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

Abstract

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-1β 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.

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

“…Skeletal muscle is not just a component of the locomotor system, but is also a tissue with a high metabolic rate, and it is the main tissue that is responsive to insulin-stimulated glucose transport [ 3 ]. Mounting evidence supports the role of skeletal muscle as a secretory organ, releasing several myokines, including IL-1β [ 7 , 16 ]. IL-1β is produced in an inactive pro-form that requires cleavage by the NLRP3 inflammasome to be released from the cell, and for its subsequent biological activity [ 9 ].…”

“…The skeletal muscle represents around 40% of the weight of an adult individual, and it has been recognized as an endocrine organ which produces and releases several myokines [ 5 , 6 ]. Recently, emerging evidence has suggested that muscle secretion of inflammatory mediators could be associated with IR development [ 7 ].…”

NLRP3 Inflammasome: Potential Role in Obesity Related Low-Grade Inflammation and Insulin Resistance in Skeletal Muscle

“…On the other hand, myocytes from obese T2D patients secreted higher levels of proinflammatory cytokines compared to non-obese non-diabetic individuals [31,32], suggesting a specific role of skeletal muscle fibers in the development of obesity-related inflammation. We have recently shown that isolated skeletal muscle fibers can express inflammatory markers in diet-induced obese mice, leading to muscle insulin resistance [33]. These data suggest that the contribution of muscle fibers to obesity-associated inflammation could complement immune cell infiltration being important factors for chronic low grade inflammation development.…”

“…Skeletal muscle represents~40% of the body weight and constitutes the body's largest organ in non-obese individuals [40]. Recent studies have shown that inflammation associated with obesity also occurs in skeletal muscle [1,[31][32][33]; however, its cause and its role in IR and T2D development is not well understood. The following evidence supports the relevance of inflammation in skeletal muscle: (i) a broad range of experimental and clinical findings show that skeletal muscle acts as a secretory organ [40]; (ii) during physiological conditions (e.g., exercise), muscle fibers produce and secrete cytokines and other peptidescollectively classified as myokines-that may exert autocrine, paracrine and endocrine effects [40,41].…”

“…High levels of eATP activate intracellular kinases associated with inflammation, triggering insulin resistance in hepatocytes and adipocytes [98,99]. Recently, it has been shown that eATP is elevated in the skeletal muscle of HFD-fed mice, and eATP is responsible for triggering an inflammatory response-with IL-1β upregulation-and insulin resistance in skeletal muscle [33]. New studies are necessary to evaluate whether elevated levels of eATP promote metabolic disturbances in skeletal muscle cells from obese individuals through ionic flux alteration and NLRP3 activation.…”