Effect of diet and cold exposure on norepinephrine turnover in brown adipose tissue of the rat.

Brown adipose tissue (BAT) is an important site of adaptive changes in thermogenesis in the rat. The sympathetic nervous system, which richly supplies BAT, is thought to play an important role in the regulation of BAT thermogenesis because catecholamines stimulate and beta adrenergic blocking agents inhibit oxygen consumption in this tissue. The present studies were carried out to assess directly sympathetic activity in BAT in response to cold exposure and to changes in dietary intake, both of which alter heat production in the rat. Sympathetic activity was determined from the rate of norepinephrine (NE) turnover in interscapular brown adipose tissue (IBAT) after preliminary experiments validated the use of NE turnover techniques in IBAT. Acute exposure to 4 degrees C increased NE turnover in IBAT 4- to 12-fold compared with ambient temperature controls, depending upon the interval over which the turnover measurement was made, while in the heart NE turnover doubled in response to the same cold stimulus. In animals exposed to cold continuously for 10 d before study, NE turnover measurements in IBAT and in the heart were elevated comparably to those obtained during acute exposure. Alterations in feeding were also associated with changes in NE turnover in IBAT. Fasting for 2 d decreased NE turnover in IBAT (-35% from 29.2+/-4.2 ng NE/h to 18.9+/-5.9) and in heart (-52%). In animals fed a "cafeteria" diet, a model of voluntary overfeeding in the rat, NE turnover was increased in both IBAT (+108% from 24.8+/-4.5 ng NE/h to 51.7+/-6.8) and heart (+66%). Because ganglionic blockade exerted a greater effect on NE turnover in IBAT in cafeteria-fed rats than in controls, the increase in NE turnover in IBAT with this overfeeding regimen reflects enhanced central sympathetic outflow. Thus NE turnover techniques can be satisfactorily applied to the assessment of sympathetic nervous system activity in IBAT. The experiments reported here demonstrate changes in sympathetic activity in IBAT that parallel known adaptive changes in heat production in the rat. These studies, therefore, support the concept that the increased thermogenesis of chronic cold exposure and of cafeteria feeding occur by similar mechanisms and imply an important role for the sympathetic nervous system, mediated in part through BAT, in the regulation of energy balance in the rat.

Leptin expression in the fetus and placenta during mouse pregnancy.

During pregnancy, leptin concentrations in the maternal circulation are elevated in both humans and rodents but decrease to pre-pregnancy levels at birth, suggesting a role for leptin in the maintenance of pregnancy. Synthesis of leptin by the human placenta is established but whether the murine placenta synthesizes leptin remains controversial. The aims of this study were to determine (a) if the mouse wild-type placenta expresses the ob gene using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and (b) whether the mouse fetus and placenta contribute to the significant increase of leptin in the maternal circulation during pregnancy. The mouse placenta did not express the ob gene at a level that could be readily detected using RT-PCR. Moreover, both maternal gain in weight and undetectable concentrations of leptin in sera in leptin-deficient ob/ob mothers bearing heterozygote (ob/+) fetuses suggested that the mouse fetus and placenta do not make a significant contribution to the dramatic increase in maternal plasma concentrations of leptin during late gestation. It is therefore concluded that neither fetal- nor placental-derived leptin modulates maternal weight gain during pregnancy.

Leptin requirement for conception, implantation, and gestation in the mouse.

The ob/ob mouse has a complete absence of circulating leptin, resulting in obesity and infertility. Using the minimum daily dose of leptin required to maintain normal body weight and sexual maturation (5 mg/kg, ip), leptin-treated ob/ob females were mated with either wild-type (+/+) or leptin-treated ob/ob males. The leptin treatment continued throughout pregnancy until weaning or was withdrawn at 0.5, 3.5, 6.5, or 14.5 d post coitum (dpc). Normal pregnancy and parturition with pups of normal weight resulted when ob/ob females were mated with +/+ males and leptin treatment was continued throughout pregnancy (6 of 8 pregnancies), to 14.5 dpc (6 of 8 pregnancies), or to 6.5 dpc (9 of 12 pregnancies). Pregnancy did not result when treatment was stopped at 3.5 dpc (1 of 7 pregnancies) or 0.5 dpc (0 of 6 pregnancies). Similar results were obtained when leptin-treated ob/ob females were mated with leptin-treated ob/ob males. The newborn pups failed to survive after birth in groups treated with leptin up to 14.5 and 6.5 dpc despite reinstating leptin at birth. This appeared to be due to a lack of development of the mammary glands. In conclusion, we have shown that leptin is essential for normal preimplantation and/or implantation processes. It is also essential for normal development of the mammary glands, but is not required for pregnancy and parturition once implantation is established.

