PI3K signalling in leptin receptor cells: Role in growth and reproduction

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149245/1/jne12685_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149245/2/jne12685.pd

Defining a novel leptin–melanocortin–kisspeptin pathway involved in the metabolic control of puberty

Objective Puberty is a key developmental phenomenon highly sensitive to metabolic modulation. Worrying trends of changes in the timing of puberty have been reported in humans. These might be linked to the escalating prevalence of childhood obesity and could have deleterious impacts on later (cardio-metabolic) health, but their underlying mechanisms remain unsolved. The neuropeptide α-MSH, made by POMC neurons, plays a key role in energy homeostasis by mediating the actions of leptin and likely participates in the control of reproduction. However, its role in the metabolic regulation of puberty and interplay with kisspeptin, an essential puberty-regulating neuropeptide encoded by Kiss1, remain largely unknown. We aim here to unveil the potential contribution of central α-MSH signaling in the metabolic control of puberty by addressing its role in mediating the pubertal effects of leptin and its potential interaction with kisspeptin. Methods Using wild type and genetically modified rodent models, we implemented pharmacological studies, expression analyses, electrophysiological recordings, and virogenetic approaches involving DREADD technology to selectively inhibit Kiss1 neurons, in order to interrogate the physiological role of a putative leptin→α-MSH→kisspeptin pathway in the metabolic control of puberty. Results Stimulation of central α-MSH signaling robustly activated the reproductive axis in pubertal rats, whereas chronic inhibition of melanocortin receptors MC3/4R, delayed puberty, and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4R or genetic elimination of kisspeptin receptors from POMC neurons did not affect kisspeptin effects. Conversely, congenital ablation of kisspeptin receptors or inducible, DREADD-mediated inhibition of arcuate nucleus (ARC) Kiss1 neurons resulted in markedly attenuated gonadotropic responses to MC3/4R activation. Furthermore, close appositions were observed between POMC fibers and ARC Kiss1 neurons while blockade of α-MSH signaling suppressed Kiss1 expression in the ARC of pubertal rats. Conclusions Our physiological, virogenetic, and functional genomic studies document a novel α-MSH→kisspeptin→GnRH neuronal signaling pathway involved in transmitting the permissive effects of leptin on pubertal maturation, which is relevant for the metabolic (and, eventually, pharmacological) regulation of puberty onsetThis work was supported by grants BFU2011-025021 & BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain; co-funded with EU funds from FEDER Program); project PIE-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain); Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain); EU research contract DEER FP7-ENV-2007-1 and the New Zealand Health Research Council. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. Senior authors are indebted with Dr. R.A. Steiner (University of Washington, Seattle, USA) and Dr. U. Boehm (University of Saarland School of Medicine, Homburg, Germany) for provision of relevant mouse lines, essential for conduction of some of the experiments included in this studyS

Análisis de rendimiento entre Oracle y Mongodb para empresas Call Centers en un contexto de Investigación Cuantitativa

Currently, companies manage an enormous amount of information. and there are several relational and non-relational database administrators, and because of this companies can choose where to manage their data, in the article it was decided to analyze the performance between Oracle (SQL) and MongoDB (NoSQL), it was based on the CRUD operations, with these results of the analysis it will be possible to provide a broader vision to these companies and they can choose DBMS according to their needs.En la actualidad, las compañías gestionan una enorme cantidad de información. y existen diversos administradores de bases de datos tanto relacionales como no relacionales, y debido a esto las empresas pueden elegir en donde administrar sus datos, en artículo se decidió analizar el rendimiento entre Oracle (SQL) y MongoDB (NoSQL), se basó en las operaciones de CRUD, con estos resultados del análisis se podrá brindar una visión más amplia a estas empresas y que puedan elegir SGBD de acuerdo con sus necesidades

Análisis de rendimiento entre Oracle y Mongodb para empresas Call Centers en un contexto de Investigación Cuantitativa

Currently, companies manage an enormous amount of information. and there are several relational and non-relational database administrators, and because of this companies can choose where to manage their data, in the article it was decided to analyze the performance between Oracle (SQL) and MongoDB (NoSQL), it was based on the CRUD operations, with these results of the analysis it will be possible to provide a broader vision to these companies and they can choose DBMS according to their needs.En la actualidad, las compañías gestionan una enorme cantidad de información. y existen diversos administradores de bases de datos tanto relacionales como no relacionales, y debido a esto las empresas pueden elegir en donde administrar sus datos, en artículo se decidió analizar el rendimiento entre Oracle (SQL) y MongoDB (NoSQL), se basó en las operaciones de CRUD, con estos resultados del análisis se podrá brindar una visión más amplia a estas empresas y que puedan elegir SGBD de acuerdo con sus necesidades

PI

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149245/1/jne12685_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149245/2/jne12685.pd

PI3K p110β subunit in leptin receptor expressing cells is required for the acute hypophagia induced by endotoxemia

