GH is one of the most important factors that regulates postnatal longitudinal growth, body weight, and body composition in mammals (18
). GH exerts anabolic actions in skeletal muscle by both promoting muscle development and facilitating nutrient uptake and utilization in the muscle, thereby coordinating global energy expenditure and body composition (46
). However, the GH and IGF-1 pathways are so intimately connected that it has been challenging to distinguish actions of GH that result secondary to GH-induced IGF-1 production, as opposed to those that might be mediated by direct GHR signaling pathways. In this study, we used a genetic approach in mice to determine the requirement of different components of the GH/IGF-1 system in skeletal muscle development and postnatal function. Understanding the precise modes and mechanisms of GH action in skeletal muscle is essential for developing the most effective therapies to treat sarcopenia in aging humans, cachexia associated with cancer or AIDS, or a number of other muscle-wasting conditions.
Our studies demonstrate that the GHR is required for normal skeletal muscle development and provide several lines of evidence that the effects of GH on skeletal muscle development are mediated by muscle production of IGF-1. First, acute exposure of myoblasts to GH did not affect their proliferative rate, whereas IGF-1 treatment increased both myoblast proliferation and fusion, even in cells lacking GHR. The signals generated in myoblasts, after exposure to either GH or IGF-1, appear to activate the NFATc2/IL-4 pathway, previously shown to be critical for myoblast fusion (6
). Therefore, while both GH and IGF-1 are capable of inducing NFAT translocation and activity (Figure , F and G), IGF-1 signaling appears to be predominate in controlling myoblast fusion and myonuclei accumulation. In accordance with this, treatment of ΔIGF-1R myoblasts with GH failed to increase myoblast fusion, while treatment with exogenous IL-4 increased the fusion index (the percentage of fused myoblasts) but not the number of nuclei per myotube (Supplemental Figure 1, I and J). This result suggests that additional pathways, either in parallel to or downstream of NFATc2 and IL-4 activation, might also be activated by IGF-1 to induce myoblast fusion and accumulation of additional myonuclei. For example, IGF-1 is known to activate the PI-3K/Akt pathway to inhibit GSK3 and activate mTOR/p70S6K to induce myotube hypertrophy (48
). Regardless of the downstream pathways activated, IGF-1 signaling appears to be vital for GH-induced myoblast fusion. Second, elimination of the IGF-1R from myoblasts abolished GH-induced myoblast fusion. Third, the skeletal muscle phenotypes resulting from disruption of GHR and IGF-1R were strikingly similar. These findings support previous observations in mice with unrestricted disruption of IGF-1R (49
) or expression of a dominant-negative IGF-1R (35
), which also exhibited hypoplastic skeletal muscle. Taken together, these data provide strong evidence that GH actions on skeletal muscle development require local IGF-1 production. It should be noted that this conclusion is the opposite of that reached by Sotiropoulos et al. (10
), who reported that GH increased skeletal muscle cell fusion but suggested that this effect was directly mediated by GH, since GH did not induce IGF-1 in their cells. The exact reasons for these different results regarding the role of IGF-1 in skeletal muscle development are unclear but may relate to the different skeletal muscle cell models and/or differences in the timing of measurements of Igf1
The histomorphometric changes observed in gastrocnemius muscles of the ΔGHR and ΔIGF-1R mice are compatible with our in vitro findings and strengthen the conclusion that GH promotes skeletal muscle development through myoblast-produced IGF-1. Skeletal muscle lacking either receptor had fewer myonuclei and smaller myofiber diameters. At 6 weeks of age, type I fiber distribution was lower in both mutants, although this defect persisted only in ΔIGF-1R mice at 16 weeks. These results suggest that GH normally functions to control type I fiber specification, likely through local IGF-1 production. Previous findings from mice globally deficient for the GHR exhibited significant reductions in soleus muscle type I fiber number and fiber diameter of soleus muscle (10
). In addition, studies in humans have demonstrated a link between GH and fiber type specification. For example, in GH-deficient humans, the number of type I myofibers are significantly reduced in the vastus lateralis (50
). These changes, together with the reduced myofiber diameters observed in the ΔGHR mice, most likely account for their reduced functional performance (grip strength and rotarod). In addition to these gross anatomical changes, it is probable that loss of GH/IGF-1 signals also compromises excitation-contraction coupling events. Consistent with this notion, preliminary assessment of showed that the force-generating capacity of single myofibers from ΔIGF-1R mice was reduced compared with that of controls (data not shown). Finally, we also created a model that lacked the IGF-1 ligand, specifically in skeletal muscle, using the same mef2c-Cre mouse used to generate the ΔGHR and ΔIGF-1R mice. Mice lacking IGF-1 had no discernible phenotype, and their body weights were indistinguishable from control littermates at all postnatal times (data not shown). We speculate that the normal muscle development of the ΔIGF-1 mice might be due to compensatory activity of local anabolic factors, including IGF-2 (51
). Together, these results indicate that loss of GHR and the attendant reduction in myoblast IGF-1/IGF-1R signaling leads to deregulated fiber type specification, reduced muscle size, and subsequent compromised muscle function.
