Diabetes is one of the most challenging health concerns facing society. Available drugs treat the symptoms but there is no cure. This presents an urgent need to better understand human diabetes in ...order to develop improved treatments or target remission. New disease models need to be developed that more accurately describe the pathology of diabetes. Organoid technology provides an opportunity to fill this knowledge gap. Organoids are 3D structures, established from pluripotent stem cells or adult stem/progenitor cells, that recapitulate key aspects of the in vivo tissues they mimic. In this review we briefly introduce organoids and their benefits; we focus on organoids generated from tissues important for glucose homeostasis and tissues associated with diabetic complications. We hope this review serves as a touchstone to demonstrate how organoid technology extends the research toolbox and can deliver a step change of discovery in the field of diabetes.
•Organoids can be generated from adult stem cells or iPSCs.•Organoids exhibit all intestinal epithelial cell types including functional EECs.•Organoids can be generated from patient specific ...biopsies.•Organoids are amenable to numerous molecular and genetic techniques.•Current organoid usage in transdifferentiation, cell fate and function of EECs.
The advent of near physiological organoid technology has produced a step change in our understanding of stem cells and has provided the research community with a powerful new cell based tool to model human physiology and disease. We review the pros and cons of intestinal organoid culture systems. The molecular and genetic tools to manipulate them and how they are being used to answer fundamental questions in metabolic research, including the function of enteroendocrine cells in health and disease.
The Glucose Tolerance Test in Mice Pedro, Patricia Fonseca; Tsakmaki, Anastasia; Bewick, Gavin A
Methods in molecular biology (Clifton, N.J.),
2020, Letnik:
2128
Journal Article
Type 2 diabetes is characterized by glucose intolerance, caused by insulin resistance in peripheral metabolic tissues and by impaired glucose-stimulated insulin secretion, the hallmark of beta-cell ...dysfunction. The glucose tolerance test is used in clinic and research to identify individuals with impaired glucose tolerance and overt type 2 diabetes. It is the most routinely used physiological test for first pass assessment of glucose homeostasis in rodents because of its simplicity. The GTT measures changes in blood glucose concentration over a 2-h period following the administration of a bolus of glucose. However, this simplicity belies several important considerations which need to be addressed, to aid reproducibility and produce interpretable data. Here, we describe in detail how to perform a GTT using four different routes of glucose administration: intraperitoneal, oral, voluntary oral, and intravenous.
Aims
Two unmet therapeutic strategies for diabetes treatment are prevention of beta‐cell death and stimulation of beta‐cell replication. Our aim was to characterize the role of neuropeptide Y ...receptors in the control of beta‐cell mass.
Materials and Methods
We used endogenous and selective agonists of the NPY receptor system to explore its role in the prevention of beta‐cell apoptosis and proliferation in islets isolated from both mouse and human donors. We further explored the intra‐cellular signalling cascades involved, using chemical inhibitors of key signalling pathways. As proof of principle we designed a long‐acting analogue of Leu31Pro34‐NPY, an agonist of the islet‐expressed Y receptors, to determine if targeting this system could preserve beta‐cell mass in vivo.
Results
Our data reveal that NPY Y1, 4 and 5 receptor activation engages a generalized and powerful anti‐apoptotic pathway that protects mouse and human islets from damage. These anti‐apoptotic effects were dependent on stimulating a Gαi‐PLC‐PKC signalling cascade, which prevented cytokine‐induced NFkB signalling. NPY receptor activation functionally protected islets by restoring glucose responsiveness following chemically induced injury in both species. NPY receptor activation attenuated beta‐cell apoptosis, preserved functional beta‐cell mass and attenuated the hyperglycaemic phenotype in a low‐dose streptozotocin model of diabetes.
Conclusion
Taken together, our observations identify the islet Y receptors as promising targets for the preservation of beta‐cell mass. As such, targeting these receptors could help to maintain beta‐cell mass in both type 1 and type 2 diabetes, and may also be useful for improving islet transplantation outcomes.
Enteroendocrine cells (EECs) survey the gut luminal environment and coordinate hormonal, immune and neuronal responses to it. They exhibit well-characterised physiological roles ranging from the ...control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in the human gut. The small molecule isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation.
We investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real-time quantitative polymerase chain reaction (RT-qPCR), fluorescent-activated cell sorting, immunostaining and single-cell RNA sequencing.
