Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice João-Paulo G. Camporeza, Max C. Petersena,b, Abulizi Abudukadiera, Gabriela V. Moreiraa, Michael J. Jurczakc, Glenn Friedmand, Christopher M. Haqqd, Kitt Falk Petersena, and Gerald I. Shulmana,b,e,1 a Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; cDivision of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261; dAtara Biotherapeutics, Westlake Village, CA 91363; and eHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
Contributed by Gerald I. Shulman, January 12, 2016 (sent for review November 29, 2015; reviewed by Se-Jin Lee and Michael O. Thorner)
Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an antimyostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease. aging
| myostatin | muscle mass | insulin resistance | sarcopenia
A
ging-associated sarcopenia affects more than 50 million people worldwide, including an estimated 50% of those more than 80 y of age (1–3). The increased weakness and fatigability that accompany sarcopenia have devastating consequences for patients, their families, and society. Sarcopenia reduces quality of life and physical activity, and markedly increases the incidence of falls and fractures. For older people in particular, declining muscle mass causes morbidity from loss of strength and mobility (4), reduced bone mineral density (5), reduced energy expenditure (6), changes in body composition favoring increased adiposity (7), increased insulin resistance (8), and increased incidence of type 2 diabetes (9). The estimated direct US healthcare costs of age-related muscle atrophy alone were $18.5 billion in 2000 (10). Despite this enormous unmet need, there are no approved, effective therapies to prevent or reverse age-associated sarcopenia. Recommended lifestyle modifications, such as physical rehabilitation, exercise, and nutritional support, suffer from low rates of adherence and variable effectiveness. The lack of available therapies capable of increasing muscle mass and strength highlights the need for development of effective pharmacological agents that, in turn, should be guided by increasing knowledge of the fundamental mechanisms regulating skeletal muscle growth and aging. One such mechanism regulating muscle mass and strength is signaling by myostatin. Myostatin is a member of the TGF-β superfamily of secreted growth factors. Unique among the TGF-β superfamily, it is expressed almost exclusively in skeletal muscle (11). Myostatin signaling is operative during both development and adulthood. Its cellular effects are mediated in an autocrine/paracrine manner through binding to type II activin receptors A and B (ActRIIA and ActRIIB), which recruit and activate type I activin receptors 4 and 5 (Alk4 and Alk5). These events stimulate phosphorylation of Smad2 and Smad3, which join with Smad4 into a Smad2/3/4 complex that triggers gene transcription (12). Myostatin is considered a master regulator of skeletal muscle growth; its inactivation leads to impressive muscle hypertrophy (11) and its overexpression induces muscle atrophy (13). Interestingly, myostatin 2212–2217 | PNAS | February 23, 2016 | vol. 113 | no. 8
expression has been reported to be increased in several human diseases associated with skeletal muscle wasting, such as cancer cachexia, AIDS, and heart failure (14). Here, we show that treatment with an anti-myostatin antibody (ATA 842) for 4 wk reverses age-associated sarcopenia, which was associated with increased muscle strength, reduced intramuscular triglyceride content, and improved muscle insulin sensitivity in old mice. Results ATA 842 Treatment Increases Muscle Mass and Strength. To assess
the potential effect of ATA 842 on skeletal muscle mass, young (10 wk) male mice fed a regular chow diet (RC) or high-fat diet (HFD), and old (22 mo) male mice fed RC were treated with ATA 842 or vehicle for 4 wk and body composition was measured by 1H-NMR spectroscopy. ATA 842 treatment increased the body weight of young mice fed RC by ∼8%, accounted for by a ∼9% increase in lean body mass without any change in body fat (Fig. 1 A–C). Young mice fed an HFD did not display changes in body weight after ATA 842 treatment (Fig. 1D). However, they displayed altered body composition, with a ∼7% increase in lean body mass (Fig. 1E) and a ∼35% decrease in adiposity (Fig. 1F). Old mice treated with ATA 842 also displayed a ∼7% increase in body weight (Fig. 1G), accounted for by a ∼7% increase in lean body mass (Fig. 1H) without any change in adiposity (Fig. 1I). Significance Sarcopenia, or aging-associated muscle atrophy, increases the risk of falls and fractures and is associated with metabolic disease. Because skeletal muscle is a major contributor to glucose handling after a meal, sarcopenia has significant effects on whole-body glucose metabolism. Despite the high prevalence and potentially devastating consequences of sarcopenia, no effective therapies are available. Here, we show that treatment of mice with an anti-myostatin antibody for just 4 wk increased muscle mass and strength in both young and old mice. In old mice, this increase in muscle mass was accompanied by an improvement in muscle insulin sensitivity. These data provide support for myostatin inhibition as a potential therapeutic strategy for aging-associated sarcopenia and insulin resistance. Author contributions: J.-P.G.C., M.C.P., A.A., G.V.M., M.J.J., G.F., C.M.H., K.F.P., and G.I.S. designed research; J.-P.G.C., M.C.P., A.A., G.V.M., and M.J.J. performed research; G.F. and C.M.H. contributed new reagents/analytic tools; J.-P.G.C., M.C.P., A.A., G.V.M., M.J.J., K.F.P., and G.I.S. analyzed data; and J.-P.G.C., M.C.P., A.A., G.V.M., M.J.J., G.F., C.M.H., K.F.P., and G.I.S. wrote the paper. Reviewers: S.-J.L., Johns Hopkins University; and M.O.T., University of Virginia Health Sciences Center. Conflict of interest statement: These studies were funded in part by an investigator initiated grant to K.F.P. from Atara Biotherapeutics, the manufacturer of ATA 842. G.F. and C.M.H. are employees of Atara and may own stock in the company. Freely available online through the PNAS open access option. 1
To whom correspondence should be addressed. Email:
[email protected].
