Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-d-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.
Obesity has grown as one of the most important epidemiological problems in modern societies (1,2). The increased consumption of high calorie foods, associated with a progressive reduction in physical activity, accounts for most cases of obesity (3,4). In recent years, an impressive progress has been made in the characterization of the hypothalamic mechanisms involved in the control of food intake and energy expenditure (5,6). According to data obtained from experimental models, weight gain is paralleled by a phenomenon of hypothalamic resistance to the anorexigenic and thermogenic inputs delivered by leptin and insulin (7,8). This resistance leads to the disruption of a tightly regulated coupling between energy intake and expenditure.
As a rule, treating obesity through dietetic approaches leads to a limited loss of body mass, which is accounted, mostly, by the establishment of a novel set point in the coupling between energy acquisition and wastage (9). Likewise, the use of the currently available drugs to treat this medical condition produces, at most, a 15% drop in body mass (10), which is rapidly recovered after the interruption of the treatment.
The complete characterization of the hypothalamic mechanisms involved in the control of the coupling between food intake and thermogenesis may reveal novel potential targets for drug therapy in treating obesity. Here, we evaluated the effect of two distinct thermogenesis-inducing conditions upon the expression of 1,176 genes in the hypothalamus of rats. The hypothesis behind this approach is that, should certain genes be similarly regulated by two distinct thermogenic approaches, they may participate in important events of the thermogenic response and, thus, may become attractive candidate targets for the therapeutics of obesity.
Our approach revealed that only 0.17% of the analyzed genes respond simultaneously to the distinct stimuli, which reinforces the complexity of the system. Nevertheless, the two genes identified herein are potential targets that shall be further studied for their roles in the regulation of thermogenesis.
Methods and Procedures
This investigation adhered to the University guidelines for using animals in experimental studies and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23 revised 1996). The University's Ethical Committee approved all the protocols (Ethical Committee approval certificate #503-2).
Antibodies, chemicals, and buffers
For protein detection by immunoblot, species-specific antibodies against insulin receptor (IR) (sc-711, rabbit polyclonal), leptin receptor (sc-1834, goat polyclonal), janus kinase-2 (sc-7229, rabbit polyclonal), Akt(sc-1618, goat polyclonal), IR substrate-1 (IRS-1) (sc-560, rabbit polyclonal), IRS-2 (sc-8299, rabbit polyclonal), extracellular signal-regulated kinase (sc-94, rabbit polyclonal), signal transducer and activator of transcription-3 (sc-482, rabbit polyclonal), and corticotropin-releasing hormone (CRH) (sc-10718, rabbit polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Richmond, CA). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and bovine serum albumin (fraction V) were from Sigma (St. Luis, MO). 125I-protein A and nitrocellulose paper (BA85, 0.2 μm) were from Amersham (Aylesbury, UK). Sodium thiopental was from Lilly (Indianapolis, IN).
Cold exposure protocols
Male Wistar rats (56–70 days old/200–260 g) from the University of Campinas Central Animal Breeding Center were used in the experiments. The animals were maintained on a 12:12 h artificial light-dark cycle and kept in individual cages supplied with standard rodent chow and water ad libitum. After an acclimatizing period (3 days), rats were transferred to metabolic cages and randomly assigned to two treatment conditions: control and cold exposure (T+4 °C). Rats of the control group were maintained at 23 ± 1 °C, whereas rats of the T+4 °C group were housed at 4 ± 1 °C for 16 days, as described previously (11).
High-fat feeding protocols
Male Wistar rats (56–70 days old/200–260 g) from the University of Campinas Central Animal Breeding Center were used in the experiments. The animals were maintained on a 12:12 h artificial light-dark cycle and kept in individual cages supplied with standard rodent chow and water ad libitum. After an acclimatizing period (3 days), rats were transferred to metabolic cages and randomly assigned to two treatment conditions: control and high-fat (HF) feeding. Control rats were fed standard rodent chow whereas HF rats were fed a hyperlipidic-hypercaloric diet (Table 1) for 16 days. For both cold exposure and HF feeding protocols, the control of food intake was performed every day and the control of body mass every second day, the hormone and biochemical determinations were performed in samples collected from eight rats of each group and analyzed in duplicates. For the cDNA preparation of macroarray analysis, mRNA extracted from the hypothalami of three rats from each group were pooled, reverse transcribed, and used in the hybridization protocol; three pools from each condition were evaluated, thus, the mRNA of nine distinct rats from each group was included in the macroarray study. For protein extract preparation, the hypothalami of four rats from each group were obtained.
