RESUMO
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Assuntos
Tecido Adiposo Marrom/metabolismo , Receptor Constitutivo de Androstano/metabolismo , Lipólise , Receptores Acoplados a Proteínas G/metabolismo , Termogênese , Adipócitos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Temperatura Baixa , Gorduras na Dieta/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Sistema Nervoso Simpático/metabolismo , Transcrição GênicaRESUMO
OBJECTIVE: Dietary fibres are essential for maintaining microbial diversity and the gut microbiota can modulate host physiology by metabolising the fibres. Here, we investigated whether the soluble dietary fibre oligofructose improves host metabolism by modulating bacterial transformation of secondary bile acids in mice fed western-style diet. DESIGN: To assess the impact of dietary fibre supplementation on bile acid transformation by gut bacteria, we fed conventional wild-type and TGR5 knockout mice western-style diet enriched or not with cellulose or oligofructose. In addition, we used germ-free mice and in vitro cultures to evaluate the activity of bacteria to transform bile acids in the caecal content of mice fed with western-style diet enriched with oligofructose. Finally, we treated wild-type and TGR5 knockout mice orally with hyodeoxycholic acid to assess its antidiabetic effects. RESULTS: We show that oligofructose sustains the production of 6α-hydroxylated bile acids from primary bile acids by gut bacteria when fed western-style diet. Mechanistically, we demonstrated that the effects of oligofructose on 6α-hydroxylated bile acids were microbiota dependent and specifically required functional TGR5 signalling to reduce body weight gain and improve glucose metabolism. Furthermore, we show that the 6α-hydroxylated bile acid hyodeoxycholic acid stimulates TGR5 signalling, in vitro and in vivo, and increases GLP-1R activity to improve host glucose metabolism. CONCLUSION: Modulation of the gut microbiota with oligofructose enriches bacteria involved in 6α-hydroxylated bile acid production and leads to TGR5-GLP1R axis activation to improve body weight and metabolism under western-style diet feeding in mice.
Assuntos
Ácidos e Sais Biliares , Dieta Ocidental , Fibras na Dieta , Suplementos Nutricionais , Microbioma Gastrointestinal , Glucose , Receptores Acoplados a Proteínas G , Animais , Camundongos , Ácidos e Sais Biliares/metabolismo , Peso Corporal , Glucose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fibras na Dieta/administração & dosagemRESUMO
BACKGROUND: Intercellular communication mediated by cationic fluxes through the Connexin family of gap junctions regulates glucose-stimulated insulin secretion and beta cell defense against inflammatory stress. Rotigaptide (RG, ZP123) is a peptide analog that increases intercellular conductance in cardiac muscle cells by the prevention of dephosphorylation and thereby uncoupling of Connexin-43 (Cx43), possibly via action on unidentified protein phosphatases. For this reason, it is being studied in human arrhythmias. It is unknown if RG protects islet cell function and viability against inflammatory or metabolic stress, a question of considerable translational interest for the treatment of diabetes. METHODS: Apoptosis was measured in human islets shown to express Cx43, treated with RG or the control peptide ZP119 and exposed to glucolipotoxicity or IL-1ß + IFNÉ£. INS-1 cells shown to lack Cx43 were used to examine if RG protected human islet cells via Cx43 coupling. To study the mechanisms of action of Cx43-independent effects of RG, NO, IkBα degradation, mitochondrial activity, ROS, and insulin mRNA levels were determined. RESULTS: RG reduced cytokine-induced apoptosis ~40% in human islets. In Cx43-deficient INS-1 cells, this protective effect was markedly blunted as expected, but unexpectedly, RG still modestly reduced apoptosis, and improved mitochondrial function, insulin-2 gene levels, and accumulated insulin release. RG reduced NO production in Cx43-deficient INS-1 cells associated with reduced iNOS expression, suggesting that RG blunts cytokine-induced NF-κB signaling in insulin-producing cells in a Cx43-independent manner. CONCLUSION: RG reduces cytokine-induced cell death in human islets. The protective action in Cx43-deficient INS-1 cells suggests a novel inhibitory mechanism of action of RG on NF-κB signaling.
