(Received for publication, July 31, 1996, and in revised form, October 23, 1996)
From the Molecular Nutrition Unit, Department of
Nutrition, University of Montreal, H3C 3J7 Montréal,
Québec, Canada, the § Department of Biochimie
Médicale, Centre Médical Universitaire, University of
Geneva, 1121 Geneva 4, Switzerland, the ¶ Departments of Medicine
and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire
03755-3834, the
Diabetes and Metabolism Unit, Boston University
Medical Center, Boston, Massachusetts 75235-9135, and ** INSERM,
CJF-9313, Hôpital Robert Debré, 75019 Paris, France
Chronic elevation in glucose has pleiotropic
effects on the pancreatic -cell including a high rate of insulin
secretion at low glucose,
-cell hypertrophy, and hyperplasia. These
actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be
identified. To further investigate the molecular mechanisms implicated
in these adaptation processes to hyperglycemia, we have studied the
regulation of genes encoding key glycolytic enzymes in the
glucose-responsive
-cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and
L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression
level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts
remained unchanged. Following a 3-day exposure to elevated glucose, a
similar induction was observed at the protein level for PFK-1 (isoforms
C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased
slightly. The study of the mechanism of GAPDH induction indicated that
glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to
GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is
involved in GAPDH gene induction. These changes in glycolytic enzyme
expression were associated with a 2-3-fold increase in insulin
secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the
artificial electron acceptor
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. A marked
deposition of glycogen, which was readily mobilized upon lowering of
the ambient glucose, and increased DNA replication were also observed
in cells exposed to elevated glucose. The results suggest that a
coordinated induction of key glycolytic enzymes as well as massive
glycogen deposition are implicated in the adaptation process of the
-cell to hyperglycemia to allow for chronically elevated glucose
metabolism, which, in this particular fuel-sensitive cell, is linked to
metabolic coupling factor production and cell activation.
The adaptation of the pancreatic -cell to chronic hyperglycemia
is characterized by an augmented secretory function, hypertrophy, and
hyperplasia (1, 2). These pleiotropic actions promoted by elevated
glucose are expected to be associated with the induction of a number of
glucose-regulated genes that must be identified. Elevated circulating
glucose has gained recognition over the last few years as a factor
contributing to
-cell dysfunction and the subsequent development of
type 2 diabetes (2-4). However, the link between sustained
hyperglycemia and long-term alterations in
-cell function is not
well understood. In animal models, prolonged hyperglycemia causes both
an impaired glucose-induced insulin secretion and the development of
peripheral insulin resistance (5, 6). These alterations can be reversed
in part by lowering glucose to normal circulating levels, as occurs
following treatment with the glycosuric agent phlorizin (7). These
observations support the notion that high circulating glucose
concentrations contribute to the development of pathologies associated
with diabetes. Other factors in addition to glucose, such as elevated
circulating free fatty acids, may also participate in the
-cell
secretory defect (4-6, 8).
Previous work has shown that an important component of the -cell
adaptation process to hyperglycemia is an increase in glucokinase activity (9, 10), whereas the sugar does not modify the expression of
the Gk1 gene (8-11). Nonetheless, the
action of elevated glucose on the
-cell likely involves many
additional proteins aside from Gk. Noteworthy are the results obtained
in hexokinase (Hk) and Gk gene transfer experiments in the
-cell
showing that other steps in glucose metabolism become rate-limiting
after only modest increases in glucose-phosphorylating activity (8, 12,
13). This suggests that in vivo the activities of enzymes
downstream of GK may not be as far in excess as previously thought from
experiments using cell homogenates and particular assay conditions that
do not match the cell situation.
One major step in linking hyperglycemia to long term phenotypic changes
in pancreatic -cells is the recognition of glucose as a main
modulator of gene expression (5). Indeed, the sugar activates insulin
gene transcription and pro-insulin mRNA translation (14-17).
Glucose also induces the accumulation of Glut-2 (18), L-pyruvate kinase
(L-PK) (11), and acetyl-CoA carboxylase (ACC) (19) mRNAs in the
-cell. The 5
-CACGTG-3
motif, which has been found in the promotor
region of a number of glucose-responsive genes, has been identified as
the element conferring carbohydrate responsiveness to the L-PK and S-14
genes in hepatocytes (20, 21). This motif is similar to the consensus
sequence that binds the MLTF (major late transcription factor), a
member of the c-myc family of transcription factors (20).
Overexpression of c-myc in transgenic mice increases the
hepatic expression of the L-PK gene (22). It remains to be determined
which particular transcription factor(s) of the c-myc/MLTF
family mediate(s) glucose induction of the L-PK and S-14 genes. On the
other hand, Sp1 binding motifs found in the second promoter of the ACC
gene, and not the MLTF/c-myc binding motif, may be
responsible for glucose inducibility of the ACC gene in mouse
preadipocytes (23). Thus, there appear to exist at least two
transcription factors and distinct mechanisms by which glucose
modulates metabolic enzyme gene expression in higher eukaryotic
cells.
