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INTRODUCTION |
The glucose-6-phosphatase enzyme complex catalyzes the final step
of gluconeogenesis. The complex is composed of a catalytic subunit of
36 kDa (P36)1
sequestered within the endoplasmic reticulum (ER), a 46-kDa
glucose-6-phosphate translocase known as P46 or T1 that delivers
glucose 6-phosphate to the catalytic subunit, and putative ER glucose
and inorganic phosphate (Pi) transporters (T2 and T3) that
move the reaction products back into the cytosol (1-3). However, only
P36 and P46 have been clearly identified and cloned (4-7). It remains
uncertain whether Pi transport is embodied in the function
of P46 or encoded by a separate protein (8). Furthermore, an earlier
report describing an ER-localized glucose transporter, GLUT-7 (9), has
recently been retracted (10).
Mutations in P36 and P46 have both been linked to glycogen storage
diseases in human subjects (5, 11). Patients with type Ia glycogen
storage disease have mutations in the P36 gene (5) and a complete
deficiency of glucose-6-phosphatase enzymatic activity, regardless of
whether the assay is performed in intact or detergent-disrupted
microsomal preparations (12). Patients with type Ib glycogen storage
disease have mutations in the P46 gene (11) and have absent or reduced
glucose-6-phosphatase enzymatic activity in intact microsomes but
normal or increased activity in detergent-disrupted preparations
(13).
Whereas the human genetic studies clearly demonstrate that both the P36
and P46 gene products are required for normal glucose 6-phosphate
(Glc-6-P) hydrolysis, the relative contributions made by these proteins
to the hydrolytic rate, and to overall regulation of carbohydrate
metabolism, are incompletely understood. One way of gaining insight
into this issue is to overexpress selectively single components of the
glucose-6-phosphatase complex. Thus, our laboratory has previously used
recombinant adenovirus to overexpress P36 in INS-1 insulinoma cells
(14), primary hepatocytes (15), or liver of normal rats (16).
Expression of P36 caused clear increases in glucose 6-phosphate
hydrolysis in both cultured cell models (14, 15), and in intact rats,
resulted in glucose intolerance, mild hyperinsulinism, and a 50%
decrease in hepatic glycogen stores (16). However, the extent of
overexpression of P36 in the INS-1 study (10-fold) was larger than the
increment in glucose production measured by a
3H2O incorporation assay (4-fold), raising the
possibility that other components of the complex such as P46 might
contribute to the overall rate at which Glc-6-P is hydrolyzed.
The purpose of the current study was to investigate the potential
regulatory role of P46 in the glucose-6-phosphatase complex by
adenovirus-mediated overexpression of the protein in rat hepatocytes. We find that overexpression of P46 increases Glc-6-P hydrolysis in
intact microsomes, although not to the same extent as overexpression of
the catalytic subunit. Overexpression of P46 also causes pronounced inhibition of glycogen synthesis and activation of glycogenolysis, but
has only small effects on glycolysis, whereas overexpression of P36 has
potent effects on both pathways. The preferential effect of P46
overexpression on glycogen metabolism may be related to its capacity to
enhance the hydrolysis of a hexose phosphate intermediate of glycogen
metabolism, glucose 1-phosphate.
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MATERIALS AND METHODS |
Preparation of Recombinant Adenoviruses--
A 2.04-kilobase
pair fragment of the human P46 cDNA (7), including the entire
protein coding region, was cloned into the pACMV.pLpA vector (17) and
used to prepare a recombinant adenovirus (AdCMV-P46), using previously
described methods (18). The preparation and testing of adenoviruses
containing the Escherichia coli
-galactosidase gene
(AdCMV-
Gal) (19), the cDNA encoders, the catalytic subunit of
glucose-6-phosphatase, P36 (AdCMV-P36, previously known as AdCMV-Glc-6-Pase) (14), or the cDNA encoding rat liver glucokinase (AdCMV-GKL) (20) has been described previously.
Hepatocyte Isolation, Culture, and Viral
Treatment--
Hepatocytes were isolated from ad libitum
fed or overnight (18 h) fasted male Wistar rats (180-225 g) using
collagenase perfusion (21). All reagents were purchased from Sigma
unless stated otherwise. Cells were suspended in Dulbecco's modified
Eagle's medium supplemented with 25 mM glucose, 10% fetal
bovine serum, 100 nM insulin (Life Technologies, Inc.), 100 nM dexamethasone, and 2% penicillin/streptomycin (attachment medium) and plated at a density of 7 × 104 cells/cm2 in either 12-well or 6-cm tissue
culture plates pre-coated with 0.1% collagen solution at 37 °C for
2 h. For studies of transgene expression and P46 subcellular
localization or studies of glycogen synthesis, lactate production, or
glucose usage, hepatocytes from overnight fasted rats were treated with
various adenoviruses immediately after attachment or were left
untreated and then incubated with culture medium (consisting of
Dulbecco's modified Eagle's medium supplemented with 0.2% bovine
fraction V albumin, 1 nM insulin, 10 nM
dexamethasone, 2% penicillin/streptomycin) containing either 5 or 25 mM glucose for 8 or 24 h. For studies on glycogen
degradation, hepatocytes from ad libitum fed rats were
incubated with culture medium containing 25 mM glucose for
24 h and were then incubated with adenoviruses added singly or in
combinations or were left untreated for 2 h at 37 °C.
