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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI3K and mTOR Yanling Wang, Masaaki Sato1, Yuan Guo2, Tore Bengtsson, Jan Nedergaardn Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

article information

abstract

Article Chronology:

The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes

Received 11 April 2014

during the process of tissue recruitment. In a primary culture system, cAMP mediates these

Received in revised form

adrenergic effects. In the present study, we demonstrated that, in contrast to other systems

18 July 2014

where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors,

Accepted 22 July 2014

especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and

Available online 4 August 2014

insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2

Keywords:

family of MAPK, the PI3K system and the mTOR complexes were all activated by cAMP, but these

Brown adipose tissue

activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform

Cell proliferation

may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally

Adrenergic effects

acknowledged cellular mediators for induction of cell proliferation are not involved in this

cAMP

process in the brown preadipocyte system; this conclusion may be of relevance both for

Protein kinase A

examination of mechanisms for induction of brown adipose tissue recruitment but also for

Erk 1/2

understanding the mechanism behind e.g. certain endocrine neoplasias.

PI3K

& 2014 Elsevier Inc. All rights reserved.

mTOR

Introduction Brown adipocytes are unique in that norepinephrine, physiologically released from sympathetic nerves, promotes three levels of cellular activity, normally thought to be controlled by distinct external factors: the acute function (i.e. thermogenesis), differentiation, and cell proliferation [12]. Particularly, cell proliferation in response to sympathetic nervous stimulation constitutes a major part of the brown adipose tissue recruitment that occurs when mammals adapt to chronic cold stress [9,11,30,31,38,60]. n

We, and others, have earlier shown that even in primary brown preadipocytes grown in culture, adrenergic stimulation can promote cell proliferation, via a β1-adrenergic and thus likely a cAMPinduced process [6,28]. However, the signalling mechanism mediating this mitogenic effect still remains enigmatic. cAMP is known to affect cell proliferation in a cell type-dependent way. An inhibitory effect is well established in fibroblasts, smooth muscle cells and a series of transformed cell lines [34,40,56,63]. However, in a variety of other cell types, particularly endocrine cells, activation of a cAMP signalling pathway has a stimulatory effect on

Correspondence to: The Arrhenius Laboratories F3n, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail address: [email protected] (J. Nedergaard). 1 Present address: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, 399 Royal Palace, Parkville VIC 3052, Australia. 2 Present address: Basic Educational Unit, Stockholm University, Stockholm, Sweden. http://dx.doi.org/10.1016/j.yexcr.2014.07.029 0014-4827/& 2014 Elsevier Inc. All rights reserved.

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cell proliferation [21,44,45,46,52,55,61,71]. Moreover, endocrine syndromes featuring neoplasia of endocrine cells correlate with various mutations (e.g. in gsp (the α-subunit of the stimulatory G protein) and in the phosphodiesterases PDE8B and PDE11A) that cause constitutive activation of the cAMP-signalling pathway [33,36,37,43], leading to abnormal increases in cAMP and ensuing abnormal cell proliferation. With the recent realisation of the evidence for the presence of active brown adipose tissue in adult humans [54] and the subsequent confirmation of this in a series of studies [19,62,74,67,73], the issue of how to promote brown adipose tissue hyperplasia as a means to potentially augment its activity (and through this possibly counteract the development of obesity and co-morbidities) has gained even translational interest. Therefore, clarification of the cellular signalling mechanisms leading from adrenergic activation of brown preadipocytes to increased cell proliferation could be an important avenue towards enabling therapeutic promotion of brown adipose tissue recruitment. In the present study, we have investigated to what extent accepted signalling pathways, activated in brown adipocytes by adrenergic stimulation and generally discussed in control of cell proliferation – such as ERK1/2, PI3K/Akt, mTOR and protein kinase C – are involved in mediating cAMP-induced cell proliferation. We conclude that only protein kinase C may be a relevant component in the mediation – a result that may also be of significance for understanding the mediation of cAMP-induced cell proliferation in other cell types, physiological as well as pathological.

Materials and methods

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culture medium as above was added but with only 2% newborn calf serum. If not otherwise indicated, the brown adipocyte cultures were washed on day-3 with a 50:50 mix of DMEM/Ham's F12 FAM nutrient mixture (HyClone, Thermo Scientific), then fresh serumfree medium was added (exceptions are indicated in figure legends), consisting of DMEM/Ham's F12 FAM nutrient mixture (50:50), supplemented with 0.5% (w/v) fatty acid-free bovine serum albumin (fraction V, Roche), 4 nM insulin, 10 mM Hepes, 4 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin and 25 μg/ml sodium ascorbate. Cells were incubated in this serumfree medium for 16–24 h, before stimulation as detailed in the specific experiments. We refer to these cells as serum-deprived day-4 cells. For inguinal adipocyte culture, the same protocol was principally followed.