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats.

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the beta3-adrenoceptor agonists; (e) BAT from genetically obese (falfa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on beta3-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.

The measurement of food and energy intake in man-an evaluation of some techniques.

A dietary survey was carried out on an Antarctic base over a period of 6 to 12 months on twelve individuals. Three methods of determining individual food and energy intake were used-weighing and recording of food as eaten in combination with food tables, dietary recall, and bomb calorimetry of duplicate meals. Use of weighed food intakes and food composition tables underestimated energy intake by 7% when compared to analysis of duplicate meals by bomb calorimetry. One week was found to be the most practical period over which intake should be measured to determine "habitual" food intake.

The measurement of daily energy expenditure--an evaluation of some techniques.

An energy balance survey was conducted on an Antarctic base on 12 individuals for periods varying between 6 and 12 months. Energy expenditure was estimated using two activity/dairy card techniques and mean 24-hr heart rate used in conjunction with a variety of derived heart rate/energy expenditure regression equations. The accuracy of the various techniques was determined by comparison with estimates obtained using energy intakes and changes in body fat. No method of measuring energy expenditure was accurate enough to predict fat gain or loss. Errors in individuals were large and random but the mean error in the estimation of energy expenditure of the community was reasonable. When using the dairy card there was no difference between using measured values for energy costs of specific activities and using values taken from the literature. Using heart rate was no less accurate than the diary card if energy expenditure was predicted from a regression line derived from heart rate and log energy expenditure, and the technique was more acceptable to the subjects.

A longitudinal study of body weight and body fat changes in Antarctica.

Body weight and body fat changes (calculated from skinfold thickness) that occurred in a male population spending a year on an Antarctic base are described. There was a marked individual variation in patterns of weight and fat changes between seasons but mean values for the whole community showed little change over the year. Combining skinfold and body weight measurements revealed probable individual differences in fat gain or loss with weight gain and loss, but the role of changes in total body water or fat-free mass could not be determined. The possibility of combining long-term measurements of body fat and energy intake to provide an estimate of energy expenditure is discussed.

Thermogenic effects of sibutramine in humans.

BACKGROUND: Sibutramine is an effective compound for the treatment of obesity, acting both on serotonergic and noradrenergic pathways. Animal studies have shown that sibutramine exerts its effect by enhancing satiety as well as by increasing thermogenesis. OBJECTIVE: We tried to compare the acute thermogenic effect of a single 30-mg dose of sibutramine with placebo on basal energy expenditure (EE) and diet-induced thermogenesis. DESIGN: The study was randomized, double-blind, and placebo controlled. Eleven healthy, normal-weight men underwent 4 distinct treatment regimens separated by washout periods of 6-10 d. EE was measured by indirect calorimetry before and for 5.5 h after sibutramine or placebo administration with or without a 2.1-MJ breakfast. Visual analogue scales for assessment of appetite were completed hourly. RESULTS: Sibutramine caused a significant increase in EE above that for placebo (over 5.5 h) during both the fed (34%, 0.15 kJ/min) and fasted (183%, 0.20 kJ/min) states (P < 0.02) as well as during the last 3.5 h of this 5.5-h period and in the fed (87%, 0.26 kJ/min) and fasted (152%, 0.22 kJ/min) states, respectively (P < 0.01). The sibutramine-induced increase in EE was accompanied by an increase in plasma epinephrine (P < 0.01), heart rate (P < 0.001), blood pressure (P < 0.05), and plasma glucose (P < 0.02). About 25% of the increased heart rate with sibutramine could be explained by increased thermogenesis. Sibutramine increased satiety more than did placebo (5-h area under the curve, P < 0.05). CONCLUSIONS: Sibutramine caused a significant increase in both EE and satiety, which may both contribute to its weight-reducing properties.