Mitochondrial oxidative events with oscillatory changes in the
concentrations of ATP and ADP, in conjunction with accelerated anaplerotic input into the citric acid cycle and a rise in malonyl-CoA, are thought to be essential factors implicated in -cell metabolic signaling (5, 6, 8). Increased glycolytic flux itself may also provide
a signal mediating KATP channel closure (24) and a Ca2+
rise independently of glucose-derived pyruvate metabolism in the
mitochondria (6, 8, 25). There is experimental evidence indicating that
accelerated NADH production by glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), linked to an effective glycerol 3-phosphate shuttle
transferring cytosolic reducing equivalents to the mitochondria (26,
27), mediates this action of glucose (25). Therefore, metabolic enzymes
in the glycolytic, anaplerotic, and lipogenic pathways can be
considered components of the transducing machinery, which links
nutrient metabolism to
-cell activation. Since increased glucose
metabolism in this particular fuel-sensitive cell is linked to signal
transduction, it is thus attractive to hypothesize that sustained
exposure to high glucose concentrations may alter the expression of
genes coding for metabolic enzymes involved in glucose metabolism
leading to changes in insulin secretion and
-cell growth. With
respect to
-cell proliferation, it is noteworthy that glucose
increases mitogen-activated protein kinase activity in INS-1 cells
(28).
In order to test this hypothesis and identify the glucose-modulated
genes in these pathways, we attempted to study the expression of genes
coding for various glycolytic, anaplerotic, and lipogenic enzymes in
INS-1 cells (29). In this paper, we show that three genes encoding key
glycolytic enzymes are induced by glucose in (INS-1) cells. These
enzymes are phosphofructokinase-1 (PFK-1), which participates in the
control of the glycolytic flux and metabolic oscillations (30); GAPDH,
which provides cytosolic NADH which may act as a coupling factor (25);
and L-PK, which catalyzes the formation of glycolytic-derived ATP.
Changes in the expression level of these enzymes are associated with
exaggerated glucose metabolism and insulin release at low
concentrations of the sugar and an increase in
-cell
proliferation.
INS-1 cells were seeded in 21-cm2 Petri dishes (1.4 × 106 cells/dish) and grown as described previously (29). When cells reached 80% confluence after approximately 7 days, they were washed twice with PBS (phosphate-buffered saline) at 37 °C and preincubated for 48 h in culture medium containing 5 mM glucose. Cells were then washed with PBS and incubated in culture medium at various glucose concentrations for the indicated times.
mRNA AnalysisPoly(A+) RNA was isolated
from total RNA (31) by loading on an oligo(dT)-cellulose column
(Pharmacia Biotech Inc.) in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 M NaCl, 0.1% SDS, and then eluted
with autoclaved bidistilled water and ethanol precipitation. mRNA
was analyzed from total RNA by Northern blotting hybridization using
the following [32P]cDNA probes: a 0.74-kb
EcoRI-BamHI fragment (positions 1040-1780) of
mouse rRNA 18 S cDNA subcloned in pUC830; a 0.864-kb
EcoRI-BamHI fragment (positions 18-882) of rat
Glut-2 cDNA subcloned in pGEM-4 (Promega) (kindly provided by Dr.
B. Thorens, University of Lausanne, Switzerland); a 2.216-kb
EcoRI-EcoRI fragment (positions 1-2216) of rat
glucokinase cDNA subcloned in pBSSK; a 1.4-kb
EcoRI-SspI fragment of rat
6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase subcloned in pBSKS
(kindly provided by Dr. K. Crepin, Louvain University, Belgium); a
1.289-kb PstI-PstI fragment (positions 1-1289)
of rat GAPDH cDNA subcloned in pBSKS (Stratagene); a 0.618-kb NcoI-XbaI fragment (positions 518-1136) of rat
L-pyruvate kinase cDNA subcloned in pSL301 (Invitrogen) (kindly
provided by Dr. B. Thorens). PFK-1-C mRNA was analyzed from
isolated poly(A+) using a 20-mer, which specifically
recognizes the C-isoform mRNA of PFK-1 (32). The oligonucleotide
was end-labeled using terminal transferase T4 polynucleotide kinase and
[32P]ATP as outlined in Ref. 33. Membranes were exposed
for autoradiography at 70 °C using x-ray films (Fuji). The
autoradiograms were analyzed by laser densitometer scanning (LKB
Bromma, Ultroscan XL) and the mRNA values were normalized to those
of the 18 S rRNA, which did not vary under our experimental
conditions.
Samples of 25 µg of INS cell protein extracts, obtained in the presence of 50 mM Tris (pH 7.5), 5 mM EDTA, 5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml antipain, and 10 mM mercaptoethanol, were resolved on 10% SDS-polyacrylamide gels and electrotransferred to nitrocellulose membranes (Bio-Rad). The membranes were incubated with a rabbit anti-rat antibody raised against the C-, M-, and L-PFK-1 isoforms (30). GAPDH was detected with an anti-rabbit monoclonal antibody (kindly provided by Dr. E. Knecht, Instituto de Investigaciones Biomédicas, Valencia, Spain). The L-PK and M-PK isoforms were detected with specific antibodies (kindly provided by Dr. J. Blair, University of West Virginia). The enzymatic activities of low Km Hk and Gk were measured in INS cells extracts as described previously (34).
