National de la Santé et de la Recherche Médicale U471, Université Bordeaux, Bordeaux, France
Submitted 22 February 2004 ; accepted in final form 9 September 2004
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ABSTRACT |
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fa/fa rats; metabolic rate; sympathetic nervous system; food deprivation; refeeding; thyrotropin-releasing hormone; tyrosine hydroxylase; corticotropin-releasing hormone
The fa/fa rat is characterized by very high circulating levels of corticosterone due to the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis (13). High levels of adrenocorticotropic hormone (ACTH) have been reported in the fa/fa rat, which also exhibits an increase in the clearance of corticosterone (59). In addition, the fa/fa rat has been reported to overexpress corticotropin-releasing hormone (CRH) when faced with stressful experimental conditions (13, 37, 57). Furthermore, we (52) have recently demonstrated that food deprivation induces a strong stress response in obese, but not in lean, Zucker rats. In fact, the hypophysiotropic CRH neurons in the parvocellular part of the paraventricular hypothalamic (PVH) nucleus are strongly activated during food deprivation in obese, but not in lean, rats (51). Besides the observed increase in hypophysiotropic CRH activity in obese animals, there is also evidence suggesting a reduction in CRH system activity (33, 35). The central administration of CRH, a neuropeptide with well-established thermogenic and anorectic effects, prevents weight gain in obese Zucker rats with few effects in lean animals (40). In obese rats, a decrease in the expression of the CRH type 2 receptor in the ventromedial hypothalamic nucleus and an increase in the expression of the CRH-binding protein in the hypothalamus and amygdala may contribute to a reduction of the activity of some components of the CRH system (37, 49).
The present study was designed to investigate, in an integrative approach, the role of the enhanced HPA axis activity in obese rats on the basal and postprandial metabolic rates. We also examined the involvement of corticosterone on the central expression of thyrotropin-releasing hormone (TRH), a neuropeptide known to play a role in the regulation of energy balance through central and peripheral effects. Finally, we also intended to clarify whether high levels of corticosterone in obese rats were responsible for the induction of stress reaction to food deprivation in these animals.
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MATERIAL AND METHODS |
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Lean (Fa/?) and obese (fa/fa) male Zucker rats, aged 6 wk, were purchased from the Canadian Breeding Laboratories (St-Constant, QC, Canada). All rats were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals, and the present protocol was approved by our institutional animal care committee. The animals were housed individually in metabolic chambers (experiment 1) or in wire-bottom cages suspended above absorbent paper (experiment 2). They were, unless otherwise specified, fed ad libitum with a stock diet (Agway Prolab, Rat/Mouse/Hamster 1000 Formula). The rats were subjected to a 14001000 (24-h clock) dark-light cycle (lights on between 0900 and 1900) and kept under an ambient temperature of 23 ± 1°C. One week after their arrival, the rats were sham operated or adrenalectomized (ADX). Bilateral removal of the adrenals was achieved through two small lateral skin incisions made under isoflurane anesthesia. Sham-operated animals were treated in the same way as ADX animals except that the adrenals were not excised. The total surgery was completed within 15 min. After surgery, the rats were provided with drinking water supplemented with NaCl (0.9%).
Experiment 1
Each rat was housed in a 4-liter Plexiglas metabolic chamber and allowed to adapt to the new environment for 48 h before the beginning of oxygen consumption (
O2) measurements.
O2 determinations were achieved using an open-circuit system (36). Ambient air was drawn through the chamber at a flow rate of 1,250 ml/min, and O2 concentration was measured using an O2 analyzer (Applied Electrochemistry, model S3A1) coupled to a CO2 sensor (Applied Electrochemistry, model N22M). An automatic valve-driver interface allowed alternate sampling in eight metabolic chambers. Between the intermittent measurements, all chambers, except that from which air was sampled for gas analyses, were ventilated at an airflow rate equal to that passing through the sampling chamber. Values of
O2 and
CO2 were corrected to express the results per kilogram to the power of 0.75 ml·min1·kg0.75, according to Refs. 17 and 34, to account for the differences in body weight. Respiratory quotient (RQ) values were calculated as the CO2 production-to-O2 consumption ratio. Lipid oxidation was determined as a function of the
O2 and
CO2 according to the equation reported by Livesey and Elia (26).
