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Cited by 135Muscle Physiology and DisordersMuscle metabolism and nutritionRegulation of Appetite and Obesity

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Leptin Administration Favors Muscle Mass Accretion by Decreasing FoxO3a and Increasing PGC-1α in ob/ob Mice

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Sáinz, Neira, Rodríguez, Amaia, Catalán, Victoria, Becerril, Sara, Ramírez, Beatriz, Gómez-Ambrosi, Javier, Frühbeck, Gema·2009·PLoS ONE
DOI 10.1371/journal.pone.0006808PMC PMC2733298
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Abstract

Absence of leptin has been associated with reduced skeletal muscle mass in leptin-deficient ob/ob mice. The aim of our study was to examine the effect of leptin on the catabolic and anabolic pathways regulating muscle mass. Gastrocnemius, extensor digitorum longus and soleus muscle mass as well as fiber size were significantly lower in ob/ob mice compared to wild type littermates, being significantly increased by leptin administration (P<0.001). This effect was associated with an inactivation of the muscle atrophy-related transcription factor forkhead box class O3 (FoxO3a) (P<0.05), and with a decrease in the protein expression levels of the E3 ubiquitin-ligases muscle atrophy F-box (MAFbx) (P<0.05) and muscle RING finger 1 (MuRF1) (P<0.05). Moreover, leptin increased (P<0.01) protein expression levels of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), a regulator of muscle fiber type, and decreased (P<0.05) myostatin protein, a negative regulator of muscle growth. Leptin administration also activated (P<0.01) the regulators of cell cycle progression proliferating cell nuclear antigen (PCNA) and cyclin D1, and increased (P<0.01) myofibrillar protein troponin T. The present study provides evidence that leptin treatment may increase muscle mass of ob/ob mice by inhibiting myofibrillar protein degradation as well as enhancing muscle cell proliferation.

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Figures

Figure 1 Leptin Increases Muscle Mass and Muscle Fiber Size of ob/ob Mice. (A) Gastrocnemius (GAS), extensor digitorum longus (EDL) and soleus (SOL) muscle weights of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 9–10 per group). *P<0.05, **P<0.01 and ***P<0.001. (B) Cross-sectional area (CSA) of GAS, EDL and SOL muscle fibers of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (approximately 100 fibers/muscle from 3 mice/group). **P<0.001 and ***P<0.0001. (C) Representative histological sections of hematoxylin-eosin-stained GAS, EDL and SOL muscles of wild type and ob/ob mice. Scale bars = 200 µm. Results are presented as mean±SEM. G: genotype, T: treatment.
Figure 1 Leptin Increases Muscle Mass and Muscle Fiber Size of ob/ob Mice. (A) Gastrocnemius (GAS), extensor digitorum longus (EDL) and soleus (SOL) muscle weights of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 9–10 per group). *P<0.05, **P<0.01 and ***P<0.001. (B) Cross-sectional area (CSA) of GAS, EDL and SOL muscle fibers of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (approximately 100 fibers/muscle from 3 mice/group). **P<0.001 and ***P<0.0001. (C) Representative histological sections of hematoxylin-eosin-stained GAS, EDL and SOL muscles of wild type and ob/ob mice. Scale bars = 200 µm. Results are presented as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central
Figure 2 Leptin Decreases FoxO3a Activity and Increases PGC-1α Protein in Gastrocnemius Muscle. (A) Representative Western blot analyses of forkhead box class O (FoxO3a) and phospho-FoxO3a proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). (B) Representative Western blot analyses of Akt activity evidenced by phosphorylated-Akt and Akt proteins ratio of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 8 per group). (C) Representative Western blot analyses of AMP-activated protein kinase (AMPK) activity evidenced by phosphorylated-AMPK and AMPK proteins ratio of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown (n = 5 per group). (D) Representative Western blot analyses of AMP- (PGC-1α) of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). Data are expressed as mean±SEM. G: genotype, T: treatment.
Figure 2 Leptin Decreases FoxO3a Activity and Increases PGC-1α Protein in Gastrocnemius Muscle. (A) Representative Western blot analyses of forkhead box class O (FoxO3a) and phospho-FoxO3a proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). (B) Representative Western blot analyses of Akt activity evidenced by phosphorylated-Akt and Akt proteins ratio of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 8 per group). (C) Representative Western blot analyses of AMP-activated protein kinase (AMPK) activity evidenced by phosphorylated-AMPK and AMPK proteins ratio of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown (n = 5 per group). (D) Representative Western blot analyses of AMP- (PGC-1α) of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). Data are expressed as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central
Figure 3 Leptin Decreases MAFbx and MuRF1 Protein Expression in Gastrocnemius Muscle of Wild Type and ob/ob Mice. (A) Representative Western blot analyses of muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). *P<0.05. (B) Immunostaining for MAFbx and MuRF1 proteins was assessed by optic microscopy. Representative images are shown for gastrocnemius muscle. Scale bars = 50 µm. Data are presented as mean±SEM. G: genotype, T: treatment.
Figure 3 Leptin Decreases MAFbx and MuRF1 Protein Expression in Gastrocnemius Muscle of Wild Type and ob/ob Mice. (A) Representative Western blot analyses of muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). *P<0.05. (B) Immunostaining for MAFbx and MuRF1 proteins was assessed by optic microscopy. Representative images are shown for gastrocnemius muscle. Scale bars = 50 µm. Data are presented as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central
Figure 4 Leptin Regulates Muscle Growth and Cell Cycle Factors. (A) Representative Western blot analyses of myostatin protein of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 2–6 per group). The myostatin band was detected only in 2 samples of wild type groups.*P<0.05. (B) Representative Western blot analyses of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin-dependent kinase inhibitor 1B (Cdkn1b/p27Kip1) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). *P<0.05, **P<0.01 and ***P<0.001. Data are expressed as mean±SEM. G: genotype, T: treatment.
Figure 4 Leptin Regulates Muscle Growth and Cell Cycle Factors. (A) Representative Western blot analyses of myostatin protein of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 2–6 per group). The myostatin band was detected only in 2 samples of wild type groups.*P<0.05. (B) Representative Western blot analyses of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin-dependent kinase inhibitor 1B (Cdkn1b/p27Kip1) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). *P<0.05, **P<0.01 and ***P<0.001. Data are expressed as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central
Figure 5 Leptin Enhances Muscle Cell Proliferation. (A) Immunohistochemical staining for dystrophin and PCNA of gastrocnemious muscle of wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. (B) Immunohistochemical staining and relative stained nuclei number for PCNA evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. **P<0.01. (C) Immunohistochemical staining and relative stained nuclei number for cyclin D1 evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. (D) Immunohistochemical staining and relative stained nuclei number for p27Kip1 evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. *P<0.05 and **P<0.01. Data are expressed as mean±SEM. G: genotype, T: treatment.
Figure 5 Leptin Enhances Muscle Cell Proliferation. (A) Immunohistochemical staining for dystrophin and PCNA of gastrocnemious muscle of wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. (B) Immunohistochemical staining and relative stained nuclei number for PCNA evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. **P<0.01. (C) Immunohistochemical staining and relative stained nuclei number for cyclin D1 evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. (D) Immunohistochemical staining and relative stained nuclei number for p27Kip1 evaluated among 500 cells in gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice (n = 3 per group). Scale bar = 50 µm. *P<0.05 and **P<0.01. Data are expressed as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central
Figure 6 Leptin Increases the Expression of Contractile Proteins in Skeletal Muscle. (A) Representative Western blot analyses of myosin heavy chain type I (MHC I), myosin heavy chain type II (MHC II), slow and fast troponin T (TnT) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). (B) Immunohistochemical staining for slow and fast troponin T (TnT) of gastrocnemius muscle of wild type and ob/ob mice. Scale bars = 100 µm. Data are expressed as mean±SEM. G: genotype, T: treatment.
Figure 6 Leptin Increases the Expression of Contractile Proteins in Skeletal Muscle. (A) Representative Western blot analyses of myosin heavy chain type I (MHC I), myosin heavy chain type II (MHC II), slow and fast troponin T (TnT) proteins of gastrocnemius muscle of PBS (open), pair-fed (gray) and leptin-treated (closed) wild type and ob/ob mice are shown. β-actin was used as a loading control (n = 8 per group). (B) Immunohistochemical staining for slow and fast troponin T (TnT) of gastrocnemius muscle of wild type and ob/ob mice. Scale bars = 100 µm. Data are expressed as mean±SEM. G: genotype, T: treatment.CC BY · PubMed Central

