Development of brown fat cells in monolayer culture

Nechad, M.; Kuusela, Pertti; Carneheim, C.; Björntorp, Per; Nedergaard, Jan; Cannon, Barbara

doi:10.1016/0014-4827(83)90384-1

Cited by 127 publications

(34 citation statements)

References 29 publications

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“…Brown adipocyte precursor cells were isolated from pooled interscapular, axillary, and cervical brown adipose tissue, as described earlier (16,17). In brief, tissue was minced in a Hepes-buffered Ringer solution containing 0.1-0.2% (w/v) collagenase type II (Sigma) and digested for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

β3- and α1-Adrenergic Erk1/2 Activation Is Src- but Not Gi-mediated in Brown Adipocytes

Lindquist

Fredriksson

Rehnmark

et al. 2000

Journal of Biological Chemistry

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A novel signaling pathway for mediation of ␤ 3 -adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G i -proteins and G␤␥-subunits, distinct from the classical cAMP pathway of ␤-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express ␤ 3 -adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both ␤ 3 receptors and ␣ 1 receptors but not via ␣ 2 receptors. Forskolin induced Erk1/2 activation similarly to ␤ 3 activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G i -pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that G␤␥ activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both ␤ 3 -and ␣ 1 -induced Erk1/2 activation. Thus, the proposed novel G i pathway for ␤ 3 mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed ␤ 3 receptors examined here. Here, the ␤ 3 signal is mediated classically via cAMP/protein kinase A. ␤ 3 and ␣ 1 signals converge at Src, which thus mediates Erk1/2 activation in both pathways.

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Section: Methodsmentioning

confidence: 99%

β3- and α1-Adrenergic Erk1/2 Activation Is Src- but Not Gi-mediated in Brown Adipocytes

Lindquist

Fredriksson

Rehnmark

et al. 2000

Journal of Biological Chemistry

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“…In this cell culture system, freshly isolated precursor cells from brown adipose tissue grow and differentiate in vitro. In the preconfluent phase, the cells have the morphological and biochemical appearance of undifferentiated cells, but in the confluent phase, the cells undergo adipose conversion [27], and advance in differentiation to reach a stage where brown-fat-specific gene expression can be demonstrated [l&28]. We here present results from these two contrasting phases of cell development: preconfluent, undifferentiated cells (here: 3 days in culture) and confluent, differentiated cells (6 days).…”

Section: Clebpa and Ciebpb Expression In Primary Cultures Derived Fromentioning

confidence: 99%

Differential adrenergic regulation of C/EBPα and C/EBPβ in brown adipose tissue

et al. 1993

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"The Wenner-wren institute, The Arrhenius ~ohoratorie~ F3, Stockholm university, S-106 91 Stockholm, Sweden and bKarolinska Institute* Center for biotechnology, NOVUM, S-14f 57 ~~ddinge* Sweden Received 11 January 1993We investigated the regulation of the expression of two members of the C/EBP family of transcriptional activators, CIEBPa and CIEBPB, in brown adipose tissue in mice. Less than one hour of cold exposure led to dramatic changes in the expression of both genes, ClEBPa steady-state mRNA and protein levels were drastically and rapidly reduced whereas C/EBP/l mRNA and protein levels were induced severalfold. Also norepinephrine injection affected the expression of the transcription factors. Preconfluent cells in brown fat primary cultures responded to norepinephrine with a decrease in CIEBPa and an increase in CIEBPB mRNA; in confluent cells the expression of both factors was increased. Thus, C/EBPcl and CIEBPB gene expression is under adrenergic control both in vivo and in vitro but the type of response is directed by the degree of differentiation of the cells.

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“…Brown adipocytes in primary culture proliferate until confluence is reached, which occurs at approximately day 5-6 after inoculation (14,19,20,25,26). At this time, differentiation spontaneously occurs.…”

Section: Mouse Brown Adipocytes Constitutively Express the Vegf Genementioning

confidence: 99%

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

Fredriksson

Lindquist

Bronnikov³

et al. 2000

Journal of Biological Chemistry

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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 Ϸ 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 ␤-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 (␣ 1 -adrenoreceptors, Ca 2؉ , protein kinase C, ␣ 2 -adrenoreceptors, and pertussis toxinsensitive 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 ␤-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.

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Development of brown fat cells in monolayer culture

Cited by 127 publications

References 29 publications

β3- and α1-Adrenergic Erk1/2 Activation Is Src- but Not Gi-mediated in Brown Adipocytes

β3- and α1-Adrenergic Erk1/2 Activation Is Src- but Not Gi-mediated in Brown Adipocytes

Differential adrenergic regulation of C/EBPα and C/EBPβ in brown adipose tissue

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

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