Activation of brown fat thermogenesis in response to central injection of corticotropin releasing hormone in the rat.

Injection of CRF-41 (2-5 nmol) into the third ventricle, or the paraventricular nucleus of anaesthetized rats caused a marked rise in the temperature of the interscapular brown adipose tissue (BAT) depot (peak rise 0.8-1 degree C) which was inhibited by prior intravenous injection of propranolol. There was also a significant increase in the mitochondrial proton conductance pathway of brown adipose tissue, assessed from the binding of guanosine diphosphate (GDP) to mitochondria isolated from the interscapular (89% above control) and perirenal and para-aortic depots (130%). Acute surgical sympathectomy of interscapular brown adipose tissue immediately prior to injection of CRF significantly attenuated the increase in mitochondrial GDP-binding. Hypophysectomized (HYPX) rats showed a large (180%) increase in GDP-binding of brown adipose tissue 7 days after surgery and this was almost completely prevented by denervation of the interscapular depot prior to hypophysectomy. Acute injection of morphine also reduced the GDP-binding in hypophysectomized, but not in control rats. These data demonstrate that central injection of CRF stimulates thermogenesis in brown adipose tissue, probably by modifying sympathetic outflow. The activation of brown adipose tissue following hypophysectomy was also dependent on the sympathetic innervation and could be due to an increase in release of CRF.

Opposing effects of activation of central GABAA and GABAB receptors on brown fat thermogenesis in the rat.

Baclofen (a GABAB agonist) stimulates body temperature, metabolic rate and brown adipose tissue (BAT) in the rat through a central action, but no effects of gamma-aminobutyric acid (GABA) itself on these parameters were observed. In the present study, it was found that the central effects of (+/-)baclofen (0.5-2.0 micrograms injection i.c.v.) on the temperature (1.2 degrees C increase) and metabolic rate (44-76% increase) of brown adipose tissue were inhibited by previous treatment with the GABAA agonist, muscimol (0.05 micrograms). Injection of GABA alone (12 micrograms) did not significantly affect these parameters, but in the presence of the GABAA antagonist bicuculline (2.5 micrograms), GABA significantly increased the temperature (0.3 degrees C) and oxygen consumption (22%) of brown fat. (-)Baclofen was found to be approximately 50-times more effective in stimulating the temperature of brown adipose tissue than (+/-)baclofen. The results indicate that activation of central GABAB receptors stimulates the activity and hence metabolic rate of brown adipose tissue. However, activation of the GABAA receptors opposes the effects of GABAB stimulation on the thermogenesis of brown fat.

Activation of thermogenesis of brown fat in rats by baclofen.

Injection of baclofen (0.5-5 micrograms) into the ventromedial hypothalamus (VMH) of anaesthetized rats produced marked increases in the temperature (over 2 degrees C) and thermogenic activity of brown adipose tissue (BAT). These effects were abolished by ganglionic or beta-adrenergic blockade, or denervation of the tissue, but unaffected by hypophysectomy, adrenalectomy or vagotomy. Injections into the hypothalamus close to, but outside the ventromedial hypothalamus did not affect brown adipose tissue. Intravenous administration of baclofen produced similar increases in the temperature of brown adipose tissue, but at larger doses (50-1000 micrograms), and a subcutaneous injection stimulated the metabolic rate in conscious rats. Chronic treatment with baclofen suppressed weight gain and stimulated activity of brown adipose tissue without affecting food intake. These effects of baclofen, which were not mimicked by injections of gamma-aminobutyric acid (GABA), indicate a novel action of baclofen in the ventromedial hypothalamus, leading to marked increases in metabolic rate and body temperature by stimulating sympathetic outflow to brown fat.

