Further information on transcriptional regulation can be gained by monitoring gene transcripts related to time following insulin administration. In fact, in pre/post stimulus studies in which the transcriptional response is monitored at one specific time instant after a prolonged insulin exposure, genes showing a transient response followed by a return to the pre-stimulus expression or a systematic, but small in magnitude, change in the expression, are likely to be missed. In contrast, monitoring the dynamic response allows identifying transient responses, which might be characteristic, and,Yubeinine if common to a number of genes associated to the same functional group, might give insight into the function or functions performed by the gene circuitry. The aim of the present work is to exploit the potential of a dynamic study to investigate the transcriptional response of skeletal muscle cells during acute insulin stimulation. To this purpose, we designed and conducted the present gene-array experiment in differentiated L6 myotubes. To identify significant transcriptional temporal patterns in muscle cells treated with insulin and to characterize them from a functional point of view, here we propose a new analytical method applied to experimental data. This method aims at overcoming some drawbacks of the conventional analysis approach based on selection of differentially expressed genes, clustering and functional annotation based on Gene Ontology. The new approach that we apply in the current study 1) improves selection of differentially expressed genes by diminishing the number of false negatives while maintaining constant the false discovery rate, Peimisine the number of false positives divided by the number of selected genes; 2) clusters genes with the same transcriptional pattern without requiring the user to fix the number of clusters and 3) automatically annotates these clusters with the most specific GO terms, avoiding redundancy of the of the sampling grid: it is safer to average the signal by pooling the biological samples than to use a sparse sampling grid. All the culturing and sampling procedure described above was repeated two additional times on different days, using the same identical cell line, to obtain a complete triplicate of the experiment. In the current study we applied a gene array profiling approach to the transcriptional response of skeletal muscle myotubes treated with insulin, to identify the main temporal expression patterns associated to functional groups of differentially expressed genes.We chose skeletal muscle cell line for this study considering that frequent sampling from human and animal studies are fraught with many logistical problems including ethical issues. The results from the current study knowing the critical time points at which the transcriptional responses are altered by insulin will enable planning of future in vivo studies to assess insulin’s effect on muscle gene transcripts. A prior time course of insulin response in primary human myotubes from type 2 diabetic and non-diabetic individuals by Hansen et al. demonstrated a time dependent transcriptional responses of inflammatory and pro-angiogenic pathways in relationship to glycogen synthesis. The authors identified 102 transcripts as differentially expressed in response to insulin some of which were similar to those among the 326 gene transcripts that were differentially expressed in our analysis. For example, the angiogenic/anti-apoptotic gene transcripts, VEGF, FOS, and SRF, were up-regulated in both studies. Comparison of the above study to the current study is difficult since different species and cell types were used, different insulin concentrations were used, and the statistical analysis and the objectives of the two studies differ.