Assuntos
Conexina 43/metabolismo , Citocinas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Oligopeptídeos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Conexina 43/genética , Citocinas/genética , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse FisiológicoRESUMO
Type 1 diabetes is due to destruction of pancreatic ß-cells. Lysine deacetylase inhibitors (KDACi) protect ß-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing ß-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1ß + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.
Assuntos
Cromatina/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Células Secretoras de Insulina/citologia , Animais , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Feminino , Fator de Transcrição GATA3/metabolismo , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Fatores de Tempo , VorinostatRESUMO
BACKGROUND: Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. RESULTS: We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNß and TGFß increase the release of α-SNC from PC12 neurons. CONCLUSIONS: We implicate stress kinases of the JNK family in the regulation of exophagy and release of α-SNC following endogenous or exogenous stimulation. In a wider scope, our results imply that microglia not only inflict bystander damage to neurons in late phases of inflammatory brain disease but may also be active mediators of disease propagation.
Assuntos
MAP Quinase Quinase 4/fisiologia , Microglia/fisiologia , Neurônios/fisiologia , alfa-Sinucleína/metabolismo , Animais , Autofagia/fisiologia , Comunicação Celular/fisiologia , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Microglia/enzimologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neurônios/enzimologia , Células PC12 , Ratos , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.
Assuntos
Vesículas Revestidas por Clatrina/enzimologia , Ativação de Macrófagos/fisiologia , Macrófagos/enzimologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Ligante de CD40/genética , Ligante de CD40/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Endossomos/enzimologia , Endossomos/genética , Exocitose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Glicoproteínas de Membrana/genética , Microglia/enzimologia , NADPH Oxidase 2 , NADPH Oxidases/genética , Ratos , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
BACKGROUND: The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. METHODS: Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. RESULTS: Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. CONCLUSIONS: OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE(2).
Assuntos
Colo/metabolismo , Neoplasias Colorretais/metabolismo , AMP Cíclico/metabolismo , Mucosa Intestinal/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/metabolismo , Neoplasias Colorretais/patologia , Dinoprostona/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Células Caliciformes/metabolismo , Complexo de Golgi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismoRESUMO
The Na+;HCO3- co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.
Assuntos
Mama/citologia , Células Epiteliais/metabolismo , Rim/citologia , Proteólise , Simportadores de Sódio-Bicarbonato/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cães , Humanos , Cinética , Lisossomos/metabolismo , Células MCF-7 , Células Madin Darby de Rim Canino , Transporte Proteico , Simportadores de Sódio-Bicarbonato/químicaRESUMO
Transcriptional changes control ß-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs) protects ß cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate ß-cell fate in response to inflammatory stress. Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting a possible role in inflammation-induced ß-cell destruction. Inhibition of KDM6 demethylases using the selective inhibitor GSK-J4 protected insulin-producing cells and human and mouse islets from cytokine-induced apoptosis by blunting nuclear factor (NF)-κB signaling and endoplasmic reticulum (ER) stress response gene expression. GSK-J4 furthermore increased expression of insulin gene and glucose-stimulated insulin secretion. Expression of genes regulating purinergic and cytokine ligand-receptor interactions was downregulated following GSK-J4 exposure, while expression of genes involved in cell maintenance and survival was upregulated. These data suggest that KDMs are important regulators of inflammation-induced ß-cell dysfunction and death.
