Glycogen phosphorylase is activated in response to glucose deprivation
but is not responsible for enhanced glucose transport activity in 3T3-
L1 adipocytes
Melissa McInerney
a
, Geidy Serrano Rodriguez
a
, Wojciech Pawlina
b
,
Christopher B. Hurt
c
, Bradley S. Fletcher
c
, Philip J. Laipis
a
, Susan C. Frost
a;Ã
a
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, 1600 SW Archer Road, Gainesville,
FL 32610, USA
b
Department of Anatomy, Mayo Medical School, Rochester, MN 55905, USA
c
Department of Pharmacology, University of Florida College of Medicine, Box 100245, 1600 SW Archer Road, Gainesville, FL 32610, USA
Received 17 September 2001; received in revised form 11 December 2001; accepted 13 December 2001
Abstract
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. ß 2002 Elsevier Science B.V. All rights reserved.
Keywords: Glycogen phosphorylase ; Glucose transport ; 3T3-L1 adipocyte ; Gene expression ; Nutrient control
1. Introduction
Glucose availability controls a number of homeostatic
processes in adipocytes at the transcriptional level. Among
these are the genes for fatty acid synthase [1,2], S14 [3,4],
and mitochondrial glycerophosphate acyl transferase [5,6],
each of which play a role in triglyceride biosynthesis. Fa-
cilitating these studies was the development of cultured
adipocyte model systems, such as the 3T3-L1 cell line,
which allows the independent manipulation of nutrients
from hormones. Using this cell line, we and others have
shown that glucose deprivation increases glucose transport
activity by at least 10-fold by a process which requires
both new transcription and translation [7^9]. This process
can be blocked by non-metabolizable glucose analogues,
which suggests that glucose itself and not one of its me-
tabolites regulates the process. This feature is further em-
phasized by the fact that hexoses which do not serve as
substrates for the transporters, like fructose, do not block
or reverse the activation. We now know that the two ma-
jor transporters in 3T3-L1 adipocytes are GLUT1 and
GLUT4 [10^13]. In our hands, there is little change in
GLUT1 mRNA, protein, or compartmentation [9,14,15],
although GLUT1 is the major transporter at the cell sur-
face. GLUT4 mRNA and protein decrease in response to
glucose deprivation [16,9]. This has led to the hypothesis
that GLUT1 is activated by a novel protein whose syn-
thesis is controlled by glucose. Such an activator has not
yet been identi¢ed.
Glucose deprivation also causes a time-dependent loss
0304-4165 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.
PII: S0304-4165(02)00154-X
* Corresponding author. Fax : 352-392-2953.
E-mail address : sfrost@u£.edu (S.C. Frost).
BBAGEN 25292 11-4-02
Biochimica et Biophysica Acta 1570 (2002) 53^62
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