brown adipose tissue was studied in cell culture. Syn- thesis of mitochondrial uncoupling protein (UCP), F1-. ATPase, and cytochrome oxidase was examined.
THE JOURNAL OF BIOLOGICAL CHEMWTRY 0 1990 by The American Society for Biochemistry
Vol. 265, No. 36, Issue of December 25, pp. 22204-22209,199O Printed in V. S.A.
and Molecular Biology, Inc.
Synthesis of Mitochondrial Uncoupling Differentiated in Cell Culture*
Protein in Brown Adipocytes (Received
Jan Kopeck$, Marie Josef Houit&kQ From
the Institute
BaudyErovi,
of Physiology,
Franc0
Czechoslouak
ZanottiS,
Academy
Dagmar
of Sciences,
In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1ATPase, and cytochrome oxidase was examined by L[36S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins @ subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 MM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the /3-adrenergic receptors and elevated intracellular concentration of CAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenie mitochondria.
Thermogenic brown adipose tissue is one of the most plastic mammalian tissues with regard to the intensity of adaptive growth and differentiation in later postnatal life (reviewed in Refs. l-3). A reliable marker of terminal differentiation (4,5) of the brown fat cell is the appearance of the mitochondrial uncoupling protein (UCP).’ The UCP is a key and ratelimiting (6) component of thermogenesis in brown adipose tissue. It allows for regulatory dissipation in the form of heat of the electrochemical proton potential gradient generated * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i Permanent address: Istituto Policattedra di Biochimica Medica e C’himica Medica, Universita di Bari, Piazza G. Cesare, I-70124 Bari, Italy. 8 To whom correspondence should be addressed. Tel.: 42-24719741. 1 The abbreviations used are: UCP, mitochondrial uncoupling protein (thermogenin); F1-ATPase, catalytic part of mitochondriai ATP svnthase: HEPES. 4-(2-hvdroxvethvl)-l-ninerazineethanesulfonic acid; IEg, isoelectric foc&&g; MEM, “Eagle’szminimal essential medium containing Earle’s balanced salt solution; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; BAT, brown adipose tissue.
Videirskh
Janikovi,
Stanislav
1083,
142 20 Prague,
for publication,
May
Pavelka,
and
22, 1990)
Czechoslovakia
across the inner mitochondrial membrane by the respiratory chain enzymes (2). In accordance with a physiological uncoupling of oxidative phosphorylation, brown adipose tissue mitochondria are endowed with a high oxidative capacity (and a high content of cytochrome oxidase), whereas the content of ATP synthase is very low (7, 8). The thermogenic mechanism of brown adipose tissue is unique in nature and the expression of the UCP gene is strictly tissue-specific (9, 10). From studies on the perinatal development of brown adipose tissue (5, 11-14) and experiments with brown adipose tissue of adult animals (3,10, 15) it was concluded that both norepinephrine and 3,5,3’-triiodo-~thyronine are involved in the control of brown adipose tissue growth, differentiation, and induction of UCP synthesis. In addition, other factors (3, 13) such as glucagon might be involved in the control of the perinatal recruitment and activation of the UCP gene (10, 16). In order to study the control of brown adipose tissue development and biogenesis of the thermogenic mitochondria in uitro, several attempts were made to induce complete differentiation of brown adipose tissue in a primary cell culture (17, 18) or in a permanent cell line (19). However, catecholamine-induced expression of the UCP gene was only achieved at the mRNA level (l&$20), while induction of UCP synthesis was so far possible only in mature adipocytes isolated from mouse brown adipose tissue (21). In the present report it is described for the first time that synthesis of physiological amounts of UCP can be induced in brown adipose tissue cells differentiated in vitro, indicating formation of functional thermogenic mitochondria. The stimulation of UCP synthesis appears to be P-adrenergically regulated and exhibits marked selectivity in comparison with other mitochondrial proteins.’ MATERIALS
AND
METHODS3
RESULTS
Morphobgy
of BFOWIZ
Adipose
Tissue
Cells
Differentiating
cell Culture-The isolated stromal-vascular fraction from mouse brown adipose tissue contained several types of nonerythrocyte cells (Fig. 2A; see also “Materials and Methods” in the Miniprint). Among them the most numerous were preadipocytes and interstitial cells (50-80%), while the contribution of mature cells was significantly lower (20-50%). The cell population attached to the dish 1 day after seeding differed substantially (Fig. 2B). The preadipocytes and interstitial cells represented the majority of cells and the relative in
’ A preliminary account of these results has been presented 3 Portions of this paper (including “Materials and Methods,” 1, and Table I) are presented in miniprint at the end of this Miniprint is easily read with the aid of a standard magnifying Full size photocopies are included in the microfilm edition Journal that is available from Waverly Press.