Virus-containing medium was then removed, and cells were cultured for
an additional 24 h in culture medium containing 15 mM
glucose. For all experiments, viral particle numbers were estimated by
measurement of A260 of viral stocks, and
for each virus, 500 particles/cell were added.
RNA Blot Hybridization Analysis--
Total RNA was isolated from
hepatocytes by extraction with the TRIZOL reagent (Life Technologies,
Inc.). 10 µg of total RNA was analyzed using a procedure described
previously (22). A 1459-base pair polymerase chain reaction product
derived from the P46 cDNA (7) was randomly labeled using the Random
Primers DNA labeling system (Life Technologies, Inc.) and was used to detect P46 mRNA by blot hybridization, whereas a 989-base pair polymerase chain reaction product derived from the P36 cDNA was similarly labeled and used to detect expression of P36 mRNA.
Immunoblot Analysis--
Hepatocytes were homogenized using a
Brinkman homogenizer in homogenization (H) buffer containing 10 mM Tris/HCl (pH 7.0), 150 mM KF, 15 mM EDTA, 600 mM sucrose, 15 mM
2-mercaptoethanol, 10 µg/ml leupeptin, 1 mM benzamidine,
and 1 mM phenylmethylsulfonyl fluoride. Immunoblot analysis
was performed on the total cell homogenate or on various subcellular
fractions prepared as described previously (15). Briefly, total cell
homogenate was centrifuged at 10,000 × g for 15 min at
4 °C, and the pellet was resuspended in H buffer. The supernatant
from this spin was recovered and recentrifuged at 105,000 × g for 1 h at 4 °C. The pellet was resuspended in H
buffer as the microsomal fraction, and the supernatant was recovered as
the cytosolic fraction. 25-75 µg of protein from each fraction was
then subjected to polyacrylamide electrophoresis. Blots were incubated
with an anti-P36 antibody (23; a gift of Dr. Rebecca Taub, University
of Pennsylvania), or an anti-peptide antibody specific for human P46
(24). After incubation with the primary antibodies, blots were treated
with anti-rabbit Ig horseradish peroxidase-linked secondary antibody
from sheep (Amersham Pharmacia Biotech).
Measurement of Hexose Phosphate Hydrolysis in Hepatocyte
Microsomes--
Hepatic microsomes were collected as described above
for immunoblot analysis, except that a glass homogenizer was used for preparation of the initial hepatocyte homogenate. The resuspended microsomal fraction was incubated in the presence or absence of 0.5%
cholic acid at 4 °C for 20 min. Reaction buffer containing 60 mM sodium cacodylate and 10 mM glucose
6-phosphate, 10 mM mannose 6-phosphate, 10 mM
glucose 1-phosphate, or 10 mM fructose 6-phosphate was
added to each microsomal sample. Hydrolysis reactions were carried out
at 37 °C for 9 min, based on time course studies showing linearity
of the reaction at this time and substrate concentration (data not
shown). Reactions were terminated by addition of trichloroacetic acid
(final concentration, 4%). After centrifugation at 3000 rpm for 5 min,
the inorganic phosphate concentration was assayed in the supernatant
using a kit from Sigma. Results were normalized to the total protein
content of the microsomal sample.
Glycogen and Lactate Assays--
At the conclusion of each
experiment, culture medium was collected for measurement of lactate
production, using a kit and protocol from Sigma. Hepatocytes were
washed with Dulbecco's-phosphate-buffered saline once and
scraped into 30% KOH solution, and the extracts were incubated in
boiling water for 15 min. Glycogen content was measured as described
(25).
Glucose Usage Assay--
Glucose usage was measured as the
conversion of [3-3H]glucose to
3H2O as described previously (15, 26). Briefly,
fasted hepatocytes were treated with adenoviruses immediately after
attachment and then incubated in culture medium containing 3 mM glucose overnight. Cells were washed once with
phosphate-buffered saline and then incubated with a Hanks' balanced
salt solution containing 5 or 25 mM glucose containing
tracer [3-3H]glucose (Amersham Pharmacia Biotech, 10 Ci/nmol) at 37 °C for 4 h. At the conclusion of this
incubation, 3H2O was collected, and the
radioactivity was analyzed using a Beckman Counter. To monitor the
effect of this treatment on glycogen content, another set of
experiments was conducted under identical conditions, except that the
cells were collected for measurement of glycogen content.
Statistical Analyses--
At least three independent experiments
were performed for each assay. Data were analyzed using the two-tailed
Student's t test.
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RESULTS |
Expression of P46 with or without Co-overexpression of P36 in
Isolated Hepatocytes--
A recombinant adenovirus containing the
cDNA encoding human P46 (AdCMV-P46) was constructed and used in
conjunction with a previously prepared virus containing the cDNA
encoding P36 from rat (AdCMV-P36, previously known as AdCMV-Glc-6-Pase;
Ref. 14). As shown in Fig. 1, treatment
of isolated hepatocytes with either or both of these viruses resulted
in large increases in mRNA levels for the respective transgenes.
