The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.     

RETARDATION OF PERIPHERAL NERVE MYELINATION IN MICE TREATED WITH INHIBITORS OF CHOLESTEROL BIOSYNTHESIS : A Quantitative Electron Microscopic Study

The effect of two inhibitors of cholesterol biosynthesis, triparanol and AY 9944, on peripheral nerve myelination, was studied. Suckling mice were intraperitoneally injected with both drugs on 3 consecutive days and were sacrificed 6 hr after the last injection; others were suckled by an injected mother and sacrificed at 2½ days of age. A single mouse which had been injected with both drugs at 1, 2, and 3 days of age was sacrificed 2 wk after the last injection. Membranous and crystalline intracytoplasmic inclusions were observed in the Schwann cells of the sciatic nerves of all the experimental animals. Both the number of unmyelinated single axons and the number of myelin lamellae around each myelinating axon in the sciatic nerves were recorded for treated mice and of mice suckled by treated mothers. The sciatic nerve of the experimental mice contained a larger proportion of unmyelinated single axons and smaller numbers of myelin lamellae around the myelinating axons, when compared with age-matched controls. The results suggest that a decrease of endogenous cholesterol in suckling mice may affect peripheral nerve myelination in two ways: by retarding the "triggering" of myelination in unmyelinated axons and by decreasing the rate of myelination already in progress.     

The relationship between the thermogenic response of brown adipose tissue and age during noradrenaline infusion in the young rabbit

This work was undertaken to determine if the thermogenic activity of brown fat decreased with age in young rabbits despite the morphological evidence indicating persistence of the brown adipocytes at 4 weeks of age. Data obtained by infusing five doses of noradrenaline and measuring oxygen consumption were used to construct cumulative dose-response curves for five age groups between 3 and 32 days of age. Blood flow to brown fat and other tissues was measured by the microsphere method at 1 and 3 weeks of age. The noradrenaline-induced increase in oxygen consumption when expressed as a percentage of resting oxygen consumption in millilitres per 100 g of body weight decreased (p less than 0.05) with age. However, the absolute noradrenaline-induced increase in metabolic rate (millilitres per minute) increased with age. Total blood flow to brown fat (millilitres per minute) during noradrenaline infusion was unchanged between 1 and 3 weeks of age, but when the blood flow was expressed in millilitres per minute per gram of tissue flow decreased significantly (p less than 0.05) probably because of infiltration of brown fat with white fat. These data suggest that the amount of brown fat and its thermogenic capacity remain relatively constant between 1 and 3 weeks of age, but as a thermogenic organ, brown fat becomes proportionally less effective with age because of the large increase in body mass.     

Effect of thyroidectomy on cardiovascular responses to hypoxia and tyramine infusion in fetal sheep

Fetal sheep were thyroidectomized at 80 days' gestation and reoperated at 118-122 days for insertion of vascular catheters. The effects of hypoxaemia and intravenous tyramine infusion on plasma catecholamine concentrations, blood pressure and heart rate were then determined in experiments at 125-135 days' gestation. Age matched intact fetuses were also studied. Thyroidectomy was associated with increased concentrations of noradrenaline, adrenaline and dopamine in some thoracic and abdominal organs, increased noradrenaline concentrations in the cerebellum, and decreased adrenaline concentrations in the hypothalamus, cervical spinal cord, and superior cervical and inferior mesenteric ganglia. Arterial pressure was significantly lower in the thyroidectomized fetuses (34.0 +/- 0.15 mmHg) than in intact fetuses (44.7 +/- 0.2 mmHg; p less than 0.001). In contrast, plasma noradrenaline concentrations were significantly higher in the thyroidectomized fetuses (2.04 +/- 0.25 ng/ml) compared to the intact fetuses (0.99 +/- 0.08 ng/ml; P less than 0.001). In the intact fetuses there was a significant increase in plasma noradrenaline concentration and blood pressure during hypoxaemia, and bradycardia at the onset of hypoxaemia. In contrast, in the thyroidectomized fetuses hypoxaemia did not cause significant change in plasma catecholamine concentrations, blood pressure or heart rate. Infusion of tyramine produced a 1.9-fold increase of plasma noradrenaline in thyroidectomized fetuses compared to a 9.2-fold increase in the intact fetuses (P less than 0.05). Tyramine infusion caused a similar proportional increase of blood pressure in both thyroidectomized and intact fetuses. Heart rate decreased during the tyramine-induced hypertension in the intact fetus, but increased in the thyroidectomized fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of functional uncoupling protein in guinea pigs infused with noradrenaline. Studies with isolated brown adipocytes.

