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2.
Sci Adv ; 10(23): eadk3081, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38848367

RESUMO

Clinical outcomes for total-pancreatectomy followed by intraportal islet autotransplantation (TP-IAT) to treat chronic pancreatitis (CP) are suboptimal due to pancreas inflammation, oxidative stress during islet isolation, and harsh engraftment conditions in the liver's vasculature. We describe a thermoresponsive, antioxidant macromolecule poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to protect islet redox status and function and to enable extrahepatic omentum islet engraftment. PPCN solution transitions from a liquid to a hydrogel at body temperature. Islets entrapped in PPCN and exposed to oxidative stress remain functional and support long-term euglycemia, in contrast to islets entrapped in a plasma-thrombin biologic scaffold. In the nonhuman primate (NHP) omentum, PPCN is well-tolerated and mostly resorbed without fibrosis at 3 months after implantation. In NHPs, autologous omentum islet transplantation using PPCN restores normoglycemia with minimal exogenous insulin requirements for >100 days. This preclinical study supports TP-IAT with PPCN in patients with CP and highlights antioxidant properties as a mechanism for islet function preservation.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Omento , Estresse Oxidativo , Transplante das Ilhotas Pancreáticas/métodos , Omento/metabolismo , Animais , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Cítrico/farmacologia , Humanos , Antioxidantes/farmacologia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/cirurgia , Pancreatite Crônica/patologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Masculino , Transição de Fase
3.
BMC Nephrol ; 25(1): 156, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724923

RESUMO

BACKGROUND: Islet transplantation is an effective treatment for diabetes or even its complications. Aim of this study is to investigate efficacy of biomaterial treated islet transplantation on treating diabetic nephropathy. METHODS: Male rats were randomly divided into 6 groups; Control, diabetic control, diabetic transplanted with untreated islets, with platelet rich plasma treated islets, with pancreatic islets homogenate treated islets, or with these biomaterials combination treated islets. Islets cultured with biomaterials and transplanted to diabetic rats. After 60 days, biochemical, oxidative stress, and stereological parameters were assessed. RESULTS: Serum albumin and BUN concentration, decreased and increased respectively, Oxidative stress of kidney impaired, kidney weight, volume of kidney, cortex, medulla, glomerulus, proximal and distal tubules, collecting ducts, vessels, inflammatory, necrotic and fibrotic tissue in diabetic group increased compared to control group (p < 0.001). In treated groups, especially pancreatic islets homogenate treated islets transplanting animals, there was significant changes in kidney weight, and volume of kidney, proximal and distal tubules, Henle's loop and collecting ducts compared with diabetic group (p = 0.013 to p < 0.001). Combination treated islets animals showed significant increase in vessel volume compared to diabetic group (p < 0.001). Necrotic and fibrotic tissue significantly decreased in islets treated than untreated islet animals, it was higher in pancreatic islets homogenate, and combination treated islets groups (p = 0.001). CONCLUSIONS: Biomaterials treated islets transplanting could improve diabetic nephropathy. Improvement of oxidative stress followed by controlling glucose level, and effects of growth factors presenting in biomaterials can be considered as capable underlying mechanism of ameliorating inflammatory, necrotic and fibrotic tissue volume.


Assuntos
Materiais Biocompatíveis , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Transplante das Ilhotas Pancreáticas , Animais , Masculino , Ratos , Nefropatias Diabéticas/patologia , Transplante das Ilhotas Pancreáticas/métodos , Materiais Biocompatíveis/uso terapêutico , Ilhotas Pancreáticas/patologia , Estresse Oxidativo , Ratos Sprague-Dawley , Resultado do Tratamento
4.
Elife ; 122024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38700926

RESUMO

The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Glucagon , Glucose , Secreção de Insulina , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , Animais Recém-Nascidos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glucagon/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/genética , Ilhotas Pancreáticas/metabolismo , Mutação , Canais de Potássio/metabolismo , Canais de Potássio/genética
5.
Nat Commun ; 15(1): 3810, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714671

RESUMO

Previous studies have revealed heterogeneity in the progression to clinical type 1 diabetes in children who develop islet-specific antibodies either to insulin (IAA) or glutamic acid decarboxylase (GADA) as the first autoantibodies. Here, we test the hypothesis that children who later develop clinical disease have different early immune responses, depending on the type of the first autoantibody to appear (GADA-first or IAA-first). We use mass cytometry for deep immune profiling of peripheral blood mononuclear cell samples longitudinally collected from children who later progressed to clinical disease (IAA-first, GADA-first, ≥2 autoantibodies first groups) and matched for age, sex, and HLA controls who did not, as part of the Type 1 Diabetes Prediction and Prevention study. We identify differences in immune cell composition of children who later develop disease depending on the type of autoantibodies that appear first. Notably, we observe an increase in CD161 expression in natural killer cells of children with ≥2 autoantibodies and validate this in an independent cohort. The results highlight the importance of endotype-specific analyses and are likely to contribute to our understanding of pathogenic mechanisms underlying type 1 diabetes development.


