INCREASE IN THE BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE IN RESPONSE TO COLD EXPOSURE AND NOREPINEPHRINE IN THE RAT

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1 The Japanese Journal of Physiology 17, pp , 1967 INCREASE IN THE BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE IN RESPONSE TO COLD EXPOSURE AND NOREPINEPHRINE IN THE RAT Akihiro KUROSHIMA, Norimichi KONNO AND Shinji ITOH Department of Physiology, Hokkaido University School of Medicine, Sapporo Recently a number of reports have appeared which indicate the thermoregulatory importance of brown adipose tissue in various mammals. Although a marked hypertrophy of interscapular brown adipose tissue in rats acclimatized to cold was found by CRAMER1) in 1920, SMITH and his colleagues2-4) were the first who have thrown a new light on the physiological significance of brown adipose tissue in cold acclimation. They demonstrated a greatly increased oxygen consumption of this tissue in cold-acclimated rats and emphasized its thermogenic role. Thereafter a great deal of physiological and biochemical evidence has accumulated which implicates the brown adipose tissue as the major site of thermoregulatory heat production in response to cold. It is known that brown adipose tissue has an unusually rich blood supply. A marked increase in the blood flow through brown adipose tissue has been shown in hibernators during arousal5,6), in new-born guinea pigs7) and new-born rabbits8,9) following administration of catecholamines. Intravenous infusion of norepinephrine into cold-adapted rats was also reported to cause a marked increase in the blood flow through brown adipose tissue10). However, available data at the present time are rather qualitative, except those of HEIM and HuLL8,9) who measured the blood flow as well as oxygen consumption of brown adipose tissue in the new-born rabbit. Therefore, we have undertaken to know more quantitative changes in the blood flow through brown adipose tissue under various experimental conditions in the rat. Since the heat produced by brown adipose tissue should be rapidly transferred to the rest of body through the blood stream, it is worth-while to measure the volume of blood flow in this tissue in order to elucidate its thermoregulatory role. Received for publication January 12,

2 524 A. KUROSHIMA, N. KONNO AND S. ITOH METHODS Adult Wistar strain rats housed at a constant temperature of 20 Ž were used throughout the experiments. Rat biscuits and water were given ad libitum. Chronic cold exposure was performed by placing the animals in a cold room at 4-5 Ž. In acute experiments animals were anesthetized with Nembutal and exposed to cold at 3 Ž for 30min. Tissue blood flow and cardiac output were estimated by the Sapirstein's method11) based on fractional distribution of radioactive indicator, 86Rb, under Nembutal anesthesia. Details of the procedure were described elsewhere12). Thyroidectomized rats were allowed to be kept at ordinary room temperature for 3 days after the surgery prior to chronic cold exposure. Adrenalectomized animals were given 1% saline solution as drinking fluid after the surgery. Norepinephrine (Sankyo Pharmaceutical Co.) was infused through a canule inserted into the femoral vein at a rate of 2 ƒêg per min for 10min. Other chemicals used in this experiment were methylthiouracil (Methiosil, Chugai Pharmaceutical Co). and hexamethonium bromide (Methobromin, Yamanouchi Pharmaceutical Co.). RESULTS 1. Effect of cold exposure. TABLE 1 summarizes the effect of cold exposure on the blood flow through interscapular brown adipose tissue and right epididymal fat pad of male rats. Chronic exposure to cold at 4-5 Ž for 2 weeks brought about a marked hypertrophy of the brown adipose tissue and a significant atrophy of the white one. Cardiac output increased 37 per cent above the control level. The blood flow through brown adipose tissue was found to increase strikingly from 0.31 }0.04 to 2.02 }0.20ƒÊl/min/mg tissue, that is, 6.7-fold increment per unit weight. Since the tissue weight was approximately doubled after the cold exposure, the blood flow in whole gland showed 14.7-fold increment. On the other hand, the total blood flow in white fat pad was not altered by the chronic cold exposure. Increase of 2-fold per mg tissue is regarded to be due to the loss of stored fat in this tissue. Prolonged exposure at 4-5 Ž for 5 weeks did not cause further increase in the blood flow through brown adipose tissue beyond the level observed after 2 week exposure. Small difference in the mean values between at 2 and 5 weeks was not statistically significant. Acute exposure to cold at 3 Ž for 30min under Nembutal anesthesia also provoked a considerable increase, 2.4-fold of the control level, in the blood flow through interscapular brown adipose tissue, while no change was observed in that of white one. The cardiac output remained unchanged under the experimental conditions employed (TABLE 2). 2. Effect of norepinephrine infusion. Norepinephrine was infused at a rate of 2pg/min/rat for 10min into cold-acclimated and non-acclimated rats. Control groups were infused with saline solution in place of norepinephrine. As shown in TABLE 3, in normal rats the infusion of norepinephrine induced 2-fold rise

