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Endocrinology Vol. 144, No. 11 4831-4840
Copyright © 2003 by The Endocrine Society

Hypothalamic Melanin-Concentrating Hormone Is Induced by Cold Exposure and Participates in the Control of Energy Expenditure in Rats

Márcio Pereira-da-Silva, Márcio A. Torsoni, Hugo V. Nourani, Viviane D. Augusto, Cláudio T. Souza, Alessandra L. Gasparetti, José B. Carvalheira, Gislaine Ventrucci, Maria Cristina C. G. Marcondes, Ariovaldo P. Cruz-Neto, Mário J. A. Saad, Antonio C. Boschero, Everardo M. Carneiro and Lício A. Velloso

Departments of Internal Medicine (M.P., M.A.T., H.V.N., C.T.S., A.L.G., J.B.C., M.J.A.S., L.A.V.) and Physiology and Biophysics (V.A., G.V., M.C.C.G.M., A.C.B., E.M.C.), State University of Campinas; and Department of Zoology (A.P.C.-N.), Paulista State University, Campus Rio Claro–State University of Sao Paulo, 13083-970 Campinas SP, Brazil

Address all correspondence and requests for reprints to: Lício A. Velloso, Departamento de Clínica Médica, FCM-UNICAMP, 13083-970 Campinas SP, Brazil. E-mail: lavelloso{at}fcm.unicamp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC EXPOSURE OF homeothermic animals to cold environment leads to several physiological adaptations that are required to maintain a stable body temperature (1, 2, 3). Among these adaptations, the increase of food consumption associated with an initial fall of body weight reflects the deviation of energy flow toward heat production, through a process of nonshivering thermogenesis (4, 5). Most of the adaptation control is coordinated by integrated responses of neurons lying in different hypothalamic nuclei (6, 7, 8).

Loss of coordinated control of food ingestion and energy expenditure is thought to be a central issue in the development of obesity, one of the most prevalent and life-threatening diseases of the modern world (9). A recent study has shown that defects in signaling of several hypothalamic neurotransmitters, such as neuropeptide Y, cocaine- and amphetamine-regulated transcript, agouti-related peptide, {alpha}-MSH, pro-opiomelanocortin, melanin-concentrating hormone (MCH), and orexin (10), as well as defects of leptin and insulin signaling in hypothalamus, may lead to disturbances of feeding and weight control (11, 12).

Because, under cold exposure, high food consumption is associated with weight loss and increased thermogenesis, we decided to use cDNA-macroarray analyses to identify candidate hypothalamic proteins that are modulated in response to cold and, therefore, evaluate their participation in the processes that integrate energy expenditure and food consumption.

Among 1176 different mRNA specificities analyzed, cold exposure induced the modulation of expression of 56 different mRNA specificities in rat hypothalamus. Initial interest was drawn over the neurotransmitter MCH, a 19-amino-acid peptide, expressed mostly in neurons of the lateral hypothalamus (LH) and known to induce hyperphagia and weight gain, if intracerebroventricularly (ICV)-injected, in experimental animals (13, 14). Moreover, the lack of MCH signaling, by knocking out the expression of its receptor in the central nervous system, leads to moderate hypophagia and leanness (15, 16). Based on these facts, we decided to evaluate the role of MCH hyperexpression in the hypothalamus of cold-exposed rats by blocking its protein synthesis using a phosphorthioate-modified MCH-specific antisense oligonucleotide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals, antibodies, and oligodeoxynucleotides
Reagents for SDS/PAGE and immunoblotting were obtained from Bio-Rad (Richmond, CA). HEPES, phenylmethylsulfonylfluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Protein A-Sepharose 6MB was from Pharmacia (Uppsala, Sweden); 125I-Protein A and nitrocellulose membranes were from Amersham Corp. (Aylesbury, UK). Microcon centrifugal filter devices, YN-3, were purchased from Millipore Corp. (Bedford, MA). Antibody against MCH (chicken anti-MCH, T-1506) was from BMA Biomedicals AG (Augst, Switzerland), and antibody against uncoupling protein (UCP)1 (goat anti-UCP1, sc-6529) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies, used as bridges, antigoat (G-4018), and anti-chicken (F-8888), as well as rat MCH (M-4542), were from Sigma Chemical Co. MCH RIA kit (RK 070-47) was from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Chemicals employed in RT-PCR, primers for MCH (sense 5'-TAC GGA GCA GCA AAC A-3' and antisense 5'-ACA GCC AGA CTG AGG G-3') and ß-actin (sense 5'-ACC ACA GCT GAG AGG GAA ATC G-3' and antisense 5'-CAG TCC GCC TAG AAG CAT TTG C-3'), phosphorthioate-modified oligonucleotides for MCH (sense 5'-CCC TCA GTC TGG CTG-3' and antisense 5'-ACA GCC AGA CTG AGG-3'), and Trizol reagent were obtained from Life Technologies (GIBCO BRL, Gaithersburg, MD). Sodium amobarbital was from Eli Lilly (Indianapolis, IN). Atlas Rat 1.2 Array Kit was from Clontech Laboratories, Inc. (East Meadow Circle, Palo Alto, CA). Salmon sperm DNA was from Sigma Chemical Co., and [{alpha}-33P]dATP was from Amersham Corp.

