IL-15 Overexpression Promotes Endurance, Oxidative Energy Metabolism, and Muscle PPARδ, SIRT1, PGC-1α, and PGC-1β Expression in Male Mice

LeBris S Quinn; Barbara G Anderson; Jennifer D Conner; Tami Wolden-Hanson

doi:10.1210/en.2012-1773

. 2012 Nov 16;154(1):232–245. doi: 10.1210/en.2012-1773

IL-15 Overexpression Promotes Endurance, Oxidative Energy Metabolism, and Muscle PPARδ, SIRT1, PGC-1α, and PGC-1β Expression in Male Mice

LeBris S Quinn ^1,^✉, Barbara G Anderson ¹, Jennifer D Conner ¹, Tami Wolden-Hanson ¹

PMCID: PMC3529369 PMID: 23161867

Abstract

Endurance exercise initiates a pattern of gene expression that promotes fat oxidation, which in turn improves endurance, body composition, and insulin sensitivity. The signals from exercise that initiate these pathways have not been completely characterized. IL-15 is a cytokine that is up-regulated in skeletal muscle after exercise and correlates with leanness and insulin sensitivity. To determine whether IL-15 can induce any of the metabolic adaptations associated with exercise, substrate metabolism, endurance, and molecular expression patterns were examined in male transgenic mice with constitutively elevated muscle and circulating IL-15 levels. IL-15 transgenic mice ran twice as long as littermate control mice in a run-to-exhaustion trial and preferentially used fat for energy metabolism. Fast muscles in IL-15 transgenic mice exhibited high expression of intracellular mediators of oxidative metabolism that are induced by exercise, including sirtuin 1, peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ coactivator-1α, and PPAR-γ coactivator-1β. Muscle tissue in IL-15 transgenic mice exhibited myosin heavy chain and troponin I mRNA isoform expression patterns indicative of a more oxidative phenotype than controls. These findings support a role for IL-15 in induction of exercise endurance, oxidative metabolism, and skeletal muscle molecular adaptations induced by physical training.

Exercise is an effective preventative measure and treatment for obesity and insulin resistance (1). Endurance exercise initiates a series of metabolic adaptations that promote fat oxidation in skeletal muscle tissue, thus interfering with the deleterious effects of fatty acids on insulin sensitivity (1, 2). Recent work has shown that exercise causes acute induction of mRNAs coding for peroxisome proliferator-activated receptor δ (PPARδ), silent information regulator of transcription (sirtuin)-1 (SIRT1), and PPARγ coactivator (PGC)-1α and -1β, whose gene products promote lipid oxidation in skeletal muscle and other tissues (3, 4). Longer-term endurance training, as well as constitutive overexpression or pharmacological activation of SIRT1, PPARδ, PGC-1α, or PGC-1β, induce skeletal muscle conversion toward a more oxidative phenotype, which in turn confers greater exercise endurance (5–8). Similar changes in gene expression and metabolism can be induced by environmental stressors such as dietary restriction and exposure to cold (8, 9). The signals from physical activity or other stressors that initiate these pathways have not been completely characterized.

IL-15 is a cytokine that is highly expressed at the mRNA level in skeletal muscle tissue (10). IL-15 is part of the innate immune system, which mediates responses of organisms to environmental stress (11). In human subjects and laboratory mice, physical exercise increases muscle IL-15 mRNA expression (12–14) and is associated with transient increases in circulating IL-15 levels (15, 16), suggesting exercise induces IL-15 release from muscle tissue. In rodents, acute administration of IL-15 increases lipid oxidation and expression of PPARδ mRNA (17). Transgenic mice (Tg mice) with constitutively elevated muscle and circulating IL-15 levels (IL-15 Tg mice) exhibit resistance to diet-induced obesity, increased insulin sensitivity, and increased expression of some markers of oxidative muscle metabolism (18, 19), whereas mice in which IL-15 is deleted are obese (20). These observations suggest that IL-15 induction may play a role in at least some of the genetic and metabolic adaptations accompanying physical exercise. This study tested the hypothesis that compared with littermate control mice, IL-15 Tg mice would exhibit enhanced exercise endurance, increased whole-body oxidative metabolism, and prooxidative molecular adaptations in skeletal muscle that are characteristic of endurance-trained subjects. Our findings support a role for IL-15 in induction of oxidative metabolism and gene expression patterns induced by exercise and/or physiological stress.

Materials and Methods

Animal subjects and husbandry

Animal procedures were approved by the VA Puget Sound Institutional Animal Care and Use Committee, and complied with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. As described previously, IL-15 Tg mice overexpressed IL-15 from the skeletal muscle-specific human skeletal actin promoter and, due to manipulation of the transgene signal sequence, exhibited high circulating levels of IL-15 (18). Mice for experiments were generated from a specific pathogen-free in-house colony by mating heterozygous IL-15 Tg males with commercially purchased C57BL/6J females (The Jackson Laboratory, Bar Harbor, ME) to produce IL-15 Tg mice and littermate control mice on a C57BL/6 background. Mouse pups were genotyped as described previously (18). Male mice at 4 months of age were used.

Mice were maintained on a medium-fat/medium-calorie breeder diet (4.6 kcal/g), by kilocalories 23% protein, 22% fat, and 55% carbohydrate (PicoLab Mouse Diet 20; Purina LabDiets, St. Louis, MO). Food and water were provided ad libitum, and mice were maintained on a 12-h light, 12-h dark cycle. Except for short-term metabolic analyses, mice were housed at 21 ± 3 C in groups of two or three per cage. Naive mice (not used for running trials, metabolic analyses, or brain IL-15 analyses) were used for body composition, IL-15, mRNA, DNA, and Western blot analyses. Separate sets of mice were used for running trials and metabolic analyses. For most parameters, six to nine mice per genotype were used.

