Chemical Senses Advance Access originally published online on June 21, 2007
Chemical Senses 2007 32(7):697-710; doi:10.1093/chemse/bjm037
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Transcription Profile Analysis Reveals That OBP-1F mRNA Is Downregulated in the Olfactory Mucosa Following Food Deprivation
1 INRA, UMR1197 Neurobiologie de l'Olfaction et de la Prise Alimentaire, Récepteurs et Communication Chimique, F-78350 Jouy en Josas, France 2 Univ Paris-Sud, UMR1197, Orsay, F-91405, France 3 INRA, Unité Mathématiques et Informatique Appliquées, F-78350 Jouy en Josas, France 4 UMR INRA/CEA Radiobiologie et Etude du Génome/Centre de Ressources Biologiques pour la Génomique des Animaux d'Elevage et d'Intérêt Economique, F-78350 Jouy en Josas, France 5 Laboratoire de Neurobiologie et Diversité Cellulaire, CNRS-ESPCI UMR7637, F-75231, Paris, France
Correspondence to be sent to: C. Baly, Unité NOPA/RCC, UMR1197 INRA-Paris 11, CRJ, Domaine de Vilvert, F-78352 Jouy-en-Josas, Cedex, France. e-mail: christine.baly{at}jouy.inra.fr
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Abstract |
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Neuroanatomical data show that olfactory mucosa (OM) is a possible place for interactions between nutrition and smell. A combination of differential display mRNA analysis together with a macroarray screening was developed to identify transcripts that are differentially expressed in rat OM following food deprivation. Using this method, backed on a stringent statistical analysis, we identified molecules that fell into several Gene Ontology terms including cellular and physiological process, signal transduction, and binding. Among the 15 most differentially expressed molecules, only one was upregulated, but 14 were downregulated in the fasted state among which was, unexpectedly, odorant-binding protein 1F (OBP-1F). Because of its potential relevance to olfactory physiology, we focused our further analysis on OBP-1F using in situ hybridization, quantitative polymerase chain reaction, and western blot analysis. OBP-1F was highlighted in the lateral nasal glands, but its expression (mRNA and protein) did not change following food deprivation. Only the minor fraction of OBP-1F mRNA expressed by the OM itself was downregulated following 48 h fasting. Altogether, our results suggest that the fine transcriptional control of OBP-1F in the OM following food deprivation could be efficient only at the local level, close to its site of secretion to participate in the perireceptor events of the olfactory signal reception.
Key words: food intake, lateral nasal glands, macroarray, neurotransmission, odorant-binding protein, q-PCR
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Introduction |
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Olfaction is a major chemical sense involved in several mammal behaviors such as food intake, social interactions, or reproduction. The relations between olfaction and nutrition are multiple and have been hypothesized for a long time (Pager 1974
). Olfactory stimuli induce metabolic modifications partly through the regulation of the digestive tract activity. Food odors influence circulating glucose and insulin levels and induce saliva secretion and gastric contractions (Mattes 1997). Neuroanatomical data show that olfactory mucosa (OM) and olfactory bulb (OB) are possible places for these interactions: the hypothalamic structures, which analyze the metabolic signals and in return control food intake and energy expenditure, project themselves directly to the OB (Hardy et al. 2005). In the OM, recent studies show the presence of many peptides and their receptors related to feeding status: orexins (Caillol et al. 2003) and leptin (Baly et al. 2007), some being locally synthesized and the others being brought by the general circulation. Moreover, an orexigenic peptide, orexin A, reinitializes a starving status in a fed rat (Sakurai et al. 1998) and modulates mitral cells response of the OB (Hardy et al. 2005). If these neuroanatomical and behavioral data support a possible interaction between smell and nutrition, few molecular analyses were undertaken to extend these results at the molecular level.
