RESUMO
AIM: In previous studies, the researchers observed an increase in insulin secretion in STZ-treated diabetic rats following treatment with the hydroalcoholic extract of Securigera securidaca (HESS) seeds. This study focuses on the relationship between the antioxidant properties of HESS with changes in diabetic pancreatic tissue and the gene expression of factors that impact insulin secretion. METHODS: In this controlled experimental study, three varying doses of HESS were administered to three groups of diabetic rats induced by STZ. Oxidative stress indicators like total antioxidant capacity (TAC), total oxidant status (TOS) and malondialdehyde were assessed in both pancreatic and liver tissues. Pancreatic histology was studied post-haematoxylin staining. Insulin and FGF21 levels in the blood were measured using the ELISA method. The expression of Nrf2 and FGF21 genes in the pancreas and liver, along with MafA and PDX-1 genes in the pancreas, was quantified using real-time PCR. RESULTS: The administration of HESS in varying doses led to a dose-dependent rise in blood insulin levels and a decrease in blood glucose levels and oxidative stress. By reducing oxidative stress, HESS treatment lowered the heightened levels of NRF2 and FGF21 in the liver and pancreas of diabetic rats, improving pancreatic tissue health. As oxidative stress decreased, the expression of MafA and PDX1 genes in the pancreas approached levels seen in healthy rats. CONCLUSION: HESS elicits an increase in insulin secretion through the mitigation of oxidative stress and tissue damage, as well as the modulation of gene expression related to the insulin transcription factors PDX-1 and MafA.
Assuntos
Diabetes Mellitus Experimental , Secreção de Insulina , Insulina , Extratos Vegetais , Sementes , Regulação para Cima , Animais , Extratos Vegetais/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Ratos , Sementes/química , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Masculino , Securidaca , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Antioxidantes/farmacologia , Fígado/metabolismo , Transativadores , Proteínas de HomeodomínioRESUMO
Pancreatic cancer, one of the deadliest human malignancies, is characterized by a fibro-inflammatory tumor microenvironment and wide array of metabolic alterations. To comprehensively map metabolism in a cell type-specific manner, we harnessed a unique single-cell RNA-sequencing dataset of normal human pancreata. This was compared with human pancreatic cancer samples using a computational pipeline optimized for this study. In the cancer cells we observed enhanced biosynthetic programs. We identified downregulation of mitochondrial programs in several immune populations, relative to their normal counterparts in healthy pancreas. Although granulocytes, B cells, and CD8+ T cells all downregulated oxidative phosphorylation, the mechanisms by which this occurred were cell type specific. In fact, the expression pattern of the electron transport chain complexes was sufficient to identify immune cell types without the use of lineage markers. We also observed changes in tumor-associated macrophage (TAM) lipid metabolism, with increased expression of enzymes mediating unsaturated fatty acid synthesis and upregulation in cholesterol export. Concurrently, cancer cells exhibited upregulation of lipid/cholesterol receptor import. We thus identified a potential crosstalk whereby TAMs provide cholesterol to cancer cells. We suggest that this may be a new mechanism boosting cancer cell growth and a therapeutic target in the future.
Assuntos
Neoplasias Pancreáticas , Microambiente Tumoral , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Metabolismo dos Lipídeos , Pâncreas/metabolismo , Pâncreas/patologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Colesterol/metabolismo , Fosforilação Oxidativa , Mitocôndrias/metabolismo , Análise de Célula ÚnicaRESUMO
The field of epigenetics broadly seeks to define heritable phenotypic modifications that occur within cells without changes to the underlying DNA sequence. These modifications allow for precise control and specificity of function between cell types-ultimately creating complex organ systems that all contain the same DNA but only have access to the genes and sequences necessary for their cell-type-specific functions. The pancreas is an organ that contains varied cellular compartments with functions ranging from highly regulated glucose-stimulated insulin secretion in the ß-cell to the pancreatic ductal cells that form a tight epithelial lining for the delivery of digestive enzymes. With diabetes cases on the rise worldwide, understanding the epigenetic mechanisms driving ß-cell identity, function, and even disease is particularly valuable. In this chapter, we will discuss the known epigenetic modifications in pancreatic islet cells, how they are deposited, and the environmental and metabolic contributions to epigenetic mechanisms. We will also explore how a deeper understanding of epigenetic effectors can be used as a tool for diabetes therapeutic strategies.
