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J Appl Physiol 100: 1992-2003, 2006. First published February 9, 2006; doi:10.1152/japplphysiol.00850.2005
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Acclimatory-phase specificity of gene expression during the course of heat acclimation and superimposed hypohydration in the rat hypothalamus

Hagit Schwimmer, Luba Eli-Berchoer, and Michal Horowitz

Laboratory of Environmental Physiology, Faculty of Dental Medicine, the Hebrew University, Jerusalem, Israel

Submitted 14 July 2005 ; accepted in final form 8 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The induction of the heat-acclimated phenotype involves reprogramming the expression of genes encoding both constitutive and inducible proteins. In this investigation, we studied the global genomic response in the hypothalamus during heat acclimation, with and without combined hypohydration stress. Rats were acclimated for 2 days (STHA) or for 30 days (LTHA) at 34°C. Hypohydration (10% decrease in body weight) was attained by water deprivation. 32P-labeled RNA samples from the hypothalamus were hybridized onto cDNA Atlas array (Clontech no. 1.2) membranes. Clustering and functional analyses of the expression profile of a battery of genes representing various central regulatory functions of body homeostasis demonstrated a biphasic acclimation profile with a transient upregulation of genes encoding ion channels, transporters, and transmitter signaling upon STHA. After LTHA, most genes returned to their preacclimation expression levels. In both STHA and LTHA, genes encoding hormones and neuropeptides, linked with metabolic rate and food intake, were downregulated. This genomic profile, demonstrating an enhanced transcription of genes linked with neuronal excitability during STHA and enhanced metabolic efficiency upon LTHA, is consistent with our previously established integrative acclimation model. The response to hypohydration was characterized by an upregulation of a large number of genes primarily associated with the regulation of ion channels, cell volume, and neuronal excitability. During STHA, the response was transiently desensitized, recovering upon LTHA. We conclude that hypohydration overrides the heat acclimatory status. It is notable that STHA and hypohydration gene profiles are analogous with the physiological profile described in the response to various types of brain injury.

microarray; ion channels; molecular signaling

ACCLIMATION/ACCLIMATIZATION is the ability found in most species to adjust to prolonged environmental change. During acclimation/acclimatization, alterations in the capacity and responsiveness of preexisting physiological processes are beneficial to the organism in the new environment. When such adjustments are made in response to a hot environment, the process is called heat acclimation.

The emergence of the heat-acclimated phenotype involves a continuum of processes, first manifested in central controlling systems as increased excitability of autonomic nervous system outflow signals serving to compensate for impaired cellular performance at the onset of acclimation (short-term heat acclimation, STHA), followed by enhanced cellular functions and decreased autonomic excitability upon stable long-term heat acclimation (LTHA) (20, 25). In the latter phase, a lower hypothalamic temperature threshold for the activation of heat-dissipation mechanisms and an elevated body temperature threshold for the development of thermal injury lead to an expanded dynamic thermoregulatory range. Changes in the ratio of the thermosensitive to insensitive preoptic and anterior hypothalamus neurons after heat acclimation (38) confirm the existence of neuronal plasticity in hypothalamic regions responsible for thermoregulatory integration during acclimation.

Accruing data from studies on peripheral tissues suggest that the induction of the heat-acclimated phenotype involves reprogramming the expression of genes encoding both constitutive and inducible proteins (e.g., 8, 9, 35, 42). In a recent study on the neuro-thermo-modulatory role of ANG II and neuronal nitric oxide synthase (nNOS) (43), for example, we presented substantial evidence that biphasic changes in the transcript levels of the ANG II receptors AT1 and AT2 and of nNOS, as well as posttranslational cytosolic changes of the encoded proteins occur throughout the acclimatory process. A marked upregulation of the AT1, AT2, and nNOS genes upon STHA leads to an increased AT2-to-AT1 receptor-protein ratio and, in turn, to altered temperature thresholds for heat dissipation upon LTHA (43). Given that angiotensin signaling involves a large number of genes (43), our data advance the hypothesis that large-scale transcriptional changes in the hypothalamus contribute to the acclimatory response.

Congruent with our findings, transcriptional plasticity during long-term processes in the brain was documented during the response to environmental enrichment (namely, a different surrounding every day) (11, 44), hypoxia (48), and learning and memory (16, 39). Each of these environmental factors induced long-term modifications in behavior and physiological mechanisms that evolved, at least in part, because transcriptional changes.

Because water is essential for both evaporative cooling and replenishment of the extracellular fluid compartments during heat stress, competing requirements arise when hypohydration (Hyp) is superimposed on heat acclimation. Unequivocally, hypohydration overrides heat acclimation and abolishes the enhanced heat endurance acquired with heat acclimation (21, 43). Pharmacological evidence supports the notion that a desensitization of ANG II-AT1 signaling (43) contributes to the masking of the favorable effects of acclimation. Yet, the transcriptional processes in the hypothalamus did not reflect this profile. Relative to the euhydrated (Euh) state, the steady-state transcript levels of both AT ANG II receptor types are upregulated after LTHA-Hyp. In contrast, the finding that nNOS mRNA does not undergo changes compared with the Euh state suggests that Hyp affects hypothalamic gene expression in a selective manner.

Taken together, this large amount of new scientific evidence presents us with an opportunity to broaden our understanding of the development of the heat-acclimated phenotype by providing insights into the global genomic response in the hypothalamus. Specifically, this view is supported by 1) the recognition that neuronal plasticity is an important component of the heat acclimation repertoire, 2) the substantiated evidence that this process involves the reprogramming of large batteries of genes, and 3) the finding that hypohydration masks the adaptive acclimatory responses via changes in gene expression. Hence the global genomic responses of the hypothalamus to the process of heat acclimation are studied here.