Continuous infusion of noradrenaline over the interscapular brown fat of guinea pigs maintained at thermoneutrality (28-32 degrees C) induces changes similar to those after cold-adaptation. (1) Multilocular fat droplets appear within the brown adipocytes. (2) The number of mitochondria per adipocyte and the total number of adipocytes both increase. (3) Noradrenaline addition to isolated adipocytes causes near maximal uncontrolled respiration. (4) The cells become more sensitive to fatty acid-induced uncoupling. (5) The tissue-specific uncoupling protein per mg of mitochondrial protein is increased 5-fold. Specific alpha- and beta-agonists were also chronically infused. (6) Separate infusion of phenylephrine or isoprenaline was not able to stimulate mitochondriogenesis or hyperplasia. (7) Adipocytes from these animals could not be uncoupled by acute noradrenaline. (8) Simultaneous chronic infusion of phenylephrine and isoprenaline reproduced the effects of chronic noradrenaline infusion.     

The relationships between interphase Schwann cells and axons before myelination: a quantitative electron microscopic study

Electron micrographs of transversely sectioned sciatic nerves removed from newborn, 3-day-old, and 7-day-old rats were used to make montages of comparable areas in the marginal bundle of the posterior tibial fascicle. At each age, the number of axons, their diameter, their relationships with Schwann cell processes, and their degree of myelination were determined. Also, three-dimensional reconstructions of representative fiber groups in the newborn nerve were made from similar montages at 5 additional transverse levels. The results showed that outgrowth of axons and migration of Schwann cells continued after birth. Families of Schwann cells, each surrounded by a common basal lamina, formed the sheaths that subdivided the bundles. Axons to be myelinated appeared to progress radially from a bundle to a 1 : 1 relationship with a Schwann cell at the sheath's outer margin. Sheaths containing multiple Schwann cells became smaller and more numerous as axon bundles were subdivided. Almost all of the isolated Schwann cells, which were separated from their neighbors by collagen were myelinating single large axons.     

Effect of noradrenaline on oxygen consumption and tissue blood flow in young pigs

Metabolic rate and tissue blood flow were measured by indirect calorimetry and the radioactive microsphere technique, respectively, in anaesthetized pigs aged 10, 17, and 28 days during infusions of saline or noradrenaline. Noradrenaline produced a rise in oxygen consumption in all pigs but the magnitude of the response declined with age in Large White pigs (percent increase: 10 days old, 29.5 +/- 3.3; 17 days old, 15.3 +/- 3.2; 28 days old, 5.4 +/- 0.17) and was less in 10-day-old Minipigs (9.1 +/- 5.1). Regional blood flow per gram of tissue was similar for pigs of all ages and noradrenaline produced increases in flow to heart and skin, and small reductions in blood flow to skeletal muscle. Noradrenaline increased average blood flow to adipose tissue (four sites) by 21-fold in 10-day-old Large White pigs, but had less effect in 10-day-old Minipigs (9.5-fold) and in older Large White pigs (9-fold), where the effect on metabolic rate was diminished. It is calculated that adipose tissue could be responsible for the increase in metabolic rate during infusion of noradrenaline. Macroscopically there are no gross differences between various adipose tissue depots in the pig but those which showed the greatest response to noradrenaline correspond to areas where brown adipocytes have previously been identified by electron microscopy.     