Assuntos
Epigênese Genética , Pâncreas , Humanos , Pâncreas/embriologia , Pâncreas/metabolismo , Animais , Células Secretoras de Insulina/metabolismo , Metilação de DNA/genética , Diabetes Mellitus/genéticaRESUMO
In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-KrasG12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis.
Assuntos
Heme Oxigenase-1 , Macrófagos , Fator 2 Relacionado a NF-E2 , Neoplasias Pancreáticas , Fatores de Transcrição , Animais , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas/efeitos dos fármacos , Humanos , Pancreatite/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/genética , Camundongos Knockout , Microambiente Tumoral/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Proteínas que Contêm Bromodomínio , Proteínas de Membrana , Proteínas NuclearesRESUMO
Oleuropein (OLP) is a naturally occurring phenolic compound in olive plant with antioxidant and anti-inflammatory potential and can possibly be used in treating pancreatic injuries. This investigation aimed to follow the molecular mechanism behind the potential therapeutic effect of OLP against pancreatic injury persuaded by ischemia-reperfusion (I/R). Pancreatic I/R injury was induced by splenic artery occlusion for 60 min followed by reperfusion. Oral administration of OLP (10 and 20 mg/kg) for 2 days significantly alleviated I/R-persuaded oxidative damage and inflammatory responses in pancreatic tissue as indicated by the decreased malondialdehyde (MDA) content and increased glutathione peroxidase (GPx) activity, accompanied by the suppression of myeloperoxidase (MPO) activity and reduced levels of interleukin-1beta (IL-1ß), nuclear factor kappa B (NF-κB), and tumor necrosis factor alpha (TNF-α) in pancreatic tissues. Furthermore, OLP treatment markedly restored the serum levels of amylase, trypsinogen-activated peptide (TAP), and lipase, with concurrent improvement in pancreatic histopathological alterations. Moreover, treatment with OLP regulated the pancreatic expression of inducible nitric oxide synthase (iNOS) and high-mobility group box 1 (HMGB1) relative to rats of the pancreatic IR group. Thus, OLP treatment significantly alleviates the I/R-induced pancreatic injury by inhibiting oxidative stress and inflammation in rats through downregulation of HMGB1 and its downstream NF-κB signaling pathway.
Assuntos
Proteína HMGB1 , Glucosídeos Iridoides , Iridoides , NF-kappa B , Estresse Oxidativo , Pâncreas , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Glucosídeos Iridoides/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteína HMGB1/metabolismo , NF-kappa B/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Ratos , Iridoides/farmacologia , Iridoides/uso terapêutico , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Ratos Sprague-Dawley , Antioxidantes/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêuticoRESUMO
Severe acute pancreatitis (SAP) is an inflammatory disease with varying severity, ranging from mild local inflammation to severe systemic disease, with a high incidence rate and mortality. Current drug treatments are not ideal. Therefore, safer and more effective therapeutic drugs are urgently needed. 7α,14ß-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione DGA, a diterpenoid compound derivatized from glaucocalyxin A, exhibits anti-inflammatory activity. In this study, we demonstrated the therapeutic potential of DGA against SAP and elucidated the underlying mechanisms. Treatment with DGA markedly (1) inhibited death of RAW264.7 and J774a.1 cells induced by Nigericin and lipopolysaccharide, (2) alleviated edema, acinar cell vacuolation, necrosis, and inflammatory cell infiltration of pancreatic tissue in mice, and (3) inhibited the activity of serum lipase and the secretion of inflammatory factor IL-1ß. DGA significantly reduced the protein expression of IL-1ß and NLRP3 and inhibited the phosphorylation of NF-κB. However, DGA exhibited no inhibitory effect on the expression of caspase-1, gasdermin D (GSDMD), NF-κB, TNF-α, or apoptosis-associated speck-like protein (ASC) and on the cleavage of caspase-1 or GSDMD. Molecular docking simulation confirmed that DGA can bind to TLR4 and IL-1 receptor. In conclusion, DGA may effectively alleviate the symptoms of SAP in mice and macrophages by inhibiting the binding of TLR4 and IL-1 receptor to their ligands; therefore, DGA is a promising drug candidate for the treatment of patients with SAP.
