Régine Monnerie

Promotion of cancer cell invasiveness and metastasis emergence caused by olfactory receptor stimulation.

Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by β-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase γ dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist β-ionone significantly enhanced metastasis emergence and spreading.

Relationship between Homo-oligomerization of a Mammalian Olfactory Receptor and Its Activation State Demonstrated by Bioluminescence Resonance Energy Transfer

G-protein-coupled receptor homo-oligomerization has been increasingly reported. However, little is known regarding the relationship between activation of the receptor and its association/conformational states. The mammalian olfactory receptors (ORs) belong to the G protein-coupled receptor superfamily. In this study, the homo-oligomerization status of the human OR1740 receptor and its involvement in receptor activation upon odorant ligand binding were addressed by co-immunoprecipitation and bioluminescence resonance energy transfer approaches using crude membranes or membranes from different cellular compartments. For the first time, our data clearly show that mammalian ORs constitutively self-associate into homodimers at the plasma membrane level. This study also demonstrates that ligand binding mediates a conformational change and promotes an inactive state of the OR dimers at high ligand concentrations. These findings support and validate our previously proposed model of OR activation/inactivation based on the tripartite odorant-binding protein-odorant-OR partnership.

Melatonin and the circadian clock in mink: effects of daily injections of melatonin on circadian rhythm of locomotor activity and autoradiographic localization of melatonin binding sites.

The present study examines a putative effect of exogenous melatonin on the Circadian organization of the mink. Two approaches were used to determine first whether entrainment of free‐running rhythms of locomotor activity in constant darkness can be obtained by daily melatonin injections, thus demonstrating a control of melatonin on the clock generating Circadian rhythms, the suprachiasmatic nucleus of the hypothalamus. Entrainment was never obtained in the 8 vehicle‐injected females and 7 out of the 8 melatonin injected‐ones. In 3 females free‐running in constant darkness, a phase advance followed by a few days of transient effect was observed when melatonin injections coincided with the onset of activity. However, the comparison of the regression of the daily activity onset related to successive days by covariance analysis revealed that true entrainment was effective in only 1 female. Second, we examined the distribution of melatonin binding sites within the brain of juvenile and adult mink using an in vitro autoradiographic procedure with [125l]2‐iodomelatonin. No binding sites were observed in the suprachiasmatic nucleus of any of the animals. However, all animals displayed a high density of melatonin binding sites in the pars tuberalis of the pituitary. The relation between a modulatory control of melatonin on the Circadian clock and the presence and density of melatonin binding sites in the clock is discussed. In mink, melatonin does not seem to act as an internal Zeitgeber.

Effects of Timed Melatonin Infusions and Lesions of the Suprachiasmatic Nuclei on Prolactin and Progesterone Secretions in Pregnant or Pseudopregnant Mink (Mustela vison)

To test the hypothesis that the duration of melatonin secretion may be a critical parameter in the transduction of photoperiodic signals on prolactin and progesterone secretions, timed intravenous melatonin infusions were carried out in intact and ganglionectomized pregnant and pseudopregnant mink. To localize the target sites of melatonin, electrolytic lesions of hypothalamic nuclei were performed in females receiving melatonin infusions. As a control, the first experiment was designed to confirm that pineal denervation by bilateral ablation of the superior cervical ganglion rendered the pregnant mink totally unresponsive to the inhibitory effects of short days on progesterone secretion. In the following experiments, timed intravenous melatonin infusions were carried out in intact and ganglionectomized females from Day 12 to 32 of pregnancy or pseudopregnancy. Daily infusions of melatonin for 16 h in intact females or for 11 or 13 h in ganglionectomized females suppressed the rise in plasma prolactin and progesterone levels. In intact as in ganglionectomized females, daily infusions of melatonin for 9 h delayed the rise in plasma prolactin concentrations without affecting the secretion of progesterone. In ganglionectomized females, saline infusions for 13 h or melatonin infusions for 7h did not modify the secretions of prolactin and progesterone. In ganglionectomized females bearing lesions of the Suprachiasmatic nuclei or the retrochiasmatic area, melatonin infusions for 13 h were still able to inhibit prolactin and progesterone secretions. These results are consistent with the hypothesis postulating that the peak duration of melatonin secretion is a critical parameter for transducing photoperiodic responses in pregnant or pseudopregnant mink. Secondly, they suggest that the Suprachiasmatic nuclei and the retrochiasmatic area are not essential for the action of melatonin in the photoperiodic control of prolactin and progesterone secretions during pregnancy or pseudopregnancy in the mink.