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8 530 A. KUROSHIMA, N. KONNO AND S. ITOH in the cardiac output. The blood flow in brown adipose tissue increased from 0.29 }0.08 to 1.67 }0.19ƒÊl/min/mg, while in the white fat tissue from 0.06 }0.010 to 0.11 }0.016ƒÊl/min/mg. Similar results were also obtained in female rats (TABLE 4). In cold-acclimated rats which had been reared at an ambient temperature of 4-5 Ž for 2 weeks, the infusion of norepinephrine failed to produce any significant change in the cardiac output. The blood flow through brown adipose tissue increased to 2.67 }0.31ƒÊl/min/mg. However, this figure was not statistically significant in comparison with that in saline-infused controls. On the other hand, in white fat tissue about 2-fold increase in the blood flow was observed following norepinephrine infusion. It should be noticed here that the cardiac output and the blood flow in brown adipose tissue in saline-infused controls were as great as in the non-acclimated animals infused with norepinephrine (TABLE 3 and 5). 3. Effect of hexamethonium bromide. It has been shown that cold-induced heat production can be effectively blocked by hexamethonium bromide in young kittens, puppies13) and new-born guinea pigs7). In order to clarify whether the ganglion-blocking agent affects the blood flow in brown adipose tissue, the effect was tested in animals exposed to cold. A considerable decrease in the cardiac output from 99 }8 to 49 }6ml/min/rat and a marked reduction in the blood flow through interscapular brown adipose tissue from 0.31 }0.04 to 0.08 }0.01 ƒêl/min/mg tissue were found 45min after subcutaneous injection of hexamethonium bromide in a dose of 7.5mg/100g body weight. However, on exposure to cold (3 Ž, 30min) 15min after hexa- FIG.1. Effect of acute cold exposure on the blood flow through adipose tissues in normal and hexamethonium-treated (HMBr) rats.

9 BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE 531 methonium injection the blood flow in brown adipose tissue increased markedly as high as to 0.56 }0.05 Đl/min/mg (FIG.1). 4. Effect of thyroidectomy, methylthiouracil treatment and adrenalectomy. Endocrine influence on the response of blood flow to cold exposure and norepinephrine infusion was surveyed in thyroid hormone deficient and adrenalectomized animals. Cardiac output and blood flow in interscapular brown adipose tissue in thyroidectomized rats 10 to 14 days after the surgery (TABLE 3) and methylthiouracil-treated ones in a daily dose of 20 mg per rat for about 3 weeks (TABLE 4) were not different from the normal levels when these animals were kept at ordinary room temperature. The weight of interscapular brown adipose tissue was, however, significantly smaller in the thyroidectomized rats than in the normal ones (FIG.2). The weight of thyroid gland increased markedly FIG.2. Effect of chronic cold exposure on the weight (mg/100g) of interscapular brown adipose tissue of intact, thyroidectomized and methylthiouraciltreated (MTU) rats.

10 532 A. KUROSHIMA, N. KONNO AND S. ITOH after methylthiouracil treatment (17.2mg/100g body weight) and the blood flow in thyroid gland was 11.9 }1.1ƒÊlimin/mg in the treated male rats. These rats with thyroid hormone deficiency responded well with the increased blood flow in brown adipose tissue to cold exposure at 3 Ž for 30min (FIG.3). The mean values after cold exposure were 1.02 }0.08ƒÊl/min/mg in thyroidectomized rats and 1.22 }0.25 in methylthiouracil-treated ones. Norepinephrine also produced a significant increase in the blood flow, showing a similar response to that observed in the control group (TABLE 3 and 4). On the other hand, chronic exposure to cold for 10 to 14 days was ineffective to produce the increment of blood flow in brown adipose tissue and rather suppressed it in the thyroidectomized rats (FIG.4). Reduced cardiac output and high mortality on chronic exposure to cold were observed in this group of animals. It is note-worthy that the weight of interscapular brown adipose tissue increased significantly on chronic exposure to cold even in the thyroidectomized or methylthiouracil-treated rats, in spite of the reduced blood flow (FIG.2). Similar results were obtained in adrenalectomized rats. Acute exposure to cold (TABLE 2) and intravenous infusion of norepinephrine (TABLE 3) produced 4.4- and 4.7-fold increase, respectively, in the blood flow through FIG.3. Effect of acute cold exposure on the blood flow through interscapular brown adipose tissue of intact, thyroidectomized and methylthiouraciltreated (MTU) rats.

