RESUMO
Insulin resistance and ß-cell dysfunction are two main molecular bases yet to be further elucidated for type 2 diabetes (T2D). Accumulating evidence indicates that stimulator of interferon genes (STING) plays an important role in regulating insulin sensitivity. However, its function in ß-cells remains unknown. Herein, using global STING knockout (STING-/-) and ß-cell-specific STING knockout (STING-ßKO) mouse models, we revealed a distinct role of STING in the regulation of glucose homeostasis through peripheral tissues and ß-cells. Specially, although STING-/- beneficially alleviated insulin resistance and glucose intolerance induced by high-fat diet, it surprisingly impaired islet glucose-stimulated insulin secretion (GSIS). Importantly, STING is decreased in islets of db/db mice and patients with T2D, suggesting a possible role of STING in ß-cell dysfunction. Indeed, STING-ßKO caused glucose intolerance due to impaired GSIS, indicating that STING is required for normal ß-cell function. Islet transcriptome analysis showed that STING deficiency decreased expression of ß-cell function-related genes, including Glut2, Kcnj11, and Abcc8, contributing to impaired GSIS. Mechanistically, the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and cleavage under targets and tagmentation (CUT&Tag) analyses suggested that Pax6 was the transcription factor that might be associated with defective GSIS in STING-ßKO mice. Indeed, Pax6 messenger RNA and protein levels were down-regulated and its nuclear localization was lost in STING-ßKO ß-cells. Together, these data revealed a function of STING in the regulation of insulin secretion and established pathophysiological significance of fine-tuned STING within ß-cells and insulin target tissues for maintaining glucose homeostasis.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Intolerância à Glucose/induzido quimicamente , Glucose/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Animais , Diabetes Mellitus Experimental , Dieta Hiperlipídica/efeitos adversos , Regulação para Baixo , Regulação da Expressão Gênica , Homeostase , Humanos , Insulina/sangue , Resistência à Insulina , Células Secretoras de Insulina , Proteínas de Membrana/genética , Camundongos , Camundongos KnockoutRESUMO
Recessive mutations in IER3IP1 (immediate early response 3 interacting protein 1) cause a syndrome of microcephaly, epilepsy, and permanent neonatal diabetes (MEDS). IER3IP1 encodes an endoplasmic reticulum (ER) membrane protein, which is crucial for brain development; however, the role of IER3IP1 in ß cells remains unknown. We have generated two mouse models with either constitutive or inducible IER3IP1 deletion in ß cells, named IER3IP1-ßKO and IER3IP1-ißKO, respectively. We found that IER3IP1-ßKO causes severe early-onset, insulin-deficient diabetes. Functional studies revealed a markedly dilated ß-cell ER along with increased proinsulin misfolding and elevated expression of the ER chaperones, including PDI, ERO1, BiP, and P58IPK. Islet transcriptome analysis confirmed by qRT-PCR revealed decreased expression of genes associated with ß-cell maturation, cell cycle, and antiapoptotic genes, accompanied by increased expression of antiproliferation genes. Indeed, multiple independent approaches further demonstrated that IER3IP1-ßKO impaired ß-cell maturation and proliferation, along with increased condensation of ß-cell nuclear chromatin. Inducible ß-cell IER3IP1 deletion in adult (8-wk-old) mice induced a similar diabetic phenotype, suggesting that IER3IP1 is also critical for function and survival even after ß-cell early development. Importantly, IER3IP1 was decreased in ß cells of patients with type 2 diabetes (T2D), suggesting an association of IER3IP1 deficiency with ß-cell dysfunction in the more-common form of diabetes. These data not only uncover a critical role of IER3IP1 in ß cells but also provide insight into molecular basis of diabetes caused by IER3IP1 mutations.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Camundongos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Homeostase/genética , Glucose/metabolismoRESUMO
IL-27, a heterodimeric cytokine of the IL-12 family, has diverse influences on the development of multiple inflammatory diseases. In this study, we identified the protective role of IL-27/IL-27R in host defense against Chlamydia muridarum respiratory infection and further investigated the immunological mechanism. Our results showed that IL-27 was involved in C. muridarum infection and that IL-27R knockout mice (WSX-1-/- mice) suffered more severe disease, with greater body weight loss, higher chlamydial loads, and more severe inflammatory reactions in the lungs than C57BL/6 wild-type mice. There were excessive IL-17-producing CD4+ T cells and many more neutrophils, neutrophil-related proteins, cytokines, and chemokines in the lungs of WSX-1-/- mice than in wild-type mice following C. muridarum infection. In addition, IL-17/IL-17A-blocking Ab treatment improved disease after C. muridarum infection in WSX-1-/- mice. Overall, we conclude that IL-27/IL-27R mediates protective immunity during chlamydial respiratory infection in mice by suppressing excessive Th17 responses and reducing neutrophil inflammation.
