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1.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
2.
Proc Natl Acad Sci U S A ; 118(29)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34266954

RESUMO

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4-dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

3.
Sci Rep ; 10(1): 19908, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199824

RESUMO

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-ß (TGF-ß). TGF-ß is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3ß which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-ß-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-ß/SMAD signaling pathway by stabilizing SMAD4.


Assuntos
Acetilglucosamina/química , Neoplasias da Mama/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Pulmonares/patologia , Processamento de Proteína Pós-Traducional , Proteólise , Proteína Smad4/química , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Serina , Transdução de Sinais , Proteína Smad4/genética , Proteína Smad4/metabolismo , Treonina , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Ubiquitina/metabolismo
5.
Cell Death Dis ; 11(9): 815, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994395

RESUMO

O-GlcNAc transferase (OGT) is an enzyme that catalyzes the O-GlcNAc modification of nucleocytoplasmic proteins and is highly expressed in many types of cancer. However, the mechanism regulating its expression in cancer cells is not well understood. This study shows that OGT is a substrate of the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) which plays an important role in cancer pathogenesis. Although LSD2 histone demethylase has already been reported as an E3 ubiquitin ligase in lung cancer cells, we identified XIAP as the main E3 ubiquitin ligase in colon cancer cells. Interestingly, OGT catalyzes the O-GlcNAc modification of XIAP at serine 406 and this modification is required for the E3 ubiquitin ligase activity of XIAP toward specifically OGT. Moreover, O-GlcNAcylation of XIAP suppresses colon cancer cell growth and invasion by promoting the proteasomal degradation of OGT. Therefore, our findings regarding the reciprocal regulation of OGT and XIAP provide a novel molecular mechanism for controlling cancer growth and invasion regulated by OGT and O-GlcNAc modification.


Assuntos
Neoplasias do Colo/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Neoplasias do Colo/patologia , Glicosilação , Células HCT116 , Células HEK293 , Humanos , Invasividade Neoplásica , Transfecção , Ubiquitinação
6.
EMBO Rep ; 21(9): e50103, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32767654

RESUMO

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proliferação de Células , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Nutrientes , Fosforilação
7.
Biochem Biophys Res Commun ; 529(3): 692-698, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32736694

RESUMO

Unlike other types of glycosylation, O-GlcNAcylation is a single glycosylation which occurs exclusively in the nucleus and cytosol. O-GlcNAcylation underlie metabolic diseases, including diabetes and obesity. Furthermore, O-GlcNAcylation affects different oncogenic processes such as osteoblast differentiation, adipogenesis and hematopoiesis. Emerging evidence suggests that skeletal muscle differentiation is also regulated by O-GlcNAcylation, but the detailed molecular mechanism has not been fully elucidated. In this study, we showed that hyper-O-GlcNAcylation reduced the expression of myogenin, a transcription factor critical for terminal muscle development, in C2C12 myoblasts differentiation by O-GlcNAcylation on Thr9 of myocyte-specific enhancer factor 2c. Furthermore, we showed that O-GlcNAcylation on Mef2c inhibited its DNA binding affinity to myogenin promoter. Taken together, we demonstrated that hyper-O-GlcNAcylation attenuates skeletal muscle differentiation by increased O-GlcNAcylation on Mef2c, which downregulates its DNA binding affinity.


Assuntos
Acetilglucosamina/metabolismo , Diferenciação Celular , Desenvolvimento Muscular , Mioblastos/citologia , Acilação , Animais , Linhagem Celular , Glicosilação , Células HEK293 , Humanos , Fatores de Transcrição MEF2/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(25): 14259-14269, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513743

RESUMO

The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proliferação de Células , Células HEK293 , Homeostase , Humanos , Fosforilação
9.
Nat Commun ; 11(1): 2127, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358544

RESUMO

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in humans with type 2 diabetes (T2D); however, the underlying mechanism remains unclear. Activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and subsequent interleukin (IL)-1ß release induces atherosclerosis and heart failure. Here we show the effect of SGLT2 inhibitor empagliflozin on NLRP3 inflammasome activity. Patients with T2D and high cardiovascular risk receive SGLT2 inhibitor or sulfonylurea for 30 days, with NLRP3 inflammasome activation analyzed in macrophages. While the SGLT2 inhibitor's glucose-lowering capacity is similar to sulfonylurea, it shows a greater reduction in IL-1ß secretion compared to sulfonylurea accompanied by increased serum ß-hydroxybutyrate (BHB) and decreased serum insulin. Ex vivo experiments with macrophages verify the inhibitory effects of high BHB and low insulin levels on NLRP3 inflammasome activation. In conclusion, SGLT2 inhibitor attenuates NLRP3 inflammasome activation, which might help to explain its cardioprotective effects.


