RESUMEN
Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic ß-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic ß-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of ß-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.
Asunto(s)
Autoantígenos , Degradación Asociada con el Retículo Endoplásmico , Proinsulina , Proteolisis , Enzimas Ubiquitina-Conjugadoras , Proinsulina/metabolismo , Proinsulina/inmunología , Proinsulina/genética , Autoantígenos/metabolismo , Autoantígenos/inmunología , Humanos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Presentación de Antígeno/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/inmunologíaRESUMEN
Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.
Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Ratones , Animales , Proinsulina/genética , Proinsulina/química , Pliegue de Proteína , Insulina/química , Retículo Endoplásmico , Homeostasis , Disulfuros/químicaRESUMEN
OBJECTIVE: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood. METHODS: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs. RESULTS: we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on beta-cell mass and function after transplantation into mice. In addition to anticipated ER stress, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. CONCLUSIONS: These results highlight a novel mechanism, the loss of beta cell identity, contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.
Asunto(s)
Diabetes Mellitus , Insulina , Humanos , Animales , Ratones , Insulina/genética , Proinsulina/genética , Diabetes Mellitus/genética , Mutación/genética , Insulina Regular Humana/genéticaRESUMEN
Delayed Golgi export of proinsulin has recently been identified as an underlying mechanism leading to insulin granule loss and ß-cell secretory defects in type 2 diabetes (T2D). Because acidification of the Golgi lumen is critical for proinsulin sorting and delivery into the budding secretory granule, we reasoned that dysregulation of Golgi pH may contribute to proinsulin trafficking defects. In this report, we examined pH regulation of the Golgi and identified a partial alkalinization of the Golgi lumen in a diabetes model. To further explore this, we generated a ß-cell specific knockout (KO) of the v0a2 subunit of the v-ATPase pump, which anchors the v-ATPase to the Golgi membrane. Although loss of v0a2 partially neutralized Golgi pH and was accompanied by distension of the Golgi cisternae, proinsulin export from the Golgi and insulin granule formation were not affected. Furthermore, ß-cell function was well preserved. ß-cell v0a2 KO mice exhibited normal glucose tolerance in both sexes, no genotypic difference to diet-induced obesity, and normal insulin secretory responses. Collectively, our data demonstrate the v0a2 subunit contributes to ß-cell Golgi pH regulation but suggest that additional disturbances to Golgi structure and function contribute to proinsulin trafficking defects in diabetes.NEW & NOTEWORTHY Delayed proinsulin export from the Golgi in diabetic ß-cells contributes to decreased insulin granule formation, but the underlying mechanisms are not clear. Here, we explored if dysregulation of Golgi pH can alter Golgi function using ß-cell specific knockout (KO) of the Golgi-localized subunit of the v-ATPase, v0a2. We show that partial alkalinization of the Golgi dilates the cisternae, but does not affect proinsulin export, insulin granule formation, insulin secretion, or glucose homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animales , Femenino , Masculino , Ratones , Adenosina Trifosfatasas , Diabetes Mellitus Tipo 2/genética , Glucosa , Insulina , Proinsulina/genéticaRESUMEN
OBJECTIVE: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. METHODS: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in ß-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. RESULTS: With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with ß-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. CONCLUSION: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing ß-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating ß-cell failure.
Asunto(s)
Glucosa , Insulina , Animales , Ratones , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Glucosa/metabolismo , Insulina/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , Vesículas Secretoras/metabolismoRESUMEN
The genetic etiology of gestational diabetes mellitus (GDM) was suggested to overlap with type-2 diabetes(T2D). Transcription factor 7-like 2 (TCF7L2) and Proprotein Convertase Subtilisin/Kexin type 2 (PCSK2) are T2D susceptibility genes of the insulin synthesis/processing pathway. We analyzed associations of TCF7L2 and PCSK2 variants with GDM risk and evaluated their potential impact on impaired insulin processing in an eastern Indian population. The study included 114 GDM (case) and 228 non-GDM pregnant women (control). rs7903146, rs4132670, rs12255372 of TCF7L2, and rs2269023 of PCSK2 were genotyped by PCR-RFLP, and genotype distributions were compared between case and control. Fasting serum proinsulin and C-peptide levels were measured by ELISA and the Proinsulin/C-peptide ratio was considered an indicator of proinsulin conversion. Significantly higher frequency of risk allele (T) of rs12255372 (p = 0.02, OR = 2.0, 95%CI = 1.11-3.64) and rs4132670 (p = 0.002, OR = 2.26, 95%CI = 1.32-3.87) of TCF7L2 was found in GDM cases than non-GDM controls; TT genotype was associated with significantly increased disease risk. In rs7903146 (TCF7L2) and rs2269023 (PCSK2), although the frequency of risk allele (T) was not significantly higher in cases than controls, an association of TT for both variants remained significant with higher GDM risk in the recessive model. Increased serum pro-insulin and proinsulin:c-peptide ratio was found in GDM than non-GDM women and the phenomenon showed significant association with careers of risk alleles for TCF7L2 variants. In conclusion, TCF7L2 and PCSK2 variants are related to GDM risk in the studied population and hence may serve as potential biomarkers for assessing the disease risk. TCF7L2 variants contribute to impaired insulin processing.
