RESUMEN
Background: Endogenous insulin supplementation is essential for individuals with type 1 diabetes (T1D). However, current treatments, including pancreas transplantation, insulin injections, and oral medications, have significant limitations. The development of engineered cells that can secrete endogenous insulin offers a promising new therapeutic strategy for type 1 diabetes (T1D). This approach could potentially circumvent autoimmune responses associated with the transplantation of differentiated ß-cells or systemic delivery of viral vectors. Methods: We utilized CRISPR/Cas9 gene editing coupled with homology-directed repair (HDR) to precisely integrate a promoter-free EMCVIRES-insulin cassette into the 3' untranslated region (UTR) of the GAPDH gene in human HEK-293T cells. Subsequently quantified insulin expression levels in these engineered cells, the viability and functionality of the engineered cells when seeded on different cell vectors (GelMA and Cytopore I) were also assessed. Finally, we investigated the therapeutic potential of EMCVIRES-based insulin secretion circuits in reversing Hyperglycaemia in T1D mice. Result: Our results demonstrate that HDR-mediated gene editing successfully integrated the IRES-insulin loop into the genome of HEK-293T cells, a non-endocrine cell line, enabling the expression of human-derived insulin. Furthermore, Cytopore I microcarriers facilitated cell attachment and proliferation during in vitro culture and enhanced cell survival post-transplantation. Transplantation of these cell-laden microcarriers into mice led to the development of a stable, fat-encapsulated structure. This structure exhibited the expression of the platelet-endothelial cell adhesion molecule CD31, and no significant immune rejection was observed throughout the experiment. Diabetic mice that received the cell carriers reversed hyperglycemia, and blood glucose fluctuations under simulated feeding stimuli were very similar to those of healthy mice. Conclusion: In summary, our study demonstrates that Cytopore I microcarriers are biocompatible and promote long-term cell survival in vivo. The promoter-free EMCVIRES-insulin loop enables non-endocrine cells to secrete mature insulin, leading to a rapid reduction in glucose levels. We have presented a novel promoter-free genetic engineering strategy for insulin secretion and proposed an efficient cell transplantation method. Our findings suggest the potential to expand the range of cell sources available for the treatment of diabetes, offering new avenues for therapeutic interventions.
Asunto(s)
Diabetes Mellitus Tipo 1 , Edición Génica , Hiperglucemia , Células Secretoras de Insulina , Insulina , Humanos , Animales , Hiperglucemia/terapia , Hiperglucemia/metabolismo , Ratones , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Insulina/genética , Células HEK293 , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/genética , Edición Génica/métodos , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Sitios Internos de Entrada al Ribosoma/genética , Regiones Promotoras Genéticas , Sistemas CRISPR-CasRESUMEN
Noncoding RNA plays a pivotal role as novel regulators of endothelial cell function. Type 2 diabetes, acknowledged as a primary contributor to cardiovascular diseases, plays a vital role in vascular endothelial cell dysfunction due to induced abnormalities of glucolipid metabolism and oxidative stress. In this study, aberrant expression levels of circHMGCS1 and MIR4521 were observed in diabetes-induced human umbilical vein endothelial cell dysfunction. Persistent inhibition of MIR4521 accelerated development and exacerbated vascular endothelial dysfunction in diabetic mice. Mechanistically, circHMGCS1 upregulated arginase 1 by sponging MIR4521, leading to decrease in vascular nitric oxide secretion and inhibition of endothelial nitric oxide synthase activity, and an increase in the expression of adhesion molecules and generation of cellular reactive oxygen species, reduced vasodilation and accelerated the impairment of vascular endothelial function. Collectively, these findings illuminate the physiological role and interacting mechanisms of circHMGCS1 and MIR4521 in diabetes-induced cardiovascular diseases, suggesting that modulating the expression of circHMGCS1 and MIR4521 could serve as a potential strategy to prevent diabetes-associated cardiovascular diseases. Furthermore, our findings provide a novel technical avenue for unraveling ncRNAs regulatory roles of ncRNAs in diabetes and its associated complications.
