The anterior and intermediate lobes of the pituitary gland derive from the surface ectoderm. They provide a simple system to assess mechanisms of developmental identity established by tissue ...determinants. Each lobe contains a lineage expressing the hormone precursor pro-opiomelanocortin (POMC): the corticotropes and melanotropes. The T-box transcription factor Tpit controls terminal differentiation of both lineages. We now report on the unique role of Pax7 as a selector of intermediate lobe and melanotrope identity. Inactivation of the Pax7 gene results in loss of melanotrope gene expression and derepression of corticotrope genes. Pax7 acts by remodeling chromatin and allowing Tpit binding to a new subset of enhancers for activation of melanotrope-specific genes. Thus, the selector function of Pax7 is exerted through pioneer transcription factor activity. Genome-wide, the Pax7 pioneer activity is preferentially associated with composite binding sites that include paired and homeodomain motifs. Pax7 expression is conserved in human and dog melanotropes and defines two subtypes of pituitary adenomas causing Cushing's disease. In summary, expression of Pax7 provides a unique tissue identity to the pituitary intermediate lobe that alters Tpit-driven differentiation through pioneer and classical transcription factor activities.
The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the ...corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.
Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates ...long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.
Acyl‐CoA‐binding protein (ACBP) or diazepam‐binding inhibitor is a secreted peptide acting centrally as a GABAA allosteric modulator. Using brain slices, cortical, and hypothalamic astrocyte cultures from ACBP KO mice, we demonstrate that ACBP mainly localizes in astrocytes and regulates unsaturated but not saturated long‐chain fatty acids (LCFA) metabolism. In addition, ACBP deficiency alters FA metabolism‐related genes and results in intracellular FA accumulation while affecting their release. Our results support a novel role for ACBP in brain lipid metabolism. FA, fatty acids; KO, knockout; PL, phospholipids; TAG, triacylglycerol.
Acyl‐CoA‐binding protein (ACBP) or diazepam‐binding inhibitor is a secreted peptide acting centrally as a GABAA allosteric modulator. Using brain slices, cortical, and hypothalamic astrocyte cultures from ACBP KO mice, we demonstrate that ACBP mainly localizes in astrocytes and regulates unsaturated but not saturated long‐chain fatty acids (LCFA) metabolism. In addition, ACBP deficiency alters FA metabolism‐related genes and results in intracellular FA accumulation while affecting their release. Our results support a novel role for ACBP in brain lipid metabolism. FA, fatty acids; KO, knockout; PL, phospholipids; TAG, triacylglycerol.
Cell-specific expression of the pituitary proopiomelanocortin (POMC) gene depends on the combinatorial action of a large number of DNA-binding transcription factors (TFs). These include general and ...cell-restricted factors, as well as factors that act as effectors of signaling pathways. We have previously defined in the distal POMC promoter a composite regulatory element that contains targets for basic helix-loop-helix TFs conferring cell specificity and for NGFI-B orphan nuclear receptors that are responsive to CRH signaling and to glucocorticoid negative feedback. These factors act on neighboring regulatory elements, the Ebox(Neuro) and NurRE, respectively. Currently, the Ebox(Neuro) is thought to be the target of NeuroD1 during fetal development, but this factor may not account for activity in the adult pituitary; it is also unknown whether the NurRE and NGFI-B-related factors are active before establishment of the hypothalamic-pituitary portal system. In order to assess the importance of these regulatory elements and their cognate TFs throughout pituitary organogenesis and in the adult, we have assessed the activity of mutant POMC promoters in transgenic mice throughout development. These experiments indicate that the Ebox(Neuro) and cognate basic helix-loop-helix factors are required throughout development and in the adult gland, beyond expression of NeuroD1. Similarly, the data reveal sustained importance of the NurRE and its cognate factors throughout pituitary development. These data contrast the sustained dependence throughout development on the same regulatory elements with the highly dynamic patterns of TF expression and the modulation of their activity in response to signaling pathways.
Glial cells have emerged as key players in the central control of energy balance and etiology of obesity. Astrocytes play a central role in neural communication via the release of gliotransmitters. ...Acyl-CoA binding protein (ACBP)-derived endozepines are secreted peptides that modulate the GABAA receptor. In the hypothalamus, ACBP is enriched in arcuate nucleus (ARC) astrocytes, ependymocytes and tanycytes. Central administration of the endozepine octadecaneuropeptide (ODN) reduces feeding and improves glucose tolerance, yet the contribution of endogenous ACBP in energy homeostasis is unknown. We demonstrated that ACBP deletion in GFAP+ astrocytes, but not in Nkx2.1-lineage neural cells, promoted diet-induced hyperphagia and obesity in both male and female mice, an effect prevented by viral rescue of ACBP in ARC astrocytes. ACBP-astrocytes were observed in apposition with proopiomelanocortin (POMC) neurons and ODN selectively activated POMC neurons through the ODN-GPCR but not GABAA, and supressed feeding while increasing carbohydrate utilization via the melanocortin system. Similarly, ACBP overexpression in ARC astrocytes reduced feeding and weight gain. Finally, the ODN-GPCR agonist decreased feeding and promoted weight loss in ob/ob mice. These findings uncover ACBP as an ARC gliopeptide playing a key role in energy balance control and exerting strong anorectic effects via the central melanocortin system.
