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1.
Am J Physiol Endocrinol Metab ; 318(6): E943-E955, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369414

RESUMO

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.


Assuntos
Fibronectinas/genética , Músculo Esquelético/metabolismo , Gordura Subcutânea/metabolismo , Animais , Desoxiglucose/metabolismo , Óxido de Deutério , Eletroporação , Fibronectinas/metabolismo , Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/genética , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Membro Posterior , Masculino , Proteínas de Desacoplamento Mitocondrial/genética , Biogênese de Organelas , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ratos
2.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R96-R105, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459971

RESUMO

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.


Assuntos
Cloretos/metabolismo , Glucose/metabolismo , Glândula de Sal/metabolismo , Squalus/metabolismo , Animais , Sequência de Bases , Glucose/farmacologia , Transportador de Glucose Tipo 2/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Homeostase , Técnicas In Vitro , Cianeto de Potássio/farmacologia , Glândula de Sal/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/metabolismo
3.
PLoS One ; 15(4): e0230044, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236143

RESUMO

LEFTY2 (endometrial bleeding associated factor; EBAF or LEFTYA), a cytokine released shortly before menstrual bleeding, is a negative regulator of cell proliferation and tumour growth. LEFTY2 down-regulates Na+/H+ exchanger activity with subsequent inhibition of glycolytic flux and lactate production in endometrial cancer cells. Glucose can be utilized not only for glycolysis but also for glycogen formation. Both glycolysis and glycogen formation require cellular glucose uptake which could be accomplished by the Na+ coupled glucose transporter-1 (SGLT1; SLC5A1). The present study therefore explored whether LEFTY2 modifies endometrial SGLT1 expression and activity as well as glycogen formation. Ishikawa and HEC1a cells were exposed to LEFTY2, SGLT1 and glycogen synthase (GYS1) transcript levels determined by qRT-PCR. SGLT1, GYS1 and phospho-GYS1 protein abundance was quantified by western blotting, cellular glucose uptake from 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake, and cellular glycogen content utilizing an enzymatic assay and subsequent colorimetry. As a result, a 48-hour treatment with LEFTY2 significantly increased SGLT1 and GYS1 transcript levels as well as SGLT1 and GYS1 protein abundance in both Ishikawa and HEC1a cells. 2-NBDG uptake and cellular glycogen content were upregulated significantly in Ishikawa (type 1) but not in type 2 endometrial HEC1a cells, although there was a tendency of increased 2-NBDG uptake. Further, none of the effects were seen in human benign endometrial cells (HESCs). Interestingly, in both Ishikawa and HEC1a cells, a co-treatment with TGF-ß reduced SGLT1, GYS and phospho-GYS protein levels, and thus reduced glycogen levels and again HEC1a cells had no significant change. In conclusion, LEFTY2 up-regulates expression and activity of the Na+ coupled glucose transporter SGLT1 and glycogen synthase GYS1 in a cell line specific manner. We further show the treatment with LEFTY2 fosters cellular glucose uptake and glycogen formation and TGF-ß can negate this effect in endometrial cancer cells.


Assuntos
Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Fatores de Determinação Direita-Esquerda/fisiologia , Transportador 1 de Glucose-Sódio/metabolismo , Linhagem Celular Tumoral , Feminino , Glucose/metabolismo , Humanos , Sódio/metabolismo
4.
J Med Chem ; 63(7): 3538-3551, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32134266

RESUMO

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.


Assuntos
Inibidores Enzimáticos/química , Glicogênio Sintase/antagonistas & inibidores , Imidazóis/química , Pirazóis/química , Animais , Caenorhabditis elegans/enzimologia , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Cinética , Estrutura Molecular , Ligação Proteica , Pirazóis/síntese química , Pirazóis/metabolismo , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
5.
Mol Med Rep ; 21(3): 1572-1580, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016474