Our major aim in the present study was twofold: 1) to take a broad-scale genomic approach to studying the hypothalamic global genomic response during the course of heat acclimation, and 2) to study the gene-expression profile when hypohydration is superimposed on heat acclimation. We decided to take advantage of gene chip array technology to study the expression profile of a battery of genes representing various central regulatory functions of body homeostasis that have not yet been associated with heat acclimation.

We have found that, in the hypothalamus, heat acclimation provokes a continuum of molecular responses, characterized by 1) an initial transient profound upregulation of genes encoding ion channels, 2) a constitutive upregulation of G protein-coupled receptors pathways, and 3) a downregulation of genes linked with hormones or peptides regulating food intake and energy metabolism. The changes observed reflect the enhanced neuronal excitability at the onset of acclimation and metabolic downregulation when acclimation homeostasis has been achieved. When hypohydration is superimposed, a marked upregulation of genes associated with ion channels or currents, transport proteins, and neural transmission predominates, implying that the apparent abolishment of heat acclimation by hypohydration (interference phenomenon) is associated with enhanced ion fluxes and neural activation. Identification of these "interference" pathways will advance our understanding of central acclimatory processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and experimental protocols.   Male rats (Rattus norvegicus, Sabra strain albino variety), initially weighing 80–100 g, each were kept in a 12:12-h light-dark cycle at 24 ± 1°C, with food available (Ambar lab chows) ad libitum. Rats were divided into three groups according to their acclimation status. LTHA was attained by continuous exposure for 30 days to 34 ± 1°C. For STHA, rats were kept at normothermic temperature for 28 days and then transferred to the climatic chamber for 2 days at 34°C. The chosen acclimation temperature is the upper limit of the thermoneutral zone. For the Sabra strain the thermoneutral zone is 28–34°C (Omri M, Horowitz M, and Samueloff S, unpublished observations; Ref. 19). Our previous studies on the dynamics of acclimation verified that acclimatory homeostasis is reached after 25–30 days whereas STHA is characterized after 2–3 days of acclimation (e.g., Ref. 20). Rats held at 24 ± 1°C (normothermic temperature) served as the control group (C). Each group was further divided into Euh and Hyp rats; hypohydration was attained by water deprivation resulting in approximately a 10% reduction in body weight (24 h for LTHA and C rats and 12 h for STHA) (21, 23, 30). Previous studies have shown that such a decrease corresponds to a 21% loss in plasma volume, whereas plasma osmolarity remains unchanged (23, 28, 30). All experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Hebrew University.

The physiological evidence for the beneficial effects of heat acclimation on heat endurance and its interference by hypohydration, the basis for the present experimental design, was studied in detail in many previous investigations (e.g., Refs. 21, 43; Table 1). Notably, heat endurance of acclimated rats is 223% of that of nonacclimated rats, whereas hypohydration decreases endurance by 56 and 77% in nonacclimated and heat-acclimated rats, respectively. In contrast, the central thermoregulatory thresholds of both groups were similarly affected by hypohydration.


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  		Table 1. Evidence of the beneficial effects of heat acclimation on heat endurance in rats and the desensitization that occurs after superimposed hypohydration

 
Experimental conditions.   All experiments were conducted between 9:00 AM to 1:00 PM to avoid changes in gene expression due to circadian rhythm. The animals were killed by cervical dislocation, the whole brain was removed, and the hypothalamus, including the preoptic area, was rapidly excised by making a 2-mm-deep vertical incision at the optic chiasm and just anterior to the mammillary bodies; the hypothalamus was then placed in liquid nitrogen (46). Each animal was treated individually to ensure precise sampling. Frozen samples were stored at –80°C for subsequent analysis.

Gene expression using Clontech cDNA Atlas array.   A Clontech Rat cDNA Atlas array containing 1,187 stress genes representing a variety of functional groups of the rat genome spotted on a nylon membrane was used (Clontech Laboratory, Palo Alto, CA). Samples of RNA from all groups were used for this experimental series. TRI Reagent (Molecular Research Center) was used to extract the total RNA from hypothalamic tissue. For detection, we combined the hypothalami of two animals into pooled mRNA preparations. Three pools (6 animals) were prepared for each experimental group. The RNA samples were treated with RNase-free DNase I to avoid genomic DNA contamination, and the purity of the RNA sample was confirmed by PCR. The quantity and quality of the sample were estimated from its absorbance at 260 and 280 nm, as well as by 1% agarose gel electrophoresis (22, 35). The probes were labeled for 1 h by the reverse transcription of total RNA (3 µg) at 42°C in a primer mix (Clontech) containing (32P)dATP (Amersham Biosciences, Buckinghamshire, UK). The reaction was terminated by adding 0.1 M EDTA and 1 mg/ml glycogen (Sigma). The unincorporated 32P-labeled nucleotides were removed using Nucleo-Spin extraction columns (Clontech).

cDNA array hybridization.   The membranes were prehybridized for 1 h at 68°C in a hybridization solution (Clontech) containing 0.1 mg/ml sheared salmon testes DNA (Sigma) to block nonspecific binding. The synthesized radiolabeled cDNA probe (5 to 15 x 106 cpm) was applied to each membrane and hybridized overnight at 68°C. Cot-1 DNA (Clontech) (1 µg/ml) was added to block nonspecific binding. Each membrane was used three times after stripping by boiling in 0.5% SDS, according to the manufacturer's instructions. For analysis, the membranes were exposed for 15, 24, and 48 h to a phosphor screen and detected by using a Bio-imaging Analyzer BAS2000 (Fuji Photo Film). We used Atlas Image 2.01 software (Clontech) to record the pixel density of each spot and to perform background subtraction. The background-subtracted data were then further analyzed.

