Impaired Detoxification of Trans, Trans‐2,4‐Decadienal, an Oxidation Product from Omega‐6 Fatty Acids, Alters Insulin Signaling, Gluconeogenesis and Promotes Microvascular Disease

Abstract Omega‐6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega‐6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans‐2,4‐decadienal (tt‐DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4‐hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt‐DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt‐DDE. Loss of Aldh9a1b increased tt‐DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b−/− zebrafish. Transcriptomic and metabolomic analyses revealed that tt‐DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt‐DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt‐DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance.

b2m.Each datapoint in this figure represented 20 larvae.The bars indicate mean±SD values.Statistical analysis was performed by Student's t-test, one-way ANOVA and chi-square test, ns, not significant; *p < 0.05.

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Figure.S7 Morphological and hyaloid vascular analysis in aldh9a1b +/+ larvae treated with BAL (A) Representative unaltered microscopic images of zebrafish larvae between 2dpf and 5dpf with 0-400µmol BAL treatment.White bar,

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Figure.S8 RNA-seq analysis of aldh9a1b larvae at 5dpf (A) PCA figure showed different groups of RNA-seq.(B)KEGG enrichment analysis of differential genes showed top altered pathways in aldh9a1b -/-compared to aldh9a1b +/+ .(C) Emapplot of KEGG enrichment analysis showed the interaction of altered pathways in aldh9a1b -/-mutants.(D) KEGG enrichment analysis of differential genes showed top altered pathways in tt-DDE treated aldh9a1b +/+ larvae compared to aldh9a1b +/+ larvae.(E) Emapplot of KEGG enrichment analysis showed the interaction of altered pathways in the tt-DDE treated group.

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Figure.S10 Altered glucose metabolism in aldh9a1b -/-and tt-DDE treated zebrafish (A-C) PLS-DA scores plot showed different groups in the larvae (A), livers (B) and muscles (C) of adult zebrafish.(D-G) Fold change analysis displayed differential metabolites induced by aldh9a1b -/-or tt-DDE by comparison to aldh9ab +/+ .

Figure. S11
Figure.S11 Morphological and hyaloid vascular rescue in aldh9a1b +/+ larvae co-treated with tt-DDE and metformin (A) Representative microscopic images of zebrafish larvae between 2dpf and 5dpf co-treated with 8µmol tt-DDE and 0-50µmol MF. White bar, 200µm.(B) Quantification of survival rates showed no significant change between these groups.(C) Quantification of morphology change showed normal morphology between different groups.(D) Quantification of heat edema showed normal heart development across all the groups.(E) Representative confocal images of hyaloid vasculature showed beneficial effects in zebrafish larvae co-treated with 8 µmol tt-DDE and 0-50µmol MF at 5dpf.White scale bar=50μm.(F) Quantification of hyaloid branchpoints and sprouts formation showed increased angiogenic vasculature caused by tt-DDE treatment, which can be rescued by 1-50µmol MF.One datapoint means one hyaloid per larva.The bars indicate mean ± SEM values.Statistical analysis was performed by one-way ANOVA, two-way ANOVA and logrank test.ns = not significant, *p < 0.05.MF, metformin.

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Figure.S12 Morphological and hyaloid vascular rescue in aldh9a1b +/+ larvae co-treated with tt-DDE and rosiglitazone (A) Representative microscopic images of zebrafish larvae between 2dpf and 5dpf co-treated with 8µmol tt-DDE and 0-10µmol RG. White bar, 200µm.(B) Quantification of survival rates showed no significant change between these groups.(C) Quantification of morphology change showed no significant alterations across all the groups.(D) Quantification of heat edema showed normal heart morphology between different groups.(E) Representative confocal images of hyaloid vasculature showed rescue of vascular alterations in zebrafish larvae co-treated with 8µmol tt-DDE and 0-10µmol RG at 5dpf.White scale bar=50μm.(F) Quantification of hyaloid branchpoints and sprouts formation showed increased angiogenic vasculature caused by tt-DDE, which can be rescued by 1-10µmol rosiglitazone.One datapoint