RESUMO

Chlamydia trachomatis (C. trachomatis) is the leading cause of bacterial sexually transmitted diseases and infectious diseases that cause blindness. The pathophysiology of chlamydial infections is poorly understood, but secreted proteins have emerged as key virulence factors. C. trachomatis glycogen synthase (GlgA) is a chlamydial secretory protein, which localizes in the lumen of chlamydial inclusion bodies and the cytosol of host cells. In order to improve understanding of the roles of GlgA in chlamydial pathogenesis, four proteins that interact with GlgA, Homo sapiens CXXC finger protein 1, prohibitin (PHB), gelsolin­like actin­capping protein and apolipoprotein A­I binding protein were identified using yeast two­hybrid assays. The functions of these proteins are complex, and preliminary results suggested that PHB interacts with GlgA. However, further studies are required to determine the specific interactions of these proteins with GlgA. The findings of the present study may provide a direction and foundation for future studies focusing on the mechanism of GlgA in C. trachomatis infection.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Chlamydia trachomatis/enzimologia , Glicogênio Sintase/metabolismo , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Bactérias/genética , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Glicogênio Sintase/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Plasmídeos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Reprodutibilidade dos Testes
6.
PLoS One ; 15(2): e0228845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053662

RESUMO

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Transporte Biológico , Regulação Fúngica da Expressão Gênica/genética , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Quinases da Glicogênio Sintase/metabolismo , Quinases da Glicogênio Sintase/fisiologia , Oxigênio/metabolismo , Fosforilação , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Esteróis , Fatores de Transcrição/metabolismo , Ativação Transcricional
7.
Environ Toxicol ; 35(7): 727-737, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32073747

RESUMO

Our previous work indicated exposure of Human liver cell 7702 (HL7702) cells to Microcystin-leucine-arginine (MC-LR) for 24 hours can disrupt insulin (INS) signaling by the hyperphosphorylation of specific proteins. For further exploring the time-dependent effect posed by MC-LR on this pathway, in the current study, HL7702 cells together with mice were exposed to the MC-LR with different concentrations under short-term treatment, and then, protein phosphatase 2A (PP2A) activity and expression of proteins related to INS signaling, as well as the characteristics of their action in the liver, were investigated. The results indicated, in HL7702 cells with 0.5, 1, and 6 hours of treatment by MC-LR, PP2A activity showed an obvious decrease in a time and concentration-dependent manner. While the total protein level of Akt, glycogen synthase kinase 3 (GSK-3), and glycogen synthase remained unchanged, GSK-3 and Akt phosphorylation increased significantly. In livers of mice with 1 hour of intraperitoneal injection with MC-LR, a similar change in these proteins was observed. In addition, the levels of total IRS1 and p-IRS1 at serine sites showed decreasing and increasing trends,respectively, and the hematoxylin and eosin staining showed that liver tissues of mice in the maximum-dose group exhibited obvious hepatocyte degeneration and hemorrhage. Our results further proved that short-term treatment with MC-LR can inhibit PP2A activity and disrupt INS signaling proteins' phosphorylation level, thereby interfering with the INS pathway. Our findings provide a helpful understanding of the toxic effects posed by MC-LR on the glucose metabolism of liver via interference with the INS signaling pathway.


Assuntos
Insulina/metabolismo , Fígado/efeitos dos fármacos , Microcistinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Toxinas Marinhas , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Diabetes ; 69(4): 578-590, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31974138

RESUMO

A single bout of exercise enhances insulin action in the exercised muscle. However, not all human studies find that this translates into increased whole-body insulin action, suggesting that insulin action in rested muscle or other organs may be decreased by exercise. To investigate this, eight healthy men underwent a euglycemic-hyperinsulinemic clamp on 2 separate days: one day with prior one-legged knee-extensor exercise to local exhaustion (∼2.5 h) and another day without exercise. Whole-body glucose disposal was ∼18% lower on the exercise day as compared with the resting day due to decreased (∼37%) insulin-stimulated glucose uptake in the nonexercised muscle. Insulin signaling at the level of Akt2 was impaired in the nonexercised muscle on the exercise day, suggesting that decreased insulin action in nonexercised muscle may reduce GLUT4 translocation in response to insulin. Thus, the effect of a single bout of exercise on whole-body insulin action depends on the balance between local effects increasing and systemic effects decreasing insulin action. Physiologically, this mechanism may serve to direct glucose into the muscles in need of glycogen replenishment. For insulin-treated patients, this complex relationship may explain the difficulties in predicting the adequate insulin dose for maintaining glucose homeostasis following physical activity.


