(Received for publication, June 5, 1995; and in revised form, July 24, 1995)
From the
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been
postulated to be a metabolic signaling enzyme, which acts as a switch
between glycolysis and gluconeogenesis in mammalian liver by regulating
the level of fructose 2,6-bisphosphate. The effect of overexpressing
the bifunctional enzyme was studied in FAO cells transduced with
recombinant adenoviral constructs of either the wild-type enzyme or a
double mutant that has no bisphosphatase activity or protein kinase
phosphorylation site. With both constructs, the mRNA and protein were
overexpressed by 150- and 40-fold, respectively. Addition of cAMP to
cells overexpressing the wild-type enzyme increased the S for fructose 6-phosphate of the kinase by 1.5-fold but had no
effect on the overexpressed double mutant. When the wild-type enzyme
was overexpressed, there was a decrease in fructose 2,6-bisphosphate
levels, even though 6-phosphofructo-2-kinase maximal activity increased
more than 22-fold and was in excess of fructose-2,6-bisphosphatase
maximal activity. The kinase:bisphosphatase maximal activity ratio was
decreased, indicating that the overexpressed enzyme was phosphorylated
by cAMP-dependent protein kinase. Overexpression of the double mutant
resulted in a 28-fold increase in kinase maximal activity and a
3-4-fold increase in fructose 2,6-bisphosphate levels.
Overexpression of this form inhibited the rate of glucose production
from dihydroxyacetone by 90% and stimulated the rate of lactate plus
pyruvate production by 200%. In contrast, overexpression of the
wild-type enzyme enhanced glucose production and inhibited lactate plus
pyruvate production. These results provide direct support for fructose
2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway
flux and suggest that regulation of bifunctional enzyme activities by
covalent modification is more important than the amount of the protein.
Gluconeogenic/glycolytic pathway flux is regulated by allosteric
effectors and by covalent modification of key regulatory enzymes and/or
by modulation of gene expression of these
enzymes(1, 2) . Until recently, it has not been
possible to alter properties of an enzyme in a metabolic pathway in a
systematic manner and then test the effect on the function of the
entire pathway in an intact cell. The development of host/vector
systems containing powerful promoters has allowed transfer and
expression of normal and mutant cDNAs of proteins in mammalian cells
and evaluation of regulatory enzymes in controlling pathway
flux(3) . Hepatic
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
(6-PF-2-K/Fru-2,6-Pase) (
)is a regulatory enzyme
in the gluconeogenic/glycolytic pathway, which catalyzes both the
synthesis and degradation of the signal molecule,
Fru-2,6-P
, an allosteric activator of
6-phosphofructo-1-kinase and an inhibitor of
fructose-1,6-bisphosphatase(4, 5, 6) . The
enzyme has been postulated to provide a switching mechanism between the
glycolytic and gluconeogenic pathways in liver(7) . The kinase
and bisphosphatase activities are reciprocally regulated by
cAMP-dependent protein kinase-catalyzed phosphorylation(1) .
Gene expression of the enzyme is subject to multihormonal regulation;
insulin and glucocorticoids enhance and cAMP suppresses gene
transcription(1) . Therefore, the level of Fru-2,6-P
and, ipso facto, glycolytic and gluconeogenic flux
depend in a complex way on hormonal milieu.
A number of questions
with regard to the bifunctional enzyme and metabolic pathway flux
remain unanswered: 1) what is the relative importance of covalent
modification and the concentration of bifunctional enzyme protein in
controlling Fru-2,6-P levels; 2) what are the relative
roles of the kinase and bisphosphatase activities in determining the
level of Fru-2,6-P
; and 3) what is the role of
Fru-2,6-P
in determining gluconeogenic pathway flux? It was
the object of this study to address these questions by using
recombinant adenovirus to overexpress wild-type
6-PF-2-K/Fru-2,6-P
ase and a double mutant (S32A/H258A) of
the protein, which only possesses 6-PF-2-K activity and cannot be
down-regulated by cAMP-dependent protein kinase-catalyzed
phosphorylation. Mutation of Ser-32 to Ala prevents cAMP-dependent
phosphorylation of the enzyme(8) , while mutation of His-258 to
alanine abolishes bisphosphatase activity, since this residue is known
to mediate catalysis via a phosphohistidine
intermediate(9, 10) . Sincethey are capable of
producing glucose from 3-carbon
precursors(11, 12, 13, 14) ,
cultured rat hepatoma FAO cells were chosen to overexpress the enzyme
forms. Thus, the switching mechanism between glycolysis and
gluconeogenesis, hypothesized for the bifunctional enzyme and
Fru-2,6-P
, can be studied.