DNA amount The total amount of DNA per well was measured with the Hoechst binding assay, as described by Downs and Wilfinger [20] with minor modifications. Principally, after treatment, cells were washed twice with ice-cold PBS, whereafter 8 mM NaOH was added to lyse the cells. The cell lysate was collected with a cell lifter (Sigma) and added to 100 ng/ml Hoechst 33,258 dissolved in TEN buffer (10 mM Tris base, 1 mM EDTA, and 200 mM NaCl). Hoechst fluorescence was measured on a fluorescence spectrophotometer (excitation at 365 nm, and emission at 460 nm). A standard curve was made for every measurement, using calf thymus DNA (Sigma-Aldrich).

Brown preadipocyte culture Flow cytometry Male NMRI mice, about 3–4 weeks old (Nova, Germany, or from own breeding), were kept at 22 1C with free access to a chow diet and water for 2–3 days after arrival at the animal facility, and were then sacrificed by CO2 anaesthesia and cervical dislocation, as approved by the Animal Ethics Committee of the Northern Stockholm region. The stromal-vascular fraction of brown adipose tissue was isolated principally as previously described [53,68]. Basically, the interscapular, axillary and cervical depots of brown adipose tissue from 6 mice were pooled and minced into small pieces, which were then incubated with 0.2% collagenase type II (Sigma-Aldrich) in a Hepes-buffered solution (as specified in [53]) for 30 min at 37 1C. The cell suspension was filtered through a 250 μm nylon filter and placed on ice for 20 min, allowing the mature adipocytes to float. After the top layer of floating adipocytes and lipid was removed with a syringe, the infranatant was filtered through a 25 μm nylon filter to further remove remaining mature brown adipocytes. Preadipocytes were pelleted by 10 min centrifugation at 2400 rpm and suspended in Dulbecco's modified Eagle's medium (DMEM, Sigma). The cells were pelleted again and resuspended in DMEM cell culture medium supplemented with 10% newborn calf serum (Gibco), 2.4 nM insulin (Sigma), 10 mM Hepes, 4 mM L-glutamine (Sigma), 50 IU/ml penicillin, 50 μg/ml streptomycin (Sigma) and 25 μg/ml sodium ascorbate (Sigma) and were seeded into 12-well culture plates at a density of 5 or 6.5 wells/mouse. The cells were cultured in 1 ml of the culture medium detailed above and kept in an incubator at 37 1C with 8% CO2 in air. If not otherwise indicated, on day-1, the cells were washed with warm DMEM, and fresh cell

After serum deprivation for 16–26 h, day-4 cells were stimulated as detailed in the specific experiments. Thereafter, the cells were washed with warm (37 1C) phosphate-buffered-saline (PBS) and incubated with trypsin-EDTA (Sigma) for 15 min. Pelleted cells were washed twice with ice-cold PBS and stored in 70% ethanol until the day for flow cytometry analysis. Cells were washed with ice-cold PBS after removal of ethanol, dispersed and incubated with 50 μg/ml propidium iodide dissolved in PBS containing 0.3% Triton X-100 and 0.2 mg/ml RNase A for 30 min at room temperature. A FACS Calibur (Becton Dickinson) with laser excitation at 488 nm and emission above 620 nm (FL3) was used to determine PI fluorescence. A gate was set on the FSC-SSC dot plot and data were collected with CellQuest Pro from 10,000– 12,000 events validated as viable cells with this gate. Flowjo (Tree Star, Ashland, OR) was used for data analysis, and doublets and cell aggregates were gated out based on an FL3 width versus FL3 area dot plot. FL3-Area was used for cell cycle profile analysis. A Watson model was applied to define the Gaussian distribution of the G0/1 and G2/M phase estimation in all experiments, based on the DNA content from individual cells.

Western blot Serum-deprived day-4 cells were treated as indicated in each experiment. The medium was aspirated and 40 μl prewarmed (65 1C) 2
SDS sample buffer, consisting of 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% SDS, 50 mM dithiothreitol and 0.1%

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bromophenol blue, was added to lyse the cells. Samples were sonicated for 7 s and the total protein lysate was separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins of interest were detected with Western blotting detection reagents. The following antibodies were used: Akt, phospho-Akt (Ser 473), phospho-Akt (Thr 308), phosphop70S6K1 (Thr 389), p44/42, and phospho-p44/42; all from Cell Signalling, 1:2000 dilution in 5% bovine serum albumin dissolved in TBST). Akt, phospho-P70S6K1 (Thr 389) and phospho-Akt (Ser473) detection was carried out on the same membranes, with membrane stripping performed in between, using a stripping buffer consisting of 100 mM 2-mercaptoethanol, 50 mM NaHPO4 and 10 mM urea for 30 min at 50 1C, followed by extensive washing in TBST.

