Secretome of Adipose Tissue as the Key to Understanding the Endocrine Function of Adipose Tissue.

The prevalence of obesity has reached pandemic levels and is becoming a serious health problem in developed and developing countries. Obesity is associated with an increased prevalence of comorbidities that include type II diabetes, cardiovascular diseases and some cancers. The recognition of adipose tissue as an endocrine organ capable of secreting adipokines that influence whole-body energy homeostasis was a breakthrough leading to a better molecular understanding of obesity. Of the adipokines known to be involved in the regulation of energy metabolism, very few are considered central regulators of insulin sensitivity, metabolism and energy homeostasis, and the discovery and characterization of new adipocyte-derived factors are still ongoing. Proteomics techniques, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry, have proven to be useful tools for analyzing the secretory function of adipose tissue (the secretome), providing insights into molecular events that influence body weight. Apart from the identification of novel proteins, the considerable advantage of this approach is the ability to detect post-translational modifications that cannot be predicted in genomic studies. In this review, we summarize recent efforts to identify novel bioactive secretory factors through proteomics.

[The pituitary gland unveiled: from the stimulation-secretion coupling to cellular networks wiring]. / L'hypophyse dévoilée : du couplage stimulation-sécrétion aux réseaux cellulaires câblant la glande.

The year 2021 ended with an event of great sadness: the death of Andrée Tixier-Vidal. She was not only a pioneer in cell biology but also the charismatic promoter of stimulating and successful multidisciplinary collaborations. Her achievements led to subsequent major discoveries on both the stimulation-secretion coupling of pituitary endocrine cells and the hitherto unknown organization of these cells into multicellular 3D networks which build-up highly organized pulses of pituitary hormones controlling basic body functions such as growth and reproduction.

Title: L'hypophyse dévoilée : du couplage stimulation-sécrétion aux réseaux cellulaires câblant la glande. Abstract: L'année 2021 s'est terminée par un événement de grande tristesse : le décès d'Andrée Tixier-Vidal. Elle fut non seulement une pionnière en biologie cellulaire mais également la promotrice charismatique de fédérations collaboratives multidisciplinaires particulièrement stimulantes et fructueuses. Cette note en retrace les succès en termes de découvertes à la fois sur le couplage stimulation-sécrétion des cellules endocrines de l'hypophyse et sur l'organisation de ces cellules hypophysaires en réseaux 3D multicellulaires à l'origine des sécrétions pulsées des hormones hypophysaires qui contrôlent des fonctions de base de l'organisme comme la croissance corporelle et la reproduction.

Architecture of the Pancreatic Islets and Endocrine Cell Arrangement in the Embryonic Pancreas of the Grass Snake (Natrix natrix L.). Immunocytochemical Studies and 3D Reconstructions.

During the early developmental stages of grass snakes, within the differentiating pancreas, cords of endocrine cells are formed. They differentiate into agglomerates of large islets flanked throughout subsequent developmental stages by small groups of endocrine cells forming islets. The islets are located within the cephalic part of the dorsal pancreas. At the end of the embryonic period, the pancreatic islet agglomerates branch off, and as a result of their remodeling, surround the splenic "bulb". The stage of pancreatic endocrine ring formation is the first step in formation of intrasplenic islets characteristics for the adult specimens of the grass snake. The arrangement of endocrine cells within islets changes during pancreas differentiation. Initially, the core of islets formed from B and D cells is surrounded by a cluster of A cells. Subsequently, A, B, and D endocrine cells are mixed throughout the islets. Before grass snake hatching, A and B endocrine cells are intermingled within the islets, but D cells are arranged centrally. Moreover, the pancreatic polypeptide (PP) cells are not found within the embryonic pancreas of the grass snake. Variation in the proportions of different cell types, depending on the part of the pancreas, may affect the islet function-a higher proportion of glucagon cells is beneficial for insulin secretion.

