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1.
Nat Commun ; 13(1): 173, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013311

RESUMO

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , DNA de Neoplasias/genética , Tolerância a Medicamentos/genética , Fluoruracila/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Receptor IGF Tipo 1/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Replicação do DNA , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Halogenação , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Ribossomos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Neurosci ; 41(11): 2360-2372, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33514676

RESUMO

Human fMRI studies show that insulin influences brain activity in regions that mediate reward and motivation, including the nucleus accumbens (NAc). Insulin receptors are expressed by NAc medium spiny neurons (MSNs), and studies of cultured cortical and hippocampal neurons suggest that insulin influences excitatory transmission via presynaptic and postsynaptic mechanisms. However, nothing is known about how insulin influences excitatory transmission in the NAc. Furthermore, insulin dysregulation accompanying obesity is linked to cognitive decline, depression, anxiety, and altered motivation that rely on NAc excitatory transmission. Using whole-cell patch-clamp and biochemical approaches, we determined how insulin affects NAc glutamatergic transmission in nonobese and obese male rats and the underlying mechanisms. We find that there are concentration-dependent, bidirectional effects of insulin on excitatory transmission, with insulin receptor activation increasing and IGF receptor activation decreasing NAc excitatory transmission. Increases in excitatory transmission were mediated by activation of postsynaptic insulin receptors located on MSNs. However, this effect was due to an increase in presynaptic glutamate release. This suggested feedback from MSNs to presynaptic terminals. In additional experiments, we found that insulin-induced increases in presynaptic glutamate release are mediated by opioid receptor-dependent disinhibition. Furthermore, obesity resulted in a loss of insulin receptor-mediated increases in excitatory transmission and a reduction in NAc insulin receptor surface expression, while preserving reductions in transmission mediated by IGF receptors. These results provide the first insights into how insulin influences excitatory transmission in the adult brain, and evidence for a previously unidentified form of opioid receptor-dependent disinhibition of NAc glutamatergic transmission.SIGNIFICANCE STATEMENT Data here provide the first insights into how insulin influences excitatory transmission in the adult brain, and identify previously unknown interactions between insulin receptor activation, opioids, and glutamatergic transmission. These data contribute to our fundamental understanding of insulin's influence on brain motivational systems and have implications for the use of insulin as a cognitive enhancer and for targeting of insulin receptors and IGF receptors to alter motivation.


Assuntos
Endorfinas/farmacologia , Ácido Glutâmico/metabolismo , Insulina/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Receptor de Insulina/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Masculino , Neurônios/efeitos dos fármacos , Obesidade/genética , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/agonistas , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D2/efeitos dos fármacos
3.
Eur J Pharmacol ; 887: 173581, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949596

RESUMO

Glucagon-like peptide-1 (GLP-1) is an endogenous gut hormone and a key regulator in maintaining glucose homeostasis by stimulating insulin secretion. Its natural cleavage product GLP-1 (9-36), which was formerly considered a "bio-inactive" metabolite mainly due to its low affinity for GLP-1 receptor, possesses unique properties such as cardiovascular protection. Little is known about the effects and mechanisms of GLP-1 (9-36) in cerebral ischemia and reperfusion injury. Here, we report that systemic application of GLP-1 (9-36) in adult mice facilitated functional recovery and reduced infarct volume, astrogliosis, and neuronal apoptosis following middle cerebral artery occlusion and reperfusion. Interestingly, these effects were still observed in GLP-1 receptor knockout (Glp-1rKO) mice but were partially reversed in insulin-like growth factor 1 (IGF-1) receptor knockdown (Igf-1rKD) mice. Primary astrocytes were cultured and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), and enzyme-linked immunosorbent assay indicated that GLP-1 (9-36) pretreatment reduces tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels. This effect was not diminished in Glp-1rKO astrocytes but was reversed in Igf-1rKO astrocytes, emphasizing that the anti-inflammatory effect of GLP-1 (9-36) in astrocytes is independent of GLP-1 receptor signaling and is instead mediated by IGF-1 receptor. Immunoprecipitation experiments showed that GLP-1 (9-36) directly interacts with IGF-1 receptor in astrocytes. Western blot data indicated that GLP-1 (9-36) activates IGF-1 receptor and downstream PI3K-AKT pathway in astrocytes upon OGD/R injury, which was abrogated by preincubation with IGF-1 receptor autophosphorylation inhibitor picropodophyllin. Thus, our findings suggest that GLP-1 (9-36) improved stroke outcome by reducing inflammation in astrocytes via interaction with IGF-1 receptor.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Astrócitos/efeitos dos fármacos , Encefalite/tratamento farmacológico , Encefalite/etiologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor IGF Tipo 1/agonistas , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Comportamento Animal/efeitos dos fármacos , Hipóxia Celular , Citocinas/metabolismo , Encefalite/psicologia , Técnicas de Silenciamento de Genes , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Glucose/deficiência , Camundongos , Camundongos Knockout , Cultura Primária de Células , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/psicologia
4.
Sci Rep ; 9(1): 13634, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541165