In Vitro Transcription AssayNuclei isolation and nuclear run-on transcription assays were performed according to Ref. 35. Briefly, nascent transcripts were elongated in vitro in the presence of [32P]UTP and 2.1 mg/ml heparin. The [32P]RNAs were subjected to mild alkaline hydrolysis (30 min, 50 °C, 50 mM Na2CO3) and hybridized to 4 µg/dot of the following DNA constructions immobilized on nitrocellulose membranes: a 0.74-kb EcoRI-BamHI fragment (positions 1040-1780) of mouse rRNA 18 S cDNA subcloned in pUC830 and a 1.289-kb PstI-PstI fragment (positions 1-1289) of rat GAPDH cDNA subcloned in pBSKS.
Glucose Metabolism, Glycogen Determination, and Insulin SecretionINS cells seeded in 24-well plates were used for these studies. Cells were preincubated for 48 h in culture medium at 5 mM glucose and incubated at 5 and 25 mM glucose for 3 days. Culture medium was removed and cells were washed twice with PBS and preincubated for 30 min at 37 °C in Krebs-Ringer bicarbonate medium (KRB) containing 10 mM Hepes (pH 7.4), 0.07% bovine serum albumin, and 2.5 mM glucose. Cells were then washed twice with PBS and incubated for 30 min in KRB-Hepes medium containing 0.07% bovine serum albumin, 0.5 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), and different glucose concentrations. Glucose metabolism was followed by the MTT reduction test, which closely reflects the rate of glucose oxidation, as described previously (36). Incubation media were collected to determine insulin release (37). The cellular glycogen content was determined according to a published procedure (38).
Cell Proliferation and DNA ContentINS-1 cells were seeded (0.8 × 105 cells/well) in 24-well plates. When cells reached 50% confluence after approximately 4 days, they were washed twice with PBS and preincubated for 48 h in culture medium containing 5 mM glucose. Cells were washed again with PBS and incubated for 5 days in culture medium containing different glucose concentrations. Cells were then detached by trypsinization and resuspended in 0.8 ml/well RPMI. An aliquot of the cell suspension was diluted in 2 M NaCl, 0.05 M sodium phosphate buffer (pH 7.4), and DNA was measured as described (39). Cells were counted in a Coulter Counter.
We elected to study the
expression of various transcripts encoding key glycolytic enzymes,
which are known to play essential regulatory role in -cell signaling
or glycolysis. These include the transporter Glut-2, whose
-cell
content is decreased in diabetes (40); Gk, which acts as a glucose
sensor (9); PFK-1, which is an important determinant of glycolytic flux
and oscillations (30); 6-phosphofructo-2-kinase/fructose
2,6-bisphosphatase (PFK-2), which synthesizes fructose
2,6-bisphosphate, a regulator of glycolytic flux (41); GAPDH, which
forms NADH which may directly or indirectly (via ATP production) act as
a coupling factor (25); and pyruvate kinase, a highly regulated enzyme
that converts cytosolic ADP to ATP.
Glut-2 mRNA increased by approximately 3-fold in INS-cells
incubated for 24 h at 25 mM glucose in comparison to
those cultured at 5 mM glucose (Fig. 1).
This confirms findings made by others in pancreatic islets and various
-cell lines (18, 42-44). The Glut-2 probe detected only one
abundant transcript of 2.8 kb in INS cells, in contrast with purified
-cells, which expressed two mRNA species of 3.9 and 2.8 kb (45).
The biological significance of the larger transcript is unknown.
Using a poly(A+) mRNA fraction, it was observed that the "platelet" PFK-1-C transcript of 3.2 kb was induced by elevated glucose but was not detectable at basal (5 mM) glucose (Fig. 1). The "liver" type PFK-1-L and "muscle" type PFK-1-M transcripts were undetectable under our experimental conditions using the Northern blot technique. However, the corresponding proteins were detected using specific antibodies (see below). The GAPDH probe recognized one 1.3-kb transcript in INS cells, which was consistently induced (about 4-5-fold) by incubating INS cells at high glucose (Fig. 1). The L-PK probe has been shown to recognize three transcripts of 3.1, 2.2, and 2.0 kb in cultured hepatocytes and INS-1 cells (11, 46). The low molecular weight species are indistinguishable in our electrophoresis conditions. Fig. 1 shows that the different L-PK transcripts are markedly induced by high glucose, confirming the results of a previous study (11). The effect of glucose on the expression level of these inducible transcripts was dose-dependent (data not shown).
