Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain.

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Characterization of Hypothalamic MCH Neuron Development in a 3D Differentiation System of Mouse Embryonic Stem Cells.

Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH+CART+ neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH+CART- and MCH+CART+ neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.

Hypothalamic Contribution to Pituitary Functions Is Recapitulated In Vitro Using 3D-Cultured Human iPS Cells.

The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

Vasopressin-secreting neurons derived from human embryonic stem cells through specific induction of dorsal hypothalamic progenitors.

Arginine-vasopressin (AVP) neurons exist in the hypothalamus, a major region of the diencephalon, and play an essential role in water balance. Here, we established the differentiation method for AVP-secreting neurons from human embryonic stem cells (hESCs) by recapitulating in vitro the in vivo embryonic developmental processes of AVP neurons. At first, the differentiation efficiency was improved. That was achieved through the optimization of the culture condition for obtaining dorsal hypothalamic progenitors. Secondly, the induced AVP neurons were identified by immunohistochemistry and these neurons secreted AVP after potassium chloride stimulation. Additionally, other hypothalamic neuropeptides were also detected, such as oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone, pro-opiomelanocortin, agouti-related peptide, orexin, and melanin-concentrating hormone. This is the first report describing the generation of secretory AVP neurons derived from hESCs. This method will be applicable to research using disease models and, potentially, for regenerative medicine of the hypothalamus.

The alpha-glucosidase inhibitor miglitol affects bile acid metabolism and ameliorates obesity and insulin resistance in diabetic mice.

OBJECTIVE: Alpha-glucosidase inhibitors (α-GIs) show various anti-diabetic or anti-obesity effects in addition to the suppression of postprandial hyperglycemia. Based on recent observations that bile acids (BAs) are involved in glucose and energy homeostasis, we examined the ability of miglitol, an α-GI, to influence BA metabolism and ameliorate insulin resistance and obesity. MATERIALS/METHODS: NSY mice, representing an obese type 2 diabetic model, were fed with a high-fat diet and treated with miglitol for 4 or 12 weeks. BAs were quantified in feces, blood from the portal vein or the vena cava and in the liver. The gene expression of type 2 iodothyronine deiodinase (D2) in brown adipose tissues, gluconeogenetic enzymes in the liver and adipokines in epididymal fat was measured, and portal blood glucagon-like peptide-1 (GLP-1) levels, body weight changes, glucose tolerance along with insulin sensitivity were evaluated. RESULTS: Miglitol significantly increased BAs in both feces and portal blood while the hepatic BA level was reduced. The drug clearly enhanced active GLP-1 secretion into the portal blood and there was a good positive correlation between the active GLP-1 levels and portal blood BA concentrations. D2 expression in brown adipose tended to increase in association with the elevated BA concentrations. Miglitol ameliorated body weight gain, glucose intolerance, insulin resistance and inflammatory adipokine upregulation that were induced by a high-fat diet. CONCLUSIONS: Collectively, miglitol substantially affects BA regulation in mice and this novel finding may explain in part the known favourable effects of the drug on diabetes and obesity.

Glucocorticoids increase NPY gene expression in the arcuate nucleus by inhibiting mTOR signaling in rat hypothalamic organotypic cultures.

The mammalian target of rapamycin (mTOR) has been implicated in the regulation of physiological functions such as cell growth and proliferation, and glucocorticoids reportedly inhibit mTOR signaling in peripheral tissues. Recent studies suggest that the mTOR signaling in the hypothalamus plays a critical role in maintaining energy homeostasis. In this study, we examined whether the mTOR signaling in the hypothalamus is involved in the regulation of neuropeptide Y (Npy) gene expression in the arcuate nucleus by glucocorticoids. In the hypothalamic organotypic cultures, the incubation with rapamycin significantly inhibited the mTOR signaling which was shown by decreases in the levels of phosphorylated p70S6K1 and S6. Similar to the action of the mTOR inhibitor rapamycin, dexamethasone (DEX), a synthetic glucocorticoid, also inhibited the mTOR signaling in the hypothalamic explants. Analyses of the explants with in situ hybridization demonstrated that the DEX or rapamycin alone significantly increased Npy gene expression in the arcuate nucleus, but that there were no additive effects of DEX and rapamycin on the expression. These data suggest that glucocorticoids upregulate the Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling, at least in part.

The pharmacological properties of a novel MCH1 receptor antagonist isolated from combinatorial libraries.

Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits potent orexigenic activity. In rodents, it exerts its actions by interacting with one receptor, MCH(1) receptor which is expressed in many parts of the central nervous system (CNS). To study the physiological implications of the MCH system, we need to be able to block it locally and acutely. This necessitates the use of MCH(1) receptor antagonists. While MCH(1) receptor antagonists have been previously reported, they are mainly not accessible to academic research. We apply here a strategy that leads to the isolation of a high affinity and selective MCH(1) receptor antagonist amenable to in vivo analyses without further chemical modifications. This antagonist, TPI 1361-17, was identified through the screening of multiple non-peptide positional scanning synthetic combinatorial libraries (PS-SCL) totaling more than eight hundred thousand compounds in conditions that allow for the identification of only high-affinity compounds. TPI 1361-17 exhibited an IC(50) value of 6.1 nM for inhibition of 1 nM MCH-induced Ca(2+) mobilization and completely displaced the binding of [(125)I] MCH to rat MCH(1) receptor. TPI 1361-17 was found specific, having no affinity for a variety of other G-protein coupled receptors and channels. TPI 1361-17 was found active in vivo since it blocked MCH-induced food intake by 75%. Our results indicate that TPI 1361-17 is a novel and selective MCH(1) receptor antagonist and is an effective tool to study the physiological functions of the MCH system. These results also illustrate the successful application of combinatorial library screening to identify specific surrogate antagonists in an academic setting.

The melanin-concentrating hormone system and its physiological functions.

Melanin-concentrating hormone (MCH) is a neuropeptide that was originally isolated from salmon pituitary where it causes pigment aggregation. MCH is also abundantly present in mammalian neurons and expressed in the lateral hypothalamus and zona incerta, brain regions that are known to be at the center of feeding behavior. MCH binds to and activates two G protein-coupled receptors, MCH1R and MCH2R. Although MCH2R is non-functional in rodents, genetic and pharmacological studies have demonstrated that rodent MCH1R is involved in the regulation of feeding behavior and energy balance. Unexpectedly, some antagonists have provided evidence that MCH signaling participates in the regulation of other processes, such as emotion and stress. The discovery of MCH receptors has extensively promoted the progress of MCH studies and may represent an ideal example of how deorphanized receptors can open new directions toward more detailed physiological studies.