Running Wheel Activity Prevents Hyperphagia and Obesity in Otsuka Long-Evans Tokushima Fatty Rats: Role of Hypothalamic Signaling
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内分泌学杂志 2005年第4期
Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Dr. S. Bi, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 618, Baltimore, Maryland 21205. E-mail: sbi@jhmi.edu.
Abstract
Otsuka Long-Evans Tokushima fatty (OLETF) rats lacking cholecystokinin-A receptors are hyperphagic, obese, and diabetic. Although exercise attenuates OLETF rats’ obesity, the mechanisms underlying the effects of exercise are unclear. In this study, we determined the effects of running wheel activity on patterns of body weight gain, food intake, and hypothalamic gene expression. We demonstrate that voluntary running activity beginning at 8 wk of age normalized meal patterns, food intake, body weight, and plasma levels of glucose and leptin in OLETF rats. During the initial exercise period, corticotropin-releasing factor (CRF) mRNA expression was significantly elevated in the dorsomedial hypothalamus (DMH) but not in the paraventricular nucleus in both OLETF and control Long-Evans Tokushima rats. In response to long-term exercise, arcuate nucleus (Arc) neuropeptide Y (NPY), and proopiomelanocortin as well as DMH NPY and CRF mRNA expression were increased in Long-Evans Tokushima rats. In contrast, whereas exercising OLETF rats had increased Arc NPY and DMH CRF expression, Arc proopiomelanocortin and DMH NPY mRNA levels were not elevated. Finally, we demonstrate that the effects of exercise on body weight in OLETF rats were long lasting. Although food intake and body weight were increased in OLETF rats when running wheels were locked, weights did not return to those of sedentary OLETF rats. Together, these data suggest that the elevation of DMH CRF expression may mediate the short-term feeding inhibitory effects of exercise and that exercise limits the elevation of DMH NPY expression to account for the overall prevention of OLETF rats’ obesity.
Introduction
ALTHOUGH THE PREVALENCE of obesity continues to increase and the weight loss obtained through dieting is usually temporary, recent data suggest that exercise may provide an important adjunct to dieting for long-term weight maintenance. Studies of individuals who have maintained significant weight loss for more than a year have demonstrated that dieters who achieve long-term success are often those who engage in a regular and extensive exercise program (1). Whereas the energy expenditure aspects of such exercise surely contribute to the effects of weight maintenance, there is the suggestion that exercise may also contribute to energy balance by altering appetite and reducing food intake (2, 3).
The effects of exercise on food intake and body weight have been studied in a number of animal models of obesity, and the evidence suggests that such effects depend on the genetic cause of the obesity. For example, chronic exercise lowers the defended body weight gain and adiposity in diet-induced obese rats (4). In contrast, exercise had only minimal or no effect on the obesity of Zucker fatty or Koletsky corpulent rats, both congenitally lacking leptin receptors (5, 6, 7). Finally, exercise prevents the obesity of Otsuka Long-Evans Tokushima fatty (OLETF) rats lacking cholecystokinin (CCK)-A (or 1) receptors (CCK-AR) (8). Although it is now clear that such phenomena are specific to the individual obesity model, the mechanisms underlying the effects of exercise on food intake and body weight have not yet been identified.
We have suggested that the obesity in OLETF rats is the outcome of both a peripheral CCK satiety deficit, leading to increased meal size (9), and a central CCK signaling deficit, resulting in dysregulation of neuropeptide Y (NPY) gene expression in the dorsomedial hypothalamus (DMH) and an overall dysfunction in energy balance control (10). OLETF rats do not have primary deficits in leptin signaling or the control of leptin’s downstream mediators. Thus, NPY and proopiomelanocortin (POMC) gene expression in the arcuate nucleus (Arc) are appropriately responding to the increased food intake and body weight (10).
As noted above, exercise has been reported to attenuate the obesity in OLETF rats and has effects on body weight beyond the exercise period (11). In the present experiments, we sought to explore the behavioral and neural mechanisms underlying these phenomena. We determined food intake, body weight, running activity, and plasma levels of glucose and leptin as well as changes in hypothalamic NPY, POMC, and corticotropin-releasing factor (CRF) mRNA levels in OLETF and control Long-Evans Tokushima (LETO) rats with or without access to running wheels at multiple time points. We demonstrate that voluntary exercise prevents the hyperphagia and obesity of OLETF rats. Furthermore, the data suggest roles for DMH CRF and NPY expression in mediating the short- and long-term effects of exercise on food intake and body weight in OLETF rats.
Materials and Methods
Animals
Male OLETF and age-matched male lean LETO rats were a generous gift of the Tokushima Research Institute, Otsuka Pharmaceutical (Tokushima, Japan). Rats were 5 wk old at the time they arrived in our laboratory. On arrival, there was no difference in body weight between the two strains. Rats were individually housed in hanging wire mesh cages and maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) in a temperature-controlled colony room (22 C) with ad libitum access to standard chow and tap water for the initial week. All procedures were approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University.
Food intake and running wheel activity
At 6 wk of age, animals were transferred to individual running wheel cages that were built with a running wheel (diameter 36 cm and width 11 cm, Wahmann, Timonium, MD), an automated pellet dispenser controlled by an infrared pellet-sensing photo beam (Med Associates Inc., Georgia, VT), and a nest box (15 x 25 x 15 cm) as previously described (12). Each revolution of the running wheel activated a microswitch that was monitored and recorded in a computer. Forty-five-milligram chow pellets were automatically delivered in response to the removal of the previous pellet. The delivery of each pellet was monitored, time stamped, and stored 24 h/d in a computer (Med Associates). Meal parameters were analyzed with a meal pattern analysis program (Tongue Twister, version 1.42, Dr. T. A. Houpt, Florida State University, Tallahassee, FL). A meal was defined as the acquisition of at least five pellets preceded and followed by at least 20 min of no feeding. Meal size was defined as the number of pellets delivered during a meal (9). For the initial 2 wk, running wheels were kept in a locked position. After this 2-wk habituation period, running wheels were unlocked and animals had 24 h/d access to the running wheels. All animals had ad libitum access to food and water.
Experiment 1
Fourteen OLETF and eight LETO rats were body weight matched and randomly divided into four groups: sedentary OLETF rats (n = 6, indicated as OLETF.S), OLETF rats with access to running wheels (n = 8, indicated as OLETF.RW), sedentary LETO controls (n = 4, indicated as LETO.S), and LETO rats with access to running wheels (n = 4, indicated as LETO.RW). After the 2-wk habituation period, eight OLETF and four LETO rats had access to the running wheels, and six OLETF and four LETO rats remained sedentary. At 14 wk of age (6 wk after access to running wheels), half of OLETF.RW rats (n = 4) had their running wheels locked (indicated as OLETF.relocked) and the other half of OLETF.RW rats (n = 4) remained with running wheel access. Data for food intake and body weight were collected for an additional 6-wk period. During the entire period, all rats had ad libitum access to food and water. Body weights were recorded daily. Food intake and running activity were monitored and stored in a computer 24 h/d as described above. Data for running activity were presented as the number of revolutions per day. At the end of the experiment (20 wk of age), running wheels were locked in exercising rats at 0900 h, and all animals were killed between 0900 and 1100 h by decapitation under ether inhalation anesthesia. Interscapular brown adipose tissue and the left side of white epididymal adipose tissue were harvested and weighed. Trunk blood was taken for evaluation of plasma levels of glucose and leptin. Plasma glucose levels were measured using a blood glucose meter (Glucometer Elite, Bayer), and plasma leptin concentration was determined by using a rat leptin RIA kit (Linco Research Inc., St. Charles, MO). Brains were removed and rapidly frozen in isopentane on dry ice for subsequent analyses of hypothalamic gene expression.
Experiment 2
Based on the results of food intake and body weight of OLETF.RW, we conducted two additional experiments (i.e. experiments 2 and 3) to determine patterns of changes in hypothalamic gene expression in response to voluntary exercise: In experiment 2, eight OLETF and eight LETO rats were body weight matched and randomly divided into four groups: four OLETF.S rats, four OLETF.RW, four LETO.S controls, and four LETO.RW. After a 2-wk period of habituation as described above, animals had access to running wheels for 4 d, and then all animals were killed. Trunk blood and brains were saved for the measurements described above.
Experiment 3
After the 2-wk period of habituation as described in the first experiment, eight OLETF rats had access to running wheels for 6 wk, and then the half of them had their wheels relocked. Four LETO rats maintained without access to running wheels and served as normal controls. Four days after relocking some of the wheels, all animals were killed and tissues were collected for the measurements as described above.
In situ hybridization determination
Coronal sections (14 μm) were cut via a cryostat, mounted on superfrost/plus slides (Fisher Scientific, Fair Lawn, NJ) as a series of six slides (section 1: slide 1, section 2: slide 2, etc.; section 7: slide 1, etc.), fixed with 4% paraformaldehyde and stored at –70 C for later in situ hybridization determination. Sections for Arc NPY and POMC and DMH NPY and CRF mRNA in situ hybridization were taken at levels 3.1–3.5 mm caudal to bregma, and sections for CRF in the paraventricular nucleus (PVN) were taken at levels 1.8–2.1 mm caudal to bregma (13). One slide from each series was stained with cresyl violet acetate to allow sections to be anatomically matched among animals, and four to six sections per brain were used for each in situ hybridization determination.
As previously described (10), 35S-labeled antisense riboprobes of NPY, POMC, or CRF were transcribed from rat NPY precursor cDNA (14), mouse full-length POMC cDNA (15), or rat CRF precursor cDNA (16), respectively, by using in vitro transcription systems (Promega, Madison, WI) and purified by Quick Spin RNA columns (Roche, Indianapolis, IN).
