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PROJET ENDOMETABO

Central PTH/PTHrp signalling: New leverage axis for the treatment of anorexia/metabolic syndrome

Ce projet est porté par l’Inserm UMRS 938 – Centre de Recherche Saint-Antoine (CRSA).

CONTEXT

Obesity and correlated diseases such as hypertension, atherosclerosis, dyslipidemia, coronary diseases and diabetes mellitus are identified as a worldwide pandemic (Molavi et al., 2006). Obesity per se, as well as the constellation of associated pathophysiology defined as the metabolic syndrome is inflicting an escalating public health burden. Whereas some genetic loci have been clearly identified and extensively studied as monogenic cause for obesity, it is widely accepted that the metabolic syndrome is in essence a multifactorial disease that encloses a complex network of molecular, cellular and physiological alterations (Kahn and Flier, 2000). The urge to understand the fundamental determinant of obesity and obesity-related disease has therefore generated a frantic race in the scientific community to unravel the mechanism involved and build potential therapeutic strategies. However, approved drugs for obesity have limited efficacy and act only acutely, with patients rapidly regaining weight after terminating treatment (Yanovski and Yanovski, 2002).

Substantial evidence indicates that the brain plays a key role in controlling energy homeostasis. Specialized neuronal networks in the hypothalamus (HpT) are known as the main orchestrators of the central control of energy balance, by integrating the effects of key metabolic hormones and nutrients.  Within the HpT, the arcuate nucleus (ARC) is of particular interest, because it is located close to the median eminence, a circumventricular organ that has evolved to facilitate the access of blood-borne metabolic signals onto first order ARC neurons. In the ARC, two sub population were demonstrated to contain two opposite branches of the melanocortin signaling pathway, the neuron producing the anorectic/catabolic POMC and the neurons producing the antagonist of the MCR, Agouti related protein. AgRP and POMC neurons represent a primary substrate for the integration of circulating signals if hunger and satiety and a key neurocircuits in the control of various aspect of energy balance such as food intake, energy expenditure and peripheral substrate utilization (Elmquist et al., 1999; Schwartz et al., 2000; Spiegelman and Flier, 2001; Barsh and Schwartz, 2002; Dietrich & Horvath, 2013; Timper & Brüning, 2017). Although the role of these neurons is pivotal in the control of whole-body metabolism, understanding of this complex regulation remains largely incomplete.

Recently, we unrevealed the presence of a novel G-protein coupled receptor linked to adenylyl cyclase in the AgRP neurons, the parathyroid hormone type 1 receptor (PTH1r), which is consider as the major regulator of Ca2+ homeostasis in the body, but with unknown role in the brain. Importantly, PTH1R can bind to either the endocrine parathyroid hormone (PTH) (exclusively produce by the parathyroid glands) and the paracrine parathyroid hormone-related peptide (PTHrp) expressed in various tissues including brain (Weaver et al., 2015).  Moreover, it has been demonstrated that alterations in either circulating PTH or PTHrP levels are correlated 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) (Timper & Bruning, 2017). Taken together, these observations raise the question of whether PTH and/or PTHrP could play an important role in the central control of energy metabolism and may represent novel therapeutic targets to prevent/treat obesity and metabolic disorders.

Using genetic, physiological, metabolic and cellular/molecular approaches, we have demonstrated that circulating PTH or paracrine PTHrP acting onto PTH1R exert distinct intracellular event in AgRP neuron to independently promote change in feeding behavior, fasting-induced adaptive responses, peripheral substrate utilization and body weight gain. These findings provide novel insights in the complex underpinnings by which circulating and paracrine signals can modulate different facets of energy balance by virtue of subtle modulation and engagement of AgRP+ neurons function. More importantly, we provide the proof-of-principle that a modulation of PTH1R system in the brain is sufficient to significantly reduced body mass gain, fat mass increase and steatosis in liver in standard mouse model of obesity, paving the way for the development of promising therapeutics for metabolic disorders, such as obesity and metabolic syndrome (Siopi et al., Cell Metabolism (in revision)).

These results provide further insight into the complex communication between brain and systemic circulating factors underpinning the central control of energy metabolism, which resonates with clinical observation showing that circulating PTHrP and PTH levels correlate with the severity of obesity (Reis et al., 2007; Saab et al., 2010; Corbetta et al., 2018). The PTH1R system in the brain offers an ideal setting for the development of a peptide-based strategy (brain penetrant small interfering peptide) targeting PTH1R signalling pathway in AgRP neuron and test their therapeutic potential to treat either cancer-induced anorexia and lipolysis, metabolic defect associated with obesity i.e hyperphagia, dyslipidaemia, diabetes and body weight gain.