11 BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE 533 brown adipose tissue. 5. Effect of high ambient temperature. The blood flow in brown adipose tissue was measured after the animals were reared at 30 Ž for 2 weeks. No significant change was found in the blood flow and the weight of brown adipose tissue as compared with those values in rats kept at 20 Ž (TABLE 1). DISCUSSION Direct measurement of the temperature of brown adipose tissue in rats14,5), hibernating hamsters8) and new-born rabbits16) indicated that local heat production in brown adipose tissue is responsible for the cold-induced thermogenesis. It has been also suggested that brown adipose tissue in human new-born infants produced the heat on exposure to cold17). In the present study it was clearly demonstrated that chronic as well as acute cold exposure resulted in a marked increase in the blood flow through brown adipose tissue in the rat. Employing a direct method for measuring venous outflow from brown adipose tissue in new-born rabbits, HEIM and FIG.4. Effect of chronic cold exposure on the blood flow through interscapular brown adipose tissue of intact, thyroidectomized and methylthiouraciltreated (MTU) rats.

12 534 A. KUROSHIMA, N. KONNO AND S. ITOH HULL9) observed that during cold exposure the mean blood flow in brown adipose tissue increased from 90 to 304ml/100g tissue/min and during norepinephrine infusion from 87 to 360ml. They have shown that at rest about one tenth and during norepinephrine infusion about one quarter of the cardiac output went to brown adipose tissue in the new-born rabbit. BULLARD and FUNKHOUSER5), using the method of Sapirstein, revealed in the thirteen-lined ground squirrel that the rate of blood flow in brown adipose tissue was much higher than that of any other organ or tissue during arousal from hibernation. Similarly, JOHANSEN6) reported several times higher blood flow level in arousing Arctic ground squirrels than that in awake animals. The results obtained in the present investigation are well in accord with these previous observations. However, in the rat the total mass of brown adipose tissue is less than 1 per cent of the body weight and so the heat produced by this tissue may be too small relative to total heat requirement in animals in the cold. In this respect, SMITH and ROBERTS4) proposed that the location and vascular geometry of brown adipose tissue in the rat may contribute the counter-current heat exchange system and provide specifically the vital organs such as the brain stem and the heart with the heat produced by brown adipose tissue. Although the evidence is not more than suggestive, it is likely that the increase in blood flow through brown adipose tissue on exposure to cold reflects an efficient heat transfer to the vital organs, serving the maintenance of core temperature. HSIEH and CARLSON18) and DEPOCAs19) demonstrated that norepinephrine exerts an immediate and marked calorigenic effect in cold-acclimated rats. Cardiovascular and pulmonary effects of norepinephrine are much greater in coldacclimated rats than in warm-acclimated ones10). In new-born mammals norepinephrine causes a marked increase in the oxygen consumption and colonic temperature20). The temperature overbrown adipose tissue rose rapidly during the infusion of norepinephrine in the new-born rabbit, while the colonic and subcutaneous lumber temperatures showed a slower and smaller rise). When brown adipose tissue was surgically excised, the rise in oxygen consumption was greatly reduced21). These observations suggest that norepinephrine is the mediator of metabolic response of brown adipose tissue to cold exposure. It was found that brown adipose tissue contains a large amount of norepinephrine22,23). The local release of norepinephrine in brown adipose tissue in response to cold exposure may stimulate the heat production in this tissue. It has been reported by HEIM and HULL8,9) that the infusion of norepinephrine caused an immediate and large rise in the blood flow through brown adipose tissue of the new-born rabbit. The present study showed that this was true in the rat. Hexamethonium, which blocks transmission in autonomic ganglia, has been reported to reduce the increase in oxygen consumption caused by cold exposure13,24). However, the response to acute cold exposure was not influenced by the previous administration of hexamethonium (FIG.1).