Chemicals The following chemicals were used to treat brown preadipocytes: forskolin, PD98059, LY294002, wortmannin, phorbol 12,13-dibutyrate (PDBu), insulin, norepinephrine, isoprenaline and rapamycin from Sigma; 6'Bnz-cAMP, 8p-CPT-20 -O-Me-cAMP from Biolog, Germany; as well as Ku-0063794 (Axon Medchem), C15e (SelleckBio) and 4CM (4-cyano-3-methylisoquinoline) (Calbiochem). Insulin, norepinephrine, isoprenaline, 60 Bnz-cAMP and 8p-CPT-20 O-Me-cAMP were dissolved or diluted in H2O, the rest in DMSO.

Results and discussion Norepinephrine is in itself a competent mitogen for brown preadipocytes We, and others, have earlier shown that adrenergic stimulation can promote cell proliferation in primary brown preadipocytes grown in culture, via a β1-adrenergic process [6,28]. However, such experiments were performed in the presence of serum, and this may imply that norepinephrine may need the presence of classical growth factors to be able to induce cell proliferation. We therefore examined the necessity of serum (growth) factors for the ability of norepinephrine to induce cell proliferation in brown preadipocytes. Under our routine primary culture conditions, freshly isolated brown preadipocytes, in the constant presence of 10% newborn calf serum, pass through an initial proliferation phase and a later differentiation phase before becoming functional brown adipocytes, with thermogenic potential [58]. As shown in Fig. 1A, the cells grew at an exponential rate at the early stage (day 1–4) and decreased thereafter (Fig. 1A black line), probably due to a negative effect of cell confluence. It is under these high-serum conditions that the ability of norepinephrine to induce cell proliferation has earlier been shown [6]. Therefore, to examine the ability of norepinephrine/cAMP to induce cell proliferation in itself, we first examined the effect of lowering the serum concentration to 2% after the first day. As seen in Fig. 1A, this diminished level of serum led to a lower rate of cell proliferation. As shown in Fig. 1B, even in the presence of the reduced serum levels, norepinephrine induced a marked increase in total DNA as compared to vehicle treatment, confirming the mitogenic effect of this physiological sympathetic agent even under reduced serum conditions (principally in agreement with

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data in [29]). The norepinephrine effect was totally mimicked by the addition of the adenylyl cyclase activator forskolin (Fig. 1B). Although this in itself does not prove that the norepinephrine effect is (fully) mediated through cAMP, addition of norepinephrine does lead to a substantial increase in cAMP levels in these cells [7,8]. Therefore, the processes described below, induced by forskolin, must necessarily also be induced after norepinephrine stimulation. To avoid any possible interference from processes induced by adrenergic receptors in addition to the activation of adenylyl cyclase and increase in cytosolic cAMP levels, most further experiments were performed with forskolin as the cell proliferation-inducing agent. Complete removal of serum from day 3 further slowed down the cell growth rate (Fig. 1A). Even under these long-term serumfree conditions, the β-adrenergic agonist isoprenaline, added on day 4, was fully competent to induce cell proliferation, and again forskolin could fully mimic this effect (Fig. 1C). Thus, in brown preadipocytes, an increase in cAMP is a sufficient signal to induce cell proliferation, even in the total absence of serum growth factors. The conditions established here, i.e. day-4 cells deprived of serum, were those chosen for further investigations.

Presence of insulin is not essential for cAMP-induced cell proliferation In a number of cell types, insulin is a crucial co-factor required to observe a mitogenic effect of cAMP [10,16,66,69]. To test the dependence of the cAMP effect on insulin in brown preadipocytes, we removed insulin from the culture medium at the same time as serum was removed. As shown in Fig. 1D, the total DNA increase in response to forskolin stimulation was unchanged whether or not 4 nM insulin was present in the serum-free medium, indicating that in brown pre-adipocytes, in contrast to other cell types, forskolin/cAMP can induce a mitogenic effect that is independent of the presence of insulin. Furthermore, high concentrations of insulin (5 mM), sufficient to mimic the mitogenic effect of IGF-1, had no significant potentiating effect on the forskolin-induced total DNA increase (not shown). Thus, the mitogenic effect of cAMP does not require the presence of serum, serum growth factors in general, or insulin specifically.

cAMP promotes proliferation also in brite/beige preadipocytes Cell proliferation in classical white adipocytes is generally accepted to be inhibited by adrenergic stimulation. Particularly, the elimination of sympathetic stimulation leads to increase in cell proliferation in white adipose tissue depots [5,17,24], and similar conclusions have been reached from in-vitro experiments [39]. However, within classical white adipose tissue depots, certain cells may be induced to express UCP1 – the mitochondrial protein responsible for non-shivering thermogenesis – under β(3)adrenergic [18] or other stimulation [59], although to a rather limited extent. This is especially marked in the inguinal (white) adipose tissue in mice, where a significant proportion of the cells are able to display this “brite” or “beige” phenotype. It was therefore of interest to examine whether also precursor cells isolated from the inguinal adipose tissue depot would be able to respond to increases in cAMP by increasing cell proliferation.