Olfaction is based on the reception of odorant molecules reaching the OM. The OM is located in the posterior part of each nasal fossae (Nef 1998); it consists of an olfactory epithelium (OE) lying on the lamina propria (LP). The OE is composed of olfactory sensory neurons (OSNs) surrounded by supporting cells and of basal stem cells that ensure the continuous renewal of the neurons throughout life. Each bipolar sensory neuron extends a single dendrite with a dendritic knob bearing ciliae, where the olfactory receptors are expressed (Pelosi 1996). The odorant molecule reaches the OSN ciliae through a thin layer of mucus covering the OM. The mucus is produced at the surface of the OE by mucous secretory cells and by subepithelial glands of the LP. Major protein components of the mucus are the odorant-binding proteins (OBPs), which are members of the lipocalin family (Flower et al. 1993; Pelosi 1994; Briand et al. 2000). Among the 3 described rat OBPs, OBP1 is synthesized by the lateral nasal gland (LNG), one of the largest nasal glands in rats, located in the posterior area of the nose, just ventral and anterior to the OM (Pevsner et al. 1988). It is secreted in high amounts via a long duct to the tip of the nose to be atomized as watery secretions. Precise roles of OBP in early olfactory perireception events remain unclear.
The mucus composition, the renewal, the differentiation, and death of the cellular populations of the OM are continuously under the control of nervous and hormonal regulations. Not only do these regulations allow an accurate transmission of the olfactory signal but they can also modulate this signal according to the physiological status of the animal, such as the nutritional status.
In this paper, we studied the putative link between these 2 important physiological functions through a transcriptional profile analysis of OM of Wistar rats. We investigated the gene expression patterns using an RNA differential display analysis (ddmRNA) followed by a high-density filters (macroarray) measurement, between normally fed (fed) or 48-h food-deprived (fasted) rat OM. We identified several molecules exhibiting a modified level of expression between the 2 nutritional conditions. A single molecule, the odorant-binding protein 1F (OBP-1F) (Briand et al. 2000), was chosen for further investigations because of both its large differential expression between the 2 nutritional conditions and its broad implication in the olfactory function.
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Animals, diet, and experimental design
Male Wistar rats, 10–12 weeks old, from our breeding stock were housed in 12:12 h light/dark cycles with free access to food and water. All experiments were conducted according to the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number and the suffering of rats. Experimental conditions were based on the use of either ad libitum fed (fed) or 48-h food-deprived (fasted) rats, a restriction protocol currently used in experiments studying central effect of food deprivation (Bariohay et al. 2005). Animals were killed by carbon dioxide asphyxiation and sacrificed by decapitation 2h after the beginning of the light phase. The OM and LNG (setting down on a cartilaginous structure at the anterior part of the nose, just underneath of the OM) were quickly removed and dissected on an ice-cold plate. Nasal OM as isolated was devoid of LNG.
Independent groups of fed or fasted rats were used for ddmRNA screening (n = 3, for each nutritional status), macroarray experiments (n = 4, for each nutritional status), in situ hybridization (ISH) (n = 3, fed rat), and western blots (n = 5, for each tissue and nutritional status). For real-time polymerase chain reaction (PCR), samples of LNG (n = 5 for each nutritional status) and OM (n = 5 for each nutritional status) were collected from brother rats and prepared as described below.
Total RNA isolation
Total RNAs were prepared from OM and LNG of fasted or fed rats following the guanidium thiocyanate–phenol-chloroform extraction method (Chomczynski and Sacchi 1987) and then DNAse-I treated before quantification using spectrophotometry (macroarray) or Agilent profile (quantitative q-PCR).
For membrane screening, 50 µg of total RNA was enriched in mRNA using Clontech Atlas Pure Total RNA Labeling System, according to the manufacturer's instructions.
For the ddmRNA strategy, OM mRNA samples were pooled from 3 fed and 3 fasted rats, DNAse-I treated and reverse transcribed following the manufacturer's recommendations using the Delta Differential Display kit (Clontech, Saint-Germain-en-Laye, France). Briefly, PCRs using labeled [
33P] dATP were performed using the anchor primer in combination with series of selected primers in conditions of low stringency. Amplified cDNA of fasted or fed samples were then resolved in adjacent lanes of a high resolution 15% polyacrylamide gel, and differentially expressed bands (n = 88) were excised from the gel, eluted, cloned into pCR2.1-TOPO plasmid (TA cloning kit, Invitrogen, Cergy-Pontoise, France), transformed in Escherichia coli before sequencing (Genome Express, Paris, France), and identified by Blast against nucleotide or translated and blasted against protein databases.
Constitution of the macroarray
Each array contained 203 molecules spotted in duplicate, obtained either from the differential display screening (n = 88, see above) or from a set of 94 genes involved in neurotransmission amplified from rat brain cDNA (provided by Dr Potier, ESPCI, Paris) together with samples of interest involved in food intake regulation or olfactive functions (n = 21). The bacterial clones are available under request at the CRB GADIE (francois.piumi{at}jouy.inra.fr).
