Cardiovascular responses to caloric restriction and ... - CiteSeerX

Am J Physiol Regulatory Integrative Comp Physiol 282: R1459–R1467, 2002. First published January 24, 2002; 10.1152/ajpregu.00612.2001.

Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice T. D. WILLIAMS,1 J. B. CHAMBERS,2 R. P. HENDERSON,2 M. E. RASHOTTE,2 AND J. M. OVERTON1 1 Departments of Nutrition, Food, and Exercise Sciences and 2Psychology, Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4340 Received 10 October 2001; accepted in final form 9 January 2002

Williams, T. D., J. B. Chambers, R. P. Henderson, M. E. Rashotte, and J. M. Overton. Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice. Am J Physiol Regulatory Integrative Comp Physiol 282: R1459–R1467, 2002. First published January 24, 2002; 10.1152/ajpregu.00612.2001.—We utilized variations in caloric availability and ambient temperature (Ta) to examine interrelationships between energy expenditure and cardiovascular function in mice. Male C57BL/6J mice (n ⫽ 6) were implanted with telemetry devices and housed in metabolic chambers for measurement of mean arterial pressure ˙ O2), and locomotor (MAP), heart rate (HR), O2 consumption (V activity. Fasting (Ta ⫽ 23°C), initiated at the onset of the dark phase, resulted in large and transient depressions in ˙ O2, and locomotor activity that occurred during MAP, HR, V hours 6–17, which suggests torporlike episodes. Food restriction (14 days, 60% of baseline intake) at Ta ⫽ 23°C resulted in progressive reductions in MAP and HR across days that were coupled with an increasing occurrence of episodic tor˙ O2 (⬍1.0 porlike reductions in HR (⬍300 beats/min) and V ml/min). Exposure to thermoneutrality (Ta ⫽ 30°C, n ⫽ 6) reduced baseline light-period MAP (⫺14 ⫾ 2 mmHg) and HR (⫺184 ⫾ 12 beats/min). Caloric restriction at thermoneutrality produced further reductions in MAP and HR, but indications of torporlike episodes were absent. The results reveal that mice exhibit robust cardiovascular responses to both acute and chronic negative energy balance. Furthermore, we conclude that Ta is a very important consideration when assessing cardiovascular function in mice.

reduces heart rate (HR) and arterial blood pressure and increases HR variability (12, 33, 34, 39, 40, 43, 45); however, the mechanisms responsible for these cardiovascular adaptations remain poorly understood. Although genetically altered mice provide an opportunity to test specific hypotheses concerning the mechanisms by which negative energy balance regulates the cardiovascular system (3, 37), there is little information about the effects of acute or chronic negative energy balance in mice. Recently, Swoap (39) reported reduced HR and blood pressure

and the development of torpor during chronic food restriction in ob/ob mice. A primary purpose of the present paper was to characterize the metabolic and cardiovascular responses of C57BL/6J mice to reduced caloric intake (both acute fasting and chronic restriction) utilizing telemetry methods (7, 29, 42). As the results reveal, we immediately observed that an overnight fast produces episodes of severe transient bradycardia (HR ⬍ 300 beats/min) and hypotension that are associated with depressed metabolic and locomotor activity suggestive of torpor (13, 17, 19, 44). Given the rapid and severe nature of the cardiovascular response to fasting, we then determined the cardiovascular and metabolic responses in C57BL/6J mice to mild longterm food restriction. We hypothesized that long-term exposure to milder caloric reduction in a restricted-feeding procedure would also reduce mean arterial pressure (MAP) and HR but might not be associated with the episodic severe bradycardia and hypotension that were observed during fasting. Furthermore, we tested this hypothesis when the ambient temperature (Ta) imposed both relatively low energetic demands [thermoneutral Ta ⬵ 30°C (14)] or the higher demands associated with standard laboratory conditions (Ta ⬵ 23°C). Thermoneutrality is defined as the range of Ta at which energy expenditure is lowest and no metabolic heat is required to maintain body temperature; it is typically ⬃28–31°C for rodents (14). Standard temperatures result in activation of facultative, nonshivering thermogenesis that is mediated primarily by an increase in sympathetic activity to brown adipose tissue in rodents (18, 41). It appears that this tonic thermogenic sympathetic activity also has cardiovascular consequences. Thermoneutral housing conditions are associated with reduced HR and MAP in several rat strains (34). Therefore, we hypothesized that warming Ta to 30°C would produce compensatory reductions in MAP, HR, ˙ O2) in mice as it does in and oxygen consumption (V rats. We further hypothesized that thermoneutrality during food restriction would ameliorate the energetic demand sufficiently to reduce or prevent cardiovascu-

