IL-6 and epinephrine have divergent fiber type effects on intramuscular lipolysis

Abstract

IL-6 is an exercise-regulated myokine that has been suggested to increase lipolysis in fast-twitch skeletal muscle. However, it is not known if a similar effect is present in slow-twitch muscle. Furthermore, epinephrine increases IL-6 secretion from skeletal muscle, suggesting that IL-6 could play a role in mediating the lipolytic effects of catecholamines. The purpose of this study was to determine whether IL-6 stimulates skeletal muscle lipolysis in a fiber type dependent manner and is required for epinephrine-stimulated lipolysis in murine skeletal muscle. Soleus and extensor digitorum longus (EDL) muscles from male C57BL/6J wild-type and IL-6^−/− mice were incubated with 1 μM (183 ng/ml) epinephrine or 75 ng/ml recombinant IL-6 (rIL-6) for 60 min. IL-6 treatment increased 5′-AMP-activated protein kinase and signal transducer and activator of transcription 3 phosphorylation and glycerol release in isolated EDL but not soleus muscles from C57BL/6J mice. Conversely, epinephrine increased glycerol release in soleus but not EDL muscles from C57BL/6J mice. Basal lipolysis was elevated in soleus muscle from IL-6^−/− mice, and this was associated with increases in adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58). The increase in ATGL content does not appear to be due to a loss of IL-6's direct effects, because ex vivo treatment with IL-6 failed to alter the expression of ATGL mRNA in soleus muscle. In summary, IL-6 stimulates lipolysis in glycolytic but not oxidative muscle, whereas the opposite fiber type effect is seen with epinephrine. The absence of IL-6 indirectly upregulates lipolysis, and this is associated with increases in ATGL and its coactivator CGI-58.

circulating IL-6 levels have been shown to increase during exercise (8, 10, 31, 34, 48), and this is thought to be the result of increases in the secretion of IL-6 from skeletal muscle (43). This observation has fueled the hypothesis that IL-6 may have direct effects on muscle substrate metabolism, which indeed has been borne out by several studies (3, 11, 23, 44). For example, treatment with recombinant IL-6 (rIL-6) has been shown to increase the phosphorylation of 5′-AMP-activated protein kinase (AMPK), the so-called master regulator of energy metabolism, in both isolated rodent skeletal muscle and in human skeletal muscle in vivo (23). Moreover, IL-6 increases skeletal muscle glucose transport (11), although this effect seems to be present only in fast-twitch skeletal muscles (11, 15).

In addition to exogenous carbohydrates, intramuscular lipids serve as an important fuel source for ATP generation during exercise (50). For instance, skeletal muscle contains 10–50 μmol/g tissue of triacylglycerol (TAG) in the form of lipid droplets (16). TAG hydrolysis is governed by the activities of distinct lipases, adipose triglyceride lipase (ATGL) which preferentially hydrolyzes TAG, and hormone-sensitive lipase (HSL) which preferentially hydrolyses diacylglycerol (DAG) (19). ATGL is less well studied but is known to require the coactivator protein, comparative gene identification-58 (CGI-58), for full activation (27). HSL is better studied and is regulated via several reversible phosphorylation sites (Ser⁵⁶³, Ser⁶⁵⁹, Ser⁶⁰⁰, and Ser⁵⁶⁵) by protein kinase A, extracellular signal-regulated kinase, and AMPK, respectively (39, 49). In addition to its stimulatory effects on glucose transport in muscle, IL-6 has recently been shown to stimulate intramuscular lipolysis in rat extensor digitorum longus (EDL) muscle, as indicated by an increase in glycerol release (23). However, it is not clear whether this occurs in oxidative muscle fibers as well, i.e., in a fiber type dependent manner.