Experimental animals and cold-exposure protocols
Male Wistar rats (56–70 d old/200–260 g) from the University of Campinas Central Animal Breeding Center were used in the experiments. The University’s Ethical Committee approved the protocols. The animals were maintained on a 12-h artificial light, 12-h dark cycle and kept in individual cages supplied with standard rodent chow and water ad libitum. After an acclimatizing period (3 d), rats were transferred to metabolic cages and randomly assigned to two treatment conditions: control and cold-exposed [temperature (T) +4 C]. Rats of the control group were continuously maintained at 23 ± 1 C, whereas rats of the T +4 C group were housed at 4 ± 1 C. Protocols lasting 2 h and 2, 4, 8, and 16 d were employed. For hormone and biochemical determinations, samples were collected from eight rats of each group, and analyses were run in duplicates. For cDNA preparation for macroarray analysis, mRNA extracted from the hypothalami of three rats of each group was pooled, reverse transcribed, and used in the hybridization protocol; three pools from each condition were evaluated. For the remaining experiments, four rats of each group were analyzed, and the experiments were run in duplicates or triplicates.

ICV cannulation and ICV oligonucleotide injection
Adult male Wistar rats were stereotaxically instrumented under sodium amobarbital (15 mg/kg body weight) anesthesia with chronic unilateral 26-gauge stainless steel indwelling guide cannulas, aseptically placed into the lateral ventricle (0.2 mm posterior, 1.5 mm lateral, and 4.2 mm ventral to bregma) as previously described (13). After a 1-wk recovery period, all rats were kept in individual cages and exposed either to 23 ± 1 C (control) or to 4 ± 1 C (T +4 C) during 4 d. Sense and antisense MCH oligonucleotides were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA) and injected once a day at 1000 h, with a total vol of 2.0 µl per dose (2.0 nmol/µl), 24, 48, and 72 h after the onset of the experimental period. Rats were randomly assigned to six different treatment conditions: control without oligonucleotide treatment (control WO); cold exposed without oligonucleotide treatment (T +4 C WO); control with sense oligonucleotide treatment (control S); cold exposed with sense oligonucleotide treatment (T +4 C S); control with antisense oligonucleotide treatment (control AS); cold exposed with antisense oligonucleotide treatment (T +4 C AS).

Biochemical, hormonal, and metabolic characterization of experimental animals
During the animal model characterization, the measurements of food intake, rectal temperature, and body weight were obtained at 1400 h daily from experimental d 1 to experimental d 8, and on experimental d 16. During ICV injection protocol, the measurements were performed daily during the whole experimental period. Rectal temperature was measured with a Thermistor digital (HI-8753) high-precision thermometer (Hanna Instruments, Inc., Woonsocket, RI) inserted 1.5 cm from the anus. Plasma glucose was determined by the glucose oxidase method (17), RIA was employed to measure serum insulin according to a previous description (18), leptin concentrations were determined using a commercially available ELISA kit (Crystal Chem. Inc., Chicago, IL), and corticosterone and TSH were detected by commercially available RIA kits (Amersham Biosciences, Biotrak, Aylesbury, UK). Glycogen was measured, according to a previously described method (19), in liver and gastrocnemius muscle fragments, at experimental d 8 (during the animal model characterization) and at experimental d 4 (during ICV oligonucleotide treatment). Body carcass composition was determined as previously described (20).