Address for reprint requests and other correspondence: J. M. Overton, Dept. of Nutrition, Food and Exercise Sciences and Program in Neuroscience, 236 Biomedical Research Facility, Florida State Univ., Tallahassee, FL 32306-4340 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

blood pressure; thermogenesis; food deprivation; radiotelemetry

NEGATIVE ENERGY BALANCE

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NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

lar torpor while still permitting caloric restriction to mediate overall reductions in MAP and HR. RESEARCH METHODS

Male C57BL/6J mice (age 25 ⫾ 0.5 wks; Jackson Laboratories, Bar Harbor, ME) were anesthetized with halothane (1–2% in 95% oxygen-5% nitrogen) and instrumented with a catheter coupled with a sensor and transmitter (model TA11PA-C20, Data Sciences, St. Paul, MN) either in the descending aorta (n ⫽ 3) or the left common carotid (n ⫽ 14) for telemetric monitoring of blood pressure (7, 29, 42). For the implantation into the carotid artery, we utilized the procedures recently described by Butz and Davisson (6). Before and during recovery from surgery, mice were housed individually in standard polycarbonate mouse cages with wood-chip bedding and were provided pellet chow (Purina 5001 rodent chow). After the recovery period, which lasted ⬃10 days, animals were transferred to larger custom-built experimental cages. 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, the cages were placed within previously described environmental chambers that provided computer-controlled Ta and lighting conditions (36). The mice were given ad libitum access to deionized water at all times and were maintained on a 12:12-h light-dark schedule with Ta ⫽ 23 ⫾ 0.1°C except during the thermoneutral conditions (see Protocols). A period of chamber maintenance occurred each day soon after the 10th hour of the light phase. At that time, daily body weight and food and water consumption were measured. All body-weight data were corrected by subtracting the weight of the telemetry device (3.3 g). Indirect Calorimetry The apparatus and procedures used for determination of metabolic rate have been described previously (45). Oxygen ˙ O2) and carbon dioxide production (V ˙ CO2) were consumption (V measured every 2.5 min by open-circuit respirometry using a modification of the approach described by Bartholomew and colleagues (2) to isolate successive samples. The flow rate of ˙ O2 was adjusted for air into the chamber was set at 1 l/min. V mass (ml/min/kg0.75) (4). Telemetry Monitoring A telemetry receiver (model RPC-1, Data Sciences) was positioned under the mouse cage within the metabolic chamber used for indirect calorimetry. The average systolic blood pressure (SBP), diastolic blood pressure (DBP), and the interbeat interval (IBI) were measured for each cardiac cycle, and MAP and HR were calculated for each 30-s period of the day and stored for off-line analysis as described previously (45). Locomotor-Activity Monitoring Locomotor activity was measured using a custom-designed force platform as described previously (45). Total activity, measured in meters, was accumulated in 30-s periods and stored with a 1-mm resolution. Protocols After the mice recovered from surgery and were acclimated to housing conditions, we obtained 4 days of baseline data from all mice and initiated the following protocols: Protocol group 1: fasting at 23°C (n ⫽ 6). To begin the 48-h period of food deprivation, food was removed ⬃1–2 h before

