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1.
Mol Metab ; 69: 101678, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36690328

RESUMO

OBJECTIVE: Pancreatic ß cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in ß cells, but few studies have examined male-female differences in ß cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in ß cell and islet gene expression and function using both human and rodent samples. METHODS: In humans, we compared ß cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and ß cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress. RESULTS: Our single-cell analysis of human ß cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males. CONCLUSIONS: Our data demonstrate sex differences in ß cell gene expression in both humans and mice, and that female ß cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Feminino , Masculino , Humanos , Camundongos , Animais , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Caracteres Sexuais , Tapsigargina/metabolismo , Proteômica , Insulina/metabolismo , Glucose/metabolismo
2.
Mol Metab ; 68: 101667, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36621763

RESUMO

OBJECTIVES: Pancreatic cancer risk is elevated approximately two-fold in type 1 and type 2 diabetes. Islet amyloid polypeptide (IAPP) is an abundant beta-cell peptide hormone that declines with diabetes progression. IAPP has been reported to act as a tumour-suppressor in p53-deficient cancers capable of regressing tumour volumes. Given the decline of IAPP during diabetes development, we investigated the actions of IAPP in pancreatic ductal adenocarcinoma (PDAC; the most common form of pancreatic cancer) to determine if IAPP loss in diabetes may increase the risk of pancreatic cancer. METHODS: PANC-1, MIA PaCa-2, and H1299 cells were treated with rodent IAPP, and the IAPP analogs pramlintide and davalintide, and assayed for changes in proliferation, death, and glycolysis. An IAPP-deficient mouse model of PDAC (Iapp-/-; Kras+/LSL-G12D; Trp53flox/flox; Ptf1a+/CreER) was generated for survival analysis. RESULTS: IAPP did not impact glycolysis in MIA PaCa-2 cells, and did not impact cell death, proliferation, or glycolysis in PANC-1 cells or in H1299 cells, which were previously reported as IAPP-sensitive. Iapp deletion in Kras+/LSL-G12D; Trp53flox/flox; Ptf1a+/CreER mice had no effect on survival time to lethal tumour burden. CONCLUSIONS: In contrast to previous reports, we find that IAPP does not function as a tumour suppressor. This suggests that loss of IAPP signalling likely does not increase the risk of pancreatic cancer in individuals with diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Neoplasias Pancreáticas , Camundongos , Animais , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas
3.
Diabetes ; 71(12): 2612-2631, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36170671

RESUMO

Transcriptional and functional cellular specialization has been described for insulin-secreting ß-cells of the endocrine pancreas. However, it is not clear whether ß-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in ß-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of ß-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single ß-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH ß-cells were enriched for markers of ß-cell maturity. Ins2(GFP)HIGH ß-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human ß-cells, can be accounted for by dynamic states of insulin gene activity.


Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Humanos , Animais , Insulina/genética , Estresse do Retículo Endoplasmático , Glucose/farmacologia
4.
Nat Commun ; 13(1): 735, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136059

RESUMO

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.


Assuntos
Diabetes Mellitus Tipo 2/genética , Hiperinsulinismo/genética , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Receptor de Insulina/genética , Animais , Conjuntos de Dados como Assunto , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Glucose/metabolismo , Humanos , Hiperinsulinismo/sangue , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Receptor de Insulina/deficiência , Fatores Sexuais
5.
Endocrinology ; 161(11)2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32894758

RESUMO

The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin. Voclosporin (VCS), a next-generation calcineurin inhibitor, is reported to cause fewer incidences of NODAT but the reason is unclear. While calcineurin signaling plays important roles in pancreatic ß-cell survival, proliferation, and function, its effects on human ß-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied 2 clinically relevant doses of TAC (10 ng/mL, 30 ng/mL) and VCS (20 ng/mL, 60 ng/mL), meant to approximate the clinical trough and peak concentrations. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad, and milder, effects on gene expression. Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery.


