Excessive production of glucose is the major cause of fasting
hyperglycemia in human (1, 2) and experimental
diabetes mellitus(3, 4) . The hydrolysis of hepatic
glucose-6-phosphate is the ``final common pathway'' for the
release of glucose into the circulation. Recent experimental evidence
supports the notion that this final step is rate-determining for the
increased rate of hepatic glucose output in diabetic states. In fact,
marked changes in the rate of formation of hepatic glucose 6-phosphate
through gluconeogenesis fail to alter hepatic glucose
production(5, 6, 7, 8, 9) ,
and in experimental diabetes hepatic glucose production is markedly
elevated in the presence of a significant decrease in the
hepatic glucose 6-phosphate pool (4, 10) .
The
catalytic portion of the glucose-6-phosphatase (Glc-6-Pase) (
)complex has recently been
cloned(11, 12, 13) . Studies in FAO rat
hepatoma cells (13) indicate that insulin down-regulates
Glc-6-Pase mRNA levels, while glucocorticoid induces it. Haber et
al.(14) have recently reported a marked increase in
hepatic Glc-6-Pase mRNA and protein in diabetic BB rats. In the same
study, in vivo treatment with 0.5 unit of insulin normalized
the plasma glucose concentration and the hepatic Glc-6-Pase mRNA levels
in diabetic rats within 4 h. Similarly, Liu et al.(15) have shown increased hepatic Glc-6-Pase mRNA and
activity in streptozotocin-induced diabetic rats. Since experimental
diabetes and insulin treatment are associated with simultaneous and
opposite changes in the plasma glucose and plasma insulin
concentrations, we have attempted to discern the relative role of
glucose and insulin in the short-term regulation of the hepatic
Glc-6-Pase gene expression in (90% partially pancreatectomized)
diabetic rats.
Our results confirm that prolonged insulin deficiency
and hyperglycemia (experimental diabetes) cause a marked increase in
the hepatic Glc-6-Pase mRNA and protein and indicate that short-term
(
8 h) correction of hyperglycemia in diabetic rats leads to
normalization of the hepatic gene expression of this enzyme, regardless
of the circulating insulin concentrations.
MATERIALS AND METHODS
Animals
Four groups of male Sprague-Dawley rats
(Charles River Breeding Laboratories, Inc., Wilmington, MA) were
studied: group I, controls (n = 9); group II, 90%
partially pancreatectomized rats (n = 10);
group III, 90% partially pancreatectomized rats acutely treated with
low dose insulin (n = 10); group IV, 90% partially
pancreatectomized rats acutely treated with phlorizin (n = 16). At 3-4 weeks of age, all rats (80-100 g)
were anesthetized with pentobarbital (50 mg/kg of body weight,
intraperitoneally), and in groups II-IV, 90% of their pancreas
was removed according to the technique of Foglia(16) , as
modified by Bonner-Weir et al.(17) . Immediately after
surgery rats were housed in individual cages and subjected to a
standard light (6 a.m. to 6 p.m.)-dark (6 p.m. to 6 a.m.) cycle.
All rats received ad libitum water and standard rat laboratory
chow (Ralston-Purina, St. Louis, MO). After surgery rats were weighed
twice weekly, and tail vein blood was collected for the determination
of nonfasting plasma glucose and insulin concentrations at the same
time (8 a.m.). Five weeks following pancreatectomy rats were
anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg
of body weight), and indwelling catheters were inserted into the right
internal jugular vein and in the left carotid artery, as described
previously(18, 19) . Food intake was carefully
monitored following catheter placement, and only rats gaining weight
and eating >12 g of chow during the night prior to the study were
included in the study.
Insulin/Phlorizin Administration
Studies were
performed in awake, unstressed, chronically catheterized rats. At
7 a.m. food was removed, and rats received either a vehicle
subcutaneous injection (groups I, II, and III) and a variable
intra-arterial insulin infusion (group III) or a subcutaneous injection
of phlorizin (group IV, 0.3 g/kg of body weight) and an intra-arterial
infusion of saline (groups I, II, and IV). The insulin infusion (group
III) was designed to gradually lower the plasma glucose concentration
in a similar fashion to that observed with a single injection of
phlorizin (group IV). The average rate of insulin infusion was 1.8
± 0.3 milliunits/kg
min during the first 3 h and 0.9
± 0.3 milliunits/kg
min thereafter. To discern the effect
of the normalization of the plasma glucose concentration from the
potential direct effect of phlorizin on Glc-6-Pase gene expression, a
subgroup (n = 6) of phlorizin-treated diabetic rats
also received glucose infusions designed to maintain hyperglycemia
throughout the in vivo study. Plasma samples for determination
of plasma insulin, glucagon, and FFA concentrations were obtained at
time -30, 0, 30, 60, 90, and 120 min during the final portion of
the in vivo study. The total volume of blood withdrawn was
3.0 ml/study; to prevent volume depletion and anemia, a solution
(1:1, v/v) of
4.0 ml of fresh blood (obtained by heart puncture
from a littermate of the test animal) and heparinized saline (4
units/ml) was infused. At the end of the in vivo studies, rats
were anesthetized (pentobarbital, 60 mg/kg of body weight,
intravenously), the abdomen was quickly opened, portal vein blood was
obtained, and the liver was freeze-clamped in situ with
aluminum tongs precooled in liquid nitrogen. The time from the
injection of the anesthetic until freeze-clamping of the liver was less
than 45 s. All tissue samples were stored at -80 °C for
subsequent analysis.The study protocol was reviewed and approved by
the Institutional Animal Care and Use Committees of the Albert Einstein
College of Medicine.
