Juan Roa

David García-Galiano

Juan M Castellano

Francisco Gaytan

Leonor Pinilla

Manuel Tena-Sempere

Metabolic Control of Puberty Onset: New Players, New Mechanisms

Keywords: Puberty, energy balance, leptin, ghrelin, Kiss1, kisspeptins, GPR54, mTOR, Crtc1 GPR54, G protein-coupled receptor 54, GnRH, gonadotropin releasing hormone, LH, luteinizing hormone, FSH, follicle-stimulating hormone, ARC, arcuate nucleus, AVPV, anteroventral periventricular nucleus

ABSTRACT

Puberty, as the end-point of a complex series of maturational events affecting the components of the hypothalamic-pituitary-gonadal (HPG) axis, is gated by the state of body energy reserves and sensitive to different metabolic cues; conditions of severe metabolic stress and energy unbalance (from anorexia to morbid obesity) being commonly linked to perturbation of the onset of puberty. In the last two decades, the neuroendocrine mechanisms responsible for the tight coupling between energy homeostasis and puberty onset have begun to be deciphered. These seemingly involve a plethora of metabolic hormones and neuropeptides, which impinge and integrate (mostly) at the hypothalamic centers governing reproduction. Yet, characterization of the mechanisms of action of such regulators (and even their nature and physiological relevance) still remains incomplete. In this review, we will summarize some recent developments in our knowledge of the effects and mechanisms of action of two key metabolic hormones, leptin and ghrelin, in the control of puberty onset. In addition, the roles of the hypothalamic Kiss1 system in the metabolic gating of puberty will be reviewed, with special attention to its regulation by leptin and the recent identification of the putative roles of Crtc1 and mTOR signaling as molecular conduits for the metabolic control of Kiss1 expression. Elucidation of these novel players and regulatory mechanisms will help for a better understanding of the determinants of the timing of puberty, and its eventual alterations in adverse metabolic conditions.

Introduction

Puberty, as developmental period when full awakening of the gonadotropic axis and attainment of reproductive capacity take place, is a key maturational event, associated with important somatic and behavioral changes [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF][START_REF] Ojeda | Puberty in the rat[END_REF]. As such, the physiology of puberty onset and the potential mechanisms for its perturbation have been deeply scrutinized using different model species (from laboratory rodents to humans) and experimental/analytical approaches (from classical physiological studies to recent genome-wide association scanning) [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF][START_REF] Ojeda | Puberty in the rat[END_REF][START_REF] Ong | Genetic variation in LIN28B is associated with the timing of puberty[END_REF]. The consensus exists that the major determinants of the timing of puberty in healthy conditions are of genetic origin [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF].

However, additional modifiers of the tempo of puberty have been recognized, with prominent roles of different environmental factors (from nutrient availability to different stressors). Indeed, puberty has been envisioned as 'sensor' for the dynamic interplay between genetic determinants and environmental cues throughout development. Such delicate balance between endogenous and exogenous regulators would be ultimately responsible for the proper timing of puberty, or its deviations [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF]. Elucidating the pathways and molecular mechanisms whereby this continuous interaction is taking place remains as an appealing challenge for the future.

Among its different putative modifiers, it has been long recognized that the state of body energy reserves is a key determinant for the onset of puberty in mammals, including humans [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF]. The functional coupling between energy sufficiency and puberty onset is especially, but probably not exclusively, present in the female, where threshold fat stores are needed in order to successfully cope with the considerable metabolic drainage of pregnancy and lactation [START_REF] Casanueva | Neuroendocrine regulation and actions of leptin[END_REF]. The association between a critical fat mass and the occurrence of menarche was first proposed on a rigorous basis by Frisch and colleagues in early 1970's [START_REF] Frisch | Height and weight at menarche and a hypothesis of critical body weights and adolescent events[END_REF][START_REF] Frisch | Influences on age of menarche[END_REF], in keeping with previous analogous observations from Kennedy and co-workers in laboratory rats [START_REF] Kennedy | Body weight and food intake as initiating factors for puberty in the rat[END_REF]. This hypothesis provided the first scientific formulation for the ancient, intuitive knowledge of the existence of a close link between fatness and fertility. Recognition of such a link paved the way for the identification of the hormonal signals and neuroendocrine mechanisms involved in this phenomenon.

During the last two decades, considerable progress has been made towards the elucidation of the neurohormonal pathways responsible for the metabolic control of puberty onset and gonadotropic function. Without discussion, a major breakthrough in this field was the identification of the adipose hormone, leptin, as an essential neuroendocrine integrator responsible for the coupling of the state of body energy stores and different hormonal functions, including reproduction [START_REF] Casanueva | Neuroendocrine regulation and actions of leptin[END_REF][START_REF] Tena-Sempere | Roles of ghrelin and leptin in the control of reproductive function[END_REF]. A number of additional peripheral hormones from key metabolic tissues, such as the gastrointestinal tract, the pancreas and the adipose, have been also proposed as putative regulators of the gonadotropic axis [START_REF] Fernandez-Fernandez | Novel signals for the integration of energy balance and reproduction[END_REF]; these likely include the gut -hormone, ghrelin, whose role as functional antagonist of leptin in terms of food intake control may apply as well to the regulation of puberty [START_REF] Tena-Sempere | Ghrelin and reproduction: ghrelin as novel regulator of the gonadotropic axis[END_REF][START_REF] Tena-Sempere | Ghrelin as a pleotrophic modulator of gonadal function and reproduction[END_REF]. Similarly, different central neuropeptides have been described as potential mediators in this phenomenon; kisspeptins being the most recently identified (and likely one of the most important) players in the central control of puberty onset by metabolic cues [START_REF] Tena-Sempere | The roles of kisspeptins and G protein-coupled receptor-54 in pubertal development[END_REF][START_REF] Castellano | KiSS-1/kisspeptins and the metabolic control of reproduction: Physiologic roles and putative physiopathological implications[END_REF]. While systematic description of every molecule and mechanism reported so far in this context clearly exceeds the scope of this review, we aim to provide herein a succinct and up-dated summary of some recent developments in our knowledge of the signals and neuroendocrine pathways singled out above (leptin, ghrelin, kisspeptins), which will help to highlight and better define physiologically-relevant mechanisms for the modulation of the timing of puberty by metabolic factors in mammals.

Leptin Signaling and Puberty Onset: Recent Developments

As stated above, identification of leptin, as product of the ob gene, was a revolutionary finding in contemporary Endocrinology, as it allowed unveiling the mechanisms underlying quite diverse regulatory functions, from body weight control to metabolic gating of fertility [START_REF] Casanueva | Neuroendocrine regulation and actions of leptin[END_REF][START_REF] Tena-Sempere | Roles of ghrelin and leptin in the control of reproductive function[END_REF]. For sake of concision, description of the fundamental features of leptin as adipose signal controlling reproductive maturation and function will be omitted, as these can be found elsewhere [START_REF] Casanueva | Neuroendocrine regulation and actions of leptin[END_REF][START_REF] Tena-Sempere | Roles of ghrelin and leptin in the control of reproductive function[END_REF][START_REF] Moschos | Leptin and reproduction: a review[END_REF][START_REF] Hill | Hypothalamic pathways linking energy balance and reproduction[END_REF]. Sufficient to stress, a wealth of experimental evidence obtained in rodents and humans has conclusively demonstrated that leptin is a permissive signal for puberty to proceed (rather than a trigger itself), thus providing the neuroendocrine basis for the critical fat mass hypothesis postulated by Frisch several decades ago.

Despite the universal recognition of the essential roles of leptin in the control of puberty, some controversy has persisted on specific facets of such physiological action. Among those, the precise pathways and mechanisms whereby leptin modulates the function of the gonadotropic axis, and its activation at puberty, have been the subject of active investigation and debate. Notwithstanding, in spite of the demonstration of actions of leptin at other levels of HPG axis, such as the pituitary and the gonads [START_REF] Tena-Sempere | Roles of ghrelin and leptin in the control of reproductive function[END_REF], it has been long recognized that leptin conducts its stimulatory/permissive actions mainly by modulation of the gonadotropin-releasing hormone (GnRH) neuronal system at the hypothalamus [START_REF] Cunningham | Leptin's actions on the reproductive axis: perspectives and mechanisms[END_REF]; a hypothesis that was substantiated, among other experimental data, by studies showing that intra-hypothalamic infusion of leptin elicits GnRH release in vivo [START_REF] Watanobe | Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats[END_REF], and that leptin treatment was sufficient to normalize pulsatile GnRH/LH secretion in fasted rats [START_REF] Nagatani | Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting[END_REF]. These findings, however, could not demonstrate whether leptin actions are conducted directly upon GnRH neurons or indirectly, though intermediate afferents; an issue that has remained contentious for years. In fact, initial RT-PCR analyses detected the expression of leptin receptor mRNA in GnRH-producing GT1-7 cells [START_REF] Magni | Expression of a leptin receptor in immortalized gonadotropin-releasing hormone-secreting neurons[END_REF], which might be suggestive of direct actions on GnRH neurons. Similarly, leptin was shown to elicit GnRH secretion in enzymatically-dispersed hypothalamic tissue in a perfusion culture system (i.e., after dispersion into single cells), which might be indicative, but not a conclusive proof, of direct actions at GnRH neurons [START_REF] Woller | Leptin stimulates gonadotropin releasing hormone release from cultured intact hemihypothalami and enzymatically dispersed neurons[END_REF]. However, despite thorough testing, localization analyses in rodents and primates (by means of in situ hybridization and immunohistochemistry) systematically failed to demonstrate the presence of leptin receptors in GnRH neurons [START_REF] Finn | The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey[END_REF][START_REF] Hakansson | Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus[END_REF]; findings that suggested that leptin acts (mainly) indirectly in the control of the GnRH system. Yet, there has been a conspicuous lack of functional data for such an indirect mode of action until very recently.

