Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

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.

Combination of Ginsenosides Rb2 and Rg3 Promotes Angiogenic Phenotype of Human Endothelial Cells via PI3K/Akt and MAPK/ERK Pathways.

Shexiang Baoxin Pill (SBP) is an oral formulation of Chinese materia medica for the treatment of angina pectoris. It displays pleiotropic roles in protecting the cardiovascular system. However, the mode of action of SBP in promoting angiogenesis, and in particular the synergy between its constituents is currently not fully understood. The combination of ginsenosides Rb2 and Rg3 were studied in human umbilical vein endothelial cells (HUVECs) for their proangiogenic effects. To understand the mode of action of the combination in more mechanistic detail, RNA-Seq analysis was conducted, and differentially expressed genes (DEGs), pathway analysis and Weighted Gene Correlation Network Analysis (WGCNA) were applied to further identify important genes that a play pivotal role in the combination treatment. The effects of pathway-specific inhibitors were observed to provide further support for the hypothesized mode of action of the combination. Ginsenosides Rb2 and Rg3 synergistically promoted HUVEC proliferation and tube formation under defined culture conditions. Also, the combination of Rb2/Rg3 rescued cells from homocysteine-induced damage. mRNA expression of CXCL8, CYR61, FGF16 and FGFRL1 was significantly elevated by the Rb2/Rg3 treatment, and representative signaling pathways induced by these genes were found. The increase of protein levels of phosphorylated-Akt and ERK42/44 by the Rb2/Rg3 combination supports the notion that it promotes endothelial cell proliferation via the PI3K/Akt and MAPK/ERK signaling pathways. The present study provides the hypothesis that SBP, via ginsenosides Rb2 and Rg3, involves the CXCR1/2 CXCL8 (IL8)-mediated PI3K/Akt and MAPK/ERK signaling pathways in achieving its proangiogenic effects.

Developmental programming of appetite and growth in male rats increases hypothalamic serotonin (5-HT)5A receptor expression and sensitivity.

BACKGROUND: Though it is well established that neonatal nutrition plays a major role in lifelong offspring health, the mechanisms underpinning this have not been well defined. Early postnatal accelerated growth resulting from maternal nutritional status is associated with increased appetite and body weight. Likewise, slow growth correlates with decreased appetite and body weight. Food consumption and food-seeking behaviour are directly modulated by central serotonergic (5-hydroxytryptamine, 5-HT) pathways. This study examined the effect of a rat maternal postnatal low protein (PLP) diet on 5-HT receptor mediated food intake in offspring. METHODS: Microarray analyses, in situ hybridization or laser capture microdissection of the ARC followed by RT-PCR were used to identify genes up- or down-regulated in the arcuate nucleus of the hypothalamus (ARC) of 3-month-old male PLP rats. Third ventricle cannulation was used to identify altered sensitivity to serotonin receptor agonists and antagonists with respect to food intake. RESULTS: Male PLP offspring consumed less food and had lower growth rates up to 3 months of age compared with Control offspring from dams fed a normal diet. In total, 97 genes were upregulated including the 5-HT5A receptor (5-HT5AR) and 149 downregulated genes in PLP rats compared with Controls. The former obesity medication fenfluramine and the 5-HT receptor agonist 5-Carboxamidotryptamine (5-CT) significantly suppressed food intake in both groups, but the PLP offspring were more sensitive to d-fenfluramine and 5-CT compared with Controls. The effect of 5-CT was antagonized by the 5-HT5AR antagonist SB699551. 5-CT also reduced NPY-induced hyperphagia in both Control and PLP rats but was more effective in PLP offspring. CONCLUSIONS: Postnatal low protein programming of growth in rats enhances the central effects of serotonin on appetite by increasing hypothalamic 5-HT5AR expression and sensitivity. These findings provide insight into the possible mechanisms through which a maternal low protein diet during lactation programs reduced growth and appetite in offspring.

Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake.

Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.

Heterogeneity of hypothalamic pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing.

OBJECTIVE: Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture. METHODS: Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons. RESULTS: Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp expression and were considered "canonical" POMC neurons (P+). The other 45/163 expressed low levels of Pomc and high levels of Agrp (A+P+). Unbiased clustering analysis of P+ neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P+ neurons expressed the leptin receptor (Lepr) compared with 58% (26/45) of A+P+ neurons. In contrast, the insulin receptor (Insr) was expressed at similar frequency on P+ and A+P+ neurons (64% and 55%, respectively). CONCLUSION: These data reveal arcuate POMC neurons to be a highly heterogeneous population. Accession Numbers: GSE92707.

Genetics of obesity: can an old dog teach us new tricks?

At one level, obesity is clearly a problem of simple physics, a result of eating too much and not expending enough energy. The more complex question, however, is why do some people eat more than others? Studies of human and mouse genetics over the past two decades have uncovered a number of pathways within the brain that play a key role in the control of food intake. A prime example is the leptin-melanocortin pathway, which we now know greatly contributes to mammalian appetitive behaviour. However, genetic disruption of this pathway remains rare and does not represent the major burden of the disease that is carried by those of us with 'common obesity'. In recent years, genome-wide association studies have revealed more than 100 different candidate genes linked to BMI, with most (including many components of the melanocortin pathway) acting in the central nervous system and influencing food intake. So while severe disruption of the melanocortin pathway results in severe obesity, subtle variations in these genes influence where you might sit in the normal distribution of BMI. As we now enter this 'post-genomics' world, can this new information influence our treatment and management of obese patients?

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

5-HT2A and 5-HT2C receptors as hypothalamic targets of developmental programming in male rats.

Although obesity is a global epidemic, the physiological mechanisms involved are not well understood. Recent advances reveal that susceptibility to obesity can be programmed by maternal and neonatal nutrition. Specifically, a maternal low-protein diet during pregnancy causes decreased intrauterine growth, rapid postnatal catch-up growth and an increased risk for diet-induced obesity. Given that the synthesis of the neurotransmitter 5-hydroxytryptamine (5-HT) is nutritionally regulated and 5-HT is a trophic factor, we hypothesised that maternal diet influences fetal 5-HT exposure, which then influences development of the central appetite network and the subsequent efficacy of 5-HT to control energy balance in later life. Consistent with our hypothesis, pregnant rats fed a low-protein diet exhibited elevated serum levels of 5-HT, which was also evident in the placenta and fetal brains at embryonic day 16.5. This increase was associated with reduced levels of 5-HT2CR, the primary 5-HT receptor influencing appetite, in the fetal, neonatal and adult hypothalamus. As expected, a reduction of 5-HT2CR was associated with impaired sensitivity to 5-HT-mediated appetite suppression in adulthood. 5-HT primarily achieves effects on appetite by 5-HT2CR stimulation of pro-opiomelanocortin (POMC) peptides within the arcuate nucleus of the hypothalamus (ARC). We show that 5-HT2ARs are also anatomically positioned to influence the activity of ARC POMC neurons and that mRNA encoding 5-HT2AR is increased in the hypothalamus ofin uterogrowth-restricted offspring that underwent rapid postnatal catch-up growth. Furthermore, these animals at 3 months of age are more sensitive to appetite suppression induced by 5-HT2AR agonists. These findings not only reveal a 5-HT-mediated mechanism underlying the programming of susceptibility to obesity, but also provide a promising means to correct it, by treatment with a 5-HT2AR agonist.

The hypothalamus and metabolism: integrating signals to control energy and glucose homeostasis.

Molecules acting in the central nervous system play a critical role in the control of both energy and glucose homeostasis. The hypothalamus consists of a highly diverse collection of interconnected neurons and supporting glial cells that allow this region of the brain to sense and respond to a diverse range of hormonal and metabolic signals. We review recent advances in our understanding of the anatomical architecture and molecular mechanisms within the hypothalamus and how these facilitate the orchestration of systemic metabolic processes.

Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1ß, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

Hypothalamic-specific manipulation of Fto, the ortholog of the human obesity gene FTO, affects food intake in rats.

Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Fto's actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.

The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

Proopiomelanocortin-deficient mice are hypersensitive to the adverse metabolic effects of glucocorticoids.

Congenital lack of proopiomelanocortin (POMC) causes obesity and glucocorticoid deficiency. The responses of Pomc-/- and wild-type mice to the administration of corticosterone were compared. In study 1, mice were given corticosterone-supplemented water (CORT) for 10 days, resulting in plasma CORT levels within the physiological range, with partial suppression of hypothalamic corticotropin-releasing hormone expression to a similar degree between genotypes. Body weight, fat mass, and food intake increased in CORT-treated Pomc-/- but not wild-type mice. CORT increased plasma insulin levels 50-fold in Pomc-/- versus 14-fold in wild-type mice (P < 0.01) and increased hypothalamic agouti-related protein (AgRP) expression by more than 200% in Pomc-/- versus 40% in wild type (P < 0.05). In study 2, mice were given CORT from weaning, and Pomc-/- but not wild-type mice developed hyperglycemia, ketonuria, and hepatic steatosis by 8-12 weeks. Thus, Pomc-/- mice are hypersensitive to the adverse metabolic effects of glucocorticoids. Additionally, as the levels of plasma CORT achieved, especially in study 1, were not grossly supraphysiological, we conclude that glucocorticoid deficiency may afford Pomc-/- mice some protection from the full adverse consequences of melanocortin deficiency. This may occur through a mechanism involving the suppression of AgRP by the hypoadrenal state.