- Email: [email protected]
Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption
FEBS Letters 579 (2005) 5680–5684
FEBS 30037
Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption J. Magnus Fredriksson*, Hideki Nikami1, Jan Nedergaard The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden Received 15 August 2005; revised 14 September 2005; accepted 24 September 2005 Available online 4 October 2005 Edited by Laszlo Nagy
Abstract To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression. 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Vascular endothelial growth factor; Norepinephrine; Brown adipose tissue; Hypoxia; Uncoupling protein-1; Thermogenesis
1. Introduction Adipose tissues have a capacity to grow throughout life in response to physiological requirements: white adipose tissue primarily to store excess energy, and brown adipose tissue primarily to utilize energy for heat production [1,2]. Tissue growth requires the formation of new vasculature, i.e., angiogenesis, a process that has attracted much scientific attention over the past decade, e.g., the importance of angiogenesis in tumour growth [3]. Recent reports have demonstrated that also growth of adipose tissue is angiogenesis-dependent [4–6]. For activation of angiogenesis, it is well recognized that low levels of oxygen are an important factor, through stimulatory effects on the expression and secretion of proangiogenic factors, e.g., vascular endothelial growth factor (VEGF) [7], which is known to be induced by hypoxia in adipocytes [8,9]. As a VEGF receptor-2 blocking antibody inhibits angiogenesis
*
Corresponding author. Fax: +46 8 156756. E-mail address: [email protected] (J.M. Fredriksson). 1 Present address: Life Science Research Center, Gifu University, Yanagido, Gifu 501-1194, Japan.
Abbreviations: UCP1, uncoupling endothelial growth factor
protein-1;
VEGF,
vascular
during adipogenesis, VEGF must be involved in regulation of adipose tissue angiogenesis [10]. It is suggested that low oxygen levels that develop in clusters in the adipose tissue trigger angiogenesis through activation of VEGF expression [2,8,9,11]. In the present study, we aimed to investigate the significance of generation of low oxygen levels for physiologically induced VEGF gene expression in brown adipose tissue. During cold exposure of an animal, sympathetic nerves release norepinephrine in brown adipose tissue, which induces thermogenic processes, through activation of the mitochondrial uncoupling protein-1 (UCP1) [1], and cold-induced hyperplasia and hypertrophy [12,13]. This process also involves endothelial proliferation [14] and a marked increase in the capacity for blood perfusion [15,16], i.e., angiogenesis, leading to a tremendously dense vascularization of the tissue [17]. Cold exposure also results in a transient increase in VEGF gene expression in brown adipose tissue, induced via norepinephrine release from sympathetic nerves [18]. In cultured brown adipocytes, both hypoxia-mimicking agents and norepinephrine can stimulate VEGF gene expression [19]. It is not presently known whether the cold-induced VEGF gene expression in brown adipose tissue in situ is caused by norepinephrine acting via adrenergic signalling pathways directly on the VEGF gene or indirectly by generation of hypoxic oxygen levels resulting from the cold-induced thermogenic process. To differentiate between these possibilities, we attempted to create a situation where the tissue was physiologically activated, but where this activation did not result in a markedly increased oxygen consumption. Therefore, we acclimated mice to thermoneutral temperature that leads to a low expression of UCP1 [20] and thus to a low capacity for oxygen consumption in the tissue. However, there is still some UCP1 even in animals acclimated to thermoneutrality (about 1% of total mitochondrial protein) [20], which is apparently sufficient to cause a small but significant cold- and norepinephrine-induced brown adipose tissue thermogenesis [15,16], leading to somewhat hypoxic oxygen levels in the tissue [15]. Therefore, we additionally investigated the level of VEGF gene expression in mice with an ablation of the UCP1 gene. UCP1-ablated mice have no capacity for cold-induced brown adipose tissue thermogenesis (and thus oxygen consumption) [21,22], and brown adipocytes isolated from these mice completely lack the ability to respond to norepinephrine stimulation with an increased oxygen consumption [23]. If cold-induced VEGF gene expression is secondary to hypoxia, no increase in expression would be expected in these mice.
0014-5793/$30.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.09.044
J.M. Fredriksson et al. / FEBS Letters 579 (2005) 5680–5684
We found that even in mice acclimated to thermoneutrality, cold exposure increased VEGF gene expression in brown adipose tissue and, surprisingly, the cold-induced effect was even higher in UCP1-ablated mice. These results indicate that generation of low oxygen levels has no regulatory function in acute cold-induced stimulation of VEGF gene expression.
5681 controls with a paired t-test. The level of statistical significance is indicated with asterisks in the figures as follows: \\\: P < 0.001; \\: P < 0.01; \: P < 0.05; (\): P < 0.1. Statistical differences between genotypes were analysed in the same way and indicated with hatches.
