RESUMO
Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.
Assuntos
Artemisininas/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Receptores de GABA-A/metabolismo , Transdução de Sinais , Animais , Artemeter , Artemisininas/administração & dosagem , Proteínas de Transporte/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Ratos , Análise de Célula Única , Fatores de Transcrição/metabolismo , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismoRESUMO
The pancreatic hormone amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making amylin receptor agonists attractive for the treatment of metabolic diseases. Amylin receptors consist of heterodimerized complexes of the calcitonin receptor and receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with amylin signalling. In RAMP1/3 knockout amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of amylin response in the mouse brain and establishes the significance of RAMP1/3 receptors in relaying this response.
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Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing ß-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into ß-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into ß-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated ß-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional ß-cell mass and thereby reverse diabetes following toxin-induced ß-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.
Assuntos
Diferenciação Celular , Diabetes Mellitus Tipo 1/genética , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/genética , Animais , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glucagon/genética , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/biossíntese , Humanos , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos Transgênicos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossínteseRESUMO
OBJECTIVES: There is significant interest in uncovering the mechanisms through which exercise enhances cognition, memory, and mood, and lowers the risk of neurodegenerative diseases. In this study, we utilize forced treadmill running and distance-matched voluntary wheel running, coupled with light sheet 3D brain imaging and c-Fos immunohistochemistry, to generate a comprehensive atlas of exercise-induced brain activation in mice. METHODS: To investigate the effects of exercise on brain activity, we compared whole-brain activation profiles of mice subjected to treadmill running with mice subjected to distance-matched wheel running. Male mice were assigned to one of four groups: a) an acute bout of voluntary wheel running, b) confinement to a cage with a locked running wheel, c) forced treadmill running, or d) placement on an inactive treadmill. Immediately following each exercise or control intervention, blood samples were collected for plasma analysis, and brains were collected for whole-brain c-Fos quantification. RESULTS: Our dataset reveals 255 brain regions activated by acute exercise in mice, the majority of which have not previously been linked to exercise. We find a broad response of 140 regulated brain regions that are shared between voluntary wheel running and treadmill running, while 32 brain regions are uniquely regulated by wheel running and 83 brain regions uniquely regulated by treadmill running. In contrast to voluntary wheel running, forced treadmill running triggers activity in brain regions associated with stress, fear, and pain. CONCLUSIONS: Our findings demonstrate a significant overlap in neuronal activation signatures between voluntary wheel running and distance-matched forced treadmill running. However, our analysis also reveals notable differences and subtle nuances between these two widely used paradigms. The comprehensive dataset is accessible online at www.neuropedia.dk, with the aim of enabling future research directed towards unraveling the neurobiological response to exercise.
Assuntos
Atividade Motora , Condicionamento Físico Animal , Camundongos , Masculino , Animais , Atividade Motora/fisiologia , Encéfalo , CogniçãoRESUMO
OBJECTIVE: Metabolic Syndrome, which can be induced or exacerbated by current antipsychotic drugs (APDs), is highly prevalent in schizophrenia patients. Recent preclinical and clinical evidence suggest that agonists at trace amine-associated receptor 1 (TAAR1) have potential as a new treatment option for schizophrenia. Intriguingly, preclinical tudies have also identified TAAR1 as a novel regulator of metabolic control. Here we evaluated the effects of three TAAR1 agonists, including the clinical development candidate ulotaront, on body weight, metabolic parameters and modulation of neurocircuits implicated in homeostatic and hedonic feeding. METHODS: Effects of TAAR1 agonists (ulotaront, RO5166017 and/or RO5263397) on body weight, food intake and/or metabolic parameters were investigated in rats fed a high-fat diet (HFD) and in a mouse model of diet-induced obesity (DIO). Body weight effects were also determined in a rat and mouse model of olanzapine-, and corticosterone-induced body weight gain, respectively. Glucose tolerance was assessed in lean and diabetic db/db mice and fasting plasma glucose and insulin examined in DIO mice. Effects on gastric emptying were evaluated in lean mice and rats. Drug-induced neurocircuit modulation was evaluated in mice using whole-brain imaging of c-fos protein expression. RESULTS: TAAR1 agonists improved oral glucose tolerance by inhibiting gastric emptying. Sub-chronic administration of ulotaront in rats fed a HFD produced a dose-dependent reduction in body weight, food intake and liver triglycerides compared to vehicle controls. In addition, a more rapid reversal of olanzapine-induced weight gain and food intake was observed in HFD rats switched to ulotaront or RO5263397 treatment compared to those switched to vehicle. Chronic ulotaront administration also reduced body weight and improved glycemic control in DIO mice, and normalized corticosterone-induced body weight gain in mice. TAAR1 activation increased neuronal activity in discrete homeostatic and hedonic feeding centers located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures. CONCLUSION: The current data demonstrate that TAAR1 agonists, as a class, not only lack APD-induced metabolic liabilities but can reduce body weight and improve glycemic control in rodent models. The underlying mechanisms likely include TAAR1-mediated peripheral effects on glucose homeostasis and gastric emptying as well as central regulation of energy balance and food intake.