Figure 1:
Schematic representation of the
strategy used for construction of Ad-PF2KWT. The pACCMVpLpA plasmid
contains the transcription unit consisting of the cytomegalovirus early
gene promoter/enhancer, the rat liver 6-PF-2-K/Fru-2,6-Pase
cDNA, and the SV40 polyadenylation genome. In the plasmid, the
transcription unit is inserted in a partial deletion site (1.3/9.1 map
units) within the adenovirus (Ad5) early region 1 (0/17 map units). The
pJM17 plasmid contains the Ad5 cDNA (36 kb = 100 map units), in
which the 4.3 kb plasmid pBRX encoding amoxicillin and tetracycline
resistance was inserted at the Ad5 XbaI site. Homologous
recombination between the two plasmids in the 293 cell generates
replication-defective adenoviruses, since adenovirus early region 1 is
replaced by the cloned chimeric gene. Three recombinant viruses were
generated: AdWT, Ad-PF2KWT, and Ad-PF2KMut (see ``Experimental
Procedures''). This scheme represents the construction of the
Ad-PF2KWT.
Figure 2:
Northern blot analysis and quantification
of overexpression of wild-type or mutated
6-PF-2-K/Fru-2,6-Pase mRNA. Northern analysis of 20 µg
of RNA extracted from treated FAO cells with different adenovirus
constructs (AdWT, Ad-PF2KWT, Ad-PF2KMut), from untreated FAO, and from
refed liver. A 1.4-kb EcoRI fragment of rat liver
6-PF-2-K/Fru-2,6-P
ase cDNA was used as a probe. The
autoradiograph was obtained after a 1-h exposure with two intensifying
screens. mRNA was quantified by scanning
densitometry.
Overexpression of the wild-type and double mutant
(S32A/H258A) 6-PF-2-K/Fru-2,6-Pase mRNAs was verified by
ribonuclease protection assay (Fig. 3). As expected, a 230-base
complementary fragment was protected with rat liver RNA (lane
L) and with RNA from FAO cells treated with Ad-PF2KWT (lane
2), while a 179-base fragment was observed with RNA from FAO cells
transduced with Ad-PF2KMut (lane 3). The 297-base nucleotide
riboprobe protects a fragment that includes part of exon I specific for
the liver 6-PF-2-K/Fru 2,6-P
mRNA and part of exon I which
is shared by both the liver and skeletal muscle forms of
6-PF-2K/Fru-2,6-P
ase mRNA(25) . Moreover, the 5`
end of exon II corresponds to the sequence encoding the S32A mutation
site. Hence, the fragment protected by the rat skeletal muscle mRNA was
the same size as the fragment protected by the mutant form (S32A/H258A)
of 6-PF-2-K/Fru-2,6-P
ase mRNA, i.e. 179 bases (Fig. 3, lanes M and 3). These results provide
definitive evidence that the wild-type and mutant bifunctional enzyme
mRNAs were overexpressed.
Figure 3: RNase protection analysis. The RNase protection assay was designed as described under ``Experimental Procedures,'' and 20 µg of RNA obtained from different sources were protected with the 297-base probe. Lane Pr contains undigested RNA probe; the other lanes contain protected fragments after hybridation of the probe with RNA from different sources and digestion with RNase. FAO cells treated with either AdWT (lane 1), Ad-PF2KWT (lane 2), or Ad-PF2KMut (lane 3), rat liver (lane L), and rat skeletal muscle (40 µg) (lane M) are shown. MWSt, molecular weight standard.
Immunofluorescence studies were performed on monolayer cultures of FAO cells by exposing these cells to an anti-bifunctional enzyme antibody (27) . A high level of bifunctional protein was detected in cells treated with Ad-PF2KWT or Ad-PF2KMut, whereas the protein was undetectable in control FAO cells incubated or not incubated with AdWT (data not shown). Both forms of overexpressed protein were homogeneously detected in the cytosol of FAO cells, with no apparent localization to any subcellular structure.