Body composition

Body composition was evaluated on conscious mice by quantitative magnetic resonance using an EchoMRI-4in1/700 mouse body composition analyzer (Echo Medical Systems, Houston, TX). Total body fat and lean body mass were quantified based on the averages of triplicate measures for each animal with coefficients of variation of less than 1.2% for fat mass and less than 1% for lean mass.

IL-15 analysis

For determination of serum and muscle IL-15 levels, mice were deeply anesthetized with pentobarbital (80 mg/kg, ip) and blood collected by cardiac puncture (exsanguination), followed by tissue collection for biochemical analyses. Serum was separated and stored frozen at −20 C. In a subset of animals, brain IL-15 concentrations were determined in mice in which blood was removed by perfusion with lactated Ringer's solution (21). Tissues were homogenized in BioPlex lysis buffer (Bio-Rad, Hercules, CA), clarified by centrifugation at 4 C, and diluted to 3 mg/ml total protein, assessed using a Bio-Rad bicinchoninic acid kit. IL-15 protein levels were assessed with a Bio-Rad mouse IL-15 kit (sensitivity 6.6 pg/ml; intraassay coefficient of variation 6%) using a BioPlex protein array instrument (Bio-Rad). Further dilution of some transgenic samples was needed for accurate assessment of abundantly expressed IL-15.

Metabolism and activity assays

Metabolic parameters, physical activity, and food intake were measured using a comprehensive laboratory animal monitoring system (Columbus Instruments, Columbus, OH). Mice maintained on the breeder diet and not used for other analyses were monitored for 30 h in individual cages after 18 h of acclimation, with a 12-h light, 12-h dark cycle continued throughout the analysis. Animals were maintained near thermoneutrality (28 ± 1 C) during testing. Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were measured at 20-min intervals using open-circuit indirect calorimetry. Respiratory exchange ratio (RER), indicative of metabolic substrate utilization, was calculated by the ratio of VCO₂/VO₂, with a lower RER reflecting preferential utilization of lipid and a higher RER reflecting preferential use of carbohydrates. Energy expenditure was calculated using the Lusk equation, VO₂ × (3.815 + 1.232 RER), expressed in kilocalories per hour. Food intake was assessed continually throughout the study by disappearance of food from feeders resting upon Mettler balances. Total physical activity was measured continuously by infrared beam-breaks at a height of 1 in. above the cage floor; ambulatory activity was defined as two or more sequential horizontal beam breaks.

Running endurance

Untrained control and IL-15 Tg mice were subjected to a single run-to-exhaustion trial to assess running endurance. On the day of the trial, mice were acclimated to the testing room for 1–2 h and then placed individually in the treadmill (Eco 3/6; Columbus Instruments; inclination +5°) and acclimated to the apparatus and motor sound for 5 min. The belt was turned to a slow speed (10 m/min), raised gradually to 15–17 m/min within the initial 10 min, and the speed held constant thereafter. Maximal running speed and the total amount of time spent running at all speeds were recorded. The treadmill was not equipped with a shock apparatus and contained a stationary area at the base of the belt where mice that stopped running could rest. Mice that stopped running were encouraged to resume by gentle tapping with a tongue depressor; mice that stopped running three times in succession were deemed to have run to exhaustion. Such mice could not be coaxed to run further, and most exhibited behavioral signs of exhaustion such as labored breathing and splayed posture.

Assays of mRNA expression

Tissue samples were equilibrated in RNAlater (QIAGEN, Valencia, CA), stored at −20 C, and homogenized into Trizol (Life Technologies, Grand Island, NY) followed by RNA precipitation. Resuspended RNA was treated with deoxyribonuclease, purified on RNeasy spin columns (QIAGEN), treated with genomic DNA removal buffer, and 1.5 μg reverse transcribed into cDNA (RT² First Stand Kit; QIAGEN). Reaction quality was assessed using hypoxanthine phosphoribosyltransferase 1 (HPRT1). Real-time PCR was performed using RT² qPCR primers from QIAGEN. Primers with comparable efficiencies approaching 100% were used with RT² Real-Time SYBR Green Fluor qPCR Master Mix (QIAGEN). Cycle threshold (C_T) values were set identically for all plates, and sample C_T values were normalized to HPRT1 C_T. Similar HPRT1 expression levels allowed mRNA expression comparisons among tissues, with relative abundance of specific mRNA species calculated as 2^−ΔCT (22). For some species, relative expression in IL-15 Tg mice was presented as fold difference (2^−ΔΔCT) from expression in the same tissue in controls (22).

Western blot analyses

Polyclonal anti-IL-15 receptor-α (IL-15Rα), anti-PPARδ, and anti-PGC-1α/β were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-SIRT1 was from Abcam (Cambridge, MA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or anti-tubulin (Santa Cruz) was used as control for loading and transfer efficiency. Soleus and extensor digitorum longus (EDL) muscles were frozen in liquid N₂ and homogenized in Bio-Plex lysis buffer (Bio-Rad) containing protease inhibitors. Soluble protein fractions were assayed for protein content by the bicinchoninic acid method. Equal amounts of protein were loaded onto SDS-PAGE gels, separated, and blotted onto 0.45-μm nitrocellulose (Bio-Rad) using transfer buffer without SDS. Membranes were blocked and incubated with primary antibodies diluted to 1 μg/ml, rinsed, and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000–2500; Santa Cruz). Bands were visualized using luminol (Santa Cruz) on autoradiography film (ISC BioExpress, Kaysville, UT), and were digitized using UN-SCAN-IT gel version 5.1 (Silk Scientific, Orem, UT). Background-corrected signal (density × area) for each band was normalized to GAPDH or tubulin. PGC-1α and PGC-1β were distinguished by relative mobility (23).

Nuclear and mitochondrial DNA assays

DNA was isolated and treated with ribonuclease (QIAGEN), and quality was assessed using 230-, 260-, and 280-nm spectrophotometry. Total DNA per milligram tissue was determined using a Quant-iT PicoGreen dsDNA kit (Invitrogen, Grand Island, NY). Mitochondrial DNA copy number relative to nuclear DNA content was analyzed using primers for mitochondrial cytochrome c-oxidase II and nuclear β-globulin by real-time PCR (24). Mitochondrial genome copies per diploid nuclear DNA were calculated and standardized to tissue weight.

