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J Nedergaard

Publications and source records attributed to J Nedergaard.

At least 109 records · Page 6Linked to original sources
PubMed1986

alpha- and beta-adrenergic control of thermogenin mRNA expression in brown adipose tissue.

By the use of an earlier characterised cDNA clone, CIN-1, corresponding to a sequence of the mRNA coding for the brown-fat specific "uncoupling" protein, thermogenin, the amount of thermogenin mRNA found in the brown adipose tissue of mice was quantitatively investigated under different physiological and pharmacological conditions. It was found that a 4 hr cold stress led to a 7-fold increase in the amount of thermogenin mRNA; injection of norepinephrine had a significant but smaller effect. Most notably, isoprenaline (beta-agonist) and phenylephrine (alpha-agonist) had in themselves no effect, but when injected together were able to increase the mRNA level synergistically. In 4 hr cold-stressed mice, norepinephrine, isoprenaline and cholera toxin could all further potentiate the effect of the cold stress itself on the mRNA level. Insulin and the glucocorticoid dexamethasone both had weak stimulatory effects on the mRNA level. It is concluded that an increase in intracellular cAMP levels is a necessary and perhaps sufficient stimulus for the increase in thermogenin gene expression. However, at least under in vivo conditions, this increase requires stimulation of both alpha- and beta-adrenergic pathways.

Adipose Tissue, Brown↗
PubMed1986

'Neuropeptide tyrosine' (NPY) is co-stored with noradrenaline in vascular but not in parenchymal sympathetic nerves of brown adipose tissue.

By using immunohistochemistry it is shown that both the parenchymal and vascular sympathetic innervation in the interscapular depot of brown adipose tissue in the rat contain the catecholamine-synthesizing enzyme tyrosine-hydroxylase (TH). In contrast, 'neuropeptide tyrosine' (NPY) is selectively present in the vascular sympathetic nerves of the tissue--but not in nerves around brown fat cells. This is consistent with the presence of two populations of neurons (containing either TH alone or TH plus NPY) in the stellate ganglion, which is the probable origin of the sympathetic nerves in the interscapular brown adipose tissue. Furthermore, the perivascular NPY-positive nerves in the brown adipose tissue disappeared after 6-hydroxydopamine treatment, demonstrating their noradrenergic nature. Taken together, these findings suggest that sympathetic nerves to blood vessels and brown fat cells represent two separate subpopulations of autonomic neurons.

Adipose Tissue, Brown↗
PubMed1986

Increased respiration in skeletal muscle mitochondria from cold-acclimated ducklings: uncoupling effects of free fatty acids.

Intermyofibrillar mitochondria from skeletal muscle (m. gastrocnemius) and liver mitochondria were isolated from cold-acclimated (4 degrees C) or control (30 degrees C) 4-week old ducklings. The respiratory rate of isolated mitochondria, with Na-succinate as substrate, was followed polarographically at 25 degrees C in order to determine the basal respiratory rate, the rate of respiration in the presence of free fatty acids (FFA) (Na-palmitate), and the fully uncoupled rate, after addition of FCCP. The basal respiration (which in liver mitochondria was unaffected by acclimation to cold) was higher (+53%) in intermyofibrillar mitochondria from cold-acclimated ducklings than from controls, and the maximal FCCP-stimulated respiration was also increased (+98%) by acclimation to cold. FFA-stimulated respiration increased as a function of FFA concentration in both types of mitochondria. The increase in respiration due to FFA was about double in intermyofibrillar mitochondria from cold-acclimated ducklings than that of controls, but in liver mitochondria there was no increase due to cold. The membrane potential was estimated by the dye safranine in the absence or in the presence of FFA in the incubation medium. There were no significant differences in the basal membrane potential in the two groups and the addition of FFA led to the same depolarization in both groups. The significance of these alterations for acclimation to cold is discussed.

Acclimatization↗
PubMed1986

Partial protection against erucoyl-carnitine inhibition in hamster brown-adipose-tissue mitochondria is due to high CoA levels: a comparison with rat brown-adipose-tissue mitochondria.

Brown-adipose-tissue mitochondria isolated from golden hamsters were found to contain more CoA per mg protein than rat brown-fat mitochondria, and after incubation with erucoyl-carnitine, a higher free CoA level remained, than in rat mitochondria. In accordance with the suggestion (Alexson et al. (1985) Biochim. biophys. Acta 834, 149-158) that the inhibitory effect of erucoyl-carnitine on brown-fat mitochondrial respiration is entirely due to CoA sequestration, hamster mitochondria (with more CoA) were less sensitive to erucoyl inhibition than were rat mitochondria. Thus, increased mitochondrial CoA levels may augment the ability of animals to withstand the detrimental effects of a high erucoyl ester content of the diet.