the onset of the dark phase. After the fasting period, food was returned at the onset of the dark phase and 4 days of recovery were recorded during which baseline conditions were in effect. We observed that some mice failed to survive a 48-h fast at 23°C. Thus data for the second 24-h of fasting included 4 animals, the first recovery day included 3 animals, and recovery days 2–4 included 2 animals. Protocol group 2: food restriction at Ta ⫽ 23°C (n ⫽ 5). Each day for 14 days, 60% of baseline calories were provided and the amount left in the food hopper the next morning was measured during maintenance. Deionized water was continuously available during the food-restriction period. A recovery period of 3 days followed restricted feeding during which food was again available ad libitum. Protocol group 3: food restriction at Ta ⫽ 30°C (n ⫽ 6). For 5 days, Ta was increased from 23° to 30°C while baseline conditions of food and water availability were maintained. The change in Ta was initiated at the onset of the dark phase and required ⬃30 min to reach the higher value. Then with Ta maintained at 30°C, the mice underwent 14 days of restricted feeding in which 60% of the average daily food intake at thermoneutrality was provided, and the amount left in the food hopper the next morning was measured during maintenance. Deionized water was continuously available. A recovery period of 3 days followed restricted feeding during which food was again available ad libitum while Ta remained at 30°C. Data Analysis and Statistics Cardiovascular variables and locomotor activity data were collected and stored in 30-s bins. Metabolic data were collected and stored in 2.5-min bins. Before further analysis, all data were averaged into 10-min bins. Because the final 2 h of the light phase (during which daily chamber-maintenance procedures were performed) were excluded from analysis, average light-phase values were based on 10-h data; average dark-phase values were based on 12-h data. The effects of fasting, food restriction, Ta, and recovery were statistically assessed by repeated-measures ANOVA. Tukey’s post hoc tests were used to determine significant differences between means. Significance levels of P ⬍ 0.05 were accepted. RESULTS

Baseline data from mice in each of the three protocols are summarized in Table 1. Protocol Group 1: Effects of Fasting at Ta ⫽ 23°C Figure 1, A and B, illustrates the key features of the cardiovascular response to fasting as observed in an individual mouse. On the fast day, food was removed at approximately the beginning of the record, about 2 h before the dark phase began. Large and transient reductions in HR and MAP were observed starting at ⬃4 h after removal of food and continuing for ⬃4 h into the light phase. The episodic fasting-related reductions in HR and MAP were typically associated with periods of low locomotor activity (Fig. 1C). In the final hours of the light phase, there was a marked increase in locomotor activity on the fast day that normalized HR and increased MAP above the baseline in this animal. Averaged group data for all mice on these two days show a pattern of marked bradycardia and hypotension that developed after a few hours on the fasting day (Fig. 2,

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Table 1. Baseline data from male C57BL/6J mice with ambient temperature at 23°C for 4 days Mean 23°C Baseline Data

Body weight, g Caloric intake, kcal Water intake, g Mean arterial pressure, mmHg 12 h Dark 10 h Light Heart rate, beats/min 12 h Dark 10 h Light ˙ O2, ml/min/kg0.75 Normalized V 12 h Dark 10 h Light Locomotor activity, m 12 h Dark 10 h Light

Protocol group 1 (n ⫽ 6)



28.7 ⫾ 0.7 20.0 ⫾ 2.7 6.3 ⫾ 0.4

28.4 ⫾ 1.0 17.9 ⫾ 1.1 5.0 ⫾ 0.4

29.1 ⫾ 0.6 16.9 ⫾ 0.9 5.7 ⫾ 0.4

120 ⫾ 4 105 ⫾ 4

123 ⫾ 2 104 ⫾ 1

126 ⫾ 4 107 ⫾ 5

581.4 ⫾ 7.8 554.6 ⫾ 10.6

570.5 ⫾ 12.7 512.1 ⫾ 15.9

582.2 ⫾ 7.8 526.2 ⫾ 11.6

31.8 ⫾ 1.3 24.7 ⫾ 1.2

36.1 ⫾ 5.8 26.0 ⫾ 4.0

30.8 ⫾ 0.5 22.8 ⫾ 0.3

198.7 ⫾ 48.9 46.1 ⫾ 13.7

126.7 ⫾ 42.7 29.0 ⫾ 16.1

148.0 ⫾ 51.1 25.9 ⫾ 13.2

Values are means ⫾ SE. Group 1: fast at 23°C; group 2: food restriction at 23°C; group 3: food restriction at 30°C. Powdered food and deionized water were provided ad libitum. Body weight reflects subtraction of telemeter weight (3.3 g). Body weight and caloric and water intakes were measured daily during 2-h maintenance period just before lights off; other measures were derived from continuous data collection and separated into 12-h-dark and 10-h-light period means.

A and B), although the severe transients so evident in the individual animal data are less apparent because of the averaging. On the fasting day, group-averaged locomotor activity increased and was quite variable after about the 4th hour of the light phase (Fig. 2D); in the same period, HR was lower than baseline (Fig. 2A) and MAP was normalized (Fig. 2B).