Adenovirus-mediated transgene expression was clearly evident at 8 h after viral treatment and increased further between 8 and 24 h.
Furthermore, co-treatment of hepatocytes with the AdCMV-P46 and
AdCMV-P36 viruses resulted in robust coexpression of the two
transgenes. To control for untoward effects of addition of two viruses,
some cell batches were co-incubated with AdCMV-P46 and AdCMV-
GAL,
and such cells exhibited identical levels of P46 expression as in cells
treated with AdCMV-P46 alone.

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Fig. 1.
Overexpression of T1 translocase and
Glc-6-Pase in isolated hepatocytes. Hepatocytes isolated from
overnight fasted rats were treated with an adenovirus containing the
-galactosidase gene, AdCMV- Gal (BG), a virus
containing the cDNA encoding P46, AdCMV-P46 (P46), or an
adenovirus containing the cDNA encoding the catalytic subunit of
glucose-6-phosphatase, AdCMV-P36 (P36), or with combinations
of these viruses, such as AdCMV-P46/AdCMV- Gal (P46/BG)
and AdCMV-P46/AdCMV-P36 (P46/P36). Other groups of control
cells were not treated with virus (null). After the cells
were incubated in culture medium containing 5 or 25 mM
glucose for 8 or 24 h, total RNA was isolated for Northern blot
analysis. A radiolabeled probe specific for the P46 transcript was used
in A and C, and a probe specific for the P36
catalytic subunit mRNA was used in B and
D.
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The glucose-6-phosphatase enzyme complex resides in the ER (1-3). To
determine whether the increase in P46 mRNA shown in Fig. 1
corresponds to equivalent increases in P46 protein targeting to the ER,
we prepared microsomal fractions of AdCMV-P46-treated and control cells
and measured P46 protein by immunoblot analysis (Fig.
2A). Treatment of hepatocytes
with AdCMV-P46 resulted in 6.1-9.9-fold increases in P46 protein
levels in the microsomal preparations. We also performed subcellular
fractionation of cells treated with AdCMV-P46 or AdCMV-
GAL. As shown
in Fig. 2B, AdCMV-P46 treatment caused a clear increase in
P46 protein expression in total cell extracts (T) and in the
microsomal fraction (M), with little or no increase in the
pellet of a 10,000 × g centrifugation (P)
or in the cytosolic fraction (supernatant of 100,000 × g spin; C). These findings suggest that most of
the transgene-encoded P46 protein was correctly targeted to the ER.

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Fig. 2.
Immunoblot analysis of P46 and P36 expression
in hepatocytes. Hepatocytes isolated from overnight fasted rats
were treated with different adenoviruses as described and abbreviated
in the legend to Fig. 1, and cultured in medium containing 25 mM glucose for 24 h. A, microsomal
fractions were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotted with anti-P36 or anti-P46 antibodies, as described under
"Materials and Methods." The blot shown is representative of three
independent experiments. B, immunoblot analysis of P46
protein was performed on total cell extract (T), the pellet
from an initial 10,000 × g centrifugation
(P), cytosol-enriched fraction (C), and
microsomal fraction (M), prepared as described under
"Materials and Methods."
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A tight functional linkage between P46 and P36 has previously been
demonstrated, such that knock-out of P36 in mice results in ablation of
microsomal glucose 6-phosphate transport (27). These findings suggest
that P46 and P36 could be physically associated in the ER, raising the
possibility that overexpression of P46 might affect the stability, and
thereby the expression level, of P36. To test this, we performed
immunoblot analysis on microsomal preparations from cells treated with
AdCMV-P46, AdCMV-P36, or both viruses. As shown in Fig. 2, cells
treated with AdCMV-P46 or the combination of AdCMV-P46 + AdCMV-
GAL
had the same levels of P36 protein as untreated cells or cells treated
with AdCMV-
GAL alone. Furthermore, the increase in P36 protein was
equivalent in cells treated with AdCMV-P36 alone, AdCMV-P36 + AdCMV-
GAL, or AdCMV-P36 + AdCMV-P46. Thus, overexpression of P46 had
no effect on endogenous or overexpressed P36 protein levels.
Effect of P46 Overexpression on Glucose 6-Phosphate and Mannose
6-Phosphate Hydrolysis--
Since overexpressed P46 is clearly
targeted to hepatocyte microsomes (see Fig. 2), we next evaluated the
effect of overexpression of this protein on glucose 6-phosphate
(Glc-6-P) and mannose 6-phosphate (Man-6-P) hydrolysis via measurement
of Pi production in intact and detergent-disrupted
microsomes. Comparison of hexose phosphate hydrolysis in intact
versus detergent-treated microsomes allows us to
differentiate between the activity of the intact system, which is a
function of the combined actions of P36 and P46, compared with
detergent-treated samples, which measure total phosphohydrolase (P36)
activity. As shown in Fig. 3A,
microsomes isolated from control hepatocytes (untreated or
AdCMV-
GAL-treated) exhibited appropriate latency for Man-6-P
hydrolysis, in that Pi production in intact microsomes was
only 33-38% that in detergent-disrupted microsomes. Treatment of
cells with either AdCMV-P46 or AdCMV-P46 + AdCMV-
GAL caused a 60%
increase in Man-6-P hydrolysis compared with untreated control cells or
AdCMV-
GAL-treated controls in intact microsomes
(nondetergent-treated) but had no effect on total Man-6-P
phosphohydrolase activity in disrupted microsomes (detergent-treated).
