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1.
Biofabrication ; 11(1): 015011, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30524058

RESUMO

Cell transplantation is a promising treatment for complementing lost function by replacing new cells with a desired function, e.g. pancreatic islet transplantation for diabetics. To prevent cell obliteration, oxygen supply is critical after transplantation, especially until the graft is sufficiently re-vascularized. To supply oxygen during this period, we developed a chemical-/electrical-free implantable oxygen transporter that delivers oxygen to the hypoxic graft site from ambient air by diffusion potential. This device is simply structured using a biocompatible silicone-based body that holds islets, connected to a tube that opens outside the body. In computational simulations, the oxygen transporter increased the oxygen level to >120 mmHg within grafts; in contrast, a control device that did not transport oxygen showed <6.5 mmHg. In vitro experiments demonstrated similar results. To test the effectiveness of the oxygen transporter in vivo, we transplanted pancreatic islets, which are susceptible to hypoxia, subcutaneously into diabetic rats. Islets transplanted using the oxygen transporter showed improved graft viability and cellular function over the control device. These results indicate that our oxygen transporter, which is safe and easily fabricated, effectively supplies oxygen locally. Such a device would be suitable for multiple clinical applications, including cell transplantations that require changing a hypoxic microenvironment into an oxygen-rich site.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Transplante das Ilhotas Pancreáticas/instrumentação , Ilhotas Pancreáticas/metabolismo , Oxigênio/metabolismo , Animais , Humanos , Ilhotas Pancreáticas/química , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Oxigênio/química , Ratos Endogâmicos Lew
2.
Pancreas ; 47(9): 1093-1100, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30142118

RESUMO

OBJECTIVES: Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich ß-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection. The aim of this study was to develop a practical method of live islet imaging by using surfactants to enhance the penetration efficiency. METHODS: Surfactants (F127, Tween 20, and Triton X-100) were co-incubated with Newport Green for fluorescent imaging of live isolated human islet and nonislet tissues. Toxicity, enhancement of Newport Green fluorescence, and effects on specificity to islets were examined. RESULTS: Newport Green fluorescent intensity was increased after co-incubation with all surfactants tested (0.2-3.2 mM); however, surfactants were toxic to islets at high concentrations. Within the nontoxic range, high specificity to islets was observed when co-incubated with Tween 20 at 0.2-0.4 mM, compared with F127 and Triton X-100. This optimized range successfully distinguished islets from nonislet tissues using statistically calculated cutoff value of Newport Green fluorescent intensity. CONCLUSIONS: Surfactants, particularly Tween 20 in the optimized range, effectively and selectively enhanced Newport Green fluorescence in live islets without increasing islet toxicity.


Assuntos
Corantes Fluorescentes/química , Aumento da Imagem/métodos , Ilhotas Pancreáticas/química , Tensoativos/química , Humanos , Ilhotas Pancreáticas/diagnóstico por imagem , Medições Luminescentes/métodos , Microscopia de Fluorescência/métodos
3.
J Vis Exp ; (136)2018 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-29985314

RESUMO

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.


Assuntos
Heparina/química , Ilhotas Pancreáticas/química , Nanopartículas Metálicas/química , Animais , Camundongos
4.
Sci Rep ; 8(1): 10456, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29993021

RESUMO

Hypoglycaemia is an important complication of Plasmodium falciparum malaria infection, which can be lethal if not treated. A decrease in blood sugar (BS) level has been correlated with disease severity, parasitaemia and the use of certain antimalarial drugs. This study explored the relationship between pancreatic pathology, including the expressions of insulin and glucagon in the islets of Langerhans, and the BS levels in P. falciparum malaria patients. Pancreatic tissues from malaria patients were divided into three groups, namely those with BS < 40 mg/dl, BS = 40-120 mg/dl, and BS > 120 mg/dl. In P. falciparum malaria, pancreatic tissues showed numerous parasitised red blood cells (PRBCs) in the capillaries, oedema, acinar necrosis and the presence of inflammatory cells. The islet size and the expression of insulin were significantly increased in P. falciparum malaria patients with hypoglycaemia. In addition, insulin expression was positively correlated with islet size and negatively correlated with BS levels. This pioneer study documents an increase in insulin expression and an increase in islet size in hypoglycaemic patients with P. falciparum malaria. This could contribute to the pathogenesis of hypoglycaemia and provides evidence for the potential need to effectively manage the hypoglycaemia seen in malaria infection.


