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B Cannon

Publications and source records attributed to B Cannon.

At least 73 records · Page 4Linked to original sources
PubMed1994

Stabilization of the mRNA for the uncoupling protein thermogenin by transcriptional/translational blockade and by noradrenaline in brown adipocytes differentiated in culture: a degradation factor induced by cessation of stimulation?

The stability of the mRNA coding for the uncoupling protein thermogenin was investigated in mouse brown-fat cells differentiated in culture. After 7 days in culture, the cells were stimulated for 24 h with noradrenaline, and a high level of thermogenin mRNA was then observed. If noradrenaline treatment was continued, the mRNA level remained high, but, upon withdrawal of noradrenaline, the level decreased rapidly, with a half-life of only 2.7 h. The presence of transcriptional (actinomycin) or translational (cycloheximide) inhibitors prolonged the apparent half-life by about 50%. The presence of noradrenaline during transcriptional blockade led to a further stabilization of thermogenin mRNA. It was concluded that an induced (or short-lived) gene product is important for thermogenin mRNA degradation. Direct interaction of noradrenaline with the cultured brown adipocytes could apparently not mimic the paradoxical destabilization of thermogenin mRNA in vivo, previously observed in the cold-exposed mouse [Jacobsson, Cannon and Nedergaard (1987) FEBS Lett. 244, 353-356], indicating significant differences between the systems in vitro and in vivo.

Adipocytes↗
PubMed1994

Sulfonates are low-affinity ligands for the GDP-binding site of brown-fat mitochondria.

In order to study the function of the brown-fat specific uncoupling protein thermogenin (UCP), the effect of certain sulfonates on [3H]GDP binding to the GDP-binding site of brown adipose tissue mitochondria was studied. The affinity of [3H]GDP for the site was 1.3 microM in the normal sucrose medium, but the apparent KD was increased to approximately 20 microM in 100 mM hexanesulfonate medium. This increase in apparent KD was found to be due to a competitive binding of hexanesulfonate to the GDP-binding site; the affinity of hexanesulfonate was only 13 mM but this was sufficient to affect the apparent affinity of GDP under experimental conditions. Also in KCl-medium, the affinity of GDP was high (approximately 3 microM), but both in a benzenesulfonate medium and in a para-aminobenzenesulfonate (sulfanilate) medium, the apparent affinity was lower (approximately 12 microM); as benzenesulfonate is well transported by thermogenin but sulfanilate is not, the reduction in affinity was unrelated to transport. In agreement with earlier data (Jezek, P. and Garlid, K.D. (1990) J. Biol. Chem. 265, 19303-19311), the potency of GDP to inhibit transport was dependent on the species transported; the fact that GDP potency was lower for benzenesulfonate transport (EC50 = 324 microM) than for Cl- transport (EC50 = 32 microM) could adequately be explained by the competitive interaction of benzenesulfonate with the GDP-binding site, but this effect could only partly explain the even lower potency of GDP to inhibit hexanesulfonate transport (EC50 = 4074 microM). It was concluded that these types of substrate for thermogenin-mediated transport may directly interact with the GDP-binding site, but that this effect could only partly explain the dependence of GDP potency on substrate species.

Adipose Tissue, Brown↗
PubMed1994

Thermogenesis in brown adipocytes is inhibited by volatile anesthetic agents. A factor contributing to hypothermia in infants?

BACKGROUND: In infants, nonshivering thermogenesis from brown adipose tissue provides an important source of heat for thermoregulation. Infants are known to have a high susceptibility to hypothermia during anesthesia. To investigate whether this could be due to an inhibition of nonshivering thermogenesis by anesthetics, the effect of preincubation with volatile anesthetics on the norepinephrine-induced heat production of brown adipocytes was investigated. METHODS: Brown adipocytes from hamsters were isolated with a collagenase digestion method and preincubated with volatile anesthetics. The cells were stimulated with norepinephrine, and heat production, measured as oxygen consumption, was monitored polarographically. RESULTS: Norepinephrine addition led to a 20-fold increase in the rate of oxygen consumption (thermogenesis). However, preincubation of cells with 3% halothane reduced the response to norepinephrine by more than 70%. The potency of norepinephrine (the median effective concentration) was not affected by halothane. Full effect of halothane was reached quickly, and after halothane withdrawal, the thermogenic response recovered, although rather slowly. Halothane, isoflurane, and enflurane were approximately equipotent inhibitors of thermogenesis, with concentrations of approximately 0.7% resulting in 50% inhibition. The inhibitory effect of 1% halothane was unaffected by the presence of 74% nitrous oxide, but nitrous oxide alone also reduced thermogenesis. CONCLUSIONS: Volatile anesthetics severely attenuated the thermogenic response to norepinephrine of isolated brown-fat cells. It is inferred that brown-adipose-tissue heat production is reduced during (and probably also some time after) anesthesia. Because infants are dependent on brown-fat-derived nonshivering thermogenesis for thermal balance, the inhibition by volatile anesthetic agents of brown-adipocyte heat production may at least partly explain the susceptibility of infants to hypothermia during and after anesthesia.

Adipocytes↗
PubMed1994

Coexisting beta-adrenoceptor subtypes: significance for thermogenic process in brown fat cells.

The possible significance of the coexisting beta 1-, beta 2-, and beta 3-adrenoceptors in brown adipose tissue for the thermogenic response was investigated. Oxygen consumption of isolated hamster brown fat cells was analyzed as a measure of thermogenesis. Thermogenesis could be evoked not only by the physiological agent norepinephrine but also by BRL-37344 and CGP-12177. No evidence for biphasic inhibition curves was found with either the selective beta 1-antagonist ICI-89406, the beta 2-antagonist ICI-118551, or the beta 1/beta 2-nonselective beta-antagonist propranolol against 1 microM norepinephrine; pI50 (the negative logarithm of the inhibitory constant for an antagonist, as estimated from the dose-response curve for an antagonist vs. a constant agonist concentration) values for ICI-89406 and ICI-118551 were very low (4-5), implying nonselective inhibition; the pI50 for propranolol was approximately 6 (as expected for the beta 3-receptor). Even with suboptimal norepinephrine, no biphasic inhibition was found. CGP-12177 at concentrations where it is primarily an antagonist to the beta 1-receptor did not influence the dose-response curve for either norepinephrine or BRL-37344. BRL-37344- or CGP-12177-induced thermogenesis was inhibited by the beta-antagonists in a manner similar to norepinephrine-induced thermogenesis. Schild plots for propranolol inhibition of norepinephrine-, isoprenaline-, BRL-37344- and CGP-12177-induced thermogenesis yielded similar pA2 (the negative logarithm of the inhibitory constant for an antagonist, as calculated from a series of agonist dose-response curves at different antagonist concentrations) (approximately 5.5), for interaction with either agonist, implying that the same receptor was stimulated by all agonists. Thus, despite the fact that different beta-receptor subtypes coexist in the tissue, we find no evidence for the participation of beta 1- or beta 2-receptors in the thermogenic response. Within the resolution of the experiments, the results therefore imply that it is predominantly or solely the beta 3-receptor that is coupled to thermogenesis, and it is via this beta-adrenergic receptor that not only norepinephrine but also CGP-12177 and BRL-37344 induce thermogenesis.

Adipose Tissue, Brown↗
PubMed1994

The uncoupling protein thermogenin during acclimation: indications for pretranslational control.

To analyze the regulation of the content of the uncoupling protein thermogenin in brown adipose tissue, we have selected a physiological transition phase during which to investigate the relationship between the level of mRNA and the level of the ensuing protein product. Mice preacclimated to 28 degrees C were transferred to 4 degrees C. Cold acclimation led to the expected increases in brown fat total protein and RNA content. Two recruited proteins were analyzed: the cytosolic glycerol-3-phosphate dehydrogenase and the mitochondrial uncoupling protein thermogenin. The activity of the dehydrogenase acutely followed the level of the corresponding mRNA, indicating pretranslational control. However, for thermogenin there was a marked time delay between the establishment of the fully recruited level of thermogenin mRNA (after only approximately 4 h of cold exposure) and that of thermogenin itself (after > 3 wk). By reiterative computer simulation, it was investigated whether a model only involving pretranslational regulation could be invoked for either system. For glycerol-phosphate dehydrogenase, a plausible model could be constructed, provided the protein half-life was shorter than approximately 24 h. Despite the long time delay between full thermogenin mRNA recruitment and full thermogenin protein recruitment, a plausible pretranslational control model could also be constructed, provided that the protein half-life was approximately 5 days. This computed value was in good agreement with the half-life obtained from independent thermogenin half-life studies. It is implied that pretranslational control may suffice to explain the regulation of thermogenin content in brown adipose tissue during a warm-to-cold transition period.

