- Email: [email protected]
Nerve growth factor, β3-adrenoceptor and uncoupling protein 1 expression in rat brown fat during postnatal development
Neuroscience Letters 246 (1998) 5–8
Nerve growth factor, b3-adrenoceptor and uncoupling protein 1 expression in rat brown fat during postnatal development E. Nisoli*, C. Tonello, M.O. Carruba Centre for Study and Research on Obesity, Department of Pharmacology, Chemotherapy and Medical Toxicology, LITA Vialba, Ospedale L. Sacco, School of Medicine, University of Milan, via G.B. Grassi 74, 20157 Milano, Italy Received 10 November 1997; received in revised form 20 February 1998; accepted 20 February 1998
Abstract An analysis was made of the expression of nerve growth factor (NGF) mRNA and protein in the brown fat of rats at different ages, and the results compared with the expression of b3-adrenoceptor and uncoupling protein 1 (UCP1). NGF, b3-adrenoceptor, and UCP1 messenger RNA and protein levels were measured by means of reverse transcription-polymerase chain reaction (RTPCR) and Western blotting in the brown fat of rats at different ages (from 20-day-old fetuses (E20) to 16-month-old rats). During the perinatal period, NGF production increased and then declined to adult levels (which are comparable with fetal levels) by eight months, and remained stable thereafter. Relatively low levels of NGF were present in the brown fat of aged rats. Taken together, these results suggest that NGF may be responsible for regulating sympathetic innervation during the perinatal and adult periods. 1998 Elsevier Science Ireland Ltd.
Keywords: Brown adipose tissue; Norepinephrine; Uncoupling protein 1; b3-Adrenoceptor; Non-shivering thermogenesis; Development; Trophic factor
Brown fat is an important site of noradrenaline (NE)stimulated energy expenditure in most mammalian hibernators and humans [5]. It produces heat in response to cold exposure (non-shivering thermogenesis) or after eating (diet-induced thermogenesis). Central to this response is the activation by means of b3-adrenoceptor stimulation of uncoupling protein type 1 (UCP1), an inner-membrane mitochondrial protein found only in brown adipocytes, which exports fatty acid anions and thus uncouples mitochondrial respiration from ATP production [6]. Of all mammalian organs, brown fat is unique insofar as it is the most needed and most developed in very young neonates. Although environmental stimuli may recruit the tissue in later life, it is always present when mammalian newborns encounter the thermal conditions of extrauterine life for the first time [9]. The factors regulating brown fat cell differentiation have been extensively investigated using fetal brown adipocytes * Corresponding author. Tel.: +39 2 70146370; fax: +39 2 70146371; e-mail: [email protected]
[7] or adipose precursor cells in primary culture [15]. Accumulating evidence shows that many hormones and growth factors affect the process of adipocyte conversion [18]. Ne´chad et al. [8] demonstrated that brown fat synthesizes a neurotrophic factor, involved in modulating the sympathetic innervation of the brown adipocytes, and we have recently shown that nerve growth factor (NGF) is synthesized in and released from brown fat cells, with its production being inversely dependent on sympathetic activity under both physiological and pathophysiological conditions, and increased in animal genetic models of obesity [10,11]. Since Ne´chad et al. [8] have reported that in vitro NGF activity varied between tissues at varying developmental stages, it was of interest to investigate whether these variations occur in brown fat in vivo. In vivo observations of mouse embryos have shown that NGF synthesis in a developing target field begins at the time of onset and is very active during the phase of innervation, and then decreases in intensity [3]. To extend these observations, we studied the expression of NGF mRNA and protein in the brown fat of
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00220- 1
6
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
