INTRODUCTION

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ACUTE AND CHRONIC CALORIC restriction reduces blood pressure and heart rate in several rodent models of hypertension (15, 32, 43, 45). It is well known that one of the primary homeostatic adaptations to reduced caloric intake is a reduction in basal metabolic rate (37). It is conceivable that modulations in blood pressure, heart rate, and basal metabolic rate, which occur during restricted food availability, are related to an adaptive response to impending starvation. During periods of reduced caloric intake, norepinephrine turnover is reduced in multiple regions, including intrascapular brown adipose tissue and the heart (12, 25, 47). These observations suggest a mechanistic action of the autonomic nervous system in the coordinated regulation of cardiovascular and metabolic function during periods of reduced caloric intake. However, reduced caloric availability is also associated with large-scale behavioral changes (16, 35) that are likely to influence cardiovascular and metabolic function.

To examine how cardiovascular and metabolic function were coupled to behavioral changes during caloric restriction, we assessed cardiovascular telemetry, indirect calorimetry, and measurement of energetically relevant behaviors (eating, drinking, locomotion) in the spontaneously hypertensive rat (SHR) housed under standard photophase and ambient temperature conditions. This approach provides the opportunity to acquire and integrate multiple physiological and behavioral measures related to cardiovascular and energy balance status over an extended time period in freely moving animals. The first objective of this study was to examine the magnitude and time course of the cardiovascular, metabolic, and behavioral responses to fasting and refeeding in the unrestrained SHR using these combined methodologies.

Higher mean arterial pressures (MAP) and heart rates (HR) are observed during the rat's active, nocturnal period (10). This is not surprising, because it is well known that rats engage in several prolonged periods of ingestive behavior during the dark phase (19) and that locomotion, eating, and drinking behavior elevate HR and blood pressure (26). Clearly, the study of circadian differences in cardiovascular and metabolic function must take into account these behavioral effects. Furthermore, although basal metabolic rates can be determined using short-term placements in a calorimeter to measure oxygen consumption, recent findings have suggested that the handling of animals associated with this approach may mask starvation-induced effects on metabolism (27). Our approach allows determination of the cardiovascular and metabolic status in rats, for both light and dark phases, and also during intervals of behavioral quiescence. Therefore, the second purpose of this study was to use behavioral monitoring to quantify metabolic and cardiovascular status during inactive periods of the day both when food was available ad libitum and during 48 h of fasting.

	RESEARCH METHODS

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Eight male SHR (age 13 ± 1 wk old; Harlan Sprague Dawley, Indianapolis, IN) were anesthetized (pentobarbital sodium, 50 mg/kg) and instrumented with a catheter in the descending aorta coupled with a sensor and transmitter (TA11PA-C40; Data Sciences, St. Paul, MN) for telemetric monitoring of blood pressure. During recovery from surgery, rats were housed individually in custom-built cages (43 by 22 by 17 cm). These cages and the food and water dispensers were similar to the experimental cages, which facilitated acclimation to the experimental conditions. The experimental cage consisted of a polycarbonate base with an 8-cm lip containing animal bedding. The sides and top of the unit were constructed of wire mesh so that adequate air mixing could be achieved for the purpose of indirect calorimetry (see below). Access to powdered chow (Purina 5001; caloric value 3.3 kcal/g) contained within a food jar was provided via a modified stainless steel tunnel feeder that minimized spillage. During the experiment, rats were housed in an ambient temperature of 23 ± 0.1°C and maintained on a 12:12-h light-dark schedule.

Indirect calorimetry. The apparatus and procedures used for determination of metabolic rate have been described previously (33). Briefly, the cage unit described above was placed within an acrylic metabolic chamber (40-liter capacity). The cage unit housing the rat stayed within this metabolic chamber continuously, except for daily experimental maintenance. Metabolic chambers were individually housed within custom-constructed environmental chambers that provided precise control of ambient temperature and the light-dark phases by illumination of an overhead incandescent lamp (illumination ~60 lx of 2,250-K light).