3. Results and discussion 2. Materials and methods 2.1. Animals: acclimation to thermoneutrality and cold exposure Female 7-week old C57Bl/6 mice (B&K UNIVERSAL) and UCP1ablated mice [21] were housed for 7 days in plastic cages at 30 C with a 12-h light–dark cycle and given free access to laboratory chow and water. On the experimental day, animals were placed in single cages at 4 C for 1–4 h, after which the animals were killed and tissues were collected and frozen in liquid nitrogen. The experiment was repeated four times with one animal per time point and genotype. 2.2. Brown adipocyte primary cultures Brown adipocyte precursors were isolated from the interscapular brown adipose tissue from mice of the C57Bl/6 strain (B&K UNIVERSAL) or from UCP1-ablated mice [21] by the procedure described previously [19]. Cells were cultured in 6-well plates in 2 ml of DulbeccoÕs modified EagleÕs medium (Gibco-BRL without L -glutamine type), 4 mM L -glutamine added, supplemented with 10% newborn calf serum (PAN systems GMBH), 4 nM insulin (Actrapid Human, Novo), 10 mM HEPES (Gibco-BRL) and 50 IU penicillin, 50 lg streptomycin (GibcoBRL) and 25 lg sodium ascorbate (Kebo) per ml. The cells were incubated at 37 C in an 8% CO2 atmosphere. The medium was changed every other day until the experimental day (i.e., day 6) when cells were treated or not with norepinephrine (( )-Arterenol bitartrate; Sigma). 2.3. RNA isolation and Northern blotting Total RNA was extracted from tissues and from cultured cells using Ultraspec RNA Isolation System (Biotecx Laboratories Inc., TX, USA). The precipitate was dissolved in diethyl pyrocarbonate-treated water followed by ethanol precipitation. After quantification by UV absorbance at 260 nm, 20 lg RNA from tissues and 10 lg RNA from cultured cells were separated on a 1% agarose/formaldehyde gel and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech) by capillary blotting, crosslinked and hybridized overnight with 32 P-labeled probes (see Section 2.4) as described by Bronnikov et al. [24]. The Hybond-N membrane was then washed three times in 0.1 · SSC, 0.2% SDS at 50 C for 20 min. The membrane was sealed in a plastic envelope and exposed to a PhosphorImager screen for 1 day. The screen was analysed on a Molecular Dynamics PhosphorImager and the overall VEGF mRNA signal was quantified with the ImageQuant program. When the same Hybond-N membrane was analysed for both VEGF and UCP1 mRNA, the previous probe was removed by placing the membrane in 0.2% SDS solution at 100 C and allowing this solution to cool down to room temperature. Equal loading of the gel and the integrity of RNA was routinely verified by observing the ethidium bromide stained gel under UV-light. 2.4. cDNA probes The VEGF cDNA was a gift from Dr. Georg Breier, Max-Planck Institute, Bad Nauheim, Germany. It contained the 580 bp mouse VEGF164 cDNA, which encodes the VEGF164 isoform [25]. This cDNA had been cloned into the EcoRI–BamHI cloning sites of a pBluescript KS vector. The vector was amplified by transformation of a TG-1 Escherichia coli strain and the cDNA was isolated. The UCP1 cDNA was that earlier used [26]. Each cDNA probe was labeled with [32P]dCTP (Amersham Pharmacia Biotech) by Ready To Go DNA Labeling Beads (Amersham Pharmacia Biotech), according to the manufacturerÕs instructions. 2.5. Statistical analysis Values for cold-exposed animals were compared to start values for untreated controls with a paired t-test. For cell culture experiments, values for norepinephrine-treated cells were compared to untreated
In the present study, the aim was to investigate the significance of high oxygen consumption resulting in hypoxic tissue oxygen levels for physiological induction of VEGF gene expression in brown adipose tissue. To reduce oxygen consumption capacity, we used mice acclimated to thermoneutrality which were then acutely cold-exposed. In wild-type mice, cold exposure induced a massive increase in UCP1 gene expression (Fig. 1A, upper panel, wt lanes), previously observed in numerous reports [1], demonstrating that the cold exposure was effective in generating a physiological response. The VEGF mRNA signal in the brown adipose tissue of the mice (Fig. 1A, wt lanes) was very similar to that observed in cultured brown adipocytes from NMRI mice [19] and in other cell types and tissues [18,25]. Several transcripts were detected, which is explainable by alternative splicing of VEGF mRNA [27]. When analysed separately, the transcripts varied in parallel and they were therefore analysed together below. In thermoneutral-acclimated wild-type mice, cold exposure significantly increased VEGF gene expression (Fig. 1A (wt lanes) and B (filled circles)), principally in agreement with the cold-induced VEGF gene expression observed in room temperature-acclimated Wistar rats [18]. Thus, even in thermoneutralacclimated wild-type mice, the cold-induced effect on VEGF gene expression is present, indicating that high rates of oxygen consumption may not be important for cold-induced VEGF gene expression. However, UCP1 is present in warm-acclimated animals [20] (see also the UCP1 mRNA signal for wt 0 h in Fig. 1A), still making the animals able to generate a significant cold-induced thermogenic response in brown adipose tissue [16]. We therefore also utilized mice with the UCP1 gene ablated. UCP1-ablated mice are unable to respond to cold exposure with an increased brown