In the last months, however, Quennell and co-workers have presented conclusive experimental evidence to demonstrate that leptin regulates GnRH neuronal function via indirect mechanisms, through a series of elegant studies which combined powerful functional genomic approaches, immunohistochemical analyses and single-cell gene expression assays [START_REF] Quennell | Leptin indirectly regulates gonadotropin-releasing hormone neuronal function[END_REF]. First, activation (phosphorylation) of STAT3, the primary mediator of leptin effects at the hypothalamus, following i.c.v. injection of leptin could not be detected in GnRH neurons, but rather in other hypothalamic areas which are known to send projections to the preoptic region, where most of GnRH neurons reside. Second, while neuron-specific leptin receptor knock-out prevented the onset of puberty and caused infertility in male and female mice, selective elimination of leptin receptors in GnRH neurons was fully compatible with (grossly) preserved puberty and normal fertility in mice. Finally, using single-cell RT-PCR techniques, GnRH neurons were shown to lack detectable leptin receptor mRNA expression. Altogether, these observations back-up previous data, and firmly demonstrate that, under physiological conditions, the actions of leptin in the control of GnRH function are indirect and likely conducted via the modulation of afferent pathways.

The nature of the neuronal afferents conveying leptin effects onto GnRH neurons is yet to be fully elucidated, and different neuropeptide pathways, such as neuropeptide Y (NPY) or galanin-like peptide (GALP), have been proposed in the last years [START_REF] Gamba | Control of GnRH neuronal activity by metabolic factors: the role of leptin and insulin[END_REF][START_REF] Castellano | Effects of galanin-like peptide on luteinizing hormone secretion in the rat: sexually dimorphic responses and enhanced sensitivity at male puberty[END_REF]. Without any doubt, however, much of the efforts recently devoted in this front have focused in the characterization of the putative roles of kisspeptin neurons in transmitting the effects of leptin in the control of GnRH neuronal function.

Importantly, not only the ability of leptin to modulate Kiss1 expression has been demonstrated, but also the potential molecular mechanisms whereby this modulatory action is brought about have begun to be characterized. Some key developments in this area will be summarized in sections 4 and 5 of this review.

Ghrelin and Puberty Onset: Modulatory Actions in the Male and Female

While the role of leptin, as signal of energy abundance, in the metabolic gating of puberty was recognized (and universally accepted) shortly after its cloning in 1994, identification of additional metabolic hormones with the potential to modulate puberty onset has remained elusive. Among other possible candidates, ghrelin, as circulating orexigenic factor that signals energy insufficiency, has emerged in recent years as putative modifier of the timing of puberty [START_REF] Tena-Sempere | Ghrelin and reproduction: ghrelin as novel regulator of the gonadotropic axis[END_REF][START_REF] Tena-Sempere | Ghrelin as a pleotrophic modulator of gonadal function and reproduction[END_REF]. Description of the major features of ghrelin in terms of structure and biological actions, as well as of its widespread distribution and signaling pathways, can be found elsewhere [START_REF] Van Der Lely | Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin[END_REF][START_REF] Korbonits | Ghrelin--a hormone with multiple functions[END_REF] . For the purpose of the present review, it is stressed that ghrelin exists in circulation in two major forms: acyl-ghrelin, which is able activate the canonical receptor GHS-R1a and will be referred herein as ghrelin, and unacyl-ghrelin (UAG), which cannot activate GHS-R1a and signals though as yet unknown receptor pathways (see Figure 1). In addition, expression of ghrelin gene in mammals is likely to give rise to another peptide, termed obestatin by virtue of its (apparent) ability to antagonize ghrelin effects in terms of body weight gain [START_REF] Gualillo | One ancestor, several peptides posttranslational modifications of preproghrelin generate several peptides with antithetical effects[END_REF]. Yet, the nature of the biological actions and receptor(s) of obestatin has been the subject of intense controversy recently.

Although in this review special emphasis will be made on the experimental evidence supporting a potential role of ghrelin in the modulation of puberty, it is important to emphasize as background information that data available in the literature strongly suggest that ghrelin operates (predominantly) as inhibitory signal for gonadotropin (mostly LH) secretion in different mammalian species, including rodents, sheep, non-human primates and humans [START_REF] Tena-Sempere | Ghrelin and reproduction: ghrelin as novel regulator of the gonadotropic axis[END_REF][START_REF] Tena-Sempere | Ghrelin as a pleotrophic modulator of gonadal function and reproduction[END_REF]; a phenomenon that might contribute to the well-known suppression of gonadotropin levels in conditions of persistent negative energy balance, in which ghrelin levels are commonly elevated. Of note, rodent studies have demonstrated that the inhibitory actions of ghrelin on LH secretion in vivo can be mimicked by UAG [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF], thus suggesting that this phenomenon is, at least partially, conducted via GHS-R1a independent pathways. In clear contrast, despite extensive testing, we have been unable to demonstrate any conclusive effect of obestatin on LH or FSH secretion in rats (our unpublished observations); the latter casts doubts on the potential role of this ghrelin-related peptide in the control of gonadotropic function, at least in this species.

The experimental evidence summarized above, and the proposed function of ghrelin as signal of energy insufficiency, made it tempting to explore its potential roles in the control of puberty onset. Indeed, studies involving acute or repeated administration of ghrelin in pubertal rats do support a function of ghrelin as putative modulator of puberty, with a predominant inhibitory action in keeping with its reported effects in terms of gonadotropin secretion [START_REF] Tena-Sempere | Ghrelin and reproduction: ghrelin as novel regulator of the gonadotropic axis[END_REF][START_REF] Tena-Sempere | Ghrelin as a pleotrophic modulator of gonadal function and reproduction[END_REF]. Thus, repeated injections of ghrelin to male rats, at two different dose-regimens (low dose: 0.5 nmol/every 12-h; high dose: 1.0 nmol/every 12-h), during the pubertal transition significantly decreased serum LH and testosterone levels, and partially delayed balanopreputial separation (as external index of puberty onset) [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF][START_REF] Fernandez-Fernandez | Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies[END_REF].

Of note, chronic treatment of peripubertal female rats with an analogous protocol of low doses of ghrelin (0.5 nmol/12-h for 10 days) did not cause major changes in serum levels of gonadotropins or estradiol, neither did it significantly change the timing of puberty, as estimated by the ages of vaginal opening and first estrus [START_REF] Fernandez-Fernandez | Effects of chronic hyperghrelinemia on puberty onset and pregnancy outcome in the rat[END_REF]. In contrast, however, twice daily injection of 1 nmol ghrelin for 10 days was sufficient to delay vaginal opening and ovarian follicular development and ovulation in pubertal female rats (see Figure 2). Taken together, these data suggest that while both male and female rats are sensitive to the modulatory (inhibitory) actions of elevated levels of ghrelin on puberty onset, males appear to be more sensitive than females. This is in contrast to that reported previously for leptin, as females are more sensitive to its regulatory (permissive) effects in terms of puberty onset than males [START_REF] Fernandez-Fernandez | Novel signals for the integration of energy balance and reproduction[END_REF]. While these data have been obtained in laboratory rodents, their implications in terms of ghrelin influence on the timing of human puberty are worthy to explore. In fact, endocrine analyses have demonstrated a progressive decline in circulating ghrelin levels along puberty in humans [START_REF] Soriano-Guillen | Ghrelin levels from fetal life through early adulthood: relationship with endocrine and metabolic and anthropometric measures[END_REF]; a phenomenon which may play a permissive role on puberty onset, assuming a similar inhibitory action of ghrelin also in humans.

The mechanisms whereby ghrelin conducts the above modulatory actions on puberty onset remain to be characterized. Fragmentary evidence suggested that, in adult rats, ghrelin is able to inhibit hypothalamic GnRH secretion ex vivo [START_REF] Fernandez-Fernandez | Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies[END_REF], while systemic administration of ghrelin reduced GnRH pulse frequency in vitro [START_REF] Lebrethon | Effects of in vivo and in vitro administration of ghrelin, leptin and neuropeptide mediators on pulsatile gonadotrophin-releasing hormone secretion from male rat hypothalamus before and after puberty[END_REF]. A similar mechanism might be operative also in pubertal rodents, as a reduction in the frequency of GnRH pulses has been reported in immature rats following systemic administration of ghrelin [START_REF] Lebrethon | Effects of in vivo and in vitro administration of ghrelin, leptin and neuropeptide mediators on pulsatile gonadotrophin-releasing hormone secretion from male rat hypothalamus before and after puberty[END_REF]. Of note, the ability of ghrelin to delay puberty onset is mimicked by administration of UAG [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF], thus suggesting the involvement of GHS-R1a independent mechanisms [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF]. In contrast, we have been unable to demonstrate any discernible effect of obestatin in the timing of puberty onset in rats (our unpublished observations). Finally, even assuming a predominant action of ghrelin at the level of the GnRH pulse generator, whether this is conducted directly on GnRH neurons or through indirect regulatory pathways is yet to be determined. Circumstantial evidence, however, suggests an indirect mode of action, as ghrelin effects on LH secretion were inhibited by a non-selective antagonist of corticotrophin-releasing hormone (CRH) [START_REF] Vulliemoz | Astressin B, a nonselective corticotropin-releasing hormone receptor antagonist, prevents the inhibitory effect of ghrelin on luteinizing hormone pulse frequency in the ovariectomized rhesus monkey[END_REF], and ghrelin was able to inhibit the effect of central elicitors of GnRH/gonadotropin release, such as kisspeptin [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF]. In this sense, very recent evidence suggests that ghrelin is able to suppress Kiss1 gene expression at discrete hypothalamic areas [START_REF] Forbes | Effects of ghrelin on Kisspeptin mRNA expression in the hypothalamic medial preoptic area and pulsatile luteinising hormone secretion in the female rat[END_REF], thus providing the neuroendocrine basis for, at least part of, its inhibitory effects on puberty onset and gonadotropin secretion.

Central Pathways for the Metabolic Regulation of Puberty: The Role of Kisspeptins

Among the central signals responsible for the neuroendocrine control of the gonadotropic axis, kisspeptins, the products of Kiss1 gene that operate via the G protein-coupled receptor, GPR54, have emerged very recently as essential gatekeepers of puberty onset and fertility, by virtue of its pivotal roles in mediating key reproductive phenomena, including sexual differentiation of the brain, the pubertal activation of the GnRH system, the feedback control of gonadotropin secretion and the pre- and key physiological roles of kisspeptins in reproductive physiology can be found elsewhere [START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF][START_REF] Popa | The Role of Kisspeptins and GPR54 in the Neuroendocrine Regulation of Reproduction[END_REF], including other chapters of this Special Issue, for the purpose of this mini-review, we will briefly summarize below the experimental data supporting an important role of central kisspeptin pathways in the metabolic control of puberty onset and reproductive function. Two major lines of evidence can be highlighted in this context: (i) the demonstration of changes in Kiss1 expression in conditions of metabolic stress, and the corresponding effects of kisspeptins in such adverse metabolic situations; and (ii) functional data concerning the effects of major metabolic regulators on hypothalamic Kiss1 expression.

On the first point, data obtained in laboratory rodents have demonstrated that conditions of negative energy balance due to acute food deprivation, which are associated to variable degrees of hypogonadotropism, evoke a detectable suppression of Kiss1 mRNA levels at the hypothalamus [START_REF] Castellano | KiSS-1/kisspeptins and the metabolic control of reproduction: Physiologic roles and putative physiopathological implications[END_REF].

Of important note for this chapter, this phenomenon has been also detected in pubertal male and female rats [START_REF] Castellano | Changes[END_REF]. While the precise location for such changes awaits further clarification, studies in female rats subjected to chronic calorie restriction along puberty (20% for 7 days) demonstrated a detectable suppression of Kiss1 expression at the arcuate nucleus (ARC) [START_REF] Roa | The Mammalian Target of Rapamycin as Novel Central Regulator of Puberty Onset via Modulation of Hypothalamic Kiss1 System[END_REF], where a prominent population of kisspeptin neurons resides [START_REF] Popa | The Role of Kisspeptins and GPR54 in the Neuroendocrine Regulation of Reproduction[END_REF]. Notwithstanding, additional changes in Kiss1 expression at the anteroventral periventricular nucleus (AVPV) cannot be excluded in pubertal females under more extreme conditions of food deprivation, in keeping with analogous observations in adult female rats subjected to 48-h fasting [START_REF] Kalamatianos | Fasting reduces KiSS-1 expression in the anteroventral periventricular nucleus (AVPV): effects of fasting on the expression of KiSS-1 and neuropeptide Y in the AVPV or arcuate nucleus of female rats[END_REF]. From a functional standpoint, such a decrease in Kiss1 expression seems causative for the perturbation (delay) in the timing of puberty at conditions of persistent energy balance, as suggested by our studies in a model of chronic subnutrition during the pubertal transition in female rats, as pharmacological 'replacement' with kisspeptin at the expected time of puberty in this model, without any other metabolic intervention, was sufficient to rescue vaginal opening (in a subset of animals) and to induce potent gonadotropic and estrogenic responses, despite the prevailing suppression of circulating levels of gonadotropins due to chronic subnutrition [START_REF] Castellano | Changes[END_REF]. These findings are in good agreement with data from adult models of metabolic stress (such as uncontrolled diabetes) [START_REF] Castellano | Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats[END_REF][START_REF] Castellano | Alterations in Hypothalamic KiSS-1 System in Experimental Diabetes: Early Changes and Functional Consequences[END_REF], suggesting a conserved role of kisspeptin pathways in the metabolic regulation of the gonadotropic axis along the lifespan.

Despite the accepted role of kisspeptin neurons as conduits for transmitting metabolic information onto the centers governing the reproductive axis, the signals that are actually responsible for the metabolic regulation of the Kiss1 system remain to be fully elucidated. In this front, most of the efforts conducted so far have been directed towards characterization of the potential roles of leptin in this phenomenon. Solid evidence supports a crucial role of leptin in the regulation of Kiss1 expression at the hypothalamus. In brief, (i) Kiss1 mRNA levels at the ARC are decreased in ob/ob mice, but increase following treatment with leptin [START_REF] Smith | KiSS-1 neurones are direct targets for leptin in the ob/ob mouse[END_REF]; (ii) diabetic rats -with severe hypoleptinemia-show decreased levels of Kiss1 mRNA at the hypothalamus, which are normalized by intracerebral infusion of leptin, but not of insulin [START_REF] Castellano | Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats[END_REF][START_REF] Castellano | Alterations in Hypothalamic KiSS-1 System in Experimental Diabetes: Early Changes and Functional Consequences[END_REF]; and (iii) leptin can increase Kiss1 mRNA expression in the murine hypothalamic cell line, N6, and human GnRH-secreting neuroblasts [START_REF] Luque | Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line[END_REF][START_REF] Morelli | Sex steroids and leptin regulate the "first Kiss" (KiSS 1/G-proteincoupled receptor 54 system) in human gonadotropin-releasing-hormone-secreting neuroblasts[END_REF]. In good agreement, intracerebral administration of leptin to adult female rats induced phosphorylation of STAT3 at the AVPV [START_REF] Quennell | Leptin indirectly regulates gonadotropin-releasing hormone neuronal function[END_REF], where a prominent population of Kiss1 neurons is detected in the female. Admittedly, however, the studies summarized above have not been specifically conducted in pubertal animals/models, and thus may not be representative of physiological mechanisms during puberty. Anyhow, our preliminary evidence in models of postnatal undernutrition in female rats suggests that leptin is also an important regulator of Kiss1/kisspeptin expression at the time of puberty, as in animals subjected to restricted feeding during lactation, which are leaner and display delayed vaginal opening at the time of puberty, there is a tight correlation between (low) circulating leptin and the hypothalamic levels of Kiss1 mRNA and number of kisspeptin-positive neurons (Castellano & Tena-Sempere, manuscript in preparation). The latter is of interest, as important developmental/plastic changes have been reported for Kiss1 neurons along pubertal maturation [START_REF] Clarkson | Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons[END_REF]. This opens the possibility that, together with other factors -such as estrogens-early changes in leptin levels may contribute to the 'shaping' of kisspeptin projections during puberty.

Finally, the involvement of other neuropeptides in the metabolic control of the Kiss1 system remains virtually unexplored. Nonetheless, fragmentary evidence suggests that NPY, IGF1 and ghrelin may operate as putative regulators of Kiss1 expression [START_REF] Forbes | Effects of ghrelin on Kisspeptin mRNA expression in the hypothalamic medial preoptic area and pulsatile luteinising hormone secretion in the female rat[END_REF][START_REF] Luque | Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line[END_REF][START_REF] Hiney | Insulin-like growth factor-I activates KiSS-1 gene expression in the brain of the prepubertal female rat[END_REF]. Again, the (scarce) data so far available in this front do not come from models of puberty, which warrants specific analyses on the putative roles of these (and other) metabolic signals in the regulation of the Kiss1 system at the time of puberty.

Novel Players in the Metabolic Regulation of Kiss1: The roles of Crtc1 and mTOR

While the ability of leptin to regulate the hypothalamic expression of Kiss1 was demonstrated by initial studies, the intracellular mechanisms for such an action have remained elusive for years.

Recent findings, however, suggest the involvement of the Creb1-regulated transcription coactivator-1 (Crct1) in mediating leptin effects on the Kiss1 system at the hypothalamus. Thus, in a recent elegant study, Altarejos and co-workers demonstrated that mice engineered to lack functional Crtc1 were not only obese and hyperphagic, but also infertile [START_REF] Altarejos | The Creb1 coactivator Crtc1 is required for energy balance and fertility[END_REF]. Interestingly, in that study, leptin was also demonstrated to dephosphorylate (and activate) Crtc1, which in turn stimulated the recruitment of Crct1 to Kiss1 gene promoter and the expression of Kiss1 mRNA at the hypothalamus [START_REF] Altarejos | The Creb1 coactivator Crtc1 is required for energy balance and fertility[END_REF]. Altogether, these findings unveil a putative leptin-Crtc1-kisspeptin pathway whereby nutritional cues and body energy stores would modulate puberty onset and fertility. As call of caution, a very recent report has been unable to replicate the consequences of functional inactivation of Crtc1 on mouse fertility [START_REF] Breuillaud | Mouse fertility is not dependent on the CREB coactivator Crtc1[END_REF], suggesting that, under some specific conditions or genetic backgrounds, the function of Crtc1 in the control of Kiss1 expression and reproductive function may be compensated by redundant regulatory pathways. Generation of conditional and/or tissue-specific knock outs of Crtc1 will help to solve this issue.

In a parallel line, we have recently obtained evidence that hypothalamic signaling through mTOR (mammalian target of rapamycin) is also involved in the control of puberty onset, at least partially, via modulation of Kiss1 expression [START_REF] Roa | The Mammalian Target of Rapamycin as Novel Central Regulator of Puberty Onset via Modulation of Hypothalamic Kiss1 System[END_REF]. Of note, mTOR is a serine/threonine kinase that operates as sensor of the cellular energy status and effector for its coupling to cell growth and proliferation [START_REF] Martin | The expanding TOR signaling network[END_REF][START_REF] Schmelzle | TOR, a central controller of cell growth[END_REF]. At the ARC, mTOR signaling had been proposed as transducer for leptin effects on energy homeostasis and food intake [START_REF] Cota | Hypothalamic mTOR signaling regulates food intake[END_REF]; yet, its potential involvement in the metabolic regulation of puberty onset and fertility remained unexplored. Our recent data demonstrate that blockade of central mTOR disrupts the normal timing of puberty in female rats. More importantly, inhibition of mTOR signaling prevented the permissive effects of leptin in terms of puberty onset, and resulted in the suppression of Kiss1 mRNA levels, mainly at the ARC [START_REF] Roa | The Mammalian Target of Rapamycin as Novel Central Regulator of Puberty Onset via Modulation of Hypothalamic Kiss1 System[END_REF]. Altogether, these findings provide evidence for a leptin-mTOR-kisspeptin pathway in the metabolic control of puberty. Whether other central fuel-sensing mechanisms are also involved in the metabolic regulation of Kiss1 expression at the hypothalamus is yet to be explored. In this context, AMPK has been suggested to reciprocally cooperate with mTOR in the central control of energy homeostasis; AMPK being an appetite-stimulating factor [START_REF] Kahn | AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism[END_REF][START_REF] Cota | The role of CNS fuel sensing in energy and glucose regulation[END_REF]. In keeping with this view, our preliminary data suggest that activation of AMPK induced a partial delay of puberty onset and modestly decreased Kiss1 mRNA levels at the ARC in female rats (unpublished observations), which may be caused, at least partially, by its ability to inhibit mTOR signaling [START_REF] Tsang | Targeting mammalian target of rapamycin (mTOR) for health and diseases[END_REF][START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF].