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3 28 (2014) 143 – 155

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Fig. 1 – Mitogenic effect of cAMP in brown preadipocytes. (A) Growth curve of brown adipocytes under different serum conditions. Cells were grown in the following conditions: 10% newborn calf serum conditions from day 0 to 7 (10%/10% / 10% NCS); 10% newborn calf serum (NCS) day 0–1, then 2% NCS day 1–7 (10%/2%/2% NCS); 10% NCS day 0–1, then 2% NCS day 1–3, then day 3–7 in serum-free medium (10%/2%/0% NCS). All three curves show total DNA measurements from one experiment with parallel incubation conditions. Values are means7SEM of triplicate wells. (B) Effect of norepinephrine and forskolin on total DNA in brown preadipocytes. Cells were moved to 2% newborn calf serum from day-1 as in A (10%/2%/2%). On day 3, cells were stimulated with 1 lM norepinephrine (NE) or 1 lM forskolin (FSK) for 32 h and then harvested. DNA change is expressed as percentage of DNA in the vehicle-treated group. Values are means7SEM from 6 experiments with triplicate samples. Student's t-test used, n indicates pr 0.05. (C) Effect of forskolin and isoprenaline on brown preadipocyte proliferation under serum-free conditions as in A (10%/2%/ 0%). On day 4, cells were stimulated with 1 lM forskolin (FSK) or 1 lM isoprenaline (ISO) for 32 h. Data expressed as in B. n¼ 6 for FSK, n ¼4 for ISO. (D) Effect of insulin on forskolin-stimulated cell proliferation under serum-free conditions. From day 3 onwards, cells were moved to either normal serum-free medium (4 nM insulin present in the culture medium) or in insulin-deprived serum-free medium (ins-) for 16 h before stimulation with 1 lM forskolin for 32 h; n¼ 6. nn indicate po0.01. (E) Effect of forskolin stimulation on primary cultured precursor cells isolated from inguinal white adipose tissue. Inguinal white adipocytes in primary culture were treated as in C. After forskolin stimulation for 32 h, total DNA were analysed; n¼ 6. (F) Effect of PKA and Epac stimulation on cell proliferation under serum-free conditions. Day-4 serum-deprived cells were stimulated with 500 lM 60 BnzcAMP (60 Bnz), 200 lM 8CPT-20 -O-Me-cAMP (8p-CPT) or a combination of both (6Bþ8p) for 32 h; n¼ 8 for 6´Bnz-cAMP treatment, n¼4 for 8p-CPT and for 6Bþ8p. nnn indicate po0.001.

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As shown in Fig. 1E, in precursors from inguinal adipose tissue, forskolin stimulation induced an increase in the total DNA, of a similar magnitude as that seen in classical brown preadipocytes. Thus, adrenergic stimulation of these adipose depots may not only promote the expression of UCP1 but also promote the proliferation of the “brite” cells.

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was seen, and cells in the S phase tended to decrease. A small but significant increase was seen in the proportion of cells in the G2/M phase, as shown in Fig. 2B. This indicates an effect of cAMP in promoting cell cycle progression. Therefore, we suggest that cAMP may target S phase cell, to promote cell cycle progression. Thus, what is followed here is cell proliferation rather than an inhibition of apoptosis.

Protein kinase A, rather than Epac, mediates cAMPinduced cell proliferation Protein kinase A and Epac are the two main intracellular effectors of cAMP [4,32]. Epac is expressed in brown adipose tissue [57]. We investigated the involvement in cell proliferation of these two intracellular cAMP effectors by stimulating the cells with either 6-Bn-cAMP or 8p-CPT-cAMP, cAMP analogues selective for protein kinase A and Epac, respectively [15]. As shown in Fig. 1F, 6-Bn-cAMP induced an increase in total DNA to a similar extent as did forskolin (Fig. 1C and D), while 8p-CPT had no effect on these cells. The combined addition of the two analogues did not further increase DNA over that caused by 6-Bn-cAMP lone. Thus protein kinase A, and not Epac, mediates the mitogenic effect of cAMP in brown preadipocytes. The generality of this conclusion is supported by observations of protein kinase A mutants leading to endocrine hyperoplasias [2,23,42].

cAMP stimulates cell cycle progression in brown preadipocytes A fraction of the total cAMP-induced DNA increase could be explained by the anti-apoptotic effect of norepinephrine (forskolin) [49]. It was therefore important to analyse the cell cycle profile with flow cytometry, in order to distinguish a mitogenic effect from an anti-apoptotic effect. Cells were serum-starved for 26 h before stimulation with forskolin. To ensure that the data reflect cAMP stimulation within one cell cycle, we performed forskolin stimulation for only 16 h, i. e. shorter than a cell cycle. Representative graphs of the cell cycle profile after DMSO (as vehicle) and forskolin treatment for 16 h are shown in Fig. 2A. In vehicle-treated samples, most cells stayed in the G0/1 phase (80%), around 15% of the cells were in S phase, and around 10% of the cells in G2 or M phase. With forskolin treatment, an increase in the number of cells in the G2/M phase