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A potential consequence of muscle hypertrophy is increased anaerobic glycolysis, which could drive lactic acidemia. Indeed, such a phenotype was observed in mice with skeletal musclespecific expression of a constitutively active Akt1 (15). However, mice treated with ATA 842 did not display any increase in plasma lactate concentrations (Fig. 1J) compared with vehicletreated mice. To determine whether the increased body weight and muscle mass in ATA 842-treated mice would affect ectopic fat content, we evaluated hepatic and muscle triglycerides (TG) content. Camporez et al.
Interestingly, young ATA 842-treated mice fed a HFD displayed reduced hepatic (Fig. 1K) and muscle (Fig. 1L) TG content compared with vehicle-treated mice. Old mice treated with ATA 842 did not display any change in hepatic TG content (Fig. 1K); however, they had reduced muscle TG content (Fig. 1L) compared with vehicle-treated mice. Consistent with the observed increase in lean body mass measured by 1H-NMR, all three groups (young mice fed RC or HFD, and old mice fed RC) treated with ATA 842 displayed an increase in gastrocnemius weight relative to vehicle-treated controls (∼17%, 13%, PNAS | February 23, 2016 | vol. 113 | no. 8 | 2213
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Fig. 1. ATA 842 treatment increases body weight and muscle mass. Initial and final body weight (A), muscle mass (B), and body fat (C) of young mice fed RC. Initial and final body weight (D), muscle mass (E), and body fat (F) of young mice fed HFD. Initial and final body weight (G), muscle mass (H), and body fat (I) of old mice fed RC. Plasma lactate (J), hepatic triglycerides (K), and muscle triglycerides (L) of young mice fed RC or HFD and old mice fed RC. Data are represented as mean ± SEM (n = 10 per group).
and 19%, respectively) (Fig. 2A). To evaluate whether the increased skeletal muscle mass in ATA 842-treated animals translated to functional improvement, we measured forelimb grip strength. After 4 wk of ATA 842 treatment, young mice fed RC displayed an ∼15% increase in grip strength (Fig. 2B), young mice fed an HFD displayed an ∼20% increase in grip strength (Fig. 2C), and old mice displayed an ∼20% increase in grip strength (Fig. 2D). ATA 842 Treatment Causes a Subtle Reduction in Energy Expenditure in Old Mice. Increased skeletal muscle mass has been associated
with increased whole-body energy expenditure. In particular, myostatin knockout mice with marked skeletal muscle hypertrophy displayed increased energy expenditure (16, 17). To investigate whether myostatin inhibition would increase wholebody metabolism, we performed metabolic cage studies on mice treated with vehicle or ATA 842. Young mice treated with ATA 842 and fed either RC or HFD had similar whole-body energy expenditure, respiratory exchange ratio (RER), caloric intake, and locomotor activity compared with control mice (Table 1). In contrast, old mice treated with ATA 842 displayed a slight reduction (∼10%) in whole-body energy expenditure (Table 1), without any change in RER, caloric intake, or locomotor activity (Table 1). ATA 842 Treatment Has an Age-Dependent Effect on Insulin Sensitivity.