Assuntos
Hipoglicemia/etiologia , Malária Falciparum/patologia , Pâncreas/patologia , Glicemia/análise , Estudos de Casos e Controles , Glucagon/metabolismo , Humanos , Hipoglicemia/sangue , Hipoglicemia/patologia , Insulina/metabolismo , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/patologia , Malária Falciparum/sangue , Malária Falciparum/complicações
5.
Domest Anim Endocrinol ; 65: 56-66, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29909240

RESUMO

Nesfatin-1 is a naturally occurring 82-amino acid protein encoded in the precursor nucleobindin-2 (NUCB2) and has been implicated in multiple physiological functions, including food intake and blood glucose regulation. This study aimed to characterize nesfatin-1 in domestic species, especially cats (Felis catus), dogs (Canis lupus familiaris), and pigs (Sus scrofa). Our in silico analysis demonstrated that the NUCB2/nesfatin-1 amino acid sequence, especially the bioactive core region of the peptide, is very highly conserved (more than 90% identity) in domestic animals. Expression of mRNAs encoding NUCB2/nesfatin-1 was detected in the cat, dog, and pig stomach and pancreas. Immunohistochemistry revealed the presence of nesfatin-1 in the gastric mucosa of the stomach of dogs, cats, and pigs, and in the pancreatic islet ß-cells of dogs and pigs. No nesfatin-1 immunoreactivity was found in the cat pancreas. Nesfatin-1 was detected in the serum of dog, cat, pig, bison, cow, horse, sheep, and chicken. Circulating nesfatin-1 in male and female dogs remained unchanged at 60 min after glucose administration, suggesting a lack of meal responsiveness in nesfatin-1 secretion in this species. The presence of nesfatin-1 in the gastric and endocrine pancreatic tissues suggests possible roles for this peptide in the metabolism of domestic animals. Future research should focus on elucidating the species-specific functions and mechanisms of action of nesfatin-1 in health and disease of domestic animals.


Assuntos
Animais Domésticos/sangue , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/sangue , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/sangue , Sequência de Aminoácidos , Animais , Bison , Proteínas de Ligação ao Cálcio/genética , Gatos , Bovinos , Galinhas , Sequência Conservada , Proteínas de Ligação a DNA/genética , Cães , Feminino , Mucosa Gástrica/química , Cavalos , Imuno-Histoquímica , Ilhotas Pancreáticas/química , Masculino , Proteínas do Tecido Nervoso/genética , Nucleobindinas , Especificidade de Órgãos , RNA Mensageiro/análise , Ovinos , Sus scrofa
6.
Methods Mol Biol ; 1766: 197-208, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29605854

RESUMO

The regulatory mechanisms that ensure an accurate control of gene transcription are central to cellular function, development and disease. Such mechanisms rely largely on noncoding regulatory sequences that allow the establishment and maintenance of cell identity and tissue-specific cellular functions.The study of chromatin structure and nucleosome positioning allowed revealing transcription factor accessible genomic sites with regulatory potential, facilitating the comprehension of tissue-specific cis-regulatory networks. Recently a new technique coupled with high-throughput sequencing named Assay for Transposase Accessible Chromatin (ATAC-seq) emerged as an efficient method to chart open chromatin genome wide. The application of such technique to different cell types allowed unmasking tissue-specific regulatory elements and characterizing cis-regulatory networks. Herein we describe the implementation of the ATAC-seq method to human pancreatic islets, a tissue playing a central role in the control of glucose metabolism.