Assuntos
Inflamação/imunologia , Interleucinas/imunologia , Neutrófilos/imunologia , Receptores de Interleucina/imunologia , Animais , Chlamydia muridarum/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina/deficiência , Células Th17/imunologiaRESUMO
The IL-21/IL-21R interaction plays an important role in a variety of immune diseases; however, the roles and mechanisms in intracellular bacterial infection are not fully understood. In this study, we explored the effect of IL-21/IL-21R on chlamydial respiratory tract infection using a chlamydial respiratory infection model. The results showed that the mRNA expression of IL-21 and IL-21R was increased in Chlamydia muridarum-infected mice, which suggested that IL-21 and IL-21R were involved in host defense against C. muridarum lung infection. IL-21R-/- mice exhibited less body weight loss, a lower bacterial burden, and milder pathological changes in the lungs than wild-type (WT) mice during C. muridarum lung infection. The absolute number and activity of CD4+ T cells and the strength of Th1/Th17 responses in IL-21R-/- mice were significantly higher than those in WT mice after C. muridarum lung infection, but the Th2 response was weaker. Consistently, IL-21R-/- mice showed higher mRNA expression of Th1 transcription factors (T-bet/STAT4), IL-12p40, a Th17 transcription factor (STAT3), and IL-23. The mRNA expression of Th2 transcription factors (GATA3/STAT6), IL-4, IL-10, and TGF-ß in IL-21R-/- mice was significantly lower than that in WT mice. Furthermore, the administration of recombinant mouse IL-21 aggravated chlamydial lung infection in C57BL/6 mice and reduced Th1 and Th17 responses following C. muridarum lung infection. These findings demonstrate that IL-21/IL-21R may aggravate chlamydial lung infection by inhibiting Th1 and Th17 responses.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia muridarum/imunologia , Interleucinas/metabolismo , Pulmão/imunologia , Receptores de Interleucina-21/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Feminino , Inflamação , Espaço Intracelular , Camundongos , Receptores de Interleucina-21/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Proteínas com Domínio T/genéticaRESUMO
BACKGROUND: NKX6.1 is a transcription factor for insulin, as well as a marker for ß cell maturity. Abnormal NKX6.1 expression in ß cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for ß cell dedifferentiation. METHODS: We obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM). RESULTS: Our results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns-) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated ß cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether ß cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between ß-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated ß cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the ß cell dedifferentiation might mainly occur after T2DM was diagnosed. CONCLUSION: Our results suggested that NKX6.1 expression in ß cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns- cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that ß cell dedifferentiation might be secondary to the pathological changes in T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Estado Pré-Diabético/metabolismo , Adulto , Idoso , Autopsia , Estudos de Casos e Controles , Contagem de Células , Diferenciação Celular , Diabetes Mellitus Tipo 2/patologia , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/patologia , Fatores de RiscoRESUMO
Hypoxia affects the function of pancreatic ß cells, and the molecular mechanism underlying hypoxia-related ß cell dysfunction in human type 2 diabetes mellitus (T2DM) remains to be elucidated. In this study, by comparing the gene expression profiles of islets from nondiabetic and T2D subjects using gene chip array, we aimed to elucidate that hypoxia signaling pathways are activated in human T2DM islets. CoCl2 treatment, which was employed to mimic hypoxic stimulation in human islets, decreased insulin secretion, insulin content, and the functional gene expression of human islets. In parallel, the expression of mature ß cell-disallowed genes was upregulated by CoCl2, including progenitor cell marker NGN3, ß cell differentiation marker ALDH1A3, and genes that