RESUMEN
Insulin receptor substrate 2 (IRS-2) modulates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which controls the suppression of gluconeogenic genes; IRS-2 is also a critical node of insulin signaling. Because of the high homology between pig and human IRS-2, we investigated the expression pattern and function of porcine IRS-2. QPCR and immunoblotting were used to detect the IRS-2 expression level in different tissues. There were high IRS-2 levels in the cerebral cortex, hypothalamus, and cerebellum in the central nervous system. In peripheral tissues, IRS-2 was expressed at relatively high levels in the liver. Immunohistochemistry analysis revealed that IRS-2 was mainly distributed in the hypothalamus and cerebral cortex. Furthermore, IRS-2 knockdown porcine hepatocytes and porcine aortic endothelial cells (PAECs) were generated. The IRS-2 knockdown induced abnormal expression of genes involved in glycolipid metabolism in hepatocytes and reduced the antiatherosclerosis ability in PAECs. In addition, we disrupted IRS-2 in porcine embryonic fibroblasts (PEFs) using the CRISPR/Cas9 genome editing system, before finally generating IRS-2 knockout embryos by somatic cell nuclear transfer (SCNT). Taken together, our results indicate that IRS-2 might be a valuable target to establish diabetes and vascular disease models in the pig.
Asunto(s)
Células Endoteliales/metabolismo , Hepatocitos/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Porcinos , Animales , Aorta , Clonación Molecular , Proteínas Sustrato del Receptor de Insulina/química , Distribución TisularRESUMEN
Patients with Her2-positive breast cancer exhibit de novo resistance or develop acquired resistance in less than one year after Her2 targeting treatment, but the mechanism is not fully elucidated. Compensatory pathways such as the IGF-1R/IRS-1 pathway, are activated, leading to aberrant enhanced PI3K/Akt/mTOR pathway activity to attenuate the efficacy of trastuzumab. Cullin7 could participate in the degradation of IRS-1 in a mTOR/S6K dependent manner. Whether Cullin7 participates in trastuzumab resistance needs to be further investigated. Here, we reveals that Cullin7 is overexpressed in trastuzumab-resistant Her2 positive breast cancer cells. Knockdown of Cullin7 reduces degradation of Ser phosphorylation of IRS-1, attenuates activation of the PI3K/AKT pathway, and partly restores trastuzumab sensitivity in trastuzumab-resistant Her2 positive breast cancer cells. IGFBP-3 expression is decreased in trastuzumab-resistant Her2 positive breast cancer cells, which leads to release of the Wnt signaling pathway inhibition and an increase in Cullin7 expression, as mediated by TCF7L2. Overexpression of Cullin7 in Her2-amplified breast cancer tissues has clinical implications because it positively correlates with shorter disease-free survival (DFS) and inadequate response to trastuzumab. Thus, our results suggest a critical role for Cullin7 in response to trastuzumab, which has significant implications for selection of the optimal therapeutic strategy for Her2 positive breast cancers.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Cullin/genética , Resistencia a Antineoplásicos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Receptor ErbB-2/genética , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Femenino , Amplificación de Genes , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Ratones , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , TrastuzumabRESUMEN
Although the insulin receptor substrate (IRS) proteins IRS1 and IRS2 share considerable homology and activate common signaling pathways, their contributions to breast cancer are distinct. IRS1 has been implicated in the proliferation and survival of breast tumor cells. In contrast, IRS2 facilitates glycolysis, invasion, and metastasis. To determine the mechanistic basis for IRS2-dependent functions, we investigated unique structural features of IRS2 that are required for invasion. Our studies revealed that the ability of IRS2 to promote invasion is dependent upon upstream insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) activation and the recruitment and activation of phosphatidylinositol 3-kinase (PI3K), functions shared with IRS1. In addition, a 174-amino-acid region in the IRS2 C-terminal tail, which is not conserved in IRS1, is also required for IRS2-mediated invasion. Importantly, this "invasion (INV) region" is sufficient to confer invasion-promoting ability when swapped into IRS1. However, the INV region is not required for the IRS2-dependent regulation of glucose uptake. Bone morphogenetic protein 2-inducible kinase (BMP2K) binds to the INV region and contributes to IRS2-dependent invasion. Taken together, our data advance the mechanistic understanding of how IRS2 regulates invasion and reveal that IRS2 functions important for cancer can be independently targeted without interfering with the metabolic activities of this adaptor protein.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutación , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
