Impact of diet on gene dysfunction in Type 2 diabetes

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Billacura, M.P., 2022. Impact of diet on gene dysfunction in Type 2 diabetes. PhD, Nottingham Trent University.

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Abstract

Type 2 diabetes (T2D) is an autoinflammatory disease and metabolic disorder characterised by high blood glucose levels, failure/reduction of insulin secretion by the pancreas, and insulin resistance. The International Diabetes Foundation reported in 2019 that 9.3% (463 million) of the adult population were living with diabetes, half of which were undiagnosed. T2D is also strongly linked to obesity, which is the single most highly modifiable risk factor for T2D.

Isolated CD-1 mice islets, INS-1 rat pancreatic β-cells and skeletal muscle C2C12 mouse myotubes were hereby used as cellular models for T2D. The effects of glucolipotoxic (GLT) metabolic stress were investigated by incubating these cells in medium supplemented with chronically elevated glucose and high free fatty acid concentrations that mimic the extracellular environment typically found in patients with poorly controlled T2D. In addition, molecules potentially able to counter these effects were investigated by incubating cells in either: control medium ± 10 mM L-carnosine (or β-alanine); control medium ± 100 μM carnosine analogues; GLT medium ± 10 mM L-carnosine (or β-alanine); GLT medium ± 100 μM carnosine analogues.

The effects of glucolipotoxicity were assessed through transcriptomic analysis utilising microarray data and Illumina-HiSeq gene expression data in combination with non-biased ranked pathway analysis using MetacoreTM technology. The results generated from these methods were validated using RT-qPCR and Western blotting. Functional assays were performed to determine cell viability, reactive species scavenging, 3-nitrotyrosine (3-NT) level, 4-hydroxynonenal (4-HNE) level and Seahorse Mito Stress assay utilised to determine the impact of GLT and potential therapeutic agents on mitochondrial function.

Using the above methodologies, hepatocyte nuclear factor 4 alpha (HNF4α) was shown to be a transcription factor that is a central regulator of protein trafficking through control of expression of multiple Rab genes and syntaxin 17. Furthermore, HNF4α was shown to be sensitive to GLT metabolic stress, and targeted HNF4α knocked down inhibited insulin secretion. HNF1α expression was reduced when HNF4α was downregulated, suggesting an association between the two transcription factors. Cell adhesion pathways were also shown to be strongly downregulated by GLT, and targeted knocked down of the extracellular matrix genes encoding matrix metallopeptidase 14 (MMP14), CD44 molecule (CD44) and Erb-B2 receptor tyrosine kinase 4 (ErbB4) expression inhibited insulin secretion.

Carnosine is a physiological histidine-containing dipeptide that has recently been shown to help reduce glycated haemoglobin 1ac (HbA1c) levels in patients with T2D, although the mechanism of action associated with this reduction is poorly understood. Data presented herein show that carnosine, its analogues, and the constituent amino acid, β-alanine, effectively scavenge radical species generated by GLT. GLT was also shown to decrease maximal respiration and reduce mitochondria's ability to generate ATP. However, this decrease was effectively reversed by the supplementation of carnosine and β-alanine to GLT media. Carnosine and carnosine mimetics also consequently increased insulin secretion that was hitherto inhibited by GLT. Importantly, carnosine analogues, M8 and E3, significantly reduced the formation of 3-NT and 4-HNE adducts in both in vitro and in vivo experiments and were shown to counter obesity and T2D in a 10-week animal study. This therefore indicates a potential role for these molecules as novel therapeutic agents in the treatment and/or prevention of metabolic diseases.

Item Type:	Thesis
Creators:	Billacura, M.P.
Date:	June 2022
Rights:	This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner(s) of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	30 Nov 2022 12:16
Last Modified:	30 Nov 2022 12:16
URI:	https://irep.ntu.ac.uk/id/eprint/47558