Quantitative RT-PCR.   To confirm the results obtained from the arrays, we subjected the RNA samples used in the gene chip array analysis to a separate quantitative real-time PCR (qRT-PCR) detection system to measure the mRNA of specific genes. Reverse transcription of total RNA was carried out in a 20-µl volume containing 1 µg of the total RNA sample, according to the manufacturer's instructions (RevertAid H Minus First Strand cDNA Synthesis Kit, Fermentas).

mRNA levels of specific genes were measured by qRT-PCR with the LightCycler system (Roche Diagnostics) using a Hot Start qPCR Master Mix kit with SYBRgreen (Eurogentec). Each sample of RNA was tested three independent times. beta-Actin mRNA was measured as a reference gene (35, 43). PCR products were detected via the intercalation of the fluorescent dye SYBRgreen. The primers used are presented in Table 2. Standards were prepared using serial dilutions of cDNA from untreated, nonacclimated rats. The protocol for the PCR included 5-min denaturation at 95°C, followed by 45 cycles consisting of denaturation for 15 s at 95°C, annealing for 22 s at 55°C for AT1, AT2, and beta-actin primers, followed by an extension phase of 15 s at 72°C. Fluorescence was measured at the end of the extension phase. Melting curve analyses were carried out at the end of each reaction to ensure the specificity of each reaction. The first PCR products for each gene were detected by using an agarose gel to verify that the length of the product matched that predicted for the gene of interest. The quality of the RT-PCR products was determined by melting-point curve analysis. mRNA values were calculated and corrected by using the control sample as an internal standard in each run, followed by normalization to the beta-actin mRNA level.


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  		Table 2. Real-Time PCR primer sequences

 
Calibration and normalization.   To avoid problems related to short- or long-term exposure to the phosphorimager (resulting in underestimation or saturation effect), we recorded the intensity of phosphorimages at different exposure times (between 3 and 60 h). To standardize different recordings of the same hybridization experiment, we performed a linear regression of the separate time points and averaged their regressed values to the recorded intensity at the middle time point. The individual data sets were used for comparing different experimental groups. To make the measurements from different hybridizations comparable, we rescaled each measured expression value by the average expression of all probes in that hybridization. We then transformed these values to make the control conditions a "baseline" for estimating the relative changes in expression. Specifically, we computed the log-ratio of a gene's (rescaled) expression value in a particular hybridization to the geometric mean of that gene's rescaled expression values in all hybridizations in the control group. We used a base-2 log so that a relative expression of 1 or –1 would indicate a twofold change from average control conditions.

Correlations and comparisons.   A series of analyses was carried out. Initially, we clustered the resulting data set using an agglomerative probabilistic method that groups genes together based on similar expression profiles (10) (ScoreGenes package, http://compbio.cs.huji.ac.il/scoregenes). This method iteratively assembles the genes such that the resulting set of clusters optimizes the likelihood that its genes come from the same distribution. We clustered two sets of data: 1) for analysis 1, we used the clustered data from euhydrated control (C), STHA, and LTHA to reveal groups of correlated genes in the course of heat acclimation; and 2) for analysis 2, we added the matched hypohydrated groups to the above and clusters were reassembled. Before clustering, we removed the genes that were not considered present in any group (those not visible by the phosphorimager) to reduce the effects of noisy data. The selection of the presence or absence of a particular gene was based on 50% presence in the total number of samples in the compared group in a series. However, this selection might be too conservative for within-treatment comparison because of a stress-specific gene evocation [e.g., acclimation phase (22), hypohydration (43)]. Therefore, we conducted within-treatment comparisons in which presence or nonpresence was based on the intensity threshold (above background) as well. A threshold of 1,200 pixels, which was compatible with spot visibility, was chosen. The specific cases for which this analysis was done are stated in the RESULTS section. After the cluster analysis, to sort the genes common to all acclimation phases (or alternatively the genes appearing in a single acclimation phase only), we constructed Venn diagrams using the SpotFire package. This second analysis was only performed on genes with an altered expression of more than 1.5-fold. For an overall functional interpretation of the common responses, these genes were sorted into groups according to their gene ontology (GO) database categories for biological processes (GO: http://www.geneontology.org). This database provides annotations regarding the functions and signaling of each gene. Pie charts were then constructed to present the data. To determine (among the significantly upregulated or downregulated genes) whether the most abundant pathways were significantly enriched and not coincidental, we used the GOstat program (http://gostat.wehi.edu.au/), which automatically obtains the GO annotations from a database and generates statistics for which annotations are overrepresented in the list of analyzed genes. The data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE2890.

Statistical analysis.   For the cluster analyses, a commercial statistics package (StatView, version 5.0) was used to determine statistical significance. Proof of significance for the kinetics of the transcriptional response over the time period of acclimation or hypohydration was provided initially by analyzing genes as groups (clusters) using a two-way ANOVA followed by multiple comparisons. Acclimation was the fixed effect for the first analysis, and acclimation and hypohydration for the second. For paired single-gene comparisons, we used the Student's t-test with Bonferroni correction. The pooled animal samples were considered as random samples from the animal population. A P value of ≤0.05 was considered statistically significant. For GOstat statistics the reader is referred to Beissbarth and Speed (2).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Of the 1,187 genes in the array, 915 genes were visible in the control (C-Euh) membranes. Among the euhydrated rats, 655 of the expressed transcripts (71.5%) changed their expression level by 1.5-fold or above that of C-Euh transcripts in at least one acclimation phase. Because previous studies (22) on heat acclimation showed that a cutoff of 1.5-fold is reliable for the interpretation of acclimatory responses, we used this set of genes for analysis. Analysis 1 aimed to characterize gene-expression dynamics during acclimation. For the Hyp state, the number of genes responding by an altered expression of at least 1.5-fold was higher (894 genes) than that recorded after acclimation at the Euh state. This set of genes was used to characterize the effects of hypohydration on heat acclimation dynamics.

