INTRODUCTION

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IN MAMMALIAN INFANTS, brown adipose tissue (BAT) thermogenesis is the primary means of endogenous heat production (9, 12, 17, 21). During moderate cold exposure in infant rats, BAT thermogenesis is regulated stably (6) and has been shown to protect sleep-related behaviors and to suppress production of ultrasonic vocalizations (8, 19). The relationship between BAT thermogenesis and cardiac rate regulation has also been investigated. Specifically, we found that, during moderate cold exposure, BAT thermogenesis was activated and cardiac rate was maintained (7, 13). In contrast, when BAT thermogenesis was overwhelmed during extreme cold exposure or was blocked with a ganglionic blocker, cardiac rate decreased as a function of air temperature.

Our previous work (7, 13) provided strong evidence of a relationship between BAT thermogenesis and cardiac rate, although the results were primarily descriptive in nature. In the present experiments, BAT was activated pharmacologically in infant rats at a thermoneutral air temperature to further examine the effects of BAT thermogenesis on cardiac rate. In experiment 1, 7- to 8-day-old rats were pretreated with saline or the ganglionic blocker chlorisondamine; chlorisondamine was administered to prevent the activation of other neural mechanisms that could potentially influence cardiac rate. Pups were then injected with CL-316243 (a ₃-adrenoceptor agonist) to stimulate BAT thermogenesis, and cardiac rate was measured. In experiment 2, 8-day-old chlorisondamine-treated rats were again injected with CL-316243. After BAT thermogenesis was activated, the region of skin overlying BAT was cooled with a thermode and cardiac rate was monitored. The results of both experiments support the hypothesis that BAT thermogenesis protects cardiac rate during cold challenge.

	METHODS

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Subjects. Seventeen 7- to 8-day-old male rat pups from twelve litters were used: twelve 7- to 8-day-old pups in experiment 1 and five 8-day-old pups in experiment 2. At the time of testing, pups in experiment 1 weighed 16.7-22.2 g and pups in experiment 2 weighed 17.1-21.5 g. Pups used in both experiments were born to Harlan Sprague-Dawley female rats maintained in the animal colony at the University of Iowa. Mothers and their litters were housed in standard laboratory cages (48 × 20 × 26 cm) in which food and water were available ad libitum. Litters were culled to eight pups within 3 days after birth (day of birth = day 0). All animals were maintained on a 12:12-h light-dark schedule with lights on at 0600.

Test environment. For experiments 1 and 2, pups were placed inside a double-walled glass metabolic chamber (for dimensions and detailed description see Ref. 7). Briefly, by pumping temperature-controlled water through the walls of the chamber, air temperature was controlled. Access holes on the side of the chamber as well as connectors on the lid allowed for the passage of air throughout the chamber and connections for all physiological recordings. Once inside the chamber, the pups were placed on a polyethylene mesh platform.

Temperature measurements. Air temperature (T_a) and physiological temperatures were measured using chromel-constantan thermocouples (Omega, Stamford, CT). All thermocouples were calibrated to within 0.1°C of a mercury thermometer and fed into a computerized data acquisition system (National Instruments, Austin, TX). T_a values were obtained using the acquired computer average of two thermocouples placed beneath the mesh platform: one near the center of the chamber and one close to the outer diameter. Physiological temperatures were attained using thermocouples attached to the dorsal skin surface of the animal by use of the adhesive collodion. One thermocouple was attached in the interscapular region directly above the BAT pad, providing a measure of interscapular temperature (T_is). A second thermocouple was attached ~1 cm rostral to the base of the tail in the lumbosacral region; this thermocouple provided a measure of skin temperature (T_back) distant from the site of heat production.

Oxygen consumption measurements. Compressed air passed through a regulator and was split into two lines. One line passed through a digital flowmeter (Omega); the air in the line was then humidified and circulated through the metabolic chamber at 300 ml/min. The air was drawn from the chamber and desiccated, then it was drawn through one of two channels of an electrochemical oxygen analyzer (Ametek, Pittsburgh, PA). The second line of air flowed directly from the regulator to the second channel of the oxygen analyzer. The oxygen content of each airstream was measured simultaneously, and the percent difference in concentration was computed to within 0.001%. The percent difference was then fed into the computerized data acquisition system and transformed to oxygen consumption (O₂) in milliliters of oxygen per kilogram per minute.

