WO2025021839A1 - Method to treat metabolic disorders - Google Patents

Abstract

The present invention relates to the treatment of metabolic disorders. In this study, the inventors reveal a previously unsuspected role for PTH/PTHrP signalling a in the brain (i.e. a central role) as a physiological regulator of feeding behavior, fasting-induced adaptive responses, energy expenditure and body weight. These findings could further elucidate the complex underpinnings of the central control of metabolism and pave the way for the development of promising therapeutics for metabolic disorders, such as obesity and metabolic syndrome. Thus, the present invention relates to an PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof.

Description

METHOD TO TREAT METABOLIC DISORDERS FIELD OF THE INVENTION: The present invention relates to the treatment of metabolic disorders. BACKGROUND OF THE INVENTION: Organismal adaptation to nutrient availability relies on the ability of the brain to accurately integrate signals coming from metabolic organs and to calculate the needs of various tissues in energy fuel (Schwartz & Porte, 2005; Schwartz et al., 2010; Dietrich & Horvath, 2013; Waterson & Horvath, 2015). Among the various inter-organ communications, the bone- brain crosstalk has gained great momentum in the field of neuroendocrinology, with a number of bone-regulating hormones being revealed as drivers of energy metabolism (Ducy P. et al., 2000; Takeda et al., 2002; Takahashi & Cone, 2005; Fulzele et al., 2010; Kajimura et al., 2013; Friedman et al, 2016; Friedman et al., 2019; Mosialou et al., 2017). These findings support the notion of a coordinated regulation of bone mass and metabolism for the maintenance of energy homeostasis (Karsenty et al., 2006; Yadav et al., 2009) and prompt further exploration of the role of bone-related endocrine/paracrine systems in the central control of energy metabolism. The parathyroid hormone receptor 1 (PTH1r) is a G protein-coupled receptor (GPCR) that constitutes a major regulator of chondrogenesis and calcium homeostasis via binding of its two ligands, the parathyroid hormone (PTH) and the paracrine parathyroid-related peptide (PTHrP) (Vilardaga et al., 2011). While the parathyroid glands are the only known source of circulating PTH, PTH1R and PTHrP are expressed in a variety of tissues, including the brain (Philbrick et al., 1996; Weaver et al., 2015). This observation raises the question of whether PTH and/or PTHrP, via coupling with PTH1R in the brain, could have central functions. Consistent with this idea, it has been reported that malignancy-induced humoral hypercalcemia, a condition characterized by increased levels of PTHrP, causes an increase in the hypothalamic levels of agouti-related peptide (AgRP) (Iguchi et al., 2001; Hashimoto et al., 2007), a peptide known to induce hunger (Jain et al., 2000; Gropp et al., 2005). Interestingly, circulating PTHrP and PTH levels correlate with the severity of obesity (Reis et al., 2007; Saab et al., 2010; Corbetta et al., 2018), a metabolic disorder linked to deficits in brain networks controlling energy metabolism, namely the hypothalamus (HpT) (reviewed in: Timper & Bruning, 2017). The HpT is a major regulator of energy metabolism, by tightly controlling the balance between food intake and energy expenditure (Elmquist et al., 1999; Schwartz et al., 2000; Spiegelman and Flier, 2001; Barsh and Schwartz, 2002; Dietrich & Horvath, 2013; Timper & Brüning, 2017). Within the HpT, the arcuate nucleus (ARC) is of particular interest, because it shows a less restrictive blood–brain barrier, allowing privileged access to circulating factors that act upon first-order ARC neurons, such as AgRP-neurons (Rodríguez et al., 2010, Dietrich & Horvath, 2013). Food deprivation increases the activity of these neurons (Hahn et al., 1998; Takahashi & Cone, 2005; Aponte et al., 2010; Yang et al., 2011; Liu et al., 2012), while AgRP/NPY neuropeptide injection in the brain elicits a robust increase in food intake (Ollmann et al., 1997; Rossi et al., 1998). Importantly, accumulating evidence shows that the effective coordination between peripheral metabolism and AgRP neuron-driven food intake is regulated by cell surface G protein-coupled receptors (GPCRs), whose expression is regulated by hormonal and nutritional feedback (Cowley et al., 2003; Ren et al., 2012). However, the functional consequences of GPCR protein signaling pathways in AgRP neurons is not fully established. SUMMARY OF THE INVENTION: Here the inventors reveal a previously unsuspected role for PTH/PTHrP signalling a in the brain (i.e. a central role) as a physiological regulator of feeding behavior, fasting-induced adaptive responses, energy expenditure and body weight. These findings could further elucidate the complex underpinnings of the central control of metabolism and pave the way for the development of promising therapeutics for metabolic disorders, such as obesity and metabolic syndrome. Thus, the present invention relates to an PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof. Particularly, the invention is defined by its claims. DETAILED DESCRIPTION OF THE INVENTION: A first aspect of the invention relates to an PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof. Particularly, the invention relates to an inhibitor of PTH1r activity for use in the treatment of metabolic disorders in a subject in need thereof. Particularly, the invention relates to either a PTH/PTH1R or PTHrp/PTH1R axis inhibitors or use in the treatment of metabolic disorders in a subject in need thereof. Particularly, the invention relates to an PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof wherein the PTH/PTHrp/PTH1r axis inhibitor has a central effect and is administrated in the brain. As used herein and according to the invention, the term “metabolic disorders” has its general meaning in the art and denotes a disorder that negatively alters the body's processing and distribution of macronutrients, such as proteins, fats, and carbohydrates. Metabolic disorders can happen when abnormal chemical reactions in the body alter the normal metabolic process. It can also be defined as inherited single gene anomaly, most of which are autosomal recessive. Metabolic disorders denote for example obesity, associated metabolic disorders like metabolic syndrome, primary hyperparathyroidism (PHPT), pseudohypoparathyroidism type 1 or type 2 cancer and cachexia. As used herein and according to the invention, the term “metabolic syndrome” has is general meaning in the art and denotes a cluster of conditions that occur together, increasing your risk of heart disease, stroke, type 2 diabetes. The key sign of metabolic syndrome is central obesity, known as visceral, male-pattern or apple-shaped adiposity. It is characterized by adipose tissue accumulation predominantly in liver (steatosis). As used herein and according to the invention, the term “obesity” has is general meaning in the art and denotes a disease in which excess body fat has accumulated to such an extent that it may negatively affect health. People are classified as obese when their body mass index (BMI)—a person's weight divided by the square of the person's height—is over 30 kg/m2; the range 25–30 kg/m2 is defined as overweight. Obesity is a major cause of disability and is correlated with various diseases and conditions, particularly cardiovascular diseases, type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis. As used herein and according to the invention, the term “primary hyperparathyroidism (PHPT)” has is general meaning in the art and denotes a disease in which excess PTH levels to such an extent that it may negatively affect health and induce significant higher glucose levels (Kautzky-Willer 1992), increased HOMA index (Ayturk 2006, Khaleeli 2007, Procopio 2002) compared to controls, and induces metabolic syndrome. As used herein and according to the invention, the term “pseudohypoparathyroidism” has is general meaning in the art and denotes a hereditary disorder characterized by resistance or unresponsiveness to PTH and blunted PKA response to G protein activation. These patients exhibit elevated BMI and hyperphagia, associated to a constitutive alteration of PKA signaling and an overactivation of PKC signaling in PTH1R. In a case of pseudohypoparathyroidism, the gene of PTH1r will have some mutations. In the context of a pseudohypoparathyroidism, a cancer or a cachexia, an inhibitor of the PTHrp/PTH1r axis will be very useful. As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject according to the invention is a human. More particularly, the subject is suffering from a metabolic disorder. According to the invention, the PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the brain. Particularly, and according to the invention, the PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the hypothalamus (HpT). Particularly, and according to the invention, the PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the hypothalamic neurons. Particularly, and according to the invention, the PTH1r inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the hypothalamic neurons. More particularly, and according to the invention, the PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the arcuate nucleus (ARC) of the hypothalamus. More particularly, and according to the invention, the PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders is administrated in a subject in need thereof in the in the AgRP neurons. As used herein, the term “AgRP neurons” denotes neurons of