A quantitative electron microscopic analysis of myelination in the optic nerve of suckling rats treated with an inhibitor of cholesterol biosynthesis

Summary The effect of AY-9944, an inhibitory cholesterol biosynthesis, on the myelination of the optic nerve of rats was studied. Suckling rats were injected intraperitoneally with the drug every other day from birth, and were sacrificed at 10, 20 and 30 days of age together with littermate controls. The analysis is based on counting, at the electron-microscope level, the number of unmyelinated axons and the number of myelin lamellae surrounding each myelinating axon. The results indicate that a decrease in endogenous cholesterol by AY 9944, induced an overall retardation of the myelination process in the optic nerve: a larger proportion of myelinated axons and smaller number of myelin lamellae around the myelinating axons, when compared with the littermate controls, was observed. Exogenous cholesterol from the maternal milk did not compensate for a lack in endogenous cholesterol.Degenerating myelin sheaths were frequently seen in the experimental optic nerves at 20 and 30 days of age. Numerous membranous, intracytoplasmic drug-induced inclusions were found at all ages studied. Acknowledgements. The author is particularly indebted to Dr. B. G. Uzman and Dr. G. M. Villegas for their valuable discussion and suggestions. He wishes also to thank Mr. F. Paredes, Mr. J. Aristimuño and Miss Marcia Escala for their technical assistance; Mr. J. Bigorra for the photographic aid, and Miss Sonia Rodríguez for her secretarial help.     

Development of cutaneous innervation in the chick: ultrastructural and quantitative analysis (author's transl)

In the chick, at the thoracic level, the dorsal branches of spinal nerves form at 4 days of incubation (stage 25) and reach the skin between 5 1/2 and 6 days (stages 28-29). At 6 days, the growing nervous peripheral processes ("axons") form large bundles (200-1,000 fibers). At 10 days, young Schwann cells divide the bundles into groups of axons. The perineurium and endoneurium differentiate between 10 and 14 days (but epineurium is formed after hatching). At 14 days of incubation, the adult pattern of cutaneous innervation is established. At this same stage, myelogenesis begins but develops mainly after hatching : 1% of the axons is myelinated at 16 days of incubation, 4% at hatching, 40% in 6-week old chickens and 60% in adults. Thus, less than 10% of myelinated axons of the adult are already myelinated at hatching. Two modes of myelogenesis were observed: 1) early myelination, starting in the embryo around axons measuring about 1 micrometer in diameter: 2) delayed myelination, occurring in the older chickens after an increase in axon diameter. These observations suggest that there is, in the development of chick skin innervation, a critical stage (14-15 days of incubation) apparently corresponding to the stabilization of cutaneous nerve supply.     

Nonshivering thermogenesis and brown fat in spontaneously hypertensive rats

Oxygen consumption was measured before and during infusion of the catecholamine isoproterenol in age-matched spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats. Mass-independent rates of oxygen consumption of anesthetized 7-week-old rats were similar in the WKY and SHR rats (11.08 +/- 0.74 and 11.33 +/- 0.82 ml O2 min-1 kg-.67, respectively). Catecholamine infusion elicited increased total oxygen consumption in both WKY and SHR animals (15.0 +/- 1.0 and 14.9 +/- 1.2 ml O2 min-1 kg-.67, respectively), and the magnitude of these increases did not significantly differ. To assess whether there were changes in the metabolic state of brown adipose tissue, the major site of catecholamine-induced thermogenesis in rats, enzymes whose activity is proportional to aerobic capacity were assayed in vitro. In both the interscapular and cervical brown fat depots, maximal citrate synthase and maximal HOAD (beta-hydroxyacyl-CoA dehydrogenase) activities were similar in SHR and WKY rats. There were also no significant differences in brown fat protein content, suggesting no differential growth of this tissue in the two rat strains. Our results indicate that the nonshivering thermogenic capacity of the hypertensive SHR rats does not differ from that of the normotensive WKY animals.     

