title: RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.
abstract: Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Johannisallee 30, 04103, Leipzig, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Böttger
authors.forename: Katrin
authors.initials: K
authors.lastname: Arnold
authors.forename: Carlo
authors.initials: C
authors.lastname: Thiel
authors.forename: Christiane
authors.initials: C
authors.lastname: Rennert
authors.forename: Susanne
authors.initials: S
authors.lastname: Aleithe
authors.forename: Ute
authors.initials: U
authors.lastname: Hofmann
authors.forename: Sebastian
authors.initials: S
authors.lastname: Vlaic
authors.forename: Susanne
authors.initials: S
authors.lastname: Sales
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
authors.forename: Madlen
authors.initials: M
authors.lastname: Matz-Soja
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007202:Indicators and Reagents / Q000008:administration & dosage* / Q000737:chemistry / Q000633:toxicity; D008055:Lipids / Q000008:administration & dosage* / Q000737:chemistry / Q000633:toxicity; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D014162:Transfection
chemical_list: D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D034741:RNA, Small Interfering