Metabolic regulation of
Na+/Pi-cotransporter-1 gene expression in
H4IIE cells
Zijian
Xie1,
Hui
Li1,
Liqin
Liu1,
Barbara B.
Kahn2,
Sonia M.
Najjar1, and
Waqar
Shah1
1 Department of Pharmacology, Medical College
of Ohio, Toledo, Ohio 43614-5804; and
2 Division of Endocrinology, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02215
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ABSTRACT |
We showed that the rat
Na+/Pi cotransporter-1 (RNaPi-1) gene was
regulated by insulin and glucose in rat hepatocytes. The aim of this
work was to elucidate signaling pathways of insulin-mediated metabolic
regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased
RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1
mRNA in the absence of glucose, clearly establishing an involvement of
metabolic signals for insulin-induced upregulation of the RNaPi-1 gene.
Pyruvate and insulin increased RNaPi-1 expression but downregulated
L-pyruvate kinase, indicating the existence of gene-specific metabolic
signals. Although fructose, glycerol, and lactate could support
insulin-induced upregulation of the RNaPi-1 gene, compounds entering
metabolism beyond pyruvate oxidation, such as acetate and citrate,
could not, suggesting that RNaPi-1-specific metabolic signals are
generated at or above pyruvate oxidation. Wortmannin, LY-294002, and
rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas
expression of dominant negative Asn17 Ras and
mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor
PD-98059 exhibited no effect. Thus we herein propose that metabolic
regulation of RNaPi-1 expression by insulin is mediated through the
phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not
the Ras/MAPK pathway.
rat Na+/Pi cotransporter-1; insulin; Ras; phosphatidylinositol 3-kinase; p70 ribosomal S6 kinase
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INTRODUCTION |
A MAJOR PHYSIOLOGICAL FUNCTION of insulin is to
regulate the key enzymes and membrane transporters that are involved in
glucose and lipid metabolism (7, 27). For insulin to exert these functions, it binds to its receptor at the plasma membrane of target
cells and activates the tyrosine kinase that is associated with the
cytoplasmic tail of the receptor (7, 27, 40). This causes
phosphorylation of the receptor and many endogenous substrates, such as
insulin receptor substrate-1 and -2, and Shc (7, 27). Phosphorylated
substrates in turn engage in the formation of the signaling complexes
via phosphotyrosine-containing binding motifs with Src
homology-2 domains found in molecules like phosphatidylinositol
3-kinase (PI 3-kinase) (11). These divergent intermolecular
interactions underlie the basic mechanism for the multiple effects of
insulin on the cell. For instance, two distinct signal pathways are
involved in gene regulation by insulin: the Ras/mitogen-activating
protein kinase (MAPK) pathway, which activates many transcription
factors and leads to c-fos induction, and the PI 3-kinase
pathway, which activates many genes involved in glucose and lipid
metabolism (7, 29).
Cytosolic inorganic phosphate (Pi) plays a central role in
cellular energy metabolism and in hormone-regulated glucose homeostasis (1, 2, 9). Administration of carbohydrates increases cellular
Pi uptake, and extracellular Pi regulates
hepatic glucose output (1). The hypoglycemic effect of insulin is
markedly diminished when serum Pi levels are low (9). The
direct effects of Pi on several key metabolic enzymes have
also been well documented (2). For example, Pi activates
6-phosphofructo-1-kinase, which catalyzes the conversion of fructose
6-phosphate to fructose 1,6-diphosphate and increases glycolysis (2).
It is now clear that intracellular Pi concentrations are
tightly regulated by a variety of mechanisms including insulin (6, 12).