are typically inhibited in mature ß cells, namely, GLUT1 and LDHA, indicating that CoCl2-mimicked hypoxia induced ß cell dedifferentiation of human islets. This finding in human islets was confirmed in mouse ß cell line NIT-1. By using Dimethyloxalylglycine (DMOG) to activate hypoxia-inducible factor-1α (HIF-1α) or siRNAs to knockdown HIF-1α, we found that HIF-1α was a key regulator of hypoxia-induced dedifferentiation of ß cells by upregulating mature ß cell-disallowed genes. Our findings suggested that HIF-1α activation might be an important contributor to ß cell dedifferentiation in human T2DM islets, and HIF-1α-targeted therapies may have the potential to reverse ß cell dedifferentiation of human T2DM islets.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular , Cobalto/toxicidade , Diabetes Mellitus Tipo 2/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Transdução de SinaisRESUMO
Chlamydia is an obligate intracellular bacteria, which can infect cervix, urethra, conjunctiva, joints, lungs and so on. Neutrophils are important in host protection against microbial invasion during the early phase of infection. Here, to investigate the mechanism of IL-17A in recruiting neutrophils during Chlamydia muridarum (Cm) lung infection, we introduced IL-17A antibodies and IL-17-/- mice to confirm the effect of IL-17A on influencing neutrophil attractants expressions. From the analysis of the data, we found that showed that Cm infection could upregulate the expression of neutrophil-related chemokines such as KC, MIP-2 and IL-6, as well as adhesion molecules including ICAM-1 and VCAM-1. With blocking endogenous IL-17A, the upregulated MIP-2 and IL-6 were decreased, which induced less neutrophil recruitment in lung. Comparing to WT mice, IL-17-/- mice showed decreased infiltration of neutrophils in lung during the early phase of Cm infection, which were accordant with decreased chemokines, such as KC, MIP-2 and IL-6 expression. Whereas, the expression of adhesion molecules including ICAM and VCAM-1 in lungs were significantly increased in IL-17-/- mice comparing to WT mice during Cm lung infection. The results demonstrated that IL-17A influenced neutrophil infiltration by affecting expression of chemokines and adhesion molecules during the early phase of chlamydial lung infection.
Assuntos
Quimiocinas/metabolismo , Infecções por Chlamydia/patologia , Chlamydia muridarum/patogenicidade , Interleucina-17/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Pneumonia Bacteriana/patologia , Animais , Infecções por Chlamydia/imunologia , Chlamydia muridarum/imunologia , Modelos Animais de Doenças , Interleucina-17/antagonistas & inibidores , Interleucina-17/deficiência , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia Bacteriana/imunologiaRESUMO
The cyclooxygenase2 (COX-2) enzyme catalyzes the first step of prostanoid biosynthesis, and is known for its crucial role in the pathogenesis of several inflammatory diseases including type 2 diabetes mellitus (T2DM). Although a variety of studies revealed that COX-2 played a role in the IL-1ß induced ß cell dysfunction, the molecular mechanism remains unclear. Here, using a cDNA microarray and in silico analysis, we demonstrated that inflammatory responses were upregulated in human T2DM islets compared with non-diabetic (ND) islets. COX-2 expression was significantly enhanced in human T2DM islets, correlated with the high inflammation level. PGE2, the catalytic product of COX-2, downregulated the functional gene expression of PDX1, NKX6.1, and MAFA and blunted the glucose induced insulin secretion of human islets. Conversely, inhibition of COX-2 activity by a pharmaceutical inhibitor prevented the ß-cell dysfunction induced by IL-1ß. COX-2 inhibitor also abrogated the IL-1ß autostimulation in ß cells, which further resulted in reduced COX-2 expression in ß cells. Together, our results revealed that COX-2/PGE2 signaling was involved in the regulation of IL-1ß autostimulation, thus forming an IL-1ß/COX-2/PGE2 pathway loop, which may result in the high inflammation level in human T2DM islets and the inflammatory impairment of ß cells. Breaking this IL-1ß/COX-2/PGE2 pathway loop provides a potential therapeutic strategy to improve ß cell function in the treatment of T2DM patients.