Evidence for an atypical, or beta 3-adrenoceptor in ferret tracheal epithelium.

1. A preparation of the ferret trachea in vitro was used to examine the effects of three selective beta-adrenoceptor agonists on lysozyme secretion from submucosal gland serous cells and epithelial albumin transport into tracheal mucus following sustained, submaximal stimulation of mucus production with methacholine (20 microM). 2. Prenalterol, salbutamol and BRL 37344 all enhanced methacholine-induced albumin output. BRL 37344 was 10,000 times more potent than salbutamol, and salbutamol was slightly more potent than prenalterol. The concentrations required to increase albumin output by 100% (EC100%) were 1.4 nM, 0.7 mM and approximately 1.0 mM for BRL 37344, salbutamol and prenalterol, respectively. All three agonists inhibited methacholine-induced lysozyme output, with salbutamol being 60 times more potent than BRL 37344, and BRL 37344 being approximately 100 times more potent than prenalterol. 3. The selective beta 2-adrenoceptor antagonist, ICI 118551, inhibited the increase in albumin output produced by BRL 37344, but much more potent at inhibiting the response to salbutamol; the pA2 for ICI 118551 was 5.55 and 7.18 (P less than 0.001) when the agonist was BRL 37344 and salbutamol, respectively. ICI 118551 also attenuated the inhibition of lysozyme output produced by the two agonists, but was 10-30 times more potent at inhibiting this response than the albumin response to BRL 37344 and salbutamol. 4. The greater potency (4-5 orders of magnitude) of BRL 37344, compared to the beta 1- (prenalterol) and beta 2- (salbutamol) adrenoceptor selective agonists, in stimulating methacholine-induced albumin transport suggests that tracheal epithelium possess an atypical, or beta 3-adrenoceptor similar to that previously reported for adipocytes and gastrointestinal smooth muscle. The weak antagonism of the response to BRL 37344 by ICI 118551 would also be consistent with an atypical adrenoceptor mediating the albumin transport response. Inhibition of methacholine-induced serous cell lysozyme output would appear to be mediated predominantly by beta2-adrenoceptors.5. In view of the possible beneficial protective effects of albumin in airway surface liquid, selective beta3-agonists like BRL 37344 might have potential value in the prevention and/or treatment of inflammatory airway disease.

Acute effects of the beta 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation.

1. The acute effects of BRL 35135 (BRL) on tissue glucose utilisation index (GUI) in vivo were investigated in anaesthetized rats by use of 2-deoxy-[3H]-glucose. 2. Intravenous injection of BRL caused a dose-dependent increase in GUI in skeletal muscle, and white and brown adipose tissue; plasma insulin and fatty acid concentrations were also increased. Chronic treatment with BRL added to the diet caused a 34 fold increase in basal GUI of brown adipose tissue (BAT), but had no effect on GUI in other tissues. After chronic treatment, the acute tissue response to an intravenous maximal dose of BRL had disappeared completely in all tissues apart from the soleus muscle. 3. A high dose (20 mg kg-1) of the non-selective beta-antagonist, propranolol, inhibited the acute effect of BRL on GUI in BAT, but failed to affect GUI in muscle. A lower dose (1 mg kg-1) of the antagonist also inhibited the BAT response, but had little or no effect on the response in Type I (working) muscles such as soleus and adductor longus (ADL), and potentiated the response in Type II (non-working) muscles such as tibialis and extensor digitorium longus (EDL). 4. A low dose (1 mg kg-1) of the selective beta 1-antagonist, atenolol, had no effect on the BRL response but the same dose of the selective beta 2-antagonist, ICI 118551, potentiated significantly the effect of BRL on GUI in most muscles without altering plasma insulin levels. 5. It is concluded that: (i) the heterogeneous tissue responses of different muscle fibre types in the presence of P-antagonists indicates that BRL affects muscle GUI directly, in addition to effects mediated by increases in plasma insulin concentration; (ii) the resistance of the BRL response to conventional P-adrenoceptor antagonists implicates an atypical adrenoceptor mediating the GUI response in skeletal muscle, but this may not be identical to the adipose tissue P3-adrenoceptor; (iii) the potentiation of BRL responses by ICI 118551 indicates an inhibitory P2-adrenoceptor-mediated component in the muscle GUI response to BRL.

