THE EFFECT OF ACTISYN ON ANABOLIC HORMONAL PROFILES AND ACTIN AND MYOSIN mRNA
AND PROTEIN LEVELS IN EXERCISED RAT AND HUMAN.
Charles B. Hensley, R. Steven Davidson and Stephan N. Mayer
Human Performance Institute
A Division of Biotem Cytotechnologies
INTRODUCTION:
The goal of this study was to assess the anabolic potential of actisyn , a
proprietary supplement (SportPharma USA ) and to compare actisyn with whey
protein. To this end, we employed rat model to determine parameters for the
subsequent human study in which we measured actisyn ’s effect on five
anabolic/catabolic hormone markers (growth hormone, insulin, IGF-1,
testosterone, and cortisol) as well as skeletal muscle actin and myosin mRNA
and protein abundance. We also measured the effect of actisyn on strength and
lean body mass.
MATERIALS AND METHODS:
Supplements.
Actisyn, a proprietary supplement, was supplied by SportPharma, USA . The
placebo, a high quality whey protein, was supplied by SportPharma, USA.
Effect of supplementation on select hormone profiles in exercising rats.
Eighty male Sprague-Dawley rats (240-260 grams) were given electrical
stimulation of the quadriceps until the muscle was completely fatigued. Thirty
minutes later, the rats received one gram per kilogram body weight of either
liquefied whey protein (n=40) or liquefied actisyn (n=40). Rats were then
anesthetized and sacrificed by decapitation in-groups of ten at 0, 1, 2 and 4
hours post supplementation. Blood was collected and plasma isolated and stored
at 4oC. The plasma levels of growth hormone, insulin, IGF-1, testosterone, and
cortisol were determined by routine procedures and the results were normalized
to the zero time point.
Effect of supplementation on actin and myosin protein expression in exercising
rats.
Sixty male Sprague-Dawley rats (240-260 grams) were given electrical
stimulation of the quadriceps muscle until the muscle was fatigued to
exhaustion. Thirty minutes later, the rats received one gram per kilogram body
weight of either liquefied whey protein (n=30) or liquefied actisyn (n=30).
Rats were anesthetized and sacrificed by decapitation in-groups of ten at 0, 12
and 24 hours post supplementation. The vastus medialis muscle was excised,
quick frozen in liquid nitrogen and stored at -70oC until processed. The
skeletal muscle was homogenized in isotonic Hank’s solution using a high speed
Polytron tissue homogenizer. Skeletal muscle protein was determined on the
whole muscle homogenate by Bradford assay. Actin and myosin protein abundance
was measured by immunoblot analysis of a constant amount of skeletal muscle
homogenate resolved by SDS-PAGE and blotted onto diazotized paper as previously
described (1, 2). The blots were probed with anti rat actin and myosin specific
antisera probed with 125I-Protein A, subjected to autoradiography and
quantified by scanning densitometry. The results were normalized to the zero
time point.
Effect of supplementation on select hormone profiles in exercising humans.
Thirty males between the ages of 21 and 40 were be utilized for this phase of
the study. All of the subjects were currently on a program of resistance
training. Fifteen of the subjects were placed in the placebo group (those
receiving whey protein) and fifteen of the subjects were placed in the test
group (those receiving actisyn). A supplement washout protocol was employed in
which the subjects were instructed to stop using supplements. During this
period, blood was drawn every 4 days and plasma test hormones were monitored
until baseline levels of all test hormones were achieved. Two days after the
end of the washout period, subjects performed six sets of leg extensions with
each set terminating only when the quadriceps muscle was fatigued to
exhaustion. Thirty minutes after the exercise period, the subjects ingested 43
grams of supplement dissolved in 12 oz. Of orange juice and blood was drawn at
0, 1, 2 and 4 hours post supplementation. Plasma was isolated and stored at
4Oc. The plasma levels of growth hormone, insulin, IGF-1, testosterone, and
cortisol were determined by routine procedures and the results were normalized
to the zero time point.
Effect of supplementation on actin and myosin isoforms in skeletal muscle in
exercising humans.
Twenty males between the ages of 21 and 40 were utilized for this phase of the
study. Ten of the subjects were placed in the placebo group (those receiving
whey protein) and ten of the subjects were placed in the test group (those
receiving actisyn). Subjects were placed on a supplement washout protocol (see
the above section). Two days after the end of the washout period, subjects
performed six sets of leg extensions with each set terminating only when the
quadriceps muscle was fatigued to exhaustion. Thirty minutes after the exercise
period, the subjects ingested 43 grams of supplement dissolved in 12 oz. of
orange juice and a skeletal muscle biopsy was taken from the vastus medialis at
0 and 24 hours post supplementation. The skeletal muscle tissue was quick
frozen in liquid nitrogen and stored at -70oC until processed. Total RNA was
isolated by phenol/chloroform extraction and skeletal muscle actin and myosin
mRNA was measured by Northern analysis on constant amounts/samples of total RNA
prepared as previously described (1, 2). Blots were subjected to
autoradiography, quantified by scanning densitometry and the results were
normalized to the zero time point.
Skeletal muscle protein was determined by Bradford assay. Actin and myosin
protein abundance was measured by immunoblot analysis of a constant amount of
skeletal muscle homogenate resolved by SDS-PAGE and blotted onto diazotized
paper as previously described (1, 2). The blots were probed with anti human
actin and myosin specific antisera. The blots were probed with 125I-Protein A,
subjected to autoradiography, quantified by scanning densitometry and the
results were normalized to the zero time point.
Effect of supplementation on body composition and strength in exercising
humans.
Twenty males between the ages of 21 and 40 were utilized for the body
composition and strength portion of the study. Ten subjects were placed in the
placebo group (those receiving whey protein) and ten subjects were placed in
the test group (those receiving actisyn). All subjects participated in a
three-day split routine and received supplementation 3 times per day for 21
days. Forty three grams of either actisyn or whey protein dissolved in 12 oz.
orange juice was consumed by the subjects in the morning, 30 minutes post
workout, and once again in the early evening. Body composition and strength was
assessed every third day for 21 days. Caliper and electrical impedance measured
body composition and strength was assessed by 100% max quadriceps one rep
workload (leg extension).
RESULTS:
RAT STUDIES
The rat study shows above and beyond placebo, the rats receiving the actisyn
formula had a 12%, 85%, and 121% increase in growth hormone after 1, 2 and 4
hours respectively. There was no increase in IGF-1 but insulin was increased by
48%, 63% and 54% after 1, 2 and 4 hours. There was no change in testosterone
after 1 hour but was increased by 18% and 32% after 2 and 4 hours respectively.
Finally, there was no change in cortisol levels in rats receiving actisyn as
opposed to the 33%, 40% and 82% increase seen in the placebo group. Actin
protein levels were increased by 27% after 12 hours and myosin protein levels
were increased by 52% after 12 hours. There was no further change in protein
abundance seen at the 24-hour point.
hr Fold increase above baseline Fold increase above baseline Actisyn effect
/ whey protein effect Actisyn relative to whey protein (% effectiveness)
WHEY PROTEIN ACTISYN
GH 1 1.12 ± 0.08(12%) 1.42 ± 0.15 (42%) 3.5-fold 250%
2 1.1 ± 0.09 (10%) 1.6 ± 0.14 (60%) 6.0-fold 500%
4 1.2 ± 0.15 (20%) 1.72 ± 0.19 (72%) 3.6-fold 260%
INSULIN 1 1.07 ± 0.05 (7%) 1.5 ± 0.15 (50%) 7.14-fold 614%
2 1.07 ± 0.12 (7%) 1.52 ± 0.23 (52%) 7.42-fold 642%
4 1.1 ± 0.02 (10%) 1.18 ± 0.04 (18%) 1.8-fold 80%
IGF-1 1 1.05 ± 0.11 (5%) 1.07 ± 0.1 (7%) 1.2-fold 20%
2 1.05 ± 0.08 (5%) 1.23 ± 0.1 (23%) 4.6-fold 360%
4 1.1 ± 0.09 (10%) 1.2 ± 0.11 (20%) 2.0-fold 100%
TESTOSTERONE 1 1.1 ± 0.06 (10%) 1.23 ± 0.1 (23%) 2.3-fold 130%
2 1.1 ± 0.09 (10%) 1.27 ± 0.1 (27%) 2.7-fold 170%
4 1.08 ± 0.08 (8%) 1.2 ± 0.06 (20%) 2.5-fold 150%
Actin mRNA 24 1.07 ± 0.05 (7%) 1.22 ± 0.08 (22%) 3.14-fold 214%
Myosin mRNA 24 1.07 ± 0.05 (7%) 1.2 ± 0.06 (20%) 2.85-fold 185%
Actin protein 24 1.05 ± 0.05 (5%) 1.35 ± 0.1 (35%) 7.0-fold 600%
Myosin protein 24 1.05 ± 0.05 (5%) 1.37 ± .08 (37%) 7.4-fold 640%
TABLE 1. Relative effect of whey protein and Actisyn on human anabolic hormonal
profiles, and actin/myosin mRNA and protein abundance.
