Vol. 274, Issue 6, E984-E991, June 1998
Glycemia-lowering effect of cobalt chloride in the diabetic
rat: role of decreased gluconeogenesis
Firas
Saker1,
Juan
Ybarra2,
Patrick
Leahy3,
Richard W.
Hanson3,
Satish C.
Kalhan1, and
Faramarz
Ismail-Beigi2
Departments of 1 Pediatrics,
2 Medicine, and
3 Biochemistry, Case Western
Reserve University, Cleveland, Ohio 44106-4951
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ABSTRACT |
Results of previous
studies indicated that treatment of diabetic rats (induced by
streptozotocin) with cobalt chloride
(CoCl2) resulted in a
significant decrement in serum glucose concentration. The present study
was designed to determine the potential role of enhanced glucose uptake
vs. decreased glucose production in the above response. The rate of
systemic appearance of glucose, measured under fasting
conditions using
[3-3H]glucose tracer,
was reduced from 35.5 ± 2.5 to 17.5 ± 1.8 µmol · kg
1 · min1
in diabetic rats treated with 2 mM
CoCl2 added to the drinking water
for 10-14 days (P < 0.01).
Tissue accumulation of intravenously administered
2-deoxy-[14C]glucose
was significantly reduced in kidney and eye of diabetic rats treated
with CoCl2, whereas the uptake
remained unchanged in several other tissues including cerebrum, red and
white skeletal muscle, heart, and liver. The relative content of
phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased
3.1-fold in livers of diabetic compared with normal rats
(P < 0.001), and treatment of
diabetic rats with CoCl2 decreased
hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the
liver was decreased by 33% in
CoCl2-treated normal rats
(P < 0.05). Treatment with CoCl2 resulted in no change in
cAMP levels in the livers of either diabetic or normal rats. These
results suggest that the glycemia-lowering effect of
CoCl2 is mediated by reductions in
the rate of systemic appearance of glucose and hepatic gluconeogenesis.
glucose uptake; phosphoenolpyruvate carboxykinase mRNA; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
CELLULAR UPTAKE and metabolism of glucose are of
critical importance in the maintenance of homeostasis and energy
production. The first step in glucose metabolism is its transport
across the plasma membranes, a function that is "rate limiting"
in many cells and tissues (5, 12). Transmembrane transport of glucose
is mediated by a family of
Na+-independent glycoprotein
glucose transporter (GLUT) molecules that are expressed in a
tissue-specific manner (20, 23). Of these, GLUT-4 and GLUT-1 are
especially relevant to diabetes because they mediate insulin-stimulated
and basal (insulin-independent) uptake of glucose, respectively (20,
23).
Results of our previous studies in Clone 9 cells,
C2C12
myoblasts, and 3T3-L1 preadipocytes demonstrated that inhibition of oxidative phosphorylation by hypoxia and by inhibitors of oxidative phosphorylation such as cyanide and azide causes a stimulation of
glucose transport and an induction of GLUT-1 gene expression (2, 19,
25, 26). We have also shown in the above cells that, similar to other
"hypoxia-responsive genes" (8), GLUT-1 gene expression is
augmented by cobalt chloride
(CoCl2) employed under normoxic
conditions (2). On the basis of these findings, we recently explored
the possibility that exposure of diabetic and normal rats to
CoCl2 will similarly enhance
tissue GLUT-1 expression and tissue glucose uptake and thereby lead to
a lowering of blood glucose (31). The results showed that treatment of diabetic rats [induced by streptozotocin (STZ)] with 2 mM
CoCl2 added to the drinking water
resulted in a lowering of nonfasting serum glucose levels from 35 ± 2 to 21 ± 2 mM; a smaller decrement in glycemia of normal rats
treated with CoCl2 was not
significant. Treatment with CoCl2
was associated with a 1.3- to 2.9-fold increase in GLUT-1 mRNA content
of ventricular myocardium, renal cortex, skeletal muscle, cerebrum, and
liver of both diabetic and normal rats. Although GLUT-1 and glucose
transport were not measured in that study (31), the results are
consistent with the possibility that the glycemia-lowering effect of
CoCl2 may be mediated by enhanced
expression of GLUT-1 mRNA, GLUT-1 protein, and glucose uptake. However,
treatment with CoCl2 could also
have reduced hepatic glucose output and thus lowered blood glucose.
