Exercise Induces a Marked Increase in Plasma Follistatin: Evidence That Follistatin Is a Contraction-Induced Hepatokine

Abstract

Follistatin is a member of the TGF-β super family and inhibits the action of myostatin to regulate skeletal muscle growth. The regulation of follistatin during physical exercise is unclear but may be important because physical activity is a major intervention to prevent age-related sarcopenia. First, healthy subjects performed either bicycle or one-legged knee extensor exercise. Arterial-venous differences were assessed during the one-legged knee extensor experiment. Next, mice performed 1 h of swimming, and the expression of follistatin was examined in various tissues using quantitative PCR. Western blotting assessed follistatin protein content in the liver. IL-6 and epinephrine were investigated as drivers of follistatin secretion. After 3 h of bicycle exercise, plasma follistatin increased 3 h into recovery with a peak of 7-fold. No net release of follistatin could be detected from the exercising limb. In mice performing a bout of swimming exercise, increases in plasma follistatin as well as follistatin mRNA and protein expression in the liver were observed. IL-6 infusion to healthy young men did not affect the follistatin concentration in the circulation. When mice were stimulated with epinephrine, no increase in the hepatic mRNA of follistatin was observed. This is the first study to demonstrate that plasma follistatin is increased during exercise and most likely originates from the liver. These data introduce new perspectives regarding muscle-liver cross talk during exercise and during recovery from exercise.

Follistatin is a glycosylated plasma protein, which is a member of the TGF-β superfamily. It was first discovered in 1987 in ovarian follicular fluid but later found to be expressed in a number of different tissues including the pituitary gland, placenta, ovary, testis, and skeletal muscle (1).

Follistatin possesses the ability to suppress secretion of FSH (1), hence it’s name, and has for that reason predominantly been related to reproduction physiology. Although involved in the menstrual cycle, plasma follistatin does not vary between the different phases in the cycle, and there is no difference in plasma follistatin throughout the reproductive age (2–4).

During the last decade, follistatin has been demonstrated to bind other members of the TGF-β superfamily, including myostatin (5, 6), which has been suggested to be involved in the regulation of skeletal muscle mass. Follistatin prevents myostatin from binding to the activin IIb receptor, by binding and thereby neutralizing myostatin in the circulation (7). As a consequence, intramyocellular Smad is prevented, thereby blocking gene transcription (7). Because myostatin is a potent negative regulator of muscle growth, the inhibition of myostatin results in marked increases in muscle mass (8–10). Neutralization of myostatin by follistatin has profound effects on the skeletal muscle growth (11). Thus, in mice with follistatin overexpression, marked increases in muscle mass are observed (10). Follistatin overexpression promotes muscle hypertrophy by satellite cell activation and is caused. in part, by inhibition of myostatin, but possibly also by interaction with other regulatory proteins (5). Consequently, follistatin has been suggested as a possible therapeutic strategy in treatment of muscle wasting disorders such as Duchenne Muscular Dystrophy and age-related sarcopenia (12–14).

Interestingly, follistatin gene expression in skeletal muscle is regulated by acute resistance exercise and by long-term stretching (15, 16), which suggests a role of follistatin in remodeling of the muscle tissue. Therefore we hypothesized that follistatin is regulated by acute endurance exercise.

Materials and Methods

Systemic human experiments

In Trial 1, Five healthy male subjects performed 3 h of exercise on a bicycle ergometer (Monark Ergomedic 839 E; Monark Ltd., Varberg, Sweden). The subjects were asked to refrain from strenuous exercise 24 h before the experimental day. On the experimental day, the subjects reported at 0700 h after an overnight fast. A catheter was inserted into the antebrachial vein, and a blood samples was drawn. During the 3 h of bicycling, blood was obtained every hour, and during the following 6 h of recovery blood was drawn every 3 h. The following morning, the subjects reported fasting at the laboratory for the final blood sample. The intensity of the exercise was 50% of individual VO₂ max, and VO₂ was assessed throughout the exercise bout by indirect calorimetry (Quark b²; CosMed, Rome, Italy). VO₂ max was determined 1–2 wk before the experimental day by a VO₂ max test as described in detail elsewhere (17). During the experimental day, the subjects fasted until the last blood sample was drawn but had free access to water.

In Trial 2, six healthy male subjects who had fasted from midnight, reported to the laboratory. They received a 3-h infusion of human recombinant IL-6 (Sandoz, Basle, Switzerland) with an infusion rate of 5 μg/h in a volume of 25 ml/h. Blood samples were obtained every hour during the infusion and during the 5-h recovery period. The subjects reported fasting the following morning for the final blood sample.

