Efficient parameterisation of non-collinear energy landscapes in itinerant magnets.

Magnetic exchange interactions determine the magnetic groundstate, as well as magnetic excitations of materials and are thus essential to the emerging and fast evolving fields of spintronics and magnonics. The magnetic force theorem has been used extensively for studying magnetic exchange interactions. However, short-ranged interactions in itinerant magnetic systems are poorly described by this method and numerous strategies have been developed over the years to overcome this deficiency. The present study supplies a fully self-consistent method for systematic investigations of exchange interactions beyond the standard Heisenberg model. In order to better describe finite deviations from the magnetic ground state, an extended Heisenberg model, including multi-spin interactions, is suggested. Using cross-validation analysis, we show that this extended Heisenberg model gives a superior description for non-collinear magnetic configurations. This parameterisation method allows us to describe many different itinerant magnetic systems and can be useful for high-throughput calculations.

Incidence and characteristics of drowning in Sweden during a 15-year period.

AIM: Drowning is a global health problem and deeper knowledge about the extent and causes is of utmost importance for implementing preventative actions. The aim of this study was to describe the incidence and characteristics of drowning in Sweden over time, including both non-fatal and fatal cases. METHODS: All cases identified as drowning (ICD-10 coding) at a national level in Sweden between 2003-2017 were collected. Three sources of data from the Swedish National Board of Health and Welfare were extracted via the Cause of Death Register and the National Patient Register. RESULTS: Over 15 years, a total of 6609 cases occurred, resulting in an annual incidence of 4.66 per 100 000. The median age was 49 years (IQR 23-67) and 67% were males. Non-fatal drownings represented 51% (n = 3363), with an overall non-fatal to fatal ratio of 1:1, this being 8:1 for children (0-17 years of age). Non-fatal cases were more often female (36% vs. 30%; p < 0.001), younger 30 (IQR 10-56) vs. 60 (IQR: 45-72) (p < 0.001) and of unintentional nature (81% vs. 55%; p < 0.001). The overall incidence decreased over time from 5.6 to 4.1 per 100 000 (p < 0.001). The highest rate of 30-day survival was found in females 0-17 years (94%, 95% CI 91.1-95.5) and the lowest in males >66 years (28.7%, 95% CI 26.2-31.2). Although the incidence in children 0-4 years increased from 7.4 to 8.1 per 100 000 (p < 0.001), they demonstrated the highest non-fatal to fatal ratio (13:1). CONCLUSION: Drowning is declining but remains a consistent and underestimated public-health problem. Non-fatal drowning cases represent about half of the burden and characteristics differ from fatal drowning cases, being younger, more often female and of unintentional nature.

Life without UCP1: mitochondrial, cellular and organismal characteristics of the UCP1-ablated mice.

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline ('norepinephrine') or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1-ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.

Malnutrition in patients suffering from chronic heart failure; the nurse's care.

Chronic heart failure is associated with a bad prognosis with considerably shortened survival and repeated hospitalisations. Patients suffering from heart failure also have symptoms that can affect their food intake, for example, tiredness when strained, breathing difficulties and gastrointestinal symptoms like nausea, loss of appetite and ascites. Pharmacological therapy can lead to a loss of appetite, which will make the intake of food inadequate to fill the required energy and nutritional needs. The nurse's interest in and knowledge of diet issues can improve these patients' nutritional status. The aim of this literature review was to describe the nurse's interventions regarding malnutrition in patients suffering from chronic heart failure. The literature search gave 13 articles, which were analysed, and sentences whose content was related to the aim were identified. Three areas of content appeared; drug treatment and consequences, gastrointestinal effects, and information and education. The results show that the nutritional status of these patients can be significantly improved by means of simple nursing interventions. Future research should focus on controlled experimental studies to evaluate differences in body weight, body mass index and quality of life between patients suffering from chronic heart failure, who are taking part in a fully enriched nutrition intervention, and patients suffering from chronic heart failure, who are eating their normal diet.

Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold.

