RESUMEN

BACKGROUND: Our previous genome­wide association studies (GWAS) have suggested rs912304 in 14q12 as a suggestive risk variant for type 1 diabetes (T1D). However, the association between this risk region and T1D subgroups and related clinical risk features, the underlying causal functional variant(s), putative candidate gene(s), and related mechanisms are yet to be elucidated. METHODS: We assessed the association between variant rs912304 and T1D, as well as islet autoimmunity and islet function, stratified by the diagnosed age of 12. We used epigenome bioinformatics analyses, dual luciferase reporter assays, and expression quantitative trait loci (eQTL) analyses to prioritize the most likely functional variant and potential causal gene. We also performed functional experiments to evaluate the role of the causal gene on islet function and its related mechanisms. RESULTS: We identified rs912304 as a risk variant for T1D subgroups with diagnosed age ≥ 12 but not < 12. This variant is associated with residual islet function but not islet-specific autoantibody positivity in T1D individuals. Bioinformatics analysis indicated that rs912304 is a functional variant exhibiting spatial overlaps with enhancer active histone marks (H3K27ac and H3K4me1) and open chromatin status (ATAC-seq) in the human pancreas and islet tissues. Luciferase reporter gene assays and eQTL analyses demonstrated that the biallelic sites of rs912304 had differential allele-specific enhancer activity in beta cell lines and regulated STXBP6 expression, which was defined as the most putative causal gene based on Open Targets Genetics, GTEx v8 and Tiger database. Moreover, Stxbp6 was upregulated by T1D-related proinflammatory cytokines but not high glucose/fat. Notably, Stxbp6 over-expressed INS-1E cells exhibited decreasing insulin secretion and increasing cell apoptosis through Glut1 and Gadd45ß, respectively. CONCLUSIONS: This study expanded the genomic landscape regarding late-onset T1D risk and supported islet function mechanistically connected to T1D pathogenesis.

Asunto(s)

Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Humanos , Diabetes Mellitus Tipo 1/genética , Islotes Pancreáticos/metabolismo , Femenino , Masculino , Polimorfismo de Nucleótido Simple/genética , Predisposición Genética a la Enfermedad , Citocinas/genética , Citocinas/metabolismo , Niño , Adolescente , Sitios de Carácter Cuantitativo , Animales , Edad de Inicio , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo

RESUMEN

Type 2 diabetes (T2D) is associated with a systemic increase in the pro-inflammatory cytokine IL-1ß. While transient exposure to low IL-1ß concentrations improves insulin secretion and ß-cell proliferation in pancreatic islets, prolonged exposure leads to impaired insulin secretion and collective ß-cell death. IL-1 is secreted locally by islet-resident macrophages and ß-cells; however, it is unknown if and how the two opposing modes may emerge at single islet level. We investigated the duality of IL-1ß with a quantitative in silico model of the IL-1 regulatory network in pancreatic islets. We find that the network can produce either transient or persistent IL-1 responses when induced by pro-inflammatory and metabolic cues. This suggests that the duality of IL-1 may be regulated at the single islet level. We use two core feedbacks in the IL-1 regulation to explain both modes: First, a fast positive feedback in which IL-1 induces its own production through the IL-1R/IKK/NF-κB pathway. Second, a slow negative feedback where NF-κB upregulates inhibitors acting at different levels along the IL-1R/IKK/NF-κB pathway-IL-1 receptor antagonist and A20, among others. A transient response ensues when the two feedbacks are balanced. When the positive feedback dominates over the negative, islets transit into the persistent inflammation mode. Consistent with several observations, where the size of islets was implicated in its inflammatory state, we find that large islets and islets with high density of IL-1ß amplifying cells are more prone to transit into persistent IL-1ß mode. Our results are likely not limited to IL-1ß but are general for the combined effect of multiple pro-inflammatory cytokines and chemokines. Generalizing complex regulations in terms of two feedback mechanisms of opposing nature and acting on different time scales provides a number of testable predictions. Taking islet architecture and cellular heterogeneity into consideration, further dynamic monitoring and experimental validation in actual islet samples will be crucial to verify the model predictions and enhance its utility in clinical applications.

