Systemic IL-6 regulation of eccentric contraction-induced muscle protein synthesis

Abstract

Systemic cytokines and contractile activity are established regulators of muscle protein turnover. Paradoxically, the IL-6 cytokine family, which shares the ubiquitously expressed membrane gp130 receptor, has been implicated in skeletal muscle’s response to both contractions and cancer-induced wasting. Although we have reported that tumor-derived cachectic factors could suppress stretch-induced protein synthesis in cultured myotubes, the ability of systemic cytokines to disrupt in vivo eccentric contraction-induced protein synthesis has not been established. Therefore, we examined whether systemic IL-6 regulates basal and eccentric contraction-induced protein synthesis through muscle gp130 signaling. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and eccentric contraction-induced protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) signaling in tibialis anterior muscle of male wild-type, muscle-specific gp130 receptor knockout, and tumor-bearing Apc^Min/+ mice. Systemic IL-6 overexpression suppressed basal protein synthesis and mTORC1 signaling independently of IL-6 level, which was rescued by muscle gp130 loss. Interestingly, only high systemic IL-6 levels suppressed eccentric contraction-induced protein synthesis. Systemic IL-6 overexpression in precachectic tumor-bearing Apc^Min/+ mice accelerated cachexia development, which coincided with suppressed basal and eccentric contraction-induced muscle protein synthesis. The suppression of eccentric contraction-induced protein synthesis by IL-6 occurred independently of mTORC1 activation. Collectively, these findings demonstrate that basal protein synthesis suppression was more sensitive to circulating IL-6 compared with the induction of protein synthesis by eccentric contraction. However, systemic IL-6 can interact with the cancer environment to suppress eccentric contraction-induced protein synthesis independently of mTORC1 activation.

INTRODUCTION

Cancer cachexia, a wasting syndrome characterized by skeletal muscle depletion, contributes to increased cancer patient morbidity and mortality (19, 48). Skeletal muscle mass loss also can lead to reduced anticancer therapy tolerance, increased susceptibility to treatment toxicity, and decreased patient quality of life (6, 18, 31). Disrupted protein turnover regulation by tumor-derived factors and tumor-host immune interactions can suppress anabolic signaling and induce catabolic signaling in wasting muscle (5, 47, 48). Although our understanding of mechanisms that regulate accelerated protein degradation in cachectic muscle has advanced significantly (13), we know considerably less about the mechanisms that suppress anabolic signaling. Moreover, our current knowledge is limited to fasting or basal protein synthesis suppression (51, 52). However, habitual skeletal muscle protein synthesis regulation involves the cyclic activation of protein synthesis by a variety of stimuli throughout the day (38). Muscle contraction and feeding are two well-characterized anabolic stimuli, and their inability to induce protein synthesis (termed “anabolic resistance”) has an established role in several wasting conditions (34). Further research is warranted to determine whether the cancer environment can suppress the activation of muscle protein synthesis by anabolic stimuli, which would serve to accelerate muscle wasting.

The cytokine interleukin-6 (IL-6) has been widely investigated for its regulatory roles in muscle’s response to exercise and as a mediator of muscle wasting (9, 11, 20). Binding of IL-6 to the specific transmembrane receptor α-subunit (IL-6R/gp80) induces homodimerization of the gp130 β-subunit, which initiates intracellular signaling pathways such as JAK/STAT, Ras/ERK, and PI3K/Akt (17, 42). Systemic IL-6 and muscle gp130 signaling during cancer cachexia have been implicated in disrupted protein turnover regulation through the activation of autophagy (37), increased E3 ligase expression (4, 8, 9), and suppression of protein synthesis (51, 52). Work from our laboratory in male Apc^Min/+ (MIN) mice, an established genetic model of colorectal cancer and cachexia, has demonstrated a strong relationship between IL-6 and disrupted protein synthesis during the progression of cachexia (51, 52). Furthermore, systemic IL-6 overexpression in precachectic male MIN mice accelerated cachexia development, suppressed protein synthesis, and disrupted mammalian target of rapamycin complex 1 (mTORC1) signaling (52). Similarly, IL-6-induced STAT3 activation is associated with myotube atrophy and suppressed mTORC1 signaling (8, 52). However, whether systemic IL-6 signaling through muscle gp130 regulates basal and stimulated protein synthesis in vivo has not been clearly defined.

While the multifactorial nature of cachexia and the heterogeneity of human cancers have likely contributed to the lack of FDA-approved therapies for cancer cachexia, exercise has the potential to stimulate protein synthesis and abrogate wasting in a variety of disease conditions (25, 28). Healthy skeletal muscle can induce a robust protein synthesis response to resistance exercise (54), and exercise training can improve muscle mass and function in cancer patients (45). However, we have found that tumor-derived cachectic factors could suppress stretch-induced protein synthesis in cultured myotubes (22). Whether cachectic skeletal muscle can induce an anabolic response to contractions has not been widely examined and has clinical ramifications for the therapeutic potential of exercise after cachexia development. To address this research gap, our laboratory and others have examined cachectic muscle’s ability to respond to eccentric contractions (ECC) induced by high-frequency electrical stimulation in tumor-bearing mice (1, 26, 27). We have recently observed that muscle inflammatory signaling involving STAT3 and NF-κB attenuated basal and ECC-induced protein synthesis in tumor-bearing mice (26). However, a specific role for systemic IL-6 and muscle gp130 signaling in the regulation of protein synthesis was not investigated. Therefore, the purpose of this study was to examine whether systemic IL-6 regulates basal and ECC-induced protein synthesis through muscle gp130 signaling. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling in tibialis anterior (TA) muscle of male wild-type (WT), muscle-specific gp130 receptor knockout (KO), and tumor-bearing MIN mice. Collectively, we found that basal protein synthesis suppression was more sensitive to circulating IL-6 than the induction of protein synthesis by ECC. However, systemic IL-6 could interact with the cancer environment to suppress ECC-induced protein synthesis independently of mTORC1 activation.