Thermode. In experiment 2 the temperature of the interscapular region was manipulated using a custom-built thermode. The conductive surface and the base of the thermode were fashioned from the head of a brass flat-head screw (0.6 cm diameter). A piece of plastic tubing (1 cm long, 0.6 cm ID) was fitted over the head of the screw and sealed in place with cyanoacrylate (Surehold, Chicago, IL). The height of the thermode was stabilized with a plastic sheath that fit tightly inside the tubing above the conductive area. Two small pieces of silicone tubing (0.2 cm OD) were fit into the top of the body of the thermode, and the top was then sealed with cyanoacrylate. Two longer pieces of silicone tubing (7 cm long, 0.2 cm ID) were placed over the smaller tubes emerging from the top of the thermode. Silicone sealant was used to prevent any leaks from the thermode to the intake and outlet tubes. From the inside of the metabolic chamber the thermode was attached to a connector protruding from one of the chamber's side access holes. On the outside of the chamber a second water circulator was connected to the intake and outlet tubes of the thermode. The thermode temperature was controlled by circulating water through the body of the thermode.

Data acquisition. For both experiments, thermal and O₂ measures were acquired four times per minute by use of a customized LabView (National Instruments, Austin, TX) data acquisition program for Macintosh. Electrocardiogram (ECG) data were acquired simultaneously on a second data acquisition system by use of one of two methods. For one method, raw ECG data were acquired at a rate of 1,000/s, and the times between successive R waves were determined after the test (7). For the other method, interbeat intervals were calculated at the time of data acquisition at a rate of 30/min. These two methods yielded identical results. Finally, thermal, O₂, and ECG measures were recorded simultaneously.

Drugs. The ₃-agonist CL-316243 (Wyeth-Ayerst Research, Pearl River, NY) and chlorisondamine hydrochloride (Ciba-Geigy, Summit, NJ) were dissolved in isotonic saline before use. All drug injections were administered at a volume of 1 µl/g body wt sc.

Procedure. On the day of testing a pup was removed from its home cage. Each pup had been fed recently, as evidenced by the presence of a milk band visible through the abdominal skin. The pup was weighed and lightly anesthetized with ether (exposure 1 min). After anesthetization the pup was placed in an incubator maintained at ~35-36°C. Three ECG leads were implanted transcutaneously, and the thermocouples for physiological temperature measures were also attached; leads and thermocouples were secured to the skin with collodion. In experiment 2, after the thermocouples were attached the thermode for cooling the interscapular region was secured directly over the site of the interscapular thermocouple with collodion. The pup was then placed inside the metabolic chamber at a T_a of ~35°C and allowed to acclimate for 45 min. The ECG leads were connected to a differential amplifier (A-M Systems, Everett, WA) that filtered and amplified the signal before it was acquired by the computer.

Data analysis. Thermal, metabolic, and cardiac data were imported into StatView 4.5 for the Macintosh. Interbeat interval was converted to cardiac rate in beats per minute, and mean cardiac rates for each pup were calculated from 30 data points at each data acquisition period of the experiment. Similarly, the thermal and metabolic data for that same period were used, giving four data points averaged over a 60-s period. For experiment 1 a repeated-measures ANOVA was used to test for differences in the variables across time, and  was set at 0.05. Data were analyzed from 1-min periods immediately before and 5, 10, 15, 30, 45, and 60 min after administration of the ₃-agonist. For O₂, data were analyzed immediately before and 15, 30, 45, and 60 min after administration of the ₃-agonist; O₂ data were unavailable at the 5- and 10-min time points, because the oxygen analysis system requires 15 min to restabilize after opening. Post hoc paired t-tests were used to test for differences between the baseline value (pre-₃-agonist) and each subsequent time point. For experiment 2, paired t-tests were used to test for differences between successive experimental periods. For both experiments, a Bonferroni correction was used to adjust  for multiple comparisons ( = 0.008 and 0.0167 for experiments 1 and 2, respectively).

	RESULTS

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Experiment 1. Stimulation of BAT thermogenesis by the ₃-agonist increased T_is significantly in saline- and chlorisondamine-pretreated pups (86.8  F_6,30  142.12, P < 0.0001; Fig. 1). Administration of the drug produced rapid increases in T_is. For pups pretreated with saline, T_is increased significantly over baseline within 5 min of drug administration (t₅ = 11.7, P < 0.0001). For pups pretreated with chlorisondamine, T_is increased significantly over baseline within 10 min of drug administration (t₅ = 16.1, P < 0.0001). For both groups the increases in T_is were maintained for the remainder of the test period (10.7  t₅  19.0, P < 0.0001). The average increase in T_is after administration of the ₃-agonist was ~2.5°C higher than baseline for both groups.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Interscapular temperature (Tis), skin temperature (Tback), cardiac rate, and O2 consumption (O2) for 1-wk-old rats pretreated with saline or chlorisondamine and then injected with 3-agonist. Thermal data are presented together: , Tis; , Tback. Activation of brown adipose tissue thermogenesis by 3-agonist led to an increase in cardiac rate in both groups. bpm, Beats/min. Values are means ± SE. * Significantly different from baseline.