As previously described (10), frozen tissue sections were allowed to warm to room temperature, treated with acetic anhydride, and incubated in hybridization buffer containing 50% formamide, 0.3 M NaCl, 10 mM Tris/Cl (pH 8.0), 1 mM EDTA (pH 8.0), 1x Denhardt’s solution (Eppendorf, Netheler, Germany), 10% dextran sulfate, 10 mM dithiothreitol, 500 μg/ml yeast tRNA, and 107 cpm/ml of 35S-uridine 5-triphosphate at 55 C for NPY, POMC, and PVN CRF and 58 C for DMH CRF overnight. After hybridization, the sections were washed three times with 2x saline sodium citrate (SSC), treated with 20 μg/ml RNase A (Sigma, St. Louis, MO) at 37 C for 30 min, and then rinsed in 2x SSC twice at 55 C and washed twice in 0.1x SSC at 55 C for 15 min except 58 C for DMH CRF. Slides were dehydrated in gradient ethanol, air dried, and exposed with BMR-2 film (Kodak, Rochester, NY) for 1–3 d except 3–7 d for DMH CRF to obtain the linear range of the autoradiographs for the semiquantification of mRNA levels. All quantification was done from films on which the signals were below saturation levels.
Quantitative analysis of the in situ hybridization was done with National Institutes of Health Scion image software (Bethesda, MD). Autoradiographic images were first scanned on a professional scanner (Epson, Long Beach, CA) by an experimentally blinded observer and saved in a computer for subsequent analyses with Scion image program using autoradiographic 14C microscales (Amersham, Piscataway, NJ) as a standard. Data for each animal represented a mean of the product of hybridization area x density (background density was subtracted) obtained from four to six sections and was normalized to an average of sedentary LETO controls as 100%. Data from each group were presented as mean ± SEM.
Statistical analysis
Data were analyzed using two-way ANOVA across the four experimental groups (LETO.S, LETO.RW, OLETF.S, and OLETF.RW rats) and one-way ANOVA within the three groups of OLETF rats (OLETF.S, OLETF.RW, and OLETF.relocked) and followed by planned t comparisons. Data for running wheel activity in LETO.RW and OLETF.RW rats were analyzed using a Student’s t test. P < 0.05 was taken to be a statistically significant difference.
Results
Running activity in LETO and OLETF rats
As presented in Fig. 1, at 8 wk of age, both LETO.RW and OLETF.RW gradually increased their activity during the first 2 wk. LETO.RW rats had a peak of activity at 6400 revolutions per day (6.4 km/d) at 10 wk of age and then maintained a relatively constant level of activity at approximately 5000 revolutions per day (5.0 km/d). OLETF.RW rats were relatively hypoactive initially, running 47% less than LETO.RW rats at 8 wk of age. However, by 11 wk of age, OLETF.RW rats were hyperactive, compared with LETO.RW rats. OLETF.RW rats had a peak of running activity at 8600 revolutions per day (8.6 km/d) at 11 wk of age and continued this higher activity until 16 wk of age. After wk 16, OLETF.RW rats reduced their activity to the levels of LETO.RW rats. Both LETO and OLETF rats had similar diurnal cycle of running behavior and conducted a majority of running activity (more than 85% of daily activity) in the dark period.
FIG. 1. Running activity in LETO.RW and OLETF.RW rats. Running activity (number of revolutions per day) was lower at 8 wk of age, higher at 11 wk of age, and normalized after 17 wk of age in OLETF.RW rats, compared with exercising LETO.RW rats. Values are means ± SEM, n = 4–8/group. *, P < 0.05, compared with LETO.RW rats by a Student’s t test.
Effect of voluntary exercise on body weight of OLETF rats
Running wheel access and resulting voluntary exercise decreased body weight gain in both LETO and OLETF rats (Table 1 and Fig. 2). Although the body weights did not differ between LETO.S controls and LETO.RW rats at the time points of the onset, 6 and 12 wk of running wheel access, the amount of body weight gain was significantly reduced in LETO.RW rats, compared with LETO.S controls over the 6- and 12-wk periods of running wheel access (Table 1). Relative to LETO rats, voluntary exercise had even greater effects on body weight gain in OLETF rats. At 8 wk of age, OLETF rats were significantly heavier than LETO rats. Running wheel access limited the body weight gain of OLETF rats and normalized their body weights to levels found in the LETO rats (Table 1 and Fig. 2). ANOVA revealed a significant interaction between strain and exercise during the 6 and 12 wk of running wheel access: F (1,14) = 65.449, P < 0.001, and F (1,14) = 76.876, P < 0.001, respectively (Table 1). At 20 wk of age (the end of experiment), OLETF.S rats had gained 42% more body weight than sedentary LETO controls (Table 1). Whereas voluntary exercise resulted in 19% less body weight gain in LETO.RW rats than LETO.S controls, exercise caused 70% less body weight gain in OLETF.RW rats, compared with OLETF.S rats, and the resulting body weight of OLETF.RW rats did not differ from that of LETO.RW animals (Table 1). When running wheels were relocked, OLETF.relocked rats gained weight rapidly for the first 3 wk (Fig. 2). Overall, the body weight of OLETF.relocked rats stabilized at a level significantly below that of OLETF.S rats, and at 20 wk of age (6 wk after relocking running wheels), OLETF.relocked rat weights were intermediate between those of OLETF.RW and OLETF.S rats (Table 1 and Fig. 2).
TABLE 1. Effects of voluntary exercise on the body weight gain in OLETF and LETO rats
FIG. 2. Effects of running wheel access on body weight in LETO and OLETF rats. OLETF.S rats gained more body weight than LETO.S rats. Access to running wheels did not affect body weight of LETO.RW rats but reversed the excess body weight gain of OLETF.RW rats to the same weight as LETO rats. Relocking running wheels resulted in increased body weight (indicated as OLETF.relocked), but the weight did not reach levels of OLETF.S animals. Values are means ± SEM, n = 4–8/group.
Analyses of weights of selected fat pads revealed that at 20 wk of age. OLETF.S rats had significantly increased epididymal white fat and interscapular brown fat masses as compared with LETO.S controls. Voluntary exercise significantly reduced both epididymal white and interscapular brown fat masses in both strains (Table 2). In addition, ANOVA revealed a significant strain by exercise interaction for both epididymal fat [F (1,14) = 21.104, P < 0.001] and interscapular brown fat [F (1,14) = 37.591, P < 0.001], indicating that voluntary exercise decreased fat masses in OLETF rats to a great degree (Table 2). As compared with LETO.RW rats, OLETF.RW animals had similar amount of epididymal fat mass but maintained more interscapular brown fat mass (Table 2). Furthermore, OLETF.relocked rats had significantly increased epididymal white fat and interscapular brown fat masses, compared with OLETF.RW rats, but these increases did not reach those of OLETF.S rats (Table 2).
TABLE 2. Effects of voluntary exercise on fat mass, food intake, plasma glucose, and leptin levels in OLETF and LETO rats at the end of experiment (20 wk of age)
Effects of voluntary exercise on food intake and meal patterns in OLETF rats
As shown in Fig. 3A, OLETF.S rats were hyperphagic relative to LETO.S controls, with 40–60% more daily energy intake over the period of 7–20 wk of age. When given access to running wheels, both LETO.RW and OLETF.RW rats decreased food intake (Fig. 3, A and B). During the initial week, there were significant effects of both strain [F (1,19) = 8.509, P = 0.009] and exercise [F (1,19) = 48.000, P < 0.001] as well as a significant strain by exercise interaction [F (1,19) = 16.797, P < 0.001], indicating that running wheel activity had a greater feeding inhibitory effect in OLETF than LETO rats (51 vs. 20% reductions). Analyses of meal patterns revealed that before access to running wheels, OLETF rats consumed 47% more per meal than LETO rats (Fig. 4A). Running wheel access significantly decreased OLETF meal sizes by 42% (Fig. 4A, P < 0.05). Meal frequency was not significantly altered by exercise (Fig. 4B). There were no significant effects of exercise on meal patterns in LETO rats (Fig. 4, A and B). As a result of the exercise-induced feeding changes in OLETF rats, both OLETF.RW and LETO.RW rats had similar total daily food intake (Fig. 3, A and B) and exhibited similar meal patterns (Fig. 4). After the transient intake reduction that accompanied the initial access to running wheels, food intake was increased in both LETO.RW and OLETF.RW rats and remained stable for the duration of running wheel access (Fig. 3, A and B). Analyses of food intake at 20 wk of age (12 wk with access to running wheels) revealed a significant effect of strain [F (1,14) = 56.665, P < 0.001] and a significant strain by exercise interaction [F (1,14) = 27.703, P < 0.001] but no main effect of exercise [F (1,14) = 0.227, P > 0.05]. As presented in Table 2, OLETF.S rats had significantly greater daily energy intake than LETO.S controls. Whereas exercise resulted in highly increased food intake in LETO.RW rats, exercise resulted in slightly decreased food intake in OLETF.RW rats. This pattern of changes resulted in OLETF.RW and LETO.RW rats having similar food intake and explains the absence of an overall effect of exercise on food intake. As shown in Fig. 3B, relocking the running wheels resulted in a large increase in food intake in OLETF rats. By 20 wk of age, food intake in OLETF.relocked rats had declined to levels of OLETF.S rats (Fig. 3B and Table 2).
FIG. 3. Effects of running wheel access on food intake in LETO and OLETF rats. A, Access to running wheels initially decreased food intake in both LETO and OLETF rats. Food intake was increased in both strains and remained stable after 10 wk of age. B, Relocking running wheels resulted in a large increase in food intake (indicated as OLETF.relocked), and their food intake did not differ from baseline by 20 wk of age. Values are means ± SEM, n = 4–8/group.