Obesity and Puberty Onset

Most of the experimental evidence summarized in previous sections originates from studies in models of negative energy balance. These are enormously instrumental to unveil putative metabolic regulators of puberty onset, and very helpful to predict the patho-physiological mechanisms and consequences of puberty disruption due to (e.g.) strenuous exercise or anorexia nervosa. Admittedly, however, these may not provide a proper insight into pubertal alterations linked to conditions of energy excess, such as morbid obesity, which are much more common among adolescents across developed countries. Indeed, epidemiological studies in humans demonstrate that obesity is frequently linked to changes in the timing of puberty and decreased reproductive performance later in life; the former manifesting usually as earlier onset of puberty in obese girls [START_REF] Kaplowitz | Link between body fat and the timing of puberty[END_REF][START_REF] Ahmed | Childhood obesity and the timing of puberty[END_REF][START_REF] Jasik | Adolescent obesity and puberty: the "perfect storm[END_REF]. The mechanisms for such association, and even its causative relationship, remain unclear and have been the matter of debate, as excess of estrogen -caused by precious puberty-could contribute to fat deposition and may predispose to obesity, but excess of adiposity -due to prevailing obesity-may as well induce inappropriately high estrogen levels that may cause earlier breast development (as conventional marker of initiation of puberty).

Several studies in human populations point out, however, that earlier puberty may be caused, or at least facilitated, by a prevailing excess of fat mass; increased body fatness or a rapid elevation in BMI being predictors of earlier onset of puberty [START_REF] Kaplowitz | Link between body fat and the timing of puberty[END_REF]. A potential explanation for such a phenomenon would be that excessive production of leptin at early stages of development may accelerate the timing of puberty within a certain temporal range. This has been suggested on the basis of longitudinal studies, where elevated leptin levels clearly preceded the pubertal rise of LH and estradiol in girls, and the age at menarche was partially related to circulating leptin concentrations. This is not incompatible with the proposed permissive role of leptin in the control of puberty; while leptin per se does not operate as trigger of puberty, threshold leptin levels are mandatory for puberty to proceed. If this threshold is achieved precociously, in the presence of other positive modulators, puberty may take place earlier. The central mechanisms whereby elevated leptin may transmit the signal for earlier puberty in humans is yet to be elucidated, but data from rodent studies would suggest a role of hypothalamic kisspeptin neurons (see section 4). In fact, using a model of postnatal overfeeding in female rats, which results in higher body weight and earlier puberty onset, we have recently obtained evidence for a close correlation between the age of vaginal opening, the circulating levels of leptin, and the hypothalamic content of Kiss1 mRNA and kisspeptin-positive neurons at the time of puberty; all parameters being advanced/elevated in overweighed animals (Castellano & Tena-Sempere, manuscript in preparation). Admittedly, however, the causative nature of such association is yet to be fully demonstrated.

In addition to the excess of leptin, other mechanisms may contribute to alterations in the timing of puberty in obese individuals. These may include the state of hyperinsulinemia and insulin resistance, frequently linked to obesity, and the excess of estrogen production due to enlargement of the adipose tissue -which is an active source of aromatase activity [START_REF] Ahmed | Childhood obesity and the timing of puberty[END_REF][START_REF] Jasik | Adolescent obesity and puberty: the "perfect storm[END_REF]. In addition, recent studies in female rodents have suggested that the elevation in androgen levels, seen in models of dietinduced obesity, could be also responsible for earlier onset of puberty, as this was blocked by the anti-androgen flutamide [START_REF] Brill | Androgen Receptor Antagonism and an Insulin Sensitizer Block the Advancement of Vaginal Opening by High-Fat Diet in Mice[END_REF]. According to this model, excess of androgens in the obese female would cause a decrease in the negative feedback restrain of sex steroids on the central elements of the HPG axis, thereby making the system more prone to its precocious activation. In any event, the ultimate neuroendocrine mechanisms responsible for the acceleration in the onset of puberty in the obese female are yet to be fully deciphered. Similarly, whether obesity may also have an impact on male puberty warrants further investigation [START_REF] Ahmed | Childhood obesity and the timing of puberty[END_REF].

Conclusions

While the contention that puberty onset critically depends on adequate body energy stores has been known for Ages, the neuroendocrine substrate for such a metabolic regulation of puberty and fertility has begun to revealed only recently. Nevertheless, in the last decades, significant progress has been achieved in this area, and several peripheral hormones, with key roles in the control of metabolism, have been demonstrated to influence the timing of puberty, through the modulation of diverse central pathways that converge, mainly, at the level of the GnRH system. Among those, we have summarized herein some of the recent developments in our knowledge on the mode of action of leptin and ghrelin in the control of puberty, with emphasis being made on the indirect mechanism whereby leptin influences the function of GnRH neurons and the detailed description of the effects of ghrelin on male and female puberty. In addition, we have provided a succinct overview of the putative roles of kisspeptin neurons at the hypothalamus as conduits for the metabolic control of puberty and reproduction, with special attention to the role of leptin in the regulation of the Kiss1 system, and the recently identified roles of Crtc1 and mTOR in such a phenomenon. A tentative integral model for such a network of peripheral signals and central pathways transmitting metabolic information to the centers (GnRH neurons) driving puberty onset is provided in Figure 3. We are confident that further research efforts along these lines will help to better define the physiological basis for the regulation of the timing of puberty by metabolic factors, and to identify the pathophysiological mechanisms for its eventual perturbations in unfavorable metabolic conditions, ranging from anorexia to morbid obesity.

Legends to Figures

Figure 1: Scheme of the major peptide products of the ghrelin gene. This gene encodes a precursor protein of 117 amino acids, termed prepro-ghrelin, which after excision of the signal peptide (SP) gives rise, through differential proteolytic processing, to ghrelin (28 amino acids) and obestatin (23 amino acids). Ghrelin exists in two major forms: acyl-ghrelin, generated by octanoyl incorporation at Ser3 -a process mediated by ghrelin O-acyltransferase (GOAT), and unacylated ghrelin (UAG). Taken from ref [START_REF] Tena-Sempere | Ghrelin as a pleotrophic modulator of gonadal function and reproduction[END_REF], with minor modifications.

Figure 2:

Summary of the effects of chronic treatments of peripubertal female rats with of repeated injections of (i) ghrelin (1.0 nmol/12-h for 10 days), (ii) UAG (1.0 nmol/12-h) or (iii) a combination of both. Treatments were initiated on day 25 postpartum. Experimental procedures were as described in detail in ref [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF] for male rats . In panel A, evolution of body weights (BW) in the different treatment groups is depicted. In panel B, accumulated percentage of vaginal opening (V.O.) in the different experimental groups, during the last three days of treatment, are shown. Finally, in panel C, different indices of follicular/ovarian maturation are provided, as described in detail elsewhere [START_REF] Roa | The Mammalian Target of Rapamycin as Novel Central Regulator of Puberty Onset via Modulation of Hypothalamic Kiss1 System[END_REF]. Thus, the percentages of animals of each group not showing signs of ovulation are indicated. In addition, the most advanced stage of follicular/corpus luteum (CL)development for each group is indicated; for this calculation, ovarian sections from each animal were scored from -5 to -1 when showing follicle classes 1-5 as the most advanced stage, respectively, or from +1 to +4 when showing 1-to 4-day-old CL, respectively. Finally, ovarian weights from each group at the end of treatments are presented (** P<0.01; ANOVA followed by Student-Newman-Keuls multiple range test). Overall, these data demonstrate that persistently elevated ghrelin levels across puberty has an inhibitory impact on puberty onset in the female rat, as evidenced by delayed V.O., delayed follicular maturation and ovulation and decreased ovarian weight. for leptin effects is reinforced by the fact that leptin receptors are not expressed in GnRH neurons, which excludes the possibility of direct effects of leptin at this site. Notwithstanding, it remains plausible that part of leptin actions on Kiss1 neurons may be indirectly conducted via intermediary neuronal pathways, whose nature and physiological relevance is yet to be elucidated. In addition, the potential involvement of mTOR and Crtc1 in transmitting metabolic (leptin) information onto Kiss1 neurons is also indicated. For sake of simplicity, discrimination between different populations of Kiss1 neurons (i.e., ARC vs. AVPV) is not provided in the scheme; yet, it remains possible that not all Kiss1 neurons are similarly regulated by different metabolic cues along puberty. For further details, see the text.

  s c r i p t Metabolic Control of Puberty 2

  s c r i p t Metabolic Control of Puberty 4

  s c r i p t Metabolic Control of Puberty 6

  A c c e p t e d M a n u s c r i p t Metabolic Control of Puberty 7

A c c e

  p t e d M a n u s c r i p t Metabolic Control of Puberty 8 ovulatory surge of gonadotropins, among others. While detailed description of the major features

  s c r i p t Metabolic Control of Puberty 10

  s c r i p t Metabolic Control of Puberty 11

  s c r i p t Metabolic Control of Puberty 12

  s c r i p t Metabolic Control of Puberty 15 in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 146, 3917-25.

  s c r i p t Metabolic Control of Puberty 17

Figure 3 :

 3 Figure 3: Tentative integral model for the metabolic control of puberty onset by the dynamic interplay between peripheral hormones and central circuits that ultimately impinge onto GnRH neurons, as final output pathway for the pubertal activation of the reproductive axis. Accordingto the focus of this chapter, the putative roles of leptin (as prominent signal for energy abundance; permissive/stimulatory factor) and ghrelin (as putative signal of energy insufficiency; inhibitory factor) are highlighted. In addition, the major nodal role of kisspeptin neurons as integrators for a wide diversity of peripheral and central signals transmitting metabolic information onto reproductive centers is also represented. Of note, Kiss1 neurons have been proposed as targets for the regulatory actions of leptin (and ghrelin), and their role as mediators

  s c r i p t Metabolic Control of Puberty 18

Figure- 1 A c c e p t e d M a n u s c

 1c Figure-1

  Figure2

Acknowledgments

The authors are indebted with the members of the research team at the Physiology Section of the University of Cordoba, who actively participated in the generation of experimental data discussed herein. The work from the authors' laboratory reviewed in this article was supported by grants BFU 2005-07446 and BFU 2008-00984 (Ministerio de Ciencia e Innovación, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), Project P08-CVI-03788 (Junta de Andalucía, Spain), and EU research contracts EDEN QLK4-CT-2002-00603 and DEER FP7-ENV-2007-1. CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).


M.D. Stephan Miehlke
email: stephan.miehlke@uniklinikum-dresden.de

Susannne Loebe

Ahmed Madisch

Eberhard Kuhlisch

Martin Laass

Dana Grossmann

Holger Knoth

Andrea Morgner

Joachim Labenz

Susanne Löbe

Martin Laas

Dana Großmann

Intragastric acidity during administration of generic omeprazole and esomeprazole -a randomized, two-way crossover study including CYP2C19 genotyping

Keywords: Acidity (intragastric) < Topics, GERD or GORD < Disease-based, Peptic ulcer disease < Disease-based, H. pylori

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Several intragastric pH studies have shown that esomeprazole at a dose of 40 mg daily is superior to standard dosages of all other available original brand PPI's in controlling intragastric acidity [START_REF] Lind | Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease[END_REF][START_REF] Röhss | Effect of esomeprazole 40 mg vs. omeprazole 40 mg on 24-hour intragastric pH in patients with symptoms of gastroesophageal reflux disease[END_REF][START_REF] Miner | Gastric acid control with esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole: a five-way crossover study[END_REF][START_REF] Wilder-Smith | Esomeprazole 40 mg provides improved intragastric acid control as compared with lansoprazole 30mg and rabeprazole 20 mg in healthy volunteers[END_REF][START_REF] Röhss | Esomeprazole 40 mg provides more effective intragastric acid control than lansoprazole 30 mg, omeprazole 20 mg, pantoprazole 40 mg and rabeprazole 20 mg, in patients with gastroesophageal reflux symptoms[END_REF], while high-dose esomeprazole was superior to high-dose pantoprazole in another study [START_REF] Miehlke | Intragastric acidity during treatment with esomeprazole 40 mg twice daily or pantoprazole 40 mg twice daily--a randomized, two-way crossover study[END_REF]. This functional superiority of esomeprazole is mirrored by significant clinical advantages over some other PPI's in healing and maintenance therapy of erosive GERD [START_REF] Kahrilas | Esomeprazole improves healing and symptom resolution as compared with omeprazole in reflux oesophagitis patients: a randomized controlled trial. The Esomeprazole Study Investigators[END_REF][START_REF] Schmitt | A multicenter, randomized, doubleblind, 8-week comparative trial of standard doses of esomeprazole (40 mg) and omeprazole (20 mg) for the treatment of erosive esophagitis[END_REF][START_REF] Lauritsen | Esomeprazole 20 mg and lansoprazole 15 mg in maintaining healed reflux oesophagitis: Metropole study results[END_REF][START_REF] Labenz | Esomeprazole 20 mg vs. pantoprazole 20 mg for maintenance therapy of healed erosive esophagitis: results from the EXPO study[END_REF] while there were no differences from omeprazole in the treatment of non-erosive GERD [START_REF] Armstrong | The role of acid suppression in patients with endoscopy-negative reflux disease: the effect of treatment with esomeprazole or omeprazole[END_REF].

Generic omeprazole has been approved and is being increasingly used for the treatment of GERD and other acid related gastrointestinal disorders. However, clinical or intragastric pH studies comparing generic omeprazole with other original PPI are sparse. A randomized study in Japanese healthy subjects demonstrated significant differences in intragastric acid control between one original and two generic omeprazole brands [START_REF] Shimatani | Acid-suppressive effects of generic omeprazole: comparison of three brands of generic omeprazole with original omeprazole[END_REF]. Finally, a comparative study of 25 non-Astra authorised omeprazole products suggests significant pharmaceutical differences between various products after storage under forced conditions [START_REF] Karlsson | An investigation of the pharmaceutical quality of non-Astra authorised omeprazole products -a comparative study[END_REF]. Due to differences in the enzymatic hepatic metabolism, the racemic omeprazole and the s-isomer esomeprazole differ in their pharmacokinetic and pharmacodynamic profile [START_REF] Hassan-Alin | A pharmacokinetic study comparing single and repeated oral doses of 20 mg and 40 mg omeprazole and its two optical isomers, S-omeprazole (esomeprazole) and R-omeprazole, in healthy subjects[END_REF][START_REF] Andersson | Pharmacokinetics and pharmacodynamics of esomeprazole, the S-isomer of omeprazole[END_REF]. Esomeprazole is less metabolized by the polymorphic cytochrome P450 2C19 (CYP2C19) than omeprazole. Several studies have shown that genetic polymorphism of CYP2C19 which encodes for different intragastric acid control by PPI's, while different PPI's may be affected differently [START_REF] Furuta | CYP2C19 genotype status and effect of omeprazole on intragastric pH in humans[END_REF][START_REF] Sagar | Effects of omeprazole on intragastric pH and plasma gastrin are dependant on the CYP2C19 polymorphism[END_REF][START_REF] Shirai | Effects of CYP2C19 genotypic differences in the metabolism of omeprazole and rabeprazole on intragastric pH[END_REF]. Accordingly, the rapid CYP2C19 metabolizer status has been recognized as a significant risk factor for treatment failure in GERD and in Helicobacter pylori eradication therapy [START_REF] Kirsch | Relevance of cytochrome P450 polymorphisms in the treatment of Helicobacter pylori infection and gastroesophageal reflux disease[END_REF].

The aim of this study was to compare the effect of generic omeprazole 20 mg per day with esomeprazole 20 mg per day on intragastric acidity, and to investigate the influence of the CYP2C19 metabolizer status.

MATERIALS & METHODS

Subjects

Healthy men and women between 18 and 60 years of age were recruited by local advertisement at the University Hospital Dresden. The subjects were considered healthy based on a detailed medical history, physical examination and routine laboratory assessment. Only subjects who were H. pylori negative based on 13 C urea breath test (INFAI, Cologne, Germany) were eligible. Subjects with past or present significant illness and those with known intolerance to PPI were excluded. Women with childbearing potential were required to use accepted birth control methods.

Further exclusion criteria were pregnancy, lactation, abnormal laboratory values at baseline, and drug or alcohol abuse. Antisecretory drugs including PPI, H2 receptor antagonists, prokinetic drugs or any other agents that could affect the pharmacokinetics or absorption of PPIs were not allowed during the study or within two weeks before randomisation. All subjects gave written informed consent prior to enrolment. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The study was approved by the local institutional review board at the University Hospital in Dresden.

Study design and procedures

In this randomized, investigator-blinded, two-way cross-over study each subject received either generic omeprazole (Omep®, Hexal AG, Holzkirchen, Germany) 20 mg once daily or esomeprazole (Nexium mups®, AstraZeneca, Wedel, Germany) 20 mg once daily for five consecutive days according to a computer-generated randomization list. Crossover administration of the study drug followed after a washout period of 14 days (+/-3 days). The study drug was placed into identically

Assessment of intragastric pH

An ambulatory continuous 24-h intragastric pH recording was performed on day 5 of each dosing period. The 24-h intragastric pH was recorded using a microelectrode (Ion sensitive field effect transistor, Veraflex®, Alpine Biomed Corp.) with an internal reference electrode linked to a Digitrapper 100 ® (Medtronic GmbH, Düsseldorf). The calibrated microelectrode was positioned 10 cm below the manometrically located lower oesophageal sphincter at 7.30 AM prior to breakfast and the administration of the study drug, and was removed the following morning at 8 AM. The electrode was calibrated before and after each recording. Intragastric pH was evaluated every 4 s and recorded on the attached Digitrapper. All pH measurements were assessed by a single gastroenterologist who was blinded to the study group.

CYP2C19 genotyping

Cytochrome P450 2C19 (CYP2C19) genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Genomic DNA was extracted from whole blood and was PCR-amplified using published allele-

Statistics

The sample size calculation was based on the data published by Lind et al. [START_REF] Lind | Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease[END_REF]. The primary endpoint of the study was the time spent with an intragastric pH >4 within 24 h. We hypothesized that for generic omeprazole the time with intragastric pH >4 would be ≥10% lower compared to original omeprazole. For esomeprazole, 12.3 to 12.7 h spent with an intragastric pH >4 was assumed (3). Based on these assumptions, a sample size of 24 subjects was considered sufficient to show a statistically significant difference in favour of esomeprazole.

We analyzed data from a two-dosing, two-period (2 x 2) cross-over design using a random effects model modelling the carryover effect form one period to the next, the treatment (drug) effect and the period effect. First the assumptions of using a crossover design were tested. The results showed that there was neither a carryover effect from one period to the next nor a period effect for all of the investigated measures. P values <0.05 were considered significant. Results are given as averaged means and their differences, including the 95% confidence interval.

RESULTS

Subject characteristics

Thirty-two healthy Caucasian subjects were screened. Eight subjects were excluded due to lack of adequate availability (n=4), pregnancy (n=1) or technical problems (n=3). Twenty-four subjects were randomized and available for complete analysis ( 13females, mean age 24.7 years, mean body mass index 22.1 kg/m 2 ).

