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Reactive oxygen species (ROS) are well-established signaling molecules implicated in a wide range of cellular processes, including both oxidative stress and intracellular redox signaling. In the context of insulin action within its target tissues, ROS have been reported to exert both positive and negative regulatory effects. However, the precise molecular mechanisms underlying this duality remain unclear. This Review examines the complex role of ROS in insulin action, with a particular focus on skeletal muscle. We aim to address three critical aspects: (a) the proposed intracellular pro-oxidative redox shift elicited by insulin, (b) the evidence supporting that redox-sensitive cysteine modifications impact insulin signaling and action, and (c) cellular mechanisms underlying how ROS can paradoxically act as both enhancers and inhibitors of insulin action. This Review underscores the urgent need for more systematic research to identify specific reactive species, redox targets, and the physiological significance of redox signaling in maintaining insulin action and metabolic health, with a particular emphasis on human skeletal muscle.

Preclinical models suggest mitochondria-derived oxidative stress as an underlying cause of insulin resistance. However, it remains unknown whether this pathophysiological mechanism is conserved in humans. Here, we used an invasive in vivo mechanistic approach to interrogate muscle insulin action while selectively manipulating the mitochondrial redox state in humans. To this end, we conducted insulin clamp studies combining intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mitoquinone). Under lipid overload, selective modulation of mitochondrial redox state by mitoquinone enhanced insulin-stimulated glucose uptake in skeletal muscle. Mechanistically, mitoquinone did not affect canonical insulin signaling but augmented insulin-stimulated glucose transporter type 4 (GLUT4) translocation while reducing the mitochondrial oxidative burden under lipid oversupply. Complementary ex vivo studies in human muscle fibers exposed to high intracellular lipid levels revealed that mitoquinone improves features of mitochondrial bioenergetics, including diminished mitochondrial H2O2 emission. These findings provide translational and mechanistic evidence implicating mitochondrial oxidants in the development of lipid-induced muscle insulin resistance in humans.

Aim: The present study aimed to investigate the effects of a single bout of resistance exercise on mitophagy in human skeletal muscle (SkM). Methods: Eight healthy men were recruited to complete an acute bout of one-leg resistance exercise. SkM biopsies were obtained one hour after exercise in the resting leg (Rest-leg) and the contracting leg (Ex-leg). Mitophagy was assessed using protein-related abundance, transmission electron microscopy (TEM), and fluorescence microscopy. Results: Our results show that acute resistance exercise increased pro-fission protein phosphorylation (DRP1Ser616) and decreased mitophagy markers such as PARKIN and BNIP3L/NIX protein abundance in the Ex-leg. Additionally, mitochondrial complex IV decreased in the Ex-leg when compared to the Rest-leg. In the Ex-leg, TEM and immunofluorescence images showed mitochondrial cristae abnormalities, a mitochondrial fission phenotype, and increased mitophagosome-like structures in both subsarcolemmal and intermyofibrillar mitochondria. We also observed increased mitophagosome-like structures on the subsarcolemmal cleft and mitochondria in the extracellular space of SkM in the Ex-leg. We stimulated human primary myotubes with CCCP, which mimics mitophagy induction in the Ex-leg, and found that BNIP3L/NIX protein abundance decreased independently of lysosomal degradation. Finally, in another human cohort, we found a negative association between BNIP3L/NIX protein abundance with both mitophagosome-like structures and mitochondrial cristae density in the SkM. Conclusion: The findings suggest that a single bout of resistance exercise can initiate mitophagy, potentially involving mitochondrial ejection, in human skeletal muscle. BNIP3L/NIX is proposed as a sensitive marker for assessing mitophagy flux in SkM.

Pre-clinical models indicate a causative nexus between mitochondrial oxidative stress and insulin resistance. However, the pathophysiological significance of this mechanism in humans remains unproven. Herein, we employed an invasive in vivo mechanistic approach in humans to manipulate mitochondrial redox state while assessing insulin action. To this end, we combined intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mtAO) in conjunction with insulin clamp studies. During lipid overload, insulin-stimulated muscle glucose uptake, as determined by the femoral arteriovenous balance technique, was increased by mtAO. At the muscle molecular level, mtAO did not affect canonical insulin signaling but enhanced insulin-stimulated GLUT4 translocation while decreasing the mitochondrial oxidative burden under lipid oversupply. Ex vivo studies revealed that mtAO ameliorated features of mitochondrial bioenergetics, including diminished mitochondrial H2O2 emission, in muscle fibres exposed to high intracellular lipid levels. These findings implicate mitochondrial oxidants in the development of lipid-induced insulin resistance in humans.