Assuntos
Macrófagos , Pancreatite , Piroptose , Animais , Piroptose/efeitos dos fármacos , Camundongos , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Pancreatite/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Células RAW 264.7 , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/farmacologia , Camundongos Endogâmicos C57BL , Interleucina-1beta/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Diterpenos do Tipo Caurano/farmacologia , Diterpenos do Tipo Caurano/uso terapêutico , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Lipopolissacarídeos , Simulação de Acoplamento Molecular , Linhagem Celular , Lipase/metabolismoRESUMO
Obesity and sedentarism are associated with increased liver and pancreatic fat content (LFC and PFC, respectively) as well as impaired organ metabolism. Exercise training is known to decrease organ ectopic fat but its effects on organ metabolism are unclear. Genetic background affects susceptibility to obesity and the response to training. We studied the effects of regular exercise training on LFC, PFC, and metabolism in monozygotic twin pairs discordant for BMI. We recruited 12 BMI-discordant monozygotic twin pairs (age 40.4, SD 4.5 years; BMI 32.9, SD 7.6, 8 female pairs). Ten pairs completed six months of training intervention. We measured hepatic insulin-stimulated glucose uptake using [18F]FDG-PET and fat content using magnetic resonance spectroscopy before and after the intervention. At baseline LFC, PFC, gamma-glutamyl transferase (GT), and hepatic glucose uptake were significantly higher in the heavier twins compared to the leaner co-twins (p = 0.018, p = 0.02 and p = 0.01, respectively). Response to training in liver glucose uptake and GT differed between the twins (Time*group p = 0.04 and p = 0.004, respectively). Liver glucose uptake tended to decrease, and GT decreased only in the heavier twins (p = 0.032). In BMI-discordant twins, heavier twins showed higher LFC and PFC, which may underlie the observed increase in liver glucose uptake and GT. These alterations were mitigated by exercise. The small number of participants makes the results preliminary, and future research with a larger pool of participants is warranted.
Assuntos
Índice de Massa Corporal , Exercício Físico , Glucose , Metabolismo dos Lipídeos , Fígado , Obesidade , Pâncreas , Tomografia por Emissão de Pósitrons , Humanos , Feminino , Fígado/metabolismo , Fígado/diagnóstico por imagem , Adulto , Obesidade/metabolismo , Obesidade/genética , Glucose/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Masculino , Pâncreas/metabolismo , Pâncreas/diagnóstico por imagem , Gêmeos Monozigóticos , Pessoa de Meia-IdadeRESUMO
Stem cell-derived ß-cells (SC-BCs) represent a potential source for curing diabetes. To date, in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation, indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here, we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ß-cells in vitro. To this aim, a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ß-cell markers, but did not improve ß-cell identity. In conclusion, the study describes the production of a PanMa hydrogel, which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs.
