Effects of hypothyroidism and hyperthyroidism on thermogenic responses to selective and nonselective beta-adrenergic agonists in rats

Oxygen consumption (VO2) and mitochondrial guanosine diphosphate (GDP) binding of interscapular brown adipose tissue (BAT) were measured in hypothyroid, hyperthyroid and euthyroid rats after stimulations with selective and nonselective beta-adrenoceptor agonists: BRL 35135A (BRL) and Isoprenaline (ISO). Resting VO2, VO2 increment and mitochondrial GDP binding after beta-adrenergic stimulations were lower in hypothyroid rats than in the euthyroid group. The reduced responses were more marked for ISO than for BRL. Restion VO2 and VO2 increment after beta-adrenergic stimulations were higher in hyperthyroid rats than in the eurthyroid group; the increment was more marked for BRL than for ISO. In hyperthyroidism, mitochondrial GDP binding after BRL and after ISO was in the same magnitude; it was higher in the hyperthyroid than in the euthyroid group after BRL but not after ISO. The different thermogenic responses after ISO and BRL stimulations suggest that BRL is acting on a beta-adrenoceptor differing from the beta-1 and beta-2 adrenoceptors responsible for the effects of ISO. Activation of thermogenesis via the beta-3 adrenoceptor seems to be less dependent on the permissive levels of thyroid hormones than activation via beta-1 and/or beta-2 adrenoceptors. The beta-3 adrenoceptor may be more sensitive to increased levels of thyroid hormones.     

Leanness of Lou/C rats does not require higher thermogenic capacity of brown adipose tissue

Lou/C rats, an inbred strain of Wistar origin, remain lean throughout life and therefore represent a remarkable model of obesity resistance. To date, the exact mechanisms responsible for the leanness of Lou/C rats remain unknown. The aim of the present study was to investigate whether the leanness of Lou/C rats relies on increased thermogenic capacities in brown adipose tissue (BAT).Results showed that although daily energy expenditure was higher in Lou/C than in Wistar rats, BAT thermogenic capacity was not enhanced in Lou/C rats kept at thermoneutrality as demonstrated by reduced thermogenic response to norepinephrine in vivo, similar oxidative activity of BAT isolated mitochondria in vitro, similar levels of UCP1 mRNA and lower abundance of UCP1 protein in interscapular BAT depots. Relative abundance of β₃-adrenergic receptor mRNA was lower in Lou/C BAT while that of GLUT4, FABP or CPT1 was not altered. Activity-related energy expenditure was however considerably increased at thermoneutrality as Lou/C rats demonstrated an impressively high spontaneous running activity in voluntary running wheels. Prolonged cold-exposure (4 °C) depressed the spontaneous running activity of Lou/C rats while BAT thermogenic capacity was increased as reflected by rises in BAT mass, oxidative activity and UCP1 expression.It is concluded that the leanness of Lou/C rats cannot be ascribed to higher thermogenic capacity of brown fat but rather to, at least in part, increased locomotor activity. BAT is not deficient in this rat strain as it can be stimulated by cold exposure when locomotor activity is reduced suggesting some substitution between these thermogenic processes.     

Brown adipose tissue of rats with obesity-inducing ventromedial hypothalamic lesions

Male and female Holtzman rats were made hyperphagic and obese with bilateral radiofrequency heat lesions of the ventromedial hypothalamic (VMH) area. When VMH rats were maintained at 28 degrees C, their brown adipose tissue (BAT) DNA, protein, and cytochrome oxidase contents were normal although more stored lipid was present, as judged from a threefold increase in wet weight. Thermogenic activity of BAT mitochondria was normal in male VMH rats, as judged from the unchanged level of guanosine diphosphate (GDP) binding (known to be a sensitive index of the functional activity of the thermogenic proton conductance pathway), and reduced in female VMH rats. When rats with VMH lesions were exposed to cold (4 degrees C for 24 h), the visible hyperemia of their BAT and normal large increase in mitochondrial GDP binding indicated normal thermogenic responsiveness. We conclude that the medial nuclei of the hypothalamus and associated afferent or efferent nerve tracts do not represent an essential central nervous system link for cold-induced, sympathetic-mediated activation of BAT thermogenesis. It is possible, however, that diet-induced, sympathetic-mediated activation of BAT function and growth might require an intact VMH region because no enhancement of BAT mitochondrial function normally associated with hyperphagia was detected in these hyperphagic VMH-lesioned animals.     