To evaluate whether the increased muscle mass observed in ATA 842-treated mice improved whole-body insulin action, we performed hyperinsulinemic-euglycemic clamp studies in young and old mice treated with vehicle or ATA 842. Basal characteristics of study mice are listed in Table 1. Consistent with previous studies, HFD-fed mice displayed reduced whole-body insulin sensitivity compared with RC-fed mice (Fig. 3 A and B), as evidenced by a lower glucose infusion rate (GIR) required to maintain euglycemia (Fig. 3 A and B) (18, 19). The whole-body insulin resistance displayed by HFD-fed mice could be attributed to both reduced insulin-stimulated peripheral glucose uptake (Fig. 3C) and reduced suppression of endogenous glucose production (EGP) during the hyperinsulinemic-euglycemic clamp (Fig. 3 D and E). However, ATA 842 treatment was not associated with any improvement in insulin action in either RC- or HFD-fed young mice (Fig. 3). In contrast to the results in young mice, ATA 842-treated old mice displayed a modest increase in whole-body insulin sensitivity as reflected by a 16% increase in the glucose infusion rate required to maintain euglycemia during the hyperinsulinemic-euglycemic clamp (Fig. 4 A and B). This increase in glucose infusion rate was accounted for entirely by increased insulin-stimulated peripheral glucose uptake (Fig. 4C), without significant effects on EGP in either the basal or the clamped state (Fig. 4D). Tissue-specific measurements of [1-14C]-2-deoxyglucose uptake revealed that the increased whole-body glucose uptake during the clamp could be attributed to increased insulin-stimulated skeletal muscle glucose uptake (Fig. 4E). Discussion To our knowledge, this is the first study to investigate the effects of myostatin inhibition on muscle hypertrophy and functionality along with whole-body insulin action in a mouse model of aging. Our results demonstrate that, in mice, inhibition of myostatin by ATA 842 treatment for a relatively short period (4 wk) led to increases in skeletal muscle mass and grip strength. These effects were observed in all groups studied: young adult mice fed either a RC or HFD and old mice fed RC diet. It was also found that the antisarcopenic effects of ATA 842 were associated with increased insulin-stimulated whole-body metabolism in the old mice. Myostatin is a known inhibitor of muscle growth and development. Myostatin knockout mice display two- to threefold greater muscle mass compared with their wild-type littermates, owing to increases in both myofibril number and myofibrillar cross-sectional area (11). In 2214 | www.pnas.org/cgi/doi/10.1073/pnas.1525795113
Fig. 2. ATA 842 treatment increases gastrocnemius weight and grip strength. Gastrocnemius weight of young mice fed RC or HFD and old mice fed RC (A). Initial and final grip strength of young mice fed RC (B) or HFD (C) and old mice (D) fed RC. Data are represented as mean ± SEM (n = 10 per group).
addition, naturally occurring mutations in myostatin result in a hypertrophic, muscle-bound phenotype in several species such as cows, dogs, and even humans (20–22). Beyond its developmental effects, myostatin also regulates muscle mass throughout the lifespan. Myostatin inhibition in postnatal life increases muscle mass (23, 24), Camporez et al.
Table 1. Basal characterization of animals Young RC mice Parameters
Vehicle
ATA 842
Young HFD mice Vehicle
ATA 842
Old RC mice Vehicle
ATA 842
Fasting glucose, mg/dL 110 ± 4.5 117 ± 10 139 ± 9.8 135 ± 7.1 90.8 ± 2.8 90.0 ± 7.3 Fasting insulin, μU/mL 3.6 ± 0.4 4.5 ± 1.2 13.9 ± 3.2 7.8 ± 1.2 8.0 ± 1.2 5.7 ± 1.3 Fasting NEFA, mmoL/L 1.29 ± 0.05 1.21 ± 0.07 1.23 ± 0.13 1.18 ± 0.04 1.16 ± 0.06 1.21 ± 0.07 Postclamp NEFA, mmoL/L 0.20 ± 0.01 0.16 ± 0.01 0.41 ± 0.04 0.47 ± 0.05 0.24 ± 0.03 0.14 ± 0.01* Fasting plasma TG, mg/dL 55.9 ± 3.4 55.6 ± 3.7 54.6 ± 3.4 49.7 ± 3.9 51.2 ± 2.7 57.6 ± 4.0 Fasting plasma IGF-I, ng/mL 147.9 ± 5.7 152.7 ± 15.6 158.2 ± 8.4 167.1 ± 10.6 180.4 ± 20.1 182.5 ± 6.1 Whole body VO2, mL·kg−1·h−1 3,318 ± 87 3,117 ± 62 3,390 ± 129 3,340 ± 115 2,999 ± 104 2,709 ± 79* Whole body VCO2, mL·kg−1·h−1 3,032 ± 73 2,850 ± 61 2,605 ± 97 2,667 ± 90 2,728 ± 96 2,475 ± 70* RER, VCO2/VO2 0.91 ± 0.01 0.91 ± 0.01 0.76 ± 0.003 0.77 ± 0.004 0.90 ± 0.007 0.91 ± 0.007 Energy expenditure, kcal·kg−1·h−1 16.3 ± 0.4 15.4 ± 0.3 15.8 ± 0.4 16.1 ± 0.3 14.7 ± 0.5 13.2 ± 0.4* Caloric intake, kcal·kg−1·h−1 19.1 ± 1.5 16.7 ± 1.5 15.7 ± 1.6 15.3 ± 1.1 15.4 ± 1.8 13.6 ± 1.8 Drinking, mL·kg−1·h−1 1.2 ± 0.1 1.1 ± 0.1 1.1 ± 0.05 1.2 ± 0.10 1.0 ± 0.1 0.9 ± 0.1 Activity, counts per h 167 ± 23 167 ± 11 154 ± 12 125 ± 9 98 ± 17 111 ± 16 Vehicle or ATA 842 treated mice for 4 wk. NEFA, nonesterified fatty acids; RER, respiratory exchange ratio; TG, triglycerides; VCO2, whole-body carbon dioxide production; VO2, whole-body oxygen consumption. *P < 0.05 compared with old vehicle-treated mice. Data are expressed as mean ± SEM.
