In previous studies we have identified the class II histone deacetylase HDAC5 as a key regulator of MEF2 activity and GLUT4 expression in human skeletal muscle in response to acute exercise, but the relevant histone acetyltransferase is unknown. Pathway analysis of results from exercise-induced genome-wide histone modifications using ChIP for two key histone marks (H3K9/14 and H3K36) identified p300 as a key target. In the present study we used transgenic mice to investigate the role of p300 and CBP in skeletal muscle GLUT4 expression.
Mice expressing a tamoxifen-inducible Cre recombinase under the human α-skeletal actin promoter were bred with mice having LoxP sites flanking exons 9 of both the Ep300 and the Crebbp genes. This resulted in mice with a skeletal muscle-specific double-knockout of Ep300 and Crebbp (mPCKO) or skeletal muscle-specific knockout of either Ep300 (PKO) or CBP (CKO). Further breeding of these lines yielded mice lacking Crebbp, but heterozygous for Ep300 (mCKO-PZ) and lacking Ep300, but heterozygous for Crebbp (mPKO-CZ). For all mouse lines, Cre negative littermates served as the experimental control (WT). Tamoxifen (2 mg) was administered to all mice via oral gavage for five consecutive days, starting at 13–15 weeks of age. At day 1, 3, or 5 after starting tamoxifen treatment, mice were fasted (4 h), anesthetised and the gastrocnemius was excised and snap frozen in liquid nitrogen. Mice were kept in a conventional facility with a 12 h light/12 h dark cycle and had free access to food and water unless otherwise noted. RNA was isolated from snap-frozen gastrocnemius using TRIzol Reagent (ThermoFisher Scientific, Waltham, MA, USA) and 1 μg of RNA was used for cDNA synthesis. Semi quantitative real-time PCR analysis was performed using iTaqTM SYBR Green master mix on a CFX384 Touch real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression Glut4 was calculated with the ΔΔCt method, using Tbp as housekeeping gene.
GLUT4 mRNA levels were not different between mice at day 1, but were reduced (P<0.05) 75 and 100% in mPCKO relative to WT on days 3 and 5 respectively. In contrast, there were no changes in GLUT4 mRNA levels with tamoxifen treatment in mCKO-PZ or mPKO-CZ at any time. In mPCKO, GLUT4 protein levels were reduced ~55% on day 5.
These results indicate that either p300 and CBP are critical for skeletal muscle GLUT4 expression, but the lack of both impairs GLUT4 transcription.