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Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.

Publication ,  Journal Article
Rohlfs, EM; Daniel, KW; Premont, RT; Kozak, LP; Collins, S
Published in: J Biol Chem
May 5, 1995

Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three beta-adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antagonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by beta-adrenergic agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective beta-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the beta-adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three beta-adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that beta-adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of beta-adrenergic receptor expression and function, and the control of Ucp expression and activity.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 5, 1995

Volume

270

Issue

18

Start / End Page

10723 / 10732

Location

United States

Related Subject Headings

  • Uncoupling Protein 1
  • Receptors, Adrenergic, beta
  • RNA, Messenger
  • Propranolol
  • Norepinephrine
  • Molecular Sequence Data
  • Mitochondrial Proteins
  • Mice, Inbred C57BL
  • Mice
  • Membrane Proteins
 

Citation

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Rohlfs, E. M., Daniel, K. W., Premont, R. T., Kozak, L. P., & Collins, S. (1995). Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines. J Biol Chem, 270(18), 10723–10732. https://doi.org/10.1074/jbc.270.18.10723
Rohlfs, E. M., K. W. Daniel, R. T. Premont, L. P. Kozak, and S. Collins. “Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.J Biol Chem 270, no. 18 (May 5, 1995): 10723–32. https://doi.org/10.1074/jbc.270.18.10723.
Rohlfs EM, Daniel KW, Premont RT, Kozak LP, Collins S. Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines. J Biol Chem. 1995 May 5;270(18):10723–32.
Rohlfs, E. M., et al. “Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.J Biol Chem, vol. 270, no. 18, May 1995, pp. 10723–32. Pubmed, doi:10.1074/jbc.270.18.10723.
Rohlfs EM, Daniel KW, Premont RT, Kozak LP, Collins S. Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines. J Biol Chem. 1995 May 5;270(18):10723–10732.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 5, 1995

Volume

270

Issue

18

Start / End Page

10723 / 10732

Location

United States

Related Subject Headings

  • Uncoupling Protein 1
  • Receptors, Adrenergic, beta
  • RNA, Messenger
  • Propranolol
  • Norepinephrine
  • Molecular Sequence Data
  • Mitochondrial Proteins
  • Mice, Inbred C57BL
  • Mice
  • Membrane Proteins