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Diabetes Mellitus Tipo 2 , Endotelio Vascular , Hidroximetilglutaril-CoA Sintasa , MicroARNs , ARN Circular , Animales , Humanos , Masculino , Ratones , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones Endogámicos C57BL , MicroARNs/metabolismo , MicroARNs/genética , ARN Circular/genética , ARN Circular/metabolismo , Hidroximetilglutaril-CoA Sintasa/genéticaRESUMEN
Myocardial fibrosis can trigger heart failure in diabetic cardiomyopathy (DCM), and irisin, an exercise-induced myokine, may have a beneficial effect on cardiac function. However, the specific molecular mechanism between exercise and irisin in the diabetic heart remains not fully explored. This study aimed to investigate how miR-34a mediates exercise-induced irisin to ameliorate myocardial fibrosis and its underlying mechanisms. Type 2 diabetes mellitus (T2DM) with DCM was induced in adult male rats with high-fat diet and streptozotocin injection. The DCM rats were subjected to swimming (60 min/d) and recombinant irisin (r-irisin, 500 µg/kg/d) interventions for 8 weeks, respectively. Cardiac function, cardiomyocyte structure, myocardial fibrosis and its correlated gene and protein expression were analyzed. Swimming intervention alleviated insulin resistance, myocardial fibrosis, and myocardial hypertrophy, and promoted blood glucose homeostasis in T2DM model rats. This improvement was associated with irisin upregulation and miR-34a downregulation in the myocardium, thus enhancing cardiac function. Similar efficacy was observed via intraperitoneal injection of exogenous recombinant irisin. Inhibition of miR-34a in vivo exhibited an anti-myocardial fibrotic effect by promoting irisin secretion through activating sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α)/fibronectin type III domain-containing protein 5 (FNDC5) signal pathway and downregulating myocardial fibrosis markers (collagen I, collagen III, and transforming growth factor-ß1). Therefore, swimming-induced irisin has the potential therapeutic effect on diabetic myocardial fibrosis through activating the miR-34a-mediated SIRT1/PGC-1α/FNDC5 signal pathway.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Cardiomiopatías Diabéticas , Fibronectinas , Fibrosis , MicroARNs , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal , Sirtuina 1 , Natación , Animales , Sirtuina 1/metabolismo , Sirtuina 1/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Fibronectinas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Masculino , Ratas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/etiología , Ratas Sprague-Dawley , Miocardio/metabolismo , Miocardio/patologíaRESUMEN
Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.
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Diferenciación Celular , Proteínas Asociadas a Microtúbulos , Osteoblastos , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Línea Celular , Cilios/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteogénesis/genética , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
PURPOSE: This study comprises an investigation of the role of meteorin-like (Metrnl) in an experimental model of diabetic kidney disease (DKD). METHODS: Twenty-four db/db mice were randomly assigned to one of the following groups: DKD, DKD + Metrnl-/-, and DKD + Metrnl+/+. Plasma Metrnl concentrations were measured using ELISA. Kidney tissues were examined via western blotting, qRT-PCR, and immunohistochemistry to determine the expression levels of inflammatory factors. Electron microscopy was employed to observe stained kidney sections. RESULTS: Compared with the NC group, FBG, BW, and UACR were elevated in the DKD and Metrnl-/- groups, with severe renal pathological injury, decreased serum Metrnl concentration, decreased renal Metrnl expression, and increased expression levels of TNF-α, TGF-ß1, TGF-R1, pSmad2, pSmad3, and α-SMA. In contrast, the Metrnl+/+ group showed decreased FBG and UACR, BUN, TC and TG, increased HDL-C and serum Metrnl concentration, increased renal Metrnl expression, and decreased expression of TNF-α, TGF-ß1, TGF-R1, pSmad2, pSmad3, and α-SMA, compared to the DKD and Metrnl-/- groups. A Pearson bivariate correlation analysis revealed a negative correlation between UACR and Metrnl, and a positive correlation between UACR and TGF-ß1. CONCLUSION: Upregulation of renal Metrnl expression can improve renal injury by downregulating the expression of molecules in the TGF-ß1/Smads signaling pathway in the renal tissues of type 2 diabetic mice; and by reducing the production of fibrotic molecules such as α-SMA.
