RESUMO
Dedifferentiation of insulin-secreting ß cells in the islets of Langerhans has been proposed to be a major mechanism of ß-cell dysfunction. Whether dedifferentiated ß cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study ß-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with ß-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in ß cells and restores maturation and function for diabetes remission. Additional ß-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases ß-cell survival and regeneration. GLP-1-oestrogen also protects human ß cells against cytokine-induced dysfunction. This study not only describes mechanisms of ß-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated ß cells for diabetes remission.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/patologia , Insulina/uso terapêutico , Animais , Diabetes Mellitus Experimental/patologia , Estrogênios/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Homeostase , Humanos , Camundongos , Polifarmacologia , Indução de Remissão , EstreptozocinaRESUMO
Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.
Assuntos
Histidina/metabolismo , Pirofosfatase Inorgânica/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Humanos , Pirofosfatase Inorgânica/deficiência , Pirofosfatase Inorgânica/genética , Masculino , Camundongos , Fosforilação , Proteômica , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K(+) increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.
Assuntos
Regulação da Expressão Gênica , Glutamina/metabolismo , Fator de Crescimento Insulin-Like II/biossíntese , Células Secretoras de Insulina/citologia , Receptor IGF Tipo 1/metabolismo , Animais , Apoptose , Proliferação de Células , Glucose/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Compostos de Sulfonilureia/químicaRESUMO
The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.
Assuntos
Temperatura Corporal/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Atividade Motora/fisiologia , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Silenciamento de Genes , Glutamina/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that is part of two structurally and functionally distinct complexes, TORC1 and TORC2. In multicellular organisms, TOR regulates cell growth and metabolism in response to nutrients, growth factors and cellular energy. Deregulation of TOR signaling alters whole body metabolism and causes age-related disease. This review describes the most recent advances in TOR signaling with a particular focus on mammalian TOR (mTOR) in metabolic tissues vis-a-vis aging, obesity, type 2 diabetes, and cancer.
Assuntos
Envelhecimento/genética , Neoplasias/genética , Serina-Treonina Quinases TOR/genética , Envelhecimento/metabolismo , Animais , Proliferação de Células , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Obesidade/genética , Obesidade/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional ß cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory ß cell mass expansion is largely unknown. We found in rodents that ß cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in ß cells using specific anti-miR molecules mimicked gene expression changes occurring during ß cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory ß cell mass expansion occurring under different insulin resistance states.
Assuntos
Adaptação Fisiológica/fisiologia , Resistência à Insulina/fisiologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , MicroRNAs/fisiologia , Obesidade/patologia , Gravidez/fisiologia , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Citocinas/biossíntese , Citocinas/genética , Estradiol/análogos & derivados , Estradiol/farmacologia , Estradiol/fisiologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Regulação da Expressão Gênica/fisiologia , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Mutantes , MicroRNAs/biossíntese , MicroRNAs/genética , Obesidade/fisiopatologia , Tamanho do Órgão/efeitos dos fármacos , Período Pós-Parto/metabolismo , Gravidez/metabolismo , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores de Glucagon/agonistas , Receptores de Glucagon/deficiência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.
Assuntos
Glucoquinase/metabolismo , Fígado/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transativadores/metabolismo , Animais , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glucoquinase/genética , Gluconeogênese , Glucose/genética , Glucose/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Glicólise , Hepatócitos/metabolismo , Homeostase , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Insulina/genética , Insulina/metabolismo , Metabolismo dos Lipídeos , Lipogênese , Fígado/enzimologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Knockout , Fosforilação , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Transativadores/deficiência , Transativadores/genética , Fatores de TranscriçãoRESUMO
OBJECTIVE: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in ß-cells. RESEARCH DESIGN AND METHODS: Glucose homeostasis and ß-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and ß-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated ß-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1ß transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of ß-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.
Assuntos
Proteínas de Homeodomínio/metabolismo , Hiperglicemia/metabolismo , Hiperglicemia/prevenção & controle , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Estresse Fisiológico/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/genética , Hiperglicemia/induzido quimicamente , Immunoblotting , Imuno-Histoquímica , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição Box Pareados/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Estreptozocina/toxicidadeRESUMO
Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição GênicaRESUMO
GLP-1 protects ß-cells against apoptosis by still incompletely understood mechanisms. In a recent study, we searched for novel anti-apoptotic pathways by performing comparative transcriptomic analysis of islets from Gipr-/-;Glp-1r-/- mice, which show increased susceptibility to cytokine-induced apoptosis. We observed a strong reduction in IGF-1R expression in the knockout islets suggesting a link between the gluco-incretin and IGF-1R signaling pathways. Using MIN6 and primary islet cells, we demonstrated that GLP-1 strongly stimulates IGF-1R expression and that activation of the IGF-1R/Akt signaling pathway required active secretion of IGF-2 by the ß-cells. We showed that inactivation of the IGF-1 receptor gene in ß-cells or preventing its up-regulation by GLP-1, as well as suppressing IGF-2 expression or action, blocked the protective effect of GLP-1 against cytokine-induced apoptosis. Thus, an IGF-2/IGF-1 receptor autocrine loop operates in ß-cells and GLP-1 increases its activity by enhancing IGF-1R expression and by stimulating IGF-2 secretion. This mechanism is required for GLP-1 to protect ß-cells against apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Comunicação Autócrina/fisiologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Animais , Apoptose/genética , Comunicação Autócrina/genética , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Receptor do Peptídeo Semelhante ao Glucagon 1 , Fator de Crescimento Insulin-Like II/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Camundongos , Camundongos Knockout , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/fisiologia , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/fisiologia , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Receptores de Glucagon/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.
Assuntos
Células Secretoras de Insulina/fisiologia , Interleucina-1beta/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Exenatida , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Ilhotas Pancreáticas/citologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Camundongos , RatosRESUMO
Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.
Assuntos
Apoptose , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Ativadoras de ras GTPase/química , Animais , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Inflamação , Secreção de Insulina , Insulinoma/metabolismo , Lentivirus/genética , Camundongos , Microscopia de Fluorescência , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Fatores de TempoRESUMO
We report here that apelin (65-77) activates p70 S6 kinase (p70S6K), not only in CHO cells that have been stably transfected with the apelin receptor, but also in umbilical endothelial cells (HUVEC), which express it endogenously. Apelin (65-77) induces a time-dependent phosphorylation of p70S6K at residues T421/S424 and T389. This dual phosphorylation is associated with two transduction cascades, involving a PI3K pathway and an ERK pathway, respectively. The PI3K pathway, which can be blocked by wortmannin, leads to phosphorylation of Akt at residues T308 or S473, which then promotes the phosphorylation of p70S6K at T421/S424 and T389. The ERK pathway is blocked by PD 098059, a MEK inhibitor, and results in the phosphorylation of p70S6K at T421/S424. Phosphorylation both of Akt and p70S6K is abrogated by pretreatment with pertussis toxin (PTX) and an inhibitor of atypical PKCs. In addition, we demonstrate that apelin (65-77) also increases the enzymatic activity of p70S6K and that the effects of the previously mentioned inhibitors on the level of T389 phosphorylation correlate with their action on enzyme activity. Interestingly, the main findings were reproduced in umbilical endothelial cells and apelin (65-77) promoted thymidine incorporation into DNA of these cells, revealing that apelin is a new mitogenic peptide for the endothelial cell.