Immunoblotting Analysis
Microsomes were prepared
according to Lange et al.(20) . Briefly, liver tissue
(100 mg) was homogenized in 10 volumes of a
Tris/sucrose/phenylmethylsulfonyl fluoride buffer (50 mM Tris
buffer, pH 7.3, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride). This homogenate was centrifuged
for 10 min at 10,000
g; the cytosol was then
centrifuged for 1 h at 100,000
g, and the pellet was
resuspended in 1 ml of Tris/sucrose/phenylmethylsulfonyl fluoride
buffer. The resuspended pellet was incubated at 4 °C for 30 min in
the presence of Triton X-100 at a final concentration of 0.1%. Protein
content was measured by the Bio-Rad assay kit using bovine serum
albumin as standard. Equal amounts of proteins (20 µg) were
subjected to a 10% SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred to nitrocellulose membranes (Schleicher
& Schuell). After blocking, the membranes were incubated with a
1:1500 dilution of affinity-purified anti-Glc-6-Pase antibody (a gift
of Dr. Rebecca Taub, University of Pennsylvania) followed by a 1:1500
dilution of goat anti-rabbit horseradish peroxidase-conjugated
secondary antibodies kit (ECL, Amersham Corp.).
Northern Blot Analysis
Total RNA was isolated on
freeze-clamped liver tissues according to the RNA-STAT kit (Tel-TEST
``B'' Inc., Friendswood, TX). The isolated RNA was assessed
for purity by the 260/280 ratio absorbance. Twenty µg of total RNA
were electrophoresed on a 1.2% formaldehyde-denatured agarose gel in 1
MOPS running buffer. The RNA was visualized with ethidium
bromide and transferred to a Hybond-N membrane (Amersham Corp.). The
Glc-6-Pase cDNA was a 1.25-kilobase pair EcoRI-HindIII fragment (kindly provided by Dr. R.
Taub, University of Pennsylvania), and tubulin cDNA probes were labeled
with
P using the Megaprime labeling system kit (Amersham
Corp.). Prehybridization and hybridization were carried out using the
rapid hybridization buffer (Amersham Corp.). The filters were then
exposed to Fuji x-ray films for 12-48 h at -80 °C with
intensifying screens. Quantification of Glc-6-Pase was done by scanning
densitometry, normalized for ribosomal RNA signal to correct for
loading irregularities.
Analytical Procedures
Plasma glucose was measured
by the glucose oxidase method (Glucose Analyzer II, Beckman
Instruments, Inc.) and plasma insulin by radioimmunoassay using rat and
porcine insulin standards. Plasma non-esterified fatty acid
concentrations were determined by an enzymatic method with an automated
kit according to the manufacturer's specifications (Waco Pure
Chemical Industries, Osaka, Japan). Differences between groups were
determined by analysis of variance.
RESULTS AND DISCUSSION
Studies of the physiologic regulation of hepatic Glc-6-Pase
have been largely limited to the assessment of its activity in
microsomal
preparations(4, 10, 21, 22, 23) .
Increased activity has been consistently demonstrated in experimental
diabetes
mellitus(4, 10, 21, 22, 23) .
This increase correlates with the plasma glucose concentrations in
these animals (10, 15) and can be decreased or
normalized by insulin treatment(10, 22, 23) .
The recent cloning of the human, rat, and murine Glc-6-Pase cDNAs (11, 12, 13, 14) allows one to
examine the physiologic regulation of its gene expression. Although
insulin per se can decrease Glc-6-Pase mRNA in FAO rat
hepatoma cells (13) and in BB diabetic rats(14) , the
physiologic in vivo regulation of Glc-6-Pase gene expression
remains to be delineated. In fact, though in two recent reports
insulinopenic diabetes was associated with a marked increase in
Glc-6-Pase mRNA in the liver(14, 15) , the potential
regulatory role of prolonged hyperglycemia per se on
Glc-6-Pase gene expression has not been studied. Thus, we quantitated
the hepatic mRNA and protein levels of Glc-6-Pase in diabetic rats
following normalization of the plasma glucose concentration, with or
without insulin administration.