Adaptive nonshivering thermogenesis may have profound effects on energy balance and is therefore therefore is a potential mechanism for counteracting the development of obesity. The molecular basis for adaptive nonshivering thermogenesis has remained a challenge that sparked acute interest with the identification of proteins (UCP2, UCP3, etc.) with high-sequence similarity to the original uncoupling protein-1 (UCP1), which is localized only in brown adipose tissue. Using UCP1-ablated mice, we examined whether any adaptive nonshivering thermogenesis could be recruited by acclimation to cold. Remarkably, by successive acclimation, the UCP1-ablated mice could be made to subsist for several weeks at 4C during which they had to constantly produce heat at four times their resting levels. Despite these extreme requirements for adaptive nonshivering thermogenesis, however, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, although the existence of, for example, muscular mechanisms for adaptive nonshivering thermogenesis has recurrently been implied, we did not find any indication of such thermogenesis. Not even during prolonged and enhanced demand for extra heat production was any endogenous hormone or neurotransmitter able to recruit any UCP1-independent adaptive nonshivering thermogenic process in muscle or in any other organ, and no proteins other than UCP1-not even UCP2 or UCP3-therefore have the ability to mediate adaptive nonshivering thermogenesis in the cold.

UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency.

The uniqueness of UCP1 (as compared to UCP2/UCP3) is evident from expression analysis and ablation studies. UCP1 expression is positively correlated with metabolic inefficiency, being increased by cold acclimation (in adults or perinatally) and overfeeding, and reduced in fasting and genetic obesity. Such a simple relationship is not observable for UCP2/UCP3. Studies with UCP1-ablated animals substantiate the unique role of UCP1: the phenomenon of adaptive adrenergic non-shivering thermogenesis in the intact animal is fully dependent on the presence of UCP1, and so is any kind of cold acclimation-recruited non-shivering thermogenesis; thus UCP2/UCP3 (or any other proteins or metabolic processes) cannot substitute for UCP1 physiologically, irrespective of their demonstrated ability to show uncoupling in reconstituted systems or when ectopically expressed. Norepinephrine-induced thermogenesis in brown-fat cells is absolutely dependent on UCP1, as is the uncoupled state and the recoupling by purine nucleotides in isolated brown-fat mitochondria. Although very high UCP2/UCP3 mRNA levels are observed in brown adipose tissue of UCP1-ablated mice, there is no indication that the isolated brown-fat mitochondria are uncoupled; thus, high expression of UCP2/UCP3 does not necessarily confer to the mitochondria of a tissue a propensity for being innately uncoupled. Whereas the thermogenic effect of fatty acids in brown-fat cells is fully UCP1-dependent, this is not the case in brown-fat mitochondria; this adds complexity to the issues concerning the mechanisms of UCP1 function and the pathway from beta(3)-adrenoceptor stimulation to UCP1 activation and thermogenesis. In addition to amino acid sequences conserved in all UCPs as part of the tripartite structure, all UCPs contain certain residues associated with nucleotide binding. However, conserved amongst all UCP1s so far sequenced, and without parallel in all UCP2/UCP3, are two sequences: 144SHLHGIKP and the C-terminal sequence RQTVDC(A/T)T; these sequences may therefore be essential for the unique thermogenic function of UCP1. The level of UCP1 in the organism is basically regulated at the transcriptional level (physiologically probably mainly through the beta(3)-adrenoceptor/CREB pathway), with influences from UCP1 mRNA stability and from the delay caused by translation. It is concluded that UCP1 is unique amongst the uncoupling proteins and is the only protein able to mediate adaptive non-shivering thermogenesis and the ensuing metabolic inefficiency.

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis.

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.

Cig30 and Pitx3 genes are arranged in a partially overlapping tail-to-tail array resulting in complementary transcripts.