Neuropeptide Y Enhances Olfactory Mucosa Responses to Odorant in Hungry Rats

Neuropeptide Y (NPY) plays an important role in regulating appetite and hunger in vertebrates. In the hypothalamus, NPY stimulates food intake under the control of the nutritional status. Previous studies have shown the presence of NPY and receptors in rodent olfactory system, and suggested a neuroproliferative role. Interestingly, NPY was also shown to directly modulate olfactory responses evoked by a food-related odorant in hungry axolotls. We have recently demonstrated that another nutritional cue, insulin, modulates the odorant responses of the rat olfactory mucosa (OM). Therefore, the aim of the present study was to investigate the potential effect of NPY on rat OM responses to odorants, in relation to the animals nutritional state. We measured the potential NPY modulation of OM responses to odorant, using electro-olfactogram (EOG) recordings, in fed and fasted adult rats. NPY application significantly and transiently increased EOG amplitudes in fasted but not in fed rats. The effects of specific NPY-receptor agonists were similarly quantified, showing that NPY operated mainly through Y1 receptors. These receptors appeared as heterogeneously expressed by olfactory neurons in the OM, and western blot analysis showed that they were overexpressed in fasted rats. These data provide the first evidence that NPY modulates the initial events of odorant detection in the rat OM. Because this modulation depends on the nutritional status of the animal, and is ascribed to NPY, the most potent orexigenic peptide in the central nervous system, it evidences a strong supplementary physiological link between olfaction and nutritional processes.

Vasoactive Intestinal Polypeptide in the Suprachiasmatic Nucleus of the Mink (Mustela vison) Could Play a Key Role in Photic Induction

The present study was conducted to visualize neuropeptides in the SCN of a mustelid, the American mink in which seasonal cycles of reproduction rely totally on the annual changes in day length. At this time, data in mustelids are lacking. Results were obtained with in situ hybridization (ISH) using synthetic oligonucleotide vasopressin (AVP) and somatostatin (SOM) and with single and dual immunohistochemistry (IHC) performed with antisera against AVP, SOM, vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and met‐enkephalin (Met‐ENK) in untreated (AVP and VIP) or colchicine (SOM, Met‐ENK and GRP) treated adult male and female mink. The most striking result, evidenced by ISH as well as IHC was the lack of AVP, SOM and Met‐ENK immunoreactive (ir)‐neurons in the SCN. In contrast, strongly VIP ir‐perikarya were widely distributed within the SCN and gave rise to a dense network of fibres extending within the periventricular (peVA) and subparaventricular (subPVA) areas. Weakly GRP ir‐perikarya were also observed in the median part of the SCN. Dual IHC revealed that the magnocellular neurons located just dorsal to the SCN, in the peVA and subPVA co‐stored AVP with VIP, SOM or Met‐ENK. The lack of SCN AVP and SOM ir‐neurons, reported for the first time in a mammalian species, raises the question of their implication in the functions of the circadian pacemaker and its entrainment by the light/dark cycle in other species. The significance of the large neurons co‐storing peptides in the terminal field of VlPergic fibres originating in the SCN has also to be determined. These results suggest that VIP could be of major importance in processing photic information mediating circadian entrainment and consequently annual rhythms.

Long-Lasting Metabolic Imbalance Related to Obesity Alters Olfactory Tissue Homeostasis and Impairs Olfactory-Driven Behaviors

Obesity is associated with chronic food intake disorders and binge eating. Food intake relies on the interaction between homeostatic regulation and hedonic signals among which, olfaction is a major sensory determinant. However, its potential modulation at the peripheral level by a chronic energy imbalance associated to obese status remains a matter of debate. We further investigated the olfactory function in a rodent model relevant to the situation encountered in obese humans, where genetic susceptibility is juxtaposed on chronic eating disorders. Using several olfactory-driven tests, we compared the behaviors of obesity-prone Sprague-Dawley rats (OP) fed with a high-fat/high-sugar diet with those of obese-resistant ones fed with normal chow. In OP rats, we reported 1) decreased odor threshold, but 2) poor olfactory performances, associated with learning/memory deficits, 3) decreased influence of fasting, and 4) impaired insulin control on food seeking behavior. Associated with these behavioral modifications, we found a modulation of metabolism-related factors implicated in 1) electrical olfactory signal regulation (insulin receptor), 2) cellular dynamics (glucorticoids receptors, pro- and antiapoptotic factors), and 3) homeostasis of the olfactory mucosa and bulb (monocarboxylate and glucose transporters). Such impairments might participate to the perturbed daily food intake pattern that we observed in obese animals.

