Healthy, active but untrained, men (n= 8; 24 ± 5 years; body mass index, 23 ± 2 kg m−2; mean ±s.d.) were recruited, and written and verbal information about the purpose, nature and potential risks relating to the experimental procedures was given to the subjects before they provided consent to participate. The protocol was reviewed and approved by the Deakin University Human Research Ethics Committee. One to two weeks prior to testing, subjects completed an incremental exercise test to volitional exhaustion on an electromagnetically braked cycle ergometer (Lode, Groningen, the Netherlands) to determine their peak pulmonary O2 uptake , which averaged 51 ± 2 ml kg−1 min−1 (mean ±s.e.m.). Expired air was analysed by O2 and CO2 analysers (AEI Technologies, Pittsburgh, PA, USA) and expired volume by a turbine ventilometer (Flow transformer K 520, KL Engineering, Australia). The gas analysers were calibrated against gases of known composition prior to each test.
Subjects were asked to refrain from exercise as well as caffeine, nicotine and alcohol ingestion for at least 24 h prior to the study. Subjects were provided with a standardized meal (∼80% carbohydrate) for the evening prior to testing and reported to the laboratory in the morning after an overnight fast (plasma glucose, 4.9 ± 0.1 mm). Subjects rested for at least 20 min in the supine position before a muscle sample was obtained from the vastus lateralis by percutaneous needle biopsy under local anaesthesia and immediately frozen in liquid N2. Subjects exercised for 40 min at 76 ± 1% with biopsies taken at 5 and 40 min of exercise and frozen within 20 s after the last contraction. Muscle samples were stored in liquid N2 until analysis.
All chemicals were purchased from Sigma-Aldrich (St Louis, MI, USA) unless otherwise stated. Muscle samples were homogenised as outlined previously (Rose & Hargreaves, 2003) in a homogenization buffer containing (mm): Tris-HCl 50 (pH 7.5), sucrose 250, EDTA 1, EGTA 1, phenylmethylsulfonyl fluoride 1, dithiothreitol 1, sodium flouride 5, sodium pyrophosphate 5 and benzamidine 1, with 10% glycerol, 5 μl ml−1 protease inhibitor cocktail and Nonidet P-40 (NP-40; 1%), and mixed well at 4°C. To examine protein localization and PKC activity, muscle samples (40–50 mg) were homogenised in 1: 8 volumes of homogenization buffer not containing NP-40 until no visible particles remained. These samples were spun at 350 000 g for 30 min and the resultant supernatant comprised the cytosolic fraction. The pellet was resuspended and mechanically disrupted in homogenization buffer containing NP-40 and after 30 min on ice, the pellet fraction was subjected to centrifugation at 100 000 g for 60 min, and the supernatant (particulate fraction) removed. For all samples, aliquots were taken for total protein assay (Pierce BCA, Rockford, IL, USA) and the remaining extract was stored at −80°C until required for analysis.
In preliminary experiments equal amounts of protein from both fractions were immunoprobed using antibodies for proteins specific to skeletal muscle subcellular organelles/structures. These included antibodies against sarcolemmal proteins: phospholemman (PLM; antibody provided by Professor Randall Moorman, University of Virginia, VA, USA) and Na+,K+-ATPase-α1 (NKAα1; B. Fambrough, Johns Hopkins University, MD, USA); t-tubule protein: dihydropyridine receptor-α1 (DHPRα1; Santa Cruz Biotechnology, CA, USA); sarcoplasmic reticulum protein: Ca2+-ATPase-1 (SERCA1; Santa Cruz Biotechnology); mitochondrial protein: cytochrome C-oxidase subunit I (COXI; Molecular Probes, OR, USA); and general marker: actin. The particulate fraction represents a preparation in which membranous structures are enriched, and the cytosolic fraction is devoid of membranes (Fig. 1).
Figure 1. Immunoblots of marker proteins for subcellular membrane structures in human skeletal muscle after fractionation Muscle samples (n= 2; S1, S2) were fractionated as described in the Methods, and equal amounts of protein (35 μg) were subjected to immunoblotting procedures and probed with antibodies specific to skeletal muscle subcellular/membrane structures. Blots were deliberately overexposed to detect the slightest immunoreactivity. Prefix α, anti; S, sample; P, particulate; C, cytosolic.