As previously reported (11), the Gk mRNA level did not vary in response to glucose. The expression of the transcript of glucose-6-phosphate dehydrogenase also remained similar at low and high glucose (data not shown). PFK-2 mRNA content barely varied in cells cultured at high glucose. The slight apparent decrease of the PFK-2 signal at high glucose is due to a difference in sample loading of the gel in this experiment (data not shown). The cDNA probe used to detect the PFK-2 transcript recognizes both the L- and M-isoform mRNA species. It is most likely that only the M-isoform is present in INS cells as well as in pancreatic islets. Indeed, reverse transcriptase-polymerase chain reaction experiments revealed that the mRNA of the L-isoform is expressed neither in INS cells nor in islets, while the mRNA for the M-isoform is present in both INS cells and islet tissue.2 The 18 S ribosomal mRNA content, which was used as a control for gel loading, remained unaffected by glucose (Fig. 1).
Our results indicate that glucose causes an induction of several genes coding for key glycolytic enzymes in INS-1 cells. The effect of glucose is selective, because some genes are induced, while others are not under the same experimental conditions.
The time dependence of the accumulation of the Glut-2, GAPDH and L-PK
transcript is depicted in Fig. 2. The lag time of both Glut-2 and L-PK mRNA induction was approximately 2 h, and a
maximal effect occurred at about 6 h of culture at high glucose.
In contrast, the onset of GAPDH induction was much slower. Increased
expression of GAPDH mRNA in response to elevated glucose required
at least 6 h, and a maximal effect was observed at 24 h. We
previously reported that ACC mRNA induction by high glucose in INS
cells requires about 3 h (19). The reason for which GAPDH mRNA
induction displays a relatively long lag time is not known. It possibly reflects a distinct mechanism of gene induction by glucose (see below).
Glucose Increases the Expression Level of GAPDH, L-PK, and Three PFK-1 Isoforms
Experiments were carried out to determine whether the accumulation of the inducible mRNAs is associated with a similar increased expression of the corresponding glycolytic enzymes. Since many glycolytic enzymes such as GAPDH have a long half-life (2-3 days), the expression of the glycolytic enzymes was measured after 3-4 days of incubation at either low (5 mM) or high (25 mM) glucose.
To assess the expression of PFK-1, a polyclonal antibody which
recognizes the three PFK-1 isoforms was used. Fig. 3
shows that the C-, M-, and L-isoforms of PFK-1 were all induced by high glucose. The presence of the C-PFK-1 (86.5 kDa) and M-PFK-1 (82.5 kDa)
isoforms has been reported in rat islets and INS cells (30). Both PFK-1
subtypes were induced in parallel and by approximately the same extent
when INS cells were incubated at the elevated glucose concentration
(Fig. 3). The L-PFK-1 isoform is poorly expressed in pancreatic islets
(30) and is undetectable in INS cells incubated at 5 mM
glucose (Fig. 3). Interestingly, the L-isoform (76.7 kDa) of PFK-1 was
expressed following a 3-day exposure of INS cells to high glucose.
The cellular GAPDH content was only marginally increased after 48 h of cell exposure to 25 mM glucose (data not shown). We
therefore studied its expression at 120 h. It is apparent that the
amount of the GAPDH protein rose to an extent similar to that for its
corresponding transcript in response to high glucose (Fig. 3).
A thorough study of L-PK transcriptional activation by glucose in INS
cells has previously been carried out (11). However, the expression of
the L-PK enzymes in response to glucose was not investigated. We
therefore studied the expression of both the L- and M-isoforms of the
enzyme in INS cells incubated for 72 or 120 h at 5 or 25 mM glucose. Immunoblot analysis of L-PK using a polyclonal
antibody revealed the presence of two proteins of 55-60 kDa. Both were
induced by elevated glucose (Fig. 4A). This
result was confirmed using a monoclonal anti-L-PK antibody (Fig.
4B). The specificity of the monoclonal antibody was assessed by showing that it detected a major specific band in liver, whereas it
detected no L-PK protein in smooth muscle where this isoform is not
expressed (Fig. 4B). The two protein bands detected by the
L-PK antibodies might be different phosphorylated forms of the enzyme,
spliced variants, or translation products from different transcripts.
The results in Fig. 4 also indicate that glucose increases the
expression level of the L-isoform of PK. Thus, M-PK, which is also
expressed in INS cells and is the most abundant isoform in islets
tissue (47), was only slightly induced by the sugar (Fig.
4B). It should be pointed out that the single immunoreactive
protein band detected by the M-PK antibody is not due to a
cross-reactivity of the antibody with L-PK for the following two
reasons. First, the M-PK antibody recognized a single abundant protein
band in muscle and revealed two weak signals in liver tissue. Second,
although M-PK has a molecular weight indistinguishable from the higher
molecular weight form of L-PK, its marked variation of expression at
high versus low glucose revealed with both L-PK antibodies
was not detected with the M-PK antibody. The results in Fig. 3 are
furthermore in agreement with the absence of 5-CACGTG-3
or
5
-CACGGG-3
carbohydrate response elements in the promoter of the rat
M-PK gene as compared to the L-PK gene.