After the original discovery that Pi transport across the
luminal brush-border membrane of mammalian kidney proximal tubule cells
was carrier mediated and Na+ dependent (16, 26),
Na+/Pi cotransporters have been found in many
different cells, including rat hepatocytes (21, 22). On the basis of
amino acid sequence similarity, three types of
Na+/Pi cotransporters have been cloned recently
(26). Rat Na+/Pi cotransporter-1-related type I
cotransporters are primarily expressed in the liver and kidney, whereas
NaPi-2-related type II cotransporters are kidney specific and play a
key role in kidney Pi reabsorption (22, 26). Recently,
expression study in Xenopus oocytes indicates that RNaPi-1 type
cotransporters may also act as Cl
channels (3). The
surface receptor for amphotropic murine retrovirus (Ram-1)-related type
III cotransporters expresses in a variety of cells and may represent a
housekeeping type of transporter (18). Although Pi
depletion regulates both type II and type III cotransporters (32, 39),
we have shown that expression of RNaPi-1 in rat hepatocytes, like many
other genes of glycolytic and lipogenic enzymes (23-25), is
regulated by insulin and glucose and that metabolic signals are
involved in this regulation (21). In this study, we aimed to identify
the metabolic components and the signaling pathways that mediate the
effects of insulin and glucose on RNaPi-1 gene expression in H4IIE rat
hepatoma cells that are devoid of endogenous gluconeogenic activity
(28, 34, 37).
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EXPERIMENTAL PROCEDURES |
Materials.
Chemicals of the highest purity available were from Sigma (St. Louis,
MO) and Boehringer Mannheim (Indianapolis, IN). TRI reagent for RNA
isolation was from Molecular Research Center (Cincinnati, OH), and
32P-labeled radionucleotides (~3,000 Ci/mmol) were from
Du Pont NEN (Boston, MA). Rabbit polyclonal anti-ACTIVE MAPK pAb and
anti-p42/44 antibodies were obtained from Promega (Madison, WI) and New
England Biolabs (Beverly, MA), respectively. An enhanced
chemiluminescence kit was from Amersham (Cleveland, OH). All protein
kinase inhibitors were purchased from Calbiochem (San Diego, CA).
Optitran and Nytran membranes were obtained from Schleicher and Schuell
(Keene, NH).
Cell culture.
H4IIE cells were cultured in DMEM supplemented with 10% fetal bovine
serum, 0.1 µM dexamethasone, and penicillin (100 U/ml) and
streptomycin (0.1 mg/ml) antibiotics. Cells were incubated in 95%
air-5% CO2 in humidified incubators at 37°C.
Approximately 14 h before treatment with effectors, the cultures were
refed glucose- and serum-free DMEM supplemented with 0.1 µM
dexamethasone and antibiotics as before.
Northern blot analysis.
Total RNA was isolated from cultured H4IIE cells using TRI reagent, as
we previously described (21). RNA was electrophoresed through 1%
agarose gels containing 1.9% formaldehyde, transferred to nylon
membranes, and fixed by ultraviolet (UV) cross-linking. RNaPi-1 cDNA
probe was made as previously described (21). Human
-actin and rat
Ram-1 clones were kindly provided by Dr. Craig Thompson (University of
Michigan, Ann Arbor, MI) and Dr. A. Dusty Miller (Fred Hutchinson
Cancer Research Center, Seattle, WA), respectively. Rat L-type pyruvate
kinase (L-PK) cDNA was PCR-cloned from rat liver mRNA and confirmed by
DNA sequence. The cDNA probes were random labeled with
-[32P]dATP. The intensities of
autoradiograms were scanned using a Bio-Rad imaging densitometer (model
GS-670), and areas were quantified. For quantitation of the relative
amount of mRNAs, the same blots were used for hybridizations with
-actin. The amounts of the RNaPi-1 and other mRNAs were normalized
to the corresponding
-actin mRNA, as previously described (21).
Nuclear run-on transcription assay.
Nuclei were isolated from H4IIE cells as previously described (17).
Briefly, cells were collected with ice-cold saline solution and
pelleted at 700 g for 5 min. After two washes with the same saline solution, cells were suspended in 40 ml of lysis buffer [0.5 M sucrose, 50 mM NaCl, 0.5 mM spermidine, 0.15 mM
phenylmethylsulfonyl fluoride (PMSF), 1% aprotinin, 7 mM
-mercaptoethanol, and 0.25% Nonidet P-40] and incubated on
ice for 3 min. Nuclei were collected by centrifugation at 1,000 g for 5 min and washed twice with the same lysis buffer without
Nonidet P-40. The nuclei pellet was resuspended in glycerol buffer
(50% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 50 mM
Tris · HCl, pH 7.5). After counting, nuclei were
frozen at
80°C at a concentration of 2-4 × 108/ml. Nuclei (10 × 106) were labeled
with
-[32P]UTP as previously described (17).
