title: Regulation of lipoprotein lipase by protein kinase C alpha in 3T3-F442A adipocytes.
abstract: Lipoprotein lipase (LPL) is an important enzyme in adipocyte and lipid metabolism with complex cellular regulation. Previous studies demonstrated an inhibition of LPL activity and synthesis following depletion of protein kinase C (PKC) isoforms with long term treatment of 3T3-F442A adipocytes with 12-O-tetradecanoylphorbol-13-acetate. To identify the specific PKC isoforms involved, we treated cells with antisense oligonucleotides that block expression of specific PKC isoforms. An antisense oligonucleotide to PKC alpha inhibited LPL activity by 78 +/- 8%, whereas antisense oligonucleotides directed against PKC delta or PKC epsilon had no effect on LPL activity. The change in LPL activity was maximal at 72 h and was accompanied by a decrease in LPL protein and LPL synthetic rate but no change in LPL mRNA, suggesting regulation at the level of translation. However, PKC depletion resulted in no change in the polysome profile, indicating that translation initiation was not affected. However, the addition of cytoplasmic extracts from adipocytes treated with 12-O-tetradecanoylphorbol-13-acetate or PKC alpha antisense oligomers inhibited LPL translation in vitro. This inhibition of LPL translation in vitro was lost when the LPL mRNA transcript did not contain nucleotides 1599-3200, thus implicating the 3'-untranslated region of LPL in the regulation of translation by PKC depletion. Both LPL activity and Raf1 activity were decreased in parallel following depletion of either total PKC or specific inhibition of PKC alpha. An antisense oligonucleotide to RAF1, which inhibited RAF1 activity, also inhibited LPL activity by 48 +/- 10%, and this decrease in LPL activity was not accompanied by a change in LPL mRNA. Cells were treated with U0126, a specific inhibitor of the ERK-activating kinases MEK1 and MEK2. Although U0126 inhibited ERK1 and ERK2 phosphorylation, U0126 had no effect on LPL activity, indicating that MEK/ERK pathways were not involved in this mechanism of LPL regulation. Together, these data indicate that PKC alpha and RAF1 are important in the translational regulation of LPL in adipocytes and that the mechanism of regulation is probably through an ERK-independent pathway.
authors.affiliation: Central Arkansas Veterans HealthCare System and Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. Rangsnsthangovx1@UAMS.edu
authors.forename: Gouri
authors.initials: G
authors.lastname: Ranganathan
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.forename: Nicholas
authors.initials: N
authors.lastname: Dean
authors.forename: Brett
authors.initials: B
authors.lastname: Monia
authors.forename: Steven W
authors.initials: SW
authors.lastname: Barger
authors.forename: Philip A
authors.initials: PA
authors.lastname: Kern
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D011132:Polyribosomes / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009570:Nitriles; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C113580:U 0126; D019908:Proto-Oncogene Proteins c-raf; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020928:Mitogen-Activated Protein Kinases; D008071:Lipoprotein Lipase; D013755:Tetradecanoylphorbol Acetate