MATERIALS AND METHODS

Animals.

Male mice harboring a LoxP-flanked (floxed) IL-6ST gene (provided by Dr. Colin Stewart in collaboration with Dr. Lothar Hennighausen) were crossed with heterozygous myosin light chain 1f (Mlc)-Cre mice [provided by Dr. Steven Burden (10)]. Offspring positive for Cre were selected and crossed with IL-6ST^lox/lox to obtain breeding pairs to generate the mice used in the current study: IL-6ST^lox/lox;Mlc-Cre^+/−, which lack gp130 expression specifically in muscle cells (herein referred to as KO), and IL-6ST^lox/lox;Mlc-Cre^−/−, which express gp130 in muscle cells (herein referred to as WT). We have previously demonstrated that these mice have reduced gp130 mRNA and membrane protein expression specifically in skeletal muscle (21, 39). In addition, male Apc^Min/+ (MIN) mice were originally purchased from Jackson Laboratories (Bar Harbor, ME), bred at the University of South Carolina’s Center for Colon Cancer Research Mouse Core, and obtained from the investigator’s breeding colony for the current experiments. The male MIN mouse, an established genetic model of colorectal cancer, develops cachexia that is dependent on high circulating IL-6 levels (11). We recently reported that female mice demonstrate a decreased sensitivity to IL-6 for the induction of cachexia; therefore, male mice were used in all experiments (29, 30). Littermates lacking the Apc mutation (C57BL/6) were used as controls. All mice used in the current study were on a C57BL/6 background and genotyped by PCR analyses using tail genomic DNA, as previously described (32, 39). Mice were individually housed, kept on a 12:12-h light-dark cycle, and had access to standard rodent chow (catalog no. 8604 Rodent Diet; Harlan-Teklad) and water ad libitum. Body weight and food measurements were taken weekly, and the percentage of body weight loss from peak body weight was calculated (WT and MIN experiments). Animal experiments were approved by the University of South Carolina’s Institutional Animal Care and Use Committee.

Experimental designs.

There were no differences in animal characteristics observed between the two separate cohorts of male WT littermate mice used to generate KO or MIN mice; therefore, cohorts were combined to determine whether systemic IL-6 regulates basal and ECC-induced muscle protein synthesis. In the first experiment, male WT mice (n = 33; 12 wk of age) were subjected to either vector (n = 17) or IL-6 (n = 16) overexpression for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling. Systemic IL-6 overexpressing mice were then further stratified by median values into low (n = 8) and high (n = 8) plasma IL-6 levels to determine if the IL-6 dose affected protein synthesis and mTORC1 signaling. In the second experiment, KO mice (n = 13; 12 wk of age) were subjected to either vector (n = 6) or IL-6 (n = 7) overexpression for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling. KO mice were compared with their WT littermates (Vector: n = 7; IL-6: n = 7) when appropriate. In the third experiment, MIN mice (n = 17; 12 wk of age) were subjected to either vector (n = 8) or IL-6 (n = 9) overexpression for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling. MIN mice were compared with their WT littermates (Vector: n = 10; IL-6: n = 9) when appropriate. The number of mice used in each experiment was calculated on the basis of our previous systemic IL-6 overexpression and contraction studies (26, 40, 53). A minimum of six animals was required to detect significant changes in puromycin incorporation (±20% of mean, SD of 10%, with type I error = 5% and type II error = 10%) between groups.

Systemic IL-6 overexpression.

Intramuscular electroporation of an IL-6 overexpression plasmid in vivo was used to increase systemic plasma IL-6 levels in mice, as previously described (11, 52). The quadriceps muscle was used as a vessel to produce and secrete IL-6 into circulation and was not used for any analyses in the study. This experimental approach has been utilized by our laboratory and others to induce long-term production of secreted proteins from muscle (reviewed in Refs. 7 and 49). Briefly, mice were injected with 50 µg of the IL-6 plasmid driven by the CMV promoter, or empty control vector, into the quadriceps muscle. To accomplish this, mice were anaesthetized with a 2% mixture of isoflurane and oxygen (1 l/min), the leg was shaved, and a small incision was made over the quadriceps muscle. Fat was dissected away from the muscle, and the plasmid was injected in a 50-µl volume of phosphate-buffered saline (PBS). A series of eight 50-ms, 100-V pulses was then used to promote uptake of the plasmid into myofibers, and then the incision was closed with a wound clip. Both vector control and IL-6 groups received the appropriate plasmid starting at 12 wk of age, and a second electroporation on the opposite leg was performed at 13 wk of age to maintain systemically elevated plasma IL-6 levels. At 14 wk of age, mice were euthanized 3 h after a single ECC bout. The tibialis anterior (TA) muscle that performed ECC was not subjected to electroporation.

Eccentric contractions.

Unilateral ECC of the TA muscle was induced by high-frequency electrical stimulation of the sciatic nerve, as previously described with slight modifications (2, 27). Mice were anesthetized via isoflurane (2% in O₂ with 1.5% maintenance), the hindlimb region was shaved, and two needle electrodes were placed subcutaneously to stimulate the sciatic nerve proximal to bifurcation. Tetanic muscle contractions were generated using a Grass Stimulator (Grass Instruments) for 10 sets of 6 repetitions (100 Hz, 6–12 V, 1 ms duration). Ten seconds of rest was given between repetitions, and 50 s of rest was given between sets. The stimulation protocol recruits all motor units and results in net plantar flexion of the ankle. The dorsiflexors (TA and extensor digitorum longus) undergo ECCs while the plantar flexors (gastrocnemius, soleus, and plantaris) perform concentric muscle contractions. In all experiments, a single hindlimb was stimulated while the nonstimulated hindlimb served as intra-animal, noncontracted control. Mice were given an intraperitoneal injection of warm saline following the stimulation procedure and were returned to cages upon complete recovery. This contraction protocol has been shown to stimulate TA protein synthesis and mTORC1 signaling in rodents (26, 44, 54). The time point (3 h post-contraction) was selected on the basis of our previous work and that of others, which demonstrated that mTORC1 signaling is maximal at this time and corresponds to the activation of protein synthesis (2, 26, 36, 44).