Cold-induced and diet-induced thermogenesis in progesterone-treated rats

Both cold-acclimated rats and rats at thermoneutrality received 1.5 mg/day of progesterone over a period of 15 days by means of two subcutaneously implanted Silastic capsules. Progesterone treatment increased total food intake and body mass gain in both groups of treated animals when compared with their controls at the same ambient temperature. However, the interscapular brown adipose tissue (BAT) of the treated rats showed the same thermogenic activity (assessed by GDP-binding), mass and gross composition as that of their respective controls. If it is assumed that enhanced food intake is the physiological drive for diet-induced thermogenesis, it could be concluded that progesterone inhibits diet-induced thermogenesis at thermoneutrality, but has no effect in cold-induced thermogenesis. However, if the physiological drive for diet-induced thermogenesis is not enhanced food intake, but an imbalance in the diet, then given that the same diet was offered to all animals thoroughout the experimental period, it could be that progesterone does not affect BAT, either at thermoneutrality or in the cold.     

Inhibition of diet-induced thermogenesis during pregnancy in the rat

Both virgin and pregnant rats were maintained at two different ambient temperatures (28° C and 10° C) for 19 days. Virgin rats maintained their daily food intake and body weight at both temperatures. At 28° C pregnant rats showed a greater daily food intake and body weight than virgin ones and their brown adipose tissue suffered regressive changes in composition when compared with brown fat of virgin rats. At 10° C the increases in daily food intake and body weight of pregnant rats took place from day 15–16 of pregnancy onward and foetuses taken from these pregnant rats were smaller than those taken from pregnant rats at 28° C. It is concluded that pregnant rats at thermoneutrality, although hyperphagic, do not show diet-induced thermogenesis. However, it is proposed that pregnant rats in the cold may show BAT cold-induced thermogenesis.     

Sex-associated differences in cold-induced UCP1 synthesis in rodent brown adipose tissue

 The effects of acute and chronic acclimation to cold on uncoupling protein 1 (UCP1) levels, as well as on GDP-binding to mitochondria, cytochrome c oxidase activity and mitochondrial protein concentration in brown adipose tissue (BAT) of intact male and female rats have been analyzed. Results reveal that females rats are more sensitive to cold because their threshold temperature for the thermogenic response is set at a higher value (around 22°C) than that of males (around 18°C), hence leading to differences in BAT UCP1 levels between the sexes at different environmental temperatures. In vitro experiments showed that steroid hormones, β-estradiol, estrone and progesterone, can reduce norepinephrine-induced UCP1 synthesis in brown adipocytes differentiated in primary culture. Thus the different sex-associated response of cold-induced thermogenesis in rats does not appear to be explained by a direct action of sex steroids upon the adipocyte, implying that other factors in the thermogenic regulatory system must be involved. Received: 23 March 1998 / Received after revision: 20 May 1998 / Accepted: 21 May 1998     

Interaction between exercise training and cold acclimation in rats

Summary Five groups of 10 rats were used. Group A included sedentary rats kept at 24° C, group B exercised-trained rats and group C rats exposed at –15° C for 2 h every day and kept at 24° C for the remaining time. These 3 groups were kept on this regimen for 10 weeks. In addition group D was acclimated to cold (2 h · d^–1 at –15° C) for 6 weeks and subsequently deacclimated at 24° C for 4 weeks. Group E was also acclimated to cold for 6 weeks and during the deacclimation, at 24° C period which lasted 4 weeks, the animals were exercised 2 h per day. Following the 10 week experimental period all animals were sacrified and DNA and protein content of the IBAT as well as its total mass were measured. The results show significant increases in the cold adapted group. Exercise training which had no effect on brown adipose tissue IBAT at room temperature, caused an accelerated reduction in weight, DNA and protein content of the BAT in rats previously acclimated to cold. In spite of this, the thermogenic response to noreadrenaline was significantly enhanced in the group which exercised during the deacclimation period. It is suggested that tissues other than IBAT may explain this enhanced heat production capacity.     

