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. 2001 Mar 27;98(7):3820-5.
doi: 10.1073/pnas.061035098. Epub 2001 Mar 13.

Restoration of Insulin-Sensitive Glucose Transporter (GLUT4) Gene Expression in Muscle Cells by the Transcriptional Coactivator PGC-1

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Free PMC article

Restoration of Insulin-Sensitive Glucose Transporter (GLUT4) Gene Expression in Muscle Cells by the Transcriptional Coactivator PGC-1

L F Michael et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Figures

Figure 1
Figure 1
Induction of GLUT4 expression by PGC-1. (A) Ectopic expression of PGC-1 potentiates GLUT4 gene expression. L6 myoblasts were grown to confluence and were induced to differentiate. Myotube cultures were infected with varying moi, as indicated, of adenovirus expressing either GFP (as control) or PGC-1. The myotubes were permitted to express the proteins for 96 h, at which time total RNA or total cellular proteins were isolated. Thirty micrograms of protein were subjected to immunoblot analysis using either anti-PGC-1 or anti-cytochrome c antisera. Twenty micrograms of total RNA was subjected to Northern blot analysis using either GLUT4 or COX IV cDNA as probe. (B) PGC-1 induction of GLUT4 gene expression is cell-type dependent. Cos 1 cells were infected with varying moi, as indicated, of adenovirus expressing either GFP (as control) or PGC-1. Total RNA was subjected to Northern blot analysis using either PGC-1 or GLUT4 cDNA as probe, and anti-cytochrome c antiserum was used as described above.
Figure 2
Figure 2
PGC-1 induces glucose transport in muscle cells. (A) PGC-1 induces both basal and insulin-stimulated glucose transport. Differentiated L6 myotubes were infected either with GFP or PGC-1 expressing adenovirus at moi of 100 or they were grown in the absence of adenovirus (control) for 96 h. The cultures were serum-deprived for 6 h before the addition of 100 nM insulin (black bars) and were incubated an additional 20 min. Cells were rinsed, and glucose uptake was measured as described in Materials and Methods. Results shown are representative of more than three independent experiments that were performed in triplicate. (B) Ectopic expression of PGC-1 in myotubes increases total and plasma membrane-associated GLUT4 protein. Differentiated L6 myotubes were infected either with GFP or PGC-1 expressing adenovirus at moi of 100 or they were grown in the absence of adenovirus (control) for 96 h. The cells were stimulated with 100 nM insulin, as indicated, and cellular homogenates were fractionated either into total membranes, plasma membrane (PM), or intracellular membranes (IM). Equivalent amounts of protein were fractionated by SDS/PAGE and assayed for GLUT4 protein by immunoblotting.
Figure 3
Figure 3
PGC-1 specifically transactivates the GLUT4 promoter via a MEF2 element. (A) PGC-1 increases transcriptional activity of the GLUT4 promoter. C2C12 myoblast cells were transfected in a 24-well format with 150 ng GLUT4 2.4-kb promoter-luciferase plasmid, 50 ng CMV βgal plasmid, and 700 ng of CMV-driven expression vectors either for p300, p/CAF, or full-length PGC-1. The cells were transfected for 3 h and 10% FBS/DMEM was replaced overnight. The transfected cells were induced to differentiate for at least 24 h before lysis. Luciferase activity was normalized by β-galactosidase activity. (B) PGC-1 coactivates GLUT4 promoter activity via the MEF2 enhancer element. The MEF2 recognition sequence (-473 to -464) of the 2.4-kb GLUT4 promoter was mutated from CTAAAAATAG to CTAAGGCTAG by site-directed mutagenesis. C2C12 myoblast cells were transfected as described above. (C and D) PGC-1 preferentially coactivates MEF2C. 293 cells were transfected in a 24-well format with 150 ng 3× MEF2 recognition sequence-luciferase plasmid, 50 ng CMV β-gal plasmid, 700 ng of CMV-driven expression vectors either for p/CAF or PGC-1, and either pRC-CMV MEF2A, MEF2C, or MEF2D. Cells were grown for 36 h posttransfection, before lysis.
Figure 4
Figure 4
PGC-1 interacts specifically with MEF2C in vivo and in vitro. (A) Interaction between PGC-1 and MEF2C in cells. Expression vectors for Flag-PGC-1 and MEF2C were transfected into 293T cells, as indicated. Total cellular lysate was collected 48 h posttransfection. Coimmunoprecipitation using a Flag affinity resin and subsequent Western blot analysis was performed as described in Materials and Methods. (B) Specific interaction between PGC-1 and MEF2C in vitro. GST fusion proteins for PPARγ, MyoD, Myogenin, and MEF2C were expressed in E. coli. Purified GST fusion proteins were bound to beads and incubated with [35S]-labeled PGC-1. After extensive washing, [35S]-labeled PGC-1/activator interaction was detected by autoradiography following SDS/PAGE separation.
Figure 5
Figure 5
Interaction between PGC-1 and MEF2C maps to discrete domains. (A) The interaction domain of PGC-1 for MEF2C binding is located between aa 403 and 570. GST-MEF2C fusion protein was immobilized on glutathione beads and incubated with [35S]-labeled PGC-1 deletion fragments. After extensive washing, [35S]-labeled PGC-1 protein interaction with GST-MEF2C was detected by autoradiography following SDS/PAGE separation. (B) Functional interaction between MEF2C and PGC-1 deletion constructs. C2C12 myoblast cells were transfected in a 24-well format with 150 ng GLUT4 2.4-kb promoter-luciferase plasmid, 50 ng CMV βgal plasmid, and 700 ng of CMV-driven expression vectors either for PGC-1 aa 1–400, 1–550 or full-length PGC-1. The cells were transfected for 3 h and 10% FBS/DMEM was replaced overnight. The transfected cells were induced to differentiate for at least 24 h before lysis. Luciferase activity was normalized by β-galactosidase activity. (C) Diagram summarizing the protein fragments of PGC-1 that were examined.
Figure 6
Figure 6
MEF2C interaction domain for PGC-1. The interaction domain of MEF2C for PGC-1 binding is located between aa 93 and 174. The top panel summarizes the protein fragments of MEF2C that were examined and the results of the GST affinity chromatography experiment that is shown below. GST-PGC-1 fusion protein was immobilized on glutathione beads and incubated with [35S]-labeled MEF2C deletion fragments. After extensive washing, [35S]-labeled MEF2C protein interaction with GST-PGC-1 was detected by autoradiography following SDS/PAGE separation.

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