32P-labeled RNA was purified using TRI reagent according to
the manufacturer's instructions, and total radioactivity was
determined using scintillation counting. An equal amount of
radioactivity was used in hybridization. cDNA probes were loaded onto
nylon membrane with slit-blot apparatus and immobilized by UV
cross-linking.
Measurement of phosphorylation of p42/44 MAPKs.
Activation of p42/44 MAPKs in cultured H4IIE cells was determined by
Western blot with a rabbit polyclonal antibody raised against dually
phosphorylated p42/44 MAPKs (19). Briefly, after cells were exposed to
insulin, reaction was terminated by the replacement of medium with 200 µl of ice-cold lysis buffer (10 mM Tris · HCl, pH
7.4, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1 mM EGTA, 1 mM PMSF, 50 mM tetrasodium pyrophosphate, 10 nM okadaic acid,
1% Triton X-100, 0.25% sodium deoxycholate, 10 µg/ml aprotinin, and
10 µg/ml leupeptin). For Western blot analysis, cell lysates (60 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels and
transferred to an Optitran membrane. The membranes were probed with an
anti-ACTIVE MAPK pAb that detects p42/44 MAPKs only when they are
activated by phosphorylation at T202 and Y204. To ensure equal loading
and protein transfer, the same blots were stripped and probed with a
polyclonal antibody recognizing both phosphorylated and
nonphosphorylated p42/44 MAPKs.
Preparation of replication-defective adenovirus Asn17
Ras and adenovirus infection of H4IIE cells.
A replication-defective adenovirus expressing dominant negative
Asn17 Ras was generated as previously described (15, 19).
Virus was amplified in human kidney 293 cells, and the viral particles were purified from 293 cell lysates by cesium chloride gradient ultracentrifugation and then desalted by dialysis (15, 19). The
concentration of recombinant adenovirus was determined on the basis of
absorbance at 260 nm, where 1 optical density unit corresponds to
1012 particles/ml. An identical adenovirus containing the
-galactosidase gene instead of the Asn17 Ras gene was
used as a virus control.
Acetate incorporation assay.
To determine whether H4IIE cells utilize acetate, cells were incubated
with [3H]acetate for various times. Cells were
then washed with cold PBS and precipitated with 10% TCA. TCA-insoluble
materials were dissolved in 0.2 N NaOH and 0.1% SDS solution. Aliquots
were counted in a scintillation counter, and the total cellular protein
was determined by the Lowry method with BSA as standard.
Statistics.
Data are given as means ± SE. Statistical analysis was performed
using Student's t-test, and significance was accepted at P < 0.05.
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RESULTS |
Insulin transcriptionally regulates the RNaPi-1 gene through signals
derived from glucose metabolism.
We have demonstrated that the RNaPi-1 gene is regulated by
insulin-activated metabolic signaling pathways in rat hepatocytes in
primary culture. To further elucidate RNaPi-1-specific metabolic signaling pathways, H4IIE cells were used in the following studies. These cells, derived from a rat hepatoma cell line H35, retain the
glycolytic but lose the gluconeogenic activity of hepatocytes (28, 34).
Moreover, these cells exhibit high sensitivity to various hormones,
such as insulin and glucocorticoids (34, 35, 37). When the effects of
insulin on RNaPi-1 mRNA were measured, insulin increased steady-state
levels of RNaPi-1 mRNA in the presence of glucose but decreased RNaPi-1
mRNA in the absence of glucose (Fig.
1A), clearly establishing a
requirement of glucose for insulin-induced upregulation of RNaPi-1
mRNA. The effects of insulin on RNaPi-1 mRNA were dose dependent. For
example, a significant induction of RNaPi-1 mRNA in the presence of
glucose was observed at 0.1 nM insulin and reached maximum at 10 nM
(data not shown). The effects of insulin appeared to be specific for
the RNaPi-1 cotransporter, insofar as it did not significantly alter
the mRNA levels of Ram-1, another Na+/Pi
cotransporter (Fig. 1B). This supports our previous
observations that Na+/Pi cotransporters are
differentially regulated (21). When time-dependent changes were
measured under these experimental conditions, significant effects of
insulin on RNaPi-1 mRNA were observed after 6 h of exposure, reached
maximum after 24 h of exposure, and lasted for
36 h (Fig.