Heat acclimation dynamics: overall profile of gene expression (based on analysis 1).   Compared with the preacclimation state, 315 genes (48% of those transcripts with an altered expression of at least 1.5-fold in one acclimation phase) changed their expression (up or down) only during STHA, whereas the expression of 115 genes (17.3%) differed only after LTHA. Clustering all the visible genes during the acclimation period on the basis of their mutual expression behavior provided a dynamic profile of the global genomic response throughout the acclimation process. Division of the data sets into 15 clusters revealed three dominant prototype genomic profiles (Fig. 1): The first profile showed a marked transient up- or downregulation in the expression of the genes upon STHA, with a return to the preacclimation expression level upon LTHA. The only difference seen between the various clusters showing this profile was in the magnitude of the increase in the averaged expression level of each cluster during the STHA phase. A second prototype profile comprised genes showing inverse changes in STHA and LTHA. A large number of genes assigned to one of these two profile types encoded either ion channels, pumps, or transporters. Taken together, both the first and the second profiles pinpoint the transient nature of membranal processes during the course of heat acclimation. In contrast, the third common pattern of gene profiling consisted of genes showing a persistent or continuous change in STHA and LTHA (Fig. 1). Prototype profile 3 comprises genes linked with trafficking and metabolic activities. The detailed profiles of the genes studied, organized into clusters according to similar expression dynamics, can be seen in the supplementary data for this article (http://jap.physiology.org/cgi/content/full/00850.2005/DC1). For further analyses, Venn diagrams were constructed to sort the genes characterizing each acclimation phase and those responding throughout the entire acclimation regimen.


Figure 1
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  		Fig. 1. Prototype-expression profiles over the period of heat acclimation. Representative clusters, demonstrating the 3 different profiles described (clusters 7, 9, and 2 for profiles 1, 2, and 3, respectively), are shown. Data are presented as the average expression of the genes assigned to each cluster in logbase2 of treatment-to-C-Euh ratio. *Significant difference vs. C-Euh (P < 0.05). C, control; Euh, untreated euhydration state; STHA, short-term heat acclimation; LTHA, long-term heat acclimation.

 
Genes expressed only during STHA.   Upon STHA, 134 of the 315 genes showing a transient change in their expression compared with the nonacclimated state (C-Euh) were upregulated. Functional annotation emphasized that 47% of the 134 genes encode ion channels, various transporters, or proteins associated with neurotransmission (e.g., synaptic transmission), signaling, or metabolism of transmitters (Fig. 2A). Among the genes of functional significance are the neurotransmitter transporters dopamine (M80570 [GenBank] ) and GABA (M95762 [GenBank] ); and neurotransmitter-receptors for acetylcholine (e.g., the nicotinic receptor X74835 [GenBank] and the muscarinic M4 M16409 [GenBank] and M2 J03025 [GenBank] receptor), {alpha}-adrenergic (M32061 [GenBank] ), serotonin (U59672 [GenBank] ), GABA (L08493 [GenBank] ), and glutamate (X95579 [GenBank] ) receptors, and ion channels such as potassium (M84203 [GenBank] , X83581 [GenBank] ), sodium (X70521 [GenBank] , M22253 [GenBank] ), and calcium channels (M57682 [GenBank] , M30581 [GenBank] ), all involved with ion currents and cellular transmission. This finding was further validated when GOstat software was used for the identification of significant enriched pathways (categorized by biological processes). Among the first 50 highly significant GO pathways delineated for this group of STHA-specific upregulated genes, 31 pathways are linked with transport activity, predominantly ions, and 10 pathways are associated with neural transmission. For representative enriched pathways comprising up- or downregulated genes vs. the nonacclimated C-Euh group, see Table 3.


Figure 2
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  		Fig. 2. Euhydrated state. Distribution of genes showing significant changes in their expression level (≥1.5, ≤–1.5) according to functional categories. STHA (A, D), genes changing expression solely upon short-term heat acclimation. LTHA (B, E), genes changing expression solely upon long-term heat acclimation. ST/LT-HA (C, F), genes showing constitutive change in expression throughout the entire acclimation process. Up, upregulated genes; Down, downregulated genes. Pie chart area corresponds with the number of genes up- or downregulated by more than 1.5-fold relative to the untreated control C-Euh. i, Immune response, ch, ion channels; transporters, pumps; t, trafficking; m, metabolism; r, receptors; o, kinases and proteases.

 

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  		Table 3. Heat acclimation dynamics: representative enriched pathways (up-or downregulated) over the course of acclimation

 
During STHA, 181 genes downregulated their transcript levels and were functionally different from upregulated genes (Fig. 2D). Approximately half of the downregulated genes code for hormones and other G-protein-coupled receptors or trafficking proteins. Among the downregulating genes are those that also participate in intracellular protein transport, such as ADP-ribosylation factors (L12384 [GenBank] , L12381 [GenBank] ) Janus kinases phosphodiesterase (JANKa-U13396, D28508 [GenBank] ), adrenergic receptors (D32045 [GenBank] , L31771 [GenBank] ), kinase adenylate cyclase (L01115 [GenBank] ), and adenosine A receptors (M64299 [GenBank] ). These genes have a role in cell maintenance and protein catabolism and phosphorylation (for examples, see Refs. 1, 12). Hence their downregulation is linked with perturbations in cellular performance. For significant enriched pathways, see Table 3.