Assuntos
Glicemia/metabolismo , Exercício Físico/fisiologia , Insulina/farmacologia , Fadiga Muscular/fisiologia , Músculo Esquelético/metabolismo , Adulto , Técnica Clamp de Glucose , Glicogênio Sintase/metabolismo , Humanos , Masculino , Músculo Esquelético/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-31954198

RESUMO

This study investigated the effects of low salinity exposure on glycogen and its metabolism biomarkers, glycogen synthase (GS) and glycogen phosphorylase (GP), representing glycogen synthesis and catabolism, respectively, in the gills and liver of great blue-spotted mudskippers (Boleophthalmus pectinirostris). The fish were accumulated at 10‰ salinity seawater for 1 week, then 270 healthy great blue-spotted mudskippers with similar size were randomly transferred to 10‰ (control group) or 3‰ (low salinity group) seawater for 72-hour stress experiment. Fish significantly elevated their blood glucose levels 12 h after low salinity challenge. At the end of experiments, a decrease in liver glycogen contents was observed in both the control and low salinity groups, the latter showing a pronounced decrease, while the gill glycogen contents were not changed for either group. The mRNA abundance and enzyme activity of GS and GP were both elevated in gill tissues, showing a rising glycogen synthesis and catabolism, probably resulting in the unchanging gill glycogen content. While in liver tissues, the mRNA abundance and enzyme activity were decreased for GS and increased for GP, showing a net increase for breaking down glycogen in liver, probably for supplying a sufficient glucose level for gills and other tissues/organs involved in the response to salinity changes.


Assuntos
Peixes/metabolismo , Brânquias/metabolismo , Glicogênio/metabolismo , Fígado/metabolismo , Estresse Salino/fisiologia , Animais , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Salinidade
10.
Adv Neurobiol ; 23: 83-123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31667806

RESUMO

Brain glycogen synthesis is a regulated, multi-step process that begins with glucose transport across the blood brain barrier and culminates with the actions of glycogen synthase and the glycogen branching enzyme to elongate glucose chains and introduce branch points in a growing glycogen molecule. This review focuses on the synthesis of glycogen in the brain, with an emphasis on glycogen synthase, but draws on salient studies in mammalian muscle and liver as well as baker's yeast, with the goal of providing a more comprehensive view of glycogen synthesis and highlighting potential areas for further study in the brain. In addition, deficiencies in the glycogen biosynthetic enzymes which lead to glycogen storage diseases in humans are discussed, highlighting effects on the brain and discussing findings in genetically modified animal models that recapitulate these diseases. Finally, implications of glycogen synthesis in neurodegenerative and other diseases that impact the brain are presented.


Assuntos
Encéfalo/enzimologia , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Animais , Glucose/metabolismo , Glicogênio/metabolismo , Humanos
11.
Adv Neurobiol ; 23: 311-329, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31667813

RESUMO

Brain glycogen is stored mainly in astrocytes, although neurons also have an active glycogen metabolism. Glycogen has gained relevance as a key player in brain function. In this regard, genetically modified animals have allowed researchers to unravel new roles of this polysaccharide in the brain. Remarkably, mice in which glycogen synthase is abolished in the brain, and thus devoid of brain glycogen, are viable, thereby indicating that the polysaccharide in this organ is not a requirement for survival. While there was growing evidence supporting a role of glycogen in learning and memory, these animals have now confirmed that glycogen participates in these two processes.The association of epilepsy with brain glycogen has also attracted attention. Analysis of genetically modified mice indicates that the relation between brain glycogen and epilepsy is complex. While the formation of glycogen aggregates clearly underlies epilepsy, as in Lafora Disease (LD), the absence of glycogen also favors the occurrence of seizures.LD is a rare genetic condition that affects children. It is characterized by epileptic seizures and neurodegeneration, and it develops rapidly until finally causing death. Research into this disease has unveiled new aspects of glycogen metabolism. Animal models of LD accumulate polyglucosan bodies formed by aberrant glycogen aggregates, called Lafora bodies (LBs). The abolition of glycogen synthase (GS) prevents the formation of LBs and the development of LD, thereby indicating that glycogen accumulation underlies this disease and the associated symptoms, and thus establishing a clear relation between the accumulation of glycogen aggregates and the incidence of seizures.Although it was initially accepted that LBs were essentially neuronal, it is now evident that astrocytes also accumulate polyglucosan aggregates in LD. However, the appearance and composition of these deposits differs from that observed in neurons. Of note, the astrocytic aggregates in LD models show remarkable similarities with corpora amylacea (CA), a type of polyglucosan aggregate observed in the brains of aged mice and humans. The abolition of GS in mice also impedes the formation of CA with age and at the same time prevents the formation of a number of protein aggregates associated with aging. Therefore CA may play a role in age-related neurological decline.


