The role of insulin in norepinephrine turnover and thermogenesis in brown adipose tissue after acute cold-exposure

The role of insulin in norepinephrine turnover (NE) and thermogenesis in brown adipose tissue (BAT) after acute cold-exposure was studied using streptozocin (STZ)-induced diabetic rats. NE turnover was estimated by the NE synthesis inhibition technique with alpha-methyl-p-tyrosine. BAT thermogenesis was estimated by measuring mitochondrial guanosine-5'-diphosphate (GDP), cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at an ambient temperature of 22 degrees C and during a six-hour cold-exposure at 4 degrees C. In insulin-deficient diabetic rats, the NE turnover, mitochondrial GDP binding, cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at 22 degrees C were significantly reduced, compared with those of control rats. Treatment of STZ-induced diabetic rats with insulin prevented a decrease in NE turnover and BAT thermogenesis. Acute cold-exposure increased the NE turnover of BAT in insulin-deficient diabetic rats. The BAT thermogenic response to acute cold-exposure, however, did not occur in insulin-deficient diabetic rats. These results suggest that insulin is not essential in potentiating NE turnover in BAT after acute cold-exposure, but is required for cold-induced thermogenesis.     

Effect of sympathetic de-activation on thermogenic function and membrane lipid composition in mitochondria of brown adipose tissue.

Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.     

Effects of exercise training on brown adipose tissue thermogenesis in ovariectomized obese rats

The effect of exercise training on brown adipose tissue (BAT) thermogenesis was studied by measuring cytochrome oxidase activity, as a marker of mitochondrial abundance, mitochondrial guanosine-5'-diphosphate (GDP) binding, as an indicator of thermogenic activity and oxygen consumption in BAT in ovariectomized (OVX) obese rats and sham-operated rats. Six-week exercise training significantly suppressed body weight gain in OVX rats to the level of sedentary control rats, although food intake in exercise trained OVX rats increased more than in the sedentary OVX rats. Exercise training increased cytochrome oxidase activity, mitochondrial GDP binding and oxygen consumption in BAT in OVX rats, which were reduced in a sedentary condition, as well as in the control rats. These results suggest that exercise training potentiates BAT thermogenesis, which may contribute to the reduction of body weight in OVX obese rats.     

Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

Impaired febrile response with age: role of thermogenesis in brown adipose tissue.

We demonstrated previously that in Escherichia coli-infected rats, the heat necessary for the febrile response is a result of thermogenesis in brown adipose tissue (BAT). To investigate whether senescent rats have an impaired febrile response to infection and whether such an impairment is a result of attenuated sympathetically activated thermogenesis in BAT, we assessed body temperature and the increase in mitochondrial guanosine 5'-diphosphate (GDP) binding sites in interscapular BAT in response to E. coli administration in young and senescent male F-344 rats. There was a significant delay of 2 hr in the onset of fever in the older animals. In addition, in senescent rats, the peak fever (1.0 +/- 0.1 delta degrees C vs 2.2 +/- 0.1) and the cumulative fever (383 +/- 43 delta degrees C.min vs 775 +/- 69) were significantly less than in the young rats (P less than 0.005). Baseline levels of GDP binding were the same in young and old rats. In young rats, during the rising phase of the fever, E. coli infection resulted in a 50% increase in the density of GDP binding sites in BAT mitochondria. In contrast, there was no increase in GDP binding in the older rats following infection. The failure to increase GDP binding may be a result of a reduced ability to unmask reserve GDP binding sites. Alternatively, there may be fewer total GDP binding sites (masked and unmasked) in senescent rats and these sites may already be unmasked. Collectively, these data suggest that the impaired febrile response with age is due to reduced thermogenesis in BAT.     

Exercise suppression of thermoregulatory thermogenesis in warm- and cold-acclimated rats

An evaluation was made of the effects of an acute exercise bout on nonshivering thermogenesis (NST) in cold-acclimated rats (4 degrees C for 6 weeks) and shivering thermogenesis in 24 degrees C-acclimated rats (24 degrees C for 6 weeks). Assessment techniques included indirect calorimetry during treadmill running and brown adipose tissue (BAT) mitochondrial guanosine diphosphate (GDP) binding immediately following a treadmill run. Calorimetric results for 24 degrees C-acclimated rats running at 4 degrees C indicated total substitution of shivering thermogenesis by exercise-derived heat. No difference in GDP-binding, an index of BAT nonshivering thermogenic activity, was observed between exercised and nonexercised 24 degrees C-acclimated rats. Calorimetric results for cold-acclimated rats running at 4 degrees C indicated a total suppression in the energy cost associated with NST, exercise-derived heat replacing or substituting for NST. Examining BAT properties in the exercised cold-acclimated rats revealed a significant 40% decrease in BAT mitochondrial GDP-binding. These results suggest that during running, metabolic heat due to the exercise totally replaces shivering in 24 degrees C-acclimated rats and totally replaces BAT nonshivering thermogenesis in cold-acclimated rats.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Reduced brown adipose tissue thermogenesis of obese rats after ovariectomy