13 BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE 535 The secretion of norepinephrine, therefore, may not be essential for the increase in blood flow through brown adipose tissue on exposure to cold, although the catecholamine is necessary for the rise in oxygen consumption of this tissue. As to this fact, it is interesting to note that hypoxia greatly reduced the increase in oxygen consumption but not the increase in blood flow in brown adipose tissue caused by norepinephrine infusiono9). EVONUK and HANNONl0), using the same method adopted in the present experiment, estimated the blood flow in the brown adipose tissue of warmadapted and cold-adapted rats during norepinephrine infusion. They pointed to the increased sensitivity to norepinephrine in the blood flow through brown adipose tissue of cold-adapted animals. Contrary to their results, in our study the infusion of norepinephrine did not lead to any appreciable increase in the blood flow in brown adipose tissue. It is not clear what caused the discrepancy between results by us and EVONUK and HANNON. Rather surprisingly, they found a similar level of the blood flow in brown adipose tissue in the coldadapted rats to that in warm-adapted ones, while we demonstrated a markedly increased blood flow in the brown adipose tissue of cold-adapted animals. Failure to demonstrate further increase in the blood flow in brown adipose tissue of cold-adapted rats in our experiments may be due to the maximally increased rate of blood flow by prolonged cold exposure. The thyroid hormone is well known to play an important role in the thermoregulatory heat production in animals exposed to cold. There might be a significant relation between the thyroid and brown adipose tissue in the view-point of cold acclimation. However, on this problem few studies have appeared in the past. Cold exposure is also known to stimulate the adrenocortical activity. Any conclusive data have not been yet presented on the relationship between the adrenal cortex and brown adipose tissue. It seemed of interest to observe whether thyroidectomy or adrenalectomy affects the response of blood flow in brown adipose tissue to cold exposure. The results indicated that thyroidectomized or methylthiouracil-treated rats as well as adrenalectomized ones fairly responded to acute cold exposure and norepinephrine with a marked increase in the blood flow. It may be inferred that both the thyroid and adrenocortical hormones are not involved in the response of blood flow in brown adipose tissue to cold. When thyroidectomized or methylthiouracil-treated rats were exposed to cold chronically, the blood flow in brown adipose tissue did not increase, but rather decreased in the thyroidectomized group. In this group the cardiac output also reduced significantly. It is widely recognized that the thyroid hormone is necessary for the survival of animals kept in cold. Actually in the present experiments one half the number of thyroidectomized rats died during 2 week cold exposure. Failure of the blood flow response in these animals may be attributed to the overall disturbances in physiological activities.

14 536 A. KUROSHIMA, N. KONNO AND S. ITOH However, it should be noted here that the hypertrophy of brown adipose tissue occurred on exposure to cold even in thyroid hormone deficient state (FIG.2). SUMMARY Blood flow through interscapular brown adipose tissue and white epididymal or perirnetrial fat was estimated by the Sapirstein's radioactive isotope indicator method in the rat. Chronic cold exposure at 4-5 Ž for 2 weeks as well as acute exposure at 3 Ž for 30min resulted in a marked increase in the blood flow in brown adipose tissue, 14.7-fold and 2.8-fold respectively, while no significant effect was observed in the white adipose tissue. Intravenous infusion of norepinephrine (2ƒÊg/min for 10min) into normal rats produced 6.4-fold increase in the blood flow through brown adipose tissue. Hexamethonium bromide could not prevent the increase of blood flow through brown adipose tissue caused by acute cold exposure. In cold-acclimated rats the infusion of norepinephrine failed to produce further increase in the blood flow of brown adipose tissue. Thyroidectomy, methylthiouracil treatment and adrenalectomy did not affect the response of blood flow through brown adipose tissue to acute cold exposure nor to norepinephrine infusion. Thyroidectomized and methylthiouracil-treated rats failed to respond to chronic cold exposure in increasing the blood flow through brown adipose tissue. The research reported in this document has been made possible through the support and sponsorship of the U. S. Department of Army, through the USA R & D Group (Far East) Office. REFERENCES 1) CRAMER, W.: On glandular adipose tissue and its relation to other endocrine organs and to the vitamine problem. Brit. J. Exp. Pathol. 1: 184 (1920). 2) SMITH, R. E.: Thermoregulation by brown adipose tissue in cold. Feder. Proc. 21: 221 (1962). 3) SMITH, R. E. AND Hock, R. J.: Brown fat: Thermogenic effector of arousal in hibernators. Science 140: 199 (1963). 4) SMITH, R. E. AND ROBERTS, J. C.: Thermogenesis of brown adipose tissue in coldacclimated rats. Amer. J. Physiol. 206: 143 (1964). 5) BULLARD, R. W. AND FUNKHOUSER, G. E.: Estimated regional blood flow by rubidium86 distribution during arousal from hibernation. Amer. J. Physiol. 203: 266 (1962). 6) JOHANSEN, K.: Distribution of blood in the arousing hibernator. Acta physiol. scand. 52: 379 (1962). 7) BRUCK, K.: General aspects of temperature regulation of small subjects. in "The Adaptation of the Newborn Infant to Extra-uterine Life" ed. by J. H. P. JoNxis, H. K. A. VISSER and J. A. TROELSTRA, C. C. THOMAS, Springfield, Ill. pp (1964).