OBJECTIVES

Our preliminary results point to the PTH/PTHrp signalling onto AgRP neuron as a fundamental regulator of energy homeostasis acting positively onto body weight gain in the condition of obesity, and negatively in the condition of cancer associated cachexia. In addition, we predict that generating small-peptide- based strategies to modulate PTH1R signalling onto AgRP neuron will allows to directly control feeding behaviours, and glucose metabolism through the control of peripheral organ activity. Therefore, we proposed to address 3 main objectives to identify new therapeutic targets that could directly or indirectly modulate the regulation of feeding behaviour and energy expenditure by PTH/PTHrp:

  1. Identify upstream and downstream cellular and molecular component that modulate PTH/PTHrp signalling in AgRP neurons.
  2. Establish the proof of concept that PTH1r signalling pathways in AgRP-neurons is a critical regulator of cancer-induced anorexia and lipolysis, and obesity-induced defect in nutrient partitioning, diabetes and dyslipidaemia.
  3. In silico design and in vivo validation of a peptide-based strategy to control PTH1R signalling pathway in AgRP neuron and energy homeostasis through brain penetrant small peptide design.

RESULTS

  1. A key question was to determine how a same receptor (i.e PTH1R) could regulate different HpT metabolic functions dependently of its activation by either PTH or PTHR. Using both in vitro and in vivo experiments, we now demonstrate that PTHrP and PTH can stimulate different signaling cascades of PTH1R in AgRP neurons: PTHrP activates the phospholipase Cβ/inositol trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway, while PTH activates the adenylyl cyclase/cAMP/protein kinase A (PKA) pathway. These results clearly indicate that both ligands can modulate AgRP neurons via a same G-coupled receptor but activating different signaling pathways. In light of these results, we have now generated new genetics tools to assess the dynamic and mechanisms of interactions of each ligand with PTH1R in AgRP neurons. 
  2. Investigating the cellular mechanism of action of the PTH1r system in AgRP neurons, we first found that PTH1R activation by PTHRP induces a massive reduction in the amount of lipid droplets in HpT neurons through the activation of a selective form of autophagy: lipophagy. This induction leads to an increase intra-neuronal free fatty acids (FFAs) availability, which is necessary to upregulate AgRP levels and thereby modulate food intake. Hence, suggesting that PTHrP could constitute a homeostatic mechanism allowing for a controlled availability of an endogenous pool of FFAs during starvation as an adaptive response to a constantly fluctuating environment. We are now investigating the cellular mode of action of PTH.
  3. Studying the upstream regulator of the hormonal feedback and specific circulating metabolic cues triggering this paracrine system in AgRP neurons, we found that one likely hormonal driver of hypothalamic PTHrP and PTH1R expression is ghrelin. Ghrelin is the only known circulating hormone that acts directly on AgRP neurons to induce rapid transcription of orexigenic neuropeptides (Nakazato et al., 2001). In vivo, selective knockdown of PTH1r in AgRP impaired feeding response achieved by systemic ghrelin injection, further confirming the convergence of ghrelin and PTH1R signaling onto AgRP neuron. This observation suggests that the PTH1R system may act as a key modulator of the effects of key systemic metabolic cues in AgRP neurons for the regulation of energy metabolism by the brain.
  1. We now provide the demonstration that local downregulation of either Pth1r or PTHrP in AgRP neurons in obesity-induced mouse model is sufficient to significantly reverse the BW and fat mass (NMR) alterations, blood glucose and insulin dysregulation, fat accumulation in liver. The same strategy will be now used to test the efficacy of the different 

VERBATIM

“The results obtained during this 1st year of the MSD program allowed us to i-enhance our understanding of the mode of action and pathways modulated by either PTH or PTHrP onto HpT circuits; ii-identify ghrelin as an upstream regulator of the PTH1R system in AgRP neurons; iii-provide the proof of concept of the curative potential of a PTH1R system modulation in obesity/metabolic syndrome, iv-design and generation of brain penetrant interferant peptides able to impact PTH1R signalling pathway, that will be now tested for their therapeutic potential to treat obesity and metabolic disorders. Importantly, post-doc necessary for the project were identified and recruited at each partner.”

RECENT PUBLICATIONS

  1. Siopi E., Galerne M., Rivagorda M., Saha S., Moigneu C., Moriceau S., Bigot M., Oury F*., Lledo P-M*. (2023). Gut microbiota changes require vagus nerve integrity to promote depressive-like behaviors in mice. Mol Psychiatry. (2023)doi: 10.1038/s41380-023-02071-6. Online ahead of print. 
  2. M Ramos-Brossier, D Romeo-Guitart, F Lanté, V Boitez, F Mailliet, S Saha, M Rivagorda, E Siopi, I Nemazanyy, C Leroy, S Moriceau, S Beck-Cormier, P Codogno, A Buisson, L Beck, G Friedlander, and F Oury*. Slc20a1 and Slc20a2 Regulate
  3. E Siopi*£, M Galerne*, A Rousseaud* F Liénard, F Mailliet, V Boitez, E Nedelec, S Moriceau, M Rivagorda, M Faour, j Castel, S Saha, C Schreiweiss, I Nemazanyy, D Romeo-Guitart, N Dupont, C Confavreux, S Luquet£, A Benani£ and F Oury£. Parathyroid Hormone Receptor Signaling in Hypothalamic AgRP Neurons is a Determinant of Feeding Behavior. Cell Metabolism (in revision) Neuronal Plasticity and Cognition independently of their phosphate transport ability. Cell Death & Disease (2023) in press
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