Western blot analysis (Fig. 4) confirmed that the wild-type (lanes 3 and 6) and the mutated form (lane
4) of overexpressed 6-PF-2-K/Fru-2,6-Pase were the
same size as the wild-type rat liver 6-PF-2-K/Fru-2,6-P
ase
(55 kDa) (lane 7). FAO cells incubated in the presence of 30
mM glucose contained a small quantity of the liver form of
6-PF-2-K/Fru-2,6-P
ase (lane 5), whereas no
measureable signal was obtained at the 11 mM glucose in
control cells (lane 1) or in cells treated with AdWT (lane
2). 6-PF-2-K/Fru-2,6-P
ase protein in FAO cells
incubated with either Ad-PF2KWT (lanes 3 and 6) or
Ad-PF2KMut (lane 4) was 40-fold greater than in the control (lane 5) and was not dependent on glucose concentration.
Figure 4:
Western blot analysis of overexpressed
6-PF-2-K/Fru-2,6-Pase protein. Proteins were extracted and
treated with 65% (NH
)
SO
as
described under ``Experimental Procedures'' from untreated
FAO cells (lane 1) or from transduced FAO cells with different
adenovirus constructs: AdWT (lanes 2 and 5),
Ad-PF2KWT (lanes 3 and 6), and Ad-PF2KMut (lane
4). The glucose concentration was different in incubation medium
during the 48 h after infection (30 instead of 11 mM for lanes 5 and 6). 100 µg of protein/lane were electrophoresed in 10% SDS-polyacrylamide gel and
electrophoretically transferred to a PVDF membrane as described under
``Experimental Procedures.'' The
6-PF-2-K/Fru-2,6-P
ase protein was detected with a
polyclonal antibody obtained from a rat liver
6-PF-2-K/Fru-2,6-P
ase protein extract. 0.05 µg of rat
liver 6-PF-2-K/Fru-2,6-P
ase protein was used as positive
control (lane 7). This protein was extracted and purified from
bacteria engineered for expression rat liver
6-PF-2-K/Fru-2,6-P
ase protein. Protein was quantified by
scanning densitometry. MWSt, molecular weight
standard.
Since maximal velocity values for the kinase and bisphosphatase
reflect the amount of enzyme(16) , 6-PF-2-K and
Fru-2,6-Pase activities were assayed in order to determine
whether activity correlated with the amount of protein. As expected,
when the wild-type 6-PF-2-K/Fru-2,6-P
ase was overexpressed,
both activities increased, 22-fold for the kinase and 29-fold for the
bisphosphatase (Table 1). When the double mutant was
overexpressed, kinase activity increased 28-fold, whereas
bisphosphatase activity increased 6-fold (Table 1), even though
the bisphosphatase domain was presumably inactivated. The maximal
velocities of both activities were measured under the same conditions
of pH, temperature, and P
concentration. An increase in the
kinase:bisphosphatase maximal activity ratio of 4-fold was obtained
when the mutated form (S32A/H258A) was overexpressed (Table 1).
In contrast, a significant decrease (1.7 ± 0.2 versus 3.0 ± 0.7; p < 0.05) in this ratio was obtained
when the wild-type enzyme was overexpressed.
Figure 5:
Glucose production in FAO cells.
55-cm plates of FAO cells were incubated for 1, 2, or 3 h
with 3 ml of Krebs-bicarbonate, pH 7.4, containing 2% bovine serum
albumin and 20 mM lactate plus pyruvate (10:1) (
) or
20 mM dihydroxyacetone (
). Glucose were measured in the
incubation medium of each plate as described under ``Experimental
Procedures.'' Results are the mean ± S.E. (n = 4) for dihydroxyacetone and n = 1 for
lactate plus pyruvate.