Uncoupling protein and its mRNA in brown adipose tissue of newborn rabbits

Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 +/- 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 +/- 0.017 at 3 days, 0.428 +/- 0.038 at 5 days, and 0.208 +/- 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.     

Effect of the early postnatal administration of cortisol on development of the nervous control of cardiac function in albino rats

When infant rats were treated with cortisol, in daily s.c. doses of 20 mg X kg-1, between the ages of 2 and 15 days, the noradrenaline content of their heart and spleen, between the ages of 23 and 65 days, was lower than in the controls. The decrease in the noradrenaline content did not diminish with advancing age; on the contrary, it was the most pronounced at 65 days. Cortisol treatment did not affect the noradrenaline content of skeletal muscles. The functional significance of the decrease in the noradrenaline content was studied in nervous control of the sinoatrial node of the heart. In agreement with the drop in noradrenaline concentration, transmural stimulation of the sinoatrial node region of isolated atria led to a mild, but statistically significant reduction of the function of sympathetic nerve endings, whereas parasympathetic innervation of the node showed no signs of impairment. This peripheral functional deficiency of sympathetic innervation of the node is not seen in the intact organism, where it is masked by central nervous mechanisms. Rats given cortisol postnatally had a significantly higher heart rate at 23, 33 and 44 days, because, in the presence of normal sympathetic influence, the tone of the parasympathetic nerves was reduced. The heart rate was highest at 23 days; with advancing age the difference diminished and at 65 days it was statistically nonsignificant.     

A comparison between the effects of cold exposure in vivo and of noradrenaline in vitro on the metabolism of the brown fat of new-born rabbits

1. Exposure of new-born rabbits to the cold leads to an increase in the incorporation of [(14)C]glucose into the glycerol of brown-fat triglyceride, but has no effect on [(14)C]glucose incorporation into triglyceride of white fat or liver. The effect of cold exposure on brown-fat triglyceride is abolished by cutting the cervical sympathetic nerve. 2. Brown fat incorporates very little [(14)C]glucose into triglyceride fatty acids, either in vivo or in vitro. 3. Noradrenaline added to incubations of brown fat from new-born rabbits stimulates O(2) consumption, CO(2) output and incorporation of glucose into triglyceride glycerol. The effects of noradrenaline in vitro are therefore consistent with the hypothesis that noradrenaline mediates the response of the brown fat of new-born rabbits to cold exposure. 4. Glycerokinase is present in the brown fat of new-born rabbits, but its activity is much less than that of the glycerokinase in the brown fat of adult rats. 5. Insulin has no effect on O(2) consumption, CO(2) output or glucose uptake in brown fat of new-born rabbits. 6. It is concluded that the thermogenic response of new-born rabbits to cold exposure is accompanied by a selective acceleration of the triglyceride cycle in brown fat. However, resynthesis of triglyceride would not account for more than 1% of the O(2) consumed in vitro by new-born rabbit brown fat in the presence of noradrenaline if respiration remains coupled to phosphorylation.     

TRANSPORT OF NORADRENALINE IN SYMPATHETIC NERVES AND THE EFFECT OF NERVE IMPULSES ON ITS CONTRIBUTION TO TRANSMITTER STORES