RESUMO

ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.


Assuntos
Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Receptor IGF Tipo 1/genética , Sialiltransferases/deficiência , Animais , Feminino , Regulação da Expressão Gênica , Genes Letais , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacologia , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Mutação com Perda de Função , Masculino , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único , Gravidez , Receptor IGF Tipo 1/agonistas
5.
Exp Neurol ; 312: 72-81, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30503192

RESUMO

Disruption of the blood-brain barrier results in the formation of edema and contributes to the loss of neurological function following intracerebral hemorrhage (ICH). This study examined insulin-like growth factor-1 (IGF-1) as a treatment and its mechanism of action for protecting the blood-brain barrier after ICH in mice. 171 Male CD-1 mice were subjected to ICH via collagenase or autologous blood. A dose study for recombinant human IGF-1 (rhIGF-1) was performed. Brain water content and behavioral deficits were evaluated at 24 and 72 h after the surgery, and Evans blue extravasation and hemoglobin assay were conducted at 24 h. Western blotting was performed for the mechanism study and interventions were used targeting the IGF-1R/GSK3ß/MEKK1 pathway. rhIGF-1 reduced edema and blood-brain barrier permeability, and improved neurobehavior outcomes. Western blots showed that rhIGF-1 reduced p-GSK3ß and MEKK1 expression, thereby increasing occludin and claudin-5 expression. Inhibition and knockdown of IGF-1R reversed the therapeutic benefits of rhIGF-1. The findings within suggest that stimulation of the IGF-1R is a therapeutic target for ICH which may lead to improved neurofunctional and blood-brain barrier protection.


Assuntos
Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Hemorragia Cerebral/metabolismo , Fator de Crescimento Insulin-Like I/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Hemorragia Cerebral/tratamento farmacológico , Injeções Intraventriculares , Masculino , Camundongos , RNA Interferente Pequeno/administração & dosagem , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo
6.
BMC Cancer ; 17(1): 636, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28882129

RESUMO

BACKGROUND: The insulin growth factor (IGF) pathway has been proposed as a potential therapeutic target in bladder cancer. We characterized the expression of components of the IGF pathway - insulin growth factor receptors (INSR, IGF1R, IGF2R), ligands (INS, IGF1, IGF2), and binding proteins (IGFBP1-7, IGF2BP1-3) - in bladder cancer and its correlation with IGF1R activation, and the anti-proliferative efficacy of an IGF1R kinase inhibitor in this setting. METHODS: We analyzed transcriptomic data from two independent bladder cancer datasets, corresponding to 200 tumoral and five normal urothelium samples. We evaluated the activation status of the IGF pathway in bladder tumors, by assessing IGF1R phosphorylation and evaluating its correlation with mRNA levels for IGF pathway components. We finally evaluated the correlation between inhibition of proliferation by a selective inhibitor of the IGF1R kinase (AEW541), reported in 13 bladder cancer derived cell lines by the Cancer Cell Line Encyclopedia Consortium and mRNA levels for IGF pathway components. RESULTS: IGF1R expression and activation were stronger in non-muscle-invasive than in muscle-invasive bladder tumors. There was a significant inverse correlation between IGF1R phosphorylation and IGFBP5 expression in tumors. Consistent with this finding, the inhibition of bladder cell line viability by IGF1R inhibitor was also inversely correlated with IGFBP5 expression. CONCLUSION: The IGF pathway is activated and therefore a potential therapeutic target for non muscle-invasive bladder tumors and IGFBP5 could be used as a surrogate marker for predicting tumor sensitivity to anti-IGF therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/antagonistas & inibidores , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Ligantes , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Ligação Proteica , RNA Mensageiro/genética , Transdução de Sinais , Neoplasias da Bexiga Urinária/patologia
7.
Breast Cancer Res ; 19(1): 14, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28173837