Assuntos
Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Idoso , Animais , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Interleucina-1beta/metabolismo , Cetonas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Pessoa de Meia-Idade , Inibidores do Transportador 2 de Sódio-Glicose , Compostos de Sulfonilureia/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
10.
Front Immunol ; 11: 589259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33603735

RESUMO

Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-ß. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.


Assuntos
Acetilglucosamina/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai , Acilação , Linhagem Celular , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Mitocôndrias/imunologia , Transdução de Sinais
11.
Sci Rep ; 9(1): 13653, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541118

RESUMO

Mass spectrometry-based spectral count has been a common choice of label-free proteome quantification due to the simplicity for the sample preparation and data generation. The discriminatory nature of spectral count in the MS data-dependent acquisition, however, inherently introduces the spectral count variation for low-abundance proteins in multiplicative LC-MS/MS analysis, which hampers sensitive proteome quantification. As many low-abundance proteins play important roles in cellular processes, deducing low-abundance proteins in a quantitatively reliable manner greatly expands the depth of biological insights. Here, we implemented the Moment Adjusted Imputation error model in the spectral count refinement as a post PLGEM-STN for improving sensitivity for quantitation of low-abundance proteins by reducing spectral count variability. The statistical framework, automated spectral count refinement by integrating the two statistical tools, was tested with LC-MS/MS datasets of MDA-MB468 breast cancer cells grown under normal and glucose deprivation conditions. We identified about 30% more quantifiable proteins that were found to be low-abundance proteins, which were initially filtered out by the PLGEM-STN analysis. This newly developed statistical framework provides a reliable abundance measurement of low-abundance proteins in the spectral count-based label-free proteome quantification and enabled us to detect low-abundance proteins that could be functionally important in cellular processes.


Assuntos
Neoplasias da Mama/metabolismo , Glucose/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteômica/métodos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatografia Líquida , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Modelos Estatísticos , Espectrometria de Massas em Tandem
12.
Cell Death Discov ; 5: 130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452956

RESUMO

Parkinson's disease (PD) is a chronic neurodegenerative disease with no cure. Calbindin, a Ca2+-buffering protein, has been suggested to have a neuroprotective effect in the brain tissues of PD patients and in experimental models of PD. However, the underlying mechanisms remain elusive. Here, we report that in 1-methyl-4-phenylpyridinium (MPP+)-induced culture models of PD, the buffering of cytosolic Ca2+ by calbindin-D28 overexpression or treatment with a chemical Ca2+ chelator reversed impaired autophagic flux, protecting cells against MPP+-mediated neurotoxicity. When cytosolic Ca2+ overload caused by MPP+ was ameliorated, the MPP+-induced accumulation of autophagosomes decreased and the autophagic flux significantly increased. In addition, the accumulation of damaged mitochondria and p62-positive ubiquitinated protein aggregates, following MPP+ intoxication, was alleviated by cytosolic Ca2+ buffering. We showed that MPP+ treatment suppressed autophagic degradation via raising the lysosomal pH and therefore reducing cytosolic Ca2+ elevation restored the lysosomal pH acidity and normal autophagic flux. These results support the notion that functional lysosomes are required for Ca2+-mediated cell protection against MPP+-mediated neurotoxicity. Thus, our data suggest a novel process in which the modulation of Ca2+ confers neuroprotection via the autophagy-lysosome pathway. This may have implications for the pathogenesis and future therapeutic targets of PD.

13.
Glycoconj J ; 36(4): 239-240, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31267248
14.
FASEB J ; 33(8): 9030-9043, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31199680

RESUMO

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.