Oxygen consumption (O₂) and carbon dioxide production (CO₂) were measured every 2.5 min by open-circuit respirometry (flow rate 2 l/min) using a modification of the approach described by Bartholomew et al. (3) to isolate successive samples. O₂ was adjusted for mass (ml · min¹ · kg^0.75; Ref. 6). A separate value for respiratory quotient (RQ; CO₂/O₂) was calculated for the entire 12-h dark phase and for 10-h of the light phase.

Telemetry monitoring. A telemetry receiver (RPC-1; Data Sciences) was positioned under the rat cage within the metabolic chamber. The pulses from the receiver were relayed to a calibrated pressure output adapter (R11CPA; Data Sciences) where they were converted to analog voltages representing blood pressure waveforms. The signals were amplified, filtered with a time constant of 1 ms, and relayed to a 12-bit analog-to-digital converter board in a computer that sampled the signals at 500 Hz with a resolution of 0.1 mmHg. Custom-written online software processed the blood pressure waveform and detected the systolic blood pressure (SBP), diastolic blood pressure (DBP), and the interbeat interval (IBI) for each cardiac cycle and calculated MAP and HR. The average SBP, DBP, MAP, HR, and the standard deviation of the IBI (SD of IBI) were calculated for each 30-s period of the day and stored on a floppy disk as 16-bit integers for off-line analysis. Extremely low or high IBIs and BPs resulting from transient signal interference occurred rarely and were automatically detected and excluded from the 30-s averages.

Locomotor activity monitoring. Some of the metabolic chambers were instrumented to record locomotor activity. The acrylic chamber containing the rat cage rested on a fulcrum positioned across the narrow axis of the cage. One end of the chamber was attached to a stiff strain gauge transducer (alpha load beam; BLH Electronics) that prevented rocking of the chamber on the fulcrum and allowed determination of the position of the rat within the chamber along the long axis. The electronic signal from the strain gauge was amplified, low-pass filtered with a 3-dB point of 10 Hz, and routed to an analog-to-digital converter where it was sampled at 20 Hz, thus allowing determination of the position of the animal's center of gravity along the long axis. To detect locomotor activity as the animal moved in the chamber, the rate of change in the chamber's center of gravity was low-pass filtered with an average time of 500 ms. Locomotor activity, measured in meters, was accumulated in 30-s periods and stored with a 1-mm resolution.

Ingestive behavior monitoring. Feeding behavior was monitored by a photobeam sensor across the entrance of the feeder, which contained a reservoir of powdered rat chow. Due to feeder design and the use of powdered chow, the rat had to remain at the feeder during periods of food consumption. Thus breakage of the photobeam provided an indication of feeding behavior and the duration of photobeam breakage was accumulated in 30-s periods with 50-ms resolution. Drinking behavior was also monitored by photobeam breakage in 30-s time periods. The total photobeam breakage every 30-s was quantified and stored for off-line analysis.

Protocol. After recovery from surgery and acclimation to the housing conditions, the rats were transferred to the metabolic chambers described above for an additional acclimation period of at least 4 days before data collection. Food and deionized water were available ad libitum. Food and water intake and body weight of each rat were determined daily ~2 h before lights off. When food and water intake and cardiovascular and metabolic data were judged to be stable, 3 days of baseline values were obtained. To begin the 48-h period of food deprivation, food was removed ~2 h before the onset of the dark phase. On the fasting days, water was replaced with an electrolyte solution containing 78 meq/l NaCl and 15 meq/l KCl to provide sodium and potassium. At the end of the fast, food and deionized water were returned to the cages just before lights off to ensure rats would resume food consumption at a time point consistent with normal circadian feeding behavior. (Pilot studies revealed that normal circadian behavioral patterns were disrupted for several days if food was returned during the light phase.) In the refeeding period, food was available ad libitum for 6 days, during which recovery kinetics in cardiovascular and metabolic variables was measured.