RESUMO

BACKGROUND: The insulin-like growth factor 1 (IGF1) signaling axis plays a major role in tumorigenesis. In a previous experiment, we chronically treated mice with several agonists of the IGF1 receptor (IGF1R). We found that chronic treatment with insulin analogues with high affinity towards the IGF1R (IGF1 and X10) decreased the mammary gland tumor latency time in a p53R270H/+WAPCre mouse model. Frequent injections with insulin analogues that only mildly activated the IGF1R in vivo (glargine and insulin) did not significantly decrease the tumor latency time in this mouse model. METHODS: Here, we performed next-generation RNA sequencing (40 million, 100 bp reads) on 50 mammary gland tumors to unravel the underlying mechanisms of IGF1R-promoted tumorigenesis. Mutational profiling of the individual tumors was performed to screen for treatment-specific mutations. The transcriptomic data were used to construct a support vector machine (SVM) classifier so that the phenotypic characteristics of tumors exposed to the different insulin analogue treatments could be predicted. For translational purposes, we ran the same classifiers on transcriptomic (micro-array) data of insulin analogue-exposed human breast cancer cell lines. Genome-scale metabolic modeling was performed with iMAT. RESULTS: We found that chronic X10 and IGF1 treatment resulted in tumors with an increased and sustained proliferative and invasive transcriptomic profile. Furthermore, a Warburg-like effect with increased glycolysis was observed in tumors of the X10/IGF1 groups and, to a lesser extent, also in glargine-induced tumors. A metabolic flux analysis revealed that this enhanced glycolysis programming in X10/IGF1 tumors was associated with increased biomass production programs. Although none of the treatments induced genetic instability or enhanced mutagenesis, mutations in Ezh2 and Hras were enriched in X10/IGF1 treatment tumors. CONCLUSIONS: Overall, these data suggest that the decreased mammary gland tumor latency time caused by chronic IGF1R activation is related to modulation of tumor progression rather than increased tumor initiation.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Glucose/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Biomarcadores , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Glicólise , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Transgênicos , Mutação , Prognóstico , Receptor IGF Tipo 1/agonistas , Transdução de Sinais , Transcriptoma , Carga Tumoral , Proteínas ras/genética
8.
Cardiovasc Diabetol ; 15(1): 161, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905925

