RESUMO
Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c+/-) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.
Assuntos
Biópsia , Diferenciação Celular/fisiologia , Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/citologia , Adipogenia/fisiologia , Biópsia/métodos , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , Macrófagos/citologiaRESUMO
AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Glucagon/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/fisiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangue , Eletrofisiologia , Exocitose/genética , Exocitose/fisiologia , Feminino , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Imuno-Histoquímica , Hibridização In Situ , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and multi-functional type I cell surface membrane protein, which is known to be phosphorylated by the activated platelet-derived growth factor receptor (PDGFR). The tyrosine kinase inhibitor imatinib, which inhibits PDGFR and c-Abl, and which has previously been reported to counteract ß-cell death and diabetes, has been suggested to reduce atherosclerosis by inhibiting PDGFR-induced LRP1 phosphorylation. The aim of the present study was to study LRP1 function in ß-cells and to what extent imatinib modulates LRP1 activity. LRP1 and c-Abl gene knockdown was performed by RNAi using rat INS-1 832/13 and human EndoC1-ßH1 cells. LRP1 was also antagonized by treatment with the antagonist low-density lipoprotein receptor-related protein associated protein 1 (LRPAP1). We have used PDGF-BB, a PDGFR agonist, and apolipoprotein E (ApoE), an LRP1 agonist, to stimulate the activities of PDGFR and LRP1 respectively. Knockdown or inhibition of LRP1 resulted in increased hydrogen peroxide (H2O2)- or cytokine-induced cell death, and glucose-induced insulin release was lowered in LRP1-silenced cells. These results indicate that LRP1 function is necessary for ß-cell function and that LRP1 is adversely affected by challenges to ß-cell health. PDGF-BB, or the combination of PDGF-BB+ApoE, induced phosphorylation of extracellular-signal-regulated kinase (ERK), Akt and LRP1. LRP1 silencing blocked this event. Imatinib blocked phosphorylation of LRP1 by PDGFR activation but induced phosphorylation of ERK. LRP1 silencing blocked imatinib-induced phosphorylation of ERK. Sunitinib also blocked LRP1 phosphorylation in response to PDGF-BB and induced phosphorylation of ERK, but this latter event was not affected by LRP1 knockdown. siRNA-mediated knockdown of the imatinib target c-Abl resulted in an increased ERK phosphorylation at basal conditions, with no further increase in response to imatinib. Imatinib-induced cell survival of tunicamycin-treated cells was partially mediated by ERK activation. We have concluded that imatinib promotes LRP1-dependent ERK activation, possibly via inhibition of c-Abl, and that this could contribute to the pro-survival effects of imatinib on ß-cells.
Assuntos
Benzamidas/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Técnicas de Silenciamento de Genes , Mesilato de Imatinib , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/fisiologia , RatosRESUMO
The aim of this study is to characterize cell type-specific transcriptional signatures in non-alcoholic steatohepatitis (NASH) to improve our understanding of the disease. We performed single-cell RNA sequencing on liver biopsies from 10 patients with NASH. We applied weighted gene co-expression network analysis and validated our findings using a publicly available RNA sequencing data set derived from 160 patients with non-alcoholic fatty liver disease (NAFLD) and 24 controls with normal liver histology. Our study provides a comprehensive single-cell analysis of NASH pathology in humans, describing 19,627 single-cell transcriptomes from biopsy-proven NASH patients. Our data suggest that the previous notion of "NASH-associated macrophages" can be explained by an up-regulation of normally existing subpopulations of liver macrophages. Similarly, we describe two distinct populations of activated hepatic stellate cells, associated with the level of fibrosis. Finally, we find that the expression of several circulating markers of NAFLD are co-regulated in hepatocytes together with predicted effector genes from NAFLD genome-wide association studies (GWAS), coupled to abnormalities in the complement system. In sum, our single-cell transcriptomic data set provides insights into novel cell type-specific and general biological processes associated with inflammation and fibrosis, emphasizing the importance of studying cell type-specific biological processes in human NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Biomarcadores/metabolismo , Fibrose , Estudo de Associação Genômica Ampla , Humanos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , TranscriptomaRESUMO
The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic ß-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5'-UTR of the human insulin mRNA.
