Absence of functional leptin receptor isoforms in the POUND (Lepr(db/lb)) mouse is associated with muscle atrophy and altered myoblast proliferation and differentiation

Abstract

Objective: Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr(db/lb)) to determine the role of leptin in skeletal muscle.

Methods and findings: Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30-40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion: These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.

Figure 1. POUND mice show increased body weight but decreased muscle mass.

A. Body mass of leptin receptor-deficient POUND mice is significantly greater than that of lean, wild-type (WT) mice (left panel). The image on the right shows the obese phenotype of a POUND mouse (top) compared with wild-type (bottom). B. Tibialis anterior muscle mass is significantly decreased in leptin receptor-deficient POUND mice. C. Frozen sections of the extensor digitorum longus muscle illustrate the larger muscle fibers and fiber cross-sectional areas of wild-type mice (WT, top row) compared to POUND mice (bottom row). D. Quantitative assessment of muscle fiber cross-sectional area demonstrates significantly decreased muscle fiber size in POUND mice. Mice are 16 weeks of age.

Figure 2. Primary myoblasts from POUND mice show impaired proliferation and differentiation.

A. Primary myoblasts cultures from wild-type and POUND mice show a significant decrease in the proliferation and metabolic activity of myoblasts in POUND mice compared to normal wild-type mice as measured using MTS assay (left panel). B. Myoblasts from POUND mice (right panel, top micrograph) fail to differentiate normally and after 7 days do not develop into the elongate myotubes characteristic of normal, wild-type mice (right panel, bottom micrograph). C. Real-time PCR data show that that the early marker of myoblast differentiation, MyoD (left graph), and the later differentiation marker myogenin (right graph) are both significantly downregulated in myoblasts from POUND mice.

Figure 3. Leptin increases myoblast proliferation.

A. Leptin-treatment (100 ng/ml) significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 12 months of age. B. Leptin-treatment (100 ng/ml) also significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 24 months of age. C. -treatment (100 ng/ml) significantly increases the expression of the myogenic factors MyoD and myogenin in primary myoblasts from mice 24 months of age. Leptin did not increase the expression of these factors in myoblasts from mice 12 months of age. D. Box-and-whisker plots showing ΔΔCt values (y-axis) for leptin (LEP) and leptin receptor (LEPR) expression in the soleus (SOL; top row) and extensor digitorum longus (EDL; bottom row) muscles of mice 12 and 24 months of age (x-axis). The whiskers mark the minimum and maximum values, the boxes the first and third quartiles, and the bar within the box indicates the median. Expression of the leptin receptor is not increased with age, and is significantly (P<.05) downregulated in aged soleus (SOLLEPR).

Figure 4. Altered leptin signaling in POUND mice alters IGF-1 signaling in skeletal muscle.

A. ELISA assays show that muscle-derived IGF-1 is significantly decreased in the hindlimb muscles (extensor digitorum longus) from leptin receptor-deficient POUND mice (left graph), whereas protein levels of myostatin in hindlimb muscle are significantly elevated in POUND mice (right graph). B. Integrated pathway analysis from mRNA array comparing gene expression in tibialis anterior muscles of POUND mice with that of normal mice. The vertical axis represents the probability that a particular gene is associated with a specific canonical pathway by chance, the higher the score on this axis the lower the probability the association between gene and pathway is by chance alone. The strongest association revealed by the analysis is between genes altered in POUND mice and those associated with IGF-1 signaling. The open blue boxes connected by the lines represent ratio values indicating the ratio of genes detected in the pathway to the total number of genes in that particular pathway. C. Heat map from reverse phase protein analysis comparing protein expression in hindlimb muscle of POUND mice with that of normal mice. Arrows indicate proteins including Akt, MAPK, and MEK that are highly expressed in muscle from normal mice (red) but not highly expressed in muscle from POUND mice (green). Western blots shown on the right are for total and phosphorylated Akt, MAPK, and MEK.