rats at different ages, and we compared it with the expression of b3-adrenoceptor and UCP1. The male outbred Sprague–Dawley rats were aged 1, 8 and 16 months (obtained from Charles River, Como, Italy), housed with lights on from 0700 to 1900 h, and had free access to standard laboratory chow and water. The pregnant Sprague–Dawley rats were individually housed and the litters from these rats were weaned 21 days after birth. Brown fat was obtained from 20-day-old fetuses (E20) of pregnant rats killed by decapitation. Total RNA was isolated from one half of the rat interscapular brown fat (the other half was used for protein preparations, see below), using the RNAzol method (TM Cinna Scientific, Friendswood, TX, USA). For PCR analysis, the RNA samples were treated for 1 h at 37°C with 6 U of RNase-free DNaseI per mg of RNA in 100 mM Tris–HCl, pH 7.5 and 50 mM MgCl2, in the presence of 2 U/ml of placenta RNase inhibitor. One microgram of total RNA was reverse transcribed with 200 units of Moloney murine leukemia virus reverse transcriptase. A control sample without reverse transcriptase was used in order to verify that the amplification was not due to the presence of residual genomic DNA. PCR was performed using Taq DNA polymerase (Promega, Milan, Italy) in 25 ml of a standard buffer (10 mM Tris–HCl, pH 9, 50 mM KCl, 2.5 mM MgCl2, 200 mM dNTP and 0.1% Triton X-100) containing 80 ng of starting total RNA from the tissue of rats at different ages and 40 pmol each of sense and antisense NGF, UCP1, or b3-adrenergic receptor specific oligonucleotide primers. These were: 5′-GGCAGTGTCAAGGGAATGCGAAGTT-3′ and 5′-CCAAGGGAGCAGCTTTCTATCCT-GG-3′ for NGF, 5′-GTGAGTTCGACAACTTCCGAAGTG-3′ and 5′-CATGAGGT-CATATGTCACCAGCTC-3′ for UCP1, and 5′TCCTCCGTCTCCTTCTACCTT-3′ and 5′-AGCACGTTGGCCAGAAAGAAG-3′ for b3-adrenergic receptor. The NGF and UCP1 cDNA was amplified using 30 cycles (94°C 30 s, 60°C 30 s, 72°C for 60 s, and 10-min final extension at 72°C); the b3-adrenergic receptor cDNA was amplified using 30 cycles (94°C for 30 s, at 58°C for 30 s, 72°C for 30 s, and 7-min final extension at 72°C), with 4% deionized formamide and 4% glycerol being included in the PCR mixture. The NGF, UCP1 and b3-adrenergic receptor bands were standardized against 18S rRNA, which was amplified (24 cycles) by means of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min with 18S PCR primers Refill (Ambion, TX, USA). The PCR products were identified on 1.5% agarose gel, revealed by a QuickImage-D (Canberra Packard, Milan, Italy) and densitometrically analyzed using Phoretix 1D version 3.0. Adipose tissue samples (100 mg) were cut into small pieces with a razor blade and resuspended in three volumes of lysis buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM phenylmethylsulfonyl fluoride, leupeptin (1 mg/ ml), pepstatin (1 mg/ml), and aprotinin (5 mg/ml). To analyse
the NGF protein levels in brown fat at the different ages, the Western blotting was performed exactly as described in Nisoli et al. [10]. An examination was made of developmental changes in brown fat NGF production. The presence of NGF mRNA and protein was determined in brown fat obtained from rats ranging in age from embryonic day 20 (E20) to 16 months after birth (Fig. 1A,B). Both NGF mRNA and protein were evident at E20 (Fig. 1A,B). Densitometric analysis of NGF mRNA/18S rRNA ratios and NGF protein levels showed that NGF mRNA and protein markedly increased on the first day after birth, and remained high for the first few weeks (Fig. 1C). NGF mRNA levels remained quite stable at 1 month, although the levels of NGF protein tended to decline (Fig. 1C). Subsequently, both NGF mRNA and protein decreased almost to fetal levels by eight months, and remained relatively low in the brown fat of aged rats (16 months; Fig. 1C). These findings strongly confirm and extend the previous data published by Ne´chad et al. [8], who reported that brown fat from newborn rats induces neurite outgrowth. It is interesting to note the discrepancy between the time course of the NGF mRNA and protein levels (see Fig. 1C) which is probably due to different turnover rates. Fig. 2 shows the developmental changes in b3-adrenoceptor and UCP1 mRNAs. Fetal brown fat expressed only small
Fig. 1. PCR and Western blot analysis of RNA and protein samples from brown fat of rats at different ages: E20, embryonic day 20; 1–8, postnatal day 1–8; 1–16 mo, postnatal months 1–16. (A) Representative ethidium bromide-stained agarose gels showing NGF mRNA and 18S rRNA in the brown fat. (B) Representative immunoblot obtained by separating 100 mg of protein on 15% SDS-polyacrylamide gel. (C) Densitometric quantification of NGF mRNA and protein levels. Open and closed bars represent mean ± SE of NGF mRNA/ 18S rRNA ratios and protein abundances, respectively, normalized to arbitrary units by assigning the value of 1 to NGF mRNA/18S rRNA ratio and protein abundance at postnatal day 3. The quantitations were performed in six animals per group of ages.
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
Fig. 2. PCR analysis of mRNA isolated from brown fat of rat at different ages. Representative ethidium bromide-stained agarose gels showing b3-adrenoceptor and UCP1 mRNA.
amounts of b3-adrenoceptor mRNA, but these amounts greatly increased on day 1 of postnatal life and remained high for the first 8 days. The levels only slightly declined over the next 3 weeks, but appeared to be markedly reduced at 8 and 16 months of age. UCP1 mRNA was significantly expressed in fetal brown fat, and this expression dramatically increased during the first four days after birth, reaching a peak on day four. UCP1 mRNA levels had decreased by day 8 and at the end of the first month; after 8 and 16 months of postnatal life, they were comparable with fetal levels. These results seem to confirm those previously reported by others [2,13]. Fig. 3 shows the correlations between developmental changes in NGF, UCP1, and b3-adrenoceptor mRNA levels. In addition, in order to compare these data with a functional one, Fig. 3 also reports the pattern of changes during development of non-shivering thermogenesis, measured in terms of the response of the animal to an injection of NE at thermoneutrality in order to elicit a maximal rise in oxygen consumption, as previously described by Nedergaard et al. [9]. Indeed, it is known that non-shivering thermogenesis in the altricial rat increases during the early postnatal days, reaches high levels between day three and day eight, with a peak response on days 4 to 5 (see [9] for review). Under our experimental conditions, NGF expression seemed to
7
precede the appearance of UCP1, which is an index of sympathetic stimulation and thus an indirect index of sympathetic brown fat innervation. It is interesting to note that UCP1 and b3-adrenoceptors are already present during fetal and early postnatal periods, when sympathetic innervation is still insufficiently developed. Subsequently, there is an increase in functional thermogenic activity, which is accompanied by marked increases in both UCP1 and b3adrenoceptor expression. These results seem to suggest that NGF may be involved in the control of brown fat recruitment during postnatal life. This tissue shows two different kinds of growth: one is the ontogenic growth affecting all body organs that occurs in harmony with body growth in general; but brown fat recruitment is a specific growth phase during which the relative significance of brown fat for the metabolism of the whole animal increases. Studies of adult animals have shown that cold acclimatization leads to an increase in BAT weight [14], and NE has been widely demonstrated to have a stimulatory effect on precursor brown fat cells [1], probably via b-adrenoceptor-activated cyclic AMP. It has thus become general opinion that it is chronic sympathetic stimulation of the tissue that is responsible for BAT recruitment. However, our results show that fetal brown fat (which is well developed even when its sympathetic innervation is scarse) is under a trophic control that is different from the catecholaminergic control typical of postnatal life, even if it is already capable of synthesizing NGF. Various growth factors, such as insulin-like growth factor-1, have already been shown to play a significant role in regulating the orderly progression of organ growth differentiation during fetal development [17]. Given that UCP1 expression (which is central to brown fat heat production in response to cold exposure) is under the control of the NE [1] released from the sympathetic
Fig. 3. Relative quantification of the developmental changes in NGF, UCP1, and b3-adrenoceptor mRNA levels in rat brown fat, and their correlation to non-shivering thermogenesis (NST; hatched line) measured as the response of the animal to an injection of noradrenaline at thermoneutrality [10]. NGF, UCP1 and b3-adrenergic receptor bands were standardized versus 18S rRNA, and PCR products were densitometrically quantified as described in the legend of Fig. 1. The maximal ratio for each mRNA species was put to 1 and the rest was expressed in relation to this. NGF, UCP1, and b3-adrenoceptor mRNA levels are relative, but not absolute, amounts; thus, they can not be compare each other.
8
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
nerve terminals innervating BAT cells, it was of interest to compare its expression with that of NGF at different developmental stages. Our results show that rat brown fat UCP1 mRNA was significantly expressed in the fetus, and its levels were extremely high after 1–4 days of postnatal life, with only a very slight decrease between the 8th day and 1 month. Using an immunohistochemical method and ultrastructural investigation, Giordano et al. [4] have recently shown that, even if noradrenergic innervation is present only in some nerves on the 19th day of intrauterine life, it gradually increases with the development of the organ; however, parenchymal fibres (which directly innervate brown fat cells) can only be discerned 7 days after birth. They found that UCP1 was expressed from the 19th day of intrauterine life onward and, in line with our UCP1 mRNA results, that its greatest expression was on the 4th day of postnatal life [4]. We have previously demonstrated that NE modulates UCP1 expression in brown adipocytes that are mainly differentiated in culture by means of b3-adrenoceptor stimulation [12]. It was therefore also of interest to study developmental changes in brown fat b3-adrenoceptor mRNA levels. Our results show that fetal BAT expresses only a small amount of b3-adrenoceptor mRNA, but that this markedly increases on day 1 of postnatal life, remains high for the first week, and only slightly decreases after 1 month. It is interesting to note that BAT b3-adrenoceptor mRNA expression in adult and aged animals gradually declines, while UCP1 expression is still significantly high (for example, at 16 months). This is in line with the results of studies showing a decrease in sympathetic innervation to the BAT of aged animals [16]. Finally, these results suggest a putative mechanism underlying the well known poor adaptability to cold environments of aged subjects.
[4]
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
We thank Dr. Alessandra Valerio (Brescia, Italy) for helpful advice, and Professor Paolo Mantegazza (Milan, Italy) for encouragement. This work was partly supported by an Educational Grant from Knoll to CSRO and by Italian MURST. [1] Bronnikov, G., Houstek, J. and Nedergaard, J., b-Adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via b1 but not b3 adrenoceptors, J. Biol. Chem., 267 (1992) 2006–2013. [2] Casteilla, L., Champigny, O., Bouillaud, F., Robelin, J. and Ricquier, D., Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth, Biochem. J., 257 (1989) 665– 671. [3] Davies, A.M., Nerve growth factor synthesis and nerve growth
[15]
[16]
[17]
[18]
factor expression in neural development, Int. Rev. Cytol., 128 (1991) 109–138. Giordano, A., Morroni, M., Santone, G., Marchesi, G.F. and Cinti, S., Tyrosine hydroxylase, neuropeptide Y, substance P, calcitonin gene related peptide and vasoactive intestinal peptide in nerves of rat periovarian adipose tissue: an immunohistochemical and ultrastructural investigation, J. Neurocytol., 25 (1996) 125–136. Himms-Hagen, J., Brown adipose tissue thermogenesis, FASEB J., 4 (1990) 2890–2898. Jezek, P., Hanus, J., Semrad, C. and Garlid, K.D., Photoactivated azido fatty acid irreversibly inhibits anion and proton transport through the mitochondrial uncoupling protein, J. Biol. Chem., 271 (1996) 6199–6205. Lorenzo, M., Roncero, C., Fabregat, I. and Benito, M., Hormonal regulation of rat foetal lipogenesis in brown-adipocyte primary culture, Biochem. J., 251 (1988) 617–620. Ne´chad, M., Ruka, E. and Thibault, J., Production of nerve growth factor by brown fat in culture: relation with the in vivo developmental stage of the tissue, Comp. Biochem. Physiol., 2 (1994) 381–388. Nedergaard, J., Connolly, E. and Cannon, B., Brown adipose tissue in the mammalian neonate. In P. Trayhurn and D.G. Nicholls (Eds.), Brown Adipose Tissue, Edward Arnold, London, 1986, pp. 152–213. Nisoli, E., Tonello, C., Benarese, M., Liberini, P. and Carruba, M.O., Expression of nerve growth factor in brown adipose tissue: implications for thermogenesis and obesity, Endocrinology, 137 (1996) 495–503. Nisoli, E., Tonello, C., Briscini, L., Flaim, R. and Carruba, M.O., Leptin and nerve growth factor regulate adipose tissue, Nature Med., 2 (1996) 130. Nisoli, E., Tonello, C., Landi, M. and Carruba, M.O., Functional studies of the first selective b3-adrenergic receptor antagonist SR 59230A in rat brown adipocytes, Mol. Pharmacol., 49 (1996) 7–14. Obregon, M.J., Jacobsson, A., Kirchgessner, T., Schotz, M.C., Cannon, B. and Nedergaard, J., Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase, Biochem. J., 259 (1989) 341–346. Page, E. and Babineau, L.M., The effect of cold environment on the hibernating gland of the rat, Rev. Can. Biol., 9 (1950) 202– 207. Rehnmark, S., Ne`chad, M., Herron, D., Cannon, B. and Nedergaard, J., a- and b-Adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture, J. Biol. Chem., 265 (1990) 16464– 16471. Scarpace, P.J., Mooradian, A.D. and Morley, J.E., Age-associated decrease in beta-adrenergic receptors and adenylate cyclase activity in rat brown adipose tissue, J. Gerontology, 43 (1988) B65–B70. Valverde, A.M., Benito, M. and Lorenzo, M., Proliferation of fetal brown adipocyte primary cultures: relationship with the genetic expression of glucose 6 phosphate deydrogenase, Exp. Cell. Res., 194 (1991) 232–237. Yamashita, H., Sato, Y., Kizaki, T., Oh-ishi, S., Nagasawa, J. and Ohno, H., Basic fibroblast growth factor (bFGF) contributes to the enlargement of brown adipose tissue during cold acclimation, Pflu¨ger. Arch., 428 (1994) 352–356.
Nerve growth factor, b3-adrenoceptor and uncoupling protein 1 expression in rat brown fat during postnatal development E. Nisoli*, C. Tonello, M.O. Carruba Centre for Study and Research on Obesity, Department of Pharmacology, Chemotherapy and Medical Toxicology, LITA Vialba, Ospedale L. Sacco, School of Medicine, University of Milan, via G.B. Grassi 74, 20157 Milano, Italy Received 10 November 1997; received in revised form 20 February 1998; accepted 20 February 1998
Abstract An analysis was made of the expression of nerve growth factor (NGF) mRNA and protein in the brown fat of rats at different ages, and the results compared with the expression of b3-adrenoceptor and uncoupling protein 1 (UCP1). NGF, b3-adrenoceptor, and UCP1 messenger RNA and protein levels were measured by means of reverse transcription-polymerase chain reaction (RTPCR) and Western blotting in the brown fat of rats at different ages (from 20-day-old fetuses (E20) to 16-month-old rats). During the perinatal period, NGF production increased and then declined to adult levels (which are comparable with fetal levels) by eight months, and remained stable thereafter. Relatively low levels of NGF were present in the brown fat of aged rats. Taken together, these results suggest that NGF may be responsible for regulating sympathetic innervation during the perinatal and adult periods. 1998 Elsevier Science Ireland Ltd.
Keywords: Brown adipose tissue; Norepinephrine; Uncoupling protein 1; b3-Adrenoceptor; Non-shivering thermogenesis; Development; Trophic factor
Brown fat is an important site of noradrenaline (NE)stimulated energy expenditure in most mammalian hibernators and humans [5]. It produces heat in response to cold exposure (non-shivering thermogenesis) or after eating (diet-induced thermogenesis). Central to this response is the activation by means of b3-adrenoceptor stimulation of uncoupling protein type 1 (UCP1), an inner-membrane mitochondrial protein found only in brown adipocytes, which exports fatty acid anions and thus uncouples mitochondrial respiration from ATP production [6]. Of all mammalian organs, brown fat is unique insofar as it is the most needed and most developed in very young neonates. Although environmental stimuli may recruit the tissue in later life, it is always present when mammalian newborns encounter the thermal conditions of extrauterine life for the first time [9]. The factors regulating brown fat cell differentiation have been extensively investigated using fetal brown adipocytes * Corresponding author. Tel.: +39 2 70146370; fax: +39 2 70146371; e-mail: [email protected]
[7] or adipose precursor cells in primary culture [15]. Accumulating evidence shows that many hormones and growth factors affect the process of adipocyte conversion [18]. Ne´chad et al. [8] demonstrated that brown fat synthesizes a neurotrophic factor, involved in modulating the sympathetic innervation of the brown adipocytes, and we have recently shown that nerve growth factor (NGF) is synthesized in and released from brown fat cells, with its production being inversely dependent on sympathetic activity under both physiological and pathophysiological conditions, and increased in animal genetic models of obesity [10,11]. Since Ne´chad et al. [8] have reported that in vitro NGF activity varied between tissues at varying developmental stages, it was of interest to investigate whether these variations occur in brown fat in vivo. In vivo observations of mouse embryos have shown that NGF synthesis in a developing target field begins at the time of onset and is very active during the phase of innervation, and then decreases in intensity [3]. To extend these observations, we studied the expression of NGF mRNA and protein in the brown fat of
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00220- 1
6
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
