We have shown here that cast immobilisation leads to a passive loss of skeletal muscle function, resulting in failure to maintain adequate core body temperature in a cold environment. Cast immobilization also activated BAT thermogenesis via the sympathetic nervous system and triggered systemic changes in energy metabolism associated with BAT thermogenesis. Furthermore, we found that free BCAAs derived from skeletal muscle serve as substrates for energy metabolism in BAT, and that skeletal muscle–derived IL-6 promotes this provision of BAT with amino acids from muscle. In addition, this thermoregulatory system between BAT and skeletal muscle may also be activated in response to cold temperature or acute stress.
A first aim of our study was to investigate the role of skeletal muscle in thermogenesis. In this study, mice with impairment of hind limb muscle contraction by cast immobilization were used as a model for a loss of muscle function. Exercise-induced muscle contraction generates large amounts of heat which dependents on hydrolysis of ATP, and skeletal muscle, as the first organ to be recruited for thermogenesis, plays an important role in maintenance of body temperature in endotherms (Rowland et al., 2015b). Muscle thermogenesis can account for up to 90% of systemic oxygen consumption during periods of maximal recruitment of muscle contraction, such as during exercise or an intense bout of shivering (Rowland et al., 2015b; Zurlo et al., 1990). We found that cast immobilization decreased the amounts of metabolites in TCA cycle and suppressed expression of mitochondrial-related genes from the early stage of immobilization, suggesting that metabolic rate is rapidly decreased in immobilized muscle. On the other hands, thermoregulatory system in endotherms cannot be explained by thermogenesis based on muscle contraction alone, with nonshivering thermogenesis being required as a component of the ability to tolerate cold temperatures in the long term (Tansey and Johnson, 2015). Our results now show that expression of the gene for sarcolipin, a key regulator of the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA) and an important mediator of muscle nonshivering thermogenesis, was not increased in muscle after cast immobilization. In contrast, we have now shown that UCP3 gene expression in muscle was transiently increased during the early stage of cast immobilization. Although the importance of UCP3 for mitochondrial energy metabolism is well established, (Bouillaud et al., 2016; Lombardi et al., 2019), its physiological function as a mitochondrial uncoupler remains unclear (Vidal-Puig et al., 2000; Shabalina et al., 2010). In addition, we found that several genes induced by cold stimulation in skeletal muscle were not increased in cast-immobilized mice. Our results thus show that metabolic thermogenesis in skeletal muscle is crucial for mammals, and that cast immobilization increases cold intolerance, even in BAT-enriched mammals such as rodents. Although our study showed that cold intolerance in mice was observed after just 2 hr of cast immobilization, these results could also be attributed not only to loss of skeletal muscle function but also to stress, decreased calorie reserves, or reduced systemic locomotor activity.
In contrast to skeletal muscle, BAT thermogenesis was activated via the sympathetic nervous system under room temperature even when skeletal muscle was immobilized. It is a well-recognized fact that BAT and skeletal muscle function in an orchestrated manner to maintain core body temperature in endotherms (Rowland et al., 2015a; Golozoubova et al., 2001; Janovska et al., 2023). A bit surprisingly, the capacity of the BAT itself was insufficient to maintain stable body temperature during acute cold exposure. Our results also suggest that the increase in noradrenaline concentrations in BAT or in blood are transient, suggesting that the activation of sympathetic nerve activity after cast immobilization is also transient. Consequently, the expression of thermogenic genes and metabolic changes in BAT may also have been induced to peak after 24 hr of cast immobilization. In addition, it has been reported that long-term cast immobilization increases nonshivering thermogenesis in immobilized muscle (Tomiya et al., 2019). Our results show that the expression of Ucp2 and Sln were increased in immobilized muscle after 7 days of cast immobilization. These data suggest that the maintenance of core body temperature by BAT thermogenesis may be an important system during short-term cast immobilization.
We also found that changes in systemic energy metabolism induced by cast immobilization were associated with the activation of BAT thermogenesis. Activation of BAT results in the tissue becoming a high consumer of a variety of energy substrates, including lipids, glucose, BCAAs, succinate, and lactate (Wang et al., 2021). Previous findings reported that mitochondrial BCAA catabolism in brown adipocytes promotes systemic BCAA clearance, suggesting that BCAAs may be supplied to BAT from other organs during BAT thermogenesis. Our results suggest that skeletal muscle is a source of free amino acids for BAT thermogenesis or hepatic gluconeogenesis. In response to metabolic demands imposed by starvation, exercise, or cold exposure, for example, skeletal muscle manifests metabolic flexibility in order to meet the demands of other organs and is an important determinant of metabolic homeostasis (Bertile et al., 2021; Holeček, 2018). In contrast, the reduction in metabolic rate may contribute to protein conservation and maintain skeletal muscle mass under hibernation (Bertile et al., 2021). Our findings now provide important insight into the role of skeletal muscle as a source of amino acids. However, a recent study also suggests that a highly active proteolysis system in the heart provides substantial amounts of amino acids for distribution to other organs via the bloodstream (Murashige et al., 2020). The heart may, therefore, be another source of free BCAAs for BAT thermogenesis in cast-immobilized mice.