Data obtained on each day of the experiment are shown in Fig. 3. On average, fasting reduced body weight by ⬃21% (⫺6.5 ⫾ 0.3 g; Fig. 3A) and water consumption by ⬃66% (⫺4.1 ⫾ 0.4 ml; Fig. 3B). Upon refeeding, there was no compensatory hyperphagia or polydipsia (Fig. 3B). Fasting was associated with variable increases in light-phase locomotor activity, al-

Fig. 1. Heart rate (HR), mean arterial pressure (MAP), and locomotor activity (Activity) are displayed for a single C57BL/6J mouse in 2.5-min bins over 24 h on two separate experimental days: baseline day 4 (thin line) and fasting day 1 (thick line) with ambient temperature (Ta) ⫽ 23°C. Powdered food and deionized water were provided ad libitum during baseline day. AJP-Regulatory Integrative Comp Physiol • VOL

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Fig. 2. HR, MAP, absolute oxygen con˙ O2), and locomotor activity are sumption (V displayed for C57BL/6J mice (n ⫽ 6) in 10-min bins over 22 h on baseline day 4 and fasting day 1 with Ta ⫽ 23°C. Final 2 h of the light phase during daily maintenance was removed. Powdered food and deionized water were provided ad libitum during baseline day.

though the increase did not reach statistical significance (Fig. 3C). Average dark-phase MAP and HR values were significantly below baseline on both fasting days, and there was a progressively greater decrease across days (Fig. 3, D and E). Light-phase values of HR were strongly depressed even in the first ˙ O2 values were relight-phase of fasting (Fig. 3E). V duced below baseline in the light and dark phases on day 2 of fasting (Fig. 3F). In the refeeding period, MAP and HR values returned to baseline levels, but locomotor activity was lower compared to baseline (Fig. 3C). We note that the effects reported on day 2 of fasting and in recovery are based on a reduced number of animals. Protocol Group 2: Food Restriction Effects at Ta ⫽ 23°C During the 14-day restricted-feeding protocol, body weight was reduced by 21% (⫺6.5 ⫾ 0.7 g; Fig. 4A) whereas caloric intake averaged 53% of baseline intake (Fig. 4B). After a transient decrease, water intake remained at baseline levels for the remaining foodrestriction period (Fig. 4B). No change occurred in dark-period locomotor activity, and there was a trend toward increased light-period activity over 14 days of food restriction (Fig. 4C). With restricted feeding, darkperiod MAP (Fig. 4D) and HR (both light and dark periods; Fig. 4E) values gradually decreased from days 1–9 and then appeared to stabilize during the final 5 ˙ O2 (Fig. 4F) also days of food restriction. Dark-period V gradually decreased and reached significance at the end of the restriction period. Interestingly, light-period MAP tended to increase over the course of the restricted period and was likely associated with the increase in light-period locomotor activity (Fig. 4C). Upon return to ad libitum food availability, a marked

hyperphagia and polydipsia were observed, which co˙ O2 incided with a rapid return of MAP, HR, and V values to prerestricted levels within 12 h. During the 3-day recovery period, evidence of refeeding hypertension was apparent in the light but not the dark phase. Similar to the fasting experiment, simple day and night averages do not fully represent the effects of food restriction (Fig. 5). Torporlike responses in MAP, HR, ˙ O2 begin to appear by day 3 of food restriction and and V continue each day throughout the restricted period. Protocol Group 3: Caloric Restriction at Thermoneutrality (Ta ⫽ 30°C) Warming the Ta from 23° to 30°C resulted in no change in body weight (Fig. 6A) or water intake but did induce a 23% (⫺6.6 ⫾ 0.4 kcal) decrease in caloric intake (Fig. 6B). There was an increase in dark-period locomotor activity beginning on day 3 and no effect on light-period activity (Fig. 6C). Increasing Ta to thermoneutrality caused significant and sustained reductions ˙ O2 (Fig. 6F) in MAP (Fig. 6D), HR (Fig. 6E), and V values that were evident during the first 12-h dark period and were sustained for 5 days. During the 14-day restricted-feeding protocol within Ta ⫽ 30°C, caloric intake averaged 56% of baseline intake and 44% of intake during the Ta ⫽ 23°C period (Fig. 6B). Food restriction produced reductions in body weight (Fig. 6A), MAP (Fig. 6D), and HR (Fig. 6E) beginning by days 4 and 5 of the restriction period. Restoration of ad libitum feeding quickly normalized all variables to prerestricted levels within 12 h. Although not statistically significant, there was a tendency for refeeding hypertension to be evident in both the light and dark phases. Figure 7 reveals the pattern of MAP and HR responses to caloric restriction within thermoneutrality. After consumption of the available


