Background: Skeletal muscle is highly active metabolic tissue, and with its myokine secretion an important endocrine organ. Interleukin-6 (IL-6) is a pleiotropic cytokine that is secreted from a wide spectrum of cells and tissues. Inflammatory and metabolic responses depend mostly on different physiological environment, time, and tissue specific actions of IL-6. Chronically elevated IL-6 has proinflammatory effects and is linked to negative prognosis of disease but its acute elevation under physiological conditions exerts beneficial effects on metabolism and health in general. IL-6 inhibitors are important in treating chronic inflammatory diseases.
Aim: Understanding the regulatory mechanisms of IL-6 broadens our possibilities in therapeutic optimisation and development of next generation drugs. Factors that determine the IL-6 double role are not yet well defined, therefore we are interested aim to gain the understanding of the regulation of IL-6 synthesis, secretion, and its actions. We study the mechanisms of IL-6 expression, receptor signalling and effects of therapeutic targeting of IL-6 in skeletal muscle. The main aim was to examine the effects of IL-6 inhibitors on IL-6 expression and action in skeletal muscle cell culture.
Hypotheses: 1.) TLR4 and ERK1/2 are important for basal IL-6 expression in human skeletal muscle cell culture.2.) IL-6 inhibitors do not affect IL-6 expression and secretion in human skeletal cell culture. 3.) IL-6 inhibitors and insulin reduce basal STAT3 phosphorylation in human skeletal muscle cells. 4.) The effect of IL-6 inhibitors on basal and insulin-stimulated Akt and ERK1/2 signalling pathway activity is dependent on the concentration and duration of IL-6 exposure. 5.) IL-6 inhibitors reduce AMP-activated protein kinase activity in human skeletal muscle cells.
Methods: Experiments were taken on primary human skeletal muscle cell culture. IL-6 mRNA expression in muscle cells was determined with quantitative real time PCR. IL-6 secretion was measured with the enzyme linked immunoabsorbent assay. Cellular signalling responses were assessed with western blotting by measuring the phosphorylation of key signalling proteins; STAT3. AKT, ERK1/2, AS160, GSK-3α/β, PAS in ACC.
Results: Constitutive IL-6 secretion from cultured skeletal muscle cells was measured. LPS treatment activated the receptor TLR4, which stimulated IL-6 expression in muscle cells. IL-6 expression was inhibited by TLR4 signalling inhibitors. Neither stimulation nor complete inhibition of IL-6 expression was noticed by any of the used inhibitors. Phosphorylation of metabolic signalling proteins after treatment with tocilizumab and tofacitinib was not significantly changed. Decreased insulin response was noticed after the chronic treatment of skeletal muscle cells with IL-6. Constitutive phosphorylation of STAT3 was effectively inhibited by tofacitinib. Inhibition of STAT3 phosphorylation by tocilizumab was measured only at the acute IL-6 treatment. Effect on insulin actions by IL-6 inhibitors was not detected.
Conclusions: Endogenous secretion of IL-6 under basal conditions was confirmed. The mechanisms of spontaneous TLR4 activation and ERK1/2 activity as an IL-6 positive feedback loop were excluded for this state. We conclude that IL-6 itself does not influence its own expression/secretion. Our results show no significant effect of IL-6 inhibition, or chronic IL-6 exposure, on STAT3, Akt, ERK1/2 and AMPK signalling pathways in cultured human skeletal muscle cells. Chronic exposure to IL-6 in muscle could affect insulin action.
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