FIG. 4. Effects of running wheel access on meal patterns in LETO and OLETF rats. Before access to running wheels, OLETF rats had increased meal size (shown as black bar, A) but normal meal frequency (shown as black bar, B), compared with LETO rats (shown as open bar, A and B). Running wheel access resulted in reduced meal size in OLETF rats (gray bar, A) to a level similar to that of LETO rats (striped bar, A). Running wheel access did not affect meal frequency (gray bar, B). "After" data represented mean of meal size and meal frequency on d 3 and 6 of running wheel access. Values are means ± SEM, n = 4–8/group. a, P < 0.05, compared with "before" LETO rats; b, P < 0.05, compared with "before" OLETF rats.
Effects of voluntary exercise on plasma levels of glucose and leptin in OLETF rats
As presented in Table 2, by 20 wk of age, plasma levels of glucose and leptin in OLETF.S rats were 1.6- and 1.8-fold higher than those of LETO.S rats. Twelve weeks of voluntary exercise normalized these elevated glucose and leptin levels in OLETF rats. LETO.RW rats had 67% decreases in plasma leptin and unchanged glucose levels as compared with LETO.S controls, and voluntary exercise reduced elevated plasma leptin levels by 79% and glucose levels by 35% in OLETF rats (Table 2). ANOVA demonstrated significant strain by exercise interactions [(F [1,14] = 5.906, P < 0.05) for leptin and (F [1,13] = 4.752, P < 0.05) for glucose levels], indicating that voluntary exercise had greater effects on plasma leptin and glucose levels in OLETF rats than those in LETO rats. As a result, both OLETF.RW and LETO.RW rats had equal levels of plasma leptin and glucose (Table 2). When running wheels were relocked, plasma leptin levels were significantly elevated. Planned t comparisons revealed that at 20 wk of age, OLETF.relocked rats had higher leptin levels than OLETF.RW animals, but the levels were still significantly lower than those of OLETF.S rats (Table 2). At this time point, the levels of plasma leptin in OLETF.relocked rats were close to those of sedentary LETO controls (Table 2). In contrast, plasma glucose levels were unchanged in OLETF.relocked rats as compared with OLETF.RW rats (Table 2).
These exercise-induced changes in plasma glucose and leptin levels occurred quickly. As shown in Table 3, 4 d of voluntary exercise significantly decreased plasma leptin levels in both strains. At 8 wk of age, OLETF.S rats had a 2.7-fold increase in plasma leptin levels, compared with LETO.S controls. Voluntary exercise normalized these increased leptin levels of OLETF rats to those of LETO.RW animals. The higher levels in OLETF.S rats but not in OLETF.RW rats resulted in a significant strain by exercise interaction [F (1,11) = 45.620, P < 0.001]. Four days of voluntary exercise normalized plasma glucose levels in OLETF rats. Plasma glucose levels were higher in OLETF.S rats and reduced by exercise in OLETF.RW rats to the levels of LETO.S controls (Table 3). Voluntary exercise did not affect glucose levels in LETO rats (P > 0.05) (Table 3). At 14 wk of age, relocking running wheels for 4 d resulted in increased plasma leptin levels in OLETF rats (P < 0.05), but plasma glucose levels did not change over this 4-d period (P > 0.05) (Table 3).
TABLE 3. Effects of voluntary exercise on plasma glucose and leptin levels in OLETF and LETO rats in 4 d of access to running wheels or 4 d of relocking running wheels after 6 wk of exercise
Effects of voluntary exercise on hypothalamic NPY, POMC, and CRF mRNA levels
NPY mRNA levels.
At both 8 and 20 wk of age, OLETF.S rats had decreased NPY mRNA levels in the Arc, compared with LETO.S rats (planned t comparisons, P < 0.05, Table 4). Although 4 d of running wheel access did not alter Arc NPY mRNA levels in either LETO or OLETF rats, Arc NPY mRNA expression was significantly elevated in both strains in response to 12 wk of voluntary exercise (Table 4). At the end of the 12-wk period of exercise, Arc NPY mRNA levels were increased by 61% in LETO.RW and 59% in OLETF.RW rats as compared with sedentary rats [effect of exercise, F (1,14) = 13.953, P = 0.002]. There was no significant interaction between strain and exercise (P > 0.05). Relocking the running wheels for 4 d resulted in a further 50% increase in Arc NPY mRNA levels in OLETF rats (Table 4). This effect was temporary. Arc NPY mRNA levels were decreased to baseline in OLETF rats that had their running wheels relocked for 6 wk (P > 0.05, Table 4).
TABLE 4. Effects of voluntary exercise on hypothalamic mRNA expression of NPY, POMC, and CRF in OLETF and LETO rats
Patterns of change in DMH NPY mRNA levels in response to running wheel access were similar to those of Arc NPY. Four days of running wheel access did not alter DMH NPY mRNA levels (Table 4). However, 12 wk of exercise resulted in significant changes [F (1,13) = 10.525, P = 0.006] (Table 4). Planned t comparisons revealed that the effect of exercise was mainly due to the elevation of DMH NPY mRNA levels in LETO.RW rats. Twelve weeks of voluntary exercise resulted in a 74% increase in NPY mRNA levels in the DMH of LETO rats but did not significantly affect DMH NPY mRNA levels in OLETF rats (Table 4). We did find that OLETF.S rats had elevated DMH NPY mRNA levels at 20 wk of age relative to LETO.S rats (Table 4). This alteration was not affected by running wheel access. However, relocking the running wheels reduced DMH NPY mRNA levels in OLETF rats at 20 wk of age (P < 0.05, Table 4).
POMC mRNA levels.
As compared with LETO.S controls, OLETF.S rats had significantly increased POMC mRNA levels in the Arc at both 8 and 20 wk of age (Table 4). Although 4 d of voluntary exercise did not affect Arc POMC mRNA levels in either strain, 12 wk of voluntary exercise resulted in significant increases in LETO but not OLETF rats (Table 4). Relocking running wheels had no immediate effect but resulted in a 50% increase in Arc POMC mRNA levels in OLETF.relocked rats, compared with OLETF.RW animals by the end of the 6-wk relocking period (Table 4).
CRF mRNA levels.
At the time point of 8 wk of age, PVN CRF mRNA levels were not affected by either strain or 4 d of voluntary exercise (Table 4). However, OLETF.S rats had decreased PVN CRF mRNA levels, compared with LETO.S controls by 20 wk of age (Table 4). Twelve weeks of voluntary exercise resulted in strain-specific modulation of CRF gene expression in the PVN [F (1,14) = 7.929, P = 0.014] (Table 4). Planned t comparisons revealed that 12 wk of voluntary exercise resulted in a 21% decrease in PVN CRF mRNA levels in LETO rats, whereas voluntary exercise elevated PVN CRF mRNA levels in OLETF rats (Table 4). These elevated levels of PVN CRF mRNA were reduced when running wheels were relocked for 6 wk (Table 4).
We noted that both LETO.RW and OLETF.RW rats had significantly elevated CRF mRNA levels in the DMH. This elevated CRF gene expression was specifically localized to the most dorsal part of the DMH, outside the compact subregion (Fig. 5B) and occurred at both the 4-d and 12-wk time points [(F [1,12] = 17.594, P = 0.001) and (F[1,1] = 49.213, P < 0.001), respectively] (Table 4). Although CRF mRNA levels was very low in the DMH of sedentary animals (Fig. 5A), voluntary exercise increased DMH CRF mRNA levels by 2-, 3.8-, and 5-fold in rats with 4 d, 6 wk, and 12 wk of voluntary exercise, respectively (Table 4). There was no significant difference in this elevation between LETO.RW and OLETF.RW rats. When running wheels were relocked for 4 d or 6 wk, the exercise-induced elevation of DMH CRF mRNA returned to basal levels (Table 4).
FIG. 5. In situ hybridization of CRF mRNA in the DMH with 35S-labeled CRF antisense riboprobe in rat brains at the end of the experiment (20 wk of age). A, DMH CRF mRNA expression was low in both sedentary rats. B, CRF mRNA expression was elevated in the DMH in response to access to running wheels. Arrows indicate CRF mRNA transcript signals.
Discussion
The current results demonstrate that running wheel activity normalizes food intake and prevents the obesity of OLETF rats. These changes in food intake and body weight are maintained as long as OLETF rats have access to running wheels. When wheels are relocked, OLETF rats temporarily increase their food intake and gain more weight. However, they stabilize at a lower level of body weight than sedentary OLETF rats. The findings on exercised-induced attenuation of weight gain in OLETF rats are similar to previous reports from Shima et al. (8, 11). We extended these findings with the demonstrations that voluntary exercise normalized altered feeding patterns and identified a variety of changes in hypothalamic gene expression that may underlie the effects of exercise on food intake and body weight in OLETF rats. Together, these data suggest that voluntary exercise activates hypothalamic pathways that overcome the deficits in CCK signaling that contribute to the hyperphagia and obesity in OLETF rats.
To identify how hypothalamic signaling systems involved in energy balance may underlie changes in food intake in response to exercise, we examined patterns of hypothalamic gene expression at multiple time points: 4 d after running wheel access, 4 d after relocking of the activity wheels after 6 wk of access, and at the end of the 12-wk experimental period. The data demonstrate that 4 d after initial running wheel access, a time that food intake is reduced, the major change in both LETO and OLETF rats was a significant elevation or induction of CRF mRNA expression in the DMH. mRNA levels for Arc NPY and POMC, DMH NPY, and PVN CRF were not changed in either group at this time point. These data suggest that increases in DMH CRF may mediate the short-term feeding inhibitory effects of exercise. Central CRF administration has been shown to decrease food intake and meal size and elevate locomotor activity (17, 18), and prior pharmacological experiments have identified a major role for CRF in exercise-induced anorexia (19). Administration of a CRF antagonist attenuates decreases in food intake in response to both voluntary (20) and forced exercise (19). The injection of a CRF antagonist further increased DMH CRF gene expression seen in voluntary exercised rats but did not affect PVN CRF gene expression (20). Moreover, although PVN CRF had been suggested as the major mediator of such effects (21), lesions of the PVN did not prevent anorectic effect of forced exercise (22). Thus, our current findings demonstrating significant elevations in DMH but not PVN CRF mRNA levels in both strains in response to exercise suggest that DMH CRF may be the site for such feeding inhibitory signals.
How DMH CRF and its signaling act in feeding control is not clear. DMH CRF neurons have been shown to project to the PVN (23). In addition, the activation of the DMH has been demonstrated to trigger the hypothalamic-pituitary-adrenal axis response to emotional or exteroceptive stress (24, 25, 26, 27). Hotta et al. (28) demonstrated that CRF receptor type 1 mediates emotional stress-induced inhibition of food intake and increase in locomotor activity in rats. Whether the mediation of running wheel access induced DMH CRF gene overexpression and the resulting inhibition of food intake is through the same signaling pathways as the DMH responding to emotional stress remains to be determined.
The exercise-induced changes in food intake and running activity in OLETF.RW rats are greater than those that occur in LETO.RW rats. In OLETF rats, overall intake was reduced by 50%, compared with a 20% reduction in LETO rats during the initial period of exercise. By 11 wk of age, OLETF.RW rats were hyperactive relative to LETO.RW rats and continued this higher activity until 16 wk of age. These differences may reflect a lowered CRFergic tone and resulting hypersensitivity to exercise-induced elevations in CRF expression and presumed release. Consistent with this idea, OLETF rats have been reported to exhibit an increased ACTH response to exogenous CRF administration (29).
LETO and OLETF rats had different patterns of hypothalamic gene expression that may explain the differences in their long-term responses to exercise. In LETO.RW rats, DMH CRF mRNA levels continued to increase, reaching levels that were 5-fold higher than sedentary LETO rats by 12 wk of exercise. This increase was accompanied by significant elevations in Arc NPY and POMC and DMH NPY and a reduction in PVN CRF mRNA levels. These latter findings are similar to those in previous reports showing that Arc and DMH NPY concentration are increased in exercising rats (30), hypothalamic POMC mRNA levels are elevated in rats maintained on a treadmill (31), and PVN CRF mRNA levels are decreased in mice in response to long-term voluntary exercise (32). Overall, such changes in ARC and DMH NPY and PVN CRF mRNA expression would stimulate food intake and may serve to balance an anorexic effects of increased Arc POMC and DMH CRF. Against this background, LETO rats maintain normal weight, gradually increasing their food intake to compensate for the energy demands of increased activity.
Although there was a similar increase in DMH CRF and Arc NPY in OLETF rats after 12 wk of exercise, PVN CRF was normalized and Arc POMC and DMH NPY mRNA levels were not altered. Thus, the increase in DMH CRF in OLETF rats is not balanced by increased DMH NPY or decreased PVN CRF, despite an elevation of Arc NPY mRNA expression. Against this background, food intake remains decreased and body weight is normalized to that of LETO controls. In contrast to pair-fed OLETF rats with normalized body weight that had elevated DMH NPY gene expression, we currently observe that exercised OLETF rats did not have such elevation, despite having normalized body weight. A role for DMH NPY in the regulation of energy balance has been suggested in our and other labs. DMH NPY expression is increased in response to lactation (33) and chronic food restriction (34). Moreover, there are several obese animal models in which elevated levels of DMH NPY mRNA expression have been noted. These include the lethal agouti yellow Ay, melanocortin 4 receptor knockout (35), tubby (36), diet-induced obese (37), and brown adipose tissue-deficient obese mice (38). We previously demonstrated that whereas pair feeding normalizes the obesity of OLETF rats, pair feeding results in a large increase (more than 8-fold) in DMH NPY mRNA levels, similar to levels found in young preobese OLETF rats (10). We have suggested that this elevated DMH NPY gene expression may be a direct result of a CCK signaling deficit and serve as a major contributing factor to the hyperphagia and obesity of OLETF rats (39). Consistent with this view, we have demonstrated that CCK normally plays a role in limiting DMH NPY gene expression. Local CCK administration lowers DMH NPY mRNA levels in intact rats (40). The effects of exercise may replace this lost CCK inhibitory influence on DMH NPY expression in OLETF rats limiting the elevation of DMH NPY gene expression, allowing the OLETF.RW rat to consume normal levels of food intake and maintain a much lower body weight.
Relocking the running wheels resulted in a period of increased food intake and body weight in OLETF rats. Four days after relocking the wheels, the exercised-induced elevation of DMH CRF mRNA levels were decreased to baseline, and Arc NPY mRNA expression was significantly elevated. Both of these changes would support increases in food intake and may contribute to the temporary hyperphagia after relocking the wheels. These data further support the proposed role for alterations in DMH CRF gene expression in the exercise-induced feeding inhibition.
During the 6 wk that running wheels were relocked, OLETF.relocked rats continued to maintain lower body weight than sedentary OLETF rats. At the end of the 6-wk period, mRNA expression for Arc and DMH NPY and DMH and PVN CRF were relatively normalized. However, Arc POMC gene expression was elevated compared with levels from sedentary OLETF rats. We previously demonstrated that Arc POMC mRNA expression is increased in ad libitum-fed OLETF rats and normalized by pair feeding and suggested that this elevation was in response to the increased body weight (10). The higher expression of Arc POMC mRNA in OLETF.relocked rats may account for their reduced body weight and may represent an exercise-induced alteration of body weight set point. Levin and Dunn-Meynell (4) suggested such an interpretation of an exercise induced lowered defended body weight in obesity prone rats.
Our finding of equivalent responses of DMH CRF gene expression to voluntary exercise in both strains suggests that the regulation of CRF gene expression in the DMH does not depend on CCK-ARs. The localization of CRF-expressing and CCK-AR-containing neurons in the DMH appears to be anatomically distinct. CRF gene expression was found in the dorsal part of the DMH in which CRF-containing neurons have been previously identified by immunohistochemistry (41). CCK-ARs are mainly found in the compact area of the DMH (40, 42, 43). This different distribution of DMH CRF and CCK-ARs suggests that the control of DMH CRF is independent of CCK signaling.
A role for leptin in the control of energy balance via down-regulating NPY and up-regulating POMC gene expression in the Arc has been documented (44, 45, 46). However, it is not clear that leptin plays a role in the effects of exercise on energy balance. The data demonstrating that voluntary exercise decreased plasma leptin levels as well as reduced food intake suggest that the feeding-inhibitory effects of exercise is not secondary to elevated leptin levels. Given that leptin receptors and CRF mRNA are expressed in different parts of the DMH (34, 47), it also seems unlikely that the regulation of DMH CRF is under the control of leptin. The alterations in Arc NPY and POMC mRNA levels in response to voluntary exercise or exercise withdrawal are also not consistent with leptin’s known actions. Thus, although plasma leptin levels were decreased in exercising LETO rats, Arc POMC mRNA levels were increased. When wheels were relocked and leptin levels were increasing, Arc NPY levels were elevated. These data suggest that exercise affects Arc gene expression through nonleptin pathways.
We found that plasma leptin levels were rapidly decreased after 4 d of running wheel access and rapidly increased within 4 d of relocking running wheels after 6 wk of exercise. By 4 d of running wheel access, although the body weight of OLETF.RW rats was heavier than that of LETO.RW animals, indicating more fat mass in OLETF rats, their leptin levels had dropped to those of the LETO.RW rats. Zheng et al. (48) reported that a single bout of exercise significantly decreased leptin mRNA levels in rat adipose tissue. Thus, exercise-induced reductions in plasma leptin levels seen in 4-d exercised OLETF and LETO rats may reflect lowered leptin mRNA levels but not decreased fat mass. Such an interpretation would be consistent with previous results demonstrating that reduction of circulating leptin concentration precedes fat loss from running exercise (49). When running wheels were relocked, plasma leptin levels were rapidly elevated. Together, these data suggest that acute exercise has a direct effect on plasma leptin levels.
Exercise decreased, and relocking the wheels increased, epididymal white fat mass. These findings are consistent with exercise stimulating lipolysis in adipose tissue, and the increased lipolysis results in decreased fat mass (50, 51). The changes in interscapular brown fat mass (BAT) in exercised OLETF rats are similar to white adipose tissue. In the absence of data on the activity of the brown fat in response to exercise, we cannot say whether this decrease in mass was due to an increase in BAT-mediated thermogenesis. Exercise has been variously shown to increase BAT thermogenesis (52), not alter BAT thermogenesis (53), or reduce BAT thermogenesis (54). Thus, the interpretation of the reduction in brown fat mass in rats with running wheel access remains to be determined.
In summary, the present results demonstrate that voluntary exercise prevents the hyperphagia and obesity of OLETF rats. The short-term exercise-induced anorexia found in both LETO and OLETF rats may be mediated via increases in DMH CRF expression, and this elevation of DMH CRF gene expression in the absence of compensatory changes in DMH NPY expression may play a critical role in the longer-term effects of exercise on energy balance in OLETF rats. Importantly, exercise appears to overcome the OLETF rat’s deficit in DMH NPY signaling, resulting in a significant and lasting attenuation of their hyperphagia and obesity.
Acknowledgments
The OLETF and LETO rats were a generous gift of the Tokushima Research Institute, Otsuka Pharmaceutical (Tokushima, Japan).
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Address all correspondence and requests for reprints to: Dr. S. Bi, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 618, Baltimore, Maryland 21205. E-mail: sbi@jhmi.edu.
Abstract
Otsuka Long-Evans Tokushima fatty (OLETF) rats lacking cholecystokinin-A receptors are hyperphagic, obese, and diabetic. Although exercise attenuates OLETF rats’ obesity, the mechanisms underlying the effects of exercise are unclear. In this study, we determined the effects of running wheel activity on patterns of body weight gain, food intake, and hypothalamic gene expression. We demonstrate that voluntary running activity beginning at 8 wk of age normalized meal patterns, food intake, body weight, and plasma levels of glucose and leptin in OLETF rats. During the initial exercise period, corticotropin-releasing factor (CRF) mRNA expression was significantly elevated in the dorsomedial hypothalamus (DMH) but not in the paraventricular nucleus in both OLETF and control Long-Evans Tokushima rats. In response to long-term exercise, arcuate nucleus (Arc) neuropeptide Y (NPY), and proopiomelanocortin as well as DMH NPY and CRF mRNA expression were increased in Long-Evans Tokushima rats. In contrast, whereas exercising OLETF rats had increased Arc NPY and DMH CRF expression, Arc proopiomelanocortin and DMH NPY mRNA levels were not elevated. Finally, we demonstrate that the effects of exercise on body weight in OLETF rats were long lasting. Although food intake and body weight were increased in OLETF rats when running wheels were locked, weights did not return to those of sedentary OLETF rats. Together, these data suggest that the elevation of DMH CRF expression may mediate the short-term feeding inhibitory effects of exercise and that exercise limits the elevation of DMH NPY expression to account for the overall prevention of OLETF rats’ obesity.
Introduction
ALTHOUGH THE PREVALENCE of obesity continues to increase and the weight loss obtained through dieting is usually temporary, recent data suggest that exercise may provide an important adjunct to dieting for long-term weight maintenance. Studies of individuals who have maintained significant weight loss for more than a year have demonstrated that dieters who achieve long-term success are often those who engage in a regular and extensive exercise program (1). Whereas the energy expenditure aspects of such exercise surely contribute to the effects of weight maintenance, there is the suggestion that exercise may also contribute to energy balance by altering appetite and reducing food intake (2, 3).
The effects of exercise on food intake and body weight have been studied in a number of animal models of obesity, and the evidence suggests that such effects depend on the genetic cause of the obesity. For example, chronic exercise lowers the defended body weight gain and adiposity in diet-induced obese rats (4). In contrast, exercise had only minimal or no effect on the obesity of Zucker fatty or Koletsky corpulent rats, both congenitally lacking leptin receptors (5, 6, 7). Finally, exercise prevents the obesity of Otsuka Long-Evans Tokushima fatty (OLETF) rats lacking cholecystokinin (CCK)-A (or 1) receptors (CCK-AR) (8). Although it is now clear that such phenomena are specific to the individual obesity model, the mechanisms underlying the effects of exercise on food intake and body weight have not yet been identified.
We have suggested that the obesity in OLETF rats is the outcome of both a peripheral CCK satiety deficit, leading to increased meal size (9), and a central CCK signaling deficit, resulting in dysregulation of neuropeptide Y (NPY) gene expression in the dorsomedial hypothalamus (DMH) and an overall dysfunction in energy balance control (10). OLETF rats do not have primary deficits in leptin signaling or the control of leptin’s downstream mediators. Thus, NPY and proopiomelanocortin (POMC) gene expression in the arcuate nucleus (Arc) are appropriately responding to the increased food intake and body weight (10).
As noted above, exercise has been reported to attenuate the obesity in OLETF rats and has effects on body weight beyond the exercise period (11). In the present experiments, we sought to explore the behavioral and neural mechanisms underlying these phenomena. We determined food intake, body weight, running activity, and plasma levels of glucose and leptin as well as changes in hypothalamic NPY, POMC, and corticotropin-releasing factor (CRF) mRNA levels in OLETF and control Long-Evans Tokushima (LETO) rats with or without access to running wheels at multiple time points. We demonstrate that voluntary exercise prevents the hyperphagia and obesity of OLETF rats. Furthermore, the data suggest roles for DMH CRF and NPY expression in mediating the short- and long-term effects of exercise on food intake and body weight in OLETF rats.
Materials and Methods
Animals
Male OLETF and age-matched male lean LETO rats were a generous gift of the Tokushima Research Institute, Otsuka Pharmaceutical (Tokushima, Japan). Rats were 5 wk old at the time they arrived in our laboratory. On arrival, there was no difference in body weight between the two strains. Rats were individually housed in hanging wire mesh cages and maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) in a temperature-controlled colony room (22 C) with ad libitum access to standard chow and tap water for the initial week. All procedures were approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University.
Food intake and running wheel activity
At 6 wk of age, animals were transferred to individual running wheel cages that were built with a running wheel (diameter 36 cm and width 11 cm, Wahmann, Timonium, MD), an automated pellet dispenser controlled by an infrared pellet-sensing photo beam (Med Associates Inc., Georgia, VT), and a nest box (15 x 25 x 15 cm) as previously described (12). Each revolution of the running wheel activated a microswitch that was monitored and recorded in a computer. Forty-five-milligram chow pellets were automatically delivered in response to the removal of the previous pellet. The delivery of each pellet was monitored, time stamped, and stored 24 h/d in a computer (Med Associates). Meal parameters were analyzed with a meal pattern analysis program (Tongue Twister, version 1.42, Dr. T. A. Houpt, Florida State University, Tallahassee, FL). A meal was defined as the acquisition of at least five pellets preceded and followed by at least 20 min of no feeding. Meal size was defined as the number of pellets delivered during a meal (9). For the initial 2 wk, running wheels were kept in a locked position. After this 2-wk habituation period, running wheels were unlocked and animals had 24 h/d access to the running wheels. All animals had ad libitum access to food and water.
Experiment 1
Fourteen OLETF and eight LETO rats were body weight matched and randomly divided into four groups: sedentary OLETF rats (n = 6, indicated as OLETF.S), OLETF rats with access to running wheels (n = 8, indicated as OLETF.RW), sedentary LETO controls (n = 4, indicated as LETO.S), and LETO rats with access to running wheels (n = 4, indicated as LETO.RW). After the 2-wk habituation period, eight OLETF and four LETO rats had access to the running wheels, and six OLETF and four LETO rats remained sedentary. At 14 wk of age (6 wk after access to running wheels), half of OLETF.RW rats (n = 4) had their running wheels locked (indicated as OLETF.relocked) and the other half of OLETF.RW rats (n = 4) remained with running wheel access. Data for food intake and body weight were collected for an additional 6-wk period. During the entire period, all rats had ad libitum access to food and water. Body weights were recorded daily. Food intake and running activity were monitored and stored in a computer 24 h/d as described above. Data for running activity were presented as the number of revolutions per day. At the end of the experiment (20 wk of age), running wheels were locked in exercising rats at 0900 h, and all animals were killed between 0900 and 1100 h by decapitation under ether inhalation anesthesia. Interscapular brown adipose tissue and the left side of white epididymal adipose tissue were harvested and weighed. Trunk blood was taken for evaluation of plasma levels of glucose and leptin. Plasma glucose levels were measured using a blood glucose meter (Glucometer Elite, Bayer), and plasma leptin concentration was determined by using a rat leptin RIA kit (Linco Research Inc., St. Charles, MO). Brains were removed and rapidly frozen in isopentane on dry ice for subsequent analyses of hypothalamic gene expression.
Experiment 2
Based on the results of food intake and body weight of OLETF.RW, we conducted two additional experiments (i.e. experiments 2 and 3) to determine patterns of changes in hypothalamic gene expression in response to voluntary exercise: In experiment 2, eight OLETF and eight LETO rats were body weight matched and randomly divided into four groups: four OLETF.S rats, four OLETF.RW, four LETO.S controls, and four LETO.RW. After a 2-wk period of habituation as described above, animals had access to running wheels for 4 d, and then all animals were killed. Trunk blood and brains were saved for the measurements described above.
Experiment 3
After the 2-wk period of habituation as described in the first experiment, eight OLETF rats had access to running wheels for 6 wk, and then the half of them had their wheels relocked. Four LETO rats maintained without access to running wheels and served as normal controls. Four days after relocking some of the wheels, all animals were killed and tissues were collected for the measurements as described above.
In situ hybridization determination
Coronal sections (14 μm) were cut via a cryostat, mounted on superfrost/plus slides (Fisher Scientific, Fair Lawn, NJ) as a series of six slides (section 1: slide 1, section 2: slide 2, etc.; section 7: slide 1, etc.), fixed with 4% paraformaldehyde and stored at –70 C for later in situ hybridization determination. Sections for Arc NPY and POMC and DMH NPY and CRF mRNA in situ hybridization were taken at levels 3.1–3.5 mm caudal to bregma, and sections for CRF in the paraventricular nucleus (PVN) were taken at levels 1.8–2.1 mm caudal to bregma (13). One slide from each series was stained with cresyl violet acetate to allow sections to be anatomically matched among animals, and four to six sections per brain were used for each in situ hybridization determination.
As previously described (10), 35S-labeled antisense riboprobes of NPY, POMC, or CRF were transcribed from rat NPY precursor cDNA (14), mouse full-length POMC cDNA (15), or rat CRF precursor cDNA (16), respectively, by using in vitro transcription systems (Promega, Madison, WI) and purified by Quick Spin RNA columns (Roche, Indianapolis, IN).