2, A and B). When RNaPi-1
mRNA levels were determined as a function of glucose concentration, a
dose-dependent induction was observed in the presence of insulin, and
maximal induction was reached at 5 mM glucose (data not shown).
However, glucose exhibited no significant effect on RNaPi-1 mRNA in the
absence of insulin. Because glucagon represses insulin-induced gene
expression by increasing intracellular cAMP levels (21, 37), it was of interest to determine whether cAMP regulates basal and insulin-induced RNaPi-1 expression in H4IIE cells. As depicted in Fig.
3, insulin induction of RNaPi-1 expression
in the presence of glucose was prevented by an increase in
intracellular cAMP in forskolin-treated H4IIE cells. However, forskolin
had no effect on basal levels of RNaPi-1 mRNA when cells were cultured
in the absence of insulin.


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Fig. 1.
Effects of insulin on steady-state levels of rat
Na+/Pi cotransporter-1 (RNaPi-1) mRNA.
A: representative autoradiogram of insulin effects. Cells were
exposed for 24 h either to buffer alone (lanes 1 and 3)
or to 10 nM insulin (lanes 2 and 4) in the presence
(lanes 3 and 4) or absence (lanes 1 and
2) of 10 mM glucose. Total RNA was isolated and assayed for
RNaPi-1, amphotropic murine retrovirus (Ram-1), and -actin mRNA
levels. B: combined data from 5 independent experiments. Values
of RNaPi-1 and Ram-1 mRNAs were normalized to those of corresponding
-actin measured on the same blots and expressed relative to a
control value of one. Values are means ± SE. * P < 0.05;
** P < 0.01 vs. control.
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Fig. 2.
Time course of effects of insulin and glucose on RNaPi-1 mRNA. Cells
were treated with 10 mM of glucose in the presence or absence of 10 nM
insulin for various times. Total RNA was isolated and assayed for
RNaPi-1 and -actin mRNAs as in Fig. 1. A: representative
autoradiogram of insulin and glucose effects. B: combined data
from 3 independent experiments. Values are means ± SE. * P < 0.05; ** P < 0.01 vs. time 0.
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Fig. 3.
Effects of forskolin on insulin-induced RNaPi-1 gene expression. Cells
were pretreated with 0.1 mM forskolin for 15 min before exposure for 24 h to 10 nM insulin in the presence of 10 mM glucose. Total RNA was
isolated and assayed for RNaPi-1 mRNA as in Fig. 1. Values are means ± SE of 4 experiments. ** P < 0.01 vs. insulin-treated
cells.
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To determine whether insulin increases transcription rate of the
RNaPi-1 gene in the presence of glucose, nuclear run-on experiments were performed. As shown in Fig. 4, after
24-h exposure, insulin increased newly synthesized RNaPi-1, but not
Ram-1, transcripts in the presence of glucose. These data indicate that
insulin transcriptionally regulates the RNaPi-1 gene in H4IIE cells.

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Fig. 4.
Nuclear run-on experiments showing effects of insulin and glucose on
RNaPi-1 transcription. Cells were treated with 10 nM insulin in the
presence of 10 mM glucose for 24 h before isolation and labeling of
nuclei with -[32P]UTP. An equal amount of
radioactive run-on RNA was used in the hybridization. Insert: a
representative autoradiogram. Graph, combined data from 3 experiments.
Intensities of signals of RNaPi-1 were corrected by subtracting signals
of a vector pBluescript (pBS), and they are represented in graph as
means ± SE. Insulin significantly stimulated transcription
of the RNaPi-1 gene in the presence of glucose (P < 0.01, Student's t-test).
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Metabolic signals leading to activation of the RNaPi-1 gene are
generated at or above pyruvate oxidation.
