Departament de Biologia Fonamental i Ciències de la Salut, Laboratori de Biologia Molecular, Nutrició i Biotecnologia, Universitat de les Illes Balears, Palma de Mallorca, Spain

Correspondence to: A Palou, Department de Biologia Fonamental i Ciències de la Salut, Laboratori de Biologia Molecular, Nutrició i Biotecnologia, Universitat de les Illes Balears, Ctra Valldemosa km 7.5, Palma de Mallorca 07071, Spain. dbfapoD@ps.uib.es

OBJECTIVE: To assess the effect of chronic treatment with CGP-12177 a ₃-adrenergic receptor (AR) agonist with ₂/₁-AR antagonist action, on the expression of the leptin gene and of genes coding for uncoupling proteins (ucp1, ucp2 and ucp3) in brown and white adipose tissues.

DESIGN: NMRI mice received a daily subcutaneous injection of CGP-12177 at a dose of 0.05, 0.2, 0.5 or 1 mg/kg for 15 days. The specific levels of the mRNAs of interest were analysed in interscapular brown adipose tissue (BAT) and in two white adipose tissue (WAT) depots, inguinal (IWAT) and epididymal (EWAT).

RESULTS: No changes in food intake or body weight were detected at any dose of CGP-12177. In the two WAT depots, the treatment led to enhanced expression of ucp1 and ucp3, but not of ucp2. In BAT, low doses (0.05 and 0.2 mg/kg) led to a decreased expression of the three ucp genes, whereas a slight stimulatory effect on the three ucp genes was elicited with a high dose (1 mg/kg). Treated animals displayed increased expression of leptin in BAT and, to a lesser extent, in IWAT, but not in EWAT.

CONCLUSION: The results reveal that simultaneous stimulation of the expression of certain ucp genes and the leptin gene can be achieved, and suggest that adrenergic regulation of the leptin gene and of genes of the ucp family in adipose tissues is the result of complex interactions between the different -AR pathways.

International Journal of Obesity (2000)24, 423-428

UCP; leptin; CGP-12177; -adrenergic regulation; adipose tissue

Introduction

The brown adipose tissue (BAT) uncoupling protein UCP1 was the first uncoupling protein described and has a key and well documented role in thermogenesis in this tissue.^1,2,3,4,5 Recently, genes encoding two other putative uncoupling proteins, UCP2^6,7 and UCP3,^8,9,10 have been identified that represent new potential molecular targets for the regulation of energy expenditure.

There are interesting differences among the three ucp genes concerning their tissue distribution and physiological regulation. While ucpl is expressed exclusively in BAT of small mammals and the young of larger mammals,⁵ ucp2 and ucp3 are more broadly expressed in tissues, including those of adult humans. In particular, ucp2 is expressed in a variety of tissues, including white adipose tissue (WAT), BAT, skeletal muscle and cells or the immune system,^6,7 ucp3 is expressed at high levels in skeletal muscle of humans and rodents and in BAT of rodents, and to a lesser extent in heart and WAT.^8,9,10 Concerning regulation, ucp1 transcription is induced by cold exposure, -adrenergic stimulation¾mainly through the ₃-adrenergic receptor (AR)¾ and thyroid hormone (T₃)^11,12 (reviewed in Refs ¹³ and ¹⁴). The regulation of ucp2 and ucp3 remains largely unknown. In some aspects, ucp3 regulation resembles, that of ucp1, since ucp3 was reported to be up-regulated by T₃ in muscle and BAT,^10,15 by a ₃-AR agonist in WAT¹⁰ and by long-term cold exposure in BAT.¹⁰ On the other hand, expression of ucp2 appears to be modulated by diet, being increased upon dietary fat consumption, but not by the sympathetic nervous system.⁶

Energy balance relies on the mechanisms that coordinate and co-regulate food intake and the different components of energy expenditure, including thermogenesis mediated by uncoupling proteins. In this coordination leptin, the product of the ob gene, plays a crucial role, being a circulating protein secreted by adipose tissues that functions as a major hormonal regulator of appetite and of total body fat cell mass.¹⁶ Administration of leptin to genetically obese ob/ob mice (which lack functional leptin due to mutation of the ob gene) and to diet-induced obese mice results in decreased food intake and increased energy expenditure.^17,18,19 The effects of leptin are mediated by its interaction with the leptin receptors, which are expressed, among other tissues, in hypothalamic areas involved in the control of body weigh.^19,20