adipose tissue oxygen consumption [21–23]. The UCP1-ablated mice showed signals for mRNA from the disrupted UCP1 gene at sizes different from endogenous UCP1 mRNA. Interestingly, the transcripts from the disrupted UCP1 gene was clearly induced by cold exposure, similarly to the case for the wild-type transcript (Fig. 1A; upper panel, / lanes). Surprisingly, in thermoneutral-acclimated UCP1-ablated mice, cold-induced VEGF gene expression (Fig. 1A, / lanes, and B, open circles) was significantly higher than that seen in thermoneutral-acclimated wild-type mice, for all times of cold exposure, with a very significant 2.4-fold increase already after 1 h. The increase caused by cold exposure was 3-fold higher in UCP1-ablated mice than in wild-type mice. The effects in both wild-type and in UCP1-ablated mice were strongest at 1 h cold exposure, starting from identical basal levels, and induced levels remained elevated over 4 h (Fig. 1B). These results from UCP1-ablated mice clearly demonstrate that low oxygen levels arising from thermogenic processes in brown adipose tissue did not contribute to the cold-induced VEGF gene expression. To investigate whether cold exposure and UCP1 gene ablation induced VEGF gene expression also in other highly
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J.M. Fredriksson et al. / FEBS Letters 579 (2005) 5680–5684
cold-induced VEGF gene expression is specific for brown adipose tissue in mice, in agreement with data from rats [18]. No effect of UCP1 gene ablation was observed on VEGF gene expression in these tissues, demonstrating that UCP1 gene ablation does not lead to a general alteration in inducibility of the VEGF gene in mouse tissues. The higher increase in brown adipose tissue VEGF gene expression in UCP1-ablated mice could be due to an increased responsiveness to norepinephrine in the brown adipocytes of UCP1-ablated mice. To investigate this possibility, we isolated brown adipocyte precursors from wild-type and from UCP1-ablated mice, and used these precursors for cell culture experiments. In cell culture, UCP1-ablated cells grow and differentiate equally well as do wild-type cells, and genes associated with adipocyte differentiation are unaltered by UCP1-ablation (our unpublished observations). After addition of norepinephrine to the cultured wild-type cells, VEGF gene expression levels increased significantly to about 3-fold over control levels, peaking after 1 h and thereafter decreasing to about 50% of maximally induced levels after 4 h (Fig. 3), in agreement with data from NMRI mice brown adipocyte cultures [19]. In brown adipocytes from UCP1-ablated animals, norepinephrine also significantly induced VEGF gene expression (Fig. 3). However, in contrast to the in vivo data from UCP1-ablated mice (Fig. 1B), brown adipocytes from UCP1-ablated mice demonstrated no enhanced responsiveness to norepinephrine stimulation, but demonstrated identical basal and norepinephrine-induced levels compared to wild-type cells (Fig. 3). Thus, these results demonstrate that UCP1 ablation does not alter the norepinephrine responsiveness of VEGF gene expression in brown adipocytes and is not the explanation for the higher induction seen in vivo.
vascularized and sympathetically innervated mouse tissues, we analysed VEGF mRNA levels in heart and kidney from coldexposed wild-type and UCP1-ablated mice. Cold exposure had no acute effect on VEGF gene expression in heart or kidney either in wild-type or in UCP1-ablated mice (Fig. 2). Thus,
UCP1(-/-) heart wt heart UCP1(-/-) kidney wt kidney
160 140 VEGF mRNA levels
Fig. 1. Cold-induced VEGF gene expression in brown adipose tissue. C57Bl/6 wild-type mice and UCP1-ablated mice were first acclimated to thermoneutrality and then cold-exposed at 4 C for the times indicated, after which the animals were killed and interscapular brown adipose tissue was dissected out. Total RNA was isolated and mRNA levels were determined by Northern blot analysis, as described in Section 2. For VEGF mRNA quantification, all visible transcripts were analysed together. (A) Northern blot of RNA from brown adipose tissue from wild-type mice (wt) and from UCP1-ablated mice ( / ) showing signals for UCP1 mRNA (upper panel (wt) and the UCP1 minigene in the UCP1-ablated mice ( / )), VEGF mRNA (middle panel) and ethidium bromide staining of the gel visualized under UV-light (lower panel). (B) VEGF mRNA levels in brown adipose tissue after the indicated times of cold exposure. Filled circles, wild-type mice (wt); open circles, UCP1-ablated mice (UCP1( / )). Values are means ± S.E. from four experiments performed as described in Section 2. The value for UCP1( / ) 1 h was set to 100% in each experimental series. Asterisks indicate the level of statistical difference versus the start value (0 h) and hatches indicate the level of statistical difference between genotypes for each time of cold exposure.
120 100
Heart
80 60 40 Kidney
20 0 0
1 2 3 Time of cold exposure (h)
4
Fig. 2. Cold exposure does not affect VEGF gene expression in heart or kidney. Heart and kidney tissues were dissected out from wild-type and UCP1-ablated mice in two of the experiments used in Fig. 1 and VEGF mRNA levels were determined as in Fig. 1. The value for heart UCP1( / ) 1 h was set to 100% in each experimental series and the values are presented as means ± S.E. Filled symbols, wild-type mice (wt); open symbols, UCP1-ablated mice (UCP1( / )).
J.M. Fredriksson et al. / FEBS Letters 579 (2005) 5680–5684
120
* * *
100 VEGF mRNA levels
UCP1(-/-) wt
* * *
80
40 20 0 1 2 3 Time of NE treatment (h)
Acknowledgements: This investigation was supported by grants from the Swedish Cancer Society and the Swedish Science Research Council. We thank Barbara Cannon for valuable discussions and Georg Breier for providing the VEGF cDNA clone.
References
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Fig. 3. Norepinephrine-induced VEGF gene expression in cultured brown adipocytes. Brown adipocytes were isolated from C57Bl/6 wildtype mice and from UCP1-ablated mice and cultured for six days as described in Section 2. Cells were then treated with 10 lM norepinephrine (NE) for the indicated times, after which total RNA was isolated and VEGF mRNA levels were determined by Northern blot analysis, as described in Section 2. All visible VEGF mRNA transcripts were quantified together. Filled circles, cells from wild-type mice (wt); open circles, cells from UCP1-ablated mice (UCP1( / )). Values are means ± S.E. from three experiments with duplicate or quadruplicate wells for each treatment. The value for UCP1( / ) 1 h was set to 100% in each experimental series. Asterisks indicate the level of statistical difference versus 0 h controls. There were no significant differences between genotypes.
Norepinephrine levels in brown adipose tissue from both wild-type and UCP1-ablated mice were recently analysed in our laboratory. UCP1-ablated mice had 2.5-fold higher norepinephrine levels, a more than 3-fold higher norepinephrine turnover rate and thus an estimated 8-fold higher sympathetic activity in brown adipose tissue compared to wild-type (Golozoubova et al., unpublished results). Apparently, the absence of a thermogenic response from brown adipose tissue results in a lack of inhibitory feedback regulation of sympathetic norepinephrine release, and thus leads to hyperactive sympathetic activity and elevated norepinephrine levels in the tissue. As wild-type animals demonstrated only about a 50% increase in VEGF mRNA levels in response to cold exposure (Fig. 1A and B), norepinephrine levels in the wild-type brown adipose tissue may not be sufficiently elevated to induce a maximal response. Thus, the markedly higher sympathetic activity observed in UCP1-ablated mice is likely the explanation for the 3-fold stronger effect of cold exposure on VEGF gene expression in these animals (Fig. 1A and B). The maximal norepinephrine effect in cell culture was also about a 3-fold increase in VEGF mRNA [19] (Fig. 3). Taken together, these results imply that although VEGF gene expression in brown adipocytes can be increased by hypoxia-mimics [19], it is norepinephrine that is the direct mediator of cold-induced VEGF gene expression in brown adipose tissue via the identified adrenergic signalling transduction pathways [19]. Generation of low oxygen levels in the tissue through thermogenic processes does not contribute to activation of the VEGF gene in this situation.
[1] Cannon, B. and Nedergaard, J. (2004) Brown adipose tissue: function and physiological significance. Physiol. Rev. 84, 277–359. [2] Trayhurn, P. (2005) The biology of obesity. Proc. Nutr. Soc. 64, 31–38. [3] Folkman, J. (2004) Endogenous angiogenesis inhibitors. APMIS 112, 496–507. [4] Rupnick, M.A., Panigrahy, D., Zhang, C., Dallabrida, S.M., Lowell, B.B., Langer, R. and Folkman, J. (2002) Adipose tissue mass can be regulated through the vasculature. Proc. Natl. Acad. Sci. USA 99, 10730–10735. [5] Bra˚kenhielm, E., Cao, R., Gao, B., Angelin, B., Cannon, B., Parini, P. and Cao, Y. (2004) Angiogenesis inhibitor, TNP-470, prevents diet-induced and genetic obesity in mice. Circ. Res. 94, 1579–1588. [6] Hausman, G.J. and Richardson, R.L. (2004) Adipose tissue angiogenesis. J. Anim. Sci. 82, 925–934. [7] Ferrara, N. (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611. [8] Zhang, Q.-X., Magovern, C.J., Mack, C.A., Budenbender, K.T., Ko, W. and Rosengart, T.K. (1997) Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis. J. Surg. Res. 67, 147–154. [9] Lolmede, K., Durand de Saint Front, V., Galitzky, J., Lafontan, M. and Bouloumie, A. (2003) Effects of hypoxia on the expression of proangiogenic factors in differentiated 3T3-F442A adipocytes. Int. J. Obes. 27, 1187–1195. [10] Fukumura, D., Ushiyama, A., Duda, D.G., Xu, L., Tam, J., Krishna, V., Chatterjee, K., Garkavtsev, I. and Jain, R.K. (2003) Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ. Res. 93, 88–97. [11] Trayhurn, P. and Wood, I.S. (2004) Adipokines: inflammation and the pleitropic role of white adipose tissue. Br. J. Nutr. 92, 347–355. [12] Rehnmark, S. and Nedergaard, J. (1989) DNA synthesis in mouse brown adipose tissue is under b-adrenergic control. Exp. Cell Res. 180, 574–579. [13] Nedergaard, J., Herron, D., Jacobsson, A., Rehnmark, S. and Cannon, B. (1995) Norepinephrine as a morphogen? – its unique interaction with brown adipose tissue. Int. J. Dev. Biol. 39, 827– 837. [14] Bukowiecki, L.J., Collet, A.J., Follea, N., Guay, G. and Jahjah, L. (1982) Brown adipose tissue hyperplasia: a fundamental mechanism of adaptation to cold and hyperphagia. Am. J. Physiol. 242, E353–E359. [15] Foster, D.O. and Frydman, M.L. (1978) Nonshivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Can. J. Physiol. Pharmacol. 56, 110–122. [16] Foster, D.O. and Frydman, M.L. (1979) Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats reevaluated from changes in tissue blood flow: the dominant role of brown adipose tissue in the replacement of shivering by nonshivering thermogenesis. Can. J. Physiol. Pharmacol. 57, 257–270. [17] Smith, R.E. and Horwitz, B.A. (1969) Brown fat and thermogenesis. Physiol. Rev. 49, 330–425. [18] Asano, A., Morimatsu, M., Nikami, H., Yoshida, T. and Saito, M. (1997) Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis. Biochem. J. 328, 179–183. [19] Fredriksson, J.M., Lindquist, J.M., Bronnikov, G.E. and Nedergaard, J. (2000) Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a badrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J. Biol. Chem. 275, 13802–13811.