In H4IIE cells that are devoid of gluconeogenesis, glucokinase (GK) is
replaced by other hexokinases capable of phosphorylating glucose
independently of insulin (34). Because glucose had no effect on RNaPi-1
mRNA in the absence of insulin (Fig. 2), it appears that glucose
6-phosphate is not a proximate regulator of the RNaPi-1 gene. To
evaluate this hypothesis, cells were treated with 2-deoxyglucose and
fructose in the presence and absence of insulin. Like glucose, fructose
supported insulin-induced RNaPi-1 expression, whereas 2-deoxyglucose
decreased RNaPi-1 mRNA (Fig. 5). Because
2-deoxyglucose can only be phosphorylated, but not further metabolized,
and because fructose is metabolized to trioses, but not to glucose
6-phosphate in these cells, these data confirm that glucose 6-phosphate
was not a proximate regulator of the RNaPi-1 gene. Because fructose had
no effect on RNaPi-1 expression in the absence of insulin, the signal
must be generated through triose metabolism. This was supported by the
fact that glycerol, lactate, and pyruvate induced RNaPi-1 expression in
the presence of insulin (Table 1).

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Fig. 5.
Effects of fructose and 2-deoxyglucose on RNaPi-1 mRNA levels. Cells
were cultured in glucose-free medium and treated with either 10 mM
fructose (Fruc) or 10 mM 2-deoxyglucose in the presence or absence of
10 nM insulin (Ins) for 24 h. RNaPi-1 mRNA levels were measured as in
Fig. 1. Values are means ± SE of 4 different experiments. * P
< 0.05; ** P < 0.01 vs. control.
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To determine whether compounds that enter metabolism beyond pyruvate
oxidation can support insulin-induced RNaPi-1 expression, cells were
cultured in the glucose-free medium and treated with insulin in the
presence of either acetate or citrate. As depicted in Table 1, neither
acetate nor citrate could support insulin-induced RNaPi-1 expression.
In the absence of insulin, none of the compounds induced RNaPi-1
expression (Table 1). To ensure that these cells could use acetate,
effects of insulin on [3H]acetate incorporation
were determined, as shown in Fig. 6. The data clearly demonstrate that H4IIE cells can use acetate in an insulin-dependent manner.

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Fig. 6.
Time-dependent [3H]acetate incorporation. Cells
were cultured in glucose-free medium and exposed to 5 mM acetate and 1 µCi [3H]acetate for various times.
Incorporation of [3H]acetate was measured as
described in EXPERIMENTAL PROCEDURES. Activity was
expressed as
counts · min 1 · 100 µg protein 1. Insert: effects of insulin on
[3H]acetate incorporation. Cells were treated
with 10 nM insulin for various times, as indicated, and then exposed to
[3H]acetate for 2 h. Values are means ± SE of
4 measurements.
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RNaPi-1 and L-PK use different metabolic signals.
Because the L-PK gene is regulated by metabolic signals generated above
pyruvate metabolism, to corroborate the above findings it was of
interest for us to determine whether pyruvate regulates L-PK and
RNaPi-1 differently in H4IIE cells. In the experiments of Fig.
7, cells were exposed to pyruvate in the
presence of insulin and assayed for RNaPi-1 and L-PK mRNAs. The data
showed that, whereas pyruvate and insulin upregulated the RNaPi-1 gene,
they downregulated L-PK. Furthermore, whereas addition of pyruvate to a
glucose-free medium showed no effect on L-PK mRNA in the absence of
insulin (data not shown), addition of glucose increased the
steady-state mRNA levels of L-PK, but not RNaPi-1, under the same
experimental conditions (Fig. 7). These data support the proposition
that L-PK and RNaPi-1 are regulated by different metabolic signals.

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Fig. 7.
Effects of glucose and pyruvate on RNaPi-1 and L-PK mRNA levels. Cells
were incubated with either 10 mM glucose or 20 mM pyruvate in the
presence or absence of 10 nM insulin for 24 h. Total RNA was isolated
and assayed for RNaPi-1 and rat L-type pyruvate kinase (L-PK) mRNAs.
Autoradiogram is representative of 4 independent experiments.
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Insulin regulates the metabolic signals through wortmannin and
rapamycin-sensitive pathways.
Insulin regulates genes through two major signaling pathways, namely,
the Ras/p42/44 MAPK pathway and the PI 3-kinase pathway in hepatocytes
(7, 27). To determine whether the Ras/MAPK pathway is involved in
insulin-induced upregulation of the RNaPi-1 gene, cells were infected
with a recombinant adenovirus expressing an Asn17 dominant
negative mutant of Ras for 12 h, as previously described (15, 19). The
virus-transduced cells were then treated with insulin in the presence
of glucose and measured for RNaPi-1 mRNA. Adenovirus
-galactosidase-infected cells served as control. As depicted in Fig.