In ob/ob mice, it was shown that leptin increases sympathetic outflow to BAT,²¹ as well as oxygen consumption and ucp1 expression in BAT,²² suggesting that one of the mechanisms by which leptin increases energy expenditure is through activation of BAT thermogenesis. On the other hand, conditions known to activate BAT thermogenesis, such as cold exposure or the administration of certain -AR agonists, were found to decrease ob gene expression both in adipose tissues of intact animals^23,24 and in cultured adipocyte cells.²⁵ From these findings, it was suggested the existence of a retroregulatory loop between the ventromedial hypothalamus and adipose tissues by which ob gene expression is inhibited when the sympathetic nervous system is stimulated, thus contributing to maintaining a constant energy balance.^25,26 It would seem that stimulation of thermogenesis and of leptin expression in a given cell are mutually exclusive phenomena, although some physiological stimuli, such as food intake, stimulate the expression of both genes, ucpl (in BAT) and ob (in WAT).

To further study the regulation of ob gene expression and its relationship with that of genes of the ucp family (ucp1, ucp2 and ucp3), we selected a treatment with CGP-12177, a ₃-AR agonist that we previously showed can induce the ectopic expression of UCP1 protein in non-BAT adipose tissues of mice,²⁷ thus providing a suitable system for the simultaneous analysis of the expression of these genes in both brown and white adipose depots.

Methods

Chemicals

(±)CGP-12177 was obtained from RBI (Natick, MA, USA). The RNA isolation reagent (Tripure), Hybond nylon membranes, and most reagents for Northern blotting (digoxigenin-labelled probes, Dig-Easy Hyb, blocking reagent, anti-digoxigenin antibodies and CDP-Star^TM) were from Boehringer Mannheim (Barcelona, Spain). Other reagents were purchased from Sigma (Madrid, Spain), and routine chemicals used were from Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain).

Animals

Four-week-old male NMRI mice (CRIFFA, Barcelona, Spain) were acclimated to 22°C, with a 12 h light/12 h dark cycle and free access to a standard chow diet (Panlab, Barcelona, Spain). Groups of six animals were subcutaneously injected daily a dose of CGP-12177 (0.05, 0.2, 0.5 or 1 mg/kg) in 100 µl of saline; control animals received saline only. After 15 days of treatment (and 16 h after the last CGP-12 177 injection), the mice were killed with C0₂, followed by cervical dislocation. Interscapular BAT, inguinal (subcutaneous) WAT and epididymal WAT depots were rapidly removed, frozen in liquid nitrogen and stored at -70°C until RNA analysis.

RNA isolation and Northern blotting

Tissue samples were homogenized in Tripure reagent and total RNA extracted following the instructions provided by Boehringer Mannheim. Twenty micrograms of total RNA, denatured with formamide/formaldehyde, were fractionated by agarose gel electrophoresis as described.²⁸ The RNA was then transferred onto a Hybond Nylon membrane in 20´SSC (saline sodium citrate buffer: 1´SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) by capillary blotting for 16 h,²⁸ and fixed with UV light.