In accord with results obtained with rat islets (48), long term exposure of INS cells to elevated glucose increased Gk activity but did not change low Km Hk enzymatic activity. The following values were detected in INS cells at 5 mM glucose for 3 days: Hk, 1.3 ± 0.1, Gk, 4.2 ± 0.3 milliunits/mg of protein. At 25 mM glucose the activities were: Hk, 1.2 ± 0.2, Gk, 5.7 ± 0.2 (p < 0.001 versus control). Results are means ± S.E. from four to five experiments.
Hence, the glucose induction of the PFK-1, GAPDH, and L-PK transcripts is associated with similar changes at the protein level, which possibly contribute to the late phenotypic alterations in INS cells exposed to an elevated glucose concentration.
Transcriptional Activation by Glucose of Genes Encoding Glycolytic EnzymesAn increased transcriptional rate of the Glut-2 (18) and
L-PK (11) genes has been documented in INS cells challenged with high
glucose. We sought to determine whether a transcriptional effect is
also implicated in the increased expression of GAPDH. Run-on
transcriptional assays were initially carried out following a 2- or 4-h
incubation period at either 5 or 25 mM glucose. In three
separate experiments, a similar transcriptional activity of the GAPDH
gene at the two glucose concentrations was measured (data not shown).
However, when the assays were carried out at 8 or 24 h with
elevated glucose, a 1.7-fold increased transcriptional rate was
observed in response to high glucose (Fig. 5). Thus, the
enhanced transcriptional activity of the GAPDH gene is delayed in
comparison to the Glut-2 (18) and L-PK (11) genes and accounts for
about half of the accumulation of the GAPDH mRNA or protein in
response to elevated glucose. These results suggest that additional mechanism(s) are implicated in the glucose regulation of GAPDH expression. These may include altered mRNA stability or
differential processing of the transcript.
We addressed the first possibility by stimulating INS cells at 25 mM glucose for 24 h and incubating them afterward for 1-8 h in the presence of high (25 mM) or low (5 mM) glucose in the presence of the transcription inhibitor actinomycin D. The results indicated that the GAPDH transcript has a long half-life, since its content was reduced by only 20% following an 8-h incubation period in the presence of the inhibitor. Longer times were not tested because the drug affected cell viability beyond 8 h. The results obtained showed no apparent difference in the GAPDH mRNA decay at low versus high glucose (data not shown). Nonetheless, these results do not entirely discount a possible action of glucose in modulating GAPDH mRNA stability. Thus, due to the limitation imposed by the cell toxicity of actinomycin D, longer incubation periods could not be tested to rigorously assess this issue. It is noteworthy that the transcriptional induction of the Glut-2 gene also does not fully account for the glucose increased expression of Glut-2 mRNA (18). Whether glucose modulates the stability of transcripts encoding glycolytic enzymes remains to be determined.
Mechanism of Glucose Regulation of GAPDH Gene ExpressionPrevious work documented that the non-metabolizable
glucose analog 2-deoxyglucose (2-DOG) induces the L-PK (11) and ACC (19) genes in INS cells. This suggested that the metabolism of the
sugar beyond the Gk step is not required for the induction of some
genes encoding metabolic enzymes (11, 19, 20). To gain insight into the
mechanism of GAPDH induction, we tested several glucose analogs as well
as nutrient stimuli and pharmacological agents, which activate various
signaling pathways and insulin secretion (Fig. 6).
Mannose, the epimer in position 2 of glucose, which is well metabolized
in the -cell (6), increased the expression level of GAPDH mRNA.
The effect of glucose was suppressed by the Gk inhibitor mannoheptulose
and was not mimicked by 2-DOG, which is phosphorylated but is not
metabolized beyond this step (49). 6-Deoxyglucose and
3-O-methylglucose, two analogs that enter mammalian cells
but cannot be phosphorylated, were ineffective. High concentrations of
pyruvate and glutamine plus leucine, which efficiently promote insulin
secretion (6, 50), did not alter the expression level of the GAPDH
transcript. Finally, elevated K+, phorbol 12-myristate
13-acetate, and forskolin, which are good secretagogues and,
respectively, activate the Ca2+, protein kinase C, and cAMP
signaling systems (51), barely modified the GAPDH mRNA content
(Fig. 6). 10
7 M insulin (autocrine control)
and 30 mM sucrose (osmolarity control) had no effect on the
expression level of Glut-2 and GAPDH mRNAs (data not shown).
The results demonstrate that glucose needs to be metabolized beyond the glucokinase step to induce the GAPDH gene and that the Ca2+, cAMP, and protein kinase C transduction systems are not implicated in this process.
MTT Reduction, Glycogen Deposition, Glucose-induced Insulin Secretion, and DNA Synthesis in INS Cells Exposed during Culture to High Glucose ConcentrationsTo examine whether the
glucose-induced changes in the expression level of key glycolytic
enzymes in (INS-1) cells were associated with similar phenotypic
changes already observed in pancreatic islets (1, 2, 8, 9, 45, 51, 52),
which might in part result from these modifications, we measured
several parameters of
-cell activation.