Protein synthesis measurement.

The Surface Sensing of Translation (SUnSET) technique was used to determine estimated muscle protein synthesis rates, as previously described (24, 35). Briefly, puromycin (EMD Millipore, catalog no. 540222) was dissolved in sterile saline and delivered by intraperitoneal injection (0.04 µmol/g body wt) 30 min before euthanasia. Puromycin incorporation into newly synthesized peptides, reflecting estimated global protein synthesis rates, was analyzed by Western blot.

Tissue collection.

Mice were anesthetized with a subcutaneous injection of ketamine-xylazine-acepromazine cocktail (1.4 ml/kg body wt) at the time of euthanasia. Muscles and organs were rapidly excised, cleared of excess connective tissue, rinsed in PBS, dried on blotting paper, weighed, and snap-frozen in liquid nitrogen. Immediately before dissection, blood was collected via retroorbital sinus with heparinized capillary tubes, placed on ice, and centrifuged (10,000 g for 10 min at 4°C). The supernatant was removed and stored for plasma IL-6 analysis. Plasma and tissue samples were stored at −80°C until analysis.

Western blotting.

Western blot analysis was performed as previously described (26). Briefly, frozen TA muscle was homogenized in Mueller buffer (50 mM HEPES, pH 7.4, 0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM Na₄P₂O₇, 100 mM β-glycerophosphate, 25 mM NaF, 1 mM Na₃VO₄, 0.5 μg/ml leupeptin, 0.5 μg/ml pepstatin, and 0.3 μg/ml aprotinin) and fractionated into soluble and insoluble fractions by centrifugation, and the protein concentration was determined by the Bradford method. Crude TA muscle extracts (10–60 µg) were fractionated on 7–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were stained with Ponceau red to verify equal loading and transfer. Membranes were then blocked at room temperature (RT) for 1 h in 5% nonfat milk-Tris-buffered saline with 0.1% Tween 20 (TBST). Primary antibodies for puromycin (EMD Millipore, catalog no. MABE343, 1:2,000), phospho-p70S6K (T389, catalog no. 9205, 1:1,000), total p70S6K (catalog no. 2708, 1:1,000), rpS6 (S240/244, catalog no. 2215, 1:500), total rpS6 (catalog no. 2217, 1:1000), phospho-Akt (S473, catalog no. 4060, 1:1,000), total Akt (catalog no. 9272, 1:2,000), phospho-NF-κB (S563, catalog no. 3033, 1:500), total NF-κB (catalog no. 4764, 1:2,000), phospho-STAT3 (Y750, catalog no. 9145, 1:1,000), total STAT3 (catalog no. 4904, 1:2,000), phospho-AMPK (T172, catalog no. 2535, 1:2,000), total AMPK (catalog no. 2532, 1:1,000), phospho-acetyl-CoA carboxylase (ACC; S59, catalog no. 3661, 1:1,000), total ACC (catalog no. 3662), gp130 (M-20; catalog no. sc-656; Santa Cruz Biotechnology), and GAPDH (catalog no. 2118, 1:10,000) were dissolved in TBST containing 1% BSA. After overnight incubation at 4°C, membranes were washed in TBST and incubated in either 5% milk-TBST containing anti-rabbit (catalog no. 7074, 1:5,000) or anti-mouse (catalog no. 7076, 1:5,000) IgG horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. The only exception was that horseradish peroxidase-conjugated rabbit anti-mouse IgG2a antibody (Life Technologies, catalog no. 610220, 1:5,000) in 5% milk-TBST was used for puromycin. Antibodies were from Cell Signaling Technology unless otherwise stated. We had previously validated and optimized experimental conditions for these antibodies in mouse skeletal muscle following eccentric contractions (26). In addition, we had validated the specificity of membrane gp130 expression in crude muscle extracts through comparisons with membrane and cytosolic fractions in both WT and KO mice. The nonspecific band (white arrow, Fig. 3A) is not observed in muscle membrane fractions or C₂C₁₂ myotube protein extractions (21). TA protein extracts from a mouse that did not receive puromycin at euthanasia was included on all puromycin gels as a negative control. Enhanced chemiluminescence (ECL; GE Healthcare Life Sciences) was used to visualize the antibody-antigen interactions. Immunoblot images were collected using a digital imager (SynGene GBox) and quantified by densitometry using imaging software (ImageJ, NIH, Bethesda, MD). Each gel contained control samples for normalization.

Fig. 3.

Role of muscle gp130 signaling in protein synthesis regulation by systemic IL-6. A: muscle gp130 protein expression. Validation of specific (closed arrow) and nonspecific (open arrow) binding in crude muscle extracts was previously performed by our laboratory using membrane and cytosolic fractions from WT and gp130 knockout (KO) muscle (21). B: muscle protein synthesis regulation by systemic IL-6 in KO mice. C: muscle mTORC1 signaling regulation by systemic IL-6 in KO mice. D: eccentric contraction (ECC)-induced muscle protein synthesis regulation by systemic IL-6 in KO mice. E: ECC-induced muscle mTORC1 signaling regulation in KO mice. The SUnSET technique was used to determine estimated muscle protein synthesis rates. Activation of signaling molecules was determined by the phosphorylated and total ratio when appropriate. All samples were run on the same gel and were normalized to Control values. Dotted lines represent blots from the same gel and were cropped for representative images; n = 6 (KO Vector) and 7 (KO IL-6). Data are means ± SE. Student’s t-test was used to determine differences between two groups when appropriate. Statistical significance was set at P < 0.05. *Significantly different from WT Vector; #significantly different from gp130 KO; ‡significantly different from Control leg within treatment group.

