Glycogen phosphorylase is activated in response to glucose deprivation but is not responsible for enhanced glucose transport activity in 3T3-L1 adipocytes.
We have previously shown that glucose deprivation activates glucose transport in a time- and protein synthesis-dependent fashion in 3T3-L1 adipocytes, a mouse cell line. Coincident with this is loss of glycogen. Because glycogen phosphorylase (GP) is responsible for glycogen degradation, we have examined its regulation to determine the relationship between transport activation and glycogen turnover. We first cloned the adipose GP cDNA and found sequence similarity to rat and human liver GP. Because the mouse liver GP cDNA sequence was unavailable, we cloned this cDNA as well and showed 100% identity between mouse adipose and liver sequences. A 3.1 kb transcript was readily observed in total RNA isolated from mouse liver or adipose by Northern blot analysis but, surprisingly, not in either total or poly(A) selected RNA from 3T3-L1 adipocytes. To evaluate regulation in 3T3-L1 adipocytes, we amplified GP mRNA from total RNA using multiplex, semi-quantitative PCR but found that expression did not change in response to deprivation. GP protein levels did not change either. However, endogenous GP activity from glucose-deprived cells was significantly elevated relative to controls, due to an increase in the phosphorylated form of GP (GPa). Finally, we overexpressed GP to determine its direct influence on the glucose transport system. These results were negative, which suggests that the nutrient control of glucose transport and GP occurs independently despite kinetic similarities in transport activation and glycogen turnover.
McInerney
Melissa
M
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, 1600 SW Archer Road, Gainesville, FL 32610, USA.
Serrano Rodriguez
Geidy
G
Pawlina
Wojciech
W
Hurt
Christopher B
CB
Fletcher
Bradley S
BS
Laipis
Philip J
PJ
Frost
Susan C
SC
D016475:3T3 Cells; D017667:Adipocytes / Q000201:enzymology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D005947:Glucose / Q000172:deficiency* / Q000378:metabolism; D051272:Glucose Transporter Type 1; D024981:Glycogen Phosphorylase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D024981:Glycogen Phosphorylase; D005947:Glucose