Intragastric pH

The mean number of hours with available pH recordings on day 5 was 23.89 h (23.3-24.0) with generic omeprazole and 23.84 h (22.6-24.0) with esomeprazole. There were no statistically significant differences in intragastric pH values on day 5 between generic omeprazole 20 mg per day and esomeprazole 20 mg per day (Table 1).

Overall, there were no significant differences between generic omeprazole and esomeprazole with respect to different intragastric pH thresholds (Figure 1).

CYP2C19 status

By CYP2C19 genotyping, 16 subjects were classified as having an RM status and 8 as having an IM status. There were no subjects with a PM status. With respect to the median intragastric pH and the time spent with an intragastric pH >4 time, there were no significant differences between generic omeprazole and esomeprazole subjects with an RM and IM status, respectively (Table 2). However, the percentage of time with intragastric pH >3.0 and pH >3.5 was significantly higher with esomeprazole compared to generic omeprazole in subjects with an RM status (∆ 7.5% (p=0.046) and ∆ 8.7% (p=0.026), respectively). Otherwise, there were no significant differences between the two study drugs with respect to other intragastric pH thresholds (Figure 2).

Side effects

In total, only 4 subjects reported mild adverse events. There were no serious adverse events. With generic omeprazole, diarrhea, flatulence and bloating was reported once each. With esomeprazole, headache was reported twice, and nausea once. In our study population, the level of intragastric acid suppression with esomeprazole 20 mg daily was comparable to that reported by Lind et al. [START_REF] Lind | Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease[END_REF].

Moreover, the level of intragastric acid suppression obtained with generic omeprazole 20 mg daily was also in the range of data reported by other pH studies using original brand omeprazole [START_REF] Lind | Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease[END_REF][START_REF] Miner | Gastric acid control with esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole: a five-way crossover study[END_REF][START_REF] Röhss | Esomeprazole 40 mg provides more effective intragastric acid control than lansoprazole 30 mg, omeprazole 20 mg, pantoprazole 40 mg and rabeprazole 20 mg, in patients with gastroesophageal reflux symptoms[END_REF]. Given the validity of the comparator in our study (esomeprazole), it appears that the generic omeprazole used in our study provides a similar degree of intragastric acid suppression. Esomeprazole 20 mg daily has been proven effective for maintenance therapy of erosive GERD [START_REF] Lauritsen | Esomeprazole 20 mg and lansoprazole 15 mg in maintaining healed reflux oesophagitis: Metropole study results[END_REF][START_REF] Labenz | Esomeprazole 20 mg vs. pantoprazole 20 mg for maintenance therapy of healed erosive esophagitis: results from the EXPO study[END_REF], for on-demand therapy in non-erosive GERD [START_REF] Tsai | Esomeprazole 20 mg on demand is more acceptable to patients than continuous lansoprazole 15 mg in the long-term maintenance of endoscopy-negative gastroesophageal reflux patients: the COMMAND Study[END_REF][START_REF] Morgner-Miehlke | Esomeprazole: potent acid suppression in the treatment of acid-related disorders[END_REF] and for prevention of gastroduodenal ulcers in patients with non-steroidal antiinflammatory drugs [START_REF] Morgner | Esomeprazole: prevention and treatment of NSAID-induced symptoms and ulcers[END_REF]. Since other intragastric pH studies and pharmaceutical studies have suggested significant differences between different generic omeprazole products [START_REF] Shimatani | Acid-suppressive effects of generic omeprazole: comparison of three brands of generic omeprazole with original omeprazole[END_REF][START_REF] Karlsson | An investigation of the pharmaceutical quality of non-Astra authorised omeprazole products -a comparative study[END_REF], the question remains whether generic omeprazole can provide a similar clinical efficacy as other original PPI brands. Although the results of our intragastric pH study can not be directly transferred to clinical outcomes, we speculate, that the generic omeprazole used in our study with a daily dose of 20 mg might offer a similar clinical benefit in the treatment of acid-related gastroduodenal disorders as other original brand PPIs. Ideally, this hypothesis should be tested by adequately sized randomized clinical trials which are, of course, unlikely to be conducted. However, two randomized controlled trials have shown that omeprazole 20 mg daily was comparable to esomeprazole 20 mg daily in the treatment of non-erosive and erosive gastroesophageal reflux disease [START_REF] Armstrong | The role of acid suppression in patients with endoscopy-negative reflux disease: the effect of treatment with esomeprazole or omeprazole[END_REF][START_REF] Lightdale | A multicenter, randomized, doubleblind, 8-week comparative trial of low-dose esomeprazole (20 mg) and standarddose omeprazole (20 mg) in patients with erosive esophagitis[END_REF]. The CYP2C19 metabolizer status has been shown to affect intragastric acid suppression by PPI's, as well as the clinical efficacy of PPI's in the treatment of GERD and in Helicobacter pylori eradication therapy [START_REF] Kirsch | Relevance of cytochrome P450 polymorphisms in the treatment of Helicobacter pylori infection and gastroesophageal reflux disease[END_REF]. In particular, the rapid metabolizer status has been identified as significant risk factor for treatment failure in Asian patient populations. In our study, which was not powered to detect differences between the two drugs in subgroups, we found nevertheless a better acid control with esomeprazole compared to generic omeprazole in subjects with an rapid metabolizer status, the time spent with an intragastric pH above 3.0 and 3.5, respectively, being significantly longer with esomeprazole than with generic omeprazole. The pH threshold of 3 is of clinical relevance with respect to healing of duodenal ulcers [START_REF] Burget | Is there an optimal degree of acid suppression for healing of duodenal ulcers? A model of the relationship between ulcer healing and acid suppression[END_REF].

Whether this finding is also clinically relevant in patients with GERD remains unclear, since an intragastric pH threshold of 4 appears to be critical for the effectiveness of PPI in the treatment of erosive GERD [START_REF] Armstrong | Review article: gastric pH -the most relevant predictor of benefit in reflux disease?[END_REF]. This finding does not come as a surprise, since the S-isomer esomeprazole is metabolized to a greater extent via the cytochrome 3A4 subunit than the racemic omeprazole [START_REF] Hassan-Alin | A pharmacokinetic study comparing single and repeated oral doses of 20 mg and 40 mg omeprazole and its two optical isomers, S-omeprazole (esomeprazole) and R-omeprazole, in healthy subjects[END_REF][START_REF] Andersson | Pharmacokinetics and pharmacodynamics of esomeprazole, the S-isomer of omeprazole[END_REF]. Accordingly, data from randomized clinical studies in Caucasian GERD patients at least suggest that the efficacy of esomeprazole is not affected by the CYP2C19 metabolizer status [START_REF] Schwab | Esomeprazole-induced healing of gastroesophageal reflux disease is unrelated to the genotype of CYP2C19: evidence from clinical and pharmacokinetic data[END_REF].

In addition, the proportion of individuals with a rapid, intermediate or poor CYP2C19 metabolizer may vary between different ethnic populations, and different PPI's may be affected differently [START_REF] Kirsch | Relevance of cytochrome P450 polymorphisms in the treatment of Helicobacter pylori infection and gastroesophageal reflux disease[END_REF]. For example, in Asian populations the frequency of poor metabolizer is around 22% while in among Caucasians only about 3% harbor a poor metabolizer status [START_REF] Xie | Allelic, genotypic and phenotypic distributions of the S-mephenytoin 4'-hydroxylase (CYP2C19) in healthy Caucasian populations of European descent throughout the world[END_REF][START_REF] Bertilsson | Geographical/interracial differences in polymorphic drug oxidation. Current state of knowledge of cytochromes P450 (CYP) 2D6 and 2C19[END_REF]. Therefore, the determination of the

INTRODUCTION

  Proton pump inhibitors (PPI) are the treatment of choice in gastroesophageal reflux disease (GERD) and other acid-related conditions of the upper gastrointestinal tract[START_REF] Olbe | A proton-pump inhibitor expedition: the case histories of omeprazole and esomeprazole[END_REF][START_REF] Sachs | Review article: the clinical pharmacology of proton pump inhibitors[END_REF]. Depending on the clinical indication, approved PPI are usually prescribed in their full or half standard dosages.

  rapid, intermediate and poor metabolizer), affects

6 looking

 6 white screw-cap plastic container. Each tablet was swallowed unchewed by the study subject between 7.30 and 8 AM together with 200 ml of water 30 minutes before meal for five consecutive days under direct supervision of the study physician, thereby providing 100% compliance. Subjects were instructed to avoid smoking and alcohol consumption during the entire treatment period and to lie down only at nighttime hours between 10 PM and 6 AM. They were also encouraged to report adverse events spontaneously. Tolerability of the study drug was assessed each day during the treatment period.

  . PCR products were digested using the restriction endonucleases SmaI and BamHI, and visualized by 3% agarose gel electrophoresis. Based on the PCR-RFLP result, subjects were classified as rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer (PM). Appropriate controls were included in each PCR analysis set.

10 DISCUSSION

 10 To our knowledge, this is the first study to compare equivalent dosages of generic omeprazole and esomeprazole in healthy Caucasian subjects. In our view, the results of the study are clinically relevant, since generic omeprazole brands are increasingly used for the treatment of acid related upper gastrointestinal disorders, while clinical data to support this strategy are clearly limited and controversial.

12 CYP2C19

 12 metabolizer status may only be of limited value for clinical practice.in Caucasian patients.In summary, our study suggests that generic omeprazole 20 mg once daily provides a similar intragastric acid control as esomeprazole 20 mg once daily in healthy volunteers. However, esomeprazole provides better acid control in the subgroup of CYP2C19 rapid metabolizers. Further studies are needed to evaluate whether this advantage translates into clinical effectiveness.

19 TABLESTable 1 : 20 Table 2 : 21 LEGENDSFigure 1 :Figure 2 :

 1912022112 Figure 1: Mean percentage of time above intragastric pH tresholds in rapid (RM) and

ACKNOWLEDGEMENTS

The study was not supported by a pharmaceutical company. We are grateful to Christian Kirsch and Renate Beckmann for their assistance in the pH studies and the data management.