15 BLOOD FLOW THROUGH BROWN ADIPOSE TISSUE 537 8) HEIM, T. AND HULL, D.: Blood flow in brown adipose tissue. J. Physiol. 181: 60P (1965). 9) HEIM, T. AND HULL, D.: The blood flow and oxygen consumption of brown adipose tissue in the new-born rabbit. J. Physiol. 186: 42 (1966). 10) EVONUK, E. AND HANNON, J. P.: Cardiovascular and pulmonary effects of noradrenaline in the cold-acclimatized rat. Feder. Proc. 22: 911 (1963). 11) SAPIRSTEIN, L. A.: Regional blood flow by fractional distribution of indicators. Amer. J. Physiol. 193: 161 (1958). 12) ARIMURA, A., YAMAGUCHI, T., YOSHIMURA, K., IMAZEKI, T. AND ITOH, S.: Role of the neurohypophysis in the release of adrenocorticotrophic hormone in the rat. Jap. J. Physiol. 15: 278 (1965). 13) MOORE, R. E. AND UNDERWOOD, M. C.: Hexamethonium, hypoxia and heat production in new-born and infant kittens and puppies. J. Physiol. 161: 30 (1962). 14) DONHOFFER, S., SARDY, F. AND SZEGVARI, G.: Brown adipose tissue and thermoregulatory heat production in the rat. Nature 203: 765 (1964). 15) DONHOFFER, S. AND SZELENYI, Z.: The role of brown adipose tissue in the non cold-adapted adult rat, guinea pig, ground squirrel and in the young rabbit. Acta physiol. Acad. Sci. Hung. 28: 349 (1965). 16) DAWKINS, M. J. R. AND HULL, D.: Brown adipose tissue and the response of new-born rabbits to cold. J. Physiol. 172: 216 (1964). 17) AHERNE, W. AND HULL, D.: Brown adipose tissue and heat production in the newborn infant. J. Path. Bact. 91: 223 (1966). 18) HSIEH, A. C. L. AND CARLSON, L. D.: Role of adrenaline and noradrenaline in chemical regulation of heat production. Amer. J. Physiol. 190: 243 (1957). 19) DEPOCAS, F.: The calorigenic response of cold-acclimated white rats to infused noradrenaline. Canad. J. Biochem. Physiol. 38: 107 (1960). 20) MOORE, R. E. AND UNDERWOOD, M. C.: Possible role of noradrenaline in control of heat production in the newborn mammals. Lancet 1: 1277 (1960). 21) HULL, D. AND SEGALL, M. M.: The contribution of brown adipose tissue to heat production in the new-born rabbit. J. Physiol. 181 : 449 (1965). 22) SIDMAN, R. L., PERKINS, M. AND WEINER, N.: Noradrenaline and adrenaline content of adipose tissue. Nature 193: 36 (1962). 23) STOCK, K. AND WESTERMANN, E. 0.: Concentration of norepinephrine, serotonin, and histamine, and of amine-metabolizing enzymes in mammalian adipose tissue. J. Lipid Res. 4: 297 (1963). 24) DAMES, G. S. AND MESTYAN, G.: Changes in the oxygen consumption of newborn guinea-pigs and rabbits on exposure to cold. J. Physiol. 168: 22 (1963).