The study considers the Suppressor of cytokine signaling 1 (SOCS1) protein as a novel Type 2 diabetes mellitus (T2DM) drug target. T2DM in human beings is also triggered by the over expression of SOCS proteins. The SOCS1 acts as a ubiquitin ligase (E3), degrades Insulin Receptor Substrate 1 and 2 (IRS1 and IRS2) proteins, and causes insulin resistance. Therefore, the structure of the SOCS1 protein was evaluated using homology-modeling and molecular dynamics methods and validated using standard computational protocols. The Protein-Protein docking study of SOCS1 with its natural substrates, IRS1 and IRS2, and subsequent solvent accessible surface area analysis gave insight into the binding region of the SOCS1 protein. The in silico active site prediction tools highlight the residues Val155 to Ile211 in SOCS1 being implicated in the ubiquitin mediated protein degradation of the proteins IRS1 and IRS2. Virtual screening in the active site region, using large structural databases, results in selective lead structures with 3-Pyridinol, Xanthine, and Alanine moieties as Pharmacophore. The virtual screening study shows that the residues Glu149, Gly187, Arg188, Leu191, and Ser205 of the SOCS1 are important for binding. The docking study with current anti-diabetic therapeutics shows that the drugs Glibenclamide and Glyclopyramide have a partial affinity towards SOCS1. The predicted ADMET and IC50 properties for the identified ligands are within the acceptable range with drug-like properties. The structural data of SOCS1, its active site, and the identified lead structures are expedient in the development of new T2DM therapeutics.
Asunto(s)
Hipoglucemiantes/química , Proteínas Sustrato del Receptor de Insulina/química , Proteína 1 Supresora de la Señalización de Citocinas/química , Secuencia de Aminoácidos , Dominio Catalítico , Diabetes Mellitus Tipo 2 , Gliburida/química , Gliburida/metabolismo , Humanos , Hipoglucemiantes/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Piridonas/química , Piridonas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , TermodinámicaRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by Aß plaque deposition in the brain, which is related to the disorder of autophagosome maturation, transport, and formation of autolysosome. Notably, abnormal insulin signaling is connected with cognitive dysfunction in AD. In this study, using APP/PS1 transgenic mice as AD model, we investigated the mechanism by which S14G-humanin (HNG) improved autophagy and insulin signaling in AD brain. Immunohistochemistry was used to determine the levels of mTOR and Aß deposition, and Western blot analysis was used to determine IRS-1, IRS-1 pSEr636, ULK1, p62, LC3 I/LC3 II protein levels. Our results demonstrated that HNG could improve the learning ability and memory in APP/PS1 transgenic mice, possibly through decreasing IRS-1 Ser636 phosphorylation and mTOR protein expression in the hippocampus, thus improving insulin resistance in the brain. In addition, HNG increased ULK1 expression, decreased p62 and LC3 I/LC3 II protein levels, thus enhancing autophagy and decreasing Aß deposition in the brain. Taken together, our results suggest that through the regulation of IRS-1/mTOR insulin signaling in the hippocampus, HNG increases the activity of autophagy and decreases Aß deposition in the brain, and improves learning ability and memory of AD mice.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/genética , Resistencia a la Insulina , Neuronas/citología , Péptidos/administración & dosificación , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides/metabolismo , Animales , Autofagia , Modelos Animales de Enfermedad , Femenino , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Aprendizaje/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Type 2 diabetes mellitus (T2DM) is a chronic and progressive disease that is strongly associated with hyperglycemia (high blood sugar) related to either insulin resistance or insufficient insulin production. Among the various molecular events and players implicated in the manifestation and development of diabetes mellitus, proteins play several important roles. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database has information on 34 human proteins experimentally shown to be related to the T2DM pathogenesis. It is known that many proteins associated with different human maladies are intrinsically disordered as a whole, or contain intrinsically disordered regions. The presented study shows that T2DM is not an exception to this rule, and many proteins known to be associated with pathogenesis of this malady are intrinsically disordered. The multiparametric bioinformatics analysis utilizing several computational tools for the intrinsic disorder characterization revealed that IRS1, IRS2, IRS4, MAFA, PDX1, ADIPO, PIK3R2, PIK3R5, SoCS1, and SoCS3 are expected to be highly disordered, whereas VDCC, SoCS2, SoCS4, JNK9, PRKCZ, PRKCE, insulin, GCK, JNK8, JNK10, PYK, INSR, TNF-α, MAPK3, and Kir6.2 are classified as moderately disordered proteins, and GLUT2, GLUT4, mTOR, SUR1, MAPK1, IKKA, PRKCD, PIK3CB, and PIK3CA are predicted as mostly ordered. More focused computational analyses and intensive literature mining were conducted for a set of highly disordered proteins related to T2DM. The resulting work represents a comprehensive survey describing the major biological functions of these proteins and functional roles of their intrinsically disordered regions, which are frequently engaged in protein-protein interactions, and contain sites of various posttranslational modifications (PTMs). It is also shown that intrinsic disorder-associated PTMs may play important roles in controlling the functions of these proteins. Consideration of the T2DM proteins from the perspective of intrinsic disorder provides useful information that can potentially lead to future experimental studies that may uncover latent and novel pathways associated with the disease.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biología Computacional/métodos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Expresión Génica , Ontología de Genes , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Anotación de Secuencia Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteoma/química , Proteoma/clasificación , Proteoma/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Animales , Sitios de Unión , Biomarcadores , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/química , Línea Celular , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Sustrato del Receptor de Insulina/química , Ligandos , Ratones , Modelos Biológicos , Desarrollo de Músculos , Mioblastos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-akt/química , Ratas , Proteínas Smad/química , UbiquitinaciónRESUMEN
Insulin Receptor Substrate (IRS) proteins are the main cytoplasmic adaptor molecules involved in transducing extracellular signals from receptors to downstream proteins. This protein family have pivotal roles on maintenance, distribution and regulation of signaling networks. Since IRS1/2 interact with and transmits signals from the receptors of insulin, Insulin Like Growth Factor 1 (IGF1), prolactin, growth hormone (GH), leptin, Vascular Endothelial Growth Factor (VEGF), TrkB, ALK and integrins this promoted scientist to think that IRS1 may have functions in cell proliferation, tumorigenesis and metastasis. Therefore, over the past decade, studies on IRS proteins and their functions in cancer has been increased and these studies provided valuable results claiming the involvement of IRS1/2 in cancer development. In this review, we discuss the function and contributions of IRS1 and IRS2 in development of breast cancer.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Transducción de SeñalRESUMEN
Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[14C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia.
Asunto(s)
Fructosa/farmacología , Músculo Liso Vascular/citología , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Aorta/citología , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína S6 Ribosómica/metabolismo , Serina/metabolismoRESUMEN
The present study aimed to decipher the mechanism of action of selected anti-diabetic plants extracts on palmitic acid mediated insulin resistance in muscle cells. Our results showed that extract from Peganum harmala seeds, Eucalyptus camaldulensis and Syzygium aromaticum leaves, showed significant antioxidant activity. We found that these extracts were able to affect stress signalling by reducing p-38 MAP kinase phosphorylation. They also reduced phosphorylation of substrate for insulin receptor (IRS) at serine residues and increased its phosphorylation at tyrosine residues and also enhanced PKB phosphorylation. Glucose uptake was also enhanced in muscle cells after treatment with these extracts. Extracts from Lantana camara, Psidium gujava fruit and different parts of Cassia alata did not affect FFA mediated down-regulation of insulin signalling. The study conclude that seeds of Peganum harmala and leaves of Eucalyptus camaldulensis and Syzygium aromaticum enhanced insulin signal transduction and glucose uptake in muscle cells via reducing oxidative stress. As a result, these herbal extracts may be considered useful to protect from insulin resistance.
