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1.
Nat Commun ; 11(1): 296, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941883

RESUMO

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in ß cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- ß cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.


Assuntos
Insulina/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Ferro/metabolismo , RNA de Transferência de Lisina/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Intolerância à Glucose/genética , Homeostase , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteínas com Ferro-Enxofre/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , RNA de Transferência de Lisina/genética , Ratos , Resposta a Proteínas não Dobradas/genética , tRNA Metiltransferases/genética
2.
Endocrinology ; 160(11): 2759-2772, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504428

RESUMO

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.


Assuntos
Glucose/metabolismo , Iodeto Peroxidase/deficiência , Envelhecimento/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Homeostase , Hiperglicemia/genética , Iodeto Peroxidase/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Masculino , Proglucagon/metabolismo , Proinsulina/metabolismo , Receptor de Insulina/metabolismo , Receptores de Glucagon/metabolismo , Peixe-Zebra
3.
Gene ; 706: 52-61, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31039435

RESUMO

Insulin is a peptide hormone responsible for stable glycemia, is entirely secreting from pancreatic ß cells at the core of glucose homeostatic regulation. Upon synthesis as preproinsulin on rough endoplasmic reticulum (rER), proinsulin is directed to trans Golgi apparatus. Subsequently, proinsulin packaging into secretory granules occurs in a dynamic and highly efficient process. During maturation stage of secretory granules, proinsulin undergoes cleavage and produces insulin and C-peptide upon acidification of the granules due to the activation of ATP-deriven proton pump. Fusion of the insulin containing secretory granules with the plasma membranes takes place after an increase in intracellular Ca2+. Finally, insulin is co-secreting with other components that are present in the secretory granules, including C-peptide, ATP, γ-aminobutyric acid (GABA), ghrelin and amylin. The other accompanying components of the insulin vesicles play important roles in the insulin secretion, insulin receptor activation and other homeostatic effects.. Responding to the glucose stimulation or increases in cytoplasmic Ca2+ levels, insulin secretion is immediately starts. Whereas, the second phase of insulin secretion is slow and continued, which reaches a plateau within 1-3 hours and lasts for longer period. In contrast to the first phase, the second phase of insulin secretion is independent of the extracellular glucose level. Finally, sequential or compound exocytosis of insulin is repressed to prevent sugar crash arising from excessive and sudden insulin secretion. In this paper we have reviewed the recent progress of molecular scenarios which are behind insulin biogenesis, intracellular sorting and exocytosis events.


Assuntos
Exocitose/fisiologia , Insulina/metabolismo , Insulina/fisiologia , Animais , Transporte Biológico , Glicemia/metabolismo , Glucose/metabolismo , Complexo de Golgi/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico/fisiologia
4.
Cell Immunol ; 338: 32-42, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30910218