ISX-9 increased the number of neurogenin3-RFP (Ngn3)-positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors that play roles in mouse EEC specification. Single-cell analysis showed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased toward serotonin-producing enterochromaffin (EC) cells. Further, we observed enrichment of organoids with functional EC cells that was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types.
Our data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to the mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.
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•ISX-9 promotes flux through the Ngn3 lineage and enriches it with enterochromaffin cells.•ISX-9 engages an enterochromaffin biased transcriptional programme in endocrine fated cells.•Enterochromaffin bias is partly dependent on calcium signalling in progenitor cells.•ISX-9 reveals conserved gut endocrine specification between mouse and human.•Pax4 overexpression in human ileum organoids mimics the effects of ISX-9 on EC bias.
Enteroendocrine (EEC) cells are hormone-producing cells that constitute 1% of the intestinal epithelium. Their hormones act locally within the gut epithelium, by signalling to its nervous system, or ...are released into the blood stream where they can influence distant organs such as the pancreas or brain. These signals have well-defined physiological roles including appetite regulation, pancreatic hormone secretion, intestinal motility and gastric emptying. However, knowledge regarding EEC differentiation in the human intestine is scarce. Understanding EEC specification may unveil targets that allow the manipulation of EEC fate offering control over the density of specific EEC populations. Dependent on the EEC population targeted, this strategy could form the basis of novel treatments for metabolic, inflammatory or cognitive disorders. ISX-9 is a small molecule, notable for inducing neuronal and beta-cell differentiation due to its ability to increase NeuroD1 expression. These cell types share similar developmental transcriptional pathways with EEC cells. The potential of ISX-9 to control EEC identity and specification was explored in mouse and human intestinal organoids derived from intestinal adult stem cells. ISX-9 upregulated NeuroD1 and increased the expression of the EEC-associated transcription factors Ngn3 and Pax4, highlighting a potential early mechanism for enterochromaffin biased fate. Analysis of mature EEC transcripts revealed exclusive upregulation of Cck and enterochromaffin cell markers ChgA, Tac1 and Tph1, but not other mature EEC markers. Double immunostaining for CHGA and serotonin revealed that the double-positive population was significantly increased by ISX-9 exposure. The newly generated enterochromaffin cells were biologically relevant and secreted more serotonin than control cells in response to stimulation. Induction of neuronal differentiation by ISX-9 is dependent on intracellular calcium signalling, suggesting this could be a mechanism by which it induces EEC differentiation. KN93, an inhibitor of CaMKII, an important calcium signalling node, partially inhibited the ISX-9-induced expansion of the enterochromaffin cell population, suggesting calcium increases were in part responsible for ISX-9 effects on EEC differentiation. Inhibition of two key epithelial signalling pathways, Notch and MEK, has been shown to stimulate EEC differentiation. Combining inhibition of these pathways with ISX-9 led to considerable increase in enterochromaffin cells, exposing an extremely powerful method for inducing enterochromaffin cells. The scRNA-seq data identified Pax4 as a potential key factor in biasing EEC fate towards enterochromaffin specification. Pax4 overexpression inhibited EEC differentiation and arrested cells in a progenitor-like state. Contrastingly, levels of Pax4 equivalent to the ones observed following ISX-9 treatment promoted comparable expression of CCK and enterochromaffin markers. These data validate Pax4 as an important factor for enterochromaffin specification and offer a mechanism for ISX-9-biased enterochromaffin differentiation. This work provides proof of concept that the manipulation of the intestine epithelium towards specific EEC populations is possible and that small molecules can be used to this effect. The potential to modulate the population of serotonin-producing cells may present a novel target in the treatment of diseases including depression and gut motility disorders, and as an adjuvant therapy in the modulation of liver regeneration and immune responses, however the physiological relevance and translation into a clinical setting will need to be determined.
The cover image, by Zara J. Franklin et al., is based on the Original Article
Islet neuropeptide Y receptors are functionally conserved and novel targets for the preservation of beta‐cell mass
, DOI:
...10.1111/dom.13119
.
