p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes
Vitor F. Martins, Samuel A. LaBarge, Alexandra Stanley, Kristoffer Svensson, Chao-Wei Hung, Omer Keinan, Theodore P. Ciaraldi, Dion Banoian, Ji E. Park, Christina Ha, Byron Hetrick, Gretchen A. Meyer, Andrew Philp, Larry L. David, Robert R. Henry, Joseph E. Aslan, Alan R. Saltiel, Carrie. E. McCurdy, Simon Schenk
Vitor F. Martins, Samuel A. LaBarge, Alexandra Stanley, Kristoffer Svensson, Chao-Wei Hung, Omer Keinan, Theodore P. Ciaraldi, Dion Banoian, Ji E. Park, Christina Ha, Byron Hetrick, Gretchen A. Meyer, Andrew Philp, Larry L. David, Robert R. Henry, Joseph E. Aslan, Alan R. Saltiel, Carrie. E. McCurdy, Simon Schenk
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Research Article Endocrinology Muscle biology

p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes

Abstract

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Authors

Vitor F. Martins, Samuel A. LaBarge, Alexandra Stanley, Kristoffer Svensson, Chao-Wei Hung, Omer Keinan, Theodore P. Ciaraldi, Dion Banoian, Ji E. Park, Christina Ha, Byron Hetrick, Gretchen A. Meyer, Andrew Philp, Larry L. David, Robert R. Henry, Joseph E. Aslan, Alan R. Saltiel, Carrie. E. McCurdy, Simon Schenk

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Figure 5

p300/CBP activity is required for insulin-stimulated acetylation and GLUT4 trafficking.

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p300/CBP activity is required for insulin-stimulated acetylation and GLU...
(A) Representative experiment and (B) normalized mean fluorescence intensity for total acetylated proteins (Ac-Lys) in L6 myoblasts in unstained, stained without saponin, DMSO, 100 nM insulin, or insulin cotreated with 10 μM C646 conditions, as analyzed by flow cytometry. Different letters signify P < 0.05 1-way ANOVA, Tukey’s multiple-comparison test. All results are representative of 3 independent experiments. Normalized basal and insulin (100 nM) 2DOGU in (C) L6 myotubes, (D) 3T3-L1 adipocytes, and (E) human skeletal muscle myotubes pretreated for 1 hour with DMSO or C646. *, P < 0.05 2-way ANOVA with Sidak’s multiple comparison versus basal within C646 concentration. #, P < 0.05 2-way ANOVA with Sidak’s multiple comparison versus DMSO within insulin treatment. All results are representative of 3 independent experiments. (F) Representative experiment and (G) normalized mean fluorescence intensity for plasma membrane localized GLUT4 in L6-GLUT4-myc myoblasts in unstained, and with or without 25 μM C646 or 100 nM insulin, as analyzed by flow cytometry. *, P < 0.05 2-way ANOVA with Sidak’s multiple comparison versus basal within treatment. Results are representative of 4 independent experiments. (H) Representative total internal reflection fluorescence imaging, using a 60× oil objective (n.a. 1.45), of Myc7-Glut4-eGFP in 3T3-L1 adipocytes 20 minutes after insulin administration and (I and J) its normalized quantitation. (I) *, P < 0.05 2-way ANOVA with Sidak’s multiple comparison versus basal within treatment. (J) *, P < 0.05 t test versus DMSO. ^, P > 0.05 1-sample t test versus “1.” Results are representative of 3 independent experiments with 6–11 cells per experiment. Data reported as mean ± SEM.