Objective: Hypophagia and increased energy expenditure under inflammatory conditions, such as that observed after bacterial lipopolysaccharide (LPS) administration, are associated with leptin secretion. The hypophagic effect of leptin depends in part on the activation of PI3K signaling pathway. However, the role of PI3K in the endotoxemia-induced hypophagia has not been determined. Methods: In an attempt to examine the functional contribution of the PI3K pathway in hypophagia and weight loss induced by LPS (100 ug/Kg, ip), we performed a central pharmacological PI3K inhibition (LY294002). Additionally, to gain mechanistic insights on the role of the catalytic PI3K p110α subunit in leptin responsive cells, mice expressing Cre-recombinase driven by the Lepr promoter (LepR-Cre) were crossed with mice carrying a loxP-modified p110α allele (Pi3kca gene) (LepRΔp110α). As studies have suggested that the PI3K p110β subunit has a dominant role over p110α in energy homeostasis, we further crossed LepR-Cre mice with loxP-modified p110α and p110β (Pi3kcb gene) alleles (LepRΔp110α+β). In order to verify the requirement of leptin in PI3K effects on food intake, we also used leptin-deficient ob/ob mice. Results: We found that LPS stimulates PI3K and STAT3 signaling pathways in cells expressing the leptin receptor. Central PI3K inhibition prevented LPS-induced hypophagia and weight loss. Genetic deletion of p110α subunit selectively in LepR cells had no effect on LPS-induced hypophagia and weight loss. However, p110α and p110β double deletion in LepR cells prevented LPS-induced hypophagia and partially reversed the weight loss. Leptin deficiency blunted LPS-induced acute pAKT and pSTAT3 phosphorylation and the acute suppression of food intake. Conclusions: Our studies show that the PI3K p110β subunit in LepR cells is required for acute endotoxemic hypophagia. The data provide promising approaches for PI3K inhibition in preventing low energy balance and cachectic states during inflammatory challenges. Keywords: LPS, Metabolism, Leptin, Hypothalamus, Inflammatio

Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification

Summary: Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol.For complete details on the use and execution of this protocol, please refer to Han et al. (2020)

Association analysis between follicle status and the presence of resting follicle neighbors in human ovaries.

<p>Association analysis between follicle status and the presence of resting follicle neighbors in human ovaries.</p

Crowding and Follicular Fate: Spatial Determinants of Follicular Reserve and Activation of Follicular Growth in the Mammalian Ovary

<div><p>Initiation of growth of resting ovarian follicles is a key phenomenon for providing an adequate number of mature oocytes in each ovulation, while preventing premature exhaustion of primordial follicle reserve during the reproductive lifespan. Resting follicle dynamics strongly suggest that primordial follicles are under constant inhibitory influences, by mechanisms and factors whose nature remains ill defined. In this work, we aimed to assess the influence of spatial determinants, with special attention to clustering patterns and crowding, on the fate of early follicles in the adult mouse and human ovary. To this end, detailed histological and morphometric analyses, targeting resting and early growing follicles, were conducted in ovaries from mice, either wild type (WT) or genetically modified to lack kisspeptin receptor expression (Kiss1r KO), and healthy adult women. Kiss1r KO mice were studied as model of persistent hypogonadotropism and anovulation. Different qualitative and quantitative indices of the patterns of spatial distribution of resting and early growing follicles in the mouse and human ovary, including the Morisita’s index of clustering, were obtained. Our results show that resting primordial follicles display a clear-cut clustered pattern of spatial distribution in adult mouse and human ovaries, and that resting follicle aggrupation is inversely correlated with the proportion of follicles initiating growth and entering into the growing pool. As a whole, our data suggest that resting follicle <i>crowding</i>, defined by changes in density and clustered pattern of distribution, is a major determinant of follicular activation and the fate of ovarian reserve. Uneven follicle crowding would constitute the structural counterpart of the major humoral regulators of early follicular growth, with potential implications in ovarian ageing and pathophysiology.</p></div

In Panel A, the proportion of small (early) growing follicles is plotted against the number of remaining resting follicles (RF) per ovary.

<p>In addition, mean changes in the proportion of resting follicles with at least one close neighbor with respect to age are shown in Panel B, while the proportion of resting follicles (RF) with at least one neighbor are plotted against the number of remaining resting follicles (RF) in Panel C. In addition, in Panel D the correlation between the proportion of early growing follicles and the percentage of follicles with close neighbors is displayed. In all figures, data from WT and Kiss1r KO mice are shown, as blue and red dots, respectively. Yet, regression analyses were applied only to WT data. Similarly, the confidence intervals (CI, presented as blue dotted lines) and the predictive intervals (PI, presented as grey dotted lines) were calculated for the regression slopes obtained from WT individuals. In Panel E, a schematic figure is presented to illustrate of the method used to evaluate the presence of resting follicle neighbors (at a distance of ≤ 60 μm) for resting and early growing follicles. An early growing follicle (GF) lacking close neighbors is highlighted in green, whereas each of the four resting follicles (RF), which have each three neighbors, is marked in red. In Panel F, a distribution plot of frequencies of the number of neighbors in resting and early growing follicles is presented for 3 moth-old WT mice. Data represent the mean ± SEM for n = 5/group. Finally, in Panel G, the Morisita’s index of clustering in WT and Kiss1r KO mice is shown. Values above the dotted line indicate clustered spatial pattern. Different superscript letters indicate statistically significant differences between age-points within each genotype (P<0.05 ANOVA followed by Student-Newman-Keuls test); *, P<0.05 vs. corresponding WT values at the same age-point (Student t-test). Regression analyses, including r² and P values, as well as CI and PI calculations, were conducted using Graph-Pad Prism software. DF: Degrees of freedom.</p