After 2 days of adaptation to the calorimeter and after baseline
O2 measurements, rats were either ADX or sham operated and immediately after surgery returned into the metabolic rooms. Monitoring of the metabolic state was undertaken in ad libitum-fed rats during 6 consecutive days. Afterward, all rats (46 animals per group) were food deprived for 12 h and refed for 1 h. During 6 days following adrenal surgery, rats were weighed daily (between 0900 and 0930). Food intake was determined twice daily, i.e., at the end of the dark period (between 0900 and 0930) and 10 h later at the end of the light period (between 1900 and 1930). Food intake was measured by subtracting the amount of food uneaten from the fixed amount provided to the rat. Food spillage was carefully calculated and accounted for in the measurements.
The influence of the sympathetic nervous system (SNS) on the 1-h metabolic response to refeeding was also assessed. Some ADX and sham-operated lean and obese Zucker rats were intraperitoneally injected with propanolol (25 mg/kg) or saline 60 min before onset of refeeding. Each group included 46 rats.
Experiment 2
Lean and obese Zucker rats, housed individually in wire-bottom cages, were ADX or sham operated. One week after adrenal surgery, a first cohort of rats was killed after ad libitum overnight access to food, after a 12-h period of overnight food deprivation, or after 1 h of ad libitum refeeding following the 12-h fast. Each group comprised six rats. The time of death was adjusted so that all rats were killed at the beginning of the light period to allow comparison of groups at the same period of the circadian glucocorticoid rhythm. A second cohort of ADX and sham-operated lean and obese rats (4 animals per group) was killed 1 wk after adrenal surgery when fed ad libitum and used for carcass determination.
Carcass Determination
Energy, protein, and fat contents of the rats were determined as previously described (38). On the day the rats were killed, they were exsanguinated by decapitation between 1030 and 1200. Carcasses were autoclaved at 125 kPa for 15 min. This procedure, which has been reported not to affect energy yield (27), was used to soften hard tissues. Once autoclaved, carcasses were homogenized in a volume of water corresponding to two times their weight. The homogenized carcasses were then freeze-dried pending the determination of their energy and nitrogen contents. Carcass energy content was determined by adiabatic bomb calorimetry, whereas carcass nitrogen was determined in 250- to 300-mg samples of dehydrated carcasses by use of the Kjeldahl procedure. Carcass protein content was computed by multiplying the carcass nitrogen content by 6.25. The energy as protein was subtracted from total carcass energy to determine energy as nonprotein matter. Because carbohydrate represents a negligible part of carcass total energy, energy from nonprotein matter was assumed to be essentially that of fat. Such an assumption has been confirmed by studies in which energy, fat, and protein were directly determined (2). Values of 23.5 and 39.3 kJ/g were used for the calculation of the energy content of protein and fat, respectively (58). The body weight densities in energy (kJ energy/g body wt), protein (g protein/g body wt), and fat (g fat/g body wt) were then computed and averaged.
Plasma Determination
Plasma glucose was determined by the glucose oxidase method using a glucose analyzer (Beckman, Palo Alto, CA). Serum corticosterone was determined by a competitive protein-binding assay (sensitivity, 0.05 nmol/l; interassay coefficient of variation, 9.0%) using plasma from a dexamethasone-treated female rhesus monkey as a source of transcortin (30). Insulin was determined by RIA with a reagent kit from Linco Research (St. Charles, MO) using rat insulin as a standard.
Brain Preparation
Rats were anesthetized with 1.5 ml of a mixture containing 20 mg/ml ketamine and 2.5 mg/ml xylazine. Without delay, they were perfused intracardially with 200 ml of ice-cold isotonic saline followed by 500 ml of a paraformaldehyde (4%) solution. Brains were removed at the end of perfusion and kept in paraformaldehyde for an additional period of 7 days. They were then transferred to a solution containing paraformaldehyde and sucrose (10%) before being cut 24 h later using a sliding microtome (Histoslide 2000; Reichert-Jung, Heidelberg, Germany). Brain sections were taken from the olfactory bulb to the brain stem. Thirty-micrometer-thick sections were collected and stored at 30°C in a cold, sterile cryoprotecting solution containing sodium phosphate buffer (50 mM), ethylene glycol (30%), and glycerol (20%).
