Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice

T. D. Williams, J. B. Chambers, R. P. Henderson, M. E. Rashotte and J. M. Overton

Am J Physiol Regul Integr Comp Physiol 282:R1459-R1467, 2002. First published 24 January 2002; doi: 10.1152/ajpregu.00612.2001 You might find this additional info useful... This article has been cited by 25 other HighWire-hosted articles: http://ajpregu.physiology.org/content/282/5/R1459#cited-by Updated information and services including high resolution figures, can be found at: http://ajpregu.physiology.org/content/282/5/R1459.full Additional material and information about American Journal of Physiology - Regulatory, Integrative and Comparative Physiology can be found at: http://www.the-aps.org/publications/ajpregu

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

This information is current as of February 11, 2013.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology publishes original investigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2002 the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at http://www.the-aps.org/.

Am J Physiol Regulatory Integrative Comp Physiol 282: R1459R1467, 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 nal 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: R1459R1467, 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 23C), 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 617, which suggests torporlike episodes. Food restriction (14 days, 60% of baseline intake) at Ta 23C 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 30C, 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. blood pressure; thermogenesis; food deprivation; radiotelemetry

NEGATIVE ENERGY BALANCE

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 specic 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 30C (14)] or the higher demands associated with standard laboratory conditions (Ta 23C). Thermoneutrality is dened as the range of Ta at which energy expenditure is lowest and no metabolic heat is required to maintain body temperature; it is typically 2831C 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 30C 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 sufciently to reduce or prevent cardiovascuThe 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. R1459

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

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: moverton@mailer.fsu.edu). http://www.ajpregu.org

0363-6119/02 $5.00 Copyright 2002 the American Physiological Society

R1460

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 modied 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 0.1C 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 modication of the approach described by Bartholomew and colleagues (2) to isolate successive samples. The ow 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 23C (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 23C. Thus data for the second 24-h of fasting included 4 animals, the rst recovery day included 3 animals, and recovery days 2 4 included 2 animals. Protocol group 2: food restriction at Ta 23C (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 30C (n 6). For 5 days, Ta was increased from 23 to 30C 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 30C, 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 30C. 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 nal 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. Tukeys post hoc tests were used to determine signicant differences between means. Signicance levels of P 0.05 were accepted.
RESULTS

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

Baseline data from mice in each of the three protocols are summarized in Table 1. Protocol Group 1: Effects of Fasting at Ta 23C 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 nal 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,
282 MAY 2002

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

R1461

Table 1. Baseline data from male C57BL/6J mice with ambient temperature at 23C for 4 days
Mean 23C Baseline Data Protocol group 1 (n 6) Protocol group 2 (n 5) Protocol group 3 (n 6)

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

28.7 0.7 20.0 2.7 6.3 0.4 120 4 105 4 581.4 7.8 554.6 10.6 31.8 1.3 24.7 1.2 198.7 48.9 46.1 13.7

28.4 1.0 17.9 1.1 5.0 0.4 123 2 104 1 570.5 12.7 512.1 15.9 36.1 5.8 26.0 4.0 126.7 42.7 29.0 16.1

29.1 0.6 16.9 0.9 5.7 0.4 126 4 107 5 582.2 7.8 526.2 11.6 30.8 0.5 22.8 0.3 148.0 51.1 25.9 13.2

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

Values are means SE. Group 1: fast at 23C; group 2: food restriction at 23C; group 3: food restriction at 30C. Powdered food and deionized water were provided ad libitum. Body weight reects 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) 23C. Powdered food and deionized water were provided ad libitum during baseline day. AJP-Regulatory Integrative Comp Physiol VOL
282 MAY 2002

www.ajpregu.org

R1462

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

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 23C. Final 2 h of the light phase during daily maintenance was removed. Powdered food and deionized water were provided ad libitum during baseline day.

