Biomedical subjects

J Nedergaard

Publications and source records attributed to J Nedergaard.

At least 91 records · Page 5Linked to original sources

PubMed1989

Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine.

Expression of the gene for the brown-fat specific uncoupling protein thermogenin was investigated in cell cultures by hybridization of isolated RNA with a cDNA clone corresponding to mouse thermogenin. The RNA was isolated 3-4 days after confluence from cells differentiated in culture from precursors isolated from the interscapular brown adipose tissue of 5-week-old mice. Very low thermogenin mRNA levels were found in cells derived from untreated mice, and there was only little effect of added norepinephrine on thermogenin gene expression in these cells. However, in cells derived from hypothyroid (methimazole-treated) mice there was a higher expression of thermogenin, and norepinephrine had a marked augmenting effect on the thermogenin mRNA level in these cells. These effects of thermogenin mRNA levels were specific, in that they contrasted with the effects of hypothyroidism and norepinephrine on the level of other mRNA species in these cells (coding for beta-actin, lipoprotein lipase, cytochrome-c oxidase, and glycerol-3-phosphate dehydrogenase). It was concluded that brown-fat cells in culture can reach a differentiated state, sufficiently advanced that the unique properties of these cells can be expressed, and that thermogenin gene expression (i.e., the level of thermogenin mRNA) is under direct control of norepinephrine.

Actins↗

PubMed1989

Comparison of the pharmacological profiles of adrenergic drugs (including BRL-agonists) at [3H]prazosin and [3H]CGP-12177 binding sites in brown adipose tissue.

1. In order to determine the selectivity of classical and novel adrenergic agents for alpha 1- and beta-adrenergic receptors in brown adipose tissue, the ability of these agents to compete for binding sites labelled with [3H]prazosin and [3H]CGP-12177, respectively, was investigated. 2. The beta-antagonist propranolol, known to inhibit norepinephrine-induced respiration in micromolar concentrations, bound to the [3H]CGP-12177 site with nanomolar affinity. 3. Among agonists, only isoprenaline showed high selectivity for beta-receptors, and only oxymetazoline for alpha 1-receptors. 4. Unexpectedly, the novel thermogenic agonists (BRL-agonists), shown to be potent and selective stimulators of brown fat thermogenesis, were unselective and bound only with low affinity to the [3H]CGP-12177 binding sites. 5. These results suggest that the beta-adrenergic binding site in brown adipose tissue identified here with [3H]CGP-12177 may not be the one (or not the only one) coupled to thermogenesis.

Adipose Tissue, Brown↗

PubMed1989

Effects of cholera toxin on gene expression in brown preadipocytes differentiating in culture.

To investigate the cellular control of the recruitment process in brown adipose tissue, the ability of cholera toxin to influence the differentiation of brown preadipocytes developing in culture was investigated. Stromalvascular cells obtained from the brown adipose tissue of 3-wk-old rats were grown in culture for 6-7 days in the presence or absence of cholera toxin. It was found that cholera toxin treatment decreased the expression of the actin gene (indicating an increased degree of differentiation), while at the same time promoting the expression of the genes coding for the mitochondriogenesis marker cytochrome-c oxidase and for the adipocyte conversion marker lipoprotein lipase (all followed at the mRNA level). Chronic cholera toxin treatment also increased the total amount of protein per cell in culture, and a specific cholera toxin-induced 35-kDa protein was identified. It was concluded that (in contrast to the case suggested for white preadipocytes) cholera toxin treatment of brown preadipocytes may not only affect the activity of catabolic enzymes but may also directly promote the differentiation process, indicating that this process is under beta-adrenergic control in the adapting animal.

Adipose Tissue, Brown↗

PubMed1989

Rare fatty acids in brown fat are substrates for thermogenesis during arousal from hibernation.

Because brown adipose tissue lipids are the preferred substrate for thermogenesis during arousal from hibernation, the fatty acid composition of brown fat lipids was followed during cold acclimation and during a hibernation bout. In control golden hamsters (living at 22 degrees C), the fatty acid composition of the white adipose tissue closely resembled that of the food, but brown adipose tissue contained more animal-derived fatty acids. As an effect of acclimation to cold, the fatty acid composition of brown adipose tissue changed to resemble that of the food, and no marked differences between white and brown adipose tissue were then evident. During a hibernation bout, a major part of the fatty acids accumulated in brown fat during entry into hibernation consisted of "rare" acids, such as homo-gamma-linoleic acid. Homo-gamma-linoleic, together with eicosadienoic acid and lignoceric acid, was preferentially utilized during the early phase of arousal. During this phase, "bulk" fatty acids, such as linoleic acid, were spared, whereas in late arousal, linoleic acid was the preferred substrate. It was concluded that rare fatty acids are of quantitative significance in brown adipose tissue during hibernation and arousal.

Adipose Tissue↗

PubMed1988

A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria.

Acyl-CoA hydrolase activities were studied in brown adipose tissue from hamsters. A latent activity was observed in isolated mitochondria. Two peaks of activity were clearly visible in mitochondria, one with an optimum at propionyl-CoA ("short-chain hydrolase") and one with an optimum at nonanoyl-CoA ("medium-chain hydrolase"); there was only low activity toward palmitoyl-CoA and longer-chain acyl-CoAs. In subcellular fractionation experiments, the activity of the short-chain and the medium-chain hydrolase fully followed that of the mitochondrial matrix marker enzyme 2-oxoglutarate dehydrogenase. The specific activity of the hydrolases in the mitochondrial fraction was doubled after cold acclimation. beta-NADH inhibited the short- and medium-chain hydrolases; alpha-NADH, NADPH, and NAD+ were without effect. ADP stimulated the short- and medium-chain hydrolases; ATP and AMP were practically without effect. Evidence is presented to indicate that NADH and ADP interact on the enzyme at the same site and that ADP is essential for the maintenance of the short- and medium-chain enzyme activities. A positive effect of KCl was found on the short- and medium-chain hydrolase activities. Also, the divalent ions Ca2+ and Mg2+ were stimulatory, but only Ca2+ was able to overcome NADH inhibition, possibly due to interaction directly with NADH. It is concluded that brown adipose tissue mitochondria, besides a conventional type of acyl-CoA hydrolase, contain two species of a novel type of acyl-CoA hydrolases which are characterized by being regulated by ADP and NADH (interacting at a common site) and by having an obligatory requirement for ADP.

Acclimatization↗

PubMed1988

Beta-adrenergic modulation of Ca2+ uptake by isolated brown adipocytes. Possible involvement of mitochondria.

Rapid, unidirectional Ca2+ influx was examined in isolated brown adipocytes by short incubations (30 s) with 45Ca2+. Ca2+ uptake was found to be large in the resting brown adipocyte, but was markedly inhibited when the cells were presented with norepinephrine. Specific alpha 1-adrenergic stimulation was without effect on Ca2+ uptake. The effect of norepinephrine (which had an EC50 of 140 nM) could be inhibited by beta-adrenergic blockade and could be mimicked by forskolin (an adenylate cyclase activator) and theophylline (a phosphodiesterase inhibitor). Exogenous free fatty acids such as octanoate and palmitate (classical stimulators of respiration in brown adipocytes) were also able to dramatically inhibit Ca2+ uptake by the cells. The artificial mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induced a large reduction in cellular Ca2+ uptake (even in the presence of the ATPase inhibitor oligomycin), and in the presence of FCCP the inhibitory effect of norepinephrine on Ca2+ uptake was significantly reduced. The effect of beta-adrenergic stimulation on Ca2+ uptake was not directly caused by the large increase in respiration that occurs in response to norepinephrine because the respiratory inhibitor rotenone did not affect the Ca2+ response of the cells to the hormone. The evidence suggests that beta-adrenergic stimulation of brown adipocyte metabolism leads to a partial inhibition of Ca2+ uptake into the mitochondrial Ca2+ pool and we discuss the possibility that this represents the effect of a reduced membrane potential (and thus reduced Ca2+ uniport activity) in the partially uncoupled mitochondria of the thermogenically active brown adipocyte.

Adipose Tissue, Brown↗

PubMed1988

Cold-induced beta-adrenergic recruitment of lipoprotein lipase in brown fat is due to increased transcription.

The cellular basis for the cold-induced increase in lipoprotein lipase activity in rat brown adipose tissue was investigated. Rats were treated with inhibitory agents and either exposed to cold for 4 h or injected with isoprenaline. Lipoprotein lipase activity was followed in acetone-ether extracts of the tissue. Besides cold, both the beta-adrenergic agonist isoprenaline and the adenylate cyclase activator cholera toxin were able to increase lipoprotein lipase activity in the tissue. The protein synthesis inhibitor cycloheximide fully abolished this response; the half-life of lipoprotein lipase activity was both in control and in the cold-exposed state approximately 2 h. Also the mRNA synthesis inhibitor actinomycin D fully abolished the cold-, the isoprenaline-, and the cholera toxin-induced increases in lipoprotein lipase activity; the half-life of lipoprotein lipase mRNA was estimated to be 20-30 h. However, in animals returned to control conditions after a 4-h cold stress, the decline in activity corresponded to a half-life of only 4 h. It was concluded that the increase in lipoprotein lipase activity in the brown adipose tissue of cold-exposed rats is not due to an activation of preexisting enzyme nor due to an increased half-life of functional enzyme. Rather it is suggested that in brown adipose tissue the rate of lipoprotein lipase gene transcription is positively regulated by the cellular level of cAMP and that this increase in lipoprotein lipase mRNA leads directly to an increased rate of enzyme synthesis and hence to the increase in activity.

Adipose Tissue, Brown↗

PubMed1987

Physiological activation of brown adipose tissue destabilizes thermogenin mRNA.

The amount of mRNA coding for the brown fat specific uncoupling protein thermogenin was followed in the brown adipose tissue of adult mice. As expected, cold exposure or norepinephrine injection caused an increase in the amount of thermogenin mRNA. However, contrary to expectation, the half-life of thermogenin mRNA was dramatically reduced, from about 18 h to about 3 h, when the mice were cold exposed. This destabilization of thermogenin mRNA was not related to the activity of protein synthesis. It was concluded that in brown adipose tissue an unusual mechanism operates which leads to a destabilization of thermogenin mRNA under the same physiological conditions which increase thermogenin gene expression.

Acclimatization↗

PubMed1987

Euthyroid status is essential for the perinatal increase in thermogenin mRNA in brown adipose tissue of rat pups.

The amount of mRNA coding for the brown-fat specific, uncoupling protein thermogenin was followed perinatally in fetuses and newborns from normal and hypothyroid rat dams. Although the growth of the fetuses and newborns was normal in the hypothyroid group, they had a lower amount of thermogenin mRNA already in-utero, and the dramatic postnatal increase in thermogenin mRNA was nearly completely abolished. It is concluded that the euthyroid state is essential for the regulation of the expression of the thermogenin gene.

Adipose Tissue, Brown↗

PubMed1987

Alpha 1-adrenergic inositol trisphosphate production in brown adipocytes is Na+ dependent.

In order to investigate the ionic requirements for inositol trisphosphate production, brown adipocytes were prelabelled with myo-[3H]inositol and the formation of inositol trisphosphates and inositol bisphosphates as a consequence of alpha 1-adrenergic stimulation was monitored. Omission of Ca2+ from the incubation medium diminished the norepinephrine-induced increase in inositol trisphosphate levels, but it would seem that this reduction can be fully accounted for by a decreased level of the 'inactive' isomer inositol 1,3,4-trisphosphate. Omission of Na+ fully abolished the norepinephrine-induced inositol trisphosphate response. However, it was observed that the presence of Li+ in the incubation medium could fully reconstitute the ability of the cells to yield the early response of inositol trisphosphate production; Li+ could, however, not substitute for Na+ in the entire alpha 1-adrenergic cellular pathway. It was concluded that the Na+-dependent step is found in the coupling mechanism between the alpha 1-receptor and the activation of the phosphodiesterase responsible for inositol trisphosphate production. Thus, all events in the alpha 1-adrenergic pathway which are consequences of IP3 production should appear to be Na+-dependent in these cells.

Adipose Tissue, Brown↗

PubMed1987

Apparent unmasking of [3H]GDP binding in rat brown-fat mitochondria is due to mitochondrial swelling.

The presence of and biochemical background for the so-called 'unmasking' phenomenon in rat brown-fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the 'uncoupling' protein thermogenin, without a concomitant increase in the amount of the protein). It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28 degrees C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP-binding capacity were used; however, the absolute values were higher with the filtration method. Based on observations of slower cytochrome-c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown-fat mitochondria isolated from warm-acclimated animals was smaller than that of cold-stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non-existent to about 1 microliter/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm-acclimated animals but was without significant effect in the already swollen mitochondria from cold-stressed animals or from animals adapted to a lower temperature. In mitochondria from warm-acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold-stressed animals, but it was practically without effect on GDP binding in mitochondria from cold-stressed animals or from animals acclimated to a lower temperature. It is concluded that the apparent 'unmasking' phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.

Adipose Tissue, Brown↗

PubMed1987

Distinction between mechanisms underlying alpha 1- and beta-adrenergic respiratory stimulation in brown fat cells.