Asunto(s)

Diabetes Mellitus Tipo 2 , Inflamación , Interleucina-1beta , Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Modelos Biológicos , Transducción de Señal/fisiología , FN-kappa B/metabolismo , Animales , Simulación por Computador , Retroalimentación Fisiológica/fisiología , Células Secretoras de Insulina/metabolismo

RESUMEN

The aim of regenerative medicine is to restore specific functions to damaged cells or tissues. A crucial aspect of success lies in effectively reintegrating these cells or tissues within the recipient organism. This is particularly pertinent for diabetes, where islet function relies on the close connection of beta cells to the bloodstream for glucose sensing and insulin release. Central to this approach is the need to establish a fast connection with the host's vascular system. In this review, we explore the intricate relationships between endocrine, vascular, and immune cell interactions in transplantation outcomes. We also delve into recent strategies aimed at enhancing engraftment, along with the utilization of in vitro platforms to model cellular interactions.

Asunto(s)

Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Humanos , Trasplante de Islotes Pancreáticos/inmunología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/citología , Animales , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citología , Medicina Regenerativa , Diabetes Mellitus/inmunología

RESUMEN

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Asunto(s)

Diferenciación Celular , Genes Reporteros , Células Secretoras de Insulina , Islotes Pancreáticos , Células Madre Pluripotentes , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Celular , Insulina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética

RESUMEN

Objective: Beta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity. Among the underlying mechanisms, secretion of extracellular vesicles (EVs) by beta cells has been suggested to mediate the crosstalk with the immune cell compartment. Methods: To study the role of cellular stress in the early events of T1D development, we generated a novel cellular model for constitutive ER stress by modulating the expression of HSPA5, which encodes BiP/GRP78, in EndoC-ßH1 cells. To investigate the role of EVs in the interaction between beta cells and the immune system, we characterized the EV miRNA cargo and evaluated their effect on innate immune cells. Results: Analysis of the transcriptome showed that HSPA5 knockdown resulted in the upregulation of signaling pathways involved in the unfolded protein response (UPR) and changes the miRNA content of EVs, including reduced levels of miRNAs involved in IL-1ß signaling. Treatment of primary human monocytes with EVs from stressed beta cells resulted in increased surface expression of CD11b, HLA-DR, CD40 and CD86 and upregulation of IL-1ß and IL-6. Conclusion: These findings indicate that the content of EVs derived from stressed beta cells can be a mediator of islet inflammation.

Asunto(s)

Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Vesículas Extracelulares , Células Secretoras de Insulina , MicroARNs , Monocitos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/inmunología , Humanos , Estrés del Retículo Endoplásmico/inmunología , MicroARNs/genética , Inflamación/inmunología , Inflamación/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Animales , Línea Celular , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada/inmunología

RESUMEN

Aim: Type II diabetes (T2D) stems from insulin resistance, with ß-cell dysfunction as a hallmark in its progression. Studies reveal that ß cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for ß differentiation and survival, thus may be a potential enhancer of ß-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on ß cells were examined. Results: PAX4 gene delivery significantly improved ß-cell survival, and increased ß-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves ß-cell function, thus could provide therapeutic benefits for T2D treatment.

---

Type II diabetes (T2D) results from insulin resistance, with ß-cell dysfunction playing a pivotal role in its progression. Deficits in ß-cell mass and function have been attributed primarily to ß-cell death through apoptosis; however, recent studies suggest ß-cell failure can also arise from ß-cell dedifferentiation ­ that is, ß cells undergo a loss of mature identity, adopting either progenitor-like or glucagon-producing α cell states during T2D development. Therefore, a strategy preventing ß-cell dedifferentiation while promoting its survival is beneficial for T2D treatment. In this study, we explored whether PAX4, a critical transcription factor for ß differentiation and survival, could alleviate ß-cell dysfunction in human islets derived from T2D patients. To accomplish that, human PAX4 cDNA was delivered into human islets isolated from T2D donors by an adenoviral vector-based vector, Ad5.Pax4 and its effects on ß-cell function were evaluated. The results showed PAX4 expression significantly improved ß-cell survival and increased ß-cell composition in the T2D islets. Notably, PAX4-treated T2D islets exhibited significantly higher basal insulin secretion and glucose-stimulated insulin secretion than control-treated islets. The data demonstrate that PAX4 gene delivery into T2D human islets enhances ß-cell mass and function, and thus may offer therapeutic benefits in the treatment of T2D.