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

though the increase did not reach statistical signicance (Fig. 3C). Average dark-phase MAP and HR values were signicantly 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 rst 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 23C 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 nal 5 O2 (Fig. 4F) also days of food restriction. Dark-period V gradually decreased and reached signicance 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 30C) Warming the Ta from 23 to 30C 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 signicant and sustained reductions O2 (Fig. 6F) in MAP (Fig. 6D), HR (Fig. 6E), and V values that were evident during the rst 12-h dark period and were sustained for 5 days. During the 14-day restricted-feeding protocol within Ta 30C, caloric intake averaged 56% of baseline intake and 44% of intake during the Ta 23C 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 signicant, 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
282 MAY 2002

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

R1463

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 23C. 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).

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 30C 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 leptindecient ob/ob and wild-type mice. Our work extends

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

food within the rst 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 23C) 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 rst 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 23C. 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).
282 MAY 2002

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

R1464

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

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 23C. 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.

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

these ndings 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 uctuations 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 23C (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 nding 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 23C, 5 days with Ta 30C, 14 food-restriction days (60% baseline intake), and 3 refeeding days with Ta 30C. 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. 30C day 5 (one-way repeated-measures ANOVA).
282 MAY 2002

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE

R1465

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 23C, baseline day 5 with Ta 30C, and food-restriction day 14 with Ta 30C. 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.

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

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 dened. 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 ndings 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 29C reduces MAP, HR, and V lean and obese Zucker rats (34). Thus standard housing conditions (Ta 21 24C) 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, reects 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 1C in the light phase. Temperatures lower than 23C 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 outow 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 outow in response to thermoregula282 MAY 2002

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

R1466

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE obesity-related hypertension. J Clin Invest 105: 1243 1252, 2000. Bartholomew GA, Vleck D, and Vleck CM. Instantaneous measurements of oxygen consumption during preight warm up and postight cooling in sphinged and saturniid moths. J Exp Biol 90: 17 32, 1981. Beck B. KOs and organisation of peptidergic feeding behavior mechanisms. Neurosci Biobehav Rev 25: 143 158, 2001. Blaxter K. Energy Metabolism in Animals and Man. Cambridge, UK: Cambridge University Press, 1989. Brown D, Livesey G, and Dauncey MJ. Inuence of mild cold on the components of 24 hour thermogenesis in rats. J Physiol (Lond) 441: 137 154, 1991. Butz GM and Davisson RL. Long-term telemetric measurement of cardiovascular parameters in awake mice: a physiological genomics tool. Physiol Genomics 5: 89 97, 2001. Carlson SH and Wyss JM. Long-term telemetric recording of arterial pressure and heart rate in mice fed basal and high NaCl diets. Hypertension 35: 1 5, 2000. Challet E, Solberg LC, and Turek FW. Entrainment in calorie-restricted mice: conicting zeitgebers and free-running conditions. Am J Physiol Regulatory Integrative Comp Physiol 274: R1751 R1761, 1998. Chambers JB, Williams TD, Nakamura A, Henderson RP, Overton JM, and Rashotte ME. Cardiovascular and metabolic responses of hypertensive and normotensive rats to one wk of cold exposure. Am J Physiol Regulatory Integrative Comp Physiol 279: R1486 R1494, 2000. Doring H, Schwarzer K, Nuesslein-Hildesheim B, and Schmidt I. Leptin selectively increases energy expenditure of food-restricted lean mice. Int J Obes Relat Metab Disord 22: 83 88, 1998. Duffy PH, Feuers RJ, and Hart RW. Effect of chronic caloric restriction on the circadian regulation of physiological and behavioral variables in old male B6C3F1 mice. Chronobiol Int 7: 291 303, 1990. Ernsberger P and Nelson DO. Effects of fasting and refeeding on blood pressure are determined by nutritional state, not by body weight change. Am J Hypertens 1 Suppl: 153 157, 1988. Gavrilova O, Leon LR, Marcus-Samuels B, Mason MM, Castle AL, Refetoff S, Vinson C, and Reitman ML. Torpor in mice is induced by both leptin-dependent and -independent mechanisms. Proc Natl Acad Sci USA 96: 14623 14628, 1999. Gordon CJ. Temperature Regulation in Laboratory Rodents. New York: Cambridge University Press, 1993. Grassi G, Seravalle G, Colombo M, Bolla G, Cattaneo BM, Cavagnini F, and Mancia G. Body weight reduction, sympathetic nerve trafc, and arterial baroreex in obese normotensive humans. Circulation 97: 2037 2042, 1998. Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, and Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 94: 8878 8883, 1997. Himms-Hagen J. Food restriction increases torpor and improves brown adipose tissue thermogenesis in ob/ob mice. Am J Physiol Endocrinol Metab 248: E531 E539, 1985. Himms-Hagen J. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB J 4: 2890 2898, 1990. Himms-Hagen J. Physiological roles of the leptin endocrine system: differences between mice and humans. Crit Rev Clin Lab Sci 36: 575 655, 1999. Ikeda T, Gomi T, Hirawa N, Sakurai J, and Yoshikawa N. Improvement of insulin sensitivity contributes to blood pressure reduction after weight loss in hypertensive subjects with obesity. Hypertension 27: 1180 1186, 1996. Irvine RJ, White J, and Chan R. The inuence of restraint on blood pressure in the rat. J Pharmacol Toxicol Methods 38: 157 162, 1997. Ishii K, Kuwahara M, Tsubone H, and Sugano S. The telemetric monitoring of heart rate, locomotor activity, and body temperature in mice and voles (Microtus arvalis) during ambient temperature changes. Lab Anim 30: 7 12, 1996. Janssen BJ, Leenders PJ, and Smits JF. Short-term and long-term blood pressure and heart rate variability in the mouse.
282 MAY 2002

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 23C. 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 Our ndings 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 inuence Ta has on cardiovascular function in mice. It is indisputable that current standard housing conditions (Ta 21 24C) 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 Programs 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 Afliate. REFERENCES 1. Aizawa-Abe M, Ogawa Y, Masuzaki H, Ebihara K, Satoh N, Iwai H, Matsuoka N, Hayashi T, Hosoda K, Inoue G, Yoshimasa Y, and Nakao K. Pathophysiological role of leptin in

2.

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

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

9.

10.

11.

12. 13.

14. 15.

16.

17. 18. 19. 20.

21. 22.

23.

AJP-Regulatory Integrative Comp Physiol VOL

www.ajpregu.org

NEGATIVE ENERGY BALANCE AND CV FUNCTION IN MICE Am J Physiol Regulatory Integrative Comp Physiol 278: R215 R225, 2000. Just A, Faulhaber J, and Ehmke H. Autonomic cardiovascular control in conscious mice. Am J Physiol Regulatory Integrative Comp Physiol 279: R2214 R2221, 2000. Klaus S, Munzberg H, Truloff C, and Heldmaier G. Physiology of transgenic mice with brown fat ablation: obesity is due to lowered body temperature. Am J Physiol Regulatory Integrative Comp Physiol 274: R287 R293, 1998. Koizumi A, Tsukada M, Wada Y, Masuda H, and Weindruch R. Mitotic activity in mice is suppressed by energy restriction-induced torpor. J Nutr 122: 1446 1453, 1992. Koizumi A, Wada Y, Tuskada M, Kayo T, Naruse M, Horiuchi K, Mogi T, Yoshioka M, Sasaki M, Miyamaura Y, Abe T, Ohtomo K, and Walford RL. A tumor preventive effect of dietary restriction is antagonized by a high housing temperature through deprivation of torpor. Mech Ageing Dev 92: 67 82, 1996. Kushiro T, Kobayashi F, Osada H, Tomiyama H, Satoh K, Otsuka Y, Kurumatani H, and Kajiwara N. Role of sympathetic activity in blood pressure reduction with low calorie regimen. Hypertension 17: 965 968, 1991. Mills PA, Huetteman DA, Brockway BP, Zwiers LM, Gelsema AJ, Schwartz RS, and Kramer K. A new method for measurement of blood pressure, heart rate, and activity in the mouse by radiotelemetry. J Appl Physiol 88: 1537 1544, 2000. Morhardt JE. Heart rates, breathing rates and the effects of atropine and acetylcholine on white-footed mice (Peromyscus sp.) during daily torpor. Comp Biochem Physiol 33: 441 457, 1970. Morrison SF. Differential control of sympathetic outow. Am J Physiol Regulatory Integrative Comp Physiol 281: R683 R698, 2001. Morrison SF. Differential regulation of sympathetic outows to vasoconstrictor and thermoregulatory effectors. Ann NY Acad Sci 940: 286 298, 2001. Overton JM, VanNess JM, and Casto RM. Food restriction reduces sympathetic support of blood pressure in spontaneously hypertensive rats. J Nutr 127: 655 660, 1997. Overton JM, Williams TD, Chambers JB, and Rashotte ME. Cardiovascular and metabolic responses to fasting and thermoneutrality are conserved in obese Zucker rats. Am J Physiol Regulatory Integrative Comp Physiol 280: R1007 R1015, 2001.

