Intracellular Delivery of Phosphatidylinositol (3,4,5)-Trisphosphate Causes Incorporation of Glucose Transporter 4 into the Plasma Membrane of Muscle and Fat Cells without Increasing Glucose Uptake

Abstract

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P 3 ) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P 3 intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P 3 caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phosphoAkt and GLUT4. Intracellular delivery of PI(3,4,5)P 3 using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P 3 was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P 3 elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P 3 did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P 3 to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.

Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells

“…Human insulin (Humulin R) was obtained from Eli Lilly (Toronto, ON, Canada), and recombinant human gAd produced in Escherichia coli was obtained from Peprotech (Ottawa, ON, Canada). We purchased D-[U- 14 Cell culture conditions for L6 myotubes For all experiments, L6 rat skeletal muscle cells were routinely grown in minimum essential medium alpha (α-MEM) supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. For differentiation into myotubes, the cells were grown in medium containing 2% fetal bovine serum for 5-7 days.…”

“…We employed cells transfected to stably overexpress GLUT4 harbouring a myc epitope on the first exofacial loop of the transporter (L6 GLUT4myc cells; a kind gift from A. Klip, The Hospital for Sick Children, Toronto, ON, Canada). This facilitated accurate quantitative analysis of GLUT4 translocation in intact cells [14,17,18].…”

“…Measurement of glycogen synthesis Glycogen synthesis was assessed by measuring the incorporation of D-[U- 14 C] glucose into glycogen as described previously [21]. Briefly, following 4 h of serum starvation, L6 myotubes in 6-well plates were incubated for 2 h in medium containing 0.15 μCi/ml of D-[U-14 C]glucose with or without gAd (60 μmol/ l) and/or insulin (100 nmol/l).…”

“…These stimuli are known to utilise different signalling pathways to elicit glucose uptake. Insulin stimulates GLUT4 translocation via a phosphatidylinositol 3-kinase-dependent signalling pathway [13,14], whereas AMP kinase plays an important role in mediating the effect of exercise [15]. Insulin acts to divert a large proportion of the glucose that enters muscle towards glycogen synthesis.…”

Identification of P-Rex1 as a Novel Rac1-Guanine Nucleotide Exchange Factor (GEF) That Promotes Actin Remodeling and GLUT4 Protein Trafficking in Adipocytes

“…GLUT4 trafficking from GLUT4 storage vesicles to the plasma membrane occurs in distinctly regulated steps (vesicle translocation, docking, fusion, and activation), and these events may occur in the absence of glucose uptake (62,63). Additional signals are required for fused GLUT4 vesicles to become fully functional and facilitate glucose entry, such as GLUT4 C-terminal unmasking (64).…”