Metabolic measurements
Rates of oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured by open-flow respirometry, with a set-up modified from a previously published method (21). After 14 h fasting, rats were placed individually into polyvinyl chloride respirometric chambers, which were then installed into a temperature-controlled cabinet (±0.1 C). Air was drawn over ascarite and drierite scrubbers to remove CO2 and H2O to a manifold, which divided the flow into four airstreams. Each stream was regulated at 1500–2500 ml/min, depending on the experimental temperature. The constancy of the flow was periodically checked with a Cole-Palmer (Vernon Hills, IL) flow meter, and less then 1% drift during the experiments was detected. Each stream fed the inlet port of each of the four respirometric chambers, one of which contained no experimental animal and was used as a baseline reference. Air leaving each respirometric chamber was directed into a computer-controlled multiplexer, where four solenoid vales selected which flow would be directed to a computer-controlled mass-flow meter (model 840; Sierra Instruments, Inc., Monterey, CA). The mass-flow meter was adjusted to deliver a constant flow of 300 ml/min of air, sequentially through water scrubbers, to the measurement cell of a CA-2A CO2 analyzer (Sable Systems International, Las Vegas, NV), again through water and gas scrubbers, and finally to the measurement cell of a FC-1B O2 analyzer (Sable). Each chamber with a rat was sampled for 20 min. Thus, a complete set of recording lasted for 80 min, and this sequence was repeated six times during the evaluation period. Experiments started at 1000 h and finished at 1800 h. VO2 and VCO2 were calculated by the Datacan program, based in the equations of Withers (22) and were expressed as milliliters per hour per gram. Animals were weighed immediately before and after the metabolic measurements, and the average of these two measurements was used as the body mass value for these calculations. The respiratory quotient (RQ) was calculated as: VCO2/VO2.

cDNA arrays analysis and RT-PCR
RNA preparation.
Control and cold-exposed rat hypothalamus were excised and rapidly frozen in liquid nitrogen. Total RNA was extracted using Trizol reagent, according to the manufacturer’s recommendations. Total RNA was quantified by spectrophotometry at A260 nm, and its integrity was determined from the A260:A280 nm ratio. After 24 h, isolated total RNA was rendered genomic-DNA-free by digestion with ribonuclease-free deoxyribonuclease (RQ1; Promega, Madison, WI).

Array hybridization.
The recommended protocol was followed in all steps. Briefly, 1 µg poly(A)+ RNA was converted into 33P-labeled first-strand cDNA by Moloney murine leukemia virus reverse transcriptase. Unincorporated 33P-labeled nucleotides were removed by chromatography using a NucleoSpin Extraction Spin Column (CLONTECH, Palo Alto, CA). Purified cDNA probes were hybridized to the Atlas membranes. Hybridization occurred overnight at 68 C. After washing, membranes were sealed in hybridization bags and exposed to imaging plates for 1 d. After exposure, the imaging plates were scanned using a BAS-1500 (Fujifilm, Fuji, Japan), and hybridization signals were counted. Hybridization signals of each gene were normalized by a positive control (signals of the housekeeping gene), and gene expression was compared between the preconditioning and control groups. Genes exhibiting differential expression in the preconditioning group were selected only if the hybridization signals were either increased or decreased by at least 2-fold, compared with those of the control group.

Semiquantitative RT-PCR.
Seven micrograms of total RNA, obtained from hypothalamus of rats exposed, or not, to cold during 4 d, were reverse-transcribed with SuperScript reverse transcriptase (200 U/µl) using oligo(deoxythymidine) (50 mM) in a 30-µl reaction vol [5x reverse transcription (RT) buffer, 10 mM deoxynucleotide triphosphate, and 40 U/µl ribonuclease-free inhibitor]. The RTs involved a 50-min incubation at 42 C and a 15-min incubation at 70 C. After RT, 0.75 µl of the RT product was used in each PCR to a final vol of 50 µl (10x PCR buffer, 1.0 mM deoxynucleotide triphosphate, 50 mM MgCl2, Taq polymerase, and sense and antisense primers for MCH and ß-actin). The expression of mRNA was determined by PCR, using the primers described above, and amplified a 323-bp DNA fragment of MCH and a 533-bp DNA fragment of ß-actin. Triplicate reactions were done using an initial incubation at 94 C for 5 min, denaturation at 94 C for 1 min, followed by annealing at 50 and 57 C (MCH and ß-actin, respectively) for 50 sec, extension at 72 C for 1 min, and final extension at 72 C for 7 min. After titration between 16 and 32 cycles, 22 cycles was selected for quantification of MCH and ß-actin. These PCR conditions were therefore used in all subsequent experiments. All PCR experiments included a control tube with no RT step. PCR-amplified products were run on 2% Tris/acetic acid/EDTA agarose gels, and the DNA was visualized by ethidium bromide staining. The size of the products was determined using a 1-kb plus DNA ladder (Life Technologies) as standard size markers. Images of the bands were captured using a TFX 35M UV transluminator (Life Technologies), and band intensity was quantified by digital densitometry (Scion Image software, Scion Corp., Frederick, MD). Results are expressed as the ratio of MCH/ß-actin ± SEM, in arbitrary scanning units.