Assuntos
Cromatina/efeitos dos fármacos , Cromatina/genética , Ensaios de Triagem em Larga Escala , Ilhotas Pancreáticas/enzimologia , Transposases/farmacologia , Cromatina/química , Epigenômica , Humanos , Ilhotas Pancreáticas/química , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Controle de Qualidade , Alinhamento de Sequência , Análise de Sequência de DNA , Técnicas de Cultura de Tecidos , Transcrição Genética , Transposases/química
7.
Int J Mol Sci ; 19(4)2018 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-29649109

RESUMO

ß-cell dedifferentiation has been recently suggested as an additional mechanism contributing to type-1 and to type-2 diabetes pathogenesis. Moreover, several studies demonstrated that in vitro culture of native human pancreatic islets derived from non-diabetic donors resulted in the generation of an undifferentiated cell population. Additional evidence from in vitro human ß-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from ß-cells, thus representing a potential in vitro model of ß-cell dedifferentiation. Here, we report the microRNA expression profiles analysis of in vitro dedifferentiated islet cells in comparison to mature human native pancreatic islets. We identified 13 microRNAs upregulated and 110 downregulated in islet cells upon in vitro dedifferentiation. Interestingly, among upregulated microRNAs, we observed the activation of microRNA miR-302s cluster, previously defined as pluripotency-associated. Bioinformatic analysis indicated that miR-302s are predicted to target several genes involved in the control of ß-cell/epithelial phenotype maintenance; accordingly, such genes were downregulated upon human islet in vitro dedifferentiation. Moreover, we uncovered that cell-cell contacts are needed to maintain low/null expression levels of miR-302. In conclusion, we showed that miR-302 microRNA cluster genes are involved in in vitro dedifferentiation of human pancreatic islet cells and inhibits the expression of multiple genes involved in the maintenance of ß-cell mature phenotype.


Assuntos
Perfilação da Expressão Gênica/métodos , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , MicroRNAs/genética , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Desdiferenciação Celular , Diferenciação Celular , Células Cultivadas , Humanos , Células Secretoras de Insulina/química , Ilhotas Pancreáticas/química , Pessoa de Meia-Idade
9.
Nat Commun ; 9(1): 882, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491378

RESUMO

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.


Assuntos
Ilhotas Pancreáticas/metabolismo , Proteínas/genética , Proteoma/genética , Perfilação da Expressão Gênica , Humanos , Ilhotas Pancreáticas/química , Espectrometria de Massas , Proteínas/química , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica
10.
Peptides ; 100: 212-218, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29412821

RESUMO

The endocrine pancreas is composed of islets of Langerhans, which secrete a variety of peptide hormones critical for the maintenance of glucose homeostasis. Insulin is the primary regulator of glucose and its secretion from beta-cells is tightly regulated in response to physiological demands. Direct cell-cell communication within islets is essential for glucose-induced insulin secretion. Emerging data suggest that islet connectivity is also important in the regulating the release of other islet hormones including glucagon and somatostatin. Autocrine and paracrine signals exerted by secreted peptides within the islet also play a key role. A great deal of attention has focused on classical islet peptides, namely insulin, glucagon and somatostatin. Recently, it has become clear that islets also synthesise and secrete a range of non-classical peptides, which regulate beta-cell function and insulin release. The current review summarises the roles of islet cell connectivity and islet peptide-driven autocrine and paracrine signalling in beta-cell function and survival. The potential to harness the paracrine effects of non-classical islet peptides for the treatment of type 2 diabetes is also briefly discussed.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Peptídeos/uso terapêutico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/química , Comunicação Parácrina/efeitos dos fármacos , Biossíntese Peptídica , Peptídeos/química , Peptídeos/metabolismo
11.
Endocrinology ; 159(3): 1393-1400, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29390052