Changes in tissue blood flow and beta-receptor density of skeletal muscle in rats treated with the beta2-adrenoceptor agonist clenbuterol.

Rats injected with the beta2-adrenoceptor agonist clenbuterol (2 mg kg-1 per day) for 18 days gained significantly more weight than controls. Tissue blood flow assessed 24 h after the last injection from the distribution of radiolabelled microspheres was increased in white (5 fold) and brown (3 fold) adipose tissue of clenbuterol-treated rats but was unaffected in kidney, brain and diaphragm, and was reduced by about 80% in skeletal muscle. Acute injection of clenbuterol one hour before measuring blood flow, increased blood flow to brown fat (20 fold) in both treated and control groups. Blood flow to skeletal muscle increased more in the rats treated chronically with clenbuterol (6 fold increase) than in control rats (2 fold increase), but absolute flow rates were still significantly lower in the rats treated chronically with clenbuterol. Skeletal muscle beta-adrenoceptor density and subtype were assessed from ligand binding and displacement studies using [3H]-dihydroalprenolol. Rats treated with clenbuterol for 18 days showed a 50% reduction in beta-receptor density, but the ratio of beta 1/beta 2-receptors was unaffected (15% beta 1/85% beta 2). The results indicate that, although clenbuterol produces acute increases in muscle blood flow, chronic treatment results in lower flow rates immediately (1 h) and 24 h after the previous injection. The attenuated response following chronic treatment is associated with a marked reduction in skeletal muscle beta-adrenoceptor density. The data suggest that any anabolic effects of clenbuterol on muscle which may require, or may be mediated by increases in blood supply, cannot be sustained by chronic treatment. Conversely, blood flow to white and brown adipose tissue would appear to be potentiated by chronic treatment, possibly reflecting increases in lipolytic and/or thermogenic activity.

Biphasic effects of the beta-adrenoceptor agonist, BRL 37344, on glucose utilization in rat isolated skeletal muscle.

1. The effects of the selective beta 3-adrenoceptor agonist, BRL 37344 (BRL) on glucose uptake and phosphorylation (i.e. glucose utilization; GU) and glycogen synthesis in rat isolated soleus and extensor digitorium longus (EDL) muscle preparations in vitro were investigated by use of 2-deoxy-[3H]-glucose (GU) and [U-14C]-glucose (glycogen synthesis). 2. Low concentrations of BRL (10(-11)-10(-9) M) significantly increased GU, with maximal increases of 30% in soleus and 24% in EDL at 10(-11) M. Neither the selective beta 1-adrenoceptor antagonist, atenolol (10(-8)-10(-6) M), nor the selective beta 2-adrenoceptor antagonist, ICI 118551 (10(-8)-10(-6) M) had any effect on the stimulation of GU induced by 10(-11) M BRL. 3. High concentrations of BRL (10(-6)-10(-5) M) caused significant inhibition (up to 30%) of GU in both soleus and EDL muscles. The inhibition of 10(-6) M BRL was blocked completely by 10(-6) and 10(-7) M ICI 118551 in soleus, and by 10(-6)-10(-8) M ICI 118551 in EDL; atenolol (10(-8)-10(6) M) had no effect. 4. Another selective beta 3-adrenoceptor agonist, CL 316,243, also caused a significant stimulation of muscle GU, with maximal increases of 43% at 10(-9) M in soleus and 45% at 10(-10) M in EDL. The stimulation of GU declined with further increases in the concentration of CL 316,243, but no inhibition of GU was seen, even at the highest concentration (10(-5) M) tested. 5. BRL at 10(-5) M inhibited completely insulin-stimulated glycogen synthesis in both soleus and EDL, but this inhibitory effect of BRL was abolished by 10(-6) M ICI 118551. BRL at 10(-11) M (with or without 10(-6) M ICI 118551) had no effect on insulin-stimulated glycogen synthesis. 6. It is concluded that: (i) low (< nM) concentrations of BRL stimulate GU via an atypical beta-adrenoceptor that is resistant to conventional beta 1-adrenoceptor and beta 2-adrenoceptor antagonists; (ii) the stimulation of GU is negated by the activation of beta 2-adrenoceptors that occurs at higher (> nM) concentrations of BRL; (iii) inhibition of GU via beta 2-adrenoceptor activation is associated with inhibition of glycogen synthesis, possibly due to activation of glycogenolysis; (iv) the opposing effects of beta 2-adrenoceptor and atypical beta-adrenoceptor activation on GU suggest that in skeletal muscle these adrenoceptors are linked to different post-receptor pathways.

