Implication de l'apeline hypothalamique dans la transition entre un statut sain et un statut obèse/diabétique via la mise en place d'une voie peroxyde d'hydrogène hypothalamique-système nerveux autonome-foie

L'hypothalamus est une région cérébrale impliquée dans le contrôle de l'homéostasie glucidique. En réponse à de nombreux facteurs, l'activité neuronale hypothalamique va être modifiée, générant une modification d'activité de tissus périphériques, via le recrutement du système nerveux autonome. Il en résulte un ajustement du métabolisme glucidique tandis que son altération conduit à des pathologies dont le diabète de type 2. Durant ce doctorat, nous nous sommes intéressés à l'apeline, une adipokine également libérée par les neurones hypothalamiques. Dans le cadre d'un diabète de type 2, les taux d'apeline sont augmentés dans le sang et dans l'hypothalamus. Ainsi nous avons mis en évidence que l'injection en intra-cérébroventriculaire chez des souris saines, de forts taux d'apeline similaires à ceux retrouvés chez des animaux obèses/diabétiques, génère des caractéristiques d'un diabète de type 2. Ces forts taux d'apeline entrainent une libération hypothalamique d'Espèces Actives de l'Oxygène telles que l'H2O2 génèrant une sur-activation du système nerveux orthosympathique, ciblant le foie et augmentant la production hépatique de glucose par glycogénolyse et néoglucogenèse. Cette voie de régulation H2O2 hypothalamique-système nerveux-autonome-foie est retrouvée dans un modèle de souris surexprimant l'apeline dans le noyau ventromédian. Cette voie est toujours présente et même amplifiée chez des animaux obèses/diabétiques révélant une absence de résistance à l'apeline. Ainsi ces travaux de thèse ont permis de mettre en lumière que les forts taux d'apeline hypothalamiques sont impliqués dans la mise en place d'un diabète de type 2.Hypothalamus is brain area involved in the control of glucose homeostasis. In response to many factors, hypothalamic activity will be modified, generating a change in activity of peripheral tissues via the autonomic nervous system. This loop of regulation generates an adjustment of glucose metabolism while its alteration leads to diseases such as diabetes type 2. During this thesis, we are interested in apelin, an adipokine also released by hypothalamic neurons. In the context of type 2 diabetes, apelin levels are increased in the blood and in the hypothalamus. Thus, we have demonstrated that the intracerebroventricular injection in normal mice, of high levels of apelin similar to that observed in the hypothalamus of obese and diabetic mice generates type 2 diabetes characteristics. This high level of apelin triggers hypothalamic Reactive Oxygen Species release such as H2O2 which over-activates sympathetic nervous system and targets liver increasing hepatic glucose production by glycogenolysis and gluconeogenesis. This hypothalamic H2O2 - autonomous nervous system - liver pathway is also found in a mouse model overexpressing apelin in the ventromedial nucleus. This pathway is always present and amplified in obese/diabetic mice demonstrating a lack of resistance to apelin. Thus this thesis brings to highlight that high levels of hypothalamic apelin are involved in the onset of type 2 diabetes

Reduced alfa-MSH underlies hypothalamic ER-stress-induced hepatic gluconeogenesis

Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D

Impact of hypothalamic reactive oxygen species in the regulation of energy metabolism and food intake

Hypothalamus is a key area involved in the control of metabolism and food intake via the integrations of numerous signals (hormones, neurotransmitters, metabolites) from various origins. These factors modify hypothalamic neurons activity and generate adequate molecular and behavioral responses to control energy balance. In this complex integrative system, a new concept has been developed in recent years, that includes reactive oxygen species (ROS) as a critical player in energy balance. ROS are known to act in many signaling pathways in different peripheral organs, but also in hypothalamus where they regulate food intake and metabolism by acting on different types of neurons, including proopiomelanocortin (POMC) and agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons. Hypothalamic ROS release is under the influence of different factors such as pancreatic and gut hormones, adipokines (leptin, apelin,...), neurotransmitters and nutrients (glucose, lipids,...). The sources of ROS production are multiple including NADPH oxidase, but also the mitochondria which is considered as the main ROS producer in the brain. ROS are considered as signaling molecules, but conversely impairment of this neuronal signaling ROS pathway contributes to alterations of autonomic nervous system and neuroendocrine function, leading to metabolic diseases such as obesity and type 2 diabetes.In this review we focus our attention on factors that are able to modulate hypothalamic ROS release in order to control food intake and energy metabolism, and whose deregulations could participate to the development of pathological conditions. This novel insight reveals an original mechanism in the hypothalamus that controls energy balance and identify hypothalamic ROS signaling as a potential therapeutic strategy to treat metabolic disorders

Cortical abnormalities and non-spatial learning deficits in a mouse model of CranioFrontoNasal syndrome.