the ventromedial part of the arcuate nucleus in the hypothalamus which secrete agouti-related protein (AgRP), a neuropeptide produced in the brain and regulating energy balance. As used herein, the term “PTH” for “parathyroid hormone” has its general meaning in the art and denotes a peptide hormone secreted by the parathyroid glands that regulates the serum calcium concentration through its effects on bone, kidney, and intestine. PTH influences bone remodeling, which is an ongoing process in which bone tissue is alternately resorbed and rebuilt over time. Its entrez reference number is 5741 and its Uniprot reference number is P01270. As used herein, the term “PTHrp” for “parathyroid hormone-related protein” has its general meaning in the art and denotes a proteinaceous hormone and a member of the parathyroid hormone family secreted by mesenchymal stem cells. It is occasionally secreted by cancer cells (for example, breast cancer, certain types of lung cancer including squamous-cell lung carcinoma). However, it also has normal functions in bone, teeth, vascular tissues and other tissues. PTHrP acts as an autocrine, paracrine, and intracrine factor. It regulates endochondral bone (chondrogenesis) development by maintaining the endochondral growth plate at a constant width. It also regulates epithelial–mesenchymal interactions during the formation of the mammary glands. PTHrP plays a major role in regulating calcium homeostasis in vertebrates, including sea bream, chick, and mammals. Its entrez reference number is 5740 and its Uniprot reference number is P12272. As used herein, the term “PTH1r” for “parathyroid hormone 1 receptor” has its general meaning in the art and denotes a protein that in humans is encoded by the PTH1R gene. PTH1R functions as a receptor for parathyroid hormone (PTH) and for parathyroid hormone-related protein (PTHrP), also called parathyroid hormone-like hormone (PTHLH). Its entrez reference number is 5745 and its Uniprot reference number is Q03431. It is known that the receptor PTH1r has two ligands: PTH (endocrine) and PTHrp (paracrine/autocrine). Thus, and as used herein, the term “PTH/PTHrp/PTH1r axis inhibitor” denotes a molecule or compound which will inhibit the PTHrp/PTH1r axis or the PTH/PTH1r axis. Thus, the invention also relates to a PTH/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof. The invention also relates to a PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof. As used herein, the term PTHrp/PTH1r axis inhibitor denotes a molecule or compound which will inhibit PTHrp and/or PTH1r. Inhibitor of PTHrp and/or PTH1r denotes a molecule or compound which can inhibit the function or activity of PTHrp and/or PTH1r like their role in the formation of bone or in calcium homeostasis, or a molecule or compound which destabilizes PTHrp or PTH1r. Particularly, an inhibitor of PTHrp and/or PTH1r denotes a molecule or compound which can inhibit the central role of either PTHrp/PTH1r axis and notably their role in the autophagy machinery in AgRP neurons towards lipid degradation, feeding behavior, fasting response and fat metabolism. The term “PTHrp/PTH1r inhibitor” also denotes an antagonist of these molecules which will inhibit the interaction between PTHrp with its receptor PTH1r. The term “PTHrp/PTH1r inhibitor” also denotes an inhibitor of the expression of the gene coding for the proteins PTHrp or PTH1r. As used herein, the term “PTH/PTH1r axis inhibitor” denotes a molecule or compound which will inhibit PTH and/or PTH1r. Inhibitor of PTH and/or PTH1r denotes a molecule or compound which can inhibit the function or activity of PTH and/or PTH1r like their role in the formation of bone or in calcium homeostasis, or a molecule or compound which destabilizes PTH or PTH1r. Particularly, an inhibitor of PTH and/or PTH1r denotes a molecule or compound which can inhibit the central role of either PTH/PTH1r axis and notably their role in the activation of the PKA pathway in hypothalamic neurons, energy expenditure and fat metabolism. The term “PTH/PTH1r inhibitor” also denotes an antagonist of these molecules which will inhibit the interaction between PTH with its receptor PTH1r and PTHrp with its receptor PTH1r. The term “PTH/PTH1r inhibitor” also denotes an inhibitor of the expression of the gene coding for the proteins PTHrp or PTH1r. As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). Particularly, the invention relates to a PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof wherein a decrease in food intake is obtained. Particularly, the invention relates to a PTH1r inhibitor for use in the treatment of metabolic disorders in a subject in need thereof wherein a decrease in food intake is obtained. In one embodiment, the inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da. In one embodiment, the inhibitor according to the invention is an antibody. Antibodies against PTH, PTHrp or PTH1r can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against PTH, PTHrp or PTH1r can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV- hybridoma technique (Cole et al.1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No.4,946,778) can be adapted to produce anti- PTH, anti- PTHrp or anti- PTH1r single chain antibodies. Compounds useful in practicing the present invention also include anti- PTH, anti- PTHrp or anti- PTH1r antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to PTH, PTHrp or PTH1r. Humanized anti- PTH, anti- PTHrp or anti- PTH1r antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No.4,816,397). Then, for this invention, neutralizing antibodies of PTH, PTHrp or PTH1r are selected. In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then, for this invention, neutralizing aptamers of PTH, PTHrp or PTH1r are selected. In one embodiment, the compound according to the invention is interfering peptides. In a particular embodiment the peptide is an antagonist of either PTHrp or PTH which are capable to prevent their binding to PTH1r and the activation of their respective pathways. Particularly, the peptide can be a mutated PTHrp or PTH protein or a similar protein without the function of PTHrp or PTH1r. In one embodiment, the peptide of the invention may be linked to a Blood Brain Barrier-penetrating peptide” to allow the passage of the interfering peptide throughout the Blood Brain Barrier (see for instance Patent no: 19305365.9-1120). In one embodiment, the peptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell. The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence, and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The interfering peptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of interfering peptides or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the interfering peptides of the invention. Preferably, the interfering peptides is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of an interfering peptide in a variety of different host cells are well known. When expressed in recombinant form, the interfering peptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous interfering peptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters. A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery. In another embodiment, the PTH, PTHrp or PTH1r inhibitor according to the invention is an inhibitor of PTH, PTHrp or PTH1r gene expression. Small inhibitory RNAs (siRNAs) can also function as inhibitors of PTH, PTHrp or PTH1r expression for use in the present invention. PTH, PTHrp or PTH1r gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that PTH, PTHrp or PTH1r gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos.6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). Short hairpin RNA or small hairpin RNA (shRNA/Hairpin Vector) can also function as inhibitors of PTH, PTHrp or PTH1r expression for use in the present invention. shRNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications. Ribozymes can also function as inhibitors of PTH, PTHrp or PTH1r gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of PTH, PTHrp or PTH1r mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of G-CSF gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone. Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing PTH, PTHrp or PTH1r. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non- cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUCl9, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation. In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters. In a particular embodiment, an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of PTH, PTHrp or PTH1r. Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol.339 : 823–826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol.8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.8:e2671.), plants (Mali et al., 2013, Science, Vol.339 : 823–826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707–714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol.41 : 4336–4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534/genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol.156 : 836– 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol.6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol.24 : 122–125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol.56 : 122–129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci. In some embodiment, the endonuclease is CRISPR-Cpf1 which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpf1) in Zetsche et al. (“Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13). According to the invention, the inhibitor of PTH, PTHrp or PTH1r gene expression can be administrated to the subject by a vector. As used herein, the term “vector” has its general meaning in the art and refers to the vehicle by which a nucleic acid molecule can be introduced into cells, so as to transform the cell and promote expression (e.g. transcription and/or translation) of the introduced sequence. According to the invention, vectors include viral vectors or non-viral vectors. Examples of viral vector include adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94/19478. In a particular embodiment, adeno-associated viral (AAV) vectors are employed. In another particular embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 and all variants of AAV9, including AAV PHP.B (see for example the patent application WO2015038958), AAVPHP.eB, AAV-PHP.N", and "AAV-PHP.B- DGT (see the patent application WO2017100671 or Chan Y Ken, Nat Neurosci. 