Assuntos
Autoanticorpos , Diabetes Mellitus Tipo 1 , Glutamato Descarboxilase , Imunidade Celular , Humanos , Diabetes Mellitus Tipo 1/imunologia , Autoanticorpos/imunologia , Autoanticorpos/sangue , Criança , Feminino , Masculino , Glutamato Descarboxilase/imunologia , Pré-Escolar , Adolescente , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Insulina/imunologia , Ilhotas Pancreáticas/imunologia , Progressão da Doença
6.
Cell Transplant ; 33: 9636897241251621, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38756050

RESUMO

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment. We herein investigated whether the addition of adipose tissue-derived stem cells (ADSCs) to GHNF affected the outcome. A silicone spacer sandwiched between two GHNFs with (AG group) or without (GHNF group) ADSCs, or a silicone spacer alone (Silicone group) was implanted into the subcutaneous space of healthy mice at 6 weeks before transplantation, then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Intraportal transplantation (IPO group) was also performed to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and inflammatory mediators were evaluated. The results in the subcutaneous transplantation were compared using the Silicone group as a control. The results of the IPO group were also compared with those of the AG group. The AG group showed significantly better blood glucose changes than the Silicone and the IPO groups. The cure rate of AG group (72.7%) was the highest among the groups (GHNF; 40.0%, IPO; 40.0%, Silicone; 0%). The number of vWF-positive vessels in the subcutaneous space of the AG group was significantly higher than that in other groups before transplantation (P < 0.01). Lectin angiography also showed that the same results (P < 0.05). According to the results of the ADSCs tracing, ADSCs did not exist at the transplant site (6 weeks after implantation). The positive rates for laminin and collagen III constructed around the transplanted islets did not differ among groups. Inflammatory mediators were higher in the Silicone group, followed by the AG and GHNF groups. Pretreatment using bioabsorbable scaffolds combined with ADSCs enhanced neovascularization in subcutaneous space, and subcutaneous islet transplantation using GHNF with ADSCs was superior to intraportal islet transplantation.


Assuntos
Tecido Adiposo , Gelatina , Hidrogéis , Transplante das Ilhotas Pancreáticas , Animais , Transplante das Ilhotas Pancreáticas/métodos , Tecido Adiposo/citologia , Gelatina/química , Camundongos , Hidrogéis/química , Masculino , Diabetes Mellitus Experimental/terapia , Células-Tronco/citologia , Células-Tronco/metabolismo , Ilhotas Pancreáticas/citologia , Glicemia/metabolismo , Camundongos Endogâmicos C57BL
7.
Physiol Rep ; 12(9): e16040, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38725080

RESUMO

The endocrine pancreas is composed of clusters of cell groups called pancreatic islets. These cells are responsible for the synthesis and secretion of hormones crucial for glycemic homeostasis, such as insulin and glucagon. Therefore, these cells were the targets of many studies. One method to study and/or understand endocrine pancreatic physiology is the isolation of these islets and stimulation of hormone production using different concentrations of glucose, agonists, and/or antagonists of specific secretagogues and mimicking the stimulation of hormonal synthesis and secretion. Many researchers studied pancreatic physiology in murine models due to their ease of maintenance and rapid development. However, the isolation of pancreatic islets involves meticulous processes that may vary between rodent species. The present study describes a simple and effective technical protocol for isolating intact islets from mice and rats for use as a practical guide for researchers. The method involves digestion of the acinar parenchyma by intraductal collagenase. Isolated islets are suitable for in vitro endocrine secretion analyses, microscopy techniques, and biochemical analyses.


Assuntos
Ilhotas Pancreáticas , Animais , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Camundongos , Ratos , Masculino , Camundongos Endogâmicos C57BL , Separação Celular/métodos
8.
Nat Commun ; 15(1): 3744, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702321

RESUMO

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.