22204
(22). Fig. paper. glass. of the
Brown
Fat
Uncoupling
Protein
in Cell Culture IEF
FIG. 2. Morphology of cultured cells. Isolated stromal-vascular cells of mouse brown adipose tissue used for seeding (A) and cell cultures after 1 day (B), 3 days (C), and 8 days (D) of cultivation (see Fig. 1, -O-; Miniprint) are shown. Different cell types are indicated: m, mature cells; p, preadipocytes; i, interstitial cells; and e, erythrocytes. Morphology of brown adipose tissue cells was revealed by Nomarski interference contrast microscopy. Bar, 50 Grn.
content of mature cells decreased to about 10%. Later on, the content of mature cells decreased further to less than 1% of cells on day 3 (Fig. 2C). During this period the cells proliferated rapidly (see Fig. 1 in the Miniprint) and started to differentiate (Fig. 2C) forming polyhedral cells (19, 24). At confluence, on day 6, almost all the cells present were filled with small lipid droplets and had the typical appearance of mature adipocytes. Their morphology remained unchanged on subsequent days, except for the increasing size of the lipid droplets (Fig. 20). When the confluent and postconfluent cells were exposed for 24 h to norepinephrine (l-10 PM), the physiological inducer of thermogenesis in brown adipose tissue, they became retracted and the lipid droplets decreased in size and number, indicating a stimulation of lipolysis of cellular triglycerides. Protein Synthesis in Brown Adipose Tissue Cells Differentiated in Culture and Effect of Catecholamines-Protein syn-
thesis in cultured cells was followed by incorporation of [35S] methionine. Although the specific labeling decreased with the time of cultivation, the differentiated cells in postconfluent cultures were capable of intensive protein synthesis (Table I, Miniprint). When norepinephrine or isoproterenol was present during the 4-h labeling period, the total [35S]methionine incorporation was significantly depressed already at 0.1 pM catecholamine. The maximum, near 70%, inhibition occurred at higher concentrations (l-10 pM) of norepinephrine. However, when norepinephrine was present for 24 h before cell harvesting, its inhibitory effect disappeared (Table I) even at the high (10 PM) concentration. In further experiments, two-dimensional electrophoresis of the labeled proteins followed by autoradiography was used to characterize [35S]methionine incorporation into individual cellular proteins. In Fig. 3, A and B, the same amount of protein, but 3-fold different amounts of trichloroacetic acidinsoluble radioactivity, from control cells and cells labeled in the presence of norepinephrine (4 h with 10 pM norepinephrine; see Table I in the Miniprint) were compared. The labeling pattern showed again that the synthesis of the majority of cellular proteins was suppressed by norepinephrine. However, even at the high norepinephrine concentration tested, the synthesis of several proteins became activated (spots of equal or increased intensity). In the two-dimensional
22205 IEF
FIG. 3. Two-dimensional electrophoretic analysis of [?3] methionine-labeled proteins in postconfluent cell cultures. Cells were labeled on day 8 for 4 h with [35S]methionine (see “Materials and Methods”). Protein aliquots (3 pg) of cells labeled in control dishes (A, lo5 dpm) and in the presence of 10 pM norepinephrine (B, 3 x lo4 dpm) were separated by isoelectrofocusing (IEF; using Ampholines 3-10 and 4-6 in l/l ratio, 2% concentration in the gel) in the first dimension, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (14-20% acrylamide) in the second dimension. For calibration, “C-labeled molecular weight protein markers were also analyzed in the second dimension (not shown). Radioactivity was detected by fluorography. In parallel, 3-pg protein aliquots of the labeled proteins were mixed with 8 pg of unlabeled mouse brown adipose tissue mitochondria and analyzed by isoelectrofocusing/SDSpolyacrylamide gel electrophoresis as above. After blotting to nitro&&lo&, immunodetection was performed with specific antisera to UCP or F,-ATPase. Positions of the B subunit of F,-ATPase (F,B and UCP in a two-dimensional separation pattern were detected-by autoradiography of immunoblots (not shown), and indicated in fluorographs A and B.