Epinephrine is recognized as a major regulator of intramuscular lipolysis, and an estimated 50% of free fatty acids oxidized during exercise are derivatives of catecholamine-stimulated intramuscular lipolysis (14, 32). Physiological epinephrine concentrations have been shown to induce endogenous TAG breakdown in oxidative, but not glycolytic, rat muscle (36). Epinephrine infusion also produces robust increases in plasma IL-6 in both rats (6) and humans (42) and has been shown to increase IL-6 secretion from skeletal muscle in vivo (13). Exercise intensity, arterial epinephrine concentration, and skeletal muscle IL-6 secretion have also been closely correlated (18). Taken together, these data raise the question as to whether epinephrine's effects on intramuscular lipolysis are entirely direct, or are in part mediated via stimulation of IL-6 release, which can then stimulate lipolysis through an autocrine/paracrine effect.

Therefore, the primary aims of this study were to determine 1) whether there are fiber type specific differences in the ability of IL-6 to stimulate lipolysis, and 2) whether IL-6 is required for epinephrine-stimulated TAG hydrolysis in skeletal muscle. We hypothesized that IL-6 would stimulate lipolysis in both oxidative and glycolytic skeletal muscle and that it would be required for epinephrine-mediated lipolysis. To address these questions, we utilized ex vivo incubations of soleus and EDL muscles obtained from wild-type (WT; C57BL/6J) or whole body IL-6-deficient (IL-6^−/−) mice.

METHODS

Materials and reagents.

Reagents, molecular weight markers, and nitrocellulose membranes for SDS-PAGE were purchased from Bio-Rad (Mississauga, ON, Canada). Western Lightning Plus enhanced chemiluminescence (ECL) was purchased from Perkin Elmer (NEL105001EA). The following primary antibodies were purchased from Cell Signaling Technology: phospho-HSL (Ser⁵⁶³ catalog no. 4139, Ser⁶⁶⁰ catalog no. 4126), total HSL (catalog no. 4107), ATGL (catalog no. 2138), AMPK-α (catalog no. 2532), p-AMPK Thr¹⁷² (catalog no. 2531), p-STAT3 Tyr⁷⁰⁵ (catalog no. 9138), Signal transducer and activator of transcription 3 (STAT3); (catalog no. 4904), p-AKT (virus oncogene cellular homolog) Thr³⁰⁸ (catalog no. 9275), p-AKT Ser⁴⁷³ (catalog no. 9271), and glycoprotein130 (GP130; catalog no. 3732). Antibodies against the β₂-adrenergic receptor (catalog no. sc9042) and the α-subunit of the IL-6 receptor (catalog no. sc-660) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for α-tubulin were purchased from Abcam (catalog no. ab4074) and CGI-58 from Novus Biologicals (catalog no. NB110–41576). Horseradish peroxidase-conjugated donkey anti-rabbit and goat anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Free glycerol was measured using a commercially available fluorometric kit from BioVision (catalog no. K630–100). SuperScript II reverse transcriptase, random primers, and dNTP were products from Invitrogen (Burlington, ON, Canada). Taqman gene expression assays for mouse β-actin (catalog no. 4352933E) and ATGL (catalog no. 4351372) were from Applied Biosytems (Foster City, CA). Murine rIL-6 was purchased from Peprotech (catalog no. 216–16). Epinephrine (catalog no. E4642), and all other chemicals were purchased from Sigma.

Animals.

All protocols were approved by the University of Guelph Animal Care Committee and followed Canadian Council on Animal Care guidelines. Twelve-week-old male IL-6^−/− mice (Jackson Laboratories B6.12952-IL6^tmlkopf/J) and age-matched C57BL/6J WT mice were housed two per cage, with a 12:12-h light-dark cycle and were fed standard rodent chow ad libitum. There were no differences in body weight between WT and IL-6^−/− mice (28.9 ± 0.5 g WT; 27.7 ± 0.9 g IL-6^−/−).

In vitro experiments.