Genes expressed only during LTHA.   After LTHA, changes were found in 140 genes that had not changed during STHA. In contrast to the STHA phase, only 30 genes were upregulated compared with the C-Euh, none of which encoded ion channels or transmitter receptors (Fig. 2B, top). The upregulated genes are associated with G protein-coupled receptors or their signaling pathways [e.g., receptors of arginine vasopressin (AVP V2; Z11932 [GenBank] )], glucagon-like peptide (GLP; S75952 [GenBank] ), glutamate, and acetylcholine (ACh), c-fos-induced growth factors (vascular endothelial growth factor D-AF014827) or cytokine receptors (e.g., interleukin-2 receptor-M55049), and intracellular transducers. The upregulation of genes linked with transcriptional regulation was also noted. Most of the LTHA-specific responding genes (99 genes) decreased their expression at the end of the acclimation process relative to their preacclimation basal levels (Fig. 2E). Of these, 49 genes encode proteins that are integral parts of membrane function, e.g., ion channels (X62841 [GenBank] : potassium channel, voltage gated; X16002 [GenBank] : potassium channel RCK4), transporters (e.g., glutamate, U28504 [GenBank] ), and transmitter receptors. The latter include G protein-coupled hormones and neuropeptide receptors such as AVP V1b (D45400 [GenBank] ), vasoactive intestinal polypeptide (VIP, U09631 [GenBank] ), serotonin, bradykinin (L26173 [GenBank] ), gastrin-releasing peptide (GRP, X56661 [GenBank] ), endothelin (X57764 [GenBank] ), cholecystokinin (CCK, S70690 [GenBank] ), and nicotinic receptors of ACh (L10077 [GenBank] ), as well as neuropeptide Y (NPY, U66274 [GenBank] ), beta1-adrenergic (D00634 [GenBank] ) and thyroid stimulating hormone (TSH, M34842 [GenBank] ) receptors, and the insulin growth factor (IGF, J04486 [GenBank] ). Most of the receptors and neuropeptides are involved with functions such as food intake, metabolic rate, or osmotic regulation (e.g., Refs. 5, 37). Thus the previously reported decrease in food intake and metabolic rate in LTHA rats, at the integrative level (25, 26), probably reflects changes in gene expression. For significant enriched pathways associated with the up- or downregulated genes vs. C-Euh, see Table 3.

Genes responding throughout the acclimation process.   Throughout the entire acclimation regimen, 276 genes comprising 193 upregulated and 83 downregulated vs. C-Euh altered their expression by more than 1.5-fold (Fig. 2, C and F, respectively). Of the upregulated genes, 50% are classified as immune system-associated cell-surface proteins (antigens, interleukins, transcription factors, antioxidants) and genes associated with cell maintenance [e.g., apolipoprotein, M00002 [GenBank] ; von Hippel-Lindau tumor-suppressor protein (VHL), U14746 [GenBank] ; and proteasomal proteins, e.g., proteasome subunit R-ring12, D10757 [GenBank] ; proteasome component C13 precursor, D10729 [GenBank] ]. Receptors of various transmitters (e.g., serotonin, L03202 [GenBank] ; glutamate, M85036 [GenBank] ; ACh, X74833 [GenBank] ) and cell signaling [inositol trisphosphate (IP3), X61677 [GenBank] ] were also upregulated. Among the downregulated genes, 34% code for hormone receptors associated with food intake, glucose metabolism, and in turn, body temperature (e.g., galanin, U94322 [GenBank] ; NPY, M20373 [GenBank] ; insulin, X58375 [GenBank] ; insulin growth factors, M62781 [GenBank] ; and glucagon, L04796 [GenBank] ), with an additional 45% having diverse functions including the cytokines IL-1{alpha} (D00403 [GenBank] ), IL-1beta (M98820 [GenBank] ), and EP3-r (D14869 [GenBank] ). Most genes showed a sustained and stable change in their expression throughout the entire acclimation regimen; nevertheless, the clustering of this group of genes drew our attention to several genes showing upregulation during STHA, with a subsequent decline upon LTHA, e.g., apolipoprotein (M00001 [GenBank] ) and other enriched pathways associated with lipid metabolism, APO-AIV precursor (M00002 [GenBank] ), VHL (U14746 [GenBank] ), and the NF-{kappa}B transcription factor p105 subunit (L26267 [GenBank] ) are noteworthy.

Global genomic response to hypohydration and its superimposition on heat acclimation (based on analysis 2).   For this analysis, genes were sorted by expression level in C-Hyp, STHA-Hyp, and LTHA-Hyp, respectively, vs. C-Euh. The results are presented in Figs. 3 and 4 and in Table 4. Hypohydration alone increased the number of visible genes. Among the nonacclimated rats, 445 genes were upregulated in hypohydration, whereas only 76 genes were downregulated. The main upregulated functional groups were ion channels, transporters and other trafficking proteins, hormone receptors, and immune system proteins (Fig. 3, control bars and pie charts), hormone receptors, and immune response proteins as above (e.g., Fig. 3, B and C). Downregulation was seen among genes coding for signaling molecules that are linked with metabolism/energy metabolism, kinases, and proteases and with several trafficking proteins (Fig. 3A). No difference was found between the Hyp and the Euh states in the functional composition of the clusters or in the gene expression dynamics, except that six of the analysis 1 assigned clusters now included a significantly higher number of genes. The data, organized in their clusters, are presented in supplementary file 1.


Figure 3
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  		Fig. 3. Left: tree view of all genes included in analysis 2, that changed expression in at least 1 treatment group. Each row represents 1 gene; the columns represent treatment groups. All changes were compared with the control euhydration group. Red, upregulation; green, downregulation; black, no change. C, control euhydration; CH, control hypohydration; SA, short-term heat acclimation; SH, short-term heat acclimation with hypohydration; LA, long-term heat acclimation; LH, long-term heat acclimation with hypohydration. Yellow borders assign clusters 2, 7, 1, and 9, respectively. Middle and right: changes in the average expression levels of clusters 2, 7, 1, and 9 over time and functional distribution of the genes in assigned clusters. Black bars indicate euhydration (Euh) and gray bars hypohydration (Hyp). Values are presented as logbase 2 treatment/C ratio. *Significant difference vs. C-Euh (P < 0.05). Pie chart area corresponds to the number of genes in each cluster. For pie-chart labeling see legend to Fig. 2.