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Nefropatías Diabéticas , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Regulación hacia Arriba , Animales , Masculino , Ratones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/genética , Riñón/metabolismo , Riñón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genéticaAsunto(s)
Angiopoyetina 1 , Factor A de Crecimiento Endotelial Vascular , Cicatrización de Heridas , Animales , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/genética , Ratones , Terapia Genética/métodos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Técnicas de Transferencia de Gen , HumanosRESUMEN
BACKGROUND: By identifying molecular biological markers linked to cuproptosis in diabetic retinopathy (DR), new pathobiological pathways and more accessible diagnostic markers can be developed. METHODS: The datasets related to DR were acquired from the Gene Expression Omnibus database, while genes associated with cuproptosis were sourced from previously published compilations. Consensus clustering was conducted to delineate distinct DR subclasses. Feature genes were identified utilizing weighted correlation network analysis (WGCNA). Additionally, two machine-learning algorithms were employed to refine the selection of feature genes. Finally, we conducted preliminary validation experiments to ascertain the involvement of cuproptosis in DR development and the transcriptional regulation of critical genes using both the streptozotocin-induced diabetic mouse model and the high glucose-induced BV2 model. RESULTS: In the STZ-induced diabetic mouse retinas, a decrease in the expression of cuproptosis signature proteins (FDX1, DLAT, and NDUFS8) suggested the occurrence of cuproptosis in DR. Subsequently, the expression of eight cuproptosis differential genes was validated through the STZ-induced diabetes and oxygen-induced retinopathy (OIR) models, with the key gene SLC31A1 showing upregulation in both models and dataset species. Further analyses, including weighted gene co-expression network analysis, GSVA, and immune infiltration analysis, indicated a close correlation between cuproptosis and microglia function. Additionally, validation in an in vitro model of microglia indicated the occurrence of cuproptosis in microglia under high glucose conditions, alongside abnormal expression of STAT1 with SLC31A1. CONCLUSION: Our findings suggest that STAT1/SLC31A1 may pave the way for a deeper comprehension of the mechanistic basis of DR and offer potential therapeutic avenues.
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Diabetes Mellitus Experimental , Retinopatía Diabética , Factor de Transcripción STAT1 , Animales , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Ratones , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Masculino , Retina/metabolismo , Retina/patología , Ratones Endogámicos C57BL , Regulación de la Expresión Génica , Redes Reguladoras de Genes , HumanosRESUMEN
BACKGROUND: The Lin-Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored. METHODS: In this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA. RESULTS: We uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype. CONCLUSION: Our findings highlight that chronic diabetes adversely alters HSCs' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.
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Diabetes Mellitus Tipo 2 , Células Madre Hematopoyéticas , MicroARNs , Transcriptoma , Animales , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Madre Hematopoyéticas/metabolismo , Perfilación de la Expresión Génica , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Masculino , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismoRESUMEN
Pregestational diabetes mellitus (PGDM) has an impact on fetal bone formation, but the underlying mechanism is still obscure. Although miRNAs have been extensively investigated throughout bone formation, their effects on fetal bone development caused by PGDM still need clarification. This study intends to examine the mechanism by which hyperglycemia impairs the bone formation of offspring via miR-322-5p (miR-322). In this study, miR-322 was selected by systemically screening utilizing bioinformatics and subsequent validation experiments. Using streptozotocin (STZ)-induced diabetic mice and ATDC5 cell lines, we found that miR-322 was abundantly expressed in the proliferative and hypertrophic zones of the growth plate, and its expression pattern was disturbed in the presence of hyperglycemia, suggesting that miR-322 is involved in the chondrocyte proliferation and differentiation in absence/presence of hyperglycemia. This observation was proved by manipulating miR-322 expression in ATDC5 cells by transfecting mimic and inhibitor of miR-322. Furthermore, Adamts5, Col12a1, and Cbx6 were identified as the potential target genes of miR-322, verified by the co-transfection of miR-322 inhibitor and the siRNAs, respectively. The evaluation criteria are the chondrocyte proliferation and differentiation and their relevant key gene expressions (proliferation: Sox9 and PthIh; differentiation: Runx2 and Col10a1) after manipulating the gene expressions in ATDC5 cells. This study revealed the regulative role miR-322 on chondrocyte proliferation and differentiation of growth plate by targeting Adamts5, Col12a1, and Cbx6 in hyperglycemia during pregnancy. This translational potential represents a promising avenue for advancing our understanding of bone-related complications in diabetic pregnancy and mitigating bone deficiencies in diabetic pregnant individuals, improving maternal and fetal outcomes.
