A team from Montpellier University has identified carnosol - a phenolic diterpene - as the compound that is responsible for the muscle growth stimulating activity exhibited by rosemary leaf extract.
Taken together with other recent research on carnosol, these findings suggest that the compound could play a role in the development of new strategies for limiting muscle loss associated with disease and aging.
“All these findings indicate that carnosol is a promising molecule for the treatment of skeletal muscle disorders such as aging and cachexia,” wrote the researchers in the journal Nutrients.
The researchers also elucidated the mechanism by which carnosol induces hypertrophy (increase and growth of muscle cells). They said their results strongly suggest that carnosol represses the ubiquitin-proteasome system-dependent protein degradation pathway through inhibition of the E3 ubiquitin ligase MuRF1.
No ideal solution for sarcopenia
Whilst exercise in conjunction with amino acid supplementation is currently accepted as the most effective measure for limiting muscle mass loss, physical activity is not always an option for people affected by chronic illness. Therefore, pharmaceutical companies have developed drugs such as anti-myostatin antibodies, myostatin inhibitors and modulators of the androgen receptor. However, some studies have shown that these molecules have side effects and are not very effective in humans.
Recent studies have shown that bioactive molecules found in plants or fruits could be used for the treatment or prevention of skeletal muscle atrophy by stimulating protein synthesis and / or inhibiting protein degradation. However, there is currently a lack of commercially available therapeutic agents.
This research team therefore developed a bioassay-guided fractionation approach to identify molecules with muscle growth promoting effects. First of all, they used cell cultures isolated from human skeletal muscle to determine whether rosemary leaf extract could induce muscle hypertrophy. Incubation of myotubes with the extract resulted in increased muscle cell size versus the control, showing that extracts from rosemary leaves could indeed induce muscle cell hypertrophy.
The researchers then used this bioassay to guide the purification and identification of the compound responsible for the extract’s hypertrophic activity. Six steps of purification were necessary to isolate the active compound. Analysis of spectroscopy data and comparison with the literature then enabled the researchers to identify this compound as carnosol.
Using Western blotting analysis they found that carnosol stimulates muscle hypertrophy by targeting the expression of specific proteins, namely the slow myosin heavy chain proteins that are present in slow-twitch type I muscle fibres. This is significant because by promoting type I muscle fibres, carnosol should make the muscle fatigue-resistant and more enduring. This hypothesis is currently being evaluated in animal models.
The next step was to evaluate the impact of carnosol on the different signalling pathways involved in the control of muscle cell size.
Finding the pathways
As carnosol can activate the antioxidant NRF2 pathway in several other cell types, the scientists determined whether this was the case in skeletal muscle. They found that activation of the NRF2 pathway by carnosol is not sufficient to explain its muscle growth promoting effect.
To test whether carnosol induced muscle hypertrophy by stimulating protein synthesis or by inhibiting protein degradation, they investigated its effect on the Akt/mTORC1 pathway that regulates protein synthesis. Their results strongly suggested that this pathway is not activated in response to carnosol exposure.
Next, they analysed the ubiquitin-proteasome system that controls protein degradation. They found that carnosol inhibited the muscle-specific E3 ligase MuRF1, but not MuRF2 and MAFbx. They also confirmed that silencing MuRF1 was sufficient to induce muscle hypertrophy in skeletal muscle cells.
Inhibition of MuRF1, a ubiquitin-proteasome system component, strongly suggested a decrease of proteaseome activity. The researchers confirmed this hypothesis in vitro, and used a proteosome activity inhibitor to confirm the importance of the ubiquitin-proteasome system in skeletal muscle hypertrophy. Incubation with increasing concentrations of this proteasome inhibitor led to significantly larger myotubes, indicating that inhibition of the proteasome can induce muscle hypertrophy.
“Our data suggest that the hypertrophic effect of carnosol in human muscle cells is mediated through inhibition of the E3 ubiquitin ligase MuRF1 and of the proteasome,” they said.
Further work is needed to explain the relationship between the ubiquitin-proteasome system and muscle differentiation as well as the mechanism by which carnosol controls MuRF1 expression.
Authors: Morel S, Hugon G, Vitou M, Védère M, Fons F, Rapior S, Saint N, Carnac G
“A bioassay-guided fractionation of rosemary leaf extract identifies carnosol as a major hypertrophy inducer in human skeletal muscle cells”