High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

Activation of the NLRP3 Inflammasome Increases the IL-1ß Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance.

Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1ß were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4myc-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1ß in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1ß secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.

Exploring NADPH oxidases 2 and 4 in cardiac and skeletal muscle adaptations - a cross-tissue comparison‡.

Striated muscle cells, encompassing cardiac myocytes and skeletal muscle fibers, are fundamental to athletic performance, facilitating blood circulation and coordinated movement through contraction. Despite their distinct functional roles, these muscle types exhibit similarities in cytoarchitecture, protein expression, and excitation-contraction coupling. Both muscle types also undergo molecular remodeling in energy metabolism and cell size in response to acute and repeated exercise stimuli to enhance exercise performance. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) isoforms 2 and 4 have emerged as signaling molecules that regulate exercise adaptations. This review systematically compares NOX2 and NOX4 expression, regulation, and roles in cardiac and skeletal muscle responses across exercise modalities. We highlight the many gaps in our knowledge and opportunities to let future skeletal muscle research into NOX-dependent mechanisms be inspired by cardiac muscle studies and vice versa. Understanding these processes could enhance the development of exercise routines to optimize human performance and health strategies that capitalize on the advantages of physical activity.

From workout to molecular switches: How does skeletal muscle produce, sense, and transduce subcellular redox signals?

Skeletal muscle is crucial for maintaining human health and overall quality of life. Acute exercise introduces a multifaceted intracellular stress, with numerous post-translational modifications believed to underpin the health benefits of sustained exercise training. Reactive oxygen species (ROS) are posited to serve as second messengers, triggering cytoprotective adaptations such as the upregulation of enzymatic scavenger systems. However, a significant knowledge gap exists between the generation of oxidants in muscle and the exact mechanisms driving muscle adaptations. This review delves into the current research on subcellular redox biochemistry and its role in the physiological adaptations to exercise. We propose that the subcellular regulation of specific redox modifications is key to ensuring specificity in the intracellular response.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

BACKGROUND: Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. METHODS: In vastus lateralis muscle biopsies from n = 26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n = 27, WT + LLC: n = 34, mAMPK-KiDe: n = 23, mAMPK-KiDe + LLC: n = 38). Moreover, male LLC-tumour-bearing mice were treated with (n = 10)/without (n = 9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. RESULTS: Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, ß2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, ß2 and γ1), fat-free mass (α1, ß2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance. CONCLUSIONS: Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

NOX2 deficiency exacerbates diet-induced obesity and impairs molecular training adaptations in skeletal muscle.

The production of reactive oxygen species (ROS) by NADPH oxidase (NOX) 2 has been linked to both insulin resistance and exercise training adaptations in skeletal muscle. This study explores the previously unexamined role of NOX2 in the interplay between diet-induced insulin resistance and exercise training (ET). Using a mouse model that harbors a point mutation in the essential NOX2 regulatory subunit, p47phox (Ncf1*), we investigated the impact of this mutation on various metabolic adaptations. Wild-type (WT) and Ncf1* mice were assigned to three groups: chow diet, 60% energy fat diet (HFD), and HFD with access to running wheels (HFD + E). After a 16-week intervention, a comprehensive phenotypic assessment was performed, including body composition, glucose tolerance, energy intake, muscle insulin signaling, redox-related proteins, and mitochondrial adaptations. The results revealed that NOX2 deficiency exacerbated the impact of HFD on body weight, body composition, and glucose intolerance. Moreover, in Ncf1* mice, ET did not improve glucose tolerance or increase muscle cross-sectional area. ET normalized body fat independently of genotype. The lack of NOX2 activity during ET reduced several metabolic adaptations in skeletal muscle, including insulin signaling and expression of Hexokinase II and oxidative phosphorylation complexes. In conclusion, these findings suggest that NOX2 mediates key beneficial effects of exercise training in the context of diet-induced obesity.

Modified Talk Test: a Randomized Cross-over Trial Investigating the Comparative Utility of Two "Talk Tests" for Determining Aerobic Training Zones in Overweight and Obese Patients.