Sympathetic mechanisms in diet-induced thermogenesis: modification by ciclazindol and anorectic drugs.

1 The sympathetic noradrenergic activation of brown adipose tissue and the biochemical mechanisms involved in diet-induced thermogenesis were studied in rats. 2 A close correlation was found between brown adipose tissue Na+, K+-adenosinetriphosphatase (Na+, K+-ATPase) activity in vitro and in vivo measurements of resting oxygen consumption (VO2). The effects of noradrenaline on in vitro NA+, K+-ATPase activity in brown adipose tissue and in vivo VO2 could be mimicked by a variety of agents. These included beta-adrenoceptor agonists and agents known to induce the release of noradrenaline or inhibit the noradrenaline uptake process. The pharmacological evidence suggests that dopaminergic mechanisms may also be involved in the control of thermogenesis. 3 Amphetamine did not increase VO2 in rats without causing associated increases in locomotor activity. Ciclazindol at doses of 3-30 mg/kg intraperitoneally increased VO2 but did not appear to increase locomotor activity or evoke any other signs of CNS stimulation including lengthening of time to sleep onset or stereotypy. Separation of metabolic and CNS effects occurred only at the lowest dose of mazindol used (0.3 mg/kg i.p.). These results are probably a reflection of (a) the relative abilities of these drugs to inhibit brain and brown adipose noradrenaline uptake processes and (b) the relatively high accumulation of ciclazindol in brown adipose. 4 Of the drugs tested, only ciclazindol was a more potent inhibitor of the noradrenaline uptake system in brown adipose tissue (BAT) than in brain. Kinetic analysis also revealed that the actions of ciclazindol on the NA uptake system and Na+, K+-ATPase in BAT differed from those of mazindol. 5 These findings suggest that ciclazindol may produce an energy wasting effect in rodents without causing overt CNS stimulation; the implications of these findings in terms of human obesity are discussed.

Thermogenic effects of sibutramine and its metabolites.

1. The thermogenic activity of the serotonin and noradrenaline reuptake inhibitor sibutramine (BTS 54524; Reductil) was investigated by measuring oxygen consumption (VO2) in rats treated with sibutramine or its two pharmacologically-active metabolites. 2. Sibutramine caused a dose-dependent rise in VO2, with a dose of 10 mg kg(-1) of sibutramine or its metabolites producing increases of up to 30% that were sustained for at least 6 h, and accompanied by significant increases (0.5-1.0 degrees C) in body temperature. 3. Based on the accumulation in vivo of radiolabelled 2-deoxy-[3H]-glucose, sibutramine had little or no effect on glucose utilization in most tissues, but caused an 18 fold increase in brown adipose tissue (BAT). 4. Combined high, non-selective doses (20 mg kg(-1)) of the beta-adrenoceptor antagonists, atenolol and ICI 118551, inhibited completely the VO2 response to sibutramine, but the response was unaffected by low, beta1-adrenoceptor-selective (atenolol) or beta2-adrenoceptor-selective (ICI 118551) doses (1 mg kg(-1)). 5. The ganglionic blocking agent, chlorisondamine (15 mg kg(-1)), inhibited completely the VO2 response to the metabolites of sibutramine, but had no effect on the thermogenic response to the beta3-adrenoceptor-selective agonist BRL 35135. 6. Similar thermogenic responses were produced by simultaneous injection of nisoxetine and fluoxetine at doses (30 mg kg(-1)) that had no effect on VO2 when injected individually. 7. It is concluded that stimulation of thermogenesis by sibutramine requires central reuptake inhibition of both serotonin and noradrenaline, resulting in increased efferent sympathetic activation of BAT thermogenesis via beta3-adrenoceptor, and that this contributes to the compound's activity as an anti-obesity agent.