The intracellular reduction of the tetrazolium dye MTT allows
determination of the metabolic activity of the -cell (36). The assay
measures the reduction of the tetrazolium salt into insoluble colored
formazan crystals. A very good correlation has been shown between MTT
reduction, glycolytic flux, glucose oxidation, and insulin secretion in
INS cells (26, 36). MTT is reduced both in the cytosol and the
mitochondria of living cells (26). It provides a suitable index of
overall (glycolytic plus mitochondrial) metabolism via reducing
equivalent production, simultaneously with secretion measurements (36).
INS cells were cultured for 3 days at 5 or 25 mM glucose
and then incubated for 30 min in the presence of MTT at various glucose
concentrations. In cells preincubated for 3 days at low glucose, a
subsequent variation of the sugar from 2.5 to 25 mM
elicited a dose-dependent increase in MTT reduction,
reaching a maximal value at 15 mM (Fig. 7,
upper panel). Cells incubated for 3 days at 25 mM glucose displayed a 3-fold higher MTT reduction at low
(2.5-5 mM) glucose and a 50% increased reduction of the
dye at maximal (15-20 mM) concentrations of the
carbohydrate. The data indicate that the basal metabolic activity of
INS cells previously challenged with high glucose is 3 times that of
cells exposed to a low physiological concentration of the sugar.
In the same set of experiments, we performed insulin secretion measurements in parallel (Fig. 7, lower panel). The patterns found for insulin secretion were very similar to those observed for MTT reduction. Cells cultured at low glucose subsequently exhibited a dose-dependent increase in secretion of 6-fold from 2.5 mM to 25 mM glucose. However, cells preincubated at 25 mM glucose for 3 days showed an increased basal insulin secretion and an absence of response to glucose between 5 and 25 mM glucose.
Glucose increases the glucose 6-phosphate content of rat islets (8, 9)
and INS cells (19). This glycolytic intermediate can also enter the
glycogen biosynthetic pathway leading to intracellular glycogen
accumulation, whose exaggerated deposition has been postulated to be
toxic for the -cell (53). Fig. 8 (left
panel) shows the dose dependence of glycogen deposition as a
function of the glucose concentration. A >20-fold increased
accumulation of glycogen in cells cultured at high (20 mM)
versus low (5 mM) glucose was observed. The
marked glycogen deposition could act as a glucose store, which, upon
lowering of external glucose, could be metabolized to maintain elevated
glucose metabolism and insulin secretion at basal concentrations of the
sugar. To test this hypothesis, we measured the glycogen content of INS
cells as a function of time following a lowering of glucose from 25 mM (for 3 days) to 5 mM glucose. The results in
Fig. 8 (right panel) indicate that 60% of the accumulated
glycogen was metabolized in a 6-h time period.
Since glucose acts as a growth factor for islet tissue and stimulates
the proliferation of normal -cells (1, 52, 54), we determined
whether similar action of the sugar occurs in
(INS) cells. Glucose
promoted a dose-dependent increase in DNA synthesis. The
following values were observed after 5 days exposure to various glucose
concentrations: glucose (5 mM), 4.7 ± 0.8 µg of
DNA/well; glucose (11 mM), 8.7 ± 0.4 µg of
DNA/well; glucose (25 mM), 14.8 ± 0.8 µg of
DNA/well (means ± S.E. of four wells). Cell proliferation measurements provided identical results (data not shown) and have been
reported previously (54).
To address the question of the molecular nature of the -cell
adaptation to hyperglycemia, we used the
-cell line INS (29). As
shown previously (19) and documented in greater detail in this study,
the secretory properties of INS cells incubated for a long period of
time (3 days) in the presence of elevated (25 mM) glucose
concentrations display two characteristic features: a markedly elevated
secretion at low (2-5 mM) glucose and a lack of response
to higher concentrations of the sugar. This sensitization to glucose is
similar to that observed for rat islets in vivo and in
vitro (2, 9, 55) and human islets in culture (56). In addition,
glucose within its physiological range of concentrations promotes the
proliferation of
(INS) cells as well as normal
-cells (1).
Finally, INS cells incubated for long periods of time at high glucose
accumulate massive amounts of glycogen, like islet tissue (53, 57). As
far as metabolic gene expression is concerned, like in rat islets (11,
18), glucose induces Glut-2 but not Gk mRNA in INS cells. In
addition, the C- and M-isoforms of PFK-1 (30), the M-isoform of PFK-2,
and the M-isoform of PK (47) are well expressed in both pancreatic
islets and INS cells (this study). Thus, INS cells appear to be a good
model to address the above mentioned questions and to carry on a
detailed study of metabolic enzyme expression in a system where only
-cells are studied. Indeed, normal islet tissue contains 40%
non-
-cells, which may be very different as far as metabolism is
concerned. For instance, studies in which sorted islet cells were
studied have indicated that FAD-linked glycerol phosphate dehydrogenase is elevated in the
-cell, whereas lactate dehydrogenase is very poorly expressed, and the same is true for INS cells (26). In contrast,
the reverse expression of these enzymes occurs in non-
-cells (26).