Overexpression of the mutant bifunctional enzyme had a dramatic inhibitory effect on the rate of glucose production from dihydroxyacetone, with almost zero glucose production during the third hour (0.1 nmol/min/plate for Ad-PF2KMut versus 1 nmol/min/plate for AdWT) (Fig. 6A). The effect was time-dependent; there was 10% inhibition after the first hour, 24% after the second hour, and 92% after the third hour (Fig. 6A). There was at the same time a stimulation of lactate plus pyruvate production that was also time-dependent; there was a 6% increase after the first hour, 19% after the second hour, and 220% after the third hour (Fig. 6B). As shown in Table 2, only 40% of the additional lactate plus pyruvate production could be accounted for by the inhibition of glucose production (192 versus 493 nmol).
Figure 6:
Effect of overexpression of wild-type or
mutated 6-PF-2-K/Fru-2,6-P on glucose and lactate plus
pyruvate production in FAO cells. 55-cm
plates of FAO cells
treated with different adenovirus constructs; AdWT (
),
Ad-PF2KWT (
), and Ad-PF2KMut (
) were incubated 1, 2, or 3
h with 3 ml of Krebs-bicarbonate, pH 7.4, containing 2% bovine serum
albumin and 20 mM dihydroxyacetone. Glucose (A) and
lactate plus pyruvate (B) productions were measured in the
incubation medium of each plate as described under ``Experimental
Procedures.'' 2Glc + Lac + Pyr is the sum of glucose and
lactate plus pyruvate production in 3-carbon equivalent (C). Results are the mean ± S.E., n =
4.
In contrast, overexpression of the wild-type form stimulated the rate of glucose production from dihydroxyacetone; there was a 25% increase after the first hour and a 50% increase after both the second and third hours (Fig. 6A). Lactate plus pyruvate production was simultaneously decreased (Fig. 6B). The stimulation of glucose production was almost completely accounted for by the inhibition of lactate plus pyruvate production (Table 2). After 3 h, 228 nmol more of 3-carbon precursors were used to synthesize glucose, whereas 295 nmol less of 3-carbon precursors were used to synthesize lactate plus pyruvate in Ad-PF2KWT-infected cells than in cells infected with the vector. The sum of 3-carbon equivalents of glucose and lactate plus pyruvate production was not significantly different during the incubation time for Ad-PF2KWT- versus AdWT-treated cells (Fig. 6C, 2106 ± 171 nmol/plate versus 2173 ± 118 nmol/plate, n = 4; n.s.).
The rate of glucose production was higher
during the first hour than in the last 2 h in both treated and
untreated cells, but the rate of dihydroxyacetone metabolism to glucose
or lactate plus pyruvate was constant for the first 2 h (Fig. 6C). The lack of linearity of glucose production
reflects a change in the balance between glucose and lactate plus
pyruvate production from dihydroxyacetone after the first hour. This
balance was also affected by the overexpression of both forms of the
enzyme. During the last 2 h, in AdWT-treated or untreated FAO cells,
about 30% of the 3-carbon precursors were used for glucose synthesis
and about 70% for lactate plus pyruvate formation. With overexpression
of the double mutant and the associated increase in
Fru-2,6-P level, only about 20% of dihydroxyacetone was
used for glucose formation and about 80% for lactate plus pyruvate
formation, whereas with overexpression of the wild-type enzyme and the
decrease in Fru-2,6-P
level, about 40% of dihydroxyacetone
was converted to glucose and about 60% to lactate plus pyruvate.
A relationship between Fru-2,6-P levels and
hepatic gluconeogenesis and glycolysis has been established by studies
on nutritional status and metabolic disease states, such as
diabetes(1, 37) . The role of Fru-2,6-P
in
regulating metabolic fluxes has also been studied by correlating
pathway flux with acute changes in Fru-2,6-P
level brought
about by hormones like glucagon, insulin, and vasopressin (37) . However, changes in nutritional status and/or hormones
involves modulation of other regulatory enzymes as
well(38, 39, 40) , and this has made it
difficult to determine the importance of any individual step. The
ability to overexpress wild-type 6-PF-2-K/Fru-2,6-P
ase or a
double mutant that has no bisphosphatase activity allowed us to analyze
the relative role of the kinase and bisphosphatase in regulating
Fru-2,6-P
levels and metabolic flux through the
gluconeogenic/glycolytic pathway. Overexpression of wild-type
6-PF-2-K/Fru-2,6-P
ase decreased Fru-2,6-P
levels and increased glucose production from dihydroxyacetone
while inhibiting lactate plus pyruvate production. In
contrast, overexpression of the double mutant increased Fru-2,6-P
levels and inhibited glucose production from dihydroxyacetone,
while stimulating lactate plus pyruvate production. These results
support the importance of Fru-2,6-P
as a regulator of the
gluconeogenic/glycolytic pathway in FAO cells.
