TGF-ß Induction of miR-143/145 Is Associated to Exercise Response by Influencing Differentiation and Insulin Signaling Molecules in Human Skeletal Muscle.

Physical training improves insulin sensitivity and can prevent type 2 diabetes (T2D). However, approximately 20% of individuals lack a beneficial outcome in glycemic control. TGF-ß, identified as a possible upstream regulator involved in this low response, is also a potent regulator of microRNAs (miRNAs). The aim of this study was to elucidate the potential impact of TGF-ß-driven miRNAs on individual exercise response. Non-targeted long and sncRNA sequencing analyses of TGF-ß1-treated human skeletal muscle cells corroborated the effects of TGF-ß1 on muscle cell differentiation, the induction of extracellular matrix components, and identified several TGF-ß1-regulated miRNAs. qPCR validated a potent upregulation of miR-143-3p/145-5p and miR-181a2-5p by TGF-ß1 in both human myoblasts and differentiated myotubes. Healthy subjects who were overweight or obese participated in a supervised 8-week endurance training intervention (n = 40) and were categorized as responder or low responder in glycemic control based on fold change ISIMats (≥+1.1 or <+1.1, respectively). In skeletal muscle biopsies of low responders, TGF-ß signaling and miR-143/145 cluster levels were induced by training at much higher rates than among responders. Target-mining revealed HDACs, MYHs, and insulin signaling components INSR and IRS1 as potential miR-143/145 cluster targets. All these targets were down-regulated in TGF-ß1-treated myotubes. Transfection of miR-143-3p/145-5p mimics in differentiated myotubes validated MYH1, MYH4, and IRS1 as miR-143/145 cluster targets. Elevated TGF-ß signaling and miR-143/145 cluster induction in skeletal muscle of low responders might obstruct improvements in insulin sensitivity by training in two ways: by a negative impact of miR-143-3p on muscle cell fusion and myofiber functionality and by directly impairing insulin signaling via a reduction in INSR by TGF-ß and finetuned IRS1 suppression by miR-143-3p.

DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences.

SUMMARY: Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. AVAILABILITY AND IMPLEMENTATION: Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The effect of differentiation and TGFß on mitochondrial respiration and mitochondrial enzyme abundance in cultured primary human skeletal muscle cells.

Measuring mitochondrial respiration in cultured cells is a valuable tool to investigate the influence of physiological and disease-related factors on cellular metabolism; however, the details of the experimental workflow greatly influence the informative value of the results. Working with primary cells and cell types capable of differentiation can be particularly challenging. We present a streamlined workflow optimised for investigation of primary human skeletal muscle cells. We applied the workflow to differentiated and undifferentiated cells and we investigated the effect of TGFß1 treatment. Differentiation of myoblasts to myotubes increased mitochondrial respiration and abundance of mitochondrial enzymes and mitochondrial marker proteins. Differentiation also induced qualitative changes in mitochondrial protein composition and respiration. TGFß1 reduced complex IV protein MTCO1 abundance in both myoblasts and myotubes. In myoblasts, spare electron transport system (ETS) capacity was reduced due to a reduction in maximal oxygen consumption. In TGFß1-treated myotubes, the reduction in spare ETS capacity is mainly a consequence of increased oxidative phosphorylation capacity and complex III protein UQCRC2. Taken together, our data shows that it is important to monitor muscle cell differentiation when mitochondrial function is studied. Our workflow is not only sensitive enough to detect physiological-sized differences, but also adequate to form mechanistic hypotheses.