Introduction
In humans, changes in muscle protein synthesis (MPS) and breakdown in response to food intake and/or exercise are normally assessed in
vastus lateralis muscle (Rennie et al.
1982). In rats, mice, cats and dogs, lagomorphs rates of MPS are markedly different between different muscle groups (Garlick et al.
1989). However, it is generally assumed that the metabolic behaviour of skeletal muscle tissue is the same throughout the human body and that results obtained in quadriceps muscle can be extrapolated to total skeletal muscle and, as such, whole-body protein turnover (Mittendorfer et al.
2005). As no significant correlations have been observed between type I muscle fibre content and the measured muscle protein fractional synthetic rates of triceps, soleus and vastus lateralis, it was concluded that differences in basal, resting muscle protein synthesis rates between muscle groups are relatively small (<15%) and of minor biological significance (Mittendorfer et al.
2005). However, interesting new data has emerged suggesting that resting muscle protein synthesis rates are substantially (~30%) higher in type I compared to type II muscle fibres (Dickinson et al.
2010). So far, almost no data are available on potential fibre type specific differences in the muscle protein synthetic response to anabolic stimuli such as food intake and/or exercise.
Rates of MPS increase rapidly following a single bout of resistance-type exercise (Phillips et al.
1997), and remain elevated for up to 24–48 h if the subjects are fed during post-exercise recovery (MacDougall et al.
1992; Phillips et al.
1997; Tang et al.
2008). It has been speculated that the muscle protein synthetic response to resistance-type exercise is fibre type specific (Koopman et al.
2006c; Mittendorfer et al.
2005; Trappe et al.
2004). In accordance with this hypothesis, we showed that muscle fibre hypertrophy after 12 weeks of resistance exercise training is restricted to the type II muscle fibres in human
vastus lateralis tissue of elderly men (Verdijk et al.
2009). In addition, it has been reported that a predominantly oxidative fibred muscle (
soleus) has a smaller hypertrophic response to resistance exercise than
vastus lateralis muscle (Trappe et al.
2004). In agreement, the phosphorylation status of key-regulatory proteins involved in the activation of protein synthesis (i.e. PKB (or Akt), mTOR and S6K1) seems to be more pronounced in muscle tissue containing a greater proportion of type II muscle fibres following resistance-type exercise in rats (Baar and Esser
1999; Parkington et al.
2003; Sakamoto et al.
2003). The latter was confirmed recently by our observation that S6K1 (at the T
421/S
424 phosphorylation sites) is phosphorylated in a fibre type dependent manner after a single bout of resistance exercise (Koopman et al.
2006c). These findings suggested that rates of MPS may be higher in type II than type I fibres after resistance exercise. However, so far no direct measurements of rates of MPS in human type I and II have been reported after exercise.
We hypothesised that MPS rates would be greater in type II than type I muscle fibres after a single session of resistance exercise. To test this, we conducted studies involving continuous infusions of l-[ring-13C6]phenylalanine combined with muscle biopsies taken immediately after a single bout of resistance exercise and after 6 h of post-exercise recovery. A modified ATPase staining procedure for freeze-dried muscle fibres was applied to differentiate between type I and II muscle fibres, allowing us to assess MPS rates in mixed muscle and in separated type I and II muscle fibres during post-exercise recovery.
Discussion
We have previously shown that the phosphorylation status of S6K1 (T
421/S
424) increases to a greater (30%) extent in the type II versus type I muscle fibres after resistance-type exercise (Koopman et al.
2006c). In addition, we have reported that muscle hypertrophy after a 12 week RET program is specific in increasing type II muscle fibre size in elderly men (Verdijk et al.
2009). Therefore, we hypothesised that muscle protein synthesis rates would be higher in type II than in type I muscle fibres during the acute stages of post-exercise recovery. We measured muscle protein synthetic rates after resistance exercise in type I and II muscle fibres from human
vastus lateralis muscle. To the best of our knowledge, this study is the first to compare MPS rates in type I and II muscle fibres following resistance-type exercise within a single muscle. Fractional muscle protein synthetic rates during post-exercise recovery were 8% higher in the type I compared with the type II muscle fibres.
Recently published data suggest that MPS in human
vastus lateralis muscle is markedly (33%) higher in type I compared with type IIa muscle fibres following an overnight fast (Dickinson et al.
2010). We extend on these data by assessing muscle fibre type specific protein synthesis rates following resistance-type exercise. We measured protein-bound
l-[ring-
13C
6]phenylalanine labelling in type I and II muscle fibres dissected (and stained for ATPase activity) from freeze-dried muscle biopsies collected immediately after cessation of exercise and again after 6 h of post-exercise recovery. We did not observe any differences in protein-bound
l-[ring-
13C
6]phenylalanine labelling between the different muscle fibre types immediately after cessation of exercise. Although we did not aim to assess basal/resting rates of fibre type specific MPS, the lack of difference in protein-bound enrichment between muscle fibre types in biopsies taken immediately after cessation of exercise strongly suggests that MPS rates in the fed state (at rest and during exercise) do not differ between the type I and type II muscle fibres, as suggested by previous work on muscles composed of predominantly one of the fibre types (Mittendorfer et al.
2005). During the 6 h post-exercise recovery period, protein-bound
l-[ring-
13C
6]phenylalanine enrichments increased to a greater extend in the type I compared with the type II muscle fibres (
P < 0.01). Muscle protein synthesis rates were 8 ± 2% higher in type I compared with type II muscle fibres following resistance-type exercise (Fig.