On the day of the experiment at ∼9–10 AM, mice in the fed state were anesthetized with pentobarbital sodium (5 mg/100 g body wt). Soleus and EDL muscles were chosen to make fiber type comparisons of epinephrine and IL-6 stimulated lipolysis. Murine soleus muscle is composed of ∼58% type I and 42% IIA fibers, while EDL is 49% IIB and 51% IIA (4). Muscles were removed and preincubated for 30 min at 30°C in oxygenated (95% O₂-5% CO₂) Krebs-Henseleit solution containing the following constituents (in mM): 125 NaCl, 5 KCl, 2.5 CaCl₂, 1.25 KH₂PO₄, 1.18 MgSO₄, 24 NaHCO₃, and 5 glucose. BSA (2%, fatty acid free) was added to media for lipolysis, but not for the signaling experiments. Soleus and EDL muscles from IL-6^−/− and WT mice were incubated in the absence or presence of 1 μM (∼183 ng/ml) epinephrine or 75 ng/ml murine rIL-6 for 60 min to measure lipolysis. Media were immediately frozen at −80°C, and free glycerol concentration was subsequently measured as an index of ex vivo lipolysis. In a separate set of experiments, soleus and EDL muscles were incubated with 1 μM epinephrine or 75 ng/ml rIL-6 for 60 min to assess signaling. Previous work examining the effect of IL-6 on lipid and glucose metabolism in isolated muscle strips used IL-6 concentrations ranging from approximately 10 to 120 ng/ml (11, 23). Based on these previous studies we chose to use a concentration of 75 ng/ml. This concentration of IL-6 is much higher than circulating levels following aerobic exercise (∼25 pg/ml) (48). However, the interstitial concentration of IL-6 surrounding muscle has been reported to increase to the nanogram per milliliter range after moderate physical activity (40). All muscles were blotted, frozen in liquid nitrogen, and stored at −80°C until analyses were performed.

To assess the effects of IL-6 on ATGL and SOCS3 gene expression in soleus, muscles were excised from C57BL/6J mice and incubated in Krebs-Henseleit buffer with or without 75 ng/ml rIL-6 for 6 h. Samples were intermittently gassed to maintain viability. All muscles were blotted, frozen in liquid nitrogen, and stored at −80°C until analyses were performed.

Free glycerol assay.

Glycerol concentration in the incubation media was measured using a fluorometric assay on a black 96-well plate. Briefly, a glycerol standard curve was prepared by diluting a 1 mM kit standard to a range of 0.1–1.2 nmol/well. All standards and samples were loaded in triplicate and wells were adjusted to 50 μl total volume with assay buffer; and 50 μl enzyme reaction mix (46 μl assay buffer, 2 μl glycerol probe, 2 μl glycerol enzyme mix) were added to samples and standards. Reactions were incubated at room temperature for 30 min and were protected from light. Concentrations were determined fluorometrically at excitation/emission wavelengths of 535/590 nm. Glycerol concentration was normalized to tissue weight (nmol/g wet weight).

Western blotting.

Soleus and EDL muscles were homogenized in a 25:1 volume-to-weight ratios of ice-cold cell lysis buffer supplemented with PMSF and protease inhibitor cocktail (Sigma catalog nos. 78830 and 9599). Samples were homogenized in a FastPrep-24 instrument (MP Biomedicals catalog no. 116004500) for two 30-s intervals and then centrifuged at 1,500 g for 15 min. Protein concentration of the supernatant was determined using the bicinchoninic acid method (41), and equal amounts of protein were separated on 10% gels to assess the protein content of ATGL, p-HSL Ser⁶⁶⁰, p-HSL Ser⁵⁶³, HSL, p-AMPK Thr¹⁷², AMPK, p-STAT3 Tyr⁷⁰⁵, STAT3, CGI-58, p-AKT Thr³⁰⁸, p-AKT Ser⁴⁷³, total AKT, GP130, β₂-adrenergic receptor, and IL-6 receptor. Proteins were transferred to nitrocellulose membranes at a constant 200 mA per tank and subsequently blocked in Tris-buffered saline-0.01% Tween (TBST) supplemented with 5% nonfat dry milk for 1 h at room temperature with gentle shaking. Membranes were incubated at 4°C overnight in primary antibodies diluted 1:1,000 in TBST with 5% BSA. The following day blots were washed with TBST and then incubated in TBST-1% nonfat dry milk supplemented with 1:2,000 horseradish peroxidase conjugated goat ant-rabbit or anti-mouse secondary antibody for 1 h at room temperature. Bands were visualized using ECL and quantified using Alpha Innotech software.