Assuntos
Corticosterona , Controle Glicêmico , Receptores Acoplados a Proteínas G , Humanos , Ratos , Camundongos , Animais , Olanzapina , Peso Corporal , Aumento de Peso , Modelos Animais de Doenças , GlucoseRESUMO
High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused alpha-cells to adopt several morphological and gene expression features of a beta-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a beta-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.
Assuntos
Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Insulina/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica/efeitos dos fármacos , Células Secretoras de Glucagon/citologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Quinolonas/química , Interferência de RNA , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Técnicas de Cultura de TecidosRESUMO
Magnetic resonance imaging (MRI) and light-sheet fluorescence microscopy (LSFM) are technologies that enable non-disruptive 3-dimensional imaging of whole mouse brains. A combination of complementary information from both modalities is desirable for studying neuroscience in general, disease progression and drug efficacy. Although both technologies rely on atlas mapping for quantitative analyses, the translation of LSFM recorded data to MRI templates has been complicated by the morphological changes inflicted by tissue clearing and the enormous size of the raw data sets. Consequently, there is an unmet need for tools that will facilitate fast and accurate translation of LSFM recorded brains to in vivo, non-distorted templates. In this study, we have developed a bidirectional multimodal atlas framework that includes brain templates based on both imaging modalities, region delineations from the Allen's Common Coordinate Framework, and a skull-derived stereotaxic coordinate system. The framework also provides algorithms for bidirectional transformation of results obtained using either MR or LSFM (iDISCO cleared) mouse brain imaging while the coordinate system enables users to easily assign in vivo coordinates across the different brain templates.
Assuntos
Encéfalo , Imageamento por Ressonância Magnética , Animais , Camundongos , Encéfalo/diagnóstico por imagem , Encéfalo/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Imageamento Tridimensional/métodos , Mapeamento Encefálico/métodos , Crânio/diagnóstico por imagemRESUMO
Peptide-based drug development for CNS disorders is challenged by poor blood-brain barrier (BBB) penetrability of peptides. While acylation protractions (lipidation) have been successfully applied to increase circulating half-life of therapeutic peptides, little is known about the CNS accessibility of lipidated peptide drugs. Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method to visualize whole-brain 3D distribution of fluorescently labelled therapeutic peptides at single-cell resolution. Here, we applied LSFM to map CNS distribution of the clinically relevant GLP-1 receptor agonist (GLP-1RA) exendin-4 (Ex4) and lipidated analogues following peripheral administration. Mice received an intravenous dose (100 nmol/kg) of IR800 fluorophore-labelled Ex4 (Ex4), Ex4 acylated with a C16-monoacid (Ex4_C16MA) or C18-diacid (Ex4_C18DA). Other mice were administered C16MA-acylated exendin 9-39 (Ex9-39_C16MA), a selective GLP-1R antagonist, serving as negative control for GLP-1R mediated agonist internalization. Two hours post-dosing, brain distribution of Ex4 and analogues was predominantly restricted to the circumventricular organs, notably area postrema and nucleus of the solitary tract. However, Ex4_C16MA and Ex9-39_C16MA also distributed to the paraventricular hypothalamic nucleus and medial habenula. Notably, Ex4_C18DA was detected in deeper-lying brain structures such as dorsomedial/ventromedial hypothalamic nuclei and the dentate gyrus. Similar CNS distribution maps of Ex4_C16MA and Ex9-39_C16MA suggest that brain access of lipidated Ex4 analogues is independent on GLP-1 receptor internalization. The cerebrovasculature was devoid of specific labelling, hence not supporting a direct role of GLP-1 RAs in BBB function. In conclusion, peptide lipidation increases CNS accessibility of Ex4. Our fully automated LSFM pipeline is suitable for mapping whole-brain distribution of fluorescently labelled drugs.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Peçonhas , Camundongos , Animais , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Peçonhas/farmacologia , Peçonhas/química , Peptídeos/química , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