RESUMO

Autoantigen-specific immunotherapy promises effective treatment for devastating tissue specific autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D). Because activated dendritic cells (DCs) stimulate the differentiation of autoreactive T cells involved in the initiation of autoimmunity, blocking the activation of DCs may be an effective strategy for inhibiting tissue specific autoimmunity. Following this approach, immature DCs were shown to remain inactive after treatment with chimeric fusion proteins composed of the cholera toxin B subunit adjuvant linked to autoantigens like proinsulin (CTB-INS). Mass spectrometer analysis of human DCs treated with CTB-INS suggest that upregulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) is responsible for inhibiting DC activation thereby resulting in a state of immunological tolerance within the DC. Here we show that the fusion protein CTB-INS inhibits human monocyte derived DC (moDC) activation through stimulation of IDO1 biosynthesis and that the resultant state of DC tolerance can be further enhanced by the presence of residual E. coli lipopolysaccharide (LPS) present in partially purified CTB-INS preparations. Additional experiments showed that LPS enhancement of DC tolerance was dependent upon stimulation of IDO1 biosynthesis. LPS stimulation of increased levels of IDO1 in the DC resulted in increased secretion of kynurenines, tryptophan degradation products known to suppress DC mediated pro-inflammatory T cell differentiation and to stimulate the proliferation of regulatory T cells (Tregs). Further, the presence of LPS in CTB-INS treated DCs stimulated the biosynthesis of costimulatory factors CD80 and CD86 but failed to upregulate maturation factor CD83, suggesting CTB-INS treated DCs may be maintained in a state of semi-activation. While treatment of moDCs with increasing amounts of LPS free CTB-INS was shown to increase DC secretion of the anti-inflammatory cytokine IL-10, the presence of residual LPS in partially purified CTB-INS preparations dramatically increased IL-10 secretion, suggesting that CTB-INS may enhance DC mediated immunological tolerance by stimulating the proliferation of anti-inflammatory T cells. While the extraction of LPS from bacterial generated CTB-INS may remove additional unknown factors that may contribute to the regulation of IDO1 levels, together, our experimental data suggest that LPS stimulates the ability of CTB-INS to induce IDO1 and IL-10 important factors required for establishment of a state of functional immunological tolerance in human DCs. Regulation of the ratio of LPS to CTB-INS may prove to be an effective method for optimization of readily available "off the shelf" CTB-INS mediated immune-therapy for tissue specific autoimmune diseases including type 1 diabetes.


Assuntos
Autoantígenos/metabolismo , Doenças Autoimunes/terapia , Toxina da Cólera/metabolismo , Células Dendríticas/imunologia , Imunoterapia/métodos , Proinsulina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Doenças Autoimunes/imunologia , Diferenciação Celular , Células Cultivadas , Toxina da Cólera/genética , Humanos , Tolerância Imunológica , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , Proinsulina/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética
5.
Diabetes ; 68(5): 1002-1013, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30796028

RESUMO

Insulin is a major autoantigen in type 1 diabetes, targeted by both CD8 and CD4 T cells. We studied an insulin-reactive T-cell receptor (TCR) α-chain transgenic NOD mouse on a TCRCα and proinsulin 2 (PI2)-deficient background, designated as A22Cα-/-PI2-/- NOD mice. These mice develop a low incidence of autoimmune diabetes. To test the role of gut microbiota on diabetes development in this model system, we treated the A22Cα-/-PI2-/- NOD mice with enrofloxacin, a broad-spectrum antibiotic. The treatment led to male mice developing accelerated diabetes. We found that enrofloxacin increased the frequency of the insulin-reactive CD8+ T cells and activated the cells in the Peyer's patches and pancreatic lymph nodes, together with induction of immunological effects on the antigen-presenting cell populations. The composition of gut microbiota differed between the enrofloxacin-treated and untreated mice and also between the enrofloxacin-treated mice that developed diabetes compared with those that remained normoglycemic. Our results provide evidence that the composition of the gut microbiota is important for determining the expansion and activation of insulin-reactive CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Microbioma Gastrointestinal/fisiologia , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Enrofloxacina/uso terapêutico , Microbioma Gastrointestinal/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proinsulina/genética , Proinsulina/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
6.
Diabetes ; 68(4): 747-760, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670477

RESUMO

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within ß-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in ß-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proinsulina/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Exocitose/fisiologia , Humanos , Insulina/metabolismo , Dobramento de Proteína , Ratos , eIF-2 Quinase/metabolismo
7.
J Clin Endocrinol Metab ; 104(4): 1090-1098, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649496