Trial 3 was a control trial in which six healthy male subjects reported to the laboratory after fasting from midnight. The participants rested supine in a bed and fasted throughout the experiment. A catheter was inserted into the antebrachial vein, and blood was obtained every hour. The characteristics of the three groups are presented in Table 1.

TABLE 1.

Subject characteristics of the participants in the human experiments

	Trial 1 (n = 5)	Trial 2 (n = 7)	Trial 3 (n = 7)	Trial 4 (n = 9)
Age (yr)	28.8 ± 2.1	27.4 ± 2.2	24.3 ± 1.3	20.9 ± 0.5
Weight (kg)	79.5 ± 7.4	81.4 ± 3.4	73.9 ± 3.0	72.6 ± 2.9
Height (cm)	182.5 ± 4.4	182.0 ± 2.4	179.9 ± 2.9	179.2 ± 1.9
Body mass index (kg/m2)	23.7 ± 1.3	24.5 ± 0.7	22.8 ± 0.5	22.6 ± 0.8
Systolic BP (mm Hg)	128.4 ± 2.3	122.1 ± 1.5	121.4 ± 2.0	132.9 ± 3.7
Diastolic BP (mm Hg)	76.4 ± 2.0	78.6 ± 2.8	66.6 ± 2.0	79.0 ± 2.6
Hemoglobin (mmol/liter)	8.8 ± 0.2	9.0 ± 0.1	8.4 ± 0.1	9.6 ± 0.2
Leukocytes (bill/liter)	4.6 ± 0.3	4.8 ± 0.6	6.7 ± 0.4	5.9 ± 0.4
Trial performance (W)	137.0 ± 9.8			31.7 ± 2.0

Values are means ± sem. Trial 1, 3 h bicycling at 50% VO₂ max; trial 2, infusion of recombinant human IL-6; trial 3, control, subjects resting in supine position; and trial 4, one-legged knee extensor exercise for 2 h.

Human arterial-venous exercise experiment

In Trial 4, nine healthy male subjects participated in the study. They underwent a medical examination and a standard set of blood analysis, which all were normal. The subjects were normally physically active without participating in any competitive sports. They were informed, both orally and in writing, about risks and discomfort associated with the experimental protocol. The subjects were asked to refrain from strenuous exercise 24 h before the experimental day. On the experimental day, the subjects reported to the laboratory at 0700 h after an overnight fast. Catheters were inserted in both femoral veins and the femoral artery of the resting leg. After baseline blood samples were drawn from the three catheters, biopsies were obtained from the vastus lateralis muscle on both legs. The subjects performed 2 h of one-legged knee extensor exercise at 50% of maximum workload on a modified Krogh ergometer. The maximum workload was determined by a pretrial test of maximum workload (18), after the subjects were familiarized with the exercise model. Blood was obtained every half hour during exercise, and at time points 0.5, 1, and 3 h during the recovery period. Biopsies were obtained in pairs from both legs after the 2 h of exercise and additionally 3 h later. The catheters were removed 3 h after the exercise had ceased, and the subjects were allowed to eat and drink ad libitum. The following morning the subjects reported at the laboratory after an overnight fast, a blood sample was drawn from a brachial vein, and percutaneous needle biopsies from the vastus lateralis muscle in both legs were obtained.

Exercising and epinephrine-injected mice

Thirty-two female NMRI (Taconic Farms, Inc., Bomholtgaard, Denmark) mice (age 8–10 wk) were divided into four groups. After 2 h of fasting, eight mice were killed before exercise, eight mice were killed immediately after 1 h of swimming exercise, eight mice were killed 2 h after the exercise bout, and finally eight mice was killed 5 h after the exercise bout. The following tissues were obtained after the mice were killed: gastrocnemius and soleus muscle, subcutaneous (inguinal) and visceral (gonadal) adipose tissue, heart, liver, kidney, and spleen. The tissues were immediately frozen in liquid nitrogen.

Twenty NMRI female mice were used to test whether epinephrine was the exercise-induced systemic factor promoting follistatin expression. Ten mice received epinephrine 2 mg/kg (Sygehus Apotekerne i Danmark, Copenhagen, Denmark), and 10 mice received placebo (saline) intraperitoneally; after 30 min the mice were killed, and the livers were stored for later analysis. The plasma glucose concentration was measured before and after the administration of either saline or epinephrine using a HemoCue Glucose 201+ system (HemoCue AB, Ängelholm, Sweden).