RESUMO

Pancreatic islets are endocrine micro-organs scattered throughout the exocrine pancreas. Islets are surrounded by a network of vasculature, ducts, neurons, and extracellular matrix. Three-dimensional imaging is critical for such structural analyses. We have adapted transparent tissue tomography to develop a method to image thick pancreatic tissue slices (1 mm) with multifluorescent channels. This method takes only 2 to 3 days from specimen preparation and immunohistochemical staining to clearing tissues and imaging. Reconstruction of the intact pancreas visualizes islets with ß, α, and δ cells together with their surrounding networks. Capturing several hundred islets at once ensures sufficient power for statistical analyses. Further surface rendering provides clear views of the anatomical relationship between islets and their microenvironment as well as the basis for volumetric quantification. As a proof-of-principle demonstration, we show an islet size-dependent increase of intraislet capillary density and an inverse decrease in sphericity.


Assuntos
Imageamento Tridimensional/métodos , Ilhotas Pancreáticas/diagnóstico por imagem , Pâncreas/diagnóstico por imagem , Animais , Anticorpos Monoclonais , Arteríolas/diagnóstico por imagem , Capilares/diagnóstico por imagem , Secções Congeladas , Glucagon/análise , Humanos , Imuno-Histoquímica , Insulina/análise , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/química , Camundongos , Pâncreas/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Somatostatina/análise
12.
Appl Spectrosc ; 72(5): 706-714, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29350550

RESUMO

Primary pancreatic α, ß, δ, and pancreatic polypeptide (PP) cells are reliable cell models for diabetes research. However, the separation and purification of these cells in living conditions remains an obstacle for researchers. The interaction of visible light with cellular molecules can produce Raman scattering, which can be analyzed to obtain cellular intrinsic molecular fingerprints. It has been speculated that primary pancreatic α, ß, δ, and PP cells can be identified and separated from each other according to their spectral differences. To test this hypothesis, Raman spectra detection was performed on rat islet cells. Single islet cells identified by Raman scattering under living conditions were verified using immunohistochemistry. Thus, Raman data were acquired from a pure line of islet cells as a training sample and then used to establish the discriminant function. Then, using the principal component analysis-linear discriminate analysis (PCA-LDA) method, the four types of islet cells could be identified and discriminated by Raman spectroscopy. This study provides a label-free and noninvasive method for discriminating islet cell types in a randomly distributed mixed islet cell population via their physical properties rather than by using antibodies or fluorescence labeling.


Assuntos
Separação Celular/métodos , Ilhotas Pancreáticas/citologia , Análise Espectral Raman/métodos , Animais , Análise Discriminante , Ilhotas Pancreáticas/química , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley
13.
Drug Deliv Transl Res ; 8(3): 857-862, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29372538

RESUMO

The ability of glucagon-like peptide-1 analogs to enhance glucose-dependent insulin secretion and to inhibit ß cell apoptosis could be of potential benefit for islet transplantation. In this study, we investigated the effect of sustained local delivery of exenatide, a synthetic exendin-4, on the in vitro viability and function of encapsulated porcine islets. Prior to encapsulation, we fabricated exenatide-loaded poly(latic-co-glycolic acid) microspheres, and investigated their release behavior with different initial drug-loading amounts. Exenatide-loaded microspheres, exhibiting a sustained release over 21 days, were subsequently chosen and co-encapsulated with porcine islets in alginate microcapsules. During the 21-day period, the islets co-encapsulated with the exenatide-loaded microspheres exhibited improved survival and glucose-stimulated insulin secretion, compared to those without. This suggested that the intracapsular sustained delivery of exenatide via microspheres could be a promising strategy for improving survival and function of microencapsulated porcine islets for islet xenotransplantation.


