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The following is the abstract of the article discussed in the subsequent letter:
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ABSTRACT |
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McMillen, J., C. M. Donovan, J. I. Messer, and W. T. Willis. Energetic driving forces are maintained in resting rat skeletal muscle
after dietary creatine supplementation. J Appl Physiol
90: 62-66, 2001.
The total creatine (TCr) pool of skeletal muscle
is composed of creatine (Cr) and phosphocreatine (PCr). In resting
skeletal muscle, the ratio of PCr to TCr (PCr/TCr; PCr energy charge)
is ~0.6-0.8, depending on the fiber type. PCr/TCr is linked to
the cellular free energy of ATP hydrolysis by the Cr kinase
equilibrium. Dietary Cr supplementation increases TCr in skeletal
muscle. However, many previous studies have reported data indicating
that PCr/TCr falls after supplementation, which would suggest that Cr
supplementation alters the resting energetic state of myocytes. This
study investigated the effect of Cr supplementation on the energy
phosphates of resting skeletal muscle. Male rats were fed either rodent
chow (control) or chow supplemented with 2% (wt/wt) Cr. After 2 wk on
the diet, the gastrocnemius and soleus muscles were freeze clamped and
removed from anesthetized animals. Cr supplementation increased TCr,
PCr, and Cr levels in the gastrocnemius by 20, 22, and 17%,
respectively (P < 0.05). A numerical 6% higher mean
soleus TCr in Cr-supplemented rats was not statistically significant.
All other energy phosphate concentrations, free energy of ATP
hydrolysis, and PCr/TCr were not different between the two groups in
either muscle. We conclude that Cr supplementation simply increased TCr
in fast-twitch rat skeletal muscle but did not otherwise alter resting
cellular energetic state.
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LETTER |
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Look Before You Leap
To the Editor: In the article by McMillen et al. (5), the authors set out "to evaluate the effect of dietary creatine supplementation on the energy phosphate status of resting soleus and gastrocnemius muscles in the rat." They hypothesized "that dietary creatine supplementation would not alter the phosphocreatine/total creatine ratio in resting type I or type II skeletal muscle," having cited, but subsequently dismissed, a whole series of independent studies involving healthy human volunteers showing the contrary [i.e., the ratio declined because most of the increase in total creatine (TCr) being in the form of free creatine (Cr)].The authors reported that feeding rats chow containing 2% (wt/wt) Cr for 2 wk had, first, no effect on soleus muscle free Cr, phosphocreatine (PCr), and TCr contents. Second, it increased free Cr, PCr, and TCr contents in the gastrocnemius muscle but did not change the PCr-to-TCr ratio. The authors concluded, "Cr supplementation increased TCr in rat fast-twitch skeletal muscle. Because no change in the PCr/TCr occurred, Cr supplementation simply provided an increased TCr content and metabolic capacitance in skeletal muscle."
It is the opinion of this reader that the authors have neglected to compare their own muscle metabolite data with analogous published work; as a result, they have arrived at a spurious conclusion. In particular, comparison of the data presented in Fig. 1 (Ref. 5) with the literature shows that the free Cr concentration of the gastrocnemius (~7.5 µmol/g wet wt) was ~50% lower than that routinely reported for white gastrocnemius and ~30% lower than that reported for red gastrocnemius (e.g., Refs. 3 and 7; the authors do not report whether they analyzed red or white gastrocnemius). Furthermore, the gastrocnemius free Cr concentration shown in Fig. 1 is depicted to be the same as that measured in soleus, which is also contrary to what has been widely published previously (it is ~40-75% higher in red and white gastrocnemius, respectively; Ref. 3).
With this information in mind, and given that ATP and PCr contents of the gastrocnemius depicted in Table 1 and Fig. 1 were comparable with that shown in published literature for red gastrocnemius (3, 7), it seems that the authors found the PCr-to-TCr ratio to be unchanged by Cr supplementation simply because they failed to measure muscle free Cr concentration correctly. This could be at least partly attributable to the authors failing to use independent methods for PCr and Cr determination.
The authors state in their discussion that they were not surprised by their findings because they can think of no compelling reasons to suspect that Cr supplementation would compromise the forces or flows generated by mitochondria. This statement reflects a bias on the part of the authors because they failed to refer to published evidence in support of the existence of the "creatine-phosphocreatine circuit" in the manuscript. Indeed, contrary to the view of McMillen et al. (5), there is substantial evidence to support the view that a change in the cellular Cr concentration is an important regulator of cellular energetics in vivo (1, 6, 8, 9).
It is also important to appreciate that the lack of an effect of Cr supplementation on Cr and PCr concentrations in rat soleus muscle reported by McMillen et al. (5) illustrates that it is unwise for investigators to use an animal model that will not allow the hypothesis under scrutiny to be tested.
Finally, the authors cite their own animal-based study, and that of Brannon et al. (2), to indicate that type II muscle becomes better suited for burst activity following Cr supplementation. However, they failed to mention that this was clearly shown to be the case and moreover was shown in human type II fibers several years previously (4).
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Creatine supplementation favorably affects performance and muscle metabolism during maximal intensity exercise in humans.
Am J Physiol Endocrinol Metab
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McMillen, JC,
Donovan CM,
Messer JI,
and
Willis WT.
Energetic driving forces are maintained in resting rat skeletal muscle after dietary creatine supplementation.
J Appl Physiol
90:
62-66,
2001
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Saks, VA,
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The phosphocreatine circuit and creatine supplementation, both come of age!