8, expression of dominant negative Ras
showed no effects on insulin-stimulated RNaPi-1 expression in H4IIE
cells. To further elucidate whether p42/44 MAPKs are involved in
regulation of RNaPi-1, the effects of insulin on RNaPi-1 mRNA were
determined in the presence of MEK inhibitor PD-98059. The data showed
that 30 µM PD-98059 exhibited no effect on insulin-induced RNaPi-1 expression (Fig. 9). As previously reported
(15), control experiments showed that expression of dominant negative
Ras and MEK inhibitor PD-98059 blocked insulin-induced activation of
p42/44 MAPK in H4IIE cells under the same experimental conditions.
Insulin caused a 2.2 ± 0.4-fold increase in p42 MAPK phosphorylation
and preincubation with 30 µM PD-98059 for 15 min decreased
insulin-induced phosphorylation to 1.1 ± 0.2-fold over control. Like
PD-98059, expression of Asn17 Ras reduced the stimulation
to 1.2 ± 0.3-fold.

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Fig. 8.
Effects of expression of Asn17 dominant negative mutant of
Ras on insulin-induced RNaPi-1 expression. Cells were infected with
Asn17 Ras adenovirus at a concentration of 2,000 particles/cell for 12 h, washed, and then exposed to 10 nM insulin in
the presence of 10 mM glucose for 24 h. The -galactosidase
virus-infected cells served as control. Total RNA was isolated and
measured for RNaPi-1 mRNA as in Fig. 1. Values are means ± SE of 3 independent experiments. ** P < 0.01 vs. control.
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Fig. 9.
Effects of PD-98059, wortmannin, and LY-294002 on insulin-induced
RNaPi-1 expression. Cells were pretreated with 30 µM PD-98059 (PD) or
100 nM wortmannin (Wort.) or 50 µM LY-294002 (LY) for 15 min before
exposure to 10 nM insulin in the presence of 10 mM glucose for 16 h.
During treatment, cells were refed with wortmannin every 4 h as
previously described (29). Total RNA was isolated and assayed for
RNaPi-1 mRNA as in Fig. 1. Values are means ± SE of 3 experiments.
* P < 0.05; ** P < 0.01 vs. control.
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To determine whether insulin-stimulated metabolic regulation of RNaPi-1
is mediated through the PI 3-kinase pathway, the effects of insulin on
RNaPi-1 mRNA were determined in the presence of PI 3-kinase inhibitors
(100 nM wortmannin and 50 µM LY-294002). As shown in Fig. 9,
insulin-induced RNaPi-1 expression was significantly blocked when the
cells were treated with either wortmannin or LY-294002. One of the
signaling pathways downstream from PI 3-kinase requires activation of
p70s6k (15, 29, 35). To determine whether this pathway is
involved in insulin regulation of the RNaPi-1 gene, rapamycin, a
p70s6k inhibitor, was used in experiments shown in Fig.
10. The data showed that rapamycin
inhibited insulin-induced RNaPi-1 expression in H4IIE cells in a
dose-dependent manner. At high concentration it also significantly
decreased basal levels of RNaPi-1 mRNA.

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Fig. 10.
Effects of rapamycin (Rap) on RNaPi-1 expression. Cells were pretreated
with different concentrations of rapamycin for 15 min before exposure
to 10 nM insulin in the presence of 10 mM glucose for 16 h. RNaPi-1
mRNA was assayed as in Fig. 1. Values are means ± SE of 3-5
experiments. ** P < 0.01 vs. control.
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DISCUSSION |
The PI 3-kinase signal pathway mediates insulin-activated metabolic
regulation of the RNaPi-1 gene.
Although the expression of NaPi-2-type transporters and Ram-1 is
regulated by Pi depletion through Ca2+
signaling (32, 39), that of RNaPi-1 is regulated by insulin and glucose
(21). In this study, we further demonstrated that insulin regulated
RNaPi-1 mRNA through a transcriptional mechanism in H4IIE cells.
