Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4 (Glucose Transporter 4) Translocation by Preventing the Formation of Phosphatidylinositol 3,4,5-Trisphosphate in 3T3L1 Adipocytes

Abstract

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.

The Role of Ca2+ in Insulin-stimulated Glucose Transport in 3T3-L1 Cells

“…Taken together, the above data suggest that Ca 2ϩ , presumably via its effects on calmodulin, plays an important role in the insulin-signaling cascade at the level of PI 3-kinase activation and are consistent with previous reports (17). It may also be inferred from the above data that inhibition of insulin-stimulated Akt phosphorylation by either BAPTA-AM or W13 may be at least partly responsible for the observed inhibition of GLUT4 translocation and glucose uptake.…”

“…Glucose Uptake-Previous studies have suggested that the ubiquitously expressed Ca 2ϩ -binding protein calmodulin is required for the efficient activation of PI 3-kinase by insulin and subsequent activation of Akt (17). Consistent with this, we found that pretreatment of cells with the calmodulin antagonist W13 inhibited insulin-stimulated Akt phosphorylation by 70% (Fig.…”

“…Intuitively one might imagine at least two loci where Ca 2ϩ might be involved in mediating the effects of insulin on glucose uptake. Firstly, Ca 2ϩ /calmodulin has been implicated in mediating insulin activation of PI 3-kinase and Akt in rat hepatocytes and in 3T3-L1 adipocytes (16,17). Secondly, several recent studies have reported a key role for Ca 2ϩ /calmodulin in the late stages of vesicle docking/fusion (18 -21).…”

Calmodulin Mediates Brain-derived Neurotrophic Factor Cell Survival Signaling Upstream of Akt Kinase in Embryonic Neocortical Neurons

“…Based upon recent studies of the involvement of calmodulin in insulin signaling, a site of action of calmodulin in cell survival signaling by BDNF downstream of receptor activation, but upstream of PI3K activation, seems likely. Thus, it was shown that calmodulin antagonists have no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate-1, but completely abolish insulin-induced activation of PI3K (51). Further analyses in the latter study showed that calmodulin is essential for the formation of phosphatidylinositol 3,4,5-triphosphate.…”

“…The Ca 2ϩ -binding requirements for the biological activities of calmodulin in neurons had not been studied previously, although it had been shown that occupancy of all four Ca 2ϩ -binding domains is required for full activity of calmodulin in other cell types (14,51,56,57). To determine the roles of Ca 2ϩ binding in the neuronal survival-promoting function of calmodulin, we mutated critical asparagine residues in each of the Ca 2ϩ -binding domains of calmodulin.…”