Beta secretase regulation and inflammation in pancreatic β-cells: the potential role of Rooibos

Burger, Joleen ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology. (2018-12)

Thesis (MScMedSc)--Stellenbosch University, 2018.

Thesis

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).

Inleiding Kroniese, lae graad ontsteking is ‘n kenmerk van insulienweerstandigheid (IR) en is onderliggend aan pankreatiese beta-sel wanfunksie en die ontwikkeling van tipe 2 diabetes (T2D), 'n siekte wat gekenmerk word deur 'n vermindering in β-sel funksionele massa. Terapeutiese middels, wat β-selle direk teen stressors soos inflammasie en oksidatiewe stres beskerm, is egter beperk. Amilien, ‘n neuro-hormoon, en insulien word gesamentlik deur β-selle afgeskei; daarom word hipersekresie van insulien geassosieer met verhoogde sekresie van amilien. Die toename in amilien sekresie word geassosieer met die ophoping van amiloïed, wat ‘n nadelige effek op β -sel funksie het. In die pankreaseilande moduleer beta-sekretase (BACE) die sitotoksiese ophoping van amiloïed, wat ontsteking en oksidatiewe stres aanstig; en maak dus BACE-inhibisie 'n terapeutiese teiken. Insig oor die onderliggende meganismes betrokke by pankreaseiland-inflammasie asook die geassosieerde effekte van BACE, kan moontlik 'n geleentheid onthul vir die ontwikkeling van nuwe terapieë om β-selle te beskerm en te bewaar tydens T2D. Benewens hul antidiabetiese eienskappe kan aspalatien (Asp), Z-2-(β-D-glukopiranoksieloksie)-3-fenielpropeniensuur (PPAG) en 'n ongefermenteerde Rooibosekstrak (GRT), anti-inflammatoriese effekte in β-selle hê, en moontlik β-selle beskerm deur BACE-aktiwiteit te moduleer. Doelstelling Om te bepaal of GRT, en twee van sy bioaktiewe polifenole, ontsteking in pankreatiese β-selle (INS1) kan verminder en ook BACE inhibisie eienskappe besit. Metodes 'n Model van matige ontsteking is in INS1-selle gevestig met behulp van 'n sitokien-mengsel van tumornekrosefaktor-α (TNF-α), interleukien-1β (IL-1β) en interferon-γ (IFN-γ). Sel-lewensvatbaarheid, funksie, en oksidatiewe stres was bepaal in reaksie tot die sitokien mengsel na Asp, PPAG, en GRT behandeling, deur intrasellulêre adenosietrifosfaat (ATP), sel-dood via apoptose en nekrose, selproliferasie tempo, insulien/amiliensekresie en -inhoud, reaktiewe suurstofspesies (ROS) produksie en nitraat (NO2-) generasie, te meet. Beta sekretase inhibisie deur GRT, Asp, en PPAG was bepaal met behulp van fluoressensie resonansie energie-oordrag (FRET) in 'n gesuiwerde ensiem-toets, gevolg deur kinetiese assessering van BACE aktiviteite in vitro. Resultate Die sitokien-mengsel het na 24 uur 'n toestand van matige ontsteking veroorsaak, hoofsaaklik deur IL-1β. 'n Neiging tot BACE inhibisie, vergeleke met die kontrole (56.20% ± 2.05, p<0.0001 en 39,80% ± 0.55, p<0.0001), is teen die hoër konsentrasies van Asp en GRT gesien. Hoë konsentrasies Asp (1000 μM) en tot 'n groter mate, GRT (1 mg/ml) het lewensvatbaarheid van ontsteekte INS1 selle verminder (19.02% ± 2.98, p<0,0001; 36,88% ± 1,53, p<0,0001), terwyl PPAG geen meetbare effek getoon het nie. Laer konsentrasies van Asp (0.1–10 μM), PPAG (0.1–10 μM), en GRT (0.0001-0.01 mg/ml) het sel-lewensvatbaarheid en funksie verbeter met gepaardgaande verlaging in oksidatiewe stress na 24 uur (97.66% ± 3.5, p<0.66; 95.54% ± 5.61, p<0.52; 96.80% ± 3.67, p<0.55). Gevolgtrekking Aspalathin, en veral GRT, het teen laer konsentrasies die β-sel in ‘n mate teen sitokien-geïnduseerde ontsteking beskerm, terwyl hoër konsentrasies, op 'n dosis-afhanklike manier, BACE-aktiwiteit matig moduleer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/104928
This item appears in the following collections: