A novel growth factor-dependent thermogenic brown adipocyte cell line from defined precursor cells

AbstractMolecular pathways regulating brown adipocyte formation and metabolism can be exploited as targets for the treatment of obesity and disorders of glucose and lipid metabolism such as type-2 diabetes. Investigations in this direction require adequate cell models for brown adipocytes and their precursors. We report the establishment of a novel clonal cell line derived from defined Lin−Sca1+ adipocyte precursors from murine interscapular brown fat. In contrast to most currently available lines, immortalization was achieved by serial passaging without viral or genetic manipulation. Instead, the media were supplemented with basic fibroblast growth factor, which was required for the maintenance of stable long-term growth and immature morphology. BATkl2 cells differentiated to adipocytes with high efficiency upon standard adipogenic induction independently of PPARg agonists and even at higher passage numbers. BATkl2 adipocytes showed readily detectable Uncoupling protein 1 (Ucp1) protein expression and acutely responded to norepinephrine with increased Ucp1 mRNA expression, lipolysis and uncoupled mitochondrial respiration. Highly efficient siRNA-mediated knockdown was demonstrated in the growth state as well as in differentiating adipocytes, whereas plasmid DNA transfection was achieved in immature cells. These features make the BATkl2 cell line an attractive brown (pre)-adipocyte cell model.

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Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.16.7561 ◽

1992 ◽

Vol 89 (16) ◽

pp. 7561-7565 ◽

Cited By ~ 77

Author(s):

S. R. Ross ◽

L. Choy ◽

R. A. Graves ◽

N. Fox ◽

V. Solevjeva ◽

...

Keyword(s):

Transgenic Mice ◽

Cell Line ◽

Protein Gene ◽

Uncoupling Protein ◽

Brown Adipocyte ◽

Adipocyte Cell ◽

Uncoupling Protein Gene

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Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression

Journal of Cell Science ◽

10.1242/jcs.107.1.313 ◽

1994 ◽

Vol 107 (1) ◽

pp. 313-319 ◽

Cited By ~ 4

Author(s):

S. Klaus ◽

L. Choy ◽

O. Champigny ◽

A.M. Cassard-Doulcier ◽

S. Ross ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Adrenergic Receptors ◽

Uncoupling Protein ◽

Primary Cultures ◽

Brown Adipocyte ◽

Mrna Levels ◽

Adrenergic Agonists ◽

Brown Adipocytes ◽

Adipocyte Cell

The HIB 1B cell line, derived from a brown fat tumor of a transgenic mouse, is the first established brown adipocyte cell line capable of expressing the brown fat-specific mitochondrial uncoupling protein (UCP). UCP gene expression, which was virtually undetectable under basic conditions, was stimulated by acute catecholamine or cyclic AMP treatment to levels comparable to primary cultures of brown adipocytes. Elevation of UCP mRNA levels following stimulation was very rapid but transient, decreasing after about 4 hours with a half-life between 9 and 13 hours. Immunoblotting showed the presence of UCP in HIB 1B mitochondria, but expression was much lower than observed in BAT or primary cultures of brown adipocytes. Upon transfection of HIB 1B cells with a reporter gene containing the UCP promoter, the activity of the transgene was regulatable by cAMP and norepinephrine. Investigation of the possible adrenergic receptors involved in UCP stimulation showed that specific beta 3-adrenergic agonists were much less effective than nonspecific beta-adrenergic agonists and that mRNA levels of the atypical, fat-specific beta 3-adrenoceptor were lower than those observed in brown adipocytes differentiated in primary culture. From pharmacological evidence we conclude that beta 3-adrenergic receptors account for approximately 30–40% of catecholamine induced UCP gene stimulation, whereas about 60–70% is stimulated via the classical beta 1/2 adrenergic pathway. We conclude that HIB 1B cells represent a functional system for the study of mechanisms related to brown adipose thermogenesis.

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Growth factor regulation of uncoupling protein-1 mRNA expression in brown adipocytes

AJP Cell Physiology ◽

10.1152/ajpcell.01396.2000 ◽

2002 ◽

Vol 282 (1) ◽

pp. C105-C112 ◽

Cited By ~ 22

Author(s):

Bibian García ◽

Maria-Jesús Obregón

Keyword(s):