Asunto(s)
Diabetes Mellitus Tipo 2 , Diabetes Gestacional , Humanos , Femenino , Embarazo , Diabetes Gestacional/genética , Proinsulina/genética , Proinsulina/metabolismo , Péptido C/genética , Polimorfismo de Nucleótido Simple , Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad , Proteína 2 Similar al Factor de Transcripción 7/genética , Proproteína Convertasa 2/genéticaRESUMEN
Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.
Asunto(s)
Diabetes Mellitus Tipo 1 , Nanopartículas del Metal , Humanos , Autoantígenos , Proinsulina/genética , Oro , Inyecciones Intradérmicas , Análisis de Expresión Génica de una Sola Célula , Péptidos/genética , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.
Asunto(s)
Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Animales , Proinsulina/genética , Proinsulina/metabolismo , Carboxipeptidasa H/genética , Carboxipeptidasa H/metabolismo , Proteómica , Proproteína Convertasa 2/genética , Proproteína Convertasa 2/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones NoqueadosRESUMEN
AIMS/HYPOTHESIS: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. METHODS: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (ßS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. RESULTS: ßS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in ßS2KO islets. Islets isolated from ßS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of ßS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in ßS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. CONCLUSIONS/INTERPRETATION: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. DATA AVAILABILITY: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Humanos , Animales , Proinsulina/genética , Proinsulina/metabolismo , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Insulina/metabolismo , Glucosa/metabolismo , Islotes Pancreáticos/metabolismoRESUMEN
Immunomodulation combined with antigen therapy holds great promise to arrest autoimmune type 1 diabetes, but clinical translation is hampered by a lack of prognostic biomarkers. Low-dose anti-CD3 plus Lactococcus lactis bacteria secreting proinsulin and IL-10 reversed new-onset disease in nonobese diabetic (NOD) mice, yet some mice were resistant to the therapy. Using miRNA profiling, six miRNAs (i.e., miR-34a-5p, miR-125a-3p, miR-193b-3p, miR-328, miR-365-3p, and miR-671-3p) were identified as differentially expressed in plasma of responder versus nonresponder mice before study entry. After validation and stratification in an independent cohort, plasma miR-193b-3p and miR-365-3p, combined with age and glycemic status at study entry, had the best power to predict, with high sensitivity and specificity, poor response to the therapy. These miRNAs were highly abundant in pancreas-infiltrating neutrophils and basophils with a proinflammatory and activated phenotype. Here, a set of miRNAs and disease-associated parameters are presented as a predictive signature for the L. lactis-based immunotherapy outcome in new-onset type 1 diabetes, hence allowing targeted recruitment of trial participants and accelerated trial execution. ARTICLE HIGHLIGHTS: Low-dose anti-CD3 combined with oral gavage of genetically modified Lactococcus lactis bacteria secreting human proinsulin and IL-10 holds great promise to arrest autoimmune type 1 diabetes, but the absence of biomarkers predicting therapeutic success hampers clinical translation. A set of cell-free circulation miRNAs together with age and glycemia at baseline predicts a poor response after L. lactis-based immunotherapy in nonobese mice with new-onset diabetes. Pancreas-infiltrating neutrophils and basophils are identified as potential cellular sources of discovered miRNAs. The prognostic signature could guide targeted recruitment of patients with newly diagnosed type 1 diabetes in clinical trials with the L. lactis-based immunotherapy.
