Energy Expenditure and Body Composition of Chronically Maintained Decerebrate Rats in the Fed and Fasted Condition
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《内分泌学杂志》
Department of Foods and Nutrition, Dawson Hall (R.B.S.H., E.W.K., W.P.P.), University of Georgia, Athens, Georgia 30602
Department of Biology & Center for Behavioral Neuroscience (T.J.B.), Georgia State University, Atlanta, Georgia 30302-4010
Department of Psychology (H.J.G.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
The contribution of the caudal brainstem to adaptation to starvation was tested using chronically maintained decerebrate (CD) and neurologically intact controls. All rats were gavage fed an amount of diet that maintained weight gain in controls. CD rats were subjected to a two-stage surgery to produce a complete transection of the neuroaxis at the mesodiencephalic juncture. One week later, the rats were housed in an indirect calorimeter, and 24 h energy expenditure was measured for 4 d. One half of each of the CD and control groups was then starved for 48 h. Fed CD rats maintained a lower body temperature (35 C), a similar energy expenditure per unit fat-free mass but an elevated respiratory quotient compared with controls. They gained less weight, had 20% less lean tissue, and had 60% more fat than controls. Circulating leptin, adiponectin, and insulin were elevated, glucose was normal, but testosterone was dramatically reduced. Responses to starvation were similar in CD and controls; they reduced energy expenditure, decreased respiratory quotient, indicating lipid utilization, defended body temperature, mobilized fat, decreased serum leptin and insulin, and regulated plasma glucose. These data clearly demonstrate that the isolated caudal brainstem is sufficient to mediate many aspects of the energetic response to starvation. In intact animals, these responses may be refined by a contribution by more rostral brain areas or by communication between fore- and hind-brain. In the absence of communication from the forebrain, the caudal brainstem is inadequate for maintenance of testosterone levels or lean tissue in fed or fasted animals.
Introduction
ANIMALS, INCLUDING humans, exposed to conditions of energy deficit make compensatory changes in efficiency of energy utilization and activity to conserve energy for essential functions and thereby maintain homeostasis (1). In addition to changing whole body energy expenditure, metabolism at the cellular level also is modified in response to activation of neural and hormonal regulatory systems that include down-regulation of sympathetic activity in major body organs such as the heart (2, 3), increased sympathetic outflow to white adipose tissue to mobilize lipid (4), inhibition of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes, and increased activity of the hypothalamic-pituitary-adrenal axis (5). Some of these metabolic changes result in the mobilization of endogenous carbohydrate and lipid stores to provide energy substrates for different organ systems and to protect body protein (6, 7).
The central mechanisms that orchestrate and integrate these responses to energy deficit have not been clearly delineated, with the majority of studies focused on the hypothalamus as a site that detects the energy deficit and then coordinates neural, physiological, and behavioral responses to the deficiency (8, 9, 10). Recent studies examining the increased hunger and food seeking behavior and the decreased energy expenditure that are apparent in conditions of food restriction or starvation have focused on the roles played by neuropeptides such as neuropeptide Y (NPY), and the melanocortins. In conditions of food deprivation, NPY protein concentration is increased in the arcuate nucleus, the paraventricular nucleus of the hypothalamus and the medial preoptic area of the hypothalamus (11). Exogenous NPY injected into the paraventricular nucleus region is associated with hyperphagia (12) and an inhibition of brown fat thermogenesis (13, 14) in rats. Gene expression of agouti-related protein, an endogenous antagonist of melanocortin receptors that is coexpressed with NPY in neurons of the arcuate nucleus, also is increased in conditions of food restriction (15). In addition, agouti-related protein contributes to sensations of hunger and may also decrease energy expenditure in this condition (16, 17). It has been suggested that the decrease in circulating concentrations of leptin that occurs rapidly during food deprivation (18) may be important in regulating the changes in arcuate nucleus gene expression (19) and many of the endocrine and metabolic responses to fasting (20, 21), although leptin does not appear to determine the decline in energy expenditure observed during short-term fasting in humans (21).
Despite the focus on hypothalamic nuclei as critical sites of regulation in the maintenance of energy balance, the caudal brainstem contains receptors for many of the neuropeptides that are known to be important in the control of food intake (see Ref.22 for review) and has been shown to be a principal component of the circuitry of sympathetic outflow to peripheral tissues (23, 24, 25). The chronic decerebrate (CD) rat model (26), in which the caudal brainstem is surgically isolated from the forebrain, has been critical in establishing feeding responses that are functional in the absence of neural input from the forebrain. Although these animals are not able to eat or drink spontaneously, studies in which food is infused directly into the oral cavity (intra-oral feeding) have allowed investigation of ingestive behavior (27). CD rats show normal acceptance and rejection responses to oral chemical stimulation (28) and demonstrate intact short-term regulation of meal size (see Ref.22 for review). In addition, CD rats show a robust sympathoadrenal hyperglycemia in response to 2-deoxyglucose treatment (29), indicating that the caudal brainstem is adequate for detection and neurogenic responses to glucoprivation. By contrast, several experiments examining the long-term regulation of energy intake fail to reveal similarities in the response of CD and control rats (30, 31) and thereby suggest that the isolated caudal brainstem is not sufficient for this aspect of energy balance control. The ability of these animals to regulate energy expenditure has, however, not previously been tested directly. Body temperature of CD rats is reduced, especially immediately after surgery, suggesting an inability to regulate heat production and/or retention (32) but, in contrast, fourth ventricle administration of the melanocortin receptor agonist MTII stimulates uncoupling protein I expression in intrascapular brown adipose tissue (IBAT) in CD rats (32), suggesting the potential for some caudal brainstem regulation of thermogenesis independently of the forebrain.
In this study, we measured energy expenditure of intact and CD rats in fed conditions and then evaluated their energetic response to 48 h of food deprivation using indirect calorimetry. The objective was to determine whether the CD rats could make the appropriate compensatory changes in energy expenditure and nutrient utilization in conditions of food deprivation, thus identifying the importance of the caudal brainstem in mediating metabolic responses to an energy deficit.
Materials and Methods
Two cohorts of 17 male Sprague Dawley rats (275–300 g; Harlan, Indianapolis, IN) were housed in individual cages in a room maintained at 23 C with lights on for 12 h each day from 0700 h. All of the experimental procedures described here were approved by the Institutional Animal Care and Use Committee of the University of Georgia and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Initially, all of the rats had free access to rodent chow (Rodent Chow 5001, Purina Mills, St. Louis, MO) and water for 1 wk of adaptation. They were then tube fed a suspendible AIN 76A rodent diet (L1001, Research Diets, New Brunswick, NJ) in three meals each day fed at 0700, 1300, and 1900 h. The volume of tube-fed meals was gradually increased from 9 to 12 ml over 4 d to deliver a total of 79 kcal/d. This volume of food maintained weight gain in control rats and also provided an adequate daily water intake for the animals.
Body weights were recorded for 1 wk and then the rats were divided into two weight-matched groups: Control (n = 14) and CD (n = 20). The CD rats were anesthetized with ketamine/xylazine given as an ip injection (90 mg/kg ketamine, 10 mg/kg xylazine). Each of these animals was then placed in a stereotaxic apparatus, a midline incision was made in the skin to expose the skull, and a fine cut was made across the skull at a distance 40% from bregma to lambda. A hemisection of the neuraxis was made at the mesodiencephalic juncture using a hand-held specially designed, blunt L-shaped spatula. The skin incision was closed. Each rat received a sc injection of ketoprofen (2 mg/kg) and was allowed to recover for 1 wk before the procedure was repeated to produce a complete sectioning of the neuraxis. This procedure has been described in detail previously (33). At the time of the second surgery on the CD rats, the control rats also were anesthetized but were not subjected to any surgical procedures. A total of 13 CD rats survived the two surgeries. After the second surgery, the rectal temperatures of CD rats were recorded with a thermistor probe (Temp 4, Thermistor Thermometer, Cole Parmer Instrument Co., Vernon Hills, IL) each time that they were fed. If body temperature fell to below 34 C, the rats were placed on a warming pad until temperature recovered. A low body temperature was most common at 0700 h (lights on) in the days immediately after the second surgery, but within 5 d of the surgery all of the CD rats were maintaining their temperature above 34 C at all times that it was measured.