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Diferenciación Celular , Proliferación Celular , Condrocitos , Diabetes Mellitus Experimental , Diabetes Gestacional , Placa de Crecimiento , MicroARNs , Animales , Femenino , Ratones , Embarazo , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Condrocitos/metabolismo , Condrocitos/patología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Gestacional/metabolismo , Diabetes Gestacional/genética , Diabetes Gestacional/patología , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
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.
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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/metabolismoRESUMEN
Islet transplantation is a promising therapy for diabetes treatment. However, the molecular underpinnings governing the immune response, particularly T-cell dynamics in syngeneic and allogeneic transplant settings, remain poorly understood. Understanding these T cell dynamics is crucial for enhancing graft acceptance and managing diabetes treatment more effectively. This study aimed to elucidate the molecular mechanisms, gene expression differences, biological pathway alterations, and intercellular communication patterns among T-cell subpopulations after syngeneic and allogeneic islet transplantation. Using single-cell RNA sequencing, we analyzed cellular heterogeneity and gene expression profiles using the Seurat package for quality control and dimensionality reduction through t-SNE. Differentially expressed genes (DEGs) were analyzed among different T cell subtypes. GSEA was conducted utilizing the HALLMARK gene sets from MSigDB, while CellChat was used to infer and visualize cell-cell communication networks. Our findings revealed genetic variations within T-cell subpopulations between syngeneic and allogeneic islet transplants. We identified significant DEGs across these conditions, highlighting molecular discrepancies that may underpin rejection or other immune responses. GSEA indicated activation of the interferon-alpha response in memory T cells and suppression in CD4+ helper and γδ T cells, whereas TNFα signaling via NFκB was particularly active in regulatory T cells, γδ T cells, proliferating T cells, and activated CD8+ T cells. CellChat analysis revealed complex communication patterns within T-cell subsets, notably between proliferating T cells and activated CD8+ T cells. In conclusion, our study provides a comprehensive molecular landscape of T-cell diversity in islet transplantation. The insights into specific gene upregulation in xenotransplants suggest potential targets for improving graft tolerance. The differential pathway activation across T-cell subsets underscores their distinct roles in immune responses posttransplantation.
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Trasplante de Islotes Pancreáticos , Análisis de la Célula Individual , Trasplante Homólogo , Animales , Ratones , Análisis de la Célula Individual/métodos , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Transcriptoma , Trasplante Isogénico , Perfilación de la Expresión Génica , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/genética , Masculino , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Linfocitos T/metabolismo , Supervivencia de Injerto/inmunología , Supervivencia de Injerto/genéticaRESUMEN
Diabetic osteoporosis (DO) presents significant clinical challenges. This study aimed to investigate the potential of magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells (BMSCs) to deliver miR-15b-5p, thereby targeting and downregulating glial fibrillary acidic protein (GFAP) expression in rat DO models. Data was sourced from DO-related RNA-seq datasets combined with GEO and GeneCards databases. Rat primary BMSCs, bone marrow-derived macrophages (BMMs), and osteoclasts were isolated and cultured. EVs were separated, and GMNPE targeting EVs were synthesized. Bioinformatic analysis revealed a high GFAP expression in DO-related RNA-seq and GSE26168 datasets for disease models. Experimental results confirmed elevated GFAP in rat DO bone tissues, promoting osteoclast differentiation. miR-15b-5p was identified as a GFAP inhibitor, but was significantly downregulated in DO and enriched in BMSC-derived EVs. In vitro experiments showed that GMNPE-EVs could transfer miR-15b-5p to osteoclasts, downregulating GFAP and inhibiting osteoclast differentiation. In vivo tests confirmed the therapeutic potential of this approach in alleviating rat DO. Collectively, GMNPE-EVs can effectively deliver miR-15b-5p to osteoclasts, downregulating GFAP expression, and hence, offering a therapeutic strategy for rat DO.