Rat strains with different metabolic statuses differ in food olfactory-driven behavior

In most species, food intake is influenced by olfactory cues and metabolic status can affect the olfactory function of animals and regulate feeding-related behaviors. We investigated whether modulation of the endocrine system that regulates or modifies energy balance affected the olfactory system by examining four rat strains, obese Zucker and obesity-resistant Lou/C rats and their counterparts. Such models were chosen because they differ largely in their energy status and in their insulin and leptin blood levels, two hormones known to impact olfactory behaviors. After evaluation of the main metabolic parameters, we analyzed the food-driven olfactory behaviors of the four strains by measuring general activity time and sniffing time in response to food cues together with food reward localization performances in fed and fasted states. In fed conditions, obese Zucker and Wistar rats exhibited a great interest for food odor, which was not enhanced by fasting, in contrast to Lou/C and Zucker lean rats. All strains, except Lou/C, showed decreased latencies to find a hidden food reward with time, whereas a 24-h fasting was necessary to improve food search performances in Lou/C. These metabolic and behavioral changes were partly associated with variations in the transcription profiles of leptin, insulin and orexin and their receptors in the hypothalamus and olfactory system. The results show that variations in metabolic-related genes expression along the olfactory pathways comes with obesity in influencing food odors-driven behaviors. Our data indicate that food-olfactory driven behaviors are clearly affected by the long-term metabolic status.

Chronic restricted access to food leading to undernutrition affects rat neuroendocrine status and olfactory-driven behaviors ☆

Previous studies have demonstrated that olfactory-driven behaviors in rats are influenced by short-term caloric restriction, partly through the modulation of olfactory sensitivity by appetite-modulating hormones or peptides such as insulin and leptin. Here, we addressed the issue of a long-term modulation of their neuroendocrine status by evaluating the effect of chronic food restriction in rats following a limitation of the duration of daily food intake to 2 h (SF) instead of 8 h (LF) on the expression of insulin and leptin system in the olfactory mucosa and bulb and on olfactory behaviors. This restriction resulted in a one-third reduction in the daily food intake and a 25% reduction in the body weight of SF rats when compared to controls, and was accompanied by lower levels of triglycerides, glucose, insulin and leptin in SF rats. Under these conditions, we observed a modulation of olfactory-mediated behaviors regarding food odors. In addition, restriction had a differential effect on the expression of insulin receptors, but not that of leptin receptors, in the olfactory mucosa, whereas no transcriptional change was observed at the upper level of the olfactory bulb. Overall, these data demonstrated that long-term changes in nutritional status modulate olfactory-mediated behaviors. Modulation of insulin system expression in the olfactory mucosa of food restricted rats suggests that this hormone could be part of this process.

Insulin but not leptin protects olfactory mucosa from apoptosis.

The mammalian olfactory mucosa (OM) is continually renewed throughout life. Owing to their position in the nasal cavity, OM cells are exposed to multiple insults, including high levels of odourants that can induce their death. OM regeneration is therefore essential to maintain olfactory function, and requires the tight control of both cell death and proliferation. Apoptosis has been implicated in OM cell death. Olfaction is one of the senses involved in food intake and depends on individual nutritional status. We have previously reported the influence of hormones related to nutritional status on odour perception and have shown that the OM is a target of insulin and leptin, two hormones known for their anti‐apoptotic properties. In the present study, we investigated the potential anti‐apoptotic effect of these metabolic hormones on OM cells. Both Odora cells (an olfactive cell line) and OM cells treated with etoposide, a p53 activity inducer, exhibited mitochondrial‐dependent apoptosis that was inhibited by the pan‐caspase inhibitor zVAD‐fmk. Insulin, but not leptin, impaired this apoptotic effect. Insulin addition to the culture medium reduced p53 phosphorylation, caspase‐3 and caspase‐9 cleavage, and caspase‐3 enzymatic activity induced by etoposide. The apoptotic wave observed in the OM after interruption of the neuronal connections between the OM and the olfactory bulb by bulbectomy was impaired by intranasal insulin treatment. These findings suggest that insulin may be involved in OM cellular dynamics, through endocrine and/or paracrine‐autocrine effects of circulating or local insulin, respectively.