Pasquale Pace
email: ppace@deis.unical.it

Valeria Loscrí
email: vloscri@deis.unical.it

Enrico Natalizio
email: enrico.natalizio@inria.fr

Tahiry Razafindralambo
email: tahiry.razafindralambo@inria.fr

Nodes Placement for reducing Energy Consumption in Multimedia Transmissions

Keywords: Wireless Sensor Networks, Multimedia, Energy Saving, Quality of Experience

Power consumption is an essential issue in wireless multimedia sensor networks (WMSNs) due to the elevated processing capabilities requested by the video acquisition hardware installed on the generic sensor node. Hence, node placement scheme in WMSNs greatly impacts the overall network lifetime. In this context, the paper first proposes a suitable hardware architecture to implement a feasible WMS node based on off-the-shelf technology, then it shows how the energy spaced placement of the wireless nodes reduces the energy consumption in comparison with the classical evenly spaced placement, without affecting the video quality of multimedia traffic.

I. INTRODUCTION

A wireless sensor network (WSN) is a collection of small and constrained devices that communicate via wireless links in a multihop fashion. In recent years, the research on wireless sensor networks has received a lot of attentions, due to their potential applications in various areas such as emergency/rescue operations, military operations, environment monitoring [START_REF] Hart | Environmental Sensor Networks: A revolution in the earth system science?[END_REF], [START_REF] Barrenetxea | The hitchhiker's guide to successful wireless sensor network deployments[END_REF].

In most of the deployments, sensors are expected to send few amount of data (temperature, humidity, etc.) either regularly or upon the detection of a specific event to a central entity, called sink (or base station). Due to resource limitation, the base station may not be reachable directly by each sensor. Thanks to the wireless communication capabilities and the protocols developed in the literature, multi-hop transmissions can be used to route data from a sensor to the sink when no direct connection is available. Moreover, sensors are supposed to be static or quasi-static.

Recently, a new generation of sensors appeared. These sensors have much more capabilities than the low-capacity (processor, memories, battery) sensors used so far. They are equipped with low cost cameras and/or microphones to be able to acquire and transmit multimedia contents to the base station. The multimedia sensing capabilities, provided by this new generation of sensors, raise some new issues in the field of wireless sensor networks due to their uniqueness [START_REF] Almalkawi | Wireless Multimedia Sensor Networks: Current Trends and Future Directions[END_REF], [START_REF] Davis | On the designand implementation of wireless multimedia sensor networks[END_REF]. These new sensors produce a huge amount of data with Quality of Service (QoS) restrictions and requirements such as end-toend delay. Furthermore, constraints such as energy, delay, loss ratio and throughput are still an issue.

Dimensioning a multimedia sensor network should be done by anticipating users' needs and issues. Unfortunately, this is not always feasible. It is not easy to increase wireless link capacity or performance (delay, bandwidth, etc.) since sensors and wireless links are finite resources. In the context of wireless networks and wireless multimedia networks, Akyildiz et al. stated in Elsevier Computer Communication 2007 [START_REF] Akyildiz | A survey on wireless multimedia sensor networks[END_REF]: "[...] capacity and delay attainable on each link are location dependent, vary continuously, and may be bursty in nature, thus, making quality of service (QoS) provisioning a challenging task [...]" In this paper, we evaluate the quality of service provided at the application layer for a multimedia traffic. We show how nodes' placement can impact the performance by increasing or decreasing the multimedia quality. We use a video evaluation framework to extract multimedia metrics such as MOS (Mean Opinion Score) [START_REF]Subjective video quality assessment methods for multimedia applications[END_REF], VQM (Video Quality Meter) [START_REF]Final report from the video quality experts group on the validation of objective models of video quality assessment, phase II[END_REF] and try different node placements (energy spaced, evenly spaced) to show that small differences in the placement (± 7% in distance) have a remarkable impact on the energy saving by extending the lifetime of the network up to more than 15% while preserving video quality.

The rest of the paper is organized as follow. Section II gives the state of the art of sensor placement schemes for multimedia traffic. Section III describes the placement schemes used for the evaluation in this paper. Section IV is devoted to the system model. Simulation results are provided in Section V before concluding the paper in Section VI.

II. RELATED WORKS

The placement of the nodes can highly affect the performance of the network in terms of routing, capacity, delay, throughput, energy consumption, etc. [START_REF] Akyildiz | A survey on wireless multimedia sensor networks[END_REF], [START_REF] Cao | Node Placement of Linear Wireless Multimedia Sensor Networks for Maximum Network Lifetime[END_REF]. At the best of our knowledge, no works have been proposed to study the effect of nodes placements on both the quality of a multimedia transmission and the energy consumption of the network.

The work proposed by So et al in [START_REF] So | Effect of relaying on capacity improvement in wireless local area networks[END_REF], shows the effect of relay nodes' placement in wireless local area networks. The authors study how the number of relay nodes and the characteristics of the traffic affect the optimal relay node placement and the expected throughput capacity of the network. The authors do not give any outline on how to place the nodes to increase the capacity or reduce the energy consumption.

In [START_REF] Shirazi | Outage optimal node placement in ultrawideband sensor networks[END_REF], the authors show the effect of node placement in ultra wideband sensor networks by evaluating the performances of communication reliability at the MAC layer.

Cao et al. in [START_REF] Cao | Node Placement of Linear Wireless Multimedia Sensor Networks for Maximum Network Lifetime[END_REF] make a comparison among placements of nodes on the straight line between source and destination, but they do not consider any multimedia aspect.

III. PLACEMENTS USED

In this work we consider two schemes of placement. The first is referred as Evenly or Uniform placement and is taken from [START_REF] Goldenberg | Towards mobility as a network control primitive[END_REF]. Specifically, in [START_REF] Goldenberg | Towards mobility as a network control primitive[END_REF], authors show that the best configuration for a monodirectional data flow in terms of energy consumption is to place relay nodes in the evenly spaced positions on the straight line between the source and the destination node. This result is based on the intrinsic assumption that nodes have all the same residual energy. Once a more realistic assumption is taken into consideration, namely nodes can have different residual energies, the best placement for the energy consumption is not anymore the evenly spaced but the energy spaced [START_REF] Natalizio | Optimal Placement of Wireless Nodes for maximizing Network Lifetime[END_REF]. Specifically, authors derive a close formula to mathematically compute the position of each node depending on the residual energy of the nodes involved in the data flow, which we recall in (1):

v i = v 1 + i-1 k=1 E k P rec T P L u, i = 2, . . . , n -1. (1)
where the position of node i (v i ) is determined incrementally from the positions of all the previous nodes by taking into consideration their residual energy. An inherent property of the placement considered in [START_REF] Natalizio | Optimal Placement of Wireless Nodes for maximizing Network Lifetime[END_REF] is that nodes relaying the same data flow will completely deplete their batteries all in the same moment. In fact, from (1), we have that the pathlifetime can be found from

T P L = 1 P rec v n -v 1 2 n-1 i=1 E i 2
and the following property is verified:

T P L = T 1 = T 2 = • • • = T n-1 .
This is a very interesting property in wireless sensor networks, since when the batteries of nodes have different durations, the first node that dies affects the whole data flow. In fact, energy will be wasted to send control packets in order to build a new path or to restore the previous one. Moreover, before being notified that the path is broken, the source will keep sending packets on the broken path causing many packet losses. In this work, if we consider the specific characteristics of multimedia data that we want to transmit, the choice of a similar placement seems to be suitable for the purpose. In Fig. 1 we show the two placements. Even if the positions of nodes are similar for the two schemes, in Section V we will show the impact of the different placement on the quality of the multimedia transmission and the energy consumption.

IV. SYSTEM MODEL

A. Hardware choices and energy consumption evaluation

The main objective of this work consists in evaluating the energy consumption of video transmissions in MWSN by using different placements. Hence, a feasible hardware implementation of the generic wireless multimedia sensor node will be detailed in this section by illustrating the specific energy consumption of each module of the sensor node. This analysis is mainly finalized to discover the "realistic" values of energy absorbed by the hardware components, in order to use them during the simulation phase. In particular, we propose to design a multimedia sensor node constituted by two different modules: a new low energy wireless transmitter called W iF ly GX [START_REF]Rowing Networks wireless for less[END_REF] • the W iF ly GX is perfect for mobile wireless applications such as asset monitoring, GPS tracking and portable devices because of its small size and extremely low power consumption;

• the CM U cam3 implements a fully programmable embedded computer vision sensor and it guarantees a good video quality for light applications. An additional motivation for us to use these hardware modules to implement a MWS node is given by the fact that they represent a well documented off-the-shelf technology. They can assess the average energy consumption in terms of transmission power, receiver sensitivity and supply voltage as resumed in Table I.

Regarding the initial maximum energy value, each MWS node can be equipped with four AA batteries of 1100mAh that generate an initial energy equal to 23760Joule. To complete our consumption analysis, we need to take into account a supply voltage of 3V that turns into the following power consumptions for the W iF ly GX module:

T XP ower W iF ly = 110mA * 3V = 330mW RXP ower W iF ly = 40mA * 3V = 120mW IdleP ower W iF ly = 35mA * 3V = 105mW
Mixing together all the operating parameters discussed so far, we can obtain the overall consumption values for the proposed MWS node architecture as shown in Table II. In the Table T XP ower S represents the transmission power consumption of the source node whilst T XP ower R is the transmission power consumption of the generic relay or destination node in which the CM U Cam3 module is in the disable state.

B. Simulation Framework and Multimedia Traffic

Upon the determination of the energy consumption parameters for feasible MWS nodes, we used these parameters in the simulation of different placement strategies by using EvalVid [START_REF] Ke | An Evaluation Framework for More Realistic Simulations of MPEG Video Transmission[END_REF]. EvalVid is a complete framework and tool-set for quality evaluation of videos transmitted over a real or simulated communication network. This framework is able to measure QoS parameters of the underlying network, such as packet loss, delays and jitter. Moreover, it supports a subjective video quality evaluation of the received video based on the frame-by-frame PSNR (Peak Signal Noise Ratio) calculation. It has a modular and network independent structure, which permits all the interactions with the network via several trace files. EvalVid can be combined with the well known NS2 simulator in order to obtain a robust and reliable framework for evaluating the perceived quality of service for multimedia video content. Concerning the multimedia traffic, we chose to use four different video sequences of commonly used video test contents in the 4:2:0 YUV format [START_REF]YUV Video Sequences[END_REF]. These video sequences have the same CIF resolution, which is compatible with the presented hardware, and different properties in terms of data rate and change of scene. In particular, we coded the uncompressed YUV files using an MPEG-4 codec in order to create compressed raw videos with different data rate as shown in figure 3.