Mouse gastric mucosal endocrine cells are sources and sites of action of Phoenixin-20.

Energy homeostasis is is determined by food intake and energy expenditure, which are partly regulated by the cross-talk between central and peripheral hormonal signals. Phoenixin (PNX) is a recently discovered pleiotropic neuropeptide with isoforms of 14 (PNX-14) and 20 (PNX-20) amino acids. It is a potent reproductive peptide in vertebrates, regulating the hypothalamo-pituitary-gonadal axis (HPG). It has been identified as a regulator of food intake during light phase when injected intracerebroventricularly in rats. In addition, plasma levels of PNX also increased after food intake in rats, suggesting that it might have possible roles in energy homeostasis. We hypothesized that gut is a source and site of action of PNX in mice. Immunoreactivity for PNX and its putative receptor, super-conserved receptor expressed in brain (SREB3; also known as the G-protein coupled receptor 173/GPR 173) was found in the stomach and intestine of male C57/BL6 J mice, and in MGN3-1 (mouse stomach endocrine) cells and STC-1 (mouse enteroendocrine) cells. In MGN3-1 cells, PNX-20 significantly upregulated ghrelin (10 nM) and ghrelin-O-acyl transferase (GOAT) mRNAs (1000 nM) at 6 h. In STC-1 cells, it significantly suppressed CCK (100 nM) at 2 h. No effects were found on other intestinal hormones tested (glucagon like peptide-1, glucose dependent insulinotropic polypeptide, and peptide YY). Together, these results indicate that PNX-20 is produced in the gut, and it could act directly on gut cells to regulate metabolic hormones.

The sodium leak channel NALCN regulates cell excitability of pituitary endocrine cells.

Anterior pituitary endocrine cells that release hormones such as growth hormone and prolactin are excitable and fire action potentials. In these cells, several studies previously showed that extracellular sodium (Na+ ) removal resulted in a negative shift of the resting membrane potential (RMP) and a subsequent inhibition of the spontaneous firing of action potentials, suggesting the contribution of a Na+ background conductance. Here, we show that the Na+ leak channel NALCN conducts a Ca2+ - Gd3+ -sensitive and TTX-resistant Na+ background conductance in the GH3 cell line, a cell model of pituitary endocrine cells. NALCN knockdown hyperpolarized the RMP, altered GH3 cell electrical properties and inhibited prolactin secretion. Conversely, the overexpression of NALCN depolarized the RMP, also reshaping the electrical properties of GH3 cells. Overall, our results indicate that NALCN is functional in GH3 cells and involved in endocrine cell excitability as well as in hormone secretion. Indeed, the GH3 cell line suitably models native pituitary cells that display a similar Na+ background conductance and appears as a proper cellular model to study the role of NALCN in cellular excitability.

Enrichment analyses of diseases and pathways associated with precocious puberty using PrecocityDB.

Precocious puberty (PP) is an important endocrine disorder affecting children globally. Several genes, SNPs and comorbidities are reported to be associated with PP; however, this data is scattered across scientific literature and has not been systematically collated and analysed. In this study, we present PrecocityDB as the first manually curated online database on genes and their ontology terms, SNPs, and pathways associated with PP. A tool for visualizing SNP coordinates and allelic variation on each chromosome, for genes associated with PP is also incorporated in PrecocityDB. Pathway enrichment analysis of PP-associated genes revealed that endocrine and cancer-related pathways are highly enriched. Disease enrichment analysis indicated that individuals with PP seem to be highly likely to suffer from reproductive and metabolic disorders such as PCOS, hypogonadism, and insulin resistance. PrecocityDB is a useful resource for identification of comorbid conditions and disease risks due to shared genes in PP. PrecocityDB is freely accessible at http://www.precocity.bicnirrh.res.in . The database source code and content can be downloaded through GitHub ( https://github.com/bic-nirrh/precocity ).

Characterisation of a mural cell network in the murine pituitary gland.