Statistical procedures

Statistical analyses were performed using SigmaPlot version 11.0 software (Systat Software, San Jose, CA). Differences between transgenic and control mice for most parameters were compared by Student's t tests. For metabolic and activity parameters, the significance of genotype and photoperiod (light or dark phase) effects and interactions of these factors were analyzed by two-way ANOVAs. Post hoc pairwise multiple comparisons were performed using Bonferroni t tests. Significant differences (P < 0.05) are noted in the table and figures.

Results

Phenotypic characteristics of IL-15 Tg and control mice

Baseline phenotypic characteristics were compared in untrained IL-15 Tg and littermate control mice. As reported previously (18), IL-15 Tg mice exhibited greatly elevated muscle and circulating IL-15 levels compared with controls (Table 1). However, IL-15 levels in brain tissue rinsed of vascular blood contamination were not significantly different in IL-15 Tg and control mice (Table 1). IL-15 levels in the predominately slow/oxidative soleus and predominately fast EDL muscle of control mice were similar, whereas in Tg mice, the EDL contained significantly more IL-15 than the soleus (Table 1). These values were reflective of IL-15 mRNA expression levels in the EDL and soleus muscles in each genotype (not shown).

Table 1.

Characteristics of control and IL-15 Tg mice

Parameter	Control	IL-15 Tg
Serum [IL-15] (pg/ml)	86.0 ± 7.5	103,029.6 ± 3,623.3^c
Soleus [IL-15] (pg/mg protein)^a	8.2 ± 0.78	1,747.8 ± 102.9^c
EDL [IL-15] (pg/mg protein)^a	8.2 ± 1.23	10,963.9 ± 1,014.9^c
Brain [IL-15] (pg/mg)^a	14.4 ± 0.3	18.8 ± 3.2
Body weight (g)	33.8 ± 1.2	31.9 ± 0.7
Lean body mass (g)	24.0 ± 0.4	23.1 ± 0.3
Total body fat (g)	8.2 ± 0.9	7.0 ± 0.4
Percent body fat	24.0 ± 1.9	21.9 ± 1.1
RP fat pad mass (mg)	171.3 ± 15.9	121.2 ± 12.6^b
IBAT mass (mg)	92.7 ± 7.3	100.7 ± 7.1
Soleus muscle mass (mg)	15.1 ± 4.7	12.4 ± 5.9
EDL muscle mass (mg)	19.0 ± 1.0	15.9 ± 0.5^b
Gastrocnemius muscle mass (mg)	144.7 ± 4.3	111.3 ± 4.2^c

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Tissue [IL-15] is expressed as picograms of IL-15 per milligram of soluble protein. Control soleus and EDL [IL-15] were not significantly different; Tg soleus and EDL [IL-15] were significantly different at P < 0.001. Significance of differences between groups for each parameter was determined by t tests.

P < 0.05.

P < 0.001.

Also as reported previously (18, 19), IL-15 Tg mice exhibited a trend toward lower overall body mass, lean body mass, total body fat, and percent body fat compared with controls, which was not significant in this cohort of mice (Table 1). Retroperitoneal (RP) fat pad mass, a measure of visceral adiposity, was significantly lower in IL-15 Tg mice, whereas intrascapular brown adipose tissue (IBAT) mass did not differ (Table 1). The mass of the slow/oxidative soleus muscle did not differ between genotypes, but masses of the fast EDL and the mixed-fiber-type gastrocnemius muscles were significantly lower in IL-15 Tg mice compared with controls (Table 1).

IL-15 Tg mice exhibit increased fat oxidation, energy expenditure, and running endurance

Whole-body metabolic parameters were assessed in untrained IL-15 Tg and control mice by indirect calorimetry. RER, a measure of metabolic substrate use, was significantly lower in IL-15 Tg mice, indicating preferential oxidation of fat as a substrate for energy metabolism (Fig. 1A). IL-15 Tg and control mice consumed similar quantities of food (Fig. 1B), indicating the lower RER measured in IL-15 Tg mice was not due to decreased energy intake. Total and ambulatory activity levels of IL-15 Tg mice were approximately double those of control mice in the dark (active) phase, consistent with increased endurance conferred by a more oxidative phenotype (Fig. 1, C and D). IL-15 Tg mice also exhibited higher energy expenditure in both light and dark phases, indicating alterations in energy metabolism independent of activity (Fig. 1E).

Fig. 1. — Energy metabolism, activity, and running endurance in control (CON) and IL-15 Tg (TG) mice. A, RER was significantly lower in TG mice, indicating preferential use of fat for energy metabolism. B, Food intake (FI) did not differ between genotypes. C and D, Total (C) and ambulatory (D) activity were higher in TG than in CON mice in the dark (active) phase. E, Energy expenditure was higher in TG in both light and dark phases. In A–E, significance was determined by two-way ANOVA, with different *letters* denoting groups that differ at P < 0.05 (n = 8 CON/6 TG). F, Running endurance in untrained CON and TG mice subjected to a single run-to-exhaustion trial. TG mice run about twice as long as littermate controls before reaching exhaustion. ***, Significant difference at P < 0.001 (t test). Maximal run speed was not significantly different; (n = 9 CON/8 TG). *Bars* represent means ± sem in all panels.

To test exercise endurance directly, untrained IL-15 Tg and control mice were challenged with a single run-to-exhaustion trial. IL-15 Tg mice ran approximately twice as long as controls (P < 0.001) before reaching exhaustion (Fig. 1F). The observation of increased exercise endurance in IL-15 Tg mice is consistent with the metabolic analyses that indicated IL-15 Tg mice exhibited increased oxidative metabolism, as well as greater ambulatory activity in the active phase, compared with control mice.