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Vesículas Extracelulares , Proteína Ácida Fibrilar de la Glía , Células Madre Mesenquimatosas , MicroARNs , Osteoclastos , Osteoporosis , Ratas Sprague-Dawley , Animales , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Mesenquimatosas/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Osteoporosis/metabolismo , Osteoporosis/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Ratas , Osteoclastos/metabolismo , Masculino , Diferenciación Celular , Nanopartículas de Magnetita , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Complicaciones de la Diabetes/metabolismo , Complicaciones de la Diabetes/genéticaRESUMEN
It has been demonstrated that circular RNAs (circRNAs) are associated with the development of diabetic retinopathy (DR). Nevertheless, the function of circSLC16A10 in the development of DR remains unclear. In order to investigate the role of circSLC16A10, we employed cell and animal models of DR. An analysis of a public database revealed that hsa_circSLC16A10 was expressed at lower levels in DR patients than in diabetic patients without DR or healthy controls. Additionally, the level of hsa_circSLC16A10 was lower in high glucose (HG)-exposed ARPE-19 cells and diabetic mice. hsa_circSLC16A10 was observed to be mainly distributed in the cytoplasm. Moreover, overexpression of hsa_circSLC16A10 alleviated HG-induced endoplasmic reticulum stress and cell apoptosis in vitro. Furthermore, overexpression of hsa_circSLC16A10 ameliorated HG-induced mitochondrial dysfunction, as evidenced by improvements in mitochondrial structure and function. hsa_circSLC16A10 acted as a hsa-miR-761-5p sponge to increase MFN2 expression. MFN2 knockdown or hsa-miR-761-5p overexpression partially reversed the protective effect of hsa_circSLC16A10 in vitro. The protective effect of mmu_circSLC16A10 against DR was confirmed in an animal model of DR. These findings indicate that circSLC16A10 may regulate DR progression by improving mitochondrial function via the miR-761-5p/MFN2 axis.
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Retinopatía Diabética , GTP Fosfohidrolasas , MicroARNs , Mitocondrias , ARN Circular , Animales , Humanos , Masculino , Ratones , Apoptosis , Línea Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Estrés del Retículo Endoplásmico , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Ratones Endogámicos C57BL , MicroARNs/metabolismo , MicroARNs/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , ARN Circular/genética , ARN Circular/metabolismoRESUMEN
The poor healing characteristics of diabetic foot ulcers are partially attributed to diabetes-induced pro-inflammatory wounds. Our previous study reported that both miR-146a-5p and miR-200b-3p decrease endothelial inflammation in human aortic endothelial cells and db/db diabetic mice. Although miR-146a-5p has been reported to improve diabetic wound healing, the role of miR-200b-3p is not clear. This study compared the roles of these miRNAs in diabetic wound healing. Two 8-mm full-thickness wounds were created in 12-week-old male db/db mice on the left and right back. After surgery, 100 ng miR-146a-5p, miR-200b-3p, or miR-negative control (NC) was injected in each wound. Full-thickness skin samples were harvested from mice at the 14th day for real-time polymerase chain reaction and immunohistochemistry analyses. At the 14th day, the miR-200b-3p group showed better wound healing and greater granulation tissue thickness than the miR-146a-5p group. The miR-200b-3p group showed a significant decrease of IL-6 and IL-1ß gene expression and a significant increase of Col3α1 gene expression compared to those in the miR-NC group. The miR-200b-3p group had the lowest gene expression of TGF-ß1, followed by the miR-146a-5p and miR-NC groups. Our findings suggest that the miR-200b-3p group had better healing characteristics than the other two groups. Immunohistochemical staining revealed that CD68 immunoreactivity was significantly decreased in both the miR-146a-5p and miR-200b-3p groups compared with that in the miR-NC group. In addition, CD31 immunoreactivity was significantly higher in the miR-200b-3p group than in the miR-146a-5p group. In conclusion, these results suggest that miR-200b-3p is more effective than miR-146a-5p in promoting diabetic wound healing through its anti-inflammatory and pro-angiogenic effects.