Basal Metabolic Parameters
There were no significant
differences in the mean body weights between the control and the
diabetic groups (Table 1). The diabetic animals were carefully
matched for their metabolic parameters prior to assignment to the three
experimental protocols. In fact, the non-fasting plasma glucose
concentrations during the 2-week period prior to the in vivo studies were significantly and similarly elevated in the diabetic
groups, compared with the control group (p < 0.01). The
non-fasting plasma insulin concentrations were significantly decreased
in all diabetic rats (p < 0.01), while the plasma glucagon
concentrations were unchanged.
Effect of Short-term Correction of Hyperglycemia on
Hormonal and Biochemical Parameters
As depicted in Fig. 1, the infusion of insulin and the injection of phlorizin
resulted in a progressive and similar lowering of the plasma glucose
concentration in diabetic rats in groups III and IV, respectively. Table 2shows plasma glucose, FFA, insulin, and glucagon
concentrations after
8 h of vehicle (saline, insulin, or
phlorizin) administration. The mean plasma glucose concentration,
significantly higher in the diabetic rats as compared with the normal
rats, was restored to normal values after acute insulin and phlorizin
treatments (Table 2). In groups III and IV, the steady state
plasma glucose concentration remained at normoglycemic levels (
9
mM) throughout the study protocol (Fig. 1). The
administration of insulin, but not of phlorizin, normalized the plasma
insulin levels. Plasma glucagon was not affected by the insulin
infusion; however, the acute normalization of the plasma glucose
concentration with phlorizin moderately increased the plasma glucagon
concentration (Table 2). The circulating FFA levels were
increased in the diabetic rats compared with control rats.
Normalization of glycemia with insulin, but not with phlorizin
treatment, restored the plasma FFA concentrations to the normal values (Table 2). Thus, this in vivo experimental manipulation
allows us to examine diabetic rats in the presence of high glucose and
low insulin (group II), low glucose and high insulin (group III), and
low glucose and low insulin (group IV). To investigate the potential
direct effect of phlorizin administration on Glc-6-Pase mRNA and
protein, a subgroup of diabetic rats received phlorizin injection and
variable glucose infusions designed to maintain the plasma glucose
concentration at the diabetic level. In this group an average rate of
30.9 ± 2.5 mg/kg
min was required to maintain the plasma
glucose concentration at 20.4 ± 2.2 mM. The latter
value was similar to the plasma glucose concentrations at base line (Table 1) and during the in vivo studies (Table 2)
in the untreated diabetic rats (group II).
Figure 1:
Plasma glucose concentrations in
diabetic conscious rats treated with insulin (Ins, open
circles) and phlorizin (Phlo, closed triangles). Diabetic
rats (plasma glucose concentration,
22 mM) were randomly
assigned to receive a variable insulin infusion or a subcutaneous
injection of phlorizin (0.3 g/kg). The plasma glucose concentrations
were slowly decreased to
9 mM within 8 h. Rats were
sacrificed and liver freeze-clamped in situ following 2 h at
the desired plasma glucose levels (displayed in the right graph of the figure and in Table 2). All tissue determinations
were performed on samples obtained at the completion of these in
vivo studies. The average rate of insulin infusion was 1.8
± 0.3 milliunits/kg
min during the first 3 h and 0.9
± 0.3 milliunits/kg
min during the last 3 h of the in
vivo study. The plasma glucose concentration in the control (group
I) and diabetic (group II) groups averaged 8.2 ± 0.2 and 21.0
± 1.3 mM, respectively. The mean rate of plasma glucose
decline was
2 mM/h in both groups. Panx,
pancreatectomized.
Effect of Short-term Correction of Hyperglycemia on
Hepatic Glc-6-Pase mRNA Levels
The relative abundance of
Glc-6-Pase mRNA was determined by Northern blot analysis of total RNA
obtained from liver samples at the end of the in vivo studies.
We used a 1.25-kilobase pair cDNA probe that recognized the region
encoding the catalytic portion of the Glc-6-Pase. Glc-6-Pase mRNA
concentrations in liver of non-diabetic rats were used as control (set
at 100%). Multiple densitometric scanning of different Northern blots
(example is shown in Fig. 2) shows that the diabetic rats
manifested a >5-fold increase in Glc-6-Pase mRNA concentrations as
compared with non-diabetic rats. As expected, low dose insulin
administration almost completely reversed this marked increase in the
Glc-6-Pase messenger RNA. However, since the infusion of insulin
concomitantly decreases the plasma glucose and FFA concentrations and
increases the plasma insulin concentration, we wished to examine the
effect of the normalization of blood glucose concentration per se on hepatic Glc-6-Pase gene expression. Thus, we induced a similar
gradual decline in the plasma glucose concentration with the glycosuric
agent, phlorizin, which normalized the plasma glucose but not the
plasma insulin and FFA levels in diabetic rats. Phlorizin treatment
causes the same marked reduction in Glc-6-Pase mRNA as seen with
insulin. When glucose was infused to prevent the decline in the plasma
glucose concentration, phlorizin administration did not alter the
Glc-6-Pase mRNA in diabetic liver (not shown). These results suggest
that correction of hyperglycemia per se is able to suppress
the marked diabetes-induced increase in Glc-6-Pase mRNA.
Figure 2:
Effect of diabetes and of the
normalization of plasma glucose concentrations with either insulin or
phlorizin on hepatic Glc-6-Pase mRNA. A, Northern analysis of
Glc-6-Pase mRNA in liver freeze-clamped in situ at the
completion of the in vivo studies. Northern blots were probed
with
P-labeled Glc-6-Pase cDNA. 20 µg of each liver
RNA sample were used. B, equal loading of RNA per lane as confirmed by ethidium-stained ribosomal RNA bands. The figure
depicts Glc-6-Pase mRNA in groups (n = 3) of samples
obtained from control rats (CON), untreated diabetic rats (PANX), insulin-treated diabetic rats (INS), and
phlorizin-treated diabetic rats (PHLO). Rats were sacrificed
and liver freeze-clamped in situ following 2 h at the desired
plasma glucose levels (as displayed in Table 2). All tissue
determinations were performed on samples obtained at the completion of
these in vivo studies. Analysis was performed several times
for all rats included in Table 1and Table 2with similar
results.
Effect of Short-term Correction of Hyperglycemia on
Hepatic Glc-6-Pase Protein Levels
We next examined whether this
marked changes in mRNA levels were reflected in a comparable decrease
in the protein concentration (Fig. 3, A and B). The amount of Glc-6-Pase enzyme in liver microsomes was
assessed using polyclonal antibodies against the catalytic portion of
Glc-6-Pase. Diabetic rats show larger than a 3-fold increase in
Glc-6-Pase protein levels, as compared with the control rats.
Normalization of the plasma glucose concentration with either insulin
or phlorizin similarly decreases the hepatic Glc-6-Pase protein levels.
However, maintenance of hyperglycemia following phlorizin
administration completely prevented the marked decrease in the hepatic
Glc-6-Pase protein level.
Figure 3:
Effect of diabetes and of the
normalization of plasma glucose concentrations with either insulin or
phlorizin on hepatic Glc-6-Pase protein. A, immunoblot of
liver microsomal fraction of control rats (CON), untreated
diabetic rats (PANX), insulin-treated diabetic rats (INS), and phlorizin-treated diabetic rats (PHLO). B, immunoblot of liver microsomal fraction of
phlorizin-treated diabetic rats in which the plasma glucose
concentration is maintained at diabetic levels of
21
mM (Glc +) or decreased and maintained at
normoglycemic levels (Glc -). Rats were sacrificed and
liver freeze-clamped in situ following 2 h at the desired
plasma glucose levels (as displayed in Table 2). All tissue
determinations were performed on samples obtained at the completion of
these in vivo studies. Analysis was performed several times
for all rats included in Table 1and Table 2with similar
results.
Thus, although the changes in hepatic
Glc-6-Pase mRNA observed with diabetes and following normalization of
the plasma glucose levels tend to be more dramatic than those observed
in the protein levels, there is an excellent correspondence of mRNA and
protein levels in all experimental conditions. The finding of such a
marked effect of extracellular glucose on the mRNA and protein levels
of this key hepatic enzyme should not be interpreted as contradictory
to the previously demonstrated regulation by
insulin(13, 14) . In fact, it is likely that both
hyperinsulinemia and the restoration of normoglycemia may independently
contribute to the physiologic regulation of Glc-6-Pase gene expression
in the diabetic liver. Regarding the mechanism by which changes in the
plasma glucose concentration may regulate gene expression, it is
noteworthy that several key enzymes in carbohydrate and lipid metabolic
pathways are transcriptionally regulated by extracellular
glucose(24, 25, 26, 27) . However,
it is likely that such regulation requires changes in the pool of
phosphorylated glucose and may better correlate with the concentrations
of glucose 6-phosphate or of other intracellular metabolites rather
than with the extracellular glucose levels per
se(25, 26, 27) . Finally, it should be
emphasized that the Glc-6-Pase complex is regulated at multiple
levels(10, 28, 29, 30, 31) ,
which include, but are not limited to, the gene expression of its
catalytic subunit.
In conclusion, we provide evidence for a
stimulatory effect of hyperglycemia per se on hepatic
Glc-6-Pase gene expression in diabetic rats. This may represent a
molecular mechanism by which prolonged hyperglycemia favors the
persistence of excessive hepatic glucose output in diabetes mellitus.