In contrast, treatment of hepatocytes with AdCMV-P36 resulted in a
3.6-fold increase in Man-6-P hydrolysis in intact microsomes relative
to either control group, but also a 4.1-4.8-fold increase in total
Man-6-P phosphohydrolase activity in disrupted microsomes, such that
the percentage of Man-6-P hydrolyzed by inact microsomes was still only
30% that hydrolyzed by disrupted microsomes. Finally,
co-overexpression of P46 and P36 did not activate Man-6-P hydrolysis
further in either intact or disrupted microsomes relative to cells with
overexpression of P36 alone.

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Fig. 3.
Mannose 6-phosphate and glucose 6-phosphate
hydrolysis in intact and detergent-disrupted hepatic microsomes.
Hepatocytes isolated from overnight fasted rats were left untreated
(null) or were treated with AdCMV- Gal (BG),
AdCMV-P46 (P46), or AdCMV-P36 (P36) alone, or
with combinations of viruses such as AdCMV-P46/AdCMV- Gal
(P46/BG) and AdCMV-P46/AdCMV-P36 (P46/P36).
Microsomes were prepared from overnight fasted rats and incubated in
the absence (intact) or presence (detergent-disrupted) of 0.5% cholic
acid. These preparations were then incubated in the presence of 10 mM mannose 6-phosphate (A) or 10 mM
glucose 6-phosphate (B) for 9 min at 37 °C.
Pi production was measured as an index of hydrolysis of
these hexose phosphates, normalized to total microsomal protein in each
sample. Data represent the mean ± S.E. for six independent
experiments. The symbols * and # indicate those samples for which
mannose 6-phosphate or glucose 6-phosphate hydrolysis was increased
relative to the corresponding AdCMV- GAL-treated control groups, with
p < 0.05.
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A different pattern was noted when Glc-6-P was used as substrate
instead of Man-6-P (Fig. 3B). In the untreated and
AdCMV-
GAL-treated control groups, Glc-6-P hydrolysis in intact
microsomes approached that of disrupted microsomes (75%), due to the
specificity of P46 for Glc-6-P. In cells treated with either AdCMV-P46
or AdCMV-P46 + AdCMV-
GAL, Glc-6-P hydrolysis in intact microsomes
was increased by 33-58% relative to either control group, with no
change in total Glc-6-P phosphohydrolase activity as measured in
disrupted microsomes. Interestingly, these changes mean that Glc-6-P
hydrolysis in intact microsomes from AdCMV-P46-treated cells tended to
be higher than in disrupted microsomes. Treatment of cells with
AdCMV-P36 increased Glc-6-P hydrolysis by 3.6- and 4.3-fold in intact
and disrupted microsomes, respectively, relative to preparations from untreated or AdCMV-
GAL-treated control cells. Finally, co-treatment of hepatocytes with AdCMV-P46 + AdCMV-P36 caused an additional 23%
increase in Glc-6-P hydrolysis in intact microsomes relative to cells
treated with AdCMV-P36 alone, with no effect on total phosphohydrolase
activity. Taken together, these experiments demonstrate that P46
overexpression in hepatocytes causes a clear increase in Man-6-P and
and Glc-6-P hydrolysis in intact but not disrupted microsomal preparations.
Effect of P46 Overexpression on Glycogen Synthesis--
To begin
to assess the metabolic impact of overexpression of P46, hepatocytes
isolated from fasted rats were treated with the various combinations of
adenoviruses described in Fig. 1 and then incubated in the presence of
5 or 25 mM glucose for 8 or 24 h prior to measurement
of glycogen content. Fasted rats with depleted hepatic glycogen stores
were used in these experiments to allow measurement of glycogen
synthesis during the period of cell culture. As shown in Fig.
4, overexpression of P36, P46, or both
proteins had no significant effect on glycogen storage at 5 mM glucose at either 8 or 24 h. However, in cells
incubated at 25 mM glucose for 8 h, each of the 4 experimental groups (AdCMV-P46 alone, AdCMV-P36 alone, AdCMV-P46 + AdCMV-
GAL, and AdCMV-P46 + AdCMV-P36) had similar decreases
(31-37%) in glycogen stores relative to the two control groups
(untreated hepatocytes or hepatocytes treated with AdCMV-
GAL alone),
although these decreases only achieved statistical significance in the
AdCMV-P46 and the AdCMV-P46 + AdCMV-
GAL-treated groups. Results were
more dramatic after 24 h of culture at 25 mM glucose.