The mRNA for leptin was detected by a chemiluminescence-based procedure, utilizing a 33-mer antisense oligonucleotide probe (5'-GGTCTGAGGCA-GGGAGCAGCTCTTGGAGAAGGC-3'),²⁴ which was synthesized commercially (Boehringer Mann-heim), labelled at both ends with a single digoxigenin ligand. Pre-hybridization was at 42°C for 15 min in DIG-Easy Hyb. Hybridization was at 42°C overnight in DIG-Easy Hyb containing the oligonucleotide probe (34 ng/ml). Hybridized membranes were submitted to 2´15 min washes at room temperature with 2´SSC/0.1% SDS (sodium dodecyl sulphate), followed by 2´15 min washes at 48°C with 0.1´SSC/0.l% SDS. After blocking, the membranes were incubated first with an anti-digoxigenin-alkaline phosphatase conjugate and then with the chemiluminescent substrate CDP-star^TM, essentially as in the protocols provided by Boehringer Mannheim. Finally, membranes were exposed to Hyperfilm^TM ECL (Amersham, Buckinghamshire, UK); bands in films were analysed by scanner photodensitometry and quantified using the BioImage program (Millipore, Bedford, MA, USA). Blots were sequentially stripped by exposure to boiling 0.1% SDS and re-probed for UCP1, UCP2 and UCP3 mRNA, using the same chemiluminescence-based procedure and probe concentration as for leptin mRNA. The probes for UCP1, UCP2 and UCP3 mRNA were: 5'-GTTGGTTTTATTCGTGGTCTCCCAGCATAG-3', 5'-GGCAGAGTTCATGTATCTCGTCTTGACCAC-3' and 5'-GACTCCTTCCCTGGCGATGGTTCTGTAGG-3', respectively. Probes for UCPs were designed for 100% homology with the nucleotide sequences of rat and mouse for each UCP cDNA, and without cross reactivity between the different UCPs. Finally, blots were stripped and re-probed for 18 S rRNA, to check the loading and transfer of RNA during blotting. For 18 S rRNA detection, the 31-mer digoxigenin-labelled antisense oligonucleotide 5'-CGCCTGCTGCCTTCCTTGGATGTGGTAGCCG-3' at a concentration of 70 pg/ml was used.²⁴

Statistics

All data are expressed as the means±s.e.m. The statistical significance of differences was assessed by Student's t-test.

Results

Expression of the ucp genes in brown adipose tissue

Expression of the mRNAs for UCP1, UCP2 and UCP3 in BAT showed a similar, biphasic response to chronic treatment with increasing doses of CGP-12177, with a significant decrease at the lower doses used compared to the expression in control animals, and a return to levels which were slightly above control levels at the higher doses used (Figure 1).

Expression of the ucp genes in white adipose tissues

CGP-12177 treatment markedly enhanced UCP1 mRNA expression in the two white adipose tissue depots examined, in a dose-dependent manner, from low (and rather variable) levels of expression in the IWAT of control animals (Figure 2) and undetectable levels in the corresponding EWAT (Figure 3). A comparison of the relative expression of ucpl in the depots of WAT and BAT of control animals is shown in Figure 4. UCP3 mRNA expression in white fat depots was also up-regulated after CGP-12177 treatment, especially in EWAT (Figure 3) and, to a lesser extent, in IWAT (Figure 2). In contrast UCP2 mRNA expression was not up-regulated by CGP-12177 in any of the white fat depots examined and even a decreased expression was found at doses lower than 1 mg/kg, both in IWAT and EWAT (Figure 3).

Leptin gene expression in brown and white adipose tissues

As shown in Figure 4, in our control animals leptin mRNA was highly expressed in EWAT and, to a lesser extent, in IWAT; we also detected leptin mRNA in BAT, although at rather low levels (about 10% the levels in EWAT and 40% the levels in IWAT), in agreement with previous reports.^23,29

The effect of chronic CGP-12177 treatment on leptin mRNA levels was tissue-dependent. No effect was detected at any dose in EWAT (Figure 3), whereas a three-fold increase of leptin mRNA levels was found in BAT at CGP-12177 doses of 0.5 and 1 mg/kg (Figure 1). The IWAT showed an intermediate profile since some stimulation (a maximum of 2-fold at a dose of 0.5 mg/kg) was detected (Figure 2).

Lack of effect of chronic treatment with CGP-12177 on food intake and body weight

Chronic treatment with CGP-12177 did not have any apparent effect on food intake, body weight or the weight of the fat depots analysed, at any dose tested. Failure of chronic CGP-12177 treatment to diminish food intake is in line with previous results showing that the acute food-reducing effect of the agent had worn off 4 h after administration and also indicating refractoriness of the food-reducing effect to show up upon a second injection of CGP-12177.³⁰

Discussion

This work aimed to analyse the effect of CGP-12177 on the expression of the leptin gene and its relationship to the expression of the ucp gene family (ucpl, ucp2 and ucp3) in brown and white adipose tissues of mice.

