Abstract

The Journal of PhysiologyVolume 588, Issue 10 p. 1779-1790 Free Access Exercise intensity-dependent regulation of peroxisome proliferator-activated receptor γ coactivator-1α mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle Brendan Egan, Brendan Egan School of Health and Human PerformanceSearch for more papers by this authorBrian P. Carson, Brian P. Carson School of Health and Human PerformanceSearch for more papers by this authorPablo M. Garcia-Roves, Pablo M. Garcia-Roves Department of Physiology & Pharmacology, Karolinska Institute, SE-171 77 Stockholm, SwedenSearch for more papers by this authorAlexander V. Chibalin, Alexander V. Chibalin Department of Molecular Medicine & Surgery, Section of Integrative PhysiologySearch for more papers by this authorFiona M. Sarsfield, Fiona M. Sarsfield School of Health and Human PerformanceSearch for more papers by this authorNiall Barron, Niall Barron Centre for Preventive Medicine National Institute for Cellular Biotechnology, Dublin City University, IrelandSearch for more papers by this authorNoel McCaffrey, Noel McCaffrey School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this authorNiall M. Moyna, Niall M. Moyna School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this authorJuleen R. Zierath, Juleen R. Zierath Department of Molecular Medicine & Surgery, Section of Integrative Physiology Department of Physiology & Pharmacology, Karolinska Institute, SE-171 77 Stockholm, SwedenSearch for more papers by this authorDonal J. O’Gorman, Donal J. O’Gorman School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this author Brendan Egan, Brendan Egan School of Health and Human PerformanceSearch for more papers by this authorBrian P. Carson, Brian P. Carson School of Health and Human PerformanceSearch for more papers by this authorPablo M. Garcia-Roves, Pablo M. Garcia-Roves Department of Physiology & Pharmacology, Karolinska Institute, SE-171 77 Stockholm, SwedenSearch for more papers by this authorAlexander V. Chibalin, Alexander V. Chibalin Department of Molecular Medicine & Surgery, Section of Integrative PhysiologySearch for more papers by this authorFiona M. Sarsfield, Fiona M. Sarsfield School of Health and Human PerformanceSearch for more papers by this authorNiall Barron, Niall Barron Centre for Preventive Medicine National Institute for Cellular Biotechnology, Dublin City University, IrelandSearch for more papers by this authorNoel McCaffrey, Noel McCaffrey School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this authorNiall M. Moyna, Niall M. Moyna School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this authorJuleen R. Zierath, Juleen R. Zierath Department of Molecular Medicine & Surgery, Section of Integrative Physiology Department of Physiology & Pharmacology, Karolinska Institute, SE-171 77 Stockholm, SwedenSearch for more papers by this authorDonal J. O’Gorman, Donal J. O’Gorman School of Health and Human Performance Centre for Preventive MedicineSearch for more papers by this author First published: 14 May 2010 https://doi.org/10.1113/jphysiol.2010.188011Citations: 254 Corresponding author D. J. O’Gorman: School of Health and Human Performance, Dublin City University, Dublin 9, Ireland. Email: donal.ogorman@dcu.ie AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1α expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1α transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 ± 4.0 min; HI, 36.0 ± 2.2 min, P 1.8) and A260/A230 (>1.5) ratios. RNA (1 μg) was reverse transcribed to cDNA using the Reverse Transcription System (Promega, Madison, WI, USA) primed with oligo-dT(15) as per the manufacturer's instructions. The cDNA template was stored at −20°C until subsequent analysis. mRNA abundance (30 ng cDNA template per reaction) was determined using quantitative real-time PCR (ABI Prism 7500, Applied Biosystems, Foster City, CA, USA) using