Adipose Tissue, Brown↗
PubMed1985

Effects of guanine nucleotides and cations on agonist affinity of alpha 1-adrenoceptors in brown adipose tissue.

In order to investigate the coupling mechanism of alpha 1-adrenoceptors in brown adipose tissue, the effects of guanine nucleotides and cations on agonist binding were studied with a membrane fraction obtained from hamsters. The affinity of the alpha 1-receptor for the adrenergic agent was followed in competition experiments with [3H]prazosin. It was found that the addition of GTP diminished the affinity of the alpha 1-receptor for norepinephrine but not for the alpha-antagonist phentolamine. This effect seemed to be dependent upon the presence of Mg2+ and could not be observed in isolated cells, indicating an intracellular site of action. A reduction of the Mg2+ concentration from the conventional 10 mM to the more physiological level of 1 mM markedly increased the affinity of the receptor for norepinephrine; this effect again was agonist-specific. The addition of Na+ (150 mM) decreased the agonist affinity of the receptor. It is suggested that the (not adenylate cyclase-coupled) alpha 1-adrenergic pathway in brown adipose tissue also is regulated by guanine nucleotides and modulated by the cations Mg2+ and Na+, indicating an involvement of a guanine nucleotide binding protein. Such a system may therefore be a general mechanism for the transduction of hormone stimulation over the cell membrane.

Adipose Tissue, Brown↗
PubMed1985

Inhibition of acetyl-carnitine oxidation in rat brown-adipose-tissue mitochondria by erucoyl-carnitine is due to sequestration of CoA.

The cause underlying the inhibitory effect of erucoyl-carnitine on acetyl-carnitine oxidation in rat brown-adipose-tissue mitochondria was investigated. The inhibition was shown to be of a noncompetitive nature, with an I50 of about 10 microM erucoyl-carnitine. Erucoyl-carnitine did not inhibit the respiratory chain or the citric acid cycle to a significant degree. Incubation of mitochondria with erucoyl-carnitine led to about 2/3 of all CoA being sequestered in the form of acid-insoluble esters (probably erucoyl-CoA). This sequestration had an apparent Km of about 10 microM. Erucoyl-carnitine also inhibited pyruvate oxidation with an I50 of about 10 microM. When added at this concentration, it also inhibited the oxidation of a wide variety of acyl-carnitines by about 50%, despite very different oxidation rates of these different acyl-carnitines. It was concluded that the inhibitory effect of erucoyl-carnitine on all CoA-dependent substrates could be adequately explained by the suggestion that erucoyl sequesters a significant fraction of mitochondrial matrix CoA as slowly metabolizable erucoyl-CoA esters. Possible physiological effects of this sequestration for brown adipose tissue thermogenesis are discussed.

Acetylcarnitine↗
PubMed1985

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of alpha 1-adrenergic stimulation.

We have previously demonstrated mobilization of Ca2+ in and efflux of Rb+ (K+) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of the alpha 1-subtype. Further, we have found that the Rb+ (K+) efflux was dependent upon a primary Ca2+ mobilization event in response to the alpha 1-adrenergic stimulation, since the Rb+ efflux could also be demonstrated by the addition of the Ca2+ ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb+ effluxes were both inhibited by the Ca2+-dependent K+-channel blocker apamin. Apamin also significantly attenuated Ca2+ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that alpha 1-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca2+ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca2+ concentration which, in turn, activates a Ca2+-dependent K+ channel, leading to a K+ release from these cells. A possible role for this channel to sustain and augment the response to alpha 1-adrenergic stimulation is discussed.

Adipose Tissue, Brown↗
PubMed1985

[3H]GDP binding and thermogenin amount in brown adipose tissue mitochondria from cold-exposed rats.

Brown fat mitochondria were isolated from cold-exposed and control rats, and their content of the brown-fat-specific 32-kDa "uncoupling" protein thermogenin determined both by the traditional [3H]GDP-binding method and by the recently developed enzyme-linked immunosorbent assay (ELISA). In mitochondria isolated from both cold-acclimated (3 wk at 4 degrees C) and cold-exposed rats (24 h), an increase in thermogenin content was observable, both when estimated by the [3H]GDP-binding method and by the ELISA assay, and there was no statistically significant difference in the magnitude of these increases in the two methods. In 1 h cold-exposed rats there was no increase in [3H]GDP binding or in the ELISA reaction. When the amount of thermogenin was plotted against [3H]GDP binding in the different states, a relationship of 75,000 g thermogenin per mole GDP bound was obtained. Based on the resolution of these two methods, and under the three conditions investigated, it was concluded that there was no reason to postulate the existence of a "masked" form of thermogenin or of an "unmasking" process and that thermogenin in the mitochondria, as in the isolated state, has apparently one GDP binding site per dimer.