RESUMO

BACKGROUND: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic process. In a previous work, we demonstrated that the insulin receptor isoform A (IRA) and its association with the insulin-like growth factor-I receptor (IGF-IR) confer a proliferative advantage to VSMCs. However, the role of IR and IGF-IR in VSMC migration remains poorly understood. METHODS: Wound healing assays were performed in VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR-/- VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs). To study the role of IR isoforms and IGF-IR in experimental atherosclerosis, we used ApoE-/- mice at 8, 12, 18 and 24 weeks of age. Finally, we analyzed the mRNA expression of total IR, IRB isoform, IGF-IR and IGFs by qRT-PCR in the medial layer of human aortas. RESULTS: IGF-I strongly induced migration of the four cell lines through IGF-IR. In contrast, insulin and IGF-II only caused a significant increase of IRA VSMC migration which might be favored by the formation of IRA/IGF-IR receptors. Additionally, a specific IGF-IR inhibitor, picropodophyllin, completely abolished insulin- and IGF-II-induced migration in IRB, but not in IRA VSMCs. A significant increase of IRA and IGF-IR, and VSMC migration were observed in fibrous plaques from 24-week-old ApoE-/- mice. Finally, we observed a marked increase of IGF-IR, IGF-I and IGF-II in media from fatty streaks as compared with both healthy aortas and fibrolipidic lesions, favoring the ability of medial VSMCs to migrate into the intima. CONCLUSIONS: Our data suggest that overexpression of IGF-IR or IRA isoform, as homodimers or as part of IRA/IGF-IR hybrid receptors, confers a stronger migratory capability to VSMCs as might occur in early stages of atherosclerotic process.


Assuntos
Aterosclerose/metabolismo , Movimento Celular , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Dieta Ocidental , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Isoformas de Proteínas , Receptor Cross-Talk/efeitos dos fármacos , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/genética , Receptor de Insulina/agonistas , Receptor de Insulina/genética , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Fatores de Tempo
9.
Endocrinology ; 157(1): 61-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26556536

RESUMO

In comparison with young females, middle-aged female rats sustain greater cerebral infarction and worse functional recovery after stroke. These poorer stroke outcomes in middle-aged females are associated with an age-related reduction in IGF-I levels. Poststroke IGF-I treatment decreases infarct volume in older females and lowers the expression of cytokines in the ischemic hemisphere. IGF-I also reduces transfer of Evans blue dye to the brain, suggesting that this peptide may also promote blood-brain barrier function. To test the hypothesis that IGF-I may act at the blood-brain barrier in ischemic stroke, 2 approaches were used. In the first approach, middle-aged female rats were subjected to middle cerebral artery occlusion and treated with IGF-I after reperfusion. Mononuclear cells from the ischemic hemisphere were stained for CD4 or triple-labeled for CD4/CD25/FoxP3 and subjected to flow analyses. Both cohorts of cells were significantly reduced in IGF-I-treated animals compared with those in vehicle controls. Reduced trafficking of immune cells to the ischemic site suggests that blood-brain barrier integrity is better maintained in IGF-I-treated animals. The second approach directly tested the effect of IGF-I on barrier function of aging endothelial cells. Accordingly, brain microvascular endothelial cells from middle-aged female rats were cultured ex vivo and subjected to ischemic conditions (oxygen-glucose deprivation). IGF-I treatment significantly reduced the transfer of fluorescently labeled BSA across the endothelial monolayer as well as cellular internalization of fluorescein isothiocyanate-BSA compared with those in vehicle-treated cultures, Collectively, these data support the hypothesis that IGF-I improves blood-brain barrier function in middle-aged females.


Assuntos
Envelhecimento , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/uso terapêutico , Receptor IGF Tipo 1/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Isquemia Encefálica/imunologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Permeabilidade Capilar/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Cérebro/efeitos dos fármacos , Cérebro/imunologia , Cérebro/metabolismo , Cérebro/patologia , Implantes de Medicamento , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Hipoglicemia/etiologia , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Microvasos/efeitos dos fármacos , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/patologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Ratos Sprague-Dawley , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
10.
Endocrinology ; 157(1): 336-45, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26469138