separation, individual proteins could be unequivocally distinguished by immunodetection on a nitrocellulose blot with specific antibodies. A spot corresponding to a protein of molecular mass close to 32 kDa and isoelectric point at pH 6.0-6.6 was identified as the UCP. Incorporation of [35S] methionine into this component showed that UCP was synthesized at a significant rate both in control cells (Fig. 3A) and in norepinephrine-treated cells (Fig. 3B). In contrast, the synthesis of F1, another key component of mitochondrial energy conversion, was decreased by norepinephrine, as documented by the incorporation of [35S]methionine into the p subunit of the enzyme (Fig. 3, A and B). Content of UCP, F,, and Cytochrome Oxidase in Cultured Brown Adipose Tissue Cells-In further experiments the
amount of mitochondrial UCP, F1, and cytochrome oxidase were evaluated by Western blotting in whole homogenates or cultured cells. A very small amount or no UCP was found 3 days after inoculation (not shown). Later on, UCP synthesis was spontaneously induced. A maximum level of UCP was reached around confluence on day 6 and decreased afterward (Table II). This indicates that UCP present in confluent and postconfluent cells is produced by newly differentiating cells in culture (compare with Fig. 2). In fact, the low and varying content of UCP on day 3 reflects slight and irregular contamination of the culture by surviving isolated mature adipocytes which involute on subsequent days (19). In postconfluent cells on day 8 (Fig. 4) significant amounts of UCP were still detected. When a higher inoculum was used for cultivation (4 x 104/cm2), an even higher amount of UCP, comparable to the UCP content in the isolated stromalvascular cells, was spontaneously formed. Immunoblotting data further showed that the levels of F1-ATPase were markedly higher in postconfluent cells than in the stromal-vascular cells, while the specific content of cytochrome oxidase was significantly less elevated in cultured cells (Fig. 4; note the difference in the amount of proteins applied in individual slots). In contrast to UCP, changes in the inoculation density did not affect the content of F1-ATPase and cytochrome
Brown Fat Uncoupling TABLE
Protein in Cell Culture
II
Specific content of UCP in cultured brown adipose tissue cells UCP was immunodetected in aliquots of total proteins (as in Fig. 4) from cells cultured under standard conditions. Where indicated, 0.1 or 10 pM norepinephrine (Ne) was present for 24 h before harvesting. The specific content of UCP was estimated as described
under “Materials and Methods” and expressedas percent of the specific content of UCP of a homogenate of mouse brown adipose tissue,used as a standard (animals maintained at 22 “C). Values are means + S.E. of several(n) indeDendentexDeriments. Days after
plating
Conditions
6 8 8 8
Control Control 10
pM
Specific content of UCP
n
-8
-7
-6
-5
0
8 16 Time(h)
1% he] PI)
Ne
FIG. 5. Dose-response rine-induced increase
0.1 pM Ne
y’
4h
control
24 h
-II
curve and of UCP-specific
time
course content.
24
of norepineph-
A, cellscultured for 8 days were exposed for the last 24 h to the indicated concentration of norepinephrine (Ne, single dose); B, cells were exposed to 0.1 pM norepinephrine for the times indicated. Untreated cells served as control. lo-rg protein aliquots of cells harvested by scraping were analyzed by immunoblotting for the content of UCP. The points represent the means of two independent experiments performed in
duplicate. The increasein the specificcontent of UCP was expressed in percent of the mean increase at 0.1 pM norepinephrine.
