Rac1 is a novel regulator of exercise‐induced glucose uptake

Exercise is widely known to lead to beneficial adaptions in whole body health, including improvements in indices of glucose homeostasis. During exercise, muscle contraction stimulates an increase in skeletal muscle glucose uptake in order to provide substrate for energy production. The provision of glucose to skeletal muscle is mediated by three factors: (1) the delivery of glucose to muscle; (2) the transport of glucose across the plasma membrane; and (3) the intracellular metabolism of glucose (Richter & Hargreaves, 2013). The delivery of glucose to working skeletal muscle is regulated by changes in blood flow and capillary recruitment, both of which increase with exercise. The transport of glucose across the plasma membrane is increased during exercise, which occurs by translocation of glucose transporter type 4 (GLUT4) from intracellular sites to the plasma membrane (sarcolemma and T-tubules), allowing for facilitated diffusion. Exercise increases the flux of glucose through glycolysis, and the enzyme hexokinase II (HKII) controls this process. Once inside the muscle cell, HKII phosphorylates glucose for commitment to glycolysis or for storage as glycogen. It is the coordination of delivery, transport and metabolism of glucose that leads to increases in glucose uptake during exercise (Richter & Hargreaves, 2013); however, the molecular mechanisms that mediate this process are not fully understood. Recently the Rho family GTPase Rac1 has emerged as having an important role in the regulation of skeletal muscle glucose uptake. Pioneering work from Drs Klip (JeBailey et al. 2004; Thong et al. 2007) and Ueda and Satoh (Ueda et al. 2010) revealed the role for Rac1 in this process, and a recent series of studies from Sylow and colleagues have further advanced our understanding. They found that Rac1 was required for insulin-induced ex vivo glucose uptake in murine soleus and extensor digitorum longus (EDL) muscles (Sylow et al. 2013a). Using pharmacological inhibition and an in vivo inducible muscle specific Rac1 knockout, they showed that Rac1 plays a critical role in electrically induced contraction-stimulated glucose transport (Sylow et al. 2013b). Finally, using a similar approach, they showed an attenuated response to stretch-induced glucose uptake in ex vivo soleus and EDL (Sylow et al. 2015). Despite this, the role of Rac1 in in vivo exercise-induced glucose uptake and GLUT4 translocation is not known. In a paper published in The Journal of Physiology, Sylow et al. (2016) demonstrate that Rac1 is a critical regulator of glucose uptake during exercise. To define the role of Rac1 in vivo, a Rac1 muscle-specific knockout (mKO) was established by breeding Rac1 floxed mice with mice carrying a muscle specific Cre recombinase. Rac1 mKO mice were induced at 10–14 weeks of age by a 3 week exposure to doxycycline in drinking water, followed by a 3 week washout period to reverse potential mitochondrial and gene expression changes induced by this treatment. This led to a reduction in the protein content of Rac1 in whole muscle homogenate of soleus, gastrocnemius, and quadriceps by 70–90% in comparison to wild-type (WT) controls. This indicates an incomplete muscle knockout of Rac1, and the authors propose that the residual Rac1 protein content detected via Western blot analysis is contamination from non-muscle tissue in the whole muscle homogenate. It is possible that an isolated muscle fibre preparation, as opposed to whole muscle, may indicate a complete muscle knockout. To test the effects of Rac1 mKO on exercise performance, a maximal running capacity test was conducted. This revealed similar maximal running speed in WT and Rac1 mKO mice. In contrast, when this test was performed in a glycogen-depleted state, induced by a single prior bout of exercise (30 min at 75% max. intensity) followed by a 2.5 h recovery, Rac1 mKO had reduced maximal running speed in comparison to WT mice. This occurred in parallel with an attenuated decrease in blood glucose, indicating that Rac1 may play a role in exercise-induced glucose uptake. To test this hypothesis, the authors measured in vivo muscle glucose uptake following an acute bout of exercise at 65% maximal intensity. Rac1 mKO had an attenuated increase in glucose uptake in the soleus and gastrocnemius, with a non-significant effect in quadriceps. As glucose uptake is influenced by exercise intensity (Richter & Hargreaves, 2013), they repeated these experiments at 85% max. intensity, and observed a similar attenuation in glucose uptake in Rac1 mKO mice. Together, these results show that although glucose uptake is impaired in the Rac1 mKO mice, this only influences exercise capacity in a glycogen depleted state. To determine how Rac1 may be regulating this process, they assessed three factors involved in the provision