As previously described (10), frozen tissue sections were allowed to warm to room temperature, treated with acetic anhydride, and incubated in hybridization buffer containing 50% formamide, 0.3 M NaCl, 10 mM Tris/Cl (pH 8.0), 1 mM EDTA (pH 8.0), 1x Denhardt’s solution (Eppendorf, Netheler, Germany), 10% dextran sulfate, 10 mM dithiothreitol, 500 μg/ml yeast tRNA, and 107 cpm/ml of 35S-uridine 5-triphosphate at 55 C for NPY, POMC, and PVN CRF and 58 C for DMH CRF overnight. After hybridization, the sections were washed three times with 2x saline sodium citrate (SSC), treated with 20 μg/ml RNase A (Sigma, St. Louis, MO) at 37 C for 30 min, and then rinsed in 2x SSC twice at 55 C and washed twice in 0.1x SSC at 55 C for 15 min except 58 C for DMH CRF. Slides were dehydrated in gradient ethanol, air dried, and exposed with BMR-2 film (Kodak, Rochester, NY) for 1–3 d except 3–7 d for DMH CRF to obtain the linear range of the autoradiographs for the semiquantification of mRNA levels. All quantification was done from films on which the signals were below saturation levels.
Quantitative analysis of the in situ hybridization was done with National Institutes of Health Scion image software (Bethesda, MD). Autoradiographic images were first scanned on a professional scanner (Epson, Long Beach, CA) by an experimentally blinded observer and saved in a computer for subsequent analyses with Scion image program using autoradiographic 14C microscales (Amersham, Piscataway, NJ) as a standard. Data for each animal represented a mean of the product of hybridization area x density (background density was subtracted) obtained from four to six sections and was normalized to an average of sedentary LETO controls as 100%. Data from each group were presented as mean ± SEM.
Statistical analysis
Data were analyzed using two-way ANOVA across the four experimental groups (LETO.S, LETO.RW, OLETF.S, and OLETF.RW rats) and one-way ANOVA within the three groups of OLETF rats (OLETF.S, OLETF.RW, and OLETF.relocked) and followed by planned t comparisons. Data for running wheel activity in LETO.RW and OLETF.RW rats were analyzed using a Student’s t test. P < 0.05 was taken to be a statistically significant difference.
Results
Running activity in LETO and OLETF rats
As presented in Fig. 1, at 8 wk of age, both LETO.RW and OLETF.RW gradually increased their activity during the first 2 wk. LETO.RW rats had a peak of activity at 6400 revolutions per day (6.4 km/d) at 10 wk of age and then maintained a relatively constant level of activity at approximately 5000 revolutions per day (5.0 km/d). OLETF.RW rats were relatively hypoactive initially, running 47% less than LETO.RW rats at 8 wk of age. However, by 11 wk of age, OLETF.RW rats were hyperactive, compared with LETO.RW rats. OLETF.RW rats had a peak of running activity at 8600 revolutions per day (8.6 km/d) at 11 wk of age and continued this higher activity until 16 wk of age. After wk 16, OLETF.RW rats reduced their activity to the levels of LETO.RW rats. Both LETO and OLETF rats had similar diurnal cycle of running behavior and conducted a majority of running activity (more than 85% of daily activity) in the dark period.
FIG. 1. Running activity in LETO.RW and OLETF.RW rats. Running activity (number of revolutions per day) was lower at 8 wk of age, higher at 11 wk of age, and normalized after 17 wk of age in OLETF.RW rats, compared with exercising LETO.RW rats. Values are means ± SEM, n = 4–8/group. *, P < 0.05, compared with LETO.RW rats by a Student’s t test.
Effect of voluntary exercise on body weight of OLETF rats
Running wheel access and resulting voluntary exercise decreased body weight gain in both LETO and OLETF rats (Table 1 and Fig. 2). Although the body weights did not differ between LETO.S controls and LETO.RW rats at the time points of the onset, 6 and 12 wk of running wheel access, the amount of body weight gain was significantly reduced in LETO.RW rats, compared with LETO.S controls over the 6- and 12-wk periods of running wheel access (Table 1). Relative to LETO rats, voluntary exercise had even greater effects on body weight gain in OLETF rats. At 8 wk of age, OLETF rats were significantly heavier than LETO rats. Running wheel access limited the body weight gain of OLETF rats and normalized their body weights to levels found in the LETO rats (Table 1 and Fig. 2). ANOVA revealed a significant interaction between strain and exercise during the 6 and 12 wk of running wheel access: F (1,14) = 65.449, P < 0.001, and F (1,14) = 76.876, P < 0.001, respectively (Table 1). At 20 wk of age (the end of experiment), OLETF.S rats had gained 42% more body weight than sedentary LETO controls (Table 1). Whereas voluntary exercise resulted in 19% less body weight gain in LETO.RW rats than LETO.S controls, exercise caused 70% less body weight gain in OLETF.RW rats, compared with OLETF.S rats, and the resulting body weight of OLETF.RW rats did not differ from that of LETO.RW animals (Table 1). When running wheels were relocked, OLETF.relocked rats gained weight rapidly for the first 3 wk (Fig. 2). Overall, the body weight of OLETF.relocked rats stabilized at a level significantly below that of OLETF.S rats, and at 20 wk of age (6 wk after relocking running wheels), OLETF.relocked rat weights were intermediate between those of OLETF.RW and OLETF.S rats (Table 1 and Fig. 2).
TABLE 1. Effects of voluntary exercise on the body weight gain in OLETF and LETO rats
FIG. 2. Effects of running wheel access on body weight in LETO and OLETF rats. OLETF.S rats gained more body weight than LETO.S rats. Access to running wheels did not affect body weight of LETO.RW rats but reversed the excess body weight gain of OLETF.RW rats to the same weight as LETO rats. Relocking running wheels resulted in increased body weight (indicated as OLETF.relocked), but the weight did not reach levels of OLETF.S animals. Values are means ± SEM, n = 4–8/group.
Analyses of weights of selected fat pads revealed that at 20 wk of age. OLETF.S rats had significantly increased epididymal white fat and interscapular brown fat masses as compared with LETO.S controls. Voluntary exercise significantly reduced both epididymal white and interscapular brown fat masses in both strains (Table 2). In addition, ANOVA revealed a significant strain by exercise interaction for both epididymal fat [F (1,14) = 21.104, P < 0.001] and interscapular brown fat [F (1,14) = 37.591, P < 0.001], indicating that voluntary exercise decreased fat masses in OLETF rats to a great degree (Table 2). As compared with LETO.RW rats, OLETF.RW animals had similar amount of epididymal fat mass but maintained more interscapular brown fat mass (Table 2). Furthermore, OLETF.relocked rats had significantly increased epididymal white fat and interscapular brown fat masses, compared with OLETF.RW rats, but these increases did not reach those of OLETF.S rats (Table 2).
TABLE 2. Effects of voluntary exercise on fat mass, food intake, plasma glucose, and leptin levels in OLETF and LETO rats at the end of experiment (20 wk of age)
Effects of voluntary exercise on food intake and meal patterns in OLETF rats
As shown in Fig. 3A, OLETF.S rats were hyperphagic relative to LETO.S controls, with 40–60% more daily energy intake over the period of 7–20 wk of age. When given access to running wheels, both LETO.RW and OLETF.RW rats decreased food intake (Fig. 3, A and B). During the initial week, there were significant effects of both strain [F (1,19) = 8.509, P = 0.009] and exercise [F (1,19) = 48.000, P < 0.001] as well as a significant strain by exercise interaction [F (1,19) = 16.797, P < 0.001], indicating that running wheel activity had a greater feeding inhibitory effect in OLETF than LETO rats (51 vs. 20% reductions). Analyses of meal patterns revealed that before access to running wheels, OLETF rats consumed 47% more per meal than LETO rats (Fig. 4A). Running wheel access significantly decreased OLETF meal sizes by 42% (Fig. 4A, P < 0.05). Meal frequency was not significantly altered by exercise (Fig. 4B). There were no significant effects of exercise on meal patterns in LETO rats (Fig. 4, A and B). As a result of the exercise-induced feeding changes in OLETF rats, both OLETF.RW and LETO.RW rats had similar total daily food intake (Fig. 3, A and B) and exhibited similar meal patterns (Fig. 4). After the transient intake reduction that accompanied the initial access to running wheels, food intake was increased in both LETO.RW and OLETF.RW rats and remained stable for the duration of running wheel access (Fig. 3, A and B). Analyses of food intake at 20 wk of age (12 wk with access to running wheels) revealed a significant effect of strain [F (1,14) = 56.665, P < 0.001] and a significant strain by exercise interaction [F (1,14) = 27.703, P < 0.001] but no main effect of exercise [F (1,14) = 0.227, P > 0.05]. As presented in Table 2, OLETF.S rats had significantly greater daily energy intake than LETO.S controls. Whereas exercise resulted in highly increased food intake in LETO.RW rats, exercise resulted in slightly decreased food intake in OLETF.RW rats. This pattern of changes resulted in OLETF.RW and LETO.RW rats having similar food intake and explains the absence of an overall effect of exercise on food intake. As shown in Fig. 3B, relocking the running wheels resulted in a large increase in food intake in OLETF rats. By 20 wk of age, food intake in OLETF.relocked rats had declined to levels of OLETF.S rats (Fig. 3B and Table 2).
FIG. 3. Effects of running wheel access on food intake in LETO and OLETF rats. A, Access to running wheels initially decreased food intake in both LETO and OLETF rats. Food intake was increased in both strains and remained stable after 10 wk of age. B, Relocking running wheels resulted in a large increase in food intake (indicated as OLETF.relocked), and their food intake did not differ from baseline by 20 wk of age. Values are means ± SEM, n = 4–8/group.