Cross adaption between stress and cold in rats

Three-hour immobilization stress was imposed on male adult rats of Wistar strain by restraining them on a board 6 days a week for 1–8 weeks. The stressed rats showed less body weight gain during the experiment compared to the controls. These stressed animals manifested an improved cold tolerance as shown by no significant fall in colonic temperature in the cold at –5° C for 300 min during the experimental period, while the colonic temperature of the controls fell progressively. Nonshivering thermogenesis as assessed by noradrenaline-induced increase in oxygen consumption was significantly potentiated in the stressed rats. The weight and protein content of the intercapsular brown adipose tissue (BAT) increased and BAT mitochondria were more packed in the stressed rats. Plasma insulin, insulin/glucagon molar ratio and thyroxine levels were lowered in the stressed rats, while the plasma triiodothyronine level remained unchanged. Removal of interscapular BAT led to a loss of improved cold tolerance and a significant reduction of nonshivering thermogenesis in the stressed rats.These results indicate that repetitive stress may induce cross adaptation between stress and cold through an enhanced capacity of nonshivering thermogenesis mediated, at least in part, via stimulation of BAT function.     

Effect of exercise training on the disappearance of cold adaptability in rats

Summary Following the transfer of cold-adapted rats to a warm environment at 25‡ C, enhanced nonshivering thermogenesis and enlarged interscapular brown adipose tissue (BAT) decreased gradually and reached a steady state after 4 weeks of de-adaptation. Animals that were exercised in the process of de-adaptation, however, showed no decrease in enhanced nonshivering thermogenesis, but did show a decrease in BAT weight as compared with sedentarily de-adapted animals. Triiodothyronine (T₃), the physiologically most active thyroid hormone, was at a higher plasma level in cold-adapted rats than in de-adapted animals with or without exercise loads. Although the resting level of T₃ in running-trained rats was not higher than that in sedentary rats, some fluctuations of T₃ level were observed during running. The part of this work using radioisotopes was performed in the Central Institute of Isotope Science, Hokkaido University     

Effect of immobilization stress on in vitro and in vivo thermogenesis of brown adipose tissue.

Repetitive intermittent stress such as immobilization has been shown to induce an improved cold tolerance through an enhanced capacity of nonshivering thermogenesis (NST), causing positive cross adaptation between nonthermal stress and cold. In the present study, effect of 3-h-daily immobilization stress for 4-5 weeks was investigated on in vitro and in vivo thermogenesis of interscapular brown adipose tissue (BAT). In vitro thermogenesis was measured in the minced tissue blocks incubated in Krebs-Ringer phosphate buffer with glucose and albumin at 37 degrees C, using a Clark-type oxygen electrode. The stressed rats showed less body weight gain during the experiment. The BAT weight, its protein and DNA contents were significantly greater in the stressed rats. Basal, noradrenaline- and glucagon-stimulated oxygen consumptions were significantly greater in the stressed rats. In vivo thermogenesis was assessed by the changes of temperatures in colon (Tcol), BAT (TBAT), and tail skin (Tsk) induced by noradrenaline or glucagon infusion in the anesthetized rats. Noradrenaline and glucagon increased the TBAT and the extent of increase was greater in the stressed rats. These results indicate that cross adaptation between nonthermal stress and cold may be mediated through an enhanced thermogenic activity of BAT.     