IL-15 Tg mice exhibit increased expression of intracellular mediators of oxidative metabolism

To examine the basis of the increased oxidative metabolism in IL-15 Tg mice, expression of intracellular mediators of oxidative metabolism were first compared at the mRNA level in multiple tissues involved in energy metabolism in untrained IL-15 Tg and control mice. For skeletal muscle, the EDL and soleus were used as examples of muscles with respective fast (glycolytic and oxidative) and slow/oxidative fiber-type predominance. Expression levels of SIRT1, PPARδ, and PGC-1β mRNA were significantly higher in EDL muscles of IL-15 Tg mice (Fig. 2, A, C, and E). The increases in SIRT1 and PGC-1β mRNA expression were specific to the EDL muscle and did not differ in the soleus muscle, liver, IBAT, or RP fat of the two genotypes; these changes are most easily noted when expressed as fold difference (Fig. 2, B and F). Although expressed in low abundance in liver tissue, PPARδ mRNA expression was also significantly increased in liver tissue of IL-15 Tg mice (Fig. 2, C and D). Similarly, PGC-1α, PPARγ, and pyruvate dehydrogenase kinase (PDK4, an inhibitor of glycolytic metabolism) mRNAs were expressed in relatively low abundance in liver but were nevertheless up-regulated in IL-15 Tg liver, but not muscle, compared with controls (Fig. 3). Subcutaneous adipose tissue (sampled from the inguinal fat depot) in IL-15 Tg mice expressed significantly lower levels of PGC-1α, PGC-1β, PPARγ, and PDK4 mRNA than in control mice (Figs. 2 and 3).

Fig. 2. — Expression of selected mRNA species involved in fat oxidation in several tissues involved in energy metabolism in untrained control (CON) and IL-15 Tg (TG) mice. Data for each species are expressed as abundance (A, C, and E) and fold difference between genotypes (B, D, and F), indicating preferential up-regulation of these species in fast skeletal muscle tissue. A and B, SIRT1 expression; C and D, PPARδ expression; E and F, PGC-1β expression. EDL, Fast/glycolytic EDL muscle; SOL, slow/oxidative soleus muscle; SQ fat, sc adipose tissue. *Bars* represent means ± sem (n = 6–9 per group depending on tissue). Significant differences between CON and TG in each tissue are denoted by *asterisks*: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3. — Differential expression of PGC-1α, PPARγ, and PDK4 in liver and sc fat of control (CON) and IL-15 Tg (TG) mice. Data for each species are expressed as abundance (A, C, and E) and fold difference between genotypes (B, D, and F). A and B, PGC-1α expression; C and D, PPARγ expression; E and F, PDK4 expression. SOL, Soleus muscle; SQ fat, sc adipose tissue. *Bars* represent means ± sem (n = 6–9 per group depending on tissue). Significant differences between CON and TG in each tissue are denoted by *asterisks*: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Western blot analysis was undertaken to confirm these findings at the protein level in the EDL and soleus muscles (Fig. 4). SIRT1 protein levels were increased in both the EDL and soleus of IL-15 Tg mice compared with controls (Fig. 4A). PPARδ and PGC-1β protein levels were increased in EDL muscle specifically, consistent with the mRNA analyses (Fig. 4, B and C). Additionally, PGC-1α protein levels were increased in the EDL muscle but not in the soleus muscle in IL-15 Tg mice (Fig. 4D). Although slight differences between the results for mRNA and protein levels were noted, in general, these findings suggest IL-15 induces oxidative metabolism by stimulating a pathway involving SIRT1, PPARδ, PGC-1β, and PGC-1α expression, particularly in fast skeletal muscle tissue. Increases in expression of these factors have been associated with exercise training, induction of oxidative metabolism, and increased endurance (3, 4, 6–8) and are consistent with the metabolic phenotype and increased running endurance observed for IL-15 Tg mice (Fig. 1).

No evidence for increased BAT formation in IL-15 Tg mice

SIRT1, PPARγ, PGC-1α, and PGC-1β are involved in promotion of highly oxidative BAT, which can also form in ectopic locations such as muscle (8, 9, 14, 25, 26). Therefore, two markers for BAT formation, uncoupling protein-1 (UCP1) and the BAT differentiation factor PRD1-BF1-RIZ1 homologous domain containing 16 (PRDM16), were examined in multiple tissues in IL-15 Tg and control mice (Supplemental Fig. 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). Using these markers, no evidence for increased BAT biogenesis or ectopic BAT formation in IL-15 Tg mice was found, consistent with a lack of difference in IBAT weight (Table 1) as well as no induction of SIRT1, PPARγ, PGC-1α, or PGC-1β in that tissue (Figs. 2 and 3).

Increased oxidative muscle phenotype in IL-15 Tg mice

Constitutive expression or activation of SIRT1, PPARδ, PGC-1α, or PGC-1β induce conversion of skeletal muscle toward a more oxidative phenotype (6–8). Therefore, several markers of skeletal muscle phenotype were compared in the fast EDL, slow/oxidative soleus, and the mixed gastrocnemius muscles of IL-15 Tg and control mice. All three muscle types exhibited increased expression of the slow mRNA isoform of troponin I, characteristic of slow/oxidative muscle fibers, in IL-15 Tg mice compared with controls (Fig. 5A). Soleus and gastrocnemius also exhibited down-regulation of the fast mRNA isoform of troponin I (Fig. 5B). Similarly, myosin heavy chain (MHC)-I mRNA, typically expressed in slow/oxidative muscle fibers, was up-regulated in all three muscles of IL-15 Tg mice (Fig. 5C). Additionally, expression of the MHC-IIx mRNA isoform, expressed in fast/oxidative muscle fibers and specifically regulated by PGC-1β (7), was increased in EDL muscles of IL-15 Tg mice, albeit decreased in IL-15 Tg soleus (Fig. 5D). Fast MHC-IIa mRNA, typically expressed in fast/oxidative-glycolytic fibers, was also significantly (P < 0.001) down-regulated in soleus muscles of IL-15 Tg mice, whereas expression of MHC-IIb (expressed in fast/glycolytic fibers) did not differ (not shown). These findings suggest IL-15 is involved in conversion of skeletal muscle toward a more oxidative phenotype and are consistent with up-regulated SIRT1, PPARδ, PGC-1β, and PGC-1α expression as well as the greater running endurance exhibited by IL-15 Tg mice.