RESUMO

CONTEXT: Primary dysregulation of adipose tissue lipolysis caused by genetic variation and independent of insulin resistance could explain unhealthy body fat distribution and its metabolic consequences. OBJECTIVE: To analyze common single nucleotide polymorphisms (SNPs) in 48 lipolysis-, but not insulin-signaling-related genes, to form polygenic risk scores of lipolysis-associated SNPs, and to investigate their effects on body fat distribution, glycemia, insulin sensitivity, insulin secretion, and proinsulin conversion. STUDY DESIGN, PARTICIPANTS, AND METHODS: SNP array, anthropometric, and metabolic data were available from up to 2789 participants without diabetes of the Tübingen Family study of type 2 diabetes characterized by oral glucose tolerance tests. In a subgroup (n = 942), magnetic resonance measurements of body fat stores were available. RESULTS: We identified insulin-sensitivity-independent nominal associations (P < 0.05) of SNPs in 10 genes with plasma free fatty acids (FFAs), in 7 genes with plasma glycerol and in 6 genes with both, plasma FFAs and glycerol. A score formed of the latter SNPs (in ADCY4, CIDEA, GNAS, PDE8B, PRKAA1, PRKAG2) was associated with plasma FFA and glycerol measurements (1.4*10-9 ≤ P ≤ 1.2*10-5), visceral adipose tissue mass (P = 0.0326), and proinsulin conversion (P ≤ 0.0272). The more lipolysis-increasing alleles a subject had, the lower was the visceral fat mass and the lower the proinsulin conversion. CONCLUSIONS: We found evidence for a genetic basis of adipose tissue lipolysis resulting from common SNPs in CIDEA, AMP-activated protein kinase subunits, and cAMP signaling components. A genetic score of lipolysis-increasing alleles determined lower visceral fat mass and lower proinsulin conversion.


Assuntos
Gordura Intra-Abdominal/diagnóstico por imagem , Lipólise/genética , Redes e Vias Metabólicas/genética , Proinsulina/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Alelos , Proteínas Reguladoras de Apoptose/metabolismo , AMP Cíclico/metabolismo , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/metabolismo , Feminino , Alemanha , Teste de Tolerância a Glucose , Glicerol/sangue , Glicerol/metabolismo , Humanos , Gordura Intra-Abdominal/metabolismo , Imagem por Ressonância Magnética , Masculino , Metabolômica , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Medição de Risco
8.
Dev Cell ; 48(1): 115-125.e4, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30503750

RESUMO

Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of "extreme" beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.


Assuntos
Heterogeneidade Genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina/fisiologia , Camundongos Transgênicos , Proinsulina/metabolismo
9.
Cell Immunol ; 335: 68-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30428974

RESUMO

CD4+ T cell responses are thought to play a role in type 1 diabetes (T1D). However, detection and characterization of T cells that respond to beta cell epitopes in subjects with T1D has been limited by technical obstacles, including the inherently low frequencies in peripheral blood and variable responsiveness of individual subjects to single epitopes. We implemented a multicolor staining approach that allows direct ex vivo characterization of multiple CD4+ T cell specificities in a single sample. Here we demonstrate and apply that multicolor approach to directly measure the frequency and phenotype of beta cell specific CD4+ T cells in T1D patients and HLA matched controls. For this work we utilized five DR0401 restricted peptides from proinsulin, GAD65, IA-2, and IGRP, which were previously reported as disease relevant epitopes. Surprisingly, although responses to each of these peptides can be readily detected after in vitro expansion, our results indicated that only proinsulin specific T cells were consistently detectable at moderate frequencies in subjects with T1D. Characterization of beta cell specific CD4+ T cells revealed only modest differences between subjects with T1D and healthy controls. Subjects with T1D did have higher proportions of CD45RA negative epitope specific T cells than controls. In patients epitope specific T cells were often CXCR3 positive and a substantial proportion were CCR7 negative, suggesting a Th1-like effector phenotype. Finally, we demonstrated that our multicolor staining approach is compatible with class I multimer analysis, facilitating the characterization of self-reactive CD4+ and CD8+ T cells using a single sample.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Células Secretoras de Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Proinsulina/imunologia , Proinsulina/metabolismo
10.
Diabetes Care ; 42(2): 258-264, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30530850

