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

Abstract

We have examined the requirement for Ca 2؉ in the signaling and trafficking pathways involved in insulinstimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca 2؉ , using 1,2-bis (o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70 -75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4°C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca 2؉ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca 2؉

DOC2B: A Novel Syntaxin-4 Binding Protein Mediating Insulin-Regulated GLUT4 Vesicle Fusion in Adipocytes

“…Similar mechanisms result in GLUT4 vesicle fusion in adipocytes and muscle cells. Whitehead et al (8) demonstrated, and we confirmed, that reduction of intracellular Ca 2ϩ ([Ca 2ϩ ] i ) using the membrane-permeable Ca 2ϩ -chelating agent BAPTA-AM diminished insulinstimulated glucose transport, whereas this reagent did not inhibit GLUT4 translocation to the plasma membrane (i.e., GLUT4 vesicle trafficking was not impaired) (8) (N.F., M.E., unpublished observation). These observations suggest that an appropriate intracellular Ca 2ϩ level may be required for the final docking/fusion steps of GLUT4 vesicles in adipocytes.…”

“…In 2001, Whitehead et al (8) first reported that a calcium chelator, BAPTA-AM, inhibited GLUT4 externalization and glucose uptake. However, the precise mechanism underlying calcium-dependent GLUT4 vesicle fusion remains unknown because no studies have as yet focused on Ca 2ϩ -sensing proteins in adipocytes.…”

Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes

“…However, it is unclear whether impairment of the insulin signaling pathway is a decisive factor for determining defective glucose uptake and utilization. Insulin-stimulated glucose uptake was achieved by activating only 10-20% of insulin receptor substrate-1 (IRS-1) and Akt in one study (Whitehead et al, 2001), and insulinstimulated Akt activation was not severely impaired in obese individuals with insulin resistance in another study (Storgaard et al, 2004), indicating that factors other than an impaired insulin signaling pathway may be involved in the induction of insulin resistance. Additionally, a report that palmitate induces insulin resistance without a defect in insulin-stimulated Akt phosphorylation in the soleus muscle (Alkhateeb et al, 2007) suspects a role for impairment of the insulin signaling pathway in PIIR.…”

The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes

“…Because calcium and phosphoinositides are known to regulate insulinstimulated glucose transport (15)(16)(17)(18)(19), it would be interesting to test whether changes in Ca 2ϩ produced by insulin signaling affect the ability of E-Syt1 to bind phospholipids and how this might affect the interaction between E-Syt1 and GLUT4.…”