Congenital hypogonadotropic hypogonadism (CHH) is a condition characterized by absent puberty and infertility due to gonadotropin releasing hormone (GnRH) deficiency, which is often associated with anosmia (Kallmann syndrome, KS). We identified loss-of-function heterozygous mutations in anti-Müllerian hormone (AMH) and its receptor, AMHR2, in 3% of CHH probands using whole-exome sequencing. We showed that during embryonic development, AMH is expressed in migratory GnRH neurons in both mouse and human fetuses and unconvered a novel function of AMH as a pro-motility factor for GnRH neurons. Pathohistological analysis of Amhr2-deficient mice showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in reduced fertility in adults. Our findings highlight a novel role for AMH in the development and function of GnRH neurons and indicate that AMH signaling insufficiency contributes to the pathogenesis of CHH in humans.

The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

The hormone GDF15 is secreted in response to cellular stressors. Metformin elevates circulating levels of GDF15, an action important for the drug's beneficial effects on body weight. Metformin can also inhibit mammalian respiratory complex I, leading to decreases in ATP:AMP ratio, activation of AMP Kinase (AMPK), and increased GDF15 production. We undertook studies using a range of mice with tissue-specific loss of Gdf15 (namely gut, liver and global deletion) to determine the relative contributions of two classical metformin target tissues, the gut and liver, to the elevation of GDF15 seen with metformin. In addition, we performed comparative studies with another pharmacological agent, the AMP kinase pan-activator, MK-8722. Deletion of Gdf15 from the intestinal epithelium significantly reduced the circulating GDF15 response to oral metformin, whereas deletion of Gdf15 from the liver had no effect. In contrast, deletion of Gdf15 from the liver, but not the gut, markedly reduced circulating GDF15 responses to MK-8722. Further, our data show that, while GDF15 restricts high-fat diet-induced weight gain, the intestinal production of GDF15 is not necessary for this effect. These findings add to the body of evidence implicating the intestinal epithelium in key aspects of the pharmacology of metformin action.

Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin's effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.

Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

Opposite-sex attraction in most mammals depends on the fine-tuned integration of pheromonal stimuli with gonadal hormones in the brain circuits underlying sexual behaviour. Neural activity in these circuits is regulated by sensory processing in the accessory olfactory bulb (AOB), the first central station of the vomeronasal system. Recent evidence indicates adult neurogenesis in the AOB is involved in sex behaviour; however, the mechanisms underlying this function are unknown. By using Semaphorin 7A knockout (Sema7A ko) mice, which show a reduced number of gonadotropin-releasing-hormone neurons, small testicles and subfertility, and wild-type males castrated during adulthood, we demonstrate that the level of circulating testosterone regulates the sex-specific control of AOB neurogenesis and the vomeronasal system activation, which influences opposite-sex cue preference/attraction in mice. Overall, these data highlight adult neurogenesis as a hub for the integration of pheromonal and hormonal cues that control sex-specific responses in brain circuits.

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15-/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies.

Reproductive competence in mammals depends on the projection of gonadotropin-releasing hormone (GnRH) neurons to the hypothalamic median eminence (ME) and the timely release of GnRH into the hypothalamic-pituitary-gonadal axis. In adult rodents, GnRH neurons and the specialized glial cells named tanycytes periodically undergo cytoskeletal plasticity. However, the mechanisms that regulate this plasticity are still largely unknown. We demonstrate that Semaphorin7A, expressed by tanycytes, plays a dual role, inducing the retraction of GnRH terminals and promoting their ensheathment by tanycytic end feet via the receptors PlexinC1 and Itgb1, respectively. Moreover, Semaphorin7A expression is regulated during the oestrous cycle by the fluctuating levels of gonadal steroids. Genetic invalidation of Semaphorin7A receptors in mice induces neuronal and glial rearrangements in the ME and abolishes normal oestrous cyclicity and fertility. These results show a role for Semaphorin7A signalling in mediating periodic neuroglial remodelling in the adult ME during the ovarian cycle.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.