The cDNA inserts were amplified, purified, quantified, and spotted in duplicate on Hybond N+ nylon membranes (8 cm x 12 cm, Amersham Biosciences, Orsay, France) using the "Qpix" robot (Genetix, Hampshire, UK). After spotting, the DNA was denatured (NaOH 0.5 M, NaCl 1.5 M; 5 min), neutralized (Tris 0.5 M, NaCl 1.5 M; 5 min), and fixed onto the membrane (2 h, 80 °C). The membranes were washed with 2x sodium saline citrate (SSC), air dried, and stored at room temperature until use.
Functional information about encoded proteins was gathered from comparisons between these sequences and the rat or mouse Gene Index (Rat Release 13.0 as 10 May 2004 and Mouse release 15.0 as 17 February 2005, http://www.tigr.org). The Institute for Genomic Research gene indices provided links from TC (Tentative Consensus sequences) to Gene Ontology (GO) terms. A comprehensive scheme of the constituted membrane is depicted in Figure 1. The complete list of gene fragments is given in Table 1.
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Synthesis of [
33P]-labeled cDNA target
One microgram of OM mRNA isolated from 8 rats (n = 4 for each experimental conditions) was reverse transcribed using Superscript First-Strand Synthesis System for RT-PCR Kit (Invitrogen) with 500 ng of hexamers (Promega, Charbonnieres, France) and 50 U Superscript II (Invitrogen) in the presence of 10 mM of each dCTP, dGTP, and dTTP and [33P] dATP (50 µCi).
Hybridization and posthybridization processing
Prehybridization was carried out in ExpressHyb Hybridization Solution (Clontech) during 30 min at 65 °C. Each complex target was denatured in boiling water for 5 min and hybridized in parallel with 8 distinct arrays overnight at 65 °C in rotating tubes. After a series of washing steps with high stringency, the hybridized arrays were exposed to a phosphor screen that was scanned using FLA 3000 system (Fujifilm, Düsseldorf, Germany). Spots treatment and signal quantification were done using AIDA software (Fujifilm). As expected, controls corresponding to water and vegetal-specific spots were negative.
Data analysis
Individual spot's intensities were corrected for background level and normalized for differences in probe labeling using the average intensity of spots. Changes in gene expression were calculated by the ratio of the spot intensity in fasted versus fed experimental conditions. The ratios (fold-factor) were independently calculated for each experimental replication, and genes whose expression ratio was more than twice or less than half between fed and fasted conditions were selected as candidates. The differential expression of candidates was validated by statistical analysis using 2 different algorithms in "R" language (Team 2004). A first program determined whether the effective gene selection could have been the result of a random selection. The second one computed the probability, for each candidate, that the expression ratio between the 2 nutritional conditions was randomly distributed. The level of differential expression and the resulting statistical probabilities (P-values) were assigned to each candidate. The expression level between duplicate spots from the membranes hybridized with targets made from the same nutritional status was always highly correlated (r > 0.99).
RT–real-time qPCR
cDNA (60 ng) was obtained by reverse transcription of 1 µg of mRNA for each sample, using Superscript First-Strand Synthesis and mixed with 10 µl Power SYBR Green PCR Master Mix (Applied Biosystems), 300 nM from each primer complementary either to OBP-1F (forward primer: 5'-CAAGTGTCTGTGGGCACCAA-3'; reverse primer: 5'-GCTGGCTGAGGAATATAATATTCCA-3'; PCR product = 63 bp) or ß-actin (forward primer: 5'-GACCCAGATCATGTTTGAGACCTT-3'; reverse primer: 5'-CACAGCCTGGATGGCTACGT-3'; PCR product = 61 bp) in 20 µl total volume. ß-Actin was chosen as the reference based on its constant expression following food deprivation. The reaction mixture was finally transferred into a 96-well optical reaction plate, sealed with appropriate optical caps, and ran on the ABI Prism 7900HT (Applied Biosystems) apparatus under standard conditions. Standard controls of both specificity and efficiency of the qPCR assays were performed. All expression data were normalized to ß-actin expression level from the same individual sample. All results are given as mean ± SEM. Comparison between groups was made using Mann–Whitney test. Statistical significance was taken as P < 0.05.