Plasma interleukin-6 concentration.

Plasma IL-6 concentrations were determined as previously described (26). A commercially available IL-6 enzyme-linked immunosorbent assay kit was obtained from BD Biosciences, and the manufacturer’s protocol was followed. Briefly, clear 96-well plates were coated and incubated overnight with an IL-6 capture antibody. The next morning, the plate was blocked with assay diluent buffer and washed, and then equal volumes of standards and plasma samples were added in duplicate. After a 2-h incubation, the plate was washed and sAV-HRP reagent was added to each well. After several washes, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added, and the reaction was developed for 20 min. The reaction was stopped with sulfuric acid, and absorbance was read at 450 nm using an iMark microplate absorbance reader (Bio-Rad Laboratories).

Statistical analysis.

Results are reported as means ± SE. Data were analyzed by repeated-measures two-way ANOVA (e.g., treatment × time) with Student-Newman-Keuls post hoc methods when appropriate. Student’s t-test was used to determine differences between two groups (e.g., Vector vs. IL-6) when appropriate. The accepted level of significance was set at P < 0.05 for all analyses. Statistical analysis was performed using GraphPad (Prism 5 for Mac OS X, La Jolla, CA).

RESULTS

Basal protein synthesis regulation by systemic IL-6.

We have previously found that tumor-derived factors can suppress basal and stretch-induced protein synthesis in myotubes (22); therefore, we extended these studies to determine whether systemic IL-6 in the absence of a cancer environment could disrupt basal protein synthesis. Systemic IL-6 overexpression was performed for 2 wk, and we then examined protein synthesis and mTORC1 signaling in wild-type (WT) mice (Fig. 1A). Systemic IL-6 overexpression did not alter normal body weight gain or daily food intake (Table 1) and did not induce TA muscle atrophy, epididymal fat mass loss, or seminal vesicle atrophy (Table 1). However, systemic IL-6 overexpression activated TA muscle inflammatory (STAT3, NF-κB) and metabolic (AMPK, ACC) signaling pathways (Fig. 1, B and C; P < 0.01), and suppressed basal muscle protein synthesis (Fig. 1D; P < 0.0001). We also stratified systemic IL-6 overexpression in mice by median values into low (88 ± 7 pg/ml, n = 8) and high (223 ± 34 pg/ml, n = 8) plasma IL-6 levels (Table 2). The high IL-6 overexpression group had greater suppression of basal muscle protein synthesis compared with the low IL-6 group (Fig. 1E; P < 0.01), demonstrating an IL-6 sensitivity to dose. Interestingly, IL-6 overexpression induced muscle Akt phosphorylation, whereas downstream mTORC1 targets p70S6K and rpS6 phosphorylation were suppressed (Fig. 1F; P < 0.001). However, neither Akt activation nor mTORC1 suppression (p70S6K, rpS6) was altered by plasma IL-6 level (Fig. 1G). Collectively, these results demonstrated that muscle protein synthesis was sensitive to varying plasma IL-6 levels and that systemic IL-6 can disrupt Akt/mTORC1 signaling independently of muscle wasting.

Fig. 1.

Basal protein synthesis regulation by systemic IL-6 in wild-type (WT) mice. A: experimental design. B: muscle inflammatory signaling regulation by systemic IL-6. C: muscle metabolic signaling regulation by systemic IL-6. D: muscle protein synthesis regulation by systemic IL-6. E: muscle protein synthesis regulation by low and high systemic IL-6. F: muscle mammalian target of rapamycin complex 1 (mTORC1) signaling regulation by systemic IL-6. G: muscle mTORC1 signaling regulation by low and high systemic IL-6. The Surface Sensing of Translation (SUnSET) technique was used to determine estimated muscle protein synthesis rates. Activation of signaling molecules was determined by the phosphorylated and total ratio when appropriate. All samples were run on the same gel and normalized to Control values. Dotted lines represent blots from the same gel and were cropped for representative images; n = 17 (Vector), 16 (IL-6), 8 (Low IL-6), and 8 (High IL-6). Data are means ± SE. Student’s t-test was used to determine differences between two groups when appropriate. Statistical significance was set at P < 0.05. *Significantly different from WT Vector; †significantly different from Low IL-6.

Table 1.

Animal characteristics of WT mice following 2 wk of systemic IL-6 overexpression

	WT
	Vector	IL-6
No. of mice	17	16
Body weight, g
Pre	24.8 ± 0.3	25.0 ± 0.4
Post	26.0 ± 0.2*	25.7 ± 0.4*
%Change from Pre	4.7 ± 0.9	2.8 ± 0.5
Tibialis anterior, mg	47 ± 0.8	46 ± 0.9
Epididymal fat, mg	291 ± 11	286 ± 15
Spleen, mg	80 ± 6	132 ± 4†
Testes, mg	192 ± 5	202 ± 5
LABC, mg	86 ± 2	82 ± 2
Seminal vesicle, mg	247 ± 9	240 ± 10
Plasma IL-6, pg/ml	0 ± 0	156 ± 24†
Food intake, g/day	3.7 ± 0.1	3.6 ± 0.1
Tibia length, mm	16.9 ± 0.1	16.9 ± 0.1

Data are means ± SE. A two-way ANOVA (IL-6 × time) was used to determine differences in body weight over time. Student’s t-test was performed to determine difference between all other variables. Statistical significance was set at P < 0.05. WT, wild type; LABC, levator ani/bulbocavernosus muscle.

