Chapter 005, Serotonin: The Central Link between Bone Mass and Energy Metabolism

As a matter of fact, leptin does not appear during evolution with appetite, energy expenditure, or reproduction. Worms and flies can eat more or less, and certainly flies can be fertile or sterile, yet neither worms nor flies have leptin. Leptin is a vertebrate invention and, in fact, appears during evolution with bones 6. This could, of course, be a coincidence; in that case, leptin would not regulate bone mass.

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If on the other hand leptin does regulate bone remodeling, this suggests that it may have been invented during evolution to allow a coregulation of bone mass and energy metabolism in the same direction. Notwithstanding its possible involvement in bone remodeling, leptin is an attractive hormone to study for several reasons: Hence, conceivably there was still much more to be learned about it.

Analysis of loss-of-function models, i. Indeed, these two mutant mice that are hypogonadic had a high bone mass phenotype affecting trabecular bone, and that worsened over time. When taken at face value, these data because they were demonstrated through loss-of-function mutations demonstrated that leptin is a powerful inhibitor of bone mass accrual, a notion now widely accepted 11 , This was literally an out of the ordinary phenotype because they were and still are the only animal model in which there is a coexistence of hypogonadism and high bone mass 11 , The very fact that the absence of leptin signaling results in a high bone mass despite hypogonadism already suggested that the regulation of bone mass was a critical function of leptin.

This was confirmed through the analysis of a gain-of-function model in leptin signaling 28 , We present this model in this review because it was a model obtained through a homologous recombination in embryonic stem cells and was based on the molecular knowledge surrounding the leptin receptor. The long and signaling form of the leptin receptor is linked to the Jak 2 tyrosine kinase 30 , Leptin binding to its receptor activates Jak 2, resulting in the phosphorylation of several tyrosine residues on the leptin receptor, and one of these tyrosine residues Y binds Socs3 to attenuate leptin signaling.

Hence, substitution in vivo of Y of the leptin receptor by an amino acid that is not phosphorylated, e. The fact that bone mass accrual is the only function of leptin that is affected by a partial gain-of-function mutation in leptin signaling, while appetite and energy expenditure are not, indicates that the threshold of leptin signaling needed to affect bone mass accrual is lower than the one needed to affect appetite and energy expenditure. As such, it is consistent with the notion that the regulation of bone mass is a critical function of leptin. Overall, this first group of studies verified the initial hypothesis.

Yet, by identifying peripheral mediators linking, specifically for the regulation of bone mass accrual, leptin signaling in the brain to bone cells in the periphery, these studies also raised a critical question: For the longest time and for a constellation of excellent reasons, it was assumed that leptin was signaling in the hypothalamus to achieve its regulatory function By way of chronology, the first very convincing argument supporting this assumption was that when chemicals were used in the s to lesion neurons of the ventromedial hypothalamic VMH nuclei or of the arcuate hypothalamic nuclei, rats became hyperphagic, as animals lacking leptin signaling are, and as a result became obese Second, upon identification of the leptin receptor, it was shown to be highly expressed in neurons of the VMH and arcuate nuclei and more generally in the hypothalamus 25 , the part of the brain orchestrating many if not all homeostatic functions.

Although it is always easy with hindsight to question any reasoning, we must emphasize again that those were quite convincing arguments. As will become apparent later in this review, the destruction through chemical means of neurons of the VMH and arcuate nuclei turned out to be a key experiment whose importance cannot be overstated. Based on the results of these classical chemical-lesioning experiments, we decided to study where leptin signals the brain to regulate bone mass accrual.

What we observed is that lesioning of VMH neurons in wild-type mice resulted in a high bone mass phenotype that could not be corrected after intracerebroventricular ICV infusion of leptin Based on these two experiments and a series of appropriate controls, we concluded that VMH neurons were leptin-sensitive antiosteogenic neurons.

As it turns out now, this conclusion was both wrong and right. There was obviously an apparent contradiction between these results and ours. A first interpretation of these two experiments could be based on favoring one technical tool over the other. Because a genetic approach is by far more accurate, sensitive, and specific than chemical lesioning, such an approach would thus tend to ignore the chemical lesioning results and embrace only the genetic results.