The anterior and intermediate lobes of the pituitary are composed of endocrine cells, as well as vasculature and supporting cells, such as folliculostellate cells. Folliculostellate cells form a network with several postulated roles in the pituitary, including production of paracrine signalling molecules and cytokines, coordination of endocrine cell hormone release, phagocytosis, and structural support. Folliculostellate cells in rats are characterised by expression of S100B protein, and in humans by glial fibrillary acid protein. However, there is evidence for another network of supporting cells in the anterior pituitary that has properties of mural cells, such as vascular smooth muscle cells and pericytes. The present study aims to characterise the distribution of cells that express the mural cell marker platelet derived growth factor receptor beta (PDGFRß) in the mouse pituitary and establish whether these cells are folliculostellate. By immunohistochemical localisation, we determine that approximately 80% of PDGFRß+ cells in the mouse pituitary have a non-perivascular location and 20% are pericytes. Investigation of gene expression in a magnetic cell sorted population of PDGFRß+ cells shows that, despite a mostly non-perivascular location, this population is enriched for mural cell markers but not enriched for rat or human folliculostellate cell markers. This is confirmed by immunohistochemistry. The present study concludes that a mural cell network is present throughout the anterior pituitary of the mouse and that this population does not express well-characterised human or rat folliculostellate cell markers.

The role of non-neuronal cells in hypogonadotropic hypogonadism.

The hypothalamic-pituitary-gonadal axis is controlled by gonadotropin-releasing hormone (GnRH) released by the hypothalamus. Disruption of this system leads to impaired reproductive maturation and function, a condition known as hypogonadotropic hypogonadism (HH). Most studies to date have focused on genetic causes of HH that impact neuronal development and function. However, variants may also impact the functioning of non-neuronal cells known as glia. Glial cells make up 50% of brain cells of humans, primates, and rodents. They include radial glial cells, microglia, astrocytes, tanycytes, oligodendrocytes, and oligodendrocyte precursor cells. Many of these cells influence the hypothalamic neuroendocrine system controlling fertility. Indeed, glia regulate GnRH neuronal activity and secretion, acting both at their cell bodies and their nerve endings. Recent work has also made clear that these interactions are an essential aspect of how the HPG axis integrates endocrine, metabolic, and environmental signals to control fertility. Recognition of the clinical importance of interactions between glia and the GnRH network may pave the way for the development of new treatment strategies for dysfunctions of puberty and adult fertility.

Mathematical modeling approaches of cellular endocrinology within the hypothalamo-pituitary-gonadal axis.

The reproductive neuroendocrine axis, or hypothalamo-pituitary-gonadal (HPG) axis, is a paragon of complex biological system involving numerous cell types, spread over several anatomical levels communicating through entangled endocrine feedback loops. The HPG axis exhibits remarkable dynamic behaviors on multiple time and space scales, which are an inexhaustible source of studies for mathematical and computational biology. In this review, we will describe a variety of modeling approaches of the HPG axis from a cellular endocrinology viewpoint. We will in particular investigate the questions raised by some of the most striking features of the HPG axis: (i) the pulsatile secretion of hypothalamic and pituitary hormones, and its counterpart, the cell signaling induced by frequency-encoded hormonal signals, and (ii) the dual, gametogenic and glandular function of the gonads, which relies on the tight control of the somatic cell populations ensuring the proper maturation and timely release of the germ cells.

Ion channel noise shapes the electrical activity of endocrine cells.

Endocrine cells in the pituitary gland typically display either spiking or bursting electrical activity, which is related to the level of hormone secretion. Recent work, which combines mathematical modelling with dynamic clamp experiments, suggests the difference is due to the presence or absence of a few large-conductance potassium channels. Since endocrine cells only contain a handful of these channels, it is likely that stochastic effects play an important role in the pattern of electrical activity. Here, for the first time, we explicitly determine the effect of such noise by studying a mathematical model that includes the realistic noisy opening and closing of ion channels. This allows us to investigate how noise affects the electrical activity, examine the origin of spiking and bursting, and determine which channel types are responsible for the greatest noise. Further, for the first time, we address the role of cell size in endocrine cell electrical activity, finding that larger cells typically display more bursting, while the smallest cells almost always only exhibit spiking behaviour.