2017 Aug;20(8):1172-1179.), AAV3B, AAV-2i8, Rh74, AAV capB10, AAVMacPNS1 or AAVMacPNS2 or any other serotypes of AAV that can infect human, monkeys or other species. Accordingly, one object of the present invention relates to a method of treating in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an PTH/PTHrp/PTH1r axis inhibitor. In order to test the functionality of a putative PTHrp, PTH or PTH1r inhibitor a test is necessary. For that purpose, for PTHrp and PTH1r inhibitor, we will measure at the cellular level, the Blockage of PKC phosphorylation and autophagy induction in hypothalamic neurons and at the functional level, we will measure appetite using metabolic cages. For PTH and PTH1r inhibitor, we will measure at the cellular level, the blockage of PKA phosphorylation in hypothalamic neurons and at the functional level, we will measure energy expenditure using metabolic cages. Therapeutic composition In another aspect, the invention relates to a therapeutic composition comprising a PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof. According to the invention, the PTH, PTHrp or PTH1r inhibitor or the therapeutic composition of the invention are administrated in a therapeutically effective amount. Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required. For example, the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically and for example, the ability of a compound to inhibit a metabolic disorder may, for example, be evaluated in an animal model system predictive of efficacy in human. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease for example obesity, or otherwise ameliorate symptoms in a subject suffering from a metabolic disorder. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. Administration may be intravenous, intramuscular, intraperitoneal, intraparenchymal, intracisternal, intrathecal, intraventricular, intracerebral or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of the inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intraparenchymal, intracisternal, intrathecal, intraventricular or subcutaneous administration and the like. Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions. In particular, these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions. In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used. In each of the embodiments of the present invention, the inhibitor of the invention is delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the inhibitor of the invention administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 µm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Combination and kit of part In another aspect of the invention, the inhibitor of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent. According to the invention, anti-metabolic disorders agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention. Particularly, anti-metabolic disorders agents like anti-obesity, anti-cancer or anti metabolic syndrome can be used according to the invention. Another aspect of the present invention relates to i) a PTH/PTHrp/PTH1r axis inhibitor, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of metabolic disorders in a subject in need thereof. As used herein, the term “simultaneous use” denotes the use of a PTHrp/PTH1r axis inhibitor and at least one therapeutic active agent occurring at the same time. As used herein, the term “separate use” denotes the use of a PTHrp/PTH1r axis inhibitor and at least one therapeutic active agent not occurring at the same time. As used herein, the term “sequential use” denotes the use of a PTHrp/PTH1r axis inhibitor and at least one therapeutic active agent occurring by following an order. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: PTH1R is abundantly expressed in the Arcuate nuclei (ARC) of the hypothalamus (HpT) where it affects feeding behavior. A) Pth1r and Pthrp relative gene expression (RT-qPCR performed in triplicate for each sample) in muscle, bone and various parts of the brain (cortex, hippocampus (HpC), hypothalamus (HpT), and cerebellum (CB)) of 3-month-old (3Mo) WT mice (n = 5). Quantification of mRNA expression is relative to muscle. B) Representative images of mice 3 weeks following injection with either AAV-U6-shRNA-Pth1r or Scramble. C) Body composition measurements (by NMR analyzer) assessing relative body fat mass, lean mass and fluid volume. D) Bar graphs representing (overall mean and day-night means) food intake of U6-sh-Pth1r and Scramble mice, measured in automated metabolic cages over 5 days, following 2 days of habituation. Data are expressed as mean ± s.e.m. For analysis of metabolic cage parameters, the Mann–Whitney test was used. For body weight analysis, the two-way ANOVA was used followed by a Sidak pos-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 2: PTH/PTHrP have distinct roles in the central regulation of energy metabolism. A) Graphs representing overall and day-night means for energy expenditure (heat) in mice that received PTH (40ng/H) via osmotic pumps (i.c.v., for 2 weeks). B) Graphs representing downregulated for Pthrp in the ARC. For longitudinal body weight gain analysis, the two-way ANOVA was used followed by a Sidak pos-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3: PTH1R and PTHrP are expressed in AgRP neurons and their coupling affects AgRP neuronal activity and feeding behavior. Graphs representing mice with Agrp-specific downregulation of Pthrp or Pth1r in the ARC. Results are shown as mean ± s.e.m. For body weight gain analysis, two-way ANOVA followed by a Sidak pos-hoc test was used. For the rest of the analysis, one-way ANOVA followed by a post-hoc Tuckey’s test was used. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 4: The PTHrP/PTH1r system participates in fasting responses by regulating AgRP neuropeptide levels in the HpT. A) Food intake measurement in the re-feeding after fasting experiment, using mice that sustained injections of AAV-Agrp-shRNAs for Pth1r or Pthrp. B) Protein quantification (bar plots), performed using HpT lysates from Agrp-sh-Pth1r, Agrp-sh-Pthrp or Scramble mice that were either fasted (F) or non-fasted (NF). Results are shown as mean ± s.e.m. For the behavioral analysis and western blot analysis of hypothalamic lysates of Agrp-sh-RNA-injected mice (F and NF), Kruskall-Wallis repeated measures followed by Dunn’s post-hoc was used. Otherwise, one-way ANOVA followed by Tuckey’s post-hoc was used. *P < 0.05, **P < 0.01, ***P < 0.001. NS: not statistically significant. Figure 5: PTHrP upregulates AgRP levels via a modulation of lipophagy. Representative images of SQSTM1/p62 immunofluorescence staining (scale bars: 20 μm) and puncta quantification performed on primary HpT neurons transfected with a lenti- shRNA-Pth1r and treated with PTHrP (100nM) (n=3 independent neuronal cultures). Analysis was performed on n=5 experimental replicates; n ≥ 8 individual neurons per group used for each experimental replicate (each neuronal preparation was derived from at least 12 embryonic HpT). The quantification was performed versus the vehicle group. Results are given as mean ± s.e.m. Kruskal-Wallis repeated measures followed by Dunn’s post-hoc and Mann–Whitney tests were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001. NS: non significant. Figure 6: AgRP-borne Pth1r downregulation decrease fat accumulation in HFD- induced obesity mice. A) Scheme of the experimental design. Mice were fed with HFD for 8 weeks before the injection of either AAV9-Agrp-GFP-Scramble-shRNAmir in mice under control-diet (CD) (n=10) or high-fat-diet (HFD) (n=10), or AAV9-Agrp-GFP-Pth1r-shRNAmir in HFD mice (n=10). B) Body weight gain of 11-weeks injected CD mice stereotactically injected with AAV9-Agrp-GFP-Scramble-shRNAmir (n=10) or HFD mice stereotactically injected with either AAV9-Agrp-GFP-Scramble-shRNAmir (n=8) or AAV9-Agrp-GFP-Pth1r-shRNAmir (n=10). C) Body weight gain of CD mice after injection with AAV9-Agrp-GFP-Scramble- shRNAmir (n=10) or HFD mice after injection with either AAV9-Agrp-GFP-Scramble- shRNAmir (n=8) or AAV9-Agrp-GFP-Pth1r-shRNAmir (n=10). D) Representative picture of HFD mice injected with either AAV9-Agrp-GFP-Scramble-shRNAmir in the left or AAV9- Agrp-GFP-Pth1r-shRNAmir in the right. E-F) Body composition measurements assessing fat mass (E) or lean mass (F) of mice injected with AAV9-Agrp-GFP-Scramble-shRNAmir fed with control diet (n=8) or HFD (n=7) and mice injected with AAV9-Agrp-GFP-Pth1r- shRNAmir fed with HFD (n=10). G-H) Representative images of adipocytes in white (WAT) and brown (BAT) adipose tissue and of liver fat deposit in 11-weeks CD mice stereotactically injected with AAV9-Agrp-GFP-Scramble-shRNAmir (n=10) or