Serem, June Cheptoo2024-12-122024-12-122025-042024-10-22*A2025http://hdl.handle.net/2263/99903Dissertation (MSc (Anatomy))--University of Pretoria, 2024.Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder with an increasing prevalence globally. Consequently, there is an increase in individuals suffering from hyperglycaemia, which contributes to oxidative stress and inflammatory responses, causing kidney, liver, and heart diseases. There is increasingly a need for new drugs that are multitargeting, having beneficial effects on several dysfunctional features of T2DM. Peptides have been identified as attractive molecules for developing new therapies that target multiple pathways and processes associated with T2DM, including oxidative stress, inflammation, and inhibiting key enzymes. Recent studies have identified four novel peptides, YPG, a synthetic peptide, SQSPA, derived from silkworm pupae, STYV extracted from the fungus Aspergillus awamori, and STY, a digest of STYV that inhibits key diabetic enzyme drug targets and has antioxidant and anti-inflammation properties. This study aimed to further investigate the potential antidiabetic properties of known antidiabetic drugs (acarbose, diprotin A and metformin) and these peptides in relevant cellular models. This aim was achieved using HepG2 hepatocarcinoma cells, differentiated 3T3-L1 adipocytes, and the murine macrophage cell line, RAW 264.7. Firstly, the cytotoxicity of known antidiabetic drugs and the peptides was investigated using the Crystal Violet (CV) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cellular morphology following exposure was evaluated with light microscopy of CV-stained cells and polarisation optical differential interference contrast (PlasDIC) microscopy. Further antioxidant analysis was conducted with the oxygen radical absorbance capacity (ORAC) assay and the cellular model the dichlorofluorescein diacetate (DCFH-DA) assay in HepG2 cells. Anti-inflammatory effects were evaluated in the LPS/RAW 246.7 model and the effects on nitric oxide (NO) and tumour necrosis factor- (TNF-) levels were determined with the Griess assay and a sandwich enzyme-linked immunosorbent assay (ELISA) respectively. In addition, the effect on lipid accumulation and morphology in 3T3-L1 adipocytes and HepG2 cells was investigated with Oil Red O (ORO) assay and staining. Lastly, the effects on glucose uptake were evaluated in the 3T3-L1 adipocytes and HepG2 cells with the 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) fluorescence assay. A 48-hour exposure to the antidiabetic drugs and peptides did not cause a significant decrease in the percentage cell number and viability of the HepG2, differentiated 3T3-L1 adipocytes and the RAW 264.7 macrophages even at the highest concentration, of 100 µM. The ORAC assay revealed that the antidiabetic drugs and SQSPA lacked antioxidant activity, while in contrast YPG, STY, and STYV effectively scavenged peroxyl radicals. The antidiabetic drugs, acarbose and metformin as well as the peptide except STY, presented with cellular antioxidant activity. The antidiabetic drugs scavenged cellular NO and reduced TNF- levels, while the peptides lacked NO scavenging activity and did not reduce TNF- levels. The antidiabetic drugs reduce the % lipid accumulation in both the 3T3-L1 adipocytes and the HepG2 hepatocytes. Exposure to the antidiabetic drugs and peptides to cells with accumulated lipids had variable effects. In the HepG2 cells, the antidiabetic drugs had no effect while diprotin A significantly reduced lipid accumulation in the 3T3-L1 adipocytes. For the peptides, 100 µM YPG reduced lipid accumulation in HepG2 cells and 50 μM of STY, and 50 μM and 100 µM STYV significantly reduced lipid accumulation in differentiating adipocytes. Neither insulin, metformin nor the peptides increased glucose uptake in HepG2 and 3T3-L1 cells. Findings of this study found that of the peptides, STYV and SQSPA are the most promising with potential multitargeting therapeutic potential that includes antioxidant, anti-inflammatory, and anti-lipid accumulation. These can be further evaluated in future studies to better evaluate the effect of these peptides on the advanced glycation end product (AGE), protein kinase C, polyol, and hexosamine biosynthesis pathways.en© 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDSustainable development goals (SDGs)Type 2 diabetes mellitus (T2DM)PeptideAntioxidantAnti-inflammationDissertationu18008268Type 2 diabetes mellitus, T2DM, peptides, cytotoxicity, antioxidant, anti-inflammation, lipid accumulation, glucose uptake, HepG2, 3T3-L1, RAW 264.7