Table 1. Macronutrient composition of diets
Hypothalamic specimen preparation
Dissections of hypothalami were exhaustively optimized following a method published previously (12). During optimization, the limits of the dissected specimens were evaluated by microscopy. The limits for dissection were: anterior, the limit of the optic chiasm; superior, the anterior commissure; posterior, the medium mammillary nucleus.
Frontal cortex and hippocampus specimen preparation
Fragments from frontal cortex were obtained at the surface of the brain region located at bregma +1.6 mm, 2.5 mm lateral from the sagittal line, whereas fragments from hippocampus were obtained at bregma −4.50 mm, 4.0 mm lateral from the sagittal line, and 3.5 mm deep (according to ref. 13).
RNA preparation for macroarray and RT-PCR
Total RNA was extracted from hypothalmus specimens using Trizol (Life Technologies, Gaithersburg, MD) reagent, according to the manufacturer's recommendations. Total RNA was rendered genomic DNA-free by digestion with RNAse-free DNAse (RQ1; Promega, Madison, WI).
Rat 1.2 Atlas Array was from Clontech (Palo Alto, CA). One μg of 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. 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 day. After exposure, the imaging plates were scanned using a BAS-1500 (Tokyo, 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 treatment and control groups. Genes exhibiting differential expression in the treatment group were selected only if the hybridization signals were either increased or decreased by at least two-fold, compared to those of the control group.
Reverse transcription was carried out with 3.0 μg total RNA using SuperScript reverse transcriptase (200 U/μl) and oligo (dT) (50 mmol/l) in a 30 μl reaction volume (5× RT buffer, 10 mmol/l dNTP, and 40 U/μl RNAse-free inhibitor). The reverse transcriptions involved a 50 min incubation at 42 °C and a 15 min incubation at 70 °C. The PCR products were submitted to 1.5% agarose gel electrophoresis containing ethidium bromide and visualized by excitation under UV light. Photodocumentation was performed using the Nucleovision System (NucleoTech, San Mateo, CA) and band quantification was performed using the Gel Expert Software (NucleoTech). The sequences of the primers for the N-methyl-d-aspartate (NMDA) receptor 2B (GI:6980984), guanosine triphosphate (GTP)-binding protein G-alpha-i1(GI:203168), melanin-concentrating hormone (MCH) (GI:6981373) and β-actin (GI:42475962) were 5′-GCA CGG GAA GAA GAT TAA TGG-3′(sense) and 5′-ATA GCC GGT AGA AGC AAA GAC-3′ (antisense) (product, 831 base pair (bp)); 5′-ACA CCA TCC AGT CCA TCA TTG-3′ (sense) and 5′-GAG GTC TTC AAA CTG ACA CTG-3′(antisense) (product, 703 bp); 5′-TAC GGA GCA GCA AAC A-3′(sense) and 5′-ACA GCC AGA CTG AGG G-3′ (antisense) (product, 323 bp); and 5′-ACC ACA GCT GAG AGG GAA ATC G-3′(sense) and 5′-CAG TCC GCC TAG AAG CAT TTG C-3′(antisense) (product, 533 bp), respectively. Control amplifications were carried out in the presence of RNA template and polymerase, but in the absence of reverse transcriptase. The RPS 29 mRNA was amplified in all samples as control for quality and amount of RNA using the primers 5′-AGG CAA GAT GGG TCA CCA GC-3′(sense) and 5′-AGT CGA ATC ATC CAT TCA GGT CG-3′(antisense) (product, 202 bp).
Protein expression evaluation by immunoblotting
Experiments were performed as described previously (14,15) with minor modifications, hypothalami were excised and immediately homogenized in solubilization buffer at 4 °C (1% Triton X-100, 100 mmol/l Tris-HCl (pH 7.4), 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium orthovanadate, 2.0 mmol/l phenylmethylsulfonyl fluoride and 0.1 mg aprotinin/ml) with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY). Insoluble material was removed by centrifugation for 40 min at 11,000 rpm on a 70. Ti rotor (Beckman) at 4 °C. The protein concentration of the supernatants was determined using the Bradford dye binding method. Aliquots of the resulting supernatants containing 0.2 mg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blotted with specific antibodies. Bands corresponding to the analyzed proteins were labeled with 125I-protein A and visualization was performed through exposure of the membranes to RX-films.
