Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures

  • Zhao, Lulu (Institute of Feed Research, Chinese Academy of Agricultural Sciences) ;
  • Cai, Hongying (Institute of Feed Research, Chinese Academy of Agricultural Sciences) ;
  • Wu, Yongbao (Institute of Feed Research, Chinese Academy of Agricultural Sciences) ;
  • Tian, Changfu (State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University) ;
  • Wen, Zhiguo (Institute of Feed Research, Chinese Academy of Agricultural Sciences) ;
  • Yang, Peilong (Institute of Feed Research, Chinese Academy of Agricultural Sciences)
  • Received : 2022.02.03
  • Accepted : 2022.04.18
  • Published : 2022.11.01
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Abstract

Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

Keywords

Acknowledgement

Conceptualization, Lulu Zhao, Zhiguo Wen, and Peilong Yang; Data curation, Lulu Zhao and Changfu Tian; Funding acquisition, Zhiguo Wen and Peilong Yang; Investigation, Lulu Zhao; Methodology, Lulu Zhao, Hongying Cai and Yongbao Wu; Project administration, Zhiguo Wen and Peilong Yang; Resources, Hongying Cai, Yongbao Wu and Changfu Tian; Writing - original draft, Lulu Zhao; Writing - review & editing, Zhiguo Wen and Peilong Yang. We acknowledge the editors and anonymous reviewers for insightful suggestions on this work.

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