Glucagon-like peptide-1 receptor (GLP-1R) agonists promote nicotine avoidance. Here, we show that the crosstalk between GLP-1 and nicotine extends beyond effects on nicotine self-administration and can be exploited pharmacologically to amplify the anti-obesity effects of both signals. Accordingly, combined treatment with nicotine and the GLP-1R agonist, liraglutide, inhibits food intake and increases energy expenditure to lower body weight in obese mice. Co-treatment with nicotine and liraglutide gives rise to neuronal activity in multiple brain regions, and we demonstrate that GLP-1R agonism increases excitability of hypothalamic proopiomelanocortin (POMC) neurons and dopaminergic neurons in the ventral tegmental area (VTA). Further, using a genetically encoded dopamine sensor, we reveal that liraglutide suppresses nicotine-induced dopamine release in the nucleus accumbens in freely behaving mice. These data support the pursuit of GLP-1R-based therapies for nicotine dependence and encourage further evaluation of combined treatment with GLP-1R agonists and nicotinic receptor agonists for weight loss.
Assuntos
Peptídeo 1 Semelhante ao Glucagon , Liraglutida , Camundongos , Animais , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Liraglutida/farmacologia , Nicotina/farmacologia , Dopamina , Obesidade/tratamento farmacológico , Obesidade/metabolismoRESUMO
The mammalian brain is by far the most advanced organ to have evolved and the underlying biology is extremely complex. However, with aging populations and sedentary lifestyles, the prevalence of neurological disorders is increasing around the world. Consequently, there is a dire need for technologies that can help researchers to better understand the complexity of the brain and thereby accelerate therapies for diseases with origin in the central nervous system. One such technology is light-sheet fluorescence microscopy (LSFM) which in combination with whole organ immunolabelling has made it possible to visualize an intact mouse brain with single cell resolution. However, the price for this level of detail comes in form of enormous datasets that often challenges extraction of quantitative information. One approach for analyzing whole brain data is to align the scanned brains to a reference brain atlas. Having a fixed spatial reference provides each voxel of the sample brains with x-, y-, z-coordinates from which it is possible to obtain anatomical information on the observed fluorescence signal. An additional and important benefit of aligning light sheet data to a reference brain is that the aligned data provides a digital map of gene expression or cell counts which can be deposited in databases or shared with other scientists. This review focuses on the emerging field of virtual neuroscience using digital brain maps and discusses some of challenges incurred when registering LSFM recorded data to a standardized brain template.
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Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treatment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of insulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanistically, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selectively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related signals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.
Assuntos
Glicemia , Fator A de Crescimento do Endotélio Vascular , Humanos , Glicemia/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Insulina/metabolismo , Homeostase , Encéfalo/metabolismoRESUMO
OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Obesidade/metabolismo , Peptídeo YY/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Energia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Derivação Gástrica , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hipotálamo , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/fisiopatologia , Peptídeo YY/fisiologia , Redução de PesoRESUMO
Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.