Asunto(s)

Diabetes Mellitus Tipo 2 , Proteínas de Homeodominio , Células Secretoras de Insulina , Insulina , Factores de Transcripción Paired Box , Humanos , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción Paired Box/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/metabolismo , Secreción de Insulina , Técnicas de Transferencia de Gen , Supervivencia Celular , Islotes Pancreáticos/metabolismo , Terapia Genética/métodos

RESUMEN

The experimental study and transplantation of pancreatic islets requires their isolation from the surrounding tissue, and therefore, from the vasculature. Under these conditions, avascular islets rely on the diffusion of peripheral oxygen and nutrients to comply with the requirements of islet cells while responding to changes in body glucose. As a complement to the experimental work, computational models have been widely used to estimate how avascular islets would be affected by the hypoxic conditions found both in culture and transplant sites. However, previous models have been based on simplified representations of pancreatic islets which has limited the reach of the simulations performed. Aiming to contribute with a more realistic model of avascular human islets, in this work we used architectures of human islets reconstructed from experimental data to simulate the availability of oxygen for α, ß and δ-cells, emulating culture and transplant conditions at different glucose concentrations. The modeling approach proposed allowed us to quantitatively estimate how the loss of cells due to severe hypoxia would impact interactions between islet cells, ultimately segregating the islet into disconnected subnetworks. According to the simulations performed, islet encapsulation, by reducing the oxygen available within the islets, could severely compromise cell viability. Moreover, our model suggests that even without encapsulation, only microislets composed of less than 100 cells would remain viable in oxygenation conditions found in transplant sites. Overall, in this article we delineate a novel modeling methodology to simulate detailed avascular islets in experimental and transplant conditions with potential applications in the field of islet encapsulation.

Asunto(s)

Supervivencia Celular , Simulación por Computador , Glucosa , Islotes Pancreáticos , Modelos Biológicos , Oxígeno , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/irrigación sanguínea , Oxígeno/metabolismo , Glucosa/metabolismo , Supervivencia Celular/fisiología , Biología Computacional , Trasplante de Islotes Pancreáticos/métodos , Hipoxia de la Célula/fisiología

RESUMEN

We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization. Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue-derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein-expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system. In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence-positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule. Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.

Asunto(s)

Tejido Adiposo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Neovascularización Fisiológica , Animales , Ratas , Trasplante de Islotes Pancreáticos/métodos , Humanos , Islotes Pancreáticos/inmunología , Trasplante de Células Madre Mesenquimatosas/métodos , Tejido Adiposo/citología , Masculino , Células Cultivadas , Células Endoteliales , Páncreas , Matriz Extracelular Descelularizada

RESUMEN

BACKGROUND/AIM: Although studies on senescence-related genes using human islets of Langerhans have been performed, the expression of senescence-related genes and their association with functional genes in islets remain insufficiently investigated. We aimed to determine whether and what types of senescent-related genes are expressed in islets and identify their correlations with pancreatic function-related genes by using islets isolated for transplantation from individuals of various ages. MATERIALS AND METHODS: Islets from deceased donors of both sexes and different ages were used for analysis. The expression status of senescence-related genes (glutaminase 1, interleukin 6, interleukin 8, cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1A, and senescence-associated beta-galactosidase) and pancreatic function-related genes (glucagon and insulin) was examined by reverse transcription-quantitative polymerase chain reaction, and their relationships with age were investigated. RESULTS: We obtained isolated human islets from 18 deceased multiorgan donors. There was no correlation between donor age and expression of any of the senescence-related genes. Regarding correlations between donor age and pancreatic function-related genes, age was positively correlated only with INS (r=0.49, p=0.03). INS expression was not correlated with that of GLS1 (r=0.23, p=0.34), IL6 (r=-0.06, p=0.79), or IL8 (r=-0.1, p=0.12), but positively related with p16 (r=0.89, p<0.0001), p21 (r=0.51, p=0.02), and SA-ß-gal (r=0.52, p=0.02). CONCLUSION: We showed the functional potential even of aged islets, which were originally thought to be functionally impaired. We were unable to identify any senescence-related genes expressed in islets from donors of different ages. Therefore, a new index is needed to evaluate not only actual chronological age but also organ- and cell-specific age.

Asunto(s)

Senescencia Celular , Islotes Pancreáticos , Donantes de Tejidos , Humanos , Islotes Pancreáticos/metabolismo , Femenino , Masculino , Adulto , Persona de Mediana Edad , Senescencia Celular/genética , Anciano , Envejecimiento/genética , Adulto Joven , Regulación de la Expresión Génica , Factores de Edad , Insulina/metabolismo , Insulina/genética

RESUMEN

Replacement of beta cells through transplantation is a potential therapeutic approach for individuals with pancreas removal or poorly controllable type 1 diabetes. However, stress and death of beta cells pose significant challenges. Circulating miRNA has emerged as potential biomarkers reflecting early beta cell stress and death, allowing for timely intervention. The aim of this study was to identify miRNAs as potential biomarkers for beta cell health. Literature review combined with small RNA sequencing was employed to select islet-enriched miRNA. The release of those miRNA was assessed by RT-qPCR in vivo, using a streptozotocin induced diabetes mouse model and in vitro, through mouse and human islets exposed to varying degrees of hypoxic and cytokine stressors. Utilizing the streptozotocin induced model, we identified 18 miRNAs out of 39 candidate islet-enriched miRNA to be released upon islet stress in vivo. In vitro analysis of culture supernatants from cytokine and/or hypoxia stressed islets identified the release of 45 miRNAs from mouse and 8 miRNAs from human islets. Investigation into the biological pathways targeted by the cytokine- and/or hypoxia-induced miRNA suggested the involvement of MAPK and PI3K-Akt signaling pathways in both mouse and human islets. We have identified miRNAs associated with beta cell health and stress. The findings allowed us to propose a panel of 47 islet-related human miRNA that is potentially valuable for application in clinical contexts of beta cell transplantation and presymptomatic early-stage type 1 diabetes.

Asunto(s)

Diabetes Mellitus Experimental , Islotes Pancreáticos , MicroARNs , Animales , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Ratones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Islotes Pancreáticos/metabolismo , Células Secretoras de Insulina/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estrés Fisiológico/genética , Masculino , RNA-Seq/métodos , Ratones Endogámicos C57BL , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo

RESUMEN

Background: Type II diabetes mellitus (DM) is an increasing health problem that has negative impacts on patients and healthcare systems, worldwide. The development of new therapies with better efficacy, fewer side effects, and lower prices are urgently needed to treat this disease. Aim: To evaluate and compare the therapeutic effects of Nigella sativa (N. sativa) seed and oil on the biochemical parameters and regeneration of pancreatic islets (or islets of Langerhans) of streptozotocin (STZ)-induced diabetic rats. Materials and Methods: The diabetic rat model was prepared by administering a single dose of STZ (35 mg/kg body weight). The whole seed or the oil of N. sativa was administered to the diabetic and control groups for a period of 28 days, but not to the negative and STZ controls. Serum blood glucose, liver enzymes, lipid profile, and renal function tests (uric acid, albumin, total protein, urea, and creatinine) were measured in all groups. After the rats were euthanized, their pancreases were extracted, and then sectioned and fixed on slides in preparation before staining with H&E stain and immunohistochemical study. Results: Treatment of STZ-diabetic rats with N. sativa seeds or oil significantly improved their serum glucose levels, lipid profiles, and liver and renal functions as well as preserved the integrity of pancreatic ß cells. Conclusion: N. sativa seeds and oil demonstrate significant therapeutic improvement effects on DM and its related complications including effective protection of islets of Langerhans. The therapeutic benefits of N. sativa seeds and oil on DM and its related complications are comparable.