Assuntos
Diferenciação Celular , Matriz Extracelular , Hidrogéis , Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Hidrogéis/química , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Matriz Extracelular/metabolismo , Animais , Suínos , Pâncreas/citologia , Pâncreas/metabolismo , Técnicas de Cultura de Células/métodos , Células CultivadasRESUMO
Herbs have been used as medicines since antiquity, and it has been discovered that the human body responds well to herbal remedies. Research on the effect of butin was conducted in the current study in the alloxan-induced diabetic rat paradigm. A total of 30 Wistar rats were randomly assigned into the following groups (n = 6): I-Normal; II-Alloxan-induced (50 mg/kg); III-Alloxan + butin 25 mg/kg; IV-Alloxan + butin 50 mg/kg; V-Butin per se 50 mg/kg. Various diabetic parameters (blood glucose, insulin, HbA1c), lipid profile, inflammatory (TNF-α, IL-1ß, IL-6 and NF-κB), antioxidant enzymes (CAT, SOD and GSH), oxidative stress indicators (MDA), apoptosis marker (caspase-3), hepatic markers (ALT and AST), and histopathological changes were assessed. Additionally, molecular docking and dynamics were performed to evaluate the interaction of butin with target proteins. Butin treatment, at both doses, significantly restored biochemical parameters and preserved pancreatic histopathology in diabetic rats. It effectively modulated blood parameters, lipid profiles, inflammatory markers, apoptosis, antioxidant enzyme activity, oxidative stress, and hepatic markers. Molecular docking revealed that butin binds to proteins such as caspase-3 (1NME), NF-κB (1SVC), and serum insulin (4IBM) with binding affinities of - 7.4, - 6.5, and - 8.2 kcal/mol, respectively. Molecular dynamics simulations further suggested that butin induces significant conformational changes in these proteins. Butin exhibits potential effects against alloxan-induced diabetic rats by restoring biochemical balance, reducing inflammation, and protecting pancreatic tissue. Its binding to key proteins involved in apoptosis and inflammation highlights its therapeutic potential in diabetes management.
Assuntos
Aloxano , Diabetes Mellitus Experimental , Simulação de Acoplamento Molecular , Ratos Wistar , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Ratos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Glicemia/metabolismo , Pâncreas/patologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Apoptose/efeitos dos fármacos , Insulina/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/química , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Simulação de Dinâmica MolecularRESUMO
Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.
Assuntos
Insulina , Camundongos Endogâmicos C57BL , Pâncreas , Proteômica , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP , Animais , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Camundongos , Proteômica/métodos , Insulina/metabolismo , Pâncreas/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos Knockout , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Secreção de Insulina , Masculino , Glucose/metabolismoRESUMO
Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.
Assuntos
COVID-19 , Modelos Animais de Doenças , Hiperglicemia , Fígado , SARS-CoV-2 , Animais , Hiperglicemia/imunologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/sangue , Chlorocebus aethiops , SARS-CoV-2/imunologia , Fígado/virologia , Fígado/metabolismo , Fígado/imunologia , Glicogênio/metabolismo , Glicemia/metabolismo , Humanos , Masculino , Pâncreas/virologia , Pâncreas/imunologia , Pâncreas/patologia , Pâncreas/metabolismo , Quimiocinas/metabolismo , Quimiocinas/sangue , Feminino , Replicação ViralRESUMO
The cold climates in autumn and winter threatens human health. The aim of this study was to reveal the effects of prolonged cold exposure on the liver and pancreas based on GLP-1R signaling, oxidative stress, endoplasmic reticulum (ER) stress and ferroptosis by Yorkshire pig models. Yorkshire pigs were divided into the control group and chronic cold stress (CCS) group. The results showed that CCS induced oxidative stress injury, activated Nrf2 pathway and inhibited the expression of GLP-1R in the liver and pancreas (P < 0.05). The toll-like receptor 4 (TLR4) pathway was activated in the liver and pancreas, accompanied by the enrichment of IL-1ß and TNF-α during CCS (P < 0.05). Moreover, the kinase RNA-like endoplasmic reticulum kinase (PERK), inositol requiring kinase 1 (IRE1), X-box-binding protein 1 (XBP1) and eukaryotic initiation factor 2α (eIF2α) expression in the liver and pancreas was up-regulated during CCS (P < 0.05). In addition, CCS promoted the prostaglandin-endoperoxide synthase 2 (PTGS2) expression and inhibited the ferritin H (FtH) expression in the liver. Summarily, CCS promotes inflammation, ER stress and apoptosis by inhibiting the GLP-1R signaling and inducing oxidative stress, and exacerbates the risk of ferroptosis in the liver and pancreas.
Assuntos
Estresse do Retículo Endoplasmático , Ferroptose , Inflamação , Fígado , Pâncreas , Transdução de Sinais , Animais , Fígado/metabolismo , Pâncreas/metabolismo , Suínos , Inflamação/metabolismo , Estresse Oxidativo , Resposta ao Choque FrioRESUMO
BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.