Asunto(s)
Diabetes Mellitus Tipo 1 , Lactococcus lactis , MicroARNs , Humanos , Animales , Ratones , Diabetes Mellitus Tipo 1/terapia , Interleucina-10 , Lactococcus lactis/genética , Proinsulina/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Biomarcadores , Ratones Endogámicos NOD , InmunoterapiaRESUMEN
The discovery of the insulin hormone over 100 years ago, and its subsequent therapeutic application, marked a key landmark in the history of medicine and medical research. The many roles insulin plays in cell metabolism and growth have been revealed by extensive investigations into the structure and function of insulin, the insulin tyrosine kinase receptor (IR), as well as the signalling cascades, which occur upon insulin binding to the IR. In this review, the insulin gene mutations identified as causing disease and the structural implications of these mutations will be discussed. Over 100 studies were evaluated by one reviewing author, and over 70 insulin gene mutations were identified. Mutations may impair insulin gene transcription and translation, preproinsulin trafficking and proinsulin sorting, or insulin-IR interactions. A better understanding of insulin gene mutations and the resultant pathophysiology can give essential insight into the molecular mechanisms underlying impaired insulin biosynthesis and insulin-IR interaction.
Asunto(s)
Células Secretoras de Insulina , Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Mutación , Proinsulina/genética , Proinsulina/metabolismo , Transporte de Proteínas , Insulina/genéticaRESUMEN
Introduction: Immune-checkpoint inhibitors are effective in various advanced cancers. Type 1 diabetes mellitus induced by them (ICI-T1DM) is a serious complication requiring prompt insulin treatment, but the immunological mechanism behind it is unclear. Methods: We examined amino acid polymorphisms in human histocompatibility leukocyte antigen (HLA) molecules and investigated proinsulin epitope binding affinities to HLA molecules. Results and Discussion: Twelve patients with ICI-T1DM and 35 patients in a control group without ICI-T1DM were enrolled in the study. Allele and haplotype frequencies of HLA-DRB1*04:05, DQB1*04:01, and most importantly DPB1*05:01 were significantly increased in patients with ICI-T1DM. In addition, novel amino acid polymorphisms in HLA-DR (4 polymorphisms), in DQ (12 polymorphisms), and in DP molecules (9 polymorphisms) were identified. These amino acid polymorphisms might be associated with the development of ICI-T1DM. Moreover, novel human proinsulin epitope clusters in insulin A and B chains were discovered in silico and in vitro peptide binding assays to HLA-DP5. In conclusion, significant amino acid polymorphisms in HLA-class II molecules, and conformational alterations in the peptide-binding groove of the HLA-DP molecules were considered likely to influence the immunogenicity of proinsulin epitopes in ICI-T1DM. These amino acid polymorphisms and HLA-DP5 may be predictive genetic factors for ICI-T1DM.
Asunto(s)
Diabetes Mellitus Tipo 1 , Humanos , Diabetes Mellitus Tipo 1/genética , Proinsulina/genética , Inhibidores de Puntos de Control Inmunológico , Aminoácidos , Epítopos , Cadenas beta de HLA-DQ/genética , Antígenos de Histocompatibilidad Clase I/genética , Insulina , Cadenas HLA-DRB1/genéticaRESUMEN
The co-occurrence of migraine and glycemic traits has long been reported in observational epidemiological studies, but it has remained unknown how they are linked genetically. We used large-scale GWAS summary statistics on migraine, headache, and nine glycemic traits in European populations to perform cross-trait analyses to estimate genetic correlation, identify shared genomic regions, loci, genes, and pathways, and test for causal relationships. Out of the nine glycemic traits, significant genetic correlation was observed for fasting insulin (FI) and glycated haemoglobin (HbA1c) with both migraine and headache, while 2-h glucose was genetically correlated only with migraine. Among 1703 linkage disequilibrium (LD) independent regions of the genome, we found pleiotropic regions between migraine and FI, fasting glucose (FG), and HbA1c, and pleiotropic regions between headache and glucose, FI, HbA1c, and fasting proinsulin. Cross-trait GWAS meta-analysis with glycemic traits, identified six novel genome-wide significant lead SNPs with migraine, and six novel lead SNPs with headache (Pmeta < 5.0 × 10-8 and Psingle-trait < 1 × 10-4), all of which were LD-independent. Genes with a nominal gene-based association (Pgene ≤ 0.05) were significantly enriched (overlapping) across the migraine, headache, and glycemic traits. Mendelian randomisation analyses produced intriguing, but inconsistent, evidence for a causal relationship between migraine and headache with multiple glycemic traits; and consistent evidence suggesting increased fasting proinsulin levels may causally decrease the risk of headache. Our findings indicate that migraine, headache, and glycemic traits share a common genetic etiology and provide genetic insights into the molecular mechanisms contributing to their comorbid relationship.