Protein analyses by Western blot and dot-blot
UCP1 was measured in brown adipose tissue (BAT) of rats submitted to ICV cannulation and treated according to the following protocols: control WO; T +4 C WO; control S; T +4 C S; control AS; and T +4 C AS. Western blot analyses followed previously described methods (23, 24) using a specific anti-UCP1 polyclonal antibody. The intensity of protein bands corresponding to UCP1 was quantified by digital densitometry (Scion Image software) of the developed autoradiographs. MCH was measured in hypothalamus of rats exposed, or not, to cold (without previous stereotaxic instrumentation), or in rats submitted to ICV cannulation and treated according to the same protocols as stated above for UCP1 measurements. For the detection of MCH in hypothalamic protein extracts, a preparatory step of protein purification by filtration was performed using a Microcon YN-3 device. A method for specific peptide detection by dot-blot, previously described (25), was used. The signal obtained was measured by digital densitometry (Scion Image software) of the developed autoradiographs. Highly purified MCH was used as control.

Determination of hypothalamic MCH by RIA
For hypothalamic MCH determination by RIA, hypothalami of rats of each experimental group were homogenized in ice-cold buffer containing 100 mM Tris (pH 7.6), 1% (vol/vol) Triton X-100, 150 mM NaCl, 0.1 mg/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA, using a PTA 20S polytron generator (Brinkmann Instruments, Westbury, NY). Homogenates were then centrifuged at 12,000 x g for 20 min at +4 C. Protein concentration of supernatants was determined by the Bradford method (26). Samples containing 2.0 mg/ml protein were evaluated for the concentration of MCH using a RIA kit, following the recommendations of the manufacturer (Phoenix Pharmaceuticals, Inc.) (27).

Data presentation and statistical analysis
All numerical results are expressed as the mean ± SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands or dots in autoradiographs and quantified by densitometry using the Scion Image software. Data were analyzed by the two-tailed unpaired Student’s t test or by repeat-measures ANOVA (one-way or two-way ANOVA) followed by post hoc analysis of significance (Bonferroni test) when appropriate, comparing experimental and control groups. The level of significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolic and hormonal characteristics of rats exposed to cold
Exposure of homeothermic animals to cold promotes changes in several metabolic parameters that reflect physiological adaptations to the novel external environment. For decades, a number of studies have characterized those adaptations; and in the present study, we repeated some of those measurements to evidence the reliability of the model employed. The results obtained during the evaluation of the metabolic parameters are summarized in Tables 1Go and 2Go. As a whole, the animal model herein matches previous characterizations of cold-exposed rats (28, 29). Of greater importance for the objectives of the present study, we emphasize that mean daily food intake was approximately 70% increased in cold-exposed rats (P < 0.05), whereas no significant changes in body temperature were detected. Cumulative body weight variation was positive in the control group, whereas a negative variation on body weight was detected in cold-exposed animals. From d 6 on, a stepwise recovery of body weight was observed in cold-exposed rats; but at d 16, it was still lower than at d 0.


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  		TABLE 1. Animal model characterization

 

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  		TABLE 2. Cumulative body weight variation (g)

 
cDNA-macroarray analyses
Poly (A)+ RNA samples from the hypothalamus of control and 4-d cold-exposed rats were prepared and used for cDNA expression array hybridization. Individual gene signal intensities were normalized to that of the housekeeping genes, according to the supplier’s instructions. To minimize an animal-specific expression pattern, we combined equal amounts of RNA from three different animals and analyzed six independent hybridization signals from three different arrays per experimental condition. Table 3Go presents the data of all genes that had their mRNA expression significantly modulated by cold exposure. Although several of the genes found to be regulated by cold exposure might play important roles in energy homeostasis, we elected MCH as the first to be studied, because of its known participation in control of food ingestion and body weight (12).


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  		TABLE 3. Results of macroarray analyses

 
Hypothalamic MCH expression in rats exposed to cold
To confirm the results obtained with the cDNA-macroarray analyses, rats were housed individually, during 4 d, at room temperature or at +4 C; and at the end of the experimental period, they were anesthetized and had their hypothalamus removed for protein or RNA extraction. MCH expression was evaluated at the protein level by dot-blot analyses using a specific MCH antibody, and by RIA. The mRNA level of MCH was determined by RT-PCR using specific primers. As shown in Fig. 1Go, cold exposure induced an increase of approximately 60% (n = 4, P < 0.05) on MCH peptide expression as detected by dot-blot (Fig. 1AGo, upper panel) and by RIA (Fig. 1AGo, lower panel), and an increase of approximately 80% (n = 4, P < 0.05) on MCH mRNA expression (Fig. 1BGo). Thus, a ratification of the results obtained with the cDNA macroarray was obtained.



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  		FIG. 1. Effects of cold exposure on MCH protein and mRNA expression in hypothalamus. Rats exposed (T +4 C), or not (Control), to cold during 4 d were analyzed for MCH content in hypothalamic extracts. The protein amount of hypothalamic MCH (A, upper panel) was detected by dot-blot and is expressed as scanning units of specific blots. The protein amount of hypothalamic MCH (A, lower panel) was determined by RIA and is expressed as picomoles per milligram of total protein in hypothalamic protein extract. MCH mRNA (B) was evaluated by RT-PCR, and data obtained are depicted as a ratio: MCH/ß-actin densitometric scanning units of specific bands in 2% agarose gels (typical gels are depicted in the upper panel of B). For both experiments, n = 4; *, P < 0.05 vs. Control.

 
Effects of MCH protein synthesis blockade on body weight, body temperature, and food intake of rats exposed to cold
ICV infusion of MCH, or hypothalamic recombinant hyperexpression of MCH promotes significant increases in food ingestion and weight gain. Homeothermic animals exposed to cold present a significant increase of food intake, which is driven through yet-unknown hypothalamic signaling events. To investigate whether elevated MCH hypothalamic expression participates in the central control of food ingestion during cold exposure, we used an ICV-infused 15-mer phosphorthioate modified MCH antisense oligonucleotide to block MCH synthesis; and we evaluated food intake, body temperature, and body weight during 4 d. Based on the Rattus norvegicus MCH mRNA sequence (accession M62641 at NCBI GenBank) three different sequences were designed and tested by ICV injection of 2.0 µl per dose (2.0 nmol/µl) antisense or respective sense oligonucleotide and were evaluated for its property of blocking MCH synthesis, by measuring MCH protein content by dot-blot and RIA of hypothalamic protein extracts. The sequence 5'-ACA GCC AGA CTG AGG-3' presented the most effective and reproducible results and was used in all experiments thereafter, in parallel with its respective sense sequence as control. This antisense oligonucleotide sequence was submitted to BLAST analyses (at www.ncbi.nlm.nih.gov) and matched only for Rattus norvegicus MCH coding sequence. Figure 2Go shows that ICV infusion of MCH antisense oligonucleotide leads to approximately 25% (n = 4, P < 0.05) reduction of MCH peptide expression in hypothalamus of control rats, whereas a reduction of approximately 45% (n = 4, P < 0.05) was detected in the hypothalamus of rats exposed to cold. In such animals, no significant variation of food intake and body temperature was induced by the blockade of MCH expression; however, a significantly greater fall in body weight was detected in MCH-blocked, cold-exposed rats (~100%, P < 0.05). Moreover, an unexpectedly significant fall of body weight (P < 0.05) was also observed in rats maintained at room temperature that received MCH antisense oligonucleotide (Fig. 2DGo).



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  		FIG. 2. Effects of ICV MCH antisense oligonucleotide treatment on MCH protein expression, food intake, body temperature, and body weight variation. Rats were ICV cannulated and, after a recovery period, were treated once a day, at 1000 h, with sense (S) or antisense (AS) MCH oligonucleotide or a similar volume of saline (WO), with a total vol of 2.0 µl per dose (2.0 nmol/µl), 24, 48, and 72-h after the onset of the experimental period. The experimental animals were randomly assigned to two groups, exposed (T +4 C) or not exposed (Control) to cold. Hypothalamic MCH protein content (A, upper panel) was detected by dot-blot and is expressed as scanning units of specific blots. The protein amount of hypothalamic MCH (A, lower panel) was determined by RIA and is expressed as picomoles per milligram of total protein in hypothalamic protein extract. Mean daily food intake (B) is expressed in grams, body temperature at experimental d 4 (C) is expressed in Celsius degrees, and body weight variation during the experimental period is depicted in grams. In all evaluations, n = 4; *, P < 0.05 vs. T +4 C WO; #, P < 0.05 vs. Control WO.

