Rac1: an emerging player in stretch‐stimulated glucose transport

Muscle contraction–relaxation and insulin signalling are the two major stimuli for glucose transport. Mechanical stress is an integral component of the muscle contraction–relaxation cycle. Several lines of evidence have demonstrated that mechanical stress regulates muscle function and muscle adaptation in different ways. The signals generated by mechanical stress are converted into molecular events that regulate multiple metabolic processes, leading to cell proliferation, repair and regeneration. Understanding the precise molecular events controlling glucose transport within skeletal muscle is vital, because impairment at various stages of the glucose-uptake signalling cascade could trigger anomalies in the intricate glucoregulatory system. There has been increasing evidence to suggest that mechanical stress could be an essential component for complete activation of glucose transport and stress-stimulated signalling within the muscle cell. However, the contributions of novel molecular mediators that regulate glucose uptake during skeletal muscle adaptation in response to mechanical stress are not yet fully understood. In recent years, a plethora of studies have been conducted to identify the molecular pathways that underlie skeletal muscle glucose transport in response to mechanical stress. In particular, the role of small Rho family guanosine triphosphatases (GTPases) has been increasingly considered and well documented (Chiu et al. 2011). Rho GTPase belongs to the Ras superfamily of GTPases, which are evolutionarily conserved proteins involved in diverse multifaceted cellular processes. Growing evidence supports the participation of the activated small Rho family GTPase Rac1 in insulin-induced and contraction-stimulated glucose transport. Pioneering studies have reported that Rac1 could be activated through mechanical stress and by passive stretching of the skeletal muscle (Kawamura et al. 2003). In addition to this role, Rac1 was also shown to play a critical role in reorganization of the actin cytoskeleton for insulin-stimulated glucose transport. In accordance, several studies have confirmed the significance of Rac1 by demonstrating decreased glucose transport upon pharmacological disruption of the actin cytoskeleton (Brozinick et al. 2004). Nevertheless, the importance of Rac1 and the actin cytoskeleton has never been studied in the context of stretch-stimulated glucose transport in skeletal muscle. In a recent study published in The Journal of Physiology, Sylow et al. (2015) investigated the involvement of Rac1 and intact actin filament organization in stretch-induced glucose transport in skeletal muscle. The authors hypothesized that Rac1 activation and a functional actin cytoskeleton would be critically involved in stretch-stimulated glucose transport within skeletal muscle. To test their hypothesis, Sylow and colleagues used Rac1 inhibitors or Rac1 knockout (KO) mice. They demonstrated that at a concentration of 15 μm, the Rac1 inhibitor II reduced stretch-induced glucose transport by ∼30 and ∼50% in soleus and extensor digitorum longus muscles, respectively, as calculated using a 2-deoxyglucose uptake assay. Immunoblotting analysis also confirmed these results, suggesting that Rac1 is a crucial regulator of glucose transport in skeletal muscle induced by mechanical stress. Several molecules have been associated with muscle stretching; however, the exact underlying mechanisms underlying glucose transport have not been clarified. The authors have previously reported that passive stretching of mouse muscle activated Rac1 and, in the present study, they showed that stretching increased phosphorylation of p21-activated protein kinase (PAK1/2), the downstream target of Rac1. This phosphorylation event was abolished by the Rac1 inhibitor II, confirming the role of Rac1 in the phosphorylation of PAK1/2 upon passive stretching. These findings are in agreement with studies from another group showing that Rac1 co-localized with PAK in response to passive stretching in skeletal muscle. Importantly, the authors also showed that AMPK or Akt-mediated signalling had no role in regulating stretch-stimulated glucose transport. To provide further confirmation of the effect of Rac1 inhibition on stretch-induced glucose transport, the authors evaluated glucose transport and downstream signa-lling by the use of inducible muscle-specific Rac1 KO mice. After verifying the true KO feature of the mice by determining Rac1 content, the authors demonstrated that stretch-stimulated glucose transport was reduced by ∼40% in soleus and by ∼30% in extensor digitorum longus in the KO mice. Upon analysis of PAK1/2 signalling, it was observed that the stretch-induced PAK1/2 phosphorylation was sign-ificantly diminished in Rac1 KO mice compared with the wild-type littermates. Collectively, these results were