The mouse Cig30 gene codes for a 30-kDa membrane glycoprotein, which appears to have a role in the recruitment of brown adipose tissue. To elucidate the structure of the Cig30 gene, we have isolated a lambda phage genomic DNA clone containing the entire mouse gene and found that Cig30 consists of four exons that are spread over 4 kilobase pairs of genomic sequence. Using a fluorescence in situ hybridization assay and interspecific backcross panel mapping, we have localized the Cig30 locus to the distal region of mouse chromosome 19, between the Tlx1 and Ins1 loci. Sequencing of the corresponding lambda clone to completion revealed that the insert contained yet another gene in the opposite orientation. It turned out to be the newly identified homeobox gene Pitx3. Interestingly, the genes are very tightly linked, so that the 3' ends of their transcripts are complementary. Thus, our results provide evidence for bidirectional transcription of a several hundred base pair-long DNA region as a result of the extremely tight linkage between Cig30 and Pitx3.

Development of adrenergic and cholinergic cardiac control in larvae of the African clawed frog Xenopus laevis.

Cardiac responses (heart rate, stroke volume, and cardiac output) to cholinergic and adrenergic receptor stimulation were investigated in developing larvae of Xenopus laevis from Nieuwkoop and Faber (NF) stage 33/34 (newly hatched) to NF stage 53 (22 d after hatching). Effects on heart rate (fH), stroke volume (SV), and cardiac output (CO) were analyzed using in situ preparations and video-microscopic techniques to record the continually beating heart. The results show that administration of acetylcholine to the heart decreases heart rate as early as NF stage 40. A significant reduction in SV and CO following acetylcholine administration to the heart was found at NF stages 45-53. Epinephrine had no significant effect on fH, SV, or CO at any of the stages investigated. However, an adrenergic tonus on the heart is present already at NF stage 40 (11%). This tonus increases up to a maximum (44%) at NF stages 45-47, when the maximal heart rate is found during development of X. laevis. We conclude that acetylcholine has a negative chronotropic and possibly also inotropic effect on the heart very early in development of X. laevis. We also hypothesize that the high adrenergic tonus found at NF stages 45-47 is responsible, at least in part, for the peak in heart rate seen at these stages.

UCP1: the original uncoupling protein--and perhaps the only one? New perspectives on UCP1, UCP2, and UCP3 in the light of the bioenergetics of the UCP1-ablated mice.

The availability of a UCP1-ablated mouse has enabled critical studies of the function of UCP1, UCP2, and UCP3. Concerning UCP1, its presence in brown-fat mitochondria is associated with innate uncoupling, high GDP-binding capacity, and GDP-inhibitable Cl- permeability and uncoupling--but the high fatty acid sensitivity found in these mitochondria is observed even in the absence of UCP1. The absence of UCP1 leads to low cold tolerance but not to obesity. UCP1 ablation also leads to an augmented expression of UCP2 and UCP3 in brown adipose tissue, making this tissue probably the one that boasts the highest expression of these UCPs. However, these very high expression levels are not associated with any inherent uncoupling, or with a specific GDP-binding capacity, or with a GDP-sensitive Cl- permeability, or with any effect of GDP on mitochondrial membrane potential, or with an increased basal metabolism of cells, or with the presence of norepinephrine- or fatty acid-induced thermogenesis in cells, and not with a cold-acclimation recruited, norepinephrine-induced thermogenic response in the intact animal. Therefore, it can be discussed whether any uncoupling effect is associated with UCP2 or UCP3 when they are endogenously expressed and, consequently, whether (loss of) uncoupling (thermogenic) effects of UCP2 or UCP3 can be invoked to explain metabolic phenomena, such as obesity.

Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese.

The mitochondrial uncoupling protein (UCP) in the mitochondrial inner membrane of mammalian brown adipose tissue generates heat by uncoupling oxidative phosphorylation. This process protects against cold and regulates energy balance. Manipulation of thermogenesis could be an effective strategy against obesity. Here we determine the role of UCP in the regulation of body mass by targeted inactivation of the gene encoding it. We find that UCP-deficient mice consume less oxygen after treatment with a beta3-adrenergic-receptor agonist and that they are sensitive to cold, indicating that their thermoregulation is defective. However, this deficiency caused neither hyperphagia nor obesity in mice fed on either a standard or a high-fat diet. We propose that the loss of UCP may be compensated by UCP2, a newly discovered homologue of UCP; this gene is ubiquitously expressed and is induced in the brown fat of UCP-deficient mice.

Contrasting adrenergic effects on lipoprotein lipase gene expression in the brown adipose tissue of intact mice and in cultured brown adipocytes from mice.

To examine the regulation of lipoprotein lipase (LPL) gene expression, LPL mRNA levels in the brown adipose tissue of intact mice and in mouse brown adipocyte cultures were examined. In intact mice, exposure to cold resulted in a rapid, transient, 5-fold increase in LPL mRNA level. Norepinephrine (NE) injection could fully mimic the effect of acute exposure to cold, and LPL mRNA and enzymatic activity were increased in parallel after NE injection. These results indicated positive adrenergic control of LPL gene expression in the brown adipose tissue of intact mice. In cultured mouse brown adipocytes, the level of spontaneously expressed LPL mRNA decreased in parallel with the progression of brown adipocyte differentiation. NE treatment of undifferentiated cells led to a decrease in LPL mRNA levels. In brown adipocytes that had reached a mature state, NE had a small negative or no effect on LPL mRNA levels, irrespective of whether the experiment was performed in the presence or absence of insulin or of newborn-calf serum. It was concluded that LPL gene expression in brown adipose tissue in intact mice is under adrenergic control but that this gene is not under positive adrenergic control in cultured brown adipocytes from mice, although these cells are otherwise adrenergically sensitive. The presence of additional factors may be necessary to confer adrenergic sensitivity to the LPL gene in the cultured brown adipocytes; alternatively, cells other than the mature brown adipocytes may confer the positive adrenergic sensitivity to the brown adipose tissue depots in situ.

Adrenergic stimulation of lipoprotein lipase gene expression in rat brown adipocytes differentiated in culture: mediation via beta3- and alpha1-adrenergic receptors.

In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control.

Cig30, a mouse member of a novel membrane protein gene family, is involved in the recruitment of brown adipose tissue.

We have identified a previously uncharacterized gene that is implicated in the thermogenic function of brown adipose tissue of mice. This gene, termed Cig30, is the first mammalian member of a novel gene family comprising several nematode and yeast genes, such as SUR4 and FEN1, mutation of which is associated with highly pleiotropic phenotypes. It codes for a 30-kDa plasma membrane glycoprotein with five putative transmembrane domains. The Cig30 mRNA was readily detected only in brown fat and liver. When animals were exposed to a 3-day cold stress, the Cig30 expression was selectively elevated in brown fat more than 200-fold. Similar increases were brought about in two other conditions of brown fat recruitment, namely during perinatal development and after cafeteria diet. The magnitude of Cig30 mRNA induction in the cold could be mimicked by chronic norepinephrine treatment in vivo. However, in primary cultures of brown adipocytes, a synergistic action of norepinephrine and dexamethasone was required for full expression of the gene, indicating that both catecholamines and glucocorticoids are required for the induction of Cig30. We propose that the CIG30 protein is involved in a pathway connected with brown fat hyperplasia.

Signal transduction in brown adipose tissue recruitment: noradrenaline and beyond.

The classical effect of noradrenaline on brown adipose tissue is stimulation of heat production. However, it is likely that noradrenaline is also the major regulator of proliferation and differentiation. The adrenergic receptors involved include at least beta 1, beta 3, alpha 2 and alpha 1. Heat production is mainly stimulated via beta 3 receptors and cAMP. Cell proliferation is mainly stimulated via beta 1 receptors and cAMP. Cell differentiation is also adrenergically promoted; at least the expression of the gene for the tissue-specific uncoupling protein thermogenin is controlled via beta 3 receptors and cAMP. There is a switch in beta-receptor endowment between young (beta 1) and mature (beta 3) cells. The expression of several transcription factors is also under adrenergic control: c-Fos gene expression depends synergistically on beta- and alpha 1-stimulation mediated via cAMP and [Ca2+]i increases. C/EBP beta gene expression is regulated only via beta-receptors, but the expression of the C/EBP alpha gene shows a switch during differentiation: in young cells, the expression is represented through both beta- and alpha 1-receptors; in mature cells, the expression is stimulated via b-receptors. It is likely that noradrenaline exerts its proliferation- and differentiation-promoting action through alterations in the expression of these or other transcription factors.