HFD mice stereotactically injected with either AAV9-Agrp-GFP-Scramble-shRNAmir (n=8) or AAV9-Agrp-GFP-Pth1r- shRNAmir (n=10) (G) and quantification of white adipocytes diameter (H). Data are expressed as mean ± s.e.m. The two-way ANOVA test followed by Dunnet’s post-hoc was used for statistical analysis or one-way ANOVA followed by Dunnet’s post-hoc test. * vs. CD-Scramble, # vs. HFD-Scramble. *P < 0.05, **P < 0.01, ***P < 0.001. EXAMPLE 1: Material & Methods Animals Experiments were performed using adult male C57BL/6j mice purchased from Janvier labs (St Berthevin, France). All mice were 3 months of age at the start of experiments. For all experiments, we used littermates as controls. Upon arrival, mice were housed in groups of five in polycarbonate cages (35.5 x 18 x 12.5) and and maintained in standard conditions (controlled room temperature and humidity, 12h/12h light/dark cycle, with lights on at 8:00 AM, ad libitum access to dry food pellets and water) at the Institut Necker’s animal care facility, officially registered for experimental studies on rodents (Approval number for animal care facilities: A 75-15-01-62014-720). All studies were performed in compliance with the French legislation (Decree of 1 February 2013, 2013-118) and the European Communities Council Directive of 22 September 2010 (2010/63/EEC). All animal experiments were designed according to the 3R’s rules and approved by the French Ministry of Research (APAFIS#17131- 2018041214074198v3). Transgenic AgRPCre:tdTomato and POMCCre:tdTomato mice were obtained by crossing AgRPCre (Stock No.012899) or PomcCre (Stock No.005965) with tdTomatoflox/flox (Stock No. 007914) mice, which were all purchased from Jackson Laboratories. Experiments were made on 6/9-week old male that were heterozygous for AgRP (cre+/−) or POMC (cre+/−), and homozygous for tdTomato. Animals were housed at 22.5 ± 1°C on a 12h-12h reversed light- dark cycle (light off at 10h30; light on at 22h30). Before experiments, animals had free access to standard diet (A04, SAFE) and water. All protocols including animals were reviewed by our local ethic committee in strict accordance with European Community guidelines and agreed by the French Ministry of Higher Education and Research (accreditation No. 00853.01).POMC Cre; PTH1Rflox/flox were obtained by crossing POMCCre (Stock No. 005965;, EUCOMM- EM:07507) with PTH1R flox/flox. Experiments were performed on 3 month-old male mice using PTH1Rflox/flox as control littermates. Viral Vectors The viruses used in these studies were either obtained from Vector Biolabs or form the Viral vector and gene transfer platform in SFR Necker (see Key Resources Table for further information). For the functional and subsequent immonohistological and molecular analyses, AAV9-GFP-U6-m-PTH1R-shRNA, AAV9-GFP-U6-m-PTHLH-shRNA, AAV9-AgRp- eGFP-mPTH1R-shRNAmir and AAV9-AgRp-eGFP-mPTHLH-shRNAmir were used. For viral manipulation of primary hypothalamic neurons, lentiviral shRNA targeting mouse PTH1r was purchased from Origen (TR501787B) and cloned into pGFP-V-RS-PTH1r-B vector VSV- G pseudotyped. The plasmid containing the pMXs mRFP-GFP-LC3 sequence was purchased from Addgene (plasmid #117413) and subcloned into pSicoR (Plasmid #11579). Neurons were transfected at DIV12. Stereotaxic Surgery Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (20mg/ml BW; 1000 Virbac) and xylazine (1000mg/ml BW; Rompun 2%; Bayer). Coordinates were identified using the Paxinos mouse brain atlas. Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The area around the incision was trimmed and an iodine solution was applied (Vétédine). Mice were injected with 1.0 mL virus in each side of the ARC using the following coordinates relative to lambda: (0,4 mm ML, -1,48 mm AP, +/- 5,69 mm DV). The skin was closed with sutures. All DV coordinates listed are relative to the pial surface. For the acute PTHrP administration experiments, mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (20mg/ml BW; 1000 Virbac) and xylazine (1000mg/ml BW; Rompun 2%; Bayer) and injected with 1.0 µL PTHrp (40ng/µL) in each side of the ARC using the same coordinates as previously described. The entire HpT was carefully dissected 1 hr after the injections. Osmotic pumps and treatments Alzet micro-osmotic pumps (model 1002) were loaded with saline, PTHrP (40ng/hr; Bachem) or PTH (40ng/hr; Bachem). Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (20mg/ml BW; 1000 Virbac) and xylazine (1000mg/ml BW; Rompun 2%; Bayer). Osmotic pumps were surgically installed subcutaneously in the back of the mice. PTHrP (1-34) (40ng/hr; Bachem) or PTH (1-84) (40ng/hr; Bachem) were delivered for a total of 28 days. Behavioral and functional parameters were assessed 2 weeks after osmotic pump installation. Indirect calorimetry measurements Indirect calorimetry was performed using automated metabolic cages (Labmaster, TSE Systems GmbH, Bad Homburg,Germany) where mice are individually housed for consecutive days and which include bedding and food and water ad libitum. Food and drink consumption was measured by highly sensitive sensors for automated online measurements located in the feeding and water ports. Energy expenditure (EE), O2 consumption and CO2 production, respiratory exchange rate (RER=VCO2/VO2) and locomotor activity are also measured. O2 and CO2 volumes are measured at the inlet ports of each cage through which a predetermined airflow is ventilated (0.4 L min−1) and compared regularly with a reference empty cage. EE was calculated using the Weir equation for respiratory gas exchange measurements. Ambulatory activity was measured with the help of an infrared light beam-based activity monitoring system. All sensors operate efficiently in both light and dark phases, allowing for continuous recording. Parameters were recorded every 15 min throughout the experiment. Data analysis was performed using extracted raw values of ̇VO2 consumed (expressed in mL h−1), ̇VCO2 production (expressed inmL h−1), energy expenditure (kcal/h), activity (cm), food intake (g) and drink (mL). Body composition analysis Body mass composition was analysed using an EchoMedical systems’ EchoMRI (RMN Analyzer minispeq mq7.5; Bruker Optik, Germany) in accordance with the manufacturer’s instructions. Readings of body composition were given within 1 min. Measurements were performed 3 weeks after stereotactic injection of the AAVs. Overnight fasting and re-feeding Molecular and immunofluorescence analyses were assessed using mouse brains dissected following overnight fasting (18H, 6 p.m.-12 a.m.). The non-fasted group (NF) was killed at the same timepoint as the fasted group. The re-feeding testing arena consisted of a regular housing cage (35.5 x 18 x 12.5 cm). Mice were exposed to pre-weighted chow pellets and food intake was measured for 30 min. Tissue collection Mice allocated to immunofluorescence studies were deeply anesthetized with a mixture of ketamine hydrochloride (20mg/ml BW; 1000 Virbac) and xylazine (1000mg/ml BW; Rompun 2%; Bayer) and were perfused transcardially with a solution containing 0.9% NaCl at 37°C, followed by 4% paraformaldehyde (PFA) in phosphate buffer (pH 7.3). Animals were sacrificed in randomized order to minimize experimental bias. The mice were then decapitated, and the brain was carefully removed. Mice allocated to molecular biology analysis were killed by cervical dislocation. The whole HpT was carefully dissected, immediately snap-frozen in liquid-nitrogen, and maintained at 80°C until further processing. The white adipose tissue (WAT) and brown adipose tissue (BAT) were harvested and were maintained in PFA 4% for 24 h before being sectioned and used for histological analysis. Slice Electrophysiology Electrophysiological recordings were performed in non-fasted 6-9 week old AgRPCre:tdTomato mice. Mice were intracardially perfused under deep anesthesia (ketamine/xylazine, 100/10 mg/kg) with an ice-cold oxygenated (95% O2/5% CO2) perfusion solution that contained (in mM) 200 sucrose, 28 NaHCO3, 2.5 KCl, 7 MgCl2, 1.25 NaH2PO4, 0.5 CaCl2, 1 L-ascorbate, 3 Na-pyruvate and 8 D-glucose (pH 7.4). The brain was quickly removed and immersed in the same ice-cold oxygenated perfusion solution. Three to five 250 μm coronal slices containing the arcuate nucleus were obtained with a vibroslice (Leica VT1000S) and placed for 1 h at room temperature in an oxygenated recovery ACSF solution containing (in mM): 118 NaCl, 5 KCl, 1 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, 1.5 CaCl2, 5 HEPES, 2.5 D-glucose and 15 sucrose (osmolarity adjusted to 310 mOsM with sucrose, pH 7.4). After recovery, slices were perfused with the same ACSF oxygenated media in a recording chamber placed under a microscope (Slicescope, Scientifica) outfitted for fluorescence and IR- DIC videomicroscopy. Viable arcuate AgRP neurons were visualized with a fluorescence video camera (Rolera Bolt Qimaging camera). For cell-attached recordings, borosilicate pipettes (4- 6 M; 1.5 mm OD, Sutter Instrument) were filled with filtered extracellular medium. For the measurement of the firing rate of AgRP neurons in response to PTHrp (100nM), action potential frequency was quantified after a stable baseline was established before (control: over the last 120 to 60 sec before PTHrp application), during (stimulated by 100 nM PTHrp 5- 7 min: over the last 60 sec of PTHrp application) and after (reversal 10 min: over 60 sec, 10 min after PTHrp application) PTHrp application, at room temperature. Recordings were made using a EPC10 Heka amplifier including data acquisition LIH 8+8 AD/DA interface and acquired