Adipose Tissue↗
PubMed1984

Increased alpha 1-adrenergic receptor density in brown adipose tissue of cold-acclimated rats and hamsters.

Binding sites for [3H]prazosin were characterized in crude membrane fractions from rat brown adipose tissue. Based on agonist (norepinephrine approximately equal to phenylephrine much greater than isoprenaline) and antagonist (prazosin much greater than yohimbine greater than propranolol) potencies to compete with [3H]prazosin, the binding sites were identified as alpha 1-receptors, not previously described in rat brown adipose tissue. As the [3H]prazosin binding sites could be observed in isolated brown fat cell preparations, they were probably postsynaptic. The effect of cold acclimation was studied in crude membrane fractions from control and cold-acclimated (4 degrees C) rats and hamsters. Cold acclimation did not change the affinity of the receptor for agonists and antagonists, but there was a significant increase in the number of alpha 1-receptors (per mg protein), both in rat (100% increase) and hamster (40% increase) brown fat. Based on these results and on earlier results on beta-receptors from this and other laboratories, it is suggested that activation of brown adipose tissue is associated with an increase in the relative density of alpha 1-receptors (i.e. in the alpha 1/beta ratio) and an increased significance of alpha 1-adrenergic pathways for the function of the tissue.

Acclimatization↗
PubMed1984

Na+-dependent regulation of extramitochondrial Ca2+ by rat-liver mitochondria.

The presence and significance of Na+-induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique. Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of 'basal' release of Ca2+ was seen (0.3 nmol Ca2+ X min-1 X mg-1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria. The addition of Na+ in 'cytosolic' levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+ X min-1 X mg-1. This effect was specific for Na+. The significance of this Na+-induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady-state level (set point) induced by Na+. A five-fold increase in this level, from less than 0.2 microM to more than 1 microM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic-anion buffer as in a sucrose buffer. Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+-controlled level, was regained. It was concluded that liver mitochondria should be considered Na+-sensitive, that the capacity of the Na+-induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+-dependent regulators of extramitochondrial ('cytosolic') Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably alpha 1-adrenergic) and changes in this extramitochondrial ('cytosolic') Ca2+ steady state level.

Animals↗
PubMed1984

Metabolic consequences of limited substrate anion permeability in brown fat mitochondria from a hibernator, the golden hamster.

Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4-5 degrees C), brown fat is probably able to combust fatty acids totally.

Adipose Tissue, Brown↗
PubMed1984

Parallel increases in amount of (3H)GDP binding and thermogenin antigen in brown-adipose-tissue mitochondria of cafeteria-fed rats.

In order to investigate the possible existence of a 'masked' (i.e. non-GDP-binding) form of thermogenin (the brown-adipose-tissue specific, 32 000 Da so-called "uncoupling" protein), rats were fed a routine pellet diet or, in addition to this, a cafeteria diet. Brown-adipose-tissue mitochondria isolated from the cafeteria-fed animals showed as expected an increased (3H)GDP binding capacity (from 0.26 to 0.41 nmol/mg protein; an increase of 57%). However, when analysed by a quantitative enzyme-linked immuno-assay system for thermogenin, the mitochondria also showed an increased content of thermogenin (from 14.9 to 20.5 micrograms per mg; an increase of 38%). The ratio between thermogenin and GDP binding was 61 000 and 53 000 g/mol in the two cases; these values were not significantly different and were in good agreement with suggestions that thermogenin binds 1 GDP per thermogenin dimer. It was concluded that under the conditions investigated, there was no reason to assume the existence of a masked form of thermogenin.

Adipose Tissue, Brown↗
PubMed1984

Alpha-adrenergic effects on 86Rb+ (K+) potentials and fluxes in brown fat cells.