Thus, AdCMV-P46 or AdCMV-P46 + AdCMV-
GAL-treated cells had
statistically significant 48 and 49% reductions in glycogen content
relative to the untreated or AdCMV-
Gal-treated control groups,
respectively, whereas treatment with AdCMV-P36 caused slightly larger
(71 and 62%) decreases. Interestingly, combined treatment with
AdCMV-P36 + AdCMV-P46 appeared to have an additive effect, causing 84 and 80% decreases in glycogen content relative to the untreated and
AdCMV-
GAL controls, respectively.

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Fig. 4.
Effect of P46 overexpression on glycogen
synthesis. Hepatocytes isolated from overnight fasted rats were
left untreated (null), or were treated with AdCMV- Gal
(BG), AdCMV-P46 (P46), or AdCMV-P36
(P36) alone, or with combinations of viruses such as
AdCMV-P46/AdCMV- Gal (P46/BG) and AdCMV-P46/AdCMV-P36
(P46/P36). After incubation in culture medium containing 5 or 25 mM glucose for 8 or 24 h, cells were collected
for measurement of glycogen content. Data represent the mean ± S.E. for five independent experiments. The various experimental groups
were compared with the AdCMV- Gal-treated control group and were
found to be different with the following levels of significance: *,
p < 0.05; #, p < 0.005; and @,
p < 0.0005.
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Effect of P46 Overexpression on Glycogen Degradation--
To
determine whether overexpression of components of the
glucose-6-phosphatase enzyme complex are capable of enhancing the rate
of glycogen degradation, glycogen-replete hepatocytes were isolated
from fed rats, treated with the various recombinant adenoviruses, and
cultured for 24 h in the presence of 25 mM glucose.
Cells were then cultured an additional 24 h in 15 mM
glucose, whereupon they were collected for measurement of glycogen
content. Importantly, treatment of hepatocytes with the AdCMV-
GAL
virus and culture for 24 h at 15 mM glucose did not
cause lowering of glycogen levels relative to untreated cells cultured
for 24 h at 25 mM glucose, showing that treatment with
a control virus does not activate glycogenolysis in response to
lowering of glucose from 25 to 15 mM (Fig.
5). In contrast, cells treated with
AdCMV-P46 or AdCMV-P46 + AdCMV-
GAL exhibited a 46% decrease in
glycogen content relative to either control group, whereas cells
treated with AdCMV-P36 or AdCMV-P36 + AdCMV-
GAL had decreases of
73%. Finally, cells treated with the combination of AdCMV-P36 and
AdCMV-P46 had the largest (80%) decline in glycogen content. Thus,
both P46 and P36 overexpression stimulate glycogen degradation in
glycogen-replete cells, and some additivity of the two effects can be
demonstrated.

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Fig. 5.
Effect of P46 overexpression on glycogen
degradation. Hepatocytes isolated from fed rats were incubated in
culture medium containing 25 mM glucose for 24 h and
then treated with AdCMV- Gal (BG), AdCMV-P46
(P46), or AdCMV-P36 (P36) alone or with
combinations of viruses such as AdCMV-P46/AdCMV- Gal
(P46/BG), AdCMV-P36/AdCMV- Gal (P36/BG), and
AdCMV-P46/AdCMV-P36 (P46/P36). After an additional 24 h
incubation in culture medium containing 15 mM glucose,
cells were collected for measurement of glycogen content. The cells
were also collected for glycogen analysis after the first 24-h
incubation (24 h). Data represent the mean ± S.E. for
three independent experiments. The various experimental groups were
compared with the AdCMV- Gal-treated control group and were found to
be different with the following levels of significance: *,
p < 0.05; **, p < 0.005; and ***,
p < 0.0001.
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Effect of P46 Overexpression on Lactate Production in
Hepatocytes--
We next tested whether overexpression of specific
elements of the glucose-6-phosphatase enzyme complex can inhibit
lactate production in hepatocytes from fasted rats. At 8 h after
viral treatment and in the presence of 25 mM glucose, the
experimental and control groups had produced the same amount of lactate
(Fig. 6). At 24 h, AdCMV-P46 or
AdCMV-P46 + AdCMV-
GAL-treated cells appeared to accumulate slightly
less lactate than controls, but statistical significance was not
achieved. In contrast, treatment with AdCMV-P36 alone caused a
statistically significant 33% decrease in lactate output relative to
controls, whereas the combination of AdCMV-P36 + AdCMV-P46 treatment
resulted in a 44% decrease. Thus, the effects of P46 overexpression on
glycogen synthesis are clearly more dramatic than its effects on
glycolytic flux, as assessed by measurement of lactate production.

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Fig. 6.
Effect of P46 overexpression on lactate
production. Hepatocytes isolated from overnight fasted rats were
treated as described in the legend for Fig. 4. The culture media were
collected and analyzed for lactate concentration. Data represent the
mean ± S.E. for five independent experiments. The various
experimental groups were compared with the AdCMV- Gal-treated control
group and were found to be different with the following levels of
significance: *, p < 0.05, and **, p < 0.0005.