R1467

24. 25.

26. 27.

28.

29.

30. 31. 32. 33. 34.

35. Overton JM, Williams TD, Chambers JB, and Rashotte ME. Central leptin infusion attenuates the cardiovascular and metabolic effects of fasting in rats. Hypertension 37: 663 669, 2001. 36. Rashotte ME, Basco PS, and Henderson RP. Daily cycles in body temperature, metabolic rate, and substrate utilization in pigeons: inuence of amount and timing of food consumption. Physiol Behav 57: 731 746, 1995. 37. Robinson SW, Dinulescu DM, and Cone RD. Genetic models of obesity and energy balance in the mouse. Annu Rev Genet 34: 687 745, 2000. 38. Rohrer DK, Chruscinski A, Schauble EH, Bernstein D, and Kobilka BK. Cardiovascular and metabolic alterations in mice lacking both 1- and 2-adrenergic receptors. J Biol Chem 274: 16701 16708, 1999. 39. Swoap SJ. Altered leptin signaling is sufcient, but not required, for hypotension associated with caloric restriction. Am J Physiol Heart Circ Physiol 281: H2473 H2479, 2001. 40. Swoap SJ, Boddell P, and Baldwin KM. Interaction of hypertension and caloric restriction on cardiac mass and isomyosin expression. Am J Physiol Regulatory Integrative Comp Physiol 268: R33 R39, 1995. 41. Talan MI, Kirov SA, and Kosheleva NA. Nonshivering thermogenesis in adult and aged C57BL/6J mice housed at 22 degrees C and at 29 degrees C. Exp Gerontol 31: 687 698, 1996. 42. Van Vliet BN, Chafe LL, Antic V, Schnyder-Candrian S, and Montani JP. Direct and indirect methods used to study arterial blood pressure. J Pharmacol Toxicol Methods 44: 361 373, 2000. 43. Van Ness JM, Casto RM, and Overton JM. Antihypertensive effects of food-intake restriction in aortic coarctation hypertension. J Hypertens 15: 1253 1262, 1997. 44. Webb GP, Jagot SA, and Jakobson ME. Fasting-induced torpor in Mus musculus and its implications in the use of murine models for human obesity studies. Comp Biochem Physiol A Physiol 72: 211 219, 1982. 45. Williams TD, Chambers JB, May OL, Henderson RP, Rashotte ME, and Overton JM. Concurrent reductions in blood pressure and metabolic rate during fasting in the unrestrained SHR. Am J Physiol Regulatory Integrative Comp Physiol 278: R255 R262, 2000.

Downloaded from http://ajpregu.physiology.org/ by guest on February 11, 2013

AJP-Regulatory Integrative Comp Physiol VOL

282 MAY 2002

www.ajpregu.org