Experimental conditions are described for selective alpha 1- and beta-adrenergic stimulation of the respiration of brown fat cells. The dual agonist norepinephrine was unsuitable as a selective alpha 1-agonist, since unacceptably high concentrations of propranolol were needed to abolish the beta-response. Phenylephrine at 50 microM, in the presence of 5 microM dl-propranolol, was shown to lead to a maximal, selective alpha 1-stimulation, whereas maximal, selective beta-stimulation was achieved with 1 microM isoproterenol in the presence of 5 microM prazosin. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to further increase respiration that was already maximally alpha 1-stimulated, but when added before the alpha 1-stimulation, FCCP totally abolished the response. In contrast, FCCP had no effect on the beta-stimulated response. Similarly, oligomycin (an inhibitor of mitochondrial ATP synthesis) inhibited alpha 1-respiration but had a much smaller effect on beta-respiration. Ouabain (an inhibitor of the Na+-K+-ATPase) halved alpha 1-respiration but only induced a small inhibition of beta-respiration. It is concluded that only a small fraction of thermogenesis from beta-adrenergic processes is due to oxidative phosphorylation, whereas alpha 1-respiration is largely due to the oxygen cost of mitochondrial ATP synthesis, and a large fraction of this ATP is apparently used for the restoration of ion gradients.

Adipose Tissue, Brown↗

PubMed1987

Noradrenergic stimulation of mitochondriogenesis in brown adipocytes differentiating in culture.

The ability of adrenergic agents to promote the differentiation and especially the mitochondriogenesis of brown fat precursor cells, grown in culture, was investigated. These cells begin to differentiate during the days preceding confluence. We found here that, already during the early growth phase, the cultures (essentially precursor cells and preadipocytes at this stage) show increased cyclic AMP (cAMP) levels when acutely stimulated with norepinephrine (NE). The cultured cells were therefore chronically treated with NE up to the time of confluence, and their cytochrome-c oxidase activity was measured as an index of mitochondriogenesis. Chronic NE treatment resulted in an increased cytochrome-c oxidase activity of the cells at confluence. This effect was reproduced by selective activation of adenylate cyclase with cholera toxin, suggesting that the NE effect was exerted through an increase in cAMP. Ascorbate (added with NE as an antioxidant) had in itself a positive effect, both on final cell number and on cytochrome-c oxidase activity. It is concluded that NE, working through beta-adrenergic receptors, can stimulate mitochondriogenesis in brown fat cells through a direct effect on the cells, in accordance with the suggestion (based on in vivo experiments) that NE accelerates, rather than initiates, the differentiation process.

Adipose Tissue, Brown↗

PubMed1987

Thermogenin amount and activity in hamster brown fat mitochondria: effect of cold acclimation.

To investigate the acclimation process in a hibernator, four different parameters of thermogenin amount and activity were investigated in brown adipose tissue mitochondria from cold-exposed and cold-acclimated Syrian hamsters. Hamsters, which are hibernators, have been considered to be "primed" for thermogenesis and thus not to show cold-acclimation effects, but here a significant increase in [3H]GDP-binding capacity was observed (from 0.5 nmol in control to 0.9 nmol GDP/mg in cold-acclimated hamsters), and this increase was paralleled by an increase in thermogenin antigen amount, as measured in an enzyme-linked immunosorbent assay. The transient nature of the effect of cold exposure on [3H]GDP binding, characteristically observed with rat mitochondria, was not observed with hamster mitochondria, and the increase in [3H]GDP binding occurred without a change in the dissociation constant (0.7 microM). The increase in thermogenin amount was paralleled by an increase both in GDP-sensitive Cl- permeability of the mitochondria and in GDP-sensitive respiration. It was established that it is the maximal activity of thermogenin that is rate limiting for thermogenesis in isolated mitochondria, provided that an optimal substrate is used (such as palmitoyl carnitine). Cold acclimation also increased the total amount of mitochondria in the tissue, leading totally to a sixfold increase in thermogenin content of the hamster. It is concluded that (contrary to the general view) hamsters show the expected physiological, pharmacological, and biochemical signs of cold acclimation (i.e., an increased capacity for nonshivering thermogenesis).

Acclimatization↗

PubMed1987

Cold-induced changes in Ca2+ transport in duckling skeletal muscle mitochondria.

Intermyofibrillar mitochondria were isolated from skeletal muscle (gastrocnemius) of cold-acclimated (4 degrees C) or control (30 degrees C) 4-wk-old ducklings. Ca2+ transport in the mitochondria was studied with the Ca2+-sensitive dye arsenazo III. The mitochondria actively took up Ca2+ but at a lower rate in mitochondria from cold-acclimated than from control ducklings. After addition of the Ca2+ uptake inhibitor ruthenium red, a low rate of Ca2+ release was revealed; this rate was, however, higher in the cold-acclimated than in the control ducklings. Furthermore, these nonmammalian mitochondria were also found to possess a mechanism for Na+-stimulated Ca2+ efflux. This mechanism was specific for Na+ (no effects with choline+ or K+, a small effect with Li+). The effect was maximal with 20 mM Na+, which led to a fourfold increase in rate of Ca2+ efflux. Cold acclimation led to a doubling of the rate of this Na+-stimulated Ca2+ release. Due to these alterations in Ca2+ fluxes caused by cold acclimation, the resultant free extramitochondrial Ca2+ levels were higher in mitochondria from cold-acclimated than from control ducklings. The basal level was doubled (from 0.26 to 0.50 microM) as was the increase in steady-state level caused by 10 mM Na+ (from +0.4 to +0.8 microM). The significance of these alterations for acclimation to cold is discussed.