Assuntos
Ciclo-Oxigenase 2/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dinoprostona/fisiologia , Interleucina-1beta/fisiologia , Ilhotas Pancreáticas/fisiopatologia , Adulto , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/patologia , Dinoprostona/metabolismo , Retroalimentação Fisiológica/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais/fisiologiaRESUMO
Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of ß cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.
Assuntos
Autoanticorpos/metabolismo , Carbodi-Imidas/química , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/terapia , Etilenos/química , Baço/citologia , Baço/transplante , Linfócitos T Reguladores/patologia , Animais , Antígenos/metabolismo , Reagentes de Ligações Cruzadas/química , Diabetes Mellitus Experimental/patologia , Regulação para Baixo/imunologia , Feminino , Tolerância Imunológica/fisiologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Endogâmicos NOD , Baço/imunologia , Baço/metabolismo , Fixação de TecidosRESUMO
Our previous studies showed that γδ T cells provided immune protection against Chlamydial muridarum (Cm), an obligate intracellular strain of chlamydia trachomatis, lung infection by producing abundant IL-17. In this study, we investigated the proliferation and activation of lung γδ T cell subsets, specifically the IL-17 and IFNγ production by them following Cm lung infection. Our results found that five γδ T cell subsets, Vγ1+ T, Vγ2+ T, Vγ4+ T, Vγ5+ T, and Vγ6+ T, expressed in lungs of naïve mice, while Cm lung infection mainly induced the proliferation and activation of Vγ4+ T cells at day 3 p.i., following Vγ1+ T cells at day 7 p.i. Cytokine detection showed that Cm lung infection induced IFNγ secretion firstly by Vγ4+ T cells at very early stage (day 3) and changed to Vγ1+ T cells at midstage (day 7). Furthermore, Vγ4+ T cell is the main γδ T cell subset that secretes IL-17 at the very early stage of Cm lung infection and Vγ1+ T cell did not secrete IL-17 during the infection. These findings provide in vivo evidence that Vγ4+T cells are the major IL-17 and IFNγ-producing γδ T cell subsets at the early period of Cm lung infection.
Assuntos
Citocinas/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Linfócitos T/metabolismo , Animais , Pulmão/metabolismo , Pulmão/patologia , CamundongosRESUMO
BACKGROUND: Islet transplantation is a promising therapy for patients with type 1 diabetes. However, ischemic injury to the donor islets during cold preservation leads to reduced islet quality and compromises transplant outcome. Several studies imply that liraglutide, a glucagon-like peptide-1 receptor agonist, has a positive effect on promoting islet survival, but its impact on islet cold-ischemic injury remains unexplored. Therefore, the aim of this study was to investigate whether liraglutide can improve islet transplantation efficacy by inhibiting cold-ischemic injury and to explore the underlying mechanisms. METHODS: Liraglutide was applied in a mouse pancreas preservation model and a human islets cold-preservation model, and islet viability, function, oxidative stress levels were evaluated. Furthermore, islet transplantation was performed in a syngeneic mouse model and a human-to-nude mouse islet xenotransplantation model. RESULTS: The supplementation of liraglutide in preservation solution improved islet viability, function, and reduced cell apoptosis. Liraglutide inhibited the oxidative stress of cold-preserved pancreas or islets through upregulating the antioxidant enzyme glutathione levels, inhibiting reactive oxygen species accumulation, and maintaining the mitochondrial membrane integrity, which is associated with the activation of Akt signaling. Furthermore, the addition of liraglutide during cold preservation of donor pancreas or donor islets significantly improved the subsequent transplant outcomes in both syngeneic mouse islet transplantation model and human-to-nude mouse islet xenotransplantation model. CONCLUSIONS: Liraglutide protects islets from cold ischemia-related oxidative stress during preservation and hence improved islet transplantation outcomes, and this protective effect of liraglutide in islets is associated with the activation of Akt signaling.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Liraglutida , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Liraglutida/farmacologia , Animais , Estresse Oxidativo/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/métodos , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Isquemia Fria/efeitos adversos , Masculino , Camundongos , Camundongos Nus , Sobrevivência de Enxerto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Apoptose/efeitos dos fármacos , Transplante Heterólogo , Criopreservação , Preservação de Órgãos/métodos , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/metabolismoRESUMO