RESUMO

OBJECTIVE: Abnormally elevated proinsulin secretion has been reported in type 2 and early type 1 diabetes when significant C-peptide is present. We questioned whether individuals with long-standing type 1 diabetes and low or absent C-peptide secretory capacity retained the ability to make proinsulin. RESEARCH DESIGN AND METHODS: C-peptide and proinsulin were measured in fasting and stimulated sera from 319 subjects with long-standing type 1 diabetes (≥3 years) and 12 control subjects without diabetes. We considered three categories of stimulated C-peptide: 1) C-peptide positive, with high stimulated values ≥0.2 nmol/L; 2) C-peptide positive, with low stimulated values ≥0.017 but <0.2 nmol/L; and 3) C-peptide <0.017 nmol/L. Longitudinal samples were analyzed from C-peptide-positive subjects with diabetes after 1, 2, and 4 years. RESULTS: Of individuals with long-standing type 1 diabetes, 95.9% had detectable serum proinsulin (>3.1 pmol/L), while 89.9% of participants with stimulated C-peptide values below the limit of detection (<0.017 nmol/L; n = 99) had measurable proinsulin. Proinsulin levels remained stable over 4 years of follow-up, while C-peptide decreased slowly during longitudinal analysis. Correlations between proinsulin with C-peptide and mixed-meal stimulation of proinsulin were found only in subjects with high stimulated C-peptide values (≥0.2 nmol/L). Specifically, increases in proinsulin with mixed-meal stimulation were present only in the group with high stimulated C-peptide values, with no increases observed among subjects with low or undetectable (<0.017 nmol/L) residual C-peptide. CONCLUSIONS: In individuals with long-duration type 1 diabetes, the ability to secrete proinsulin persists, even in those with undetectable serum C-peptide.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proinsulina/metabolismo , Adolescente , Adulto , Peptídeo C/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 1/sangue , Jejum/metabolismo , Feminino , Humanos , Insulina/sangue , Masculino , Refeições , Pessoa de Meia-Idade , Proinsulina/sangue , Fatores de Tempo , Adulto Jovem
11.
J Immunol ; 201(12): 3524-3533, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30455401

RESUMO

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.


Assuntos
Autoantígenos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/metabolismo , Peptídeos/metabolismo , Proinsulina/metabolismo , Linfócitos T/imunologia , Motivos de Aminoácidos/genética , Apresentação do Antígeno , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Predisposição Genética para Doença , Glutamato Descarboxilase/metabolismo , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/metabolismo , Humanos , Ativação Linfocitária , Modelos Químicos , Peptídeos/genética , Proinsulina/genética
12.
PLoS One ; 13(11): e0207065, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30412637

RESUMO

AIMS: Increased proinsulin (PI) compared to C-peptide (CP) concentrations have been reported, both prior to type 1 diabetes mellitus (T1D) onset, as well as early in disease. In this pilot study, we sought to define the normal PI secretion in a healthy cohort and compare this to a local T1D cohort and a separate well-defined nationally representative T1D cohort with measurable CP. METHODS: Thirteen healthy subjects and 12 T1D subjects with T1D >3 years from the local T1D cohort completed mixed meal tolerance tests (MMTT) with PI and CP measured over 90 and 240 minutes. The change in CP (maximum versus baseline, ΔCP) during MMTT in the T1D Exchange T1D cohort was stratified according to non-fasting PI concentrations, based on a fasting PI threshold, as defined by the healthy control group. RESULTS: The maximum fasting PI in the control group was 6 pmol/L. Individuals from the T1D Exchange with a non-fasting PI ≥ 6 pmol/L had a lower ΔCP during a MMTT, compared to those with a PI < 6 pmol/L. While only three individuals from the local T1D cohort had measurable CP and PI during the MMTT, those with a greater ΔCP had lower PI secretion. CONCLUSION: While all T1D subjects from the T1D Exchange secreted measurable non-fasting PI, those with a greater non-fasting PI demonstrated a decrease in ΔCP during the MMTT. PI may be preferentially secreted compared to CP in some individuals with long standing T1D.