Tissue preparation and ISH
ISH was carried out on OM sections from 2-month-old normally fed Wistar rats. After deep anesthesia (intraperitoneal injection of pentobarbital, Sanofi Synthelabo, France), 3 adult rats were perfused transcardially with 200 ml saline and then with 300 ml of a freshly prepared fixative solution of 4% PFA in PBS. Bones of the skull were discarded from the lateral and dorsal walls of the nose, and the nasal mucosa was removed as a block. Tissues were postfixed in the same fixative for 3 h at room temperature, cryoprotected with sucrose (30%), and cut in a cryostat in serial anteroposterior nasal mucosa sections of 14 µm thickness (Figure 2).
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OBP-1F mRNA probe was obtained after in vitro transcription (IVT) of the recombinant 2.1 TOPO plasmid (Invitrogen) containing the PCR product generated after amplification using customized random primers (Delta Differential Display kit, Clontech) and corresponding to a fragment of rat OBP-1F sequence (NM_138903; position 462–756). The sense and antisense probes were synthesized after plasmid linearization and IVT using T7 or Sp6 RNA polymerase in the presence of digoxigenin–dUTP (DIG-dUTP) using DIG RNA labeling kit (Roche, Meylan, France) according to manufacturer's recommendations. Before hybridization, the slides were treated with proteinase K (10 µg/ml; 5 min), triethanolamine (0.1 M; 10 min), and anhydrous acetic acid (2.6%). Sections were dehydrated by successive baths in ethanol with increasing concentration (50–100%). The labeled probes (0.1 µg/µl) in hybridization mix were denatured beforehand at 68 °C during 10 min and hybridized overnight at 55 °C. After 3 washes in 2x SSC for 30 min each, RNAse A treatment (20 µg/ml) for 30 min, immunodetection was performed using DIG Nucleic Acid Detection kit (Roche) following supplier's recommendations. Revelation time was 10 min for section 1, 40 min for section 2, and 5 h for sections 3 and 4. Slides were mounted in Vectashield (Vector AbCys, Paris, France).
Phase-contrast images were acquired on an upright Leica DMR HC microscope equipped with a color Olympus DP 50 camera using ViewFinder lite dedicated software for acquisition and Olympus Bio System CellF-dedicated software for 2D reconstruction. All images were adjusted for contrast and brightness to equilibrate light levels. Images were cropped, resized, and rotated for presentation's sake. The images were not software modified in any case.
Western blot analysis
In order to quantify OBP-1F protein, we collected the mucus by direct application of sample discs (Wescor, Logan, UT) on undamaged tissues in different regions of the OE: between septum and turbinates and on both external sides of the olfactory turbinates, or inside LNG, or inside the respiratory trachea as a negative control. Anyway, bloody samples were discarded from the study. Humidified sample discs were kept for 30 min in cold buffer (10 mM HEPES–NaOH, 150 mM NaCl, 1 mM EGTA), 1% phenylmethylsulfonyl fluoride, and a cocktail of anti-proteases (Complete, Roche Diagnostics) on a rotating wheel at 4 °C, and then centrifuged at 5000 r.p.m., 10 min. Supernatants were collected and protein levels quantified using BCA protein assay (Pierce, Perbio Science, Brébières, France). To test the presence of OBP-1F, we performed an 8% SDS-PAGE analysis of boiled aliquots (5 min) of 25 µg of total proteins from each extracts. Following electrotransfer, membranes were blocked with 5% no-fat milk at room temperature for 1 h, then incubated overnight at 4 °C in 4.5% no-fat milk containing a custom-made rabbit polyclonal antibody (1:500) developed by Eurogentec (Belgium), and directed against recombinant OBP-1F (Briand et al. 2000). Control using a preabsorbed antibody (1:1 in equimolar concentration) was negative (see Results). After extensive washing in PBS–0.5% milk (3 x 15 min each), the membranes were incubated with goat anti-rabbit secondary antibody coupled with horseradish peroxidase (1:5000, Sigma Aldrich, l'Isle-d'Abeau, France). The targeted proteins were detected using ECL Western Blotting Detection kit (Amersham Biosciences). The integrated density of protein level of each band on the blots was determined by densitometry using the ImageJ software (ImageJ).