at 2 kHz using patchmaster (2x91 (Harvard Bioscience, Inc., HEKA Elektronik GmbH). All acquired data were analyzed with Clampfit software (Molecular Devices, Inc.). Cultures of primary hypothalamic neurons Hypothalamic neurons were isolated from mouse embryos (embryonic day 16.5). After dissections, hypothalamus was digested with trypsin 0.05% and EDTA 0.02% for 15 min at 37°C. After three washes with DMEM (61965059: Thermo Fisher Scientific), supplemented 10% FBS, 100 U/ml penicillin-streptomycin, cells were dissociated and then plated. The dissociated cells were plated onto poly-L-lysine-coated plates or glass coverslips for microscopic examination.24h after plating, the medium was replaced with Neurobasal medium (Thermo Fisher Scientific) containing B27 supplement (Thermo Fisher Scientific), GlutaMAX and Mycozap (Lonza), and was renewed two times per week while neurons were maintained in 5% CO2 and 37°C. To decrease the proportion of glial cells present in the culture, 2 µM of Ara- C inhibitor (C1768, Sigma) was added in pre-warmed complete Neurobasal medium, starting at Day In Vitro (DIV) 5. Experiments were performed at DIV12. Treatment of primary hypothalamic neurons All treatments were administered in the neurobasal medium. PTHrP (100nM) and Bafilomycin A1 (1mM) were administered for 4H, and Lalistat-1 (10µM) was administered 20H before PTHrP treatment onset. After treatment, neurons dedicated to molecular biology were rinsed in PBS, proteins were extracted in Laemmli buffer (1X) containing phosphatase and protease inhibitors and maintained at -80°C until further processing. Neurons dedicated to immunofluorescence analysis were fixed in 4% PFA / 4% sucrose for 20 min at room temperature, then rinsed 3 times with PBS and stored at 4°C. Quantitative RT-qPCR Brain tissues were immediately flash-frozen after dissection and total RNA was isolated with TRIzol Reagent using a homogenizer. Single-strand cDNA was synthesized from total RNA (2 μg) by using SuperScript II Reverse Transcriptase. qRT–PCR was performed using iTAQ SYBR Green (BioRad). Real-Time PCR System. Relative expression of Pthrp and PTH1r was calculated using the 2(11C.t/) method. Conditions for real-time PCR were: initial denaturation for 10 min at 95°C, followed by amplification cycles with 15 s at 95°C, and 1 min at 60°C. Western blots Brain tissue was homogenized in RIPA lysis buffer (25mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce Thermo Scientific), supplemented with protease (cOmplete, Sigma) and phosphatase (phosSTOP, Sigma) inhibitors. Protein concentration was measured with Pierce BCA protein Assay Kit (ThermoFischer Scientific) prior to the Western blot assay. Tissue lysates were mixed with 4x NuPage LDS loading buffer (Invitrogen) and reducing agent (Invitrogen NP0004), and proteins were separated on a 4–12% SDS-polyacrylamide gradient gel (Invitrogen NP0329) and subsequently transferred by semi-dry or liquid transfer onto a PVDF membrane (Trans-blot Turbo Mini PVDF, Biorad). The blots were blocked in 6% milk in Tris-buffered saline with Tween (TBS- T) and incubated with the primary antibodies, including rabbit polyclonal anti-AgRP (1/500, abcam ab254558), mouse monoclonal anti-ATG5 (1/1000, Nanotools 0262-100), mouse monoclonal anti-Beclin (1/1000, BD Transduction Laboratories AB_399484), rabbit polyclonal anti-LC3 (1/5000, Sigma L7543), guinea pig polyclonal anti-p62 (1/1000, Progen GP62-C), rabbit polyclonal anti-PTH1r (1/1000, abcam ab180762), mouse polyclonal anti-PTHrp (1/50, Santa-Cruz sc-53936), mouse polyclonal anti-ATG5/12 (1:500, Nanotools, 0262-100), rabbit polyclonal anti-VPS34 (1/1000, Cell Signaling 4273) and mouse monoclonal anti-β-actin (1/5000, Sigma A5316). To detect protein signal, the following Horseradish peroxidase– conjugated secondary antibodies were used: anti-rabbit IgG HRP-linked (1:5000, Cell Signaling 7074), anti-mouse IgG HRP-linked (1/5000, Cell Signaling 7076) and anti-guinea pig IgG HRP-linked antibody (1/5000, Sigma A5545) revealed using an ECL kit (Clarity Western ECL Substrate: BioRad) for protein detection with a Chemidoc Imaging System (Biorad). Bands were quantified using the Image Lab software. Hematoxylin and eosin staining After 24h of incubation in PFA 4%, BAT and WAT samples were washed with ethanol 50% and embedded in paraffin. Pictures of the sections were taken under light microscopy with a slide scanner (Nanozoomer 2.0 HT, Hamamatsu) using 20X objective. The images were then analyzed using the Adiposoft plug-in of Fiji software. Immunofluorescence Perfused brains were cut at 40-micron thick coronal sections using a vibrating microtome (VT1000S, Leica). Immunostaining was performed on free-floating sections. Non- specific staining was blocked by 0.2% Triton and 10% donkey serum albumin (Sigma-Aldrich). Sections were incubated with the following primary antibodies at 4°C: rabbit polyclonal anti- AgRP (1/200, abcam ab254558), rabbit polyclonal aniti-LC3 (1/200, Sigma L7543), guinea pig polyclonal anti-p62 (1/200, Progen GP62-C), rabbit polyclonal anti-PTH1r (1/200, abcam ab180762), mouse polyclonal anti-PTHrp (1/50, Santa-Cruz sc-53936), MAP2 (1/200, Millipore MAB364). Sections were incubated with secondary antibodies (Alexa-conjugated secondary antibodies at 1:800 dilution, ThermoFischer) for 2h at room temperature. Fluorescent sections were stained with the nuclear dye Hoechst and then mounted using Fluoromount aqueous mounting medium (Sigma-Aldrich). Imaging and quantification Images were acquired using an Apotome with the Zen Imaging software (Zeiss). Z- stacks were obtained (step size: 0.5 μm) using sequential scanning. Cell counting was performed either manually or by using the Icy open-source platform (http://www.icy.bioimageanalysis.org). Values are expressed as the mean number of immune- positive cell counts. The number of subjects, brain slices, primary neuron preparations included in each counting are mentioned in the corresponding legends. Metabolic data analysis For metabolomic analysis, the extraction solution was composed of 50% methanol, 30% acetonitrile (ACN) and 20% water. The volume of the extraction solution was adjusted to cell number (1 ml per 1E7 cells). After addition of extraction solution, samples were vortexed for 5 min at 4 °C and centrifuged at 16,000g for 15 min at 4 °C. The supernatants were collected and stored at −80 °C until analysis. LC/MS analyses were conducted on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe coupled to a Dionex UltiMate 3000 uHPLC system (Thermo). External mass calibration was performed using a standard calibration mixture every seven days, as recommended by the manufacturer. The 5 µl samples were injected onto a ZIC-pHILIC column (150 mm × 2.1 mm; i.d.5 µm) with a guard column (20 mm × 2.1 mm; i.d. 5 µm) (Millipore) for LC separation. Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide (pH 9.2), and buffer B was ACN. The chromatographic gradient was run at a flow rate of 0.200 µl min−1 as follows: 0–20 min, linear gradient from 80% to 20% of buffer B; 20–20.5 min, linear gradient from 20% to 80% of buffer B; 20.5–28 min, 80% buffer B. The mass spectrometer was operated in full scan, polarity switching mode with the spray voltage set to 2.5 kV and the heated capillary held at 320 °C. The sheath gas flow was set to 20 units, the auxiliary gas flow to 5 units and the sweep gas flow to 0 units. The metabolites were detected across a mass range of 75–1,000 m/z at a resolution of 35,000 (at 200 m/z) with the automatic gain control target at 106 and the maximum injection time at 250 ms. Lock masses were used to ensure mass accuracy below 5 ppm. Data were acquired with Thermo Xcalibur software (Thermo). The peak areas of metabolites were determined using Thermo TraceFinder software (Thermo), identified by the exact mass of each singly charged ion and by the known retention time on the HPLC column. Metabolomic data can contain a large portion of missing values, largely affecting the attribution of the metabolic features. A percentile transformation function (percentize in R) was used to normalize the data, assigning a rank to the value of the metabolite. In order to avoid direct assignment of 0 to highly variant data points, rank based normalization preserves the broad trends in the experiment compared to controls. For dimensional reduction, we performed a principal component analysis (PCA) on the normalized metabolite reads. We used the euclidean distance to determine orthogonal eigen vectors. prcomp function in R was used, and the first two principal components explaining the largest statistical variability in the data (approximately 70%) was plotted in a biplot representation with controls (Vehicle) and experimental samples (PTHrp). A heatmap representation of the metabolite was generated using the heatmap function in R. Furthermore, we clustered the data using a K-means clustering algorithm (function kmeans in R) to observe segregation of metabolites into distinct clusters. The number of k medians was set to 6, while the maximal iterations was at 10000. A “MacQueen” algorithm was used to cluster the data in order to reduce the sum of squared errors. Points are assigned to nearest centroid according to the Euclidean distance and the centroids are updated using the mean of the points in the group. The algorithm iterates till the minimum sum of squared errors is achieved. Statistical analysis All data were expressed as mean ± SEM. Statistical analyses were performed using the Prism software (GraphPad, version 6), with p < 0.05 considered statistically significant. Data were analyzed using unpaired two-tailed Student’s t-test, unpaired one-tailed Mann-Whitney, unpaired Kruskall-Wallis followed by Dunn post-hoc test, one-way analysis of variance (ANOVA), or two-way ANOVA followed by Dunn post-hoc test. Results PTH1R receptor is highly expressed in the ARC where it affects feeding behavior We investigated the expression patterns of Pth1r and Pthrp in different brain structures by using the muscle as negative control and the bone, which is reported to highly express both transcripts, as positive control. We found that among the brain regions analyzed, both Pth1r and Pthrp are predominantly expressed in the HpT (Figure 1A), the major integrative centre of metabolic feedback and an important regulator of energy balance. Within the HpT, we identified by immunofluorescence a high presence of both proteins in the ARC (data not shown), localized in the ventral part of the mediobasal HpT adjacent to the third ventricle (3V). The ARC is known to integrate circulating metabolic signals and to drive feeding behavior and energy expenditure (Rodríguez et al., 2010; Dietrich & Horvath, 2013; Timper & Brüning, 2017 Ahima et al.2000, Barsh and Schwartz 2002, Elmquist et al.1999, Friedman and Halaas 1998, Schwartz et al.2000, Spiegelman and Flier 2001). In this regard, we sought to examine whether Pth1r signaling in the ARC could impact energy metabolism. To address this question, we downregulated Pth1r expression in the ARC, via bilateral stereotactic injection of an adeno- associated virus (AAV) expressing short-hairpin RNA (shRNA) against Pth1r under the control of the ubiquitous U6 promoter (data not shown). A decrease of up to 70% in Pth1r expression was validated in the HpT 3 weeks after injection (data not shown). Mice were then subjected to a series of physiological and functional analyses, including longitudinal body weight measurement, screening of body composition and metabolic cage phenotyping. We found that selective Pth1r downregulation in the ARC led to a drastic decrease in body weight gain in mice fed with normal chow (Figure 1B). This lean phenotype was associated with a decrease in white fat pad mass (data not shown). This was further confirmed by nuclear magnetic resonance spectroscopy, showing a drastic diminution of fat mass and a slight increase of lean mass, while fluid volume was not significantly affected (Figure 1C). Histology of epididymal white adipose tissue and interscapular brown adipose tissue revealed a decrease in lipid vacuole size in adipocytes by Pth1r downregulation in the ARC (data not shown). To elucidate the reason behind the significant difference in body weight gain between U6-sh-Pth1r and Scramble mice, we subjected the animals to metabolic cage phenotyping, where food/drink intake measurements and indirect calorimetry parameters were measured. We found that Pth1r downregulation in the ARC induced a decrease in food intake (Figure 1D) that was not associated with any changes in water intake (data not shown). We also observed a slight albeit not statistically significant increase in energy expenditure (EE) (data not shown). Other metabolic parameters involved in the control of energy balance, such as the respiratory exchange ratio (RER; indicative of the energy substrates used: RER = ∼1 for carbohydrates, RER = ∼0.7 for lipids), O2 consumption and CO2 production, were unchanged (data not shown). Likewise, ambulatory activity was unaffected (data not shown). Altogether these findings indicate that PTH1R in the ARC regulates energy metabolism by promoting feeding behavior. Distinct roles of PTH and PTHrP signaling in the ARC on energy metabolism PTH1R can be potentially activated by its two ligands, the circulating PTH, which has been reported to cross the blood-brain barrier (Joborn et al., 1991; Bühler at al 1997; Lourida et al., 2015), and PTHrP, which we showed here to be abundantly expressed in the ARC. Therefore, we next asked which ligand(s) is/are responsible for the prominent effects on feeding behavior observed following selective downregulation of Pth1r in the ARC. To explore independently the implication of each ligand, we first employed gain-of- function paradigms for PTHrP and PTH. Mice were administered PTH by intracerebroventricular infusion (i.c.v.). We found that brain PTH infusion did not change food intake (data not shown) but significantly decreased EE (Figure 2A). Water intake, locomotor activity, VO2, VCO2 and RER were not affected (data not shown). Conversely, loss of function model for PTH (PTHKO) show alteration in energy expenditure when compared to control littermates. Importantly, we found that brain PTHrP (i.c.v.) administration increases food intake without changes in EE (data not shown). These results were further corroborated by experiments using mice where Pthrp was specifically downregulated in the ARC (stereotactic injection of AAV-U6-Pthrp-shRNA, data not shown), after confirming a downregulation of 70% in the HpT (data not shown). Indeed, Pthrp downregulation mimicked the phenotypic and metabolic effects of Pth1r downregulation in the ARC, namely a decrease in body weight gain and fat mass (Figure 2B) and a decrease in food and water intake (data not shown). As with U6- sh-Pth1r mice, U6-sh-Pthrp mice did not display any significant changes in EE and RER (data not shown), VO2 consumption, VCO2 production (data not shown) and ambulatory activity (data not shown). Loss of function model for PTH (PTHKO) show alteration in energy expenditure when compared to control littermates. Altogether, these results indicate that the effects of the ARC-borne PTH1R on feeding behavior are mediated by its paracrine ligand PTHrP and not by its endocrine ligand, PTH. By contrast, brain delivery of PTH can modulate EE. PTHrP affects feeding behavior via coupling with PTH1R on AgRP neurons The important decrease in food intake following downregulation of either PTHrP or PTH1R in the ARC raised the question regarding the cellular identity of this system in the ARC and the specific mechanisms conveying its effects. The ARC contains two major, well-characterized neuronal populations that integrate hormonal and nutrient signals and that drive feeding behavior in an antagonistic fashion, the orexigenic agouti-related peptide (AgRP) and anorexigenic proopiomelanocortin (POMC) expressing neurons (Cone, 2005). We performed a series of immunofluorescence analyses using transgenic mice expressing the reporter td-Tomato in AgRP neurons (AgRPCre:tdTomato) or POMC neurons (POMCCre:tdTomato). We found that both PTH1R and PTHrP are widely expressed in AgRP neurons (data not shown), while only a scarce number of POMC neurons express these proteins (data not shown). Of note, we also detected PTH1R expression in cells of the median eminence and along the walls of the 3V. We then examined the acute effect of PTHrP on the activity of AgRP neurons using patch-clamp recordings. Fresh brain slices were prepared from transgenic AgRPCre:tdTomato mice killed at the beginning of the dark period in a calorie-deficient state. AgRPCre:tdTomato neurons were detected by fluorescence and electrical activity was recorded. Bath-applied PTHrP (100 nM) increased firing rate by 87% in approximately 90% of active AgRPCre:tdTomato neurons (n = 12 out of 13 neurons; basal: 1.72±0.46 Hz versus PTHrP: 3.21±0.75 Hz) (data not shown). Most of PTHrP-stimulated AgRPCre:tdTomato neurons returned to their basal firing rate 10 minutes after PTHrP application (i.e. during wash-out) (data not shown). Since activation of AgRP neurons is known to induce food foraging, we asked whether the AgRP-borne PTHrP/PTH1R system could play a role in this function. We stereotactically injected in the ARC an AAV expressing shRNA for either Pthrp or Pth1r under the control of the Agrp promoter (data not shown), and subjected mice to systematic body weight measurements and metabolic cage phenotyping. We found that both Agrp-shRNA-Pth1r and Agrp-shRNA-Pthrp mice gained significantly less weight compared to scramble-injected mice (Figure 3). Nuclear magnetic resonance spectroscopy showed a diminution of fat mass in Agrp-shRNA-Pth1r and Agrp-shRNA-Pthrp mice and an increase in lean mass, without changes in fluid volume (data not shown). Histological examination of BAT and WAT tissues showed that AgRP-specific downregulation of Pth1r and Pthrp induced fat droplet shrinkage, caused by a decrease in lipid vesicle size (data not shown). Metabolic cage phenotyping revealed that this lean phenotype could be explained by a decrease in food intake in both Agrp-shRNA-Pth1r and Agrp-shRNA-Pthrp mice (data not shown). Consistent with the notion that PTH1R is also a receptor for PTH, we observed a slight albeit not statistically significant increase in EE for Agrp-shRNA-Pth1r mice (data not shown). Other metabolic parameters were not affected in either group (data not shown). To rule out any participation of POMC neurons in the observed metabolic effects, we subjected Pomc-cre; Pth1rfl/fl mice to metabolic phenotyping and found no significant changes in body weight, feeding behavior, drink intake, RER, VO2, VCO2, locomotor activity and EE (data not shown), indicating that POMC neurons do not participate in the metabolic effects of PTH1R in the ARC. Collectively, these data indicate that coupling of PTHrP with PTH1R in AgRP-neurons affects feeding behavior. PTHrP/PTH1R coupling in AgRP neurons regulates AgRP levels and fasting-induced re-feeding If PTHrP signaling through PTH1R is involved in AgRP neuron-mediated feeding responses, then