Glucose tolerance tests.

Intraperitoneal glucose tolerance tests were performed as an assessment of whole body glucose homeostasis. Mice were fasted for 6 h prior to an intraperitoneal injection of glucose (2 g/kg body wt). Blood glucose levels were determined by tail vein sampling at the indicated intervals using a glucometer. Changes in glucose over time were plotted, and the area under the curve was calculated.

Real time PCR.

RNA was isolated from soleus tissue using the Qiagen RNeasy kit according to the manufacturer's instructions. cDNA was synthesized by using 1 μg of RNA and SuperScript II reverse transcriptase, random primers, and dNTP. Real time PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems), as described previously (9). Each well (20 μl total volume) contained 1 μl cDNA template, 8 μl RNase free water, 1 μl gene expression assay, and 10 μl Taqman Fast Universal PCR Master Mix. Results for ATGL expression were normalized to the mRNA expression of β-actin. The expression of β-actin did not change with treatment [mean cycle threshold (CT) values: 22.84 ± 0.33 vehicle vs. 23.46 ± 0.77 IL-6 treated]. Relative differences between control and IL-6-treated samples were determined using the 2^−ΔΔCT method (28). The amplification efficiencies of the gene of interest and the housekeeping gene were equivalent.

Statistical analysis.

Comparisons between groups within a given genotype were made using a paired, two-tailed Student's t-test. Differences between genotypes was assessed using an unpaired t-test. Statistical significance was established at P < 0.05.

RESULTS

IL-6 increases lipolysis in glycolytic but not oxidative muscle.

Previous work has shown that IL-6 stimulates lipolysis in adipocytes (35), rat EDL muscle (23), L6 myotubes (37), primary human myocytes (1), and in humans during whole body infusions (44). However, it is not known whether IL-6 induces lipolysis in oxidative skeletal muscle or if fiber type differences exist. To address this question we treated isolated mouse EDL and soleus muscles with either epinephrine or IL-6 ex vivo. The protein content of the IL-6 receptor, GP130, and the β₂-adrenergic receptor was not different in EDL and soleus muscles harvested directly from the mouse compared with muscles that had been incubated (data not shown). As shown in Fig. 1, IL-6 failed to stimulate lipolysis in isolated mouse soleus muscle. Similarly, there were no changes in the phosphorylation of STAT3 or AMPK in response to IL-6 (Fig. 1D). A 60-min treatment with 75 ng/ml IL-6 tended to increase glycerol release from isolated mouse EDL muscle by ∼33% compared with vehicle controls (P = 0.07). In conjunction with these findings, 60 min of IL-6 exposure increased the phosphorylation of STAT3 and AMPK by ∼40% and 54%, respectively (P < 0.05) in EDL muscle (Fig. 1D).

Fig. 1.

IL-6 induces lipolysis in soleus but not extensor digitorum longus (EDL) mouse skeletal muscle. IL-6 (75 ng/ml) treatment for 60 min increased lipolysis in EDL (A) but not soleus (B) isolated muscle. The phosphorylation of STAT3 and 5′-AMP-activated protein kinase (AMPK) was increased in EDL (C) but not soleus muscle (D) following treatment with 75 ng/ml IL-6 for 60 min. Data are means ± SE. Quantified Western blot data are expressed relative to the vehicle control, n = 6 for soleus and n = 10 for EDL muscle (*P < 0.05). Representative Western blots are shown to the right of the quantified data in C and D.

Basal lipolysis is elevated in oxidative skeletal muscle from IL-6^−/− mice and not further increased by epinephrine.

