Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures

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 <i>in vitro</i> 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 <i>alb</i> and <i>afp</i> 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.

Embryonic growth and effect of embryonic age on quantitative and functional characteristics of duck primary hepatocytes

“…Due to the small number of hepatocytes isolated from E20 and E22 and the limited quantity of RNA extracted after 24-h culture, both EA could not perform further operations. Therefore, the qPCR results mainly focused on E6 to E18, which still supported the similar trend of increasing Alb expression in isolated hepatocytes as observed in Zhao's study ( Zhao et al, 2022 ). Alb was also determined by immunofluorescence method.…”

“…Hepatocytes account for over 80% of liver cells ( Li et al, 2022 ), and have been widely utilized for studying liver functions ( Vilei et al, 2001 ; Funaki et al, 2002 ; Zhang et al, 2018 ), including nutrient metabolism and disease-resistance mechanisms ( Mason et al, 2008 ; Ye et al, 2009 ; Zhai et al, 2019 ). The collagenase perfusion method is the classic approach for HI, which commonly employed for obtaining adult mammalian hepatocytes due to their abundance and high purity in cells, but less frequently used in poultry ( Figueiredo et al, 2021 ; Zhao et al, 2022 ). Considering the size of the liver and blood vessels, this method is not applicable for HI in duck embryos.…”

“…The hepatocyte suspension was determined using an automatic cell counter (Nexcelom, Mini - 004 - 0200, Boston, MA), and cell viability was assessed using the trypan blue rejection method ( Zhao et al, 2022 ). To determine viability, the cell suspension was mixed with trypan blue (Solarbio, Beijing, China) in a ratio of 9 to 1 and stained for 3 min.…”

“…Previous studies have primarily focused on livestock and poultry liver to investigate feed nutrition and disease models, resulting in a large number of animals being slaughtered or dying. However, primary hepatocytes ( PH ) could be utilized to study metabolic and disease-resistance mechanisms, potentially reducing costs associated with animal research ( Sardi et al, 2006 ; Dhawan et al, 2010 ; Awde et al, 2013 ; Soldatow et al, 2013 ; Zhao et al, 2015 ; Kim et al, 2017 ; Zhao et al, 2022 ).…”

“…However, this method has drawbacks, such as high operational difficulty and special equipment requirements ( Li et al, 2010 ). Another commonly used method is enzymatic digestion, which includes trypsin digestion and collagenase digestion ( Zhao et al, 2022 ). The collagenase digestion method is relatively mild and easier to control, resulting in a higher yield of cells in good condition ( Seglen, 1973b ).…”