Assuntos
Fator de Iniciação 4A em Eucariotos/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Biossíntese de Proteínas/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Células Cultivadas , Glucose/farmacologia , Humanos , Insulina/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Esteróis/farmacologia , Triazenos/farmacologiaRESUMO
A dynamic production of insulin is necessary for proper glucose homeostasis. In order to generate enough insulin available for exocytosis in response to the demands of the organism, the level of preproinsulin mRNA in the pancreatic beta-cell needs to fluctuate. In animal models for type 2 diabetes the contents of preproinsulin mRNA are lowered, which might suggest that an impaired metabolism of preproinsulin mRNA contributes to the development of glucose intolerance and diabetes. Thus, it is of importance to understand the mechanisms by which preproinsulin mRNA levels are regulated. Although extensively studied, there are aspects of the regulation of insulin gene expression that still remain enigmatic. Our understanding of insulin gene transcription has improved considerably the last 20 years, but less effort has been invested into the control of preproinsulin mRNA stability. The preproinsulin mRNA has a long half-life and changes in preproinsulin mRNA stability, induced by glucose, are likely to be regulated through specific mechanisms. Recent findings indicate that the polypyrimidine tract-binding protein (PTB), also named hnRNP I, by binding to the 3'-UTR (untranslated region) of the preproinsulin mRNA molecule, stabilizes the messenger, thereby participating in the glucose-induced increase in preproinsulin mRNA. This review will focus both on recent findings pertinent to PTB function in general, and on the specific role of PTB on the production of insulin in beta-cells. We will also discuss the putative co-operativity between PTB and other proteins in the control of preproinsulin mRNA stability, and review beta-cell signaling events that may control the mRNA stabilizing effect of PTB.
Assuntos
Insulina/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Precursores de Proteínas/metabolismo , Estabilidade de RNA , Animais , Diabetes Mellitus/metabolismo , Humanos , Insulina/biossíntese , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Transdução de SinaisRESUMO
BACKGROUND: Enhanced IAPP production may contribute to islet amyloid formation in type 2 diabetes. The objective of this study was to determine the effects of the saturated fatty acid palmitate on IAPP levels in human ß-cells. METHODS: EndoC-ßH1 cells and human islets were cultured in the presence of sodium palmitate. Effects on IAPP/insulin mRNA expression and secretion were determined using real-time qPCR/ELISA. Pharmacological activators and/or inhibitors and RNAi were used to determine the underlying mechanisms. RESULTS: We observed that EndoC-ßH1 cells exposed to palmitate for 72 h displayed decreased expression of Pdx-1 and MafA and increased expression of thioredoxin-interacting protein (TXNIP), reduced insulin mRNA expression and glucose-induced insulin secretion, as well as increased IAPP mRNA expression and secretion. Further, these effects were independent of fatty acid oxidation, but abolished in response to GPR40 inhibition/downregulation. In human islets both a high glucose concentration and palmitate promoted increased IAPP mRNA levels, resulting in an augmented IAPP/insulin mRNA ratio. This was paralleled by elevated IAPP/insulin protein secretion and content ratios. CONCLUSIONS: Addition of exogenous palmitate to human ß-cells increased the IAPP/insulin expression ratio, an effect contributed to by activation of GPR40. These findings may be pertinent to our understanding of the islet amyloid formation process.
Assuntos
Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ácido Palmítico/farmacologia , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Insulina/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Oxirredução , Proteína Quinase C/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVES: Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. METHODS AND RESULTS: To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5'- and the 3'-untranslated regions (UTRs) of rat and human insulin mRNA in vitro. We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-ßH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. CONCLUSIONS: These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.
RESUMO
The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy.
