Abstract
The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.
Highlights
A major physiologic action of insulin in peripheral tissues is to stimulate the translocation of the facilitative glucose transporter (GLUT4) to the cell surface, leading to enhanced glucose uptake and storage in these tissues
To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HAGLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes
Studies examining the role of the actin cytoskeleton in this process in L6 myotubes, 3T3-L1 adipocytes, and rat adipocytes have shown that disruption of the actin cytoskeleton by either cytochalasin D or latrunculin B inhibits insulin-mediated GLUT4 translocation (19 –22)
Summary
A major physiologic action of insulin in peripheral tissues is to stimulate the translocation of the facilitative glucose transporter (GLUT4) to the cell surface, leading to enhanced glucose uptake and storage in these tissues. The cortical actin network itself undergoes rearrangement when 3T3-L1 adipocytes are treated with insulin and inhibition of actin remodeling inhibits GLUT4 translocation, suggesting that the reorganization of actin facilitates GLUT4 translocation [22, 28, 29]. The purpose of these experiments is to determine whether the actin network is required for insulin-mediated GLUT4 translocation prior to activation of Akt/PKB1 or following Akt/. A requirement for the actin network at a step prior to Akt/PKB activation would be entirely consistent with a role for actin in organization of the insulin-signaling complex. Our data support a role for the actin network in the organization of the insulin signal leading to Akt/PKB activation, supporting a role for actin in signal transduction
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