Asunto(s)

Diabetes Mellitus Experimental , Nigella sativa , Aceites de Plantas , Semillas , Estreptozocina , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Semillas/química , Ratas , Aceites de Plantas/farmacología , Aceites de Plantas/uso terapéutico , Nigella sativa/química , Masculino , Ratas Wistar , Inmunohistoquímica , Glucemia/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Carum

RESUMEN

Human islets from deceased organ donors have made important contributions to our understanding of pancreatic endocrine function and continue to be an important resource for research studies aimed at understanding, treating, and preventing diabetes. Understanding the impacts of isolation and culture upon the yield of human islets for research is important for planning research studies and islet distribution to distant laboratories. Here, we examine islet isolation and cell culture outcomes at the Alberta Diabetes Institute (ADI) IsletCore (n = 197). Research-focused isolations typically have a lower yield of islet equivalents (IEQ), with a median of 252,876 IEQ, but a higher purity (median 85%) than clinically focused isolations before culture. The median recovery of IEQs after culture was 75%, suggesting some loss. This was associated with a shift toward smaller islet particles, indicating possible islet fragmentation, and occurred within 24 h with no further loss after longer periods of culture (up to 136 h). No overall change in stimulation index as a measure of islet function was seen with culture time. These findings were replicated in a representative cohort of clinical islet preparations from the Clinical Islet Transplant Program at the University of Alberta. Thus, loss of islets occurs within 24 h of isolation, and there is no further impact of extended culture prior to islet distribution for research.

Asunto(s)

Técnicas de Cultivo de Célula , Islotes Pancreáticos , Humanos , Islotes Pancreáticos/citología , Alberta , Masculino , Técnicas de Cultivo de Célula/métodos , Femenino , Adulto , Trasplante de Islotes Pancreáticos/métodos , Persona de Mediana Edad , Células Cultivadas , Anciano , Adulto Joven , Separación Celular/métodos , Adolescente

RESUMEN

The sympathetic nervous system is crucial for the regulation of visceral organ function. For instance, the activation of the sympathetic nervous system promotes glycogenolysis in the liver and modulates glucagon and insulin release from the pancreas, thereby raising blood glucose levels. A decrease in sympathetic nerve activity has the opposite effect. Although such acute effects of sympathetic activity changes have been studied, their long-term outcomes have not been previously examined. In this study, we removed the celiac/superior mesenteric ganglia, where sympathetic postganglionic neurons innervating pancreas and liver locate, and examined its effects on glucose homeostasis and islet size several weeks after surgery. Consistent with the reduction in gluconeogenesis, glucose tolerance improved in gangliectomized mice. However, contrary to our expectation that the inhibition of pancreatic function by sympathetic nerves would be relieved with gangliectomy, insulin or C-peptide release did not increase. Examining the size distribution of pancreatic islets, we identified that the gangliectomy led to a size reduction in large islets and a decrease in the proportion of α and ß cells within each islet, as analyzed by immunostaining for insulin and glucagon, respectively. These results indicate that the absence of sympathetic nerve activity reduces the size of the pancreatic islets within a few weeks to reinstate the homeostatic mechanism of blood glucose levels.