Such an interpretation therefore rules out VMH neurons as the location where leptin signals in the brain to control bone mass. It does not provide any alternative mechanism to explain this well-documented function of leptin. Another interpretation of these two observations does not discard the validity of the chemical-lesioning experiments that are, after all, the founding experiments in the field of the central control of appetite.

If we consider that, at least technically, both genetic and lesioning experiments are valid, then what do they teach us and how do we reconcile them? A conservative view of these two experiments states, and in fact states only, that leptin needs the integrity of VMH and arcuate neurons of the hypothalamus to regulate bone mass accrual and appetite but needs not to signal itself in these neurons to fulfill these two functions. In other words, it may be that leptin needs the integrity of VMH and arcuate neurons to regulate bone mass and energy metabolism but that it needs not to signal itself in these neurons to do so.

Before explaining how we set out to test this hypothesis, it is critical to underscore how fortunate it was that both types of experiments, the chemical lesioning and the cell-specific gene inactivation, were performed. Indeed, if only the gene inactivation experiments had been done, one would not have been in a position to question their results. Indirectly, this illustrates that no technique in biology is above any other and that the conjunction of assays is always the most powerful approach.

To test the aforementioned hypothesis, we relied heavily on clinical information.

Coregulation of Bone Remodeling and Energy Metabolism

In particular, we were intrigued by the fact that patients chronically taking serotonin reuptake inhibitors, a class of drugs supposed to increase the concentration of serotonin in the extracellular milieu, were prone to developing low bone mass, hyperphagia, and weight gain 41 — This clinical information indicates that brain serotonin regulates, directly or indirectly, bone remodeling and appetite. Serotonin is a bioamine that is synthesized in the brain by neurons of the dorsal and ventral raphe in the brainstem.

A remarkable feature of serotonin is that, although it can by synthesized in the brain or in the duodenum, it does not cross the blood-brain barrier. Hence, an ablation of brain serotonin does not affect the function of serotonin in the periphery and vice versa. The biosynthesis of serotonin is initiated by two different enzymes in brainstem neurons and duodenal cells: Therefore, inactivating Tph2 should generate a mouse model lacking serotonin only in the brain.

This is the strategy we used to study the role of serotonin in the regulation of bone mass accrual and energy metabolism. Notwithstanding behavior and mood abnormalities, Tph2 -null, i. They were osteopenic, anorectic, and had an increase in energy expenditure The existence of these phenotypic abnormalities established that brain serotonin is a regulator of bone remodeling and energy metabolism.

To determine how serotonin controls these functions, we first focused on the regulation of bone mass by brain serotonin. Brain-derived serotonin is a neurotransmitter synthesized in Raphe nuclei neurons of the brainstem. The serotonergic neurons project to the ventromedial hypothalamus VMH and arcuate Arc neurons of the hypothalamus. Brain-derived serotonin regulates bone mass accrual positively after its binding to Htr2c receptors in neurons of VMH, whereas after its binding to the Htr1a and Htr2b receptors on neurons of the arcuate nuclei, serotonin favors appetite.

How could the positive regulation of bone mass accrual exerted by brain serotonin be related to the negative regulation exerted by leptin signaling in the brain? In agreement with this notion, we were able to show through double immunohistochemistry that the long form of the leptin receptor is expressed in Tph2 -expressing, i. Obviously, the formal proof, however, had to come from in vivo manipulations. This is exactly what was observed. Conversely, and with all the limitations that everybody using this technology accepts, cell-specific deletion of the leptin receptor should affect bone mass only when it takes place in brainstem neurons.

Again, this is exactly what was observed Leptin, an adipocyte-derived hormone, regulates bone mass accrual and appetite by inhibiting serotonin synthesis and release by the raphe nuclei neurons of the brainstem. Leptin acts through its binding to its receptor ObRb in the serotonergic neurons of the raphe nuclei.

Given the fact that hypothalamic deletion of the leptin receptor does not affect these two functions, the problem was the same as for the regulation of bone mass by leptin Then, if the problem was the same, the hypothesis could be the same. And the first suspect was already the same because patients chronically taking serotonin reuptake inhibitors often develop hyperphagia and obesity This hypothesis was tested using the brain-derived serotonin-deficient mice we had generated. These mutant mice demonstrated a severe anorexia and an early increase in energy expenditure.

Hence, brain serotonin is in vertebrates, as it is in invertebrates, a positive and powerful regulator of appetite 50 , Axon guidance experiments showed the existence of connections between serotonergic neurons and arcuate neurons of the hypothalamus that are widely seen as the main hypothalamic site of the regulation of appetite.

From these experiments one can draw a model that fits the discrepancies highlighted earlier as well as the evolutionary pattern of expression of leptin. In this model, serotonin is an ancestral molecule favoring appetite from worms to humans; when bone appeared, serotonin acquired another function that is to favor bone mass accrual. For instance serotonin, like leptin, uses the sympathetic tone to regulate bone mass and, also like leptin, it requires VMH neurons integrity to achieve this function.

As shown below, multiple lines of evidence indicate that it is by inhibiting BDS synthesis that leptin prevents bone mass accrual. These data suggest a model whereby leptin regulates bone mass accrual through a double inhibitory loop. Leptin inhibits synthesis of BDS, which in turn reduces, by signaling in VMH neurons, the sympathetic tone; as a result leptin prevents bone mass accrual. A In situ hybridization analysis and co-immunolocalization of ObRb expression in serotonergic neurons. J Representative traces of action potentials recorded from WT mice before, during and after the application of leptin nM.

The mediation of peripheral hormone action on the output of the brain relies on altered circuit activity. Interaction between neuronal circuits hinges on electric properties of neurons, particularly on the generation of action potentials. Thus to test whether leptin directly alters serotonin output from brainstem neurons, we analyzed the responses of serotonin-producing cells to leptin with whole cell patch clamp recording in brain slices containing dorsal raphe DR Supplemental methods.

Serotonergic neurons were identified according to their unique properties long-duration action potential, activation by norepinephrine and inhibition by serotonin itself Liu et al. These data show that leptin can alter directly activity of serotonergic neurons in the brainstem and that this effect of leptin is mediated by ObRb expressed on these neurons. This surprising observation led us to analyze in greater details energy metabolism in these mutant mice. F Analysis of axonal projections emanating from the serotonergic neurons. Cross of Sert-Cre and Rosa26REcfp mice identified projections reaching arcuate Arc nuclei in the hypothalamus through Ecfp immunohistochemistry colocalized to molecular markers of arcuate neurons Pomc-1 and Npy by in situ hybridization.

G In situ hybridization analysis of Htr1a , Htr2b in Pomc1 -expressing arcuate neurons of the hypothalamus. This observation along with the fact that the control of appetite and energy expenditure requires the integrity of the arcuate nuclei of the hypothalamus raised the prospect that axonal projections emanating from Tph2 -expressing neurons reach arcuate nuclei to regulate these functions. Taken together these results indicate that BDS regulates appetite and energy expenditure and that for the control of appetite this mediation occurs through the Htr1a and Htr2b receptors and involves melanocortin signaling.

The first one is that the conjunction of a decrease in appetite and an increase in energy expenditure is the mirror image of what is seen in mice lacking leptin signaling; the second one is that leptin inhibition of serotonin synthesis in the brainstem is the mechanism used by this hormone to inhibit bone mass accrual; the third one is that no molecular mechanisms has been identified so far to explain the common control of bone mass and energy metabolism. Next, to establish that serotonegic neurons of the brainstem and BDS are a critically important entry point and target of leptin in the brain, we analyzed bone mass, appetite and energy expenditure in mouse strains lacking ObRb in distinct neuronal populations in the brain Figure S7A—B.

This analysis was performed on mice fed a normal diet since leptin signaling-deficient mice develop a massive obesity on this diet. The specificity of Cre expression was verified for each mouse line by crossing them with RosaR26 mice and by in situ hybridization Soriano, Figure S7A—B. K Model of the leptin-dependent regulation of bone mass and appetite.

Leptin inhibits release of brainstem-derived serotonin, which favors bone mass accrual and appetite. Adipocytes are in yellow; serotonergic neurons are in pink; VMH is in blue and arcuate is in green. ObRb deletion in Tph2 -expressing neurons also had an organizational effect on Pomc -expressing neurons of the arcuate nuclei. The results presented here demonstrate that in order to regulate bone mass accrual, appetite and energy expenditure leptin needs to inhibit the electrical activity and serotonin synthesis in Tph2 -expressing neurons of the brainstem Figure 7K.