However, it remains to be determined whether insulin increases RNaPi-1
protein levels as well as Na+-dependent Pi
cotransport activity in these cells. It is well established that
binding of insulin to its receptor results in phosphorylation of
endogenous substrates that allow for intermolecular interactions
between phosphoproteins and SH-2 domain-containing proteins (7, 27). In
H4IIE cells, insulin activated both Ras/p42/44 MAPK and PI 3-kinase
pathways (15, 35). Insulin also increases cell volume, which in turn
can further activate PI 3-kinase (20). Although activation of the
Ras/MAPK pathway plays a key role in the effects of insulin on
mitogenesis and c-fos expression, activation of the PI 3-kinase
pathway leads to insulin regulation of many metabolic enzymes, such as
phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase II
(HKII) (15, 29, 35). To determine how insulin generates
RNaPi-1-specific signals, a recombinant adenoviral vector and several
well characterized pathway-specific protein-kinase inhibitors were used
in this study (15, 19). Although expression of dominant negative Ras
and MEK inhibitor PD-98059 blocked insulin-induced activation of p42/44
MAPK, they failed to repress insulin-induced RNaPi-1 expression (Figs.
8 and 9), suggesting that activation of a Ras/MAPK pathway is not required for insulin regulation of the RNaPi-1 gene. In contrast, when
cells were treated with PI 3-kinase inhibitors wortmannin and
LY-294002, insulin-induced RNaPi-1 expression was abolished (Fig. 9),
indicating that the PI 3-kinase pathway is involved in insulin
regulation of the RNaPi-1 gene. Interestingly, a recent study has
demonstrated that synthesis of a NaPi-2-type transporter was also
mediated by activation of PI 3-kinase in OK cells (30). Two downstream
signaling pathways have been described for PI 3-kinase, Akt-GSK3 and
p70s6k, which can be distinguished by their sensitivity to
rapamycin (7, 8, 31). It has been demonstrated that rapamycin
specifically blocks insulin-induced activation of p70s6k in
H4IIE cells (35). p70s6k controls the ribosomal protein S6
phosphorylation in response to mitogens and plays an essential role in
controlling the translation machinery (4, 31). Because significant
increases in RNaPi-1 expression occurred after the cells were exposed
to insulin for 6 h (Fig. 2), it is quite possible that the induction of
RNaPi-1 requires increased protein synthesis. Accordingly, specific
inhibition of p70s6k by rapamycin was found to be
sufficient to block insulin-induced upregulation of RNaPi-1 expression
(Fig. 10). These data suggest that activation of p70s6k by
insulin may increase translation of the proteins that are involved in
generation of RNaPi-1-specific metabolic signals. However, it remains
to be determined as to which proteins are involved in regulation of
RNaPi-1 and how they work. Interestingly, like RNaPi-1, insulin
regulation of HIIK is also sensitive to rapamycin (29). On the other
hand, insulin-induced downregulation of PEPCK and translocation of
glucose transporter 4 are sensitive only to wortmannin, but not
rapamycin (13, 35). These data point out that, downstream from PI
3-kinase, both p70s6k-dependent and
p70s6k-independent pathways are used by insulin in
regulation of metabolic enzymes and transporters.
RNaPi-1-specific metabolic signals may be generated at or above
pyruvate oxidation.
Many metabolic enzymes are regulated by insulin in a glucose-dependent
manner (36, 38). As shown in Figs. 1 and 2, the stimulatory effects of
insulin on the RNaPi-1 gene in H4IIE cells were clearly glucose
dependent. Because insulin most likely augmented glucose metabolism in
H4IIE cells, we postulated that, like many other proteins,
insulin-mediated transcriptional upregulation of RNaPi-1 was mediated
by metabolic signals generated from glucose and other carbohydrates
(10, 14, 33). Interestingly, in addition to this glucose-dependent
pathway, an apparent glucose-independent pathway was also involved in
regulation of RNaPi-1 mRNA in rat hepatocytes in primary culture,
because insulin increased RNaPi-1 mRNA in the absence of glucose (21).
However, it is quite possible that the glucose-independent regulation
of RNaPi-1 is also mediated by metabolic signals. As discussed below,
insulin-induced metabolic signals are generated at or above pyruvate
oxidation. Because rat hepatocytes in primary culture exhibit high
gluconeogenic activity, glucose metabolites, especially those not
heavily controlled by insulin (e.g., the production of pyruvate), may
accumulate through the gluconeogenic process. These metabolites may
then suffice to mediate insulin induction of RNaPi-1 expression in rat
hepatocytes. In contrast, because H4IIE cells lost gluconeogenic capacity (28, 34, 37), they cannot accumulate glucose and glucose
metabolites at levels sufficient to mediate an insulin effect.