Women with polycystic ovary syndrome (PCOS) have been shown to be less insulin sensitive compared with control (CON) women, independent of BMI. Training is associated with molecular adaptations in skeletal muscle, improving glucose uptake and metabolism in both healthy individuals and patients type 2 diabetes. In the current study, lean hyperandrogenic women with PCOS (n = 9) and healthy CON women (n = 9) completed 14 weeks of controlled and supervised exercise training. In CON, the training intervention increased whole body insulin action by 26% and insulin-stimulated leg glucose uptake by 53%, together with increased insulin-stimulated leg blood flow and a more oxidative muscle fiber type distribution. In PCOS, no such changes were found, despite similar training intensity and improvements in VO2max. In skeletal muscle of CON but not PCOS, training increased GLUT4 and HKII mRNA and protein expressions. These data suggest that the impaired increase in whole-body insulin action in women with PCOS with training is caused by an impaired ability to upregulate key glucose-handling proteins for insulin-stimulated glucose uptake in skeletal muscle and insulin-stimulated leg blood flow. Still, other important benefits of exercise training appeared in women with PCOS, including an improvement of the hyperandrogenic state.

The small molecule kinase inhibitor SBI-0206965 was originally described as a specific inhibitor of ULK1/2. More recently, it was reported to effectively inhibit AMPK and several studies now report its use as an AMPK inhibitor. Currently, we investigated the specificity of SBI-0206965 in incubated mouse skeletal muscle, measuring the effect on analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated AMPK-dependent glucose transport and insulin-stimulated AMPK-independent glucose uptake. Pre-treatment with 10 µM SBI-0206965 for 50 min potently suppressed AICAR-stimulated glucose transport in both the extensor digitorum longus (EDL) and soleus muscle. This was despite only a modest lowering of AICAR-stimulated AMPK activation measured as ACC2 Ser212, while ULK1/2 Ser555 phosphorylation was prevented. Insulin-stimulated glucose transport was also potently inhibited by SBI-0206965 in soleus. No major changes were observed on insulin-stimulated cell signaling. No general effect of SBI-0206965 on intracellular membrane morphology was observed by transmission electron microscopy. As insulin is known to neither activate AMPK nor require AMPK to stimulate glucose transport, and insulin inhibits ULK1/2 activity, these data strongly suggest that SBI-0206965 has a non-specific off-target inhibitory effect on muscle glucose transport. Thus, SBI-0206965 is not a specific inhibitor of the AMPK/ULK-signaling axis in skeletal muscle, and data generated with this inhibitor must be interpreted with caution.

Significance: Skeletal muscle is a crucial tissue to whole-body locomotion and metabolic health. Reactive oxygen species (ROS) have emerged as intracellular messengers participating in both physiological and pathological adaptations in skeletal muscle. A complex interplay between ROS-producing enzymes and antioxidant networks exists in different subcellular compartments of mature skeletal muscle. Recent evidence suggests that NADPH oxidases (NOX) are a major source of contraction- and insulin-stimulated oxidants production, but may paradoxically also contribute to muscle insulin resistance and atrophy.Recent advances: Pharmacological and molecular biological tools, including redox-sensitive probes and transgenic mouse models, have generated novel insights into compartmentalized redox signaling and suggested that NOX2 contributes to redox control of skeletal muscle metabolism. Critical issues: Major outstanding questions in skeletal muscle include where NOX2 activation occurs under different conditions in health and disease, how NOX2 activation is regulated, how superoxide/ H2O2 generated by NOX2 reaches the cytosol, what the signaling mediators are downstream of NOX2, and the role of NOX2 for different physiological and pathophysiological processes. Future directions: Future research should utilize and expand the current redox-signaling toolbox to clarify the NOX2-dependent mechanisms in skeletal muscle and determine whether the proposed functions of NOX2 in cells and animal models are conserved into man.

Skeletal muscle is described as an endocrine organ, constitutively or intermittently secreting bioactive molecules. The signaling pathways by which these molecules mediate changes in skeletal muscle and regulate interorgan crosstalk are only partly understood. Lactate is widely described as a signaling molecule in different cells, but the role of lactate as a signaling molecule in mature skeletal muscle has not been fully unveiled. The aim of this study was to determine the role of lactate on activation of signaling pathways in adult mouse skeletal muscle. Male mice were injected intraperitoneally with lactate or saline, and tissues were dissected after 40 min. Phosphorylation levels of relevant proteins in muscle were assessed by Western blotting. After lactate administration, we found an increase in p-ERK1/2Thr202/Tyr204 (3.5-fold; P = 0.004) and p-p70S6KThr389 (1.9-fold; P = 0.01) in quadriceps; and an increase in p-rpS6Ser235/236 in both quadriceps (6.3-fold; P = 0.01) and EDL (2.3-fold; P = 0.01), without changes in soleus. There was a tendency toward an increase in p-AMPKThr172 (1.7-fold; P = 0.08), with a significant increase in p-ACCSer79 (1.5-fold; P = 0.04) in soleus, without changes in quadriceps and EDL. These results support the hypothesis that lactate plays a role in the molecular signaling related to hypertrophy and to oxidative metabolism on adult skeletal muscle and suggest that this activation depends on the skeletal muscle type. The mechanisms that underlie the effect of lactate in mature skeletal muscles remain to be established.