RESUMO
The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.
Assuntos
Apoptose , Autoantígenos/fisiologia , Movimento Celular , Mitose , Ribonucleoproteínas/fisiologia , Actinas/análise , Autoantígenos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Células HeLa , Humanos , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a Poli(A)/metabolismo , Pseudópodes/química , Pseudópodes/ultraestrutura , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
OBJECTIVE: Maturity-onset diabetes of the young (MODY) due to variants in HNF1A is the most common type of monogenic diabetes. Frequent misdiagnosis results in missed opportunity to use sulfonylureas as first-line treatment. A nongenetic biomarker could improve selection of subjects for genetic testing and increase diagnosis rates. We previously reported that plasma levels of antennary fucosylated N-glycans and high-sensitivity C-reactive protein (hs-CRP) are reduced in individuals with HNF1A-MODY. In this study, we examined the potential use of N-glycans and hs-CRP in discriminating individuals with damaging HNF1A alleles from those without HNF1A variants in an unselected population of young adults with nonautoimmune diabetes. RESEARCH DESIGN AND METHODS: We analyzed the plasma N-glycan profile, measured hs-CRP, and sequenced HNF1A in 989 individuals with diabetes diagnosed when younger than age 45, persistent endogenous insulin production, and absence of pancreatic autoimmunity. Systematic assessment of rare HNF1A variants was performed. RESULTS: We identified 29 individuals harboring 25 rare HNF1A alleles, of which 3 were novel, and 12 (in 16 probands) were considered pathogenic. Antennary fucosylated N-glycans and hs-CRP were able to differentiate subjects with damaging HNF1A alleles from those without rare HNF1A alleles. Glycan GP30 had a receiver operating characteristic curve area under the curve (AUC) of 0.90 (88% sensitivity, 80% specificity, cutoff 0.70%), whereas hs-CRP had an AUC of 0.83 (88% sensitivity, 69% specificity, cutoff 0.81 mg/L). CONCLUSIONS: Half of rare HNF1A sequence variants do not cause MODY. N-glycan profile and hs-CRP could both be used as tools, alone or as adjuncts to existing pathways, for identifying individuals at high risk of carrying a damaging HNF1A allele.
Assuntos
Proteína C-Reativa/metabolismo , Diabetes Mellitus Tipo 2/sangue , Fator 1-alfa Nuclear de Hepatócito/sangue , Polissacarídeos/sangue , Adolescente , Adulto , Alelos , Biomarcadores/sangue , Colesterol/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Insulina/sangue , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Análise de Sequência de DNA , Triglicerídeos/sangue , Adulto JovemRESUMO
INTRODUCTION: Maturity onset diabetes of the young due to HNF1A mutations (HNF1A-MODY) is the most frequent form of monogenic diabetes in adults. It is often misdiagnosed as type 1 or type 2 diabetes, but establishing genetic diagnosis is important, as treatment differs from the common types of diabetes. HNF1A-MODY has not been investigated in Croatia before due to limited access to genetic testing. In this study we aimed to describe the characteristics of young adults diagnosed with diabetes before the age of 45 years, who have rare HNF1A allele variants, and estimate the prevalence of HNF1A-MODY in Croatia. MATERIALS AND METHODS: We recruited 477 C-peptide positive and beta cell antibody negative subjects through the Croatian Diabetes Registry. HNF1A was sequenced for all participants and systematic assessment of the variants found was performed. The prevalence of HNF1A-MODY was calculated in the study group and results extrapolated to estimate the proportion of diabetic individuals with HNF1A-MODY in Croatia and the population prevalence. RESULTS: Our study identified 13 individuals harbouring rare HNF1A allelic variants. After systematic assessment, 8 were assigned a diagnosis of HNF1A-MODY. Two individuals were able to discontinue insulin treatment following the diagnosis. We estimated that HNF1A-MODY in Croatia has a prevalence of 66 (95% CI 61 - 72) cases per million. CONCLUSIONS: The estimated prevalence of HNF1A-MODY in Croatia is similar to that reported in other European countries. Finding cases lead to important treatment changes for patients. This strongly supports the introduction of diagnostic genetic testing for monogenic diabetes in Croatia.
Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Fator 1-alfa Nuclear de Hepatócito/genética , Mutação , Sistema de Registros , Adolescente , Adulto , Idoso , Alelos , Autoanticorpos/sangue , Biomarcadores/sangue , Croácia/epidemiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Feminino , Expressão Gênica , Frequência do Gene , Testes Genéticos , Fator 1-alfa Nuclear de Hepatócito/imunologia , Humanos , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Masculino , Pessoa de Meia-Idade , Prevalência , Análise de Sequência de DNARESUMO
Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-ßH1 and EndoC-ßH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca2+]i, membrane depolarisation and increased action potential firing. Similar to human primary beta cells, KATP channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the KATP channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-ßH1 and -ßH2 cells share many features of primary human ß-cells and thus represent a useful experimental model.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Exocitose , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Edulcorantes/farmacologiaRESUMO
Human genetic studies have emphasised the dominant contribution of pancreatic islet dysfunction to development of Type 2 Diabetes (T2D). However, limited annotation of the islet epigenome has constrained efforts to define the molecular mechanisms mediating the, largely regulatory, signals revealed by Genome-Wide Association Studies (GWAS). We characterised patterns of chromatin accessibility (ATAC-seq, n = 17) and DNA methylation (whole-genome bisulphite sequencing, n = 10) in human islets, generating high-resolution chromatin state maps through integration with established ChIP-seq marks. We found enrichment of GWAS signals for T2D and fasting glucose was concentrated in subsets of islet enhancers characterised by open chromatin and hypomethylation, with the former annotation predominant. At several loci (including CDC123, ADCY5, KLHDC5) the combination of fine-mapping genetic data and chromatin state enrichment maps, supplemented by allelic imbalance in chromatin accessibility pinpointed likely causal variants. The combination of increasingly-precise genetic and islet epigenomic information accelerates definition of causal mechanisms implicated in T2D pathogenesis.
Assuntos
Diabetes Mellitus Tipo 2/genética , Epigênese Genética , Estudo de Associação Genômica Ampla , Ilhotas Pancreáticas/fisiopatologia , Cromatina/metabolismo , Metilação de DNA , Humanos , População BrancaRESUMO
Most genetic association signals for type 2 diabetes risk are located in noncoding regions of the genome, hindering translation into molecular mechanisms. Physiological studies have shown a majority of disease-associated variants to exert their effects through pancreatic islet dysfunction. Systematically characterizing the role of regional transcripts in ß-cell function could identify the underlying disease-causing genes, but large-scale studies in human cellular models have previously been impractical. We developed a robust and scalable strategy based on arrayed gene silencing in the human ß-cell line EndoC-ßH1. In a screen of 300 positional candidates selected from 75 type 2 diabetes regions, each gene was assayed for effects on multiple disease-relevant phenotypes, including insulin secretion and cellular proliferation. We identified a total of 45 genes involved in ß-cell function, pointing to possible causal mechanisms at 37 disease-associated loci. The results showed a strong enrichment for genes implicated in monogenic diabetes. Selected effects were validated in a follow-up study, including several genes (ARL15, ZMIZ1, and THADA) with previously unknown or poorly described roles in ß-cell biology. We have demonstrated the feasibility of systematic functional screening in a human ß-cell model and successfully prioritized plausible disease-causing genes at more than half of the regions investigated.
Assuntos
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Seguimentos , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Fatores de RiscoRESUMO
Repopulating hematopoietic cell compartments after myeloablative chemotherapy remains a key factor in a successful chemotherapy program. Modified and chimeric cytokines have been developed to help reduce inflammation, fever and hospitalization time for patients. A chimeric cytokine, progenipoietin-1 (ProGP-1), containing the G-CSF and FL receptor agonists binds both the G-CSF receptor and FLT-3. It also stimulates the growth of dendritic cells, which play an important role in immunotherapy. While in vivo effects of ProGP-1 are well described, the mechanisms by which it stimulates growth are not well understood. We have investigated the effects of ProGP-1 on prevention of apoptosis in the human hematopoietic cell line OCI-AML.5. ProGP-1 promoted cellular proliferation better than G-CSF or FL separately but stimulated proliferation similar to their co-addition as demonstrated by growth curves and [3H]-thymidine incorporation. ProGP-1 prevented apoptosis to a greater degree than G-CSF or FL alone as determined by annexin V/propidium iodide binding and TUNEL assays. ProGP-1 promoted maintenance of the mitochondrial membrane potential better than G-CSF or FL alone. In addition, Pro-GP promoted a lower redox potential as higher levels of free radicals were detected after cytokine treatment than in cytokine-deprived cells implying increased respiration. These data indicate that ProGP-1 promotes the proliferation and prevents the apoptosis of human hematopoietic cells better than FL or G-CSF alone, and to a similar extent as their co-addition. Thus, ProGP-1 can be used to repopulate certain hematopoietic cells as a single entity rather than the introduction of two different cytokines.
Assuntos
Apoptose/fisiologia , Fatores Estimuladores de Colônias/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Laranja de Acridina , Anexina A5 , Apoptose/efeitos dos fármacos , Fatores Estimuladores de Colônias/farmacologia , Etídio , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Propídio , Proteínas RecombinantesRESUMO
We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signaling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signaling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A (PKA), Rho associated kinase (ROCK), and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho, and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.