FIG. 4. Effects of running wheel access on meal patterns in LETO and OLETF rats. Before access to running wheels, OLETF rats had increased meal size (shown as black bar, A) but normal meal frequency (shown as black bar, B), compared with LETO rats (shown as open bar, A and B). Running wheel access resulted in reduced meal size in OLETF rats (gray bar, A) to a level similar to that of LETO rats (striped bar, A). Running wheel access did not affect meal frequency (gray bar, B). "After" data represented mean of meal size and meal frequency on d 3 and 6 of running wheel access. Values are means ± SEM, n = 4–8/group. a, P < 0.05, compared with "before" LETO rats; b, P < 0.05, compared with "before" OLETF rats.
Effects of voluntary exercise on plasma levels of glucose and leptin in OLETF rats
As presented in Table 2, by 20 wk of age, plasma levels of glucose and leptin in OLETF.S rats were 1.6- and 1.8-fold higher than those of LETO.S rats. Twelve weeks of voluntary exercise normalized these elevated glucose and leptin levels in OLETF rats. LETO.RW rats had 67% decreases in plasma leptin and unchanged glucose levels as compared with LETO.S controls, and voluntary exercise reduced elevated plasma leptin levels by 79% and glucose levels by 35% in OLETF rats (Table 2). ANOVA demonstrated significant strain by exercise interactions [(F [1,14] = 5.906, P < 0.05) for leptin and (F [1,13] = 4.752, P < 0.05) for glucose levels], indicating that voluntary exercise had greater effects on plasma leptin and glucose levels in OLETF rats than those in LETO rats. As a result, both OLETF.RW and LETO.RW rats had equal levels of plasma leptin and glucose (Table 2). When running wheels were relocked, plasma leptin levels were significantly elevated. Planned t comparisons revealed that at 20 wk of age, OLETF.relocked rats had higher leptin levels than OLETF.RW animals, but the levels were still significantly lower than those of OLETF.S rats (Table 2). At this time point, the levels of plasma leptin in OLETF.relocked rats were close to those of sedentary LETO controls (Table 2). In contrast, plasma glucose levels were unchanged in OLETF.relocked rats as compared with OLETF.RW rats (Table 2).
These exercise-induced changes in plasma glucose and leptin levels occurred quickly. As shown in Table 3, 4 d of voluntary exercise significantly decreased plasma leptin levels in both strains. At 8 wk of age, OLETF.S rats had a 2.7-fold increase in plasma leptin levels, compared with LETO.S controls. Voluntary exercise normalized these increased leptin levels of OLETF rats to those of LETO.RW animals. The higher levels in OLETF.S rats but not in OLETF.RW rats resulted in a significant strain by exercise interaction [F (1,11) = 45.620, P < 0.001]. Four days of voluntary exercise normalized plasma glucose levels in OLETF rats. Plasma glucose levels were higher in OLETF.S rats and reduced by exercise in OLETF.RW rats to the levels of LETO.S controls (Table 3). Voluntary exercise did not affect glucose levels in LETO rats (P > 0.05) (Table 3). At 14 wk of age, relocking running wheels for 4 d resulted in increased plasma leptin levels in OLETF rats (P < 0.05), but plasma glucose levels did not change over this 4-d period (P > 0.05) (Table 3).
TABLE 3. Effects of voluntary exercise on plasma glucose and leptin levels in OLETF and LETO rats in 4 d of access to running wheels or 4 d of relocking running wheels after 6 wk of exercise
Effects of voluntary exercise on hypothalamic NPY, POMC, and CRF mRNA levels
NPY mRNA levels.
At both 8 and 20 wk of age, OLETF.S rats had decreased NPY mRNA levels in the Arc, compared with LETO.S rats (planned t comparisons, P < 0.05, Table 4). Although 4 d of running wheel access did not alter Arc NPY mRNA levels in either LETO or OLETF rats, Arc NPY mRNA expression was significantly elevated in both strains in response to 12 wk of voluntary exercise (Table 4). At the end of the 12-wk period of exercise, Arc NPY mRNA levels were increased by 61% in LETO.RW and 59% in OLETF.RW rats as compared with sedentary rats [effect of exercise, F (1,14) = 13.953, P = 0.002]. There was no significant interaction between strain and exercise (P > 0.05). Relocking the running wheels for 4 d resulted in a further 50% increase in Arc NPY mRNA levels in OLETF rats (Table 4). This effect was temporary. Arc NPY mRNA levels were decreased to baseline in OLETF rats that had their running wheels relocked for 6 wk (P > 0.05, Table 4).
TABLE 4. Effects of voluntary exercise on hypothalamic mRNA expression of NPY, POMC, and CRF in OLETF and LETO rats
Patterns of change in DMH NPY mRNA levels in response to running wheel access were similar to those of Arc NPY. Four days of running wheel access did not alter DMH NPY mRNA levels (Table 4). However, 12 wk of exercise resulted in significant changes [F (1,13) = 10.525, P = 0.006] (Table 4). Planned t comparisons revealed that the effect of exercise was mainly due to the elevation of DMH NPY mRNA levels in LETO.RW rats. Twelve weeks of voluntary exercise resulted in a 74% increase in NPY mRNA levels in the DMH of LETO rats but did not significantly affect DMH NPY mRNA levels in OLETF rats (Table 4). We did find that OLETF.S rats had elevated DMH NPY mRNA levels at 20 wk of age relative to LETO.S rats (Table 4). This alteration was not affected by running wheel access. However, relocking the running wheels reduced DMH NPY mRNA levels in OLETF rats at 20 wk of age (P < 0.05, Table 4).
POMC mRNA levels.
As compared with LETO.S controls, OLETF.S rats had significantly increased POMC mRNA levels in the Arc at both 8 and 20 wk of age (Table 4). Although 4 d of voluntary exercise did not affect Arc POMC mRNA levels in either strain, 12 wk of voluntary exercise resulted in significant increases in LETO but not OLETF rats (Table 4). Relocking running wheels had no immediate effect but resulted in a 50% increase in Arc POMC mRNA levels in OLETF.relocked rats, compared with OLETF.RW animals by the end of the 6-wk relocking period (Table 4).
CRF mRNA levels.
At the time point of 8 wk of age, PVN CRF mRNA levels were not affected by either strain or 4 d of voluntary exercise (Table 4). However, OLETF.S rats had decreased PVN CRF mRNA levels, compared with LETO.S controls by 20 wk of age (Table 4). Twelve weeks of voluntary exercise resulted in strain-specific modulation of CRF gene expression in the PVN [F (1,14) = 7.929, P = 0.014] (Table 4). Planned t comparisons revealed that 12 wk of voluntary exercise resulted in a 21% decrease in PVN CRF mRNA levels in LETO rats, whereas voluntary exercise elevated PVN CRF mRNA levels in OLETF rats (Table 4). These elevated levels of PVN CRF mRNA were reduced when running wheels were relocked for 6 wk (Table 4).
We noted that both LETO.RW and OLETF.RW rats had significantly elevated CRF mRNA levels in the DMH. This elevated CRF gene expression was specifically localized to the most dorsal part of the DMH, outside the compact subregion (Fig. 5B) and occurred at both the 4-d and 12-wk time points [(F [1,12] = 17.594, P = 0.001) and (F[1,1] = 49.213, P < 0.001), respectively] (Table 4). Although CRF mRNA levels was very low in the DMH of sedentary animals (Fig. 5A), voluntary exercise increased DMH CRF mRNA levels by 2-, 3.8-, and 5-fold in rats with 4 d, 6 wk, and 12 wk of voluntary exercise, respectively (Table 4). There was no significant difference in this elevation between LETO.RW and OLETF.RW rats. When running wheels were relocked for 4 d or 6 wk, the exercise-induced elevation of DMH CRF mRNA returned to basal levels (Table 4).
FIG. 5. In situ hybridization of CRF mRNA in the DMH with 35S-labeled CRF antisense riboprobe in rat brains at the end of the experiment (20 wk of age). A, DMH CRF mRNA expression was low in both sedentary rats. B, CRF mRNA expression was elevated in the DMH in response to access to running wheels. Arrows indicate CRF mRNA transcript signals.
Discussion
The current results demonstrate that running wheel activity normalizes food intake and prevents the obesity of OLETF rats. These changes in food intake and body weight are maintained as long as OLETF rats have access to running wheels. When wheels are relocked, OLETF rats temporarily increase their food intake and gain more weight. However, they stabilize at a lower level of body weight than sedentary OLETF rats. The findings on exercised-induced attenuation of weight gain in OLETF rats are similar to previous reports from Shima et al. (8, 11). We extended these findings with the demonstrations that voluntary exercise normalized altered feeding patterns and identified a variety of changes in hypothalamic gene expression that may underlie the effects of exercise on food intake and body weight in OLETF rats. Together, these data suggest that voluntary exercise activates hypothalamic pathways that overcome the deficits in CCK signaling that contribute to the hyperphagia and obesity in OLETF rats.
To identify how hypothalamic signaling systems involved in energy balance may underlie changes in food intake in response to exercise, we examined patterns of hypothalamic gene expression at multiple time points: 4 d after running wheel access, 4 d after relocking of the activity wheels after 6 wk of access, and at the end of the 12-wk experimental period. The data demonstrate that 4 d after initial running wheel access, a time that food intake is reduced, the major change in both LETO and OLETF rats was a significant elevation or induction of CRF mRNA expression in the DMH. mRNA levels for Arc NPY and POMC, DMH NPY, and PVN CRF were not changed in either group at this time point. These data suggest that increases in DMH CRF may mediate the short-term feeding inhibitory effects of exercise. Central CRF administration has been shown to decrease food intake and meal size and elevate locomotor activity (17, 18), and prior pharmacological experiments have identified a major role for CRF in exercise-induced anorexia (19). Administration of a CRF antagonist attenuates decreases in food intake in response to both voluntary (20) and forced exercise (19). The injection of a CRF antagonist further increased DMH CRF gene expression seen in voluntary exercised rats but did not affect PVN CRF gene expression (20). Moreover, although PVN CRF had been suggested as the major mediator of such effects (21), lesions of the PVN did not prevent anorectic effect of forced exercise (22). Thus, our current findings demonstrating significant elevations in DMH but not PVN CRF mRNA levels in both strains in response to exercise suggest that DMH CRF may be the site for such feeding inhibitory signals.