The present study was therefore conducted to determine the potential
role of enhanced tissue glucose uptake vs. decreased systemic glucose
production in mediating the glycemia-lowering effect of
CoCl2 in diabetic rats. The rate
of systemic appearance (Ra) of
glucose was measured under fasting conditions by tracer dilution in
diabetic and normal rats treated or not treated with CoCl2. We also determined the
effect of CoCl2 on the relative abundance of hepatic phosphoenolpyruvate carboxykinase (PEPCK) mRNA and on the concentration of cAMP in the liver as a measure of
changes in hepatic gluconeogenesis. The results indicate that the
dominant effect of CoCl2 in
reducing the glycemia of diabetic rats results from a reduction in the
Ra of glucose and decreased hepatic gluconeogenesis.
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MATERIALS AND METHODS |
Materials.
CoCl2 and other standard chemicals
were obtained from Sigma Chemical (St. Louis, MO). Reagent-grade ether
was obtained from Fisher (Pittsburgh, PA). The cAMP kit was obtained
from Amersham (Arlington Heights, IL).
[3-3H]glucose (10.4 Ci/mmol),
2-deoxy-[U-14C]glucose
(294 Ci/mmol), and
deoxy-[
-32P]cytidine
(3,000 Ci/mmol) were obtained from DuPont-NEN Research Products
(Boston, MA). PE-50 polyethylene nontoxic tubing (ID, 0.58 mm; OD,
0.965 mm) was obtained from Becton-Dickinson (Sparks, MD). The rodent
sling jacket was obtained from Harvard Bioscience (South Natick, MA).
QuickPrep total RNA extraction kit and Quikhyb were purchased from
Pharmacia Biotech (Piscataway, NJ) and Stratagene (La Jolla, CA),
respectively. GeneScreen Plus hybridization membrane was obtained from
DuPont-NEN Life Sciences. The cDNA probe for the cytosolic form of
PEPCK was a 1.1-kb Pst
I/Pst I fragment from the 3' end
of the rat PEPCK cDNA, pPCK 10 (32). Kits for measurement of cAMP were
obtained from Amersham.
Animals.
Male Sprague-Dawley rats weighing 225-250 g were obtained from
Zivic Miller (Zelienople, PA). The animals were kept in a controlled environment that meets the requirements of the American Association for
the Accreditation of Laboratory Animals. They had free access to rat
chow (Purina), and water was administered on a 12:12-h light-dark
cycle. The protocol was approved by the Institutional Animal Care and
Use Committee.
Diabetes was induced by injection of a freshly prepared solution of STZ
in saline at 60 mg/kg body wt in the tail vein (31). After 1 wk, a
sample of blood was obtained from the tail vein to ensure the presence
of diabetes (serum glucose >25 mM). One-half of the group of normal
rats and one-half of the diabetic group were then placed on 2 mM
CoCl2 in the drinking water for
12-16 days. During the ~2-wk period, normal and diabetic rats
gained ~30 and ~15 g of weight, respectively, and treatment with
CoCl2 resulted in no change in
weight gained by diabetic rats. In a previous study employing various
concentrations of CoCl2, we found that a similar group of diabetic rats treated with up to 4 mM CoCl2 for 7 wk gained ~100 g in
body weight, a rate that was identical to that in diabetic rats not
treated with CoCl2 (31). On the basis of the daily water intake, we estimate that diabetic and normal
rats received a total dose of ~2 and ~1 mmol of
CoCl2, respectively. Values for
serum glucose, electrolytes, and other constituents summarized in Table
1 were measured in the
hospital laboratory.
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Table 1.
Effect of treatment with CoCl2 on serum concentration of
certain constituents in diabetic and normal rats
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Measurement of Ra of glucose and of
glucose uptake by tissues.
These experiments were performed in different sets of animals from the
ones described above; diabetes and treatment with
CoCl2 were as indicated above.
Indwelling arterial and venous catheters were placed 3-4 days
before the tracer study. The animals were anesthetized with an
intramuscular injection (0.1 ml/100 g body wt) of ketamine-acepromazine
mixture (90 mg ketamine and 1 mg acepromazine per ml). PE-50 catheters
were inserted in the right external jugular and left carotid artery
under sterile conditions. The catheters were filled with an
anticoagulant mixture of polyvinylpyrrolidone and heparin in isotonic
saline (polyvinylpyrrolidone, 0.75 g; heparin, 25 units in 1 ml of
isotonic saline). This polymer allows catheter patency for a week or
more. The catheters were tunneled subcutaneously, and the distal end of
the catheter was sutured to the dorsum of the rat's neck. The free end
of the catheter was sealed, and the rat was placed in a Harvard rodent
jacket sling, thus allowing the animal to move freely during the
postoperative period. After surgery, rats were housed in individual
cages and had free access to a standard diet (Purina rat chow) and
water or CoCl2 solution as
indicated.