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Metadata

Authors
Sáinz, N., Rodríguez, A., Catalán, V., Becerril, S., Ramírez, B., Gómez-Ambrosi, J., & Frühbeck, G.
Affiliations
Instituto de Salud Carlos III (ES)
Corresponding author
Frühbeck, Gema
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-09-04
Volume
4
Issue
9
Pages
e6808
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0006808
PMC
PMC2733298
OpenAlex
W2002233417
Cited by
135
References
92
Citations by year
2026: 3 · 2025: 8 · 2024: 3 · 2023: 7 · 2022: 5 · 2021: 7 · 2020: 6 · 2019: 7 · 2018: 6 · 2017: 10 · 2016: 6 · 2015: 7 · 2014: 13 · 2013: 15 · 2012: 11
FWCI
4.30
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Deposito Adademico Digital Universidad De Navarra (University of Navarra) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Centro de Investigación Biomédica en Red-Fisiopatología de la Obesidad y Nutrición
  • Gobierno de Navarra
  • Instituto de Salud Carlos III
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Nursing › Nutrition and Dietetics › Muscle metabolism and nutrition
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Regulation of Appetite and Obesity
Keywords
leptin, ob/ob mice, FOXO3, PGC-1α, atrogin-1, MuRF1, myostatin, muscle atrophy, muscle fiber type, myofibrillar protein degradation, muscle cell proliferation, cyclin D1, proliferating cell nuclear antigen, troponin T, skeletal muscle
MeSH terms
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha, Forkhead Box Protein O3, Animals, Male, Mice, Inbred C57BL, Muscles, Transcription Factors, Trans-Activators, Proliferating Cell Nuclear Antigen, Cyclin D1, Troponin T, Oligonucleotide Array Sequence Analysis, Leptin, Gene Expression Profiling, Cell Proliferation, Mice, Forkhead Transcription Factors, Myostatin

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