Fig. 5. — Expression of mRNA markers for muscle phenotype in control (CON) and IL-15 Tg (TG) mice in mixed gastrocnemius muscle (GASTROC), EDL, and soleus muscle. A, Troponin I slow; B, Troponin I fast; C, MHC-1, typically expressed in slow/oxidative fibers; D, MHC-IIx, typically expressed in fast/oxidative fibers; E, MHC-IIa, typically expressed in fast/oxidative-glycolytic fibers. MHC-IIb (expressed in fast/glycolytic fibers) was not different (not shown). *Bars* represent means ± sem (n = 6 per group). Significant differences between CON and TG in each muscle are denoted by *asterisks:* *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Markers of mitochondrial function and biogenesis

Markers of mitochondrial activity and/or biogenesis were examined in the EDL and soleus muscles, liver, and IBAT in IL-15 Tg and control mice. Total (predominately nuclear) DNA content was higher in EDL muscles of IL-15 Tg mice (Fig. 6A), consistent with a conversion toward a more oxidative muscle phenotype (27). In contrast, mitochondrial DNA content was not increased in any of the tissues examined (Fig. 6B). However, expression of two mRNA species coding for mitochondrial proteins involved in lipid metabolism was altered in IL-15 Tg mice (Fig. 6, C and D). The muscle isoform of carnitine palmitoyltransferase-1B (CPT1B), which promotes fatty acid oxidation (28), was significantly elevated in EDL muscle, albeit decreased in the soleus muscle (Fig. 6C). Additionally, significantly higher expression of UCP2, another modulator of fatty acid metabolism (29), was observed in EDL, liver, and IBAT of IL-15 Tg mice (Fig. 6D).

Fig. 6. — Nuclear and mitochondrial (Mt) DNA, and markers of Mt activity, in selected tissues of control (CON) and IL-15 Tg (TG) mice. A, Total (predominately nuclear) DNA per milligram tissue; B, Mt DNA per tissue weight; C, CPT1B mRNA expression; D, UCP2 mRNA expression. Significant differences, determined by t tests, between CON and TG in each muscle are denoted by *asterisks*: *, P < 0.05; **, P < 0.01; n = 6–9 mice per group depending on tissue.

IL-15Rα is not down-regulated in IL-15 Tg mice

Several of the metabolic characteristics of IL-15 Tg mice were similar to those reported for mice in which the IL-15-specific α subunit of the heterotrimeric IL-15Rα was deleted (30). It was possible that high IL-15 levels in IL-15 Tg mice could lead to down-regulated IL-15Rα expression. Therefore, expression of IL-15Rα in control and IL-15 Tg mice was examined. No differences in IL-15Rα mRNA expression were detected in any tissues examined (Fig. 7A). As reported previously (19), the β- and γ-subunits of the IL-15R (IL-2Rβ and -γ) were also not down-regulated at the mRNA level in IL-15 Tg mice (not shown). At the protein level, expression of IL-15Rα did not differ between control and IL-15 Tg mice in the EDL and soleus muscles (Fig. 7B).

Fig. 7. — IL-15Rα expression in control (CON) and IL-15 Tg (TG) mice. A, IL-15Rα mRNA expression (n = 6–9 mice per group); B, IL-15Rα protein levels in muscle tissue, determined by Western blot (n = 4 CON and 5 TG). Signal in each blot was normalized to GAPDH. SOL, Soleus muscle; SQ fat, sc adipose tissue. No significant differences between TG and CON were detected by t tests at P < 0.05.

Discussion

The present study examined exercise endurance, substrate metabolism, and expression of prooxidative intracellular factors in Tg mice with constitutively elevated circulating IL-15. Previous studies have shown that circulating IL-15 levels are elevated after exercise (15, 16) and that IL-15 increases fat oxidation and gene expression markers for oxidative metabolism (17, 19). Therefore, we hypothesized that IL-15 Tg mice would exhibit at least some of the metabolic and molecular adaptations characteristic of endurance exercise-trained subjects. When tested in a run-to-exhaustion protocol, untrained IL-15 Tg mice ran approximately twice as long as untrained littermate control mice. Moreover, indirect calorimetry indicated IL-15 Tg mice exhibited a lower RER, indicative of preferential use of fat as a substrate for energy metabolism. At the molecular level, the predominately fast EDL muscle of IL-15 Tg mice displayed significantly higher expression of intracellular factors that are up-regulated after exercise and that promote increased endurance and oxidative skeletal muscle metabolism, specifically SIRT1, PPARδ, PGC-1β, and PGC-1α (3, 4). Muscle tissue in IL-15 Tg mice also exhibited troponin I and MHC mRNA isoform expression patterns indicative of a conversion of muscle to a more oxidative phenotype. Additionally, although mitochondrial density was not increased, mediators of mitochondrial lipid oxidation such as CPT1B and UCP2 were up-regulated in IL-15 Tg mice. Inasmuch as such features are characteristic of endurance exercise-trained subjects, these findings support a role for IL-15 in induction of several of the molecular and metabolic adaptations associated with exercise.