Data analysis and statistics. Cardiovascular data, time spent in ingestive behaviors, and locomotor activity data were collected and stored in 30-s bins. Metabolic data were collected and stored in 2.5-min bins. The collection of all data was synchronized so that the metabolic and cardiovascular consequences of behavior could be evaluated. Before further analysis, all data were averaged into 10-min bins, with feeding and drinking photobeam breakage accumulated in 10-min bins. The final 2 h of the light phase (during which daily chamber maintenance procedures were performed) was excluded from analysis, resulting in 12-h averages for the dark phase and 10-h averages for the light phase. The effects of fasting and refeeding on these values were statistically assessed by repeated-measures ANOVA.

We determined inactive period values for MAP, HR, O₂, and SD of IBI separately for the light and dark phases during the last day of baseline and on the second day of fasting. Inactive periods were identified as 10-min bins with no feeding or drinking and accumulated locomotor activity <1.0 m. All 10-min bins of data meeting these criteria were separately averaged in the dark and light phases to assess cardiovascular and metabolic status when behavioral influences were minimal. Due to equipment limitations, this analysis could be performed on data from four of the eight animals studied. MAP, HR, O₂, and SD of IBI during inactive periods were compared with values calculated over the entire dark and light phases to assess the influence of behavior on these measures both in ad libitum and fasting conditions. Statistical comparisons were made using two-way repeated-measures ANOVA. Tukey post hoc tests were used to determine significant differences between means. Significance levels of P < 0.05 were accepted.

	RESULTS

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Integration of indirect calorimetry, telemetry, and behavioral monitoring data. Figure 1 illustrates the profound effects of behavior on cardiovascular function and energy expenditure during the 24-h day of an SHR housed in standard laboratory conditions. The figure reveals that the rat spends many hours of the dark phase engaged in discrete periods of food consumption (indicated by shaded areas), during which there are substantial increases in MAP, HR, and O₂. There is typically very little ingestive behavior or locomotor activity during the light phase, and, as expected, MAP, HR, and O₂ recorded during these times were substantially lower. RQ values remained close to 1.0 throughout the day, indicating that dietary energy provided the substrate for energy metabolism.

View larger version (78K): [in this window] [in a new window]   Fig. 1.   Integrated multivariable plots of 2.5-min averaged data over 24-h time period during baseline conditions for a single male spontaneously hypertensive rat (SHR; food and water available ad libitum). Mean arterial pressure (MAP) and heart rate (HR) were derived from implanted telemetry devices. Time spent feeding (Eat) and drinking (Drink) was determined by photobeam breakage. Locomotor activity was determined using a load sensor. Oxygen consumption (O2) and respiratory quotient (RQ) were determined by indirect calorimetry. Shaded areas represent periods of feeding behavior. Removal of rat from metabolic box during daily maintenance is indicated between 8:00 and 9:00 AM. Hours of light and dark phases are indicated at top and bottom of figure.

Caloric intake, fluid intake, and body weight. Over the 3 baseline days, caloric intake before fasting averaged 89 ± 3 kcal/day (Fig. 2A), fluid intake averaged 39 ± 3 ml/day, and sodium intake averaged 4.7 ± 0.1 mmol/day. Baseline body weight averaged 353 ± 16 g and fell 46 ± 3 g (13%) during fasting (Fig. 2B). During fasting, fluid intake averaged 47 ± 1 ml/day and sodium intake averaged 3.6 ± 0.1 mmol/day. Upon refeeding, caloric intake was elevated relative to baseline on the first day. Body weight remained below baseline levels for the first 3 recovery days.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Mean (±SE) of caloric intake (A; n = 4) and body weight (B; n = 8) on 3 baseline days, 2 fasting days, and 6 refeeding days. * P < 0.05 vs. baseline.