RESUMO

IGF-1 receptor (IGF-1R) signaling is implicated in cardiac hypertrophy and longevity. However, the role of IGF-1R in age-related cardiac remodeling is only partially understood. We therefore sought to determine whether the deletion of the IGF-1R in cardiomyocytes might delay the development of aging-associated myocardial pathologies by examining 2-year-old male cardiomyocyte-specific IGF-1R knockout (CIGF1RKO) mice. Aging was associated with the induction of IGF-1R expression in hearts. Cardiomyocytes hypertrophied with age in wild-type (WT) mice. In contrast, the cardiac hypertrophic response associated with aging was blunted in CIGF1RKO mice. Concomitantly, fibrosis was reduced in aged CIGF1RKO compared with aged WT hearts. Expression of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and receptor activator of nuclear factor-κB ligand was increased in aged WT hearts, but this increase was attenuated in aged CIGF1RKO hearts. Phosphorylation of Akt was increased in aged WT, but not in aged CIGF1RKO, hearts. In cultured cardiomyocytes, IGF-1 induced senescence as demonstrated by increased senescence-associated ß-galactosidase staining, and a phosphoinositide 3-kinase inhibitor inhibited this effect. Furthermore, inhibition of phosphoinositide 3-kinase significantly prevented the increase in IL-1α, IL-1ß, receptor activator of nuclear factor-κB ligand, and p21 protein expression by IGF-1. These data reveal an essential role for the IGF-1-IGF-1R-Akt pathway in mediating cardiomyocyte senescence.


Assuntos
Envelhecimento , Cardiomegalia/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Receptor IGF Tipo 1/metabolismo , Remodelação Ventricular , Animais , Biomarcadores/metabolismo , Cardiomegalia/imunologia , Cardiomegalia/patologia , Cardiomegalia/prevenção & controle , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Fibrose , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/imunologia , Ventrículos do Coração/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
11.
J Biol Chem ; 291(9): 4547-60, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26702053

RESUMO

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1ß blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteína Fosfatase 1/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática , Feminino , Células da Granulosa/citologia , Humanos , Proteínas Substratos do Receptor de Insulina/agonistas , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/genética , Mutação , Fosfatidilinositol 3-Quinase/química , Fosforilação , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Processamento de Proteína Pós-Traducional , Interferência de RNA , Ratos Sprague-Dawley , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Tirosina/metabolismo
12.
Diabetes ; 64(12): 4148-57, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26384384

RESUMO

Insulin-like growth factor 2 (IGF2), produced and secreted by adult ß-cells, functions as an autocrine activator of the ß-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in ß-cell and whole-body physiology is not known. Here, we studied mice with ß-cell-specific inactivation of Igf2 (ßIGF2KO mice) and assessed ß-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female ßIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female ßIGF2KO mice displayed lower GSIS compared with control mice, but reduced ß-cell mass was observed only in female ßIGF2KO mice. During pregnancy, there was no increase in ß-cell proliferation and mass in ßIGF2KO mice. Finally, ß-cell mass expansion in response to acute induction of insulin resistance was lower in ßIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult ß-cell mass and function to preserve in vivo GSIS in aging and to adapt ß-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.


Assuntos
Envelhecimento , Resistência à Insulina , Fator de Crescimento Insulin-Like II/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptor IGF Tipo 1/agonistas , Transdução de Sinais , Alostase , Animais , Apoptose , Proliferação de Células , Cruzamentos Genéticos , Dieta Hiperlipídica/efeitos adversos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Intolerância à Glucose/etiologia , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Secreção de Insulina , Fator de Crescimento Insulin-Like II/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Masculino , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Caracteres Sexuais , Técnicas de Cultura de Tecidos
13.
Arch Physiol Biochem ; 121(1): 32-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25897878

RESUMO

BACKGROUND: We have previously shown that both insulin and IGF1 lead to increased proliferation of keratinocytes. However, whereas insulin supports keratinocytes differentiation, IGF1 inhibits this process. The aim of the present study was to examine the proliferative and differentiative effects of insulin analogues (glargine, detemir, lispro and aspart) in primary keratinocytes in comparison with insulin and IGF1. METHODS: Primary keratinocytes cultures were produced from newborn BALB/c mice skin. Proliferation rates were assessed by [(3)H]-thymidine incorporation and XTT assays and differentiation was evaluated by Western blots analysis. Insulin receptor and IGF1 receptor phosphorylation was assessed by immunoprecipitation assays. RESULTS: Treatment with glargine or detemir resulted in an insulin-like effect on the differentiation process whereas lispro and aspart treatment led to an IGF1-like effect. In addition, treatment of keratinocytes with aspart led to a rapid phosphorylation of the IGF1 receptor. CONCLUSIONS: Our study provides evidence that insulin analogues elicit atypical actions in the skin.


