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In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Myocardial infarction (MI) is a major atherosclerotic cardiovascular disease which represents a leading cause of death worldwide. Kruppel-like factor 5 (KLF5) is a member of the kruppel-like transcription factor family which has been reported with pro-apoptotic functions in myocardial cells. This work focuses on the function of KLF5 in the pathogenesis of MI and the molecules involved. A mouse model with MI was established. Hypoxia/reoxygenation (H/R)-treated H9C2 cells were applied for in vitro experiments. A KLF5-specific inhibitor ML264 was administrated in cell and animal models. ML264 significantly reduced apoptosis, expression of fibrosis-related markers, reactive oxygen species in the H/R-treated H9C2 cells, and it reduced myocardial injury, infarct size, apoptosis and fibrosis in the myocardial tissues in model mice through specific downregulation of KLF5. A microRNA (miRNA) microarray analysis was performed, which suggested miR-27a as the most upregulated miRNA in the H/R-treated cells after ML264 treatment. miR-27a mimic reduced apoptosis and fibrosis in H/R-treated cells, while miR-27a inhibition blocked the protective roles of ML264. The integrated bioinformatic analyses and luciferase assays confirmed glutamine fructose-6-phosphate transaminase 2 (GFPT2) mRNA as a target of miR-27a. Overexpression of GFPT2 counteracted the protective functions of miR-27a against MI through the activation of the TGF-β/Smad2/3 signaling pathway. To conclude, this study evidenced that KLF5 possibly induces cell and tissue damage in MI through downregulation of miR-27a and the subsequent activation of GFPT2/TGF-β/Smad2/3 axis. This study may offer novel thoughts into MI treatment.

The hexosamine biosynthetic pathway (HBP), a branch of glucose metabolism, provides a substrate for glycosylation modification, which has a wide-ranging effect on cellular functions. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) has been reported to regulate the HBP as the first and rate-limiting enzyme. Given the inverse association between GFPT2 expression and survival of patients with serous ovarian cancer (SOC) observed in The Cancer Genome Atlas (TCGA) database, we attempted to investigate the role of GFPT2 and its related mechanisms in SOC. The results showed that GFPT2 was over-expressed in SOC tissues, and positive correlations with advanced stage (FIGO III/IV), suboptimal removal rate and poor survival were observed in 90 SOC patients. Cell migration and invasion were also inhibited in GFPT2 knockdown SKOV3 and HEY cells. The levels of O-linked β-N-acetylglucosamine (O-GlcNAc) and intranuclear β-catenin were evaluated and the observed increase in O-GlcNAcylation induced by GFPT2 may contribute to epithelial-mesenchymal transition (EMT). These data provide novel insights into the function of GFPT2 and O-GlcNAcylation in the EMT and thus the invasiveness SOC., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Purpose: Asthenozoospermia (ASZ) is a condition characterized by reduced sperm motility in semen affecting approximately 19% of infertile men. Major risk factors, particularly gene mutations, still remain unknown. The main aim of the present study was to identify novel genes and mutations that may influence human sperm motility., Methods: Whole-exome sequencing (WES) was performed on a large pedigree of infertile men (n = 5) followed by bioinformatics analyses. Candidate pathogenic variants were screened in a control cohort of 400 ancestry-matched Iranian fertile men, 30 unrelated men with idiopathic ASZ, and public databases., Results: A rare mutation in GFPT2 gene (c.1097G > A; p.Arg366Gln) located in the SIS 1 domain was segregated with the phenotype and was consistent with autosomal recessive inheritance. The in silico analyses revealed that the mutation might affect the function of SIS 1 domain and abolish its carbohydrate-binding ability., Conclusion: Homozygosity of the GFPT2 p.Arg366Gln mutation was associated with increased levels of reactive oxygen species (ROS) in spermatozoa and decreased sperm motility., (Copyright © 2019 Elsevier B.V. All rights reserved.)

PPP2R2A is one of the regulatory subunits of the PP2A phosphatase complexes, and previous studies showed that its upregulation promotes cancer cell survival and growth. In this research, we used the tandem affinity purification and the HPLC-Chip-ESI/MS/MS mass spectrometry to screen the PPP2R2A-binding proteins and the results indicated that the GFPT-1/-2 were the potential partners of PPP2R2A. We further validated the interaction between PPP2R2A and GFPT-1/-2 through GST Pull-down, co-immunoprecipitation and immunofluorescence assays. And we found that knockdown of PPP2R2A by lentivirus-mediated shRNA enhanced the phosphorylation of GFPT2, whereas the phosphorylation of GFPT1 had no significant change. GFPT2 is a rate-limiting enzyme in the hexosamine pathway. Our results showed that the knockdown of PPP2R2A promoted the total cellular O-GlcNAcylation in MDA-MB-231 breast cancer cells. These results suggest that PPP2R2A interacts with GFPT1/2, and leads to the phosphorylation of GFPT2, which can regulate the cellular O-GlcNAcylation.