MAP. Baseline MAP averaged 143.7 ± 3.7 mmHg during the dark phase and 140.1 ± 3.7 mmHg during the light phase. MAP was reduced in the dark phase of the second fasting day, but in the light phase on both fasting days (Fig. 3A). In comparison to baseline values, MAP on the second day of fasting was reduced by 9.2 ± 1.6 mmHg (6%) in the dark phase and 8.6 ± 1.8 mmHg (6%) during the light phase. Refeeding was associated with an immediate return of MAP to baseline levels in the dark phase. In the light phase, however, MAP did not reach baseline levels until day 3 of refeeding.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Mean MAP (A), HR (B), standard deviation of interbeat interval (SD of IBI; C), O2 (D), RQ (E), and locomotor activity (F) during 12-h dark () and 10-h light () phases on 3 baseline days, 2 fasting days, and 6 refeeding days (n = 8; except locomotor activity in F, n = 4). * P < 0.05 vs. baseline; + P < 0.05 vs. fast day 1 (1-way repeated-measure ANOVA).

We used off-line analysis to identify all 10-min bins of data that met our criteria for inactive periods for the last baseline day and second fasting day. On the baseline day, 20% of bins (140 min) comprising the dark phase and 68% of the bins (400 min) comprising the light phase met these criteria. During the second day of fasting, 41% of bins (290 min) in the dark phase and 59% of bins (350 min) in the light phase met these criteria. These bins were used to calculate MAP during inactive periods (Fig. 4A). Compared with total phase data, MAP was lower in inactive periods irrespective of circadian or dietary conditions (Fig. 4A). Although the magnitude of fasting-induced reductions in inactive period MAP were similar for both dark and light phases, significant decreases were only observed during the light phase. Interestingly, there were no day-night differences in total phase or inactive period MAP in either the baseline or fasting conditions.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Comparison of MAP (A), HR (B), SD of IBI (C), and O2 (D) for total dark and light phases and for inactive periods of phases during final baseline day (Base) and second day of fasting (Fast). Statistical significance: a total-phase vs. inactive periods, b final baseline day vs. fast day 2, c dark vs. light phase. All comparisons: n = 4; P < 0.05.

HR. Baseline HR in the 3 days before fasting averaged 330.0 ± 5.2 beats/min during the dark and 294.0 ± 5.2 beats/min during the light phase (Fig. 3B). Fasting was associated with reduced HR during the dark and light phases on both days of fasting. Relative to baseline values, HR on the second day of fasting was reduced by 43.4 ± 5.2 beats/min (13%) in the dark phase and by 27.4 ± 5.2 beats/min (9%) in the light phase. Refeeding was associated with a rapid return to the baseline HR level, but in the dark phase HR was lower than baseline on the second and fourth refeeding days. HR during inactive periods was lower than total dark and light phases both in baseline and fasting conditions (Fig. 4B). Fasting was associated with reductions in inactive period HR in both the light and dark phases. Finally, there were day-night differences in both total phase and inactive period HR values obtained during baseline and during the second day of fasting (Fig. 4B).

SD of IBI. Baseline SD of IBI averaged 6.0 ± 0.3 ms during the dark and 6.7 ± 0.2 ms during the light phase (Fig. 3C). Fasting was associated with increases in SD of IBI during both light phases and the final dark phase. There was an increase in SD of IBI between the first and second light phase of the fast. Inactive period levels of the SD of IBI were not different from total values for either the dark or light phases (Fig. 4C). There were no day-night differences in SD of IBI.

O₂. Baseline O₂ averaged 20.6 ± 0.3 ml · min¹ · kg^0.75 during the dark phase and 14.9 ± 0.2 ml · min¹ · kg^0.75 during the light phase. Fasting produced reductions in metabolic rate in both phases and on both fasting days (Fig. 3D). In comparison to baseline values, metabolic rate on the second day of fasting was reduced by 5.0 ± 0.6 ml · min¹ · kg ^0.75 (24%) in the dark phase and by 2.7 ± 0.2 ml · min¹ · kg ^0.75 (18%) in the light phase. O₂ during inactive periods was lower than the respective total phase dark and light in both the baseline and fasting conditions (Fig. 4D). Furthermore, fasting lowered both total phase and inactive period metabolic rate in the dark and light phases. Although there were day-night differences in total phase metabolic rate, there were no comparable differences in inactive period O₂ (Fig. 4D).

