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1.
Redox Biol ; 59: 102576, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36535130

RESUMO

Glyoxalase 2 is the second enzyme of the glyoxalase system, catalyzing the detoxification of methylglyoxal to d-lactate via SD-Lactoylglutathione. Recent in vitro studies have suggested Glo2 as a regulator of glycolysis, but if Glo2 regulates glucose homeostasis and related organ specific functions in vivo has not yet been evaluated. Therefore, a CRISPR-Cas9 knockout of glo2 in zebrafish was created and analyzed. Consistent with its function in methylglyoxal detoxification, SD-Lactoylglutathione, but not methylglyoxal accumulated in glo2-/- larvae, without altering the glutathione metabolism or affecting longevity. Adult glo2-/- livers displayed a reduced hexose concentration and a reduced postprandial P70-S6 kinase activation, but upstream postprandial AKT phosphorylation remained unchanged. In contrast, glo2-/- skeletal muscle remained metabolically intact, possibly compensating for the dysfunctional liver through increased glucose uptake and glycolytic activity. glo2-/- zebrafish maintained euglycemia and showed no damage of the retinal vasculature, kidney, liver and skeletal muscle. In conclusion, the data identified Glo2 as a regulator of cellular energy metabolism in liver and skeletal muscle, but the redox state and reactive metabolite accumulation were not affected by the loss of Glo2.


Assuntos
Lactoilglutationa Liase , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Aldeído Pirúvico/metabolismo , Ácido Láctico , Glucose , Tioléster Hidrolases/metabolismo
2.
J Bacteriol ; 194(5): 941-55, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22178972

RESUMO

Expression profiling of Corynebacterium glutamicum in comparison to a derivative deficient in the transcriptional regulator AtlR (previously known as SucR or MtlR) revealed eight genes showing more than 4-fold higher mRNA levels in the mutant. Four of these genes are located in the direct vicinity of the atlR gene, i.e., xylB, rbtT, mtlD, and sixA, annotated as encoding xylulokinase, the ribitol transporter, mannitol 2-dehydrogenase, and phosphohistidine phosphatase, respectively. Transcriptional analysis indicated that atlR and the four genes are organized as atlR-xylB and rbtT-mtlD-sixA operons. Growth experiments with C. glutamicum and C. glutamicum ΔatlR, ΔxylB, ΔrbtT, ΔmtlD, and ΔsixA derivatives with sugar alcohols revealed that (i) wild-type C. glutamicum grows on D-arabitol but not on other sugar alcohols, (ii) growth in the presence of D-arabitol allows subsequent growth on D-mannitol, (iii) D-arabitol is cometabolized with glucose and preferentially utilized over D-mannitol, (iv) RbtT and XylB are involved in D-arabitol but not in D-mannitol metabolism, (v) MtlD is required for D-arabitol and D-mannitol metabolism, and (vi) SixA is not required for growth on any of the substrates tested. Furthermore, we show that MtlD confers D-arabitol and D-mannitol dehydrogenase activities, that the levels of these and also xylulokinase activities are generally high in the C. glutamicum ΔatlR mutant, whereas in the parental strain, they were high when cells were grown in the presence of D-arabitol and very low when cells were grown in its absence. Our results show that the XylB, RbtT, and MtlD proteins allow the growth of C. glutamicum on D-arabitol and that D-arabitol metabolism is subject to arabitol-dependent derepression by AtlR.


Assuntos
Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Álcoois Açúcares/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Deleção de Genes , Perfilação da Expressão Gênica , Glucose/metabolismo , Manitol/metabolismo , Óperon , Proteínas Repressoras/genética
3.
Cell Cycle ; 18(20): 2683-2696, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31451030

RESUMO

Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified epoa being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for epoa have not been described so far. In order to compare a transient knockdown vs. permanent knockout for epoa in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate epoa knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to epoa morphants, epoa-/- zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named epob, was identified and upregulated in epoa-/- mutants. epob knockdown altered pronephros development, which was further aggravated in epoa-/- mutants. Likewise, epoa and epob morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of epoa during embryonic development can be compensated by epob leading to phenotypical discrepancies in epoa knockdown and knockout zebrafish embryos.


Assuntos
Eritropoetina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Organogênese/genética , Pronefro/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Eritropoetina/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Heterozigoto , Homozigoto , Microscopia Eletrônica , Morfolinos/genética , Pronefro/anormalidades , Pronefro/metabolismo , Proteínas Recombinantes/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
4.
JCI Insight ; 4(12)2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31217350

RESUMO

The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1-/- zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced-obesity zebrafish model was used to analyze glo1-/- zebrafish under high nutrient intake. Glo1-/- zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1-/- zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1-/- zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake.


Assuntos
Hiperglicemia/etiologia , Lactoilglutationa Liase/fisiologia , Obesidade/complicações , Animais , Sistemas CRISPR-Cas , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/genética , Dieta , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Glucose/metabolismo , Hiperglicemia/genética , Resistência à Insulina , Lactoilglutationa Liase/genética , Fígado/metabolismo , Masculino , Aldeído Pirúvico/metabolismo , Retina/patologia , Peixe-Zebra/crescimento & desenvolvimento
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