Work by other groups has shown that IL-15 is up-regulated transiently after physical exercise. Tamura et al. (16) demonstrated significant increases in serum IL-15 levels within 10 min after treadmill running in untrained male human subjects, which returned to preexercise baselines by 180 min. Similar findings of increased plasma IL-15 immediately after intensive exercise in both trained and untrained human subjects have been reported (15). Although it is likely the increased circulating IL-15 was released from skeletal muscle due to its high expression in that tissue and association with exercise, these studies did not demonstrate rigorously that muscle was the tissue of origin. In this regard, it is suggestive that large postexercise elevations in IL-15 mRNA expression have been reported in skeletal muscle (12–14). Transient postexercise increases in SIRT1, PGC-1α, PGC-1β, and PPARδ expression occur in a time course (within 4 h) that is consistent with the transient postexercise increases in circulating IL-15 expression (3, 4). Because circulating IL-15 levels in the IL-15 Tg model employed here are elevated constitutively, our study does not demonstrate rigorously that an exercise-induced rise in circulating IL-15 levels is an acute trigger for these metabolic adaptations. However, acute administration of recombinant IL-15 into rats increases whole-body fat oxidation and muscle PPARδ mRNA expression (17). Thus, when considered in conjunction with studies showing acute postexercise increases in circulating IL-15 levels, our findings are consistent with a role for IL-15 release in induction of several of the molecular and metabolic adaptations induced by exercise.

IL-15 is distinct from a recently identified factor, irisin, which is induced by prolonged exercise protocols and PGC-1α overexpression (14). Irisin induces brown fat development within white adipose tissue, whereas our study found no evidence for such activity by IL-15. However, both irisin and IL-15 are up-regulated in transgenic mice with constitutively elevated PGC-1α expression (14). Calcium mobilization via the Ca²⁺-activated calcineurin complex has also been implicated in the metabolic and molecular adaptations to acute exercise (5, 31). The effects of calcineurin activation are similar to those of IL-15 (5, 31), but the relationship between these two factors was not addressed in the present study.

In this study, littermate controls and IL-15 Tg mice had similar body composition, with a trend toward lower body weight and adiposity, as well as significantly lower visceral fat mass, in IL-15 Tg mice. Although there were no significant differences in overall lean mass, the weights of both the fast EDL and mixed gastrocnemius muscles of IL-15 Tg mice were significantly lower than those of control mice. This is likely due to conversion of skeletal muscle to the more oxidative phenotype indicated by MHC and troponin I mRNA isoform expression patterns, because oxidative skeletal muscle fibers are much smaller than glycolytic fibers (27). In the comprehensive laboratory animal monitoring system analysis, food intake did not differ between IL-15 Tg and control mice, consistent with several previous studies using different diets that found no effects of both chronic and acute IL-15 administration on food intake in rodents (18, 32, 33). IL-15 Tg mice nevertheless exhibited higher activity levels and energy expenditure, consistent with the greater work efficiency of oxidative muscle fibers (34).

The increased endurance and prooxidative metabolic profile of IL-15 Tg mice is similar to mouse models in which SIRT1, PGC-1α, PGC-1β, and PPARδ expression are elevated constitutively (5–8). The relationships among these factors are complex, but PPARδ may be pivotal in the effects of IL-15 on muscle metabolism, because PPARδ can induce SIRT1 and PGC-1 mRNA expression (23, 35). In this regard, IL-15-induced metabolic changes in cultured skeletal muscle cell lines were shown to be dependent on PPARδ signaling (36). Although both PGC-1α and PGC-1β protein levels were elevated in IL-15 Tg mice, we observed increased PGC-1β, but not PGC-1α, expression at the mRNA level. SIRT1 deacetylates PGC-1α, which may stabilize as well as activate this molecule posttranslationally (8). PGC-1α and PCG-1β have similar, but not completely overlapping, activity and expression patterns (3, 23). Although both promote oxidative metabolism, PGC-1α induces expression of MHC-I (characteristic of slow/oxidative muscle fibers), whereas PGC-1β specifically induces MHC-IIx, a marker of fast/oxidative fibers (5, 7). In this study, increased expression of both these forms of MHC was observed in IL-15 Tg muscles, suggesting both isoforms of PGC-1 were active in IL-15 Tg mice. These MHC expression patterns, as well as those of troponin I, indicated constitutive expression of IL-15 induced a shift toward a more oxidative skeletal muscle phenotype, which may be largely responsible for the increased whole-body oxidative metabolic profile of IL-15 Tg mice revealed by indirect calorimetry.

Due to the muscle-specific promoter employed, fast, slow, and mixed (18) muscles in IL-15 Tg mice express extremely high levels of IL-15. However, this study revealed that transgenic EDL muscles contained significantly higher levels of IL-15 compared with transgenic soleus muscles. We cannot exclude the possibility that this difference was responsible for the more pronounced effects of the IL-15 transgene on SIRT1 mRNA, PPARδ, and PGC-1 expression in the EDL muscle. However, both muscles expressed very high levels of IL-15 compared with controls and also presumably had access to the high circulating levels of IL-15 in these mice. Additionally, we observed changes in SIRT1 protein and expression of slow troponin I and myosin isoforms in the soleus as well as the EDL muscle in IL-15 Tg mice. These observations suggest differences in response to IL-15 were more likely fiber-type specific, because SIRT1 mRNA, PGC-1β mRNA and protein, and PGC-1α protein were up-regulated in IL-15 Tg EDL muscles to levels similar to those in control and transgenic soleus muscles.

Oxidative muscle fibers generally exhibit increased nuclear and mitochondrial density (27). Although increased muscle nuclear density was observed in the EDL muscle from IL-15 Tg mice, increased muscle mitochondrial density was not observed in these mice. However, fast muscle tissue in IL-15 Tg mice exhibited increased expression of CPT1B and UCP2 mRNA, which code for proteins that promote lipid oxidation (28, 29). CPT1B is rate limiting for the entry of long-chain fatty acids into mitochondria, and up-regulating CPT1B increases lipid oxidation (28). UCP2, which is not involved in BAT formation but rather modulates mitochondrial reactive oxygen species formation and fatty acid metabolism (29), was up-regulated in transgenic EDL muscle, liver, and IBAT. Both CPT1B and UCP2 expression are stimulated by PPARδ (37), consistent with the hypothesis that PPARδ induction may be central to the metabolic actions of IL-15. Our findings are consistent with several studies that indicate PPARδ, SIRT1, and PGC-1 can promote fatty acid metabolism independently from mitochondrial biogenesis (8, 23, 38, 39).