Asunto(s)
Hipoglucemiantes/administración & dosificación , Resistencia a la Insulina , Ácido Palmítico/administración & dosificación , Peganum/química , Extractos Vegetales/administración & dosificación , Syzygium/química , Animales , Línea Celular , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: The study aims to find regulatory microRNA(s) responsible for down-regulated insulin receptor (InsR) in the liver of HFD-MetS E3 rats with insulin resistance. METHODS: Firstly, hepatic insulin resistance in HFD-MetS E3 rats was evaluated by RT-qPCR, western blotting, immunohistochemistry and PAS staining. Secondly, the candidate miRNAs targeting rat InsR were predicted through online softwares and detected in the liver of HFD-MetS E3 rats with insulin resistance. Then, the expression of InsR, phosphorylated IRS-1 (pIRS-1) at Tyr632, phosphorylated AKTs (pAKTs) at Ser473 and Thr308, phosphorylated GSK-3ß (p GSK-3ß) at Ser9, phosphorylated GS (pGS) at Ser641 and the glycogen content were detected in CBRH-7919 cells treated with 100 nM insulin for different time periods by western blotting or PAS staining respectively, after transient transfection with miR-497 mimics or inhibitors for 24 h. Lastly, the relation between miR-497 and InsR was further determined using dual luciferase reporter assay. RESULTS: Elevated miR-497 was negatively related with down-regulated InsR in the liver of HFD-MetS E3 rats with insulin resistance. Comparing with the mNC group, glycogen content and the expression of InsR, pIRS-1 (Tyr632), pAKTs (Ser473 and Thr308) and pGSK-3ß (Ser9) decreased significantly in CBRH-7919 cells, while pGS (Ser641) increased significantly, after transient transfection with miR-497 mimics for 24 h and treatment with 100 nM insulin for corresponding time periods, counter to those results in CBRH-7919 cells after similar procedures with miR-497 inhibitors and insulin. In addition, dual luciferase reporter assay further confirmed that miR-497 can bind to the 3'UTR of rat InsR. CONCLUSION: Insulin receptor is the target gene of miR-497, and elevated miR-497 might induce hepatic insulin resistance in HFD-MetS E3 Rats through inhibiting the expression of insulin receptor and confining the activation of IRS-1/PI3K/Akt/GSK-3ß/GS pathway to insulin.
Asunto(s)
Resistencia a la Insulina/genética , Hígado/metabolismo , Síndrome Metabólico/metabolismo , MicroARNs/metabolismo , Receptor de Insulina/genética , Regiones no Traducidas 3' , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Glucógeno Sintasa/química , Glucógeno Sintasa/metabolismo , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Regulación hacia ArribaRESUMEN
BACKGROUND: Insulin resistance is characterized by deficient responses to insulin in its target tissues. In the present study, we examined the effects of L-Citrulline (L-Cit) on insulin sensitivity and signaling cascades in rat hepatoma H4IIE cells and SHRSP.Z-Leprfa/IzmDmcr rats. METHODS: H4IIE cells were pretreated in the presence or absence of 250 µM L-Cit in serum-free medium and then incubated in the presence or absence of 0.1 nM insulin. Rats were allocated into 2 groups; a control group (not treated) and L-Cit group (2 g/kg/day, L-Cit) and treated for 8 weeks. RESULTS: L-Cit enhanced the insulin-induced phosphorylation of Akt in H4IIE cells. Moreover, the inhibited expression of Dex/cAMP-induced PEPCK mRNA by insulin was enhanced by the L-Cit treatment. The phosphorylation of tyrosine, which is upstream of Akt, in insulin receptor substrate-1 (IRS-1) was increased by the L-Cit treatment. The L-Cit-induced enhancement in insulin signaling was not related to the binding affinity of insulin to the insulin receptor or to the expression of the insulin receptor, but to a decrease in the phosphorylation of serine 1101 in IRS-1. These results were also confirmed in animal experiments. In the livers of L-Cit-treated rats, PI3K/Akt signaling was improved by decreases in the phosphorylation of serine 1101. CONCLUSIONS: We herein demonstrated for the first time the beneficial effects of L-Cit on improved insulin resistance associated with enhanced insulin sensitivity. These results may have clinical applications for insulin resistance and the treatment of type-2 diabetes.
