Browsing by Author "Burger, Joleen"

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    Beta secretase regulation and inflammation in pancreatic β-cells: the potential role of Rooibos
    (Stellenbosch : Stellenbosch University, 2018-12) Burger, Joleen; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology.; Chellan, Nireshni; Lopes, John; Muller, Christo J.F.
    Introduction Chronic, low-grade inflammation is a hallmark of insulin resistance (IR) and underlies pancreatic β-cell failure and the development of type 2 diabetes (T2D), a disease characterized by a reduction in β-cell functional mass. However, therapeutics that directly protect β-cells from stressors, such as inflammation and oxidative stress, are limited. Amylin, a neuro-hormone, is co-secreted with insulin, therefore hyperinsulinemia often coincides with hyper secretion of amylin. Increased secretion of amylin is associated amyloid deposition which have detrimental effects on β-cell function. In pancreatic islets, β-secretase (BACE) modulates the deposition of cytotoxic islet amyloid, an initiator of intra-islet inflammation and oxidative stress, making BACE inhibition a therapeutic target. Insights into mechanisms involved in islet inflammation and the associated effect(s) of BACE, may reveal an opportunity to develop novel therapeutics to protect and preserve β-cells in T2D. In addition to their antidiabetic properties, aspalathin (Asp), Z-2-(β-D-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), and an unfermented Rooibos extract (GRT) may have anti-inflammatory effects in β-cells and may additionally modulate BACE activity. Aim To determine if GRT or two of its most bioactive polyphenols have BACE inhibition activity and can reduce pro-inflammatory effects in rat insulinoma (INS1) pancreatic β-cells. Methods A model of moderate inflammation was established in INS1 cells using a cytokine cocktail, containing tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Rat insulinoma cell viability, function, and oxidative stress was measured in response to the cytokine cocktail and exposure to Asp, PPAG, and GRT by quantifying cellular adenosine triphosphate (ATP) production, cell death via apoptosis and necrosis, proliferation rate, insulin/amylin secretion and content, reactive oxygen species (ROS) production and nitrate (NO2-) generation. Beta secretase inhibition profiling of GRT, Asp, and PPAG was assessed using fluorescence resonance energy transfer (FRET) in a purified enzyme assay, followed by kinetically assessing BACE activity in vitro. Results The cytokine cocktail induced a state of moderate inflammation after 24 hours, mainly through IL-1β. High concentrations of Asp (1000 μM) and moreso, GRT (1 mg/ml), showed a trend towards BACE inhibition compared to controls (56.20% ± 2.05, p<0.0001 and 39.80% ± 0.55, p<0.0001), but reduced mitochondrial dehydrogenase activity in inflamed INS1 cells (19.02% ± 2.98, p<0.0001; 36.88% ± 1.53, p<0.0001), while PPAG showed no measurable effect. Lower concentrations of Asp (0.1–10 μM), PPAG (0.1–10 μM), and GRT (0.0001-0.01 mg/ml) may have a protective effect on inflamed pancreatic β-cells as an increase, albeit not significant, in overall cell viability and function was observed, with a concomitant decrease in oxidative stress after 24 hours (97.66% ± 3.5, p<0.66; 95.54% ± 5.61, p<0.52; 96.80% ± 3.67, p<0.55).