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MicroARNs , Cicatrización de Heridas , MicroARNs/genética , MicroARNs/metabolismo , Animales , Cicatrización de Heridas/genética , Masculino , Ratones , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Pie Diabético/genética , Pie Diabético/metabolismo , Pie Diabético/patología , Neovascularización Fisiológica/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Antígenos CD/genética , Antígenos CD/metabolismo , Piel/metabolismo , Piel/patología , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Ratones Endogámicos C57BL , Molécula CD68RESUMEN
BACKGROUND: Diabetes mellitus (DM)-induced testicular damage is associated with sexual dysfunction and male infertility in DM patients. However, the pathogenesis of DM-induced testicular damage remains largely undefined. METHODS: A streptozotocin (STZ)-induced diabetic model and high glucose (HG)-treated in vitro diabetic model were established. The histological changes of testes were assessed by H&E staining. Serum testosterone, iron, MDA and GSH levels were detected using commercial kits. Cell viability and lipid peroxidation was monitored by MTT assay and BODIPY 581/591 C11 staining, respectively. qRT-PCR, immunohistochemistry (IHC) or Western blotting were employed to detect the levels of BRD7, Clusterin, EZH2 and AMPK signaling molecules. The associations among BRD7, EZH2 and DNMT3a were detected by co-IP, and the transcriptional regulation of Clusterin was monitored by methylation-specific PCR (MSP) and ChIP assay. RESULTS: Ferroptosis was associated with DM-induced testicular damage in STZ mice and HG-treated GC-1spg cells, and this was accompanied with the upregulation of BRD7. Knockdown of BRD7 suppressed HG-induced ferroptosis, as well as HG-induced Clusterin promoter methylation and HG-inactivated AMPK signaling in GC-1spg cells. Mechanistical studies revealed that BRD7 directly bound to EZH2 and regulated Clusterin promoter methylation via recruiting DNMT3a. Knockdown of Clusterin or inactivation of AMPK signaling reverses BRD7 silencing-suppressed ferroptosis in GC-1spg cells. In vivo findings showed that lack of BRD7 protected against diabetes-induced testicular damage and ferroptosis via increasing Clusterin expression and activating AMPK signaling. CONCLUSION: BRD7 suppressed Clusterin expression via modulating Clusterin promoter hypermethylation in an EZH2 dependent manner, thereby suppressing AMPK signaling to facilitate ferroptosis and induce diabetes-associated testicular damage.