 
Effects of MCH protein synthesis blockade on liver and muscle glycogen contents and carcass composition
Because the blockade of hypothalamic MCH in cold-exposed rats produced a significantly increased fall of body weight, in the absence of major variations of food intake and body temperature, we decided to evaluate the relative amounts of body glycogen, fat, and lean mass stocks in control and cold-exposed rats, treated or not with MCH antisense oligonucleotide during 4 d. As depicted in Fig. 3Go, the exposure of rats to cold produced increases in liver (~400%, P < 0.05) and muscle (~50%, not significant) glycogen contents; however, if the rats exposed to cold were treated with antisense oligonucleotide, lower levels of liver (~120%, P < 0.05, vs. T +4 C WO) and muscle (~20%, not significant) glycogen increments were detected. On the other hand, relative body fat was significantly reduced in rats exposed to cold; if cold-exposed rats were treated with antisense MCH oligonucleotide, higher amounts of relative body fat were encountered (P < 0.05, vs. T +4 C WO). Finally, lean body mass was unaffected by cold exposure; however, under MCH blockade, cold-exposed animals consumed significantly higher amounts of relative lean body mass, which differed from that observed in cold-exposed non-antisense-MCH-oligonucleotide-treated rats (P < 0.05).



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  		FIG. 3. Effects of hypothalamic MCH blockade on liver and muscle glycogen content and relative fat and lean body mass. Rats were ICV cannulated and, after a recovery period, were treated once a day at 1000 h, with antisense (AS) MCH oligonucleotide with a total vol of 2.0 µl per dose (2.0 nmol/µl), 24, 48, and 72 h after the onset of the experimental period, or with a similar volume of saline (WO). The experimental animals were randomly assigned to two groups, exposed (T +4 C) or not exposed (Control) to cold. Liver (A) and muscle (B) glycogen are expressed as milligrams per 100 grams of tissue. Relative body fat (C) and lean mass (D) are expressed as percent of body weight. In all measurements, n = 4; *, P < 0.05 vs. T +4 C WO.

 
Effects of MCH protein synthesis blockade on oxygen consumption, carbon dioxide production, and RQ
Cold-exposed rats exhibited a significantly higher O2 consumption (3.24 ± 0.26 vs. 1.54 ± 0.02 ml/g·h for cold-exposed and control rats, respectively) and CO2 production (2.50 ± 0.22 vs. 1.21 ± 0.04 ml/g·h for cold-exposed and control rats, respectively) than control rats. However, no major change in RQ was detected among the groups (0.77 ± 0.01 vs. 0.78 ± 0.03 for cold-exposed and control rats, respectively). The blockade of MCH expression with antisense oligonucleotide promoted no significant changes in O2 consumption, CO2 production, and RQ in both groups studied.

Effects of MCH protein synthesis blockade on BAT UCP1 expression
Prolonged survival of homeothermic animals exposed to cold depends on efficient means of maintaining a stable body temperature. In rodents, much of this function is exerted by BAT located mostly in interscapular space and perirenal area. Heat production in BAT is, at least in part, driven by a leak of the mitochondria electron transport chain, and UCP1 is directly involved in this process (30). To investigate the participation of MCH on cold-induced UCP1 induction, Western blot analyses were employed to measure interscapular UCP1 expression in cold-exposed MCH-blocked rats and compare them with respective controls. As shown in Fig. 4Go, cold exposure induced an increase of approximately 30% (P < 0.05) in UCP1 expression in BAT. Blockade of hypothalamic MCH promoted a further significant increase in UCP1 expression in cold-exposed rats (P < 0.05 vs. cold exposed with no oligonucleotide treatment); and, in fact, even in non-cold-exposed rats, the treatment with MCH antisense oligonucleotide induced a nonsignificant increase (P = 0.058) in UCP1 expression in BAT, as compared with control. The same period of MCH blockade promoted a significant increase in relative [BAT weight (g)/body weight (g)] interscapular BAT weight (0.099 ± 0.004 vs. 0.083 ± 0.003% for cold-exposed, antisense MCH-treated and cold-exposed, non-oligonucleotide-treated rats, respectively).



