Hyperglycaemic-induced regulation of SIRT3 and downstream antioxidant profile.

Doctor of Philosophy in Medical Biochemistry. University of KwaZulu-Natal, Medical School 2015.Hyperglycaemia increases reactive oxygen species (ROS) production and mitochondrial dysfunction which are involved in metabolic disorders. Sirtuin 3 (SIRT3) is a primary mitochondrial deacetylase that regulates mitochondrial function and antioxidant (AO) defence. We investigated the role of SIRT3 in AO defence under hyperglycaemic conditions in HepG2 cells in the presence and absence of metformin and curcumin. We also examined cell protective mechanisms that counterbalance apoptotic stress under these oxidative conditions. HepG2 cells were cultured with 5mM (control), 19.9mM mannitol (OC), 10mM glucose, 30mM glucose (hyperglycaemic), 10mM nicotinamide (NAM) at 24hr and 72hr time points in the absence or presence of curcumin (5μM and 10μM) or metformin (3mM). Increased expressions of SIRT3, peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), mitochondrial AO enzymes glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), uncoupling protein 2 (UCP2) and mtDNA repair enzyme 7, 8-dihydro-8-oxoguanine (OGG1) were observed under hyperglycaemic conditions. The same trend was observed for all parameters following metformin and curcumin treatment. In addition, curcumin also increased expressions of nuclear factor-kappa B (NF-κB), lon protease (Lon) and heat shock protein 70 (Hsp70). These were optimally expressed in the 10μM curcumin-treated groups. We also showed that under hyperglycaemic conditions, apoptosis was initiated but may not have been fully executed due to the induction of stress proteins (heat shock protein 27, nuclear factor erythroid-derived 2-like 2) and AO defence that counterbalance apoptotic stress. The results suggest that SIRT3 modulates AO defence and confers resistance to oxidative stress (OS)- induced damage under hyperglycaemic conditions in HepG2 cells. Our data also suggests that metformin and curcumin may work synergistically with SIRT3, or through SIRT3-mediated mechanisms, to improve AO defence. Our model shows that hyperglycaemia may induce apoptosis; however, apoptotic stress may be counterbalanced by cell survival mechanisms that include stress response proteins and the downstream activation of AO defence. Mitochondria are susceptible to OS, which is involved in metabolic disorders. SIRT3 may, therefore, be therapeutically targeted as a potential cyto-protective factor. Modulation of SIRT3 function, by chemical or natural therapeutics, may also improve disease outcomes

Association of -308 TNF-alpha promoter polymorphism with viral load and CD4 T-helper cell apoptosis in HIV-1 infected black South Africans

Objective. To determine whether the -308 TNF-α promoter polymorphism is associated with markers of HIV progression in the South African population. Methods. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the -308 TNF-α polymorphism in 75 patients and 76 healthy controls. Serum TNF-α concentrations were measured using ELISA in each cohort. CD4+ T cell apoptosis and HIV-1 RNA viral load were determined using Annexin-V-FITC assay and Nuclisens Easy Q HIV-1 assay respectively. CD4 + T cell counts were measured flow cytometrically. Results. The frequency of -308 G allele was similar in the HIV-1 and control cohorts. The -308GG genotype was associated with lower TNF-α concentrations and markers of increased HIV progression indicated by higher TH lymphocyte apoptosis, lower TH lymphocyte count and higher plasma viral load, irrespective of treatment. Conclusion. The presence of the TNF-α -308 G allele in HIV-1 patients may be associated with increased risk of HIV-1 progression. Further research is required to investigate the nature of this association. S Afr J HIV Med 2012;13(2):72-77