Type 2 diabetes mellitus (T2D) affects 463 million individuals worldwide. ß-cell dysfunction and relatively inadequate ß-cell mass has been implicated in the pathogenesis of T2D. Primary human islets from T2D patients can reveal the islet dysfunction and the underlying mechanisms and thus have become valued resources for diabetes research. Our center (Human Islet Resource Center, China) has prepared a number of batches of human islets from T2D organ donors. The present study aims to characterize islet isolation processes, islet yields, and qualities of T2D pancreases by comparing with non-diabetic (ND) ones. Overall, 24 T2D and 80 ND pancreases were obtained with informed research consents. The digestion time, islet purity, yield, size distribution, islet morphology score, viability, and function in each islet preparation were analyzed. We found that at digestion stage, T2D pancreases need significantly longer digestion duration and have worse digestion rates and lower gross islet yields. At purification stage, T2D pancreases have poorer purity, purification rate, morphology score, and islet yields after purification. Functional evaluation by GSI assay showed that the human T2D islets have significantly lower glucose stimulated insulin secretion ability. In conclusion, the features of longer digestion duration, lower yields and quality, and impaired insulin secretion in T2D group are consistent with the pathological condition of this disease. Both islet yields and islet function evaluation results did not support human T2D islets as clinical transplantation resources. However, they could serve as good research models for T2D disease studies and promote the advancement of diabetes research.
Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Pâncreas , Doadores de Tecidos , Pesquisa , BioensaioRESUMO
The FK506-binding protein 51 (FKBP51, encoded by FKBP5 gene) has emerged as a critical regulator of mammalian endocrine stress responses and as a potential pharmacological target for metabolic disorders, including type 2 diabetes (T2D). However, in ß cells, which secrete the only glucose-lowering hormone-insulin, the expression and function of FKBP5 has not been documented. Here, using human pancreatic tissue and primary human islets, we demonstrated the abundant expression of FKBP5 in ß cells, which displayed an responsive induction upon acute inflammatory stress mimicked by in vitro treatment with a cocktail of inflammatory cytokines (IL-1ß, IFN-γ, and TNF-α). To explore its function, siRNAs targeting FKBP5 and pharmacological inhibitor SAFit2 were applied both in clonal NIT-1 cells and primary human/mice islets. We found that FKBP5 inhibition promoted ß-cell survival, improved insulin secretion, and upregulated ß-cell functional gene expressions (MAFA and NKX6.1) in acute-inflammation stressed ß cells. In primary human and mice islets, which constitutively suffer from inflammation stress during isolation and culture, FKBP5 inhibition also presented decent performance in improving islet function, in accordance with its protective effect against inflammation. Molecular studies found that FKBP5 is an important regulator for FOXO1 phosphorylation at Serine 256, and silencing of FOXO1 abrogated the protective effect of FKBP5 inhibition, suggesting that it is the key downstream effector of FKBP5 in ß cells. At last, in situ detection of FKBP5 protein expression on human and mice pancreases revealed a reduction of FKBP5 expression in ß cells in human T2D patients, as well as T2D mice model (db/db), which may indicate a FKBP5-inhibition-mediated pro-survival mechanism against the complex stresses in T2D milieus.
