In muscle cells, insulin elicits recruitment of the glucose transporter GLUT4 to the plasma membrane. This process engages sequential signaling from insulin receptor substrate (IRS)-1 to phosphatidylinositol (PI) 3-kinase and the serine/threonine kinase Akt. GLUT4 translocation also requires an Akt-independent but PI 3-kinase-and Racdependent remodeling of filamentous actin. Although IRS-1 phosphorylation is often reduced in insulin-resistant states in vivo, several conditions eliciting insulin resistance in cell culture spare this early step. Here, we show that insulin-dependent Rac activation and its consequent actin remodeling were abolished upon exposure of L6 myotubes beginning at doses of C2-ceramide or oxidant-producing glucose oxidase as low as 12.5 mol/l and 12.5 mU/ml, respectively. At 25 mol/l and 25 mU/ml, glucose oxidase and C2-ceramide markedly reduced GLUT4 translocation and glucose uptake and lowered Akt phosphorylation on Ser473 and Thr308, yet they affected neither IRS-1 tyrosine phosphorylation nor its association with p85 and PI 3-kinase activity. Small interfering RNA-dependent Rac1 knockdown prevented actin remodeling and GLUT4 translocation but spared Akt phosphorylation, suggesting that Rac and actin remodeling do not contribute to overall Akt activation. We propose that ceramide and oxidative stress can each affect two independent arms of insulin signaling to GLUT4 at distinct steps, Rac-GTP loading and Akt phosphorylation. Diabetes 56:394 -403, 2007 I nsulin promotes dietary glucose disposal into skeletal muscle through recruitment of GLUT4-containing vesicles to the cell surface. Signaling to GLUT4 requires tyrosine phosphorylation of the insulin receptor substrate (IRS)-1 isoform, which recruits and activates phosphatidylinositol (PI) 3-kinase (1). The latter triggers activation of several serine/threonine kinases, notably Akt, which, through its substrate AS160, regulates GLUT4 vesicle mobilization to and/or fusion with the plasma membrane (2).Along with proper signaling, GLUT4 translocation and stimulation of glucose uptake require dynamic changes in the actin cytoskeleton. Insulin induces actin filament remodeling in mature skeletal muscle (3) and muscle cells in culture (4) that manifest as mesh-like structures beneath the plasma membrane. Actin filament-disrupting drugs (e.g., cytochalasin D and latrunculin B) and actin-stabilizing drugs (e.g., jasplakinolide) inhibit GLUT4 translocation and its consequent glucose uptake in muscle (4,5) and adipose (6,7) cells, as do toxins that inhibit Rho family GTPases that control actin dynamics (8). Interestingly, under these conditions, IRS-1 phosphorylation and PI 3-kinase activity remain unaffected (9 -11). In muscle cells, actin remodeling was also prevented by wortmannin or expression of a dominant-negative mutant of the p85 subunit of class I PI 3-kinase (5), but not by a dominantnegative Akt mutant (12). Hence, insulin signaling bifurcates downstream of PI 3-kinase, one arm leading to actin remodeling and another to Akt activation, bo...

BackgroundThe newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardized format. In this study the relationship between the β-values provided by the Illumina, Inc. array for each individual CpG dinucleotide and the quantitative methylation levels obtained by pyrosequencing were analyzed. In addition, the representation of microRNA genes and imprinted loci on the Illumina, Inc. array was assessed in detail. Genomic DNA from 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PMC42) and 18 human breast cancer specimens as well as 4 normal mammary epithelial fractions was analyzed on 450k DNA methylation arrays. The β-values for 692 individual CpG sites from 62 different genes were cross-validated using conventional quantitative pyrosequencing.FindingsThe newly released 450k methylation array from Illumina, Inc. shows a high concordance with quantitative pyrosequencing if identical CpG sites are analyzed in cell lines (Spearman r = 0.88, p ≪ 0.0001), which is somewhat reduced in primary tumor specimens (Spearman r = 0.86, p ≪ 0.0001). 80.7% of the CpG sites show an absolute difference in methylation level of less than 15 percentage points. If different CpG sites in the same CpG islands are targeted the concordance is lower (r = 0.83 in cell lines and r = 0.7 in primary tumors). The number of CpG sites representing microRNA genes and imprinted loci is very heterogeneous (range: 1 – 70 CpG sites for microRNAs and 1 – 288 for imprinted loci).ConclusionsThe newly released 450k methylation array from Illumina, Inc. provides a genome-wide quantitative representation of DNA methylation aberrations in a convenient format. Overall, the congruence with pyrosequencing data is very good. However, for individual loci one should be careful to translate the β-values directly into percent methylation levels.

These results depict a clear difference between genetically and epigenetically unstable breast carcinomas indicating different ways of tumor progression and/or initiation, which strongly supports the association of CIMP with the lobular subtype and provide new options for detection and therapy.

DNA methylation is an epigenetic mark playing an important role in development and disease. Aberrant DNA methylation was identified as an alternative mechanism for gene inactivation complementing deletions and mutations in cancer initiation and progression. However, to accurately compare differences in DNA methylation among various tissue types, adequate quantitative approaches are required. Pyrosequencing(®), as a sequencing-by-synthesis method, allows such quantification with single CpG resolution and the ability for threshold determination. This book chapter provides a detailed protocol for DNA methylation analysis by Pyrosequencing, including information on assay design and practical procedure. Additionally, emphasis is placed on the discussion of strengths and weaknesses of the methodology.