Assuntos
Insulina Aspart/farmacologia , Insulina Detemir/farmacologia , Insulina Glargina/farmacologia , Insulina Lispro/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Queratinócitos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Diferenciação Celular , Proliferação de Células , Expressão Gênica , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo
14.
Cell Signal ; 27(7): 1297-304, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25817573

RESUMO

Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit ß-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gß/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gß/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that ß-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Butadienos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neuropeptídeo Y/farmacologia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 2/antagonistas & inibidores , Receptor IGF Tipo 2/genética , Receptores de Neuropeptídeo Y/genética , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Tirfostinas/farmacologia
15.
J Biol Chem ; 290(1): 467-77, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25391655

RESUMO

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE(-/-) mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca(2+)/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.


Assuntos
Indutores da Angiogênese/farmacologia , Ginsenosídeos/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Receptor IGF Tipo 1/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
16.
Diabetes Obes Metab ; 16 Suppl 1: 16-20, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25200291

RESUMO

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.


Assuntos
Retroalimentação Fisiológica , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Modelos Neurológicos , Neurogênese , Transdução de Sinais , Animais , Antígenos CD/metabolismo , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Homeostase , Humanos , Secreção de Insulina , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/agonistas , Receptor de Insulina/metabolismo
17.
Growth Horm IGF Res ; 24(5): 157-63, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25002025

RESUMO

It is virtually undisputed that IGF-I promotes cell growth and survival. However, the presence of several IGF-I isoforms, vast numbers of intracellular signaling components, and multiple receptors results in a complex and highly regulated system by which IGF-I actions are mediated. IGF-I has long been recognized as one of the critical factors for coordinating muscle growth, enhancing muscle repair, and increasing muscle mass and strength. How to optimize this panoply of pathways to drive anabolic processes in muscle as opposed to aberrant growth in other tissues is an area that deserves focus. This review will address how advances in the bioavailability, potency, and tissue response of IGF-I can provide new potential directions for skeletal muscle therapeutics.


Assuntos
Fator de Crescimento Insulin-Like I/uso terapêutico , Músculo Esquelético , Doenças Musculares/tratamento farmacológico , Animais , Humanos , Fator de Crescimento Insulin-Like I/química , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Isoformas de Proteínas , Receptor IGF Tipo 1/agonistas , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/fisiologia
18.
PLoS Pathog ; 10(6): e1004165, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24967908

RESUMO

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.


Assuntos
Arginase/metabolismo , Leishmaniose Visceral/imunologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor IGF Tipo 1/agonistas , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Células Th2/imunologia , Animais , Arginase/genética , Linhagem Celular , Células Cultivadas , Progressão da Doença , Indução Enzimática/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interleucina-4/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Mesocricetus , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT6/agonistas , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Transdução de Sinais/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Células Th2/parasitologia
19.
J Clin Endocrinol Metab ; 99(7): E1183-90, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24758182