I
I
I
I
14
6
4
1
Protein w
2
Inoculation (~10~~ viable
FIG. 4. Immunodetection of chrome oxidase in total proteins (SV) cells and of postconfluent
density cells/cm2)
F1-ATPase, UCP, and cytoof isolated stromal-vascular cells. F1-ATPase,UCP, and
with time for 24 h (Fig. 5B), showing a somewhat lower rate of increase during the first few hours. When exposed to norepinephrine for more than 24 h, cells started to detach from the dishes. A similar time dependence was observed at 1 and 10 pM norepinephrine (not shown). Under suboptimal conditions (10 pM norepinephrine) the specific content of UCP in cultured cells reached in 24 h about one-half the UCP content in the homogenate of brown adipose tissue from mice kept at 22 “C (Table II), whereas at 0.1 PM norepinephrine the specific UCP content of cultured cells was practically equal to the content in brown adipose tissue homogenate. All the UCP formed in cell cultures was associated with the crude mitochondrial fraction (see Fig. 7) and no immunoreactive UCP was detected in the 12,000 x g supernatant (not shown), thus indicating a quantitative insertion of UCP into mitochondria. Accordingly, the specific content of UCP found in the isolated mitochondrial fraction was about two times higher than in the total cell proteins.
cytochrome oxidase (COX) antigens were detected by immunoblotCharacterization of Adrenergic Receptors Involved in the ting in aliquots of SV cells (left, 6 and 14 pg of protein, respectively) with 0.1 pM norepiand of cellscultured for 8 days(right, 15 rg of protein; cellsharvested Control of UCP Synthesis-Stimulation in the presenceof SDS; see “Materials and Methods”). Cells were nephrine for 24 h of the UCP synthesis in postconfluent cells inoculated at a standard or high density, and norepinephrine (Ne) was largely abolished by 10 pM and almost completely by 50 was present for 4 or 24 h before harvesting, as indicated. FM propranolol, the p-selective antagonist (Fig. 6). The alselective antagonist prazosin (50 pM) was significantly less oxidase (Fig. 4), indicating that the content of UCP, F1, and inhibitory than propranolol. Isoproterenol (0.1 PM), the /3selective agonist, was found to be similarly effective as norcytochrome oxidase can be changed quite specifically and that different factors influence their synthesis in brown adipose epinephrine in elevation of the UCP content. As further shown in Fig. 6, a pronounced increase in the specific content tissue cells. The content of immunoreactive UCP significantly in- of UCP was also induced by modulating the intracellular creased after 4 h of incubation with 10 or 0.1 PM norepinephconcentration of CAMP. After addition of 1 pM forskolin, an rine (Fig. 4). When the hormone was present for 24 h, even activator of adenylate cyclase, the UCP synthesis increased higher levels of UCP were detected (Fig. 4). In contrast to to approximately 75% and stimulation with 1 mM dibutyryl induction of UCP, nonsignificant and irreproducible changes CAMP amounted to 95% of the norepinephrine-induced value. (several experiments) in F1-ATPase and cytochrome oxidase Fast Loss of Mitochondrial UCP-A spontaneously formed content were found after addition of norepinephrine (Fig. 4), UCP always reached a maximum level on day 6 and decreased indicating that the effect of catecholamine on the synthesis afterward as apparent from its content in homogenate (Table of UCP is rather specific. II) and crude mitochondrial fraction (Fig. 7) prepared from The dose-response curve for the effect of norepinephrine cultured cells. The decrease was rather fast and was usually on UCP synthesis and the time course of the norepinephrinegreater (down to 20% in 48 h) in the presence of 5% rather activated UCP synthesis are shown in Fig. 5. The maximum than 10% fetal calf serum. The fate of the other mitochondrial increase of the specific content of UCP was found at 0.1 pM proteins, F1-ATPase and cytochrome oxidase (Fig. 7) was norepinephrine (Fig. 5A). After addition of 0.1 pM norepidifferent. Cytochrome oxidase did not change at all, the pnephrine the specific content of UCP continuously increased subunit of FL-ATPase decreased only slightly and, interest-