Adaptation, Physiological↗

PubMed1986

Requirement of gene transcription and protein synthesis for cold- and norepinephrine-induced stimulation of thyroxine deiodinase in rat brown adipose tissue.

The increase in propylthiouracil-insensitive 'type II' thyroxine 5'-deiodinase activity of brown adipose tissue was investigated in rats exposed to acute cold stress or single-dose norepinephrine injection. The 20-fold cold-induced increase in enzyme activity showed a 2-h lag phase and reached a maximum after only 8 h; reacclimation occurred with a 2-h time lag and a half-life of 2.2 h. 4 h after a single norepinephrine injection, the deiodinase activity was almost identical to that after a 4-h cold stress; norepinephrine could not potentiate the effect of the cold stress. Treatment with the protein synthesis inhibitor cycloheximide before exposure to cold or before norepinephrine injection totally blocked the increase in deiodinase activity, suggesting that the increase is due to de novo protein synthesis. The half-life of the enzyme in vivo was estimated to be 0.7 h. Treatment with the RNA synthesis inhibitor actinomycin D totally abolished the cold- and norepinephrine-induced increases, indicating that the increase requires mRNA synthesis. It was concluded that the dramatic cold-induced increase in thyroxine deiodinase activity in brown adipose tissue was not due to activation of preexisting enzyme but was fully due to a norepinephrine-induced increase in expression of the gene and subsequent synthesis of the protein.

Adipose Tissue, Brown↗

PubMed1986

Norepinephrine-induced Na+ influx in brown adipocytes is cyclic AMP-mediated.

To examine the involvement of Na+ ions in adrenergic responses in brown adipose tissue, a method is described for measuring Na+ influx into isolated brown adipocytes, using short (30 s) incubations with 22Na+, followed by a two-step centrifugation recovery procedure. Using this method, a clear norepinephrine-stimulated accumulation of intracellular 22Na+ was observed, which was enhanced by the addition of ouabain, was insensitive to amiloride (a Na+/H+ exchange blocker), and could not be mimicked by the total removal of oxygen from the incubation medium. The norepinephrine-stimulated Na+ influx was dose-dependent for the hormone with an EC50 of 250 nM, was blocked by the beta-antagonist propranolol but not by the alpha 1-antagonist prazosin, and could be induced by adrenergic agonists with the order of potency: isoproterenol greater than norepinephrine greater than phenylephrine, indicating a beta-receptor-mediated process. The Na+ influx was found to be cAMP-dependent since it could be induced by both theophylline (a phosphodiesterase inhibitor) and forskolin (an adenylate cyclase activator), but it was independent of other known cellular cAMP-dependent responses since neither addition of fatty acid substrates (octanoate or palmitate), nor of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone could induce the phenomenon, despite having significant stimulatory effects on cellular respiration. Furthermore, total respiratory inhibition with rotenone, or total oxygen depletion of the medium with dithionite, did not prevent the normal norepinephrine-induced Na+ influx. The possibility that this beta-mediated norepinephrine-stimulated Na+ influx plays an important physiological role in brown adipose tissue activity is discussed, perhaps as one of the, as yet undefined, signals initiating tissue growth in the chronically beta-stimulated tissue of animals facing long-term increases in thermogenic demands.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1986

Beta-adrenergic stimulation of fatty acid release from brown fat cells differentiated in monolayer culture.

The ability of brown adipocytes differentiated in monolayer culture to respond to norepinephrine was investigated. It was found that fatty acid release in confluent brown adipocytes in monolayer culture was induced by norepinephrine, thus these cells were hormone-sensitive. After confluence, the rate of fatty acid release successively declined. The norepinephrine-stimulated fatty acid release was inhibited by propranolol, but not by phentolamine, indicating a mediation via beta-adrenergic receptors. It was concluded that there exist in the brown adipose tissue of nonfetal rats preadipocytes which possess the ability to express in culture a fully developed beta-adrenergic lipolytic response.

Adipose Tissue, Brown↗