Net K+ fluxes in isolated hamster brown fat cells were studied by the use of the K+ analogue 86Rb+. In isolated cells, cold-stored overnight to diminish K+ gradients, an equilibrium 86Rb+ (K+) clearance value of 27 microliter/million cells was obtained after 30 min incubation at 37 degrees C. This corresponds to a 10-fold K+ gradient over the plasma membrane, and a K+ potential of about -60 mV. The attainment of this equilibrium was dependent upon the presence of Na+ in the extracellular medium, and the uptake was fully inhibited by the (Na+ + K+)-ATPase inhibitor ouabain. Ouabain had, however, no significant acute effect on the maximal rate of thermogenesis achieved after norepinephrine stimulation of the cells, but if the restoration of ionic equilibrium was inhibited by ouabain in prolonged incubations, a decreased thermogenesis was observed. This was probably due to the low cytosolic K+ content then encountered, and the resulting inhibition of lipolysis. The addition of norepinephrine to cells in which 86Rb+ (K+) equilibrium had been attained resulted in a rapid efflux of 86Rb+ and the establishment of a new equilibrium value, at about 65% of the unstimulated value. This corresponds to a decrease in K+ potential of about 15 mV. The effect of norepinephrine was stereospecific and reversible, and had an EC50 value of about 10 nM. As catecholamine effects were much more sensitive to phentolamine than to propranolol, the adrenergically-induced efflux was classified as predominantly alpha-adrenergic. It is suggested that the norepinephrine-induced K+ efflux is due to a (probably Ca2+-mediated) opening of K+ channels in the cell membrane, and that this effect occurs secondarily to the alpha-adrenergically induced membrane depolarization (and increase in cytosolic Ca2+). The increased PK over the cell membrane would counteract further depolarization, and the K+ gradient would then approach the Nernst equilibrium under the new steady-state conditions.

Adipose Tissue, Brown↗
PubMed1984

Na+-dependent, alpha-adrenergic mobilization of intracellular (mitochondrial) Ca2+ in brown adipocytes.

The existence and significance of a hormone-sensitive, rapidly mobilizable intracellular pool of Ca2+ in hamster brown-fat cells was investigated with 45Ca2+-labelling techniques. It was shown that such a pool existed and was probably located within the abundant mitochondria. It was rapidly mobilized by norepinephrine (median effective concentration 50 nM) through alpha-adrenergic mechanisms. The mobilization of Ca2+ from the intracellular stores (mitochondria) required the presence of extracellular Na+, but not of Ca2+, K+ or Mg2+. It is concluded that the experiments are in agreement with a hypothesis linking the mobilization of intracellular Ca2+ pools with an alpha-adrenergically-induced increase in plasma membrane Na+ permeability (observed as a membrane depolarization), and a subsequent activation of the mitochondrial Na+/Ca2+ exchange, leading to mobilization of mitochondrial Ca2+ and the mediation of alpha-adrenergic effects as a result of an elevated cytosolic Ca2+ level.

Adipose Tissue, Brown↗
PubMed1984

Increased alpha 1-adrenergic receptor density in brown adipose tissue of cafeteria-fed rats.

A possible general corollary between alpha 1-receptor density in brown adipose tissue and the degree of activation of the tissue was investigated. For this purpose, the effect of cafeteria feeding on alpha 1-adrenergic receptors in brown adipose tissue of seven-week-old female rats was studied by the use of the alpha 1-antagonist (3H)-prazosin. In cafeteria-fed rats, the KD of the alpha 1-receptor for (3H)prazosin was unchanged (about 0.35 nM), but the receptor density was doubled (up to 40 fmol per mg of membrane protein). This was also observed when the results were expressed per unit of a plasma-membrane marker (5'-nucleotidase). It was concluded that an increased alpha 1-receptor density is seen not only in cold-acclimated rats, but also in other conditions where brown fat is activated, and a possible general physiological significance of alpha 1-adrenergic pathways in brown adipose tissue is discussed.

Adipose Tissue, Brown↗
PubMed1984

Enzyme-linked immunosorbent assay (ELISA) studies of the interaction between mammalian and avian anti-thermogenin antibodies and brown-adipose-tissue mitochondria from different species.

Three different antibody preparations, rabbit anti-hamster and rabbit anti-rat thermogenin sera and chicken anti-rat thermogenin IgG, were tested for cross-reactivity towards isolated thermogenin and BAT mitochondria from different mammalian species using an ELISA-technique. It was found that the antibody preparations readily cross-reacted with different species, but that the affinity of the antibody preparations was greater towards the homologous species than the other species. The reactivity of an antibody preparation towards mitochondria from different tissues from the homologous species was also tested, and the exclusive occurrence of thermogenin in BAT could be confirmed.

Acclimatization↗