In vitro studies of muscle cells

For myocyte cell culture experiments, murine C2C12 myoblasts (<7 passages; American Type Culture Collection, Manassas, VA) were maintained at 37 C, 5% CO₂ in DMEM containing 10% fetal bovine serum, and penicillin/streptomycin (100 U/ml, 100 μg/ml) (all from Invitrogen). To induce differentiation, the serum content of the cell culture media was swapped to 2% horse serum for 7–8 d. For in vitro electrical pulse stimulation (EPS) experiments, fully formed myotubes seeded in six- or four-well plates (NUNC, Rochester, NY) were placed in a cell culture stimulator (Ionoptix, Milton, MA), following a fresh media change and maintained at 37 C, 5% CO₂. On completion of experiments, mRNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA), as per kit instructions. After deoxyribonuclease treatment (Invitrogen) and cDNA synthesis (Taqman Reverse transcriptase reagents; Applied Biosystems, Carlsbad, CA), murine follistatin gene expression was measured by RT-PCR (Applied Biosystems) using preinventoried primer sets (Mm 03023987_ml, Assay on demand; Applied Biosystems). Follistatin was measured in the culture media of EPS-activated C2C12 myotubes by ELISA with an intraassay CV of 8.8% (R&D Systems).

Fully differentiated C2C12 myotubes were subjected to 4 h of EPS (40 V/60 mm, 1 Hz, 10 msec) during which contraction of myotubes was observed (see Ref. 21 for a description of this model’s characteristics). The conditioned media were immediately removed from EPS and control plates and placed on fully confluent AML 12 liver cells for 1 and 4 h. Fresh media (media only) served as a further negative control along with 10 ng/ml TGF-β (positive control).

Ethical comity approvals

All experimental protocols were approved by local ethical comity and were in accordance with the Declaration of Helsinki; KF-11-067/02, H-D-2007-0125, H-D-2007-0127.

Plasma analysis

All blood samples were obtained in tubes containing EDTA and were immediately spun at 4 C at 2500 × g for 15 min. The plasma fractions were stored at −80 C until analyzed. Human and mouse plasma follistatin were measured using a commercially available ELISA kits with an intraassay coefficient of variation (CV) of 1.9% (R&D Systems, Minneapolis, MN). All samples were run in duplicate in accordance with the protocol from the manufacturer. Plasma IL-6 was measured by high-sensitivity ELISA with an intraassay CV of 9.2% (R&D Systems). All samples were analyzed in duplicate.

Real-time quantitative PCR

The tissues were analyzed for the mRNA content by use of real-time PCR. Total RNA extraction from both human and mouse tissues followed by reversed transcription of total RNA was done using TRIzol Reagent (Invitrogen) and Taqman Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions as primers as previously described (19). Real-time PCR was performed using an ABI 7900 Sequence detection system (Applied Biosystems). Human and mouse follistatin, and the endogenous control, 18S RNA, were designed for SYBR Green. The sequences used were as follows: human follistatin, forward: 5′-GGA AAA CCT ACC GCA ATG AA-3′; reverse, 5′-GAG CTG CCT GGA CAG AAA AC-3′; mouse follistatin: forward: 5′-TAC TCC AGC GCC TGC CAC CT-3′; reverse, 5′-TCC CGC CGC CAC ACT GGA TA-3′. The mRNA content of both target gene and 18S was calculated from the cycle threshold values using a standard curve, and the ratio between target gene and 18S was calculated.

Western blot

We determined follistatin protein content in liver samples from exercising mice (pre, 0 h, 2 h, and 5 h) by Western blotting. Liver lysate preparation, SDS-PAGE and immunoblotting were performed as previously described (20). Briefly, the tissue was homogenized, and the samples were then diluted with 5× sample buffer [1/5 loading buffer (0.313 m Tris-HCl, 10% sodium dodecyl sulfate, 50% glycerol, and 0.05% bromophenol blue) and 1/20 reducing agent (2 m dithiothreitol)]. After boiling the samples for 5 min at 95 C, each well was loaded with 40 μg protein. Proteins were separated by SDS-PAGE (4–12% bis-Tris gel; Invitrogen, Carlsbad, CA), and immunoblotted to polyvinylidene difluoride membranes (iBlot gel, Invitrogen). The membranes were blocked in 5% Topblock (LuBioScience, Lucerne, Switzerland) for 1 h at room temperature and then washed in buffer (Tris-buffered saline with 0.1% Tween 20). The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO). The following morning the membranes were washed, incubated for 1 h at room temperature with a secondary antibody (Polyclonal Rabbit Antigoat; Dako Corp., Carpenteria, CA) using skim milk at a 1:5000 dilution and subsequently washed in wash buffer. Supersignal West Femto (Pierce Chemical Co, Rockford, IL) was used to detect the bands, and quantification was done by using a charge-coupled device camera (ChemiDocXRS; Bio-Rad Laboratories, Inc., Hercules, CA) and software (Quantity One, Bio-Rad).