Assuntos
Biomarcadores , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Modelos Animais de Doenças , Células Estreladas do Pâncreas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Animais , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Ovinos , Pâncreas/metabolismo , Pâncreas/patologia , Gravidez , Pancreatopatias/genética , Pancreatopatias/metabolismo , Pancreatopatias/patologia , Transcriptoma , Humanos , Perfilação da Expressão GênicaRESUMO
Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, â¼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
Assuntos
Diferenciação Celular , Elementos Facilitadores Genéticos , Pâncreas , Humanos , Elementos Facilitadores Genéticos/genética , Diferenciação Celular/genética , Pâncreas/metabolismo , Pâncreas/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genéticaRESUMO
BACKGROUND: Ferroptosis is a newly recognized form of regulatory cell death characterized by severe lipid peroxidation triggered by iron overload and the production of reactive oxygen species (ROS). However, the role of ferroptosis in severe acute pancreatitis(SAP) has not been fully elucidated. METHODS: We established four severe acute pancreatitis models of rats including the sham control group, the SAP group, the Fer -1-treated SAP (SAP + Fer-1) group, the 3-MA-treated SAP (SAP + 3-MA) group. The SAP group was induced by retrograde injection of sodium taurocholate into the pancreatic duct. The other two groups were intraperitoneally injected with ferroptosis inhibitor (Fer-1) and autophagy inhibitor (3-MA), respectively. The model of severe acute pancreatitis with amylase crest-related inflammatory factors was successfully established. Then we detected ferroptosis (GPX4, SLC7A1 etc.) and autophagy-related factors (LC3II, p62 ect.) to further clarify the relationship between ferroptosis and autophagy. RESULTS: Our study found that ferroptosis occurs during the development of SAP, such as iron and lipid peroxidation in pancreatic tissues, decreased levels of reduced glutathione peroxidase 4 (GPX 4) and glutathione (GSH), and increased malondialdehyde(MDA) and significant mitochondrial damage. In addition, ferroptosis related proteins such as GPX4, solute carrier family 7 member 11(SLC7A11) and ferritin heavy chain 1(FTH1) were significantly decreased. Next, the pathogenesis of ferroptosis in SAP was studied. First, treatment with the ferroptosis inhibitor ferrostatin-1(Fer-1) significantly alleviated ferroptosis in SAP. Interestingly, autophagy occurs during the pathogenesis of SAP, and autophagy promotes the occurrence of ferroptosis in SAP. Moreover, 3-methyladenine (3-MA) inhibition of autophagy can significantly reduce iron overload and ferroptosis in SAP. CONCLUSIONS: Our results suggest that ferroptosis is a novel pathogenesis of SAP and is dependent on autophagy. This study provides a new theoretical basis for the study of SAP.
Assuntos
Autofagia , Modelos Animais de Doenças , Ferroptose , Peroxidação de Lipídeos , Pancreatite , Ratos Sprague-Dawley , Animais , Pancreatite/metabolismo , Pancreatite/patologia , Ratos , Masculino , Adenina/análogos & derivados , Adenina/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Ácido Taurocólico , Cicloexilaminas/farmacologia , Pâncreas/patologia , Pâncreas/metabolismo , Fenilenodiaminas/farmacologia , Malondialdeído/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença Aguda , Glutationa/metabolismo , Ferro/metabolismoRESUMO
There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (â¼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.