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Fig. 3. Body weight (A), food and water intake (B), locomotor activity ˙ O2 (F) in male C57BL/6J mice (C), MAP (D), HR (E), and normalized V (n ⫽ 6) during 4 baseline days, 2 fasting days, and 4 refeeding days with Ta ⫽ 23°C. Powdered food and deionized water were provided ad libitum during baseline and refeeding periods. All body-weight data have been corrected by subtracting the weight of the telemetry device (3.3 g). Due to severity of fasting challenge, animals died during study: fast day 2 (n ⫽ 4), refeed day 1 (n ⫽ 3), and refeed days 2 and 3 (n ⫽ 2). *P ⬍ 0.05 vs. baseline (one-way repeated-measures ANOVA).

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restriction, these torporlike cardiovascular and metabolic responses were followed by marked increases in anticipatory locomotor activity late in the light phase. In addition to the effects of negative energy balance, these experiments reveal the very important role of Ta in the regulation of HR and MAP in mice. We observed that increasing Ta from 23° to 30°C reduced lightphase MAP by ⬃15 mmHg and light-phase HR by ⬃175 beats/min. Finally, this study demonstrates that C57BL/6J mice respond to long-term caloric restriction while housed at thermoneutrality with further reductions in MAP and HR values, which suggests an additive effect of temperature and caloric challenges. Taken together, these results illustrate important roles of energy balance on the control of cardiovascular function in mice. To date, there is minimal information concerning the cardiovascular responses to reduced caloric availability in mice (1, 39). In one study, 2 wk of caloric restriction reduced tail-cuff SBP in agouti mice but had no effect on SBP of wild-type mice (1). The limitations of the tail-cuff method for assessment of blood pressure in rats and mice are well established (21, 42). Very recently, it was reported (39) that 1 wk of 50% caloric restriction reduces HR and MAP values in both leptindeficient ob/ob and wild-type mice. Our work extends

food within the first few hours of the dark cycle, further ˙ O2 values were observed reductions in HR, MAP, and V relative to the already lower levels produced by exposure to thermoneutrality. In fact, food restriction within thermoneutrality produced the lowest values with HR levels consistently below 300 beats/min and MAP levels below 80 mmHg. DISCUSSION

These experiments illustrate the powerful cardiovascular effects of both reduced caloric intake and exposure to thermoneutrality in male C57BL/6J mice. Mice vigorously respond to both an acute fasting challenge as well as to long-term mild food restriction within standard laboratory conditions (Ta ⫽ 23°C) by reducing ˙ O2. Although examinabody weight, MAP, HR, and V tion of mean cardiovascular values over the course of the 10–12-h day-night averages gives the initial impression that these responses are similar to those previously observed in rats (34, 45), analysis of within-day cardiovascular and behavioral patterns in C57BL/6J mice demonstrates very large and transient reductions ˙ O2 values during the late dark phase and in HR and V early light phase. This is likely associated with periods of shallow torpor (13, 17, 19, 39, 44), although we do not have simultaneous body-temperature measures. On the first day of fasting or after 3–4 days of caloric

Fig. 4. Body weight (A), food and water intake (B), locomotor activity ˙ O2 (F) in male C57BL/6J mice (C), MAP (D), HR (E), and normalized V (n ⫽ 5) during 4 baseline days, 14 food-restriction days (60% baseline intake) and 3 refeeding days with Ta ⫽ 23°C. Powdered food and deionized water were provided ad libitum during baseline and refeeding periods. All body-weight data have been corrected by subtracting the weight of the telemetry device (3.3 g). * P ⬍ 0.05 vs. baseline (one-way repeated-measures ANOVA).