Abstract— —A study has been made of the contribution of noradrenaline transport along sympathetic nerves to their terminal stores of transmitter by ligating the splenic nerves of the cat, and measuring both the noradrenaline that accumulates above the constriction, and the noradrenaline content of the spleen. The biochemical estimations were supplemented by fluorescence histochemistry. The effect of abolishing efferent impulses in the splenic nerves was examined by cutting their preganglionic nerve supply.
The proximo-distal flow rate for noradrenaline was calculated as 1.4-3.3 mm/hr assuming that all the noradrenaline that accumulates is derived from the cell bodies in the ganglion without net addition or loss in the axons. The process was not dependent on impulse traffic in the nerves, since decentralization did not significantly effect the accumulation. The amount of noradrenaline arrested by the constriction in 24 hr was only 1 per cent of the stores in the terminals of those nerves, and consequently no change was detected in the spleen's noradrenaline content as a result of constricting its nerve supply.
In the presence of an intact reflex pathway to the spleen, the stress of the operative procedure produced a marked constriction of the spleen, and depletion of its noradrenaline content. These changes could be prevented either by section of the preganglionic splanchnic nerves, or by ligation of the splenic nerves, thereby blocking the conduction of efferent nerve impulses.
The evidence favours a proximo-distal flow of noradrenaline in sympathetic nerves, independent of nerve impulses, which makes, however, a negligible quantitative contribution to the terminal stores of transmitter.     

Brown and white fat during the life of the rabbit

The authors examined brown and white fat of rabbits of all ages for its sympathetic nerve content. The results of chemical analyses and fluorescent histochemistry showed that the catecholamines of brown fat of the young rabbit slowly disappear with age. This gradual process occurred at the same time as brown fat changed to white fat.     

Effect of prolonged glucose infusion into fetal sheep on body growth, fat deposition and gestation length

Eleven Merino sheep fetuses were supplemented with glucose by direct continuous intravenous infusion of 50% dextrose into the fetus from day 115 of gestation until spontaneous delivery. Infusion rates of 15 or 25 g/day per kg were used and equivalent volumes of saline were infused into 11 control fetuses. Infusion periods approximated 27 days in both groups. Fetal plasma glucose concentrations were significantly (P less than 0.001) elevated throughout glucose infusion and resulted in variable but consistently higher plasma insulin concentrations in the glucose than in the saline-infused fetuses. Glucose-infused fetuses were significantly heavier than controls (mean +/- SEM; 3.86 +/- 0.16 vs 3.28 +/- 0.24 kg, P less than 0.05) and body fat depots (in g/kg body wt.) were larger in glucose-infused than control fetuses (9.91 +/- 0.65 vs 6.73 +/- 0.37, P less than 0.005, for internal brown fat depots; 1.25 +/- 0.44 vs 0.27 + 0.13, P less than 0.05, for subcutaneous white adipose tissue). The results indicate that growth and lipid deposition in the sheep fetus are responsive to increased glucose supply, an effect which may be mediated through the actions of insulin. Mean gestation length was 146.60 +/- 1.45 days for controls and 144.18 +/- 1.23 days for glucose-infused animals (normal term 150 days).     

NEUROCHEMICAL PROPERTIES OF ADRENERGIC NERVES REGENERATED AFTER 6-HYDROXYDOPAMINE

Abstract— The uptake-storage properties and synthesis of noradrenaline, and fluorescence morphology of adrenergic nerves which have been allowed to regenerate for 4 weeks after a chemical sympathectomy produced by 6-hydroxydopamine have been investigated in mouse iris and atrium. The regenerated nerve terminals displayed a lower formaldehyde-induced fluorescence intensity whereas the non-terminal axons exhibited a stronger fluorescence intensity and a more beaded appearance compared with mature nerves. The endogenous noradrenaline concentration after 6-hydroxydopamine was 30% in iris and 45% in atrium compared to control values. Recovery of [³H]noradrenaline uptake was found to be more rapid than that of endogenous noradrenaline concentration after the 6-hydroxydopamine treatment. [³H]Noradrenaline uptake in regenerating and adult mature nerves both obeyed Michaelis-Menten kinetics having identical K_m values. There was a close correlation between [³H]noradrenaline uptake and nerve density of adrenergic nerves regenerated after 6-hydroxydopamine. These results show that [³H]noradrenaline uptake is a better index for the number of regenerated nerve terminals than is the endogenous noradrenaline concentration. The retention of [³H]noradrenaline taken up and accumulated in vitro was about the same in regenerated and mature nerves, although a slight tendency to less effective retention was observed in the regenerated nerves. Subcellular distribution studies showed that relatively less [³H]noradrenaline was recovered in the microsomal fraction after 6-hydroxydopamine treatment. The formation of ¹⁴C-labelled catecholamines from [¹⁴C]DOPA was higher in regenerating nerves than indicated by the endogenous noradrenaline concentration but lower than that indicated by the [³H]noradrenaline. It is concluded that the regenerating nerves contain less endogenous noradrenaline than adult mature nerves and that the uptake mechanism develops promptly, whereas the development of the storage mechanism lags behind.     