Changes in body composition, brown adipose tissue activity and thermogenic capacity in BN/BiRij rats undergoing senescence.

Metabolic rate, thermogenesis, brown adipose tissue (BAT) activity, and body composition were followed in ageing rats (female BN/BiRij) at 3 to 35.5 months of age. Colonic temperatures were similar in rats at 3 to 23 months of age (37.1-37.6 degrees C), but significantly reduced (36.3 degrees C) in those aged 36 months. Resting oxygen consumption (VO2), corrected for body size, was comparable in all groups, but the thermogenic response to noradrenaline was significantly reduced with age. BAT mass was unaffected by age, but brown fat protein content, specific mitochondrial cytochrome oxidase activity, and thermogenic activity (assessed from mitochondrial purine nucleotide binding) all declined markedly with age. Carcass analysis revealed a fall in body protein in very old (35.5 month) rats, but body fat content increased up to 23 months of age and thereafter declined.

Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat.

Young male rats were treated with vehicle or ciglitazone (150 mg/kg/day, intragastric) for 8 or 14 days. Drug treatment did not affect food intake but reduced body weight and energy gains over 14 days, and significantly depressed energetic efficiency. Energy expenditure and resting oxygen consumption (VO2), when corrected for body size, were elevated in ciglitazone-treated rats, but the difference in VO2 was abolished by treatment of the animals with a beta-adrenergic antagonist (propranolol). The acute thermic response (postprandial rise in VO2) to a fat meal was similar for both groups, but the response to carbohydrate ingestion was greater in ciglitazone-treated rats (18%) than controls (11.5%). The mass of interscapular brown adipose tissue was not affected by drug treatment, but its protein content was increased and its thermogenic activity (mitochondrial purine nucleotide binding) was elevated by 25% after chronic treatment with ciglitazone. These results indicate that ciglitazone enhances thermogenesis via sympathetic activation of brown adipose tissue, probably as a result of improved insulin sensitivity.

Effect of selective beta-adrenoceptor stimulation on UCP synthesis in primary cultures of brown adipocytes.

Given the co-existence of the three beta-adrenoceptor (beta AR) subtypes (beta 1AR, beta 2AR and beta 3AR) in brown adipocytes, the present study was undertaken to determine the relative importance of these in the induction of UCP synthesis in mouse BAT precursor cells in primary culture. Cells at different stages of differentiation were exposed to different beta AR agonists: prenalterol (a selective beta 1AR agonist), salbutamol or clenbuterol (selective beta 2AR agonists), or BRL 37344 (a selective beta 3AR agonist). As with the endogenous agonist, noradrenaline, and the non-selective beta AR agonist, isoprenaline, all four beta AR agonists induced UCP in the confluent stage of the cells, but with different potencies, and with the highest induction being seen after clenbuterol or BRL 37344 treatment. Cells in the confluent stage of development were the most sensitive to the effects of the agonists, although clenbuterol and BRL 37344 induced a weak UCP synthesis in pre-confluent cells. None of these beta AR agonists were able to induce UCP synthesis in the post-confluent period. The responses to prenalterol and salbutamol were inhibited by propranolol at relatively low concentrations, suggesting their effects were mediated by beta 1AR and beta 2AR, respectively. However, propranolol was a particularly weak antagonist of BRL 37344 and, unexpectedly, of the clenbuterol UCP responses, which suggests that both induce UCP synthesis via the beta 3AR. In summary, the beta 3AR is the most important adrenoceptor coupled to the induction of UCP synthesis, although both beta 1AR and beta 2AR activation may make a contribution. However, all three beta AR subtypes do not become fully functional until cultured cells become confluent.