Experiment 2. As in experiment 1, T_is increased 2°C over baseline values after administration of the ₃-agonist (t₄ = 6.5, P < 0.005; Fig. 2). When cool water was circulated through the thermode, even with BAT thermogenesis stimulated, the interscapular region was cooled significantly (t₄ = 13.1, P < 0.0005). When circulation of water through the thermode was terminated, T_is again increased significantly (t₄ = 19.2, P < 0.0001).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Tis/thermode temperature (Tthermode), Tback, cardiac rate, and O2 for 1-wk-old rats after pretreatment with chlorisondamine, after administration of 3-agonist, during interscapular cooling with thermode, and after thermode was turned off and pups were allowed to reheat. Activation of brown adipose tissue thermogenesis by 3-agonist increased cardiac rate, and this effect was counteracted by focal cooling of interscapular region. Values are means ± SE. * Significantly different from previous time period.

	DISCUSSION

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Many years ago, it was demonstrated that cardiac rate is responsive to changes in temperature in vivo and in vitro (1, 11, 14). When the thermogenic function of BAT was elucidated, anatomic studies led to the suggestion that BAT thermogenesis provides heat focally to vital organs in the thoracic cavity (16, 18). Previous work from our laboratory built on these earlier studies to demonstrate that, in infant rats, BAT thermogenesis protects against bradycardia during moderate, but not extreme, cold exposure (7, 13). In addition, when pups were injected with a ganglionic blocker and thus BAT thermogenesis was inhibited, cardiac rate fell in lock step with decreasing T_is (7). These results provided the first evidence in support of the hypothesis that interscapular BAT thermogenesis protects cardiac function by supplying warmed venous blood to the heart.

The results of the present study provide further support for the hypothesized link between BAT thermogenesis and the maintenance of cardiac rate during cold exposure. In experiment 1, pharmacological activation of BAT thermogenesis by use of a selective ₃-agonist led to an increase in cardiac rate in pups tested in a thermoneutral environment. Moreover, this tachycardia was observed even in pups pretreated with a ganglionic blocker, indicating that changes in cardiac rate during BAT thermogenesis do not require neural control of the heart. Because the dose of chlorisondamine used here was identical to that used in previous experiments to completely block BAT thermogenesis (7, 20), we are confident that the sympathetic blockade was complete in the present experiments. Experiment 2 showed that cooling of the interscapular region reverses the tachycardia produced by pharmacological activation of BAT thermogenesis. It should be stressed that these results should not be interpreted to mean that thermal manipulation of other areas of the body could not have produced similar results. Rather, the present experiment was designed only to determine whether cooling the region that overlies the interscapular BAT pad is sufficient to reverse the tachycardia induced by the ₃-agonist. Again, because this effect of focal cooling was observed in ganglionically blocked animals, it is apparent that neural mechanisms are not required. The results of both experiments provide strong evidence that manipulation of T_is, by the activation of BAT thermogenesis or by cooling of the overlying skin, modulates cardiac rate.

Cardiac rate can be increased by the nonselective stimulation of -adrenoceptors. However, CL-316243 has a high selectivity for ₃-adrenoceptors and low affinity for ₁- and ₂-receptors, precluding direct effects on cardiac rate or secondary effects mediated through the activation of baroreceptors (4). Tavernier et al. (22) and Berlan et al. (3) found that the ₃-adrenoceptor agonists BRL-37344 and CGP-12177 increase cardiac rate in adult dogs. However, both agents induced hypotension, and their ability to increase cardiac rate was eliminated after sinoaortic denervation, indicating that the tachycardia was produced by activation of baroreceptor mechanisms. Because, in the present study, increases in cardiac rate after CL-316243 administration were similar in ganglionically blocked and nonblocked animals, the alterations in cardiac rate could not have been due to a baroreceptor-mediated increase in sympathetic outflow. In addition, preweanling rats do not develop effective baroreceptor mechanisms regulating cardiac rate until ~12-15 days of age (2, 10). Therefore, in the present study it is not likely that the increases in cardiac rate after activation of BAT were mediated by the nonselective activation of -adrenoceptors.

It is apparent that cardiac rate is determined by a combination of factors that includes cardiac temperature and autonomic activation (7). With respect to the determinants of cardiac temperature, interscapular BAT is ideally suited for the efficient delivery of warmed blood to the heart (17). Nonetheless, it must be stressed that cardiac temperature can be influenced by the flow of venous blood from all regions of the body and that the temperature of interscapular BAT will be more or less important for the determination of cardiac temperature, depending on the ability of infant rats to regulate blood flow to and from the extremities.

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