ATP Released from Skeletal Myofibers to Extracellular Medium Induces an Inflammatory State Linked to Insulin Resistance in Obese Mice
Authors: Jorquera Gonzalo , Meneses‐Valdés Roberto , Rosales‐Soto Giovanni , Llanos Paola , Casas Mariana , Jaimovich Enrique 
Published: June 2021

Skeletal muscle is a key target organ for insulin actions and a main glycemia regulator. In obese individuals there is a chronic low‐grade inflammatory state affecting adipose tissue, liver, and skeletal muscle. This inflammatory response has been associated to the development of insulin resistance. We have previously described adult myofibers can release ATP to extracellular medium (ECM) through pannexin channels. It is also known that high concentration of ATP in ECM can be a potent inflammatory signal. We propose myofibers from obese subjects release high ATP levels, through pannexin channels, promoting activation of gene expression associated to inflammation and insulin resistance.

We fed C57BL/6 mice with regular or high fat diet (HFD) for 8–12 weeks and we isolated myofibers derived from Flexor Digitorium Brevis (FDB) muscle. For obtaining pannexin‐1 knockdown FDB we electroporated in vivo a sh‐pannexin1/mcherry or just mcherry codifying plasmids. We analyzed ATP release in a luciferine/luciferase assay. Gene expression was studied with RT‐qPCR. Protein expression was evaluated by WB. Insulin sensitivity was analyzed in a 2‐NBDG (fluorescent glucose analog) uptake assay.

Mice fed with a HFD showed an important weight gain compared with control diet fed animals. In addition, HFD‐fed mice showed an altered glucose tolerance test, elevated fasting glycemia and insulinemia. We found TLR4, IL‐6, IL‐1β, and TNF‐α genes, which are associated to inflammation and insulin resistance, were upregulated in obese mice FDB muscle. We observed higher ATP release in myofibers from obese mice, in basal state and after electrical stimulation, compared to control ones. Oleamide 100 μM, a pannexin inhibitor, blocked ATP release in obese mice myofibers. Gene expression linked to inflammation was inhibited using oleamide and apyrase 2 U/ml, a nucleotidase enzyme. Using a molecular biology approach, we knockdowned pannexin channel expression in FDB. In knockdown myofibers we observed a diminished ATP release and inflammatory gene expression was also inhibited. Finally, we were able to induce insulin resistance with a single pulse of 300 μM ATP incubated for 12 hours in fibers from control mice. Incubation with pannexin blocker, carbenoxolone 5 μM, increases 2‐NBDG uptake in HFD fibers after insulin stimulation, rescuing insulin resistance condition.

In this work we propose a novel mechanism for the development of inflammation associated to insulin resistance in skeletal muscle. We have found that ATP is released to ECM in an uncontrolled way, by pannexin channels, leading to a high concentration of ATP in the extracellular compartment. This stimulus is able to induce an inflammatory state leading to insulin resistance in skeletal muscle from obese individuals.

Support or Funding Information

Funded by Fondecyt Postdoc 3170194 (GJ) and Fondecyt 1151293 (EJ)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.