Regulation of lipoprotein lipase by protein kinase Cα in 3T3-F442A adipocytes

Gouri Ranganathan, Wei Song, Nicholas Dean, Brett Monia, Steven W. Barger, Philip A. Kern

Research output: Contribution to journalArticle

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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 PKCα inhibited LPL activity by 78 ± 8%, whereas antisense oligonucleotides directed against PKCδ or PKCε 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 PKCα 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 PKCα. 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 PKCα 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.

Original languageEnglish
Pages (from-to)38669-38675
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number41
DOIs
StatePublished - Oct 11 2002
Externally publishedYes

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Lipoprotein Lipase
Adipocytes
Protein Kinase C
Antisense Oligonucleotides
Protein Isoforms
MAP Kinase Signaling System
Tetradecanoylphorbol Acetate
Messenger RNA
Acetate Kinase
Phosphorylation
Polyribosomes
Mitogen-Activated Protein Kinase Kinases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ranganathan, G., Song, W., Dean, N., Monia, B., Barger, S. W., & Kern, P. A. (2002). Regulation of lipoprotein lipase by protein kinase Cα in 3T3-F442A adipocytes. Journal of Biological Chemistry, 277(41), 38669-38675. https://doi.org/10.1074/jbc.M206917200

Regulation of lipoprotein lipase by protein kinase Cα in 3T3-F442A adipocytes. / Ranganathan, Gouri; Song, Wei; Dean, Nicholas; Monia, Brett; Barger, Steven W.; Kern, Philip A.

In: Journal of Biological Chemistry, Vol. 277, No. 41, 11.10.2002, p. 38669-38675.

Research output: Contribution to journalArticle

Ranganathan, G, Song, W, Dean, N, Monia, B, Barger, SW & Kern, PA 2002, 'Regulation of lipoprotein lipase by protein kinase Cα in 3T3-F442A adipocytes', Journal of Biological Chemistry, vol. 277, no. 41, pp. 38669-38675. https://doi.org/10.1074/jbc.M206917200
Ranganathan, Gouri ; Song, Wei ; Dean, Nicholas ; Monia, Brett ; Barger, Steven W. ; Kern, Philip A. / Regulation of lipoprotein lipase by protein kinase Cα in 3T3-F442A adipocytes. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 41. pp. 38669-38675.
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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 PKCα inhibited LPL activity by 78 ± 8{\%}, whereas antisense oligonucleotides directed against PKCδ or PKCε 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 PKCα 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 PKCα. 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 PKCα 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.",
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