^*Main effect of post time point;

^†different from Vector.

Table 2.

Animal characteristics of WT mice with low and high plasma IL-6 levels following 2 wk of systemic IL-6 overexpression

	WT
	Low	High
No. of mice	8	8
Body weight, g
Pre	24.9 ± 0.7	25.1 ± 0.4
Post	25.8 ± 0.7*	25.7 ± 0.4*
%Change from Pre	3.2 ± 0.9	2.4 ± 0.5
Tibialis anterior, mg	46 ± 0.9	47 ± 1.6
Epididymal fat, mg	290 ± 17	283 ± 26
Spleen, mg	128 ± 3	137 ± 7
Testes, mg	211 ± 7	194 ± 5
LABC, mg	82 ± 2	83 ± 4
Seminal vesicle, mg	228 ± 15	250 ± 12
Plasma IL-6, pg/ml	88 ± 7	223 ± 34†
Tibia length, mm	16.9 ± 0.1	16.9 ± 0.1

Data are means ± SE. A two-way ANOVA ([IL-6] × time) was used to determine differences in body weight over time with Student-Newman-Keuls post hoc methods performed when appropriate. Student’s t-test was performed to determine difference between all other variables. Statistical significance was set at P < 0.05. LABC, levator ani/bulbocavernosus muscle; WT, wild type.

^*Main effect of post time point;

^†different from Vector.

ECC-induced protein synthesis regulation by systemic IL-6.

We next examined whether systemic IL-6 could disrupt ECC-induced muscle protein synthesis. Systemic IL-6 overexpression was performed for 2 wk, and we then examined protein synthesis and mTORC1 signaling 3 h after a single ECC bout in WT mice (Fig. 2A). This time point was chosen on the basis of our previous work and that of others which have demonstrated that mTORC1 signaling is maximal at this time and corresponds to the activation of protein synthesis (26, 36). When all mice undergoing systemic IL-6 overexpression were examined collectively, we observed no effect of IL-6 on ECC-induced protein synthesis (Fig. 2B). However, when the mice were stratified by low and high IL-6 levels, the ECC-induced protein synthesis response was attenuated by high IL-6 overexpression (Fig. 2C; P < 0.001). Interestingly, the ECC induction of mTORC1 signaling was greater following systemic IL-6 overexpression (Fig. 2D; P < 0.01); however, this response was not affected by plasma IL-6 level (Fig. 2E). These findings demonstrate that ECC-induced protein synthesis was sensitive to systemic IL-6 levels. Interestingly, ECC-induced mTORC1 signaling was resistant to varying plasma IL-6 levels and was disassociated from protein synthesis suppression at high IL-6 levels.

Fig. 2.

Eccentric contraction (ECC)-induced protein synthesis regulation by systemic IL-6 in WT mice. A: experimental design. B: ECC-induced muscle protein synthesis regulation by systemic IL-6. C: ECC-induced muscle protein synthesis regulation by low and high systemic IL-6. D: ECC-induced muscle mTORC1 signaling regulation by systemic IL-6. E: ECC-induced muscle mTORC1 signaling regulation by low and high systemic IL-6. The SUnSET technique was used to determine estimated muscle protein synthesis rates. Activation of signaling molecules was determined by the phosphorylated and total ratio when appropriate. All samples were run on the same gel and normalized to Control values. Dotted lines represent blots from the same gel and were cropped for representative images; n = 17 (Vector), 16 (IL-6), 8 (Low IL-6), and 8 (High IL-6). Data are means ± SE. Student’s t-test or one-way ANOVA was used to determine differences when appropriate. Statistical significance was set at P < 0.05. *Significantly different from WT Vector; †significantly different from Low IL-6; ‡significantly different from Control leg within treatment group.

Role of muscle gp130 signaling in protein synthesis regulation by systemic IL-6.

IL-6 initiation of intracellular signaling requires the membrane gp130 receptor (17, 42). Thus, we examined whether the muscle gp130 receptor was necessary for systemic IL-6 regulation of protein synthesis. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling in mice lacking the gp130 receptor specifically in skeletal muscle (KO). The phenotype of the mice lacking muscle gp130 has been previously characterized by our laboratory (21, 39), and in the current study we found that gp130 loss combined with systemic IL-6 overexpression had no additional effects on the mouse phenotype (Table 3). IL-6 overexpression in KO mice did not affect body weight gain, TA muscle mass, epididymal fat mass, or seminal vesicle mass (Table 3). Reduced TA gp130 protein expression was observed in KO mice regardless of systemic IL-6 overexpression (Fig. 3A; P < 0.0001). Muscle gp130 loss blocked IL-6 suppression of basal protein synthesis and mTORC1 signaling (Fig. 3, B and C; P < 0.01). While ECC induced protein synthesis in KO mice regardless of systemic IL-6 overexpression (Fig. 3D; P < 0.01), the induction by ECC was attenuated compared with WT mice (WT: 1.45 ± 0.07, KO: 1.14 ± 0.04, P = 0.01). Systemic IL-6 overexpression in KO mice did not disrupt ECC-induced mTORC1 signaling (Fig. 3E; P < 0.01). Collectively, these results demonstrate that systemic IL-6 through muscle gp130 regulates basal protein synthesis and mTORC1 signaling. However, IL-6 regulation of ECC-induced protein synthesis occurs independently of gp130 signaling.

Table 3.

