Biomedical subjects

Barbara Cannon

Publications and source records attributed to Barbara Cannon.

At least 19 recordsLinked to original sources

PubMed2006

Adrenergic receptor density in brown adipose tissue of active and hibernating hamsters and ground squirrels.

The ligand-binding characteristics (B(max) and K(D)) of alpha(1)- and beta(1)/beta(2)-adrenoceptors were investigated in membranes prepared from brown adipose tissue (BAT) of warm-acclimated, cold-acclimated, hibernating and arousing ground squirrels (Spermophillus undulatus) and hamsters (Mesocricetus auratus) by specific binding of [(3)H]prazosin and [(3)H]CGP-12177, respectively. The physiological state did not change the affinity for the adrenoceptors in the BAT of ground squirrels and hamsters. There was a significant decrease in alpha(1)-receptor density in arousing ground squirrels and a significant decrease in beta(1)/beta(2) density in hibernating ground squirrels. The level of alpha(1)-receptors was in all conditions higher than that of beta(1)/beta(2) receptors. The results indicate a possible change in balance of adrenoceptor density in the processes of cold acclimation, hibernation and arousal. The balance between the various adrenoceptor subtypes may be important for the final effect of catecholamines in BAT in different physiological states.

Acclimatization↗

PubMed2006

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively.

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

Adenine Nucleotide Translocator 1↗

PubMed2006

Decreased brown adipocyte recruitment and thermogenic capacity in mice with impaired peroxisome proliferator-activated receptor (P465L PPARgamma) function.

Mice with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutation (P465L) unexpectedly had normal amounts of adipose tissue. Here, we investigate the adipose tissue of the PPARgamma P465L mouse in detail. Microscopic analysis of interscapular adipose tissue of P465L PPARgamma mice revealed brown adipocytes with larger unilocular lipid droplets, indicative of reduced thermogenic capacity. Under conditions of cold exposure, the brown adipose tissue of the PPARgamma P465L mice was less active, a fact reflected in decreased uncoupling protein 1 levels. Analysis of the white adipocytes confirmed their normal cytoarchitecture and development, yet classical white adipose depots of the P465L PPARgamma mice had a striking reduction in brown adipocyte recruitment, a finding supported by reduced expression of UCP1 in the perigonadal adipose depot. Taken together, these data suggest that whole animal impairment of PPARgamma alters the cellular composition of the adipose organ to a more "white" adipose phenotype. Physiologically, this impairment in brown adipocyte recruitment is associated with decreased nonshivering thermogenic capacity after cold acclimation as revealed by norepinephrine responsiveness. Our results indicate that maintenance of oxidative brown-like adipose tissue is more dependent on PPARgamma function for development than white adipose tissue, an observation that may be relevant when considering PPARgamma-dependent strategies for the treatment of obesity.

Acclimatization↗

PubMed2006

Uncoupling proteins: a role in protection against reactive oxygen species--or not?

A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the "novel" UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.

Aldehydes↗

PubMed2006

UCP1 is essential for adaptive adrenergic nonshivering thermogenesis.

Participation of brown adipose tissue [through the action of the uncoupling protein-1 (UCP1)] in adaptive adrenergic nonshivering thermogenesis is recognized, but the existence of a response to adrenergic stimulation in UCP1-ablated mice implies that a mechanism for an alternative adaptive adrenergic thermogenesis may exist. Here, we have used UCP1-ablated mice to examine the existence of an alternative adaptive adrenergic nonshivering thermogenesis, examined as the oxygen consumption response to systemically injected norepinephrine into anesthetized or conscious mice acclimated to different temperatures. We confirm that UCP1-dependent adrenergic nonshivering thermogenesis is adaptive, but we demonstrate that the adrenergic UCP1-independent thermogenesis is not recruitable by cold acclimation. Thus, at least in the mouse, no other proteins or enzymatic pathways exist that can participate in or with time take over the UCP1 mediation of adaptive adrenergic nonshivering thermogenesis, even in the total absence of UCP1. UCP1 is thus the only protein capable of mediating cold acclimation-recruited adaptive adrenergic nonshivering thermogenesis.

