Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: OBJECTIVE – Oxidative stress increases flux through the hexosamine biosynthetic pathway
(HBP) resulting in greater O-GlcNAcylation of target proteins. Since increased oxidative stress
and HBP flux are associated with insulin resistance, we hypothesized that its activation leads to
greater O-GlcNAcylation of BAD (pro-apoptotic) and increased myocardial apoptosis.
RESEARCH DESIGN AND METHODS – To investigate our hypothesis, we employed two
experimental models: 1) H9c2 cardiomyoblasts exposed to high glucose (33 mM glucose) ± HBP
modulators ± antioxidant treatment vs. matched controls (5.5 mM glucose); and 2) a rat model of
high fat diet-induced insulin resistance and hyperglycemia. We evaluated apoptosis in vitro by
Hoechst nuclear staining, Annexin-V staining, caspase activity measurements and
immunoblotting while in vivo apoptosis was assessed by immunoblotting. In vitro reactive
oxygen species (ROS) levels were quantified by H2DCFDA staining (fluorescence microscopy,
flow cytometry). We determined overall and BAD O-GlcNAcylation, both by immunoblotting
and immunofluorescence microscopy. As BAD-Bcl-2 dimer formation enhances apoptosis, we
performed immunoprecipitation analysis and immunofluorescence microscopy (co-localization)
to determine BAD-cl-2 dimerization. In vivo overall O-GlcNAcylation, BAD O-GlcNAcylation
and BAD-Bcl-2 dimerization was determined by immunoprecipitation and immunoblotting.
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RESULTS – High glucose treatment of cells significantly increased the degree of apoptosis as
revealed by Hoechst nuclear staining (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V staining (43 ±
5%), caspase activity assay (26 ± 2%) and immunoblotting. In parallel, overall OGlcNAcylation
(p<0.001 vs. 5.5 mM), BAD O-GlcNAcylation (p<0.05 vs. 5.5 mM) and ROS
levels were increased (fluorescence microscopy – p<0.05 vs. 5.5 mM; flow cytometry – p<0.001
vs. 5.5 mM). HBP inhibition using DON and antioxidant treatment (α-OHCA) attenuated these
effects while HBP activation by PUGNAc exacerbated it. Likewise, insulin resistant rat hearts
exhibited significantly higher caspase-3 (p<0.05 vs. controls), overall O-GlcNAcylation (p<0.05
vs. controls) and BAD O-GlcNAcylation levels (p<0.05 vs. 5.5 mM). BAD-Bcl-2 dimer
formation was increased in cells exposed to hyperglycemia [immunoprecipitation analysis and
co-localization] and in insulin resistant hearts.
CONCLUSIONS - Our study identified a novel pathway whereby hyperglycemia results in
greater oxidative stress, resulting in increased HBP activation and increased BAD OGlcNAcylation.
We also found greater BAD-Bcl-2 dimerization increasing myocardial
apoptosis, suggesting that this pathway may play a crucial role in the onset of the diabetic
cardiomyopathy. / AFRIKAANSE OPSOMMING: DOELWIT – Oksidatiewe stres verhoog fluks deur die heksosamien biosintetiese weg (HBW)
wat in „n groter O-GlcNAsetilering van teiken proteïene resulteer. Weens die feit dat verhoogde
oksidatiewe stres en HBW fluks verband hou met insulienweerstandigheid, hipotetiseer ons dat
die aktivering hiervan tot groter O-GlcNAsetilering van BAD (pro-aptoptoties) en verhoogde
miokardiale apoptose lei.
NAVORSINGS ONTWERP EN METODES – Om die hipotese te ondersoek het ons twee modelle
ontplooi: 1) H9c2 kardiomioblaste is blootgestel aan hoë glukose konsentrasie (33mM glucose) ±
HBW moduleerders ± antioksidant behandeling vs. gepaarde kontrole (5.5mM glucose); en 2) „n
hoë vet dieetgeïnduseerde insulienweerstandige rotmodel en hiperglukemie. Ons het apoptose in
vitro deur middel van Hoescht nukleuskleuring geëvalueer, kasapase aktiwiteit bepalings en
immunoblotting terwyl apoptose in vivo getoets is deur immunoblotting. Reaktiewe
suurstofspesie (RSS) vlakke is deur middel van H2DCFDA verkleuring (fluoresensie
mikroskopie, vloeisitometrie) bepaal. Algehele en BAD O-GlcNAsetilering is beide deur
immunoblotting en immunofluoresensie mikroskopie bepaal. BAD-Bcl-2 dimeervorming
bevorder apoptose, om BAD-cl-2 dimerisasie te bepaal is daar van immunopresipitering analise
en immunofluoresensie mikroskopie (ko-lokalisasie) gebruik gemaak. In vivo is algehele OGlcNAsetiliering,
BAD O-GlcNAsetiliering en BAD-Bcl-2 dimerisasie deur immunopresipitasie
en immunoblotting bepaal.
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RESULTE – Hoë glukose behandeling van selle het die graad van apotpose betekenisvol verhoog
soos blootgelê deur Hoechst nukleuskleuring (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V kleuring
(43 ± 5%), kaspase aktiviteit assay (26 ± 2%) en immunoblotting. In parallel, algehele OGlcNAsetilering
(p<0.001 vs. 5.5 mM), BAD O-GlcNAsetilering (p<0.05 vs. 5.5 mM) en RSS
vlakke is verhoog (fluoresensie mikroskopie– p<0.05 vs. 5.5 mM; vloeisitometrie– p<0.001 vs.
5.5 mM). HBW inhibering deur van DON en van antioksidant behandeling gebruik te maak (α-
OHCA) het hierdie effekte verlaag terwyl HBW aktivering deur PUGNAc dit verhoog het.
Netso, het insulienweerstandige rotharte betekenisvolle hoë kaspase -3 (p<0.05 vs. kontrole),
algeheel O-GlcNAsetilering (p<0.05 vs. kontrole) en BAD O-GlcNAsetiliering vlakke (p<0.05
vs. 5.5 mM) getoon. BAD-Bcl-2 dimeervorming is verhoog in hiperglukemies blootgestelde selle
[immunopresipitering analise en ko-lokalisering] en in insulienweerstandige harte.
GEVOLGTREKKINGS – Ons studie het „n nuwe weg geïdenifiseer waar hiperglukemie in groter
oksidatiewe stres resulteer wat weer HBW aktivering verhoog en BAD O-GlcNAsetilering
verhoog het. Ons het verder bevind dat groter BAD-Bcl-2 dimerisasie miokardiale apoptose
verhoog wat voorstel dat hierdie weg „n belangrike rol in diabetiese kardiomiopatie speel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1142 |
| Date | 12 1900 |
| Creators | Rajamani, Uthra |
| Contributors | Essop, M. Faadiel, University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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