The results of the present study suggest direct modulation of IGF-I availability within muscle of control mice is capable of acutely regulating the phosphorylation of proteins central to maintaining protein synthesis. In this regard, increasing IGF-I bioavailability by the intramuscular injection of Leu24
IGF-I appeared to increase mTOR activity as evidenced by enhanced phosphorylation of both 4E–BP1 and S6K1 [27
]. These data extend previous work showing the addition of Leu24
IGF-I to cultured myocytes increases protein synthesis and that this stimulation resulted from a displacement of native IGF-I from various IGFBPs and not from its direct interaction with the IGF-I receptor [28
]. Conversely, sequestration of bioactive IGF-I within muscle after the intramuscular injection of IGFBP-1 reduced phosphorylation of 4E–BP1. These data are also consistent with the reported reduction in muscle protein synthesis observed when IGFBP-1 was infused intravenously at a dose sufficient to reduce the circulating concentration of free IGF-I [14
] or when myocytes were cultured with excess IGFBP-1 [29
]. In the present study, the intramuscular and intraperitoneal injection of either Leu24
IGF-I or IGFBP-1 produced qualitatively similar changes in 4E–BP1 phosphorylation. However, we cannot draw conclusions regarding the sensitivity of muscle protein synthesis to these two different routes of administration because complete dose response curves were not generated. Regardless, our data clearly demonstrate local regulation of IGF-I has the potential to acutely impact muscle protein balance.
In preliminary studies, repeated intramuscular injections of Leu24Ala31 IGF-I were performed in an attempt to produce a sustained effect over several days. However, multiple injections into the muscle produced muscle inflammation as manifested by an increase in inflammatory cytokine mRNA content and by altered phosphorylation of 4E–BP1 (Lang unpublished observations) Hence, we replaced this invasive method in subsequent studies by the implantation of a time-release pellet in close proximity to the gastrocnemius. Muscle from the contralateral leg implanted with the placebo pellet had cytokine mRNA levels as well as surrogate markers of protein synthesis and degradation which were not different from muscle of naive control mice, suggesting the implantation procedure per se did not alter muscle protein balance. Furthermore, based on the circulating concentrations of IGF-I, IGFBP-1 and insulin as well as the heart weight, all of which were not different between sham and pellet-implanted mice, the effect of the IGF-I containing pellet on the target muscle appears to result solely from an elevation in the local muscle concentration of IGF-I.
Muscle-directed IGF-I increased the gastrocnemius mass in both control and septic mice, and the increment in weight compared to the contralateral muscle was not different between groups. The IGF-I pellet successfully restored IGF-I protein content in the target muscle of septic mice to basal control levels. In contrast, the IGF-I pellet did not significantly increase IGF-I protein in muscle of control mice, although a trend was noted. This differential effect between control and septic mice in response to IGF-I appears to result from a reduction in IGF-I synthesis (e.g., as evidenced by the decrease in IGF-I mRNA content) in muscle of control but not septic mice. The ability of locally directed IGF-I to prevent the sepsis-indued atrophy in gastrocnemius led to a corresponding increase in muscle protein. These results differ from those of Criswell et al [30
] where muscle-specific over-expression of IGF-I failed to prevent atrophy produced by disuse. Although the reason for this difference is unknown, it is noteworthy that the atrophic response produced by disuse was not associated with a reduction in muscle IGF-I, whereas the IGF-I content was markedly reduced by sepsis. The ability of muscle-directed IGF-I to increase muscle protein and mass in septic animals is consistent with similar responses produced in various catabolic conditions when IGF-I is administered intravenously [31
]. One limitation of the current study is that we did not determine whether locally administered IGF-I increased the number or size of myofibers. However, IGF-I is known to stimulate both cellular proliferation and differentiation s22 [8
]. Moreover, the local infusion of IGF-I in adult rats produces a proportional increase in the muscle DNA and protein suggesting a hypertrophic response, possibly via stimulation of satellite cells [11
Under basal conditions, sepsis and endotoxin deplete muscle protein in part via a decrease in mTOR activity and inhibition of mRNA translation [4
]. In the current study this is evidenced by the reduction in 4E–BP1 phosphorylation and the redistribution of eIF4E from the active eIF4E·eIF4G complex to the inactive eIF4E·4EBP1 complex. Although protein synthesis was not directly determined, the changes in 4E–BP1 phosphorylation and eIF4 distribution are consistent with other studies which report a significant correlation between these parameters and protein synthesis per se in the same muscle [1
]. Muscle-directed IGF-I increased the phosphorylation of 4E–BP1 (and tended to increase S6K1 phosphorylation), suggesting an increased mTOR activity. Furthermore, locally administered IGF-I also shifted a larger amount of eIF4E into the active eIF4E·eIF4G complex. While these IGF-I produced changes were relatively smaller than those seen in muscle from nonseptic mice exposed to IGF-I, they nonetheless maintained all of these endpoints at values comparable to those seen in control muscle under basal conditions. The exact mechanism for this blunted response in septic muscle was not determined but may be related to the increased concentration of IGFBP-1 in the blood and, presumably, in the muscle.