Norepinephrine as a morphogen?: its unique interaction with brown adipose tissue.

Norepinephrine is normally considered a neurotransmitter mediating acute metabolic effects in target cells. However, analysis of the regulation of the recruitment process in brown adipose tissue has indicated that norepinephrine may interact with this tissue in such a way that it could be considered a morphogen for this tissue. Besides stimulating the acute thermogenic processes, norepinephrine can induce the expression of tissue-specific proteins such as the uncoupling protein, induce expression of non-tissue specific proteins necessary of the thermogenic processes (e.g. lipoprotein lipase) and repress the expression of non-essential proteins (e.g. subunit c of the ATP-synthase). Upon chronic adrenergic stimulation, the general differentiation state of the tissue is advanced, indicating that the expression of factors with a more general effect on brown adipocyte differentiation is also under adrenergic control. It may even be discussed that norepinephrine may be involved early in the embryonal determination process directing cell clones into this line. The molecular basis for these effects of norepinephrine are only poorly known at present, but adrenergic effects on the expression level of many transcription factors, such as C/EBPalpha, C/EBPbeta, and PPARgamma 2, have been noted. These collective recruitment effects of norepinephrine are well suited to allow the tissue to grow or atrophy in response to the physiological needs of the organism.

Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region.

The product of the c-myc proto-oncogene, Myc, has been implicated in the transcriptional regulation of several genes, acting either as an activator or repressor of gene expression. To determine whether Myc is involved in the modulation of the expression of the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene, we used both stable cell lines overexpressing Myc and transient co-transfection assays. We show that the endogenous C/EBP alpha protein level is repressed in stable cell lines overexpressing Myc. We also show that enforced expression of Myc in mouse hibernoma HIB-1B cells dramatically repressed the expression of C/EBP alpha--promoter-reporter fusion genes. This effect of Myc was mediated through the core promoter region. Mutation of the initiator site could not abolish this affect, indicating that Myc may interact with some component(s) of the basal transcription machinery.

Norepinephrine utilizes alpha 1- and beta-adrenoreceptors synergistically to maximally induce c-fos expression in brown adipocytes.

In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat cells, we have investigated the ability of brown fat cells to respond to norepinephrine stimulation with an increase in the expression of the proto-oncogene c-fos. Stimulation of brown fat precursor cells (isolated from young mice and grown for 4 days in culture) with norepinephrine led to a marked but transient (maximal approximately 30 min) induction of c-fos expression. The magnitude of this induction was similar in pre- and postconfluent cells. The norepinephrine effect could be blocked by both alpha 1- and beta-adrenergic antagonists. Forskolin had a small inductive ability, as had the selective alpha 1-agonist cirazoline, but with both together a high induction was obtained. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) could in itself induce c-fos expression, but pretreatment with TPA did not abolish the ability of norepinephrine to induce c-fos expression, indicating that TPA-sensitive protein kinase C was not a primary mediator in this pathway. Also the Ca2+ ionophore A23187 had in itself an inductive ability, but A23187 in combination with forskolin led to a large increase in c-fos expression, indicating synergistic interaction between a cAMP pathway and a [Ca2+]i pathway. This interaction was not proximal, i.e. alpha 1 stimulation or increase in [Ca2+]i by A23187 did not augment forskolin-induced cAMP levels, and beta stimulation or forskolin did not affect [Ca2+]i levels; and it did not require protein synthesis. It was concluded that norepinephrine, in agreement with its fundamental role in the control of brown fat cell growth and development, was able to induce c-fos expression, that this induction was not exclusively linked to promotion of either proliferation or differentiation, and that the induction was mediated via a distal synergism between beta/cAMP and alpha 1/[Ca2+]i pathways, thus conferring to the alpha 1-adrenoreceptors on the cell a potentially significant role in the control of cell growth and development.