Twenty-four to thirty hours before the infusion, food was removed from
the animal cages.
[3-3H]glucose in
isotonic saline was given as a prime constant-rate infusion using a
Harvard pump, starting at 11:00 AM. The priming dose was 1.5-2.0
µCi, and tracer was infused at the rate of 0.05-0.07 µCi · kg body
wt1 · min1.
Four arterial samples (two 0.5- and two 0.2-ml samples) were obtained
at 10-min intervals between 80 and 120 min. Two hours after the
[3-3H]glucose
infusion, a bolus dose (20 µCi) of
2-deoxy-[U-14C]glucose
was administered. Heparinized blood samples of 0.2 ml were obtained at
1, 3, 5, 7, 10, 15, 20, 30, and 50 min. Total volume of blood drawn
from each animal was ~2.8 ml and was replaced with fresh blood from a
donor rat. At the end of the study, the animals were anesthetized using
intravenous pentobarbital sodium, and tissues including cerebrum, heart
(ventricle), kidney (cortex), skeletal muscle (red and white
gastrocnemius), and eye orbits were rapidly obtained, rinsed in
isotonic cold saline to wash off blood, blotted, and frozen in liquid
nitrogen.
For measurement of specific activity of glucose, plasma samples were
deproteinated with 0.3 M barium hydroxide and 0.3 M zinc sulfate. The
neutralized supernatant was evaporated to dryness to remove any
radioactive water formed during glycolysis. The dry samples were
reconstituted with water and passed through a mixed-bed ion-exchange
column (16). Glucose was eluted using 5 ml of water, evaporated to
dryness, and used to measure glucose concentration (Beckman glucose
analyzer) as well as radioactivity (Packard Instruments, Downers Grove,
IL). The amount of
2-deoxy-[14C]glucose
6-phosphate in various tissues was measured as described by Ferré
et al. (6). A weighed amount of tissue was homogenized in 1.0 ml of 1.0 M sodium hydroxide and heated at 60°C for 2 h or until total
digestion. The hydrolyzed homogenate was neutralized with 1 ml of 1.0 M
HCl. The accumulation of
2-deoxy-[14C]glucose
6-phosphate was measured from the difference between total
radioactivity
(2-deoxy-[14C]glucose + 2-deoxy-[14C]glucose
6-phosphate) and that due to
2-deoxy-[14C]glucose
(6).
The Ra of glucose was calculated
during isotopic steady state using the tracer dilution equation
Ra (µmol/min) = I/(sp act), where I is the rate of infusion of tracer isotope (dpm/min) and sp act
is the specific activity of plasma glucose (dpm/µmol). Tissue uptake
of glucose (nmol · g
tissue1 · min1)
was calculated as the ratio of tissue
2-deoxy-[14C]glucose
6-phosphate content to the integral of the specific activity
of
2-deoxy-[14C]glucose
in the plasma from 0 to 50 min, where the numerator represents the
2-deoxyglucose 6-phosphate radioactivity present in each tissue sample
(dpm/g tissue) (6). The integral of the specific activity of
2-deoxy-[14C]glucose
in the plasma was calculated by establishing the best-fit exponential
curve for the plasma specific activity measurements. No correction was
made for lump constant, the correction factor for the discrimination
against
2-deoxy-[14C]glucose
in glucose transport and phosphorylation pathways (6). It was assumed
that the lump constant will be the same in control and experimental
animals.
Measurement of cAMP levels in the
liver.
Groups of CoCl2-treated and
untreated normal and diabetic rats separate from those used above were
employed. Rats were decapitated after
CO2-inhalation anesthesia. Livers
were removed promptly and frozen immediately using liquid nitrogen or
dry ice. cAMP determinations are based on a competition enzyme
immunoassay as described by the manufacturer (Amersham). Approximately
100 mg of frozen liver were homogenized in 10 volumes of ice-cold 6% (wt/vol) trichloroacetic acid by means of a Teflon-glass homogenizer. After centrifugation at 2,000 g for 15 min at 4°C, a 250-µl aliquot of the supernatant was extracted
five times with 1 ml of water-saturated diethyl ether. Two-hundred
microliters of the aqueous phase were lyophilized and resuspended in 1 ml of the assay buffer supplied with the kit. cAMP determinations were
carried out using 100-µl aliquots of samples, with standards ranging
from 12.5 to 3,200 fmol/well.
Isolation of RNA and Northern blot
analysis.