The mRNA content for atrogin-1 and MuRF1, which are collectively referred to as “atrogenes,” is upregulated in various atrophic conditions [23
]. Although some reports question the causal relationship between atrogin-1 and MuRF1 mRNA expression and protein degradation per se [39
], an increased mRNA content for these muscle-specific atrogenes appears to be directly proportional to increased proteolysis in many conditions. Our present data indicate both atrogin-1 and MuRF1 mRNAs were increased 5 days after induction of sepsis and these findings are consistent with previous reports indicating an upregulation at earlier times points in response to sepsis and endotoxin [40
]. Muscle-directed IGF-I selectively decreased the sepsis-induced increase in atrogin-1 mRNA. In contrast, IGF-I failed to decrease MuRF1 mRNA in septic mice. As a result of this divergent response between the two atrogenes, it is not possible to unequivocally conclude whether the ability of locally directed IGF-I to prevent sepsis-induced wasting is mediated in part by a reduction in muscle proteolysis. In this regard, while a sustained intravenous administration of IGF-I has been reported to decrease muscle proteolysis in some catabolic conditions, such as burns and aging [42
], it appears relatively ineffective at preventing the increased degradation produced by sepsis [44
]. Therefore, additional studies which directly quantitate protein breakdown are required to definitively determine whether the ability of locally directed IGF-I to ameliorate the sepsis-induced decrease in muscle mass is mediated solely by its stimulatory effect on protein synthesis or in part by slowing the accelerated rate of protein degradation.
Elevations in one or more of the inflammatory cytokines, TNFα, IL-1 or IL-6, either directly or indirectly have been implicated in the muscle atrophy produced in a variety of conditions [25
]. In general, the increase in the circulating and tissue content of these cytokines occurs relatively early (4–24 h) after infection and wanes with time [19
]. Of the three inflammatory cytokines studied (e.g., TNFα, IL-1 and IL-6), only the latter was significantly elevated in skeletal muscle at the 5-day time point used in the current study, However, based on information in the literature, it is likely that all of these cytokines were elevated in both liver and muscle at earlier points (e.g., < 24 h) during the septic insult [48
]. In contrast, in the current study, the hepatic expression for all three cytokines was not different from basal nonseptic levels at this relatively late time point. Due to technical problems, plasma cytokine levels were not assessed in our study and therefore we cannot determine whether the elevation in muscle IL-6 mRNA resulted in an elevation in its circulating concentration. The ability of the IGF-I pellet to prevent the sepsis-induced atrophy in adjacent muscle was associated with a reduction in IL-6 mRNA content. Such a reduction in cytokine production is consistent with previous reports that the systemic administration of IGF-I can attenuate the elevation in circulating TNFα, IL-1 and IL-6 levels produced by sepsis and burns [49
]. The mechanism for the suppressive effect of IGF-I on muscle IL-6 mRNA content has not been elucidated but our preliminary studies would suggest an indirect effect of the growth factor because pre-incubation of C2C12 myoblasts with different concentrations of IGF-I did not prevent or ameliorate the endotoxin-induced increase in IL-6 mRNA content (preliminary data, Frost and Lang). Furthermore, these data are consistent with the growth defect and reduced IGF-I levels observed in transgenic mice over expressing IL-6 [52
], the ability of IL-6 to decrease blood borne IGF-I [53
], and the ability of locally infused IL-6 to decrease muscle protein synthesis [54
]. Collectively, our data suggest it is possible to modulate IGF-I bioavailability within muscle per se and that agents increasing the availability of IGF-I within muscle might be effective in ameliorating the sepsis-induce loss of muscle mass without having undesirable effects on metabolic processes in distant organs.