In: Guanidino Compounds 5, edited by Mori A,
Ishida M,
and Clark JF.. Tokyo, Japan: Blackwell Science, 1999, p. 117-129.
|
Paul Greenhaff, School of Biomedical Sciences University Medical School Queen's Medical Centre Nottingham NG7 2UH, United Kingdom |
To The Editor: Greenhaff and his
collaborators have indeed published "a whole series of independent
studies" reporting decreased phosphocreatine (PCr) to total creatine
(TCr) ratios in resting human skeletal muscle in response to dietary
creatine (Cr) supplementation (Cr-Sup). However, they have seemingly
dismissed the rather profound implications this purported finding would
bring on the resting metabolic rate of creatine-loaded skeletal muscle.
We are not biased against the "Cr-PCr energy circuit" as
Greenhaff suggests. We studied resting skeletal muscle. There is general agreement that the creatine kinase reaction maintains thermodynamic equilibrium in resting muscle (14). Under this condition,
the energy phosphate system possesses only one degree of freedom (5);
thus all kinetic (4, 13, 18) and energetic (6, 16) models of
respiratory control reduce to the same prediction: a fall in PCr/TCr
should result in an elevated rate of mitochondrial oxidative
phosphorylation. Thus the bias that Greenhaff seems to believe is
present in our report would have no bearing on our conclusions in any
case because the molecular signals of all models are linked by the
equilibrium. Greenhaff and his co-authors must include themselves in
this consensus view, as evidenced by their use of the creatine kinase
equilibrium to calculate the free ADP concentration
([ADP]f) of resting skeletal muscle (11). With their
calculation, they reported that the fall in PCr/TCr after Cr-Sup
indicated a ~24% increase in [ADP]f in resting
myocytes, to quote (11) "our findings point to Cr supplementation
having a substantial impact on cellular energetics." Substantial
indeed, especially since the mitochondrial response to ADP has been
shown to be at least second order (13). For example, using an
S0.5 for ADP of 44 µM (13), the most conservative Hill
coefficient of two, and the values for resting [ADP]f
reported by Green et al. (11) would predict that Cr-Sup increases the resting ATP turnover of myocytes by >40%. We were curious as to what
cellular energy-conserving or energy-dissipating reactions might
accommodate such an impressive increase in mitochondrial energy
delivery in resting Cr-Sup cells and why such profound implications of
decreased PCr/TCr appeared to be of no concern. On the other hand, it
was conceivable that Cr-Sup impaired mitochondrial sensitivity to the
signal(s); thus resting metabolic rate would remain unchanged in the
face of a depressed energy phosphate status. A final possibility was
that Cr-Sup precipitated a phosphate (Pi) limitation.
Because we are interested in the control of mitochondrial respiration,
we carried out a complete assessment of the impact of Cr-Sup on muscle
energy phosphates, including enzymatically assayed Pi. We
found that a modest 20% rise in TCr resulted in neither any predicted
rise in resting metabolic rate nor any impairment in the ability of
mitochondria to maintain the resting cellular energy state, an outcome
we sincerely consider unsurprising. Thus we agree that "creatine
concentration is an important regulator of cellular energetics in
vivo." This is why we did the study and why a Cr term appears in
three of the five equations of our paper. As we discussed (15),
unchanged PCr/TCr in human skeletal muscle after Cr-Sup has been
reported by Febbraio et al. (10).
In response to Greenhaff's criticism of our assay, we would point out
that the colorimetric assay for Cr and PCr we employed (8) has been
widely used for decades (e.g., Refs. 7, 17, 19). The colorimetric assay correlates with the enzymatic
assay demonstrating a slope of unity and r = 0.997 (1),
achieves ~100% recovery of Cr added to tissue samples (9), and has
no interfering molecules in skeletal muscle (9). Our own pilot data
confirmed all of these published reports. In addition, the colorimetric
assay provides an extinction coefficient about twice that of
NAD+-linked assays, all Cr and PCr assays were run in
triplicate, and the coefficient of variation of the assay in our hands
is <3%. Our assay indicated that dietary Cr-Sup increased TCr in rat
whole gastrocnemius muscle by 20%, and the unchanged PCr/TCr we
measured before and after dietary Cr-Sup is very close to, but slightly
below, published values (5). This directionality is opposite to the
criticism advanced by Greenhaff.
In the past, Greenhaff et al. (3) ascribed little relevance to type I
fiber creatine status, yet he now expresses concern that the rat soleus
fails to exhibit increased TCr in response to Cr-Sup (2, 15). However,
if we had defined "Cr-responders" as only those Cr-Sup solei
demonstrating TCr above the mean value of the control group (a strategy
inspired by the human study in Ref. 12), then Cr-Sup would
have yielded 28% higher TCr in our soleus "responders" but still
no change in PCr/TCr.
REPLY
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Effects of creatine loading and training on running performance and biochemical properties of rat skeletal muscle.
Med Sci Sports Exerc
29:
489-495,
1997.
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Casey, A,
Constantin-Teodosiu D,
Howell S,
Hultman E,
and
Greenhaff PL.
Creatine ingestion favorably affects performance and muscle metabolism during maximal exercise in humans.
Am J Physiol Endocrinol Metab
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E31-E37,
1996.
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McMillen, J,
Donovan CM,
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and
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Energetic driving forces are maintained in resting rat skeletal muscle after dietary creatine supplementation.
J Appl Physiol
90:
62-66,
2001.
16.
Meyer, RA.
A linear model of muscle respiration explains monoexponential phosphocreatine changes.
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and
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Biochem J
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Wayne Willis, Jennifer McMillen, Exercise and Sport Research Institute Arizona State University Tempe, Arizona 85287-0404 waynewillis{at}asu.edu | ||||||||||||
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Casey Donovan, Department of Kinesiology University of Southern California Los Angeles, California 90089-0652 |
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