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Fig. 2 – cAMP effect on cell cycle progression in brown preadipocytes under serum-deprived conditions. Day-4 serumdeprived cells were stimulated with forskolin (1 μM) or vehicle (DMSO) for 16 h. Cells were stained with propidium iodide. Flow cytometry was used for cell cycle analysis, as described in Materials and Methods. (A) A representative cell cycle profile created with Flowjo layout editor showing the percentage of cells in G0/1, S and G2/M phase. Upper panel: vehicle treatment for 16 h (DMSO, 16 h); lower panel: forskolin treatment for 16 h (FSK, 16 h). (B) Compilation of cell cycle profile of samples from experiments as in A. Percentage of cells in S and G2 phase after 16 h treatment with vehicle or 1 lM forskolin is shown. Values are means7SEM, n¼ 5. Student's t-test, nnn, pr0.001.

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cAMP-stimulated cell proliferation is independent of the PI3K pathway Since the data above demonstrate that Erk1/2 activation does not mediate cAMP-induced cell proliferation, we have examined other possible pathways. In the thyroid system, signalling via PI3K (phosphoinositol 3 kinase) is involved in the transmission of mitogenic signals [16,72]. In brown adipocytes, the PI3K pathway is adrenergically activated, downstream of cAMP and protein

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Erk1/2 activation is generally accepted to be involved in stimulation of mitogenesis (e.g. Meloche et al. (2007) [51]). Also in brown preadipocytes, Erk1/2 activation is induced by the classical growth factors (EGF and PDGF), and the increase in Erk1/2 activation correlates with increased DNA synthesis (3H-thymidine incorporation) [35]. Furthermore, inhibition of Erk1/2 activation with an Erk1/2 inhibitor (PD98059) also inhibits EGF- and PDGFinduced DNA synthesis [35]. Since stimulation of β-adrenergic pathways induces Erk1/2 activation [25,26,48,49,50,65,68], as well as cell proliferation in brown preadipocytes, a possibility would be that also in this case Erk1/2 activation mediated the norepinephrine/cAMP-induced cell growth. This could be thought to occur through transactivation of the EGF receptor, as has been discussed for many G-protein-coupled receptors in other systems (for review see [47]). However, in brown preadipocytes and brown adipocytes, EGF receptor transactivation does not mediate the adrenergically induced Erk1/2 activation [68], and adrenergic stimulation of cell proliferation can therefore not be understood as occurring simply by evoking the EGF receptor signalling pathway. However, this does not exclude that Erk1/2 activation could in itself mediate the cAMP-induced cell proliferation. We therefore examined whether cAMP-induced Erk1/2 activation is part of the process mediating the mitogenic signal onwards from cAMP. The ability of cAMP to induce Erk1/2 activation was confirmed by following Erk1/2 phosphorylation. Cells in serum-free conditions were stimulated with forskolin for 5 min, which led to a marked Erk1/2 activation (Fig. 3, lower panel). Pretreatment for 45 min with either PD98059 or U0126, both reported to be specific inhibitors of ERK1/2 [1,22], fully inhibited Erk1/2 activation (Fig. 3). To examine the significance of this cAMP-induced Erk1/2 activation for cell proliferation, we tested the effect of PD98059 or U0126 on the forskolin-stimulated cell proliferation. Neither PD98059 nor U0126 – that both fully inhibited Erk1/2 activation – had any inhibitory effect on the forskolin-induced DNA increase (Fig. 3). Thus, Erk1/2 activation does not mediate cAMP-induced cell proliferation. Therefore, whereas the coupling between classical growth factor activation, Erk1/2 activation and cell proliferation in brown preadipocytes proceeds as expected, the pathway for cAMPinduced cell proliferation does not utilise the Erk1/2 pathway. Although cAMP induces Erk1/2 activation, this activation is not involved in cell proliferation. This implies, in its turn, that growthfactor-induced cell proliferation in brown preadipocytes requires not only Erk1/2 activation but also a further signal, and this signal is not initiated when the cells are stimulated with norepinephrine/cAMP.