Animal characteristics of muscle gp130 KO mice following 2 wk of systemic IL-6 overexpression

	WT	KO
	Vector	Vector	IL-6
No. of mice	7	6	7
Body weight, g
Pre	24.8 ± 0.5	26.3 ± 0.8	25.6 ± 0.6
Post	25.7 ± 0.4	27.0 ± 0.8*	26.4 ± 0.5*
%Change from Pre	3.9 ± 1.9	2.8 ± 0.6	2.8 ± 0.6
Tibialis anterior, mg	47 ± 0.6	49 ± 1.4	49 ± 1.0
Epididymal fat, mg	274 ± 9	295 ± 26	284 ± 23
Spleen, mg	87 ± 5	76 ± 4	137 ± 3†
Testes, mg	185 ± 9	211 ± 4‡	222 ± 6
LABC, mg	90 ± 2	85 ± 2	84 ± 1
Seminal vesicle, mg	259 ± 12	257 ± 15	273 ± 16
Plasma IL-6, pg/ml	0 ± 0	0 ± 0	200 ± 34†
Tibia length, mm	16.8 ± 0.1	17.0 ± 0.1	16.9 ± 0.1

Data are means ± SE. Wild-type (WT) and knockout (KO) littermates subjected to control Vector electroporation were compared by Student’s t-test to determine differences with gp130 loss. A two-way ANOVA (IL-6 × time) was used to determine differences in body weight over time between Vector and IL-6 within KO mice. Student’s t-test was performed to determine differences in all other variables between Vector and IL-6 within KO mice. Statistical significance was set at P < 0.05. LABC, levator ani/bulbocavernosus muscle.

^*Main effect of post time point;

^†different from KO Vector;

^‡different from WT Vector.

Basal and ECC-induced protein synthesis regulation by systemic IL-6 in tumor-bearing mice.

We previously reported that systemic IL-6 overexpression in precachectic male MIN mice can accelerate cachexia development (3, 41, 52). We extended this work in the current studies to examine whether systemic IL-6 within the context of a cancer environment could disrupt basal and ECC-induced muscle protein synthesis. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and ECC-induced protein synthesis and mTORC1 signaling in MIN mice. As expected, systemic IL-6 overexpression in MIN mice increased plasma IL-6 levels and accelerated cachexia development (Table 4; P < 0.01). MIN mice overexpressing IL-6 displayed several key characteristics of cachexia, including body weight loss, TA muscle atrophy, epididymal fat depletion, and hypogonadal features [levator ani/bulbocavernosus (LABC) and seminal vesicle atrophy; Fig. 4A and Table 4; P < 0.05]. Plasma IL-6 levels were negatively correlated with TA muscle mass in MIN mice overexpressing IL-6 (Fig. 4B; P = 0.01). Systemic IL-6 overexpression activated signaling pathways related to inflammation (STAT3, NF-κB) and metabolism (AMPK, ACC) (Fig. 4, C and D; P < 0.01), as we observed during the natural progression of cachexia in male MIN mice (52). Furthermore, systemic IL-6 overexpression also suppressed basal protein synthesis and disrupted mTORC1 signaling (Fig. 4, E and F; P < 0.05). These findings demonstrate that systemic IL-6 overexpression accelerated cachexia development, which coincided with suppressed basal protein synthesis and mTORC1 signaling in male MIN mice.

Table 4.

Animal characteristics of Apc^Min/+ mice following 2 wk of systemic IL-6 overexpression

	WT	MIN
	Vector	Vector	IL-6
No. of mice	10	8	9
Body weight, g
Pre	24.7 ± 0.3	23.1 ± 0.7	22.0 ± 0.6
Post	26.1 ± 0.3	22.9 ± 0.8*†	20.2 ± 0.6†‡
%Change from Pre	5.3 ± 0.7	−1.0 ± 1.7*	−9.0 ± 1.2‡
Tibialis anterior, mg	47 ± 1.4	39 ± 1.9*	32 ± 2.0‡
Epididymal fat, mg	298 ± 15	207 ± 29*	100 ± 37‡
Spleen, mg	77 ± 8	299 ± 45*	428 ± 34‡
Testes, mg	197 ± 5	192 ± 7*	154 ± 13‡
LABC, mg	84 ± 2	67 ± 5*	48 ± 5‡
Seminal vesicle, mg	239 ± 12	154 ± 23*	86 ± 20‡
Plasma IL-6, pg/ml	0 ± 0	31 ± 5*	130 ± 26‡
Food intake, g/day	3.7 ± 0.1	3.2 ± 0.1*	3.1 ± 0.1
Tibia length, mm	16.9 ± 0.1	16.7 ± 0.1*	16.6 ± 0.1

Data are means ± SE. Wild-type (WT) and Apc^Min/+ (MIN) littermates subjected to control Vector electroporation were compared by Student’s t-test to determine differences with tumor presence. A two-way ANOVA (IL-6 × time) was used to determine differences in body weight over time between Vector and IL-6 within MIN mice. Student’s t-test was performed to determine differences in all other variables between Vector and IL-6 within MIN mice. Statistical significance was set at P < 0.05. LABC, levator ani/bulbocavernosus muscle.

^*Different from WT Vector;

^†main effect of post time point;

^‡different from MIN Vector.

Fig. 4.