5684 [20] Ashwell, M., Jennings, G., Richard, D., Stirling, D.M. and Trayhurn, P. (1983) Effect of acclimation temperature on the concentration of the mitochondrial ‘‘uncoupling’’ protein measured by radioimmunoassay in mouse brown adipose tissue. FEBS Lett. 161, 108–112. [21] Enerba¨ck, S., Jacobsson, A., Simpson, E.M., Guerra, C., Yamashita, H., Harper, M.-E. and Kozak, L.P. (1997) Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 387, 90–94. [22] Golozoubova, V., Hohtola, E., Matthias, A., Jacobsson, A., Cannon, B. and Nedergaard, J. (2001) Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold. FASEB J. 15, 2048–2050. [23] Matthias, A., Ohlson, K.E.B., Fredriksson, J.M., Jacobsson, A., Nedergaard, J. and Cannon, B. (2000) Thermogenic responses in brown-fat cells are fully UCP1-dependent: UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty-acid induced thermogenesis. J. Biol. Chem. 275, 25073–25081.
J.M. Fredriksson et al. / FEBS Letters 579 (2005) 5680–5684 [24] Bronnikov, G., Bengtsson, T., Kramarova, L., Golozoubova, V., Cannon, B. and Nedergaard, J. (1999) b1 to b3 switch in control of cAMP during brown adipocyte development explains distinct badrenoceptor subtype mediation of proliferation and differentiation. Endocrinology 140, 4185–4197. [25] Breier, G., Albrecht, U., Sterrer, S. and Risau, W. (1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114, 521–532. [26] Rehnmark, S., Ne´chad, M., Herron, D., Cannon, B. and Nedergaard, J. (1990) a- and b-Adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture. J. Biol. Chem. 265, 16464– 16471. [27] Robinson, C.J. and Stringer, S.E. (2001) The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J. Cell Sci. 114, 853–865.
FEBS 30037
Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption J. Magnus Fredriksson*, Hideki Nikami1, Jan Nedergaard The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden Received 15 August 2005; revised 14 September 2005; accepted 24 September 2005 Available online 4 October 2005 Edited by Laszlo Nagy
Abstract To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression. 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Vascular endothelial growth factor; Norepinephrine; Brown adipose tissue; Hypoxia; Uncoupling protein-1; Thermogenesis
1. Introduction Adipose tissues have a capacity to grow throughout life in response to physiological requirements: white adipose tissue primarily to store excess energy, and brown adipose tissue primarily to utilize energy for heat production [1,2]. Tissue growth requires the formation of new vasculature, i.e., angiogenesis, a process that has attracted much scientific attention over the past decade, e.g., the importance of angiogenesis in tumour growth [3]. Recent reports have demonstrated that also growth of adipose tissue is angiogenesis-dependent [4–6]. For activation of angiogenesis, it is well recognized that low levels of oxygen are an important factor, through stimulatory effects on the expression and secretion of proangiogenic factors, e.g., vascular endothelial growth factor (VEGF) [7], which is known to be induced by hypoxia in adipocytes [8,9]. As a VEGF receptor-2 blocking antibody inhibits angiogenesis
*
Corresponding author. Fax: +46 8 156756. E-mail address: [email protected] (J.M. Fredriksson). 1 Present address: Life Science Research Center, Gifu University, Yanagido, Gifu 501-1194, Japan.