BACKGROUND: To validate the traditional talk test (TTT) and an alternative talk test (ATT; using a visual analog scale) in overweight/obese (OW-OB) patients and to establish its accuracy in determining the aerobic training zones. METHODS: We recruited 19 subjects aged 34.9 ± 6.7 years, diagnosed with overweight/obesity (BMI 31.8 ± 5.7). Every subject underwent incremental cycloergometric tests for maximal oxygen consumption, and TTT in a randomized order. At the end of each stage during the TTT, each subject read out loud a 40 words text and then had to identify the comfort to talk in two modalities: TTT which consisted in answering "Yes," "I don't know," or "No" to the question Was talking comfortable?, or ATT through a 1 to 10 numeric perception scale (visual analog scale (VAS)). The magnitude of differences was interpreted in comparison to the smallest worthwhile change and was used to determine agreement. RESULTS: There was an agreement between the power output at the VAS 2-3 of ATT and the power output at the ventilatory threshold 1 (VT1) (very likely equivalent; mean difference - 1.3 W, 90% confidence limit (CL) (- 8.2; 5.6), percent chances for higher/similar/lower values of 0.7/99.1/0.2%). Also, there was an agreement between the power output at the VAS 6-7 of ATT and the power output at the ventilatory threshold 2 (VT2) (very likely equivalent; mean difference 11.1 W, 90% CL (2.8; 19.2), percent chances for higher/similar/lower values of 0.0/97.6/2.4%). CONCLUSIONS: ATT is a tool to determine exercise intensity and to establish aerobic training zones for exercise prescription in OW-OB patients.

Compartmentalized muscle redox signals controlling exercise metabolism - Current state, future challenges.

Exercise imposes cellular stress on contracting skeletal muscle fibers, forcing them to complete molecular adaptations to maintain homeostasis. There is mounting evidence that redox signaling by reactive oxygen species (ROS) is vital for skeletal muscle exercise adaptations across many different exercise modalities. The study of redox signaling is moving towards a growing appreciation that these ROS do not signal in a global unspecific way, but rather elicit their effects in distinct subcellular compartments. This short review will first outline the sources of ROS in exercising skeletal muscle and then discuss some examples of exercise adaptations, which are evidenced to be regulated by compartmentalized redox signaling. We speculate that knowledge of these redox pathways might one day allow targeted manipulation to increase redox-signaling in specific compartments to augment the exercise-hormetic response in health and disease.

Effects of Polarized Training on Cardiometabolic Risk Factors in Young Overweight and Obese Women: A Randomized-Controlled Trial.

Introduction: Volume and intensity are major variables governing exercise training-mediated beneficial effects in both athletes and patients. Although polarized endurance training optimizes and maximizes physiological gains in highly trained individuals, its cardiometabolic protective-effects have not been established. The purpose of the present single site, randomized-controlled trial was to compare the effects of 12-weeks of high-intensity interval training (HIIT), moderate-intensity continuous training (MICT), and polarized volume training (POL) programs on cardiometabolic risk factors in young overweight and obese women. Materials and Methods: A total of 64 overweight/obese young women (age 23.3 ± 3.8 years, body mass index 33.8 ± 3.8 kg/m2) were randomly assigned to four groups: control group (CTRL), polarized volume training group, moderate-intensity endurance training group, and HIIT group. The cardiorespiratory capacity, glycemic and lipid profiles, whole-body substrate utilization, and body composition were assessed before and after the intervention. Results: After the intervention, VO2peak and power output at VO2peak increased in all exercised-groups (time effect: p < 0.0001). Power output at VT1 was increased only in the POL group compared to the CTRL group (p = 0.019). Relative fold changes in fasting plasma glucose concentrations decreased only in POL group (p = 0.002). Training induced a significant increase in relative fat oxidation in all the groups (time effect: p < 0.001). Relative fat oxidation increased only in the POL group compared to the CTRL group (training effect: p = 0.032). Conclusion: Twelve-weeks of polarized volume training showed overall superior effects on cardiorespiratory fitness, basal glycemic control, and substrate oxidation in comparison to MICT and HIIT training modalities. These data suggest that polarized volume training is an effective non-pharmacological treatment strategy for reducing cardiovascular disease risk factors in young overweight and obese women. The trial is registered at ISRCTN, number ISRCTN34421723.