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Metabolic Effects of P46 and P36 Overexpression in Hepatocytes with
Overexpressed Glucokinase--
Isolated hepatocytes are known to
rapidly lose differentiated function, including expression of
glucokinase (28). Decreased expression of glucokinase can in turn
impact the expression of other glycolytic and gluconeogenic enzymes
(29, 30). We therefore performed a final set of studies in which
glucokinase expression was maintained in hepatocytes isolated from
fasted rats at a high constant level via adenovirus-mediated expression
of the enzyme (AdCMV-GKL virus, Ref. 20). Cells prepared in this way
were then treated with the combinations of viruses encoding components of the glucose-6-phosphatase complex and were used for analysis of
glycogen content and glycolytic flux via measurement of
[3-3H]glucose usage. As shown in Fig.
7, treatment of hepatocytes with
AdCMV-GKL or AdCMV-GKL + AdCMV-
GAL resulted in an approximate doubling (217% increase) in[3-3H]glucose usage relative
to cells treated with AdCMV-
GAL alone, consistent with our previous
findings (15). Combined treatment of hepatocytes with AdCMV-GKL and
AdCMV-P46 did not result in a significant decrease in glucose usage
relative to cells treated with AdCMV-GKL + AdCMV-
GAL, whereas
co-treatment with AdCMV-GKL + AdCMV-P36 resulted in a 39% decrease.
Thus, overexpression of P36, but not P46, partially counteracts the
stimulatory effect of glucokinase overexpression on glucose usage in
fasted hepatocytes.

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Fig. 7.
Effect of P46 overexpression on glucose
usage. Hepatocytes isolated from overnight fasted rats were
treated with AdCMV- Gal (BG), AdCMV-P46 (P46),
AdCMV-P36 (P36), or an adenovirus containing the cDNA
encoding glucokinase, AdCMV-GKL (GKL) alone, or with
combinations of viruses such as AdCMV-P46/AdCMV- Gal (P46/BG),
AdCMV-P36/AdCMV- Gal (P36/BG), AdCMV-GKL/AdCMV- Gal
(GKL/BG), AdCMV-P46/AdCMV-P36 (P46/P36),
AdCMV-P46/AdCMV-GKL (P46/GKL), and AdCMV-P36/AdCMV-GKL
(P36/GKL). Cells were incubated in culture medium containing
3 mM glucose for 18 h, prior to assay of
[3-3H]glucose usage. Data represent the mean ± S.E.
for five independent experiments. The symbol * indicates statistical
significance at the level of p < 0.05 between the
indicated groups and the AdCMV- Gal-treated group. The symbol # indicates statistical significance at the level of p < 0.05 between the indicated group and the AdCMV-GKL-treated group.
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To investigate the effects of P46 and P36 expression on glycogen
metabolism in cells with constant glucokinase expression, cells
subjected to the same treatment as described for Fig. 7 were used for
measurement of glycogen content. As shown in Fig. 8, treatment of hepatocytes with
AdCMV-GKL alone or AdCMV-GKL + AdCMV-
GAL resulted in 15.6- and
11.3-fold increases in glycogen content relative to cells treated with
AdCMV-
GAL alone. Co-treatment of cells with AdCMV-GKL + AdCMV-P46
resulted in a small (13.4%, p = 0.09) decrease in
glycogen storage relative to the AdCMV-GKL + AdCMV-
GAL-treated
group. In contrast, co-treatment of cells with AdCMV-GKL + AdCMV-P36
completely prevented the glucokinase-induced increase in glycogen
storage. Thus, whereas P46 expression exerts a counteractive effect on
glucokinase-mediated stimulation of glycogen synthesis, this effect is
small compared with that achieved by overexpression of P36.

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Fig. 8.
Effect of P46 overexpression on glycogen
content in cells used for the glucose usage assay. Hepatocytes
isolated from overnight fasted rats were treated as described in the
legend for Fig. 7. Cells were collected for measurement of glycogen
content. Data represent the mean ± S.E. for five independent
experiments. The symbol * indicates statistical significance at the
level of p < 0.05 between the indicated groups and the
AdCMV- Gal treated group. The symbol # indicates statistical
significance at the level of p < 0.05 between the
indicated group and the AdCMV-GKL-treated group.
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P46 Overexpression Activates Glucose 1-Phosphate
Hydrolysis--
Our data showing that P46 overexpression increases the
rate of Man-6-P hydrolysis led us to investigate whether hydrolysis of
other hexose phosphates is also increased. Glucose 1-phosphate (Glc-1-P) is an intermediate involved in both glycogen synthesis and
glycogen degradation. Hydrolysis of Glc-1-P as a consequence of
overexpression of P46 could explain both the reduced glycogen accumulation in hepatocytes exposed to high glucose and the increased rate of glycogen degradation in hepatocytes exposed to a decline in
glucose concentration (the latter via alteration of the equilibrium of
the glycogen phosphorylase reaction).