One week after the second surgery, the rats were housed in an indirect calorimetry system that has been described in detail previously (34, 35). The temperature, light cycles, and feeding schedule were maintained as before. The system measured O2 consumption and CO2 production from each cage once every 20 min for 24 h a day except when the cages were cleaned between 1230 h and 1300 h. This coincided with the second tube-fed meal of the day. The feeding of each rat for the first and last meal of the day was timed in such a way as to avoid losing any measures of energy expenditure. The rats were housed in the calorimeter for 24 h before any data were collected. Baseline measures of expenditure and respiratory quotient (RQ) were then recorded for 4 d. At the end of the baseline period, half of the controls and half of the CD rats were deprived of food for 48 h before they were killed. The starved rats were gavaged with 9 ml of water at each feeding time. Rectal temperatures of all CD rats were recorded at each feeding time, and temperatures of both control and CD rats were recorded at the last three feeding times of the study.
Rats were decapitated and trunk blood was collected for subsequent measurement of serum glucose (Ascesion Elite blood glucose strips; Bayer Corp., Mishawaka, IN), free fatty acids (FFA: NEFA C kit; Wako Chemicals, Richmond, VA), glycerol (free glycerol reagent F6428; Sigma-Aldrich, St. Louis, MO), triglycerides (L-Type TG H kit; Wako Chemicals), leptin (rat leptin RIA kit, Linco Research Inc., St. Charles, MO), insulin (rat insulin RIA kit; Linco Research), adiponectin (mouse adiponectin RIA kit; Linco Research), testosterone (testosterone RIA kit; Diagnostic Systems Laboratories, Webster, TX), T3 (total T3 RIA kit: Diagnostic Systems Laboratories), and corticosterone (DA Corticosterone 125I kit; MP Biomedicals, Orangeburg, NY). Inguinal (Ing), epididymal (Epi), and mesenteric (Mes) white fat and IBAT were dissected and weighed. Small (50 mg) samples of Epi and Ing fat were fixed in osmium tetroxide for measurement of cell number and size distribution by Coulter Counter and Channelizer (Beckman Coulter, Inc., Fullerton, CA), as described previously (36). The IBAT was snap frozen and stored at –80 C. Portions of the brown fat were analyzed for norepinephrine content by HPLC as described previously (37). Total RNA also was extracted using TriZol reagent (Invitrogen, Carlsbad, CA) and real-time PCR was used to measure uncoupling protein 1 (UCP1) mRNA expression, as described below. The carcasses, less gastrointestinal tract, were analyzed for body composition, as described previously (38). Brains of CD rats were examined histologically to confirm the completeness of the transection.
Statistical analysis of data was performed using Statistica software (StatSoft, Tulsa, OK). Daily body weights, energy expenditure, and RQ measured across 24 h and cell size distribution were compared by repeated measures ANOVA. Measures at specific time points were analyzed by post hoc Duncan’s multiple range test. Measures of body composition, fat pad weight, serum metabolite, and hormone concentrations and tissue mRNA expression were compared by two-way ANOVA and post hoc Duncan’s multiple range test. Differences were considered significant at P < 0.05, but in the interest of space and clarity the details of each analysis are not included in the text.
Real-time quantitative PCR (Q-PCR)
After total RNA extraction, 1 μg of RNA was treated with deoxyribonuclease I (Invitrogen) and incubated at room temperature for 15 min. Deoxyribonuclease I was inactivated by addition of 1 μl of 25 mM EDTA and heating at 65 C for 10 min. cDNA was prepared from 500 ng total RNA by RT (Promega Reverse Transcription System; Promega Corp., Madison, WI). A negative control containing no avian myeloblastosis virus reverse transcriptase was used to ensure specificity of the PCR amplification. After RT, cDNA was treated with ribonuclease H (Invitrogen). First-strand cDNA was then diluted to 50 μl using nuclease-free water, yielding a concentration of 25 ng/μl. PCR primers were designed using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and purchased from Invitrogen. Q-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol using the Bio-Rad iCycler iQ system. Amplification conditions were as follows: initial denaturation, 95 C for 3 min; 40 cycles of 95 C for 30 sec, 58 C for 30 sec, and 72 C for 30 sec. Melt curve analysis was performed immediately after Q-PCR amplification. Triplicate determinations of UCP1 mRNA and 18S rRNA expression were made for each sample. The primer sequences were: 18s forward primer ACG GAA GGG CAC CAC CAG GA, 18s reverse primer CAC CAC CAC CCA CGG AAT CG, UCP1 forward primer TCC CTC AGG ATT GGC CTC TAC, UCP-1 reverse primer GTC ATC AAG CCA GCC GAG AT. UCP1 mRNA expression was calculated using the comparative threshold (CT) method (http://www.dorak.info/genetics/realtime.html), calculating the difference between threshold cycle for 18S and UCP1, therefore, the greater the value of CT, the lower the expression of UCP1. The average CT for control-fed rats was designated a value of 1.0, and the values of the other groups are expressed in relation to this arbitrary value.
Results
Histology confirmed complete transection in all of the CD rats. A representative midsaggital section depicting the lesion is shown in Fig. 1. The liquid diet regimen maintained a weight gain of 2.9 ± 0.1 g/d in control rats (Fig. 2A). Immediately after the first surgical procedure, weight gain was inhibited in CD rats (0.8 ± 0.2 g/d) even though they were fed the same amount of food as the controls. After the second surgery, the rectal temperature of CD rats fell to approximately 34 C (Fig. 2B). After about 5 d, rectal temperature was maintained at approximately 35 C. As seen in Fig. 2B, temperature of CD rats increased at successive meals during each day and then fell each night during the 12 h when they received no food. Energy expenditure, expressed on a per animal basis, was significantly lower in CD than control rats at all times of the day (Fig. 3A: CD, P < 0.0001; Time, P < 0.0001; Int, P < 0.0001). Control rats showed an increased rate of expenditure at the start of the dark period, but this increase was muted in CD rats. The energy expenditure of both CD and control rats declined progressively during the dark phase when there was the longest interval between tube-fed meals. RQ was significantly higher in CD than control rats (Fig. 3B: CD, P < 0.0001; Time, P < 0.0001; Int, P < 0.0001) at all times except the last 3 h of the dark period. During the light period and the first half of the dark period, the RQ of CD rats was greater than 1.0, indicative of lipid deposition, whereas the control rats maintained an RQ of about 1.0, which is typical for carbohydrate oxidation. During the dark period, when there was a 12-h interval between meals, the RQ of all of the rats declined to about 0.8, indicating a high rate of fatty acid and/or protein oxidation (40). Average 24-h energy expenditure and RQ for control and CD rats is shown in Fig. 3C and illustrates the decreased expenditure but increased RQ for CD rats compared with controls when it was expressed on a per animal basis. When expenditure was expressed per unit fat-free mass, there was no longer any effect of decerebration on energy expenditure, although a reduction due to starvation was still apparent (see Fig. 6C; P < 0.065).
Both Control and CD rats lost weight in response to 48 h of starvation (Table 1). Body composition measured at the end of the experiment indicated that although CD rats were lighter than controls they were significantly fatter and had less lean tissue (protein plus water). The increase in carcass fat of CD rats was associated with an increase in the size of all of the depots measured, although the difference did not reach statistical significance for Epi depots (P < 0.059; Fig. 4), and this was due to an increase in adipocyte size rather than number (Fig. 5). The weight loss caused by starvation was predominantly water in the controls but was mainly fat in the CD rats (Table 1). Mes and Ing fat but not Epi fat showed a significant decrease in size in the starved CD rats, and this was associated with a reduction in cell size rather than number (Fig. 5). Although there was some decrease in the size of white fat depots in starved controls, none of the differences were statistically significant (P < 0.1). Energy expenditure of both control and CD rats was decreased during the 48 h of starvation (Fig. 6, A and B: CD, P < 0.001; Starvation, P < 0.0001; Time, P < 0.0001; CD x Starvation, NS; CD x Time, P < 0.0001; Starvation x Time, P < 0.0001; CD x Starvation x Time, NS). The starved control rats continued to show an increase in expenditure at the start of the dark period, whereas there was no effect of the dark phase on expenditure of the starved CD rats. Figure 6C shows average 24 h expenditure during the 48 h of starvation, and the significant inhibition of expenditure in both control and CD rats when expressed per rat but no difference between groups if expenditure was expressed based on fat-free mass (Starvation, P < 0.065). The RQ of both control and CD rats fell to 0.8 after about 10 h of food deprivation, indicating that both groups of rats were using lipid and/or protein (Fig. 7A: CD, P < 0.0001; Starvation, P < 0.0001; Time, P < 0.0001; CD x Starvation, P < 0.0001; CD x Time, P < 0.0001; Starvation x Time, P < 0.0001; CD x Starvation x Time, P < 0.0001). Average 48-h RQ was significantly increased for fed CD rats compared with fed controls, but it was low and not different between the groups of starved rats. Despite the reduction in energy expenditure, both the control and CD starved rats maintained the same body temperature as their fed counterparts (Fig. 8). IBAT weight was significantly increased in fed CD rats compared with fed controls, but it was the same weight in starved controls and starved CD rats (Table 2). The norepinephrine content of IBAT was reduced in fed CD rats compared with controls and was not changed by starvation in either group of rats (Table 2). There were no differences in UCP1 mRNA expression (Table 2).
Starvation caused a significant drop in blood glucose concentrations for both control and CD rats, but there was no significant effect of decerebration on glucose in either fed or starved conditions (Table 1). Similarly, there were no significant differences in serum FFA, triglyceride, or glycerol concentrations between fed control and CD rats. Starvation caused a significant decrease in circulating triglycerides and a significant increase in FFA in all rats but had no effect on glycerol concentrations. In contrast, the obese, fed CD rats were hyperinsulinemic, hyperadiponectinemic, and hyperleptinemic compared with fed controls. Starvation caused a significant fall in the concentration of both insulin and leptin in CD rats so that there was no difference in either leptin or insulin concentrations of starved CD and control rats, but adiponectin remained elevated in the CD rats. There was no effect of either decerebration or starvation on serum concentrations of corticosterone. Total T3 was not different between control and CD rats but was decreased by starvation in both groups. Serum testosterone was extremely low in CD rats, being undetectable in eight of the 13 animals. There was no effect of starvation on testosterone concentrations in control rats.
Discussion
This is the first study to directly evaluate the long-term regulation of energy balance in CD rats by exposing them to an energy deficit caused by 2 d of starvation. The measurement of energy expenditure and body composition of fed CD rats also provides new information on the metabolic state of rats that are dependent only upon the caudal brainstem for the neural control of physiologic function under normal and energetically challenging conditions. Although we assume that regulatory responses observed in CD rats are mediated by the brainstem, we cannot exclude the possibility of communication and control by humoral factors initiated in the forebrain. In order for the forebrain to respond to a change in energetic conditions, however, it also would have to be responding to humoral signals because rostral coursing projections from peripheral afferents and from the caudal brainstem itself would have been destroyed. In this experiment, fed CD rats had a lower body temperature, a lower energy expenditure, a higher RQ, markedly less lean body mass and more body fat compared with fed intact controls. Fed CD rats also had elevated circulating concentrations of leptin, adiponectin, and insulin but had normal blood glucose, corticosterone, and total T3 concentrations compared with fed intact controls. In contrast, circulating concentrations of testosterone were extremely low. The lower energy expenditure of these rats was associated with a reduced fat-free mass and an apparent decrease in voluntary activity. The CD rats did not exhibit exploratory behavior, as described previously (41), and they did not show the typical increase in expenditure associated with handling and feeding. This is particularly apparent in Fig. 6, A and B, during the period of starvation, when there was a significant elevation in expenditure of the starved control rats as they were removed from their cages and handled to be tube-fed water. In contrast, the CD rats showed no elevation in expenditure in response to these stimuli. There were similar differences in 24-h energy expenditure of the two treatment groups during the period of starvation at the end of the study when the data were expressed on a per rat basis, but these differences disappeared if energy expenditure was expressed per unit fat-free mass. Therefore, the reduced lean body mass had a major impact on the heat production of the CD rats. Despite using a similar amount of energy per unit fat-free mass and using less energy for activity, the rats were maintaining a reduced body temperature, implying that they were using energy inefficiently and unable to retain heat effectively.
We compared expenditure on a per rat basis because the large differences in body composition of the two treatment groups did not justify the use of kg0.75 as a unit of metabolic body size. Immediately after the first surgery, the rate of weight gain of CD rats was inhibited, and this slow rate of gain was maintained throughout the experiment suggesting that the failure to retain body protein may have started after only a partial sectioning of the brain because accumulation of body energy as fat represents a smaller gain in body weight than deposition of an equivalent amount of energy as lean tissue, which includes an obligatory significant amount of water. A similar loss of lean mass has been reported for rats with lesions of the lateral hypothalamus (42), although it is not known whether the loss of lean tissue in these two conditions is caused by the same mechanism. Because releasing hormones were not measured in this study, it is not known whether the pituitary was functioning normally; however, the observation that serum corticosterone and total T3 were the same in control and CD rats suggests that at least some aspects of pituitary function do not rely on input from the forebrain. By contrast, circulating concentrations of testosterone were significantly changed in the absence of neural communication between the forebrain and caudal brainstem. The loss of lean mass demonstrates a critical role for higher brain areas in regulating protein retention and accretion, either directly or through control of growth factors. Further studies are needed to demonstrate both the degree of brain function that is needed to maintain normal protein metabolism and the deficits in metabolism that cause the rapid and substantial reduction in lean tissue of CD rats. One such factor that is known to affect lean body mass, especially protein, that is markedly reduced in CD rats is serum testosterone. Loss of testosterone due to castration triggers a decrease in body weight in rats (43), accompanied by decreases in carcass protein and increases in carcass lipid that are reversible with testosterone administration (44). Therefore, it seems likely that a significant portion of the decrease in lean body mass and some of the increased fat accretion in CD rats resulted from the functional castration that was initiated by decerebration. A second factor that contributed to the increased adiposity of the CD rats was that they were essentially overfed. Control and CD rats were tube fed the same daily energy intake even though the CD rats had a significantly lower energy expenditure. It is likely that overfeeding accounted for their elevated RQ of greater than 1.0, which is indicative of energy storage and has been used as a marker for overfeeding (45); however, because the rats were being overfed it is even more surprising that they were not able to at least maintain their lean body mass.
All of the rats in this study received their daily food and water intake in three tube-fed meals each day that were adequate for maintenance of a normal rate of growth for the controls. Meals were delivered over a 12-h period, which meant that the rats were subjected to 12 h without food during the dark cycle. By the end of this period, all of the rats had an RQ of approximately 0.8, a value that is indicative of food deprivation (45) and that fatty acids or protein are being oxidized (40). There was no difference in RQ of CD and intact controls at this time, suggesting that the high RQ of CD rats during the light phase was due to an excess energy intake, rather than an inability to oxidize fat or carbohydrate. The energy expenditure of fed CD rats appeared to decline more during the dark phase than that of the fed control rats (at 1030 h, Control = 0.97 ± 0.03, CD = 0.89 ± 0.03; at 0630 h, Control = 0.86 ± 0.03, CD = 0.56 ± 0.03 kcal/rat·h) and body temperatures of CD rats were at their nadir before the first meal of the day, suggesting a possible deficit in their ability to regulate energy utilization and/or partitioning. After the first meal of the day, the body temperatures of the CD rats increased and their RQ almost immediately went above 1.0, indicating that although some metabolic heat was trapped and raised body temperature, a large portion of the ingested energy was stored as fat. In contrast, the RQ of the control rats remained around 1.0 after the first meal, and they showed a more pronounced increase in energy expenditure after the meal due to either nutrient processing and utilization (46) or to the thermic effect of food (47).
Because of the increased adiposity of CD rats, it is impossible to determine whether their hyperinsulinemia and hyperleptinemia were secondary to obesity or were the result of a lack of central control of pancreatic function or adipose leptin expression. The normal circulating concentrations of glucose indicate that the rats were insensitive to insulin but were not insulin resistant. Again, this may have been secondary to the overfeeding and adiposity rather than a direct result of loss of neural control by the forebrain. The elevated levels of adiponectin in fed CD rats was surprising because this adipokine usually is decreased in conditions of obesity and diabetes (48) and increased with weight loss (49). The high concentrations of adiponectin in CD rats is, however, consistent with a recent report that resting metabolic rate is a strong predictor of adiponectin concentration in humans and that a low resting metabolic rate is associated with elevated adiponectin (50). Others have reported that stimulation of -adrenergic receptors inhibits adiponectin production by 3T3L1 cells (51), and it is possible that the high adiponectin levels in the CD rats was due to reduced sympathetic tone on white adipose tissue and that the low metabolic rate is partially due to low sympathetic tone in other major organs.
When CD rats were subjected to 48 h of starvation, they appeared to make essentially the same energetic and hormonal responses to the challenge that were made by intact, starved control rats. Even though the daily expenditure of the CD rats already was lower than that of the controls, both the CD and control rats reduced their daily energy expenditure. Despite this low energy expenditure, the CD rats maintained body temperature at the same level as during feeding, demonstrating that the isolated caudal brainstem is sufficient for regulating body temperature in both the fed and fasted state, albeit at a less than optimal level. RQ declined rapidly in both groups of animals to approximately 0.8, indicating that they were oxidizing fatty acids and/or protein for energy. The CD rats had no obvious difficulty in mobilizing lipid from their white fat depots, whereas there were only small, nonsignificant changes in the fat content of starved control rats. Because energy expenditure expressed per unit fat-free mass was not different between the groups, and because expenditure per rat was lower in CD than control rats, it is difficult to explain how they could have mobilized more fat than controls. It is unlikely that the CD rats were able to breakdown triglycerides but not oxidize the FFA because circulating FFA were similar in the two groups. One possible explanation for this apparent anomaly of energy balance is that the difference in fat between fed and fasted CD rats was due in part to continued accumulation of body fat in the fed CD rats during the 48-h starvation period, which exaggerated the amount of fat that appeared to be mobilized in response to starvation.
The difference in carcass fat content of fed and starved rats was fat depot specific because both Ing and Mes pads were reduced by starvation, whereas the weight of the Epi pads did not change. The Epi pads in rats show a dampened lipid mobilization during fasting compared with other fat pads (52), and this may be due to reduced sympathetic drive on this fat depot in response to energetic challenges (53). The obesity of the fed CD rats was associated with a significant increase in the size of adipocytes rather than an increase in cell number and the loss of fat during starvation was associated with a reduction in cell size, rather than number. It is tempting to speculate that one of the factors contributing to adiposity in fed CD rats was low sympathetic tone in white adipose tissue; however, enlargement of white fat depots after denervation has been attributed to an increase in cell number rather than size (54, 55). In addition, if lipid mobilization during starvation is predominantly mediated by increased sympathetic drive (56, 57), then it is surprising that the CD rats were able to mobilize fat so effectively unless brainstem circuits are sufficient for this response. Although lipid mobilization in response to fasting is largely independent of adrenal medullary catecholamines (58), we did not measure circulating concentrations of catecholamines in this study. Others have reported that the lipolytic response to fasting is not impaired in mice that are deficient in -adrenoceptors (59); therefore, it also is possible that factors such as the dramatic change in insulin (60) or potential increase in glucagons (61) promoted fatty acid mobilization and oxidation in the starved CD rats.
Similar to humans and intact rodents (18, 62), the starved CD rats showed a rapid and exaggerated fall in serum leptin concentration. The dramatic reductions in insulin and leptin in starved compared with fed CD rats suggests that the hyperleptinemia and hyperinsulinemia were associated with overfeeding, rather than adiposity per se, because the starved CD rats were still significantly fatter than fed controls. It has been hypothesized that the substantial fall in circulating concentrations of leptin that occur during the early stages of food deprivation signals the hypothalamus to initiate a coordinated response to the energy deficit (20, 21). The results from this study demonstrate that efferent neural signals from the hypothalamus or other areas of the forebrain are not required for the fall in energy expenditure, or regulation of metabolism to maintain blood glucose or body temperature during conditions of energy deficit. The responses seen in CDs must be initiated by signals that can be received, integrated, and responded to by circuits endemic to the caudal brainstem and is consistent with a previous report that the caudal brainstem is adequate for sensing and responding to the metabolic challenge produced by 2-deoxyglucose administration (29). The specific sites responsible for this remain to be defined, but there is ample evidence that multiple nuclei in the brainstem can make a significant contribution to different aspects of the regulation of energy balance (see Refs.22 and 63 for review).
In many animals, BAT is an important site of thermogenesis that is primarily under control of the sympathetic nervous system. The IBAT in fed CD rats from this study had a significantly lower norepinephrine (NE) content, compared with fed intact controls, indicative of a reduced level of activation and heat production, but there was no difference in UCP1 mRNA levels between groups. If the NE content of the tissue can be considered an indirect indicator of sympathetic activity in the tissue, then reduced activation of IBAT in CD rats may have contributed to the lower daily energy expenditure of the CD rats compared with controls. A previous study has shown that stimulation of melanocortin receptors in the brainstem of CD rats causes a significant increase in IBAT UCP1 mRNA expression (32). The observation that tissue NE content was low in animals in this study may indicate that the maintenance of basal sympathetic tone to IBAT requires input from the forebrain. There was no significant effect of starvation on IBAT NE or UCP1 mRNA content, although it would be anticipated that both of these factors would decrease as energy was conserved. It is possible that these changes would have been observed, at least in control rats, if the period of deprivation had been extended.
The objective of this study was to determine whether CD rats were able to make appropriate adaptations to a period of starvation. The results demonstrate that functional neural control by the caudal brainstem is sufficient for rats to respond to an energy deficit by reducing energy expenditure and increasing fatty acid oxidation to maintain body temperature. In normal animals, metabolic changes during the early stages of starvation protect lean body mass (7); in CD rats, however, there appears to be a severe disruption of the systems regulating protein metabolism even during conditions of energy excess, and this may largely be because the animals are functionally castrated, having undetectable levels of testosterone. During starvation, however, CD rats rely predominantly on lipid mobilization for energy. Measures in fed CD rats indicate that more rostral brain areas are required for the rats to maintain an appropriate lean body mass, metabolic rate, and body temperature. Energy expenditure associated with activity also seems to be diminished due to a failure to respond to environmental stimuli. The importance of these factors in maintaining homeostasis in baseline conditions in CD rats remains to be determined. For example, the reduced body temperature may promote accumulation of body fat when the energy intake of the rats is fixed by tube feeding, and, as noted above, the substantial decline in circulating testosterone undoubtedly contributed to the failure to maintain lean body mass. Over the long-term, reduced voluntary activity of the animals also may contribute to a loss of lean tissue. The impact of these impairments on the response to starvation, however, appeared minimal because the CD rats showed the same energetic responses to 48 h of food deprivation as the intact controls. The reduction in energy expenditure associated with a low body temperature and reduced voluntary activity may, in fact, be beneficial in conditions of energy deficit. It is well established that testosterone concentrations in intact rats are reduced by about 80% after 4 d of starvation (39, 64); therefore, it is unlikely that the preexisting hypogonadism of CD rats in this study influenced their response to starvation. The testosterone concentrations in intact control rats in this experiment decreased by about 30% after 2 d of starvation, but this difference was not statistically significant. Thus, it appears that although the caudal brainstem is adequate for making gross responses to energetic challenges such as starvation, input from the forebrain is needed to refine these responses and to maintain an optimal physiologic and hormonal environment under baseline conditions when energy intake is adequate.
Acknowledgments
The authors thank Joyce Power and Jessica Davenport at University of Georgia for their expert technical assistance, Kate Nautiyal at University of Pennsylvania for her technical advice, and Dr. Kay Song, Georgia State University for photomicroscopy.
Footnotes
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants SCRO DK-21397 (awarded to H.J.G., T.J.B., and R.B.S.H.), R01 DK-53903 (awarded to R.B.S.H.), and R01 DK-35254 (awarded to T.J.B.).
First Published Online December 15, 2005
Abbreviations: CD, Chronic decerebrate; CT, comparative threshold; Epi, epididymal; IBAT, intrascapular brown adipose tissue; Ing, inguinal; NE, norepinephrine; NPY, neuropeptide Y; Q-PCR, quantitative PCR; RQ, respiratory quotient; UCP1, uncoupling protein 1.
Accepted for publication December 5, 2005.
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Department of Biology & Center for Behavioral Neuroscience (T.J.B.), Georgia State University, Atlanta, Georgia 30302-4010
Department of Psychology (H.J.G.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
The contribution of the caudal brainstem to adaptation to starvation was tested using chronically maintained decerebrate (CD) and neurologically intact controls. All rats were gavage fed an amount of diet that maintained weight gain in controls. CD rats were subjected to a two-stage surgery to produce a complete transection of the neuroaxis at the mesodiencephalic juncture. One week later, the rats were housed in an indirect calorimeter, and 24 h energy expenditure was measured for 4 d. One half of each of the CD and control groups was then starved for 48 h. Fed CD rats maintained a lower body temperature (35 C), a similar energy expenditure per unit fat-free mass but an elevated respiratory quotient compared with controls. They gained less weight, had 20% less lean tissue, and had 60% more fat than controls. Circulating leptin, adiponectin, and insulin were elevated, glucose was normal, but testosterone was dramatically reduced. Responses to starvation were similar in CD and controls; they reduced energy expenditure, decreased respiratory quotient, indicating lipid utilization, defended body temperature, mobilized fat, decreased serum leptin and insulin, and regulated plasma glucose. These data clearly demonstrate that the isolated caudal brainstem is sufficient to mediate many aspects of the energetic response to starvation. In intact animals, these responses may be refined by a contribution by more rostral brain areas or by communication between fore- and hind-brain. In the absence of communication from the forebrain, the caudal brainstem is inadequate for maintenance of testosterone levels or lean tissue in fed or fasted animals.
Introduction
ANIMALS, INCLUDING humans, exposed to conditions of energy deficit make compensatory changes in efficiency of energy utilization and activity to conserve energy for essential functions and thereby maintain homeostasis (1). In addition to changing whole body energy expenditure, metabolism at the cellular level also is modified in response to activation of neural and hormonal regulatory systems that include down-regulation of sympathetic activity in major body organs such as the heart (2, 3), increased sympathetic outflow to white adipose tissue to mobilize lipid (4), inhibition of the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes, and increased activity of the hypothalamic-pituitary-adrenal axis (5). Some of these metabolic changes result in the mobilization of endogenous carbohydrate and lipid stores to provide energy substrates for different organ systems and to protect body protein (6, 7).
The central mechanisms that orchestrate and integrate these responses to energy deficit have not been clearly delineated, with the majority of studies focused on the hypothalamus as a site that detects the energy deficit and then coordinates neural, physiological, and behavioral responses to the deficiency (8, 9, 10). Recent studies examining the increased hunger and food seeking behavior and the decreased energy expenditure that are apparent in conditions of food restriction or starvation have focused on the roles played by neuropeptides such as neuropeptide Y (NPY), and the melanocortins. In conditions of food deprivation, NPY protein concentration is increased in the arcuate nucleus, the paraventricular nucleus of the hypothalamus and the medial preoptic area of the hypothalamus (11). Exogenous NPY injected into the paraventricular nucleus region is associated with hyperphagia (12) and an inhibition of brown fat thermogenesis (13, 14) in rats. Gene expression of agouti-related protein, an endogenous antagonist of melanocortin receptors that is coexpressed with NPY in neurons of the arcuate nucleus, also is increased in conditions of food restriction (15). In addition, agouti-related protein contributes to sensations of hunger and may also decrease energy expenditure in this condition (16, 17). It has been suggested that the decrease in circulating concentrations of leptin that occurs rapidly during food deprivation (18) may be important in regulating the changes in arcuate nucleus gene expression (19) and many of the endocrine and metabolic responses to fasting (20, 21), although leptin does not appear to determine the decline in energy expenditure observed during short-term fasting in humans (21).
Despite the focus on hypothalamic nuclei as critical sites of regulation in the maintenance of energy balance, the caudal brainstem contains receptors for many of the neuropeptides that are known to be important in the control of food intake (see Ref.22 for review) and has been shown to be a principal component of the circuitry of sympathetic outflow to peripheral tissues (23, 24, 25). The chronic decerebrate (CD) rat model (26), in which the caudal brainstem is surgically isolated from the forebrain, has been critical in establishing feeding responses that are functional in the absence of neural input from the forebrain. Although these animals are not able to eat or drink spontaneously, studies in which food is infused directly into the oral cavity (intra-oral feeding) have allowed investigation of ingestive behavior (27). CD rats show normal acceptance and rejection responses to oral chemical stimulation (28) and demonstrate intact short-term regulation of meal size (see Ref.22 for review). In addition, CD rats show a robust sympathoadrenal hyperglycemia in response to 2-deoxyglucose treatment (29), indicating that the caudal brainstem is adequate for detection and neurogenic responses to glucoprivation. By contrast, several experiments examining the long-term regulation of energy intake fail to reveal similarities in the response of CD and control rats (30, 31) and thereby suggest that the isolated caudal brainstem is not sufficient for this aspect of energy balance control. The ability of these animals to regulate energy expenditure has, however, not previously been tested directly. Body temperature of CD rats is reduced, especially immediately after surgery, suggesting an inability to regulate heat production and/or retention (32) but, in contrast, fourth ventricle administration of the melanocortin receptor agonist MTII stimulates uncoupling protein I expression in intrascapular brown adipose tissue (IBAT) in CD rats (32), suggesting the potential for some caudal brainstem regulation of thermogenesis independently of the forebrain.
In this study, we measured energy expenditure of intact and CD rats in fed conditions and then evaluated their energetic response to 48 h of food deprivation using indirect calorimetry. The objective was to determine whether the CD rats could make the appropriate compensatory changes in energy expenditure and nutrient utilization in conditions of food deprivation, thus identifying the importance of the caudal brainstem in mediating metabolic responses to an energy deficit.
Materials and Methods
Two cohorts of 17 male Sprague Dawley rats (275–300 g; Harlan, Indianapolis, IN) were housed in individual cages in a room maintained at 23 C with lights on for 12 h each day from 0700 h. All of the experimental procedures described here were approved by the Institutional Animal Care and Use Committee of the University of Georgia and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Initially, all of the rats had free access to rodent chow (Rodent Chow 5001, Purina Mills, St. Louis, MO) and water for 1 wk of adaptation. They were then tube fed a suspendible AIN 76A rodent diet (L1001, Research Diets, New Brunswick, NJ) in three meals each day fed at 0700, 1300, and 1900 h. The volume of tube-fed meals was gradually increased from 9 to 12 ml over 4 d to deliver a total of 79 kcal/d. This volume of food maintained weight gain in control rats and also provided an adequate daily water intake for the animals.
Body weights were recorded for 1 wk and then the rats were divided into two weight-matched groups: Control (n = 14) and CD (n = 20). The CD rats were anesthetized with ketamine/xylazine given as an ip injection (90 mg/kg ketamine, 10 mg/kg xylazine). Each of these animals was then placed in a stereotaxic apparatus, a midline incision was made in the skin to expose the skull, and a fine cut was made across the skull at a distance 40% from bregma to lambda. A hemisection of the neuraxis was made at the mesodiencephalic juncture using a hand-held specially designed, blunt L-shaped spatula. The skin incision was closed. Each rat received a sc injection of ketoprofen (2 mg/kg) and was allowed to recover for 1 wk before the procedure was repeated to produce a complete sectioning of the neuraxis. This procedure has been described in detail previously (33). At the time of the second surgery on the CD rats, the control rats also were anesthetized but were not subjected to any surgical procedures. A total of 13 CD rats survived the two surgeries. After the second surgery, the rectal temperatures of CD rats were recorded with a thermistor probe (Temp 4, Thermistor Thermometer, Cole Parmer Instrument Co., Vernon Hills, IL) each time that they were fed. If body temperature fell to below 34 C, the rats were placed on a warming pad until temperature recovered. A low body temperature was most common at 0700 h (lights on) in the days immediately after the second surgery, but within 5 d of the surgery all of the CD rats were maintaining their temperature above 34 C at all times that it was measured.
One week after the second surgery, the rats were housed in an indirect calorimetry system that has been described in detail previously (34, 35). The temperature, light cycles, and feeding schedule were maintained as before. The system measured O2 consumption and CO2 production from each cage once every 20 min for 24 h a day except when the cages were cleaned between 1230 h and 1300 h. This coincided with the second tube-fed meal of the day. The feeding of each rat for the first and last meal of the day was timed in such a way as to avoid losing any measures of energy expenditure. The rats were housed in the calorimeter for 24 h before any data were collected. Baseline measures of expenditure and respiratory quotient (RQ) were then recorded for 4 d. At the end of the baseline period, half of the controls and half of the CD rats were deprived of food for 48 h before they were killed. The starved rats were gavaged with 9 ml of water at each feeding time. Rectal temperatures of all CD rats were recorded at each feeding time, and temperatures of both control and CD rats were recorded at the last three feeding times of the study.
Rats were decapitated and trunk blood was collected for subsequent measurement of serum glucose (Ascesion Elite blood glucose strips; Bayer Corp., Mishawaka, IN), free fatty acids (FFA: NEFA C kit; Wako Chemicals, Richmond, VA), glycerol (free glycerol reagent F6428; Sigma-Aldrich, St. Louis, MO), triglycerides (L-Type TG H kit; Wako Chemicals), leptin (rat leptin RIA kit, Linco Research Inc., St. Charles, MO), insulin (rat insulin RIA kit; Linco Research), adiponectin (mouse adiponectin RIA kit; Linco Research), testosterone (testosterone RIA kit; Diagnostic Systems Laboratories, Webster, TX), T3 (total T3 RIA kit: Diagnostic Systems Laboratories), and corticosterone (DA Corticosterone 125I kit; MP Biomedicals, Orangeburg, NY). Inguinal (Ing), epididymal (Epi), and mesenteric (Mes) white fat and IBAT were dissected and weighed. Small (50 mg) samples of Epi and Ing fat were fixed in osmium tetroxide for measurement of cell number and size distribution by Coulter Counter and Channelizer (Beckman Coulter, Inc., Fullerton, CA), as described previously (36). The IBAT was snap frozen and stored at –80 C. Portions of the brown fat were analyzed for norepinephrine content by HPLC as described previously (37). Total RNA also was extracted using TriZol reagent (Invitrogen, Carlsbad, CA) and real-time PCR was used to measure uncoupling protein 1 (UCP1) mRNA expression, as described below. The carcasses, less gastrointestinal tract, were analyzed for body composition, as described previously (38). Brains of CD rats were examined histologically to confirm the completeness of the transection.
Statistical analysis of data was performed using Statistica software (StatSoft, Tulsa, OK). Daily body weights, energy expenditure, and RQ measured across 24 h and cell size distribution were compared by repeated measures ANOVA. Measures at specific time points were analyzed by post hoc Duncan’s multiple range test. Measures of body composition, fat pad weight, serum metabolite, and hormone concentrations and tissue mRNA expression were compared by two-way ANOVA and post hoc Duncan’s multiple range test. Differences were considered significant at P < 0.05, but in the interest of space and clarity the details of each analysis are not included in the text.
Real-time quantitative PCR (Q-PCR)
After total RNA extraction, 1 μg of RNA was treated with deoxyribonuclease I (Invitrogen) and incubated at room temperature for 15 min. Deoxyribonuclease I was inactivated by addition of 1 μl of 25 mM EDTA and heating at 65 C for 10 min. cDNA was prepared from 500 ng total RNA by RT (Promega Reverse Transcription System; Promega Corp., Madison, WI). A negative control containing no avian myeloblastosis virus reverse transcriptase was used to ensure specificity of the PCR amplification. After RT, cDNA was treated with ribonuclease H (Invitrogen). First-strand cDNA was then diluted to 50 μl using nuclease-free water, yielding a concentration of 25 ng/μl. PCR primers were designed using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and purchased from Invitrogen. Q-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol using the Bio-Rad iCycler iQ system. Amplification conditions were as follows: initial denaturation, 95 C for 3 min; 40 cycles of 95 C for 30 sec, 58 C for 30 sec, and 72 C for 30 sec. Melt curve analysis was performed immediately after Q-PCR amplification. Triplicate determinations of UCP1 mRNA and 18S rRNA expression were made for each sample. The primer sequences were: 18s forward primer ACG GAA GGG CAC CAC CAG GA, 18s reverse primer CAC CAC CAC CCA CGG AAT CG, UCP1 forward primer TCC CTC AGG ATT GGC CTC TAC, UCP-1 reverse primer GTC ATC AAG CCA GCC GAG AT. UCP1 mRNA expression was calculated using the comparative threshold (CT) method (http://www.dorak.info/genetics/realtime.html), calculating the difference between threshold cycle for 18S and UCP1, therefore, the greater the value of CT, the lower the expression of UCP1. The average CT for control-fed rats was designated a value of 1.0, and the values of the other groups are expressed in relation to this arbitrary value.
Results
Histology confirmed complete transection in all of the CD rats. A representative midsaggital section depicting the lesion is shown in Fig. 1. The liquid diet regimen maintained a weight gain of 2.9 ± 0.1 g/d in control rats (Fig. 2A). Immediately after the first surgical procedure, weight gain was inhibited in CD rats (0.8 ± 0.2 g/d) even though they were fed the same amount of food as the controls. After the second surgery, the rectal temperature of CD rats fell to approximately 34 C (Fig. 2B). After about 5 d, rectal temperature was maintained at approximately 35 C. As seen in Fig. 2B, temperature of CD rats increased at successive meals during each day and then fell each night during the 12 h when they received no food. Energy expenditure, expressed on a per animal basis, was significantly lower in CD than control rats at all times of the day (Fig. 3A: CD, P < 0.0001; Time, P < 0.0001; Int, P < 0.0001). Control rats showed an increased rate of expenditure at the start of the dark period, but this increase was muted in CD rats. The energy expenditure of both CD and control rats declined progressively during the dark phase when there was the longest interval between tube-fed meals. RQ was significantly higher in CD than control rats (Fig. 3B: CD, P < 0.0001; Time, P < 0.0001; Int, P < 0.0001) at all times except the last 3 h of the dark period. During the light period and the first half of the dark period, the RQ of CD rats was greater than 1.0, indicative of lipid deposition, whereas the control rats maintained an RQ of about 1.0, which is typical for carbohydrate oxidation. During the dark period, when there was a 12-h interval between meals, the RQ of all of the rats declined to about 0.8, indicating a high rate of fatty acid and/or protein oxidation (40). Average 24-h energy expenditure and RQ for control and CD rats is shown in Fig. 3C and illustrates the decreased expenditure but increased RQ for CD rats compared with controls when it was expressed on a per animal basis. When expenditure was expressed per unit fat-free mass, there was no longer any effect of decerebration on energy expenditure, although a reduction due to starvation was still apparent (see Fig. 6C; P < 0.065).
Both Control and CD rats lost weight in response to 48 h of starvation (Table 1). Body composition measured at the end of the experiment indicated that although CD rats were lighter than controls they were significantly fatter and had less lean tissue (protein plus water). The increase in carcass fat of CD rats was associated with an increase in the size of all of the depots measured, although the difference did not reach statistical significance for Epi depots (P < 0.059; Fig. 4), and this was due to an increase in adipocyte size rather than number (Fig. 5). The weight loss caused by starvation was predominantly water in the controls but was mainly fat in the CD rats (Table 1). Mes and Ing fat but not Epi fat showed a significant decrease in size in the starved CD rats, and this was associated with a reduction in cell size rather than number (Fig. 5). Although there was some decrease in the size of white fat depots in starved controls, none of the differences were statistically significant (P < 0.1). Energy expenditure of both control and CD rats was decreased during the 48 h of starvation (Fig. 6, A and B: CD, P < 0.001; Starvation, P < 0.0001; Time, P < 0.0001; CD x Starvation, NS; CD x Time, P < 0.0001; Starvation x Time, P < 0.0001; CD x Starvation x Time, NS). The starved control rats continued to show an increase in expenditure at the start of the dark period, whereas there was no effect of the dark phase on expenditure of the starved CD rats. Figure 6C shows average 24 h expenditure during the 48 h of starvation, and the significant inhibition of expenditure in both control and CD rats when expressed per rat but no difference between groups if expenditure was expressed based on fat-free mass (Starvation, P < 0.065). The RQ of both control and CD rats fell to 0.8 after about 10 h of food deprivation, indicating that both groups of rats were using lipid and/or protein (Fig. 7A: CD, P < 0.0001; Starvation, P < 0.0001; Time, P < 0.0001; CD x Starvation, P < 0.0001; CD x Time, P < 0.0001; Starvation x Time, P < 0.0001; CD x Starvation x Time, P < 0.0001). Average 48-h RQ was significantly increased for fed CD rats compared with fed controls, but it was low and not different between the groups of starved rats. Despite the reduction in energy expenditure, both the control and CD starved rats maintained the same body temperature as their fed counterparts (Fig. 8). IBAT weight was significantly increased in fed CD rats compared with fed controls, but it was the same weight in starved controls and starved CD rats (Table 2). The norepinephrine content of IBAT was reduced in fed CD rats compared with controls and was not changed by starvation in either group of rats (Table 2). There were no differences in UCP1 mRNA expression (Table 2).