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Results |
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Identification of genes differentially expressed following food deprivation
To study the differential expression of genes in the OM of rats following food deprivation, we spotted a nylon membrane with a mixed collection of samples (Table 1). It included gene fragments (n = 88) isolated from a differential display screening between fed and fasted rats. Among them, 77 samples matched with already annotated sequences from the rat and mouse gene index (Table 1), whereas 11 were unidentified in the available databases. A collection of genes involved in neurotransmission (n = 94) and some samples of interest (n = 21) completed this array (Table 1). Altogether, almost half of the total 203 cDNA spotted on the macroarray (n = 92) were linked to a GO term. Those molecules included functions related to cellular and physiological processes, binding, and signal transduction activities (indent 2 of the GO tree; Figure 1). The spotted macroarray was probed with labeled retrotranscribed RNA either from OM of normally fed (fed, n = 4) or from 48-h food-deprived rats (fasted, n = 4) rats. Normalization and statistical treatment of hybridization signals between fed and fasted rats revealed that 15 molecules were differentially expressed, by at least a 2 fold-factor (P < 0.05 in each case): 14 were downregulated in fasted state and only one was up regulated, the ribosomal 18S RNA (Table 2). Out of the 15 sequences, 11 matched known proteins in databases and were classified depending on their biological functions. Four neurotransmitter receptors, but also molecules relevant to the OM function (OBP, OMP, or glial fibrillary acidic protein [GFAP]), were obtained with scores of gene repression ranging from 2.36 to 5.43. Among the unknown molecules, RP23-125 corresponds to a Riken cDNA of mouse, RP23-81P12 is homologous to the human SEC14L3 gene (tocopherol-associated protein 2) (Kempna et al. 2003), BAC CH230-19C12 corresponds to a repeated sequence already described in rodents, and finally, the 82C sequence has no match in the databases.
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Among these 15 candidates, we selected one molecule, the OBP-1F because it displayed the highest gene repression (5.43) and the lowest P-value. Moreover, taking into account that this molecule is mainly produced in the LNG, that is, outside the OM, its expression by the OM was rather unexpected.
OBP-1F transcript is evidenced in LNGs of the rat nasal mucosa by ISH
We explored the tissue distribution of the OBP-1F transcript in the nasal mucosa by ISH of serial sections (1–4) in normally fed 2-month-old Wistar rats (Figure 2A). A schematic representation of each section structure is given in Figure 2L–O. Digoxigenin-labeled antisense probe revealed a strong signal for OBP-1F mRNA, largely confined to LNG structures on sections 1 and 2 in the lateral anterior region of the nasal mucosa (Figure 2G–I), with a clear labeling of acini (arrows) as already described (Pevsner et al. 1988). Some of the other multiple nasal glands were devoid of labeling (data not shown), suggesting an heterogeneity toward the expression of OBF-1F between the different nasal glands (Bojsen-Moller 1964) and between acini composing the LNG (Figure 2I). In the 2 last anteroposterior sections 3 and 4 (even after long exposure time), no labeling was observed (Figure 2J–K). Negative controls using a sense probe, under identical conditions, showed no staining at all (Figure 2B–F). Therefore, although histological data confirmed transcription of OBP-1F in the LNG as already published (Pevsner et al. 1988), this first survey was unable to disclose the site of OBP-1F expression in the OM.
OBP-1F gene is transcribed both by LNG and OM of rats
In order to clarify the occurrence of OBP-1F mRNA expression in OM itself, we carefully dissected apart each OM and LNG structures from fed adult rats to quantify OBP-1F gene expression levels by qPCR in both tissues (Figure 3A). The relative messenger levels were expressed as a mean normalized to an endogenous reference, ß-actin. The OM expressed low, albeit detectable levels of OBP-1F mRNA, with a relative expression level 
500 fold less than in the LNG (n = 5; P < 0.05, Figure 3A). Therefore, a contribution of OM in OBP-1F mRNA transcription inside the nasal mucosa had to be considered in addition to the main site already characterized, that is, the LNG.
