A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

Abstract

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.

Fig. 1.

Acute inhibition (30 min) of glycogen synthase kinase-3 (GSK-3) activity with lithium chloride (20 mM) or SB-216763 (10 μM) caused increased [³H]-2-deoxy-glucose (2-DOG) uptake in A7r5 cells (*P < 0.05 vs. control) (A). More chronic GSK-3 inhibition (24 h) induced glucose transporter type 1 (GLUT1) protein expression (n = 6, *P < 0.05 vs. control) (B) as well as 2-DOG uptake (n = 8, **P < 0.01) (C).

Fig. 2.

GSK-3 inhibition with 20 mM lithium chloride (A) or 10 μM SB-216763 (B) in rat aortic explants resulted in increased GLUT1 protein expression. SMA, smooth muscle actin.

Fig. 3.

Adenoviral infection with a constitutively active GSK-3β for 48 h decreased GLUT1 expression (A) and 2-DOG uptake (B) in vascular smooth muscle cells (n = 7 each, *P < 0.05; **P < 0.01 vs. control).

Fig. 4.

2-DOG uptake was increased in embryonic fibroblast cells (EEF8) and renal cells (LEF) lacking functional tuberous sclerosis complex subunit 2 (TSC2) compared with wild-type cells (EEF4) or TSC2 mutant cells in which a wild-type TSC2 construct was stably expressed (LEF+TSC2) (n = 12 each, *P < 0.05) (A). Similarly, 2-DOG uptake was increased in TSC2 mutant cells acutely infected with a retrovirus expressing wild-type TSC2 (EEF8+TSC2; n = 14) vs. the same cells infected with a control vector (EEF8; n = 11) for 24 h (*P < 0.05 vs. TSC2-infected cells) (B).

Fig. 5.

GLUT1 protein expression was increased in the absence of functional TSC2 in LEF cells (n = 12, **P < 0.01) (A) and in EEF8 cells (n = 3, *P < 0.05) (B) when compared with cells with stable (LEF) or transient (EEF8) expression of a wild-type TSC2 construct. GLUT1 RNA expression was also increased in cells that do not express functional TSC2 (n = 6, *P < 0.05 vs. LEF+TSC2) (C).

Fig. 6.

LEF cells that lack functional TSC2 exhibit increased S6 kinase (SK6) phosphorylation on mammalian target of rapamycin (mTOR) C1-specific residue, T389 (n = 8, *P < 0.05 vs. LEF+TSC2).

Fig. 7.

GSK-3 inhibition (24 h) with SB-216763 (10 μM) increased GLUT1 expression (n = 9, **P < 0.01 vs. LEF+TSC2 control) (A), increased 2-DOG uptake (n = 12 *P < 0.05 vs. LEF+TSC2 control) (B), and increased [³H]-3-O-methyl-glucose uptake (n = 8, **P < 0.01, *P < 0.05 vs. LEF+TSC2 control) (C) only in the presence of functional TSC2.

Fig. 8.

Wild-type, but not mutant, TSC2 expression resulted in decreased 2-DOG uptake in EEF8 cells infected (24 h) with either a wild-type TSC2 construct (TSC2), empty control vector (Empty), or a TSC2 construct mutated at three GSK-3 phosphorylation sites (TSC2–3A). GSK-3 inhibition (10 nM Glycogen Synthase Kinase-3β Inhibitor II, 8 h) abrogated the wild-type TSC2 effect (n = 5, *P < 0.05, vs. TSC2 control).

Fig. 9.

Rapamycin treatment (20 nM, 8 h) resulted in reduced GLUT1 expression (n = 3, *P < 0.05, vs. LEF control) (A) and 2-DOG uptake (n = 12, *P < 0.05 vs. LEF control) (B) only in cells lacking functional TSC2.