To assess the interaction between IL-6 and epinephrine in stimulating skeletal muscle lipolysis, we assessed basal and epinephrine-stimulated lipolysis in skeletal muscle from WT and IL-6^−/− mice. As some have shown that IL-6^−/− mice have impaired glucose homeostasis (29), and given that this could impact lipolysis, we measured glucose tolerance in chow-fed WT and IL-6^−/− mice. Glucose tolerance (Fig. 2) and body weights were similar between genotypes. Basal lipolysis was elevated by ∼40% (P < 0.05) in soleus muscle from IL-6^−/− mice (Fig. 3A). Epinephrine significantly increased glycerol release ∼50% in WT soleus muscle (10.7 ± 0.72 vehicle, 16.07 ± 2.51 nM·gram tissue⁻¹·h⁻¹ epinephrine) but had no effect in IL-6^−/− mice (Fig. 3B). Thus, despite comparable absolute lipolytic rates with epinephrine stimulation, the fold change from basal to epinephrine-mediated lipolysis was significantly higher in WT mice (Fig. 3C). Basal glycerol release was not different in EDL muscle from WT and IL-6^−/− mice (Fig. 3D). In contrast to soleus muscle, epinephrine did not stimulate lipolysis in glycolytic skeletal muscle in either genotype (Fig. 3, E and F).

Fig. 2.

Glucose tolerance is normal in IL-6^−/− (KO) mice. Mice were injected intraperitoneally with 2 g/kg body wt glucose and changes in blood glucose over time determined. Data are means ± SE for 8–10 animals per group. WT, wild type.

Fig. 3.

Basal lipolysis is elevated in soleus muscle from IL-6^−/− mice. A: basal lipolysis is elevated in soleus muscle from IL-6^−/− compared with WT mice. B: epinephrine treatment (1 μM, 60 min) does not cause further increases in lipolysis. C: change in (epinephrine minus basal from muscles from the same animal) glycerol release. Basal (D), epinephrine (E), and change in glycerol release (F) in isolated EDL muscle from WT and IL-6^−/− mice. Data are means ± SE for 6–10 muscles per group. *P < 0.05 compared with corresponding group in the same condition.

ATGL and CGI-58 protein content are elevated in soleus muscle from IL-6^−/− mice.

Protein content of both ATGL and its coactivator CGI-58 was significantly higher in soleus muscle from IL-6^−/− compared with WT mice (Fig. 4). There were no genotype differences in total or phosphorylated HSL Ser⁵⁶³ or Ser⁶⁶⁰ residues. Similarly, the phosphorylation of AKT on serine and threonine residues was also comparable.

Fig. 4.

Basal adipose triglyceride lipase (ATGL) and comparative gene identification-58 (CGI-58) are elevated in soleus muscle from IL-6^−/− mice. The protein abundance and/or phosphorylation of hormone-sensitive lipase (HSL) and AKT were not different in soleus muscle from WT vs. IL-6^−/− mice; however, ATGL and CGI-58 were significantly increased. Data are means ± SE for 10 muscles per group and are expressed relative to WT mice. Representative blots are shown to the right of the quantified data. *P < 0.05 compared with WT.

IL-6 does not directly alter expression of ATGL in isolated soleus muscle.

Since elevated ATGL protein expression was associated with increases in basal lipolysis in IL-6^−/− soleus muscle, we questioned whether IL-6 could directly regulate ATGL mRNA in mouse soleus muscle. Treatment of isolated soleus muscle with 75 ng/ml IL-6 for 6 h did not alter the mRNA expression of ATGL (Fig. 5) or the mRNA expression of SOCS3, a marker of IL-6 signaling.

Fig. 5.

IL-6 does not induce changes in ATGL gene expression in isolated soleus muscles. Soleus muscles from C57BL/6J mice were treated with or without 75 ng/ml IL-6 for 6 h and the mRNA expression of suppressor of cytokine signaling 3 (SOCS3) and ATGL determined. Data are means ± SE and quantified relative to β-actin.

