Increased flux through the hexosamine biosynthetic pathway leads to the induction of acetol-CoA caboxylase gene expression in the heart

[摘要] ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is inducedin a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor ofmitochondrial fatty acid uptake. Previous studies show that increased flux through thehexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions maycontribute to the development of insulin resistance. In light of this, we hypothesisedthat increased HBP flux induces cardiac ACCb gene expression thereby contributingto the onset of insulin resistance.We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2myoblasts with a 1,317 bp human ACCb promoter-luciferase construct (pPIIb-1317)and an expression construct encoding the rate-limiting step of the HBP i.e. glutamine:fructose 6-phosphate amidotransferase (GFAT). Overexpression of GFAT increasedACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfectionexperiments were repeated in the presence of varying concentrations ofL-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activitywas dose-dependently increased. To further corroborate these findings, weemployed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucinewere administered to transfected cells for a period of 24 hours. Here bothazaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity.In agreement, co-transfections with two dominant negative GFAT constructs alsodiminished ACCb gene promoter activity. We next inhibited two enzymes of the HBPacting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase usingalloxan (0.1 mM, 1 mM and 2 mM) and streptozotocin (5 mM and 10 mM), respectively, for a period of 24 hours. Addition of alloxan attenuated ACCb genepromoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increasedactivity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 astranscriptional regulatory candidates for HBP-induced ACCβ promoter regulation.Our data implicates USF2 as an important transcriptional regulator of HBP-inducedACCβ promoter regulation.In summary, this study demonstrates that increased flux through the hexosaminebiosynthetic pathway induces ACCb gene promoter activity. We further propose thatsuch an induction would reduce cardiac fatty acid oxidation, thereby leading tointracellular lipid accumulation due to a mismatch between sarcolemmal FA uptakeand mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia).

[发布机构] Stellenbosch University