BAT expresses the three ucp genes and we found a common biphasic response of the three to increasing concentrations of CGP-12177. This biphasic response can be explained considering that CGP-12177 is both a low affinity ₃-AR agonist and a high affinity ₁/₂-AR antagonist, so that the latter effects will predominate at low concentrations of the agent.^31,32 It should be pointed out that the animals were maintained at 22°C¾a sufficiently low temperature to stimulate the BAT¾which could explain the basal expression of ucp1 in BAT. Thus, in the case of ucp1, inhibition by the lower doses is in agreement with the reported partial contribution of the ₁-AR to ucp1 expression,¹² a contribution that could be magnified under submaximal (basal) catecholamine stimulation, while stimulation by the higher doses is in agreement with the main role of the ₃-AR in mediating sympathetic stimulation of BAT thermogenesis and with previous results showing that CGP-12177 can increase UCPl protein levels in BAT²⁷ and stimulate oxygen consumption in whole animals.^31,32

We found changes in the expression of ucp2 and ucp3 in BAT after CGP-12177 treatment that paralleled that of ucp1. This is in apparent contrast to results of Gong et al,¹⁰ who reported a lack of stimulatory effect of CL316243, a potent ₃-AR agonist, on ucp2 and ucp3 expression in BAT of rats under conditions that promoted up-regulation of ucp1 expression in this tissue. It is possible that, in our system, the ₁/₂-antagonist effects of CGP-12177 might be neutralizing basal catecholamine stimulation, and by so doing, allow us to better detect ₃-agonist enhancing effects of the agent (at higher doses) on the expression of the three ucp genes. When considered together, our results and those of Gong et al¹⁰ suggest that catecholamine stimulation is important for basal ucp2 and ucp3 expression in BAT.

Interestingly, we found up-regulation of ucp1 and ucp3, but not ucp2, expression in epididymal and inguinal white fat depots after CGP-12177 treatment. Expression or activity of UCP1 in white fat pads after treatment with ₃-AR agonists has been previously described, although considerable differences were obtained depending on the animals (species and strains) and treatment conditions.^10,27,33,34 Parallel regulation of ucpl and ucp3 is not surprising, since ucp3 has been reported to be highly responsive to regulatory signals of energy balance that also modu-late BAT ucp1; thus, ucp3 expression is up-regulated by T₃ in muscle and BAT,^10,15 by the ₃-AR agonist CL316243 in WAT¹⁰ and by long-term cold exposure in BAT.¹⁵ Lack of stimulatory effect of CGP-12177 on ucp2 expression, on the other hand, is consistent with previous results showing modulation of this gene by diet but not by the sympathetic nervous system,⁶ although inhibition of expression at low CGP-12177 doses (Figure 2 and Figure 3) needs further clarification. Our results indicate that, under particular -adrenergic stimulation conditions, WAT can assume some BAT-like characteristics, increasing the expression of two ucp genes that are normally exclusively (ucp1) or highly expressed (ucp3) in BAT, and may make a greater contribution to thermogenesis.

Leptin mRNA levels were analysed to investigate how ob gene expression is modulated under a condition that can stimulate ucp1 and ucp3 expression. We found leptin gene expression unchanged in EWAT and up-regulated in BAT and, to a lesser extent, in IWAT after CGP-12177 treatment. Up-regulation of leptin expression was an unexpected finding, since it contrasts with the reported inhibition of this parameter under conditions of ₃-adrenergic stimulation, both in vivo^23,24 and in vitro.²⁵ We believe that the up-regulating effect of CGP-12177 on leptin expression is attributable to the ₁- and/or ₂-AR antagonist actions of the agent, and thus it reflects the importance of ₁- and/or ₂-AR in mediating the inhibitory effect of catecholamines on leptin expression. In fact, results in cultured brown adipocytes sustain the involvement of the three -AR subtypes in the latter effect.²⁵ In addition, it has been suggested ²⁶ that repression of leptin expression in BAT by basal levels of circulating catecholamines occurs in vivo mainly through the ₁- and/or ₂-AR, rather than through the ₃-AR, since the former two subtypes display a higher affinity for catecholamines than the latter. However, as the functional affinity of an agent is dependent on mote steps than that of the ligand binding, it is therefore difficult to generalize as to what are relevant affinities under the present circumstances. Also, in this context, it may be noted that the ₁/₂-antagonist effect of CGP-12177 is observed at very low doses compared to those needed to evoke its ₃-agonist effect; thus, even when the agonist effect has ceased, it is likely that the antagonist action would persist for some time.³² Lack of stimulatory effect of CGP-12177 on leptin expression in EWAT, on the other hand, is consistent with results showing that administration of this particular agent prevents the normal rise of leptin mRNA in various visceral white fat depots, including the epididymal depot, upon refeeding of fasted rats.³⁰ In addition to the ₁- ₂-antagonist effects of CGP-12177, the apparent differences between this study and some of those published previously may be in part a reflection of the length of time of treatment with the agent. In fact, Li et al³⁰ reported that, when rats were treated with a single dose of CGP-12177, a significant reduction in leptin mRNA levels in WAT was found 2 h after the treatment. However, when CGP-12177 was administered twice (at 0 and 2 h), the previously observed effects on leptin expression had worn off at 4 h.

Although animals chronically treated with high doses of CGP-12177 overexpressed leptin, ucpl and ucp3 in certain adipose depots, the agent did not have any apparent effect on food intake or body weight. It must be emphasized that a simple relationship between food intake, body weight and the expression of the genes studied cannot be directly established. For example, it is possible that the contribution to total circulating leptin of those tissues in which leptin is up-regulated in response to CGP-12177 (such as BAT and IWAT) is relatively small as compared to the contribution of those tissues where leptin expression remains unaffected (such as EWAT) or may be even down-regulated. In addition, it is conceivable that increased thermogenesis by itself may help to counterbalance the effects of leptin on food intake and energy expenditure. Many other factors can be involved as well.

Our results reveal that adrenergic regulation of leptin and genes of the ucp family in adipose tissues can not be described as a single process of down-regulation (as usually considered for ob) or up-regulation (as usually considered for ucp1), but is the result of more complex interactions between the different -AR pathways, the presence and activity of which is different depending on the specific adipose depot considered.

Acknowledgements

This work was funded by Dirección General de Enseñanza Superior e Investigación Científica, Ministerio de Educación y Ciencia, Spain (grant PM97-0094 to A.P.) and by the European Commission DGXII (contract no. ERBCHRX^ÓCT94O49O and COST 918).

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Figure 1 Northern blotting analysis of mRNAs for UCP1, UCP2, UCP3 and leptin in interscapular brown adipose tissue of control and CGP-12177-treated mice. Animals received a daily subcutaneous injection of CGP-12177 (0.02, 0.05, 0.5 or 1 mg/kg) in 100 µl of saline for 15 days. Total RNA (20 µg/lane) was analysed. Upper panel: representative blots. Bottom panel: means±s.e.m. (n=6) of ratios of specific mRNA levels to I8 S rRNA. The resulting values in vehicle controls were set to 100%, and the other values expressed relative to them. *Statistically significant effects of the CGP treatment vs control (P <0.05, Student's t-test).

Figure 2 Northern blotting analysis of mRNAs for UCPI, UCP2, UCP3 and leptin in inguinal white adipose tissue of control and CGP-12l77-treated mice. Animals received a daily subcutaneous injection of CGP-12177 (0.02, 0.05, 0.5 or 1 mg/kg) in 100 µl of saline for 15 days. Total RNA (20 µg/lane) was analysed. Upper panel: representative blots. Bottom panel: means±s.e.m. (n=6) of ratios of specific mRNA levels to 18 S rRNA. The resulting values in vehicle controls were set to 100%, and the other values expressed relative to them. *Statistically significant effects of the CGP treatment vs control (P <0.05, Student's t-test).

Figure 3 Northern blotting analysis of mRNAs for UCPI, UCP2, UCP3 and leptin in epididymal white adipose tissue of control and CGP-12177-treated mice. Animals received a daily subcutaneous injection of CGP-12177 (0.02, 0.05, 0.5 or 1 mg/kg) in 100 µl of saline for 15 days. Total RNA (20 µg/lane) was analysed, Upper panel: representative blots. Bottom panel: means±s.e.m. (n=6) of ratios of specific mRNA levels to 18 S rRNA. The resulting values in vehicle controls were set to 100%, and the other values expressed relative to them. Curve for UCP1 mRNA has not been included since no ucp1 expression was found in control animals. *Statistically significant effects of the CGP treatment vs control (P <0.05, Student's t-test).

Figure 4 Representative northern blots for UCP1 and leptin mRNAs in epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT) and interscapular brown adipose tissue (BAT) of control mice. Total RNA (20 µg/lane) was analysed. The corresponding blot for the 18 S rRNA is also shown.