Starvation caused a significant drop in blood glucose concentrations for both control and CD rats, but there was no significant effect of decerebration on glucose in either fed or starved conditions (Table 1). Similarly, there were no significant differences in serum FFA, triglyceride, or glycerol concentrations between fed control and CD rats. Starvation caused a significant decrease in circulating triglycerides and a significant increase in FFA in all rats but had no effect on glycerol concentrations. In contrast, the obese, fed CD rats were hyperinsulinemic, hyperadiponectinemic, and hyperleptinemic compared with fed controls. Starvation caused a significant fall in the concentration of both insulin and leptin in CD rats so that there was no difference in either leptin or insulin concentrations of starved CD and control rats, but adiponectin remained elevated in the CD rats. There was no effect of either decerebration or starvation on serum concentrations of corticosterone. Total T3 was not different between control and CD rats but was decreased by starvation in both groups. Serum testosterone was extremely low in CD rats, being undetectable in eight of the 13 animals. There was no effect of starvation on testosterone concentrations in control rats.
Discussion
This is the first study to directly evaluate the long-term regulation of energy balance in CD rats by exposing them to an energy deficit caused by 2 d of starvation. The measurement of energy expenditure and body composition of fed CD rats also provides new information on the metabolic state of rats that are dependent only upon the caudal brainstem for the neural control of physiologic function under normal and energetically challenging conditions. Although we assume that regulatory responses observed in CD rats are mediated by the brainstem, we cannot exclude the possibility of communication and control by humoral factors initiated in the forebrain. In order for the forebrain to respond to a change in energetic conditions, however, it also would have to be responding to humoral signals because rostral coursing projections from peripheral afferents and from the caudal brainstem itself would have been destroyed. In this experiment, fed CD rats had a lower body temperature, a lower energy expenditure, a higher RQ, markedly less lean body mass and more body fat compared with fed intact controls. Fed CD rats also had elevated circulating concentrations of leptin, adiponectin, and insulin but had normal blood glucose, corticosterone, and total T3 concentrations compared with fed intact controls. In contrast, circulating concentrations of testosterone were extremely low. The lower energy expenditure of these rats was associated with a reduced fat-free mass and an apparent decrease in voluntary activity. The CD rats did not exhibit exploratory behavior, as described previously (41), and they did not show the typical increase in expenditure associated with handling and feeding. This is particularly apparent in Fig. 6, A and B, during the period of starvation, when there was a significant elevation in expenditure of the starved control rats as they were removed from their cages and handled to be tube-fed water. In contrast, the CD rats showed no elevation in expenditure in response to these stimuli. There were similar differences in 24-h energy expenditure of the two treatment groups during the period of starvation at the end of the study when the data were expressed on a per rat basis, but these differences disappeared if energy expenditure was expressed per unit fat-free mass. Therefore, the reduced lean body mass had a major impact on the heat production of the CD rats. Despite using a similar amount of energy per unit fat-free mass and using less energy for activity, the rats were maintaining a reduced body temperature, implying that they were using energy inefficiently and unable to retain heat effectively.
We compared expenditure on a per rat basis because the large differences in body composition of the two treatment groups did not justify the use of kg0.75 as a unit of metabolic body size. Immediately after the first surgery, the rate of weight gain of CD rats was inhibited, and this slow rate of gain was maintained throughout the experiment suggesting that the failure to retain body protein may have started after only a partial sectioning of the brain because accumulation of body energy as fat represents a smaller gain in body weight than deposition of an equivalent amount of energy as lean tissue, which includes an obligatory significant amount of water. A similar loss of lean mass has been reported for rats with lesions of the lateral hypothalamus (42), although it is not known whether the loss of lean tissue in these two conditions is caused by the same mechanism. Because releasing hormones were not measured in this study, it is not known whether the pituitary was functioning normally; however, the observation that serum corticosterone and total T3 were the same in control and CD rats suggests that at least some aspects of pituitary function do not rely on input from the forebrain. By contrast, circulating concentrations of testosterone were significantly changed in the absence of neural communication between the forebrain and caudal brainstem. The loss of lean mass demonstrates a critical role for higher brain areas in regulating protein retention and accretion, either directly or through control of growth factors. Further studies are needed to demonstrate both the degree of brain function that is needed to maintain normal protein metabolism and the deficits in metabolism that cause the rapid and substantial reduction in lean tissue of CD rats. One such factor that is known to affect lean body mass, especially protein, that is markedly reduced in CD rats is serum testosterone. Loss of testosterone due to castration triggers a decrease in body weight in rats (43), accompanied by decreases in carcass protein and increases in carcass lipid that are reversible with testosterone administration (44). Therefore, it seems likely that a significant portion of the decrease in lean body mass and some of the increased fat accretion in CD rats resulted from the functional castration that was initiated by decerebration. A second factor that contributed to the increased adiposity of the CD rats was that they were essentially overfed. Control and CD rats were tube fed the same daily energy intake even though the CD rats had a significantly lower energy expenditure. It is likely that overfeeding accounted for their elevated RQ of greater than 1.0, which is indicative of energy storage and has been used as a marker for overfeeding (45); however, because the rats were being overfed it is even more surprising that they were not able to at least maintain their lean body mass.
All of the rats in this study received their daily food and water intake in three tube-fed meals each day that were adequate for maintenance of a normal rate of growth for the controls. Meals were delivered over a 12-h period, which meant that the rats were subjected to 12 h without food during the dark cycle. By the end of this period, all of the rats had an RQ of approximately 0.8, a value that is indicative of food deprivation (45) and that fatty acids or protein are being oxidized (40). There was no difference in RQ of CD and intact controls at this time, suggesting that the high RQ of CD rats during the light phase was due to an excess energy intake, rather than an inability to oxidize fat or carbohydrate. The energy expenditure of fed CD rats appeared to decline more during the dark phase than that of the fed control rats (at 1030 h, Control = 0.97 ± 0.03, CD = 0.89 ± 0.03; at 0630 h, Control = 0.86 ± 0.03, CD = 0.56 ± 0.03 kcal/rat·h) and body temperatures of CD rats were at their nadir before the first meal of the day, suggesting a possible deficit in their ability to regulate energy utilization and/or partitioning. After the first meal of the day, the body temperatures of the CD rats increased and their RQ almost immediately went above 1.0, indicating that although some metabolic heat was trapped and raised body temperature, a large portion of the ingested energy was stored as fat. In contrast, the RQ of the control rats remained around 1.0 after the first meal, and they showed a more pronounced increase in energy expenditure after the meal due to either nutrient processing and utilization (46) or to the thermic effect of food (47).
Because of the increased adiposity of CD rats, it is impossible to determine whether their hyperinsulinemia and hyperleptinemia were secondary to obesity or were the result of a lack of central control of pancreatic function or adipose leptin expression. The normal circulating concentrations of glucose indicate that the rats were insensitive to insulin but were not insulin resistant. Again, this may have been secondary to the overfeeding and adiposity rather than a direct result of loss of neural control by the forebrain. The elevated levels of adiponectin in fed CD rats was surprising because this adipokine usually is decreased in conditions of obesity and diabetes (48) and increased with weight loss (49). The high concentrations of adiponectin in CD rats is, however, consistent with a recent report that resting metabolic rate is a strong predictor of adiponectin concentration in humans and that a low resting metabolic rate is associated with elevated adiponectin (50). Others have reported that stimulation of -adrenergic receptors inhibits adiponectin production by 3T3L1 cells (51), and it is possible that the high adiponectin levels in the CD rats was due to reduced sympathetic tone on white adipose tissue and that the low metabolic rate is partially due to low sympathetic tone in other major organs.