Assuntos
Ciclosporina/farmacologia , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Tacrolimo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glucose/farmacologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Fatores de Transcrição NFATC/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos
6.
J Am Coll Nutr ; 38(6): 493-498, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30620684

RESUMO

Objective: Obesity is growing at epidemic proportions worldwide. Natural compounds curcumin and α-lipoic acid have been shown to reduce body-weight gain in both preclinical and clinical studies. This study examined the effect of a combination of curcumin and α-lipoic acid on weight gain and adiposity in high-fat-diet (HFD)-fed mice. Methods: C57BL6 mice (7 weeks old) were randomly assigned to receive either HFD (60% fat) or a normal diet (ND, 10% fat) for a 12-week period, following which the mice receiving HFD were further assigned to supplemental curcumin (0.07%), α-lipoic acid (0.2%), or a combination of curcumin and α-lipoic acid formulated into the HFD for a further 12 weeks. Food intake and body mass were determined on a weekly basis. Body fat composition was determined by dual energy X-ray absorptiometry. Results: Treatment with both curcumin and α-lipoic acid significantly reduced body weight gain in HFD-treated mice, and the combination was more effective in attenuating body weight compared to the individual agents. Food intake and caloric intake were significantly lower in the mice that received α-lipoic acid. Percentage body fat and fat mass and lean body mass, which were increased following HFD feeding, were attenuated in the mice receiving curcumin and the combination. Lean mass was also elevated in the mice that were subjected to an HFD, which was unaltered by curcumin or the combination. Conclusions: Taken together, the combination of curcumin and α-lipoic acid exhibits an additive effect in reducing weight gain and adiposity in response to high-fat feeding.


Assuntos
Adiposidade/efeitos dos fármacos , Curcumina/farmacologia , Dieta Hiperlipídica , Ácido Tióctico/farmacologia , Aumento de Peso/efeitos dos fármacos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
J Biol Inorg Chem ; 21(3): 369-81, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26898644

RESUMO

While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr(3+) to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.


Assuntos
Cromo/farmacologia , Oligopeptídeos/farmacologia , Animais , Células Cultivadas , Cromo/administração & dosagem , Cromo/química , Glucose/administração & dosagem , Glucose/metabolismo , Teste de Tolerância a Glucose , Injeções Intravenosas , Resistência à Insulina , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Espectrometria de Fluorescência , Espectrofotometria Infravermelho
9.
Cell Metab ; 23(1): 179-93, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26626461

RESUMO

Pancreatic ß cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive ß cells into more pliable states with reduced cellular insulin levels, increased ß cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, ß cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses ß cell proliferation in a cell-autonomous manner.


Assuntos
Proliferação de Células , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/fisiologia , Insulina/biossíntese , Animais , Células Cultivadas , Metaboloma , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mapas de Interação de Proteínas , Proteoma/metabolismo , Transdução de Sinais , Transcriptoma
10.
Biochim Biophys Acta ; 1852(2): 299-309, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25018087

RESUMO

Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca²âº release as well as prolonged duration of relengthening and intracellular Ca²âº decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Dieta Hiperlipídica , Deleção de Genes , Miocárdio/patologia , Obesidade/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Western Blotting , Cardiomegalia/complicações , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Comportamento Alimentar , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Obesidade/complicações , Obesidade/patologia , Obesidade/fisiopatologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Serina-Treonina Quinases TOR/metabolismo
11.
J Pharmacol Exp Ther ; 349(2): 248-57, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24549372

RESUMO

Type 2 diabetes is growing at epidemic proportions, and pharmacological interventions are being actively sought. This study examined the effect of a novel neuroprotective curcuminoid, CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol], on glucose intolerance and insulin signaling in high-fat diet (HFD)-fed mice. C57BL6 mice (5-6 weeks old) were randomly assigned to receive either a HFD (45% fat) or a low-fat diet (LFD, 10% fat) for 24 weeks, together with CNB-001 (40 mg/kg i.p. per day). Glucose tolerance test revealed that the area under the curve of postchallenge glucose concentration was elevated on HF-feeding, which was attenuated by CNB-001. CNB-001 attenuated body weight gain, serum triglycerides, and IL-6, and augmented insulin signaling [elevated phosphoprotein kinase B (p-Akt), and phosphoinsulin receptor (p-IR)ß, lowered endoplasmic reticulum (ER) stress, protein-tyrosine phosphatase 1B (PTP1B)] and glucose uptake in gastrocnemius muscle of HFD-fed mice. Respiratory quotient, measured using a metabolic chamber, was elevated in HFD-fed mice, which was unaltered by CNB-001, although CNB-001 treatment resulted in higher energy expenditure. In cultured myotubes, CNB-001 reversed palmitate-induced impairment of insulin signaling and glucose uptake. Docking studies suggest a potential interaction between CNB-001 and PTP1B. Taken together, CNB-001 alleviates obesity-induced glucose intolerance and represents a potential candidate for further development as an antidiabetic agent.