Assuntos
Diabetes Mellitus Experimental , Desenvolvimento Fetal , Gravidez em Diabéticas , Receptor para Produtos Finais de Glicação Avançada/genética , Animais , Glicemia/análise , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Dinoprosta/análogos & derivados , Dinoprosta/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Reabsorção do Feto , Expressão Gênica , Fígado/embriologia , Fígado/metabolismo , Lisina/análogos & derivados , Lisina/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Gravidez , Gravidez em Diabéticas/sangue , Gravidez em Diabéticas/genética , Aldeído Pirúvico/sangueRESUMO
The aim of the present investigation was to delineate cytokine-induced signaling and death using the EndoC-ßH1 cells as a model for primary human beta-cells. The cytokines IL-1ß and IFN-γ induced a rapid and transient activation of NF-κB, STAT-1, ERK, JNK and eIF-2α signaling. The EndoC-ßH1 cells died rapidly when exposed to IL-1ß + IFN-γ, and this occurred also in the presence of the actinomycin D. Inhibition of NF-κB and STAT-1 did not protect against cell death, nor did the cytokines activate iNOS expression. Instead, cytokines promoted a rapid decrease in EndoC-ßH1 cell respiration and ATP levels, and we observed protection by the AMPK activator AICAR against cytokine-induced cell death. It is concluded that EndoC-ßH1 cell death can be prevented by AMPK activation, which suggests a role for ATP depletion in cytokine-induced human beta-cell death.
Assuntos
Adenilato Quinase/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Fragmentos de Peptídeos/farmacologia , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Camundongos , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: It has been proposed that the histamine 1 (H(1)) receptor not only promotes allergic reactions but also modulates autoimmune diseases, such as type 1 diabetes. In line with this, it has recently been reported that the H(1)-receptor antagonist cetirizine can counteract the activation of signals/factors pertinent to the pathogenesis of type 1 diabetes and cytokine-induced ß-cell destruction. Therefore, the overall aim of this study was to determine whether H(1)-receptor antagonists affect cytokine-induced ß-cell death and signaling in vitro. METHODS: The insulin-producing cell line ß-TC6 was exposed to the proinflammatory cytokines interleukin 1ß(+) interferon γ, or hydrogen peroxide. The H(1)-receptor antagonists desloratadine and cetirizine were added to the cell cultures and cell viability; macrophage inhibitory factor levels, c-Jun N-terminal kinase phosphorylation, c-Jun expression, and ß-catenin levels were analyzed by flow cytometry, real-time polymerase chain reaction, and immunoblotting. RESULTS: Cetirizine protected partially against both cytokine- and hydrogen peroxide-induced cell death. This effect was paralleled by an inhibition of cytokine-induced c-Jun N-terminal kinase phosphorylation, c-Jun induction, and a restoration of macrophage inhibitory factor contents. Cetirizine also increased the ß-TC6 cell contents of ß-catenin at basal conditions. CONCLUSIONS: Our results indicate a protective effect of a specific H(1)-receptor antagonist.
Assuntos
Cetirizina/farmacologia , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/toxicidade , Interleucina-1beta/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Relação Dose-Resposta a Droga , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Oxirredutases Intramoleculares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.
Assuntos
Glucose/farmacologia , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , MicroRNAs/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/genética , Ilhotas Pancreáticas/efeitos dos fármacos , MicroRNAs/genética , Ácido Palmítico/farmacologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The aim of this study was to further elucidate the role of the polypyrimidine tract binding protein (PTB) in the control of insulin mRNA stability. We observed that the glucose- or interleukin-1beta-induced increase in insulin mRNA was paralleled by an increase in PTB mRNA. To further test the hypothesis that PTB controls insulin gene expression, betaTC-6 cells were treated with a PTB-specific siRNA to modify the beta-cell content of PTB. Surprisingly, we observed an increase in PTB mRNA and PTB protein levels in response to the siRNA treatment. In addition, the PTB-siRNA treatment also increased insulin mRNA. We conclude that expression of the PTB gene controls insulin production.