When CD rats were subjected to 48 h of starvation, they appeared to make essentially the same energetic and hormonal responses to the challenge that were made by intact, starved control rats. Even though the daily expenditure of the CD rats already was lower than that of the controls, both the CD and control rats reduced their daily energy expenditure. Despite this low energy expenditure, the CD rats maintained body temperature at the same level as during feeding, demonstrating that the isolated caudal brainstem is sufficient for regulating body temperature in both the fed and fasted state, albeit at a less than optimal level. RQ declined rapidly in both groups of animals to approximately 0.8, indicating that they were oxidizing fatty acids and/or protein for energy. The CD rats had no obvious difficulty in mobilizing lipid from their white fat depots, whereas there were only small, nonsignificant changes in the fat content of starved control rats. Because energy expenditure expressed per unit fat-free mass was not different between the groups, and because expenditure per rat was lower in CD than control rats, it is difficult to explain how they could have mobilized more fat than controls. It is unlikely that the CD rats were able to breakdown triglycerides but not oxidize the FFA because circulating FFA were similar in the two groups. One possible explanation for this apparent anomaly of energy balance is that the difference in fat between fed and fasted CD rats was due in part to continued accumulation of body fat in the fed CD rats during the 48-h starvation period, which exaggerated the amount of fat that appeared to be mobilized in response to starvation.
The difference in carcass fat content of fed and starved rats was fat depot specific because both Ing and Mes pads were reduced by starvation, whereas the weight of the Epi pads did not change. The Epi pads in rats show a dampened lipid mobilization during fasting compared with other fat pads (52), and this may be due to reduced sympathetic drive on this fat depot in response to energetic challenges (53). The obesity of the fed CD rats was associated with a significant increase in the size of adipocytes rather than an increase in cell number and the loss of fat during starvation was associated with a reduction in cell size, rather than number. It is tempting to speculate that one of the factors contributing to adiposity in fed CD rats was low sympathetic tone in white adipose tissue; however, enlargement of white fat depots after denervation has been attributed to an increase in cell number rather than size (54, 55). In addition, if lipid mobilization during starvation is predominantly mediated by increased sympathetic drive (56, 57), then it is surprising that the CD rats were able to mobilize fat so effectively unless brainstem circuits are sufficient for this response. Although lipid mobilization in response to fasting is largely independent of adrenal medullary catecholamines (58), we did not measure circulating concentrations of catecholamines in this study. Others have reported that the lipolytic response to fasting is not impaired in mice that are deficient in -adrenoceptors (59); therefore, it also is possible that factors such as the dramatic change in insulin (60) or potential increase in glucagons (61) promoted fatty acid mobilization and oxidation in the starved CD rats.
Similar to humans and intact rodents (18, 62), the starved CD rats showed a rapid and exaggerated fall in serum leptin concentration. The dramatic reductions in insulin and leptin in starved compared with fed CD rats suggests that the hyperleptinemia and hyperinsulinemia were associated with overfeeding, rather than adiposity per se, because the starved CD rats were still significantly fatter than fed controls. It has been hypothesized that the substantial fall in circulating concentrations of leptin that occur during the early stages of food deprivation signals the hypothalamus to initiate a coordinated response to the energy deficit (20, 21). The results from this study demonstrate that efferent neural signals from the hypothalamus or other areas of the forebrain are not required for the fall in energy expenditure, or regulation of metabolism to maintain blood glucose or body temperature during conditions of energy deficit. The responses seen in CDs must be initiated by signals that can be received, integrated, and responded to by circuits endemic to the caudal brainstem and is consistent with a previous report that the caudal brainstem is adequate for sensing and responding to the metabolic challenge produced by 2-deoxyglucose administration (29). The specific sites responsible for this remain to be defined, but there is ample evidence that multiple nuclei in the brainstem can make a significant contribution to different aspects of the regulation of energy balance (see Refs.22 and 63 for review).
In many animals, BAT is an important site of thermogenesis that is primarily under control of the sympathetic nervous system. The IBAT in fed CD rats from this study had a significantly lower norepinephrine (NE) content, compared with fed intact controls, indicative of a reduced level of activation and heat production, but there was no difference in UCP1 mRNA levels between groups. If the NE content of the tissue can be considered an indirect indicator of sympathetic activity in the tissue, then reduced activation of IBAT in CD rats may have contributed to the lower daily energy expenditure of the CD rats compared with controls. A previous study has shown that stimulation of melanocortin receptors in the brainstem of CD rats causes a significant increase in IBAT UCP1 mRNA expression (32). The observation that tissue NE content was low in animals in this study may indicate that the maintenance of basal sympathetic tone to IBAT requires input from the forebrain. There was no significant effect of starvation on IBAT NE or UCP1 mRNA content, although it would be anticipated that both of these factors would decrease as energy was conserved. It is possible that these changes would have been observed, at least in control rats, if the period of deprivation had been extended.
The objective of this study was to determine whether CD rats were able to make appropriate adaptations to a period of starvation. The results demonstrate that functional neural control by the caudal brainstem is sufficient for rats to respond to an energy deficit by reducing energy expenditure and increasing fatty acid oxidation to maintain body temperature. In normal animals, metabolic changes during the early stages of starvation protect lean body mass (7); in CD rats, however, there appears to be a severe disruption of the systems regulating protein metabolism even during conditions of energy excess, and this may largely be because the animals are functionally castrated, having undetectable levels of testosterone. During starvation, however, CD rats rely predominantly on lipid mobilization for energy. Measures in fed CD rats indicate that more rostral brain areas are required for the rats to maintain an appropriate lean body mass, metabolic rate, and body temperature. Energy expenditure associated with activity also seems to be diminished due to a failure to respond to environmental stimuli. The importance of these factors in maintaining homeostasis in baseline conditions in CD rats remains to be determined. For example, the reduced body temperature may promote accumulation of body fat when the energy intake of the rats is fixed by tube feeding, and, as noted above, the substantial decline in circulating testosterone undoubtedly contributed to the failure to maintain lean body mass. Over the long-term, reduced voluntary activity of the animals also may contribute to a loss of lean tissue. The impact of these impairments on the response to starvation, however, appeared minimal because the CD rats showed the same energetic responses to 48 h of food deprivation as the intact controls. The reduction in energy expenditure associated with a low body temperature and reduced voluntary activity may, in fact, be beneficial in conditions of energy deficit. It is well established that testosterone concentrations in intact rats are reduced by about 80% after 4 d of starvation (39, 64); therefore, it is unlikely that the preexisting hypogonadism of CD rats in this study influenced their response to starvation. The testosterone concentrations in intact control rats in this experiment decreased by about 30% after 2 d of starvation, but this difference was not statistically significant. Thus, it appears that although the caudal brainstem is adequate for making gross responses to energetic challenges such as starvation, input from the forebrain is needed to refine these responses and to maintain an optimal physiologic and hormonal environment under baseline conditions when energy intake is adequate.
Acknowledgments
The authors thank Joyce Power and Jessica Davenport at University of Georgia for their expert technical assistance, Kate Nautiyal at University of Pennsylvania for her technical advice, and Dr. Kay Song, Georgia State University for photomicroscopy.
Footnotes
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants SCRO DK-21397 (awarded to H.J.G., T.J.B., and R.B.S.H.), R01 DK-53903 (awarded to R.B.S.H.), and R01 DK-35254 (awarded to T.J.B.).
First Published Online December 15, 2005
Abbreviations: CD, Chronic decerebrate; CT, comparative threshold; Epi, epididymal; IBAT, intrascapular brown adipose tissue; Ing, inguinal; NE, norepinephrine; NPY, neuropeptide Y; Q-PCR, quantitative PCR; RQ, respiratory quotient; UCP1, uncoupling protein 1.
Accepted for publication December 5, 2005.
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