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˙ O2), and locoFig. 5. HR, MAP, absolute (V motor activity are displayed for C57BL/6J mice (n ⫽ 5) in 10-min bins over 22 h on baseline day 4 and food-restriction day 14 with Ta ⫽ 23°C. Final 2 h of the light phase during daily maintenance was removed. Powdered food and deionized water were provided ad libitum during baseline day and 60% of baseline chow intake and ad libitum water was provided on food-restriction day.

these findings by clearly demonstrating that either acute fasting or chronic caloric restriction for several days reduces MAP and HR values in C57BL/6J mice. Himms-Hagen has noted (19) that “when food is scarce, the mouse is living on the brink of disaster.” Physiological responses to reduced caloric availability observed in mice and some hamsters, but not in rats, include concurrent torpor and hypothermia (11, 13, 17, 26, 30, 44). We suspect that the large, transient fluctuations in MAP and HR that were evident during the late dark phase and early light phase are coincident with torpor in mice that are in negative energy balance. Comparable HR responses have been recorded during daily cold-induced torpor in the white-footed mouse (Peromyscus, Ref. 30) and during chronic caloric restriction in wild-type and ob/ob mice (39). Our experiments reveal that torporlike patterns of marked bradycardia become evident within 6 h of fasting (see Fig. 2) and 3–4 days of caloric restriction while living at Ta ⫽ 23°C (data not shown). We did not measure body temperature in these studies, but Swoap (39) has recently observed reductions in daily body temperature coincident with hypotension and bradycardia during chronic food restriction in mice. This finding suggests that the daily bradycardia and hypotension that corresponds to reductions in metabolic rate in our studies is part of the torpor response to caloric deprivation in these mice. Several lines of evidence suggest that both reduced sympathetic activity and increased vagal activity contribute to the cardiovascular effects of reduced caloric intake (15, 20, 28, 33, 43). Given that sympathetic blockade and ␤-receptor deletion generally produce a HR ⬇ 450–500 beats/min in mice (23, 24, 38), it seems likely that the bradycardia associated with torpor requires either marked vagal activation or reduction in

Fig. 6. Body weight (A), food and water intake (B), locomotor activity ˙ O2 (F) in male C57BL/6J mice (C), MAP (D), HR (E), and normalized V (n ⫽ 6) during 4 baseline days with Ta ⫽ 23°C, 5 days with Ta ⫽ 30°C, 14 food-restriction days (60% baseline intake), and 3 refeeding days with Ta ⫽ 30°C. Powdered food and deionized water were provided ad libitum during baseline and refeeding periods. All body-weight data have been corrected by subtracting the weight of the telemetry device (3.3 g). * P ⬍ 0.05 vs. baseline; ⫹P ⬍ 0.05 vs. 30°C day 5 (one-way repeated-measures ANOVA).


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˙ O2 (C), Fig. 7. HR (A), MAP (B), absolute V and locomotor activity (D) are displayed for C57BL/6J mice (n ⫽ 6) in 10-min bins over 22 h on baseline day 4 with Ta ⫽ 23°C, baseline day 5 with Ta ⫽ 30°C, and food-restriction day 14 with Ta ⫽ 30°C. Final 2 h of the light phase during daily maintenance was removed. Powdered food and deionized water were provided ad libitum during baseline days and 60% of baseline chow intake and ad libitum water were provided on food-restriction day.

the intrinsic rate. The role of increased vagal activity is supported by the observation that atropine administration prevents the bradycardia during entrance into hibernation in white-footed mice (30). Nonetheless, the mechanisms linking autonomic and cardiovascular function with caloric availability remain poorly defined. Although the mechanisms responsible for these cardiovascular responses were not studied, it should be noted that leptin administration generally prevents food-restriction-induced reductions in metabolic rate, body temperature, and HR (10, 13, 16, 35). Our findings demonstrate that circadian factors must be considered when evaluating the cardiovascular responses to fasting and food restriction in mice. We observed consistent reductions in mean dark-phase MAP and HR values; however, in the light phase, the reductions in HR and MAP values were generally less in magnitude, thereby resulting in a loss of circadian variation in mice subjected to caloric restriction. The smaller effect on HR and MAP during the light phase is likely due to an augmentation of light-phase locomotor activity. Caloric restriction produces an increase in anticipatory activity before the time when food is provided (8). Interestingly, the magnitude of this anticipatory activity in response to caloric restriction was less in mice studied in thermoneutrality even though these mice were subjected to caloric restriction of a similar magnitude (60% of baseline caloric intake). We have recently demonstrated that increasing Ta ˙ O2 values in from 23° to 29°C reduces MAP, HR, and V lean and obese Zucker rats (34). Thus standard housing conditions (Ta ⫽ 21–24°C) are a form of mild cold stress for rats that results in a tonic elevation of MAP and HR above those observed at thermoneutrality. Given that the transition to thermoneutrality reduces ˙ O2 by 50% in mice (25), we were not surprised that V