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

The osteocyte: A multifunctional cell within the bone.

The knowledge of bone biology has largely changed in the last few decades. Osteocytes are multifunctional bone cells that are surrounded by mineralized bone matrix and for decades it was considered that they might be relatively inactive cells. However, nowadays it is known that osteocytes are highly active cells which are indispensable for the normal function of the skeleton, playing main roles in several physiological processes, both within and beyond the bone microenvironment. This review highlights and updates the current state of knowledge of the osteocyte and focuses on its roles in bone remodeling and mineral homeostasis, and also reviews its recently discovered endocrine function. Osteocytes secrete sclerostin (a protein that works as a negative regulator of bone mass), and FGF-23, the most important osteocyte-secreted endocrine factor, since it is able to regulate the phosphate metabolism. Moreover, osteocytes can act as mechanosensory cells, transforming the mechanical strain into chemical signaling towards the effector cells (osteoblasts and osteoclasts). Therefore, the osteocyte plays an important role in bone biology, specifically in the remodeling process, since it regulates both the osteoblast and osteoclast activity. Finally, the paper discusses the clinical application of the bone biology, updating the new therapies against bone-loss disorders.

Modeling Spatial Correlation of Transcripts with Application to Developing Pancreas.

Recently high-throughput image-based transcriptomic methods were developed and enabled researchers to spatially resolve gene expression variation at the molecular level for the first time. In this work, we develop a general analysis tool to quantitatively study the spatial correlations of gene expression in fixed tissue sections. As an illustration, we analyze the spatial distribution of single mRNA molecules measured by in situ sequencing on human fetal pancreas at three developmental time points-80, 87 and 117 days post-fertilization. We develop a density profile-based method to capture the spatial relationship between gene expression and other morphological features of the tissue sample such as position of nuclei and endocrine cells of the pancreas. In addition, we build a statistical model to characterize correlations in the spatial distribution of the expression level among different genes. This model enables us to infer the inhibitory and clustering effects throughout different time points. Our analysis framework is applicable to a wide variety of spatially-resolved transcriptomic data to derive biological insights.

Deletion of Cdkn1b in ACI rats leads to increased proliferation and pregnancy-associated changes in the mammary gland due to perturbed systemic endocrine environment.

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived ß cells.

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature ß cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived ß cells that resemble their endogenous counterparts.

Retrospective Analysis of Endocrine Dysfunctions in a Population of Adult Polytransfused Patients: Correlation of GH-IGF1 Axis Alteration with Cardiac Performance.

Endocrine complications of haemochromatosis and heart failure mostly affect morbidity and mortality in polytransfused patients. This study analyzes endocrine dysfunctions and the impact of GH-IGF-1 axis alteration on cardiac performance in a population of 31 patients. A retrospective study on 31 Caucasian polytransfused outpatients, 27 adults and 4 pediatric, residing in Apulia, Italy, followed from 2005 to 2016, was conducted. Patients underwent basal and dynamic hormonal evaluation. GHRH plus arginine test was performed in 21 patients (19 adults and 2 children). Among them, 9 patients were affected by left ventricle diastolic dysfunction and/or atrial or ventricular dilatation (HD group) and 12 patients did not have cardiovascular disease (non-HD group). Twenty-nine out of 31 patients (94%) had at least one endocrinopathy. We found severe or mild GH deficit (GHD) in all HD patients versus 3 patients in the non-HD group (p=0.001). Mean IGF-1 levels were significantly lower in the HD group than in non-HD subjects (53±30 versus 122±91 µg/L, p=0.04). Our study confirms the need to perform a dynamic evaluation of the GH-IGF1 axis in polytransfused patients, especially when heart dysfunction emerges. An intervention study with GH replacement therapy in a larger randomized adult population will clarify the role of GH/IGF axis on cardiovascular outcomes in this patient population.

