Protein Kinase Cζ Mediates Insulin-induced Glucose Transport through Actin Remodeling in L6 Muscle Cells

Abstract

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,...

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

“…[64]. These findings are consistent with studies suggesting that PKC is an important component of insulin signaling pathways leading to GLUt4 translocation [65,66]. At present, the basis for the divergent genetic, biochemical and cell biological data concerning the functional role of the Munc18 proteins and particularly Munc18c in GLUT4 trafficking remains an open question.…”

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

“…Our studies offer insight into a unique pathway that would lead to preferential regulation of LKB1 function in response to oxidative stress. PKC is implicated in regulation of various cellular functions (37), including metabolism (38,39) and polarity (40,41), which are similar to the functions of LKB1. Because PKC can phosphorylate both LKB1 L and LKB1 S , this pathway might play an important physiological role in various cell defense mechanisms and pathophysiological conditions characterized by oxidative stress.…”

Prostaglandin E2 regulates melanocyte dendrite formation through activation of PKCζ

“…PKCζ activation in response to lysophosphatidylcholine in melanocytes is upstream of Rac, and Rac activation is upstream of PKCζ activation in other cell types [21,[51][52][53]. Because our data show that PGE 2 has no effect on Rac activity, an alternative mechanism for PGE 2 dependent PKCζ activation is operative in melanocytes.…”