RESUMO
Brain-specific serine/threonine-protein kinase 2 (BRSK2) plays critical roles in insulin secretion and ß-cell biology. However, whether BRSK2 is associated with human type 2 diabetes mellitus (T2DM) has not been determined. Here, we report that BRSK2 genetic variants are closely related to worsening glucose metabolism due to hyperinsulinemia and insulin resistance in the Chinese population. BRSK2 protein levels are significantly elevated in ß cells from T2DM patients and high-fat diet (HFD)-fed mice due to enhanced protein stability. Mice with inducible ß-cell-specific Brsk2 knockout (ßKO) exhibit normal metabolism with a high potential for insulin secretion under chow-diet conditions. Moreover, ßKO mice are protected from HFD-induced hyperinsulinemia, obesity, insulin resistance, and glucose intolerance. Conversely, gain-of-function BRSK2 in mature ß cells reversibly triggers hyperglycemia due to ß-cell hypersecretion-coupled insulin resistance. Mechanistically, BRSK2 senses lipid signals and induces basal insulin secretion in a kinase-dependent manner. The enhanced basal insulin secretion drives insulin resistance and ß-cell exhaustion and thus the onset of T2DM in mice fed an HFD or with gain-of-function BRSK2 in ß cells. These findings reveal that BRSK2 links hyperinsulinemia to systematic insulin resistance via interplay between ß cells and insulin-sensitive tissues in the populations carrying human genetic variants or under nutrient-overload conditions.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperinsulinismo , Resistência à Insulina , Células Secretoras de Insulina , Humanos , Camundongos , Animais , Resistência à Insulina/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Dieta HiperlipídicaRESUMO
ß cells suffer from hypoxia due to the rapid metabolic rate to supply insulin production. Mechanistic study of ß cell survival under hypoxia may shed light on the ß cell mass loss in type 2 diabetes mellitus (T2DM). Here, we found that the expressions of LC3 and p62/SQSTM1, two key autophagy regulators, were significantly higher in ß cells than that in non-ß endocrine cells in both non-diabetic and T2DM human pancreases, and the autophagy process was accelerated upon Cobalt Chloride (CoCl2) treatment in ex vivo cultured primary human islets. Meanwhile, CoCl2 induced the upregulation of FOXO1 in human islets, where HIF-1α played a key role. CoCl2 treatment caused the increase of ß cell apoptosis, yet inhibiting autophagy by Chloroquine or by FOXO1 knockdown further aggravated apoptosis, suggesting that FOXO1-regulated autophagy is protective for ß cell survival under hypoxia. Immunofluorescence staining showed that LC3 and p62/SQSTM1 expressions were significantly decreased in T2DM patients and negatively correlated with HbA1c, indicating that the autophagy capacity of ß cells is impaired along with the progression of the disease. Our study revealed that HIF-1α/FOXO1 regulated autophagy benefits ß cell survival under hypoxia and autophagy dysregulation may account for ß cell mass loss in T2DM. BRIEF SUMMARY: Our study revealed that HIF-1α/FOXO1 regulated autophagy benefits ß cell survival under hypoxia and autophagy dysregulation may account for ß cell mass loss in T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , Autofagia , Hipóxia Celular , Sobrevivência Celular , Cobalto/farmacologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Hipóxia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Human pluripotent stem-cell-derived islets (hPSC-islets) are a promising cell resource for diabetes treatment1,2. However, this therapeutic strategy has not been systematically assessed in large animal models physiologically similar to humans, such as non-human primates3. In this study, we generated islets from human chemically induced pluripotent stem cells (hCiPSC-islets) and show that a one-dose intraportal infusion of hCiPSC-islets into diabetic non-human primates effectively restored endogenous insulin secretion and improved glycemic control. Fasting and average pre-prandial blood glucose levels significantly decreased in all recipients, accompanied by meal or glucose-responsive C-peptide release and overall increase in body weight. Notably, in the four long-term follow-up macaques, average hemoglobin A1c dropped by over 2% compared with peak values, whereas the average exogenous insulin requirement reduced by 49% 15 weeks after transplantation. Collectively, our findings show the feasibility of hPSC-islets for diabetic treatment in a preclinical context, marking a substantial step forward in clinical translation of hPSC-islets.
Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Glicemia , Diabetes Mellitus Experimental/terapia , Humanos , Insulina , Transplante das Ilhotas Pancreáticas/fisiologia , PrimatasRESUMO
OBJECTIVE: Progressive beta-cell dysfunction is a hallmark of type 2 diabetes (T2D). Increasing evidence indicates that over-stimulating proinsulin synthesis causes proinsulin misfolding and impairs insulin maturation and storage in db/db mice. However, defective insulin maturation in patients with T2D remains unknown. METHODS: We examined intra-islet and intra-cellular distributions of proinsulin and insulin and proinsulin to insulin ratio in the islets of patients with T2D. The expression of transcription factor NKX6.1 and dedifferentiation marker ALDH1A3, as well as glucagon, were detected by immunofluorescence. RESULTS: We identified a novel subgroup of beta cells expressing only proinsulin but not insulin. Importantly, significantly increased proinsulin positive and insulin negative (PI+/INS-) cells were evident in T2D, and this increase was strongly correlated with levels of hemoglobin A1C (HbA1c) in T2D and prediabetes. The percentages of beta cells expressing prohormone convertase 1/3 and carboxypeptidase E were not reduced. Indeed, while proinsulin displayed a higher degree of co-localization with the golgi markers GM130/TGN46 in control beta cells, it appeared to be more diffused within the cytoplasm and less co-localized with GM130/TGN46 in PI+/INS- cells. Furthermore, the key functional transcription factor NKX6.1 markedly decreased in the islets of T2D, especially in the cells with PI+/INS-. The decreased NKX6.1+/PI+/INS+ was strongly correlated with levels of HbA1c in T2D. Almost all PI+/INS- cells showed absence of NKX6.1. Moreover, the percentages of PI+/INS- cells expressing ALDH1A3 were elevated along with an increased acquisition of glucagon immunostaining. CONCLUSION: Our data demonstrate defective insulin maturation in patients with T2D.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proinsulina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Adulto , Aldeído Oxirredutases/metabolismo , Estudos de Casos e Controles , Desdiferenciação Celular/fisiologia , China , Diabetes Mellitus Tipo 2/patologia , Feminino , Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/patologiaRESUMO
During the process of human islet isolation a cascade of stressful events are triggered and negatively influence islet yield, viability, and function, including the production of proinflammatory cytokines and activation of apoptosis. Carbon monoxide-releasing molecule 2 (CORM-2) is a donor of carbon monoxide (CO) and can release CO spontaneously. Accumulating studies suggest that CORM-2 exerts cytoprotective and anti-inflammatory properties. However, the effect of CORM-2 on islet isolation is still unclear. In this study, we found that CORM-2 pretreatment significantly decreased the expression of critical inflammatory genes, including tissue factor, intercellular adhesion molecule-1, chemokine (C-C motif) ligand 2, C-X-C motif chemokine 10, Toll-like receptor 4, interleukin-1ß, interleukin-6, and tumor necrosis factor-α (TNF-α). The isolated islets of the CORM-2 pretreatment group showed reduced apoptotic rate, improved viability, and higher glucose-stimulated insulin secretion, and functional gene expression in comparison to control group. Importantly, CORM-2 pretreatment prevented the impairment caused by TNF-α, evidenced by the improved glucose-stimulated index and transplantation outcomes. The present study demonstrated the anti-inflammatory property of CORM-2 during human islet isolation, and we suggest that CORM-2 pretreatment is an appealing treatment to mitigate inflammation-mediated islet dysfunction during isolation and culture ex vivo and to preserve long-term islet survival and function.
Assuntos
Inflamação/tratamento farmacológico , Compostos Organometálicos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Citometria de Fluxo , Teste de Tolerância a Glucose , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos Endogâmicos BALB C , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: A physiological hallmark of patients with type 2 diabetes mellitus (T2DM) is ß cell dysfunction. Despite adequate treatment, it is an irreversible process that follows disease progression. Therefore, the development of novel therapies that restore ß cell function is of utmost importance. METHODS: This study aims to unveil the mechanistic action of mesenchymal stem cells (MSCs) by investigating its impact on isolated human T2DM islets ex vivo and in vivo. FINDINGS: We propose that MSCs can attenuate ß cell dysfunction by reversing ß cell dedifferentiation in an IL-1Ra-mediated manner. In response to the elevated expression of proinflammatory cytokines in human T2DM islet cells, we observed that MSCs was activated to secret IL-1R antagonist (IL-1Ra) which acted on the inflammed islets and reversed ß cell dedifferentiation, suggesting a crosstalk between MSCs and human T2DM islets. The co-transplantation of MSCs with human T2DM islets in diabetic SCID mice and intravenous infusion of MSCs in db/db mice revealed the reversal of ß cell dedifferentiation and improved glycaemic control in the latter. INTERPRETATION: This evidence highlights the potential of MSCs in future cell-based therapies regarding the amelioration of ß cell dysfunction.