C. PQoS evaluation: PSNR and VQM

Since MWSNs are well suited for video content provisioning, the best way to evaluate the system performance consists in using video quality indexes based on human vision and real PQoS (Perceived Quality of Service) or QoE (Quality of Experience). Over the last years, emphasis has been put on developing methods and techniques for evaluating the perceived quality of digital video content. These methods are mainly categorized into two classes: subjective and objective.

The subjective test methods involve an audience of people, who watch a video sequence and score its quality as perceived by them, under specific and controlled watching conditions. The arithmetic mean of all the opinion scores collected is the MOS (Mean Opinion Score) standardized by ITU-T [START_REF]Subjective video quality assessment methods for multimedia applications[END_REF]. Since the preparation and execution of subjective methods is costly and time consuming, researchers have turned to simple error measures such as MSE (Mean-Squared Error) or PSNR, suggesting that they would be equally valid.

The EvalVid tool integrates the module for computing the PSNR value of a transmitted video, so that we can use this feature to evaluate the goodness of the multimedia transmission according to different nodes placements. Moreover, according to the work conducted in [START_REF] Klaue | EvalVid -A framework for video transmission and quality evaluation[END_REF] it is possible to match few ranges of PSNR values with the corresponding MOS index in order to use both subjective and objective quality metrics (see Table IV).

Unfortunately, the PSNR metric does not take into account human vision; thus, it cannot be a reliable predictor of perceived visual quality because this simple objective measure operates solely on the basis of pixel-wise differences neglecting the impact of viewing conditions on the actual visibility of videos. For this reason, in our study, we also considered the VQM (Video Quality Meter) index that represents a more general and robust purpose video quality model taking into account specific issues of human vision [START_REF]Final report from the video quality experts group on the validation of objective models of video quality assessment, phase II[END_REF] [19].

V. RESULTS

In this section, we will show the results obtained by simulating the behavior of the two aforementioned nodes placements in the integrated simulation framework EvalVid+NS2, when multimedia data has to be delivered from a source node to a destination node. In Table III, we reported the values of the parameters used throughout the simulation.

The performance parameters considered in this work are: PSNR and VQM as explained in section IV-C. Furthermore, since the main objective of the paper is to investigate the energy consumption issue of nodes when they relay multimedia traffic, we also consider the lifetime of the network.

A. Simulation Campaigns 1) Nodes Number:

A first simulation campaign has been conducted to determine the impact of the number of relay nodes on the measured PSRN. The placement used in this first simulation campaign is the evenly spaced. In Fig. 4 we reported the value of PSNR when the number of nodes involved in the multimedia transmission increases. With the aim of highlighting the decrease in video quality due to multihop, the basic configuration is firstly constituted only by source and destination, progressively the number of nodes increases from 2 to 7 and, consequently, the number of relay nodes from 0 to 5. Since in our simulations the distance between nodes is kept constant, the field area dimension has been increased accordingly from 66m× 66m considering only two nodes, to 400m × 400m for the configuration with seven evenly spaced nodes.

In Table IV, we recalled the values used to classify the quality of a video through the usage of the PSNR metric. We can see that the number of nodes involved in the flow does not affect the transmission of the videos with the two lowest bit rates, whose quality is always excellent. In this case, the capacity of the channel is large enough to permit a very high quality transmission. In order to appreciate the effect of a multihop transmission for such low bit rate videos, we will investigate the behavior of the placements for multiflow transmission as future works. When the bit rate of the transmitted video increases and saturates the capacity of the channel, the quality decreases drastically, especially for a higher number of nodes. In fact, the quality of "Hall" degrades from excellent to fair, and that of "Coastguard" from good to bad in the longest multihop path. If we pay particular attention to this last video we can argue that, even if the difference in video quality between the scenario with 4 nodes and the one with 7 nodes is very small (i.e., about 3dB), the high transmission rate required by this specific video, makes the fruition of the video content almost impossible in extended multihop scenarios.

In the rest of the simulation we will use the scenario with 7 nodes.

2) Comparison of placements:

The first comparison that we propose regards again the value of PSNR for both energy spaced placement and evenly spaced placement. In fact, it is important to notice that the energy spaced placement has been introduced in order to face the issue of energy consumption. Therefore, it could show a bad behavior in terms of video quality in respect of a more evenly distributed placement.

In Fig. 5 we show that, even if relay nodes are placed in different positions, the performance of the two placement schemes in terms of PSNR is the same. In fact, both the schemes obtain excellent quality for the two lowest bit rate videos, whereas they incur in a fair and a poor quality for the highest bit rate videos, respectively.

Since the only valuable information that was possible to extract from the previous plot was that the energy spaced placement obtains the same results of the evenly spaced in terms of PSNR, we decided to compare the two placements schemes according to the metrics explained in Section IV-C. As we said in the mentioned Section, the VQM is a more precise index of the quality perceived by the observer. VQM has to be read in the opposite way in respect of the PSNR, higher values mean lower qualities. In fact, we can see in Fig. 6 that the two lowest bit rate videos perform very well and that highest bit rate videos experience a slightly better quality for the evenly spaced placement (≈3% and ≈1% for "Hall" and "Coastguard", respectively). Since the main focus of the paper is on the energy consumption of the two placements, we need to understand how much the inappreciable improvement in terms of VQM is paid back in terms of energy by the evenly spaced placement. In Fig. 7 we can see that the energy spaced placement always obtain a lifetime that is at least 10% longer than the evenly spaced scheme, and in case of "Coastguard", energy spaced outperforms evenly spaced of about 15%. In absolute terms, it means that energy spaced makes multimedia nodes last from 1.5 up to 2.25 hours longer. This significant improvement in terms of energy saving supports the hypothesis to implement new energy-aware algorithms for multimedia sensor nodes based on controlled-mobility strategies.

VI. CONCLUSION

In this paper we first evaluated the energy consumption of a feasible WMS node specifically designed to exploit the potential of the current off-the-shelf hardware technology; afterwards, we validated further the results obtained from the energy spaced placement of wireless sensor nodes by extending the analysis to multimedia traffic sources. Future research directions will address the issues related to multiflow transmission of multimedia contents on different paths throughout energy-aware routing protocols.

Fig. 1 .

 1 Fig. 1. Nodes' positions with the Energy and Evenly placements.

  based on WiFi standard (fig.2.a), and a well known CM U cam3 video camera (fig.2.b) able to send CIF files format (352 × 288 pixels)[14]. The two modules can work together once mounted on top of a four lithium cell AA batteries. Specifically:

Fig. 2 .

 2 Fig. 2. Hardware modules of a generic MWS node: a) WiFly GX and b) CMUCam3.

Fig. 3 .

 3 Fig. 3. Multimedia Video Contents and Average Data Rates.

Fig. 4 .

 4 Fig. 4. PSNR when number of nodes varies in the transmission of the 4 videos of Figure 3.

Fig. 5 .Fig. 6 .

 56 Fig. 5. PSNR achieved by the two different placements for the scenario with 7 nodes.

Fig. 7 .

 7 Fig. 7. Lifetime achieved by the two different placements.

TABLE I MWS NODE HARDWARE CHARACTERISTICS Radio Characteristics of WiFly GX module

 I XP ower S = T Xantenna+T X W iF ly +active Cam 0.893W T XP ower R = T Xantenna+T X W iF ly +disable Cam 0.693W RXP ower = RX W iF ly + disable Cam 0.420W IdleP ower = Idle W iF ly + disable Cam

	Receive sensitivity	-85dBm
	Antenna TX Power	18dBm = 63.09mW
	Electrical Characteristics of WiFly GX module
	Supply Voltage	[2 -3.3]V
	WiFly GX Power Consumption
	Standby/Idle	35mA
	Connected (RX)	40mA
	Connected (TX)	110mA
	CMUcam3 General Characteristics
	max resolution	CIF 352 × 288
	color depth	8bits per pixel
	frame rate	26FPS
	CMUcam3 Power Consumption [15]
	Active Cam	500mW
	Disable Cam	300mW
		TABLE II
	MWS NODE OVERALL POWER CONSUMPTION
			0.405W
	CST hresh = receive Sensitivity = -85dBm	3.162e -12 W
	RXT hresh (transmission range 100m)	6.232e -10 W

T

TABLE III

 III

	SIMULATION PARAMETERS
	Field Area (L × L)	400m × 400m
	Source Position (x S , y S )	(0,0)
	Destination Position (x D , y D )	(400,400)
	Initial Maximum Residual Energy (Emax)	23760 J
	Initial Minimum Residual Energy (E min )	80% of Emax
	Maximum Transmission Range (r)	100 m
	Transmission Rate (r T )	1, 2, 5.5, 11 M b/s
	Actual Videos Transmission Rate (rv T )	see figure 3
	Propagation Error-Free Model	Two-Ray ground
	MAC Protocol	Standard 802.11b
	Routing Protocol	AODV
	Number of run for each scenario	100
	Interval confidence for average	95%

TABLE IV PSNR AND VIDEO QUALITY PSNR Value MOS Value (Quality)

 IV

	> 37dB	5 (Excellent)
	31 ÷ 37dB	4 (Good)
	25 ÷ 31dB	3 (Fair)
	20 ÷ 25dB	2 (Poor)
	< 20dB	1 (Bad)

ACKNOWLEDGMENTS

This work is partially funded by the French National Research Agency (ANR) under the project ANR VERSO RES-CUE (ANR-10-VERS-003) and the French Research National Institute of Computer Science and Automation (INRIA) under the ARC MISSION project.