Assay-On-Demand primer pairs and probes (P/N 4331182, Assay IDs PGC-1α Hs00173304_m1, PDK4 Hs00176875_m1; Taqman Gene Expression Assays, Applied Biosystems) and Taqman Universal PCR Master Mix (Applied Biosystems). GAPDH mRNA (4333764F, Applied Biosystems) was stable across all time points and used as the housekeeping gene to which target mRNA expression was normalized. The average CT values of the unknown samples were converted to relative expression data using an appropriate standard curve. Protein quantification Approximately 25 mg of crude muscle was homogenized in 1 ml of ice-cold homogenization buffer (20 mm Tris (pH 7.8), 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 1% Triton X-100, 10% (w/v) glycerol, 10 mm NaF, 1 mm EDTA, 5 mm sodium pyrophosphate, 0.5 mm Na3VO4, 1 μg ml−1 leupeptin, 0.2 mm phenylmethyl sulfonyl fluoride, 1 μg ml−1 aprotinin, 1 mm dithiothreitol, 1 mm benzamidine, 1 μm microcystin) using a motorized pestle. Homogenates were rotated end-over-end for 60 min at 4°C and centrifuged (12,000 g for 15 min at 4°C), and the protein content of the supernatant was determined by a commercially available detergent-compatible colorimetric assay (Bio-Rad Laboratories, Hercules, CA, USA). An aliquot of muscle homogenate (50 μg protein) was mixed with Laemmli buffer (20% glycerol, 62.5 mmol l−1 Tris-HCl, 2% SDS, 0.00125% bromophenol blue, 2%β-mercaptoethanol). The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Non-specific binding was blocked in a 5% milk/TBS-t (10 mmol l−1 Tris pH 7.5, 100 mmol l−1 NaCl, 0.1% Tween 20) for 2 h at room temperature. Membranes were incubated overnight with primary antibodies directed towards phospho-AMPKα Thr172 (1:1000; Cell Signaling Technology, Beverly, MA; no. 2531), AMPKα (1:1000; Cell Signaling; no. 2532), phospho-acetyl-CoA carboxylase (ACC) Ser79 (1:500; Cell Signaling; no. 3661), ACC (1:1000; Millipore, Billerica, MA, USA; 07-439), phospho-ATF-2 Thr71 (1:500; Cell Signaling; no. 9221), ATF-2 (1:1000; Millipore; 06-326), phospho-CaMKII Thr286 (1:1000; Cell Signaling; no. 3361), CaMKII (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-13082), phospho-CREB Ser133 (1:1000; Cell Signaling; no. 9191), CREB (1:1000; Cell Signaling; no. 9197), phospho-HDAC4/5/7 Ser632/Ser498/Ser486 (1:1000; Cell Signaling; no. 3424), HDAC4 (1:1000; Cell Signaling; no. 2072), HDAC5 (1:1000; Cell Signaling; no. 2082), phospho-p38 MAPK Thr180/Tyr182 (1:1000; Cell Signaling; no. 9211), p38 MAPK (1:1000; Cell Signaling; no. 9212), and GAPDH (1:4000; Santa Cruz; sc-25778). Membranes were washed in TBS-t and incubated with appropriate secondary horseradish peroxidase-conjugated antibodies (1:20000; Bio-Rad), visualized by enhanced chemiluminescence (ECL; GE Healthcare, Arlington Heights, IL, USA) and quantified by densitometry (GS800 Calibrated Imaging Densitometer, Bio-Rad). Samples for each subject from the respective exercise trials were compared in parallel on the same gel, and representative blots are included. GAPDH and total protein abundance of each phosphorylated protein were used for normalization where appropriate. A representative blot for each protein analysed is presented in Fig. 1. Figure 1Open in figure viewerPowerPoint Representative immunoblots Representative immunoblots corresponding to phosphorylated protein, total protein expression and loading control (GAPDH) measured before (Pre), immediately after (+0 h) and after 3 (+3 h) and 19 h (+19 h) of recovery from isocaloric (400 kcal) stationary cycle ergometer exercise at either 40% (low intensity, LO) or 80% (high intensity, HI) . See text for abbreviations and antibody descriptions. Statistical analysis Experimental data are presented as means ±s.e.m. Data were evaluated using the SigmaStat for Windows v3.11 software package (Systat Software, Inc, San Jose, CA, USA). Two-way (trial × time) repeated measures ANOVA, with Student–Neuman–Keuls post-hoc