Protein kinase C (PKC) has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKC on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKC translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKC with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKC distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKC pseudosubstrate peptide. In stable transfected cells, overexpression of PKC caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKC overexpression were abolished by cell-permeable PKC pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKC to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKC translocation. Together, these results suggest that PKC mediates insulin effect on glucose transport through actin remodeling in muscle cells. INTRODUCTIONInsulin stimulates glucose uptake into skeletal muscle tissue mainly through GLUT4 translocation from intracellular pools to the plasma membrane (Klip et al., 1993;Bryant et al., 2002;Saltiel and Pessin, 2002). Tyrosine phosphorylation of insulin receptor substrate-1 by insulin activates phosphatidylinositol 3-kinase (PI3-K) and induces activation of downstream signal molecules protein kinase B (PKB/Akt) (Kohn et al., 1996;Tanti et al., 1997;Hill et al., 1999;Wang et al., 1999) and atypical PKCs (aPKCs) and / (Bandyopadhyay et al., 1997a(Bandyopadhyay et al., ,b, 1999Standaert et al., 1997;Kotani et al., 1998). aPKCs have been implicated in insulin action in adipocytes and muscle tissues (Kotani et al., 1998;Kim et al., 1999;Sajan et al., 2004), although this notion is not supported consistently by some studies (Tsuru et al., 2002). Evidence indicates that activation of aPKCs by insulin in skeletal muscles is defective in type 2 diabetic patients, monkeys, and rodents, and this defect seems to contribute significantly to the diminution in insulin-stimulated glucose disposal and muscle-dependent insulin resistance seen in these diabetic states (Bandyopadhyay et al., 1997a(Bandyopadhyay et al., , 1999Standaert et al., 2002;Beeson et al., 2003;Kim et al., 2003).We have previously shown that insulin causes a rapid and dynamic remodeling of actin into a cortical mesh (Khayat et al., 2000;Tong et al., 2001). Within the submembrane mesh, insulin-effective molecules such as glucose transporter (GLUT) isoform 4, vesicle-associated membrane protein (VAMP) 2,...

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Type 2 diabetes (T2D) is a very heterogeneous and multifactorial disease. The pathophysiology of T2D is presumed to occur with an alteration in the levels of plasma proteins. To identify these differentially expressed proteins, plasma samples from normal and T2D humans were subjected to two-dimensional gel electrophoresis, quantitative densitometry, and mass spectrometry. Up to 200 protein spots were visible on each gel, of which 57 appeared modulated in diabetic individuals. Subsequently, 31 spots with > or =2-fold change in their expression were analyzed by MALDI-TOF mass spectrometry leading to the identification of 11 proteins with average sequence coverage of approximately 38%. The expression of apolipoprotein A-I was reduced by 4.2-fold, and galectin-1 was increased 4.8 times in diabetic samples. Induction of galectin-1 in T2D samples was confirmed by ELISA. In addition, the dose-dependent treatment of rat L6 skeletal muscle cells with glucose resulted in an upregulation of galectin-1. These data implicate the association of galectin-1 with the pathophysiology of diabetes and identify galectin-1 as a novel diagnostic marker protein in T2D patients.

BackgroundReactive oxygen species arise in the mitochondria as byproducts of respiration and oxidase activity and have important roles in many physiological and pathophysiological conditions. The level of reactive oxygen species is regulated by a number of enzymes and physiological antioxidants, including HO-1, Sod2, catalase and COX-2, etc. And HO-1 against oxidative stress requires an increase in stress-responsive genes, such as Sod2 and catalase. Especially for the activity of HO-1, cobalt protoporphyrin is known to be a potent and effective inducer in many tissues. The transcription factor, FOXO1 is resistant to oxidative stress through downregulating reactive oxygen species production. Previous study showed that FOXO1 induces HO-1 expression by binding to HO-1 promoter. The question whether cobalt protoporphyrin induces HO-1 expression mediated by FOXO1 and subsequently lessens reactive oxygen species production remains to be elucidated.ResultsCobalt protoporphyrin enhances the expression of FOXO1 and facilitates FOXO1 binding to HO-1 promoter and increasing its transcriptional activity without influencing the FOXO1 protein stability. CoPP induces HO-1 and other oxidative stress-responsive genes expression, such as catalase, cytochrome c, Sod2, and COX-2, and decreases mitochondria-derived reactive oxygen species production, which are mediated partially by FOXO1.ConclusionsCobalt protoporphyrin induces HO-1 and other oxidative stress-responsive genes expression mediated partially by FOXO1, and has an important role in reducing cellular reactive oxygen species level. Cobalt protoporphyrin may be a more promising therapeutic agent to upregulate some antioxidantive genes.