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A 48-h fasting downregulates OBP-1F mRNA expression in the nasal mucosa
Based on the above data, we undertook a further analysis of OBP-1F mRNA expression to refine the influence of the nutritional status on both structures. Quantitative PCR was performed for OBP-1F gene expression in nasal mucosa isolated either from fed or 48-h fasted rats and then normalized to ß-actin gene expression. As depicted in Figure 3B, OBP-1F expression in the OM was decreased almost 3-fold in fasted rats compared with fed rats (n = 5 for each; relative expression: 5.3 ± 1 vs. 1.9 ± 1, P < 0.05). This result is consistent with macroarray data, which showed a more than 5-fold decrease in fasted rats compared with fed ones. A similar quantification of OBP-1F mRNA expression levels in LNG, where mRNA levels are 500-fold more expressed than in OM, revealed no significant difference of mRNA level between fed and fasted rats (Figure 3C). Therefore, only the fraction of OBP-1F mRNA expressed in OM appeared regulated by the nutritional status, suggesting a tissue-specific OBP-1F gene transcription control in response to a 48-h fasting.
OBP-1F protein ratio between LNG and OM is correlated to the mRNA expression ratio
As aforementioned, the differential expression of OBP-1F mRNA between LNG and OM reached a relative expression level of 500 fold. To see whether this ratio was also evidenced at the protein level, we carefully dissected out OM and LNG from fed adult rats and then absorbed the mucus either in contact with the OM or inside the LNG structures. Immunoblot analysis of total protein extracts from these mucus samples (n = 5 for each tissue) was performed. Samples blotted were analyzed using anti-OBP-1F antibody and revealed a single specific 19-kDa band corresponding to known OBP-1F apparent molecular mass (Figure 4A). Quantitative analysis showed that OM mucus contains 17-fold less OBP-1F protein when compared with LNG mucus (Figure 4B, n = 5). Although the protein ratio between LNG and OM was still correlated to the mRNA expression ratio, the trend is less striking. This decreased ratio can be ascribed to the topology of the nasal mucosa because the extracted mucus in contact with the OM is fully exposed to OBP-1F atomization via the LNG duct. Thus, the OBP-1F contribution from the LNG might lower the differential expression ratio between these 2 tissues. To validate this hypothesis, a differential expression study of OBP-1F between fed and fasted rats was undertaken by immunoblot analysis of mucus either in contact with LNG (Figure 4C) or OM (Figure 4D). As expected, this study revealed no significant difference between the 2 experimental conditions either at the LNG or OM level.
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Discussion |
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We demonstrated here that starvation is followed by transcriptional changes in the OM. We identified several genes whose expression was regulated by the nutritional status in the OM of rats.
The combination of our differential display analysis together with the screening of a macroarray was well adapted to study this situation where few samples had to be analyzed. After ddmRNA, we found a relatively low number of unknown genes (11) following annotation on the rat, mouse, or human genomes. They might have resulted from cDNA sequence modifications during the whole complex molecular biology procedure, resulting in some limitations in the use of ddmRNA alone. In addition, the macroarray screening identified only 7% of potential candidates of 203 molecules selected by ddmRNA, confirming that the latter approach generates a high number of false-positive results and requires additional screening (Matz and Lukyanov 1998). The 2-fold change criteria used for transcriptomic studies together with our highly stringent statistical analysis rejected certainly candidates from the ddmRNA screening; however, this enables us to affirm that selected candidates were highly relevant.
It cannot be ruled out that part of the changes might result from the enhanced oxidative stress with fasting as previously described (Sorensen et al. 2006). However, none of the molecular systems known to be involved in stress response have been highlighted during the ddmRNA screening.
Due to the structure of the nasal tissue, a small amount of intermingled respiratory mucosa partly participates in the OM sampling during the extraction procedure, possibly leading to a biased analysis. However, results of the macroarray assay indicate that the samples are homogenous in terms of ratio between olfactory and respiratory mucosae. Indeed, some of the screened molecules, such as one olfactory receptor (NM_001000932) or the neuronal nitric oxide synthase, Nos1 (NM_052799), are neuron specific and do not display any change of their expression level (Broillet and Firestein 1996). In addition, among the 94 molecules involved in neurotransmission, 53 of them exhibiting a median spot intensity 2-fold up to the median background keep a constant expression level between nutritional conditions (not shown).