pair-wise comparisons was performed to identify differences between the two intensities of exercise for variables with serial measurements. Student's paired t test was performed to identify differences between trials for variables with single measurements. The significance level was set at α= 0.05 for all statistical tests. Results Energy expenditure and substrate utilization Total energy expenditure was similar between trials, despite the difference in exercise intensity and total exercise time (P < 0.05) (Table 1). The difference in the rate of energy expenditure (P < 0.05) resulted in a greater reliance on the relative (P < 0.05) and absolute (P < 0.05) contribution of carbohydrate oxidation during the high intensity trial. As expected, the contribution of fat oxidation to total energy expenditure was lower during the high intensity trial (P < 0.05). Table 1. Energy expenditure and metabolic responses during isocaloric low and high intensity exercise trials LO HI Total EE (kcal) 412 ± 11 403 ± 1 Rate of EE (kcal min−1) 6.0 ± 0.3 11.5 ± 0.7* Exercise intensity (%) 38.8 ± 0.4 79.4 ± 1.5* Exercise time (min) 69.9 ± 4.0 36.0 ± 2.2* RER 0.90 ± 0.01 0.98 ± 0.01* CHO oxidation rate (g min−1) 0.9 ± 0.1 2.5 ± 0.2* Total carbohydrate oxidized (g) 64 ± 2 89 ± 3* Rate of fat oxidation (g min−1) 0.23 ± 0.02 0.12 ± 0.04* Total fat oxidized (g) 15 ± 1 4 ± 1* Rate of glycogen utilization (mmol (kg dw)−1 min−1) 1.3 ± 0.2 3.1 ± 1.0* Plasma lactate at rest (mm) 1.12 ± 0.16 0.99 ± 0.14 Plasma lactate at termination (mm) 1.22 ± 0.11 7.23 ± 1.07* Values are mean ±s.e.m. *Significantly different from low intensity trial (P < 0.05). EE, energy expenditure; CHO, carbohydrate; RER, respiratory exchange ratio. Muscle glycogen content was similar at baseline (259 ± 17 vs. 249 ± 19 mmol (kg dw)−1 for the LO and HI trials, respectively) and decreased following both exercise trials (176 ± 22 vs. 128 ± 34 mmol (kg dw)−1 for the LO and HI trials, respectively; P < 0.05), but returned to baseline the following morning. The net rate of glycogen utilization was higher during the high intensity trial (1.3 ± 0.2 vs. 3.1 ± 1.0 mmol (kg dw)−1 min−1, for the LO and HI trials respectively, P < 0.05). Plasma lactate concentration was similar at rest (LO, 1.12 ± 0.16 mm; HI, 0.99 ± 0.14 mm), and was unchanged from baseline during LO, but increased to 7.23 ± 1.07 mm at the end of HI (P < 0.05, compared to both baseline and LO). PGC-1α mRNA abundance PGC-1α mRNA abundance was elevated 3.8- and 10.2-fold 3 h after LO and HI, respectively (P < 0.05), with an effect of exercise intensity observed between trials (P < 0.05) (Fig. 2). PDK4 mRNA abundance was elevated 6.8–7.2-fold at +3 h in both trials (data not shown), suggesting that intensity-dependent effects on mRNA abundance are gene-specific rather than a generalized exercise effect. Figure 2Open in figure viewerPowerPoint Intensity-dependent regulation of PGC-1α gene expression by a single bout of exercise The effect of exercise intensity on PGC-1α mRNA abundance immediately after (+0 h) and during recovery (+3 h and +19 h) from isocaloric (400 kcal) exercise bouts. Open bars represent low intensity trial, LO; filled bars represent high intensity trial, HI. PGC-1α mRNA was normalized using the housekeeping gene GAPDH. Values are means ±s.e.m., n= 8. *Significantly different from baseline within same trial (P < 0.05); †significantly different from LO at same time point (P < 0.05). Contraction-activated signal cascades AMPK phosphorylation (Fig. 3A) was similar at baseline, but increased 2.8-fold at +0 h following exercise in the high (P < 0.05) but not low intensity trial, resulting in a difference between trials (P < 0.05). Acetyl-CoA carboxylase β (ACCβ) phosphorylation (Fig. 3B) increased 2.3- and 5.2-fold immediately following exercise for LO and HI, respectively (P < 0.05), resulting in a difference between trials at this time point (P < 0.05). Figure 3Open in figure viewerPowerPoint Greater activation of AMPK signalling by a single bout of high intensity exercise The effect of exercise intensity on phosphorylation of AMPK (A) and ACCβ (B) protein immediately after (+0 h) and during recovery (+3 h and +19 h) from isocaloric (400 kcal) exercise bouts. Open bars represent low intensity trial, LO; filled bars represent high intensity trial, HI. Representative immunoblots are shown in Fig. 1. Phosphorylated protein is normalised to total protein content (con) of the respective protein. Values are means ±s.e.m., n= 8. *Significantly different from baseline within same trial (P < 0.05); †significantly different from LO at same time point (P < 0.05). p38 MAPK phosphorylation (Fig. 4A) was increased at +0 h in both exercise trials (∼2.0-fold, P < 0.05), but the phosphorylation of a downstream transcription factor, ATF-2, a regulator of PGC-1α expression, was increased only in HI (2.4-fold, P < 0.05; Fig. 4B), indicating intensity-dependent regulation of this pathway. Figure 4Open in figure viewerPowerPoint Similar activation of p38 MAPK, but greater activation of ATF-2, by a single bout of high intensity compared to low intensity exercise The effect of exercise intensity on phosphorylation of p38 MAPK (A) and ATF-2 (B) protein immediately after (+0 h) and during recovery (+3 h and +19 h) from isocaloric (400 kcal) exercise bouts. Open bars represent low intensity trial, LO; filled bars represent high intensity trial, HI. Representative immunoblots are shown in Fig. 1. Phosphorylated protein is normalised to total protein content (con) of the respective protein. Values are means ±s.e.m., n= 8. *Significantly different from baseline within same trial (P < 0.05); †significantly different from LO at same time point (P < 0.05). Total CaMKII phosphorylation (summation of βM and γ/δ isoforms; Rose et al. 2006) increased immediately following the high (84%, P < 0.05), but not low intensity trial, with a difference between trials (Fig. 5A; P < 0.05). Phosphorylation of the calcium-dependent transcription factor CREB was reduced at +0 h in HI (−64%, P < 0.05), but also tended to be reduced in LO (−36%, P= 0.104). However, CREB phosphorylation was subsequently elevated at +3 h after both trials (∼80%, P < 0.05; Fig. 5B). Phosphorylation of class IIa HDACs, purported downstream targets of both AMPK and CaMKII signalling and negative regulators of gene transcription, was measured by HDAC4/5/7 phosphorylation (Fig. 5C) and also increased only in HI at +0 h (2.0-fold, P < 0.05), consistent with the intensity-dependent activation of these upstream kinases. Figure 5Open in figure viewerPowerPoint Greater activation of CaMKII and HDACs, but not CREB, by a single bout of high intensity exercise The effect of exercise intensity on phosphorylation of CaMKII (A), CREB (B) and class IIa HDAC (C) protein immediately after (+0 h) and during recovery (+3 h and +19 h) from isocaloric (400 kcal) exercise bouts. Open bars represent low intensity trial, LO; filled bars represent high intensity trial, HI. Representative immunoblots are shown in Fig. 1. Phosphorylated protein is normalised to total protein content (con) of the respective protein. Phosphorylated HDAC4/5/7 is normalised to the summed total protein content of HDAC4 and 5. Values are means ±s.e.m., n= 8. *Significantly different from baseline within same trial (P < 0.05); †significantly different from LO at same time point (P < 0.05). Discussion In the present study, differential expression of PGC-1α mRNA following an acute bout of isocaloric exercise was associated with intensity-dependent regulation of intracellular signalling cascades. Skeletal muscle contraction activates AMPK, CaMKII and p38 MAPK signalling cascades in an intensity-dependent manner as a result of increased bioenergetic processes, calcium flux and cellular stress (Widegren et al. 2