To test this idea, we compared the hydrolysis of glucose 6-phosphate,
glucose 1-phosphate (Glc-1-P), fructose 6-phosphate (Fru-6-P), and
mannose 6-phosphate in intact and disrupted microsomes in hepatocytes
with or without overexpressed P46. Interestingly, distinct results were
obtained with each of these sugar phosphates (Fig. 9). Thus, Glc-6-P and Man-6-P were
handled much as described in Fig. 3. Glc-6-P hydrolysis was nearly
equal in intact and disrupted microsomes and was increased
significantly in intact microsomes by overexpression of P46. Man-6-P
hydrolysis was much lower in intact than disrupted microsomes, and
activity in intact microsomes was significantly increased by P46
overexpression. Surprisingly, both Glc-1-P and Fru-6-P were hydrolyzed
to equal extent by intact and disrupted microsomes prepared from
control hepatocytes, similar to the pattern with Glc-6-P. However,
Glc-1-P hydrolysis, but not Fru-6-P hydrolysis, was increased in intact
microsomes by P46 overexpression. Thus, these data are consistent with
a model in which P46 overexpression had preferential effects on
glycogen deposition because of its specific ability to stimulate
hydrolysis of a hexose phosphate intermediate of glycogen
metabolism.

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Fig. 9.
Hydrolysis of glucose 1-phosphate, but not
fructose 6-phosphate, is increased by P46 overexpression.
Hepatocytes isolated from 18-h fasted rats were treated with AdCMV-P46
(P46), AdCMV- GAL (BG), or were left untreated
(null). Cells were incubated in culture medium containing 3 mM glucose for 18 h, prior to harvesting of
microsomes. Microsomes were incubated in the absence
(intact) or presence (detergent-disrupted) of
0.5% cholic acid. These preparations were then incubated in the
presence of 10 mM glucose 6-phosphate (G6P), 10 mM mannose 6-phosphate (M6P), 10 mM glucose
1-phosphate (G1P), or 10 mM fructose 6-phosphate (F6P) for
9 min at 37 °C. Pi production was measured as an index
of hydrolysis of these hexose phosphates, normalized to total
microsomal protein in each sample. Data represent the mean ± S.E.
for five independent experiments. The symbol * indicates those samples
for which hexose phosphate hydrolysis was increased relative to the
corresponding AdCMV- GAL-treated control group, with
p < 0.05.
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DISCUSSION |
The cDNA encoding the T1 translocase/P46 component of the
glucose-6-phosphatase complex was cloned originally on the basis of its
homology to the bacterial gene products UhpT, GlpT, and UhpC, which are
hexose phosphate transporter, glycerol phosphate transporter, and
hexose phosphate receptor proteins, respectively (7, 8). The transcript
corresponding to the putative P46 clone was found to be expressed at
highest levels in liver and kidney, consistent with a role in the
gluconeogenic glucose-6-phosphatase complex. The strongest evidence
that the newly cloned gene was a component of the mammalian
glucose-6-phosphatase enzyme complex was the identification of
mutations in the gene in two human subjects with type 1b glycogen
storage disease (11). However, the precise biochemical function of P46
in mammalian cells has not been elucidated. Therefore, the purpose of
the current study was to provide insight into the metabolic and
biochemical properties of this protein through its adenovirus-mediated
overexpression in isolated rat hepatocytes.
Type 1b glycogen storage disease is characterized by an absence of
glucose-6-phosphatase enzymatic activity in intact liver microsomes but
normal or elevated activity in detergent-disrupted preparations (12).
The biochemical phenotype of type 1b glycogen storage disease patients
lends support to the substrate-transport model for the
glucose-6-phosphatase complex, in which the role of P46 is suggested to
be as an ER-localized transporter for glucose 6-phosphate, allowing the
luminally oriented Glc-6-Pase catalytic subunit to gain access to its
substrate (2). The substrate-transport model in its original form held
that the translocase function was rate-limiting for the system, based
on higher rates of glucose 6-phosphate hydrolysis in
detergent-disrupted compared with intact microsomes. However,
subsequent rapid kinetic studies revealed that in the first 10 s,
rates of glucose 6-phosphate hydrolysis are equal in intact and
disrupted microsomes, whereas at longer time points the rate decreases
in the intact preparations but maintains linearity in the disrupted
ones (31). This was interpreted to indicate a close interaction of the
translocase and phosphohydrolase components of the
glucose-6-phosphatase complex, with regulation of activity through a
conformational change in one or both interacting proteins (3, 31). The
tight association of the two proteins is supported by recent studies in
which knock-out of the Glc-6-Pase catalytic subunit causes loss of
glucose 6-phosphate transport into microsomes (27).