Contribution of skeletal muscle to the regulatory non-shivering thermogenesis in small mammals

Energy dissipation () was measured by direct microcalorimetry in perifused resting soleus muscles from cold adapted, euthyroid, hypothyroid and hyperthyroid mice, before and during exposure to noradrenaline (NA), lipid substrates or ouabain. The thermogenic effect of NA on the muscle was transitory and it did not exceed 5% of basal, in all groups of preparations. The substrate effects were larger than that of NA and were sustained. They were the largest in hypothyroid animals and were not potentiated by NA. Basal and the thermogenic effects of NA and the lipid substrates were identical in preparations from mice adapted to 23°C and to 8°C. The inhibitory effect of ouabain in resting muscles was very small, but it was increased by adaptation to the lower temperature. Experiments performed on rat muscles perfused in situ showed much larger thermogenic effects of NA than that observed in perifused mouse muscles. It is suggested that the NA thermogenic effect in resting muscles from small mammals is essentially mediated by hemodynamic changes which tend to suppress a hypoxic and acidotic restriction of the metabolic rate, rather than by any direct effect of NA on skeleral muscle cells.     

Nitric oxide and thermogenic function of brown adipose tissue in rats

To clarify the effects of cold acclimation and immobilization stress adaptation of rats on nitric oxide (NO) activity in interscapular brown adipose tissue (BAT), we incubated neatly diced (1-mm(3) blocks) BAT in a metabolic chamber for respiration, measured oxygen consumption using a Clark electrode, and estimated NO release in the buffer medium by measuring nitrite plus nitrate (NO(x)) using the Griess method (diazotization reaction). The production of NO(x) in the buffer medium confirmed that BAT releases NO, as there is no other source of NO(x) in the system. The NO activity was observed in the basal condition and increased with noradrenaline stimulation, showing a correlation with oxygen consumption in the warm (25 degrees C)-acclimated control rats. Cold acclimation (5 degrees C, 5 weeks) or immobilization stress adaptation (3 h daily, 25 degrees C, 5 weeks) caused enhanced NO activity in the basal condition in comparison with the control. We suggest that NO is involved in enhancement of the thermogenic functions of BAT in rats.     

Guanethidine sympathectomy does not prevent meal-induced increases in the weight or oxygen consumption of brown fat

The interscapular brown adipose tissue (BAT) of adult rats that were neonatally sympathectomized with guanethidine (GUA) consumed less oxygen but weighed the same as BAT from intact controls. In response to a 2-hr mixed-constituent meal, BAT from sympathectomized and control rats showed similar increases in oxygen uptake and weight. These data suggest that some functions of BAT can be maintained even without sympathetic stimulation.     

Effect of acute cold-exposure on norepinephrine turnover and thermogenesis in brown adipose tissue and metabolic rate in MSG-induced obese mice

Mice treated neonatally with monosodium-L-glutamate (MSG) are known to develop into obese adults without hyperphagia, which are characterized by the reduced levels in the resting metabolic rate (RMR) and the thermogenesis of brown adipose tissue (BAT) in the thermoneutral environment. The present study revealed that an acute cold-exposure (5 degrees C, 1 h) of these animals resulted in the increase in norepinephrine turnover and mitochondrial-5'-diphosphate (GDP) binding in the interscapular BAT as well as the guanosine RMR, suggesting a normal thermogenic responsiveness of BAT to cold.     

Brown adipose tissue response to cafeteria diet-feeding involves induction of the UCP2 gene and is impaired in female rats as compared to males