RQ. Baseline RQ on the 3 days before fasting averaged 0.99 ± 0.01 during the dark phase and 0.96 ± 0.01 during the light phase. These values indicate that carbohydrate metabolism (substrate from food consumption) was occurring in both phases of the day. Fasting was associated with reduced RQ (Fig. 3E), indicating that fat reserves were being metabolized. On the second day of fasting, RQ in the dark phase was lower relative to the first day. RQ rapidly returned to control levels upon refeeding.

Locomotor activity. Baseline locomotor activity averaged 216 ± 48 m during the dark phase and 74 ± 16 m during the light phase (Fig. 3F). Locomotor activity tended to be reduced in the dark phase during fasting, increased during the first two nights of refeeding (not significant), and returned to baseline levels thereafter.

	DISCUSSION

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Two days of fasting resulted in significant reductions in body weight, MAP, HR, O₂, and RQ in male SHR during both the light and dark phases of the daily cycle. Refeeding generally produced a rapid normalization of these measures. By removing the influence of ingestive behavior and locomotor activity, we were able to determine that inactive period levels of MAP, HR, and O₂ during both the light and the dark phases were lower than when values were computed over the entire phases. Furthermore, fasting reduced inactive period values for MAP, HR, and O₂. Thus the cardiovascular and metabolic adaptations to fasting in the SHR occur independent of changes in feeding, drinking, and locomotor activity.

Cardiovascular responses to reduced caloric intake. The powerful effects of long-term caloric deprivation on cardiovascular function have been recognized for more than 50 years (9). More recently, several studies indicate that during the first few days of negative energy balance, blood pressure falls rapidly despite minimal changes in body weight (15, 42). Previous studies using both tail cuff and acute tethering methods have demonstrated that fasting reduces blood pressure and heart rate in the SHR (13, 22, 48). Therefore, we chose to focus the current study on a detailed analysis of the cardiovascular and metabolic responses to fasting in the male SHR using combined telemetry and calorimetry. It is important to note that the magnitude of the cardiovascular and metabolic responses to caloric deprivation may depend on several factors, including age, gender, strain, and species of animal studied.

It is now clear from telemetric measurements of blood pressure and HR that acutely catheterized animals may exhibit elevated MAP and HR (4, 17). Our telemetry data reveal that in the light phase for the adult male SHR, MAP during behavioral quiescence is ~140 mmHg and HR is ~300 beats/min. These values are consistent with previous reports using telemetry (4, 10, 31) and, in fact, are consistent with values obtained using chronic tethering of SHR (7, 21). Importantly, we found that fasting reduces MAP and HR from even these low levels obtained using telemetry, providing further evidence for an important interaction between regulation of energy balance and cardiovascular function.

Although telemetric methods provide the distinct advantage of long-term monitoring of cardiovascular function of laboratory animals in a home cage environment, it is not typical that behavioral influences are partitioned to obtain a basal assessment of cardiovascular status. Rats housed in standard laboratory conditions engage in several sustained periods of ingestive behavior (see Fig. 1; and reviewed in Ref. 46), and it is well recognized that ingestive behavior and locomotor activity can produce substantial effects on BP and HR (34). Furthermore, the SHR is known to be hyperresponsive to environmental stimuli and, in fact, has a greater pressor response to drinking (26). Therefore, we considered the possibility that fasting-induced reductions in MAP and HR could simply result from diminished periods of ingestive behavior and locomotor activity. However, we found that inactive period MAP and HR, determined during periods of no ingestion and low locomotor activity, were significantly reduced during caloric deprivation. Forty-eight hours of fasting lowered inactive period MAP to 123 ± 4 mmHg and HR to 245 ± 5 beats/min during the light phase (Fig. 4, A and B). Although the magnitude of these reductions in MAP and HR may be different in other rat strains, it is clear that in the SHR, cardiovascular parameters are reduced independent of energetically relevant behaviors during food deprivation.