Asunto(s)
Trastornos Migrañosos , Proinsulina , Humanos , Proinsulina/genética , Hemoglobina Glucada , Estudio de Asociación del Genoma Completo , Trastornos Migrañosos/genética , Insulina , Ayuno , Cefalea , Glucosa , Polimorfismo de Nucleótido SimpleRESUMEN
Insulin secretion is critical for glucose homeostasis, and increased levels of the precursor proinsulin relative to insulin indicate pancreatic islet beta-cell stress and insufficient insulin secretory capacity in the setting of insulin resistance. We conducted meta-analyses of genome-wide association results for fasting proinsulin from 16 European-ancestry studies in 45,861 individuals. We found 36 independent signals at 30 loci (p value < 5 × 10-8), which validated 12 previously reported loci for proinsulin and ten additional loci previously identified for another glycemic trait. Half of the alleles associated with higher proinsulin showed higher rather than lower effects on glucose levels, corresponding to different mechanisms. Proinsulin loci included genes that affect prohormone convertases, beta-cell dysfunction, vesicle trafficking, beta-cell transcriptional regulation, and lysosomes/autophagy processes. We colocalized 11 proinsulin signals with islet expression quantitative trait locus (eQTL) data, suggesting candidate genes, including ARSG, WIPI1, SLC7A14, and SIX3. The NKX6-3/ANK1 proinsulin signal colocalized with a T2D signal and an adipose ANK1 eQTL signal but not the islet NKX6-3 eQTL. Signals were enriched for islet enhancers, and we showed a plausible islet regulatory mechanism for the lead signal in the MADD locus. These results show how detailed genetic studies of an intermediate phenotype can elucidate mechanisms that may predispose one to disease.
Asunto(s)
Diabetes Mellitus Tipo 2 , Proinsulina , Humanos , Proinsulina/genética , Proinsulina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Insulina/genética , Insulina/metabolismo , Glucosa , Factores de Transcripción/genética , Proteínas de Homeodominio/genéticaRESUMEN
Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.
Asunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Proinsulina/genética , Proinsulina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Esfingolípidos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Homeostasis , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismoRESUMEN
BACKGROUND: CDK5 regulatory subunit associated protein 1 like 1 (CDKAL1) encodes a tRNA modifying enzyme involved in the proper protein translation and regulation of insulin production encoded by the CDKL gene. Sequence variations in the CDKAL1 gene lead to the misreading of the Lys codon in proinsulin, resulting in decreased glucose-stimulated proinsulin production. Various polymorphic sequence variants of the CDKAL1 gene such as rs7754840, rs7756992, rs9465871, and rs10946398 are reported to be associated with type 2 diabetes mellitus and gestational diabetes mellitus (GDM) incidence. One of these single nucleotide polymorphisms i.e., rs10946398 has been reported to impact the risk of GDM and its outcomes in pregnant women of different ethnicities i.e., Egypt, Chinese, Korean, Indian, Arab, and Malaysian. Numerous findings have shown that rs10946398 overturns the regulation of CDKAL1 expression, resulting in decreased insulin production and elevated risk of GDM. However, there is no data regarding rs10946398 genotype association with GDM incidence in our population. METHODOLOGY: In this study, 47 GDM patients and 40 age-matched controls were genotyped for rs10946398 CDKAL1 variant using Tetra primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra ARMS-PCR). RESULTS: Analysis of the results showed the significant association of the C allele of CDKAL1 SNP rs10946398 (χ2 = 0.02 p = 0.001) with the risk of GDM development. Conclusively, the results support the role of SNP i.e., rs10946398 of CDKAL1 gene in GDM development in Pakistani female patients. However, future large-scale studies are needed to functionally authenticate the role of variant genotypes in the disease pathogenesis and progression.
