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Abstract

Background and Aim: The increased formation of methylglyoxal (MG) under hyperglycaemia is associated with the development of microvascular complications in patients with diabetes. However, in zebrafish, a permanent knock out of glyoxalase 1(Glo1), the central MG detoxifying system, only led to a two-fold elevation of endogenous MG levels. Importantly, a two-fold increase in aldehyde dehydrogenases (ALDH) activity, a group of enzymes which catalyse the oxidation of aldehydes, was observed. Besides, RT-qPCR based results identified elevated aldh3a1 mRNA level in glo1-/- mutants. Thus, this dissertation aims to generate Aldh3a1 knockout zebrafish and analyse its function in glucose homeostasis and diabetes.

Materials and Methods: aldh3a1-/- zebrafish were generated by using CRISPR/Cas9. Vasculature, pancreatic and β-Cell mass area size were analysed in Tg(fli1:EGFP), Tg(hb9:GFP) and Tg(ins:nfsB-mCherry)zebrafish larvae. mRNA expression was examined by RT-qPCR and RNA-seq analysis. 4-Hydroxynonenal (4-HNE) amount was measured by ELISA (BioVision) and whole-body glucose was measured by Glucose Assay Kit (Merck).

Results: Aldh3a1 knockout zebrafish were successfully generated and validated by significantly decreased ALDH activity. In Tg(fli1:EGFP) zebrafish larvae, loss of Aldh3a1 increased abnormal intersegmental vessels formation in the trunk and widened branch diameters in retina hyaloid vasculature, which can be further enhanced via pancreatic and duodenal homeobox 1 (pdx1) expression silencing. A combination of Aldh3a1 knockout and knockdown strategies identified a decreased pancreatic size in Tg(hb9:GFP) and reduced β-Cell mass dimension in Tg(ins:nfsB-mCherry) zebrafish larvae. Also, mRNA expression of pdx1 and insulin were decreased and RNA-seq data have further confirmed disruption of the endocrine pancreas development in the alda3a1 mutants. Consequently, aldh3a1 –/– larvae exhibited hyperglycaemia by 40% whole-body glucose elevation. Moreover, disruption of the pancreas in alda3a1mutants is driven by an increased 4-HNE amount and external 4-HNE in wild type zebrafish larvae mimics the phenotype in aldh3a1 mutants.

Conclusion: Overall, this dissertation provided patent evidence for the contribution of deficient 4-HNE detoxification and subsequent increased 4-HNE concentration to the development of hyperglycaemia via pancreas dysfunction in aldh3a1 mutants, as a novel direction for future research regarding diabetic pathophysiology and therapy.