Anorexia nervosa (AN) is a serious disease characterized by decreased food intake and body weight, associated with excessive physical activity (PA) and a preference for protein-enriched diet (PED) that is known to decrease hunger. AN patients maintain euglycemia and minimal body weight, despite low energy intake and increased PA, suggesting that adaptations should take place at the level of energy metabolism and of endogenous glucose production (EGP). EGP is activated during fasting, with an increased contribution of renal (RGN) and intestinal gluconeogenesis (IGN). Since food restriction (FR) is featured by partial fasting state, we hypothesize a change in EGP characteristic of fasting in FR, i.e. increased RGN and IGN. Since IGN curbs hunger via a signal to the brain, we hypothesize that this could help to endure food restriction in AN. Since PED induces IGN, this could explain why AN patients adopt this kind of regimen. At last, since the portal glucose signal initiated by IGN induces food preference, we hypothesize that increased IGN could enhance the preference for PED in AN patients or FR mice (aim1). PA is matched with fasting and PED under several aspects. We hypothesize that PA should activate IGN, which could enhance the behaviour of PA via IGN action on the reward system (aim 2.) We will then raise the question of the adaptation of energy expenditure and metabolism in FR associated with physical activity, which might explain the plateauing of body weight in this situation (aim 3). Aims 1 to 3 will be assessed using a mouse model of FR/AN recently developed. In parallel, the hypothesis that food preference for PED and PA could be correlated will be assessed in a cohort of patients suffering from AN using dedicated questionnaires (aim 4). At last, the microbiota composition could have a role in the metabolic/behavioral adaptations in AN, and in food restriction and PA. his hypothesis will be assessed in our AN model of mouse and our cohort of patients (aim 5).

Obesity and diabetes are characterized by deregulations of endogenous glucose production. Three organs, the liver, kidney and intestine can produce glucose in the blood because they have the key enzyme: glucose-6 phosphatase (G6Pase). While hepatic glucose production is deleterious, intestinal glucose production is beneficial, since it sends a signal to the brain and exerts anti-obesity and anti-diabetes mediated by the hypothalamus. This especially takes place when intestinal glucose production is induced by diets enriched in protein or in soluble fiber fermented by the gut microbiota. We here want to make the proof of concept that intestinal glucose production per se may exert beneficial effects, i.e. regardless of any nutritional regulation. With this aim, we shall characterize new original animal models developed by the laboratory. We shall study the protection of mice constitutively overexpressing intestinal G6Pase against the development of obesity and diabetes induced by a deleterious “westernized” diet (rich in fat and sucrose). Using a procedure of overexpression inducible (by tamoxifen), we shall evaluate the capacity of intestinal glucose production to combat obesity and the deregulation of glucose control under conditions of pre-established obesity/diabetes. As an alternative to mimic intestinal glucose production and its benefits, we shall study the effect of infusions of glucose into the portal vein (via a catheter) in rodent models of obesity, pre-diabetes or diabetes. This will include genetic models of obesity/diabetes, such as Ob/Ob and Db/Db mice, and the Zucker Diabetic Fatty rat. In parallel, we shall deepen the molecular mechanisms involved in the central signal initiated by intestinal glucose production. This will include the identification of the nervous routes of transmission of the portal glucose signal, using an approach of electrophysiology on isolated gastrointestinal nerves and on central nuclei in vivo, and of the molecular mechanisms taking place in the hypothalamus, focusing on the original hypothesis of an interaction between the portal glucose signal and the hypothalamic leptin signalling. This will be studied by comparing the mechanisms taking place in mice deficient in intestinal G6Pase and mice overexpressing intestinal G6Pase. A better knowledge of the mechanisms initiated by intestinal glucose production and its metabolic benefits could pave the way for future approaches of prevention or treatment of metabolic diseases.