Asunto(s)

Ganglios Simpáticos , Glucagón , Islotes Pancreáticos , Animales , Islotes Pancreáticos/metabolismo , Ganglios Simpáticos/metabolismo , Glucagón/metabolismo , Masculino , Glucemia/metabolismo , Insulina/metabolismo , Ratones Endogámicos C57BL , Ratones , Tamaño de los Órganos , Prueba de Tolerancia a la Glucosa , Ganglionectomía/métodos

RESUMEN

Dietary carbohydrates raise blood glucose levels, and limiting carbohydrate intake improves glycemia in patients with type 2 diabetes. Low carbohydrate intake (< 25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty acid oxidation, the liver produces ketones to serve as an alternative energy source. ß-Hydroxybutyrate (ßHB) is the most abundant ketone. While ßHB has a wide range of functions outside of the pancreas, its direct effects on islet cell function remain understudied. We examined human islet secretory response to acute racemic ßHB treatment and observed increased insulin secretion at a low glucose concentration of 3 mM. Because ßHB is a chiral molecule, existing as both R and S forms, we further studied insulin and glucagon secretion following acute treatment with individual ßHB enantiomers in human and C57BL/6J mouse islets. We found that acute treatment with R-ßHB increased insulin secretion and decreased glucagon secretion at physiological glucose concentrations in both human and mouse islets. Proteomic analysis of human islets treated with R-ßHB over 72 hours showed altered abundance of proteins that may promote islet cell health and survival. Collectively, our data show that physiological concentrations of ßHB influence hormone secretion and signaling within pancreatic islets.

Asunto(s)

Ácido 3-Hidroxibutírico , Glucagón , Secreción de Insulina , Insulina , Islotes Pancreáticos , Ratones Endogámicos C57BL , Ácido 3-Hidroxibutírico/farmacología , Animales , Humanos , Glucagón/metabolismo , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratones , Insulina/metabolismo , Masculino , Glucosa/metabolismo , Femenino

RESUMEN

OBJECTIVES: This study aimed to evaluate the efficacy of a purification method developed for isolating alpha, beta, and delta cells from pancreatic islets of adult mice, extending its application to islets from newborn and aged mice. Furthermore, it sought to examine transcriptome dynamics in mouse pancreatic endocrine islet cells throughout postnatal development and to validate age-related alterations within these cell populations. METHODS: We leveraged the high surface expression of CD71 on beta cells and CD24 on delta cells to FACS-purify alpha, beta, and delta cells from newborn (1-week-old), adult (12-week-old), and old (18-month-old) mice. Bulk RNA sequencing was conducted on these purified cell populations, and subsequent bioinformatic analyses included differential gene expression, overrepresentation, and intersection analysis. RESULTS: Alpha, beta, and delta cells from newborn and aged mice were successfully FACS-purified using the same method employed for adult mice. Our analysis of the age-related transcriptional changes in alpha, beta, and delta cell populations revealed a decrease in cell cycling and an increase in neuron-like features processes during the transition from newborn to adult mice. Progressing from adult to old mice, we identified an inflammatory gene signature related to aging (inflammaging) encompassing an increase in ß-2 microglobulin and major histocompatibility complex (MHC) Class I expression. CONCLUSIONS: Our study demonstrates the effectiveness of our cell sorting technique in purifying endocrine subsets from mouse islets at different ages. We provide a valuable resource for better understanding endocrine pancreas aging and identified an inflammaging gene signature with increased ß-2 microglobulin and MHC Class I expression as a common hallmark of old alpha, beta, and delta cells, with potential implications for immune response regulation and age-related diabetes.

Asunto(s)

Senescencia Celular , Células Secretoras de Glucagón , Células Secretoras de Insulina , Transcriptoma , Animales , Ratones , Células Secretoras de Insulina/metabolismo , Senescencia Celular/genética , Células Secretoras de Glucagón/metabolismo , Ratones Endogámicos C57BL , Regulación hacia Arriba , Células Secretoras de Somatostatina/metabolismo , Masculino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Islotes Pancreáticos/metabolismo , Animales Recién Nacidos , Antígenos CD/metabolismo , Antígenos CD/genética

