Administration of chitosan-tripolyphosphate-DNA nanoparticles overexpressing key enzymes to improve omega-3 long-chain polyunsaturated fatty acid synthesis in gilthead sea bream (Sparus aurata)
[eng] Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) known to prevent atherosclerosis, stroke, obesity, type-2 diabetes, inflammation and autoimmune disease, among others. Few organisms, such as Caenorhabd...
| Autor: | |
|---|---|
| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Recursos: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/199462 |
| Acesso em linha: | https://hdl.handle.net/2445/199462 http://hdl.handle.net/10803/688476 |
| Access Level: | acceso abierto |
| Palavra-chave: | Teràpia genètica Nanopartícules Àcids grassos insaturats Orada Quitosan Gene therapy Nanoparticles Unsaturated fatty acids Sparus aurata Chitosan |
| Resumo: | [eng] Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) known to prevent atherosclerosis, stroke, obesity, type-2 diabetes, inflammation and autoimmune disease, among others. Few organisms, such as Caenorhabditis elegans and some invertebrates, can synthesize the n-3 fatty acid series in significant amounts, and marine fish and shellfish, which acquire pre-formed LC-PUFA by trophic transfer, are the major sources of n-3 LC-PUFA in the human diet. However, substitution of fish oil by vegetable oils in aquafeeds reduces healthy n-3 LC-PUFA in cultured fish, while increases proinflammatory n-6 fatty acids. The present study aimed to empower Sparus aurata to boost endogenous synthesis of n-3 LC-PUFA by transient expression of fish codon-optimized Caenorhabditis elegans Δ12/n-6 (FAT-2) and Δ15/n-3 (FAT-1) fatty acid desaturases using chitosan-tripolyphosphate (TPP) nanoparticles as DNA delivery system. Growth performance, body composition, serum metabolites, fatty acid profile and expression of key enzymes in intermediary metabolism were evaluated in Sparus aurata juveniles 72 hours after a single intraperitoneal injection of chitosan-TPP nanoparticles encapsulated with expression plasmids encoding fish codon-optimized C. elegans FAT-1 and FAT-2 (short-term effect) and in 70-day treated fish that were periodically administered with chitosan-TPP-DNA nanoparticles (long- term sustained effect). For the short-term study, increased levels of FAT-1 and FAT-2 mRNAs (> 10-fold) were detected 72 hours post-injection in the S. aurata liver. Expression of FAT-1 elevated hepatic EPA and total n-3 PUFA. In addition to reduced serum triglycerides, co-expression of FAT-1 and FAT-2 also induced proportions of DHA and PUFA in the S. aurata liver. Compared to control fish, treatment with FAT-1, FAT-2 and FAT-1 + FAT-2 downregulated expression of hepatic key genes in glycolysis and lipogenesis, including g6pd, pfk1, pk, pfkfb1, acaca, acacb, fasn, scd1a and fads2. In addition, co-expression of FAT-1 and FAT-2 significantly upregulated the activity of rate-limiting enzymes in glycolysis and the pentose phosphate pathway. Fish expressing FAT-2 and FAT-1 + FAT-2 showed lower hepatic expression of pparg and srebf1, and higher of hnf4a. Treatment with FAT-1 and FAT-2 alone downregulated srebf1 and upregulated ppara, respectively, in the S. aurata liver. To study long-term sustained expression of FAT-1, FAT-2 and FAT-1 + FAT-2, chitosan-TPP-DNA nanoparticles were provided to fish every 4 weeks (3 doses in total). After 70 days of treatment, tissue distribution analysis showed high expression levels for FAT-1 and FAT-2 in the liver (>200-fold) followed by the intestine (10 to 25-fold), while no differential expression occurred in the skeletal muscle and brain. Expression of FAT-1 and FAT-1 + FAT-2 increased weight gain. Fatty acid methyl esters assay revealed that co-expression of FAT-1 and FAT-2 increased liver production and muscle accumulation of EPA, DHA and total n-3 LC-PUFA, while decreased the n-6/n-3 ratio. Co-expression of FAT-1 and FAT-2 downregulated srebf1 and genes encoding rate-limiting enzymes for de novo lipogenesis in the liver, leading to decreased circulating triglycerides and cholesterol. In contrast, FAT-2 and FAT-1 + FAT-2 upregulated hepatic hnf4a, nr1h3 and key enzymes in glycolysis and the pentose phosphate pathway. Our findings demonstrate that administration of chitosan-TPP-DNA nanoparticles, a methodology that circumvents obtention of genetically modified organisms, is a proper gene delivery method for sustained expression of exogenous enzymes in the liver of Sparus aurata. Specifically, co-expression of FAT-1 and FAT-2 enabled the production of functional fish for human consumption, rich in n-3 LC-PUFA, notably EPA and DHA, and with decreased n-6/n-3 fatty acids ratio. In addition, co- expression of FAT-1 and FAT-2 increased weight gain and the specific growth rate in Sparus aurata. |
|---|