Abstract
The transcription factor ZAC1 is expressed in a variety of tissues including the developing heart, but its physiological role is unclear. We examined the role of ZAC1 in regulating expression of the insulin-responsive glucose transporter GLUT4 and whether ZAC1 expression is altered in cardiomyocyte hypertrophy. We demonstrated expression of Zac1 mRNA and protein in rat cardiomyocytes by PCR and Western blotting, respectively. Using a combination of chromatin immunoprecipitation and luciferase assays, we showed that ZAC1 regulates Glut4 expression via a specific binding site in the Glut4 promoter. Overexpression of ZAC1 increased Glut4 mRNA and protein expression and resulted in increased glucose uptake in cardiomyocytes as determined by a fluorescent analog uptake assay. Induction of hypertrophy by phenylephrine or isoproterenol resulted in increased Zac1 expression. We identified a novel putative promoter in the Zac1 gene and demonstrated increased binding of MEF2 to this promoter in response to hypertrophic stimulation. MEF2 regulated transactivation of the Zac1 promoter and ZAC1 protein expression. This work identifies ZAC1 as a novel and previously unknown regulator of cardiomyocyte Glut4 expression and glucose uptake. Our results also implicate MEF2 as a regulator of ZAC1 expression in response to induction of hypertrophy.
Highlights
To accommodate this metabolic flexibility, cardiac fuel uptake and anabolism are tightly regulated by transcriptional, translational, and post-translational mechanisms
We hypothesized that ZAC1 may regulate genes involved in glucose metabolism such as the insulinresponsive glucose transporter Glut4
We report that ZAC1 directly regulates expression of the insulinresponsive glucose transporter Glut4 via a highly conserved G4N12G4 motif upstream of the transcription start site
Summary
Cell Culture and Cardiomyocyte Isolation—COS7 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine. Cells were transfected (Lipofectamine 2000, Invitrogen) with reporter plasmid (human GLUT4 promoter luciferase reporter hG4-luc [10]; T3GN12G4-hG4-luc bearing a mutated ZAC1-binding site in hG4-luc; mZac1pr-pGL3; or mutMEF2-mZac1pr-pGL3), pCMVlacZ for transfection normalization, and expression vectors as required (pcDNA1-mycMEF2C, pcDNA1-mycMEF2A, pcDNA1mycMEF2D, pcDNA3.1-FLAG-HDAC5S/A, pSG5-HA-mZac1b (mZAC1b is a functionally identical splice variant of ZAC1 containing 11 additional amino acids [11]), and pSG4-HA-GRIP1). Western Blot Analysis of Proteins—Protein lysates were isolated as described [9] and assayed using a Coomassie stainbased kit (Pierce) and resolved on 12% SDS-polyacrylamide gels and wet-transferred onto polyvinylidene difluoride (Bio-Rad). After blocking (2% milk powder in phosphate-buffered saline plus 0.1% Tween 20), cells were incubated with anti-␣-actinin (Sigma) or antiGLUT4 antibodies (Abcam), followed by Alexa Fluor 488-conjugated goat-anti-mouse or Alexa Fluor 594-conjugated goatanti-rabbit secondary antibody (Molecular Probes).
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