RESUMEN

The pancreatic islet is the functional and structural unit of the pancreatic endocrine portion. Islet remodeling occurs in both normal development and pathogenesis of type 1 (T1D) and type 2 diabetes (T2D). However, accurately quantifying changes in islet cellular makeup and hormone expressions poses significant challenges due to large intra- and inter-donor heterogeneity and the limited scalability of traditional methods such as immunostaining. The cytometry by time-of-flight (CyTOF) technology enables simultaneous quantification of more than 30 protein markers at single-cell resolution in a high-throughput fashion. Moreover, with distinct DNA and viability markers, single live cells can be explicitly selected in CyTOF. Here, leveraging the CyTOF data generated by the Human Pancreas Analysis Program, we characterized more than 12 million islet cells from 71 donors. Our data revealed continued age-related changes in islet endocrine cell compositions, but the maturity of endocrine cells is reached by 3 years of age. We also observed significant changes in beta cell numbers and key protein expressions, along with a significant increase in bihormonal cells in T1D donors. In contrast, T2D donors exhibited minimal islet remodeling events. Our data shine a light on the islet dynamics during development and diabetes pathogenesis and suggest divergent pathogenesis processes of T1D and T2D. Our comprehensive approach not only elucidates islet plasticity but also establishes a foundation for integrated CyTOF analysis in islet biology and beyond.

Asunto(s)

Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , Humanos , Islotes Pancreáticos/patología , Islotes Pancreáticos/metabolismo , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/metabolismo , Adulto , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/metabolismo , Masculino , Femenino , Adolescente , Persona de Mediana Edad , Niño , Adulto Joven , Preescolar , Citometría de Flujo/métodos , Lactante , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Células Secretoras de Insulina/citología , Anciano , Análisis de la Célula Individual/métodos

RESUMEN

Lipid nanoparticles (LNPs) have recently been used as nanocarriers in drug delivery systems for nucleic acid drugs. Their practical applications are currently primarily limited to the liver and specific organs. However, altering the type and composition ratio of phospholipids improves their distribution in organs other than the liver, such as the spleen and lungs. This study aimed to elucidate the effects of LNP components and particle size on in vivo distribution through systemic circulation to pancreatic islets to achieve better targeting of islets, which are a fundamental therapeutic target for diabetes. Fluorescence-labeled LNPs were prepared using three phospholipids: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), with particle sizes of 30-160 nm (diameter) using a microfluidic device. Baffled-structured iLiNP devices with adjusted flow-rate ratios and total flow rates were used. After the intravenous administration of LNPs to C57BL/6 J mice, the distribution of each LNP type to the major organs, including the pancreas and pancreatic islets, was compared using ex vivo fluorescence imaging and observation of pancreatic tissue sections. DSPC-LNPs- and DOPE-LNPs showed the highest distribution in the spleen and liver, respectively. In contrast, the DOPC-LNPs showed the highest distribution in the pancreas and the lowest distribution in the liver and spleen. In addition, smaller particles showed better distribution throughout the pancreas. The most significant LNP distribution in the islets was observed for DOPC-LNPs with a particle size of 160 nm. Furthermore, larger LNPs tended to be distributed in the islets, whereas smaller LNPs tended to be distributed in the exocrine glands. DOPC-LNPs were distributed in the islets at all cholesterol concentrations, with a high distribution observed at >40% cholesterol and > 3% PEG and the distribution was higher at 24 h than at 4 h. Thus, LNP composition and particle size significantly affected islet distribution characteristics, indicating that DOPC-LNPs may be a drug delivery system for effectively targeting the pancreas and islets.

Asunto(s)

Islotes Pancreáticos , Ratones Endogámicos C57BL , Nanopartículas , Tamaño de la Partícula , Fosfatidilcolinas , Fosfolípidos , Animales , Nanopartículas/química , Nanopartículas/administración & dosificación , Islotes Pancreáticos/metabolismo , Fosfatidilcolinas/química , Fosfolípidos/química , Distribución Tisular , Fosfatidiletanolaminas/química , Masculino , Ratones

RESUMEN

Type 1 diabetes recipients of intrahepatic islet transplantation exhibit glucose-dependent suppression of insulin and activation of glucagon secretion in response to insulin-induced hypoglycemia associated with clinical protection from hypoglycemia. Whether sympathetic activation of adrenergic receptors on transplanted islets is required for these responses in defense against hypoglycemia is not known. To evaluate the adrenergic contribution to posttransplant glucose counterregulation, we performed a randomized, double-blind crossover study of responses during a hyperinsulinemic euglycemic-hypoglycemic clamp under phentolamine (α-adrenergic blockage), propranolol (ß-adrenergic blockage), or placebo infusion. Characteristics of participants (5 females/4 males) were as follows: median (range) age 53 (34-63) yr, diabetes duration 29 (18-56) yr, posttransplant 7.0 (1.9-8.4) yr, HbA1c 5.8 (4.5-6.8)%, insulin in-/dependent 5/4, all on tacrolimus-based immunosuppression. During the clamp, blood pressure was lower with phentolamine and heart rate was lower with propranolol versus placebo (P < 0.05). There was no difference in the suppression of endogenous insulin secretion (derived from C-peptide measurements) during the euglycemic or hypoglycemic phases, and although levels of glucagon were similar with phentolamine or propranolol vs. placebo, the increase in glucagon from eu- to hypoglycemia was greater with propranolol vs. placebo (P < 0.05). Pancreatic polypeptide was greater with phentolamine versus placebo during the euglycemic phase (P < 0.05), and free fatty acids were lower and the glucose infusion rate was higher with propranolol versus placebo during the hypoglycemic phase (P < 0.05 for both). These results indicate that neither physiological α- nor ß-adrenergic blockade attenuates transplanted islet responses to hypoglycemia, suggesting sympathetic reinnervation of the islet graft is not necessary for posttransplant glucose counterregulation.NEW & NOTEWORTHY Whether adrenergic input to islets is necessary for glucose homeostasis in humans is debated. Here, the adrenergic contribution to intrahepatically transplanted islet cell responses to hypoglycemia in individuals with type 1 diabetes was investigated through α- or ß-adrenergic receptor blockade during hyperinsulinemic euglycemic-hypoglycemic clamps. Neither α- nor ß-adrenergic blockage affected the suppression of endogenous insulin or activation of glucagon secretion, suggesting that sympathetic reinnervation of islet grafts is not required for posttransplant defense against hypoglycemia.

Asunto(s)

Antagonistas Adrenérgicos beta , Estudios Cruzados , Diabetes Mellitus Tipo 1 , Técnica de Clampeo de la Glucosa , Hipoglucemia , Trasplante de Islotes Pancreáticos , Fentolamina , Propranolol , Humanos , Femenino , Masculino , Diabetes Mellitus Tipo 1/metabolismo , Persona de Mediana Edad , Adulto , Trasplante de Islotes Pancreáticos/efectos adversos , Hipoglucemia/inducido químicamente , Hipoglucemia/metabolismo , Método Doble Ciego , Antagonistas Adrenérgicos beta/farmacología , Fentolamina/farmacología , Propranolol/farmacología , Glucemia/metabolismo , Glucemia/efectos de los fármacos , Antagonistas Adrenérgicos alfa/farmacología , Insulina/metabolismo , Glucagón/metabolismo , Glucagón/sangre , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo

RESUMEN

Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.

Asunto(s)

Estrés del Retículo Endoplásmico , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Estrés del Retículo Endoplásmico/genética , Islotes Pancreáticos/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Análisis de la Célula Individual , Células Secretoras de Glucagón/metabolismo , Análisis de Secuencia de ARN , Transcriptoma , Estrés Fisiológico

RESUMEN

Autoreactive lymphocytes that infiltrate the pancreatic islet environment and target ß cells are primary drivers of type 1 diabetes. In this issue of Immunity, Srivastava et al.1 examine the role of the islet microenvironment in autoimmunity and find that the scavenging receptor CXCL16 on islet-resident macrophages uptakes oxidized low-density lipoproteins and promotes the differentiation and survival of infiltrating pathogenic CD8+ T cells.

Asunto(s)

Autoinmunidad , Linfocitos T CD8-positivos , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Macrófagos , Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Humanos , Animales , Macrófagos/inmunología , Macrófagos/metabolismo , Linfocitos T CD8-positivos/inmunología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/inmunología