Assuntos
Peptídeos , Proteoma , Proteômica , Humanos , Proteoma/análise , Proteômica/métodos , Peptídeos/análise , Peptídeos/química , Pâncreas/metabolismo , Pâncreas/química , Nanotecnologia , Técnicas Analíticas Microfluídicas/instrumentação , Microdissecção e Captura a Laser/métodosRESUMO
Single-cell RNA sequencing (scRNA-seq) technologies have become essential tools for characterizing cellular landscapes within complex tissues. Large-scale single-cell transcriptomics holds great potential for identifying rare cell types critical to the pathogenesis of diseases and biological processes. Existing methods for identifying rare cell types often rely on one-time clustering using partial or global gene expression. However, these rare cell types may be overlooked during the clustering phase, posing challenges for their accurate identification. In this paper, we propose a Cluster decomposition-based Anomaly Detection method (scCAD), which iteratively decomposes clusters based on the most differential signals in each cluster to effectively separate rare cell types and achieve accurate identification. We benchmark scCAD on 25 real-world scRNA-seq datasets, demonstrating its superior performance compared to 10 state-of-the-art methods. In-depth case studies across diverse datasets, including mouse airway, brain, intestine, human pancreas, immunology data, and clear cell renal cell carcinoma, showcase scCAD's efficiency in identifying rare cell types in complex biological scenarios. Furthermore, scCAD can correct the annotation of rare cell types and identify immune cell subtypes associated with disease, thereby offering valuable insights into disease progression.
Assuntos
Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Camundongos , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Algoritmos , Transcriptoma , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas/citologia , RNA-Seq/métodos , Biologia Computacional/métodosRESUMO
Regeneration of insulin-producing ß-cells is an alternative avenue to manage diabetes, and it is crucial to unravel this process in vivo during physiological responses to the lack of ß-cells. Here, we aimed to characterize how hepatocytes can contribute to ß-cell regeneration, either directly or indirectly via secreted proteins or metabolites, in a zebrafish model of ß-cell loss. Using lineage tracing, we show that hepatocytes do not directly convert into ß-cells even under extreme ß-cell ablation conditions. A transcriptomic analysis of isolated hepatocytes after ß-cell ablation displayed altered lipid- and glucose-related processes. Based on the transcriptomics, we performed a genetic screen that uncovers a potential role of the molybdenum cofactor (Moco) biosynthetic pathway in ß-cell regeneration and glucose metabolism in zebrafish. Consistently, molybdenum cofactor synthesis 2 (Mocs2) haploinsufficiency in mice indicated dysregulated glucose metabolism and liver function. Together, our study sheds light on the liver-pancreas crosstalk and suggests that the molybdenum cofactor biosynthesis pathway should be further studied in relation to glucose metabolism and diabetes.
Assuntos
Coenzimas , Glucose , Hepatócitos , Células Secretoras de Insulina , Fígado , Metaloproteínas , Cofatores de Molibdênio , Pteridinas , Peixe-Zebra , Animais , Células Secretoras de Insulina/metabolismo , Pteridinas/metabolismo , Coenzimas/metabolismo , Camundongos , Fígado/metabolismo , Fígado/citologia , Metaloproteínas/metabolismo , Metaloproteínas/genética , Hepatócitos/metabolismo , Glucose/metabolismo , Regeneração/genética , Pâncreas/metabolismo , Pâncreas/citologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The unsustainable use of manmade chemicals poses significant threats to biodiversity and human health. Emerging evidence highlights the potential of certain chemicals to cause transgenerational impacts on metabolic health. Here, we investigate male transmitted epigenetic transgenerational effects of the anti-androgenic herbicide linuron in the pancreas of Xenopus tropicalis frogs, and their association with metabolic phenotypes. Reduced representation bisulfite sequencing (RRBS) was used to assess genome-wide DNA methylation patterns in the pancreas of adult male F2 generation ancestrally exposed to environmentally relevant linuron levels (44 ± 4.7 µg/L). We identified 1117 differentially methylated regions (DMRs) distributed across the X. tropicalis genome, revealing potential regulatory mechanisms underlying metabolic disturbances. DMRs were identified in genes crucial for pancreatic function, including calcium signalling (clstn2, cacna1d and cadps2), genes associated with type 2 diabetes (tcf7l2 and adcy5) and a biomarker for pancreatic ductal adenocarcinoma (plec). Correlation analysis revealed associations between DNA methylation levels in these genes and metabolic phenotypes, indicating epigenetic regulation of glucose metabolism. Moreover, differential methylation in genes related to histone modifications suggests alterations in the epigenetic machinery. These findings underscore the long-term consequences of environmental contamination on pancreatic function and raise concerns about the health risks associated with transgenerational effects of pesticides.