In Situ Hybridization Histochemistry
In situ hybridization histochemistry was used to localize CRH mRNA, pro-TRH mRNA, and c-fos mRNA on tissue sections taken from the entire brain. The protocol used was largely adapted from the technique described by Simmons et al. (45). Briefly, one of every five brain sections was mounted onto poly-L-lysine-coated slides and allowed to desiccate overnight under vacuum. The sections were then successively fixed for 20 min in paraformaldehyde (4%), digested for 30 min at 37°C with proteinase K (10 µg/l in 100 mM Tris·HCl containing 50 mM EDTA, pH 8.0), acetylated with acetic anhydride (0.25% in 0.1 M trietholamine, pH 8.0), and dehydrated through graded concentrations (50, 70, 95, and 100%) of alcohol. After vacuum drying for 2 h, 90 µl of the hybridization mixture, which contained an antisense 35S-labeled cRNA probe (107 cpm/ml), were spotted on each slide. The slides were sealed under a coverslip and incubated overnight at 60°C in a slide warmer. The next day, the coverslips were removed and the slides rinsed four times with 4x SCC (0.6 M NaCl, 60 mM sodium citrate buffer, pH 7.0), digested for 30 min at 37°C with RNase-A (20 µg/ml in 10 mM Tris-500 mM NaCl containing 1 mM EDTA), washed in descending concentrations of SSC (2x, 10 min; 1x, 5 min; 0.1x, 30 min at 60°C), and dehydrated through graded concentrations of alcohol. After a 2-h period of vacuum drying, the slides were exposed on an X-ray film (Eastman Kodak, Rochester, NY) for periods varying between 12 and 24 h, depending on the nature of the probes used. Once removed from the autoradiography cassettes, the slides were exposed from 7 to 14 days before being developed in D19 developer (Eastman Kodak) for 3.5 min at 1415°C and fixed in rapid fixer (Eastman Kodak) for 5 min. Finally, tissues were rinsed in running distilled water for 1 or 2 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX.
Combination of Immunocytochemistry with In Situ Hybridization
Immunocytochemical detection of Fos, the protein encoded by the oncogene c-fos, was combined with detection of CRH mRNA to determine whether CRH cells were activated during food deprivation or refeeding. Brain sections were first processed for immunochemical detection of Fos by means of a conventional avidin-biotin-immunoperoxidase method. Briefly, brain slices were washed in sterile 0.05 M potassium phosphate-buffered saline (KPBS) that was treated with diethyl pyrocarbonate water. They were then incubated for 24 h at 4°C with a Fos antibody (rabbit polyclonal IgG; Oncogene Science, New York, NY). The Fos antibody was used at a 1:50,000 dilution in KPBS (50 mM) with heparin (0.25%), Triton X-100 (0.4%), and bovine serum albumin (2%). Twenty-four hours after incubation at 4°C with the first antibody, the brain slices were rinsed in sterile KPBS and incubated with a mixture of KPBS, Triton X-100, heparin, and biotinylated goat anti-rabbit IgG (1:1,500 dilution; Vector Laboratories, Burlingame, CA) for 90 min. Sections were then rinsed with KPBS and incubated at room temperature for 60 min with an avidin-biotin-peroxidase complex (Vectastain ABC Elite Kit; Vector Laboratories). After several rinses in sterile KPBS, the brain slices were allowed to react in a mixture containing sterile KPBS, the chromagen 3,3'-diaminobenzidine tetrachloride (0.05%), and 1% hydrogen peroxide. Thereafter, tissues were rinsed in sterile KPBS, mounted onto poly-L-lysine-coated slides, desiccated overnight under vacuum, fixed in paraformaldehyde (4%) for 30 min, and digested for 30 min at 37°C with proteinase K (10 µg/ml in 100 mM Tris·HCl containing 50 mM EDTA, pH 8.0). Prehybridization, hybridization, and posthybridization steps were performed as described above except for the dehydration step, which was shortened to avoid decolorization of Fos-immunoreactive (Fos-ir) cells. After being vacuum dried for 2 h, sections were exposed onto X-ray film, defatted in xylene, and dipped in NTB2 nuclear emulsion. Slides were exposed for 7 days, developed in D19 developer for 3.5 min at 15°C, and fixed in rapid fixer for 5 min. Thereafter, tissues were rinsed in running distilled water for 1 or 2 h, rapidly dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX.
