IL6 and LIF mRNA expression in skeletal muscle is regulated by AMPK and the transcription factors NFYC, ZBTB14, and SP1

Adenosine monophosphate-activated protein kinase (AMPK) controls glucose and lipid metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells, and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce IL6 or leukemia inhibitory factor (Lif) mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors that may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors nuclear transcription factor Y subunit c (Nfyc), specificity protein 1 (Spl), and zinc finger and BTB domain containing 14 (Zbtb14), or AMPK alpha 1/alpha 2 subunits, increased constitutive levels of IL6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK. Nfyc, Spl and Zbtb14 as novel regulators of immunometabolic signals from skeletal muscle.