Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. T... more Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNFa-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharideelicited production of nitrite ðNO À 2 Þ by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO À 2 production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFNc. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNFa, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNFa synthesis. Additionally, PD144795 did not block NF-jB nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug. Ó
Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The pur... more Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The purpose of this study was to explore the link between NO and the survival pathway triggered by insulin-like growth factor-1 (IGF-1) and insulin in insulin producing RINm5F cells and in pancreatic islets. Results show that exposure of cells to IGF-1/insulin protects against serum deprivation-induced apoptosis. This action is prevented with inhibitors of NO generation, PI3K and Akt. Moreover, transfection with the negative dominant form of the tyrosine kinase c-Src abrogates the effect of IGF-1 and insulin on DNA fragmentation. An increase in the expression level of NOS3 protein and in the enzyme activity is observed following exposure of serum-deprived RINm5F cells to IGF-1 and insulin. Phosphorylation of IRS-1, IRS-2 and to less extent IRS-3 takes place when serum-deprived RINm5F cells and rat pancreatic islets are exposed to either IGF-1, insulin, or diethylenetriamine nitric oxide adduct (DETA/NO). In human islets, IRS-1 and IRS-2 proteins are present and tyrosine phosphorylated upon exposure to IGF-1, insulin and DETA/NO. Both rat and human pancreatic islets undergo DNA fragmentation when cultured in serum-free medium and IGF-1, insulin and DETA/NO protect efficiently from this damage. We then conclude that generation of NO participates in the activation of survival pathways by IGF-1 and insulin in beta cells.
Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor that regulates the embryonic ... more Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation towards beta cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESC) to high concentrations of NO donor diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of Polycomb Repressive Complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent marks of histone H3K27me3 and H3K4me3, site specific changes in DNA methylation, and no change in H3 acetylation. Based on these findings, we developed a protocol to differentiate mESC towards insulin producing cells consisting of sequential exposure to DETA-NO, valproic acid and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to fo...
Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cel... more Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cells. The best outcome of the refined protocols became apparent in the first clinical trial announced by ViaCyte, based on the implantation of pancreatic progenitors that would further mature into functional insulin-producing cells inside the patient's body. Areas covered: Several groups, including ours, have contributed to improve strategies to generate insulin-producing cells. Of note, the latest results have gained a substantial amount of interest as a method to create a potentially functional and limitless supply of β-cell to revert diabetes mellitus. This review analyzes the accomplishments that have taken place over the last few decades, summarizes the state-of-art methods for β-cell replacement therapies based on the differentiation of embryonic stem cells into glucose-responsive and insulin-producing cells in a dish and discusses alternative approaches to obtain new sources of insulin-producing cells. Expert opinion: Undoubtedly, recent events preface the beginning of a new era in diabetes therapy. However, in our opinion, a number of significant hurdles still stand in the way of clinical application. We believe that the combination of the private and public sectors will accelerate the process of obtaining the desired safe and functional β-cell surrogates.
Mechanisms involved in the protective action of nitric oxide (NO) in insulin producing cells are ... more Mechanisms involved in the protective action of nitric oxide (NO) in insulin producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO (Cell Signal 13:809-817, 2001). In this study, using insulin producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of IRS-1, PI3K, Akt and Bad phosphorylation conform a substantial component of the survival route since pharmacological inhibition of PI3K and Akt cancelled the antiapototic effects of NO. Studies performed with the PKG inhibitor KT-5823 revealed that NOdependent activation of c-Src/ IRS-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO following overexpression of eNOS in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous DETA/NO. In addition, we found that NO produces c-Src/PI3K-and PKG-dependent activation of Erk 1/2. The MEK inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine and taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation.
The function of pluripotency genes in differentiation is a matter of investigation. We report her... more The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.
Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human... more Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic beta cells, but the protein profile of this action remains to be determined. We have applied a two dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediat...
Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to ... more Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2–20 μM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the
Diabetes mellitus (DM) is considered a global pandemic, and the incidence of DM continues to grow... more Diabetes mellitus (DM) is considered a global pandemic, and the incidence of DM continues to grow worldwide. Nutrients and dietary patterns are central issues in the prevention, development and treatment of this disease. The pathogenesis of DM is not completely understood, but nutrient-gene interactions at different levels, genetic predisposition and dietary factors appear to be involved. Nutritional genomics studies generally focus on dietary patterns according to genetic variations, the role of gene-nutrient interactions, genediet-phenotype interactions and epigenetic modifications caused by nutrients; these studies will facilitate an understanding of the early molecular events that occur in DM and will contribute to the identification of better biomarkers and diagnostics tools. In particular, this approach will help to develop tailored diets that maximize the use of nutrients and other functional ingredients present in food, which will aid in the prevention and delay of DM and its complications. This review discusses the current state of nutrigenetics, nutrigenomics and epigenomics research on DM. Here, we provide an overview of the role of gene variants and nutrient interactions, the importance of nutrients and dietary patterns on gene expression,
Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. T... more Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNFa-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharideelicited production of nitrite ðNO À 2 Þ by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO À 2 production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFNc. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNFa, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNFa synthesis. Additionally, PD144795 did not block NF-jB nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug. Ó
Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a g... more Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 5 0 -aza-2 0 -deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(b-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 lM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and b-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis.
Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to ... more Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2-20 lM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the antiapoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.
ABSTRACT Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed ... more ABSTRACT Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed as a source of undifferentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson’s disease, Fanconi’s anemia and diabetes. In addition to their potential in regenerative therapy, an understanding of the mechanisms by which these cells differentiate into any functional cell type will provide valuable information about basic biology. Screens for small compounds that can drive self-renewal maintenance or differentiation protocols are relevant to this goal. Nitric oxide (NO) is a diffusible second messenger implicated in numerous physiological functions in mammals. This molecule plays an important role in the maintenance of key features required for embryonic development and extension in ES cells. The goal of this chapter is to discuss recent advances concerning the ways in which NO signaling pathways mediate diverse mechanisms involved in the differentiation of ES cells toward multiple lineages. This chapter will also discuss the mechanisms by which NO can modify tissue-specific gene expression thorough chromatin remodeling and post-translational modification of transcription factors.
Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric ... more Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; ER stress, endoplasmic reticulum stress; T1D, type 1 diabetes mellitus; T2D, type 2 diabetes mellitus; TF, transcription factor; UPR, unfolded protein response; MAC, mitochondrial apoptosis-induced channel; VDAC, voltagedependent anion channel; MPT pore, mitochondrial permeability transition pore; MOMP, mitochondrial outer membrane permeabilisation; GCK, glucokinase; GC, guanylate cyclase; PKG, cGMP-dependent protein kinase hand, the natural history of type 2 diabetes mellitus (T2D) is highly variable. Here, we focus on those factors that contribute to T2D associated with increased NO production. Glucotoxicity and lipotoxicity negatively affect pancreatic β-cells. 12,13 Both of them enhance NO production by inducing iNOS expression. Additionally, pancreatic β-cells constitutively express two NOS isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), which produce homeostatic levels of NO necessary for their adequate function. Several activities related to cell survival are activated by NO, such as activation of signaling pathways and regulation of gene expression. This review therefore examines the role of NO in both scenarios. Further, the latest research about NO and its properties are introduced. Finally, we discuss the potential application of NO in cell therapy with pancreatic islets.
Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. T... more Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNFa-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharideelicited production of nitrite ðNO À 2 Þ by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO À 2 production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFNc. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNFa, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNFa synthesis. Additionally, PD144795 did not block NF-jB nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug. Ó
Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The pur... more Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The purpose of this study was to explore the link between NO and the survival pathway triggered by insulin-like growth factor-1 (IGF-1) and insulin in insulin producing RINm5F cells and in pancreatic islets. Results show that exposure of cells to IGF-1/insulin protects against serum deprivation-induced apoptosis. This action is prevented with inhibitors of NO generation, PI3K and Akt. Moreover, transfection with the negative dominant form of the tyrosine kinase c-Src abrogates the effect of IGF-1 and insulin on DNA fragmentation. An increase in the expression level of NOS3 protein and in the enzyme activity is observed following exposure of serum-deprived RINm5F cells to IGF-1 and insulin. Phosphorylation of IRS-1, IRS-2 and to less extent IRS-3 takes place when serum-deprived RINm5F cells and rat pancreatic islets are exposed to either IGF-1, insulin, or diethylenetriamine nitric oxide adduct (DETA/NO). In human islets, IRS-1 and IRS-2 proteins are present and tyrosine phosphorylated upon exposure to IGF-1, insulin and DETA/NO. Both rat and human pancreatic islets undergo DNA fragmentation when cultured in serum-free medium and IGF-1, insulin and DETA/NO protect efficiently from this damage. We then conclude that generation of NO participates in the activation of survival pathways by IGF-1 and insulin in beta cells.
Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor that regulates the embryonic ... more Pdx1 (Pancreatic and duodenal homeobox 1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation towards beta cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESC) to high concentrations of NO donor diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of Polycomb Repressive Complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent marks of histone H3K27me3 and H3K4me3, site specific changes in DNA methylation, and no change in H3 acetylation. Based on these findings, we developed a protocol to differentiate mESC towards insulin producing cells consisting of sequential exposure to DETA-NO, valproic acid and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to fo...
Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cel... more Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cells. The best outcome of the refined protocols became apparent in the first clinical trial announced by ViaCyte, based on the implantation of pancreatic progenitors that would further mature into functional insulin-producing cells inside the patient's body. Areas covered: Several groups, including ours, have contributed to improve strategies to generate insulin-producing cells. Of note, the latest results have gained a substantial amount of interest as a method to create a potentially functional and limitless supply of β-cell to revert diabetes mellitus. This review analyzes the accomplishments that have taken place over the last few decades, summarizes the state-of-art methods for β-cell replacement therapies based on the differentiation of embryonic stem cells into glucose-responsive and insulin-producing cells in a dish and discusses alternative approaches to obtain new sources of insulin-producing cells. Expert opinion: Undoubtedly, recent events preface the beginning of a new era in diabetes therapy. However, in our opinion, a number of significant hurdles still stand in the way of clinical application. We believe that the combination of the private and public sectors will accelerate the process of obtaining the desired safe and functional β-cell surrogates.