Assuntos
Curcumina/análogos & derivados , Hipoglicemiantes/farmacologia , Resistência à Insulina , Fármacos Neuroprotetores/farmacologia , Obesidade/metabolismo , Pirazóis/farmacologia , Adiposidade/efeitos dos fármacos , Animais , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Curcumina/farmacologia , Curcumina/uso terapêutico , Gorduras na Dieta/administração & dosagem , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metabolismo Energético , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/metabolismo , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Obesidade/tratamento farmacológico , Obesidade/fisiopatologia , Ácido Palmítico/administração & dosagem , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Pirazóis/uso terapêutico , Transdução de Sinais , Aumento de Peso/efeitos dos fármacos
12.
Am J Physiol Cell Physiol ; 306(7): C648-58, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24500281

RESUMO

Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.


Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/enzimologia , Pulmão/enzimologia , Fatores de Crescimento Neural/metabolismo , Proteína Quinase C-delta/metabolismo , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Anticorpos Neutralizantes/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Complexo de Golgi/enzimologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Midkina , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/imunologia , Fenótipo , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Vimentina/metabolismo
13.
PLoS One ; 8(10): e77228, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24204775

RESUMO

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [(3)H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.


Assuntos
Estresse do Retículo Endoplasmático/genética , Resistência à Insulina/genética , NF-kappa B/genética , Obesidade/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Dieta Hiperlipídica , Gorduras na Dieta/efeitos adversos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Regulação da Expressão Gênica , Intolerância à Glucose , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , NF-kappa B/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tunicamicina/farmacologia
14.
Dev Neurosci ; 35(4): 293-305, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23751520

RESUMO

Reactive oxygen species (ROS) have been reported to affect neural stem cell self-renewal and therefore may be important for normal development and may influence neurodegenerative processes when ROS activity is elevated. To determine if increasing production of superoxide, via activation of NADPH oxidase (Nox), increases neural stem cell proliferation, 100 nM angiotensin II (Ang II) - a strong stimulator of Nox - was applied to cultures of a murine neural stem cell line, C17.2. Twelve hours following a single treatment with Ang II, there was a doubling of the number of neural stem cells. This increase in neural stem cell numbers was preceded by a gradual elevation of superoxide levels (detected by dihydroethidium fluorescence) from the steady state at 0, 5, and 30 min and gradually increasing from 1 h to the maximum at 12 h, and returning to baseline at 24 h. Ang II-dependent proliferation was blocked by the antioxidant N-acetyl-L-cysteine. Confocal microscopy revealed the presence of two sources of intracellular ROS in C17.2 cells: (i) mitochondrial and (ii) extramitochondrial; the latter indicative of the involvement of one or more specific isoforms of Nox. Of the Nox family, mRNA expression for one member, Nox4, is abundant in neural stem cell cultures, and Ang II treatment resulted in elevation of the relative levels of Nox4 protein. SiRNA targeting of Nox4 mRNA reduced both the constitutive and Ang II-induced Nox4 protein levels and attenuated Ang II-driven increases in superoxide levels and stem cell proliferation. Our findings are consistent with our hypothesis that Ang II-induced proliferation of neural stem cells occurs via Nox4-generated superoxide, suggesting that an Ang II/Nox4 axis is an important regulator of neural stem cell self-renewal and as such may fine-tune normal, stress- or disease-modifying neurogenesis.


Assuntos
Angiotensina II/farmacologia , Proliferação de Células/efeitos dos fármacos , NADPH Oxidases/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Western Blotting , Contagem de Células , Células Cultivadas , Interpretação Estatística de Dados , Camundongos , Microscopia Confocal , NADPH Oxidase 4 , NADPH Oxidases/genética , Células-Tronco Neurais/ultraestrutura , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
15.
Cardiovasc Res ; 95(3): 356-65, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22735370

