Alain Théophile Sané1, Ernest Seidman1, Noel Peretti1, Marie Laure Kleme1, Edgard Delvin1, Colette Deslandres1, Carole Garofalo1, Schohraya Spahis1, Emile Levy2. 1. From the CHU Sainte-Justine Research Centre (A.T.S., M.L.K., E.D., C.D., C.G., S.S., E.L.), Department of Nutrition (M.L.K., S.S., E.L.), and Department of Pediatrics (C.D.), Université de Montréal, Quebec, Canada; Division of Gastroenterology, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada (E.S.); and Centre de recherche Rhône-Alpes en nutrition humaine, Hôpital Edouard-Herriot, Université de Lyon 1, France (N.P.). 2. From the CHU Sainte-Justine Research Centre (A.T.S., M.L.K., E.D., C.D., C.G., S.S., E.L.), Department of Nutrition (M.L.K., S.S., E.L.), and Department of Pediatrics (C.D.), Université de Montréal, Quebec, Canada; Division of Gastroenterology, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada (E.S.); and Centre de recherche Rhône-Alpes en nutrition humaine, Hôpital Edouard-Herriot, Université de Lyon 1, France (N.P.). emile.levy@recherche-ste-Justine.qc.ca.
Abstract
BACKGROUND: Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog. OBJECTIVE: The central aim of this work is to examine the cause-effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis. APPROACH AND RESULTS: The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1). CONCLUSIONS: These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout.
BACKGROUND: Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog. OBJECTIVE: The central aim of this work is to examine the cause-effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis. APPROACH AND RESULTS: The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1). CONCLUSIONS: These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout.
Authors: Hong S Lu; Ann Marie Schmidt; Robert A Hegele; Nigel Mackman; Daniel J Rader; Christian Weber; Alan Daugherty Journal: Arterioscler Thromb Vasc Biol Date: 2018-10 Impact factor: 8.311
Authors: Aldi T Kraja; Chunyu Liu; Jessica L Fetterman; Mariaelisa Graff; Christian Theil Have; Charles Gu; Lisa R Yanek; Mary F Feitosa; Dan E Arking; Daniel I Chasman; Kristin Young; Symen Ligthart; W David Hill; Stefan Weiss; Jian'an Luan; Franco Giulianini; Ruifang Li-Gao; Fernando P Hartwig; Shiow J Lin; Lihua Wang; Tom G Richardson; Jie Yao; Eliana P Fernandez; Mohsen Ghanbari; Mary K Wojczynski; Wen-Jane Lee; Maria Argos; Sebastian M Armasu; Ruteja A Barve; Kathleen A Ryan; Ping An; Thomas J Baranski; Suzette J Bielinski; Donald W Bowden; Ulrich Broeckel; Kaare Christensen; Audrey Y Chu; Janie Corley; Simon R Cox; Andre G Uitterlinden; Fernando Rivadeneira; Cheryl D Cropp; E Warwick Daw; Diana van Heemst; Lisa de Las Fuentes; He Gao; Ioanna Tzoulaki; Tarunveer S Ahluwalia; Renée de Mutsert; Leslie S Emery; A Mesut Erzurumluoglu; James A Perry; Mao Fu; Nita G Forouhi; Zhenglong Gu; Yang Hai; Sarah E Harris; Gibran Hemani; Steven C Hunt; Marguerite R Irvin; Anna E Jonsson; Anne E Justice; Nicola D Kerrison; Nicholas B Larson; Keng-Hung Lin; Latisha D Love-Gregory; Rasika A Mathias; Joseph H Lee; Matthias Nauck; Raymond Noordam; Ken K Ong; James Pankow; Amit Patki; Alison Pattie; Astrid Petersmann; Qibin Qi; Rasmus Ribel-Madsen; Rebecca Rohde; Kevin Sandow; Theresia M Schnurr; Tamar Sofer; John M Starr; Adele M Taylor; Alexander Teumer; Nicholas J Timpson; Hugoline G de Haan; Yujie Wang; Peter E Weeke; Christine Williams; Hongsheng Wu; Wei Yang; Donglin Zeng; Daniel R Witte; Bruce S Weir; Nicholas J Wareham; Henrik Vestergaard; Stephen T Turner; Christian Torp-Pedersen; Evie Stergiakouli; Wayne Huey-Herng Sheu; Frits R Rosendaal; M Arfan Ikram; Oscar H Franco; Paul M Ridker; Thomas T Perls; Oluf Pedersen; Ellen A Nohr; Anne B Newman; Allan Linneberg; Claudia Langenberg; Tuomas O Kilpeläinen; Sharon L R Kardia; Marit E Jørgensen; Torben Jørgensen; Thorkild I A Sørensen; Georg Homuth; Torben Hansen; Mark O Goodarzi; Ian J Deary; Cramer Christensen; Yii-Der Ida Chen; Aravinda Chakravarti; Ivan Brandslund; Klaus Bonnelykke; Kent D Taylor; James G Wilson; Santiago Rodriguez; Gail Davies; Bernardo L Horta; Bharat Thyagarajan; D C Rao; Niels Grarup; Victor G Davila-Roman; Gavin Hudson; Xiuqing Guo; Donna K Arnett; Caroline Hayward; Dhananjay Vaidya; Dennis O Mook-Kanamori; Hemant K Tiwari; Daniel Levy; Ruth J F Loos; Abbas Dehghan; Paul Elliott; Afshan N Malik; Robert A Scott; Diane M Becker; Mariza de Andrade; Michael A Province; James B Meigs; Jerome I Rotter; Kari E North Journal: Am J Hum Genet Date: 2018-12-27 Impact factor: 11.025