Asunto(s)
Diabetes Mellitus Tipo 2 , Diabetes Gestacional , Humanos , Femenino , Embarazo , Diabetes Gestacional/genética , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Proinsulina/genética , Pakistán , Polimorfismo de Nucleótido Simple/genética , Genotipo , Insulina/genética , Insulina/metabolismo , Predisposición Genética a la Enfermedad , ARNt Metiltransferasas/genéticaRESUMEN
Monogenetic diabetes mellitus (DM) describes a collection of single-gene diseases marked by hyperglycemia presenting in childhood or adulthood and the absence of immunological markers of type 1 DM. Mutations in the human insulin gene INS give rise to two separate clinical syndromes: permanent neonatal DM, type 4 (PNDM4), and maturity-onset diabetes of youth, type 10 (MODY10); the former presents shortly after birth and the latter presents in childhood and adulthood. We describe a 40-year-old man in a kindred with high prevalence of DM who presented with severe hyperglycemia but not ketoacidosis or hypertriglyceridemia. Twelve years after initial presentation, the patient had elevated proinsulin and normal plasma C-peptide when nearly euglycemic on treatment with insulin glargine. A novel INS mutation, Gln65Arg, within the C-peptide region was identified. The INS (p.Gln65Arg) mutation may cause MODY10 by disrupting proinsulin maturation.
Asunto(s)
Diabetes Mellitus Tipo 2 , Hiperglucemia , Masculino , Recién Nacido , Humanos , Adolescente , Adulto , Proinsulina/genética , Péptido C/genética , Diabetes Mellitus Tipo 2/genética , Insulina/uso terapéutico , MutaciónRESUMEN
Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in ß cells. SEL1L-HRD1 ERAD deficiency in ß cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in ß cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and ß cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in ß cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and ß cell function.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Endorribonucleasas , Ratones , Animales , Endorribonucleasas/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas/metabolismoRESUMEN
BACKGROUND: Non-coding genetic variants that influence gene transcription in pancreatic islets play a major role in the susceptibility to type 2 diabetes (T2D), and likely also contribute to type 1 diabetes (T1D) risk. For many loci, however, the mechanisms through which non-coding variants influence diabetes susceptibility are unknown. RESULTS: We examine splicing QTLs (sQTLs) in pancreatic islets from 399 human donors and observe that common genetic variation has a widespread influence on the splicing of genes with established roles in islet biology and diabetes. In parallel, we profile expression QTLs (eQTLs) and use transcriptome-wide association as well as genetic co-localization studies to assign islet sQTLs or eQTLs to T2D and T1D susceptibility signals, many of which lack candidate effector genes. This analysis reveals biologically plausible mechanisms, including the association of T2D with an sQTL that creates a nonsense isoform in ERO1B, a regulator of ER-stress and proinsulin biosynthesis. The expanded list of T2D risk effector genes reveals overrepresented pathways, including regulators of G-protein-mediated cAMP production. The analysis of sQTLs also reveals candidate effector genes for T1D susceptibility such as DCLRE1B, a senescence regulator, and lncRNA MEG3. CONCLUSIONS: These data expose widespread effects of common genetic variants on RNA splicing in pancreatic islets. The results support a role for splicing variation in diabetes susceptibility, and offer a new set of genetic targets with potential therapeutic benefit.
Asunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , ARN Largo no Codificante , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Islotes Pancreáticos/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , Isoformas de Proteínas/genética , Empalme del ARN , ARN Largo no Codificante/metabolismoRESUMEN
Defective insulin processing is associated with obesity and diabetes. Prohormone convertase 1/3 (PC1/3) is an endopeptidase required for the processing of neurotransmitters and hormones. PC1/3 deficiency and genome-wide association studies relate PC1/3 with early onset obesity. Here, we find that deletion of PC1/3 in obesity-related neuronal cells expressing proopiomelanocortin mildly and transiently change body weight and fail to produce a phenotype when targeted to Agouti-related peptide- or nestin-expressing tissues. In contrast, pancreatic ß cell-specific PC1/3 ablation induces hyperphagia with consecutive obesity despite uncontrolled diabetes with glucosuria. Obesity develops not due to impaired pro-islet amyloid polypeptide processing but due to impaired insulin maturation. Proinsulin crosses the blood-brain-barrier but does not induce central satiety. Accordingly, insulin therapy prevents hyperphagia. Further, islet PC1/3 expression levels negatively correlate with body mass index in humans. In this work, we show that impaired PC1/3-mediated proinsulin processing, as observed in human prediabetes, promotes hyperphagic obesity.