Assuntos
Peptídeo C/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Proinsulina/metabolismo , Adolescente , Adulto , Idoso , Glicemia/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto Jovem
13.
Cytotherapy ; 20(11): 1355-1370, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30340983

RESUMO

BACKGROUND: Antigen-specific regulatory T cells (Tregs) have proven to be effective in reversing established autoimmunity in type 1 diabetes (T1D). Cord blood (CB) can serve as an efficient and safe source for Tregs for antigen-specific immunomodulation in T1D, a strategy that is yet to be explored. Therefore, we assessed the potential of CB in generation of proinsulin (PI)-specific Tregs by using HLA class II tetramers. METHODS: We analyzed the frequency of PI-specific natural Tregs (nTregs) and induced Tregs (iTregs) derived from the CB as well as peripheral blood (PB) of patients with T1D and healthy control subjects. For this, CD4+CD25+CD127low and CD4+CD25-T cells were cultured in the presence of PI-derived peptides, transforming growth factor (TGF)-ß and rapamycin. PI-specific Tregs were then selected using allele-specific HLA II tetramers loaded with PI-derived peptides, followed by suppression assays. RESULTS: Following stimulation, we observed that CB harbors a significantly higher frequency of PI-specific Tregs than PB of subjects with T1D (P = 0.0003). Further, the proportion of PI-specific Tregs was significantly higher in both the nTreg (P = 0.01) and iTreg (P = 0.0003) compartments of CB as compared with PB of subjects with T1D. In co-culture experiments, the PI-specific Tregs suppressed the proliferation of effector T cells significantly (P = 0.0006). The expanded nTregs were able to retain hypomethylation status at their Tregs-specific demethylated region (TSDR), whereas iTregs were unable to acquire the characteristic demethylation pattern. CONCLUSION: Our study demonstrates that CB can serve as an excellent source for generation of functional antigen-specific Tregs for immunotherapeutic approaches in subjects with T1D.


Assuntos
Sangue Fetal/citologia , Proinsulina/metabolismo , Linfócitos T Reguladores/imunologia , Cordão Umbilical/citologia , Adulto , Linfócitos T CD4-Positivos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Diabetes Mellitus Tipo 1/terapia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunomodulação , Recém-Nascido , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Fator de Crescimento Transformador beta/farmacologia
14.
Diabetes Obes Metab ; 20 Suppl 2: 64-76, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30230179

RESUMO

Biosynthesis of peptide hormones by pancreatic islet endocrine cells is a tightly orchestrated process that is critical for metabolic homeostasis. Like neuroendocrine peptides, insulin and other islet hormones are first synthesized as larger precursor molecules that are processed to their mature secreted products through a series of proteolytic cleavages, mediated by the prohormone convertases Pc1/3 and Pc2, and carboxypeptidase E. Additional posttranslational modifications including C-terminal amidation of the ß-cell peptide islet amyloid polypeptide (IAPP) by peptidyl-glycine α-amidating monooxygenase (Pam) may also occur. Genome-wide association studies (GWAS) have showed genetic linkage of these processing enzymes to obesity, ß-cell dysfunction, and type 2 diabetes (T2D), pointing to their important roles in metabolism and blood glucose regulation. In both type 1 diabetes (T1D) and T2D, and in the face of metabolic or inflammatory stresses, islet prohormone processing may become impaired; indeed elevated proinsulin:insulin (PI:I) ratios are a hallmark of the ß-cell dysfunction in T2D. Recent studies suggest that genetic or acquired defects in proIAPP processing may lead to the production and secretion of incompletely processed forms of proIAPP that could contribute to T2D pathogenesis, and additionally that impaired processing of both PI and proIAPP may be characteristic of ß-cell dysfunction in T1D. In islet α-cells, the prohormone proglucagon is normally processed to bioactive glucagon by Pc2 but may express Pc1/3 under certain conditions leading to production of GLP-1(7-36NH2 ). A better understanding of how ß-cell processing of PI and proIAPP, as well as α-cell processing of proglucagon, are impacted by genetic susceptibility and in the face of diabetogenic stresses, may lead to new therapeutic approaches for improving islet function in diabetes.