Assuntos
Astrócitos/patologia , Astrócitos/fisiologia , Glicogênio/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Astrócitos/metabolismo , Glicogênio/fisiologia , Glicogênio Sintase/metabolismo , Humanos , Doença de Lafora/metabolismo , Doença de Lafora/patologia , Neurônios/metabolismo
12.
EBioMedicine ; 48: 386-404, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31594754

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. METHODS: Using real-time PCR, western blotting, immunohistochemistry, and In situ hybridization, we detected the expression of miR-BART22 and MAP2K4 in tissues and cells, as well as evaluated their clinical relevance in NPC patients. The effects of miR-BART22 on cell metastasis, stemness and DDP chemoresistance were examined by sphere formation assay, side population analysis, transwell, boyden, in vivo xenograft tumor mouse model et al. Western blotting, immunofluorescence staining, luciferase reporter assay, ChIP, EMSA and Co-IP assay et al. were performed to explore the detailed molecular mechanism of EBV-miR-BART22 in NPC. Finally, we estimated the effects and molecular basis of Cinobufotalin on EBV-miR-BART22-overexpressing NPC cells in vitro and in vivo assays. FINDINGS: We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analysis indicated that EBV-miR-BART22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3ß(GSK3ß) protein and induced its ubiquitin degradation by activating PI3K/AKT/c-Jun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3ß protein. Reductions in the GSK3ß protein thus promoted ß-catenin expression and nuclear translocation, which induced tumor stemness and the epithelial-to-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3ß/ß-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients. INTERPRETATION: Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC.


Assuntos
Bufanolídeos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Herpesvirus Humano 4/genética , MicroRNAs/genética , RNA Viral , Linhagem Celular Tumoral , Glicogênio Sintase/metabolismo , Humanos , MAP Quinase Quinase 4/genética , Cadeias Pesadas de Miosina/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , beta Catenina/metabolismo
13.
Biochem J ; 476(14): 2059-2092, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366571

RESUMO

The evolution of metabolic pathways is a major force behind natural selection. In the spotlight of such process lies the structural evolution of the enzymatic machinery responsible for the central energy metabolism. Specifically, glycogen metabolism has emerged to allow organisms to save available environmental surplus of carbon and energy, using dedicated glucose polymers as a storage compartment that can be mobilized at future demand. The origins of such adaptive advantage rely on the acquisition of an enzymatic system for the biosynthesis and degradation of glycogen, along with mechanisms to balance the assembly and disassembly rate of this polysaccharide, in order to store and recover glucose according to cell energy needs. The first step in the classical bacterial glycogen biosynthetic pathway is carried out by the adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase. This allosteric enzyme synthesizes ADP-glucose and acts as a point of regulation. The second step is carried out by the glycogen synthase, an enzyme that generates linear α-(1→4)-linked glucose chains, whereas the third step catalyzed by the branching enzyme produces α-(1→6)-linked glucan branches in the polymer. Two enzymes facilitate glycogen degradation: glycogen phosphorylase, which functions as an α-(1→4)-depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of α-(1→6)-linked ramifications. In this work, we rationalize the structural basis of glycogen metabolism in bacteria to the light of the current knowledge. We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Regulação Alostérica
14.
Mol Med Rep ; 19(6): 5105-5114, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059076

RESUMO

Portal hypertension is the primary cause of complications in patients with chronic liver diseases, and markedly impacts metabolism within the nervous system. Until recently, the role of portal hypertension in hepatocellular metabolism was unclear. The present study demonstrated that an increase in extracellular pressure significantly decreased hepatocellular glycogen concentrations in HepG2 and HL­7702 cells. In addition, it reduced glycogen synthase activity, by inhibiting the phosphorylation of glycogen synthase 1. RNA­seq analysis revealed that mechanical pressure suppressed glycogen synthesis by activating the p53/phosphatase and tensin homolog pathway, further suppressing glycogen synthase activity. The present study revealed an association between mechanical pressure and hepatocellular glycogen metabolism, and identified the regulatory mechanism of glycogen synthesis under pressure.


Assuntos
Glicogênio/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fosforilação , Pressão , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
15.
Proc Natl Acad Sci U S A ; 116(21): 10435-10440, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31048503

RESUMO

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.


