Thesis (MSc)--Stellenbosch University, 2008. / 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).
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21459 |
| Date | 03 1900 |
| Creators | Imbriolo, Jamie |
| Contributors | Essop, M. Faadiel, Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | viii, 115 leaves : ill. |
| Rights | Stellenbosch University |
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