Brown adipose tissue (BAT) thermogenesis was assessed by measuring mitochondrial guanosine diphosphate (GDP) binding, cytochrome oxidase activity and oxygen consumption in ovariectomized (OVX) and sham-operated rats. The food intake and body weight of OVX rats increased more than those of controls and OVX rats became obese. Mitochondrial GDP binding, as an indicator of thermogenic activity, cytochrome oxidase activity, as a marker of mitochondrial abundance, and mitochondrial respiration of BAT in OVX rats were significantly reduced compared with those in controls. And, also, even when OVX rats were restricted in food intake (pair-gained) to produce comparable changes in body weight with sham-controls, or matched in food intake (pair-fed) with sham-controls, these parameters in both pair-gained and pair-fed OVX groups were decreased markedly compared to those in sham-controls. As expected, body weight in pair-fed OVX rats increased significantly more than that in sham-controls. In response to cold exposure, these parameters of OVX rats increased as much as those of controls did. These results suggest that reduced brown adipose tissue thermogenesis might be one of the important factors that are responsible for the development of obesity after OVX.     

Thermogenesis in brown adipose tissue of genetically obese, diabetic (KKAY) mice

Thermogenesis of brown adipose tissue (BAT) of genetically obese mice, KKAY mice, was examined by measuring the BAT mitochondrial guanosine diphosphate (GDP) binding as an index of thermogenesis and comparing it with that of normal C57BL mice. No great difference in GDP binding was observed in KKAY and C57BL mice fed a stock diet. However, when they were given a sucrose solution, the increase in BAT mitochondrial GDP binding of KKAY mice (+22%) was much lower than that of C57BL mice (+106%). A high fat diet increased BAT mitochondrial GDP binding in KKAY mice to the same extent (+82%) as in C57BL mice. When the mice were fasted for 48 h, BAT mitochondrial GDP binding of C57BL mice decreased by 70%, while that of KKAY mice showed no change. Both acute exposure to cold and norepinephrine injections increased GDP binding in KKAY mice by 90% and 131%, respectively. These results indicate that low BAT thermogenesis in response to sucrose intake may be a cause of obesity in KKAY mice, and this may be brought about by defects in the central nervous system.     

Brown fat activity in fasted and refed rats

Four days of fasting in the rat reduced brown-adipose-tissue (BAT) mass, mitochondrial protein, and tissue protein content. Specific binding of guanosine diposphate (GDP) to BAT mitochondria was depressed by 55% in 4d-fasted rats. Rats fasted for 3 d, and then refed a single carbohydrate meal (40 kJ), showed a significant increase in specific GDP-binding (27% above fasted) 24 h later, and a large increase in total binding. Specific activities of cytochrome oxidase and -glycerophosphate dehydrogenase in BAT mitochondria were not significantly affected by fasting or refeeding. These results suggest that BAT may be partly responsible for the fall in metabolic rate associated with fasting and the delayed increase after carbo-hydrate refeeding. These effects may be due to changes in the mitochondrial proton-conductance pathway in brown fat.     

The effects of essential fatty acid deficiency on brown adipose tissue activity in rats maintained at thermal neutrality

1. The consequences of essential fatty acid (EFA) deficiency on the resting metabolism, food efficiency and brown adipose tissue (BAT) thermogenic activity were examined in rats maintained at thermal neutrality (28 C). 2. Weanling male Long-Evans rats were fed a hypolipidic semi-purified diet (control diet: 2% sunflower oil; EFA-deficient diet: 2% hydrogenated coconut oil) for 9 weeks. 3. They were kept at 28 C for the last 5 weeks. Compared to controls, in EFA-deficient rats the growth shortfall reached 21% at killing. 4. As food intake was the same in EFA-deficient and control rats, food efficiency was thus decreased by 40%. 5. Resting metabolism expressed per surface unit was 15% increased. 6. Non-renal water loss was increased by 88%. 7. BAT weight was 28% decreased but total and mitochondrial proteins were not modified. 8. Heat production capacity, tested by GDP binding per BAT was 69% increased in BAT of deficient rats. 9. The stimulation of BAT was established by two other tests: GDP inhibition of mitochondrial O2 consumption and swelling of mitochondria. 10. It is suggested that the observed enhancement of resting metabolism in EFA-deficient rats is, in part, due to an activation of heat production in BAT.     

Variations in energization parameters and proton conductance induced by cold adaptation and essential fatty acid deficiency in mitochondria of brown adipose tissue in the rat

Male weanling Long-Evans rats were fed on a low-fat semipurified diet (control diet, 2% sunflower oil; essential fatty acid (EFA) deficient diet, 2% hydrogenated coconut oil) for 9 weeks. In order to modulate need for non-shivering thermogenesis, groups of rats on each diet were exposed at 28 degrees C (thermoneutrality) and at 5 degrees C (cold acclimation) for the last 5 weeks. In brown adipose tissue (BAT) mitochondria, several parameters of mitochondrial energization, protonmotive force (delta p) and its components delta pH and membrane potential, delta psi, were investigated. Simultaneous measurement of oxygen consumption and delta psi (the main component of delta p) was performed by varying alpha-glycerophosphate concentration and the force/flux relationship of the mitochondria was established by comparison of proton conductance, CmH+, over the whole range of protonmotive force. delta p. In the absence of GDP, at 28 degrees C, EFA deficiency induced a marked increase in CmH+. Cold acclimation led to comparable enhanced CmH+ in control and EFA-deficient mitochondria. In the presence of GDP which binds and inhibits the BAT 32 kDa uncoupling protein, CmH+ was the same in 28 degrees C and 5 degrees C control mitochondria, but EFA deficiency led to an enhanced GDP independent CmH+ at 28 degrees C and to a lesser extent at 5 degrees C. These results are discussed with reference to substantial changes in mitochondrial lipid composition induced by the deficiency.     

Time-course variations of effective proton conductance and GDP binding in brown adipose tissue mitochondria of rats during prolonged cold exposure.

1. Time-course variations of the thermogenic pathway in rat brown adipose tissue (BAT) mitochondria were examined. 2. Several parameters of mitochondrial energization, protonmotive force and its components pH gradient and membrane potential were investigated. The specific binding of GDP was compared with the effective proton conductance (CmH+) of the membrane. 3. Ten-days cold exposure led to maximal GDP binding and GDP-dependent CmH+. 4. The subsequent relative decrease in GDP binding observed during prolonged cold exposure (40 days) was functional and led to a lower GDP-dependent CmH+. CmH+ showed greater variation than GDP binding. 5. The CmH+ decrease was not due to a masking of active sites of the uncoupling protein. 6. Basal GDP-independent CmH+ was not modified. 7. Results are discussed with reference to the significance of biochemical measures and to the physiological regulation of BAT thermogenesis.     

Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats

The control of uncoupling protein-1, -2 and -3 (UCP-1, UCP-2, UCP-3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT-PCR assays. In rats reared at thermoneutrality (25 degrees C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP-1 mRNA level (-38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP-2 and -3 mRNA levels. Cold exposure (7 days, 4 degrees C) markedly increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold-induced rise in BAT UCP-1 and UCP-2 mRNAs, but not that in BAT UCP-3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP-1, UCP-2 and UCP-3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.     

Disparate effects of fenfluramine on thermogenesis in brown adipose tissue in the rat.

It has been suggested that fenfluramine, a clinically used appetite suppressant, can also promote weight loss by augmenting energy expenditure, as indicated by increased whole-body O2 consumption (VO2) and mitochondrial GDP binding in brown adipose tissue (BAT) of fenfluramine-treated rats. To further investigate a possible involvement of BAT in the drug's metabolic effects, 113Sn-labelled microspheres were injected into the left cardiac ventricle of conscious rats 70-80 min after intraperitoneal delivery of 20 mg/kg fenfluramine (DL-mixture) or saline vehicle. At 28 degrees C ambient temperature, fenfluramine augmented resting whole-body VO2 and increased the microsphere entrapment in BAT, indicating enhanced blood flow and metabolism. At 20 degrees C ambient temperature, the expected increase in BAT blood flow associated with nonshivering thermogenesis was observed in control rats, but in fenfluramine-treated rats the increase in BAT blood flow was severely attenuated, and VO2 and body temperature were reduced. The stimulatory effect of fenfluramine on BAT metabolism was not prevented by urethane anesthesia but did not occur if the tissue was denervated. These blood flow measurements corroborate previous reports, based on GDP-binding assays, that fenfluramine treatment can augment thermogenesis in BAT by effects mediated through the innervation of the tissue. However, the data also indicate that this calorigenic effect is dependent on ambient temperature being near thermoneutrality and that in a cool environment the drug inhibits BAT thermogenesis.     