We found that muscle-derived IL-6 directly increased the abundance of BCAAs in skeletal muscle after cast immobilization. Previous studies have shown that excessive IL-6 induces amino acid catabolism in skeletal muscle, (Bonetto et al., 2012; Zanders et al., 2022), and that IL-6 is one of the factors responsible for the induction of muscle atrophy in cast-immobilized mice (Hirata et al., 2022). IL-6 is a secreted factor induced by several mechanisms, including muscle contraction, intracellular calcium signaling, and inflammation. (Pedersen and Febbraio, 2008; Bustamante et al., 2014) However, inflammation associated with macrophage infiltration is not thought to be induced in the early stages of muscle atrophy (Kawanishi et al., 2018), and thus inflammation-induced IL-6 expression may, therefore, not contribute to the changes in amino acid metabolism in cast-immobilized muscle. In addition, whereas intracellular calcium concentrations may increase after muscle immobilization for 2 weeks (Tomiya et al., 2019), they appear to decrease in response to short-term cast immobilization (Hirata et al., 2022). Cast immobilization-induced Il6 expression may, therefore, also not be regulated by calcium signaling. Upregulation of IL-6 expression by cast immobilization was previously shown to be mediated by a Piezo1-KLF15 axis (Hirata et al., 2022), whereas our observation that Il6 expression were increased in non-immobilized forelimb muscles. Furthermore, acute cold exposure and short-term restraint stress tended to increase Il6 expression in skeletal muscle, suggesting that muscle-derived IL-6 for thermoregulatory system may also be regulated by the central nervous system. A recent study showed that motor circuits modulate the production of neutrophil-induced chemokines in skeletal muscle after acute stress (Poller et al., 2022). The regulation of IL-6 production in muscle by the central nervous system warrants further investigation.
The central nervous system tightly controls core body temperature through integrates information about external temperature, humidity, and thermal sensation to induce an adaptive response (Tansey and Johnson, 2015). Disappointingly, our study was inconclusive as to whether the trigger for BAT thermogenesis after cast immobilization was hypothermia associated with loss of skeletal muscle function or stress. However, we found that acute cold exposure and short-term restraint stress may also recruits substrate supply from skeletal muscle for BAT thermogenesis. Possibly, thermoregulatory system through amino acid metabolism in skeletal muscle and BAT may also be an important metabolic strategy even in the case of fever response induced by stress or infection. Furthermore, inflammation, infection, and acute stress trigger rapid increases in the circulating IL-6 concentration (Qing et al., 2020; Cheng et al., 2015) and induce a fever response by promoting PGE2 synthesis. Mitochondrial BCAA oxidation in BAT was recently shown to be increased to support thermogenesis induced by PGE2 (Yoneshiro et al., 2019). We found that BAT-derived IL-6 increased blood IL-6 levels after cast immobilization and administration of exogenous IL-6 ameliorated cold intolerance in cast-immobilized IL-6 KO mice via the sympathetic nervous system. It may modulate thermal and energy homeostasis through the fever response and metabolic regulation in multiple organs.
In conclusion, we have shown that cast immobilization-induced thermogenesis in BAT that was dependent on the utilization of free amino acids derived from skeletal muscle, and that muscle-derived IL-6 stimulated BCAA metabolism in skeletal muscle. Although some effects, such as activation of BAT thermogenesis and changes in energy metabolic dynamics, observed with cast immobilization were modest in magnitude, thermoregulatory system through amino acid metabolism in skeletal muscle and BAT may be an important metabolic strategy even under cold temperature or acute stress. Our findings may provide new insights into the significance of skeletal muscle as a large reservoir of amino acids in the regulation of body temperature. In addition, given that circulating levels of IL-6 or BCAAs are associated with obesity and diabetes,(Wallenius et al., 2002; Wang et al., 2011) IL-6–mediated BCAA metabolism in skeletal muscle and BAT may also be associated with muscle atrophy in metabolic diseases. Further investigation of IL-6–dependent amino acid metabolism in BAT and skeletal muscle may inform the development of preventive or therapeutic interventions for some forms of muscle atrophy.
Limitations of the study
In our cast immobilization strategy, we were unable to determine which components of muscle thermogenesis are actually inhibited by cast immobilization, and what is the relative heat contribution. This study does not experiment that directly tests whether BCAAs derived from adipose tissue are used for thermogenesis, which would require more robust tracing experiments.
In addition, given that rodents are BAT-enriched mammals, whether BAT thermogenesis is similarly activated in humans after skeletal muscle immobilization remains to be investigated. Our study also did not determine the mechanism underlying the induction of Il6 expression in skeletal muscle by cast immobilization. In addition, whereas muscle-derived IL-6 may stimulate protein catabolism in immobilized muscle, the mechanism by which IL-6 increases BCAA concentrations in skeletal muscle remains unclear. In further investigation, RNA-seq profiling of BAT and muscle tissues should be used to rigorously determine the mechanisms of BCAA metabolism stimulated by muscle-derived IL-6.