Surprisingly, loss of GHR in skeletal muscle was accompanied by a progressive increase in body weight and accumulation of peripheral fat. While nighttime voluntary locomotion was decreased in ΔGHR mice, food intake decreased and metabolic rate tended to increase in the mutants. We therefore assumed that the decreased nocturnal activity was likely not the causative factor for the observed increase in fat mass. Rather, we suspected that loss of GHR from skeletal muscle caused alterations of global nutrient metabolism. Indeed, serum glucose in the fed state was elevated in ΔGHR mutants, and the development of insulin resistance in these mice was confirmed by GTTs and ITTs. This finding might seem paradoxical, given that global GH resistance in mice that lack GHR/GH binding protein results in increased insulin sensitivity (53
). However, GHR/GH binding protein mutant mice exhibit increased liver IR abundance and enhanced hepatic insulin-stimulated IR tyrosine phosphorylation (59
), which likely accounts for their increased insulin sensitivity. Therefore, it would appear that the different metabolic profile exhibited by our ΔGHR mice, which lack GHR exclusively in skeletal muscle, is explained partially by maintenance of insulin action in nonskeletal muscle tissues in which GHR remains intact. It should also be noted that serum insulin remained unchanged in ΔGHR mice at all time points examined, further supporting normal insulin action and regulation in other insulin-responsive tissues. Taken together, the increased serum glucose and triglyceride levels observed in ΔGHR mice, in the face of normal insulin levels, suggests a failure of nutrient uptake by skeletal muscle. We predict that the increased fat mass observed in ΔGHR mutants results from redistribution of this nutrient supply from skeletal muscle to GHR-intact adipose tissue.
Importantly, the body composition and metabolic alterations seen in ΔGHR mice were not observed in ΔIGF-1R mice, which had reduced body weights, decreased peripheral fat, and normal, nonfasting serum glucose levels. These findings clearly suggest that the changes in body composition and insulin resistance seen in the ΔGHR mice are not due to loss of IGF-1 signaling in muscle. Rather, the actions of IGF-1 seem to be restricted primarily to anatomical development of the skeletal muscle. Interestingly, mice rendered IGF-1 resistant in skeletal muscle by targeting overexpression of a dominant-negative IGF-1R in skeletal muscle (MKR mice), exhibited metabolic disturbances similar to those observed in our ΔGHR mice, including increased peripheral fat and insulin resistance (62
). In the MKR mice, expression of the dominant-negative IGF-1R effectively disrupts both IGF-1R and IR signaling by inducing formation of nonfunctioning hybrids between the mutant and the endogenous IGF-1 and IRs. By contrast, we propose that insulin signaling is attenuated in the ΔGHR mice due to decreased IR abundance and increased phosphorylation of IRS-1 on Ser 1101 (39
), which together inhibit insulin signaling. Thus, the MKR mice and ΔGHR mice share a similar metabolic phenotype as a result of compromised IR signaling in skeletal muscle, which is manifested through distinct mechanisms. These defects in insulin signaling most likely represent the primary reason for the development of insulin resistance and increased peripheral adiposity in both models.
Our studies also shed light on the molecular mechanisms responsible for the development of insulin resistance in skeletal muscle from the ΔGHR mice. Since myoblasts lacking STAT5ab were also less sensitive to insulin-induced glucose uptake, it would appear that GH modulates insulin sensitivity, at least in part, through STAT5. Indeed, a number of genes modulating glucose and triglyceride uptake and metabolism are known to be regulated through GH-induced STAT5 activation (63
). Accordingly, mice lacking STAT5ab in skeletal muscle also had increased fat mass, further suggesting the involvement of GH-induced STAT5-mediated genes in the metabolic effects seen in our ΔGHR mice (64
). However, the reduction of insulin-induced glucose uptake seen in ΔSTAT5ab myoblasts was not as pronounced as that of ΔGHR myoblasts, indicating the contribution additional pathways in the development of insulin resistance. As mentioned above, insulin resistance in myotubes lacking GHR appears to derive from changes at a number of proximal and distal points along the insulin signaling pathway, including decreased IR abundance, reduced basal Akt (Thr 308) and Erk phosphorylation, and the increased Ser 1101 phosphorylation of IRS-1 already discussed. Basal phosphorylation of other known inhibitory IRS-1 residues (Ser 612 and Ser 636/639) was reduced, at least in the context of our in vitro model system. However, it remains possible that the hyperglycemic, hypertriglyceridemic, and likely inflammatory conditions experienced in vivo may further exacerbate insulin resistance in skeletal muscle through pathways known to act on these inhibitory IRS-1 residues (66
). Since IR signals primarily through the PI-3K pathway, it is more likely that the reduction in basal Erk activation is a result of GHR disruption. However, loss of GH-mediated Erk activity could have indirect effects to further suppress insulin-induced glucose uptake in ΔGHR myoblasts. Erk interacts with TSC2 (68
) to relieve its suppression of mTOR, allowing GLUT expression and glucose uptake (69
), and the ability of GH to alter insulin sensitivity via nutrient-sensing pathways, like PI3K/mTOR/S6K (70
), is currently being investigated.
In conclusion, the results presented in this paper indicate 2 circumscribed roles for GHR signaling is skeletal muscle. First, by regulating myoblast production of IGF-1, GH promotes normal myofiber type specification, myonuclei accumulation, and expansion of myofiber diameter, processes which are required for development of fully functional skeletal muscle. Second, GH functions independent of IGF-1R signaling to facilitate normal insulin action in skeletal muscle, which ultimately impacts global nutrient metabolism. Precisely how these distinct GH-generated signals are compartmentalized within skeletal muscle is a question currently under investigation in our laboratory. Further elucidation of these mechanisms should guide more informed use of GH or GH analogs for promoting muscle development and attenuating muscle loss as well as provide alternative means to affect insulin sensitivity and global nutrient metabolism across a range of metabolic syndromes.