Among the 15 sequences selected as being regulated by the nutritional status, some belong to olfactory or neuronal tissue–specific class, such as the olfactory marker protein or one OBP, whereas other are more related to metabolic cues (18S RNA or glucose-6-phosphate dehydrogenase), which was rather expected (Wensley et al. 1995). Other sequences, such as RP23-81P12, a rat sequence homologous to the human gene SEC14L3 (tocopherol-associated protein 2, TAP2), which encodes a transcription factor depending on tocopherol (vitamin E) (Kempna et al. 2003), might be considered. Actually, a vitamin E deprivation is depicted as one of the diseases that cause olfactory dysfunction (Henkin and Hoetker 2003). Thus, TAP2 might play a role in olfactory modulation through its downregulation following food deprivation.
Some candidates belonging to the neurotransmitter receptors family also exhibit a downregulation in OM of fasted rats: 2 metabotropic glutamate receptors (Grm2, Grm6) and 2 cholinergic receptors, one nicotinic (Chrnb2) and one muscarinic (Chrm2). The cholinergic receptor subtypes were previously identified in the OM of amphibian and fish (Getchell ML and Getchell TV 1992; Drescher et al. 2004), whereas the G-protein–coupled metabotropic glutamate receptors were only described at the OB level (Sahara et al. 2001). A potential target might be the olfactory ensheathing cells (OECs) located in the LP that wrap olfactory neuron axons in the olfactory nerve. Those metabotropic glutamate receptors have been suggested to modulate synaptic transmission via both pre- and postsynaptic effects (Sahara et al. 2001). Interestingly, 3 of our candidates (Grm2, Grm6, and Chrm2) are coupled to the Gi protein, which results in the inhibition of adenylyl cyclase (Pin and Duvoisin 1995; Fryer and Jacoby 1998). Thus, according to the nutritional status, Grm2 and Grm6 might modulate the olfactory signal transmission toward the OB, by acting at the presynaptic level of the synaptic junction between olfactory neurons and mitral cells. Indeed, metabotropic receptors subtypes expressed by glial cells participate in the communication with neurons in the developing OB through calcium signaling (Rieger et al. 2006). However, only the cellular distribution of those receptors at the light and electron microscopic level would help to clarify their potential roles in olfactory neuromodulation.
In our study, we show that the GFAP mRNA, which is expressed by OECs of the LP, is downregulated by the nutritional status, possibly by changes in circulating hormonal levels. Indeed, GFAP was already shown as being modulated by cytokines, steroid hormones, or growth factors (Laping et al. 1994). More interestingly, Dennis et al. (2005) reported that GFAP was downregulated in OECs extracted from OM of type-1 diabetic rats, thus linking a diminution of insulin receptor signaling to glial cell function changes. The resulting changes of GFAP might also disturb the neuron–glia interactions, as suggested above for the metabotropic receptors.
More interestingly, we highlighted one OBP, OBP-1F, as displaying the 2 characteristics of 1) being physiologically relevant to olfaction and 2) presenting the highest repression upon food restriction.
Our macroarray, western blot, and q-PCR data pointed out the OM as an OBP-1F-secreting tissue, in addition to its main site, that is, the LNGs (Pevsner et al. 1988). This contribution seems to be minor because OBP-1F mRNA expression is about 500-fold less expressed in OM than in the LNG's acini. This may result either from the presence of discrete synthesis sites spread all over the OM or from numerous cells transcribing low levels of OBP-1F gene. If nasal glands were described to be the unique site of synthesis for 2 OBP-related proteins in mouse (Pes et al. 1998), few data showed an expression of OBP by Bowman's glands in bovine or Xenopus species (Pevsner et al. 1986; Millery et al. 2005). However, our present study failed to detect any labeling of those secreting glands either by ISH or ICC in the rat OM.
More interestingly, our work indicated that OBP-1F gene expression was downregulated by a 48-h food deprivation only in the OM, that is, in a tissue-specific way. Despite their close anatomical localization, each secretory structure was differentially affected by the nutritional status in terms of the transcriptional control of OBP-1F gene expression. Recently, a microarray-based study of OBP genes expression in the mosquito showed that whereas some OBPs were overexpressed in the female after a blood feeding, most of them were downregulated (Biessmann et al. 2005). In addition, olfactometer assays demonstrated that host seeking was drastically inhibited after a blood meal in the Anopheles gambiae (Takken et al. 2001). However, none of the 2 other known OBP in the mucus of the rat species was highlighted by our ddmRNA analysis. Contrary to the insect where interactions between odorant ligands and members of the large family of OBPs are quite specific, the lack of specificity between a mammalian OBP and an odorant class prevents further hypotheses on having selected OBP-1F only.