Specific protein bands present in the blots were quantified by digital densitometry (ScionCorp, Frederick, MD). Mean values ± s.e.m obtained from densitometric scans, and the values for all metabolic and biochemical measurements were compared utilizing a one-way ANOVA with post-hoc test (Tukey). For reverse-transcription PCR (RT-PCR), values obtained from the scanning of bands of target sequences are presented as a ratio of the control sequence (RPS 29) and compared utilizing a one-way ANOVA with post-hoc test (Tukey). A P value of <0.05 was accepted as statistically significant.
Metabolic and biochemical characterization of the experimental animals
As shown in Table 2, 16 days of exposure to a cold environment promoted significant changes in food intake (increase) and blood levels of insulin and leptin (decrease). There was a discrete fall in the body mass of the cold-exposed rats, while the control rats presented an increase in this parameter; however, the difference between groups was not significant. Rats fed on the HF diet presented a significant increase in body mass, food intake, blood nonesterified fatty acid, insulin, and leptin. As a rule, the metabolic and biochemical characteristics of our models match those of previous reports (7,11,16).
Table 2. Metabolic characteristics of the experimental animals
Cold exposure led to a significant modulation of the expression of 47 mRNA species (43 increased; 4 decreased) (Table 3). This corresponds to 3.99% of the analyzed genes. Among the modulated mRNA species, there were sequences encoding for proteins related to RNA processing, intracellular and extracellular protein processing, mitochondrial function, cell structure, ion channels, membrane receptors, serine-threonine and tyrosine kinases, neurotransmitters and hormones, among others. Only a few of these proteins are known to play roles in the control of food intake and thermogenesis, such as MCH and CRH (16,17,18). With regard to HF feeding, there was a modulation of 168 mRNA species (90 increased; 78 decreased) (Table 3). This corresponds to 14.28% of the analyzed genes. In this case, there was a modulation of mRNAs encoding proteins related to immune response, hormone action, growth factors, transcription factors, lipid metabolism, and membrane and nuclear receptors, among other functions. Interestingly, only two mRNA species—the NMDA receptor 2B and GTP-binding protein G-alpha-i1—were simultaneously modulated (increased) by both experimental conditions (Table 3), this corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes.
Table 3. Genes significantly modulated by the experimental conditions
Evaluation of selected protein/mRNA expression by immunoblot or semiquantitative RT-PCR
In Figure 1a–c, we present the results of immunoblot or semiquantitative RT-PCR evaluation of some proteins or mRNAs belonging to signal transduction pathways and neurotransmitter systems known to be involved in the regulation of thermogenesis. As shown in Figure 1a, there was a significant increase in IR expression in the hypothalamus of rats fed HF and a significant increase in extracellular signal-regulated kinase expression in rats exposed to cold (T4). The remainder of the proteins of the insulin signaling pathway evaluated, IRS-1, IRS-2, and Akt were not modulated by either condition tested. These results are in accordance with the microarray data. Accordingly, regarding proteins belonging to the leptin signaling pathway (Figure 1b), there was a significant increase in leptin receptor expression in HF rats and a significant decrease in janus kinase-2 expression in HF rats. No significant changes in signal transducer and activator of transcription-3 expression were detected in either experimental condition. Again, these results are in accordance with the findings in the macroarray. The expression of the neurotransmitter CRH was decreased in T4, whereas the expression of MCH was increased in T4 (Figure 1c), similar to the macroarray results. In Figure 1d, the expression of NMDA receptor 2B and GTP-binding protein G-alpha-i1 were evaluated by RT-PCR in samples from rat hypothalamus. Confirming the results of the array, both mRNA species were significantly increased by both experimental conditions. The anatomical specificity of the phenomenon was further tested by determining the expressions of NMDA receptor 2B and GTP-binding protein G-alpha-i1 genes in other regions of the brain. As shown in Figure 1e, no changes in either mRNA specificity expression were detected under both the experimental conditions in the frontal cortex and hippocampus.
In this study, we employed cDNA macroarray analysis to evaluate the effect of two distinct thermogenic conditions on the expression of 1,176 mRNA species in the hypothalamus of rats. The experimental conditions were chosen based on a number of studies that have thoroughly evaluated the capacity of hypercaloric feeding and cold exposure to induce increased thermogenesis in mammals (19,20). These conditions produce different outcomes, being the modification of body mass the most evident. We hypothesized that, should such different thermogenic conditions modulate similar genes in the hypothalamus, these might encode important proteins for the control of thermogenesis. According to our results, the consumption of a HF diet modulates the expression of 14.28% of the analyzed mRNAs, whereas cold exposure modulates the expression of 3.99%. Some of the mRNAs induced by either condition have been previously reported to play a role in the regulation of, or to respond to, thermogenic conditions. These genes/mRNAS include MCH (16,17), CRH (17), leptin receptor (21), and IR (22), among others; however, none of these well-known determinants or targets of thermogenesis have been simultaneously regulated by both the experimental conditions tested. To our surprise, only two mRNAs, corresponding to 0.17% of the analyzed genes, were modulated simultaneously by cold and hypercaloric feeding. The products of these mRNAs are the proteins, NMDA receptor 2B, and GTP-binding protein G-alpha-i1.