These results modify the map of leptin signaling in the brain and indicate that the serotonergic neuronal circuitry exerts a more fundamental influence on several homeostatic functions than previously thought. Moreover, they identify BDS as the long sought-after molecular basis for the common control of bone mass and energy metabolism. This effort to complete our understanding of the regulation of bone mass by serotonin led to two unexpected results.

The first one is that, depending on its site of synthesis, serotonin regulates bone mass accrual in opposite directions: To our knowledge this is the first example of a molecule exerting different influences on bone remodeling depending on its site of synthesis. The central function of serotonin is mediated through the Htr2c receptor expressed in VMH neurons. Given what is known about the molecular signaling of serotonin in osteoblasts Yadav et al.

Since BDS synthesis is regulated by leptin these results infer that leptin regulation of bone mass is more important than the one exerted by gut-derived serotonin Yadav et al. Patients taking chronically serotonin reuptake inhibitors SSRIs have an increased risk of osteoporotic fractures Richards et al.

Consistent with this observation an animal model reproducing this chronic use of SSRIs, namely mice lacking 5-hydroxytryptamine transporter 5HTT , a molecule responsible for serotonin reuptake by cells, also develop a low bone mass phenotype Warden et al.

Surprisingly however, this low bone mass phenotype cannot be ascribed to an increase in circulating serotonin levels i. The study of BDS functions led to other unexpected findings beyond the control of bone mass. Indeed the deletion of Tph2 shows that BDS is a powerful orexigenic molecule in vertebrates. This function of BDS is similar to the function of serotonin in invertebrates Nonogaki et al. Genetic analysis showed that the orexigenic effect of serotonin occurs through Htr1a and Htr2b receptors, in a Mc4r -dependent manner Heisler et al. This role of Htr1a in the regulation of appetite is consistent with the orexigenic action of Htr1a agonists Gilbert et al.

The demonstration that it is through its expression in arcuate neurons that Htr1a regulates appetite will need generation of a cell-specific deletion of this gene. The fact that removal of the ligand and addition of a pharmacological agonist of a receptor result in the same phenotype, i.

For instance, it is possible that signaling through some serotonin receptors may antagonize signaling through others. Likewise, it should be noted that in most settings the effect of pharmacologic agents are acute while by definition the effect of gene deletion is chronic.

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Lastly, it is also possible that different serotonin receptors regulate differently food intake possibly even on the same neuron Xu et al. Several lines of evidence suggested initially that leptin regulates bone mass, appetite and energy expenditure following binding to its receptors located on hypothalamic neurons.

Chief among those are the facts that the hypothalamus is a known regulator of most homeostatic functions and that ObRb is highly expressed in various hypothalamic neuronal populations Elmquist et al. Surprisingly however, deletion of ObRb in those hypothalamic neuronal populations did not affect any of these three functions Balthasar et al. These latter findings suggested the following hypothesis: That serotonin is the initial target in the brain of leptin regulation of bone mass accrual was an incentive to test whether it could also be implicated in the leptin regulation of appetite and energy expenditure.

We show here, through cell-specific gene deletion of Tph2 or the leptin receptor, that BDS enhances appetite and decreases energy expenditure and that leptin regulates these functions by inhibiting BDS synthesis. Thus leptin does use the same mechanism to regulate bone mass accrual, appetite and energy expenditure.

Relation of Serum Serotonin Levels to Bone Density and Structural Parameters in Women

We remain aware however, that these data do not exclude formally the possibility that leptin acts also on hypothalamic neurons at a level not detectable using cell-specific gene deletion experiments. For instance, our study does not rule out an involvement of Agrp -expressing neurons in the control of various aspects of energy metabolism van de Wall et al.

By identifying brainstem serotonergic neurons as an initial target of leptin this study provides a cellular and molecular explanation for the apparent contradiction between the fact that chemical lesioning of hypothalamic neurons hampers leptin signaling while inactivating ObRb in these neurons does not. A question raised by our work is to know what are the functions of the leptin receptor expressed in hypothalamic neurons? At the present time it seems that ObRb expression on these neurons is mainly needed for the regulation of insulin secretion and glucose metabolism Coppari et al.