Asunto(s)
Citrulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Insulina/metabolismo , Hígado , Serina/metabolismo , Animales , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/efectos de los fármacos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Fosforilación/efectos de los fármacos , RatasRESUMEN
Inflammation induced by exposure to the common food additive carrageenan leads to insulin resistance by increase in Ser(P)(307)-insulin receptor substrate 1 (IRS1) and subsequent decline in the insulin-stimulated increase in Ser(P)(473)-AKT. Inhibition of carrageenan-induced inflammation reversed the increase in Ser(P)(307)-IRS1 but did not completely reverse the carrageenan-induced decline in Ser(P)(473)-AKT. To identify the additional mechanism responsible for the decrease in Ser(P)(473)-AKT, studies were performed in human HepG2 cells and in C57BL/6J mice. Following carrageenan, expression of GRB10 (growth factor receptor-bound 10 protein), an adaptor protein that binds to the insulin receptor and inhibits insulin signaling, increased significantly. GRB10 silencing blocked the carrageenan-induced reduction of the insulin-stimulated increase in Tyr(P)-IRS1 and partially reversed the decline in Ser(P)(473)-AKT. The combination of GRB10 silencing with BCL10 silencing and the reactive oxygen species inhibitor Tempol completely reversed the decline in Ser(P)(473)-AKT. After carrageenan, GRB10 promoter activity was enhanced because of activation by GATA2. A direct correlation between Ser(P)(473)-AKT and Ser(P)(401)-GATA2 was evident, and inhibition of AKT phosphorylation by the PI3K inhibitor LY294002 blocked Ser(401)-GATA2 phosphorylation and the increase in GRB10 expression. Studies indicated that carrageenan inhibited insulin signaling by two mechanisms: through the inflammation-mediated increase in Ser(P)(307)-IRS1, a negative regulator of insulin signaling, and through a transcriptional mechanism leading to increase in GRB10 expression and GRB10-inhibition of Tyr(P)-IRS1, a positive regulator of insulin signaling. These mechanisms converge to inhibit the insulin-induced increase in Ser(P)(473)-AKT. They provide internal feedback, mediated by Ser(P)(473)-AKT, Ser(P)(401)-GATA2, and nuclear GATA2, which links the opposing effects of serine and tyrosine phosphorylations of IRS1 and can modulate insulin responsiveness.
Asunto(s)
Carragenina/toxicidad , Proteína Adaptadora GRB10/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Insulina/metabolismo , Animales , Factor de Transcripción GATA2/metabolismo , Proteína Adaptadora GRB10/química , Proteína Adaptadora GRB10/genética , Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Estadísticos , Fosforilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Serina/química , Transducción de Señal , Tirosina/químicaRESUMEN
A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucocorticoides/efectos adversos , Músculo Esquelético/metabolismo , Péptidos/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Sistema Libre de Células , Femenino , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/inducido químicamente , Ácido Mirístico/química , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitina/químicaRESUMEN
Several models that predict where post-translational modifications are likely to occur and formulate the corresponding association rules are available to analyze the functional potential of a protein sequence, but an algorithm incorporating the functional groups of the involved amino acids in the sequence analyses process is not yet available. In its previous version, MAPRes was utilized to investigate the influence of the surrounding amino acids of post- translationally and co-translationally modifiable sites. The MAPRes has been upgraded to take into account the different biophysical and biochemical properties of the amino acids that have the potential to influence different post- translational modifications (PTMs). In the present study, the upgraded version of MAPRes was implemented on phosphorylated Ser/Thr/Tyr data by considering the polarity and charge of the surrounding amino acids. The patterns mined by MAPRes incorporating structural information on polarity and charge of amino acids suggest distinct structure-function relationships for phosphorylated serines in a multifunctional protein such as the insulin-receptor substrate-1 (IRS-1) protein. The new version of MAPRes is freely available at http://www.imsb.edu.pk/Database.htm.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Análisis de Secuencia de Proteína , Programas Informáticos , Secuencia de Aminoácidos , Minería de Datos , Bases de Datos de Proteínas , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Fosforilación , Fosfoserina/metabolismo , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: To establish the key insulin receptor substrate 1 (IRS-1) structural elements required in this insulin regulatory pathway, we investigated the effects of substituting alanine for serine 307 in IRS-1 on the ability of tumor necrosis factor-α (TNF-α) and a related mediator, suppressor of cytokine signaling 3 (SOCS3), to phosphorylate IRS-1 and regulate insulin signaling in the rat retinal Müller cell (rMC-1) cell line. METHODS: rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose medium and transfected with either normal IRS-1(Ser307)plasmid or a mutated IRS-1(Ser307Ala) plasmid. Cells were also treated with recombinant TNF-α or SOCS3 to induce increased levels of these proteins. RESULTS: In cells with IRS-1(Ser307Ala), TNF-α and SOCS3 failed to phosphorylate IRS-1. Likewise, resulting downstream effects, including changes in phosphorylation of insulin receptor(Tyr960), antiapoptotic Akt phosphorylation, and proapoptotic cleavage of caspase 3 were also blocked. We also report for the first time that SOCS3 and TNF-α are reciprocally stimulatory leading to a mutual enhancement of levels of both factors, thus forming a potential positive feedback loop that contributes to insulin receptor resistance. CONCLUSIONS: Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina.