Noradrenaline-dependent brown adipose tissue (BAT) thermogenesis is activated by the cold and excess energy intake, largely depends on the activity of the uncoupling protein 1 (UCP1), and is mediated mainly through the beta3-adrenoceptor (beta3-AR). We investigated the expression of ucp2, a gene that encodes a putative UCP1-like uncoupling protein, along with that of ucp1 and beta3-ar, in the interscapular BAT (IBAT) of male and female rats chronically fed a cafeteria diet. After 3 months on this diet, male rats attained a 34% excess body mass and showed IBAT hypertrophy and increased IBAT thermogenic potential, in terms of both UCP1 and UCP2 mRNA expression (both by 1.6-fold), UCP1 protein expression (by 1.75-fold) and GDP binding to IBAT mitochondria (by 2.2-fold); female rats attained a larger excess body weight (50%) and their IBAT, although hypertrophied, showed no signs of increased thermogenic potential per gram of tissue. Interestingly, the IBAT of female rats was already activated compared to males. Treatment of mouse brown adipocytes in primary culture with noradrenaline also triggered a dose-dependent increase of the levels of UCP1 mRNA and UCP2 mRNA. Retroregulatory down-regulation of the beta3-AR mRNA levels was found in the two models used. The results support a physiological role for UCP2, along with UCP1, in rodent BAT thermogenesis.     

Effects of cold and immobilization stress on noradrenaline turnover in brown adipose tissue of rat

Noradrenaline (NA) turnover of the interscapular brown adipose tissue (BAT) was determined in order to evaluate a role of sympathetic NA of this tissue in an enhanced nonshivering thermogenesis which had been previously evidenced in the repetitively stressed rats by immobilization (daily 3-h immobilization for 4 weeks) and the cold-acclimated ones (5 degrees C, 4 weeks). The disappearance rate of NA from the BAT following blockade of NA synthesis with alpha-methyl-p-tyrosine was adopted for estimation of NA turnover of the tissue. Cold acclimation increased both fractional turnover rate (%/h) (k) and turnover rate (ng/(g BAT.h)). Repetitive immobilization stress also elevated turnover rate, but not k. In the warm non-stressed controls acute cold exposure to -5 degrees C and acute immobilization stress elevated the turnover rate. The effect of cold exposure was significantly greater than that of immobilization stress for both indices of NA turnover. In the cold-acclimated rats acute cold exposure increased k as well as turnover rate, but not acute immobilization stress. In the repetitively immobilized rats both acute cold exposure and acute immobilization stress elevated k and turnover rate. These results indicate that immobilization enhances sympathetic activity of thermogenic tissue, BAT. The results also suggest that the extent of sympathetic participation is not necessarily the same between the cold-acclimated and the stressed rats.     

Suppression of norepinephrine-induced thermogenesis in brown adipose tissue by fasting

Fasting-induced changes in thermogenic responses to norepinephrine (NE, 4.0 micrograms X kg-1 X min-1 iv) were studied in anesthetized rats previously cold acclimated. The rats were divided into five groups at the end of 30-40 days of cold acclimation (5 degrees C). The five groups were kept for 5 days at 25 degrees C and fed (intact fed), fasted (intact fasted), fasted with daily treatment with thyroxine (T4, 2 micrograms/kg sc), thyroidectomized and fed, or thyroidectomized and fasted. In the intact fasted group, in which the weight of brown adipose tissue decreased, NE-induced increases in oxygen consumption, colonic temperature (T col), and temperature of the interscapular brown adipose tissue (TBAT) were markedly suppressed. The two thyroidectomized groups also showed a reduction in thermogenic response. In these three groups, TBAT was lower than Tcol throughout NE infusion. In the T4-treated fasted group, fasting-induced suppression of thermogenic response to NE was largely prevented. In the intact fed and the T4-treated fasted groups, TBAT attained higher values than Tcol during NE infusion. Plasma levels of thyroid hormones were significantly lower in the intact fasted group than in the intact fed or the T4-treated fasted group. These results suggest that fasting-induced suppression of the thermogenic response to NE is largely due to the reduced thermogenic response of brown adipose tissue to NE. The lowering of the levels of the thyroid hormones induced by fasting may be one of a number of causes of the reduction in the thermogenic response of brown adipose tissue.     