Abbreviations: UCP1, uncoupling endothelial growth factor
protein-1;
VEGF,
vascular
during adipogenesis, VEGF must be involved in regulation of adipose tissue angiogenesis [10]. It is suggested that low oxygen levels that develop in clusters in the adipose tissue trigger angiogenesis through activation of VEGF expression [2,8,9,11]. In the present study, we aimed to investigate the significance of generation of low oxygen levels for physiologically induced VEGF gene expression in brown adipose tissue. During cold exposure of an animal, sympathetic nerves release norepinephrine in brown adipose tissue, which induces thermogenic processes, through activation of the mitochondrial uncoupling protein-1 (UCP1) [1], and cold-induced hyperplasia and hypertrophy [12,13]. This process also involves endothelial proliferation [14] and a marked increase in the capacity for blood perfusion [15,16], i.e., angiogenesis, leading to a tremendously dense vascularization of the tissue [17]. Cold exposure also results in a transient increase in VEGF gene expression in brown adipose tissue, induced via norepinephrine release from sympathetic nerves [18]. In cultured brown adipocytes, both hypoxia-mimicking agents and norepinephrine can stimulate VEGF gene expression [19]. It is not presently known whether the cold-induced VEGF gene expression in brown adipose tissue in situ is caused by norepinephrine acting via adrenergic signalling pathways directly on the VEGF gene or indirectly by generation of hypoxic oxygen levels resulting from the cold-induced thermogenic process. To differentiate between these possibilities, we attempted to create a situation where the tissue was physiologically activated, but where this activation did not result in a markedly increased oxygen consumption. Therefore, we acclimated mice to thermoneutral temperature that leads to a low expression of UCP1 [20] and thus to a low capacity for oxygen consumption in the tissue. However, there is still some UCP1 even in animals acclimated to thermoneutrality (about 1% of total mitochondrial protein) [20], which is apparently sufficient to cause a small but significant cold- and norepinephrine-induced brown adipose tissue thermogenesis [15,16], leading to somewhat hypoxic oxygen levels in the tissue [15]. Therefore, we additionally investigated the level of VEGF gene expression in mice with an ablation of the UCP1 gene. UCP1-ablated mice have no capacity for cold-induced brown adipose tissue thermogenesis (and thus oxygen consumption) [21,22], and brown adipocytes isolated from these mice completely lack the ability to respond to norepinephrine stimulation with an increased oxygen consumption [23]. If cold-induced VEGF gene expression is secondary to hypoxia, no increase in expression would be expected in these mice.
0014-5793/$30.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.09.044
J.M. Fredriksson et al. / FEBS Letters 579 (2005) 5680–5684
We found that even in mice acclimated to thermoneutrality, cold exposure increased VEGF gene expression in brown adipose tissue and, surprisingly, the cold-induced effect was even higher in UCP1-ablated mice. These results indicate that generation of low oxygen levels has no regulatory function in acute cold-induced stimulation of VEGF gene expression.
5681 controls with a paired t-test. The level of statistical significance is indicated with asterisks in the figures as follows: \\\: P < 0.001; \\: P < 0.01; \: P < 0.05; (\): P < 0.1. Statistical differences between genotypes were analysed in the same way and indicated with hatches.
3. Results and discussion 2. Materials and methods 2.1. Animals: acclimation to thermoneutrality and cold exposure Female 7-week old C57Bl/6 mice (B&K UNIVERSAL) and UCP1ablated mice [21] were housed for 7 days in plastic cages at 30 C with a 12-h light–dark cycle and given free access to laboratory chow and water. On the experimental day, animals were placed in single cages at 4 C for 1–4 h, after which the animals were killed and tissues were collected and frozen in liquid nitrogen. The experiment was repeated four times with one animal per time point and genotype. 2.2. Brown adipocyte primary cultures Brown adipocyte precursors were isolated from the interscapular brown adipose tissue from mice of the C57Bl/6 strain (B&K UNIVERSAL) or from UCP1-ablated mice [21] by the procedure described previously [19]. Cells were cultured in 6-well plates in 2 ml of DulbeccoÕs modified EagleÕs medium (Gibco-BRL without L -glutamine type), 4 mM L -glutamine added, supplemented with 10% newborn calf serum (PAN systems GMBH), 4 nM insulin (Actrapid Human, Novo), 10 mM HEPES (Gibco-BRL) and 50 IU penicillin, 50 lg streptomycin (GibcoBRL) and 25 lg sodium ascorbate (Kebo) per ml. The cells were incubated at 37 C in an 8% CO2 atmosphere. The medium was changed every other day until the experimental day (i.e., day 6) when cells were treated or not with norepinephrine (( )-Arterenol bitartrate; Sigma). 2.3. RNA isolation and Northern blotting Total RNA was extracted from tissues and from cultured cells using Ultraspec RNA Isolation System (Biotecx Laboratories Inc., TX, USA). The precipitate was dissolved in diethyl pyrocarbonate-treated water followed by ethanol precipitation. After quantification by UV absorbance at 260 nm, 20 lg RNA from tissues and 10 lg RNA from cultured cells were separated on a 1% agarose/formaldehyde gel and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech) by capillary blotting, crosslinked and hybridized overnight with 32 P-labeled probes (see Section 2.4) as described by Bronnikov et al. [24]. The Hybond-N membrane was then washed three times in 0.1 · SSC, 0.2% SDS at 50 C for 20 min. The membrane was sealed in a plastic envelope and exposed to a PhosphorImager screen for 1 day. The screen was analysed on a Molecular Dynamics PhosphorImager and the overall VEGF mRNA signal was quantified with the ImageQuant program. When the same Hybond-N membrane was analysed for both VEGF and UCP1 mRNA, the previous probe was removed by placing the membrane in 0.2% SDS solution at 100 C and allowing this solution to cool down to room temperature. Equal loading of the gel and the integrity of RNA was routinely verified by observing the ethidium bromide stained gel under UV-light. 