3,
P < 0.01). Consequently, our data indicate that muscle protein synthesis rates are only marginally higher in type I compared with type II muscle fibres during recovery following resistance-type exercise.
To be able to determine muscle fibre type specific muscle protein synthesis rates, we collected as much as 200–250 mg muscle tissue to have sufficient tissue available to adequately detect protein-bound
l-[ring-
13C
6]phenylalanine enrichment both in mixed muscle and in specifically prepared type I and II muscle fibres. This required us to take biopsies from both legs at each time-point, restricting the total number of time points where biopsies could be collected. Furthermore, as muscle fibre type specific protein synthesis rates following an overnight fast have been reported previously (Dickinson et al.
2010), we did not assess basal MPS rates. Muscle sample preparation requires separation of each individual muscle fibre, followed by ATPase staining, and subsequent separation of the type I (black) and II (white/grey) muscle fibres (Fig.
1) until sufficient (2–4 mg dry weight) fibres are separated to allow accurate detection of protein-bound
l-[ring-
13C
6]phenylalanine labelling using GC-IRMS. The coefficient of variance (CV) of the measurements of protein-bound
l-[ring-
13C
6]phenylalanine labelling in the type I and type II muscle fibres was similar to the CV (~1%) we normally observe for mixed muscle tissue samples. We must, however, recognise that it is theoretically possible that there are small (short lived) differences in MPS rates within the 6 h recovery period from exercise, which might not have been detected by the applied protocol. Short-lived responses of feeding and resistance exercise on MPS have previously been observed (Rennie et al.
2002; Kumar et al.
2009). However, given the lack of evidence of significant differences in the feeding responses of MPS in muscles containing predominantly type 1 or type 2 muscle fibres (Mittendorfer et al.
2005) made over a 3 h period, this seems unlikely.
The observation that type II muscle fibres do not show a greater post-exercise muscle protein synthesis rates when compared with the type I muscle fibres seems to be at odds with previous findings from our laboratory (Koopman et al.
2006c) as well as others (Tannerstedt et al.
2009). Tannerstedt et al. (
2009) have recently shown that the phosphorylation status of S6K1 (T
389) and S6 (but not Akt and mTOR) are increased to a greater extend in type II compared with type I fibres following maximal lengthening contractions. These data are in line with our previous findings that S6K1 phosphorylation at T
421/S
424 increases to a greater extend in type II compared with type I fibres following resistance exercise (when exercise is performed in the fasted state) (Koopman et al.
2006c). The latter suggests that the anabolic response to resistance-type exercise is greater in the type II versus type I muscle fibres. We will highlight a few mechanisms that may explain the described discrepancy between our protein synthesis rates and previously published signalling data.
Glycogen utilisation during resistance-type exercise is higher in type II than in type I muscle fibres (Koopman et al.
2006c). AMP-activated protein kinase (AMPK) has a glycogen-binding domain (Sakamoto et al.
2004), and has been shown to be substantially activated when muscle glycogen concentrations are low (Wojtaszewski et al.
2003). Therefore, it could be speculated that AMPK activity is increased to a greater extend in type II than in type I fibres during/following resistance exercise. As a result, the rise in 4E-BP1 phosphorylation (Dreyer et al.
2006; Drummond et al.
2008; Koopman et al.
2006c), and MPS could be more attenuated specifically in type II muscle fibres following exercise. In addition, synthesis rates of myofibrillar and mitochondrial protein are increased following a single session of resistance-type exercise (Wilkinson et al.
2008). As muscle fibre mitochondrial content is up to 2-fold higher in type I versus II muscle fibres (Koopman et al.
2006a) and mitochondrial protein synthesis rates are ~50% higher than that of myofibrillar protein (Wilkinson et al.
2008) it can be speculated that the observed small differences in post-exercise mixed muscle protein FSR between fibre types are due to differences in mitochondrial protein content/turnover. Alternatively, the fact that we observed only marginal differences between fibre types may also be attributed to the fact that food was ingested prior to exercise. Free phenylalanine concentrations do not differ between type I and II muscle fibres prior to exercise and during post-exercise recovery (Blomstrand and Essen-Gustavsson
2009), suggesting that amino acid-induced changes in muscle protein synthesis are similar between fibre types. In line with these observations, we have shown that S6 phosphorylation increases to a similar extend in both type I and II muscle fibres during post-exercise recovery when food is ingested prior to and/or after exercise (Koopman et al.
2007b).
As recent studies have highlighted the discrepancies between anabolic signalling and muscle protein synthesis (Greenhaff et al.
2008), it is apparent that studies are warranted that investigate fibre type specific muscle protein synthesis at rest and following exercise with and without food intake using contemporary stable isotope methodology (i.e. using
13C-labelled phenylalanine). To the best of our knowledge, our study is the first to compare muscle protein synthesis rates in type I and II muscle fibres within one specific muscle group, showing that post-exercise muscle protein synthesis rates are only marginally higher in type I compared with type II muscle fibres. Based on the minor differences between muscle protein synthesis rates in the type I versus type II muscle fibres, it seems reasonable to rely on the measurement of mixed muscle protein synthesis rates when aiming to study the combined effects of physical activity and food intake on muscle protein synthesis.