Statistical analysis

All analyses were performed using SAS software, version 9.1, (SAS institute, Cary, NC). Data are presented as mean and sem. Two groups were compared by Student’s t test. For comparisons over time, a one-way ANOVA was performed. When the dataset contained two groups and a time course, a two-way ANOVA was applied. The two-sided P values <0.05 were considered significant.

Results

Systemic plasma follistatin increased after 3 h of bicycle exercise in human healthy males. As depicted in Fig. 1A, plasma follistatin increased 7-fold (P < 0.05), peaking 3 h into recovery after an acute bout of exercise. The plasma level was reduced to preexercise levels the following morning.

Fig. 1.

A, Plasma follistatin in relation to 3 h of bicycling exercise (n = 5). The shaded area marks the 3 h period of exercise. B, Arterial and venous follistatin plasma concentrations during 2 h of one-legged knee-extensor exercise and the following recovery period (n = 9). The shaded area marks the 2-h period of exercise. C, Follistatin mRNA expression in skeletal muscle. D, Follistatin mRNA expression in C2C12 muscle cells in response to EPS. E, Follistatin protein release from C2C12 muscle cells in response to EPS. *, P < 0.05.

To evaluate whether the plasma follistatin originated from the working muscle tissue, we measured the arterial venous difference across the working leg. As shown in Fig. 1B, when performing 2 h of one-legged knee extensor exercise, a 2-fold (P < 0.05) increase in systemic concentrations of plasma follistatin was observed. However, no arterial-venous difference could be detected over the exercising leg (Fig. 1B). This was in accordance with the mRNA content of follistatin in the skeletal muscle tissue from the resting and exercising leg (Fig. 1C), where no change was observed between legs or over time. This was confirmed in vitro. To determine whether follistatin is produced in skeletal muscle cells, we adopted an EPS protocol that we have previously validated (21). Neither follistatin mRNA (Fig. 1D) nor follistatin protein content (Fig. 1E) in the cell culture media was increased by EPS.

Mice were investigated in relation to 1 h of swimming exercise to identify the tissue(s) responsible for the exercise-induced follistatin production. Plasma follistatin increased 3-fold (P < 0.05) 2 h after the swimming exercise (Fig. 2A). Eight tissues were obtained: soleus and gastrocnemius muscle, subcutaneous (inguinal) and visceral (gonadal) adipose tissue, heart, kidney, liver, and spleen. Follistatin mRNA was detected in all tissues at all time points. Consistent with the humane data, no change was observed in the skeletal muscle regarding the follistatin mRNA content. An increase of 2.8-fold in visceral adipose tissue was borderline significant (one-way ANOVA, P = 0.053). However, a 21.5-fold (P < 0.05) increase was observed in the liver follistatin mRNA content immediately after the exercise bout (Fig. 2B). The increase in liver follistatin was confirmed on the protein level by Western blotting. In Fig. 2C, time points 1 h, 3 h, and 6 h are depicted as the relative proportion of the preexercise levels. The follistatin protein content is moderately elevated at all time points (P < 0.05).

Fig. 2.

A, Plasma follistatin in mice in relation to 1 h of swimming and the following recovery period (n = 8 at each time point). B, Regulation of follistatin mRNA expression in mice in relation to 1 h of swimming and the following recovery period (n = 8 at each time-point). The following tissues were analyzed: spleen, liver, subcutaneous (SubQ) and visceral fat (Vis), soleus, and gastrocnemius (Gastro) muscle, heart, and kidney. C, Western blot on liver tissue from mice performing 1 h of swimming exercise. Time points 1 h, 3 h, and 6 h are depicted as the relative proportion of the 0 h levels. *, P < 0.05.

