RESUMO
Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.
Assuntos
Proteínas Quinases Ativadas por AMP , Acetilglucosamina , Diabetes Mellitus Experimental , Retinopatia Diabética , N-Acetilglucosaminiltransferases , Retinopatia Diabética/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Animais , Camundongos , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , Apoptose/efeitos dos fármacos , Metformina/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Retina/metabolismo , Retina/patologia , Retina/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
Neutral pullulanases, having a good application prospect in trehalose production, showed a limited expression level. In order to address this issue, two approaches were utilized to enhance the yield of a new neutral pullulanase variant (PulA3E) in B. subtilis. One involved using multiple copies of genome integration to increase its expression level and fermentation stability. The other focused on enhancing the PulA-type atypical secretion pathway to further improve the secretory expression of PulA3E. Several strains with different numbers of genome integrations, ranging from one to four copies, were constructed. The four-copy genome integration strain PD showed the highest extracellular pullulanase activity. Additionally, the integration sites ytxE, ytrF, and trpP were selected based on their ability to enhance the PulA-type atypical secretion pathway. Furthermore, overexpressing the predicated regulatory genes comEA and yvbW of the PulA-type atypical secretion pathway in PD further improved its extracellular expression. Three-liter fermenter scale-up production of PD and PD-ARY yielded extracellular pullulanase activity of 1767.1 U/mL at 54 h and 2465.1 U/mL at 78 h, respectively. Finally, supplementing PulA3E with 40 U/g maltodextrin in the multi-enzyme catalyzed system resulted in the highest trehalose production of 166 g/L and the substrate conversion rate of 83%, indicating its potential for industrial application.