DISCUSSION

The role of IL-6 as a regulator of skeletal muscle lipolysis has not been extensively examined. Kelly et al. (23) have previously shown that 60 min of treatment with 120 ng/ml IL-6 induces lipolysis in isolated rat glycolytic (EDL) muscle. In our current study, we have extended these findings to demonstrate a distinct fiber type response. Lipolysis tended to increase in glycolytic EDL, but not in oxidative soleus muscle, during incubation with IL-6, and indices of IL-6 signaling, p-STAT3 and p-AMPK, were also increased after IL-6 treatment in EDL, but not in soleus muscle. IL-6 signal transduction is initiated by the binding of IL-6 to the IL-6 receptor GP130 complex (38), with some evidence to suggest that the soluble IL-6 receptor potentiates the effects of IL-6 (15). The blunted responsiveness to IL-6 of soleus compared with EDL muscle is not explained by differences in the protein content of the IL-6 receptor or GP130. In fact we found that GP130 protein content was similar between muscles, whereas IL-6 receptor α-protein content was actually higher in soleus compared with EDL muscle (data not shown). IL-6 signaling is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). SOCS3 binds and inhibits janus kinases, critical components of the proximal IL-6 signaling pathway (2). Recent data have shown an association between increases in SOCS3 protein content and attenuated IL-6 signaling in human skeletal muscle, independent of reductions in the content of the IL-6 receptor and GP130 (21). Thus it seems plausible that differences in the content or cellular localization of SOCS3 could explain the marked differences in IL-6 responsiveness between soleus and EDL muscles ex vivo.

As with IL-6, we also found fiber type specific differences in the response to epinephrine. However, in this instance epinephrine stimulated lipolysis in oxidative soleus but not glycolytic EDL muscle. These findings are consistent with previous work from Peters et al. (36) who demonstrated that physiological doses of epinephrine (0.1, 2.5, 10 nM) activated intramuscular triglyceride (IMTG) hydrolysis but not glycogenolysis in isolated rat soleus muscle (36). The lack of a lipolytic response to epinephrine in EDL muscle is unlikely to be related to a limitation in IMTG content, as we and Kelly et al. (23) have shown that IL-6 can stimulate lipolysis in rat and mouse EDL muscle. It is possible that the absence of an epinephrine effect in EDL could be explained by lower expression of β-adrenergic receptors in glycolytic muscle (20).

It should be acknowledged that we are measuring the net accumulation of glycerol in the incubation medium as an index of lipolysis following treatment with IL-6 or epinephrine. Several groups have shown that glycerol is taken up by skeletal muscle (17, 47); thus the accumulation of glycerol in the incubation buffer in our experiments could be a function of both release and uptake. However, we feel that, in our ex vivo experiments, glycerol uptake was unlikely to be a significant confounding factor. First, given the relatively large volume for the glycerol to dissipate, uptake would likely be minimized. Second, if glycerol uptake was significant and was different in oxidative and glycolytic fibers, this would be difficult to reconcile with our observation of opposite patterns of hormone-activated lipolysis, i.e., glycerol accumulation tended to be increased following IL-6 treatment in glycolytic muscle, but was increased in oxidative muscle with epinephrine. Thus we would attribute change in net glycerol accumulation largely to changes in glycerol efflux.

The fiber type differences observed with both IL-6 and epinephrine-mediated lipolysis may lend insight into the hormonal regulation of IMTG hydrolysis during exercise. During low- to moderate-intensity exercise (<65% V̇o_2max), slow-twitch fibers are predominantly recruited, circulating epinephrine increases, but increases in plasma IL-6 are negligible. As exercise intensity increases, fast-twitch fibers are recruited and robust increases in IL-6 are observed (22, 33). Taken in the context of our ex vivo data, it seems reasonable to speculate that, during low-intensity exercise, lipolysis in slow-twitch muscle fibers is stimulated primarily by catecholamines, whereas, during higher-intensity exercise, IL-6 increases lipolysis in the recruited fast-twitch muscle fibers.