There have been only
a limited number of reports of overexpression of enzymes of mammalian
glucose metabolism with concomitant analysis of the consequences on
pathway
flux(19, 41, 42, 43, 44) . ()In several instances there was not a perfect quantitative
correlation between overexpression of an enzyme and the predicted
metabolic consequences of that overexpression. For example, a
10-100-fold overexpression of hexokinase I in a pancreatic
-cell line (MIN 6) (41) or in isolated islets of
Langerhans (42) enhanced glucose utilization or insulin
secretion by only 2-fold. Overexpression of glycogen phosphorylase by
46-fold in primary hepatocytes did not change glycogen content in the
basal state, although preferential activation of glycogenolysis was
evident upon treatment with pharmacologic agents(19) .
Overexpression of glucokinase in islets had no effect on glucose
utilization or insulin secretion.
These results suggest
that other steps in a pathway may become rate-limiting when one
enzymatic step is enhanced by overproduction of the protein and/or that
other as yet unrecognized regulatory mechanisms may be revealed by
overexpression. On the other hand, overexpression of
phosphoenolpyruvate carboxykinase in H4IIE-C3 cells (43) or of
glucokinase in the same cell line or in FTO-2B cells (44) was
quantitatively correlated with enhanced pathway fluxes,
gluconeogenesis, and glycolysis, respectively.
The changes in
Fru-2,6-P levels were better correlated with the change in
the kinase:bisphosphatase maximal activity ratio than with the kinase
maximal activity, which was enhanced 22-28-fold in the case of
overexpression of the wild-type and double mutant enzymes. However,
there were a number of unexpected findings: 1) mRNA abundances of the
wild-type enzyme and its double mutant were increased to a greater
extent than the protein level. This discrepancy may be due to a higher
rate of expression of the mRNA relative to the FAO cells' ability
to translate and/or process the mRNA. The apparent discrepancy between
massive overexpression of the enzyme and relatively modest effects on
Fru-2,6-P
concentration may reflect the heterogeneity of
the FAO cells since the efficiency of gene transfer was 70%. 2)
Overexpression of the wild-type enzyme resulted in a decrease in
Fru-2,6-P
level. 3) Overexpression of the double mutant
resulted in a nearly 6-fold increase in bisphosphatase, even though
this enzyme form is devoid of significant bisphosphatase
activity(10) .
The paradoxical drop in Fru-2,6-P argues for covalent modification of the overexpressed wild-type
enzyme in FAO cells. Cyclic AMP-dependent protein kinase-catalyzed
phosphorylation increases the kinase S
for Fru-6-P and
has no effect on the kinase maximal velocity but enhances the
bisphosphatase maximal velocity 2-3-fold(1) .
Phosphorylation of the overexpressed wild-type enzyme is supported by
both the higher S
for Fru-6-P of the 6-PF-2-K, compared
with the double mutant, and the decrease in the kinase:bisphosphatase
maximal activity ratio compared with the ratio for the enzyme in
untreated or vector-treated cells. (
)The question remains as
to why the overexpressed enzyme is phosphorylated to a greater extent
than the endogenous enzyme. In untreated FAO cells, the enzyme would be
predicted to have a low phosphate content, since the concentration of
6-PF-2-K/Fru-2,6-P
ase was estimated to be less than 1
µM by Western blot analysis (data not shown), which is far
below its K
(10 µM) for
phosphorylation by cAMP-dependent protein kinase(45) . An
increase in bifunctional enzyme concentration to about 40 µM after overexpression would be predicted to enhance its extent of
phosphorylation in FAO cells. However, the possibility that an
unrecognized covalent modification of the enzyme inhibits maximal
kinase activity cannot be ruled out. For example, it has recently been
demonstrated that in vitro ADP-ribosylation of the liver
enzyme inactivates the kinase, but has no effect on the
bisphosphatase(46) .