2.4. cDNA probes The VEGF cDNA was a gift from Dr. Georg Breier, Max-Planck Institute, Bad Nauheim, Germany. It contained the 580 bp mouse VEGF164 cDNA, which encodes the VEGF164 isoform [25]. This cDNA had been cloned into the EcoRI–BamHI cloning sites of a pBluescript KS vector. The vector was amplified by transformation of a TG-1 Escherichia coli strain and the cDNA was isolated. The UCP1 cDNA was that earlier used [26]. Each cDNA probe was labeled with [32P]dCTP (Amersham Pharmacia Biotech) by Ready To Go DNA Labeling Beads (Amersham Pharmacia Biotech), according to the manufacturerÕs instructions. 2.5. Statistical analysis Values for cold-exposed animals were compared to start values for untreated controls with a paired t-test. For cell culture experiments, values for norepinephrine-treated cells were compared to untreated
In the present study, the aim was to investigate the significance of high oxygen consumption resulting in hypoxic tissue oxygen levels for physiological induction of VEGF gene expression in brown adipose tissue. To reduce oxygen consumption capacity, we used mice acclimated to thermoneutrality which were then acutely cold-exposed. In wild-type mice, cold exposure induced a massive increase in UCP1 gene expression (Fig. 1A, upper panel, wt lanes), previously observed in numerous reports [1], demonstrating that the cold exposure was effective in generating a physiological response. The VEGF mRNA signal in the brown adipose tissue of the mice (Fig. 1A, wt lanes) was very similar to that observed in cultured brown adipocytes from NMRI mice [19] and in other cell types and tissues [18,25]. Several transcripts were detected, which is explainable by alternative splicing of VEGF mRNA [27]. When analysed separately, the transcripts varied in parallel and they were therefore analysed together below. In thermoneutral-acclimated wild-type mice, cold exposure significantly increased VEGF gene expression (Fig. 1A (wt lanes) and B (filled circles)), principally in agreement with the cold-induced VEGF gene expression observed in room temperature-acclimated Wistar rats [18]. Thus, even in thermoneutralacclimated wild-type mice, the cold-induced effect on VEGF gene expression is present, indicating that high rates of oxygen consumption may not be important for cold-induced VEGF gene expression. However, UCP1 is present in warm-acclimated animals [20] (see also the UCP1 mRNA signal for wt 0 h in Fig. 1A), still making the animals able to generate a significant cold-induced thermogenic response in brown adipose tissue [16]. We therefore also utilized mice with the UCP1 gene ablated. UCP1-ablated mice are unable to respond to cold exposure with an increased brown adipose tissue oxygen consumption [21–23]. The UCP1-ablated mice showed signals for mRNA from the disrupted UCP1 gene at sizes different from endogenous UCP1 mRNA. Interestingly, the transcripts from the disrupted UCP1 gene was clearly induced by cold exposure, similarly to the case for the wild-type transcript (Fig. 1A; upper panel, / lanes). Surprisingly, in thermoneutral-acclimated UCP1-ablated mice, cold-induced VEGF gene expression (Fig. 1A, / lanes, and B, open circles) was significantly higher than that seen in thermoneutral-acclimated wild-type mice, for all times of cold exposure, with a very significant 2.4-fold increase already after 1 h. The increase caused by cold exposure was 3-fold higher in UCP1-ablated mice than in wild-type mice. The effects in both wild-type and in UCP1-ablated mice were strongest at 1 h cold exposure, starting from identical basal levels, and induced levels remained elevated over 4 h (Fig. 1B). These results from UCP1-ablated mice clearly demonstrate that low oxygen levels arising from thermogenic processes in brown adipose tissue did not contribute to the cold-induced VEGF gene expression. To investigate whether cold exposure and UCP1 gene ablation induced VEGF gene expression also in other highly
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cold-induced VEGF gene expression is specific for brown adipose tissue in mice, in agreement with data from rats [18]. No effect of UCP1 gene ablation was observed on VEGF gene expression in these tissues, demonstrating that UCP1 gene ablation does not lead to a general alteration in inducibility of the VEGF gene in mouse tissues. The higher increase in brown adipose tissue VEGF gene expression in UCP1-ablated mice could be due to an increased responsiveness to norepinephrine in the brown adipocytes of UCP1-ablated mice. To investigate this possibility, we isolated brown adipocyte precursors from wild-type and from UCP1-ablated mice, and used these precursors for cell culture experiments. In cell culture, UCP1-ablated cells grow and differentiate equally well as do wild-type cells, and genes associated with adipocyte differentiation are unaltered by UCP1-ablation (our unpublished observations). After addition of norepinephrine to the cultured wild-type cells, VEGF gene expression levels increased significantly to about 3-fold over control levels, peaking after 1 h and thereafter decreasing to about 50% of maximally induced levels after 4 h (Fig. 3), in agreement with data from NMRI mice brown adipocyte cultures [19]. In brown adipocytes from UCP1-ablated animals, norepinephrine also significantly induced VEGF gene expression (Fig. 3). However, in contrast to the in vivo data from UCP1-ablated mice (Fig. 1B), brown adipocytes from UCP1-ablated mice demonstrated no enhanced responsiveness to norepinephrine stimulation, but demonstrated identical basal and norepinephrine-induced levels compared to wild-type cells (Fig. 3). Thus, these results demonstrate that UCP1 ablation does not alter the norepinephrine responsiveness of VEGF gene expression in brown adipocytes and is not the explanation for the higher induction seen in vivo.