Metabolic and locomotor responses to reduced caloric intake. Previous studies have demonstrated that caloric restriction or deprivation can produce progressive reductions in metabolic rate over the course of several days in rodents (5, 16, 27, 28, 30, 41). The magnitude of the reductions observed during the 24- and 48-h fast for the SHR are similar to those previously reported for Sprague-Dawley rats (41). The 48-h period of food deprivation is associated with a 13% reduction in body weight. Although we did not determine lean body mass in these studies, we corrected oxygen consumption for metabolic body size to account for decreases in total body mass (6). Although this approach may not be ideal, it is clear that the reduction in metabolic rate reflects a homeostatic response to reduced caloric intake, rather than simply a reduction in mass.

Similar to the cardiovascular effects of fasting, we considered the possibility that reduced ingestive behavior and locomotor activity could partially explain the observed reductions in metabolic rate. Recently, it was determined that fasting-induced reductions in metabolic rate are easier to detect when long-term measurements of metabolism are used, rather than short-term measures obtained after animal handling (27). The effects of reduced caloric availability on rodent activity appear to depend on housing conditions. When allowed access to running wheels, locomotor activity is increased during periods of reduced caloric availability (35). However, locomotor activity may decrease in the SHR during fasting when animals are housed in standard cage conditions (16). We observed a tendency for reductions in locomotor activity during food restriction (see Fig. 3F). Nonetheless, fasting significantly reduced inactive period metabolic rate, providing further support for the idea that decreases in metabolic rate reflect a homeostatic response to caloric deprivation.

Coordinated regulation of metabolic and cardiovascular responses to fasting. There are several lines of evidence that support the hypothesis that the autonomic nervous system plays a major role in the homeostatic response to reduced energy intake. Caloric deprivation produces significant reductions in sympathetic activity as indicated by decreased cardiac, liver, renal, and brown adipose tissue norepinephrine turnover (12, 25, 47). Starvation also reduces directly measured sympathetic nerve activity to brown adipose tissue (36). We recently reported that fasting reduces sympathetic support of blood pressure as determined by the depressor responses to ganglionic blockade (22). In humans, reductions in urinary and plasma catecholamine levels, as well as reductions in directly measured muscle sympathetic nerve activity, have been demonstrated after various periods of reduced caloric intake (1, 20, 23, 24). In addition, weight reduction produces decreases in cardiac sympathetic tone and increases in parasympathetic tone in humans (2). In the current study, we observed increases in the SD of IBI during fasting, indicating either increased parasympathetic tone or decreased sympathetic tone (44). Taken together, these observations are consistent with the hypothesis that the hypotensive and bradycardic responses to fasting may be mediated by the autonomic nervous system.

It is common in the literature to discuss hypothalamic mechanisms that are linked to altering appetite and sympathetic nerve activity in a scheme describing homeostatic regulation of energy balance (37-39, 46). It is now clear that leptin plays a key role in the regulation of food intake and body weight (14). Furthermore, a growing body of evidence indicates that in addition to inhibition of food intake, leptin has sympathoexcitatory and cardiovascular actions (11, 18, 40). For example, we recently demonstrated that acute central administration of leptin normalizes MAP and HR in the fasted, normotensive Sprague-Dawley rat (11). Thus we hypothesize that fasting-associated reductions in plasma leptin activate central neural pathways that produce a coordinated sequalae of events. These responses include increased appetite and decreased sympathetic outflow, which is likely a major mechanism for the reduction in MAP, HR, and metabolic rate.

In conclusion, we combined telemetric assessment of cardiovascular function with continuous indirect calorimetry to test the hypothesis that caloric deprivation produces concurrent reductions in MAP, HR, and metabolic rate in the SHR. We found that these parameters were decreased in both the light and dark photoperiods by fasting and that the reductions were not dependent on behavioral changes. We hypothesize that the metabolic and cardiovascular responses to caloric deprivation are interrelated and regulated to a large extent by leptin actions at the level of the hypothalamus. Future studies will rigorously examine this hypothesis.

Perspectives