 

Figure 4
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  		Fig. 4. Hypohydrated state. Distribution of genes showing significant changes in expression levels (≥1.5, ≤–1.5) with acclimation according to functional categories. A and D: STHA. B and E: LTHA. C and F: ST/LT-HA. Pie chart area corresponds with the number of genes up- or downregulated by more than 1.5-fold in relation to C-Euh. For symbols, of pie chart labeling see legend to Fig. 2.

 

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  		Table 4. Hypohydration superimposed on heat acclimation

 
The global genomic response to hypohydration differed at each heat acclimation phase. Figure 3 shows that, compared with STHA-Euh, a desensitized response (e.g., cluster 2), or even silencing of many genes occurred after STHA-Hyp. Upon LTHA, a recovery of the hypohydration genomic response was noted, seen by the return of the number of genes responding, and the resemblance in composition of each functional group to that of the C-Hyp state. Thus the biphasic profile characterizing a large number of genes during acclimation (see also Fig. 1, profiles 1 and 2) was maintained, although in a reciprocal manner, as demonstrated in Fig. 3 (left: C-Hyp and LTHA-Hyp vs. STHA-Hyp Treeview bars and, e.g., Fig. 3C, cluster 1 profile). In cluster 1, the average expression of C-Hyp and LTHA-Hyp (2.7- and 1.8-fold, respectively) is reciprocal to that of the STHA-Hyp (–3.7-fold). Of the genes in this cluster, 45% encode hormone receptors, neurotransmitters, interleukins, and other genes related to water intake. A large number of additional clusters (not shown) shared a similar profile. For further details, see supplementary file 1.

Venn analysis (Fig. 4) confirmed that upon hypohydration, the number of the responding genes vary with heat acclimation and that the response is desensitized upon STHA. Hypohydration per se induces a marked increase in the expression of genes coding for ion channels and other ion transporting mechanisms [e.g., voltage-gated ion channels (M68880 [GenBank] , L48619 [GenBank] ), ATP-dependent channels (M92848 [GenBank] ), cation and anion transporters (AB000113 [GenBank] , L19031 [GenBank] ), and Ca2+ regulation proteins] as well as an upregulation of genes linked with neural transmission [e.g., serotonin, dopamine, IP3 receptors, and antioxidation (Cyt p450 units, J05156 [GenBank] , U09540 [GenBank] )]. This striking phenomenon was observed in all groups (C-Hyp, STHA-Hyp and LTHA-Hyp), both in the acclimatory phase-specific genes and among those genes responding throughout the course of acclimation (shared genes): STHA 26%, LTHA 39%, shared 62%. Consequently, the gene-expression profile of the LTHA-Hyp hypothalami resembles that of STHA-Euh rather than that of LTHA-Euh (Fig. 2A vs. Fig. 4B, top). These findings were confirmed when GOstat software for identification of abundant enriched pathways was used (Table 4).

Taken together, the number of downregulated transcripts was significantly lower than that of the upregulated genes (Fig. 4, bottom). The downregulated genes were from completely different functional groups than the upregulated genes. A dominant function in all experimental groups was that of trafficking, particularly in LTHA-Hyp specific (50%) and STHA-Hyp, the LTHA-Hyp shared (36%) genes. Hormones and neurotransmitter receptors were abundant among the STHA-Hyp (41%) genes but not in LTHA-Hyp or shared (7 and 9%, respectively) genes.

Confirmatory analysis.   To validate the results obtained in the gene chip array (22), on the basis of the hybridization outcome in the various acclimatory phases, we used real-time PCR to measure the expression of four genes with significant differences in their expression compared with the C group and representing separate functional biological categories: 1) cellular activation markers, c-fos proto-oncogene; 2) signal transduction, inositol 1,4,5-triphosphate receptor; 3) humoral pathway, galanin receptor; and 4) peptide YY precursor. Basal as well as LTHA and LTHA-Hyp stress levels were measured. Congruence was found between the results obtained for the individual genes mRNA using qRT-PCR and their expression on the array membranes (Fig. 5).


Figure 5
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  		Fig. 5. Changes in expression level of confirmatory representative genes, c-fos proto-oncogene (c-fos), inositol 1,4,5-triphosphate receptor (ip3), galanin receptor (galanin), and peptide YY precursor (pyy), compared with a standard control sample in response to untreated hypohydration (C-Hyp), LTHA, and combined LTHA-Hyp, using real-time PCR. Each bar represents the average of 3 separate PCR products from 3 samples. *Significant difference compared with the untreated C-Euh.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The hypothalamus is a key regulator of body homeostasis and an integrator of diverse functions such as circadian rhythm, food and water uptake, and energy balance. Genes associated with these aspects in the rat cDNA array used here enabled the delineation of global hypothalamic genomic responses to sustained environmental heat in rats, thereby revealing how acute hypohydration stress modulates the response. Our data show 1) that the process of acclimation to environmental heat stress induces a continuum of molecular responses that temporally vary in a selective manner and 2) that an interaction with overriding hypohydration stress involves either silencing or activating specific groups of genes, regardless of the acclimation status. In the discussion below, we shall focus primarily on these issues with respect to homeostasis. Additionally we will reveal and discuss novel functional groups in heat acclimation.

Heat acclimation dynamics: a continuum of genomic responses.   The hypothalamic transcriptome map outlined here implies that the process of acclimation coincides with three major categories of genomic responses: 1) abrupt transient upregulation or downregulation of genes during STHA, followed by their return to preacclimation levels when acclimation homeostasis has been achieved (LTHA phase); 2) genes showing downregulation or upregulation only upon LTHA; and 3) genes showing a sustained change in their expression throughout the entire acclimation period.