Total RNA was extracted from livers of experimental animals using the
QuickPrep total RNA extraction kit. Seventy-five to one-hundred
milligrams of frozen liver (obtained as described above) were
homogenized and treated as per the manufacturer's instructions. RNA
samples (10 µg) were fractionated on formaldehyde-agarose gels and
transferred to GeneScreen Plus membrane by capillary action. The
membrane was hybridized with a randomly primed
32P-labeled rat PEPCK cDNA probe
(32) and washed, and the radioactivity in each band was measured by
video densitometry using a Molecular Dynamics phosphorimager. To ensure
equal RNA loading of the lanes and to control for completeness of RNA
transfer, ethidium bromide staining of ribosomal 28S and 18S bands on
the gels and on the nitrocellulose paper was monitored throughout.
To compare the relative abundance of hepatic PEPCK mRNA among different
experimental groups, the average densitometric measurement of PEPCK
mRNA in normal rats not treated with
CoCl2 was calculated and set to
1.0. Values obtained for all samples were then normalized against the
mean value obtained for the control group.
Analysis of data and statistical
methods.
Data on all four experimental groups, i.e., diabetic rats, diabetic
rats treated with CoCl2, normal
rats, and normal rats treated with
CoCl2, are presented throughout.
However, analysis of the potential effects of
CoCl2 on any given parameter was
performed in diabetic rats and normal rats treated (vs. not treated)
with the agent. This was done because diabetes itself is associated with changes in some of the parameters being examined.
All experimental results are expressed as means ± SE. Unpaired
Student's t-test was employed, and
P < 0.05 was considered significant
(27).
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RESULTS |
Effect of CoCl2 on the concentration of
glucose and selected other constituents in the blood of diabetic and
normal rats.
Normal and diabetic rats gained ~30 and ~15 g, respectively, during
the ~2 wk of study, and treatment with
CoCl2 resulted in no change in the
rate of weight gain by diabetic rats. Table 1 summarizes the effect of
10 days of treatment with CoCl2 on the concentration of several serum constituents in the four groups of
rats. Rats in both diabetic groups were not fasted before obtaining blood samples. In both groups of normal rats, with the exception of
serum glucose and insulin, values are from rats after a 24-h period of
food deprivation. Blood glucose was determined under nonfasting
conditions, because it was found in preliminary studies that blood glucose concentration in diabetic rats decreased to normal
levels after a 24- to 30-h period of food deprivation. In accordance
with our previous observations (31), treatment with
CoCl2 resulted in a dramatic
reduction of nonfasting glycemia of diabetic rats from 35.4 ± 2.8 to 21.2 ± 2.2 mM glucose (P < 0.01), whereas the agent resulted in no significant change in the serum
glucose concentration of normal rats. In diabetic rats treated with
CoCl2, there was a significant
decrease in the concentration of bicarbonate and phosphate compared
with diabetic rats not treated with the agent; the decrements in serum
cholesterol and triglyceride in
CoCl2-treated diabetic rats were
not significant, although two diabetic rats that had not been treated
with CoCl2 had lipemic serum.
Treatment of normal rats with
CoCl2 resulted in no measurable change in the concentration of any of the serum constituents. The
concentrations of insulin and glucagon in the serum of diabetic rats
treated or not treated with CoCl2
[both reported previously (31)] have also been included in
Table 1. After 10 days of treatment with
CoCl2, hematocrit values in both
normal and diabetic rats rose equally from 37 ± 1 to 40 ± 1%.
Effect of CoCl2 on the
Ra of glucose in diabetic and normal rats.
The Ra was determined in all four
experimental groups after 24-30 h of food deprivation; the fasting
period resulted in a normalization of serum glucose concentration in
both groups of diabetic rats. Serum glucose concentrations during
measurement of Ra were 6.37 ± 0.22 and 6.04 ± 0.42 mM in diabetic and
CoCl2-treated diabetic rats,
respectively. Ra, expressed either
as micromoles per minute (data not shown) or as micromoles per minute
per kilogram body weight, was ~50% lower in diabetic animals treated
with CoCl2 (35.5 ± 2.5 vs.
17.5 ± 1.8 µmol · kg1 · min1;
P < 0.01; Fig.
1). Ra
also was lower by ~35% in normal rats treated with
CoCl2 (from 47.5 ± 8.5 to 33.0 ± 3.0 µmol · kg1 · min1),
but the change was not significant (Fig. 1). Comparison of glucose
Ra values in diabetic vs. normal
rats [both groups not treated with
CoCl2] reveals no
significant difference between the two groups. Because the
concentration of glucose in the blood was constant during the 3-h
period of measurement of Ra, the
experimental results can be used to calculate the clearance of glucose
from the circulation. Glucose clearance was 0.89 ± 0.22 and 0.59 ± 0.07 ml · 100 g body
wt1 · min1
in normal and CoCl2-treated normal
rats, respectively (P > 0.1). It was
0.53 ± 0.04 and 0.26 ± 0.04 ml · 100 g body
wt1 · min1
in diabetic and CoCl2-treated
diabetic rats, respectively (P < 0.01).
