Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

Abstract

AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKB␣/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.Translocation of GLUT4 from an intracellular compartment to the plasma membrane largely accounts for the stimulation of glucose transport by insulin in skeletal muscle (16,31,38), cardiac muscle (48), and adipose cells (23,24). Two insulinresponsive cell lines also express this transporter: L6 rat skeletal myotubes (34, 40) and 3T3-L1 mouse adipocytes (24). Transfection of a molecularly engineered form of this transporter containing an exofacial epitope tag between the first and second transmembrane domains allows for the detection of surface transporters in intact cells. GLUT4 molecules with an exofacial epitope tag have been heterologously expressed in rat adipose cells (44, 51), 3T3-L1 adipocytes (26), CHO cells (12, 26), H9c2 cardiomyocytes (55), and rat 3Y1 cells (22). We have recently shown that stable expression of GLUT4myc in L6 myoblasts (L6-GLUT4myc myoblasts) mimics the response to insulin seen with endogenous GLUT4 in differentiated myotubes (29, 60).Insulin-induced translocation of GLUT4 to the plasma membrane requires the activity of phosphatidylinositol (PI) 3-kinase (47) in rat adipocytes (43, 45), 3T3-L1 adipocytes (8,9,21,27,39,51), L6 muscle cells (53), and rat skeletal muscle (62). Moreover, treatment of intact 3T3-L1 adipocytes with a cell-permeant PI 3,4,5-triphosphate [PI (3,4,5)-P 3 ] compound, which is converted into a product of PI 3-kinase once inside the cell, partly rescued the inhibition of insulin-stimulated glucose transport by wortmannin (25). It is unclear how the lipid products of PI 3-kinase relay the insulin signal to the glucose transporters, but the serine/threonine kinase protein kinase B (PKB)/Akt interacts with the lipid products of PI 3-kinase (19), and activation of PKB/Akt by insulin is prevented by inhibitors of PI 3-kinase (1). To date, three isoforms of PKB/Akt have been identified: PKB␣, -␤, and -␥ (Akt1, -2, and -3) (17). In skeletal muscle and L6 muscle cells, PKB␣ and PKB␥, but not PKB␤, are stimulated by insulin (59). Full activation of PKB/ Akt by insulin requires hierarchical phosphorylation on two residues, Thr308 (Thr309 and Thr305 in the case of PKB␤ and -␥, respectively) and Ser473 (Ser474 in the case of PKB␤; PKB␥ lacks an equivalent site) by 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and PDK-2, respectively (1-3, 14, 50).Recent reports have suggested that activation of PKB/Akt may mediate the stimulation of glucose transport by insulin, since stable overexpression of wild-type PKB␣/Akt1 or constitutively active mutants of PKB␣/Akt1 increased glucose transport and translocation of GLUT4 to levels similar to or greater than those achieved with insulin in rat adipocytes (52), 3T3-L1 adipocytes (33,56), and L6 muscle cells (20,56...

Nelfinavir-induced insulin resistance is associated with impaired plasma membrane recruitment of the PI 3-kinase effectors Akt/PKB and PKC-?

“…5d). Since the β isoform of PKB (or Akt2) was recently linked to insulin stimulation of GLUT4 translocation in adipocytes [38,39,40], we also assessed its recruitment to the plasma membrane in response to insulin using an isoform-specific antibody. In control cells, insulin stimulation resulted in a nearly six-fold increase in Akt2/PKB-β plasma membrane content (Fig.…”

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

“…In response to insulin, GLUT4myc is readily recruited to the plasma membrane, resulting in a twofold gain in surface-exposed GLUT4myc (Ueyama et al, 1999;Wang et al, 1999). The insulin effect is illustrated by immunofluorescence detection of the myc epitope in intact L6-GLUT4myc myoblasts (Figure 7A).…”

Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA

“…AKT has been implicated in the regulation of glucose transporter translocation and glucose transport [15][16][17], and some [14,[18][19][20], but not all [21,22], studies provide evidence that insulin signalling at the level of AKT is impaired in skeletal muscle from type 2 diabetic patients. Clearly, a detailed molecular analysis of the different effects of the specific IRS or AKT isoforms on metabolic and gene regulatory responses in humans poses a challenge.…”