Mechanisms involved in the protective action of nitric oxide (NO) in insulin producing cells are ... more Mechanisms involved in the protective action of nitric oxide (NO) in insulin producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO (Cell Signal 13:809-817, 2001). In this study, using insulin producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of IRS-1, PI3K, Akt and Bad phosphorylation conform a substantial component of the survival route since pharmacological inhibition of PI3K and Akt cancelled the antiapototic effects of NO. Studies performed with the PKG inhibitor KT-5823 revealed that NOdependent activation of c-Src/ IRS-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO following overexpression of eNOS in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous DETA/NO. In addition, we found that NO produces c-Src/PI3K-and PKG-dependent activation of Erk 1/2. The MEK inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine and taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation.
The function of pluripotency genes in differentiation is a matter of investigation. We report her... more The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.
Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human... more Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic beta cells, but the protein profile of this action remains to be determined. We have applied a two dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediat...
Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to ... more Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2–20 μM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the
Diabetes mellitus (DM) is considered a global pandemic, and the incidence of DM continues to grow... more Diabetes mellitus (DM) is considered a global pandemic, and the incidence of DM continues to grow worldwide. Nutrients and dietary patterns are central issues in the prevention, development and treatment of this disease. The pathogenesis of DM is not completely understood, but nutrient-gene interactions at different levels, genetic predisposition and dietary factors appear to be involved. Nutritional genomics studies generally focus on dietary patterns according to genetic variations, the role of gene-nutrient interactions, genediet-phenotype interactions and epigenetic modifications caused by nutrients; these studies will facilitate an understanding of the early molecular events that occur in DM and will contribute to the identification of better biomarkers and diagnostics tools. In particular, this approach will help to develop tailored diets that maximize the use of nutrients and other functional ingredients present in food, which will aid in the prevention and delay of DM and its complications. This review discusses the current state of nutrigenetics, nutrigenomics and epigenomics research on DM. Here, we provide an overview of the role of gene variants and nutrient interactions, the importance of nutrients and dietary patterns on gene expression,
Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. T... more Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNFa-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharideelicited production of nitrite ðNO À 2 Þ by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO À 2 production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFNc. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNFa, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNFa synthesis. Additionally, PD144795 did not block NF-jB nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug. Ó
Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a g... more Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 5 0 -aza-2 0 -deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(b-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 lM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and b-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis.
Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to ... more Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2-20 lM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the antiapoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.
ABSTRACT Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed ... more ABSTRACT Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed as a source of undifferentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson’s disease, Fanconi’s anemia and diabetes. In addition to their potential in regenerative therapy, an understanding of the mechanisms by which these cells differentiate into any functional cell type will provide valuable information about basic biology. Screens for small compounds that can drive self-renewal maintenance or differentiation protocols are relevant to this goal. Nitric oxide (NO) is a diffusible second messenger implicated in numerous physiological functions in mammals. This molecule plays an important role in the maintenance of key features required for embryonic development and extension in ES cells. The goal of this chapter is to discuss recent advances concerning the ways in which NO signaling pathways mediate diverse mechanisms involved in the differentiation of ES cells toward multiple lineages. This chapter will also discuss the mechanisms by which NO can modify tissue-specific gene expression thorough chromatin remodeling and post-translational modification of transcription factors.
Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric ... more Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; ER stress, endoplasmic reticulum stress; T1D, type 1 diabetes mellitus; T2D, type 2 diabetes mellitus; TF, transcription factor; UPR, unfolded protein response; MAC, mitochondrial apoptosis-induced channel; VDAC, voltagedependent anion channel; MPT pore, mitochondrial permeability transition pore; MOMP, mitochondrial outer membrane permeabilisation; GCK, glucokinase; GC, guanylate cyclase; PKG, cGMP-dependent protein kinase hand, the natural history of type 2 diabetes mellitus (T2D) is highly variable. Here, we focus on those factors that contribute to T2D associated with increased NO production. Glucotoxicity and lipotoxicity negatively affect pancreatic β-cells. 12,13 Both of them enhance NO production by inducing iNOS expression. Additionally, pancreatic β-cells constitutively express two NOS isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), which produce homeostatic levels of NO necessary for their adequate function. Several activities related to cell survival are activated by NO, such as activation of signaling pathways and regulation of gene expression. This review therefore examines the role of NO in both scenarios. Further, the latest research about NO and its properties are introduced. Finally, we discuss the potential application of NO in cell therapy with pancreatic islets.
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