Excitatory response of guinea-pig myometrium to intravenous noradrenaline declines towards term

Guinea-pigs were anaesthetized at three stages of pregnancy. Intrauterine pressure was recorded for a 1-h control period and during 10-min intravenous infusions of noradrenaline at rates of 1.0 and 10 micrograms/(min X kg). The mean and maximum amplitude of contractions occurring during the infusions was compared with that of contraction cycles registered in the control period. At 18-21 and 35-43 days post coitum, noradrenaline invariably evoked a rapid and sustained rise in intrauterine pressure, the amplitude of the contractions being greater than during spontaneous contraction cycles recorded in the control period. In late pregnancy, 59-68 days p.c., infusion of 1.0 micrograms noradrenaline/(min X kg) failed to elicit a clear response; contractions occurring during infusion of 10 micrograms noradrenaline/(min X kg) had amplitudes similar to those of the control period and were without a sustained contracture. The absence of denervation hypersensitivity, despite the occurrence of sympathetic denervation in the course of pregnancy, may be due to a generalized effect on excitation-contraction coupling, possibly caused by relaxin.     

Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man

Vascular effects of neuropeptide Y (NPY) and noradrenaline (NA) were studied in six human volunteers. Systemic infusion of human NPY for 40 min (5 pmol X kg-1 X min-1) increased arterial plasma NPY-like immunoreactivity (NPY-LI) from 12 +/- 2 to 356 +/- 30 pM. This concentration caused no systemic cardiovascular effects. The disappearance curve for NPY-LI was biphasic; the slopes of the two phases corresponding to half lives of 4.1 +/- 0.4 and 20 +/- 2 min respectively. Close i.a. infusion of human NPY in the forearm caused a slowly developing and dose dependent decrease in forearm blood flow (FBF) and increase in venous tone with maximal values of 44 +/- 6 and 235 +/- 81% of control respectively at 5 nmol X min-1. The corresponding values for NA (5 nmol X min-1) were 21 +/- 9 and 489 +/- 78% of control. A threshold concentration for a decrease in FBF was obtained at a plasma NPY-LI of 3.7 +/- 0.6 nM. The decrease in FBF caused by NPY was maintained for a much longer period compared to that of NA.     

Adrenergic mechanisms in the hepatic microcirculation in the rat

The in vivo hepatic microvascular bed of the rat was observed microscopically in the transilluminated liver and the diameter of the hepatic sinusoids was measured by serial photomicrography. Intraportal infusion of tyramine induced concentration-dependent constriction of the hepatic sinusoids, but also dilatation of the sinusoids when the dose was small. These effects were attributed to the release of endogenous noradrenaline which activated either alpha- or beta-adrenergic receptors and caused constriction, or dilatation, of the sinusoids respectively. Adrenaline and noradrenaline induced similar changes in the hepatic sinusoids as tyramine, while phenoxybenzamine induced dilatation, and propranolol constriction, of the sinusoids. All the above responses were abolished by pretreatment with reserpine. A possible noradrenaline-mediated basal vasomotor tone in the hepatic sinusoids for autonomic control of the blood flow in the sinusoids was postulated.