How DMH CRF and its signaling act in feeding control is not clear. DMH CRF neurons have been shown to project to the PVN (23). In addition, the activation of the DMH has been demonstrated to trigger the hypothalamic-pituitary-adrenal axis response to emotional or exteroceptive stress (24, 25, 26, 27). Hotta et al. (28) demonstrated that CRF receptor type 1 mediates emotional stress-induced inhibition of food intake and increase in locomotor activity in rats. Whether the mediation of running wheel access induced DMH CRF gene overexpression and the resulting inhibition of food intake is through the same signaling pathways as the DMH responding to emotional stress remains to be determined.
The exercise-induced changes in food intake and running activity in OLETF.RW rats are greater than those that occur in LETO.RW rats. In OLETF rats, overall intake was reduced by 50%, compared with a 20% reduction in LETO rats during the initial period of exercise. By 11 wk of age, OLETF.RW rats were hyperactive relative to LETO.RW rats and continued this higher activity until 16 wk of age. These differences may reflect a lowered CRFergic tone and resulting hypersensitivity to exercise-induced elevations in CRF expression and presumed release. Consistent with this idea, OLETF rats have been reported to exhibit an increased ACTH response to exogenous CRF administration (29).
LETO and OLETF rats had different patterns of hypothalamic gene expression that may explain the differences in their long-term responses to exercise. In LETO.RW rats, DMH CRF mRNA levels continued to increase, reaching levels that were 5-fold higher than sedentary LETO rats by 12 wk of exercise. This increase was accompanied by significant elevations in Arc NPY and POMC and DMH NPY and a reduction in PVN CRF mRNA levels. These latter findings are similar to those in previous reports showing that Arc and DMH NPY concentration are increased in exercising rats (30), hypothalamic POMC mRNA levels are elevated in rats maintained on a treadmill (31), and PVN CRF mRNA levels are decreased in mice in response to long-term voluntary exercise (32). Overall, such changes in ARC and DMH NPY and PVN CRF mRNA expression would stimulate food intake and may serve to balance an anorexic effects of increased Arc POMC and DMH CRF. Against this background, LETO rats maintain normal weight, gradually increasing their food intake to compensate for the energy demands of increased activity.
Although there was a similar increase in DMH CRF and Arc NPY in OLETF rats after 12 wk of exercise, PVN CRF was normalized and Arc POMC and DMH NPY mRNA levels were not altered. Thus, the increase in DMH CRF in OLETF rats is not balanced by increased DMH NPY or decreased PVN CRF, despite an elevation of Arc NPY mRNA expression. Against this background, food intake remains decreased and body weight is normalized to that of LETO controls. In contrast to pair-fed OLETF rats with normalized body weight that had elevated DMH NPY gene expression, we currently observe that exercised OLETF rats did not have such elevation, despite having normalized body weight. A role for DMH NPY in the regulation of energy balance has been suggested in our and other labs. DMH NPY expression is increased in response to lactation (33) and chronic food restriction (34). Moreover, there are several obese animal models in which elevated levels of DMH NPY mRNA expression have been noted. These include the lethal agouti yellow Ay, melanocortin 4 receptor knockout (35), tubby (36), diet-induced obese (37), and brown adipose tissue-deficient obese mice (38). We previously demonstrated that whereas pair feeding normalizes the obesity of OLETF rats, pair feeding results in a large increase (more than 8-fold) in DMH NPY mRNA levels, similar to levels found in young preobese OLETF rats (10). We have suggested that this elevated DMH NPY gene expression may be a direct result of a CCK signaling deficit and serve as a major contributing factor to the hyperphagia and obesity of OLETF rats (39). Consistent with this view, we have demonstrated that CCK normally plays a role in limiting DMH NPY gene expression. Local CCK administration lowers DMH NPY mRNA levels in intact rats (40). The effects of exercise may replace this lost CCK inhibitory influence on DMH NPY expression in OLETF rats limiting the elevation of DMH NPY gene expression, allowing the OLETF.RW rat to consume normal levels of food intake and maintain a much lower body weight.
Relocking the running wheels resulted in a period of increased food intake and body weight in OLETF rats. Four days after relocking the wheels, the exercised-induced elevation of DMH CRF mRNA levels were decreased to baseline, and Arc NPY mRNA expression was significantly elevated. Both of these changes would support increases in food intake and may contribute to the temporary hyperphagia after relocking the wheels. These data further support the proposed role for alterations in DMH CRF gene expression in the exercise-induced feeding inhibition.
During the 6 wk that running wheels were relocked, OLETF.relocked rats continued to maintain lower body weight than sedentary OLETF rats. At the end of the 6-wk period, mRNA expression for Arc and DMH NPY and DMH and PVN CRF were relatively normalized. However, Arc POMC gene expression was elevated compared with levels from sedentary OLETF rats. We previously demonstrated that Arc POMC mRNA expression is increased in ad libitum-fed OLETF rats and normalized by pair feeding and suggested that this elevation was in response to the increased body weight (10). The higher expression of Arc POMC mRNA in OLETF.relocked rats may account for their reduced body weight and may represent an exercise-induced alteration of body weight set point. Levin and Dunn-Meynell (4) suggested such an interpretation of an exercise induced lowered defended body weight in obesity prone rats.
Our finding of equivalent responses of DMH CRF gene expression to voluntary exercise in both strains suggests that the regulation of CRF gene expression in the DMH does not depend on CCK-ARs. The localization of CRF-expressing and CCK-AR-containing neurons in the DMH appears to be anatomically distinct. CRF gene expression was found in the dorsal part of the DMH in which CRF-containing neurons have been previously identified by immunohistochemistry (41). CCK-ARs are mainly found in the compact area of the DMH (40, 42, 43). This different distribution of DMH CRF and CCK-ARs suggests that the control of DMH CRF is independent of CCK signaling.
A role for leptin in the control of energy balance via down-regulating NPY and up-regulating POMC gene expression in the Arc has been documented (44, 45, 46). However, it is not clear that leptin plays a role in the effects of exercise on energy balance. The data demonstrating that voluntary exercise decreased plasma leptin levels as well as reduced food intake suggest that the feeding-inhibitory effects of exercise is not secondary to elevated leptin levels. Given that leptin receptors and CRF mRNA are expressed in different parts of the DMH (34, 47), it also seems unlikely that the regulation of DMH CRF is under the control of leptin. The alterations in Arc NPY and POMC mRNA levels in response to voluntary exercise or exercise withdrawal are also not consistent with leptin’s known actions. Thus, although plasma leptin levels were decreased in exercising LETO rats, Arc POMC mRNA levels were increased. When wheels were relocked and leptin levels were increasing, Arc NPY levels were elevated. These data suggest that exercise affects Arc gene expression through nonleptin pathways.
We found that plasma leptin levels were rapidly decreased after 4 d of running wheel access and rapidly increased within 4 d of relocking running wheels after 6 wk of exercise. By 4 d of running wheel access, although the body weight of OLETF.RW rats was heavier than that of LETO.RW animals, indicating more fat mass in OLETF rats, their leptin levels had dropped to those of the LETO.RW rats. Zheng et al. (48) reported that a single bout of exercise significantly decreased leptin mRNA levels in rat adipose tissue. Thus, exercise-induced reductions in plasma leptin levels seen in 4-d exercised OLETF and LETO rats may reflect lowered leptin mRNA levels but not decreased fat mass. Such an interpretation would be consistent with previous results demonstrating that reduction of circulating leptin concentration precedes fat loss from running exercise (49). When running wheels were relocked, plasma leptin levels were rapidly elevated. Together, these data suggest that acute exercise has a direct effect on plasma leptin levels.
Exercise decreased, and relocking the wheels increased, epididymal white fat mass. These findings are consistent with exercise stimulating lipolysis in adipose tissue, and the increased lipolysis results in decreased fat mass (50, 51). The changes in interscapular brown fat mass (BAT) in exercised OLETF rats are similar to white adipose tissue. In the absence of data on the activity of the brown fat in response to exercise, we cannot say whether this decrease in mass was due to an increase in BAT-mediated thermogenesis. Exercise has been variously shown to increase BAT thermogenesis (52), not alter BAT thermogenesis (53), or reduce BAT thermogenesis (54). Thus, the interpretation of the reduction in brown fat mass in rats with running wheel access remains to be determined.
In summary, the present results demonstrate that voluntary exercise prevents the hyperphagia and obesity of OLETF rats. The short-term exercise-induced anorexia found in both LETO and OLETF rats may be mediated via increases in DMH CRF expression, and this elevation of DMH CRF gene expression in the absence of compensatory changes in DMH NPY expression may play a critical role in the longer-term effects of exercise on energy balance in OLETF rats. Importantly, exercise appears to overcome the OLETF rat’s deficit in DMH NPY signaling, resulting in a significant and lasting attenuation of their hyperphagia and obesity.
Acknowledgments
The OLETF and LETO rats were a generous gift of the Tokushima Research Institute, Otsuka Pharmaceutical (Tokushima, Japan).
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