Assuntos
Camundongos/embriologia , Pâncreas/embriologia , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Pâncreas Exócrino/embriologia , Hormônios Peptídicos/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neurog3 is expressed transiently in pancreatic endocrine progenitors where it is responsible for activating a transcription factor cascade which eventually defines the mature endocrine cells. However, the mechanism by which Neurog3 regulates different aspects of the endocrine differentiation program is less clear. In this report we used in ovo electroporation to investigate how manipulation of Neurog3 protein activity affected migration, differentiation and fate determination. We found that changes in the onset of Neurog3 expression only had minor effect on differentiation. However increasing the transcriptional activity of Neurog3 by fusing it to VP16 or co-electroporating with Ep300 caused the electroporated cells to migrate rather than differentiate. In contrast, reducing the transcriptional activity of Neurog3 by deleting parts of the activation domain, by fusing Neurog3 to the engrailed repressor domain, or co-electroporating with Hdac1 greatly increased the proportion of glucagon expressing cells.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Endócrinas/citologia , Endoderma/citologia , Proteínas do Tecido Nervoso/genética , Animais , Diferenciação Celular/genética , Movimento Celular/genética , Galinhas , Eletroporação , Células Endócrinas/metabolismo , Endoderma/metabolismo , Hibridização In Situ , Microscopia Confocal , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
OBJECTIVE: The development of effective anti-obesity therapeutics relies heavily on the ability to target specific brain homeostatic and hedonic mechanisms controlling body weight. To obtain further insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes. METHODS: Chow-fed C57BL/6J mice (n = 8 per group) received acute treatment with lorcaserin (7 mg/kg; i.p.), rimonabant (10 mg/kg; i.p.), bromocriptine (10 mg/kg; i.p.), sibutramine (10 mg/kg; p.o.), semaglutide (0.04 mg/kg; s.c.) or setmelanotide (4 mg/kg; s.c.). Brains were sampled two hours post-dosing and whole-brain neuronal activation patterns were analysed at single-cell resolution using c-Fos immunohistochemistry and automated quantitative three-dimensional (3D) imaging. RESULTS: The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, a web-based 3D imaging data viewer was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system. CONCLUSIONS: Whole-brain c-Fos expression signatures of various weight-lowering drug classes point to a discrete set of brain regions and neurocircuits which could represent key neuroanatomical targets for future anti-obesity therapeutics.
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Fármacos Antiobesidade/farmacologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Peso Corporal , Ciclobutanos , Homeostase , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Angiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Ecocardiografia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Humanos , Masculino , Camundongos , Microscopia , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologiaRESUMO
In recent years, the combination of whole-brain immunolabelling, light sheet fluorescence microscopy (LSFM) and subsequent registration of data with a common reference atlas, has enabled 3D visualization and quantification of fluorescent markers or tracers in the adult mouse brain. Today, the common coordinate framework version 3 developed by the Allen's Institute of Brain Science (AIBS CCFv3), is widely used as the standard brain atlas for registration of LSFM data. However, the AIBS CCFv3 is based on histological processing and imaging modalities different from those used for LSFM imaging and consequently, the data differ in both tissue contrast and morphology. To improve the accuracy and speed by which LSFM-imaged whole-brain data can be registered and quantified, we have created an optimized digital mouse brain atlas based on immunolabelled and solvent-cleared brains. Compared to the AIBS CCFv3 atlas, our atlas resulted in faster and more accurate mapping of neuronal activity as measured by c-Fos expression, especially in the hindbrain. We further demonstrated utility of the LSFM atlas by comparing whole-brain quantitative changes in c-Fos expression following acute administration of semaglutide in lean and diet-induced obese mice. In combination with an improved algorithm for c-Fos detection, the LSFM atlas enables unbiased and computationally efficient characterization of drug effects on whole-brain neuronal activity patterns. In conclusion, we established an optimized reference atlas for more precise mapping of fluorescent markers, including c-Fos, in mouse brains processed for LSFM.
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Encéfalo , Neurônios , Algoritmos , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional , Camundongos , Microscopia de FluorescênciaRESUMO
OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.
Assuntos
Proteína Forkhead Box O1/metabolismo , Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adulto , Animais , Desdiferenciação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.
Assuntos
Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/embriologia , Polipeptídeo Pancreático/metabolismo , Fatores de Transcrição/genética , Animais , Regulação da Expressão Gênica , Glucagon/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Camundongos , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Diabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14â days with either vehicle or the insulin receptor antagonist S961 (2.4â nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14â weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.