Assuntos
Carboxipeptidase H/fisiologia , Ilhotas Pancreáticas/metabolismo , Pró-Proteína Convertase 1/fisiologia , Pró-Proteína Convertase 2/fisiologia , Amidina-Liases/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/metabolismo , Oxigenases de Função Mista/metabolismo , Proinsulina/metabolismo
15.
Diabetes ; 67(9): 1783-1794, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29950394

RESUMO

Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic ß-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that ß-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single ß-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human ß-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human ß-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.


Assuntos
Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proinsulina/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Insulina/química , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Cinética , Família Multigênica , Mapeamento de Nucleotídeos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Proinsulina/química , Proinsulina/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/genética
16.
Am J Hum Genet ; 102(4): 620-635, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625024

RESUMO

Genome-wide association studies (GWASs) and functional genomics approaches implicate enhancer disruption in islet dysfunction and type 2 diabetes (T2D) risk. We applied genetic fine-mapping and functional (epi)genomic approaches to a T2D- and proinsulin-associated 15q22.2 locus to identify a most likely causal variant, determine its direction of effect, and elucidate plausible target genes. Fine-mapping and conditional analyses of proinsulin levels of 8,635 non-diabetic individuals from the METSIM study support a single association signal represented by a cluster of 16 strongly associated (p < 10-17) variants in high linkage disequilibrium (r2 > 0.8) with the GWAS index SNP rs7172432. These variants reside in an evolutionarily and functionally conserved islet and ß cell stretch or super enhancer; the most strongly associated variant (rs7163757, p = 3 × 10-19) overlaps a conserved islet open chromatin site. DNA sequence containing the rs7163757 risk allele displayed 2-fold higher enhancer activity than the non-risk allele in reporter assays (p < 0.01) and was differentially bound by ß cell nuclear extract proteins. Transcription factor NFAT specifically potentiated risk-allele enhancer activity and altered patterns of nuclear protein binding to the risk allele in vitro, suggesting that it could be a factor mediating risk-allele effects. Finally, the rs7163757 proinsulin-raising and T2D risk allele (C) was associated with increased expression of C2CD4B, and possibly C2CD4A, both of which were induced by inflammatory cytokines, in human islets. Together, these data suggest that rs7163757 contributes to genetic risk of islet dysfunction and T2D by increasing NFAT-mediated islet enhancer activity and modulating C2CD4B, and possibly C2CD4A, expression in (patho)physiologic states.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Ilhotas Pancreáticas/patologia , Mutação/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Idoso , Alelos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Cromatina/metabolismo , Cromossomos Humanos Par 15/genética , Citocinas/metabolismo , DNA Intergênico/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/metabolismo , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único/genética , Proinsulina/metabolismo , Ratos , Fatores de Risco
17.
J Cell Biol ; 217(4): 1287-1301, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29507125

RESUMO

In mammalian pancreatic ß cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed ß cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic ß cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic ß cells.


Assuntos
Endorribonucleases/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Proinsulina/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Sítios de Ligação , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus/sangue , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Endorribonucleases/deficiência , Endorribonucleases/genética , Insulina/genética , Insulinoma/enzimologia , Insulinoma/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredução , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Fosforilação , Proinsulina/química , Proinsulina/genética , Regiões Promotoras Genéticas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína 1 de Ligação a X-Box/genética , eIF-2 Quinase/metabolismo
18.
PLoS One ; 13(3): e0193882, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29543849