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U0126 Fig. 3 – Effect of ERK1/2 inhibitors on cAMP-stimulated cell proliferation. Upper panel: Day-4 serum-deprived cells were pretreated with 25 lM PD98059 or 10 lM U0126 for 45 min before the addition of 1 lM forskolin; DNA was measured after 32 h. Values are means7SEM from 3 individual experiments with triplicate wells in each. Student's t-test, n #, significant forskolin effect in the absence (n) and presence (#) of inhibitors, pr 0.05. Left panel: DNA change following treatment: for each experiment, the change in total DNA relative to DMSO was expressed as percentage of the mean total DNA in the DMSO-treated sample. Right panel: forskolin effect compensated for the effect of inhibitor as such (i.e. for each inhibitor ((DNA amount after forskolin stimulation in the presence of inhibitor) – (DNA amount in the sole presence of the inhibitor))/(DNA amount in the sole presence of the inhibitor); no statistical effect of inhibitor. Lower panel: Effect of PD98059 and U0126 on ERK1/2 phosphorylation. Cells as above were pretreated with 25 lM PD98059 or 10 lM U0126 or vehicle (DMSO) for 45 min before stimulation with either vehicle or 1 lM forskolin for 5 min; Erk1/2 phosphorylation was followed as described in Materials and Methods.

kinase A [14]. It could therefore be suggested that PI3K could be involved in mediation of the adrenergic, cAMP-induced cell proliferation in the brown preadipocytes studied here. A possible involvement of PI3K in cAMP-stimulated cell proliferation was therefore studied by applying three different PI3K inhibitors (wortmannin, LY294002, C15e) to the brown preadipocyte cultures 45 min before forskolin stimulation. As shown in Fig. 4(A–C), all three inhibitors showed a significant (Po0.06) inhibitory effect on the basal level of total DNA (left panels). In the presence of the inhibitors, forskolin stimulation resulted in DNA levels that did not exceed or barely exceeded those in unstimulated cultures (left panels). While this could be interpreted to indicate that PI3K is involved in the mediation of cAMP-induced cell proliferation, we believe that this conclusion is misleading, due to the effect of inhibitors on the basal level of DNA. We therefore also analysed the cAMP effect versus the relevant basal

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level (black bars, right panels). From this analysis, the conclusion is that there was no inhibitory effect of any of the three PI3K inhibitors on the cAMP-induced cell proliferation and that cAMP-induced cell proliferation is thus not mediated via PI3K activation.

mTOR complexes are activated by cAMP, but are not involved in cAMP-stimulated cell proliferation Various extracellular signals, including growth factors, nutrients and energy status, affect the activity of the mTOR complexes in different cell types [64]. cAMP regulation of this activity has been reported to correlate with cAMP’s cell-type dependent effect on cell proliferation [13], i.e. activation [3,41,55] or inhibition [13,70]. We examined the activation of mTOR complexes by cAMP in the brown preadipocytes by following the phosphorylation of p70S6K1 (Thr 389) and Akt (Ser 473), downstream effectors of mTOR complex I and II, respectively. As insulin is a potent agonist of mTOR complexes [64], it was removed during these experiments in the serum-free conditions. The cells were stimulated with forskolin for 1.5 h. As shown in Fig. 5A, p70S6K1 was markedly phosphorylated after forskolin stimulation; the effect still remained even in the presence of 4 nM insulin in the culture medium (Fig. 5A, lower panel). A similar response was seen for Akt phosphorylation at Ser 473 (Fig. 5B), indicating that both mTOR complex I and mTOR complex II are activated by cAMP. To investigate the involvement of mTOR complex activation in the cAMP-stimulated cell proliferation, we applied two commonly used inhibitors for the mTOR complexes: rapamycin and Ku0063794. Pretreatment with rapamycin or Ku-0063794 fully prevented the cAMP-induced phosphorylation of p70S6K1 (Fig. 4C lower panel). DNA measurements indicated that rapamycin and Ku-0063794 both decreased the basal DNA content by some 20–40%, indicating that mTOR complexes may play a significant role in maintaining basal cell survival under these conditions. However, when analysing for cAMP-induced cell proliferation, we found that rapamycin only partly diminished the stimulatory effect (Fig. 5C), and no inhibition of forskolin-stimulated cell proliferation (Fig. 5D) was detected with Ku-0063794 treatment. Thus, mTOR activation is not necessary for the mediation of cAMP-induced cell proliferation.

Fig. 4 – Effect of PI3K inhibitors on cAMP-stimulated cell proliferation. (A-C) Cells were preincubated with 0.1 lM wortmannin (A) or 10 lM LY294003 (B) or 1 lM C15e (C) for 30 min before forskolin (1 lM) stimulation. Left panels: DNA change following treatment. Change in total DNA relative to DMSO was expressed as percentage of total DNA in DMSOtreated sample. Right panels: comparison of forskolin effect on total DNA in the presence and absence of inhibitor, calculated as in Fig. 3. Values shown are means7SEM. Student's t-test; (n) n, indicates effect of forskolin, pr0.10 and 0.05; (#), ## indicate effects of forskolin in the presence of inhibitors (Po0.10 oro0.01); ✪ indicates significant effect of inhibitors pr0.05. A, n¼2; B, n¼4; C, n¼4.