Although IL-6 alone was not sufficient to stimulate lipolysis in mouse soleus muscle, we could not discount the fact that it could be playing a permissive role in mediating the stimulatory effects of epinephrine. To address this question, we incubated soleus muscle from WT and whole body IL-6^−/− mice ex vivo with epinephrine. As expected, epinephrine induced a >50% increase in lipolysis in soleus muscle from WT mice. Surprisingly, basal lipolysis was significantly elevated in soleus muscle from IL-6^−/− relative to WT mice, but was not further increased with epinephrine. Enhanced basal lipolysis was associated with increases in the protein content of ATGL and its coactivator CGI-58. ATGL is critical for basal and catecholamine-stimulated lipolysis and requires CGI-58 for full TAG hydrolase activation (27). Adenoviral ATGL overexpression in 3T3-L1 adipocytes caused increases in lipolysis, while siRNA-mediated knockdown decreases lipolysis under basal and isoproterenol-treated conditions (25, 51). Similarly, ATGL-null mice exhibit decreased plasma free fatty acids at rest and are not able to increase lipolysis during exercise (19). Given these findings, our observation of increased basal lipolysis in soleus muscle from IL-6^−/− mice may be causally related to elevated ATGL and CGI-58 protein expression. The inability of epinephrine to further stimulate lipolysis in soleus muscle from IL-6^−/− mice initially suggests that IL-6 may be required for epinephrine-induced lipolysis. However, as discussed later, we were unable to demonstrate IL-6 signaling in soleus muscle; therefore, the lack of a further effect was most likely due to lipolysis already being elevated.

The increase in ATGL protein content in soleus muscle from IL-6^−/− mice does not appear to be due to the loss of a direct effect of IL-6, as incubating soleus muscles with a high dose of IL-6 failed to increase the phosphorylation of AMPK and STAT3 or induce SOCS3 mRNA, markers of IL-6 signaling. As would be expected given these results, IL-6 also did not directly alter ATGL mRNA expression. Although ATGL mRNA expression was not reduced, it could be argued that IL-6 mediates its effects on ATGL through a posttranscriptional event. While plausible, this would be difficult to reconcile with the apparent lack of IL-6 signaling in isolated soleus muscle.

Our understanding of the mechanisms regulating the expression of ATGL in skeletal muscle is limited. In adipose tissue, insulin decreases ATGL expression at the transcriptional level (5, 25, 26). Interestingly, some but not all, have reported that IL-6-deficient mice develop glucose intolerance (7, 29, 46). Thus an increase in ATGL protein content could be secondary to impaired insulin action in muscle. However, in the current study, glucose tolerance was nearly identical in IL-6^−/− compared with WT mice, and the phosphorylation of AKT on serine and threonine residues in soleus muscle from IL-6-deficient mice in the fed state was not impaired. Collectively, these results would argue against a role of impaired insulin action and point toward an unidentified mechanism regulating the increased expression of ATGL in soleus muscle from IL-6^−/− mice.

In summary, we have provided novel data demonstrating fiber type specific effects of IL-6 and epinephrine on lipolysis in isolated murine skeletal muscle. Although we originally speculated that IL-6 may be required for the full effects of epinephrine to stimulate muscle IMTG lipolysis, the response to the hormones was divergent. While epinephrine has been shown to preferentially stimulate lipolysis in oxidative muscle, IL-6 stimulates lipolysis in glycolytic but not oxidative muscle. Moreover, the ablation of IL-6 leads to increases in basal lipolysis and a blunting of further lipolytic activation by epinephrine only in oxidative muscle. The increase in basal lipolysis does not appear to be a consequence of the removal of a direct effect of IL-6, as we are unable to demonstrate known IL-6 signaling in the soleus muscle. Thus, through both direct (glycolytic muscle) and unidentified indirect effects (oxidative muscle), IL-6 is able to regulate skeletal muscle lipolysis.

GRANTS

This research was funded by grants from the Canadian Institutes of Health Research (D. C. Wright) and the Natural Sciences and Engineering Research Council of Canada (D. C. Wright and D. J. Dyck). D. C. Wright is a Tier II Canada Research Chair and a Canadian Diabetes Association Scholar.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: T.L.M., Z.W., D.J.D., and D.C.W. conception and design of research; T.L.M., Z.W., S.F.-C., and D.J.D. performed experiments; T.L.M., Z.W., S.F.-C., D.J.D., and D.C.W. analyzed data; T.L.M., S.F.-C., D.J.D., and D.C.W. interpreted results of experiments; T.L.M. and Z.W. prepared figures; T.L.M., D.J.D., and D.C.W. drafted manuscript; T.L.M., Z.W., D.J.D., and D.C.W. edited and revised manuscript; T.L.M., Z.W., S.F.-C., and D.C.W. approved final version of manuscript.