The current study provides the first demonstration that overexpression
of P46 is sufficient to enhance the activity of the glucose-6-phosphatase enzyme complex. Key data supporting this point
include the following. 1) The P46 transgene product is targeted to a
microsome-enriched fraction, suggesting normal delivery of the
overexpressed protein to the endoplasmic reticulum in intact cells. 2)
Overexpression of P46 enhances glucose 6-phosphate, mannose
6-phosphate, and glucose 1-phosphate hydrolysis in intact microsomes,
without an effect on total phosphohydrolase activity in disrupted
microsomes. However, the data also show that the impact of P46
overexpression on Glc-6-P hydrolysis in intact microsomes is much less
than the effect of overexpression of P36, suggesting that most of the
flux control is vested in the phosphohydrolase component of the
complex. These data do not clearly support or refute any of the
foregoing models of glucose-6-phosphatase complex function, since the
increased Glc-6-P hydrolysis observed with P46 overexpression can be
accommodated by the substrate transport model or a conformational
model. 3) Overexpression of P46 clearly inhibits glycogen accumulation
in hepatocytes from fasted rats and also causes activation of
glycogenolysis in hepatocytes from fed rats. Interestingly, P46
overexpression has minimal effects on lactate production or
[3-3H]glucose usage, in contrast to the more pronounced
effects of overexpressed P36. Overexpressed P46 is also much less
effective than overexpressed P36 at countering the enhancement of
glycolytic flux and glycogen synthesis induced by glucokinase overexpression.
Our work has uncovered a surprising heterogeneity in the handling of
various hexose phosphates by the intact glucose-6-phosphatase system.
Thus, we have shown that both Glc-1-P and Fru-6-P are hydrolyzed by
intact microsomes from control hepatocytes, although at approximately
one-third to one-half the rate of Glc-6-P when all of these hexose
phosphates are present at a concentration of 10 mM.
Moreover, overexpression of P46 increases Glc-1-P, but not Fru-6-P
hydrolysis, suggesting that the former sugar may be a real
physiological substrate for the glucose-6-phosphatase system. These
findings also suggest a mechanism by which P46 overexpression preferentially impairs glycogen metabolism relative to its minimal effects on glycolysis in hepatocytes from fasted rats, while also explaining why P46 overexpression increases glycogenolysis in glycogen-replete cells. Glc-1-P is a hexose phosphate intermediate that
is specific to glycogen metabolism. Enhanced hydrolysis of Glc-1-P
during periods of glycogen synthesis would reduce the amount of
substrate for the UDPG pyrophosphorylase reaction, whose product
UDP-glucose is the immediate precursor of glycogen synthesis. In
glycogen-replete hepatocytes with P46 overexpression subjected to a
sudden drop in glucose concentration, depletion of Glc-1-P would alter
the equilibrium of the glycogen phosphorylase reaction in favor of
glycogen degradation and Glc-1-P formation.
Although the ability of the glucose-6-phosphatase complex to hydrolyze
Glc-1-P seems to explain the specific effects of overexpressed P46 on
glycogen metabolism, we have also considered the possibility that P46
facilitates specific interactions between the glucose-6-phosphatase complex with proteins or enzymes that regulate glycogen metabolism. There is growing evidence that glucose disposal in general, and glycogen metabolism in particular, are spatially organized pathways within liver cells. For example, in the fasted state, cytosolic glucokinase enzyme activity is low due to sequestration of the enzyme
in the nucleus via its binding to an inhibitory glucokinase regulatory
protein (32-34). In the postprandial state, glucose stimulates the
translocation of glucokinase from the nucleus to the cytosol. The
enzymes of glycogen metabolism also exhibit spatial organization. Thus,
in liver cells, glycogen synthase is translocated from an intracellular
site to the cell membrane in response to glucose and insulin, resulting
in synthesis of glycogen in a gradient from the membrane surface toward
the interior of the cell (3, 35, 36). Controlled movement of glycogen
synthase within cells is complemented by targeting of protein
phosphatase-1 (PP1) to the glycogen particle. This targeting is
facilitated by glycogen targeting subunits of protein phosphatase-1,
which also appear to bind to the key enzymes of glycogen metabolism,
thereby serving as a molecular scaffold for glycogen synthesis
(reviewed in Ref. 37). The existence of this glycogen "metabolon"
(38), coupled with the remarkable organization of the
glucose-6-phosphatase complex, suggests the possibility that these two
metabolic machines could interact. Further work will be required to
determine whether such interaction actually occurs, and if so, the role
of the P46 T1 translocase in this process.
The findings described herein may also have relevance to understanding
of regulation of hepatic glucose metabolism and the control of blood
glucose homeostasis. Hepatic glycogen stores are decreased in all forms
of human diabetes (39-41), but the mechanisms responsible for this
defect are not well understood. One possibility is enhanced flux
through the glucose-6-phosphatase complex. Consistent with this idea,
it has recently been demonstrated that patients with type 2 diabetes
have a reduced capacity to regulate endogenous glucose production by
glucose per se, leading the authors to suggest that
regulation of glucose-6-phosphatase is impaired (42). To date,
investigation of the molecular basis of up-regulated
glucose-6-phosphatase activity in diabetes has focused on the
expression of the P36 catalytic subunit, leading to the finding that
P36 expression is increased in response to hyperglycemia and
hyperlipidemia in rodents and cultured cells (30, 43, 44). Recently, it
was found that P46 expression was also up-regulated in diabetes (45), mainly by hyperglycemia (46). Thus, the potential contribution of
changes in expression and activity of the P46 subunit should be
considered more carefully, particularly with regard to its effect on
hepatic glycogen storage in type 2 diabetes.