of glucose to muscle. First, GLUT4 translocation to the plasma membrane was measured using immunohistochemistry on 12 μm cryosections of tibialis anterior muscle as the amount of GLUT4 localized to the α-sarcoglycan area (i.e. plasma membrane). WT mice had a 42% increase in GLUT4 translocation, whereas this was impaired in Rac1 mKO. It is possible that this increase may actually be of greater magnitude as the resolution of the microscope may not allow for complete distinction of plasma membrane inserted GLUT4 vs. GLUT4 beneath the membrane. The cryosections were then prepared for measurement of 2-deoxyglucose uptake, which was attenuated in Rac1 mKO mice. These data suggest that Rac1 may regulate the translocation and binding of GLUT4 to the plasma membrane, and therefore glucose uptake. Second, Sylow et al. examined indices of glucose delivery through measurement of capillary density. Using cryosections of tibialis anterior muscle stained for Lectin 1, they found this was not reduced in Rac1 mKO mice. Third, alterations in indices of glycolytic and oxidative metabolism were measured in quadriceps and gastrocnemius via Western blots of HKII and the mitochondrial proteins cytochrome c, and electron transport chain complexes I and II. No differences between WT and Rac1 mKO mice were found. In addition to these measures, muscle glycogen content was measured after exercise at 65% max. intensity, and it was found that Rac1 mKO and WT mice had similar reductions, suggesting that Rac1 mKO mice do not rely more on muscle glycogen during exercise. Together, this demonstrates that Rac1 is likely to be involved in exercise-induced glucose transport through regulation of GLUT4 translocation and binding to the plasma membrane. To determine how Rac1 may be regulating exercise-induced glucose uptake and GLUT4 translocation, the authors next assessed the role of potential molecular mediators, including 5′AMP-activated protein kinase (AMPK), calcium (Ca2+), and β-adrenergic activation. The phosphorylation of AMPKThr172 was increased in quadriceps and gastrocnemius by 30% but not in soleus after a bout of exercise at 65% max. intensity; however, there was no difference between WT and Rac1 mKO mice. In addition, AMPKα2 but not AMPKα1 activity was increased after exercise in both WT and Rac1 mKO mice. These results, and their previous work (Sylow et al. 2013b), suggest that Rac1 does not act via AMPK to influence glucose uptake during exercise. However, these findings are in contrast to those of other groups that have demonstrated a role for AMPK in this process. Next, the role of calcium and β-adrenergic activation was assessed using cultured L6 myotubes. Ca2+ signalling was stimulated with caffeine, β-adrenergic activation with isoproterenol, and insulin was utilized as a known Rac1–GTP binding stimulus. There was no effect of either Ca2+ or β-adrenergic stimulation on Rac1–GTP binding, while insulin increased Rac1–GTP binding as previously demonstrated (Sylow et al. 2013a). In contrast to these results, in experiments using C2C12 myotubes, electrical pulse stimulation was able to increase Rac1–GTP binding, which is similar to their ex vivo results using electrical stimulation (Sylow et al. 2013b) and mechanical stretch (Sylow et al. 2015). These results suggest that potential molecular mediators of Rac1 during exercise-induced glucose uptake involve mechanical stretch and stress, and probably not AMPK, Ca2+, or β-adrenergic activation. In this paper, the authors demonstrate a critical role of Rac1 in regulating skeletal muscle glucose uptake during exercise. The data indicate that Rac1 is primarily involved in the regulation of GLUT4 translocation and plasma membrane binding. However, the mechanisms through which Rac1 is activated by exercise are not understood. A role of AMPK, calcium, or β-adrenergic stimuli as mediating candidates is not supported herein; however, data from this study (Sylow et al. 2015) and that published previously (Sylow et al. 2013b, 2015) suggest that pathways induced by electrical stimulation and mechanical stretch may be potential mechanistic candidates, but this remains to be completely investigated. To further unravel the role of Rac1, a future study should consider whether Rac1 exerts effects unique to skeletal muscle, or if it is involved in glucose uptake into other tissues during exercise. Overall, the findings of this study, and that of the other studies published by this group, highlight Rac1 as a novel regulator of glucose uptake in response to diverse physiological stimuli. None R.E.K.M. is funded by a PDF through the Alzheimers Society of Canada. W.T.P. is supported by the Guelph Food Technology Centre (GFTC) and Vitamin Scholarship (University of Guelph). The authors thank Dr David Wright for critical comments on this manuscript.

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