Assuntos
Alginatos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/química , Microesferas , Peptídeos/administração & dosagem , Peçonhas/administração & dosagem , Alginatos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Liberação Controlada de Fármacos , Exenatida , Ácido Glucurônico/administração & dosagem , Ácido Glucurônico/química , Ácidos Hexurônicos/administração & dosagem , Ácidos Hexurônicos/química , Hipoglicemiantes/química , Ilhotas Pancreáticas/efeitos dos fármacos , Ácido Láctico/administração & dosagem , Ácido Láctico/química , Peptídeos/química , Ácido Poliglicólico/administração & dosagem , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Suínos , Peçonhas/química
14.
J Biomed Mater Res B Appl Biomater ; 106(2): 555-568, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28240814

RESUMO

Macroencapsulation is a powerful approach to increase the efficiency of extrahepatic pancreatic islet transplant. FTY720, a small molecule that activates signaling through sphingosine-1-phosphate receptors, is immunomodulatory and pro-angiogenic upon sustained delivery from biomaterials. While FTY720 (fingolimod, Gilenya) has been explored for organ transplantation, in the present work the effect of locally released FTY720 from novel nanofiber-based macroencapsulation membranes is explored for islet transplantation. We screened islet viability during culture with FTY720 and various biodegradable polymers. Islet viability is significantly reduced by the addition of high doses (≥500 ng/mL) of soluble FTY720. Among the polymers screened, islets have the highest viability when cultured with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Therefore, PHBV was blended with polycaprolactone (PCL) for mechanical stability and electrospun into nanofibers. Islets had no detectable function ex vivo following 5 days or 12 h of subcutaneous implantation within our engineered device. Subsequently, we explored a preconditioning scheme in which islets are transplanted 2 weeks after FTY720-loaded nanofibers are implanted. This allows FTY720 to orchestrate a local regenerative milieu while preventing premature transplantation into avascular sites that contain high concentrations of FTY720. These results provide a foundation and motivation for further investigation into the use of FTY720 in preconditioning sites for efficacious islet transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 555-568, 2018.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Cloridrato de Fingolimode/administração & dosagem , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/efeitos dos fármacos , Membranas Artificiais , Animais , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Relação Dose-Resposta a Droga , Cloridrato de Fingolimode/química , Humanos , Ilhotas Pancreáticas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanofibras/química , Poliésteres/química , Poliésteres/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Estreptozocina/farmacologia
15.
Cell Tissue Bank ; 19(1): 77-85, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28916910

RESUMO

Islet transplantation has made major progress to treat patients with type 1 diabetes. Islet mass and quality are critically important to ensure successful transplantation. Currently, islet status is evaluated using insulin secretion, oxygen consumption rate, or adenosine triphosphate (ATP) measurement. These parameters are evaluated independently and do not effectively predict islet status post-transplant. Therefore, assessing human pancreatic islets by encompassing ATP, DNA, insulin, and protein content from a single tissue sample would serve as a better predictor for islet status. In this study, a single step procedure for extracting ATP, DNA, insulin, and protein content from human pancreatic islets was described and the biomolecule contents were quantified. Additionally, different mathematical calculations integrating total ATP, DNA, insulin, and protein content were randomly tested under various conditions to predict islet status. The results demonstrated that the ATP assay was efficient and the biomolecules were effectively quantified. Furthermore, the mathematical formula we developed could be optimized to predict islet status. In conclusion, our results indicate a proof-of-concept that a simple logarithmic formula can predict overall islet status for various conditions when total islet ATP, DNA, insulin, and protein content are simultaneously assessed from a single tissue sample.


Assuntos
Trifosfato de Adenosina/análise , DNA/análise , Insulina/análise , Ilhotas Pancreáticas/química , Algoritmos , Humanos , Transplante das Ilhotas Pancreáticas , Modelos Biológicos , Técnicas de Cultura de Órgãos
16.
Endocrinology ; 158(10): 3325-3338, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28977592