Skeletal muscle releases IL-6 into the circulation during exercise, and we have previously shown that IL-6 acts as a myokine, signaling from the muscle to the liver to increase liver glucose production during exercise (22). In the current study, plasma IL-6 peaked before the increase in plasma follistatin (IL-6 peaks after 3 h rising from 0.3 pg/ml to 12.2 pg/ml; data not shown). Accordingly, we tested whether acute elevations of IL-6 in plasma could induce an increase of plasma follistatin in healthy male subjects. As shown in Fig. 3A, plasma IL-6 increased to 200 pg/ml (P < 0.05) during the infusion of recombinant human IL-6; however circulating follistatin was not affected (Fig. 3B).

Fig. 3.

A, Infusion of recombinant IL-6 in healthy young men (n = 7). A, Plasma IL-6 during a 3-h IL-6-infusion study. The shaded area marks the 3-h period of IL-6 infusion. B, Plasma follistatin during the 3-h IL-6-infusion study. No regulation of plasma follistatin. *, P < 0.05. The shaded area marks the 3-h period of IL-6 infusion.

Another important exercise-induced humoral factor is epinephrine. As shown in Fig. 4A, plasma glucose increased as expected in the group receiving epinephrine (P < 0.05). However, no increase in follistatin mRNA content in the liver could be detected after epinephrine administration; in contrast, a decrease was detected as demonstrated in Fig. 4B.

Fig. 4.

A, Injection (ip) of 2 mg/kg epinephrine to mice (n = 10 in each group). Plasma glucose before and after epinephrine injection. B, Injection (ip) of 2 mg/kg epinephrine to mice (n = 10 in each group) Expression of follistatin in the liver in the control and epinephrine-stimulated groups. *, P < 0.05. C, Hepatocytes treated with EPS-conditioned media from C2C12 myotubes. CON, Media from non-EPS-stimulated myotubes; MO, negative control consisting of media only; POS, TGF-β were used as a positive control. Only TGF-β could induce an increase in follistatin mRNA expression.

Media from EPS-stimulated myotubes were not able to induce follistatin in hepatocytes, suggesting a nonmuscle stimulus for exercise-induced follistatin (Fig. 4C).

Discussion

The main finding of this study is that endurance exercise induces increased levels of follistatin in the circulation. The kinetics revealed that plasma follistatin increased markedly during the recovery after exercise both in humans and in mice. The increase in plasma follistatin after exercise appears to be dependent on both the intensity and duration of exercise. Thus, 3 h of bicycling exercise induced a 7-fold increase in plasma follistatin, whereas 2 h of one-legged knee extensor exercise only increased plasma follistatin by 2-fold. The exercise-induced increase in follistatin may also be dependent on the muscle mass recruited during the exercise bout. Plasma follistatin does not vary throughout the female cyclus (2–4), and seems to be independent of gender and species, and our data suggest that exercise induces a similar response in male humans as in female mice.

Origin of exercise-induced follistatin

Because follistatin can antagonize myostatin, several studies have investigated follistatin in skeletal muscle tissue in relation to exercise. Using in vivo human and mice models, we could demonstrate neither any regulation in the skeletal muscle tissue of follistatin nor a release from the exercising human leg to the circulation. This observation was confirmed in vitro, where electrostimulated myotubes did not changed neither expression nor release of follistatin to the cell media.

Other groups have investigated the effect of exercise on the expression of follistatin mRNA in the skeletal muscle (15, 23, 24), but the results are conflicting (15). Jensky et al. (24) recently investigated the expression of follistatin in skeletal muscle in young women in relation to both acute resistance exercise and resistance training and found that neither acute exercise nor training regulates the expression of follistatin mRNA significantly in the muscle. Furthermore, Jensky et al. (23) have investigated the expression of follistatin mRNA in the skeletal muscle in young and older men performing a single bout of resistance exercise consisting of several repetitions of maximum workload. No regulation of follistatin expression is observed in regard to exercise or training, but interestingly, a higher baseline level of follistatin mRNA in older men when compared with young men was detected (23). In general these data are in line with our finding, because we do not observe any change in follistatin mRNA content in the muscle in relation to exercise. In contrast, when the mRNA expression of follistatin in skeletal muscle was assessed in postmenopausal women in hormone replacement therapy performing eccentric exercise training, an up-regulation was observed in both the intervention group and in the control group (15). Furthermore, the expression of follistatin mRNA increases markedly when mice are subjected to a hind limb-stretching protocol (16). In previous studies of follistatin in relation to exercise (15, 23, 24), focus has been on resistance exercise with relatively few repetitions of maximum physical workload. In the present study, we evaluate follistatin expression in response to a single bout of endurance exercise at lower intensity.

