title: Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6 Myotubes Incubated With Palmitate.
abstract: The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160-/PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160-). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160-vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160-/PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373-2386, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.
authors.affiliation: Department of Physiology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Mikłosz
authors.affiliation: Department of Physiology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Bartłomiej
authors.initials: B
authors.lastname: Łukaszuk
authors.affiliation: Department of Hygiene, Epidemiology and Ergonomics, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Żendzian-Piotrowska
authors.affiliation: Department of Biology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Brańska-Januszewska
authors.affiliation: Department of Biology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Halina
authors.initials: H
authors.lastname: Ostrowska
authors.affiliation: Department of Physiology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Chabowski
mesh_terms: D000818:Animals; D001692:Biological Transport; D018955:CD36 Antigens / Q000378:metabolism; D002460:Cell Line; D050612:Fatty Acid Transport Proteins / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D007333:Insulin Resistance; D050356:Lipid Metabolism / Q000187:drug effects*; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: D018955:CD36 Antigens; C008132:Cd36 protein, rat; D050612:Fatty Acid Transport Proteins; D050556:Fatty Acid-Binding Proteins; D020690:GTPase-Activating Proteins; C515999:Got2 protein, rat; D012333:RNA, Messenger; C493570:Slc27a1 protein, rat; C542875:Slc27a4 protein, rat; C533101:TBC1D4 protein, rat; D019308:Palmitic Acid; D051057:Proto-Oncogene Proteins c-akt