There is growing concern over confounding artifacts associated with β-cell-specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible ...β-cell-specific transgenic (MIP-CreERT(1Lphi)) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT(1Lphi) mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.
•ACBP deficiency in GFAP astrocytes does not affect anxiety-like behaviour.•Anxiety is not affected in whole body ACBP knock-out mice.•Whole body ACBP knock-out mice have impaired anxiolytic ...responses to diazepam.
Diazepam is well known for its anxiolytic properties, which are mediated via activation of the GABAA receptor. Diazepam Binding Inhibitor (DBI), also called acyl-CoA binding protein (ACBP), is a ubiquitously expressed protein originally identified based on its ability to displace diazepam from its binding site on the GABAA receptor. Central administration of ACBP or its cleaved fragment, commonly referred to as endozepines, induces proconflict and anxiety-like behaviour in rodents. For this reason, ACBP is known as an anxiogenic peptide. However, the role of endogenous ACBP in anxiety-like behaviour and anxiolytic responses to diazepam has not been investigated. To address this question, we assessed anxiety behaviour and anxiolytic responses to diazepam in two complementary loss-of-function mouse models including astrocyte-specific ACBP KO (ACBPGFAP KO) and whole-body KO (ACBP KO) mice. Male and female ACBPGFAP KO and ACBP KO mice do not show significant changes in anxiety-like behaviour compared to control littermates during elevated plus maze (EPM) and open field (OF) tests. Surprisingly, ACBPGFAP KO and ACBP KO mice were unresponsive to the anxiolytic effect of a low dose of diazepam during EPM tests. In conclusion, our experiments using genetic ACBP loss-of-function models suggest that endozepines deficiency does not affect anxiety-like behaviour in mice and impairs the anxiolytic action of diazepam.
Pro-opiomelanocortin (POMC) is expressed in two lineages of the pituitary, the anterior lobe corticotrophs and the intermediate lobe melanotrophs. POMC expression in these two lineages is highly ...dependent on the cell-restricted transcription factor Tpit. As Tpit intervenes relatively late in differentiation of those lineages, we have been searching for other transcription factors that may participate in their gene expression program. On the basis of similarity with the Tpit expression profile, we identified Ets variant gene 1 (Etv1/Er81) as a putative POMC transcription factor. Using Etv1-lacZ knockin mice, we describe preferential Etv1 expression in pituitary POMC cells and also in posterior lobe pituicytes. We further show that Etv1 enhances POMC transcription on its own and in synergy with Tpit. The Ets-binding site located within the Tpit/Pitx regulatory element is necessary for Etv1 activity in POMC-expressing AtT-20 cells but dispensable for synergy with Tpit. Etv1 and Tpit interact together in coimmunoprecipitation experiments. Furthermore, Etv1 is present at the POMC promoter, and siRNA-mediated knockdown of Etv1 in AtT-20 cells produces a significant decrease in POMC expression. Etv1 knockout pituitaries show normal POMC cell distribution and normal POMC mRNA abundance, suggesting compensation by other factors. The coordinate expression of Etv1 with POMC cell differentiation and its interaction with the highly cell-restricted Tpit factor indicate that Etv1 participates in a combinatorial code for pituitary cell-specific gene expression.
There is growing concern over confounding artifacts associated with β-cell-specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible ...β-cell-specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in a-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.
As the key organ in the endocrine system, the pituitary has long been the subject of intense scientific questioning. From the characterization of pituitary hormones to the understanding of the ...mechanisms controlling their release, the study of the pituitary yielded great discoveries, some being awarded a Nobel prize. More recently, the transcriptional control of genes encoding pituitary hormones was the object of much attention. In that context, our laboratory described the function of Tpit and NeuroD1 in the control of POMC-expressing cell differentiation and POMC cell-specific transcription and regulatory mechanisms. These two important genes do not suffice however to explain every aspect of POMC cell differentiation. We thus undertook the systematic screening of the developing and adult pituitary transcriptome in a search for novel transcriptional regulators. Using state of the art bioinformatic tools, we observed the concerted variations of biologically relevant gene groups as the organ develops and matures. Using a candidate gene approach, we identified new transcription factors expressed in POMC lineages. We describe for the first time the expression of Etv1, an Ets-domain containing factor, in the pituitary. We showed that Etv1 expression is specific to POMC cells and that it is important for activation of Pomc transcription in collaboration with Tpit and Pitx1. Furthermore, we demonstrated the melanotroph-specific expression of Pax7 in the pituitary, establishing a clear distinction between the two Pomc-expressing lineages at the transcriptional level. Using Pax7 knock-out mice, we determined the impact of this transcription factor on the genetic program of Pomc cells. Pax7 plays a major role in activating melanotroph genes and repressing corticotroph genes. In summary, our bio-informatic analysis yielded extremely relevant data with regard to pituitary development, particularly through the elucidation of key critical roles for Etv1 and Pax7 in Pomc cell biology.
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