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We also analysed the cell cycle profile of cultures treated with the mTOR inhibitor Ku-0063794. Cells were pre-treated with Ku-0063794 for 45 min before stimulation with forskolin for 56 h. As shown in Fig. 5EF, Ku-0063794 on its own increased the proportion of cells in the quiescent state (cells in G0/1 phase increased from 8072% to 9072%), while the percentage of cells in both S phase (from 1272% to 571%, P¼ 0.06) and G2 phase (from

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771% to 371%, P ¼ 0.04) were significantly reduced, confirming that active mTOR complexes play crucial roles in maintaining basal brown preadipocyte proliferation. As shown above, forskolin treatment for 56 h increased the proportion of proliferating cells. In Fig. 5F, left graph, the cell number in the (SþG2/M) phases increased from 1872% in vehicle-treated cultures to 2172% in forskolin-treated cultures, confirming a cell cycle promoting effect of cAMP. In cultures

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pretreated with Ku-0063794, the cell number in the (SþG2/M) phases similarly increased from 871% in vehicle-treated to 1172% in forskolin-treated cultures (Fig. 5F, right). Thus, Ku-0063794 did not exert any negative effect on forskolininduced cell cycle progression. Based on these results, we suggest that mTOR complexes are important for brown adipocyte maintenance, rather than being directly involved in cAMP-stimulated cell proliferation.

Akt Ser 473

2

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ns

K FS

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40

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40 20 0

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20

D

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M D

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DMSO FSK

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100 85 70 20

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Protein kinase C may be involved in cAMP-stimulated cell proliferation Although activation of protein kinase C is not generally associated with activation of the β-adrenergic/cAMP pathway, there are indications that a cAMP/protein kinase A pathway also may activate protein kinase C in brown adipocytes [14]. There are also indications in brown preadipocytes that activation of protein kinase C may lead to increased DNA synthesis (3H-thymidine incorporation) [27]. However, we found that under the conditions here used, direct stimulation of protein kinase C with low concentrations of the phorbol ester PDBu (40 nM) for 32 h showed no stimulatory effect on total DNA (Fig. 6A). Long-term treatment with phorbol esters at higher concentrations leads to the degradation of the kinase and therefore inhibition of protein kinase C activity. This has commonly been used for the functional study of protein kinase C involvement. Therefore, we preincubated brown preadipocytes for 24 h with 0.2 μM PDBu (phorbol 12,13-dibutyrate) before stimulation with forskolin. We observed an inhibition of lipid accumulation when protein kinase C was inhibited with PDBu pretreatment (Fig. 6E versus 6C). This may indicate that the cells are kept in a less differentiated (and thus more proliferative) state when protein kinase C is chronically inhibited. In agreement with this, measurement of total DNA showed that treatment of the brown preadipocytes with PDBu in itself led to a marked increase in cell proliferation (Fig. 6B). There was no additional effect of forskolin on cell proliferation in the PDBupretreated cells. There are three possible interpretations of this result, two of which involve protein kinase C in the mediation of cAMP-induced cell proliferation. One interpretation is that the experiment simply shows that when protein kinase C is inhibited, cAMP can no longer induce

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cell proliferation; this means that protein kinase C activation would be part of the signalling process. A second interpretation is that the experiment shows that protein kinase C has an independent inhibitory effect on cell proliferation. When this inhibition is abolished, the brown preadipocytes increase their rate of proliferation. The further addition of forskolin cannot induce a markedly higher proliferation rate, as a cellular ceiling for proliferation rate would already have been reached. In this interpretation, protein kinase C is not involved in mediation of the cAMP-induced cell proliferation. A third interpretation would be that cAMP acts by inhibiting (the relevant) protein kinase C activity. When this has already happened (through the prolonged PDBu treatment), forskolin can no longer affect the rate of cell proliferation, and the experiment would thus show that inhibition of protein kinase C was a mediatory step in cAMP-induced cell proliferation. Thus, whereas we have found that a protein kinase C pathway cannot be ruled out from being involved in the mediation of cAMP-induced cell proliferation, clarification of this issue must await further experimentation.

Conclusions In the present study, we investigated cell signalling pathways mediating cAMP-stimulated cell proliferation in brown preadipocytes. As summarised in Fig. 7. we found that, in contrast to a series of cell types where cAMP also stimulates cell proliferation, brown preadipocytes represent a cell type in which cAMP has mitogenic effects that are independent of the presence of insulin or any serum-derived growth factors (or any other added classical growth factors). Correspondingly, the cell signalling pathways commonly activated by classical growth factors, such as Erk1/2, PI3K or mTOR were not involved in the mediation of