old) after 4 wk of treatment with an anti-myostatin antibody (PF-354), whereas another study (28) showed just increased in situ muscle strength of aged (21 mo old) mice treated with this same antibody for 14 wk. Importantly, our study showed, for the first time to our knowledge, that old mice (∼23 mo old) displayed increased muscle mass, which was associated with increased grip strength after 4 wk of treatment with ATA 842. Aging is also associated with muscle insulin resistance, and we also found that ATA 842 treatment resulted in increased insulinstimulated muscle glucose uptake in old mice. Indeed, myostatin knockout mice display increased energy expenditure (29) and protection against lipid-induced insulin resistance, glucose intolerance, and HFD-induced obesity (16, 30–32). In contrast with these studies, LeBrasseur et al. (27) did not observe an improvement in glucose tolerance and insulin sensitivity in aged
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which agrees with our observations of increased muscle mass in young and old mice treated with ATA 842. As expected, the increased muscle mass in mice produced by myostatin inhibition also increases muscle strength. Using two different approaches to evaluate muscle function, the rotarod and grip strength tests, Nakatani et al. showed that mdx Duchenne muscular dystrophy mice overexpressing the endogenous myostatin inhibitor follistatin displayed increased muscle strength (25). Further, long-term myostatin inhibition using a monoclonal antibody against myostatin (24) or a single postnatal intramuscular injection of adenoassociated virus encoding follistatin or other myostatin inhibitors (26) resulted in improvements in muscle strength. In agreement with these results, we also found that inhibition of myostatin by antibody treatment increased skeletal muscle function. However, a previous study (27) failed to show increased grip strength in aged mice (24 mo
Fig. 3. ATA 842 treatment does not increase insulin sensitivity in young mice. Time-course of plasma glucose (A) and GIR (B) during the hyperinsulinemiceuglycemic clamp. Whole-body glucose uptake (C) during the steady-state period (final 40 min) of the clamp, and basal (D) and clamped (E) EGP. Data are represented as mean ± SEM (n = 10 per group). *P < 0.01 compared with vehicle-treated mice.
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Fig. 4. ATA 842 treatment subtly improves insulin sensitivity in old mice. Time course of plasma glucose (A) and GIR (B) during the hyperinsulinemiceuglycemic clamp. Whole-body glucose uptake (C) during the steady-state period (final 40 min) of the clamp, and basal and clamped (D) EGP. Skeletal muscle and white adipose tissue 2-deoxyglucose uptake (E) during the clamp. Data are represented as mean ± SEM (n = 10 per group).