image
O cancro do ovário representa o cancro ginecológico com maior taxa de mortalidade em Portugal. O mau prognóstico explica-se pelo estádio avançado em que a maioria dos tumores é diagnosticada. Os ...tratamentos atualmente à disposição são de eficácia limitada uma vez que induzem o desenvolvimento de resistência e o seu sucesso a longo prazo é questionável, com a maioria das pacientes a desenvolver recorrências. No sentido de ultrapassar estas barreiras, foram testados novos fármacos, como é o caso dos inibidores do proteossoma, um complexo enzimático que regula o proteoma celular por degradação de proteínas desnecessárias ou anómalas. Os inibidores dos proteossomas revelaram desde o início grande eficácia em tumores hematológicos, mas o mesmo não se verificou na generalidade dos cancros sólidos, como é o caso do cancro do ovário. Esta diferença não parece ser facilmente explicada, pelo que o conhecimento das vias que intercetam a função do proteossoma pode ajudar a desvendar os mecanismos envolvidos. Neste trabalho abordámos a via Wnt/-catenina, uma via de transdução de sinal particularmente envolvida no cancro do ovário, cujo funcionamento é influenciado pelo proteossoma. Especificamente, o proteossoma regula os níveis citoplasmáticos de -catenina, limitando a sua transmigração nuclear e consequente ativação dos genes alvo.Como modelo recorremos à linha celular de cancro do ovário TOV-112D; o inibidor do proteossoma usado foi o MG262. Estudou-se o tipo de morte celular por citometria de fluxo com dupla marcação para a Anexina IV e iodeto de propídeo; avaliou-se o grau de inibição do proteossoma por citometria de fluxo após marcação celular com anticorpo dirigido a proteínas conjugadas com ubiquitina; analisou-se a ativação da via NF-KB por citometria de fluxo após marcação com anticorpo específico para o NF-KB livre, ativo; avaliou-se a ativação da via Wnt/-catenina quer por imunofluorescência, quantificando a transmigração nuclear da -catenina, quer quantificando a expressão de um dos seus genes alvo, o gene da ciclina D1 (CCND1) por qRT-PCR; para avaliar a interferência da inibição do proteossoma com a agressividade tumoral, analisaram-se a expressão da subunidade catalítica da telomerase, hTERT e a expressão do gene SNAIL, associado ao fenótipo de transição epitélio-mesênquima, também por qRT-PCR, e a mobilidade celular, pelo teste wound-healing e teste de migração em câmaras transwell.Após 48 horas de exposição ao inibidor do proteossoma MG262, houve uma diminuição da viabilidade celular dependente da concentração (P < 0.05). O IC50 para este composto foi obtido para a concentração de 14 nM (R2=0.9945), uma concentração muito superior à registada para as doenças hematológicas. O tipo de morte celular preferencial foi a necrose e apoptose/tardia necrose. A inibição proteossoma ocorreu para a concentração de 5 nM (P < 0.05) e inesperadamente, verificou-se uma activação da via NFκB (P < 0.01) desencadeada por esta inibição. Por sua vez, verificou-se um aumento da expressão da β-catenina ao nível do núcleo (P < 0.01). Contudo houve diminuição da expressão do gene alvo CCND1 (P < 0.05), atribuída a uma sobreposição do efeito inibidor da viabilidade celular do MG262. Também a análise do gene hTERT refletiu a diminuição da proliferação celular (P < 0.05). Para o gene SNAILnão foram observadas variações significativas (P < 0.05). Ocorreu ainda uma diminuição estatisticamente significativa (P < 0.001) do potencial de migração celular por exposição ao MG262.
The present paper reports the application of augmented simplex-centroid mixture design to obtain a high BET surface area activated carbon using as reactants KOH, K2CO3 and K2C2O4. The optimum mixture ...composition was 2.51 g of KOH, 0.49 g of K2CO3 and absence of K2C2O4, generating an optimized AC (ACop) with SBET value equals to 1984 m2 g−1. The results herein obtained show that low amounts of K2CO3 can catalyze the pore development in the presence of KOH, increasing the surface area. Furthermore, the fractal dimensions of ACop are greater than 2.72, indicating the material has a complex pore structure with irregularities self-similar upon variations of resolution, as seen by SEM images. The TPD curves showed that the ACop has different oxygenated molecular fragments, which agrees with the pHPZC value (5.05). The ACop was applied in the adsorption of rhodamine B (RhB) and metformin (Met) in both binary and monocomponent systems. The simultaneous adsorption at 30 °C reveals that the adsorption capacity of RhB is 630.94 mg g−1, while for Met the value is 103.83 mg g−1.
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•A simplex-centroid mixture design was applied in the production of activated carbon (AC).•The optimized AC showed specific surface area of 1984 m2 g−1.•The monocomponent and binary adsorptions showed differences in rhodamine B and metformin adsorption.•The proposed binary adsorption mechanism was confirmed by DFT-based quantum chemical descriptors.
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