Michael J. Stock

Chronic effects of beta 2-adrenergic agonists on body composition and protein synthesis in the rat

Chronic treatment of rats with the β2-adrenergic agonists clenbuterol and fenoterol over 16–19 d raised energy intake, expenditure, and body weight gain but did not affect fat or energy deposition, and body protein gain was increased by 50 and 18%, respectively. Both drugs increased the protein content and mitochondrial GDP-binding capacity of brown adipose tissue. Clenbuterol did not affect plasma insulin, growth hormone, or triiodothyronine levels, although insulin levels were reduced by fenoterol. Both drugs caused hypertrophy of skeletal muscle (gastrocnemius), and muscle protein synthesis in vivo (fractional rate) was elevated by 34 and 26% in clenbuterol and fenoteroltreated rats, respectively.

Influence of noradrenaline on blood flow to brown adipose tissue in rats exhibiting diet-induced thermogenesis.

Abstract1.The influence of noradrenaline on regional blood flow was determined using radioactive microspheres in rats maintained on either stock diet or a palatable cafeteria diet.2.Cardiac output and blood flow to brain, lungs, liver and skeletal muscle were similar for rats on the two diets.3.Blood flow to total dissectable brown adipose tissue in control and cafeteria rats represented 1 and 2% of cardiac output respectively but these values rose to 7 and 15.5% during infusion of noradrenaline.4.Arterial oxygen content was similar for all groups but the oxygen content of venous blood draining the interscapular brown adipose tissue fell to 6 ml O2/100 ml blood in control rats and 1 ml/100 ml in cafeteria rats after noradrenaline.5.The total oxygen consumption of brown adipose tissue was calculated and found to account for 42% of the response to noradrenaline in control rats and 74% in cafeteria animals. The increments in the oxygen consumption of other tissues were almost identical in both groups and so all the diet-induced changes in thermogenic capacity can be attributed to increases in brown adipose tissue metabolism.6.These findings demonstrate the quantitative importance of brown adipose tissue in diet-induced thermogenesis and confirm the similarities between diet and non-shivering thermogenesis.

Thermogenic effects of sibutramine and its metabolites

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. 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°C) in body temperature. 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). Combined high, non‐selective doses (20 mg kg−1) of the β‐adrenoceptor antagonists, atenolol and ICI 118551, inhibited completely the VO2 response to sibutramine, but the response was unaffected by low, β1‐adrenoceptor‐selective (atenolol) or β2‐adrenoceptor‐selective (ICI 118551) doses (1 mg kg−1). 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 β3‐adrenoceptor‐selective agonist BRL 35135. 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. 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 β3‐adrenoceptor, and that this contributes to the compounds activity as an anti‐obesity agent.

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.

Hyperphagia in cold-exposed rats is accompanied by decreased plasma leptin but unchanged hypothalamic NPY

Chronic cold exposure stimulates sympathetically driven thermogenesis in brown adipose tissue (BAT), resulting in fat mobilization, weight loss, and compensatory hyperphagia. Hypothalamic neuropeptide Y (NPY) neurons are implicated in stimulating food intake in starvation, but may also suppress sympathetic outflow to BAT. This study investigated whether the NPY neurons drive hyperphagia in rats that have lost weight through cold exposure. Rats exposed to 4°C for 21 days weighed 14% less than controls maintained at 22°C ( P < 0.001). Food intake increased after 3 days and remained 10% higher thereafter ( P < 0.001). Increased BAT activity was confirmed by 64, 96, and 335% increases in uncoupling protein-1 mRNA at 2, 8, and 21 days. Plasma leptin decreased during prolonged cold exposure. Cold-exposed rats showed no significant changes in NPY concentrations in any hypothalamic regions or in hypothalamic NPY mRNA at any time. We conclude that the NPY neurons are not activated during cold exposure. This is in contrast with starvation-induced hyperphagia, but is biologically appropriate since enhanced NPY release would inhibit thermogenesis causing potentially lethal hypothermia. Other neuronal pathways must therefore mediate hyperphagia in chronic cold exposure.