Growth Factor ◽

Adipocyte Differentiation ◽

Uncoupling Protein ◽

Brown Adipocyte ◽

Mrna Levels ◽

Uncoupling Protein 1 ◽

Brown Adipocytes ◽

Proliferation And Differentiation ◽

Epidermal Growth ◽

Continuous Presence

To study the effect of the mitogens epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and vasopressin on brown adipocyte differentiation, we analyzed the expression of uncoupling protein-1 (UCP-1) mRNA. Quiescent brown preadipocytes express high levels of UCP-1 mRNA in response to triiodothyronine (T3) and norepinephrine (NE). The addition of serum or the mitogenic condition aFGF + vasopressin + NE or EGF + vasopressin + NE decreases UCP-1 mRNA. A second addition of mitogens further decreases UCP-1 mRNA. Treatment with aFGF or bFGF alone increases UCP-1 mRNA, whereas the addition of EGF or vasopressin dramatically reduces UCP-1 mRNA levels. The continuous presence of T3 increases UCP-1 mRNA levels in cells treated with EGF, aFGF, or bFGF. The effect of T3 on the stimulation of DNA synthesis also was tested. T3 inhibits the mitogenic activity of aFGF and bFGF. In conclusion, mitogens like aFGF or bFGF allow brown adipocyte differentiation, whereas EGF and vasopressin inhibit the differentiation process. T3 behaves as an important hormone that regulates both brown adipocyte proliferation and differentiation.

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Beta-1 and Not Beta-3 Adrenergic Receptors May Be the Primary Regulator of Human Brown Adipocyte Metabolism

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgz298 ◽

2019 ◽

Vol 105 (4) ◽

pp. e994-e1005 ◽

Cited By ~ 8

Author(s):

Mette Ji Riis-Vestergaard ◽

Bjørn Richelsen ◽

Jens Meldgaard Bruun ◽

Wei Li ◽

Jacob B Hansen ◽

...

Keyword(s):

Adrenergic Receptors ◽

Cell Model ◽

Uncoupling Protein ◽

Brown Adipocyte ◽

Mrna Levels ◽

Glycerol Release ◽

Adrenergic Stimulation ◽

Brown Adipose ◽

Ucp1 Expression ◽

Treatment Of Obesity

Abstract Purpose Brown adipose tissue (BAT) activation in humans has gained interest as a potential target for treatment of obesity and insulin resistance. In rodents, BAT is primarily induced through beta-3 adrenergic receptor (ADRB3) stimulation, whereas the primary beta adrenergic receptors (ADRBs) involved in human BAT activation are debated. We evaluated the importance of different ADRB subtypes for uncoupling protein 1 (UCP1) induction in human brown adipocytes. Methods A human BAT cell model (TERT-hBA) was investigated for subtype-specific ADRB agonists and receptor knockdown on UCP1 mRNA levels and lipolysis (glycerol release). In addition, fresh human BAT biopsies and TERT-hBA were evaluated for expression of ADRB1, ADRB2, and ADRB3 using RT-qPCR. Results The predominant ADRB subtype in TERT-hBA adipocytes and BAT biopsies was ADRB1. In TERT-hBA, UCP1 mRNA expression was stimulated 11.0-fold by dibutyryl cAMP (dbcAMP), 8.0-fold to 8.4-fold by isoproterenol (ISO; a pan-ADRB agonist), and 6.1-fold to 12.7-fold by dobutamine (ADRB1 agonist), whereas neither procaterol (ADRB2 agonist), CL314.432, or Mirabegron (ADRB3 agonists) affected UCP1. Similarly, dbcAMP, ISO, and dobutamine stimulated glycerol release, whereas lipolysis was unaffected by ADRB2 and ADRB3 agonists. Selective knockdown of ADRB1 significantly attenuated ISO-induced UCP1 expression. Conclusion The adrenergic stimulation of UCP1 and lipolysis may mainly be mediated through ADRB1. Moreover, ADRB1 is the predominant ADRB in both TERT-hBA and human BAT biopsies. Thus, UCP1 expression in human BAT may, unlike in rodents, primarily be regulated by ADRB1. These findings may have implications for ADRB agonists as future therapeutic compounds for human BAT activation.

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In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid

Biochemical Journal ◽

10.1042/bj3170827 ◽

1996 ◽

Vol 317 (3) ◽

pp. 827-833 ◽

Cited By ~ 83

Author(s):

Pere PUIGSERVER ◽

Francisca VÁZQUEZ ◽

María L. BONET ◽

Catalina PICÓ ◽

Andreu PALOU

Keyword(s):

Retinoic Acid ◽

Cultured Cells ◽

Uncoupling Protein ◽

Primary Cultures ◽

Brown Adipocyte ◽

Brown Adipose ◽

Adipocyte Cell ◽

Differentiation Stage

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.

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The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.2.e386 ◽

2000 ◽

Vol 279 (2) ◽

pp. E386-E394 ◽

Cited By ~ 53

Author(s):

Patrice Penfornis ◽

Say Viengchareun ◽

Damien Le Menuet ◽

Françoise Cluzeaud ◽

Maria-Christina Zennaro ◽

...

Keyword(s):

Cell Line ◽

Mineralocorticoid Receptor ◽

Uncoupling Protein ◽

T Antigen ◽

Proximal Promoter ◽

Brown Adipocyte ◽

Brown Adipocytes ◽

Aldosterone Antagonists ◽

Fatty Acid Binding ◽

Gene Markers

By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10−9 M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-γ, and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

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