Therefore, the effects of insulin on RNaPi-1 are completely dependent
on glucose or other carbohydrates in H4IIE cells. Alternatively, insulin may activate different signaling pathways in hepatocytes from
those in H4IIE cells.
Several glucose metabolites have now been identified to play important
roles in regulation of a number of enzymes that are involved in glucose
and lipid metabolism (10, 23-25). An increase in
glucose-6-phosphate concentration, for example, is found to stimulate
genes such as fatty acid synthase and acetyl-CoA carboxylase (5, 14).
However, glucose-6-phosphate does not seem to act as a proximate
regulator of RNaPi-1 gene because glucose had no effect on RNaPi-1 mRNA
in the absence of insulin although insulin-dependent GK is replaced by
other hexokinases capable of phosphorylating glucose independently of
insulin in H4IIE cells (34). This notion is supported by the
observations that in the presence of insulin, fructose induces RNaPi-1
expression while it is metabolized to trioses, but not to
glucose-6-phosphate in these cells in the presence of insulin. Because
2-deoxyglucose can be phosphorylated to 2-deoxyglucose-6-phosphate, but
not further metabolized, that 2-deoxyglucose decreased RNaPi-1 mRNA
further supports that RNaPi-1 is regulated by signals generated
downstream of glucose-6-phosphate.
Recently, downstream from glucose 6-phophate, a pentose pathway
metabolite xylulose 5-phosphate has been identified as a proximate regulator of several enzymes including L-PK, PEPCK, and
glucose-6-phosphatase (10, 24). In the presence of glucose, insulin
induces GK and increases xylulose 5-phosphate concentration in rat
hepatocytes by enhancing the conversion of glucose to glucose
6-phosphate, resulting in an insulin- and glucose-dependent
upregulation of L-PK and downregulation of PEPCK (10, 24). However, the
RNaPi-1 gene appeared to be regulated by metabolic signals other than xylulose 5-phosphate, because although the RNaPi-1 gene was induced, the L-PK gene was downregulated by pyruvate and insulin (Fig. 7). The
proximate metabolic signals for RNaPi-1 appear to be generated at or
above pyruvate oxidation because glycerol, lactate, and pyruvate
induced RNaPi-1 expression in the presence of insulin (Table 1). It is
clear that pyruvate itself is not a direct regulator of the RNaPi-1
gene, because in the absence of insulin pyruvate failed to induce
RNaPi-1 gene expression. Interestingly, synthesis of malic enzyme in
hepatocytes is also regulated by signals generated from pyruvate
oxidation (23). Like malic enzyme (23), exposure of H4IIE cells to
insulin in the presence of acetate and citrate failed to increase
RNaPi-1 mRNA abundance (Table 1). Because H4IIE cells use acetate in an
insulin-dependent manner (Fig. 6), these data indicate that, once
glucose is irreversibly converted to cytosolic acetyl-CoA, its
stimulatory effects on RNaPi-1 are lost.
In summary, we have demonstrated that the RNaPi-1 gene is regulated by
insulin through a unique signaling pathway that includes insulin
activation of PI 3-kinase/p70s6k and generation of
metabolic signals (Fig. 11). It remains
to be determined which metabolic signals generated by insulin and
glucose are responsible for RNaPi-1 induction and how these metabolic signals regulate RNaPi-1 expression at gene levels.

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|
Fig. 11.
A putative model for metabolic regulation of the RNaPi-1 gene by
insulin and glucose. PI 3-kinase, phosphatidylinositol 3-kinase.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
HL-36573 and DK-43051 and by a grant-in-aid from the American Heart
Association with funds contributed in part by the American Heart
Association, Ohio-West Virginia Affiliate.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Z. Xie,
Department of Pharmacology, Medical College of Ohio, 3035 Arlington
Ave., Toledo, OH 43614-5804 (E-mail: zxie{at}mco.edu).
Received 22 July 1999; accepted in final form 8 November 1999.
 |
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