changes in the levels of all these proteins should be correlated in food deprivation, a situation known to upregulate AgRP levels (Cone, 2005). To address this, we measured the levels of PTH1R and PTHrP in fasted (F), non-fasted (NF) and fasted mice that were re-fed (F-RF) (data not shown). As expected in fasting conditions (Takahashi & Cone, 2005), there was an increase in AgRP levels that was attenuated following 2H of re-feeding (data not shown). Interestingly, the same pattern of protein increase was observed with PTHrP and PTH1R, significantly increasing by fasting and then returning to normal upon re-feeding (data not shown). Using F and NF AgRPCre:td-tomato mice, we determined that this fasting-induced increase in PTHrP levels happens specifically in AgRP neurons where PTHrP and PTH1R are expressed, while PTH1R expression also appears to increase albeit in a more diffused fashion in the ARC (data not shown). To further validate the link between PTHrP and AgRP expression, we delivered PTHrP in the ARC and found that it promptly increased AgRP levels in the HpT, 1H post-injection (data not shown). Conversely, AgRP-specific downregulation of either Pth1r or Pthrp in the ARC decreased AgRP protein levels in the HpT (data not shown). Finally, the interaction between PTHrP/PTH1R was also validated in vitro, where we observed that treatment of primary HpT neurons with PTHrP increased AgRP levels (data not shown). We then tested whether the PTHrP-induced increase in AgRP was translated into an effect on fasting-induced refeeding responses. To address this, we assessed food intake following a 18H fast in mice injected with either AAV-Agrp-shRNA-Pthrp or AAV-Agrp- shRNA-Pth1r. As shown in Figure 4A, food intake after fasting was markedly decreased in Agrp-shRNA-Pthrp and Agrp-shRNA-Pth1r mice compared to Scramble mice. Furthermore, we observed a failure in Agrp-shRNA-Pthrp mice to upregulate hypothalamic AgRP levels in response to fasting (Figure 4B). These findings indicate that PTHrP/PTH1R coupling induces an upregulation of AgRP levels, leading to increased feeding and participating in fasting- induced adaptive responses. PTHrP induces starvation-like hypothalamic lipid degradation In light of the results, we sought to determine the cellular mechanisms that would underlie these effects. We analysed by liquid chromatography-tandem mass spectrometry (LC- MS/MS) the metabolomic profile of primary HpT neurons treated or not with PTHrP. We observed an increase in free fatty acids (FFAs) upon PTHrP treatment compared to vehicle- treated (data not shown), reflecting an induction of lipid droplet degradation and increase in FFA availability. PCA biplot also indicated that vehicle and PTHrP datasets were orthogonal to each other, indicating an increase in the proportion of FFAs by PTHrP (data not shown). Fasting increases the levels of circulating FFAs that are rapidly taken up by organs and stored in the form of lipid droplets, mainly composed by triglycerides, until lipolytic mechanisms then break them down to provide energy. Importantly, intra-neuronal FFA availability has been previously described as important in the regulation of feeding behavior and fasting responses (Kaushik et al., 2011). In view of our observations, we used LipidTox, a neutral lipid dye, to measure the amount of lipid droplets in primary HpT neurons transfected with a lentivirus carrying shRNA for Pth1r. Downregulation of Pth1r significantly increased the number of lipid droplets, suggesting inhibition of lipolytic processes (data not shown). Conversely, when primary HpT neurons were treated with PTHrP, a massive reduction in the amount of lipid droplets was observed (data not shown). Moreover, acute PTHrP administration in the ARC promptly decreased the amount of LipidTox staining in the ARC (data not shown). Taken together, these findings suggest that the paracrine PTHrP/PTH1R system is a regulator of intra- neuron FFA availability. PTHrP-induced lipid degradation is driven by the autophagy machinery Lipid degradation can be the result of either enzyme-driven lipolysis in the cytosol or lipophagy. Accumulating evidence highlights the role of this selective form of autophagy in nutrient-deficient situations in various tissues, including in the HpT (Kaushik et al., 2011). To examine whether PTHrP-induced increase in hypothalamic FFA levels could be due to activation of autophagy, we treated primary HpT neurons with PTHrP and an inhibitor of autophagic flux, Bafilomycin (BAF), which blocks the autophagosome-lysosome fusion and subsequent protein degradation in lysosomes. We found that BAF abolishes the PTHrP- triggered reduction in intrinsic lipid droplets (data not shown), suggesting an implication of the autophagy process in PTHrP’s effect on intracellular lipid mobilization. Accordingly, there was a significant upregulation of key autophagy proteins, such as Beclin-1, ATG5/12, VPS34 and LC3I/II in lysates of PTHrP-treated primary HpT neurons (data not shown). We then confirmed our results in vivo, by showing that PTHrP delivery in the ARC increased the levels of ATG5/12 and LC3II, together with AgRP (data not shown). To assess more specifically autophagosome (AP) formation and autophagic flux, we used the pH-sensitive tandem mRFP-GFP-LC3 (fusion of mRFP, GFP and LC3) reporter that highlights AP as yellow puncta and autophagolysosomes (post-lysosomal fusion) as red puncta (the acidic environment inside the lysosome quenches the fluorescent signal of GFP). We found that PTHrP induced an increase in the number of both APs and autophagolysosomes (data not shown), indicative of an increase in both autophagy formation (GFP+/RFP+) and flux (GFP-/RFP+). Conversely, AgRP-specific downregulation of either Pth1r or Pthrp in the ARC resulted in a decrease in autophagy-related protein levels (data not shown) and a decrease in autophagic flux, as shown by an accumulation of p62 protein, a key cargo adaptor for the autophagic degradation by lysosomal proteases (data not shown). We then confirmed that PTHrP stimulates the autophagy machinery via its receptor PTH1R, by using PTHrP-treated primary HpT neurons transfected with a lenti-shRNA for Pth1r. We observed that while PTHrP treatment increased p62 levels, Pth1r downregulation blocked this effect (Figure 5), meaning that PTH1R is necessary for PTHrP’s effects on autophagic flux in AgRP neurons. These results demonstrate that PTHrP/PTH1R coupling harnesses the autophagy machinery in AgRP neurons towards lipid degradation. PTHrP-induced lipophagy increases FFA availability and upregulates AgRP neuropeptide expression To confirm the participation of lipophagy in PTHrP’s effects on intraneuronal lipids, we examined whether inhibition of lipophagy would block the PTHrP-induced lipid droplet degradation in HpT neurons. To that end, we treated primary HpT neurons with Lalistat-1, a potent and specific competitive inhibitor of the lysosomal acid lipase, known to block lipid droplet degradation by the lysosome. Co-treatment of PTHrP with Lalistat-1 hindered PTHrP’s effect on lipid droplet degradation (data not shown), confirming the implication of lipophagy in PTHrP’s lipolytic effects. To establish a mechanistic link between PTHrP-induced lipophagy and AgRP neuropeptide expression, we measured the amount of AgRP protein in lysates of PTHrP-treated primary HpT neurons after lipophagy inhibition with Lalistat-1. We found that Lalistat-1 blocked the PTHrP-induced increase in AgRP in these neurons. These results indicate that by stimulating the lipophagy process in AgRP neurons, PTHrP/PTH1R coupling upregulates AgRP levels and, consequently, promotes feeding behavior. EXAMPLE 2: Selective Pth1r downregulation in AgRP neurons improve the metabolic status of obesity-induced mouse model We investigated the curative therapeutic potential of a modulation of AgRP-borne PTH1R in an obesity-induced mouse model (Figure 6A). To do so, C57BL6/j mice (3 months- old) were submitted to either normal or high fat diet (HFD) (data not shown). Then, 8 weeks after the beginning of the regime, mice were stereotactically injected in the ARC with an adeno- associated virus (AAV) expressing short-hairpin RNA (shRNA) against Pth1r under the control of Agrp promoter. The site of injections and the efficacy of the downregulation were validated in the HpT, 5 weeks after AAVs injection by comparison to control mice (injected with shRNA scramble) (data not shown). Performing longitudinal analysis of the body weight, we observed that Agrp-shRNA-Pth1r mice under HFD gained significantly less weight compared to the control group (scramble-injected mice under HFD) (Figure 6B, 6C, 6D). This limited gain weight observed in Agrp-shRNA-Pth1r mice is associated to a reduction of fat mass (Figure 6E-F). Histological examination of brown adipose tissue (BAT) and white adipose (WAT) tissues showed that AgRP-specific downregulation of Pth1r induced adipocyte diameter shrinkage (Figure 6G-H), cause by a decrease of the lipid droplets. Importantly, we also observed a drastic reduction of lipid accumulation in liver induced by HFD, suggesting the potential therapeutic effect of a downregulation of AgRP-borne PTH1R on steatosis related to obesity (Figure 6G). Altogether, these findings demonstrate that a modulation of PTH1R signaling in AgRP is sufficient to improve the deleterious metabolic effects associated to HFD- induced obesity in mice. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Aponte, Y., Atasoy, D. & Sternson, SM. (2010). 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Claims