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Proteínas Quinasas Activadas por AMP , Clusterina , Metilación de ADN , Diabetes Mellitus Experimental , Ferroptosis , Regiones Promotoras Genéticas , Transducción de Señal , Testículo , Animales , Masculino , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Línea Celular , Clusterina/genética , Clusterina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/complicaciones , ADN Metiltransferasa 3A/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Ferroptosis/genética , Ratones Endogámicos C57BL , Testículo/metabolismo , Testículo/patologíaRESUMEN
Diabetic peripheral neuropathy (DPN) is a common complication associated with diabetes, and can affect quality of life considerably. Dorsal root ganglion (DRG) plays an important role in the development of DPN. However, the relationship between DRG and the pathogenesis of DPN still lacks a thorough exploration. Besides, a more in-depth understanding of the cell type composition of DRG, and the roles of different cell types in mediating DPN are needed. Here we conducted single-cell RNA-seq (scRNA-seq) for DRG tissues isolated from healthy control and DPN rats. Our results demonstrated DRG includes eight cell-type populations (e.g., neurons, satellite glial cells (SGCs), Schwann cells (SCs), endothelial cells, fibroblasts). In the heterogeneity analyses of cells, six neuron sub-types, three SGC sub-types and three SC sub-types were identified, additionally, biological functions related to cell sub-types were further revealed. Cell communication analysis showed dynamic interactions between neurons, SGCs and SCs. We also found that the aberrantly expressed transcripts in sub-types of neurons, SGCs and SCs with DPN were associated with diabetic neuropathic pain, cell apoptosis, oxidative stress, etc. In conclusion, this study provides a systematic perspective of the cellular composition and interactions of DRG tissues, and suggests that neurons, SGCs and SCs play vital roles in the progression of DPN. Our data may provide a valuable resource for future studies regarding the pathophysiological effect of particular cell type in DPN.
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Neuropatías Diabéticas , Ganglios Espinales , Perfilación de la Expresión Génica , Células de Schwann , Análisis de la Célula Individual , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Neuropatías Diabéticas/patología , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/metabolismo , Ratas , Células de Schwann/metabolismo , Células de Schwann/patología , Masculino , Transcriptoma , Neuronas/metabolismo , Neuronas/patología , Ratas Sprague-Dawley , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Análisis de Expresión Génica de una Sola CélulaRESUMEN
BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. METHODS: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac/skeletal myopathy using cellular and animal models. RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. Deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression using an adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 regulates hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.
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Tolerancia al Ejercicio , Transportador de Glucosa de Tipo 1 , Ratones Noqueados , Animales , Humanos , Masculino , Ratones , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/fisiopatología , Cardiomiopatías Diabéticas/etiología , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Hiperglucemia/metabolismo , Hiperglucemia/genética , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
Islet ß-cell dysfunction is an underlying factor for type I diabetes (T1D) development. Insulin sensing and secretion are tightly regulated in ß-cells at multiple subcellular levels. The epithelial intermediate filament (IF) protein keratin (K) 8 is the main ß-cell keratin, constituting the filament network with K18. To identify the cell-autonomous functions of K8 in ß-cells, mice with targeted deletion of ß-cell K8 (K8flox/flox; Ins-Cre) were analyzed for islet morphology, ultrastructure, and integrity, as well as blood glucose regulation and streptozotocin (STZ)-induced diabetes development. Glucose transporter 2 (GLUT2) localization was studied in ß-cells in vivo and in MIN6 cells with intact or disrupted K8/K18 filaments. Loss of ß-cell K8 leads to a major reduction in K18. Islets without ß-cell K8 are more fragile, and these ß-cells display disjointed plasma membrane organization with less membranous E-cadherin and smaller mitochondria with diffuse cristae. Lack of ß-cell K8 also leads to a reduced glucose-stimulated insulin secretion (GSIS) response in vivo, despite undisturbed systemic blood glucose regulation. K8flox/flox, Ins-Cre mice have a decreased sensitivity to STZ compared with K8 wild-type mice, which is in line with decreased membranous GLUT2 expression observed in vivo, as GLUT2 is required for STZ uptake in ß-cells. In vitro, MIN6 cell plasma membrane GLUT2 is rescued in cells overexpressing K8/K18 filaments but mistargeted in cells with disrupted K8/K18 filaments. ß-Cell K8 is required for islet and ß-cell structural integrity, normal mitochondrial morphology, and GLUT2 plasma membrane targeting, and has implications on STZ sensitivity as well as systemic insulin responses.NEW & NOTEWORTHY Keratin 8 is the main cytoskeletal protein in the cytoplasmic intermediate filament network in ß-cells. Here for the first time, we assessed the ß-cell autonomous mechanical and nonmechanical roles of keratin 8 in ß-cell function. We demonstrated the importance of keratin 8 in islet and ß-cell structural integrity, maintaining mitochondrial morphology and GLUT2 plasma membrane targeting.
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Membrana Celular , Diabetes Mellitus Experimental , Transportador de Glucosa de Tipo 2 , Células Secretoras de Insulina , Queratina-8 , Mitocondrias , Animales , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Ratones , Queratina-8/metabolismo , Queratina-8/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Glucosa/metabolismo , Insulina/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Diabetic ulcers (DUs) are characterized by chronic inflammation and delayed re-epithelialization, with a high incidence and weighty economic burden. The primary therapeutic strategies for refractory wounds include surgery, non-invasive wound therapy, and drugs, while the optimum regimen remains controversial. Sirtuin-6 (SIRT6) is a histone deacetylase and a key epigenetic factor that exerts anti-inflammatory and pro-proliferatory effects in wound healing. However, the exact function of SIRT6 in DUs remains unclear. METHODS: We generated tamoxifen-inducible SIRT6 knockout mice by crossing SIRT6flox/flox homozygous mice with UBC-creERT2+ transgenic mice. Systemic SIRT6 null mice, under either normal or diabetic conditions, were utilized to assess the effects of SIRT6 in DUs treatment. Gene and protein expressions of SIRT6 and inflammatory cytokines were measured by Western blotting and RT-qPCR. Histopathological examination confirmed the altered re-epithelialization (PCNA), inflammation (NF-κB p50 and F4/80), and angiogenesis (CD31) markers during DUs restoration. RESULTS: Knockout of SIRT6 inhibited the healing ability of DUs, presenting attenuated re-epithelialization (PCNA), exacerbated inflammation responses (NF-κB p50, F4/80, Il-1ß, Tnf-α, Il-6, Il-10, and Il-4), and hyperplasia vascular (CD31) compared with control mice. CONCLUSIONS: SIRT6 could boost impaired wound healing through improving epidermal proliferation, inflammation, and angiogenesis. Our study highlighted the therapeutic potential of the SIRT6 agonist for DUs treatment.
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Ratones Noqueados , Sirtuinas , Cicatrización de Heridas , Animales , Cicatrización de Heridas/genética , Sirtuinas/genética , Sirtuinas/metabolismo , Sirtuinas/deficiencia , Ratones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Citocinas/metabolismo , Ratones Endogámicos C57BL , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , MasculinoRESUMEN
Diabetic cardiomyopathy (DCM) is characterized by oxidative damage and inflammatory responses. Myeloid differentiation protein 1 (MD1) exhibits antioxidant and anti-inflammatory properties. However, the specific role of MD1 in DCM has yet to be elucidated. This study aims to investigate the role of MD1 in DCM and to elucidate the underlying mechanisms. We utilized a gain-of-function approach to explore the involvement of MD1 in DCM. Diabetes was induced in MD1-transgenic (MD1-TG) mice and their wild-type (WT) counterparts via streptozotocin (STZ) injection. Additionally, a diabetes cell model was established using H9c2 cells exposed to high glucose levels. We conducted comprehensive evaluations, including pathological analyses, echocardiography, electrocardiography, and molecular assessments, to elucidate the underlying mechanisms of MD1 in DCM. Notably, MD1 expression was reduced in the hearts of STZ-induced diabetic mice. Overexpression of MD1 significantly improved cardiac function and markedly inhibited ventricular pathological hypertrophy and fibrosis in these mice. Furthermore, MD1 overexpression resulted in a substantial decrease in myocardial reactive oxygen species (ROS) accumulation, mitigating myocardial oxidative stress and reducing the levels of inflammation-related markers such as IL-1ß, IL-6, and TNF-α. Mechanistically, MD1 overexpression inhibited the activation of the TLR4/STAT3 signaling pathway, as demonstrated in both in vivo and in vitro experiments. The overexpression of MD1 significantly impeded pathological cardiac remodeling and improved cardiac function in STZ-induced diabetic mice. This effect was primarily attributed to a reduction in ROS accumulation and mitigation of myocardial oxidative stress and inflammation, facilitated by the inhibition of the TLR4/STAT3 signaling pathway.