Assuntos
Relógios Circadianos , Regulação da Expressão Gênica , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Neurospora crassa/metabolismo , Proteínas Fúngicas/metabolismo , Glicogênio Sintase/genética , Neurospora crassa/genética
16.
Food Funct ; 10(5): 2828-2838, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31049543

RESUMO

This study aimed at investigating the structure, hypoglycemic activity and the underlying mechanism of a homogeneous polysaccharide (PSP-2) purified from Sargassum pallidum. Structural characterization revealed that PSP-2 with a molecular weight of 144.8 kDa was composed of fucose (21.6%), arabinose (2.5%), galactose (22.4%), glucose (2.2%), xylose (18.8%), mannose (1.2%), glucuronic acid (7.7%) and galacturonic acid (23.6%). The backbone chain of PSP-2 was composed of →1)-ß-d-Xylp-(3→, →1,3)-ß-l-Fucp-(4→, →1)-α-d-Galp-(6→, and →1)-α-d-GlcpNAc-(2→, and the side chains were composed of →1,3,6)-α-d-Galp-(2→, →3)-ß-l-Fucp-(1,4→, ß-d-GalpNAc-(1→, and α-d-Manp-(1→. In vitro hypoglycemic assays indicated that PSP-2 could significantly enhance glucose consumption, glycogen synthesis, and pyruvate kinase (PK) and hexokinase (HK) activities of insulin-resistant HepG2 cells. Furthermore, the underlying mechanistic studies revealed that PSP-2 could ameliorate insulin resistance by up-regulating the expression levels of insulin receptor substrate-1 (IRS-1), glycogen synthase (GS), phosphoinositide-3-kinase (PI3K) and glucose transporter-4 (GLUT4). These results suggested that PSP-2 may be a potential candidate for the prevention and treatment of Type 2 diabetes mellitus.


Assuntos
Hipoglicemiantes/química , Extratos Vegetais/química , Polissacarídeos/química , Sargassum/química , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Peso Molecular , Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia
17.
Biomed Pharmacother ; 112: 108715, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30970519

RESUMO

BACKGROUND: Dysregulation of glucose and glycogen metabolism are crucial mechanisms implicated in type 2 diabetes mellitus (T2DM). Centella asiatica (L.) Urban (Apiaceae) has been utilized as a traditional medicine in Africa and Asia for centuries and is commercially available as a dietary supplement. AIM: We explored for the first time, the possible efficacy of Centella asiatica (CA) extract in ameliorating T2DM-induced changes in key enzymes involved in glucose and glycogen metabolism in the rat skeletal muscle. METHODS: Diabetic rats were orally treated with vehicle, CA (500 and 1000 mg/kg) or metformin (300 mg/kg) daily for 14 days. Skeletal muscle activities of hexokinase (HK), phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBPase) were determined by spectrophotometric assays while those of glycogen synthase (GS) and glycogen phosphorylase (GP) were assayed radio-chemically. Histological examination of skeletal muscle was also performed. RESULTS: Rats with induced T2DM had reduced activities of HK (25%), PFK (88%), and GS (38%) when compared to non-diabetic rats. Treatment of diabetic rats with CA500 increased the activities of PFK (7-fold), and FBPase (23%). Further, treatment of diabetic rats with CA1000 also increased the activities of GS (27%) and GP (50%) with little change in these parameters for diabetic rats treated with CA500. These effects probably led to the reduced blood glucose level and elevated skeletal muscle glycogen content observed in CA-treated rats relative to diabetic controls. Furthermore, CA treated rats had reduced the morphological damage of skeletal muscle fibres compared to the non-treated diabetic control rats. CONCLUSION: Our findings strongly suggest that the anti-diabetic effects of CA in part target muscle glucose and glycogen metabolism and hence supporting its folkloric medical use as an anti-diabetic remedy.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/metabolismo , Glicogênio/metabolismo , Hipoglicemiantes/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Triterpenos/uso terapêutico , Animais , Glicemia/análise , Centella/química , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Medicina Tradicional , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Folhas de Planta/química , Ratos Sprague-Dawley , Estreptozocina , Triterpenos/farmacologia
18.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31010017

