Browsing by Author "Rajamani, Uthra"

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    Hyperglycemia-induced activation of the hexosamine biosynthetic pathway causes myocardial cell death
    (Stellenbosch : University of Stellenbosch, 2009-12) Rajamani, Uthra; Essop, M. Faadiel; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
    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. 4 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.
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    Oleanolic acid : a novel cardioprotective agent that blunts hyperglycemia-induced contractile dysfunction
    (Public Library of Science, 2012-10-16) Mapanga, Rudo F.; Rajamani, Uthra; Dlamini, Nonkululeko; Zungu-Edmondson, Makhosazane; Kelly-Laubscher, Roison; Shafiullah, Mohammed; Wahab, Athiq; Hasan, Mohamed Y.; Fahim, Mohamed A.; Rondeau, Phillipe; Bourdon, Emmanuel; Essop, M. Faadiel
    Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher in-hospital mortality. Previous studies implicate oxidative stress, excessive flux through the hexosamine biosynthetic pathway (HBP) and a dysfunctional ubiquitin-proteasome system (UPS) as potential mediators of this process. Since oleanolic acid (OA; a clove extract) possesses antioxidant properties, we hypothesized that it attenuates acute and chronic hyperglycemia-mediated pathophysiologic molecular events (oxidative stress, apoptosis, HBP, UPS) and thereby improves contractile function in response to ischemia-reperfusion. We employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 mM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion 6 OA treatment; 3) In vivo coronary ligations were performed on streptozotocin treated rats 6 OA administration during reperfusion; and 4) Effects of long-term OA treatment (2 weeks) on heart function was assessed in streptozotocin-treated rats. Our data demonstrate that OA treatment blunted high glucose-induced oxidative stress and apoptosis in heart cells. OA therapy also resulted in cardioprotection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia-reperfusion under hyperglycemic conditions. In parallel, we found decreased oxidative stress, apoptosis, HBP flux and proteasomal activity following ischemia-reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. These findings are promising since it may eventually result in novel therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associatedcardiovascular complications.