The PGC-1α-related coactivator promotes mitochondrial and myogenic adaptations in C2C12 myotubes

Abstract

The transcriptional coactivator PGC-1α is a potent regulator of skeletal muscle metabolism. Less is known about the structurally similar PGC-1α-related coactivator (PRC) that is enriched in myoblasts and adult skeletal muscle. The present study was designed to determine the effect of PRC on the metabolic profile of C2C12 myotubes. Overexpression of full-length PRC increased PRC gene expression by 2.7 ± 0.3-fold and protein content by 108 ± 5.3%. This modest elevation in PRC resulted in an increased rate of myoblast proliferation (61.5 ± 2.7%) and resulted in myotubes characterized by increased MyoD (18.2 ± 0.52%) and myosin heavy chain (15.4 ± 3.13%) protein. PRC overexpressing myotubes showed increases in mRNA for some-COX4 (2.6 ± 0.18-fold), ATP5B (2.7 ± 0.34-fold) cytochrome c (5.1 ± 0.68-fold)-but not all, MTCO1 (0.61 ± 0.18-fold) and HAD (0.98 ± 0.36-fold) mitochondrial genes, as well as a significant increase in cytochrome-c (28.7 ± 7.02%) protein content. The enzyme activity of the electron transport chain (ETC) complex IV (3.7 ± 0.01-fold) and citrate synthase (2.1 ± 0.14-fold) was increased by PRC, as was the mtDNA:nucDNA ratio (11 ± 0.3%). PRC increased cellular respiration (142%), basal (197%) and insulin-stimulated (253%) glucose uptake, as well as palmitate uptake (28.6 ± 3.31%) and oxidation (31.1 ± 2.17%). Associated with these changes in function, PRC overexpression increased GLUT4 mRNA (4.5 ± 0.22-fold) and protein (13.8 ± 2.08%) and CPT1 protein (28.9 ± 4.23%). Electrical stimulation of C2C12 myotubes resulted in a transient increase in PRC mRNA that was smaller (2.1 ± 0.3-fold vs. 4.4 ± 0.23-fold) and occurred earlier (3 h vs. 6 h) than PGC-1α. Collectively, our data show that PRC promotes skeletal muscle myogenesis and metabolism in vitro, thus identifying PRC as a functional skeletal muscle coactivator capable of regulating mitochondrial substrate utilization and respiration.

Fig. 1.

Overexpression of PGC-1α related coactivator (PRC) in C2C12 myotubes increased the protein content (A) and gene expression of PRC (B) with no effect on PGC-1α protein content (C). PRC increased cellular proliferation 12 and 36 h after transfection (D). Compared with empty vector (EV) controls (E), myotubes overexpressing PRC (F) displayed an altered phenotype associated with PRC overexpression. Data are representative (n = 8) of three independent transfection experiments. *Significantly different than EV control and †significantly different from 12 h (P < 0.0001).

Fig. 2.

PRC increases myosin heavy chain (MHC) and MyoD. MyoD (A), total MHC (B), fast MHC (C), and slow MHC (D) levels in C2C12 cells transfected with an EV or PRC. MyoD, total MHC, and slow MHC increase as a result of PRC overexpression. Eukaryotic initiation factor 2 (eEF2) was used as a loading control for each protein. Data are representative (n = 6) of two independent experiments. *Significantly different than EV control (P < 0.05).

Fig. 3.

PRC overexpression increases mitochondrial gene expression but not protein content. A: PRC increases cytochrome oxidase (COX) 4, β ATP synthase (ATP5B) and cytochrome-c (CytC) mRNA expression, whereas the mitochondrially encoded cytochrome oxidase 1 (MTCO1), citrate synthase (CS), and 3-hydroxyacyl-CoA dehydrogenase (HAD) mRNA expression was unchanged. B: PRC significantly increased CytC and tended to increase mitochondrial transcription factor-A (mtTFA) and succinate dehydrogenase (SDH) protein content but had modest effects on COX IV and β ATP synthase (ATP Synβ) protein content. Eukaryotic initiation factor 2 (eEF2) was used as a loading control for each protein. Data are representative (n = 6) of two independent experiments. *Significantly different than EV controls (P < 0.05).

Fig. 4.

A: PRC overexpression results in functional mitochondrial adaptation. PRC increases basal oxygen consumption rate (OCR) compared with EV controls. B: PRC increases ETC complex IV and CS activity. C: PRC increases the mitochondrial DNA (mtDNA) to nuclear DNA (nucDNA) ratio (mtDNA:nucDNA) indicative of increased mitochondrial mass. Data are representative (n = 12) of two independent experiments with *P < 0.05.

Fig. 5.

PRC increases basal and insulin-stimulated glucose uptake. A: basal (light bar) and insulin-stimulated (dark bar) glucose uptake. GLUT4 mRNA expression (B) and GLUT4 protein content (C) following PRC or EV control overexpression. C: PRC did not affect the protein content of GLUT1. Data are representative (n = 6) of two independent experiments. *Significantly different than EV control and †significantly greater than PRC basal (P < 0.05).

Fig. 6.

PRC increases palmitate uptake and oxidation. Palmitate uptake (A) and oxidation (B), as well as CPT1 protein (C) are increased following PRC transfection compared with EV control. Data are representative (n = 6) of two independent experiments. *Significantly different between EV control P < 0.05.

Fig. 7.

PRC mRNA increases following 3 h in vitro contraction. Following 3 h of stimulation (1-Hz frequency/0.3-ms pulse width), PRC mRNA (A) increased significantly. In contrast, the induction and time-course of PGC1α mRNA (B) was different postcontraction. Data are representative (n = 3) of three independent transfection experiments where * indicates P < 0.05.