Glycogen storage disease 1a (GSD1a) is a rare metabolic disease due to glucose-6 phosphatase (G6PC) deficiency and it is characterized by short fasting hypoglycemia. Since the 80’s, life expectancy of GSD1a patients has improved considerably thanks to frequent carbohydrate-rich meals and intense nutritional management. Nevertheless, these patients develop long-term chronic kidney disease, eventually leading to renal failure. Renal transplants or dialysis is the only treatment for these patients. Living with an advanced renal disease has important negative impacts on the quality of life and it represents a frequent cause of morbidity. Until recently, the molecular mechanisms involved in GSD1a nephropathy remained unclear, since the GSD1a animal models were hardly viable after weaning. Because of this, we recently developed a mouse model with a G6pc deletion specifically in the kidneys (K.G6pc-/- mice). K.G6pc-/- mice are viable and develop the same renal complications as GSD1a patients. K.G6pc-/- mice exhibit a progressive deterioration in renal functions initiated by early tubular dysfunction, later on by a filtration barrier injury, and finally by glomerulosclerosis development. Then, K.G6pc-/- mice develop renal failure and polycystic kidneys with age. The goal of this project is to improve the diagnosis and treatment of GSD1a patients, by a better understanding of the renal pathology. Using the K.G6pc-/- mice, we can: 1) decipher the molecular mechanisms responsible for the renal pathophysiology; 2) study the effect of diet on these molecular pathways, as well as the progression of the pathology; 3) identify therapeutic targets; 4) identify biomarkers specific for the early stages of the renal pathology, that will allow an early pharmacologic intervention when needed in patients; and 5) develop new gene therapy strategies, including CRISPR/cas9-based genome editing. We would also like to propose an elaboration of a register of GSD1 patients, and a creation of a bio-bank (blood and urine samples) with the agreement of the GSD1 patients registered in the “Centre de référence des maladies héréditaires du métabolisme hépatique (Paris)”, in order to follow the development of nephropathy in GSD1a patients. There are many anabolic pathways that are hyper-activated in the renal tubules due to the excess in glucose-6 phosphate. We will focus on the role of lipid accumulation (lipidomics), and their effects and consequences on the cellular defenses in K.G6pc-/- kidneys. Recently, several new early biomarkers were established in other chronic kidney diseases. We intend to test these biomarkers in K.G6pc-/- mice and in GSD1 patient samples before the microalbuminuria stage, and evaluate if we can develop a more finely-tuned diagnosis method. In order to improve and unify the nutritional advice given to patients, we will study the effect of different diets on the progression of the nephropathy and the renal metabolism as a whole. We also propose to study the effects of several pharmacological agents, alone or combined, in order to target the specific mechanisms involved in GSD1 renal pathology. Finally, the K.G6pc-/- mice are an excellent in vivo tool for testing new viral vectors for targeted gene replacement. The outcome of these studies will be translated into new therapeutic pharmacological and dietary interventions, allowing us to improve the long-term outcome and, thereby the quality of life of GSD1 patients. Identification of biomarkers that appear earlier and are more sensitive could permit a better prevention of the deterioration of renal functions in GSD1 patients.

Obesity and Type 2 Diabetes are now considered as worldwide epidemics. To prevent and/or to cure these pathologies, the regulatory mechanisms of biological processes involved in their development need to be characterized. Among these processes, endogenous glucose production (EGP) is a crucial function for glucose homeostasis. Indeed, increased hepatic glucose production (HGP) has been associated with the settle of type 2 diabetes. On the contrary, intestinal glucose production (IGP) induces beneficial effects in energy and glucose homeostasis. Indeed, in the portal vein, the sensing of glucose produced by the intestine induces a signal to the brain. The portal glucose signal produced by IGP protects against the development of obesity and type 2 diabetes by decreasing food intake and HGP and by activating energy expenditure. Tissue-specific regulations of both HGP and IGP are thus crucial processes to decipher. EGP is restricted to the liver, kidney and intestine since these organs are the only organs expressing the key enzyme glucose-6-phosphatase (G6Pase). Glucose-6 phosphatase catalyzes the production of glucose, which is then exported out of the cell by the facilitated transporter Glut2 and by a membrane-based pathway, which still needs to be fully deciphered. This project aims at characterizing this vesicular glucose transport pathway and demonstrating the role of G6Pase in such a glucose transport process. I will first identify the components of the vesicular pathway of glucose export in the liver. Our preliminary data suggested that caveolin-1 (Cav1: a key component of caveolae), and dynamins (Dyn: necessary for the fission of vesicles) are involved in glucose export. Based on in vivo experiments performed in transgenic mouse models and in vitro experiments performed in hepatocytes, I will further characterize how these proteins participate in glucose transport. The importance of the vesicular glucose transport pathway compared with the glucose transport by Glut2 will be then assessed. We recently demonstrated that G6Pase activity is regulated by a membrane-based mechanism. I will then question the involvement of G6Pase in this glucose export pathway. Particularly, I will analyze the interaction of G6Pase with the components of the vesicular glucose transport pathway identified previously. I will then specify whether the vesicular glucose transport pathway is deregulated in the context of type 2 diabetes. Finally, I will extend this study to other gluconeogenic organs (i.e the kidney and intestine). In addition to molecular studies, I will focus on intestinal glucose absorption to firmly demonstrate that G6Pase may directly participate to a glucose transport function. G6Pase might participate in intestinal glucose absorption in the absence of Glut2. I will thus assess whether the suppression of G6Pase specifically in the intestine (I.G6pc-/-) decreases intestinal glucose absorption. Thus, my project aims at identifying a new function of G6Pase linking its capacity of glucose production to vesicular glucose export. These results may possibly lead to revisit the paradigms in glucose homeostasis. In addition, deciphering the regulations of this new function, and particularly tissue-specific regulations, should provide the bases for original strategies targeting EGP to prevent and/or cure type 2 diabetes.

