Landmark studies using rodent models showed that diverse atrophy-inducing stresses (including fasting, systemic illness, and muscle disuse) generate similar patterns of changes in skeletal mRNA expression (Sacheck et al., 2007). We translated those findings to humans by determining the effect of fasting on global skeletal muscle mRNA expression in human skeletal muscle. Our fasting protocol was well tolerated by subjects, relatively simple to execute, and required only a few subjects to discern important changes in skeletal muscle mRNA expression. For example, it allowed us to demonstrate, for the first time, fasting-mediated induction of atrogin-1 and MuRF1mRNAs in human skeletal muscle.
In human skeletal muscle, fasting altered levels of >500 skeletal muscle mRNAs (approximately 3% of the total mRNAs examined). However, only a few of these mRNAs are known to play central roles in muscle atrophy in mice (includingatrogin-1, MuRF1, ZFAND5, and PGC-1α). Likewise, most mRNAs that were altered by fasting in both human and mouse muscle (which formed the basis for our first Connectivity Map query), and most mRNAs that were altered by both fasting and SCI in human muscle (which formed the basis for our second Connectivity Map query), have undefined roles in muscle atrophy. Although we do not yet know the functional roles of most mRNAs whose levels are altered by fasting or SCI, or the precise mechanisms that regulate them in the setting of an atrophy-inducing stress, we used these data to query the Connectivity Map. This unbiased approach singled out one compound as a predicted inhibitor of atrophy-inducing stress: ursolic acid.
A water-insoluble pentacyclic triterpenoid, ursolic acid is the major waxy component in apple peels (Frighetto et al., 2008). It is also found in many other edible plants. Interestingly, because it exerts beneficial effects in animal models of diabetes and hyperlipidemia (Liu, 1995,Wang et al., 2009), ursolic acid is thought to be the active component in a variety of folkloric antidiabetic herbal medicines (Liu, 1995,Liu, 2005). As predicted by the Connectivity Map, we found thatursolic acid reduced skeletal muscle atrophy in the setting of two distinct atrophy-inducing stresses (fasting and muscle denervation). A major strength of the Connectivity Map is that it takes into account positive and negative changes in mRNA expression that together constitute an authentic mRNA expression signature. Thus, by querying the Connectivity Map with signatures of muscle atrophy, we were, in effect, querying with the reciprocal signature of muscle hypertrophy. Indeed, ursolic acid not only reduced muscle atrophy but also induced muscle hypertrophy.
The strategy that led us to ursolic acid implied that ursolic acid might increase muscle mass by inhibiting atrophy-associated skeletal muscle gene expression. Indeed, we found that acute ursolic acid treatment of fasted mice reducedatrogin-1 and MuRF1 mRNAs in association with reduced muscle atrophy. Similarly, chronic ursolic acid treatment of unstressed mice reduced atrogin-1 and MuRF1 mRNAs and induced muscle hypertrophy. Interestingly, ursolic acid-induced muscle hypertrophy was also associated with induction or repression of >60 other skeletal muscle mRNAs, including IGF1 mRNA (which was induced). Although previous studies showed that increased skeletal muscle IGF1expression is sufficient to inhibit atrophy and promote hypertrophy, we noted that, following a hypertrophic stimulus such as mechanical loading, increased IGF1 gene expression is a late event (Adams et al., 1999). We therefore asked whether ursolic acid might stimulate earlier events in insulin/IGF-I signaling by examining skeletal muscle Akt activation, a critical node in the insulin and IGF-I signaling cascades. Indeed, in muscles that had hypertrophied secondary to chronic ursolic acid treatment, Akt phosphorylation was increased. Interestingly, this increase in skeletal muscle Aktactivity can potentially account for many of ursolic acid's effects, including reduced atrophy-associated gene expression, reduced muscle atrophy, increased muscle hypertrophy, and reduced adiposity (Izumiya et al., 2008,Lai et al., 2004). However, additional studies will be needed to determine whether Akt is required for the effects of ursolic acid, and whether other pathways (such as the calcineurin/NFAT and MAP kinase pathways) might also be involved.
Although ursolic acid increased skeletal muscle Akt phosphorylation in vivo, those experiments could not determine ifursolic acid acted directly on skeletal muscle, how quickly ursolic acid acted, and if the effect of ursolic acid requiredIGF-I or insulin, which are always present in healthy animals, even during fasting. To address these questions, we studied serum-starved skeletal myotubes and found that ursolic acid rapidly stimulated IGF-I receptor and insulin receptoractivity, but only if IGF-I or insulin was also present. Taken together, our data suggest that ursolic acid first enhances the capacity of pre-existing IGF-I and insulin to activate skeletal muscle IGF-I receptors and insulin receptors, respectively. This activates Akt, S6K, and ERK and alters skeletal muscle gene expression in a manner that reduces atrophy and promotes hypertrophy. Specific changes in downstream gene expression include induction of IGF1 (a feed-forward mechanism that likely contributes to ursolic acid-mediated hypertrophy), repression of atrogin-1 and MuRF1, and induction or repression of many other genes whose contributions to muscle atrophy or hypertrophy remain to be determined. Some of these changes in skeletal muscle gene expression (such as repression of atrogin-1 and MuRF1) can be explained by our finding that ursolic acid enhances IGF-I-mediated inhibition of FoxO transcription factors. However, ursolic acid might also inhibit other transcription factors that promote atrophy, such as NF-kB, and this is an important area for future investigation.
Importantly, ursolic acid alone was not sufficient to increase phosphorylation of the IGF-I receptor or the insulin receptor. Rather, its effects also required IGF-I or insulin, respectively. This suggests that ursolic acid either facilitates hormone-mediated receptor autophosphorylation or it inhibits receptor dephosphorylation. The latter possibility is supported by previous in vitro data showing that ursolic acid directly inhibits PTP1B (Zhang et al., 2006), a tyrosine phosphatase that dephosphorylates (inactivates) the IGF-I and insulin receptors (Kenner et al., 1996). However, neither global nor muscle-specific PTP1B knockout mice were found to possess increased muscle mass (Delibegovic et al., 2007,Klaman et al., 2000). This may suggest the existence of another receptor for ursolic acid, which might be closely related to PTP1B. Identifying the receptor(s) for ursolic acid is an important area for future investigation that may elucidate important mechanisms of metabolic control. Pharmacokinetic studies of ursolic acid will also be critical for fully understanding its in vivo effects.
Given the current lack of therapies for skeletal muscle atrophy, we speculate that ursolic acid might be investigated as a potential therapy for illness- and age-related muscle atrophy. It may be useful as a monotherapy or in combination with other strategies that have been considered, such as myostatin inhibition (Zhou et al., 2010). Given its capacity to reduce adiposity, fasting blood glucose, and plasma lipid levels, ursolic acid might also be investigated as a potential therapy for obesity, metabolic syndrome, and type 2 diabetes. A systematic search for ursolic acid derivatives that are more potent and/or efficacious could also be undertaken. Further work in this area may lead to new medical therapies for increasingly common metabolic diseases that reduce the absolute or relative amount of skeletal muscle.