RESUMO

Although ß-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the cystic fibrosis transmembrane conductance regulator (CFTR) channel influences islet insulin secretion remains debated. We investigated the CFTR-dependent islet-autonomous mechanisms affecting insulin secretion by using islets isolated from CFTR knockout ferrets. Total insulin content was lower in CF as compared with wild-type (WT) islets. Furthermore, glucose-stimulated insulin secretion (GSIS) was impaired in perifused neonatal CF islets, with reduced first, second, and amplifying phase secretion. Interestingly, CF islets compensated for reduced insulin content under static low-glucose conditions by secreting a larger fraction of islet insulin than WT islets, probably because of elevated SLC2A1 transcripts, increased basal inhibition of adenosine triphosphate-sensitive potassium channels (K-ATP), and elevated basal intracellular Ca2+. Interleukin (IL)-6 secretion by CF islets was higher relative to WT, and IL-6 treatment of WT ferret islets produced a CF-like phenotype with reduced islet insulin content and elevated percentage insulin secretion in low glucose. CF islets exhibited altered expression of INS, CELA3B, and several ß-cell maturation and proliferation genes. Pharmacologic inhibition of CFTR reduced GSIS by WT ferret and human islets but similarly reduced insulin secretion and intracellular Ca2+ in CFTR knockout ferret islets, indicating that the mechanism of action is not through CFTR. Single-molecule fluorescent in situ hybridization, on isolated ferret and human islets and ferret pancreas, demonstrated that CFTR RNA colocalized within KRT7+ ductal cells but not endocrine cells. These results suggest that CFTR affects ß-cell function via a paracrine mechanism involving proinflammatory factors secreted from islet-associated exocrine-derived cell types.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/análise , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Furões/genética , Técnicas de Inativação de Genes , Glucose/farmacologia , Humanos , Hibridização in Situ Fluorescente , Insulina/análise , Secreção de Insulina , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/antagonistas & inibidores , Masculino , RNA/análise
17.
Appl Spectrosc ; 71(12): 2681-2691, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28937262

RESUMO

Mammalian cells contain various macromolecules that can be investigated non-invasively with Raman spectroscopy. The particular mixture of major macromolecules present in a cell being probed are reflected in the measured Raman spectra. Determining macromolecular identities and estimating their concentrations from these mixture Raman spectra can distinguish cell types and otherwise enable biological research. However, the application of canonical multivariate methods, such as principal component analysis (PCA), to perform spectral unmixing yields mathematical solutions that can be difficult to interpret. Non-negative matrix factorization (NNMF) improves the interpretability of unmixed macromolecular components, but can be difficult to apply because ambiguities produced by overlapping Raman bands permit multiple solutions. Furthermore, theoretically sound methods can be difficult to implement in practice. Here we examined the effects of a number of empirical approaches on the quality of NNMF results. These approaches were evaluated on simulated mammalian cell Raman hyperspectra and the results were used to develop an enhanced procedure for implementing NNMF. We demonstrated the utility of this procedure using a Raman hyperspectral data set measured from human islet cells to recover the spectra of insulin and glucagon. This was compared to the relatively inferior PCA of these data.


Assuntos
Técnicas Citológicas/métodos , Análise Espectral Raman/métodos , Algoritmos , Animais , Células Cultivadas , Glucagon/análise , Glucagon/química , Humanos , Insulina/análise , Insulina/química , Ilhotas Pancreáticas/química , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/química , Análise Multivariada
18.
Nutrition ; 43-44: 47-53, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28935144

RESUMO

OBJECTIVE: Despite increasing evidence that pharmacologic concentrations of biotin modify glucose metabolism, to our knowledge there have not been any studies addressing the effects of biotin supplementation on glucagon production and secretion, considering glucagon is one of the major hormones in maintaining glucose homeostasis. The aim of this study was to investigate the effects of dietary biotin supplementation on glucagon expression, secretion, and action. METHODS: Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 wk postweaning. Glucagon gene mRNA expression was measured by the real-time polymerase chain reaction. Glucagon secretion was assessed in isolated islets and by glucagon concentration in plasma. Glucagon action was evaluated by glucagon tolerance tests, phosphoenolpyruvate carboxykinase (Pck1) mRNA expression, and glycogen degradation. RESULTS: Compared with the control group, glucagon mRNA and secretion were increased from the islets of the biotin-supplemented group. Fasting plasma glucagon levels were higher, but no differences between the groups were observed in nonfasting glucagon levels. Despite the elevated fasting glucagon levels, no differences were found in fasting blood glucose concentrations, fasting/fasting-refeeding glucagon tolerance tests, glycogen content and degradation, or mRNA expression of the hepatic gluconeogenic rate-limiting enzyme, Pck1. CONCLUSIONS: These results demonstrated that dietary biotin supplementation increased glucagon expression and secretion without affecting fasting blood glucose concentrations or glucagon tolerance and provided new insights into the effect of biotin supplementation on glucagon production and action.