Assuntos
Queratina-18/genética , Queratina-18/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Hepatopatias/genética , Hepatopatias/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Acetilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Células HT29 , Humanos , Queratina-18/química , Queratina-8/química , Corpos de Mallory/metabolismo , Metilação , Proteínas Mutantes/química , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Espectrometria de Massas em Tandem
15.
Org Lett ; 21(12): 4439-4442, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31045373

RESUMO

For both fluorescence imaging and isolation of glycosidases in cells, we prepared novel activity-based, trifunctional fluorogenic probes that consist of (1) a sugar moiety as a glycosidase substrate, (2) a fluoromethylated coumarin for fluorescent labeling, and (3) an alkyne tag for click reaction to enable isolation of the labeled enzyme. One probe, ß-GlcNAc-CM-F, was employed to fluorescently detect endogenous O-GlcNAcase in cells and to isolate the labeled enzyme by affinity chromatography.


Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Glicosídeo Hidrolases/isolamento & purificação , Imagem Óptica , Açúcares/química , Cromatografia de Afinidade , Cumarínicos/síntese química , Corantes Fluorescentes/síntese química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Células HT29 , Humanos , Estrutura Molecular , Açúcares/síntese química
16.
Diabetes Obes Metab ; 21(4): 801-811, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30407726

RESUMO

AIM: To investigate sodium-glucose cotransporter 2 inhibitor (SGLT2i)-induced changes in ketogenic enzymes and transporters in normal and diabetic mice models. MATERIALS AND METHODS: Normal mice were randomly assigned to receive either vehicle or SGLT2i (25 mg/kg/d by oral gavage) for 7 days. Diabetic mice were treated with vehicle, insulin (4.5 units/kg/d by subcutaneous injection) or SGLT2i (25 mg/kg/d by intra-peritoneal injection) for 5 weeks. Serum and tissues of ketogenic organs were analysed. RESULTS: In both normal and diabetic mice, SGLT2i increased beta-hydroxybutyrate (BHB) content in liver, kidney and colon tissue, as well as in serum and urine. In these organs, SGLT2i upregulated mRNA expression of ketogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. Similar patterns were observed in the kidney, ileum and colon for mRNA and protein expression of sodium-dependent monocarboxylate transporters (SMCTs), which mediate the cellular uptake of BHB and butyrate, an important substrate for intestinal ketogenesis. In diabetic mice under euglycaemic conditions, SGLT2i increased major ketogenic enzymes and SMCTs, while insulin suppressed ketogenesis. CONCLUSIONS: SGLT2i increased systemic and tissue BHB levels by upregulating ketogenic enzymes and transporters in the liver, kidney and intestine, suggesting the integrated physiological consequences for ketone body metabolism of SGLT2i administration.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Colo/efeitos dos fármacos , Hidroximetilglutaril-CoA Sintase/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Transportadores de Ácidos Monocarboxílicos/efeitos dos fármacos , Oxo-Ácido-Liases/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/urina , Animais , Compostos Benzidrílicos/farmacologia , Colo/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucosídeos/farmacologia , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Corpos Cetônicos/metabolismo , Rim/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Transportadores de Ácidos Monocarboxílicos/genética , Oxo-Ácido-Liases/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos
17.
Diabetes ; 68(1): 156-162, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389747

RESUMO

Senescent T cells have been implicated in chronic inflammatory and cardiovascular diseases. In this study, we explored the relationship between senescent T cells and glycemic status in a cohort of 805 participants by investigating the frequency of CD57+ or CD28null senescent T cells in peripheral blood. Participants with normal glucose tolerance (NGT) with follow-up data (N = 149) were included to determine whether hyperglycemia (prediabetes or type 2 diabetes) developed during follow-up (mean 2.3 years). CD8+CD57+ and CD8+CD28null T-cell frequencies were significantly higher in prediabetes and type 2 diabetes compared with NGT. Increased CD57+ or CD28null cells in the CD8+ T-cell subset were independently associated with hyperglycemia. Furthermore, among participants with baseline NGT, the frequency of CD8+CD57+ T cells was an independent predictor of hyperglycemia development. Immunofluorescent analyses confirmed that CD8+CD57+ T-cell infiltration was increased in visceral adipose tissue of patients with prediabetes or type 2 diabetes compared with those with NGT. Our data suggest that increased frequency of senescent CD8+ T cells in the peripheral blood is associated with development of hyperglycemia.