Brown Fat Uncoupling
I
T i i
FIG. 6. The
effect CAMP
N,’
Ne
Ne
Pro,,
P:,,,
PL
CAMP
F
of adrenergic agonists and antagonists on UCP synthesis. Cells cultured for 8
days were exposed for the last 24 h to the following agents or their combination: 0.1 jtM norepinephrine (Ne), 0.1 MM isoprenaline (Zso), 10 pM propranolol (Pro,~), 50 FM propranolol (Pro.&, 50 gM prazosin (Pm), 1 PM forskolin (F), or 1 mM dibutyryl CAMP (CAMP) as indicated. Untreated cells served as control. Specific content of UCP was determined by immunoblotting in aliquots (10 pg of protein) of cells harvested by scraping. In each experiment the change in the specific content of UCP was expressed in percent of the mean value of the increase at 0.1 gM norepinephrine. The values are the means f S.D. from three independent experiments performed in duplicate. forskolin
and
FJ~--
-q@kck
cox
FIG. 7. Changes cytochrome oxidase from cell cultures.
‘K-k
in
-
-
-UCP
Ire
the (COX)
content of UCP, F1-ATPase, and in crude mitochondrial fractions
Cultivation was performed in the presence of 5 or 10% fetal calf serum. Cells were harvested by scraping on day 6 or day 8 of cultivation, homogenized, and centrifuged for 30 min at 127000 X g. Samples (5 rg-of protein) of the crude mitochondrial fraction (sediment) were analyzed for the content of UCP, Fi-ATPase, and cytochrome oxidase antigens by immunoblotting. The recovery of the total cell protein in the 12,000 X g sediment was 25-32%. ingly, the content of (Y subunit of Fi-ATPase appeared to decrease similarly as UCP. It is noteworthy that all the mitochondrial proteins studied are membrane-bound and are completely (UCP and F1) or in part (cytochrome oxidase; subunits IV-VIII) coded for by nuclear DNA and synthesized extramitochondrially (10, 35). DISCUSSION
The exneriments nresented here demonstrate that vrecursor cells isolated from mouse brown adipose tissue can develop in primary culture into fully differentiated mature adipocytes capable of complete expression of the gene for UCP. This is apparent as norepinephrine-induced UCP synthesis of remarkable intensity resulting in formation of physiological levels of UCP within 24 h. The maximum content of UCP was found in the cells around confluence (Fig. 4, Table II). These cells (Fig. 2) also exhibited all the mornholoeical features of fullv develoved mature multilocular adipocytes found in thermogenically active brown adipose tissue (24, 25). In less developed cells the
Protein in Cell Culture
22207
synthesis was very weak and UCP was not detectable. A high content of mitochondria in cultured cells reflected in the high levels of cytochrome oxidase (Fig. 4) is also typical for brown adipose tissue (2, 7). The cytochrome oxidase content was in fact higher than in previous experiments when brown adipose tissue cells differentiating in culture were unable to synthesize UCP (19). Temporary spontaneous synthesis of UCP around confluence (Fig. 4, Table II) may indicate that at a given stage of development the cells can transiently activate expression of the UCP gene without external stimulation, or that some regulatory factors are produced in cell culture. These could be for example catecholamines released from neural cells present in heterogenous cell population of primary cultures.* Interestingly, the higher specific content of UCP was found at increased inoculation density (Fig. 4) and frequent changes of the medium during cultivation prevented spontaneous UCP synthesis (not shown). Both norepinephrine ((Ye-, P-agonist) and the pure P-agonist isoprenaline exerted similar stimulatory effects on UCP synthesis (Fig. 6), indicating that the P-adrenergic receptors were mainly involved. This was confirmed by pronounced inhibition of norepinephrine-stimulated UCP synthesis by propran0101 (Fig. 6). Also in uiuo studies (36, 37) and experiments with isolated mature adipocytes (19, 21) showed that p-adrenergic receptors can activate UCP gene expression. Significant inhibition of norepinephrine-induced UCP synthesis was also observed with prazosin (Fig. 6), suggesting that al-receptors may contribute to the control