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.     

Effects of rearing temperature on sympathoadrenal activity in young adult rats

Animals reared at 18 degrees C exhibit enhanced innervation of brown adipose tissue (BAT) and greater cold tolerance as adults, yet gain more weight when fed an enriched diet compared with rats reared at 30 degrees C. To explore this paradox, sympathoadrenal activity was examined using techniques of [(3)H]norepinephrine ([(3)H]NE) turnover and urinary catecholamine excretion in male and female rats reared until 2 mo of age at 18 or 30 degrees C. Gene expression in BAT was also analyzed for several sympathetically related proteins. Although [(3)H]NE turnover in heart did not differ between groups, [(3)H]NE turnover in BAT was consistently elevated in the 18 degrees C-reared animals, even 2 mo after removal from the cool environment. Gene expression for uncoupling proteins 1 and 3, GLUT-4, leptin, and the alpha(1A)-adrenergic receptor was more abundant in BAT and the increase in epinephrine excretion with fasting suppressed in 18 degrees C-reared animals. These studies demonstrate that obesity consequent to exposure to 18 degrees C in early life occurs despite tonic elevation of sympathetic input to BAT. Diminished adrenal epinephrine responsiveness to fasting may play a contributory role.     

Effects of neonatal sympathectomy on brown fat development and susceptibility to high fat diet induced obesity in mice.

Injections of 6-hydroxydopamine in mouse neonates caused extensive and long lasting damage to the sympathetic nervous system and impaired brown fat development. Brown adipose tissue (BAT) thermogenic capacity of sympathectomized mice (up to 120 days old) was reduced because of marked reductions in the tissue mitochondrial protein content and the mitochondrial concentration of uncoupling protein, as assessed by [3H]GDP binding and immunoassay. Neonatal sympathectomy did not affect BAT DNA content. Sympathectomized mice also had reduced epinephrine-stimulated rates of oxygen consumption. BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable high-fat dietary supplement that increased calorie intake of both normal and sympathectomized mice. The high-fat diet caused increases in body weight, carcass fat, and gonadal white fat pad weights in sympathectomized animals that were similar to those of control mice. These results show that inactivation of BAT metabolism did not accentuate the development of obesity caused by a dietary supplement rich in fat and suggest that stimulation of BAT metabolism was not very effective in counteracting the obesity-inducing effect of this diet.     

Brown adipose tissue, liver, and diet-induced thermogenesis in cafeteria diet-fed rats

Diet-induced thermogenesis (DIT) in young rats overeating a "cafeteria" (CAF) diet of palatable human foods is characterized by a chronic, propranolol-inhibitable elevation in resting metabolic rate (VO2) and is associated with various changes in brown adipose tissue (BAT) that have been taken as evidence for BAT as the effector of DIT. But direct evidence for participation of BAT in DIT has been lacking. By employing a nonocclusive cannula to sample the venous effluent of interscapular BAT (IBAT) for analysis of its O2 content and measuring tissue blood flow with microspheres, we accomplished direct determination (Fick principle) of the O2 consumption of BAT in conscious CAF rats. In comparison with normophagic controls fed chow, the CAF rats exhibited a 43% increase in metabolizable energy intake, reduced food efficiency, a 22% elevation in resting VO2 at 28 degrees C (thermoneutrality) or 24 degrees C (housing temperature), and characteristic changes in the properties of their BAT (e.g., increased mass, protein content and mitochondrial GDP binding). They also exhibited the greater metabolic response to exogenous noradrenaline characteristic of CAF rats and the near elimination by propranolol of their elevation in VO2. By the criterion of their elevated VO2, the CAF rats were exhibiting DIT at the time of the measurements of BAT blood flow and blood O2 levels. However, BAT O2 consumption was found to be no greater in the CAF rats than in the controls at either 28 or 24 degrees C. At 28 degrees C it accounted for less than 1% of whole body VO2; at 24 degrees C it increased to about 10% of overall VO2 in both diet groups.(ABSTRACT TRUNCATED AT 250 WORDS)