Mechanisms of thermogenesis induced by low protein diets

Weanling (22-day-old) rats fed a low protein (8% casein) diet consumed the same amount of energy as controls (22% casein diet), but intake corrected for body size (kJ/kg0.75) was increased in the former group. Weight gain and the efficiency of gain (g gain/MJ) were markedly reduced in low protein fed rats. Resting oxygen consumption (VO2) was elevated by 15% in the low protein group but this difference was completely abolished by beta-adrenergic blockade with propranolol. Interscapular brown adipose tissue (BAT) mass, protein content, mitochondrial yield and GDP binding were increased in low protein fed rats but mitochondrial alpha-glycophosphate shuttle activity of BAT was unaltered, although shuttle activity was elevated in liver mitochondria. Plasma triiodothyronine levels were increased by 64% in the low protein group, whereas insulin levels were markedly reduced in spite of normal blood glucose levels. Resting VO2 and BAT mass were also increased in older (55-day-old) rats fed the low protein diet, but the changes were smaller than in weanling rats. These data suggest that the decreased metabolic efficiency seen in rats fed protein deficient diets involves sympathetic activation of BAT, and is therefore similar to the thermogenic responses seen in cold adapted and cafeteria-fed animals.

Effect of chronic food restriction on energy balance, thermogenic capacity, and brown-adipose-tissue activity in the rat

Young male rats were fed a pelleted stock diet either ad libitum (control) or in restricted amounts (65% of control intake) for 17 d. Body energy gain and energy expenditure, determined from energy balance measurements, were reduced by 73 and 27% respectively compared to controls. Resting oxygen consumption was similar for both groups during the light phase but was significantly depressed in energy-restricted rats at night, and the thermogenic response to noradrenaline was also reduced in these animals. Brown-adipose-tissue mass, protein content, and mitochondrial protein were all decreased in the food-deprived rats, and specific and total depot mitochondrial GDP-binding capacity were 29 and 53% lower than controls. The reduced energy expenditure which occurs during food restriction may be due partly to a lower activity of brown adipose tissue which is associated with a decrease in thermogenic capacity.

Changes in tissue blood flow and β‐receptor density of skeletal muscle in rats treated with the β2‐adrenoceptor agonist clenbuterol

1 Rats injected with the β2‐adrenoceptor agonist clenbuterol (2 mg kg−1per day) for 18 days gained significantly more weight than controls. 2 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. 3 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. 4 Skeletal muscle β‐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 β‐receptor density, but the ratio of β1/β2‐receptors was unaffected (15% β1/85% β2). 5 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 β‐adrenoceptor density. 6 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.

Leptin directly stimulates thermogenesis in skeletal muscle

Using a method involving repeated oxygen uptake (MO2) determinations in skeletal muscle ex vivo, the addition of leptin was found to increase MO2 in soleus muscles from lean mice. These effects were found to be inhibited by phosphatidylinositol 3‐kinase inhibitors, absent in muscles from obese Leprdb mice which have the dysfunctional long form of leptin receptor, and blunted in muscles from diet‐induced obese mice in the fed state but not during fasting. These findings indicate that leptin has direct thermogenic effects in skeletal muscle, and that these effects require both the long form of leptin receptors and phosphatidylinositol 3‐kinase signalling.

Sympathetic activation of brown-adipose-tissue thermogenesis in cachexia

Tumour-bearing mice spontaneously lose weight 8–9 weeks after implantation of a human hypernephroma, in spite of a normal food intake. Resting oxygen consumption was up to 40% higher in these animals than in sham-operated controls, but was significantly reduced by 8-adrenergic blockade with propranolol in the former group. The injection of noradrenaline caused a marked stimulation of the metabolic rate in all the animals, but the greatest response was seen in the cachectic mice. The brown-adipose-tissue mass was similar for both groups, but guanosine diphosphate binding to brownadipose-tissue mitochondria (an index of thermogenic capacity) was significantly increased in turnout-bearing mice, and the injection of noradrenaline 1 h prior to sacrifice caused the greatest stimulation of binding in the cachectic group. These data suggest that the rapid weight loss of tumour-bearing animals may be due to a high metabolic rate which results from sympathetic stimulation of brown-adipose-tissue metabolism. The relevance of these results to cancer-induced cachexia in man is discussed.