Overexpression of the double mutant
increased the kinase:bisphosphatase ratio and increased Fru-2,6-P levels, as expected. However, the 6-fold increase in
bisphosphatase activity was surprising, since this mutant lacks the
histidine residue needed to form the phosphoenzyme intermediate that
mediates Fru-2,6-P
hydrolysis(9, 10) .
Despite the inability to form phosphoenzyme intermediate, the H258A
mutant retains a very low residual activity, (
)and
overexpression of the protein by 40-fold may account in part for the
increased bisphosphatase activity. Increased phosphorylation of the
overexpressed enzyme is unlikely to have contributed to the increased
bisphosphatase activity, since the phosphorylation site (Ser-32) also
was mutated to Ala. It has been reported that Ser-33 can undergo
phosphorylation in vitro but at a very low rate and with
negligible effect on the bisphosphatase(8) . It also cannot be
ruled out that the endogenous enzyme underwent phosphorylation as a
result of the large increase in total bifunctional enzyme protein,
resulting in enhanced bisphosphatase activity. However, this
phosphorylation would not fully explain the 6-fold increase in total
bisphosphatase activity. This change probably involves both residual
activity of the overexpressed protein and phosphorylation of the
endogenous enzyme.
The results reported here also support previous
work with H4IIE cells, which demonstrated that regulation of the
activities of the bifunctional enzyme and Fru-2,6-P levels
by covalent modification is more important than changes in amount of
this two-domain protein(33) . For example, dexamethasone, which
did not affect phosphorylation state, increased bifunctional enzyme
mRNA (11-fold) and protein (3-fold) but had only a small effect on
Fru-2,6-P
content. In contrast, insulin, which increased
the kinase:bisphosphatase activity ratio by causing dephosphorylation
of the enzyme, increased Fru-2,6-P
content by 15-fold.
Although the phosphorylation state of the enzyme is probably the most
important determinant of net synthesis or degradation of
Fru-2,6-P
, it is likely that the amount of the enzyme is
important in some situations, i.e. lipogenic conditions, where
elevated Fru-2,6-P
levels have been correlated with
increased enzyme amount(16) . In addition, insulin and
dexamethasone had highly synergistic effects on Fru-2,6-P
in H4IIE cells by altering both the phosphorylation state and the
amount of protein(33) .
As shown previously, FAO cells are
capable of synthesizing glucose from various 3-carbon
precursors(13) . Rates of glucose synthesis from
lactate/pyruvate (10:1) or dihydroxyacetone are between 5 and 15%
(0.3-0.7 µmol/min/dry mass) that of isolated
hepatocytes(30, 31) and perfused liver(47) ,
depending on the substrate and growth medium. The lower rate of glucose
production may also be due to differences in incubation conditions
and/or to different rate-limiting steps in hepatocytes versus hepatoma cells, since the latter contain lower
fructose-1,6-bisphosphatase activity and higher phosphoenolpyruvate
carboxykinase activity(11, 12) . However, the rate of
glucose synthesis in FAO cells was high enough to study the role of
Fru-2,6-P in regulation of glycolytic and gluconeogenic
flux, particularly with the use of dihydroxyacetone.
Tumor cells are usually thought to have high rates of glycolysis(34, 35) . However, like other rat hepatoma cell lines such as FT0-2B and H4IIE(44) , and in contrast to isolated hepatocytes, FAO cells exhibit very low rates of glycolysis from glucose but a high lactate plus pyruvate production from dihydroxyacetone. The lack of expression of the glucokinase gene in FAO cells (data not shown) and in other rat hepatoma cell lines such as FT0-2B and H4IIE (44) compared with isolated hepatocytes (7) probably accounts for the low glycolytic rate from glucose and the substrate effect (dihydroxyacetone versus glucose) on this rate.
The results of this study point to the utility of FAO
cells as a model system for glycolytic/gluconeogenic pathway
engineering. Adenoviral and/or retroviral constructs containing the
coding region for glucokinase can be used to enhance glucose
utilization in these cells and test the role of this and other enzymes
in controlling glycolytic flux, substrate cycling, and Fru-2,6-P levels. Such work is in progress.