Obesity and its associated diseases, such as type 2 diabetes, are a worldwide health concern. The most effective and durable therapies for the treatment of obesity nowadays involve surgical rather than behavioral or pharmacological interventions. Patients that undergo bariatric surgery not only lose weight, but also have rapid diabetes remission. The mechanisms leading to these beneficial metabolic changes remain largely unclear and are the focus of intense research. Besides, despite its efficacy, surgery is not a suitable therapeutic option for many obese subjects. Thus, the understanding of the underlying biological mechanisms responsible for the surgery-induced weight loss and metabolic improvements is crucial so that affected pathways can be targeted in a less-invasive, more specific manner. Bile acids have been recently recognized as an important molecular underpinning for the beneficial effects of bariatric surgery. Besides, bile acids are known to exert pleiotropic effects in peripheral organs where they regulate energy expenditure, glucose and lipid metabolism. Bile acids exert most of these metabolic effects by engaging the membrane-bound G protein-coupled bile acid receptor 1, also known as the Takeda G protein-coupled receptor 5 (TGR5), and the nuclear Farnesoid X receptor (FXR). So far, however, all studies investigating bile acids function in energy balance and metabolism have exclusively focused on their action at peripheral level. Based on solid preliminary data, here we put forward the original hypothesis that there is a long-range, hypothalamic control of bile acids effects on metabolism, which involves signaling through hypothalamic TGR5 and FXR. Thus, the main objectives of the BABrain project are: 1) to dissect the role of hypothalamic bile acids-TGR5 and FXR signaling in metabolic control; 2) to define the role of hypothalamic bile acids signaling in determining the beneficial effects of bariatric surgery. In order to reach these objectives, we will use a multidisciplinary approach including pharmacological, genetic, behavioral, metabolic and molecular studies combined with the use of animal models of diet-induced obesity (DIO) and bariatric surgery. In particular, we propose to characterize the metabolic effects of central chronic administration of TGR5 or FXR agonists (including specific bile acids) in diet-induced obese mice by performing a complete behavioral and metabolic phenotyping. “Smart” TGR5 agonists will be also tested for their ability to impact organism’s metabolic responses through a tightly controlled and selective activation of brain TGR5 receptors. Effects of TGR5 and FXR signaling on hypothalamic circuits will be investigated by using chemogenetics and ad hoc genetic animal models. Involvement of specific mechanisms underlying the phenotype, such as modulation of the autonomous nervous system and intestinal gluconeogenesis, as well as changes in bile acids metabolism and of the expression of specific molecular targets will be investigated using biochemistry, pharmacology, RNA sequencing and by employing specific genetic models. Finally, in order to evaluate the causative role of hypothalamic TGR5 or FXR in the phenotype obtained by chronic central administration of TGR5 or FXR agonists or after bariatric surgery, hypothalamic TGR5 or FXR expression will be silenced using Cre/lox and/or ShRNA-AAV strategies. We expect that the obtained findings will unambiguously establish the role of hypothalamic bile acids signaling in modulating metabolic responses and in determining potential beneficial effects of bile acids in metabolic disease.