Acclimatization↗

PubMed2006

UCP1 and defense against oxidative stress. 4-Hydroxy-2-nonenal effects on brown fat mitochondria are uncoupling protein 1-independent.

Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1-/- mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1-/- mice, irrespective of whether they were adapted to thermoneutral temperature (30 degrees C) or to the cold (4 degrees C). The absence of oxidative damage in UCP1-/- mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage.

Adipose Tissue, Brown↗

PubMed2006

Ablation of PGC-1beta results in defective mitochondrial activity, thermogenesis, hepatic function, and cardiac performance.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

Adipose Tissue, Brown↗

PubMed2005

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity.

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.

Animals↗

PubMed2005

PPARgamma in the control of brown adipocyte differentiation.

The effects of fatty acids and retinoic acid (carotene) on brown adipose tissue differentiation are mediated by activation of the transcription factors PPARgamma and PPARalpha in combination with RXR. There is good support for the idea that activated PPARgamma promotes adipogenesis also in brown adipose tissue. However, the issue is more complex concerning the full differentiation to the brown adipocyte phenotype, particularly the expression of the brown-fat-specific marker UCP1. The effect of norepinephrine on PPARgamma gene expression, at least in-vitro, is negative, PPARgamma-ablated brown adipose tissue can express UCP1, and PGC-1alpha coactivates other transcription factors (including PPARalpha); thus, the significance of PPARgamma for the physiological control of UCP1 gene expression is not settled. However, importantly, the effects of PPAR agonists demonstrate the existence of a pathway for brown adipose tissue recruitment that is not dependent on chronic adrenergic stimulation and may be active in recruitment conditions such as prenatal and prehibernation recruitment. The ability of chronic PPARgamma agonist treatment to promote the occurrence of brown-fat features in white adipose tissue-like depots implies a role in anti-obesity treatment, but this will only be effective if the extra thermogenic capacity is activated by adrenergic stimulation.

Adipose Tissue, Brown↗

PubMed2005

A human-specific role of cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) in adipocyte lipolysis and obesity.

Elevated circulating fatty acid concentration is a hallmark of insulin resistance and is at least in part attributed to the action of adipose tissue-derived tumor necrosis factor-alpha (TNF-alpha) on lipolysis. Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) belongs to a family of proapoptotic proteins that has five known members in humans and mice. The action of CIDEA is unknown, but CIDEA-null mice are resistant to obesity and diabetes. We investigated CIDEA in adipose tissue of obese and lean humans and mice. The mRNA was expressed in white human fat cells and in brown mouse adipocytes. The adipose mRNA expression of CIDEA in mice was not influenced by obesity. However, CIDEA expression was decreased twofold in obese humans and normalized after weight reduction. Low adipose CIDEA expression was associated with several features of the metabolic syndrome. Human adipocyte depletion of CIDEA by RNA interference stimulated lipolysis and increased TNF-alpha secretion by a posttranscriptional effect. Conversely, TNF-alpha treatment decreased adipocyte CIDEA expression via the mitogen-activated protein kinase c-Jun NH(2)-terminal kinase. We propose an important and human-specific role for CIDEA in lipolysis regulation and metabolic complications of obesity, which is at least in part mediated by cross-talk between CIDEA and TNF-alpha.

Adipocytes↗

PubMed2004

Noradrenaline represses PPAR (peroxisome-proliferator-activated receptor) gamma2 gene expression in brown adipocytes: intracellular signalling and effects on PPARgamma2 and PPARgamma1 protein levels.

