Volume 270,
Number 18,
Issue of May 5, pp. 10723-10732, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the
Uncoupling Protein Gene ( Ucp) by 
,
, and 
-Adrenergic Receptor Subtypes
in Immortalized Brown Adipose Cell Lines
Elizabeth M.
Rohlfs
,
Kiefer W.
Daniel
,
Richard T.
Premont
,
Leslie
P.
Kozak
,
Sheila
Collins
Immortalized brown adipocyte cell lines derived from a mouse
hibernoma express all three 
-adrenergic receptor subtypes,
including 
-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
-adrenergic receptor agonist CL316,243 by stimulating
adenylyl cyclase activity (3-10-fold over basal).
-, 
-, and
-adrenergic receptor mRNA was detected by Northern
blotting and/or reverse transcriptase-polymerase chain reaction.
Competition binding assays with the antagonists CGP20712A and
I-cyanopindolol showed the proportions of
AR and AR in immortalized cells to be
similar to brown adipocytes from tissue (cells: 35%
AR, 65% AR; brown adipocytes from
tissue: AR 41%, 59% AR). Expression
of brown fat-specific mitochondrial uncoupling protein ( Ucp)
was stimulated by -adrenergic agonists in two of the four cell
lines. The ability of individual AR subtypes to regulate Ucp expression was examined with combinations of selective
-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the -adrenergic receptor subtypes.
However, the greatest response was obtained by stimulating all three
-adrenergic receptor subtypes simultaneously (100 µM
isoproterenol). Incubation of membranes from cultured cells or brown
adipocytes from tissue with CL316,243 at an optimal concentration (5
µM) did not prevent norepinephrine from further
stimulating adenylyl cyclase activity, suggesting that the combined
activation of AR/AR, plus
AR, 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 -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
-adrenergic receptor expression and function, and the control of
Ucp expression and activity.
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