Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

Abstract

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (⌬PH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas ⌬PH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or ⌬PH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3␣/␤, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither ⌬PH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3␣/␤ pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or ⌬PH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulinstimulated glucose transport in which PDK1 to PKB or aPKC is not involved. glycogen synthesis; glucose transport; GLUT4; protein kinase B; protein kinase C INSULIN STIMULATES GLUCOSE UPTAKE into muscle and adipose tissues by redistribution of the insulin-responsive glucose transporter GLUT4 from intracellular stores to the plasma membrane. Insulin transmits its signals through a cell surface tyrosine kinase receptor, which stimulates multiple intracellular signaling events (49). Activated insulin receptors phosphorylate adapter proteins, members of the insulin receptor substrate family, which recruit and activate downstream effector molecules. Among the downstream proteins, activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin's acute glucose metabolism-regulating actions, such as acceleration of glucose transport and glycogen synthesis.PI 3-kinase (pharmacological) inhibitors, e.g., wortmannin and LY-294002, reportedly completely inhibit the insulin-induced activation of glycogen synthase in a variety of cell models (27,35,43,45). Glycogen synthase kinase 3 (GSK3) plays an important role in the regulation of glycogen synthesis via inhibitory phosphorylation of glycogen synthase (22,50). Two isoforms of GSK3, GSK3␣ and -␤, are broadly expressed and play multiple regulatory roles in development and metabolism (40). GSK3 is constitutively active in cells and is transiently inhibited after insulin treatment (17). Insulin-induced inactivation of GSK3 by insulin appears to be mediated by phosphorylation of GSK3 at...

The Major Target of the Endogenously Generated Reactive Oxygen Species in Response to Insulin Stimulation Is Phosphatase and Tensin Homolog and Not Phosphoinositide-3 Kinase (PI-3 Kinase) in the PI-3 Kinase/Akt Pathway

“…These results indicate that the insulin-mediated activation of PI-3 kinase is not sufficient to produce the levels of cellular PtdIns(3,4,5)P 3 needed to recruit PDK-1 and activate Akt and suggest that the transient increase of ROS may play a pivotal role in increasing PtdIns(3,4,5)P 3 levels. Consistent with this possibility, our data demonstrate that inhibition of insulin-mediated ROS production in SK-N-SH cells by using DPI results in a decrease of PDK-1 translocation to the membrane fraction and reduced Akt activation without significant effect on PI-3 kinase activity, although several reports showed that the subcellular localization of PDK-1 seem to be growth factor insensitive (Yamada et al, 2002;Kim et al, 2003) and that PDK-1 is activated through a PI-3 kinase-independent pathway (Kim et al, 2003). However, a recent report demonstrated that Akt cannot be activated by insulin-like growth factor-1 in homozygous knockin embryonic stem cells expressing a form of PDK-1 with a mutation in its PH domain that abolishes Membrane fractions and lysates were prepared from cells stimulated with insulin, insulin plus DPI, or insulin plus LY294002.…”

Insulin-Stimulated Protein Kinase C λ/ζ Activity Is Reduced in Skeletal Muscle of Humans With Obesity and Type 2 Diabetes

“…Support for an insulin effect comes from in vitro studies showing that insulin increases phosphorylation and catalytic activity of PDK-1 in isolated rat adipocytes (32) and skeletal muscle cells (44). However, some studies indicated that PDK-1 is a constitutively active enzyme whose catalytic activity is not stimulated by growth factors such as insulin or IGF-1 in 293 cells or CHO cells or 3T3-L1 adipocytes (45)(46)(47)(48)(49). The difference may be due to differences in the type of cells or assay methodology in these studies.…”

Diabetes and obesity during pregnancy alter insulin signalling and glucose transporter expression in maternal skeletal muscle and subcutaneous adipose tissue

“…A number of other genes have also been implicated in insulin signalling and glucose metabolism in peripheral tissues. These include glycogen synthase kinase (GSK)-3 (Cline et al 2002, Hojlund et al 2003, Patel et al 2008, phosphoinositide-dependent kinase (PDK)-1 (Yamada et al 2002, Hashimoto et al 2006, Bayascas et al 2008, insulin-like growth factor-binding protein 1 (IGFBP1; Qiu et al 2005) and phosphoenolpyruvate carboxykinase (PEPCK; Franckhauser et al 2006).…”