PPAR (peroxisome-proliferator-activated receptor) gamma is expressed in brown and white adipose tissues and is involved in the control of differentiation and proliferation. Noradrenaline stimulates brown pre-adipocyte proliferation and brown adipocyte differentiation. The aim of the present study was thus to investigate the influence of noradrenaline on PPARgamma gene expression in brown adipocytes. In primary cultures of brown adipocytes, PPARgamma2 mRNA levels were 20-fold higher than PPARgamma1 mRNA levels. PPARgamma expression occurred during both the proliferation and the differentiation phases, with the highest mRNA levels being found at the time of transition between the phases. PPARgamma2 mRNA levels were downregulated by noradrenaline treatment (EC50, 0.1 microM) in both proliferative and differentiating cells, with a lagtime of 1 h and lasting up to 4 h, after which expression gradually recovered. The down-regulation was beta-adrenoceptor-induced and intracellularly mediated via cAMP and protein kinase A; the signalling pathway did not involve phosphoinositide 3-kinase, Src, p38 mitogen-activated protein kinase or extracellular-signal-regulated kinases 1 and 2. Treatment of the cells with the protein synthesis inhibitor cycloheximide not only abolished the noradrenaline-induced down-regulation of PPARgamma2 mRNA, but also in itself induced PPARgamma2 hyperexpression. The down-regulation was probably the result of suppression of transcription. The down-regulation of PPARgamma2 mRNA resulted in similar down-regulation of PPARgamma2 and phosphoPPARgamma2 protein levels. Remarkably, the level of PPARgamma1 protein was similar to that of PPARgamma2 (despite almost no PPARgamma1 mRNA), and the down-regulation by noradrenaline demonstrated similar kinetics to that of PPARgamma2; thus PPARgamma1 was apparently translated from the PPARgamma2 template. It is suggested that beta-adrenergic stimulation via cAMP and protein kinase A represses PPARgamma gene expression, leading to reduction of PPARgamma2 mRNA levels, which is then reflected in down-regulated levels of PPARgamma2, phosphoPPARgamma2 and PPARgamma1.

Adipocytes↗

PubMed2004

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation.

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.

Adenylyl Cyclases↗

PubMed2004

Native UCP1 displays simple competitive kinetics between the regulators purine nucleotides and fatty acids.

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity approximately 30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K(m) of 0.05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a K(i) of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

Adipose Tissue, Brown↗

PubMed2004

Angiogenesis inhibitor, TNP-470, prevents diet-induced and genetic obesity in mice.

Adipose tissue growth has been proposed to involve recruitment of new blood vessels. Here, we test the hypothesis that delivery of an angiogenesis inhibitor in mice may prevent diet-induced obesity, the most common type of obesity in humans. We show that systemic administration of a selective angiogenesis inhibitor, TNP-470 (AGM-1470), prevents obesity in high caloric diet-fed wt mice as well as in genetically leptin-deficient ob/ob mice. Inhibition of obesity in mice by TNP-470 involves a reduction of vascularity in the adipose tissue. This therapeutic strategy appears to selectively affect the growth of adipose tissue as measured by the ratio between total fat and lean body mass. Interestingly, the treatment with TNP-470 results in decreased serum levels of low-density lipoprotein cholesterol. Furthermore, insulin levels are reduced, which indicates increased insulin sensitivity, suggesting that angiogenesis inhibitors may prevent the development of type II diabetes. Our findings suggest that similarly to growth and organogenesis in other tissues, adipose tissue growth is dependent on angiogenesis. Our observations may have conceptual implications for the prevention of obesity and related disorders.

3T3-L1 Cells↗

PubMed2004

IL-1 and LPS but not IL-6 inhibit differentiation and downregulate PPAR gamma in brown adipocytes.