mice treated with anti-myostatin for 4 wk, although they displayed increased muscle mass by the treatment (27). The inability of LeBrasseur et al. (27) to detect the subtle increase in insulin sensitivity, which we report here, may be explained by our use of a much more sensitive test to evaluate insulin sensitivity: the hyperinsulinemic-euglycemic clamp. Given that we observed just a small increase in whole-body insulin sensitivity in old antibody-treated mice, the less sensitive glucose tolerance test and insulin tolerance test performed by LeBrasseur et al. (27) may not be adequate to observe any differences between groups. Surprisingly, short-term myostatin inhibition in young mice did not induce a striking effect on whole-body insulin sensitivity and energy expenditure in our study, despite increases in muscle mass. Although old mice treated with ATA 842 displayed similar improvements in muscle mass to young mice, the old mice manifested improved whole-body insulin sensitivity, which could be accounted for by enhanced insulin-stimulated skeletal muscle glucose uptake. It is not surprising that the metabolic phenotype of our 4-wk ATA 842-treated mice was much subtler than the phenotype of knockout mice, given the duration and extent of myostatin inhibition in the two models. Myostatin knockout mice exhibit a >100% increase in muscle mass, but ATA 842-treated mice displayed a modest ∼7% increase in lean body mass. The subtle hypertrophic effect of 4-wk ATA 842 treatment thus appears insufficient to significantly alter whole-body insulin sensitivity. Increasing the extent of muscle hypertrophy by extending the duration of treatment and/or using a higher ATA 842 dose would reveal whether a threshold level of hypertrophy is needed to manifest improvements in whole-body insulin sensitivity. In addition to its antisarcopenic effects, these data would suggest that increasing skeletal muscle mass by myostatin inhibition may also be a promising strategy for the treatment of type 2 diabetes and related metabolic disease. Given the high prevalence of sarcopenia, muscle insulin resistance and type 2 diabetes in older people (33, 34), our findings suggest that myostatin inhibition may be a particularly useful therapeutic strategy in older adults with these common and debilitating conditions. Taken together, these data provide evidence, for the 2216 | www.pnas.org/cgi/doi/10.1073/pnas.1525795113
first time to our knowledge, that short-term myostatin inhibition can increase muscle mass and strength in adult mice of all ages, and can also improve skeletal muscle insulin sensitivity in older mice, pointing to the potential utility of this therapeutic approach for age-associated sarcopenia and metabolic disease. Experimental Procedures Animal Procedures. All experimental procedures were approved by and conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Yale University School of Medicine. For studies of young mice, 10-wk-old male mice were randomly divided into two groups: RC-fed mice or HFD-fed mice for 7 wk (60% kcal from fat, 20% kcal from carbohydrate, 20% kcal from protein, D12492; Research Diets). All mice were treated with once-weekly s.c. injections of vehicle or anti-myostatin antibody (ATA 842; 20 mg·kg−1·wk−1) (Atara Biotherapeutics) for 4 wk. For studies of old mice, 22-mo-old male mice fed RC were divided and treated with vehicle or ATA 842 for 4 wk. During all procedures, mice were in a temperature-controlled environment, with 12:12 h light:dark cycle, with free access to water and food. Fat and lean body mass were assessed by 1H-magnetic resonance spectroscopy (Bruker BioSpin). The Comprehensive Animal Metabolic Monitoring System (Columbus Instruments) was used to evaluate O2 consumption, CO2 production, energy expenditure, activity, and food consumption. Drinking was assessed by a computer system counting consumed water droplets. Forelimb grip strength was evaluated by using a triangular pull bar attached to a grip strength meter (Columbus Instruments). Each mouse was subjected to five consecutive tests to obtain the peak value, as described (35). In vitro binding studies have shown that ATA 842 binds to free myostatin and does not cross-react with GDF11. Plasma TAG concentrations were measured by using a DCL triglyceride reagent (Diagnostic Chemicals). Tissue triglycerides were extracted by using the method of Folch et al. (36) from animals fasted for 6 h and measured by using a DCL triglyceride reagent (Diagnostic Chemicals). Plasma IGF1 levels were measured by a Mouse/Rat IGF1 ELISA kit (R&D Systems). Hyperinsulinemic-Euglycemic Clamp Studies. Hyperinsulinemic-euglycemic clamps were performed as described (37). Statistical Analysis. All data are expressed as mean ± SEM. Results were assessed by using two-tailed unpaired Student’s t test or two-way ANOVA (GraphPad Prism 5). A P value less than 0.05 was considered significant.
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(ADA)-Distinguished Clinical Investigator Award (to K.F.P.), National Institutes of Health K01 DK-099402 Grant (to M.J.J.); and ADA-Merck Mentor Based Clinical/Translational Science Postdoctoral Fellowship Award 1-14-10 Merck (to G.I.S.) from the American Diabetes Association. M.C.P. is supported by National Institutes of Health Grants T32 GM-007205 and F30 DK-104596.
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ACKNOWLEDGMENTS. We thank Ali Nasiri and Gina Butrico for their skilled technical assistance. This work was funded by National Institutes of Health Grants R01 DK-40936, R24 DK-085638, U24 DK-059635, P30 DK-45735, and R01 AG-23686 (to K.F.P.); an investigator-initiated grant from Atara Pharmaceuticals (to K.F.P.); an American Diabetes Association
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