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  		FIG. 4. Effects of hypothalamic MCH blockade on BAT UCP1 protein expression. Rats were ICV cannulated and, after a recovery period, were treated once a day, at 1000 h, with sense (S) or antisense (AS) MCH oligonucleotide or a similar volume of saline (WO), with a total vol of 2.0 µl per dose (2.0 nmol/µl), 24, 48, and 72-h after the onset of the experimental period. The experimental animals were randomly assigned to two groups, exposed (T +4 C) or not exposed (Control) to cold. At the end of the experimental period, the rats were anesthetized, BAT was excised and homogenized, and 0.2 mg of total protein was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with UCP1 antibodies. Specific bands were measured by densitometry, and results obtained are depicted as scanning units. n = 4; *, P < 0.05 vs. T +4 C WO.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene array analyses are powerful tools for evaluating the complex dynamics of tissue-specific gene expression, and to compare the effects of different external stimuli on the genetic phenotype of a given tissue (31). Because the exposure of homeothermic animals to cold environment leads to a unique physiologic situation characterized by increased feeding, associated with high thermogenesis, and no weight gain, we hypothesized that, by characterizing hypothalamic proteins regulated by cold exposure, candidate proteins that participate in the control of feeding behavior and energy homeostasis would be revealed. Therefore, by evaluating 1176 genes, we detected cold-induced modulation of 56 genes expressed in rat hypothalamus. Among those, we found neurotransmitters, membrane receptors, enzymes involved in energy metabolism, proteins of the transcription and translation machinery, membrane ion-channels, and others. We believe that many of them may have direct participation in the central adaptations to cold and will certainly deserve further studies. As for the objectives of the present study, we chose evaluation of the role of the neurotransmitter MCH in cold-induced feeding behavior and energy homeostasis. For that purpose, MCH expression, which is up-regulated in cold-exposed animals, was partially abrogated by ICV phosphorthioate-modified MCH antisense oligonucleotide treatment; and several metabolic, biochemical and hormonal parameters were studied.

Since the seminal work of Qu and collaborators (12), most studies have emphasized the attribute of MCH to increase food consumption in experimental animals (10, 32, 33). Therefore, it was surprising that the partial blockade of MCH provided by ICV oligonucleotide treatment led to no significant modulation of food ingestion in cold-exposed and in control rats. Taking a closer look at the data available in the literature concerning MCH effects on feeding behavior, it becomes clear that this is not a straightforward matter. It seems that acute ICV injection of MCH promotes significant increase of food intake (12, 13, 34), whereas chronic ICV infusion does not lead to significant increase of caloric intake if rats are maintained under regular chow, but does so if they are treated with a high-fat diet (35, 36). The blockade of hypothalamic MCH activity by ICV infusion of a selective MCH1-R antagonist promotes evident acute effects on feeding inhibition and only moderate effects after chronic treatment (37). The recombinant hyperexpression of MCH also leads to increased food ingestion and obesity, but the magnitude of feeding augment is not as exuberant as in acutely MCH-treated animals (10). Finally, targeted-disruption of MCH1-R results in hyperphagia and resistance to diet-induced obesity (7, 15). Thus, it seems that the orexigenic effects of MCH in hypothalamus are dependent on the duration of stimulus and on receptor subtype, and that some of the approaches used to modulate MCH action produce substantial changes of body weight that are not accompanied by similar regulation of food intake.

Body temperature was another parameter evaluated in the present study. Normal rats exposed to cold environment (+4 C) present an early fall in body temperature, reaching a nadir of approximately 33 C (rectal) after 2–6 h and returning to euthermia after 24–48 h at most (28, 29). After 4 d of MCH blockade, no significant changes in rectal temperature were detected in cold-exposed and control rats. These findings are in accordance with previous pharmacological and recombinant approaches undertaken to study MCH function in hypothalamus (12, 13, 34, 37).

Different from the two previous parameters evaluated, body weight was significantly influenced by the partial blockade of MCH. As in the present study, most previously published data show that exposure of rats to +4 C leads to an initial loss of body weight, which is followed by gradual recovery. However, even after several weeks, cold-exposed rats do not reach the same rate of weight gain as their matched controls maintained at room temperature. In the present experiments, MCH-blocked, cold-exposed rats presented a significantly greater weight loss than their controls, and even non-cold-exposed rats treated with MCH antisense oligonucleotide underwent significant weight loss.

