P1606Glutamine-fructose-6-phosphate amidotransferase 2 mediates isoproterenol-induced cardiac hypertrophy by increasing Akt O-GlcNAcylation through hexosamine biosynthesis pathway

Abstract

Abstract Background Cardiac hypertrophy is an independent risk factor for heart failure and cardiac death. Hexosamine biosynthesis pathway (HBP), an accessory pathways of glycolysis, is known to be involved in the attachment of O-linked N-acetylglucosamine motif (O-GlcNAcylation) to proteins, a post-translational modification. However, the role of HBP has not been determined in pathological cardiac hypertrophy. Purpose The purpose of this study to examine whether glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2), a critical enzyme of HBP, mediates cardiac hypertrophy by protein O-GlcNAcylation and activating hypertrophic signaling in cardiomyocytes. Methods and results C57BL/6J mice were treated with isoproterenol (ISO: 15 mg/kg/day, 1 week) with or without 6-Diazo-5-oxo-L-norleucine (DON, an inhibitor of GFAT: 500 μg/kg/day, 1week). ISO-treated mice (ISO+vehicle) showed cardiac hypertrophy, which were attenuated in ISO and DON-treated mice (ISO+DON) (heart weight to tibial length ratio: 7.70±0.09 vs. 7.11±0.15 mg/mm, n=12, p<0.05, left ventricular wall thickness: 1.05±0.02 vs. 0.86±0.03 mm, n=6, p<0.05). Cardiomyocyte cross-sectional area was also decreased in ISO+DON compared with ISO+vehicle (309±25 vs. 252±13 mm2, n=,3 p<0.05). Whereas expression levels of GFAT2 and protein O-GlcNAcylation in the heart were increased in ISO+vehicle compared with control+vehicle by 3.3 and 1.5 folds, respectively (n=9 and n=9, p<0.05), expression levels of O-GlcNAc transferase (OGT) and the β-N-acetylglucosaminidase (OGA), other enzymes regulating O-GlcNAcylation, were not altered in both groups, indicating that ISO activated HBP by GFAT2. Protein O-GlcNAcylation in ISO+DON was lower than that in ISO+vehicle by 83% (n=9, p<0.05). In addition, phosphorylation of Akt, a critical mediator of cardiac hypertrophy, but not other mediators of cardiac hypertrophy such as ERK, JNK, or p38MAPK, was significantly decreased in ISO+DON by 76% (n=9, p<0.05). In cultured neonatal rat ventricular myocytes, treatment with ISO (1μM, 12h) increased the expression levels of GFAT2 and protein O-GlcNAcylation by 1.3 and 1.5 folds, respectively (n=6 and n=6, p<0.05), but not GFAT1. Furthermore, ISO stimulation increased a direct O-GlcNAcylation of Akt by 1.4 folds (n=3, p<0.05). Downregulation of GFAT2 by RNA silencing decreased cell size by 82% (n=6, p<0.05) and protein O-GlcNAcylation and phosphorylation of Akt by 76% and 54%, respectively (n=9 and n=9, p<0.05) in cardiomyocyte treated with ISO. Conversely, administration of glucosamine, a substrate of HBP, increased protein of O-GlcNAcylation and phosphorylation of Akt by 1.3 and 1.8 folds, respectively (n=6 and n=6, p<0.05). Conclusions GFAT2, a limiting enzyme of HBP, mediates pathological cardiac hypertrophy by Akt activation probably due to its O-GlcNAcylation. GFAT2-O-GlcNAcylation-Akt pathway might be a potential novel therapeutic target for cardiac hypertrophy.