Antisense 35S-Labeled cRNA Probes
The CRH cRNA probe was generated from the 1.2-kb EcoRI fragment of a rat CRH cDNA (Dr. K. Mayo, Northwestern University, Evanston, IL), subcloned into a pGEM4 vector (Stratagene, La Jolla, CA), and linearized with HindIII and EcoRI (Pharmacia Biotech Canada) for antisense and sense probes, respectively. The c-fos cRNA probe was generated from the EcoRI fragment of rat c-fos cDNA (Dr. I. Verma, The Salk Institute, La Jolla, CA), subcloned into pBluescript SK-1 plasmid (Stratagene), and linearized with SmaI and XhoI (Pharmacia Biotech Canada) for antisense and sense probes, respectively. The rat pro-TRH cRNA probe was generated from a 1.3-kb EcoRI-PstI fragment of the rat proTRH cDNA (Dr. S. Lee, New England Medical Center, Boston, MA), subcloned into a pSP64 plasmid (Stratagene), and linearized with BamHI and HindIII to generate antisense and sense probes, respectively. The specificity of each probe was confirmed by the absence of a positive signal in sections hybridized with sense probes. Radioactive riboprobes were synthesized by incubation of 250-ng linearized plasmid in 10 mM NaCl, 10 mM dithiothreitol, 6 mM MgCl, 40 mM Tris (pH 7.9), 0.2 mM ATP-GTP-CTP, -35S-labeled UTP, 40 U of RNasin (Promega, Madison, WI) and 20 U of SP 6, T7, or T3 RNA polymerase for CRH, c-fos, and pro-TRH antisense probes, respectively, for 60 min at 37°C. The DNA templates were treated with 100 µl of DNase solution (1 µl DNase, 5 µl of 5 mg/ml tRNA, 94 µl of 10 mM Tris-10 mM MgCl2). The preparation of the riboprobes was completed through phenol-chloroform extraction and ammonium acetate precipitation.
Quantitative Analysis of Hybridization Signals
The hybridization signals revealed on NTB2-dipped nuclear emulsion slides were analyzed and quantified under a light microscope (Olympus, BX50) equipped with a black-and-white video camera (Sony, XC-77) coupled to a Macintosh computer (Power PC 7100/66) using Image software (version 1.57 non-FPU; Wayne Rasband, National Institutes of Health, Bethesda, MD). The optical density (OD) of the hybridization signal was determined from coronal brain sections under dark-field illumination at a magnification of x25. The brain sections of the various rats were matched as closely as possible for rostrocaudal level. OD determination was performed on each side of the brain, and the two OD values obtained were averaged. This average was included in the statistical analyses as the individual score of a rat. When no hybridization signal was visible under dark-field illumination, the brain structures of interest were outlined under bright-field illumination and then subjected to densitometric analysis under dark-field illumination. The OD for each specific region was corrected for the average background signal, which was determined by sampling unlabeled areas outside of the areas of interest.
Statistical Analyses
In experiment 1, the effects of "time" (T, measurements taken on 6 consecutive days after surgery) and "daily cycle" (C, measurements taken during dark vs. light period) on body weight, food intake, and metabolic measurements and the effects of "treatment" (Tr, propanolol vs. saline) on the O2 measurements in the rats pretreated with propanolol or saline before refeeding were assessed using ANOVA. Each group used included 46 animals.
A 2 x 2 x 3 ANOVA was used to detect significant main and interaction effects of "phenotype" [P, lean (Fa/?), obese (fa/fa)], "surgery" (S, sham, ADX), and "nutrition" (N, fed ad libitum, 12-h food deprivation, 1-h refeeding) on the various dependent variables measured in a first cohort of animals in experiment 2. The variables measured in a second cohort of rats in experiment 2 were analyzed by 2 x 2 ANOVA to detect significant main and interaction effects of P and S. A posteriori comparisons were performed using the Bonferroni-Dunn multiple comparison procedure.
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RESULTS |
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Body weight. Figure 1 illustrates changes in body weight of lean and obese rats during 6 consecutive days following adrenal surgery. ANOVA revealed a significant main effect of surgery and a significant interaction of surgery and phenotype on body weight, which suggests attenuation in body weight gain after surgery in both phenotypes but with a higher effect in obese than in lean rats. In addition, a significant interaction of time, phenotype, and surgery was accounted for by the fact that lean ADX rats gained weight at a higher rate than did obese ADX animals and that this difference increased over the 6 postoperative days.