Basal protein synthesis regulation by systemic IL-6 in Apc^Min/+ (MIN) mice. A: body weight change during systemic IL-6 overexpression in MIN mice. B: relationship between tibialis anterior (TA) muscle mass and plasma IL-6 in MIN mice. C: muscle inflammatory signaling regulation by systemic IL-6 in MIN mice. D: muscle metabolic signaling regulation by systemic IL-6 in MIN mice. E: muscle protein synthesis regulation by systemic IL-6 in MIN mice. F: muscle mTORC1 signaling regulation by systemic IL-6. The SUnSET technique was used to determine estimated muscle protein synthesis rates. Activation of signaling molecules was determined by the phosphorylated and total ratio when appropriate. All samples were run on the same gel and normalized to Control values. Dotted lines represent blots from the same gel and were cropped for representative images; n = 8 (MIN Vector) and 9 (MIN IL-6). Data are means ± standard error. Student’s t-test or one-way ANOVA was used to determine differences when appropriate. Statistical significance was set at P < 0.05. * = significantly different to Apc^Min/+ Vector.

We then examined whether systemic IL-6 overexpression in male tumor-bearing MIN mice disrupted ECC-induced protein synthesis. Whereas ECC robustly activated muscle STAT3 and NF-κB signaling in control MIN mice, NF-κB was induced by ECC only in MIN mice overexpressing IL-6 (Fig. 5A; P < 0.05). ECC decreased AMPK and ACC signaling in control MIN, which did not occur in MIN mice overexpressing IL-6 (Fig. 5B; P < 0.05). As expected, ECC induced protein synthesis in control MIN mice; however, this induction was blocked by systemic IL-6 overexpression (Fig. 5C; P < 0.01). Interestingly, systemic IL-6 overexpression in MIN mice suppressed basal mTORC1 signaling without altering the induction by ECC (Fig. 5D; P < 0.01). Collectively, these findings demonstrate that systemic IL-6 can interact with the cancer environment to disrupt anabolic signaling induced by ECC.

Fig. 5.

Eccentric contraction (ECC)-induced protein synthesis regulation by systemic IL-6 in Apc^Min/+ (MIN) mice. A: muscle inflammatory signaling regulation by ECC and systemic IL-6 in MIN mice. B: muscle metabolic signaling regulation by ECC and systemic IL-6 in MIN mice. C: ECC-induced muscle protein synthesis regulation by systemic IL-6 in MIN mice. D: ECC-induced mTORC1 signaling regulation by systemic IL-6 in MIN mice. The SUnSET technique was used to determine estimated muscle protein synthesis rates. Activation of signaling molecules was determined by the phosphorylated and total ratio when appropriate. All samples were run on the same gel and normalized to Control values. Dotted lines represent blots from the same gel and were cropped for representative images. Data are means ± SE. Student’s t-test (within and between groups) was used to determine differences when appropriate. Statistical significance was set at P < 0.05. *Significantly different from Apc^Min/+ Vector; ‡significantly different from Control leg within treatment group.

DISCUSSION

We previously found that tumor-derived cachectic factors can suppress the mechanical activation of myotube protein synthesis by stretch in vitro (22). Our current study extends this prior research to examine whether systemic IL-6 through muscle gp130 overexpression in vivo can regulate basal and contraction-induced protein synthesis independently of cancer. In otherwise healthy mice, systemic IL-6 overexpression for 2 wk suppressed basal protein synthesis directly through the muscle gp130 receptor, which occurred independently of IL-6 dose and muscle mass loss. In contrast, only high circulating IL-6 levels suppressed ECC-induced protein synthesis. Interestingly, the sensitivity of ECC-induced protein synthesis to IL-6 dose was independent of mTORC1 activation. We also report that IL-6 overexpression in tumor-bearing mice accelerated cachexia development, which coincided with suppressed protein synthesis and mTORC1 signaling. Systemic IL-6 also interacted with the cancer environment to suppress ECC-induced protein synthesis, which was mTORC1 independent. Collectively, these findings demonstrate that basal protein synthesis suppression was more sensitive to the circulating IL-6 level compared with the induction of protein synthesis by ECC. However, the systemic cachectic environment in tumor-bearing mice served to further perturb the muscle’s anabolic signaling response to high circulating IL-6 levels. Future research is warranted to understand how IL-6 and the systemic environment disrupt contraction-induced protein synthesis during cancer cachexia.

Skeletal muscle is highly responsive to both the local and the systemic environment, and IL-6 signaling has been implicated in the regulation of muscle growth and metabolism (20). Paradoxically, we have found that acute IL-6 exposure in vitro can induce myotube protein synthesis and mTORC1 signaling (23), whereas chronic IL-6 exposure can suppress myotube protein synthesis and mTORC1 signaling (52). To date, significant gaps exist in our understanding of how chronically elevated systemic IL-6 regulates in vivo muscle protein synthesis, and this is especially true when muscle mass loss is not evident. Taken together, we report that systemic IL-6 differentially regulated basal and contraction-induced protein synthesis, and this regulation also extended to mTORC1 activation. Systemic IL-6 through muscle gp130 signaling suppressed basal protein synthesis and mTORC1 signaling. Interestingly, the suppression of ECC-induced protein synthesis by high IL-6 was independent of mTORC1 activity. These data demonstrate that high IL-6 levels in the absence of a cancer environment do not perturb mechanosensitive signaling pathways, which could have therapeutic implications in several wasting conditions. However, these findings also suggest that systemic IL-6 can regulate translation through mTORC1-independent mechanisms. Given that several complex and integrative cellular processes (e.g., initiation, elongation, termination, and ribosome recycling) influence the overall protein synthetic rate (43), it is likely that the systemic cachectic environment regulates translation at several levels. Indeed, myotube protein synthesis suppression by the cachectic mediator proteolysis-inducing factor (PIF) involves eukaryotic initiation factor 2α (eIF2α) phosphorylation, independent of mTORC1 signaling (15, 16). Since eIF2α activity can regulate translation initiation independently of mTORC1 and is highly responsive to energy and nutrient stress (50), further research is warranted to examine this regulation with IL-6 and contraction. Although not examined in the current study, IL-6/gp130 signaling may also serve to disrupt processes involving oxidative metabolism, which have established roles in protein synthesis regulation (28). Thus, determining the IL-6-sensitive intracellular signaling pathways that regulate protein synthesis is needed to fully understand the systemic environment’s influence on the muscle anabolic response to contraction.