The transcriptome map of genes assigned to the first category of responses emphasizes two phenomena: 1) a marked transient upregulation in transcript confined predominantly to genes encoding voltage-gated ion channels, ion pumps, or transporters, as well as hormone or transmitter receptors and cellular messengers, collectively points to enhanced membrane depolarization, leading to the release of transmitters and neuronal excitability at this acclimation phase; and 2) a transient downregulation among the groups of genes participating in intracellular protein trafficking, metabolism, or phosphorylation processes implies a perturbation in cellular maintenance (1, 12). These transient responses are similar to those that characterize stressed or traumatic brain conditions (7, 40). In contrast to the STHA phase, a decrease in the expression of specific LTHA-activated genes related to various metabolic activities, including those linked with mitochondrial energy metabolism and cellular maintenance processes, together with the resumption of preacclimation transcript levels of genes encoding proteins linked with ion movement and membrane or cellular signaling, is noteworthy. An additional significant finding is the constitutive downregulation of genes associated with energy metabolism and food intake and the marked upregulation of a large group of genes linked with the immune response.

The global transcriptional sketch delineated above is consistent with the physiological model of the acclimation process, a shift from an early transient, perturbed state (STHA) to a long-lasting "efficient" and stable acclimatory homeostasis (25). Work from our group (18) and others (15) demonstrated that during the STHA phase there is an increase in salivation, moderate dehydration, as well as a reduction in food intake and a transient increase in metabolic rate (15, 18). A loss of the circadian rhythm of activity and drinking behavior (Huebschle T, Schwimmer H, Horowitz M, and Gerstberger R, unpublished observations) suggests the occurrence of central and integrative perturbations. In earlier reports, we predicted that at this acclimation phase 1) increased autonomic excitability compensates for impaired postsynaptic signaling (cholinergic, adrenergic) and for other cellular disturbances in heat-dissipating organs (8, 24, 25, 29, 31), and 2) peripheral effectors with central feedback control the acclimatory neuronal excitability (27). Therefore, the upregulation of genes functionally associated with neuronal excitation seen in the present investigation supports our predictions.

The transcriptional changes found in the hypothalamus of STHA rats resemble the transcriptional profile of hyperosmotic stress, exemplified by the transcription of genes encoding ion pumps and channels [Na+, K+, and Cl–, and Na channels (14, 47)] and AQP3 (45) to reestablish cellular volume. The upregulation of, e.g., cytochrome c-oxidase, voltage-gated anion channels transcripts characterizing hypernatremic stress (13, 49) was also identified in our model of the STHA. Notable is the resemblance of the transcriptome map of STHA-Euh hypothalami to that of the C-Hyp group. Thus the results of this investigation suggest that stimuli induced by moderate dehydration occurring at STHA are additional players in the peripheral-central control feedback setup. Acclimation homeostasis, in contrast, is characterized by improved metabolic and physiological performance with a marked reduction in heat production (19, 26). In peripheral heat-dissipation effectors, for example, increased physiological efficiency is manifested by a greater effector output-to-activation stimulus ratio (25) or by the redistribution of wasteful vs. economic ATPase isoforms (25, 26). Such constitutive long-lasting adjustments diminish the need for the enhanced autonomic excitability characterizing the STHA phase. Indeed, the LTHA gene profile showing a decreased expression of genes associated with food intake and glucose metabolism supports the notion of an economical integrated physiological map. A molecular profile of acclimation in the hypothalamus has only been documented for three genes that are associated with the angiotensin-mediated neuromodulation of thermoregulation (43). Our present investigation extends these findings and shows, for the first time, that the acclimation process responds with a continuum of large-scale hypothalamic genomic activity. The results of the present study, together with our knowledge of global molecular acclimatory responses in the heart (22, 26), indicate a two-tier defense strategy. The first is an immediate transient response, associated with the maintenance of cellular integrity during the strain of the onset of acclimation, and the second is a sustained response correlated with adaptive, constitutive processes.

Hypohydration and heat acclimation: response interference.   Hypohydration induced several notable responses: 1) Hypohydration alone resulted in a significantly larger number of upregulated transcripts relative to the control euhydrated state. 2) With the onset of heat acclimation, the hypothalamic "hypohydration response" was desensitized. The STHA-Euh gene profile seemed to resemble that of C-Hyp rats, but superimposition of hypohydration at that acclimation phase did not induce further transcription upregulation, and gene silencing was evident. 3) After LTHA, recovery of the molecular hypohydration response maintains the biphasic acclimation profile. The finding that the gene profile of LTHA-Hyp rats resembles that of STHA-Euh rats is noteworthy because it links the impaired heat endurance occurring under these physiological conditions and the specific transcriptome profile, as discussed below.

Sorting the genes according to functional groups revealed that the predominant transcriptional activation could be categorized into genes associated with transmembrane ion transport, including those associated with potassium currents, sodium and calcium conductance, and neuronal signaling. In the two groups (C-Hyp and LTHA-Hyp) showing the full scope of the molecular hypohydration response, vasopressin and angiotensin receptors, cytochrome c oxidase, and several transporters characterizing supraoptic nucleus activation (48) were upregulated as well. Concomitantly, we noted a downregulation of a large number of gene transcripts associated with maintaining homeostatic cellular processes. Most of the activated genes were linked with cell volume regulation and neuronal excitability. This profile resembles that characterized for STHA-Euh and is analogous with the physiological profile described for various brain traumatic situations associated with cellular energy exhaustion and, in turn, with depolarization, transmitter release, etc. (e.g., 7, 40). The enhanced activation of pathways associated with the regulation of cell volume agrees with our previous findings that a hypohydration of –10% in body weight does not affect plasma osmolarity or Hct, suggesting that the main water loss under our experimental conditions is intracellular (30). Furthermore, the enhanced transcription of neurotransmitters and neurotransmitter receptors, such as the GABA or adrenergic signaling observed here, follows the findings of Di and Tasker (6). Using whole-cell recordings in hypothalamic slices to study the chronic, dehydration-induced plasticity of magnocellular neurons in the rat supraoptic nucleus, the authors showed that dehydration leads to an increase in glutamate and GABA release onto supraoptic magnocellular neurons, as well as a marked enhancement of the facilitatory effect of norepinephrine on glutamate release and an inhibitory effect on GABA release, collectively leading to increased excitability of the magnocellular neurons (6).