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RQ. Whole body RQ in ADX and sham-operated lean and obese rats was expressed as the average of diurnal (24-h) RQ as well as the light-RQ (between 0900 and 1900) and dark-RQ (between 1900 and 0900) measured over the 6 postoperative days. Compared with lean sham-operated animals, obese sham-operated rats demonstrated significantly higher levels of RQ that depend mostly on an increase in RQ levels in obese rats during the light period (Table 2). ADX resulted in a decrease in RQ in both phenotypes. ANOVA applied to RQ measurements revealed a significant phenotype x surgery interaction, suggesting that the decrease in RQ levels after ADX was more pronounced in obese animals than in lean rats. Indeed, there was no more difference in RQ values between lean and obese rats after ADX (Table 2).
When substrate utilization was considered over 24 h, a twofold higher rate of lipid oxidation was found in lean sham-operated rats compared with their obese counterparts (phenotype effect, P < 0.0001; Table 2). ADX resulted in a twofold increase in the rate of lipid oxidation in both phenotypes (surgery effect, P < 0.0001), but ADX obese rats continued to oxidize significantly less lipid than lean ADX animals. When lipid oxidation measurements were corrected for the mass exponent (kg0.75), the same trends were observed (phenotype effect, P < 0.0001; surgery effect, P < 0.0001).
Twelve hours of fasting significantly reduced RQ levels in both lean and obese rats (ad libitum vs. fasting, P < 0.001 for all groups; Fig. 3). However, lean and ADX obese rats decreased their levels of RQ significantly faster than did obese sham-operated animals (phenotype effect, P < 0.0001; surgery effect, P < 0.001). Sham-operated lean rats reached an RQ of 0.7 after 7 h of food deprivation, whereas their obese counterparts attained that value only at the end of 12 h of fasting. After ADX, obese rats decreased more rapidly their fasting RQ and as a result reached the value of RQ of food-deprived ADX lean rats as early as at the 5th h of fasting. An important increase in RQ levels following refeeding was detected in all groups except in lean ADX rats (lean ADX rats vs. other 3 groups, P < 0.001).
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Body composition. Because of the dramatic differences in body weight between lean and obese rats, the measurements of protein and lipid carcass composition were expressed in absolute values (in grams) and as the percentage of the total carcass weight (g/carcass weight x 100; Fig. 4). Both analyses demonstrated that obese rats had reduced carcass protein compared with lean animals and that ADX did not affect protein content. In contrast, obese rats had much higher levels of body fat than lean animals. ADX decreased lipid carcass content in both phenotypes (Fig. 4).
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TRH mRNA expression in the brain. ANOVA revealed a significant main effect of phenotype on the expression of the TRH mRNA in the parvocellular PVH in sham-operated and ADX lean and obese rats (Fig. 5). There was a stronger decline in TRH mRNA expression during food deprivation in obese rats compared with lean animals. The decrease in TRH mRNA levels in response to food deprivation was blunted by ADX (Figs. 5 and 6). After refeeding, the expression of TRH in the PVH was restored at the ad libitum levels in all groups.
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DISCUSSION |
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The reducing effects of ADX on energy gain were largely attributable to the hypophagic effects of ADX. ADX lean and obese rats ate less than sham-operated animals, which is consonant with the view that the removal of adrenals causes hypophagia in laboratory rodents (6). There was no marked difference in the 24-h food ingestion between ADX lean and ADX obese rats. It is noteworthy that ADX-induced alterations in food intake took place during the nocturnal phase in lean rats whereas in obese animals ADX resulted in a decrease in food consumption during both dark and light diurnal phases. Genetically obese (fa/fa) Zucker rats are known to be hyperphagic (6) and to have a characteristic diurnal feeding pattern with similar food intakes during the dark and light phases, whereas lean (Fa/?) rats exhibit a pattern of nocturnal eating (3).