The present study shows that long term exposure of INS cells to
elevated glucose causes a coordinated induction of several glycolytic
enzymes, which may play a key role in the coupling process from
nutrient metabolism to insulin release and other biological functions
including insulin biosynthesis and -cell replication (1, 5, 8, 14,
17). These enzymes are PFK-1, GAPDH, and L-PK. The increase of Glut-2,
PFK-1, GAPDH, and L-PK is associated with an exaggerated metabolic
activity of INS cells, increased secretion at low glucose, and enhanced cell proliferation. Consistent with these observations, fasting of rats
for 72-120 h caused a 20% reduction in the activity of pancreatic
islet Gk (58), PFK-1 (58, 59), and GAPDH (58), and a decrease in the
expression level of L-PK mRNA (11).
The question arises as to how the augmented expression level of each of
these particular proteins might relate to the observed phenotypic
changes. Concerning PFK-1, the M-isoform of this enzyme plays a key
role in glycolytic oscillations (5, 6, 30), which are thought to be
implicated in the oscillatory nature of -cell metabolism (60) and
insulin secretion (5, 6). The expression level of the M- and C-isoforms
are increased by glucose as is the L-isoform, which cannot be detected
at low concentrations of the sugar. The different isoforms may form
heterotetramers displaying intermediate kinetic properties (61, 62). It
is attractive to hypothesize that the induction of the L-isoform favors
the formation of new tetramers, which can lead to a different pattern
of glycolytic oscillations resulting in impaired insulin secretion. It
should also be noted that mathematical modeling of
-cell glycolysis
indicates that the metabolic flux control coefficient of Gk and PFK-1
are similar at low (around 4 mM) glucose (9). Thus, an
increased expression of PFK-1 may be required for increased
-cell
growth and for high glycolytic flux and secretion as well as to match
the augmentation of Gk activity in both rat islets (48) and INS cells
(present study), which occurs with long exposures to elevated glucose
(9, 55). It should be noted that an increase in the quantity and
activity (Vmax) of a highly regulated allosteric
enzyme like PFK-1 will influence glycolytic flux only when the enzyme
is activated as it presumably is during metabolic oscillations, since
at basal metabolite levels and ATP/ADP ratios, the enzyme is largely
inhibited. Thus, PFK-1 induction might possibly permit larger
glycolytic oscillations. Another potential role for some PFK-1 isoforms
is suggested by a recent report documenting an association between a
glucose and ATP-stimulatable Ca2+-independent phospholipase
A2 in the
-cell with a phosphofructokinase-like protein
closely related or identical to M-isoenzyme (63).
The glucose induction of GAPDH is of particular interest in view of a
novel hypothesis, proposing that a glycolytic signal which occurs at a
step catalyzed by this enzyme (8) is responsible for KATP channel
closure by glucose. Dukes and co-workers (25) have provided evidence in
support of the notion that NADH, generated at the GAPDH step, fuels
mitochondrial ATP production via the glycerol-phosphate shuttle (27).
This event might be causally linked to the action of glucose on the
KATP channel and Ca2+ influx (8, 25). Also consistent with
the idea that GAPDH deserves much more attention in -cell signaling
than previously suspected is the finding that of all glucose
metabolites, glyceraldehyde 3-phosphate is the most potent insulin
secretagogue (64). With respect to insulin exocytosis noteworthy is the
identification of a fusogenic role of a brain isoform of GAPDH, which
catalyzes an extremely rapid fusion of phospholipid vesicles (65). The concept is also emerging that some metabolic enzymes have important cellular functions in addition to their role as enzymes. In this regard, numerous non-glycolytic activities have been attributed to
GAPDH, including interaction with the cytoskeleton, nucleic acid
binding, and DNA repair (66-69). It remains to be proven whether any
of these alternative functions ascribed to GAPDH, which might be
implicated in the late phenotypic changes caused by elevated glucose,
are operative in the
-cell. A previous report showed that the GAPDH
activity of pancreatic islets in culture remains constant after a 24-h
exposure to high glucose (70). The results of this study are consistent
with this previous observation, since we also observed no change in
GAPDH activity after a 24-h period but only after 3 days of incubation
at elevated glucose.
Pancreatic islets contain the M-isoenzyme of pyruvate kinase (47) and
express the L-type PK gene at a low level (11, 71). L-PK/T antigen
transgenic mice fed a high carbohydrate diet were shown to frequently
develop endocrine pancreatic tumors (72), and reverse
transcriptase-polymerase chain reaction experiments indicated that
glucose intake increased L-PK mRNA in islet tissue (11). The
results of this study are in accordance with these observations and
furthermore show that the M-isoenzyme is constitutively expressed. By
contrast, glucose markedly induce the accumulation of the two protein
isoforms L and L encoded by the L-PK gene. Altogether, these
observations indicate that L-PK protein expression is induced by
elevated glucose in the
(INS)-cell. The significance of this
inductive process is uncertain. As stated above for PFK-1, increased
L-PK expression may be required for the accelerated glycolytic flux and
the resulting increased secretion and
-cell growth because the
expression of the M-isoenzyme is not induced by the sugar. In the liver
L-PK is a highly regulated enzyme whose activity is changed by its
phosphorylation state (72). The L-isoenzyme is allosterically activated
by fructose bisphosphate, whereas the muscle isoenzyme is not (73).