RESUMO

The rhythmic nature of insulin secretion over the 24h cycle in pancreatic islets has been mostly investigated using transcriptomics studies showing that modulation of insulin secretion over this cycle is achieved via distal stages of insulin secretion. We set out to measure ß-cell exocytosis using in depth cell physiology techniques at several time points. In agreement with the activity and feeding pattern of nocturnal rodents, we find that C57/Bl6J islets in culture for 24h exhibit higher insulin secretion during the corresponding dark phase than in the light phase (Zeitgeber Time ZT20 and ZT8, respectively, in vivo). Glucose-induced insulin secretion is increased by 21% despite normal intracellular Ca2+ transients and depolarization-evoked exocytosis, as measured by whole-cell capacitance measurements. This paradox is explained by a 1.37-fold increase in beta cell insulin content. Ultramorphological analyses show that vesicle size and density are unaltered, demonstrating that intravesicular insulin content per granule is modulated over the 24h cycle. Proinsulin levels did not change between ZT8 and ZT20. Islet glucagon content was inversely proportional to insulin content indicating that this unique feature is likely to support a physiological role. Microarray data identified the differential expression of 301 transcripts, of which 26 are miRNAs and 54 are known genes (including C2cd4b, a gene previously involved in insulin processing, and clock genes such as Bmal1 and Rev-erbα). Mouse ß-cell secretion over the full course of the 24h cycle may rely on several distinct cellular functions but late night increase in insulin secretion depends solely on granule insulin content.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretórias/metabolismo , Animais , Exocitose/fisiologia , Glucagon/metabolismo , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proinsulina/metabolismo
19.
J Biol Chem ; 293(6): 1908-1909, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29462795

RESUMO

Most newly synthesized proteins destined for the secretory pathway contain a signal peptide (SP) that triggers cotranslational translocation into the endoplasmic reticulum (ER). However, how small polypeptides undergo ER translocation is not fully understood. In this issue of JBC, Guo et al. describe a mechanism for posttranslational translocation of small secretory proteins featuring a positive charge within the SP N-terminal region. Defects in this element disrupt proper secretion and explain the effects of genetic mutations associated with one type of diabetes.


Assuntos
Peptídeos/metabolismo , Proinsulina/química , Sinais Direcionadores de Proteínas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Peptídeos/química , Peptídeos/genética , Proinsulina/genética , Proinsulina/metabolismo , Biossíntese de Proteínas , Transporte Proteico , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo
20.
J Biol Chem ; 293(14): 5134-5149, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29444822

RESUMO

Loss-of-function mutations of the protein kinase PERK (EIF2AK3) in humans and mice cause permanent neonatal diabetes and severe proinsulin aggregation in the endoplasmic reticulum (ER), highlighting the essential role of PERK in insulin production in pancreatic ß cells. As PERK is generally known as a translational regulator of the unfolded protein response (UPR), the underlying cause of these ß cell defects has often been attributed to derepression of proinsulin synthesis, resulting in proinsulin overload in the ER. Using high-resolution imaging and standard protein fractionation and immunological methods we have examined the PERK-dependent phenotype more closely. We found that whereas proinsulin aggregation requires new protein synthesis, global protein and proinsulin synthesis are down-regulated in PERK-inhibited cells, strongly arguing against proinsulin overproduction being the root cause of their aberrant ER phenotype. Furthermore, we show that PERK regulates proinsulin proteostasis by modulating ER chaperones, including BiP and ERp72. Transgenic overexpression of BiP and BiP knockdown (KD) both promoted proinsulin aggregation, whereas ERp72 overexpression and knockdown rescued it. These findings underscore the importance of ER chaperones working in concert to achieve control of insulin production and identify a role for PERK in maintaining a functional balance among these chaperones.


Assuntos
Proinsulina/metabolismo , eIF-2 Quinase/metabolismo , Animais , Diabetes Mellitus/metabolismo , Retículo Endoplasmático/fisiologia , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Proinsulina/genética , Biossíntese de Proteínas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/genética
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