Groups annotated by letters cannot be compared with groups annotated by numbers

Groups annotated by letters cannot be compared with groups annotated by numbers. identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator. A defining pathology of type 2 diabetes is impaired insulin-stimulated glucose uptake in skeletal muscle. Skeletal muscle is the largest tissue in the human body by mass and is the chief site of insulin-stimulated glucose disposal. Insulin stimulation causes translocation of GLUT4 glucose transporters from intracellular regions to the plasma membrane and t-tubule system where they function to import glucose. In individuals with type 2 diabetes, insulin fails to stimulate adequate GLUT4 translocation, resulting in impaired glucose uptake and poor glucose tolerance. Skeletal muscle is unique as an insulin-sensitive tissue because voluntary contraction Mouse monoclonal antibody to HAUSP / USP7. Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process counteredby deubiquitinating enzyme (DUB) action. Five DUB subfamilies are recognized, including theUSP, UCH, OTU, MJD and JAMM enzymes. Herpesvirus-associated ubiquitin-specific protease(HAUSP, USP7) is an important deubiquitinase belonging to USP subfamily. A key HAUSPfunction is to bind and deubiquitinate the p53 transcription factor and an associated regulatorprotein Mdm2, thereby stabilizing both proteins. In addition to regulating essential components ofthe p53 pathway, HAUSP also modifies other ubiquitinylated proteins such as members of theFoxO family of forkhead transcription factors and the mitotic stress checkpoint protein CHFR during exercise causes GLUT4 translocation completely independent of insulin signaling (1, 2). Contraction-stimulated glucose uptake is preserved in the muscle of individuals with type 2 diabetes, thus demonstrating the existence of signaling pathways that circumvent defective components of the insulin signaling pathway (3). If and where insulin- and contraction-stimulated glucose uptake pathways converge have been topics of considerable interest. Recently, the Akt substrate of 160 kDa (AS160)2 was identified as a mediator of both insulin- and contraction-stimulated glucose uptake and, therefore, a potential nexus for convergent signaling (4, 5). AS160 is a functional rab-GTPase-activating protein (rab-GAP) and is thought to restrain exocytotic GLUT4 translocation by keeping target rabs in an inactive, GDP-bound state (6-8). Phosphorylation of AS160 at Akt substrate motifs (Rfor 10 min. Lysate protein concentrations were determined by the Bradford assay (24). test, one-way analysis of variance (ANOVA) or two-way ANOVA. When differences between means were detected by one- or two-way analysis of variance, Fisher’s least significance difference test was used for post hoc testing. When data failed tests for normality or equal variance, data were rank-transformed before analysis. Data are expressed as the means S.E. The differences between groups were NS 1738 considered significant when 0.05. RESULTS = 6-8). 0.05; ?, = 0.052. Groups annotated by letters cannot be compared NS 1738 with groups annotated by numbers. #, types I and IIa myosin heavy chain were only detected in soleus muscle and excluded from the statistical analysis. were assessed by injecting mice with insulin intraperitoneally for 0 (control), 5, 10, or 20 min. PAS-160 and P-Akt were measured in soleus (= 6-8). and 0.05. were determined by injecting mice with AICAR subcutaneously for 0 (control), 10, 30, or 60 min. PAS-160 and P-AMPK were measured in soleus (= 3-8). 0.05. = 8). 0.05. and ?and3= 8). 0.05. shows that the TBC1D1 antibody does not cross-react with AS160. = 6-8). *, stimulation caused a statistically NS 1738 significant increase in TBC1D1 phosphorylation compared with controls, 0.05. by AMPK and Akt for 0, 30, or 60 min, by AMPK and Akt combined for 60 min, and buffer alone for 60 min. Replicates produced similar results. with recombinant AMPK, Akt, or AMPK plus Akt for 30 or 60 min. Incubation with AMPK, Akt, or AMPK and Akt combined resulted in similar increases in TBC1D1 PAS phosphorylation (Fig. 7(Fig. 7and Ser-231 SFS*QPGLR 5.5 0.20 28 KSFS*QPLGR 34 0.64 54 Thr-253 QDASLRRSST*F 1.3 NS 1738 9.6 0.14 Thr-499 SLT*ESLESILSR 0.0036 N/A N/A Thr-590 ANT*LSHFPVECPAPPEPAQSSPGVSQR N/A N/A N/A Ser-621 YHS*VSTETPHER 3.0 2.3 1.3 Ser-660 LNPSAS*SPNFFK 0.83 0.023 37 Ser-700 LHSSSS*VPNFLK 56 5.3 10 KLHSSSS*VPNFLK 43 0.83 52 Open in a separate window The ratio of relative peak ion intensities was NS 1738 computed to semiquantitatively compare the effects of AICAR and insulin.