Microtubule network is required for insulin-induced signal transduction and actin remodeling.

Both microtubule and actin are required for insulin-induced glucose uptake. However, the roles of these two cytoskeletons and their relationship in insulin action still remain unclear. In this work, we examined the morphological change of microtubule/actin and their involvement in insulin signal transduction using rat skeletal muscle cells. Insulin rapidly led to microtubule clustering from ventral to dorsal surface of the cell. Microtubule filaments were rearranged to create space where new actin structures formed. Disruption of microtubule prevented insulin-induced actin remodeling and distal insulin signal transduction, with reduction in surface glucose transporter isoform 4 (GLUT4) and glucose uptake. Though microtubule mediated actin remodeling through PKCζ, reorganization of microtubule depended on tyrosine phosphorylation of insulin receptor, the mechanism is different from insulin-induced actin remodeling, which relied on the activity of PI3-kinase and PKCζ. We propose that microtubule network is required for insulin-induced signal transduction and actin remodeling in skeletal muscle cells.

Fatty acids inhibit insulin-mediated glucose transport associated with actin remodeling in rat L6 muscle cells.

In skeletal muscle cells, insulin stimulates cytoskeleton actin remodeling to facilitate the translocation of glucose transporter GLUT4 to plasma membrane. Defect of insulin-induced GLUT4 translocation and actin remodeling may cause insulin resistance. Free fatty acids cause insulin resistance in skeletal muscle. The aim of this study was to investigate the effects of fatty acids on glucose transport and actin remodeling. Differentiated L6 muscle cells expressing c-myc epitope-tagged GLUT4 were treated with palmitic acid, linoleic acid and oleic acid. Surface GLUT4 and 2-deoxyglucose uptake were measured in parallel with the morphological imaging of actin remodeling and GLUT4 immunoreactivity with fluorescence, confocal and transmission electron microscopy. Differentiated L6 cells showed concentration responses of insulin-induced actin remodeling and glucose uptake. The ultrastructure of insulin-induced actin remodeling was cell projections clustered with actin and GLUT4. Acute and chronic treatment with the 3 fatty acids had no effect on insulin-induced actin remodeling and GLUT4 immunoreactivity. However, insulin-mediated glucose uptake significantly decreased by palmitic acid (25, 50, 75, 100 µmol/L), oleic acid (180, 300 µmol/L) and linoleic acid (120, 180, 300 µmol/L). Oleic acid (120, 300 µmol/L) and linoleic acid (300 µmol/L), but not palmitic acid, significantly decreased insulin-mediated GLUT4 translocation. These data suggest that fatty acids inhibit insulin-induced glucose transport associated with actin remodeling in L6 muscle cells.

The pivotal role of protein kinase C zeta (PKCzeta) in insulin- and AMP-activated protein kinase (AMPK)-mediated glucose uptake in muscle cells.

Insulin and AMP-activated protein kinase (AMPK) signal pathways are involved in the regulation of glucose uptake. The integration of signals between these two pathways to maintain glucose homeostasis remains elusive. In this work, stimulation of insulin and berberine conferred a glucose uptake or surface glucose transporter 4 (GLUT4) translocation that was less than simple summation of their effects in insulin-sensitive muscle cells. Using specific inhibitors to key kinases of both pathways and PKCzeta small interference RNA, protein kinase C zeta (PKCzeta) was found to regulate insulin-stimulated protein kinase B (PKB) activation and inhibit AMPK activity on dorsal cell surface. In the presence of berberine, PKCzeta controlled AMPK activation and AMPK blocked PKB activity in perinuclear region. The inhibition effect of PKCzeta on AMPK activation or the arrestment of PKB activity by AMPK still existed in basal condition. These results suggest that there is antagonistic regulation between insulin and AMPK signal pathways, which is mediated by the switch roles of PKCzeta.

Berberine modulates insulin signaling transduction in insulin-resistant cells.

Berberine has been shown to have insulin-sensitizing effect, but the molecular mechanism underlying remains elusive. In this work, we investigated the effect of berberine on insulin-induced signal transduction and glucose uptake in both insulin-sensitive and insulin-resistant rat skeletal muscle cells. Berberine increased the activity of AMPK and PKCzeta and AS160 phosphorylation in normal cells, but had little effect on PKB activation. In insulin-resistant state, berberine exhibited synergistic effect on insulin-induced glucose uptake and GLUT4 translocation. Berberine improved insulin-induced tyrosine-phosphorylation of IRS-1 and the recruitment of p85 to IRS-1. These changes were accompanied by enhancement in insulin-induced PKCzeta and PKB activity and actin remodeling. The ameliorated insulin signal transduction was related to the inhibition of mTOR by berberine, which attenuated serine-phosphorylation of IRS-1. These results suggest that berberine may overcome insulin resistance via modulating key molecules in insulin signaling pathway, leading to increased glucose uptake in insulin-resistant cells.

Protein kinase Czeta mediates insulin-induced glucose transport through actin remodeling in L6 muscle cells.

Protein kinase C (PKC) zeta 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 PKCzeta on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCzeta 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 PKCzeta with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCzeta distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCzeta pseudosubstrate peptide. In stable transfected cells, overexpression of PKCzeta 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 PKCzeta overexpression were abolished by cell-permeable PKCzeta pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCzeta to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCzeta translocation. Together, these results suggest that PKCzeta mediates insulin effect on glucose transport through actin remodeling in muscle cells.