RESUMO

AIMS: Pulmonary hypertension (PH) is a devastating condition for which no disease-modifying therapies exist. PH is recognized as proliferative disease of the pulmonary artery (PA). In the experimental newborn calf model of hypoxia-induced PH, adventitial fibroblasts in the PA wall exhibit a heightened replication index. Because elevated platelet-derived growth factor ß receptor (PDGFß-R) signalling is associated with PH, we tested the hypothesis that the activation of PDGFß-R contributes to fibroblast proliferation and adventitial remodelling in PH. METHODS AND RESULTS: Newborn calves were exposed to either ambient air (P(B) = 640 mmHg) (Neo-C) or high altitude (P(B) = 445 mm Hg) (Neo-PH) for 2 weeks. PDGFß-R phosphorylation was markedly elevated in PA adventitia of Neo-PH calves as well as in cultured PA fibroblasts isolated from Neo-PH animals. PDGFß-R activation with PDGF-BB stimulated higher replication in Neo-PH cells compared with that of control fibroblasts. PDGF-BB-induced proliferation was dependent on reactive oxygen species generation and extracellular signal-regulated kinase1/2 activation in both cell populations; however, only Neo-PH cell division via PDGFß-R activation displayed a unique dependence on c-Jun N-terminal kinase1 (JNK1) stimulation as the blockade of JNK1 with SP600125, a pharmacological antagonist of the JNK pathway, and JNK1-targeted siRNA selectively blunted Neo-PH cell proliferation. CONCLUSIONS: Our data strongly suggest that hypoxia-induced modified cells engage the PDGFß-R-JNK1 axis to confer distinctively heightened proliferation and adventitial remodelling in PH.


Assuntos
Túnica Adventícia/enzimologia , Proliferação de Células , Fibroblastos/enzimologia , Hipertensão Pulmonar/enzimologia , Hipóxia/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Artéria Pulmonar/enzimologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Túnica Adventícia/patologia , Altitude , Animais , Animais Recém-Nascidos , Becaplermina , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , Hipertensão Pulmonar Primária Familiar , Fibroblastos/patologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/etiologia , Hipóxia/genética , Hipóxia/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Artéria Pulmonar/patologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção
16.
Am J Pathol ; 178(1): 98-109, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21224048

RESUMO

Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1(+/+), heterozygous MKP-1(+/-), and deficient MKP-1(-/-) mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 µm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1(-/-) lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1(-/-) lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1(+/+) MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung.


Assuntos
Fosfatase 1 de Especificidade Dupla/fisiologia , Hipóxia/enzimologia , Pulmão/irrigação sanguínea , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/biossíntese , Actinas/metabolismo , Animais , Fosfatase 1 de Especificidade Dupla/genética , Hipóxia/fisiopatologia , Antígeno Ki-67/metabolismo , Pulmão/fisiologia , Camundongos , Camundongos Mutantes , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Clin Exp Pharmacol Physiol ; 37(8): 841-7, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20456427

RESUMO

1. Isoliquiritigenin (ISL) is a simple chalcone-type flavonoid derived from liquorice compounds. It has been reported to have anti-oxidative and antitumour activities. The aim of the present study was to investigate the antitumour effect of ISL on prostate cancer cells and to explore the possible signalling mechanisms involved. 2. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The fluorescent probe 2',7'-dichlorofluorescein diacetate (H(2)DCF-DA) was used to measure intracellular levels of reactive oxygen species (ROS). Mitochondrial membrane potential (Psi(m)) was measured using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1). 3. Isoliquiritigenin treatment (10-100 micromol/L for 24 h) markedly inhibited the proliferation of both C4-2 and LNCaP prostate cancer cells in a dose-dependent manner. Intriguingly, ISL treatment (10-100 micromol/L for 24 h) had no effect on the viability of IEC-6 normal epithelial cells. Treatment of C4-2 and IEC-6 cells with 87.0 micromol/L ISL significantly decreased ROS levels and the Psi(m) of C4-2 cells, but had no effect on either parameter in IEC-6 cells. Furthermore, AMP-activated protein kinase (AMPK) and extracellular-signal regulated kinase (ERK) levels were three to fourfold higher in IEC-6 cells than in C4-2 cells (P < 0.05). 4. The results of the present study suggest that ISL, a natural anti-oxidant, selectively inhibits the proliferation of prostate cancer C4-2 cells, which may be attributed, in part, to defective AMPK and ERK signalling pathways in C4-2 compared with IEC-6 cells.


Assuntos
Antineoplásicos Fitogênicos , Antioxidantes/farmacologia , Chalconas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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