Shivering and nonshivering thermogenesis in exercised cold-deacclimated rats

Summary Norepinephrine (NE)-induced increase in oxygen consumption ( ) and colonic temperature (Tc) was greater in cold-acclimated rats housed at 4° C for 4 weeks (CA) than warm-acclimated controls housed at 24° C for 4 weeks (WA). On the other hand, shivering activity measured at 4° C was less in CA than in WA, while propranolol administration eliminated the difference between these two groups by enhancing shivering in CA. Wet weight and protein content of interscapular brown adipose tissue (IBAT) were greater in CA than in WA. Following cold acclimation, CA were deacclimated at 24° C for 5 weeks. During deacclimation, half of this latter group were forced to run (15 m·min^–1 for 1 h) every day (CD-T) while the remaining rats remained sedentary (CD-S). Shivering activity assessed at 4° C 4 weeks after commencing cold deacclimation was significantly less in CD-T than in CD-S and the difference disappeared following propranolol injection. and Tc responses to NE injection measured 1, 2 and 5 weeks after commencing cold deacclimation did not differ between CD-S and CD-T. Although IBAT weight was lighter in CD-T than in CD-S, its total protein content was not different between the latter two groups of rats. These results suggest that a greater degree of NE-independent nonshivering thermogenesis (NST) is retained in rats that are exercised during the process of deacclimation as compared with animals that are sedentary. This difference in NST would not seem to be directly related to BAT thermogenic capacity.     

Effect of altered thyroid state on the in situ mechanical properties of adult cat soleus

To determine the responsiveness of cat hindlimb muscles to thyroid manipulation, adult female cats were made hypothyroid (thyroidectomy plus tapazole treatment), hyperthyroid (synthroid pellets), or maintained euthyroid. After 4 months, the hypothyroid soleus had slower time-to-peak (TPT, 80%) and half-relaxation (HRT) times, whereas the hyperthyroid soleus had faster TPT (20%) and HRT than euthyroid cats. The tension at low stimulation frequencies (5-15 Hz) was higher in hypothyroid and lower in hyperthyroid cats compared to euthyroid cats. Muscle weight, maximum twitch and tetanic (Po) tensions, and maximum rates of shortening (Vmax) were similar across groups. The soleus of hypothyroid cats was more fatigable than normal. The myosin heavy chain (MHC) composition, based on gel electrophoresis, was unaffected by thyroid hormone manipulation. Based on the reaction of monoclonal antibodies for specific MHCs, some fast fibers in the hypothyroid cats coexpressed developmental MHC. These data indicate that 4 months of an altered thyroid state result in changes in the isometric twitch speed properties of the cat soleus, but not the tension-related or isotonic properties. Further, a chronic decrease in thyroid hormone had a greater impact than a chronic increase in thyroid hormone on the mechanical properties of the adult cat soleus.     

Evaluation of oxidative phosphorylation in hearts from euthyroid, hypothyroid, and hyperthyroid rats.

The energy relationships between cytosolic and mitochondrial metabolism were studied in the hearts from euthyroid, hypothyroid, and hyperthyroid rats. Isolated mitochondria showed high respiratory control ratios and impermeability to exogenous NADH. Hypo- and hyperthyroidism, respectively, resulted in lower and higher contents of both cytochromes per mitochondrion and mitochondrial protein per gram of wet weight of heart without changes in the ratio of cytochrome c to cytochrome aa3. In isolated perfused heart, the hyperthyroid state led to an increase in work rate and thereby an elevation of Vo2, which resulted in an increase oxidation-reduction turnover number for the cytochromes. An agreement was found between [ATP]/[ADP][Pi] of cytosolic free adenine nucleotides and the value calculated from a mathematical model of mitochondrial respiration. This implies that mitochondrial respiration is controlled at the cytochrome oxidase reaction and that oxidative phosphorylation in intact tissue is tightly coupled irrespective of thyroid state. It is concluded that thyroid hormone causes an increase in the mitochondrial mass, mitochondrial cytochrome content, and respiratory rate, and consequently expands the capacity of oxidative metabolism without an uncoupling effect on oxidative phosphorylation.