Eph receptors and their ephrin ligands play critical roles in the development of the nervous system, however, less is known about their functions in the adult brain. Here, we investigated the function of ephrinB1, an ephrinB family member that is mutated in CranioFrontoNasal Syndrome. We show that ephrinB1 deficient mice (EfnB1(Y/-)) demonstrate spared spatial learning and memory but exhibit exclusive impairment in non-spatial learning and memory tasks. We established that ephrinB1 does not control learning and memory through direct modulation of synaptic plasticity in adults, since it is not expressed in the adult brain. Rather we show that the cortex of EfnB1(Y/-) mice displayed supernumerary neurons, with a particular increase in calretinin-positive interneurons. Further, the increased neuron number in EfnB1(Y/-) mutants correlated with shorter dendritic arborization and decreased spine densities of cortical pyramidal neurons. Our findings indicate that ephrinB1 plays an important role in cortical maturation and that its loss has deleterious consequences on selective cognitive functions in the adult

The apelinergic system: Sexual dimorphism and tissue-specific modulations by obesity and insulin resistance in female mice.

International audienceIt has been proposed that the apelinergic system (apelin and its receptor APJ) may be a promising therapeutic target in obesity-associated insulin resistance syndrome. However, due to the extended tissue-distribution of this system, the therapeutic use of specific ligands for APJ may target numerous tissues resulting putatively to collateral deleterious effects. To unravel specific tissular dysfunctions of this system under obesity and insulin-resistance conditions, we measured the apelinemia and gene-expression level of both apelin (APL) and APJ in 12-selected tissues of insulin-resistant obese female mice fed with a high fat (HF) diet. In a preliminary study, we compared between adult male and female mice, the circadian plasma apelin variation and the effect of fasting on apelinemia. No significant differences were found for these parameters suggesting that the apelinemia is not affected by the sex. Moreover, plasma apelin level was not modulated during the four days of the estrous cycle in females. In obese and insulin-resistant HF female mice, plasma apelin concentration after fasting was not modified but, the gene-expression level of the APL/APJ system was augmented in the white adipose tissue (WAT) and reduced in the brown adipose tissue (BAT), the liver and in kidneys. BAT apelin content was reduced in HF female mice. Our data suggest that the apelinergic system may be implicated into specific dysfunctions of these tissues under obesity and diabetes and that, pharmacologic modulations of this system may be of interest particularly in the treatment of adipose, liver and renal dysfunctions that occur during these pathologies

EphrinB1 is not expressed in the adult cortex.

<p><i>A, B.</i> Representative coronal section of the brain from wild type (<i>A</i>) and <i>EfnB1</i> mutant (<i>B</i>) adult mice showing normal gross morphology of the brain. Both mutant and wild type brains have similar sizes. A slight enlargement of the ventricles was observed in some of the mutant brains (<i>B</i>, asterisks). <i>C–D’’’.</i> Immunohistochemistry for EphrinB1 at noted postnatal (P) dates in <i>EfnB1^Y/+</i> and <i>EfnB1^Y/−</i> cortices. EphrinB1 is not detected in <i>EfnB1^Y/+</i> adult cortices (<i>C</i>), rather strong, positive signals are observed at P5 (<i>C”’</i>) and decline by P15 (<i>C’</i>) in <i>EfnB1^Y/+</i> samples. <i>E</i>. Quantitative RT-PCR of P30–150 whole brain (WB) extracts and cortical extracts were analysed for <i>EfnB1</i> expression and compared to control embryonic (E) day 16.5 cortical extracts that express <i>EfnB1.</i> n = 3/age. <i>F–G’.</i> Immunohistochemistry for ephrinB1 in <i>EfnB1^Y/+</i> and <i>EfnB1^Y/−</i> hippocampi at P5 and P150. EphrinB1 is not detected in <i>EfnB1^Y/+</i> adult hippocampi (<i>F</i>), but observed in the CA1 region at P5 (F’; inlet).</p

Changes in cell number in <i>EfnB1</i> mutant cortices.

<p><i>A, B.</i> Representative images of cortical sections from <i>EfnB1^Y/+</i> (WT; <i>A</i>) and <i>EfnB1^Y/−</i> (<i>B</i>) mice at P150 stained for NeuN. Shown are areas of the frontal cortex. <i>C.</i> Quantification of NeuN stained cells demonstrating an increase in NeuN-positive cells in P150 <i>EfnB1^Y/−</i> mice, P10 <i>EfnB1^Y/−</i> mice and not at P0 stages. * p<0.05. <i>D.</i> Ratio of cortical thickness in <i>EfnB1^Y</i>^{<b><i>/−</i></b>} mice as compared to controls. <i>E.</i> Quantification of NeuN positive cells in the CA3 and CA1 region of the hippocampus, and in the striatum and amygdala in P150 <i>EfnB1^Y/+</i> and <i>EfnB1^Y/−</i> mice.</p

Comprised dendritic arbors in ephrinB1 mutant cortical pyramidal cells.

<p><i>A, B.</i> Representative photomicrographs of golgi-cox impregnated S2 pyramidal neurons from <i>EfnB1^Y/+</i> (<i>A</i>) and <i>EfnB1^Y/−</i> (<i>B</i>) cortices. Note the presence of dendritic varicosities on dendrites from <i>EfnB1^Y/−</i> samples (arrows). <i>C.</i> Sholl analysis of the structure of dendritic arbors. Mean number of dendritic intersections of layer II/III cortical pyramidal neurons show a significant decrease in the number of intersections in pyramidal neurons from <i>EfnB1^Y/−</i> cortex at longer distances from the cell soma. Note that majority of dendritic intersections in <i>EfnB1^Y/−</i> cortex were found proximal to the soma, at a distance of ∼20–50 µm compared to <i>EfnB1^Y/+</i> cortex. <i>D.</i> Quantitative analysis of dendritic branch length shows a statistically significant decrease in the branch length of pyramidal neurons from <i>EfnB1^Y/−</i> samples compared to controls (<i>EfnB1^Y/+</i> = 125 µm±7; <i>EfnB1^Y/−</i> = 88 µm±12; p<0.01). <i>E, F.</i> Higher magnified photomicrographs of <i>EfnB1^Y/+</i> (<i>E</i>) and <i>EfnB1^Y/−</i> (<i>F</i>) pyramidal neurons in the S2 field. Note the varicosities in dendrites from <i>EfnB1^Y/−</i> samples. <i>G.</i> Quantitative analysis of the number of morphologically different spines on the basilar tree of cortical layer II/III pyramidal neurons in <i>EfnB1^Y/+</i> and <i>EfnB1^Y/−</i> samples. A significantly higher number of thin spines are observed on neurons from <i>EfnB1^Y/−</i> mice. <i>H.</i> Analysis of the total density of spines along a dendrite showed fewer spines throughout the entire length of the basal dendrites. Each dot represents 4–5 neurons per individual. n = 5/genotype. * p<0.05, **p<0.001.</p

Increased interneuron number in adult <i>EfnB1^Y/−</i> mutants.

<p><i>A–F.</i> Representative immunofluorescent images of <i>EfnB1^Y/+</i> (<i>A, C, E</i>) and <i>EfnB1^Y/−</i> (<i>B, D, F</i>) mutant somatosensory S2 cortices stained for PV (<i>A, B</i>), CR (<i>C, D</i>) and NPY (<i>E, F</i>) The percent of interneuron numbers per 100 micro(µ)m² is shown for the S2 region of <i>EfnB1^Y/+</i> versus <i>EfnB1^Y/−</i> samples. <i>G.</i> We observed an overall increase in PV+, CR+ and NPY+ neurons in <i>EfnB1^Y/−</i> mutants as compared to <i>EfnB1^Y/+</i> in all cortical regions studied. A significant increase was detected for CR+ neurons in the <i>EfnB1^Y/−</i> S2 cortical region. * p<0.05.</p

General behaviour and spatial memory are preserved in <i>EfnB1</i> mutant mice.

<p><i>A.</i> Locomotor activity and anxiety levels in <i>EfnB1</i> mutant (<i>EfnB1^Y/−</i>) mice as compared to control (WT) mice. <i>B.</i> Graph presenting swim path length as a function of individual training sessions (S1–S4). Throughout the training sessions, <i>EfnB1^Y/−</i> mice learned equally well to locate the hidden platform and exhibited decreasing swim distances over blocks of trials (F_3,45 = 12.58, <i>p</i><0.001). <i>C.</i> Number of annulus crossings during probe tests. All groups of mice showed similar preference for the target zone where the platform was located during training sessions as compared to the adjacent (Adj1 and Adj2) and opposite zones (target <i>vs</i> others, **<i>p</i><0.01; ***<i>p</i><0.001). <i>D.</i> Performances in the object location task are expressed as the group mean (± SEM) preference index. The horizontal line represents equal exploration of the two objects. <i>EfnB1^Y/−</i> and WT mice presented the same level of reaction to the object displacement (F_1,17 = 0.139, <i>p</i> = 0.714) and spent significantly more time exploring the displaced object than the non-displaced one (^###p<0.001; index <i>vs</i> chance level (50%)). NS: not significant.</p