Constitutive activation of GSK3 down-regulates glycogen synthase abundance and glycogen deposition in rat skeletal muscle cells.
The effects of inhibition or constitutive activation of glycogen synthase kinase-3 (GSK3) on glycogen synthase (GS) activity, abundance, and glycogen deposition in L6 rat skeletal muscle cells were investigated. GS protein expression increased approximately 5-fold during differentiation of L6 cells (comparing cells at the end of day 5 with those at the beginning of day 3). However, exposure of undifferentiated myoblasts (day 3) to 50 microM SB-415286, a GSK3 inhibitor, led to a significant elevation in GS protein that was not accompanied by changes in the abundance of GLUT4, another late differentiation marker. In contrast, stable expression of a constitutively active form of GSK3beta (GSK3S9A) led to a significant reduction (approximately 80%) in GS protein that was antagonized by SB-415286. Inhibition of GSK3 or expression of the constitutively active GSK3S9A did not result in any detectable changes in GS mRNA abundance. However, the increase in GS protein in undifferentiated myoblasts or that seen following incubation of cells expressing GSK3S9A with GSK3 inhibitors was blocked by cycloheximide suggesting that GSK3 influences GS abundance possibly via control of mRNA translation. Consistent with the reduction in GS protein, cells expressing GSK3S9A were severely glycogen depleted as judged using a specific glycogen-staining antibody. Inhibiting GSK3 in wild-type or GSK3S9A-expressing cells using SB-415286 resulted in an attendant activation of GS, but not that of glucose transport. However, GS activation alone was insufficient for stimulating glycogen deposition. Only when muscle cells were incubated simultaneously with insulin and SB-415286 or with lithium (which stimulates GS and glucose transport) was an increase in glycogen accretion observed. Our findings suggest that GSK3 activity is an important determinant of GS protein expression and that while glycogen deposition in muscle cells is inherently dependent upon the activity/expression of GS, glucose transport is a key rate-determining step in this process.
MacAulay
Katrina
K
Division of Molecular Physiology, Medical Sciences Institute/Wellcome Trust Building Complex, Faculty of Life Sciences, University of Dundee, DD1 5EH, United Kingdom.
Blair
Anne S
AS
Hajduch
Eric
E
Terashima
Tatsuo
T
Baba
Otto
O
Sutherland
Calum
C
Hundal
Harinder S
HS
D000627:Aminophenols / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers; D051275:Glucose Transporter Type 4; D006003:Glycogen / Q000378:metabolism*; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000627:Aminophenols; D017931:DNA Primers; D051275:Glucose Transporter Type 4; D008301:Maleimides; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; C495694:Slc2a4 protein, rat; D006003:Glycogen; D006006:Glycogen Synthase; D038362:Glycogen Synthase Kinase 3