Fig. 5 – Involvement of mTOR complexes in cAMP-stimulated cell proliferation. (A–B) cAMP activation of mTORC1 and mTORC2. On day 3, cells were cultured in serum-free medium for 24 h before treatment in the presence or absence of 4 nM of insulin. Cells were stimulated with 1 lM forskolin or vehicle (DMSO) for 1.5–2 h. Antibodies against phosphorylated p70S6K1 (Thr 389) and Akt (Ser 473) were used for detection of protein phosphorylation. Total Akt was used as loading control. Values are means7SEM from the number of experiments given below, with duplicate wells in each treatment. Student's t-test, n pr 0.05; nn pr0.01. (A) p70S6K1 phosphorylation by cAMP. Upper panel: compilation of p70S6K1 phosphorylation at Thr 389 normalised to Akt under serum-free, insulin-free conditions (n¼ 5); lower panel: a representative immunoblot of p70S6K1 phosphorylation at Thr 389 by forskolin stimulation in the presence or absence of 4 nM of insulin. (B) Akt phosphorylation by cAMP. Upper panel, a compilation of Akt phosphorylation at Ser 473 normalised to Akt under insulin-free conditions (N¼ 5); lower panel, a representative immunoblot of Akt Ser 473 in the presence or absence of 4 nM insulin. (C–D) mTOR inhibitor effect on forskolin-stimulated cell proliferation. Data presented as in Fig. 3. Values shown are means7SEM from 2–6 experiments. Upper panel: effect of inhibitor on total DNA, lower panel: effect of inhibitor on p70S6K1 phosphorylation. n, #, statistical significance against vehicle-treated samples and inhibitortreated samples respectively,✪, inhibitor effect on FSK-induced DNA increase. (C), effect of 100 nM rapamycin, n¼ 6. (D), effect of 1 lM Ku-0073794, n¼ 6. (E-F), Effect of Ku-0063794 on cell cycle progression. After serum starvation for 24 h, cells were pretreated with 1 lM Ku-0063794 for 45 min before 1 lM forskolin stimulation for 56 h. Cell cycle stages were analysed with Flowjo cell cycle analysis software. (E) Ku-0063794 effect on cell cycle progression. Cells were treated with Ku or vehicle (DMSO) for 56 h. Graph was created with FlowJo cell cycle analysis layout editor. (F) Compilation of the effect of mTOR inhibition with Ku-0063794 on forskolin-promoted cell cycle progression. Cells were pretreated with 1 lM Ku-0063794 for 45 min before forskolin stimulation for 56 h. Flow cytometry was used for cell cycle analysis. Left graph, no KU pretreatment, n¼ 3; right graph, 1 lM Ku-0063794 pretreated, n¼ 2. n, pr0.05, significant effect of forskolin in the corresponding cell phase, nn, pr0.01.

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60

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Fig. 6 – Involvement of protein kinase C in cAMP-stimulated cell proliferation. (A) Activation of protein kinase C on cell proliferation. Day-4 serum-deprived cells were stimulated with vehicle (DMSO) or 40 nM PDBu (PDBu 40 nM) for 32 h, Student´s ttest, n¼4, ns¼ not significant. (B) Effect of inhibition of protein kinase C on cAMP-stimulated cell proliferation. Day-3 cells where serum-deprived and pretreated or not with 0.2 lM PDBu for 24 h before stimulation with either vehicle or 1 lM forskolin for 32 h. Total DNA was measured and analysed as in Fig. 3. Student's t-test, n¼5. nn, #, significant effect of forskolin and inhibitor respectively, compared to vehicle treatment. ✪✪, inhibitor effect on FSK-induced DNA change, po0.01. (C–F) Effect of PDBu pretreatment on cell morphology. Cells were treated the same way as in Fig. 6B. Representative pictures of day-5 cells are shown: C, no PDBu pretreatment, vehicle stimulation; D, no PDBu pretreatment, forskolin stimulation; E, with PDBu pretreatment, vehicle stimulation; F, with PDBu pretreatment, forskolin stimulation.

cAMP-induced cell proliferation; however, a phorbol-ester sensitive protein kinase C isoform may be involved in the process. These results point to the necessity to identify novel pathways for

the mediation of cAMP-induced cell proliferation, in brown preadipocytes and probably also in other cell types where cAMP promotes cell proliferation and thus tissue hyperplasia.

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[7]

[8]

[9]

Fig. 7 – Tentative model of β1-adrenoceptor induced cell proliferation in brown preadipocytes. cAMP elevated in response to norepinephrine (NE) released from the sympathetic nervous system (SNS) activates different pathways that couple or do not couple to cell proliferation: Epac, protein kinase A (PKA), ERK1/2 family of MAPK (earlier demonstrated to inhibit apoptosis) and mTOR complexes, none of which are directly involved in cAMP-stimulated cell proliferation. There is a possible involvement of protein kinase C in the cAMP signalling pathway, regulating cell proliferation. Normal arrow: direct stimulation of downstream effector; dashed line: with unknown intermediate(s). β1-AR: β1-isoform of adrenergic receptor; AC, adenylyl cyclase.

Acknowledgements We thank Changrong Ge and Irina Shabalina for technical assistance in the FACS experiments, and Barbara Cannon for valuable discussion. This work was supported by research grants from the Swedish Cancer Society and the Swedish Research Council and the Wallenberg Foundation, as well as by a Chinese Scholarship Council grant to YW. We are members of the DIABAT program within the European Union's Seventh Program.

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