Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.

Key points: The discovery of microRNAs (miRNAs) has established new mechanisms that control health, but little is known about the regulation of skeletal muscle miRNAs in response to exercise. This study investigated components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin‐5), muscle enriched miRNAs, (miR‐1, ‐133a, ‐133b and 206), and several miRNAs dysregulated in muscle myopathies, and showed that 3 h following an acute exercise bout, Drosha, Dicer and Exportin‐5, as well as miR‐1, ‐133a, ‐133‐b and miR‐181a were all increased, while miR‐9, ‐23a, ‐23b and ‐31 were decreased. Short‐term training increased miR‐1 and miR‐29b, while miR‐31 remained decreased. Negative correlations were observed between miR‐9 and HDAC4 protein, miR‐31 and HDAC4 protein and between miR‐31 and NRF1 protein, 3 h after exercise. miR‐31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species potentially involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Abstract  The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin‐5), muscle enriched miRNAs, (miR‐1, ‐133a, ‐133b and ‐206), and several miRNAs dysregulated in muscle myopathies (miR‐9, ‐23, ‐29, ‐31 and ‐181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate‐intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin‐5, as well as miR‐1, ‐133a, ‐133‐b and ‐181a were all increased. In contrast miR‐9, ‐23a, ‐23b and ‐31 were decreased. Short‐term training increased miR‐1 and ‐29b, while miR‐31 remained decreased. Negative correlations were observed between miR‐9 and HDAC4 protein (r=−0.71; P= 0.04), miR‐31 and HDAC4 protein (r =−0.87; P= 0.026) and miR‐31 and NRF1 protein (r =−0.77; P= 0.01) 3 h following exercise. miR‐31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise‐mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health.

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.