Nucleotides Released From Palmitate-Challenged Muscle Cells Through Pannexin-3 Attract Monocytes

Abstract

Obesity-associated low-grade inflammation in metabolically relevant tissues contributes to insulin resistance. We recently reported monocyte/macrophage infiltration in mouse and human skeletal muscles. However, the molecular triggers of this infiltration are unknown, and the role of muscle cells in this context is poorly understood. Animal studies are not amenable to the specific investigation of this vectorial cellular communication. Using cell cultures, we investigated the crosstalk between myotubes and monocytes exposed to physiological levels of saturated and unsaturated fatty acids. Media from L6 myotubes treated with palmitate—but not palmitoleate—induced THP1 monocyte migration across transwells. Palmitate activated the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in myotubes and elevated cytokine expression, but the monocyte chemoattracting agent was not a polypeptide. Instead, nucleotide degradation eliminated the chemoattracting properties of the myotube-conditioned media. Moreover, palmitate-induced expression and activity of pannexin-3 channels in myotubes were mediated by TLR4-NF-κB, and TLR4-NF-κB inhibition or pannexin-3 knockdown prevented monocyte chemoattraction. In mice, the expression of pannexin channels increased in adipose tissue and skeletal muscle in response to high-fat feeding. These findings identify pannexins as new targets of saturated fatty acid–induced inflammation in myotubes, and point to nucleotides as possible mediators of immune cell chemoattraction toward muscle in the context of obesity.

Regulation of Connexin-Based Channels by Fatty Acids

“…Conversely, a PUFA as AA (20:4) reduces the macroscopic membrane current of Panx1 channels expressed in Xenopus oocytes, and reduces the release of ATP (Xiao et al, 2012; Samuels et al, 2013). With regard to Panx3, treatment of L6 myotubes with palmitate, but not palmitolate, was observed to promote the release of a macrophage chemoattracting agent likely to be ATP-released through Panx3 channels, since it was abrogated after silencing Panx3 (Pillon et al, 2014). …”

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis

“…Muscle and adipose tissue from obese humans show an elevated presence of immune cells (18) that originate from the bone marrow and cross the endothelial layer of capillaries and venules to reach the tissues where they differentiate into macrophages (19). Monocyte chemoattractant protein-1 (MCP1) and other chemokines, as well as nucleotides are recognized chemoattractants for monocytes in obesityassociated inflammation (36), but the infiltration of immune cells requires expression of adhesion molecules at the surface of endothelial cells to promote their adhesion and transmigration. This interaction is carried out at the microvascular endothelium, by members of the immunoglobulin superfamily (intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3, and vascular cell adhesion molecule VCAM-1) and selectins (E and P) that recognize their cognate ligands on the leukocyte surface.…”

Contracting C2C12 myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction

“…CCL2 and ATP are known monocyte chemoattractants (30), and postexercised skeletal muscle presents elevated numbers of macrophages and other immune cells, dependent on the intensity and type of exercise (16,27,31,46). Depletion of circulating monocytes via clodronate-containing liposome injection in mice reduced contraction-induced accumulation of macrophages in mouse skeletal muscle without affecting the number of resident macrophages in noncontracted muscles (16).…”

“…Whether CCL2 itself or other factors responding to CCL2 availability are the chemoattracting agent is unknown and difficult to discern by in vivo studies. Indeed, in palmitate-challenged cultured myotubes, nucleotides such as ATP and UTP released through pannexin channels, rather than secreted chemokines, exert chemoattraction on THP-1 monocytes (30).…”