We chose to express real-time PCR data as mRNA abundance, as well as the typically used fold difference calculation (22), allowing a more quantitative comparison of the effects of IL-15 on various tissues. Despite high systemic expression of IL-15 and robust expression of IL-15Rs among tissues, less robust differences in mRNA expression in other tissues involved in metabolism, particularly brown and visceral fat, were observed compared with those in muscle tissue. Nevertheless, IL-15 Tg mice displayed significant increases in expression of PPARδ, PGC-1α, PPARγ, and PDK4 mRNA expression in liver and also exhibited elevated levels of UCP2 mRNA in several tissues (EDL muscle, liver, and IBAT). It is unclear how much IL-15-induced modulations in these tissues contributed to the observed changes in whole-body metabolism. Additionally, sc fat tissue in IL-15 Tg mice exhibited significantly lower expression of PGC-1α, PGC-1β, PDK4, and PPARγ. Other studies have shown direct effects of IL-15 on white adipose tissue (20, 40), leading us (18, 32, 33, 40) and others (16) to suggest that IL-15 may constitute a so-called myokine, implying IL-15 has endocrine effects on other tissues. The results of the present study suggest that IL-15 may primarily exert a paracrine role in skeletal muscle tissue that can alter whole-body metabolism, with smaller direct effects on other tissues.

IL-15 and its receptors are expressed in brain and suggested to affect energy expenditure and stress responses (41, 42). IL-15 was also reported to cross the blood-brain barrier (43). However, we found no increase in brain IL-15 levels despite high circulating IL-15 levels in our transgenic model. Although localized transport cannot be ruled out, our findings suggest the effects of IL-15 in this study were largely peripheral. IL-15 signaling is canonically transduced by either a membrane-bound heterodimeric or heterotrimeric receptor complex (11). The heterotrimeric IL-15R comprises the IL-2R β- and γ-chains (IL-2Rβ and IL-2Rγ, respectively) plus the IL-15-specific IL-15Rα (11). However, the IL-15Rα chain is not necessary for IL-15 signaling, which can be transduced by a heterodimeric IL-2Rβ/IL-2Rγ complex (11). In an apparently contradictory report, mice in which the IL-15Rα is absent [IL-15R-knockout (KO) mice] exhibit phenotypic characteristics similar to those reported here for IL-15 Tg mice, including increased running endurance, prooxidative muscle fiber conversion, smaller muscle fiber sizes, and increased expression of PPARδ mRNA (30). It was possible that IL-15 overexpression in our model down-regulated expression of IL-15Rα. However, IL-15Rα expression was not significantly different between IL-15 Tg mice and controls, making such a mechanism for the phenotypic similarity of IL-15 Tg and IL-15RKO mice unlikely. Rather, because IL-15RKO mice have elevated circulating IL-15 levels (42), these observations suggest deletion of the IL-15Rα liberates cell surface-associated IL-15 to increase circulating IL-15 levels, thus creating a phenocopy of IL-15 Tg mice.

Endurance exercise has emerged as an effective preventative measure and treatment for obesity and insulin resistance (1, 2). Research at several levels has identified exercise-induced stimulation of intracellular mediators of oxidative metabolism such as PPARδ, SIRT1, PGC-1α, and PGC-1β as crucial to the beneficial effects of exercise on metabolism (1, 2). In both human subjects and laboratory rodents, IL-15 expression correlates with leanness and insulin sensitivity (44). Genetic variations in the genes coding for IL-15 and the IL-15Rα correlate with body composition, markers of type 2 diabetes, and athletic abilities in human subjects (15, 30, 44). Our findings identify IL-15 as an important regulator of oxidative metabolism, endurance, and muscle metabolic phenotype and as a novel regulator of PPARδ, SIRT1, PGC-1β, and PGC-1α expression in skeletal muscle tissue. The association of IL-15 with exercise and a prooxidative metabolic profile suggests IL-15 or an IL-15 derivative could be explored as a possible pharmacological exercise mimetic.

Supplementary Material

Supplemental Data

supp_154_1_232__index.html^{(973B, html)}

Acknowledgments

Dr. William Banks and Kristin Lynch (VA Puget Sound) provided advice and/or technical assistance. Drs. Brent Wisse (University of Washington), Charles Wilkinson (VA Puget Sound), and Diana Williams (Florida State University) provided helpful comments on the manuscript.

This work was supported by Merit Review BX001026 from the Department of Veterans Affairs (to L.S.Q.), National Institutes of Health (NIH) Grant RO1AG024136 from the National Institute on Aging (NIA) (to L.S.Q.), Seattle Institute for Biomedical and Clinical Research, and use of resources and facilities including the Rodent Metabolic and Behavioral Phenotyping Core at VA Puget Sound Health Care System, the Transgenic Resource Core at the University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging (NIA Grant 5P30AG-013280), and the University of Washington Diabetes Endocrinology Research Center (NIH Grant P30 DK-17047).

Disclosure Summary: The authors have no conflicts of interest to disclose.

Footnotes

Abbreviations:

CPT1B: Carnitine palmitoyltransferase-1B
C_T: cycle threshold
EDL: extensor digitorum longus
GAPDH: glyceraldehyde-3-phosphate dehydrogenase
HPRT1: hypoxanthine phosphoribosyltransferase 1
IBAT: intrascapular brown adipose tissue
IL-15Rα: IL-15 receptor-α
KO: knockout
MHC: myosin heavy chain
PDK4: pyruvate dehydrogenase kinase 4
PGC: PPARγ coactivator
PPARδ: peroxisome proliferator-activated receptor δ
RER: respiratory exchange ratio
RP: retroperitoneal
SIRT1: silent information regulator of transcription (sirtuin)-1
Tg mice: transgenic mice
UCP1: uncoupling protein-1.