The brain's hemodynamic response function rapidly changes under acute psychosocial stress in association with genetic and endocrine stress response markers.

Ample evidence links dysregulation of the stress response to the risk for psychiatric disorders. However, we lack an integrated understanding of mechanisms that are adaptive during the acute stress response but potentially pathogenic when dysregulated. One mechanistic link emerging from rodent studies is the interaction between stress effectors and neurovascular coupling, a process that adjusts cerebral blood flow according to local metabolic demands. Here, using task-related fMRI, we show that acute psychosocial stress rapidly impacts the peak latency of the hemodynamic response function (HRF-PL) in temporal, insular, and prefrontal regions in two independent cohorts of healthy humans. These latency effects occurred in the absence of amplitude effects and were moderated by regulatory genetic variants of KCNJ2, a known mediator of the effect of stress on vascular responsivity. Further, hippocampal HRF-PL correlated with both cortisol response and genetic variants that influence the transcriptional response to stress hormones and are associated with risk for major depression. We conclude that acute stress modulates hemodynamic response properties as part of the physiological stress response and suggest that HRF indices could serve as endophenotype of stress-related disorders.

Neurog3 misexpression unravels mouse pancreatic ductal cell plasticity.

In the context of type 1 diabetes research and the development of insulin-producing ß-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene Neurog3 in ductal cells in vivo. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary ß-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new ß-like cells and that strategies aiming at controlling the expression of Neurog3, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.

A new mode of pancreatic islet innervation revealed by live imaging in zebrafish.

Pancreatic islets are innervated by autonomic and sensory nerves that influence their function. Analyzing the innervation process should provide insight into the nerve-endocrine interactions and their roles in development and disease. Here, using in vivo time-lapse imaging and genetic analyses in zebrafish, we determined the events leading to islet innervation. Comparable neural density in the absence of vasculature indicates that it is dispensable for early pancreatic innervation. Neural crest cells are in close contact with endocrine cells early in development. We find these cells give rise to neurons that extend axons toward the islet as they surprisingly migrate away. Specific ablation of these neurons partly prevents other neurons from migrating away from the islet resulting in diminished innervation. Thus, our studies establish the zebrafish as a model to interrogate mechanisms of organ innervation, and reveal a novel mode of innervation whereby neurons establish connections with their targets before migrating away.

Tuft cells: Distribution and connections with nerves and endocrine cells in mouse intestine.

Tuft cells are gastrointestinal (GI) sensory cells recognized by their characteristic shape and their microvilli "tuft". Aims of the present study were to elucidate their regional distribution and spatial connections with satiety associated endocrine cells and nerve fibers throughout the intestinal tract. C57BL/6 J mice were used in the experiments. The small intestine was divided into five segments, and the large intestine was kept undivided. The segments were coiled into "Swiss rolls". Numbers and topographic distribution of tuft cells and possible contacts with endocrine cells and nerve fibers were estimated in the different segments, using immunocytochemistry. Tuft cells were found throughout the intestines; the highest number was in proximal small intestine. Five percent of tuft cells were found in close proximity to cholecystokinin-immunoreactive (IR) endocrine cells and up to 10% were in contact with peptide YY- and glucagon-like peptide-1-IR endocrine cells. Sixty percent of tuft cells in the small intestine and 40% in the large intestine were found in contact with nerve fibers. Calcitonin gene-related peptide-IR fibers constituted one-third of the fiber-contacts in the small intestine and two-thirds in the large intestine. These observations highlight the possibility of tuft cells as modulators of GI activities in response to luminal signaling.