Because the blockade of MCH expression produced significantly greater weight loss than in respective controls, which was not accompanied by changes in food intake and body temperature, we decided to measure body energy stocks, oxygen consumption, carbon dioxide production, RQ, and UCP1 expression in BAT, as indirect means of estimating the flow of the energy that was lost in MCH-blocked animals, and the participation of BAT in thermogenic adaptation after cold exposure and MCH signaling interference. The data herein presented reveals that blockade of MCH reduces liver and muscle glycogen content, increases relative amounts of body fat, and decreases relative amounts of lean mass of cold-exposed rats. Moreover, in MCH-blocked rats, the amount of UCP1 in BAT, which is significantly stimulated by cold exposure, suffers a further and significant increment, which is accompanied by an increase in the relative weight of BAT. All these changes occurred in the absence of major modifications of O2 consumption, CO2 production, and RQ.

BAT is a specialized organ that actively participates in the production of heat. Hypothalamic nuclei stimulate BAT activity and UCP1 expression by sympathetic inputs. In a recent study, the origin of sympathetic connections that innervate BAT has been mapped to paraventricular nucleus, LH, prefornical area, and retrochiasmatic nucleus (38). Thus, as one of the predominant neurotransmitters expressed in the LH, MCH may participate in the control of UCP1 expression and BAT activity in certain conditions. Based on the present findings, it seems that, during cold exposure, the optimal expression of UCP1, which is required for full thermogenic activity of BAT, is dependent on increased MCH expression in hypothalamus. Once MCH expression is disrupted, the animals may loose the capacity of tight regulation of energy expenditure for heat production, and increased UCP1 may be a reflection of that.

Energy expenditure by motor activity is another possible mechanism involved in MCH-dependent body weight control. According to Marsh and colleagues (15), the disruption of MCH1-R gene produces a phenotype of leanness, motor hyperactivity, and hyperphagia. Apparently, most of the increased metabolic rate of this animal model is attributable to hyperactivity, because it was detected during the dark phase of the light-dark cycle. This observation may explain why many studies, including the present one, that attempted to characterize the role of hypothalamic MCH signaling on food intake and body weight control, encountered no straight correlation between variation in caloric intake and body weight. This may also explain why, in the present study, no significant changes in O2 consumption, CO2 production, and RQ were detected, because the rats were restrained during the measurements.

Finally, a recent study (39) provides, by indirect means, further support for a role of MCH in energy expenditure control. The treatment of rodents with the fatty acid synthase inhibitor C75 was shown to induce hypophagia accompanied by a nonproportional loss of body weight, i.e. C75-treated rats lose weight to a lesser extent than food-deprived rats, although both groups have no caloric intake. These animals express less hypothalamic neuropeptide Y and Agouti-related peptide, and more pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, than their controls. However, most important, in our opinion, is the fact that they express higher levels of hypothalamic MCH. Taken together with the data herein presented, it seems that, during some situations that provoke catabolism, the levels of hypothalamic MCH are increased to provide means for minimizing energy expenditure during adversity.

In conclusion, the present study suggests that hyperexpression of MCH in hypothalamus of cold-exposed rats participates in the fine tuning of energy expenditure, providing means for optimal heat production with minimal caloric wastage. Apparently, under chronic cold exposure, the role of MCH in the induction of hyperphagia is minimal or absent. These data reinforce the potentiality of MCH as a target for pharmacological therapy of obesity and related diseases, and provide novel support for the concept of dichotomized central control of thermogenesis and food intake.


   Footnotes
 
This study was granted by Fundação de Amparo à Ciência do Estado de São Paulo. M.P.S. was a recipient of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

Abbreviations: AS, With antisense oligonucleotide treatment; BAT, brown adipose tissue; ICV, intracerebroventricular(ly); LH, lateral hypothalamus; MCH, melanin-concentrating hormone; RQ, respiratory quotient; RT, reverse transcription; S, with sense oligonucleotide treatment; T, temperature; UCP, uncoupling protein; VO2, rate of oxygen consumption; VCO2, rate of carbon dioxide production; WO, without oligonucleotide treatment.

Received February 24, 2003.

Accepted for publication July 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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