Whether L-PK is also regulated similarly in the
-cell is not known.
L-PK provides glycolytic ATP. Perhaps the enzyme activity might cause
local ATP gradients in the cytoplasm, in particular in the vicinity of
the plasma membrane to influence more directly the open state
probability of KATP channels or the activity of phospholipase
A2 (ATP-stimulatable), which liberates free arachidonic
acid, a potent stimulator of insulin secretion (74).
It must be pointed out that the action of the sugar is specific for
some genes coding for glycolytic enzymes, as not all the induced genes
are activated to the same extent and with the same kinetics. The data
also support the notion that there may exist several mechanisms
implicated in the action of glucose on glycolytic enzyme gene
expression in the -cell. A transcriptional activation is involved in
the increased expression of the Glut-2 (18), GAPDH, and L-PK (11)
genes, although additional mechanisms such as alterations in transcript
stability likely contribute to the increased Glut-2 (18) and GAPDH
mRNA accumulation at high glucose. Moreover, glucose 6-phosphate
(19, 75) or an intermediate of the pentose pathway (76) may mediate
gene activation for L-PK because 2-deoxyglucose is also effective (11).
In contrast, GAPDH induction seems to be dependent of the metabolism of
glucose because of the inability of 2-deoxyglucose to mimic the action of glucose.
The induction of key glycolytic enzymes is associated with a marked
increase in metabolic activity of INS cells. The MTT reduction experiments show an elevated -cell oxidative metabolism at all tested glucose concentrations in cells exposed to elevated glucose for
3 days. A parallel pattern is observed for insulin secretion. The
massive deposition of glycogen may contribute to the hypersecretion of
insulin at low glucose by providing glucose through glycogenolysis at
low external concentrations of the sugar. Thus, since
glucose-6-phosphatase is barely expressed in rat islets (9) and INS
cells,3 the vast majority of the glucose
mobilized from glycogen should enter the glycolytic pathway. If it can
be extended to the in vivo situation, these changes in
glycogen metabolism and
-cell glycolysis could be related to the
hyperinsulinemia developed in the first stages of
non-insulin-dependent diabetes mellitus. Increased
glycolytic enzyme gene expression and metabolic activity may also be
implicated in the stimulation of
-cell proliferation. Indeed,
glucose is a major growth factor in this particular fuel-sensitive cell
both in vivo and in vitro (1, 2). Elevated
glycolytic flux should lead to the enhanced production of various
signaling molecules and activate transduction pathways implicated in
the regulation of cell growth such as the protein kinase C,
mitogen-activated protein kinase, and Ca2+ signaling
systems. Interestingly, glucose activates mitogen-activated protein
kinases in INS cells and induces a number of immediate early response
genes implicated in cell growth regulation (28).
An important issue is to know whether the action of glucose on the
expression of genes encoding metabolic enzymes is the consequence of
glucose-directed gene regulation, increased metabolism of the sugar, or
its mitogenic action. With respect to the L-PK and ACC genes, a
"direct" action of the sugar independent of accelerated glycolytic
flux is likely. Thus, the non-metabolizable analog 2-DOG, which has no
mitogenic action, induces the L-PK (11) and ACC (19) genes. Consensus
carbohydrate response elements are present in the promoter region of
the L-PK gene (20). With respect to ACC, two short sequences that
resemble the consensus sequence are also present within its promoter
(19). It is unclear at present whether these sequences contribute to
the glucose regulation of the ACC gene in the -cell. Concerning
Glut-2, requirement of glucose metabolism has been documented in both
liver (77) and rat islets (78) to regulate the Glut-2 gene. The
induction of the GAPDH gene by glucose is relatively delayed with
respect to the other investigated genes, it is not mimicked by 2-DOG, and a consensus response element is not found in its promoter. We
therefore favor the view that GAPDH gene induction by glucose is the
consequence of accelerated glucose metabolism and possibly early
mitogenic events cause by the sugar. Nonetheless, these inductive
processes, whatever the diversity of the mechanisms involved, pinpoint
candidate genes and proteins that might be crucially involved for the
normal adaptation response to elevated glucose (i.e.
hypersecretion, hypertrophy, and hyperplasia of the
-cell). Possibly
a defect in glucose regulation, either direct or indirect, of one of
these genes could contribute to the pathogenesis of type 2 diabetes
where the adaptation of the
-cell to hyperglycemia has failed.
Finally, glucose is not the only calorigenic nutrient that may
participate in the phenotypic alterations of insulin secreting cells
during diabetic pathologies. In addition to glucose, other circulating
factors, such as elevated fat, appear to play an instrumental role (5,
6, 8, 79-81). The mechanism whereby each individual calorigenic
nutrient or combination of them participate in the etiology of diabetes
remains largely an open question.
Antibodies against liver and muscle pyruvate kinase were kindly supplied by James Blair (University of West Virginia).