References

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Associated Data

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Supplementary Materials

Supplemental Data

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[B1] 1. Benton CR, Wright DC, Bonen A. 2008. PGC-1α-mediated regulation of gene expression and metabolism: implications for nutrition and exercise prescriptions. Appl Physiol Nutr Metab 33:843–862 [DOI] [PubMed] [Google Scholar]

[B2] 2. Matsakas A, Narkar VA. 2010. Endurance exercise mimetics in skeletal muscle. Curr Sports Med Rep 9:227–232 [DOI] [PubMed] [Google Scholar]

[B3] 3. Perry CG, Lally J, Holloway GP, Heigenhauser GJ, Bonen A, Spriet LL. 2010. Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle. J Physiol 588:4795–4810 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Dumke CL, Mark Davis J, Angela Murphy E, Nieman DC, Carmichael MD, Quindry JC, Travis Triplett N, Utter AC, Gross Gowin SJ, Henson DA, McAnulty SR, McAnulty LS. 2009. Successive bouts of cycling stimulates genes associated with mitochondrial biogenesis. Eur J Appl Physiol 107:419–427 [DOI] [PubMed] [Google Scholar]

[B5] 5. Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM. 2002. Transcriptional co-activator PGC-1α drives the formation of slow-twitch muscle fibers. Nature 418:797–801 [DOI] [PubMed] [Google Scholar]

[B6] 6. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang H, Evans RM. 2004. Regulation of muscle fiber type and running endurance by PPARδ. PLoS Biol 2:e294. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Arany Z, Lebrasseur N, Morris C, Smith E, Yang W, Ma Y, Chin S, Spiegelman BM. 2007. The transcriptional coactivator PGC-1β drives the formation of oxidative type IIX fibers in skeletal muscle. Cell Metab 5:35–46 [DOI] [PubMed] [Google Scholar]

[B8] 8. Feige JN, Lagouge M, Canto C, Strehle A, Houten SM, Milne JC, Lambert PD, Mataki C, Elliott PJ, Auwerx J. 2008. Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation. Cell Metab 8:347–358 [DOI] [PubMed] [Google Scholar]

[B9] 9. Liang H, Ward WF. 2006. PGC-1α: a key regulator of energy metabolism. Adv Physiol Educ 30:145–151 [DOI] [PubMed] [Google Scholar]

[B10] 10. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh M, Johnson L, Alderson MR, Watson JD, Anderson DM, Giri J. 1994. Cloning of a T cell growth factor that interacts with the β-chain of the interleukin-2 receptor. Science 264:965–968 [DOI] [PubMed] [Google Scholar]

[B11] 11. Fehniger TA, Caligiuri MA. 2001. Interleukin-15: biology and relevance to human disease. Blood 97:14–32 [DOI] [PubMed] [Google Scholar]

[B12] 12. Nielsen AR, Mounier R, Plomgaard P, Mortensen OH, Penkowa M, Speerschneider T, Pilegaard H, Pedersen BK. 2007. Expression of interleukin-15 in human skeletal muscle effect of exercise and muscle fibre type composition. J Physiol 584:305–312 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Dieli-Conwright CM, Spektor TM, Rice JC, Sattler FR, Schroeder ET. 2009. Hormone therapy attenuates exercise-induced skeletal muscle damage in postmenopausal women. J Appl Physiol 107:853–858 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Boström P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, Rasbach KA, Boström EA, Choi JH, Long JZ, Kajimura S, Zingaretti MC, Vind BF, Tu H, Cinti S, Højlund K, Gygi SP, Spiegelman BM. 2012. A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481:463–468 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Riechman SE, Balasekaran G, Roth SM, Ferrell RE. 2004. Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses. J Appl Physiol 97:2214–2219 [DOI] [PubMed] [Google Scholar]

[B16] 16. Tamura Y, Watanabe K, Kantani T, Hayashi J, Ishida N, Kaneki M. 2011. Upregulation of circulating IL-15 by treadmill running in healthy individuals: Is IL-15 an endocrine mediator of the beneficial effects of endurance exercise? Endocr J 58:211–215 [DOI] [PubMed] [Google Scholar]

[B17] 17. Almendro V, Busquets S, Ametller E, Carbó N, Figueras M, Fuster G, Argilés JM, López-Soriano FJ. 2006. Effects of interleukin-15 on lipid oxidation. Disposal of an oral [¹⁴C]-triolein load. Bioch Biophys Acta 1761:37–42 [DOI] [PubMed] [Google Scholar]

[B18] 18. Quinn LS, Anderson BG, Strait-Bodey L, Stroud AM, Argilés JM. 2009. Oversecretion of interleukin-15 from skeletal muscle reduces adiposity. Am J Physiol Endocrinol Metab 296:E191–E202 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

IL-15 Overexpression Promotes Endurance, Oxidative Energy Metabolism, and Muscle PPARδ, SIRT1, PGC-1α, and PGC-1β Expression in Male Mice

LeBris S Quinn

Barbara G Anderson

Jennifer D Conner

Tami Wolden-Hanson

Abstract

Materials and Methods

Animal subjects and husbandry

Body composition

IL-15 analysis

Metabolism and activity assays

Running endurance

Assays of mRNA expression

Western blot analyses

Nuclear and mitochondrial DNA assays

Statistical procedures

Results

Phenotypic characteristics of IL-15 Tg and control mice

Table 1.

IL-15 Tg mice exhibit increased fat oxidation, energy expenditure, and running endurance

Fig. 1.

IL-15 Tg mice exhibit increased expression of intracellular mediators of oxidative metabolism

Fig. 2.

Fig. 3.

Fig. 4.

No evidence for increased BAT formation in IL-15 Tg mice

Increased oxidative muscle phenotype in IL-15 Tg mice

Fig. 5.

Markers of mitochondrial function and biogenesis

Fig. 6.

IL-15Rα is not down-regulated in IL-15 Tg mice

Fig. 7.

Discussion

Supplementary Material

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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