RESUMO
Insulin production in pancreatic beta cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40-48 bp from the 5' end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5'UTR, the human ppI 5'UTR conferred glucose regulation of translation. However alternative splicing of the human 5'UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.
Assuntos
Regiões 5' não Traduzidas , Glucose/metabolismo , Proinsulina/biossíntese , Proinsulina/genética , Biossíntese de Proteínas , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Processamento Alternativo , Animais , Regulação da Expressão Gênica , Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Transforming growth factor (TGF)-alpha- and epidermal growth factor (EGF)-induced signal transduction was directly compared with that of glucose and insulin-like growth factor-1 (IGF-1) in INS-1 cells. TGF-alpha/EGF transiently (<20 min) induced phosphorylation of extracellular-regulated kinase (Erk)-1/2 (>20-fold), glycogen synthase kinase (GSK)-3 (>10-fold), and protein kinase B (PKB) (Ser(473) and Thr(308)), but did not increase [(3)H]thymidine incorporation. In contrast, phosphorylation of Erk1/2, GSK-3, and PKB in response to glucose and IGF-1 was more prolonged (>24 h) and, though not as robust as TGF-alpha/EGF, did increase beta-cell proliferation. Phosphorylation of p70(S6K) was also increased by IGF-1/glucose, but not by TGF-alpha/EGF, despite upstream PKB activation. It was found that IGF-1 induced phosphatidylinositol 3-kinase (PI3K) association with insulin receptor substrate (IRS)-1 and -2 in a glucose-dependent manner, whereas TGF-alpha/EGF did not. The importance of specific IRS-2-mediated signaling events was emphasized in that adenoviral-mediated overexpression of IRS-2 further increased glucose/IGF-1-induced beta-cell proliferation (more than twofold; P < 0.05) compared with control or adenoviral-mediated IRS-1 overexpressing INS-1 cells. Neither IRS-1 nor IRS-2 overexpression induced a beta-cell proliferative response to TGF-alpha/EGF. Thus, a prolonged activation of Erk1/2 and PI3K signaling pathways is important in committing a beta-cell to a mitogenic event, and it is likely that this sustained activation is instigated by signal transduction occurring specifically through IRS-2.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador alfa/farmacologia , Adenoviridae , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA/biossíntese , Vetores Genéticos , Glucose/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/citologia , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidina/metabolismo , TransfecçãoRESUMO
IRS-2 plays a pivotal role in the control of pancreatic beta-cell growth. Here, the effect of altering IRS-2 expression levels in the pancreatic beta-cell line, INS-1, was examined. Adenoviral-mediated increased in IRS-2 protein levels protected against fatty acid (FFA)-induced apoptosis, associated with increased activation of PKB and decreased levels of activated caspase-9. Conversely, decreasing endogenous IRS-2 in INS-1 cells, using adenoviral-mediated expression of IRS-2 antisense, caused a three-fold increase in baseline apoptosis that was further enhanced in the presence of FFA. This was associated with decreased activation of PKB and increased caspase-9 activation. Although IRS-4 is not normally expressed in beta-cells, it was found that adenoviral-mediated introduction of IRS-4 into INS-1 cells enhanced glucose/IGF-1 induced mitogenesis, and protected against FFA-induced apoptosis, similarly to IRS-2. Moreover, expression of IRS-4 in INS-1 cells depleted of IRS-2 levels by IRS-2 antisense, was able to compensate for the lack of IRS-2 and reduce apoptosis in these cells back to normal. Thus, in beta-cells IRS-4 and -2 have similar biological functions. Also, this study further emphasizes the importance of IRS-2 signaling in control of beta-cell survival.
Assuntos
Apoptose , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Adenoviridae/genética , Animais , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática , Ácidos Graxos não Esterificados/toxicidade , Vetores Genéticos , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/citologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de SinaisRESUMO
IRS-2 plays an important role in the control of pancreatic beta-cell growth, however it is unclear if other IRS family members are also involved. Using recombinant adenoviruses, IRS-1, -2 and -3 expression was varied in the beta-cell line, INS-1. Increased IRS-1 expression had no appreciable effect on beta-cell growth. However, increased IRS-2 expression augmented glucose/IGF-1 induced beta-cell growth mitogenesis and decreased apoptosis due to glucose-deprivation. In contrast, increased IRS-3 expression significantly inhibited mitogenesis and increased apoptosis. IRS-3 was intransiently located to the beta-cell plasma membrane, and appeared to be inert in terms of IGF-1 induced signaling. However, increased IRS-3 expression blocked glucose/IGF-1 induced IRS-2 translocation from the cytosol to the plasma membrane, dampening IRS-2/IGF-1R interaction and subsequent activation of the PI3K/PKB/GSK3 signaling pathway. In contrast, glucose/IGF-1 induced Erk-1/-2 and p70S6K activation were unaffected by IRS-3. These data emphasize the importance of IRS-2/PI3K/PKB signal transduction for beta-cell growth and survival.
Assuntos
Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais , Animais , Apoptose , Divisão Celular , Glucose/farmacologia , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/citologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Insulin receptor substrate 2 (IRS-2) plays a critical role in pancreatic beta-cells. Increased IRS-2 expression promotes beta-cell growth and survival, whereas decreased IRS-2 levels lead to apoptosis. It was found that IRS-2 turnover in rat islet beta-cells was rapid, with mRNA and protein half-lives of approximately 90 min and approximately 2 h, respectively. However, this was countered by specific glucose-regulated IRS-2 expression mediated at the transcriptional level. Glucose (> or = 6 mM) increased IRS-2 mRNA and protein levels in a dose-dependent manner, reaching a maximum 4-fold increase in IRS-2 mRNA and a 5-6-fold increase in IRS-2 protein levels at > or = 12 mM glucose (p < or = 0.01). Glucose (15 mM) regulation of islet beta-cell IRS-2 gene expression was rapid, with a significant increase in IRS-2 mRNA levels within 2 h that reached a maximum 4-fold increase by 4 h. IRS-2 protein expression in beta-cells followed that of IRS-2 mRNA. Glucose metabolism was necessary for increased IRS-2 expression in beta-cells. Moreover, inhibition of a glucose-induced rise in islet beta-cell cytosolic [Ca2+]i prevented an increase in IRS-2 expression, indicating this was Ca2+-dependent. The glucose-induced rise in IRS-2 levels correlated with increased IRS-2 tyrosine phosphorylation and downstream activation of protein kinase B. These data indicate that fluctuations of glucose in the normal physiological range (5-15 mM) promote beta-cell survival via regulation of IRS-2 expression and a subsequent parallel protein kinase B activation. Given that the onset of type-2 diabetes is marked by loss of beta-cells, these data further the idea that controlled IRS-2 expression in beta-cells could be a therapeutic means to promote beta-cell survival and delay the onset of the disease.
Assuntos
Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Imunofluorescência , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Transcrição GênicaRESUMO
Regulation of insulin receptor substrate (IRS)-2 expression is critical to beta-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic beta-cells (INS-1), chronic exposure (>8 h) to 15 mm glucose and/or 5 nm IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTORDelta) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviral-mediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected beta-cells expressing mTORDelta, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased beta-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in beta-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased beta-cell apoptosis. This may be a contributing mechanism as to how beta-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.