Among several factors that might fluctuate following food deprivation (cytokines, growth factors) and might influence OBP-1F transcription, we should also point out leptin, a well-known circulating cytokine. Convincing facts, such as 1) leptin expression is regulated by the nutritional status in the rat OM (Baly et al. 2007), 2) leptin receptors are expressed by Bowman's glands of the OM, 3) ob/ob leptin-deficient mice display olfactory dysfunctions (Getchell et al. 2006), and 4) a perfusion with leptin induces an increase of the intestinal mucus secretion in rats (Plaisancie et al. 2006), argue that leptin might be a candidate to the olfactory modulation by the nutritional status through the control of mucus composition.
At the protein level, the mucus in contact with the OM displays 17-fold less OBP-1F protein when compared with LNG mucus, thus confirming to a less extent the variation of mRNA expression ratio. In addition to several posttranscriptional regulations that might extensively modify this protein:mRNA ratio, a simple hypothesis could be added. Taking into account the anatomical structure of the rat nose, atomization of OBP-1F from the LNG duct caused by the sniffing largely contributes to the composition of the OM mucus (Pevsner et al. 1988), thus lowering the differential expression ratio between these 2 parts of the nasal secretions. Obviously, knowing the absence of nutritional regulation of OBP-1F mRNA in the LNG, macroscopic effects on OBP-1F at the OM level could not be evidenced. However, we could speculate that the fine transcriptional control of OBP-1F in the OM, as demonstrated above, display functional consequences at the local level in relation to one (or more) of the several functions of these molecules in early olfactory perireception events: 1) reception of the odorant molecules upon their arrival inside the nasal cavity (Pelosi 1994), 2) elimination of odorous molecules in excess to prevent the saturation of the olfactory receptors (Schofield 1988; Burchell 1991), and 3) clearance of toxic substances to protect the OM against injuries (Boudjelal et al. 1996). According to our results, the OBP-1F secreted by LNG might play a role in defense mechanism by countering possible toxicity, whereas the OBP-1F produced by the OM itself might also act locally to regulate the mucus composition for the control of in vivo odorant responsiveness (Oka et al. 2006). Knowing the broad spectrum of activity found for mammalian OBP in the olfactory mucus and their high turnover rate (Pelosi 2001), a local modification of the mucus composition might rapidly affect its physicochemical or biological properties, possibly through the dynamics of dimerization of OBP-1F (Nespoulous et al. 2004). Similarly, a cytochrome P450 overexpression in the rat nasal mucosa following a 72-h fasting was proposed to improve clearance of hormones, drugs, or inhaled odorants (Longo et al. 2000).
Among the 2 aforementioned expression sites for OBP-1F mRNA, only the OM fraction is under a fine transcriptional control following food deprivation. It could regulate the local molecular environment close to its site of secretion to participate in the perireceptor events of the olfactory signal reception. In conclusion, these data are also in agreement with a possible involvement of the nutritional status in OM functions through the indirect control of secretory functions of the OM.
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We wish to thank the UEAR (Jouy-en-Josas) for animal care and Dr Loic Briand (INRA, UMR1197 NOPA, Biochimie de l'Olfaction et de la Gustation, F-78350 Jouy en Josas), for kindly providing the antibody against the OBP-1F. We thank CRB-GADIE (Centre de Ressources Biologiques pour la Génomique des Animaux d'Elevages et d'Intérêt Economique) and PICT (Plateau d'Instrumentation et de Compétences en Transcriptomique) for macroarray manufacture and qPCR analyses facilities, respectively. We are also grateful to Cedric Cabau (Agenae, Unité Mathématiques, Informatique et Génome, F-78352 Jouy-en-Josas) for the GO annotation, to Didier Durieux (INRA, UMR1197 NOPA, Récepteurs et Communication Chimique, F-78350 Jouy en Josas) for preparation of ISH sections. We are also grateful to Dr Annick Faurion (INRA, UMR1197 NOPA, Neurobiologie Sensorielle, F-78350 Jouy-en-Josas) for critical reading of the manuscript. We acknowledge the Région Ile-de-France in the framework of a Sésame contract (#2002/A01497). K.B. is financially supported by the French Agence Nationale de la Recherche (no. 59000033).
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Accepted 19 April 2007
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