An intriguing question is why hypohydration interferes with the beneficial effects conferred by heat acclimation. In vivo studies have shown that, during hypohydration, the temperature threshold for heat-dissipation mechanisms is elevated (21, 43), whereas the metabolic rate decreases (27). Despite significant differences in temperature thresholds for heat dissipation in LTHA-Hyp vs. C-Hyp and STHA-Hyp rats, their thermal tolerance markedly decreases, hence the beneficial effect of heat endurance gained by heat acclimation disappears (43). The water loss of acclimated-hypohydrated heat-stressed rats is significantly higher than that of control rats (43). Considering that the salivary glands of heat-acclimated rats (the evaporating cooling effector of this species) increase their output-to-stimulus ratio (25, 31) when acclimated, we hypothesize that the disruption of acclimatory thermal endurance in the hypohydrated state is due to a failure in adjusting secretion at the glandular level rather than to a central failure.

Unmasking novel functional groups in the heat-acclimated phenotype.   The results of the GO analysis confirm our previous findings of heat acclimation-mediated enhancement of the three cytoprotective networks: antiapoptosis, antioxidation, and heat shock responses (22), pointing to important functional groups that form the acclimated phenotype. Given the complexity and nonspecificity of many of the emerging interactions, we will now focus on two distinct functional groups that seem to play a main part in the formation of the acclimated phenotype. A prominent finding is the massive effect of acclimation on transcripts linked with membrane-associated processes. This finding calls our attention to the importance of the presence of a large number of GO pathways, implicating enriched lipid metabolism in the acclimated hypothalamus, including genes associated with cholesterol biosynthesis, apolipoproteins, lipids transporters, etc. (Table 4). Cholesterol and fatty acid synthesis associated with membrane integrity are a crucial part of the acclimatory response to temperature in poikilotherms (17). In homeotherms, this issue has been less studied. Shmeeda et al. (41) provided evidence that heat acclimation augments the level of polyunsaturated fatty acid docosohexanoic acid, together with variations in the cholesterol-to-phospholipid ratio. Membrane enrichment with docosohexanoic acid represents a strategy for the protection of membrane integrity via its interplay with cholesterol to control the membrane free volume, implying lipid-protein interactions (36) or membrane water capacitance. Taken together, our data suggest that membrane plasticity is an integral part of the acclimatory response to provide receptor and signaling integrity.

An additional finding that caught our attention is the global influence of acclimation on a large body of cytokines and various pyrogenic neuropeptides and neuropeptide receptors. To our knowledge, this acclimation effect has not yet been reported. The activation of pyrogenic cytokines is traditionally linked with fever and pathological conditions, the sequence of various heat illnesses, or strenuous exercise. An imbalance between the released inflammatory and anti-inflammatory cytokines can result in inflammation-associated injuries, refractory immunosuppression, and severe neuronal injury (4). IL-1 receptor antagonists or corticosteroids given to animals before heat stroke improve survival (33, 34). In the present investigation, the concomitant transient downregulation of pyrogenic cytokines (IL-1r, IL-4r, IL-8r, EP1-r) and decrease in temperature threshold for heat dissipation effectors during STHA, both returning to preacclimation values upon LTHA, and the constitutive downregulation of NPY, IL-1{alpha}, IL-1beta, IL-10, and EP3-r throughout the entire acclimation period imply that all these factors play a role in the normal thermoregulatory response. Nevertheless, the cross talk between heat acclimation and changes in proinflammatory cytokines has not yet been explained. Furthermore, reports concerning interactions between combined heat exposure and fever are sparse (3, 32).

In summary, we have shown that heat acclimation involves substantial but transient central genomic responses during the course of the acclimation period. STHA is characterized by an upregulation of the genes coding ion channels, transporters, and neurotransmitter signaling. We hypothesize that cell volume regulation, associated with ion fluxes, depolarizes the cell membrane and leads to the release of transmitters, with an enhanced excitability occurring at that acclimation phase. After LTHA, a downregulation of genes encoding G-protein-coupled receptors and signaling proteins is evident. Membrane lipid remodeling is likely to play a pivotal role in the latter response. Previous reports that hypohydration overrides heat acclimation were confirmed here at the genomic level. The genomic response was manifested by upregulation of genes involved with ion channels and transporters. Interestingly, during the transient STHA phase the hypohydration response was desensitized and overridden by the acclimation stress. However, when acclimatory homeostasis had been achieved the characterized hypohydration gene profile was seen again. Collectively, our overview highlights significant facilitatory and interfering pathways that are influenced by acclimation and provides a basis for exploring the specific mechanisms involved with heat acclimation and its interference.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the German Israeli Foundation Research Grant I-572-22.2/1998 to M. Horowitz and R. Gerstberger.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Horowitz, Laboratory of Environmental Physiology, Faculty of Dental Medicine, The Hebrew Univ., POB 12272, Jerusalem 91120, Israel (e-mail: horowitz{at}cc.huji.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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