One of the most appreciable changes found in obese and lean rats after ADX was an increase in whole body fat oxidation. In obese Zucker rats, which are characterized by a low fatty acid utilization (23), ADX normalized 24-h RQ by bringing the level down below that of sham-operated lean rats. ADX as well as food deprivation tended to abolish the difference in RQ between lean and obese rats. The reducing effect of ADX on RQ in obese rats was particularly apparent during the day (light phase). Indeed, during the light phase, ADX led to a 3.7-fold increase in fat oxidation in obese rats compared with a 1.7-fold increase in lean animals. This difference between lean and obese rats in the effects of ADX on fat oxidation was possibly attributable to the effects of ADX on food intake, which were also stronger in obese than in lean animals during the day. It is noteworthy that the difference in fat oxidation between lean and obese animals does not uniquely relate to phenotype difference in HPA axis activity, as a difference in fat oxidation still persists between lean and obese animals following ADX.
The present results convincingly show that the metabolic rate was higher in lean rats than in obese animals regardless of whether the variable was expressed in absolute or relative terms. This finding is consistent with some reports (4, 31, 34), but contradicts others (10, 28). The discrepancy between the results can be ascribed to differences in housing conditions, daily time of assessment, presence or absence of food during measurements, and the O2-monitoring procedure itself. Frequently,
O2 ensued from short (3060 min) and single periods of measurement, which are often associated with stress-induced changes in
O2. The present results were obtained with an approach allowing for a 24-h continuous measurement of
O2 over several days, with ad libitum access to food and water. In these conditions, lean rats demonstrated higher absolute and relative metabolic rates compared with obese rats. The basal hypometabolism in obese animals cannot be entirely explained by the lower spontaneous activity of obese Zucker rats (46), because they also demonstrated a decrease in body temperature (28). Because the metabolic rate remained lower in ADX obese rats compared with ADX lean animals, our results suggest that HPA axis hyperactivity is probably not directly involved in the decreased resting metabolic rate of the obese Zucker rat.
The reduction in the metabolic rate following ADX was likely attributable to decreases in food intake and body weight associated with ADX. The compensatory drop in energy expenditure following ADX did not, however, prevent the reduction in energy gain caused by the procedure. Given that ADX can stimulate BAT thermogenesis (22), one can reasonably argue that an increase in BAT thermogenesis potentially curbed the drop in metabolic rate associated to a deficit in energy intake.
Postprandial activation of the SNS in lean and obese Zucker rats was estimated in the present study. The acute rise in metabolic rate after a meal is known to be a consequence of the energy costs of absorption and assimilation of nutrients and of an additional increase in energy expenditure, which is probably related to the activation of BAT thermogenesis (41, 43) and can be inhibited by -adrenergic blockade. The present results provide some evidence that the
-adrenergic antagonist propanolol is less effective in blunting the postprandial
O2 in obese than it is in lean rats. This finding is in agreement with the view that SNS-mediated BAT thermogenesis is impaired in obese rats (22, 24).
We observed higher pro-TRH mRNA levels in the PVH of lean rats compared with obese animals. Although in ad libitum-fed animals this difference did not reach a level of statistical significance, the consideration of all feeding statuses revealed significantly lower TRH contents in obese rats. Decrease in pro-TRH mRNA contents in the PVH has been reported in lean rats after 3 days of starvation (53). In obese rats, we found a significant reduction in the levels of pro-TRH transcript after only 12 h of food deprivation. ADX blunted the decrease in pro-TRH during food deprivation and even led to a slight increase in pro-TRH mRNA levels in lean, food-deprived rats. Corticosteroids are known to suppress the expression of hypothalamic pro-TRH mRNA (16). Van Haasteren et al. (53) have demonstrated that starvation-induced increase in corticosterone secretion is part of the mechanism responsible for the reduced pro-TRH mRNA, TSH mRNA, and plasma TSH synthesis and release during starvation. Moreover, the presence of a glucocorticoid-responsive element in the promoter region of the pro-TRH gene (19) and the occurrence of glucocorticoid receptors in TRH-synthesizing cells in the paraventricular part of the PVH (7) argued strongly for a TRH expression regulation by glucocorticoids. Therefore, the hypercorticosteronemia of obese Zucker rats may be responsible, at least in part, for the reported functional hypothyroidism in these animals (1, 23, 42). A mechanism for TRH decrease in obesity and food deprivation is the apparent inhibition of the TRH neurons by neuropeptide Y (NPY) (14). Arcuate nucleus neurons producing NPY, which is strongly increased in both obese and food-deprived animals (14), innervate the TRH neurons in the PVH (14). Arcuate nucleus ablation prevents fasting-induced suppression of pro-TRH (21), and the administration of leptin to fasting animals has been shown to restore pro-TRH levels (20). Lack of leptin signaling, which normally decreases NPY, may be a cause of TRH suppression in obese animals via leptin-NPY signaling. The contents of the thyroid hormones were not measured in the present study, which makes difficult any speculations about the activity of the entire hypothalamic-thyroid axis. The present observations on TRH mRNA levels in fasted and ADX animals are not contradictory to the hypothesis (5) that a centrally triggered mild hypothyroidism in obese animals may contribute to the decrease in the basal energy expenditure.