Asunto(s)
Células Ependimogliales/metabolismo , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Células Ependimogliales/citología , Células Ependimogliales/efectos de los fármacos , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Glucosa/farmacología , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Serina/genética , Serina/metabolismo , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.
Asunto(s)
Cofilina 1/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Procesamiento Proteico-Postraduccional , Extensiones de la Superficie Celular/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/química , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Prolactin is the key hormone to stimulate milk synthesis in mammary epithelial cells. It signals through the Jak2-Stat5 pathway to induce the expression of ß-casein, a milk protein which is often used as a marker for mammary differentiation. Here we examined the effect of pyrrolidine dithiocarbamate (PDTC) on prolactin signaling. Our results show that PDTC downregulates prolactin receptor levels, and inhibits prolactin-induced Stat5 tyrosine phosphorylation and ß-casein expression. This is not due to its inhibitory action on NF-κB since application of another NF-κB inhibitor, BAY 11-7082, and overexpression of I-κBα super-repressor do not lead to the same results. Instead, the pro-oxidant activity of PDTC is involved as inclusion of the antioxidant N-acetylcysteine restores prolactin signaling. PDTC triggers great extents of activation of ERK and JNK in mammary epithelial cells. These do not cause suppression of prolactin signaling but confer serine phosphorylation of insulin receptor substrate-1, thereby perturbing insulin signal propagation. As insulin facilitates optimal ß-casein expression, blocking insulin signaling by PDTC might pose additional impediment to ß-casein expression. Our results thus imply that lactation will be compromised when the cellular redox balance is dysregulated, such as during mastitis.
Asunto(s)
Acetilcisteína/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Prolactina/metabolismo , Pirrolidinas/antagonistas & inhibidores , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tiocarbamatos/antagonistas & inhibidores , Tiocarbamatos/farmacología , Animales , Caseínas/genética , Bovinos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glándulas Mamarias Animales/citología , Ratones , FN-kappa B/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Embarazo , Serina/metabolismoRESUMEN
Melatonin is biosynthesized in the pineal gland and secreted into the bloodstream. Evidences indicate a role of melatonin in the regulation of glucose metabolism. The objective of this study was to investigate the effect of melatonin on insulin sensitivity in insulin resistant adipocytes. Following a preincubation with melatonin or vehicle for 30 min, insulin resistant cells of 3T3-L1 adipocytes were induced by palmitic acids (300 µM, 6 h). Our results showed that palmitic acids inhibited both the basal and insulin-stimulated uptake of [(3)H]-2-Deoxyglucose, down-regulated the levels of IRS-1 and GLUT-4. However, compared to the vehicle group, melatonin pre-treatment increased significantly the uptake of [(3)H]-2-Deoxyglucose as well as the level of GLUT-4, and decreased phosphorylated IRS-1 (Ser307) although total IRS-1 did not change significantly. These data suggest that palmitic acids impair insulin signal via down-regulating the expressions of IRS-1 and GLUT-4; whereas melatonin can ameliorate insulin sensitivity by inhibiting Ser307 phosphorylation in IRS-1 and increasing GLUT-4 expressions in insulin resistant 3T3-L1 adipocytes. We conclude that melatonin regulates the insulin sensitivity and glucose homeostasis via inhibiting Ser-phosphorylation and improving function of IRS-1.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Melatonina/farmacología , Ácido Palmítico/efectos adversos , Serina/metabolismo , Células 3T3-L1 , Animales , Transporte Biológico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas Sustrato del Receptor de Insulina/química , Ratones , Fosforilación/efectos de los fármacosRESUMEN
Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3ß (Gsk-3ß). Inhibition of Gsk-3ß by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.