rats at different ages, and we compared it with the expression of b3-adrenoceptor and UCP1. The male outbred Sprague–Dawley rats were aged 1, 8 and 16 months (obtained from Charles River, Como, Italy), housed with lights on from 0700 to 1900 h, and had free access to standard laboratory chow and water. The pregnant Sprague–Dawley rats were individually housed and the litters from these rats were weaned 21 days after birth. Brown fat was obtained from 20-day-old fetuses (E20) of pregnant rats killed by decapitation. Total RNA was isolated from one half of the rat interscapular brown fat (the other half was used for protein preparations, see below), using the RNAzol method (TM Cinna Scientific, Friendswood, TX, USA). For PCR analysis, the RNA samples were treated for 1 h at 37°C with 6 U of RNase-free DNaseI per mg of RNA in 100 mM Tris–HCl, pH 7.5 and 50 mM MgCl2, in the presence of 2 U/ml of placenta RNase inhibitor. One microgram of total RNA was reverse transcribed with 200 units of Moloney murine leukemia virus reverse transcriptase. A control sample without reverse transcriptase was used in order to verify that the amplification was not due to the presence of residual genomic DNA. PCR was performed using Taq DNA polymerase (Promega, Milan, Italy) in 25 ml of a standard buffer (10 mM Tris–HCl, pH 9, 50 mM KCl, 2.5 mM MgCl2, 200 mM dNTP and 0.1% Triton X-100) containing 80 ng of starting total RNA from the tissue of rats at different ages and 40 pmol each of sense and antisense NGF, UCP1, or b3-adrenergic receptor specific oligonucleotide primers. These were: 5′-GGCAGTGTCAAGGGAATGCGAAGTT-3′ and 5′-CCAAGGGAGCAGCTTTCTATCCT-GG-3′ for NGF, 5′-GTGAGTTCGACAACTTCCGAAGTG-3′ and 5′-CATGAGGT-CATATGTCACCAGCTC-3′ for UCP1, and 5′TCCTCCGTCTCCTTCTACCTT-3′ and 5′-AGCACGTTGGCCAGAAAGAAG-3′ for b3-adrenergic receptor. The NGF and UCP1 cDNA was amplified using 30 cycles (94°C 30 s, 60°C 30 s, 72°C for 60 s, and 10-min final extension at 72°C); the b3-adrenergic receptor cDNA was amplified using 30 cycles (94°C for 30 s, at 58°C for 30 s, 72°C for 30 s, and 7-min final extension at 72°C), with 4% deionized formamide and 4% glycerol being included in the PCR mixture. The NGF, UCP1 and b3-adrenergic receptor bands were standardized against 18S rRNA, which was amplified (24 cycles) by means of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min with 18S PCR primers Refill (Ambion, TX, USA). The PCR products were identified on 1.5% agarose gel, revealed by a QuickImage-D (Canberra Packard, Milan, Italy) and densitometrically analyzed using Phoretix 1D version 3.0. Adipose tissue samples (100 mg) were cut into small pieces with a razor blade and resuspended in three volumes of lysis buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM phenylmethylsulfonyl fluoride, leupeptin (1 mg/ ml), pepstatin (1 mg/ml), and aprotinin (5 mg/ml). To analyse
the NGF protein levels in brown fat at the different ages, the Western blotting was performed exactly as described in Nisoli et al. [10]. An examination was made of developmental changes in brown fat NGF production. The presence of NGF mRNA and protein was determined in brown fat obtained from rats ranging in age from embryonic day 20 (E20) to 16 months after birth (Fig. 1A,B). Both NGF mRNA and protein were evident at E20 (Fig. 1A,B). Densitometric analysis of NGF mRNA/18S rRNA ratios and NGF protein levels showed that NGF mRNA and protein markedly increased on the first day after birth, and remained high for the first few weeks (Fig. 1C). NGF mRNA levels remained quite stable at 1 month, although the levels of NGF protein tended to decline (Fig. 1C). Subsequently, both NGF mRNA and protein decreased almost to fetal levels by eight months, and remained relatively low in the brown fat of aged rats (16 months; Fig. 1C). These findings strongly confirm and extend the previous data published by Ne´chad et al. [8], who reported that brown fat from newborn rats induces neurite outgrowth. It is interesting to note the discrepancy between the time course of the NGF mRNA and protein levels (see Fig. 1C) which is probably due to different turnover rates. Fig. 2 shows the developmental changes in b3-adrenoceptor and UCP1 mRNAs. Fetal brown fat expressed only small
Fig. 1. PCR and Western blot analysis of RNA and protein samples from brown fat of rats at different ages: E20, embryonic day 20; 1–8, postnatal day 1–8; 1–16 mo, postnatal months 1–16. (A) Representative ethidium bromide-stained agarose gels showing NGF mRNA and 18S rRNA in the brown fat. (B) Representative immunoblot obtained by separating 100 mg of protein on 15% SDS-polyacrylamide gel. (C) Densitometric quantification of NGF mRNA and protein levels. Open and closed bars represent mean ± SE of NGF mRNA/ 18S rRNA ratios and protein abundances, respectively, normalized to arbitrary units by assigning the value of 1 to NGF mRNA/18S rRNA ratio and protein abundance at postnatal day 3. The quantitations were performed in six animals per group of ages.
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
Fig. 2. PCR analysis of mRNA isolated from brown fat of rat at different ages. Representative ethidium bromide-stained agarose gels showing b3-adrenoceptor and UCP1 mRNA.
amounts of b3-adrenoceptor mRNA, but these amounts greatly increased on day 1 of postnatal life and remained high for the first 8 days. The levels only slightly declined over the next 3 weeks, but appeared to be markedly reduced at 8 and 16 months of age. UCP1 mRNA was significantly expressed in fetal brown fat, and this expression dramatically increased during the first four days after birth, reaching a peak on day four. UCP1 mRNA levels had decreased by day 8 and at the end of the first month; after 8 and 16 months of postnatal life, they were comparable with fetal levels. These results seem to confirm those previously reported by others [2,13]. Fig. 3 shows the correlations between developmental changes in NGF, UCP1, and b3-adrenoceptor mRNA levels. In addition, in order to compare these data with a functional one, Fig. 3 also reports the pattern of changes during development of non-shivering thermogenesis, measured in terms of the response of the animal to an injection of NE at thermoneutrality in order to elicit a maximal rise in oxygen consumption, as previously described by Nedergaard et al. [9]. Indeed, it is known that non-shivering thermogenesis in the altricial rat increases during the early postnatal days, reaches high levels between day three and day eight, with a peak response on days 4 to 5 (see [9] for review). Under our experimental conditions, NGF expression seemed to
7
precede the appearance of UCP1, which is an index of sympathetic stimulation and thus an indirect index of sympathetic brown fat innervation. It is interesting to note that UCP1 and b3-adrenoceptors are already present during fetal and early postnatal periods, when sympathetic innervation is still insufficiently developed. Subsequently, there is an increase in functional thermogenic activity, which is accompanied by marked increases in both UCP1 and b3adrenoceptor expression. These results seem to suggest that NGF may be involved in the control of brown fat recruitment during postnatal life. This tissue shows two different kinds of growth: one is the ontogenic growth affecting all body organs that occurs in harmony with body growth in general; but brown fat recruitment is a specific growth phase during which the relative significance of brown fat for the metabolism of the whole animal increases. Studies of adult animals have shown that cold acclimatization leads to an increase in BAT weight [14], and NE has been widely demonstrated to have a stimulatory effect on precursor brown fat cells [1], probably via b-adrenoceptor-activated cyclic AMP. It has thus become general opinion that it is chronic sympathetic stimulation of the tissue that is responsible for BAT recruitment. However, our results show that fetal brown fat (which is well developed even when its sympathetic innervation is scarse) is under a trophic control that is different from the catecholaminergic control typical of postnatal life, even if it is already capable of synthesizing NGF. Various growth factors, such as insulin-like growth factor-1, have already been shown to play a significant role in regulating the orderly progression of organ growth differentiation during fetal development [17]. Given that UCP1 expression (which is central to brown fat heat production in response to cold exposure) is under the control of the NE [1] released from the sympathetic
Fig. 3. Relative quantification of the developmental changes in NGF, UCP1, and b3-adrenoceptor mRNA levels in rat brown fat, and their correlation to non-shivering thermogenesis (NST; hatched line) measured as the response of the animal to an injection of noradrenaline at thermoneutrality [10]. NGF, UCP1 and b3-adrenergic receptor bands were standardized versus 18S rRNA, and PCR products were densitometrically quantified as described in the legend of Fig. 1. The maximal ratio for each mRNA species was put to 1 and the rest was expressed in relation to this. NGF, UCP1, and b3-adrenoceptor mRNA levels are relative, but not absolute, amounts; thus, they can not be compare each other.
8
E. Nisoli et al. / Neuroscience Letters 246 (1998) 5–8
nerve terminals innervating BAT cells, it was of interest to compare its expression with that of NGF at different developmental stages. Our results show that rat brown fat UCP1 mRNA was significantly expressed in the fetus, and its levels were extremely high after 1–4 days of postnatal life, with only a very slight decrease between the 8th day and 1 month. Using an immunohistochemical method and ultrastructural investigation, Giordano et al. [4] have recently shown that, even if noradrenergic innervation is present only in some nerves on the 19th day of intrauterine life, it gradually increases with the development of the organ; however, parenchymal fibres (which directly innervate brown fat cells) can only be discerned 7 days after birth. They found that UCP1 was expressed from the 19th day of intrauterine life onward and, in line with our UCP1 mRNA results, that its greatest expression was on the 4th day of postnatal life [4]. We have previously demonstrated that NE modulates UCP1 expression in brown adipocytes that are mainly differentiated in culture by means of b3-adrenoceptor stimulation [12]. It was therefore also of interest to study developmental changes in brown fat b3-adrenoceptor mRNA levels. Our results show that fetal BAT expresses only a small amount of b3-adrenoceptor mRNA, but that this markedly increases on day 1 of postnatal life, remains high for the first week, and only slightly decreases after 1 month. It is interesting to note that BAT b3-adrenoceptor mRNA expression in adult and aged animals gradually declines, while UCP1 expression is still significantly high (for example, at 16 months). This is in line with the results of studies showing a decrease in sympathetic innervation to the BAT of aged animals [16]. Finally, these results suggest a putative mechanism underlying the well known poor adaptability to cold environments of aged subjects.
[4]
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
We thank Dr. Alessandra Valerio (Brescia, Italy) for helpful advice, and Professor Paolo Mantegazza (Milan, Italy) for encouragement. This work was partly supported by an Educational Grant from Knoll to CSRO and by Italian MURST. [1] Bronnikov, G., Houstek, J. and Nedergaard, J., b-Adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via b1 but not b3 adrenoceptors, J. Biol. Chem., 267 (1992) 2006–2013. [2] Casteilla, L., Champigny, O., Bouillaud, F., Robelin, J. and Ricquier, D., Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth, Biochem. J., 257 (1989) 665– 671. [3] Davies, A.M., Nerve growth factor synthesis and nerve growth
[15]
[16]
[17]
[18]
factor expression in neural development, Int. Rev. Cytol., 128 (1991) 109–138. Giordano, A., Morroni, M., Santone, G., Marchesi, G.F. and Cinti, S., Tyrosine hydroxylase, neuropeptide Y, substance P, calcitonin gene related peptide and vasoactive intestinal peptide in nerves of rat periovarian adipose tissue: an immunohistochemical and ultrastructural investigation, J. Neurocytol., 25 (1996) 125–136. Himms-Hagen, J., Brown adipose tissue thermogenesis, FASEB J., 4 (1990) 2890–2898. Jezek, P., Hanus, J., Semrad, C. and Garlid, K.D., Photoactivated azido fatty acid irreversibly inhibits anion and proton transport through the mitochondrial uncoupling protein, J. Biol. Chem., 271 (1996) 6199–6205. Lorenzo, M., Roncero, C., Fabregat, I. and Benito, M., Hormonal regulation of rat foetal lipogenesis in brown-adipocyte primary culture, Biochem. J., 251 (1988) 617–620. Ne´chad, M., Ruka, E. and Thibault, J., Production of nerve growth factor by brown fat in culture: relation with the in vivo developmental stage of the tissue, Comp. Biochem. Physiol., 2 (1994) 381–388. Nedergaard, J., Connolly, E. and Cannon, B., Brown adipose tissue in the mammalian neonate. In P. Trayhurn and D.G. Nicholls (Eds.), Brown Adipose Tissue, Edward Arnold, London, 1986, pp. 152–213. Nisoli, E., Tonello, C., Benarese, M., Liberini, P. and Carruba, M.O., Expression of nerve growth factor in brown adipose tissue: implications for thermogenesis and obesity, Endocrinology, 137 (1996) 495–503. Nisoli, E., Tonello, C., Briscini, L., Flaim, R. and Carruba, M.O., Leptin and nerve growth factor regulate adipose tissue, Nature Med., 2 (1996) 130. Nisoli, E., Tonello, C., Landi, M. and Carruba, M.O., Functional studies of the first selective b3-adrenergic receptor antagonist SR 59230A in rat brown adipocytes, Mol. Pharmacol., 49 (1996) 7–14. Obregon, M.J., Jacobsson, A., Kirchgessner, T., Schotz, M.C., Cannon, B. and Nedergaard, J., Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase, Biochem. J., 259 (1989) 341–346. Page, E. and Babineau, L.M., The effect of cold environment on the hibernating gland of the rat, Rev. Can. Biol., 9 (1950) 202– 207. Rehnmark, S., Ne`chad, M., Herron, D., Cannon, B. and Nedergaard, J., a- and b-Adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture, J. Biol. Chem., 265 (1990) 16464– 16471. Scarpace, P.J., Mooradian, A.D. and Morley, J.E., Age-associated decrease in beta-adrenergic receptors and adenylate cyclase activity in rat brown adipose tissue, J. Gerontology, 43 (1988) B65–B70. Valverde, A.M., Benito, M. and Lorenzo, M., Proliferation of fetal brown adipocyte primary cultures: relationship with the genetic expression of glucose 6 phosphate deydrogenase, Exp. Cell. Res., 194 (1991) 232–237. Yamashita, H., Sato, Y., Kizaki, T., Oh-ishi, S., Nagasawa, J. and Ohno, H., Basic fibroblast growth factor (bFGF) contributes to the enlargement of brown adipose tissue during cold acclimation, Pflu¨ger. Arch., 428 (1994) 352–356.