Assuntos
Apoptose , Benzazepinas/farmacologia , Citoproteção , Células Secretoras de Insulina/patologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Citocinas/farmacologia , Citoproteção/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de SinaisRESUMO
ß-Cells may be a source of IL-1ß that is produced as inactive pro-IL-1ß and processed into biologically-active IL-1ß by enzymatic cleavage mediated by the NLRP1-, NLRP3- and NLRC4-inflammasomes. Little is known about the ß-cell inflammasomes. NLRP1-expression was upregulated in islet-cells from T2D-patients and by IL-1ß+IFNγ in INS-1 cells in a histone-deacetylase dependent manner. NLRP3 was downregulated by cytokines in INS-1 cells. NLRC4 was barely expressed and not regulated by cytokines. High extracellular K+ reduced cytokine-induced apoptosis and NO production and restored cytokine-inhibited accumulated insulin-secretion. Basal inflammasome expression was JNK1-3 dependent. Knock-down of the ASC interaction domain common for NLRP1 and 3 improved insulin secretion and ameliorated IL-1ß and/or glucolipotoxicity-induced cell death and reduced cytokine-induced NO-production. Broad inflammasome-inhibition, but not NLRP3-selective inhibition, protected against IL-1ß-induced INS-1 cell-toxicity. We suggest that IL-1ß causes ß-cell toxicity in part by NLRP1 mediated caspase-1-activation and maturation of IL-1ß leading to an autocrine potentiation loop.
Assuntos
Apoptose , Inflamassomos/metabolismo , Células Secretoras de Insulina/metabolismo , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização CARD , Morte Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/farmacologia , Citoproteção/efeitos dos fármacos , Feminino , Glucose/toxicidade , Histona Desacetilases/metabolismo , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Interleucina-1beta/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipídeos/toxicidade , Pessoa de Meia-Idade , Potássio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Adulto JovemRESUMO
The proinflammatory cytokines interleukin-1 beta (IL-1ß) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4 are expressed in pancreatic islets and inhibit beta-cell growth and function. In this study, we describe that IL-1ß and IFN-γ induce the expression of BMP-2 suggesting a possible role for BMP-2 in mediating the effects of IL-1ß and IFN-γ on beta-cell apoptosis and dysfunction. IL-1ß increased BMP-2 mRNA levels 6- and 3-fold in isolated islets of Langerhans from neonatal rat and human. Downstream target genes of the BMP pathway were also increased by cytokine treatment and could be reversed by neutralization of endogenous BMP activity. Nuclear factor kappa B- (NFκB) binding sites were identified in the rat BMP-2 promoter, and reporter assays verified the role of NFκB in cytokine-induced BMP-2 expression. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed NFκB binding to BMP-2 promoter upon IL-1ß stimulation in beta cells. In conclusion, we suggest that NFκB stimulates BMP-2 mRNA expression in rat and human beta cells upon cytokine exposure.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Sítios de Ligação , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interferon gama/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pessoa de Meia-Idade , NF-kappa B/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , Ratos , Ratos Wistar , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in ß-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic ß-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic ß-cells from inflammatory cytokines and nutrient overload in diabetes.
Assuntos
Citoproteção/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RatosRESUMO
Pancreatic ß-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate ß-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced ß-cell apoptosis. We established insulin-producing INS1 cell lines stably expressing JNK subtype specific shRNAs to understand the differential roles of the individual JNK isoforms. JNK activity was increased after 3 h of palmitate and high glucose exposure associated with increased expression of ER and mitochondrial stress markers. JNK1 shRNA expressing INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect palmitate and high glucose induced apoptosis or ER stress markers, but increased Puma mRNA expression compared to non-sense shRNA expressing INS1 cells. Finally, JNK3 shRNA expressing INS1 cells did not induce apoptosis compared to non-sense shRNA expressing INS1 cells when exposed to palmitate and high glucose but showed increased caspase 9 and 3 cleavage associated with increased DP5 and Puma mRNA expression. These data suggest that JNK1 protects against palmitate and high glucose-induced ß-cell apoptosis associated with reduced ER and mitochondrial stress.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Ácido Palmítico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
AIM: An insufficient functional ß-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore ß-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in ß-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced ß-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to ß cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net ß-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes. MATERIALS AND METHODS: miRNA expression was manipulated using Lentiviral transduction of the ß-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. RESULTS: Overexpression of miR-21 decreased net ß-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net ß-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. CONCLUSION: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase ß-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce ß-cell death and dysfunction.
Assuntos
Apoptose/genética , Proliferação de Células/genética , Células Secretoras de Insulina/citologia , MicroRNAs/genética , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Óxido Nítrico/metabolismo , Ratos , TransfecçãoRESUMO
Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.