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

Imbriolo, Jamie (Stellenbosch : Stellenbosch University, 2008-03)

Thesis (MSc)--Stellenbosch University, 2008.

Thesis

ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is induced in a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake. Previous studies show that increased flux through the hexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions may contribute to the development of insulin resistance. In light of this, we hypothesised that increased HBP flux induces cardiac ACCb gene expression thereby contributing to the onset of insulin resistance. We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2 myoblasts 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 increased ACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfection experiments were repeated in the presence of varying concentrations of L-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activity was dose-dependently increased. To further corroborate these findings, we employed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucine were administered to transfected cells for a period of 24 hours. Here both azaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity. In agreement, co-transfections with two dominant negative GFAT constructs also diminished ACCb gene promoter activity. We next inhibited two enzymes of the HBP acting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase using alloxan (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 gene promoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increased activity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 as transcriptional regulatory candidates for HBP-induced ACCβ promoter regulation. Our data implicates USF2 as an important transcriptional regulator of HBP-induced ACCβ promoter regulation. In summary, this study demonstrates that increased flux through the hexosamine biosynthetic pathway induces ACCb gene promoter activity. We further propose that such an induction would reduce cardiac fatty acid oxidation, thereby leading to intracellular lipid accumulation due to a mismatch between sarcolemmal FA uptake and mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia).

AFRIKAANSE OPSOMMING: Geen uitdrukking van die kardiale isoform asetiel-KoA karboksilase (ACCb) word in ‘n glukose afhanklike wyse geïnduseer. ACCb produseer maloniel-KoA, ‘n kragtige inhibeerder van mitochondriale vetsuuropname. Vorige studies toon aan dat verhoogde fluks deur die heksosamien biosintestiese weg (HBW) onder hiperglukemiese toestande bydra tot die ontwikkeling van insulienweerstand. In die lig hiervan, word daar gehipotetiseer dat verhoogde HBP fluks kardiale ACCb geenuitdrukking induseer en so bydra tot die ontstaan van insulienweerstand. Ons hipotese is getoets deur die kardiale afkomstige rot H9c2 mioblaste met ‘n 1.317 bp mens ACCb-lusiferase promotor konstruk (pPII-1317) te transfekteer en ‘n uitdrukking te konstrueer wat die tempo bepalende stap van HBP i.e. glutamien: fruktose-6-fosfaat amidotransferase (GFAT) kodeer. Ooruitdrukking van GFAT verhoog ACCb geenpromotor aktiviteit deur 75 ± 23% teenoor kontrole (n=6, p<0.001). Die herhaling van ko-transfeksie eksperimente is herhaal in die teenwoordigheid van variëerbare L-glutamienkonsentrasies (0 mM, 4 mM, 8 mM), ’n substraat vir die HBP, ACCb promotor aktiwiteit is dosisafhanglik verhoog. Om die bevindinge verder te staaf, is twee inhibeerders van GFAT, i.e. 40 μM azaserien en 40 μM 6-diazo-5-oxo-L-norleusien aan transfeksie selle toegedien vir ’n tydperk van 24 uur. Beide azaserien en 6-diazo-5-oxo-L-norleusien verlaag ACCb geenpromotor aktiwiteit. In ooreenstemming met die bogenoemde het ko-transfeksies met twee dominante negatiewe GFAT konstrukte ook ACCb geenpromoter aktiwiteit verminder. Die volgende stap is om twee ensieme van die HBP wat stroomaf van GFAT aktief is, vir ‘n periode van 24 uur te inhibeer i.e. O-GlcNAc transferase en O-GlcNAcase deur alloxan (0.1 mM, 1 mM en 2 mM) and streptozotosien (5 mM en 10 mM) onderskeidelik vir ‘n 24 uur periode te gebruik. Toevoeging van alloxan het die ACCb geenpromotor aktiwiteit by 35.6 ± 1.9% (n=16, p<0.001) verlaag en streptozotosien aktiwiteit verhoog by 32 ± 12% (n=12, p<0.001). Ons het ook die USF1 en USF2 as transkripsie regulerings kandidate vir HBP-geïnduseerde ACCβ promotor regulering ondersoek. Ons data impliseer dat USF2 as ‘n belangrike transkripsie reguleerder van HBP-geïndiseerde ACCβ promotor regulering is. Samevattend het hierdie studie demonstreer dat verhoogde fluks deur die hexosamien biosintetiese weg ACCb geenpromotor aktiwiteit induseer. Ons stel verder voor dat hierdie induksie die kardiale vetsuuroksidasie verlaag wat daartoe lei dat intrasellulêre lipied akkumulasie as gevolg van onparing tussen sarkolemma vetsuuropname en mitochondriale vetsuuroksidasie in ’n insulien weerstandige situasie (i.e. hiperlipidaemia).

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/21459
This item appears in the following collections: