Inhibited insulin signaling in mouse hepatocytes is associated with increased phosphatidic acid but not diacylglycerol - PubMed (original) (raw)

Inhibited insulin signaling in mouse hepatocytes is associated with increased phosphatidic acid but not diacylglycerol

Chongben Zhang et al. J Biol Chem. 2015.

Abstract

Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes.

Keywords: Diacylglycerol Kinase; Glycerol-3-phosphate Acyltransferase; Glycerophospholipid; Insulin Resistance; Mammalian Target of Rapamycin (mTOR); Phospholipase D; Protein Kinase C (PKC).

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Overexpressing PLD1 and PLD2 impaired insulin signaling to Akt. Mouse primary hepatocytes were infected for 24 h with EGFP, Flag-GPAT1, PLD1, inactive PLD1 mutant (PLD1-m), PLD2, or inactive PLD2 mutant (PLD2-m) adenoviruses. Cells were lysed and subjected to Western blotting with appropriate antibodies (A), scraped from the dish, centrifuged to obtain total particulate preparations and assayed for PLD activity (B), or treated with or without insulin (100 n

m

) for 10 min, followed by cell lysate preparation for Western blotting (C and D). A, C, and D show representative Western blots from three independent experiments; B shows results from three independent experiments, each done in triplicate. E shows the quantitative analysis of data from D. * and #, p < 0.05 compared with EGFP basal and PMA-stimulated, respectively.

FIGURE 2.

FIGURE 2.

Overexpressing PLD1 and PLD2 increased the cellular content of total PA and di16:0 PA but did not change total or di16:0 DAG. Lipids were extracted from mouse primary hepatocytes overexpressing EGFP, PLD1, or PLD2. A, content of total PA, 16:0-containing PA, and other PA. B, content of di16:0 PA. C, content of total DAG, 16:0-containing DAG, and other DAG. D, content of di16:0 DAG. *, p < 0.05 compared with EGFP. The data are from three independent experiments, each done in triplicate.

FIGURE 3.

FIGURE 3.

Overexpressing DGKθ impaired insulin signaling to Akt in mouse hepatocytes. Mouse primary hepatocytes were infected for 24 h with GFP or DGKθ lentiviruses. Cells were lysed and subjected to Western blotting with appropriate antibodies (A), lysed and assayed for DGK activity (B), or treated with or without insulin (100 n

m

) for 10 min, followed by cell lysate preparation for Western blotting (C and D). A, C, and D show representative Western blots from three independent experiments; B shows results from three independent experiments, each done in triplicate. E shows the quantitative analysis of data from D. *, p < 0.05 compared with GFP.

FIGURE 4.

FIGURE 4.

Overexpressing DGKθ increased the cellular content of total PA and di16:0 PA and decreased total DAG and di16:0 DAG. Lipids were extracted from mouse primary hepatocytes overexpressing GFP or DGKθ, and lipid content was assayed. A, content of total PA, 16:0-containing PA, and other PA. B, content of di16:0 PA. C, content of total DAG, 16:0-containing DAG, and other DAG. D, content of di16:0 DAG. The data are from three independent experiments, each done in triplicate. *, p < 0.05 compared with GFP.

FIGURE 5.

FIGURE 5.

Overexpressed GPAT1 and GPAT4 in mouse hepatocytes, but not GPAT3, impaired insulin signaling to Akt. Mouse primary hepatocytes were infected for 24 h with EGFP, Flag-GPAT1, Flag-GPAT3, or Flag-GPAT4 adenoviruses. Cells were lysed and subjected to Western blotting with anti-Flag antibodies (A); scraped from the dish, centrifuged to obtain total particulate preparations, and assayed for total and NEM-resistant (NEM-R) GPAT activity (B); or treated with or without insulin (100 n

m

) for 10 min, followed by cell lysate preparation for Western blotting (C). A and C show representative Western blots from three independent experiments; B shows data from three independent experiments, each done in triplicate. * and #, p < 0.05 compared with EGFP total and NEM-resistant GPAT activity, respectively.

FIGURE 6.

FIGURE 6.

Overexpressing GPAT4, but not GPAT3, increased the cellular content of total PA, DAG, and di16:0 PA. Lipids were extracted from mouse primary hepatocytes overexpressing EGFP, Flag-GPAT3, or Flag-GPAT4, and lipid content was assayed. A, content of total PA, 16:0-containing PA, and other PA. B, content of di16:0 PA. C, content of total DAG, 16:0-containing DAG, and other DAG. D, content of di16:0 DAG. The data are from three independent experiments, each done in triplicate. *, p < 0.05 compared with EGFP.

FIGURE 7.

FIGURE 7.

Overexpressing PLD1, PLD2, or DGKθ diminished mTOR-rictor association and inhibited mTORC2 activity. Mouse primary hepatocytes overexpressing EGFP, PLD1, PLD2, or DGKθ were lysed and immunoprecipitated (IP) and then assayed for mTORC2 kinase activity followed by Western blotting. A, Western blots representative of three independent experiments. B and C, quantitative analysis of data from A. *, p < 0.05 compared with EGFP.

FIGURE 8.

FIGURE 8.

Overexpressing GPAT1 or GPAT4 increased the protein ratio of membrane to cytosolic PKCϵ but did not alter PKC activity. Membrane and cytosolic preparations from mouse primary hepatocytes overexpressing EGFP, Flag-GPAT1, Flag-GPAT3, or Flag-GPAT4 were subjected to Western blotting (A) and protein quantification (B) or assayed for PKC activity (C). A and B show representative Western blots and protein quantifications from three independent experiments; C shows data from three independent experiments, each done in triplicate. *, p < 0.05 compared with EGFP.

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