Brown Adipocytes In Primary Culture Research Articles

Promotion of lipid storage rather than of thermogenic competence by fetal versus newborn calf serum in primary cultures of brown adipocytes

ABSTRACTMuch current understanding of brown adipocyte development comes from in-vitro cell models. Serum type may affect the behavior of cultured cells and thus conclusions drawn. Here, we investigate effects of serum type (“fetal bovine” versus “newborn calf”) on responses to differentiation inducers (the PPARγ agonist rosiglitazone or the neurotransmitter norepinephrine) in cultured primary brown adipocytes. Lipid storage was enhanced by fetal versus newborn serum. However, molecular adipose conversion (Pparg2 and Fabp4 expression) was not affected by serum type. Rosiglitazone-induced (7-days) expression of thermogenic genes (i.e. Ucp1, Pgc1a, Dio2 and Elovl3) was not systematically affected by serum type. However, importantly, acute (2 h) norepinephrine-induced thermogenic gene expression was overall markedly higher (and adipose genes somewhat lower) in cells cultured in newborn serum. Thus, newborn serum promotes thermogenic competence, and the use of fetal serum in brown adipocyte cultures (as is often routine) counteracts adequate differentiation. Agents that counteract this inhibition may therefore confoundingly be ascribed genuine thermogenic competence-inducing properties.

11β-HSD1 Modulates the Set Point of Brown Adipose Tissue Response to Glucocorticoids in Male Mice

Glucocorticoids (GCs) are potent regulators of energy metabolism. Chronic GC exposure suppresses brown adipose tissue (BAT) thermogenic capacity in mice, with evidence for a similar effect in humans. Intracellular GC levels are regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, which can amplify circulating GC concentrations. Therefore, 11β-HSD1 could modulate the impact of GCs on BAT function. This study investigated how 11β-HSD1 regulates the molecular architecture of BAT in the context of GC excess and aging. Circulating GC excess was induced in 11β-HSD1 knockout (KO) and wild-type mice by supplementing drinking water with 100 μg/mL corticosterone, and the effects on molecular markers of BAT function and mitochondrial activity were assessed. Brown adipocyte primary cultures were used to examine cell autonomous consequences of 11β-HSD1 deficiency. Molecular markers of BAT function were also examined in aged 11β-HSD1 KO mice to model lifetime GC exposure. BAT 11β-HSD1 expression and activity were elevated in response to GC excess and with aging. 11β-HSD1 KO BAT resisted the suppression of uncoupling protein 1 (UCP1) and mitochondrial respiratory chain subunit proteins normally imposed by GC excess. Furthermore, brown adipocytes from 11β-HSD1 KO mice had elevated basal mitochondrial function and were able to resist GC-mediated repression of activity. BAT from aged 11β-HSD1 KO mice showed elevated UCP1 protein and mitochondrial content, and a favorable profile of BAT function. These data reveal a novel mechanism in which increased 11β-HSD1 expression, in the context of GC excess and aging, impairs the molecular and metabolic function of BAT.

Differences in the Response of UCP1 mRNA to Hormonal Stimulation Between Rat and Mouse Primary Cultures of Brown Adipocytes

Uncoupling protein 1 (UCP-1), the specific marker of brown adipose tissue, is transcriptionally activated in response to adrenergic stimuli and thyroid hormones are necessary for its full expression. We describe differences in the regulation of UCP-1 mRNA expression between rat and mouse brown adipocytes in culture, using norepinephrine (NE), triiodothyronine (T3), insulin and retinoic acid (RA). Results: NE and cAMP-elevating agents strongly increase UCP-1 mRNA levels in cultures of mouse adipocytes, but increases are low in those from rat. In rat adipocytes NE poorly increases UCP-1 mRNA expression and T3 markedly increases the adrenergic response of UCP-1, an effect not observed in mouse adipocytes. In the absence of insulin, T3 itself increases UCP-1 mRNA in rat adipocytes and enhances the response to NE, while in mouse adipocytes no effect of T3 is observed. RA by itself stimulates UCP-1 mRNA in mouse adipocytes, but not in those from rat. In rat cultures, RA requires the presence of NE and/or T3. Conclusions: We find important differences in the hormonal regulation of UCP-1 mRNA expression in cultured preadipocytes depending on the species used as donor; those differences are observed using identical culture conditions and should be considered when doing cultures from these species.

Norepinephrine and rosiglitazone synergistically induceElovl3expression in brown adipocytes

The Elovl3 gene, which putatively encodes for a protein involved in the elongation of saturated and monounsaturated fatty acids in the C20-C24 range, is expressed in murine liver, skin, and brown adipose tissue (BAT). In BAT, Elovl3 is dramatically upregulated during tissue activation in response to cold exposure, and functional data imply that ELOVL3 is a critical enzyme for lipid accumulation in brown adipocytes during the early phase of tissue recruitment. The activation of BAT is controlled by sympathetic nerve activity and norepinephrine release. By using primary cultures of brown adipocytes, we show here that the induced Elovl3 gene expression is synergistically regulated by norepinephrine and the peroxisome proliferator-activated receptor (PPAR) gamma ligand rosiglitazone. In addition, the potency of rosiglitazone to induce Elovl3 expression was several orders of magnitude higher than for the PPARalpha and PPARdelta ligands WY-14643 and L-165041, respectively. The maximal increase in mRNA level by norepinephrine and rosiglitazone is achieved by induced transcription as well as increased mRNA stability, and the whole process requires novel protein synthesis. We conclude that norepinehrine and PPARgamma, despite having different roles in brown adipocyte activation and differentiation, cooperate in expanding the intracellular lipid pool by synergistically stimulating Elovl3 expression.

The control of UCP1 is dissociated from that of PGC-1α or of mitochondriogenesis as revealed by a study using β-less mouse brown adipocytes in culture

In rodent brown adipose tissue, the β-adrenergic signaling is believed, by an action on PGC-1α, to control UCP1 expression and mitochondriogenesis. We addressed this hypothesis using β 1/β 2/β 3-adrenoceptor knockout (β-less) brown adipocytes in primary culture. In these cells: (a) proliferation and differentiation into multilocular cells were normal; (b) UCP1 mRNA expression was dramatically decreased (by 93%), whereas PGC-1α and mtTFA mRNA expressions were not; (c) UCP1, PGC-1α and COX IV protein expressions were decreased by 97%, 62% and 22%, respectively. Altogether the data show a dissociation between the control of UCP1, which is mostly β-adrenoceptor-dependent and that of PGC-1α and of mitochondriogenesis which are not.

Adiponectin is expressed in the brown adipose tissue and surrounding immature tissues in mouse embryos

Adiponectin is one of the adipocytokines, which are adipose-specific secretory factors. We examined its expression during embryogenesis. Transcripts of adiponectin were detected at a late stage of embryogenesis on embryonic (E) day E16.5. In situ hybridization showed that adiponectin transcripts were localized in brown adipose tissues (BATs) and surrounding immature tissues in mouse embryos. Immunohistochemistry using a specific anti-adiponectin antibody showed that the distribution of adiponectin closely parallels that of its mRNA. Adiponectin was also detected in serum at day E16.5, and its concentration peaked at birth. By contrast, transcripts of both the adiponectin receptors 1 and 2 were already expressed by day E12.5 in many tissues. Thus, their expression profile differed from that of adiponectin itself. Furthermore, experiments using primary cultures of brown adipocytes showed that adiponectin is regulated in brown adipocytes by various modulators, similar to its regulation in white adipose tissues (WATs). These data indicate that adiponectin has important roles in glucose and lipid metabolism during the perinatal period.

α1- and β1-Adrenoceptor Signaling Fully Compensates for β3-Adrenoceptor Deficiency in Brown Adipocyte Norepinephrine-Stimulated Glucose Uptake

To assess the relative roles and potential contribution of adrenergic receptor subtypes other than the beta3-adrenergic receptor in norepinephrine-mediated glucose uptake in brown adipocytes, we have here analyzed adrenergic activation of glucose uptake in primary cultures of brown adipocytes from wild-type and beta3-adrenergic receptor knockout (KO) mice. In control cells in addition to high levels of beta3-adrenergic receptor mRNA, there were relatively low alpha1A-, alpha1D-, and moderate beta1-adrenergic receptor mRNA levels with no apparent expression of other adrenergic receptors. The levels of alpha1A-, alpha1D-, and beta1-adrenergic receptor mRNA were not changed in the beta3-KO brown adipocytes, indicating that the beta3-adrenergic receptor ablation does not influence adrenergic gene expression in brown adipocytes in culture. As expected, the beta3-adrenergic receptor agonists BRL-37344 and CL-316 243 did not induce 2-deoxy-d-glucose uptake in beta3-KO brown adipocytes. Surprisingly, the endogenous adrenergic neurotransmitter norepinephrine induced the same concentration-dependent 2-deoxy-D-glucose uptake in wild-type and beta3-KO brown adipocytes. This study demonstrates that beta1-adrenergic receptors, and to a smaller degree alpha1-adrenergic receptors, functionally compensate for the lack of beta3-adrenergic receptors in glucose uptake. Beta1-adrenergic receptors activate glucose uptake through a cAMP/protein kinase A/phosphatidylinositol 3-kinase pathway, stimulating conventional and novel protein kinase Cs. The alpha1-adrenergic receptor component (that is not evident in wild-type cells) stimulates glucose uptake through a phosphatidylinositol 3-kinase and protein kinase C pathway in the beta3-KO cells.

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.

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.

Rosiglitazone and Retinoic Acid Induce Uncoupling Protein-1 (UCP-1) in a p38 Mitogen-activated Protein Kinase-dependent Manner in Fetal Primary Brown Adipocytes

Brown adipose tissue expresses the thermogenic uncoupling protein-1 (UCP-1), which is positively regulated by peroxisome proliferator-activated receptor (PPAR) agonists and retinoids through the activation of the heterodimers PPAR/retinoid X receptor (RXR) and retinoic acid receptor (RAR)/RXR and binding to specific elements in the ucp-1 enhancer. In this study we show that in fetal rat brown adipocyte primary cultures the PPARgamma agonist rosiglitazone (Rosi), as well as retinoic acids 9-cis-retinoic acid and all-trans-retinoic acid also have "extragenic" effects and induce p44/p42 and p38 mitogen-activated protein kinase (p38MAPK) activation. The latter is involved in UCP-1 gene expression, because inhibition of p38MAPK activity with PD169316 impairs the ability of Rosi and retinoids for UCP-1 induction. The inhibitory effects of PD169316 are mimicked by the antioxidant GSH, suggesting a role for reactive oxygenated species (ROS) generation in the increase of UCP-1 expression in response either to Rosi or 9-cis-retinoic acid. Thus, we propose that Rosi and retinoids act as PPAR/RXR and RAR/RXR agonists and also activate p38MAPK. These two coordinated actions could result in a high increase of transcriptional activity on the ucp-1 enhancer and hence on thermogenesis. PPARalpha and gamma agonists but not retinoids also increase UCP-3 expression in fetal brown adipocytes. However, the regulation of UCP-3, which is not involved in thermogenesis, seems to differ from UCP-1 given the fact that is not affected by p38MAPK inhibition.

A novel pathway for adrenergic stimulation of cAMP-response-element-binding protein (CREB) phosphorylation: mediation via α1-adrenoceptors and protein kinase C activation

Because of the central role of adrenergic mechanisms in the expression of crucial genes during brown adipocyte differentiation, we examined the activation (phosphorylation) of CREB (cAMP-response-element-binding protein) in mouse brown adipocytes in primary culture. We found that noradrenaline ('norepinephrine') stimulated CREB phosphorylation rapidly (maximum effect in < or =5 min with slow decay) and efficiently (EC(50), 6 nM). The increase in CREB phosphorylation coincided with increased expression of an artificial cAMP-response-element-containing reporter construct. CREB phosphorylation was partly inhibitable, both by the beta-adrenergic antagonist propranolol and by the alpha(1)-adrenergic antagonist prazosin. Adenylate cyclase hyperactivation (by forskolin) could stimulate CREB phosphorylation to the same extent as noradrenaline. The alpha(1)-adrenergic agonist cirazoline also increased CREB phosphorylation. An increase in intracellular [Ca(2+)] had, however, no effect, but protein kinase C activation by PMA was a potent stimulator. The cirazoline-stimulated (alpha(1)-adrenergic) CREB phosphorylation was inhibited by a desensitizing pretreatment with PMA, demonstrating that the alpha(1)-stimulation was mediated via protein kinase C activation; neither Src nor extracellular-signal-regulated kinases 1 and 2 activation was involved in the signalling process. We conclude that CREB phosphorylation in brown adipocytes is mediated not only through the classical beta-adrenergic/cAMP pathway but also through a novel alpha(1)-adrenergic/protein kinase C/CREB pathway, which has not been described previously in any tissue.