During cancer cachexia, the disruption of muscle protein synthesis and mTORC1 signaling coincides with increased circulating plasma IL-6 in male MIN mice (51). Moreover, systemic IL-6 overexpression accelerates cachexia development and suppresses basal muscle protein synthesis in precachectic male tumor-bearing MIN mice (52). We have extended these findings to demonstrate that systemic IL-6 suppressed ECC-induced protein synthesis in male MIN mice. Interestingly, systemic IL-6 overexpression induced cachexia development in tumor-bearing mice, which was not observed in WT mice. These findings confirm previous observations that demonstrated preserved muscle mass and myofiber area during systemic IL-6 overexpression in healthy mice (3, 12) and highlight a potential interaction between systemic IL-6 and the cancer environment for cachexia development. Since several cytokines are elevated in both cancer patients and preclinical models (9, 14), further research is warranted to determine the tumor-derived factors and host interactions that synergize with circulating IL-6 to accelerate wasting. Indeed, single-treatment modalities have not proved beneficial in preventing or treating cancer cachexia in clinical trials (13). Furthermore, the cancer environment may also induce endocrine and metabolic disturbances that increase muscle’s sensitivity to gp130/STAT3 signaling. We have recently reported sex differences in IL-6 sensitivity and cachexia development in MIN mice (29, 30). Female MIN mice are protected against IL-6-induced wasting during the initiation of cachexia; however, the eventual loss of ovarian function during severe cachexia was associated with increased IL-6 sensitivity and enhanced muscle inflammatory signaling (29). Further research is warranted to understand the interactions between circulating endocrine hormones and inflammatory cytokines that can potentially alter distinct signaling pathways regulating muscle proteostasis.

The ability of the systemic cachectic environment to disrupt skeletal muscle adaptation to contraction has significant ramifications for the cancer patient. Therefore, we have examined whether cachectic muscle retains the plasticity to induce anabolic signaling following varying types of contraction (26, 40). Recently, we have examined cachectic muscle’s signaling response 3 h after a single ECC bout, a time point that corresponds to the activation of mTORC1 signaling and protein synthesis (26, 36). We observed that cachectic muscle mechanosensitive signaling pathways were uncoupled from ECC-induced protein synthesis (26). The current study has extended these findings to report that, despite mechanical signaling being maintained in wasting muscle, the ability to induce protein synthesis is disrupted by high IL-6. Chronic skeletal muscle AMPK activation in tumor-bearing mice has been implicated in suppressed protein synthesis and mTORC1 signaling (51, 52). Although the cyclic activation of muscle AMPK is associated with several health benefits of exercise (20), chronic AMPK activation can suppress mTORC1 activity and promote protein breakdown (33). Interestingly, we found that ECC-induced protein synthesis coincided with reduced AMPK activation, which has also been reported following castration and during the natural progression of cachexia (26, 44). Pharmacological (e.g., AICAR) activation of AMPK before ECC has been shown to attenuate mTORC1 signaling in skeletal muscle (46). Similarly, we found that disrupted ECC-induced protein synthesis by IL-6 was accompanied by sustained AMPK activation in cachectic MIN muscle. Future studies are needed to determine whether sustained AMPK activation is induced by metabolic and energetic stress, as systemic IL-6 can perturb mitochondrial quality control during the development of cancer cachexia (53). Given that metabolic intermediates can serve as new biomass and metabolites for posttranslational modifications, determining whether disrupted oxidative metabolism serves to impede the anabolic response to muscle contraction should be investigated in further detail.

Conclusion.

In summary, we examined systemic IL-6 regulation of basal and ECC-induced protein synthesis and mTORC1 signaling. We report that systemic IL-6 independently of dose suppressed basal protein synthesis and mTORC1 signaling, which required the muscle gp130 receptor. In contrast, only high IL-6 suppressed ECC-induced protein synthesis and occurred independently of mTORC1 activity. Thus, basal protein synthesis demonstrated greater sensitivity to circulating IL-6 compared with the induction by ECC. Systemic IL-6 overexpression in tumor-bearing mice accelerated cachexia development, which coincided with the suppression of muscle protein synthesis and mTORC1 signaling. Elevated circulating IL-6 levels in tumor-bearing mice also suppressed ECC-induced protein synthesis independently of mTORC1 activation. Future research is warranted to understand the interaction between the systemic cachectic environment and the ability to induce protein synthesis during cancer cachexia.

GRANTS

This work was supported by National Institutes of Health Grants R01 CA-121249 (National Cancer Institute) and P20 RR-017698 (National Institute of General Medical Science) to J. A. Carson; SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina, and an American College of Sports Medicine Foundation (ACSM) Foundation Research Grant to J. P. Hardee. J. P. Hardee was supported by a Donna and Andrew Sorensen Graduate Student Fellowship in Cancer Research (University of South Carolina, Center for Colon Cancer Research).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.P.H., X.W., E.C.G., H.-J.K., and J.A.C. conceived and designed research; J.P.H. and D.K.F. performed experiments; J.P.H. and J.A.C. analyzed data; J.P.H., D.K.F., X.W., E.C.G., H.-J.K., and J.A.C. interpreted results of experiments; J.P.H. prepared figures; J.P.H. and J.A.C. drafted manuscript; J.P.H., D.K.F., X.W., E.C.G., H.-J.K., and J.A.C. edited and revised manuscript; J.P.H., D.K.F., X.W., E.C.G., H.-J.K., and J.A.C. approved final version of manuscript.