vascularized and sympathetically innervated mouse tissues, we analysed VEGF mRNA levels in heart and kidney from coldexposed wild-type and UCP1-ablated mice. Cold exposure had no acute effect on VEGF gene expression in heart or kidney either in wild-type or in UCP1-ablated mice (Fig. 2). Thus,
UCP1(-/-) heart wt heart UCP1(-/-) kidney wt kidney
160 140 VEGF mRNA levels
Fig. 1. Cold-induced VEGF gene expression in brown adipose tissue. C57Bl/6 wild-type mice and UCP1-ablated mice were first acclimated to thermoneutrality and then cold-exposed at 4 C for the times indicated, after which the animals were killed and interscapular brown adipose tissue was dissected out. Total RNA was isolated and mRNA levels were determined by Northern blot analysis, as described in Section 2. For VEGF mRNA quantification, all visible transcripts were analysed together. (A) Northern blot of RNA from brown adipose tissue from wild-type mice (wt) and from UCP1-ablated mice ( / ) showing signals for UCP1 mRNA (upper panel (wt) and the UCP1 minigene in the UCP1-ablated mice ( / )), VEGF mRNA (middle panel) and ethidium bromide staining of the gel visualized under UV-light (lower panel). (B) VEGF mRNA levels in brown adipose tissue after the indicated times of cold exposure. Filled circles, wild-type mice (wt); open circles, UCP1-ablated mice (UCP1( / )). Values are means ± S.E. from four experiments performed as described in Section 2. The value for UCP1( / ) 1 h was set to 100% in each experimental series. Asterisks indicate the level of statistical difference versus the start value (0 h) and hatches indicate the level of statistical difference between genotypes for each time of cold exposure.
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Fig. 2. Cold exposure does not affect VEGF gene expression in heart or kidney. Heart and kidney tissues were dissected out from wild-type and UCP1-ablated mice in two of the experiments used in Fig. 1 and VEGF mRNA levels were determined as in Fig. 1. The value for heart UCP1( / ) 1 h was set to 100% in each experimental series and the values are presented as means ± S.E. Filled symbols, wild-type mice (wt); open symbols, UCP1-ablated mice (UCP1( / )).
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Acknowledgements: This investigation was supported by grants from the Swedish Cancer Society and the Swedish Science Research Council. We thank Barbara Cannon for valuable discussions and Georg Breier for providing the VEGF cDNA clone.
References
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Fig. 3. Norepinephrine-induced VEGF gene expression in cultured brown adipocytes. Brown adipocytes were isolated from C57Bl/6 wildtype mice and from UCP1-ablated mice and cultured for six days as described in Section 2. Cells were then treated with 10 lM norepinephrine (NE) for the indicated times, after which total RNA was isolated and VEGF mRNA levels were determined by Northern blot analysis, as described in Section 2. All visible VEGF mRNA transcripts were quantified together. Filled circles, cells from wild-type mice (wt); open circles, cells from UCP1-ablated mice (UCP1( / )). Values are means ± S.E. from three experiments with duplicate or quadruplicate wells for each treatment. The value for UCP1( / ) 1 h was set to 100% in each experimental series. Asterisks indicate the level of statistical difference versus 0 h controls. There were no significant differences between genotypes.
Norepinephrine levels in brown adipose tissue from both wild-type and UCP1-ablated mice were recently analysed in our laboratory. UCP1-ablated mice had 2.5-fold higher norepinephrine levels, a more than 3-fold higher norepinephrine turnover rate and thus an estimated 8-fold higher sympathetic activity in brown adipose tissue compared to wild-type (Golozoubova et al., unpublished results). Apparently, the absence of a thermogenic response from brown adipose tissue results in a lack of inhibitory feedback regulation of sympathetic norepinephrine release, and thus leads to hyperactive sympathetic activity and elevated norepinephrine levels in the tissue. As wild-type animals demonstrated only about a 50% increase in VEGF mRNA levels in response to cold exposure (Fig. 1A and B), norepinephrine levels in the wild-type brown adipose tissue may not be sufficiently elevated to induce a maximal response. Thus, the markedly higher sympathetic activity observed in UCP1-ablated mice is likely the explanation for the 3-fold stronger effect of cold exposure on VEGF gene expression in these animals (Fig. 1A and B). The maximal norepinephrine effect in cell culture was also about a 3-fold increase in VEGF mRNA [19] (Fig. 3). Taken together, these results imply that although VEGF gene expression in brown adipocytes can be increased by hypoxia-mimics [19], it is norepinephrine that is the direct mediator of cold-induced VEGF gene expression in brown adipose tissue via the identified adrenergic signalling transduction pathways [19]. Generation of low oxygen levels in the tissue through thermogenic processes does not contribute to activation of the VEGF gene in this situation.
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