Acclimatization↗
PubMed1993

Attenuation of Gs alpha coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters.

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5'-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.

Acclimatization↗
PubMed1992

Induction and degradation of the uncoupling protein thermogenin in brown adipocytes in vitro and in vivo. Evidence for a rapidly degradable pool.

The induction and degradation of the brown-fat-specific uncoupling protein thermogenin in brown fat cell cultures was investigated. Cultures were initiated with undifferentiated precursor cells from young mice and the amount of thermogenin was determined by immunoblotting. High levels of thermogenin could be induced by noradrenaline treatment in cells grown for more than 5 days in culture, and in such cell cultures continuously stimulated with noradrenaline, the thermogenin level continued to increase for at least a further 5 days. In cell cultures stimulated for only 24 h, the induced thermogenin was subsequently specifically and rapidly degraded, with a half-life of 20 h. As the half-life was prolonged by cycloheximide treatment, the degradation was apparently due to the induction of specific proteins after cessation of adrenergic stimulation. In cell cultures continuously stimulated with noradrenaline for 5 days, the induced thermogenin was degraded much more slowly after noradrenaline removal, with a half-life of 70 h. This half-life was unchanged by cycloheximide treatment, and the degradation after cycloheximide was in parallel with the degradation of protein in general, and was therefore non-specific. The prolongation of the half-life of thermogenin after the chronic treatment may be related to mitochondrial incorporation of thermogenin and consequent stabilization of the protein. The half-life of thermogenin in an in vivo situation of similar experimental design (the reacclimation of mice to warm after 5 days in the cold), was also long (about 7 days), and the loss was also non-specific, as it paralleled the loss of protein. Thus different molecular events are involved in thermogenin degradation when the protein is found in different functional pools.

Adaptation, Physiological↗
PubMed1992

Regulation of expression of the lipoprotein lipase gene in brown adipose tissue.

The regulation of lipoprotein lipase gene expression in brown adipose tissue was studied. Rats were preacclimated to 21 degrees C. Exposure to cold (4 degrees C) resulted in a rapid increase in the level of lipoprotein lipase mRNA in the tissue. The level peaked (expressed per microgram total RNA) after approximately 8 h and then slowly declined. The increased lipoprotein lipase mRNA level was not due to an increased stability of the mRNA, but, in a transition event from a high to a low expression of the lipoprotein lipase gene, a transcription-dependent process was recruited that accelerated the breakdown of lipoprotein lipase mRNA. Norepinephrine injections increased lipoprotein lipase mRNA levels in the tissue; this effect was mediated via a beta-adrenergic receptor. The effect of cold could be mimicked by norepinephrine injections, and these two effects were not additive, indicating that the cold effect was mediated by norepinephrine. The lipoprotein lipase mRNA level was also increased by insulin injections (into fasted animals); thus an increase in lipoprotein lipase gene expression in brown adipose tissue may be induced via two different stimuli, which, intracellularly, would be mediated via different signaling systems. In all investigated conditions, the changes in lipoprotein lipase mRNA levels observed here were parallelled by alterations in lipoprotein lipase activity reported earlier from this laboratory. It was therefore concluded that, under the conditions studied, lipoprotein lipase activity in brown adipose tissue was primarily regulated at the transcriptional level.

Adipose Tissue, Brown↗
PubMed1992

Increased alpha 1-adrenoceptor density in brown adipose tissue indicates recruitment drive in hypothyroid rats.

The effects of hypothyroidism on whole body thermogenesis, brown adipose tissue recruitment state, and alpha 1-adrenergic receptor density were investigated. Treatment of rats with methimazole for 4-5 wk led, as expected, to reduction of growth and resting metabolic rate. The thermogenic response to norepinephrine injection was practically abolished. Generally, only small effects of hypothyroidism on brown adipose tissue were observed: total protein content, mitochondrial GDP binding capacity, and total content of the uncoupling protein thermogenin were not altered. The density of beta-adrenergic receptors (estimated with [3H]CGP-12177 as a ligand) was also unchanged. However, the density of alpha 1-adrenergic receptors (estimated with [3H]prazosin) was markedly increased; in other physiological conditions, such an increase has been associated with an increased degree of recruitment of the tissue. These data indicate that brown adipose tissue in the subthermoneutral hypothyroid animal, probably due to homeostatic mechanisms, is exposed to an increased sympathetic stimulation, leading to an increased alpha 1-adrenoceptor density. However, other features of recruitment are only poorly induced, probably due to attenuation of the beta-adrenergic signaling mechanism. The increased alpha 1-adrenergic receptor density may be responsible for certain altered features of brown adipose tissue in hypothyroid animals, such as peroxisomal recruitment and perhaps also for maintenance of the thermogenin content. The results also indicate that the increased alpha 1-adrenergic density generally seen in recruitment would not result from chronic beta-adrenergic stimulation of the tissue but may be controlled via another regulatory pathway, e.g., via the alpha 1-adrenergic pathway itself.

Adipose Tissue, Brown↗
PubMed1990

Alpha- and beta-adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture.

In order to examine the control of expression of the gene coding for the brown fat specific uncoupling protein thermogenin (UCP), brown fat cells isolated as undifferentiated precursors from the interscapular brown adipose tissue of young mice were grown in culture. In these cells, it was possible by norepinephrine (NE) addition to induce specifically the expression of the UCP gene. The effect of NE was due to activation of transcription. The ability to express the UCP gene was maximal in cells around confluence; cell cultures younger or older than this showed a lower response. The response to NE showed a sharp optimum around 0.1 microM and was linear with time over the 4-h period studied. The presence of insulin or thyroid hormones facilitated the NE response. Pharmacological analysis of the adrenergic response indicated that UCP gene expression could be induced both via beta-receptors (probably beta 3) and via alpha 1-receptors; these effects were synergistic. It was concluded that it is possible to promote these precursor cells to advance to such a state of differentiation that they can demonstrate the selective feature of the brown fat cell, i.e. the ability to express UCP. The expression of the UCP gene is regulated via interacting adrenergic mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Norepinephrine-induced synthesis of the uncoupling protein thermogenin (UCP) and its mitochondrial targeting in brown adipocytes differentiated in culture.

Synthesis of the brown adipocyte-specific mitochondrial uncoupling protein thermogenin (UCP) is demonstrated here in brown adipocytes differentiated in culture from precursor cells. By immunoblotting, no UCP was detectable in untreated multilocular adipocytes. The synthesis of UCP was stimulated by norepinephrine at physiological concentrations and was observable already after 2 h. It was evident from immunoelectron microscopy that the newly synthesised protein was targeted to the mitochondrial inner membrane, demonstrating the functional competence of these cultured cells.

Adipose Tissue, Brown↗
PubMed1990

Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M.

The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two-dimensional mapping of their tryptic peptides and suggests either a post-transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal. Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non-growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.

Amino Acid Sequence↗
PubMed1990

Mammalian hibernation.

In mammalian hibernation, the body temperature approaches that of the surroundings, allowing large savings in energy costs of basal metabolism and eliminating the need for heat production to compensate for heat loss. During entry into hibernation, heat production ceases while the body temperature set-point gradually decreases during slow-wave sleep. In the hibernating phase, the animal copes with problems concerning the maintenance of ion gradients, possible membrane phase transitions and the risk of ventricular fibrillation. In the arousal phase, the main part of the heat and practically all the necessary substrate comes from brown adipose tissue. The hibernation season is preceded by a preparatory phase. It may be concluded that hibernation is a practical, and perhaps even enviable, solution to a mammalian problem.

Animals↗
PubMed1989

Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase.

In order to investigate the postnatal recruitment process, gene expression in the brown adipose tissue of rat pups was followed during the first 20 h of life. In normal pups, the level of mRNA coding for the uncoupling protein thermogenin increased markedly but gradually within the first 24 h. Lipoprotein lipase and actin mRNA levels were relatively low and remained constant. In pups exposed to thermoneutral temperature (35 degrees C) for the first 12 h after birth, no increase in thermogenin mRNA or lipoprotein lipase mRNA was observed, whereas in pups exposed to 28 degrees C a clear increase in both thermogenin and lipoprotein lipase mRNA levels was found. Actin mRNA levels were not affected by the environmental temperature under these circumstances. It was concluded that the postnatal recruitment in brown adipose tissue is a consequence of the cold stress experienced by the newborn pups. Thus, postnatal recruitment is not ontogenically predetermined.

Actins↗
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

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↗