Download figure to PowerPoint
PKC activity was measured in skeletal muscle extracts (10–20 μg protein, 5 μl) in kinase assay buffer containing (mm): Hepes 10 (pH 7.2), MgCl2 5, EGTA 1, sodium pyrophosphate 0.1, ATP 0.1 (0.2 Ci mmol−1 5′[γ32P]ATP; Amersham Biosciences, Uppsala, Sweden); with 40 μm epidermal growth factor (EGF) receptor substrate peptide (H2N-KRTLRR-OH), 1.2 mm CaCl2, 10 μm phorbol-12-myristate-13-acetate (PMA), and 20 μg ml−1 phosphatidylserine in a final reaction volume of 25 μl. The EGF receptor peptide was chosen as it has the minimal requirements for conventional, novel and atypical PKC substrates including basic residues at −2 and +3, as well as a hydrophobic residue at the +1 position (Nishikawa et al. 1997). The reaction procedure was conducted as outlined previously (Rose & Hargreaves, 2003), except that the reaction time was 5 min.
To directly measure isoform-specific PKC activity, PKC isoforms and isotypes were immunoprecipiated (IP) from muscle extracts (500 μg) incubated with anti-PKCθ or -PKCζ/ι antibodies (2 μg; Santa Cruz Biotechnology) in a final volume of 600 μl with gentle mixing for 2 h at 4°C. Following this 40 μl of 50% (v:v) of protein-A-sepharose (Amersham Biosciences) was added and further incubated with mixing for 2 h at 4°C. To immunopurify conventional PKC (cPKC), extracts (500 μg) were incubated with agarose-conjugated PKC-MC5 antibodies (20 μl; Santa Cruz Biotechnology) in a final volume of 600 μl with gentle mixing for 2 h at 4°C. The beads were washed and resuspended (40 μl) in buffer containing (mm): Tris 10 (pH 7.2), sodium pyrophosphate 1, EGTA 1. cPKC and PKCθ activities were assayed for 10 min as described above. PKCζ/ι activity was assayed as described by Bandyopadhyay et al. (1997b) with minor modifications. Assays were performed in kinase assay buffer at 30°C with 40 μm[S25]-PKC(19–31) substrate peptide (H2N-RFARKGSLRQKNV-OH), and 40 μg ml−1 phosphatidylserine in a final reaction volume of 25 μl. Assays were conducted as previously described (Rose & Hargreaves, 2003). The assay time and PKC concentration were within a linear range for lysate and IP assays (data not shown). Preliminary experiments demonstrated that exercise did not alter the efficiency of immunoprecipitation of any of the PKC isoforms investigated (data not shown).
PKC abundance and phosphorylation were determined in equal amounts of protein in muscle extracts and IP samples using immunoblot analysis as previously described (Rose & Hargreaves, 2003) with anti-PKCα (H7), anti-PKCβII (C-18), anti-PKCδ (C-20), anti-PKCɛ (C-15), anti-PKCθ (C-19), anti-PKCζ/ι (C-20) (Santa Cruz Biotechnology), anti-pT410/403-PKCζ/ι and anti-pS-PKC substrate motif (R/K-X-SPO4-Hyd-R/K; Cell Signalling Technology, Beverly, MA, USA) antibodies.
To provide a control sample for assays, L6 skeletal myotubes were treated with PMA (0.1 μm; PKC activity) or insulin (1 μm; PKCζ/ι activity). All media solutions were purchased from Invitrogen (Carlsbad, CA, USA). L6 myotubes were prepared as previously described (Rose & Hargreaves, 2003). For PMA treatment, cells (n= 3 dishes per treatment) were treated with PMA (0.1 μm) and control cells were treated with vehicle (0.1% ethanol) for 30 min. The cells were lysed in homogenization buffer without NP-40 and collected after 10 min on ice. PKC activity in the cytosolic and particulate fractions was determined as described above. The percentage of PKC activity in the particulate fraction relative to total PKC activity was 46 ± 5% in vehicle and 64 ± 3% in PMA-treated myotubes (n= 3, P < 0.05). Furthermore, a significant translocation of PKCα from the cytosolic fraction to the particulate fraction was shown by immunoblot analysis (P < 0.01; data not shown) as observed in other studies (Braiman et al. 1999). In addition, PMA induced increases (0.7- to 1.3-fold, P < 0.05) in the density of four out of five bands detected when particulate extracts were analysed by immunoblotting with a pSer-PKC substrate motif antibody (data not shown). These data indicate that the PKC assay and immunoblot analyses of fractionated cell lysates are sensitive measures of PKC activity and localization.
Myotubes (n= 3 dishes per treatment) were treated with or without insulin (1 μm) for 5 min and cells were lysed with homogenization buffer containing NP-40 and collected after 10 min on ice. IP PKCζ/ι activity was determined as described above. An increase in aPKC activity (control, 12.3 ± 1.3 pmol mg −1min−1; insulin, 28.3 ± 1.3 pmol mg−1min−1; P < 0.001) was measured with insulin treatment when compared with control cells, as observed in other studies (Bandyopadhyay et al. 1997a).