NMDA receptors are members of the ionotropic glutamate-gated ion channels that are widely expressed in the central nervous system and play important roles in functions such as integration of convergent signals to the brain, integration of sensory and motor commands, processing of memories and thoughts, among others (23). Structurally, NMDA receptors are composed of different subunits: NR1, whose presence is mandatory; NR2A-D and, eventually, NR3A or B. The subunit composition determines the pharmacology and other functional parameters of the receptor, while alternative subunit mRNA splicing further contributes to the diversity of the pharmacological properties of these proteins (24). In the hypothalamus, the activity of NMDA receptors has been linked to several different autonomic functions, such as the control of growth hormone secretion (25), regulation of puberty surge (26), control of reproductive and adrenal axes (27,28), and the modulation of appetite and thermogenesis (29,30,31). However, only a small number of studies have been specifically devoted to characterizing the mechanisms involved in the NMDA-dependent control of thermogenesis. According to these studies, NMDA receptors, present in the raphe pallidus, mediate at least part of the excitatory neurotransmission that activates brown adipose tissue thermogenesis (30,31). This pathway is responsive, for example, to prostaglandin (31) and may depend on the disinhibition of neurons of the dorsomedial hypothalamus (30). In addition, at least two drugs currently used to treat obesity, sibutramine and rimonabant, are known to exert inhibitory actions on NMDA activity in the hypothalamus (32,33). It will be interesting to evaluate whether receptors composed by 2B subunits play an important role in this process and whether they are preferentially expressed in hypothalamic regions involved in the integrative control of thermogenesis. Should this be the case, this subclass of receptors may become interesting targets for the therapeutics of obesity.
G-proteins are heterotrimers that associate with G-protein coupled receptors to act as the initial transducers of the incoming signals (34). A number of hormones and neurotransmitters employ this class of receptors to deliver their signals to different cells and tissues (35). Some of these G-proteins have been shown to play important roles in the hypothalamic control of feeding behavior (35,36). This is of obvious interest because a number of well-known modulators of feeding and thermogenesis act through G-protein coupled receptors, such as serotonin, galanin, MCH, CRH, thyrotrophin-releasing hormone (TRH), neuropeptide Y, catecholamines, somatostatin and bombesin, among others (36,37,38). With regard to GTP-binding protein G-alpha-i1, we have found no study that has looked specifically at its role in the control of thermogenesis or feeding. However, using an antisense oligonucleotide toward GTP-binding protein G-alpha-common, Plata-Salaman and colleagues showed that the inhibition of the expression of this class of proteins is capable of significantly reducing spontaneous food intake (39). It will be interesting to characterize the anatomical distribution of different types of GTP-binding proteins G-alpha to evaluate whether they may be of any potential interest to act as targets for pharmacological approaches to treat obesity.
In our opinion, there are two main outcomes of this study. First, thermogenesis in response to distinct stimuli seems to be regulated by a predominantly divergent set of genes/proteins. This fact reinforces the complexity of the system that coordinates energy flow in mammals and may explain, for example, the common failure of a number of therapeutic approaches used to treat patients with obesity (4,9). In this context, the inhibition of energy expenditure during a program that includes the consumption of a restricted calorie diet, associated with the use of an anorexigenic/thermogenic drug, such as sibutramine (40), has an impact on body weight only for a limited period of time and is blunted due to the creation of a novel set point for body energy stores regulation.
The second important outcome of this study is the fact that two proteins, out of 1,176, simultaneously responded to cold exposure and hypercaloric feeding. This suggests that only a small number of proteins may play a pivotal role in the control of the hypothalamic mechanisms involved in the regulation of thermogenesis. If this is the case, characterization of these proteins may help to indicate the most appropriate targets for drugs aimed at treating obesity and other related metabolic diseases.
The grants for this study were obtained from Fundação de Apoio à Pesquisa do estado de São Paulo (FAPESP) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Dr Conran for editing the article for language.