Why would bone remodeling and energy metabolism need to be co-regulated in the first place? To answer this question, one needs to look at what was the original purpose of bone remodeling earlier during evolution. Through its ability to constantly renew bone the original function of bone remodeling was to repair micro and macro damages, i. This function was, early on, absolutely necessary to maintain mobility and therefore to assure survival. In addition, bone remodeling is characterized by two opposing processes: Thus, for bone remodeling to occur there must be a constant supply of energy channeled to osteoclasts and osteoblasts.

This view of bone remodeling predicts that there should be one or several hormones, appearing during evolution with this function, and regulating it and energy metabolism. To date leptin is the only known hormone fulfilling all these criteria. This view of bone remodeling also implies that the metabolic importance of the skeleton that begins to be unraveled Lee et al.

In situ hybridization on brain sections was performed as described Oury et al. Ex vivo axonal tracing was performed using Rhodamine-conjugated dextrans Molecular Probes, Eugene, Oregonaxonal; See supplemental methods for details. Bone histomorphometric analyses were performed on undecalcified sections using the Osteomeasure analysis system Osteometrics, Atlanta.

Six to 12 animals were analyzed for each group. Serotonin levels in the brain and serum were quantified as described Yadav et al. San Diego, CA used to standardize between urine samples. Genotypes of all the mice were determined by PCR. All primer sequences for genotyping and DNA probes for southern hybridization are available upon request. Brain slice preparation and electrophysiological recording were performed as described [ Liu et al. Food intake was measured using metabolic cages Nalgene, Rochester, NY and energy expenditure by indirect calorimetry method as described [ Shi et al.

Different letters indicate significant differences among groups. Patricia Ducy for critical reading of the manuscript. Special thanks to Drs. Elmquist, Streamson Chua Jr. This work was supported by grants from the NIH G.

Relation of Serum Serotonin Levels to Bone Density and Structural Parameters in Women

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See other articles in PMC that cite the published article. Associated Data Supplementary Materials Abstract Leptin inhibition of bone mass accrual requires the integrity of specific hypothalamic neurons but not expression of its receptor on these neurons. Introduction Leptin is an adipocyte-derived hormone that regulates a broad spectrum of homeostatic functions following its binding to the signaling form of its receptor, ObRb, present on neurons of the central nervous system Friedman and Halaas, ; Spiegelman and Flier, Results Low bone mass in mice deprived of serotonin in the brain Serotonin synthesis is initiated by hydroxylation of tryptophan, a rate-limiting reaction performed by the enzyme tryptophan hydroxylase 2 Tph2 in the brain Walther et al.

Open in a separate window. The influence of brain-derived serotonin on bone mass prevails over the one of gut-derived serotonin That serotonin exerts opposite influences on bone remodeling depending on its site of synthesis was unexpected. Brain-derived serotonin regulates bone mass through the hypothalamus Since the sympathetic regulation of bone mass requires the integrity of the VMH neurons of the hypothalamus Takeda et al.

Serotonin promotes bone mass through Htr2c receptors in VMH A—C Analysis of axonal projections emanating from the serotonergic neurons of the brainstem. D qPCR analysis of serotonin receptor expression in hypothalamus. Leptin inhibits bone mass accrual by decreasing brain-derived serotonin synthesis Although leptin and serotonin exert opposite influences on bone mass accrual, several features suggested that they might operate in the same pathway.

Leptin inhibits bone mass accrual by inhibiting brain-derived serotonin synthesis A In situ hybridization analysis and co-immunolocalization of ObRb expression in serotonergic neurons.

Leptin inhibits the neuronal activity of serotonergic neurons The mediation of peripheral hormone action on the output of the brain relies on altered circuit activity. Discussion The results presented here demonstrate that in order to regulate bone mass accrual, appetite and energy expenditure leptin needs to inhibit the electrical activity and serotonin synthesis in Tph2 -expressing neurons of the brainstem Figure 7K.

Brain-derived serotonin regulation of bone mass This effort to complete our understanding of the regulation of bone mass by serotonin led to two unexpected results. Serotonin regulation of appetite and energy expenditure The study of BDS functions led to other unexpected findings beyond the control of bone mass.