Regulation of Skeletal Muscle Glucose Uptake: A Focus on Nitric Oxide Synthase and Rac1 Signalling

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Kerris, Jarrod P (2020) Regulation of Skeletal Muscle Glucose Uptake: A Focus on Nitric Oxide Synthase and Rac1 Signalling. PhD thesis, Victoria University.


Muscle contractions and exercise have been shown to potently stimulate muscle glucose uptake via molecular mechanisms that are different to insulin-stimulated glucose uptake. Importantly, although insulin-stimulated glucose uptake is diminished in people with type 2 diabetes, the ability for contractions or exercise to stimulate muscle glucose uptake appears to be preserved. Activating this exercise signalling pathway with novel drugs could theoretically bypass the defective insulin-signalling pathway and lower blood glucose levels in insulin-resistant individuals. However, the exact signalling mechanisms involved require a better understanding. Several potential regulators of contraction-stimulated glucose uptake have been identified and some degree of redundancy probably exists. Nitric oxide (NO) has been suggested to regulate muscle glucose uptake in rodent and human models. People with type 2 diabetes appear to have a greater reliance on NO to regulate muscle glucose uptake during exercise compared to healthy individuals. Recently, Rac1 has been identified as a novel regulator of muscle glucose uptake during stretch, contraction, and exercise in rodent models. However, the exact mechanisms how both NO and Rac1 regulate glucose uptake are not fully understood. Interestingly, there is some evidence from cell culture studies suggesting an interaction between NO and Rac1 may exist, however, this remains untested in the context of contraction- or exercise-stimulated glucose up in mature muscle models. Therefore, in this thesis, I examined the regulation of skeletal muscle glucose uptake during stretch, contraction, and exercise with a focus on NOS and Rac1 signalling mechanisms. In particular, the potential for NOS and Rac1 to act via the same signalling pathway was explored. The pathways activated by stretch have been considered to overlap with contraction signalling. Stretch is known to increase glucose uptake via Rac1, and there is published evidence that stretch increase muscle NO levels. Therefore, in Study 1, an ex vivo muscle stretching model was used to examine whether NO also plays a role in stretch-stimulated glucose uptake. Stretch increased glucose uptake in isolated EDL muscles and treatment with the NO synthase (NOS) inhibitors L-NMMA and L-NMMA had no effect on this stretch- stimulated glucose uptake. Likewise, stretch-stimulated glucose uptake was normal in muscles from nNOSμ knockout (KO) and eNOS KO mice. A dissociation between NO and Rac1 was observed given that stretching stimulated an increase in Rac1 signalling but did not increase NOS activity above resting levels. This suggested that activation of Rac1 during stretch does not require NO to regulate glucose uptake. I was interested to further examine a potential NO and Rac1 interaction in skeletal muscle given there is evidence from previously published studies suggesting that increased levels of NO activate Rac1 in cells in culture. Therefore, in Study 2, I treated isolated muscles with a NO donor (DETA/NO) to increase NO levels, and this increased glucose uptake above resting levels. Addition of a Rac1 inhibitor (Rac1 inhibitor II) completely prevented this DETA/NO stimulation of skeletal muscle glucose uptake. This suggests that NO can stimulate glucose uptake via a pathway involving Rac1. However, when tested during muscle contraction to increase endogenous NO levels, NOS inhibition did not alter Rac1 signalling during muscle contraction. This suggests that in a more physiological setting, NO does not activate Rac1. Further evidence dissociating a NOS-Rac1 link is suggested by the observation that contraction-stimulated glucose uptake was attenuated by Rac1 inhibition but not by NOS inhibition. The finding that NOS inhibition did not attenuate glucose uptake was surprising as this contrasted with previous studies by our group. Therefore, these results provide further evidence that in regulating muscle glucose uptake, Rac1 signalling does not involve NO. Furthermore, these findings question the hypothesis that NOS plays an important role in the regulation of skeletal muscle glucose uptake during contraction. Given that findings from Study 1 and Study 2 suggest NOS does not play a role in regulating Rac1 signalling and skeletal muscle glucose uptake, I next turned attention to another promising candidate regulator of Rac1 signalling. RhoGDIα has previously been reported to negatively regulate Rac1 activity in cell culture models. It was important however to examine this in a more physiological model such as whole-body exercise where Rac1 is known to be a major regulator of skeletal muscle glucose uptake. Therefore, in Study 3, RhoGDIα was overexpressed in skeletal muscles of mice to test the hypothesis that exercise- stimulated glucose uptake would be attenuated by the negative action of RhoGDIα on Rac1 signalling. However, increased skeletal muscle RhoGDIα protein levels did not attenuate exercise-stimulated glucose uptake, and Rac1 signalling appeared to be normal. Interestingly, increased levels of Rac1 protein were observed in RhoGDIα overexpressing mice. Therefore, in contrast to previous cell culture studies where increased levels of RhoGDIα were found to reduce Rac1 signalling, such a negative role for RhoGDIα in a more physiological setting is not so clear. The increase in Rac1 protein levels in RhoGDIα overexpressing mice could have served to maintain Rac1 signalling during exercise and highlights the potential of a compensatory mechanism to protect signalling pathways regulating glucose metabolism. In summary, findings from this thesis show that during muscle stretching or contraction, NO and Rac1 are not linked. The most striking finding of this thesis is that we were unable to reproduce findings of previous work from our lab since we report that NOS inhibition does not attenuate contraction-stimulated glucose uptake. Given the observed dissociation between NOS and Rac1, another potential regulator of Rac1 signalling, RhoGDIα was examined. In contrast to previous cell culture studies, the role of RhoGDIα as a negative regulator of Rac1 in the context of exercise-stimulated glucose uptake is not clear, since RhoGDIα overexpression induced compensatory changes to other proteins including Rac1 that ultimately did not affect glucose uptake. Further work is required to unravel the complex regulation of Rac1 signalling towards glucose uptake in muscle.

Item type Thesis (PhD thesis)
Subjects Historical > FOR Classification > 0606 Physiology
Historical > FOR Classification > 1106 Human Movement and Sports Science
Historical > FOR Classification > 1116 Medical Physiology
Current > Division/Research > Institute for Health and Sport
Keywords skeletal muscle; glucose; nitric oxide synthase; Rac1 signalling; muscles; stretching; contraction; NOS inhibition; RhoGDIα; exercise
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