Brown adipose tissue (BAT) is well recognized as a heat-producing organ of rodents and generally all young mammals. There is good experimental evidence that its thermogenic activity plays a role also during development of fever and that this activation is mediated exclusively via an adrenergic pathway. However, we have shown that brown adipocytes highly express receptors for pyrogenic factors (IL-1, LPS) and that after their activation, brown adipocytes are capable of responding by production of IL-6 and IL-1alpha. In this study we examined the effect of chronic treatment of mouse brown adipocytes in primary culture with pyrogens (IL-1beta, IL-6 and LPS) on their ability to differentiate into mature adipocytes. We found that treatment with IL-1beta or LPS, but not with IL-6, inhibited the differentiation of brown adipocytes, which was accompanied by a 2.5-10-fold decrease in PPARgamma mRNA. The anti-adipogenic effect of IL-1beta and LPS could thus be mediated via downregulation of PPARgamma levels, similar to the anti-adipogenic action of another pyrogenic cytokine--TNFalpha. We also found that pretreatment of brown adipocytes with IL-1beta or LPS led to a more pronounced (2-4-fold) induction of IL-6 transcripts upon whole 24h time course of adrenergic stimulation. These results support the view that BAT functions not only as an effector but also as a mediator of the febrile response; while the thermogenic activity is associated with terminally differentiated cells, earlier developmental states of brown adipocytes seem to be able to play a mediatory role.

Adipose Tissue, Brown↗

PubMed2004

Brown adipose tissue: function and physiological significance.

The function of brown adipose tissue is to transfer energy from food into heat; physiologically, both the heat produced and the resulting decrease in metabolic efficiency can be of significance. Both the acute activity of the tissue, i.e., the heat production, and the recruitment process in the tissue (that results in a higher thermogenic capacity) are under the control of norepinephrine released from sympathetic nerves. In thermoregulatory thermogenesis, brown adipose tissue is essential for classical nonshivering thermogenesis (this phenomenon does not exist in the absence of functional brown adipose tissue), as well as for the cold acclimation-recruited norepinephrine-induced thermogenesis. Heat production from brown adipose tissue is activated whenever the organism is in need of extra heat, e.g., postnatally, during entry into a febrile state, and during arousal from hibernation, and the rate of thermogenesis is centrally controlled via a pathway initiated in the hypothalamus. Feeding as such also results in activation of brown adipose tissue; a series of diets, apparently all characterized by being low in protein, result in a leptin-dependent recruitment of the tissue; this metaboloregulatory thermogenesis is also under hypothalamic control. When the tissue is active, high amounts of lipids and glucose are combusted in the tissue. The development of brown adipose tissue with its characteristic protein, uncoupling protein-1 (UCP1), was probably determinative for the evolutionary success of mammals, as its thermogenesis enhances neonatal survival and allows for active life even in cold surroundings.

Adipose Tissue, Brown↗

PubMed2003

Depressed thermogenesis but competent brown adipose tissue recruitment in mice devoid of all hormone-binding thyroid hormone receptors.

We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression.

Adipose Tissue, Brown↗

PubMed2003

Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs.

To identify the signaling pathways that mediate the adrenergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 approximately 6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 approximately 6.9), BRL 37344 (pEC50 approximately 8.6), CL 316243 (pEC50 approximately 9.7) and CGP 12177 (pEC50 approximately 7.3) and was thus mediated by beta3-adrenergic receptors. The NE-induced effect on 2-deoxy-D-glucose uptake was mediated by adenylyl cyclase and cAMP because responses were inhibited by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. Cholera toxin and 8-bromoadenosine cAMP were both able to increase 2-deoxy-D-glucose uptake. Involvement of other adrenergic signaling pathways (alpha1-and alpha2-adrenergic receptors) were excluded. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished beta-adrenergic- or 8-bromoadenosine cAMP-stimulated 2-deoxy-D-glucose uptake, demonstrating that a cAMP-dependent PI3K-mediated pathway is positively connected to glucose uptake. Inhibition of the beta-adrenergically stimulated response with protein kinase C (PKC) inhibitors (Gö 6983, which inhibits (alpha, beta, gamma), (delta), and (zeta) isoforms and Ro-31-8220, which inhibits (alpha, beta1, beta2, gamma) and (epsilon) but not atypical isoforms) indicated that cAMP-mediated glucose uptake is stimulated via conventional and novel PKCs. These results demonstrate that adrenergic stimulation, through beta3-adrenergic receptors/cAMP/protein kinase A, recruits a PI3K pathway stimulating conventional and novel PKCs, which mediate glucose uptake in brown adipocytes.

Adipocytes↗