Assuntos
Senescência Celular/fisiologia , Hiperglicemia/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Antígenos CD57/metabolismo , Antígenos CD8/metabolismo , Senescência Celular/genética , Humanos , Hiperglicemia/genética , Imuno-Histoquímica , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Modelos Logísticos , Estudos Longitudinais , Estudos Prospectivos
18.
Artigo em Inglês | MEDLINE | ID: mdl-30532735

RESUMO

Background: The protein netrin-1 has demonstrated anti-inflammatory, tissue regeneration, and immune modulation properties. Although inflammation is a major contributing factor in the development of insulin resistance and type 2 diabetes, little is known about a possible relationship between serum netrin-1 and type 2 diabetes. Therefore, we investigated the association between circulating levels of netrin-1 and glycometabolic parameters predictive of type 2 diabetes. Methods: Serum samples were collected from 41 normal controls, 85 subjects with impaired fasting glucose (IFG), and 92 subjects with newly diagnosed type 2 diabetes. Clinical and laboratory parameters were assessed and netrin-1 levels were measured by commercial enzyme-linked immunosorbent assay. Spearman correlation analyses and multivariable-adjusted regression analyses were conducted to examine the relationship between serum netrin-1 levels and glycometabolic parameters. Results: Serum netrin-1 levels in subjects with type 2 diabetes or IFG were significantly higher compared to normal controls (441.0, 436.6, and 275.9 pg/mL, respectively; P for trend < 0.001). Serum netrin-1 levels were significantly positively correlated with fasting glucose, HbA1c, and insulin resistance index (all Ps < 0.01). Serum netrin-1 levels were independently associated with IFG or type 2 diabetes (standardized ß = 0.405, P < 0.001) after adjusting for covariates and potential confounders. In addition, the receiver operating characteristic (ROC) analysis showed that serum netrin-1 levels could identify the presence of IFG and type 2 diabetes with the area under the ROC curve (AUC) of 0.784 (P < 0.001). Conclusions: Our results suggest that elevated serum netrin-1 levels are significantly associated with the presence of IFG and type 2 diabetes.

19.
Cell Death Dis ; 9(12): 1189, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538224

RESUMO

Autophagy is a regulated, intracellular degradation process that delivers unnecessary or dysfunctional cargo to the lysosome. Autophagy has been viewed as an adaptive survival response to various stresses, whereas in other cases, it promotes cell death. Therefore, both deficient and excessive autophagy may lead to cell death. In this study, we specifically attempted to explore whether and how dysregulated autophagy contributes to caspase-dependent neuronal cell death induced by the neurotoxin 6-hydroxydopamine (6-OHDA). Ultrastructural and biochemical analyses indicated that MN9D neuronal cells and primary cultures of cortical neurons challenged with 6-OHDA displayed typical features of autophagy. Cotreatment with chloroquine and monitoring autophagic flux by a tandem mRFP-EGFP-tagged LC3 probe indicated that the autophagic phenomena were primarily caused by dysregulated autophagic flux. Consequently, cotreatment with an antioxidant but not with a pan-caspase inhibitor significantly blocked 6-OHDA-stimulated dysregulated autophagy. These results indicated that 6-OHDA-induced generation of reactive oxygen species (ROS) played a critical role in triggering neuronal death by causing dysregulated autophagy and subsequent caspase-dependent apoptosis. The results of the MTT reduction, caspase-3 activation, and TUNEL assays indicated that pharmacological inhibition of autophagy using 3-methyladenine or deletion of the autophagy-related gene Atg5 significantly inhibited 6-OHDA-induced cell death. Taken together, our results suggest that abnormal induction of autophagic flux promotes apoptotic neuronal cell death, and that the treatments limiting dysregulated autophagy may have a strong neuroprotective potential.


Assuntos
Apoptose/genética , Autofagia/genética , Caspase 3/genética , Neurônios/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/genética , Inibidores de Caspase/farmacologia , Cloroquina/farmacologia , Humanos , Camundongos , Neurônios/patologia , Oxidopamina/farmacologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Nat Commun ; 9(1): 4283, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327479

RESUMO

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/química , Interações Hospedeiro-Patógeno/fisiologia , Acetilglucosamina/metabolismo , Animais , Arginina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Feminino , Glicosilação , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Salmonella typhimurium/patogenicidade , Especificidade por Substrato , Fatores de Virulência/química
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