in accordance with the pharmacological inhibition of Rac1 previously demonstrated in ex vivo incuba-ted muscles, thus further confirming the role of Rac1 and the downstream effec-tor PAK1/2 in stretch-stimulated glucose transport. Beyond its role as a scaffolding structure, the actin cytoskeleton has also been implicated in the transduction of various extracellular signals. In addition to the classical insulin signalling cascade mediated by Akt, activation of the small Rho GTPase Rac1 and downstream actin remodelling have emerged as potential mechanisms of stretch-stimulated glucose transport. In an attempt to demonstrate that Rac1 could regulate actin cytoskeleton dynamics and thereby mediate stretch-stimulated glucose transport, Sylow et al. (2015) performed experiments using the actin-depolymerizing agent, latrunculin B. With the use of a 2-deoxyglucose uptake assay and immunoblotting analysis they showed that latrunculin B significantly reduced stretch-stimulated glucose transport by ∼50% in soleus and ∼40% in extensor digitorum longus muscle, suggesting that an intact and functional actin cytoskeleton is essential for the regulation of stretch-induced glucose transport. Notably, they also showed that disruption of the actin cytoskeleton using latrunculin B did not affect stretch-activated signalling. In contrast, the authors suggested that the actin cytoskeleton might facilitate the docking and fusion of GLUT4 (a glucose transporter protein) vesicles for the entry of glucose into the cell. Using a GLUT4 inhibitor, cytochalasin B, they showed that stretch-stimulated 2-deoxyglucose uptake was completely inhibited, indicating that glucose transport during stretching was indeed via GLUT4. Subsequently, using the ATPase inhibitors and Rac1 inhibitor, they also indicated that Rac1 could partly regulate contraction-stimulated glucose transport but only in the presence of a mechanical stress component. Overall, the authors have demonstrated that treatment of skeletal muscle with a Rac1 inhibitor or muscle-specific deletion of Rac1 significantly reduced stretch-stimulated PAK1/2 activation but had no effect on phospho-p38 MAPK level, suggesting that PAK1/2 signalling, as opposed to p38 MAPK, is likely to be downstream of Rac1. It is important to note, however, that PAK1 has been suggested to function upstream of Rac1 in other cell types via phosphorylation of Rho Guanine diphosphate dissociation inhibitor to yield activated Rac1. With this evidence, additional studies are needed to investigate the precise placement of PAK1/2 in the molecular events that take place after stretch-induced Rac1 activation. Moreover, studies employing the use of p38 MAPK inhibitors and skeletal muscle-specific p38 MAPK KO mice could be beneficial to confirm whether the observed stretch-induced glucose transport in skeletal muscle via Rac1 activation is truly independent of p38 MAPK. Future investigations should also use overexpression studies of Rac1 to provide better evidence of the molecular pathways involved in stretch-stimulated glucose transport. Lastly, exercise and muscle stretching have been shown to increase skeletal muscle production of free radicals, such as nitric oxide and reactive oxygen species (Chambers et al. 2009). Although Rac1 signalling, nitric oxide and reactive oxygen species production have been suggested to play a role in glucose transport, a direct link between Rac1 and a reactive oxygen species/nitric oxide pathway in the regulation of stretch-stimulated glucose uptake in skeletal muscle has not been investigated. Future studies are thus needed to investigate how these seemingly disparate pathways/mechanisms fit into the model described by Sylow et al. (2015). It is noteworthy that future studies on Rac1 and its downstream signalling effectors could possibly unravel other putative candidates involved in stretch-stimulated glucose transport. In conclusion, this study is the first to highlight the novel role of Rac1 and an intact actin cytoskeleton for the regulation of stretch-stimulated glucose transport in skeletal muscle (Fig. 1). Altogether, the findings presented suggest that Rac1 regulates the mechanical stress component of stretch-stimulated glucose transport, possibly via the actin cytoskeleton. Although limited, the present study by Sylow et al. (2015) clearly reinforces the functional role of Rac1 and the cortical actin cytoskeleton in mediating glucose transport. Abbreviations: GDP: Guanosine diphosphate; GTP: Guanosine triphosphate; GLUT4: Glucose transporter type 4 None declared. No sources of funding were required to prepare this manuscript. The authors would like to thank Dr Sri Nagarjun Batchu and Michael Kuliszewski (St Michael's Hospital) for reading the manuscript and would like to apologize for not being able to cite all the relevant articles due to reference limitation.

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