The present data suggest that the exercise-induced increase in plasma follistatin originates from the liver. By screening various tissues in the mouse, we found an increase in hepatic follistatin both at the mRNA and protein level. Very few known proteins are secreted from the liver in relation to an acute bout of exercise. IGF-I is produced and secreted from the liver in response to a GH stimulus. This is also observed in relation to exercise-induced GH release (25). Most studies report that plasma IGF-I increases late in recovery after exercise, with a peak values at 16–28 h after exercise (26–28); however, follistatin increases after only 3 h after an exercise bout. A related liver-derived protein, IGF-binding protein-1, has also been shown to be regulated on the mRNA level in the liver and most likely is secreted to the circulation in response to exercise (29). Interestingly IGF-I stimulates skeletal muscle protein synthesis in response to resistance training, thereby enhancing muscle hypertrophy (30). Like IGF-I, follistatin possesses the ability to promote skeletal muscle growth, because it prevents myostatin from exerting its effects on the muscle tissue (10).

Regulation of liver follistatin production

When hepatocytes were treated with conditioned media from electrostimulated myocytes, no increase in follistatin mRNA content was observed. Hence, our data do not indicate that secreted substances from skeletal muscle are involved in the regulation of exercise-induced hepatic follistatin. We have previously reported that IL-6 acts as a canonical signal between the skeletal muscle and the liver, whereby contraction-induced, muscle-derived IL-6 enhances liver glucose production (22). In addition, IL-6 possesses the ability to promote production of cortisol (31); however, based on the human IL-6 infusion experiment, it seems unlikely that cortisol is responsible for the exercise-induced increase in follistatin. Furthermore, epinephrine could not be held responsible for the exercise-induced increase in plasma follistatin when administered intraperitoneally to mice. Norepinephrine is also increased during exercise (32), but when hepatocytes in culture are stimulated with norepinephrine, no release of follistatin is observed (33). Follistatin is regulated by TGF-β; however, plasma TGF-β is only moderately affected by exercise. Heinemeier et al. (34) measured TGF-β in relation to 1 h of up-hill running and found only a moderate increase from 1100 pg/ml to 1300 pg/ml (34), making it physiologically difficult to explain the marked increase in follistatin. Taken together, it seems unlikely that the hepatic exercise-induced follistatin production is mediated by a humeral factor secreted by the exercising muscle tissue.

Effects of follistatin

During endurance exercise and recovery from exercise, a number of processes with regard to adaptation are activated including restoration of energy homeostasis and skeletal muscle remodeling. Because follistatin increases in the recovery after an exercise bout, it might be involved in the regulation of these processes. It is well documented how plasma follistatin binds and neutralizes myostatin and thereby promotes skeletal muscle hypertrophy and hyperplasia (8, 9, 10). The role of follistatin in response to exercise has mainly been investigated in resistance exercise (23). However, muscle remodeling also occurs in response to endurance exercise, and the present data could suggest that follistatin may have a regulatory role.

Considering the timing of the peak of follistatin and the fact that it is able to promote glucose uptake in other tissues (35), it may stimulate the energy turnover in skeletal muscle to meet the energy demands during recovery from exercise. Thus, although speculative, it may represent communication between the liver and skeletal muscle, a yet unappreciated phenomenon.

In conclusion, we demonstrate that follistatin is released to the circulation in response to exercise, and that the source is most likely the liver. These data introduce new perspectives to the regulation of muscle hypertrophy and may introduce new perspectives regarding regulation of energy balance, in response to exercise. Furthermore, these data might introduce new perspectives to the phenomenon of muscle-liver cross talk.

Acknowledgments

We thank Ruth Rousing, Hanne Villumsen, and Lone Nielsen (The Centre of inflammation and Metabolism, Department of Infectious Diseases and CMRC, Rigshospitalet, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark) for their technical assistance.

The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (no. 02-512-55). This study was further supported by the Danish Medical Research Council, the Commission of the European Communities (Grant Agreement no. 223576-MYOAGE), and by grants from Novo Nordisk Foundation and Danish Diabetes Association and by a Millennium Type 2 Research Grant from the Diabetes Australia Research Trust. CIM is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology and Innovation. CIM is a member of DD2 (the Danish Center for Strategic Research in Type 2 Diabetes) (the Danish Council for Strategic Research, grant no. 09-067009 and 09-075724). M.A.F. is a Principal Research Fellow of The National Health & Medical Research Council of Australia.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations

 
• CV

  Coefficient of variation

 
• EPS

  electrical pulse stimulation