RESUMO

CONTEXT: Graves' orbitopathy (GO) is caused by expansion of the orbital contents by excess adipogenesis and overproduction of hyaluronan (HA). Immunosuppressive and antiinflammatory treatments of GO are not always effective and can have side effects, whereas targeting GO-associated tissue remodeling might be a more logical therapeutic strategy. Previously we reported that signaling cascades through IGF1 receptor and thyrotropin receptor within orbital preadipocytes/fibroblasts drove adipogenesis and HA production. Our current study combined the stimulation of IGF1 receptor and thyrotropin receptor increase of HA accumulation, which we hypothesize is by activation of phosphatidylinositol 3-kinase (PI3K)-1A/PI3K1B, respectively. The central aim of this study was to investigate whether PI3K/mammalian target of rapamycin complex 1 (mTORC1) inhibitors affected adipogenesis and/or HA production within orbital preadipocyte/fibroblasts. METHODS: Human orbital preadipocytes were treated with/without inhibitors, LY294002 (PI3K1A/mTORC1), AS-605240 (PI3K1B), or PI103 (PI3K1A/mTORC1) in serum-free medium for 24 hours or cultured in adipogenic medium for 15 days. Quantitative PCR was used to measure hyaluronan synthases (HAS2) transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. RESULTS: Unlike AS-605240, both LY294002 (10 µM) and PI-103 (5 µM) significantly decreased HAS2 transcripts/HA accumulation and adipogenesis. Because PI-103 and LY294002 are dual PI3K/mTOR inhibitors, we investigated the inhibition of mTORC1 (rapamycin 100 nM), which significantly decreased adipogenesis but had no effect on HAS2 transcripts/HA, implicating PI3K-1A in the latter. CONCLUSIONS: The combined inhibition of PI3K1A and mTORC1 signaling in vitro decreased both HA accumulation and adipogenesis. Because PI3K and mTOR inhibitors are clinically used to treat other conditions, they have the potential to be repositioned to be used as an alternative nonimmunosuppressive therapy of GO.


Assuntos
Descoberta de Drogas , Oftalmopatia de Graves/terapia , Terapia de Alvo Molecular , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Células Cultivadas , Cromonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oftalmopatia de Graves/genética , Oftalmopatia de Graves/metabolismo , Humanos , Ácido Hialurônico/genética , Ácido Hialurônico/metabolismo , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Quinoxalinas/farmacologia , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/metabolismo , Receptores da Tireotropina/agonistas , Receptores da Tireotropina/metabolismo , Transdução de Sinais/genética , Tiazolidinedionas/farmacologia
20.
Endocrinology ; 155(5): 1827-37, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24617524

RESUMO

This study investigated potential mechanisms by which age and IGF-I receptor (IGF-Ir) signaling in the neuroendocrine hypothalamus affect estradiol-positive feedback effects on GnRH neuronal activation and on kisspeptin and N-methyl-D-aspartate (NMDA)-induced LH release and on the abundance of NMDA receptor subunits Nr1 and Nr2b and Kiss1r transcript and protein in the hypothalamus of young and middle-aged female rats. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-Ir, into the third ventricle of ovariectomized female rats primed with estradiol or vehicle and injected with vehicle, kisspeptin (3 or 30 nmol/kg), or NMDA (15 or 30 mg/kg). Regardless of dose, NMDA and kisspeptin resulted in significantly more LH release, GnRH/c-Fos colabeling, and c-Fos immunoreative cells in young than in middle-aged females. Estradiol priming significantly increased Kiss1r, Nr1, and Nr2b receptor transcript and protein abundance in young but not middle-aged female hypothalamus. JB-1 attenuated kisspeptin and NMDA-induced LH release, numbers of GnRH/c-Fos and c-Fos cells, and Kiss1r, Nr1, and Nr2b transcript and protein abundance in young females to levels observed in middle-aged females. IGF-I significantly enhanced NMDA and kisspeptin-induced LH release in middle-aged females without increasing numbers of GnRH/c-Fos or c-Fos immunoreactive cells. IGF-I infusion in middle-aged females also increased Kiss1r, Nr1, and Nr2b protein and transcript to levels that were equivalent to young estradiol-primed females. These findings indicate that age-related changes in estradiol-regulated responsiveness to excitatory input from glutamate and kisspeptin reflect reduced IGF-Ir signaling.


Assuntos
Envelhecimento , Fator de Crescimento Insulin-Like I/metabolismo , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Receptor IGF Tipo 1/agonistas , Receptores de N-Metil-D-Aspartato/agonistas , Transmissão Sináptica , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/crescimento & desenvolvimento , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Infusões Intraventriculares , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , N-Metilaspartato/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Neuroendócrinas/citologia , Células Neuroendócrinas/efeitos dos fármacos , Células Neuroendócrinas/metabolismo , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
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