HUMAN STUDIES
Hormonal profiles.
The human data shows that subjects receiving actisyn showed a marked increase
in the anabolic hormonal profile when compared with the whey protein (Figures
1-5 and 8; Table 1). Above and beyond placebo (whey protein), the subjects
receiving the actisyn formula had a 30% (250% better than placebo), 50% (500%
better than placebo) and 52% (260% better than placebo) increase in growth
hormone after 1, 2, and 4 hours receptively. IGF-1 was increased by 2% (20%
better than placebo), 18% (360% better than placebo) and 10% (100% better than
placebo); insulin by 43% (614% better than placebo), 45% (642% better than
placebo) and 8% (80% better than placebo); testosterone by 13% (130% better
than placebo), 17% (170% better than placebo) and 12% (150% better than
placebo). Cortisol was unchanged with controls increasing by 22%, 175 and 10%
after 1, 2, and 4 hours respectively.
Actin and Myosin mRNA levels.
Actin mRNA increased by 15% (214% better than placebo) and myosin mRNA
increased by 13% (185% better than placebo) (Figures 6 and 7; Table 1).
Actin and Myosin Protein Abundance.
Actin protein abundance increased by 30% (600% better than placebo) myosin
protein abundance increased by 32% (640% better than placebo) (Figures 6 and 7;
Table 1).
Strength and Body Composition.
Strength increased by 8.5% (37% better than placebo) and lean body mass
increased by 2.33% (105% better than placebo) (Figure 9).
DISCUSSION:
The goal of this study was to assess the anabolic potential of actisyn , a
proprietary supplement (SportPharma USAä ) and to compare actisyn with whey
protein. To this end, we defined the study parameters in the rat model and
assayed for five anabolic/catabolic hormone markers (growth hormone, insulin,
IGF-1, testosterone, and cortisol in the exercised human. We also measured the
effect of actisyn on the actin and myosyn mRNA and protein expression. In
well-trained athletes, we found that actisyn was superior to straight whey
protein supplementation in increasing each of the anabolic hormones and
decreasing the catabolic hormone, cortisol. We also found that actisyn
increased the actin and myosin mRNA levels by 15% and protein levels by 30%.
The fact that both actin and myosin protein levels increased to a greater
extent than did the actin and myosin mRNA indicates that actisyn is acting not
only at the level of transcription/RNA stability but also on
translation/protein stability as well. We also found that actisyn was superior
to whey protein in increasing strength and lean body mass in the human.
In conclusion, we find that actisyn alters the anabolic hormonal milieu and
drives both gene expression and post-transcriptional accumulation of the
skeletal muscle actin and myosin protein resulting in increased strength and
lean body mass.
LITERATURE CITED:
1. Horowitz, B. Hensley, C. B. Quintero, M. Azuma, K. K. Putnam, D. and
McDonough, A. A. Differential regulation of Na, K-ATPase alpha1, alpha2, and
beta subunit mRNA levels by thyroid hormone. J. Biol. Chem. 265: 14308-14314.
1990. 2. Lescale-Matys, L. Hensley, C. B. Crnkovic-Markovic, R. Putnam, D. and
McDonough, A. A. Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by
differentially increasing beta and not alpha subunit mRNA. J. Biol. Chem. 265.
1990.
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