As the proliferation promoter noradrenaline induces expression of ICER (induced cAMP early repressor) in proliferative brown adipocytes, ICER may not be a universal tumour suppressor

The CREM (cAMP-response-element modulator) gene product ICER (induced cAMP early repressor) has been proposed to function as a tumour (cell proliferation) suppressor. To investigate the generality of this concept, the expression pattern of ICER in brown adipocytes was followed; this was critical because brown adipocytes are one of few cell types in which cAMP is associated positively with cell proliferation but negatively with apoptosis. In response to the physiological stimulus of cold (which induces cell proliferation), ICER mRNA levels were increased in brown adipose tissue in vivo. In brown adipocytes in primary culture, ICER gene expression was induced by noradrenaline (norepinephrine) not only in the mature state (where noradrenaline potentiates differentiation), but also in the proliferative state of the cell cultures (where noradrenaline enhances cell proliferation). The induction was mediated via β-receptors and the cAMP/protein kinase A pathway. The induced ICER appeared to repress its own expression and that of the β2-adrenoceptor. It is thus evident that also in cell types in which cAMP induces proliferation, and even when these cells are in the proliferative state, ICER expression is induced by the same agents that stimulate proliferation. This can either mean that ICER is not a general tumour suppressor, or that brown adipocytes temporally or spatially avoid this role of ICER.

As the proliferation promoter noradrenaline induces expression of ICER (induced cAMP early repressor) in proliferative brown adipocytes, ICER may not be a universal tumour suppressor.

The CREM (cAMP-response-element modulator) gene product ICER (induced cAMP early repressor) has been proposed to function as a tumour (cell proliferation) suppressor. To investigate the generality of this concept, the expression pattern of ICER in brown adipocytes was followed; this was critical because brown adipocytes are one of few cell types in which cAMP is associated positively with cell proliferation but negatively with apoptosis. In response to the physiological stimulus of cold (which induces cell proliferation), ICER mRNA levels were increased in brown adipose tissue in vivo. In brown adipocytes in primary culture, ICER gene expression was induced by noradrenaline (norepinephrine) not only in the mature state (where noradrenaline potentiates differentiation), but also in the proliferative state of the cell cultures (where noradrenaline enhances cell proliferation). The induction was mediated via beta-receptors and the cAMP/protein kinase A pathway. The induced ICER appeared to repress its own expression and that of the beta2-adrenoceptor. It is thus evident that also in cell types in which cAMP induces proliferation, and even when these cells are in the proliferative state, ICER expression is induced by the same agents that stimulate proliferation. This can either mean that ICER is not a general tumour suppressor, or that brown adipocytes temporally or spatially avoid this role of ICER.

Norepinephrine Induces Vascular Endothelial Growth Factor Gene Expression in Brown Adipocytes through a β-Adrenoreceptor/cAMP/Protein Kinase A Pathway Involving Src but Independently of Erk1/2

To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose- and time-dependent manner (EC(50) approximately 90 nm). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via beta-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (alpha(1)-adrenoreceptors, Ca(2+), protein kinase C, alpha(2)-adrenoreceptors, and pertussis toxin-sensitive G(i)-proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through beta-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.

9- cis Retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9- cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all- trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9- cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

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.

Direct effects of leptin on brown and white adipose tissue.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

TNF-α induces apoptosis in rat fetal brown adipocytes in primary culture

The effect of TNF-α on cell death in rat fetal brown adipocytes maintained in primary culture was determined. TNF-α inhibited proliferation and induced apoptosis in these cells. Most of the cells undergoing apoptosis after TNF-α treatment did not express PCNA, suggesting an induction of apoptosis by TNF-α in non-proliferative cells. IGF-I but not EGF prevented TNF-α-induced apoptosis.

Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes.

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.

β-Adrenergic stimulation of interleukin-1α and interleukin-6 expression in mouse brown adipocytes

Mouse brown adipocytes in primary culture were shown to contain high levels of mRNA for interleukin-1α (IL-1α) which could be further stimulated up to 9-fold by norepinephrine (NE). Even higher stimulation by NE, up to 40-fold, was found in case of interleukin-6 (IL-6). Time-course of activation of both genes was biphasic, but the response of IL-6 gene was slower than of IL-1α gene. IL-1α mRNA level reached the maximum after 1 h and the second, lower increase, occurred after 8 h. IL-6 mRNA level showed first maximum after 2 h, but the highest level was found after 8 h. Similarly to NE, the expression of IL-1α and IL-6 genes was stimulated by selective β-adrenergic agonist isoproterenol, β 3-selective agonist CGP-12117, forskoline and db-cAMP. The activation of both genes by CGP-12177 was dose-dependent with the optimum at 100 nM concentration. Stimulation of α-adrenergic receptors by cirazoline and oxymetazoline was without any effect. When the expression of IL-6 was studied at the protein level, the stimulation of IL-6 gene via β 3-receptors resulted in secretion of IL-6 up to the concentration 10 ng/ml culture media in 24 h. The results indicate a new type of regulation of expression of IL-1α and IL-6 genes in brown adipocytes by catecholamines acting via β 3-adrenergic receptors. The resulting increase in IL-6 production by brown adipocytes could significantly contribute to systemic levels of IL-6.