of the UCP gene. This is in accordance with previous in uiuo studies (37) and contrasts with the lack of effect of the cui-agonist in isolated mature adipocytes (21). The maximum stimulation of UCP synthesis in cultured cells (Fig. 5) as well as the activation of UCP mRNA synthesis (20) was observed at 10T7 M norepinephrine which also maximally stimulated lipolysis and respiration in isolated brown adipose tissue cells (38). Elevation of intracellular CAMP concentration due to forskolin or direct addition of dibutyryl CAMP stimulated UCP synthesis to a similar extent as catecholamines (Fig. 6). This confirms the involvement of adenylate cyclase and CAMP in activation of the UCP gene and shows that the regulation of UCP may share the initial steps with the regulation of hormone-sensitive lipase and thermogenesis in brown adipose tissue. In accordance with fast activation of UCP synthesis in cold exposed animals (2, 3, 10, 11,37), in cultured cells exposed to norepinephrine a significant increase of UCP content was detected after 4 h and the UCP synthesis remained stimulated for at least 24 h (Fig. 5). In contrast, in the presence of norepinephrine the labeling of the total cellular proteins with [35S]methionine transiently decreased after 4 h (Table I, Fig. 3). Catecholamines may influence the energetic state of cultured adipocytes similarly as in isolated hamster brown adipocytes, where norepinephrine markedly lowered cytosolic ATP (39). This may be due to a simultaneous activation of ATP-consuming synthetic processes and thermogenesis which means a full uncoupling of oxidative phosphorylation. A similar suppression of protein synthesis was found in isolated brown adipose tissue cells of adult Syrian hamster,5 and energetic “insufficiency” of adipocytes would be consistent with a nonspecific inhibitory effect of norepinephrine on transcription of several mRNA species in cultured brown adipose tissue cells (18). A rather selective stimulation of UCP synthesis with respect to the other mitochondrial proteins during perinatal ’ M. BaudyHovi, manuscript in preparation. ’ J. HouStek, unpublished data.
22208
Brown Fat Uncoupling Protein in Cell Culture
development of brown adipose tissue (4, 5) appears to be reflected in a highly selective synthesis of UCP in cell culture. The complete absence of any effect of catecholamines on the content of two other mitochondrial enzymes, ATPase and cytochrome oxidase (Fig. 4), indicates that the expression of the UCP gene and of nuclear genes for other mitochondrial proteins are separately regulated. To gain more information about the control of the latter genes, cultured brown adipose tissue cells may be of advantage, since the specific content of F,-ATPase in postconfluent cells (Fig. 4) is apparently high with respect to diminished F,-ATPase in brown adipose tissue in uivo (4, 5, 7, 8). The reason for less repressed synthesis of F,-ATPase in culture than in situ is unclear and deserves further studies. The main control of UCP synthesis is expected at the transcriptional level (10). Moreover, a regulatable degradation of mRNA for UCP may be also involved in the control of UCP content (40,41). The above mechanisms, and/or a much shorter half-life of mitochondrial proteins in cultured brown adipose tissue cells than in uiuo (7-12 days; Ref. 42) should explain the fast loss of UCP detected in this report. Interestingly, also in the case of denervation-induced atrophy of brown adipose tissue in cold-acclimated mice a rapid and selective loss of UCP was found (43). However, the observed differences in decrease of several mitochondrial proteins in cell culture will require further studies. The in vitro system of brown adipose tissue cell culture described in this report represents a promising model for further investigation of biogenesis of the unique thermogenic mitochondria. Acknowledgments-We thank B. Cannon, A. Kotyk, for critical reading of the manuscript. Expert technical V. Fialovi and H. NoviEkovi is gratefully acknowledged.
and S. Kuiela assistance of
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Brown Fat Uncoupling
Protein in Cell Culture
r