Assuntos
Algoritmos , Diabetes Mellitus Tipo 1 , Pâncreas , Proteômica , Humanos , Proteômica/métodos , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Análise de Célula Única/métodos , Redes Neurais de Computação , Linfócitos T CD8-Positivos/metabolismo , Citometria por Imagem/métodos
9.
Sci Rep ; 14(1): 11640, 2024 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773268

RESUMO

Porcine islet xenotransplantation is a promising therapy for severe diabetes mellitus. Maintenance of the quality and quantity of porcine islets is important for the success of this treatment. Here, we aimed to elucidate the influence of relatively short-term (14 days) culture on adult porcine islets isolated from three micro-minipigs (P111, P112 and P121). Morphological characteristics of islets changed little after 14 days of culture. The viability of cultured islets was also maintained at a high level (> 80%). Furthermore, cultured islets exhibited similar glucose-stimulated insulin secretion and insulin content at Day 14 were preserved comparing with Day 1, while the expressions of Ins, Gcg and Sst were attenuated at Day 14. Xenotransplantation using diabetic nude mice showed no normalization of blood glucose but increased levels of plasma porcine C-peptide after the transplantation of 14 day cultured porcine islets. Histological assessment revealed that relatively short-term cultured porcine islets were successfully engrafted 56 days following transplantation. These data show that relatively short-term culture did not impair the quality of adult porcine islets in regard to function, morphology, and viability. Prevention of impairment of gene correlated with endocrine hormone is warranted for further improvement.


Assuntos
Insulina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Transplante Heterólogo , Animais , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Suínos , Transplante das Ilhotas Pancreáticas/métodos , Insulina/metabolismo , Camundongos , Camundongos Nus , Secreção de Insulina , Diabetes Mellitus Experimental/terapia , Glicemia/metabolismo , Porco Miniatura , Sobrevivência Celular , Peptídeo C/metabolismo , Peptídeo C/sangue
10.
Sci Rep ; 14(1): 12402, 2024 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-38811610

RESUMO

Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation.


Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Humanos , Animais , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Experimental/terapia , Masculino , Diabetes Mellitus Tipo 1/metabolismo , Hipóxia/metabolismo , Feminino , Hipóxia Celular , Pessoa de Meia-Idade , Glicemia/metabolismo
11.
J Cell Sci ; 137(20)2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-38804679

RESUMO

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.


Assuntos
Cílios , Ilhotas Pancreáticas , Cílios/ultraestrutura , Cílios/metabolismo , Animais , Humanos , Camundongos , Ilhotas Pancreáticas/ultraestrutura , Ilhotas Pancreáticas/metabolismo , Microscopia Eletrônica de Varredura/métodos
12.
Cells ; 13(10)2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38786050

RESUMO

Allogeneic islet transplantation has become a standard therapy for unstable type 1 diabetes. However, considering the large number of type 1 diabetic patients, the shortage of donors is a serious issue. To address this issue, clinical islet xenotransplantation is conducted. The first clinical islet xenotransplantation was performed by a Swedish team using fetal pancreatic tissue. Thereafter, clinical trials of islet xenotransplantation were conducted in New Zealand, Russia, Mexico, Argentina, and China using neonatal pig islets. In clinical trials, fetal or neonatal pancreata are used because of the established reliable islet isolation methods. These trials demonstrate the method's safety and efficacy. Currently, the limited number of source animal facilities is a problem in terms of promoting islet xenotransplantation. This limitation is due to the high cost of source animal facilities and the uncertain future of xenotransplantation. In the United States, the first xenogeneic heart transplantation has been performed, which could promote xenotransplantation. In Japan, to enhance xenotransplantation, the 'Medical Porcine Development Association' has been established. We hope that xenogeneic transplantation will become a clinical reality, serving to address the shortage of donors.


Assuntos
Transplante das Ilhotas Pancreáticas , Transplante Heterólogo , Transplante das Ilhotas Pancreáticas/métodos , Animais , Humanos , Rejeição de Enxerto , Suínos , Resultado do Tratamento , Diabetes Mellitus Tipo 1/cirurgia , Diabetes Mellitus Tipo 1/terapia , Ensaios Clínicos como Assunto , Ilhotas Pancreáticas
13.
PLoS Comput Biol ; 20(5): e1012130, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38739680

RESUMO

Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics.


Assuntos
Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Biologia Computacional/métodos , Camundongos , Insulina/metabolismo , Humanos , Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citologia , Secreção de Insulina/fisiologia , Modelos Biológicos , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia
14.
Cell Transplant ; 33: 9636897241249556, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38742734

RESUMO

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.


Assuntos
Colágeno , Ilhotas Pancreáticas , Alicerces Teciduais , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Alicerces Teciduais/química , Porosidade , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/instrumentação , Transplante das Ilhotas Pancreáticas/métodos
15.
Sci Rep ; 14(1): 10136, 2024 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698049

RESUMO

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.


Assuntos
Imageamento Tridimensional , Imagem Multimodal , Pâncreas , Animais , Imageamento Tridimensional/métodos , Pâncreas/diagnóstico por imagem , Pâncreas/irrigação sanguínea , Suínos , Imagem Multimodal/métodos , Microtomografia por Raio-X/métodos , Ilhotas Pancreáticas/diagnóstico por imagem , Ilhotas Pancreáticas/irrigação sanguínea , Tomografia Computadorizada por Raios X/métodos
16.
Mol Metab ; 84: 101955, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38704026

RESUMO

OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina , Mitocôndrias , Animais , Ratos , Humanos , Mitocôndrias/metabolismo , Células Secretoras de Insulina/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Masculino , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Camundongos , Ratos Wistar , Transporte de Elétrons
18.
J Immunol ; 212(12): 1971-1980, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38709159

RESUMO

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1ß, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.


Assuntos
Antígeno CD47 , Inflamação , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Camundongos Endogâmicos C57BL , Trombomodulina , Animais , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Camundongos , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Inflamação/imunologia , Masculino , Estreptavidina
19.
Nat Commun ; 15(1): 3740, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702347

RESUMO

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.


Assuntos
Células Acinares , Diabetes Mellitus Experimental , Células Secretoras de Insulina , Animais , Células Secretoras de Insulina/metabolismo , Camundongos , Células Acinares/metabolismo , Masculino , Insulina/metabolismo , Transdiferenciação Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Ilhotas Pancreáticas/metabolismo
20.
J Diabetes Res ; 2024: 5574968, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38800586

RESUMO

Islet transplantation (ITx) is an established and safe alternative to pancreas transplantation for type 1 diabetes mellitus (T1DM) patients. However, most ITx recipients lose insulin independence by 3 years after ITx due to early graft loss, such that multiple donors are required to achieve insulin independence. In the present study, we investigated whether skeletal myoblast cells could be beneficial for promoting angiogenesis and maintaining the differentiated phenotypes of islets. In vitro experiments showed that the myoblast cells secreted angiogenesis-related cytokines (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and stromal-derived factor-1α (SDF-1α)), contributed to maintenance of differentiated islet phenotypes, and enhanced islet cell insulin secretion capacity. To verify these findings in vivo, we transplanted islets alone or with myoblast cells under the kidney capsule of streptozotocin-induced diabetic mice. Compared with islets alone, the group bearing islets with myoblast cells had a significantly lower average blood glucose level. Histological examination revealed that transplants with islets plus myoblast cells were associated with a significantly larger insulin-positive area and significantly higher number of CD31-positive microvessels compared to islets alone. Furthermore, islets cotransplanted with myoblast cells showed JAK-STAT signaling activation. Our results suggest two possible mechanisms underlying enhancement of islet graft function with myoblast cells cotransplantation: "indirect effects" mediated by angiogenesis and "direct effects" of myoblast cells on islets via the JAK-STAT cascade. Overall, these findings suggest that skeletal myoblast cells enhance the function of transplanted islets, implying clinical potential for a novel ITx procedure involving myoblast cells for patients with diabetes.


Assuntos
Diabetes Mellitus Experimental , Insulina , Transplante das Ilhotas Pancreáticas , Mioblastos Esqueléticos , Neovascularização Fisiológica , Animais , Transplante das Ilhotas Pancreáticas/métodos , Diabetes Mellitus Experimental/metabolismo , Mioblastos Esqueléticos/transplante , Mioblastos Esqueléticos/metabolismo , Camundongos , Masculino , Insulina/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Camundongos Endogâmicos C57BL , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/irrigação sanguínea , Quimiocina CXCL12/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/cirurgia , Transdução de Sinais , Secreção de Insulina , Diferenciação Celular
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