thermoneutrality was also associated with very substantial and sustained reductions in MAP and HR values in both light and dark circadian phases (see Figs. 5 and 6). It is interesting to note that whereas cardiovascular parameters were reduced, dark-phase locomotor activity tended to increase during thermoneutrality (see Fig. 6C). A similar increase in spontaneous activity with transition from mild cold to thermoneutrality has been shown in rats (5). In mild cold, rats and mice may spend less time engaged in spontaneous locomotor activity and more time reducing heat loss by adopting a cold-defensive posture. The observation of markedly lower HR and MAP values, despite increases in dark-phase activity, reflects the strength of the effect of Ta on cardiovascular function. Furthermore, light-phase HR is reduced by 175 beats/min (from 525 to 350 beats/min) in C57BL/6J mice housed at thermoneutrality with no change in light-phase locomotor activity. Stated simplistically, we observed an effect of Ta on HR of ⬃25 beats/min per 1°C in the light phase. Temperatures lower than 23°C produce further increases in HR in both rats and mice (9, 22). However, additional studies will be useful in providing a detailed quantitative analysis of the relationship between Ta and cardiovascular function in rodents. The mechanisms responsible for the relationship between mild cold stress and cardiovascular function are not well understood but are likely the direct or indirect result of sympathetically mediated nonshivering thermogenesis. One could speculate that exposure to thermoneutrality concurrently reduces sympathetic outflow to multiple tissues, including brown adipose tissue and the heart, resulting in the concurrent reductions in ˙ O2, HR, and MAP. Morrison has made substantial V progress in delineating the central neural organization of sympathetic outflow in response to thermoregula-


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tory stimuli (31, 32). Interestingly, the thermoneutrality-induced magnitude of bradycardia is similar to that of 24 h of fasting and ⬎14 days of food restriction at 23°C. However, in contrast to fasting, the bradycardia associated with thermoneutrality is accompanied by greater reductions in MAP. Although the cardiovascular responses to fasting and thermoneutrality are both associated with reduced metabolic rate, different physiological pathways are likely involved. Housing mice at thermoneutrality minimizes torpor induced by caloric restriction (27). Thus we examined the cardiovascular responses to caloric restriction in C57BL/6J mice acutely adapted to thermoneutrality. The results demonstrate the reductions in MAP and HR values that are induced by caloric restriction are not dependent on an elevated baseline that is produced by mild cold (see Fig. 5). As illustrated in Fig. 7, we continued to observe large reductions in MAP and HR values beginning ⬃2 h after the daily feeding. HR and MAP values generally remained very low during the late dark and early light phases and then increased in association with restriction-induced anticipatory activity. The results reveal that even after adaptation to thermoneutrality, C57BL/6J mice respond to reduced caloric availability by decreasing MAP and HR. Perspectives

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3. 4. 5. 6. 7. 8.

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Our findings emphasize the need for attention to the nutritional status and temperature conditions in experimental mouse models. Transgenic mouse models that have altered energy balance regulation, such as obese agouti or lean melanin-concentrating hormoneknockout mice, may also exhibit altered responses to caloric or Ta challenges. An examination of the literature reveals that many cardiovascular phenotyping studies fail to report Ta during blood pressure and HR measurements. The issue is not trivial given the magnitude of influence Ta has on cardiovascular function in mice. It is indisputable that current standard housing conditions (Ta ⫽ 21–24°C) impose a background of tonically elevated MAP and HR, presumably related to the requirement for nonshivering thermogenesis, in rats and mice. Further, this mild cold stress is associ˙ O2, and greater ated with greater food intake, greater V oxidative stress. Many of these observations have clear implications for important research efforts in aging, obesity, and cardiovascular disease. The authors gratefully acknowledge the technical assistance of Belinda Coulter and Akiko Nakamura. The Florida State University (FSU) Neuroscience Program’s Technical Support Group provided expertise in instrumentation (Paul Hendricks and Ron Thompson) and computer programming (Chris Baker). This work was supported by National Institutes of Health Grant HL-56732 and a Program Enhancement Grant from the FSU Research Foundation. T. D. Williams is a predoctoral fellow of the American Heart Association, Florida/Puerto Rico Affiliate.

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