CLAIMS: 1. A PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof.

2. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claim 1 wherein the inhibitor is a PTH/PTH1r axis inhibitor.

3. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claim 1 wherein the inhibitor is a PTHrp/PTH1r axis inhibitor.

4. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claim 1 wherein the inhibitor is an inhibitor of PTH1r.

5. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 4 wherein metabolic disorders include obesity, associated metabolic disorders like metabolic syndrome, primary hyperparathyroidism (PHPT), pseudohypoparathyroidism type 1 or type 2, cancer and cachexia.

6. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 4 wherein metabolic disorder is obesity.

7. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 6, wherein the inhibitor is an inhibitor of PTH, PTHrp or PTH1r gene expression.

8. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 7, wherein the inhibitor is an inhibitor of PTH, PTHrp or PTH1r gene expression and the metabolic disorder is obesity.

9. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 8 wherein the inhibitor is administrated in a subject in need thereof in the brain.

10. The PTH/PTHrp/PTH1r axis inhibitor for use according to the claims 1 to 9 wherein the inhibitor is administrated in a subject in need thereof in the hypothalamus, in particular in the arcuate nucleus (ARC) of the hypothalamus.

11. A therapeutic composition comprising a PTH/PTHrp/PTH1r axis inhibitor for use in the treatment of metabolic disorders in a subject in need thereof.

12. A method of treating metabolic disorders in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an PTH/PTHrp/PTH1r axis inhibitor.