RESUMO

Polyhydroxybutyrate (PHB) is a polymer of great interest as a substitute for conventional plastics, which are becoming an enormous environmental problem. PHB can be produced directly from CO2 in photoautotrophic cyanobacteria. The model cyanobacterium Synechocystis sp. PCC 6803 produces PHB under conditions of nitrogen starvation. However, it is so far unclear which metabolic pathways provide the precursor molecules for PHB synthesis during nitrogen starvation. In this study, we investigated if PHB could be derived from the main intracellular carbon pool, glycogen. A mutant of the major glycogen phosphorylase, GlgP2 (slr1367 product), was almost completely impaired in PHB synthesis. Conversely, in the absence of glycogen synthase GlgA1 (sll0945 product), cells not only produced less PHB, but were also impaired in acclimation to nitrogen depletion. To analyze the role of the various carbon catabolic pathways (EMP, ED and OPP pathways) for PHB production, mutants of key enzymes of these pathways were analyzed, showing different impact on PHB synthesis. Together, this study clearly indicates that PHB in glycogen-producing Synechocystis sp. PCC 6803 cells is produced from this carbon-pool during nitrogen starvation periods. This knowledge can be used for metabolic engineering to get closer to the overall goal of a sustainable, carbon-neutral bioplastic production.


Assuntos
Glicogênio/metabolismo , Hidroxibutiratos/metabolismo , Nitrogênio/deficiência , Poliésteres/metabolismo , Synechocystis/metabolismo , Carbono/metabolismo , Glicogênio/biossíntese , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Redes e Vias Metabólicas , Mutação/genética
19.
Development ; 146(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30918052

RESUMO

The polysaccharide glycogen is an evolutionarily conserved storage form of glucose. However, the physiological significance of glycogen metabolism on homeostatic control throughout the animal life cycle remains incomplete. Here, we describe Drosophila mutants that have defective glycogen metabolism. Null mutants of glycogen synthase (GlyS) and glycogen phosphorylase (GlyP) displayed growth defects and larval lethality, indicating that glycogen plays a crucial role in larval development. Unexpectedly, however, a certain population of larvae developed into adults with normal morphology. Semi-lethality in glycogen mutants during the larval period can be attributed to the presence of circulating sugar trehalose. Homozygous glycogen mutants produced offspring, indicating that glycogen stored in oocytes is dispensable for embryogenesis. GlyS and GlyP mutants showed distinct metabolic defects in the levels of circulating sugars and triglycerides in a life stage-specific manner. In adults, glycogen as an energy reserve is not crucial for physical fitness and lifespan under nourished conditions, but glycogen becomes important under energy stress conditions. This study provides a fundamental understanding of the stage-specific requirements for glycogen metabolism in the fruit fly.


Assuntos
Proteínas de Drosophila/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Feminino , Glicogênio/metabolismo , Glicogênio Fosforilase/genética , Glicogênio Sintase/genética , Masculino , Trealose/metabolismo
20.
Gen Comp Endocrinol ; 277: 82-89, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902611

RESUMO

In this experiment, Genetically improved farmed Nile tilapia Oreochromis niloticus were intraperitoneally injected with 1 g glucose/kg of body weight or saline. Red and white muscle tissues were collected at 0, 1, 2, 4, 6 and 12 h after the glucose tolerance test (GTT) or saline injection, and the time course of changes in molecular and metabolic adaption of glucose metabolism of these two tissues were evaluated. The results showed that the expression of insulin-responsive glucose transporter 4 (glut4) was up-regulated at 4 h after the GTT in the red muscle, implying an increase of glucose uptake. However, the expression of glut4 in the white muscle did not change with glucose load. The glycolysis of red muscle in tilapia was stimulated during 2-4 h after the GTT, as the expression of hexokinase 1b (hk1b), hk2, phosphofructokinase muscle type a (pfkma) and pfkmb and the activity of HK and PFK increased. By contrast, only the expression of hk1b was up-regulated at 6 h after the GTT in the white muscle. The mRNA level of glycogen synthase 1 (gys1) and glycogen content increased at 2 and 6 h, respectively after the GTT in the red muscle, suggesting that glucose storage was provoked. However, glycogen content in the white muscle was not impacted by GTT. Lipogenesis was stimulated in the red muscle as reflected by up-regulated expression of acetyl-CoA carboxylase α (accα) (during 2-4 h) and accß (during 4-12 h) with GTT. In the white muscle, however, the expression of accα was not changed, and mRNA level of accß was not up-regulated until 6 h after the GTT. Taken together, it was concluded that the glycolytic and glycogen synthesis mechanisms in the red muscle were highly regulated by an acute glucose load while those in the white muscle were less responsive to this stimulus.


Assuntos
Adaptação Fisiológica , Ciclídeos/metabolismo , Glucose/metabolismo , Músculos/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Animais Geneticamente Modificados , Ciclídeos/genética , Teste de Tolerância a Glucose , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fosfofrutoquinases/metabolismo
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