Assuntos
Biotina/administração & dosagem , Glucagon/metabolismo , Glucagon/farmacologia , Animais , Dieta , Suplementos Nutricionais , Expressão Gênica/efeitos dos fármacos , Glucagon/genética , Gluconeogênese/efeitos dos fármacos , Glicogênio/metabolismo , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/análise
19.
J Phys Chem B ; 121(37): 8661-8668, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28829144

RESUMO

Amyloid aggregates are characteristic of many serious diseases such as Alzheimer's disease, Parkinson's, and type 2 diabetes and commonly involve intrinsically disordered proteins (IDPs), those that populate an ensemble of conformations rather than a single folded structure. Human islet amyloid polypeptide (hIAPP or amylin) is an amyloidogenic IDP implicated in pancreatic ß-cell death during the pathogenesis of type 2 diabetes. The target of amylin's toxic activity is thought to be the cell's lipid membrane, which may also act as a catalyst for aggregation. Since amylin is intrinsically disordered, differing environments can have a large impact on its equilibrium conformational ensemble. We apply atomistic molecular dynamics simulations on multiple systems containing a full-length amylin monomer and a lipid bilayer to study the changes induced by the membrane. We observe stabilized helical conformations structurally similar to those determined by NMR experiments conducted in similar environments. We also find that bilayers of different compositions result in greatly different equilibrium ensembles of amylin. Finally, we discuss how a mixed bilayer containing zwitterionic and anionic lipid headgroups can allow for greater preference toward conformations which are adsorbed below the membrane surface through rearrangement of lipids for more favorable protein-lipid interactions.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/química , Lipídeos de Membrana/química , Humanos , Ilhotas Pancreáticas/citologia , Simulação de Dinâmica Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica
20.
Sci Rep ; 7(1): 5024, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28694456

RESUMO

Single-cell RNA-seq (scRNA-seq) of pancreatic islets have reported on α- and ß-cell gene expression in mice and subjects of predominantly European ancestry. We aimed to assess these findings in East-Asian islet-cells. 448 islet-cells were captured from three East-Asian non-diabetic subjects for scRNA-seq. Hierarchical clustering using pancreatic cell lineage genes was used to assign cells into cell-types. Differentially expressed transcripts between α- and ß-cells were detected using ANOVA and in silico replications of mouse and human islet cell genes were performed. We identified 118 α, 105 ß, 6 δ endocrine cells and 47 exocrine cells. Besides INS and GCG, 26 genes showed differential expression between α- and ß-cells. 10 genes showed concordant expression as reported in rodents, while FAM46A was significantly discordant. Comparing our East-Asian data with data from primarily European subjects, we replicated several genes implicated in nuclear receptor activations, acute phase response pathway, glutaryl-CoA/tryptophan degradations and EIF2/AMPK/mTOR signaling. Additionally, we identified protein ubiquitination to be associated among East-Asian ß-cells. We report on East-Asian α- and ß-cell gene signatures and substantiate several genes/pathways. We identify expression signatures in East-Asian ß-cells that perhaps reflects increased susceptibility to cell-death and warrants future validations to fully appreciate their role in East-Asian diabetes pathogenesis.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Perfilação da Expressão Gênica/métodos , Ilhotas Pancreáticas/química , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Europa (Continente) , Extremo Oriente , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Secretoras de Glucagon/química , Humanos , Células Secretoras de Insulina/química , Masculino , Especificidade de Órgãos , Ubiquitinação
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