The hyperactivity of the HPA axis in obese Zucker rats results in very high levels of corticosterone in these animals (13). High levels of ACTH have also been observed in the obese Zucker rat, which also exhibits an increase in the clearance of corticosterone (59). In addition, the fa/fa rat has been reported to overexpress CRH when faced with stressful experimental conditions (13, 37, 57). Food deprivation results in a stress reaction with strong activation of the hypophysiotropic CRH neurons in obese, but not in lean, rats (52). In this study, we aimed to clarify the implication of corticosterone in CRH expression and PVH activation in the stress-like response to fasting in obese rats, and in the postprandial response in both phenotypes. We have found that ADX did not abolish the activation of hypothysiotropic parvocellular CRH neurons in obese food-deprived rats. This may suggest that stress activation of the central nervous system in obese rats in response to food deprivation is independent of the increased corticosterone levels in these animals and occurs via specific neural pathways, including sensory, anticipatory, and limbic system-linked emotional circuits. In this, our observations are consistent with those demonstrating that ADX did not alter the magnitude of the Fos response to swim stress (15, 25).
Recently, we reported a particular pattern of neuronal activation in the PVH during refeeding (50). Postprandial response resulted in moderate activation of the hypophysiotropic CRH neurons in the parvocellular PVH, with higher Fos expression in the ventral and dorsal autonomic parvocellular PVH and very strong activation of the magnocellular PVH, including Fos expression in all arginine vasopressin (AVP) neurons. After ADX, we found a certain shift in activation from the magnocellular to the parvocellular part of the PVH. Fos expression in the parvocellular PVH and within the CRH PVH neurons was significantly higher in ADX refed rats compared with sham-operated refed animals. This increase in the postprandial expression of Fos in the parvocellular PVH after ADX was accompanied by an enhanced CRH expression that was mainly prominent in lean rats. Therefore, we may conclude that, although corticosterone levels are decreased during refeeding, the inherent content of circulating corticosterone is still important to negative feedback control of the postprandial activity of CRH hypophysiotropic neurons. The implication of the corticosterone in the regulation of postprandial activation of the magnocellular part of the PVH is less obvious, because corticosterone is known to decrease the expression of AVP in nonstressed as well as stressed animals (18, 47, 56). In contrast, we observed a decrease in activation in response to refeeding in magnocellular neurons after corticosterone elimination by ADX. The shift in sodium homeostasis seems rather to be responsible for a decrease in activation of magnocellular neurons after ADX. The ADX animals, even when maintained on saline drinking, are hyponatremic (60). The hyponatremic animals fail to respond to ether exposure and osmotic and volemic stimuli with increased secretion of AVP (11, 54, 55), and the synthesis rate of this peptide in magnocellular neurons is also reduced (39).
In conclusion, the present study demonstrates a decrease in body fat and food intake in obese and lean Zucker rats after ADX. At the level of the central nervous system, ADX reversed a decrease in TRH mRNA expression in the PVH detected in fasting animals. ADX resulted in enhanced postprandial activation of the parvocellular PVH. In contrast, the magnocellular part of the PVH was less responsive to refeeding in ADX animals. Finally, ADX failed to prevent the stress response of obese rats to food deprivation, suggesting that the high corticosterone levels in obese rats is not responsible for the fasting-induced stress reaction in these animals. The present results provide evidence that the removal of adrenals resolves some of the metabolic defects encountered in the obese Zucker rats. They also demonstrate that not all the abnormalities of the obese Zucker rats are attributable to the hyperactivity of the HPA axis.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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