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Fig. 1.
Effect of cobalt chloride
(CoCl2) on rate of systemic
appearance (Ra) of glucose in
diabetic and normal rats. Jugular vein and carotid artery indwelling
catheters were placed 72-96 h before study. Rats were deprived of
food for 24-30 h before experimentation.
Ra, measured as described under
MATERIALS AND METHODS, is expressed as
µmol glucose · kg body
wt1 · min1.
Results are expressed as means ± SE;
n = 5 rats/group.
Left: diabetic rats weighed 270 ± 10 g, and CoCl2-treated diabetic
rats weighed 270 ± 8 g; serum glucose concentrations during
measurement of Ra were 6.37 ± 0.22 and 6.04 ± 0.42 mM, respectively. Nonfasting serum glucose
concentrations before placement of catheters were 37.5 ± 3.0 and
20.6 ± 2.6 mM in diabetic rats and
CoCl2-treated diabetic rats,
respectively. * P < 0.05 compared with respective
CoCl2-untreated group.
Right: normal rats weighed 290 ± 20 g, and CoCl2-treated normal
rats weighed 260 ± 5 g; serum glucose concentrations during
measurement of Ra were 5.54 ± 0.18 and 5.64 ± 0.49 mM, respectively. Changes in
Ra resulting from
CoCl2 treatment are not
significant.
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Effect of CoCl2 on the rate of glucose
uptake by selected tissues in diabetic and normal rats.
After determination of Ra, glucose
uptake by several tissues of the same rats was measured by previously
described methods (6). Figure 2 summarizes
the amount of
2-deoxy-[14C]glucose
6-phosphate accumulated in tissues of diabetic rats not treated or
treated with CoCl2. It should be
noted that accumulation of glucose in tissues is a composite function
of transport and phosphorylation steps acting sequentially. Among the
tissues examined in the diabetic rat, heart and cerebrum exhibited high
rates of uptake (expressed as nmol · g tissue
wt1 · min1).
Because only a small fraction of the eye represents the metabolically highly active cells of the retina, this tissue probably manifests the
highest rate of glucose uptake and metabolism. The kidney had an
intermediate rate of uptake, whereas the red and white gastrocnemius
muscle exhibited lower rates. Treatment of diabetic rats with
CoCl2 tended to increase the rate
of uptake of
2-deoxy-[14C]glucose
by heart and liver, whereas the uptake by cerebrum, kidney, muscle, and
eye was decreased; of the aforementioned changes, only the decrements
in uptake by the kidney and eye were significant.
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Fig. 2.
Effect of CoCl2 on
2-deoxy-[14C]glucose
uptake in several tissues of diabetic rats. Glucose uptake was
estimated by accumulation of
2-deoxy-[14C]glucose
6-phosphate in various tissues. Experimental animals are same as those
in Fig. 1. Results are expressed as means ± SE;
n = 5 rats/group. Ventr, ventricle;
Gast R, red gastrocnemius; Gast W, white gastrocnemius.
* Significant decreases in uptake in kidney and eye
(P < 0.05 compared with respective
CoCl2-untreated group).
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Results of measurement of
2-deoxy-[14C]glucose
uptake by the indicated tissues in normal rats not treated or treated
with CoCl2 are summarized in Fig.
3. Compared with diabetic rats, rates of glucose uptake in normal rats are somewhat higher in cerebrum, liver,
and muscle and lower in heart, kidney, and eye; none of the changes are
significant. Treatment of normal rats with
CoCl2 resulted in a slight to
moderate decrease in the rate of glucose uptake by all the tissues
examined, although none of the changes reached significance. It is
probable, however, that the sum of uptake values by all the tissues
combined is reduced as a result of treatment with
CoCl2.
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Fig. 3.
Effect of CoCl2 on
2-deoxy-[14C]glucose
uptake in several tissues of normal rats. Glucose uptake was measured
as described in legend to Fig. 2. Experimental animals are same as
those in Fig. 1.
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Effect of CoCl2 on the content of PEPCK
mRNA in the livers of diabetic and normal rats.
We next examined the possibility that the activity of PEPCK, the key
enzyme in the control of gluconeogenesis, might be suppressed as a
result of exposure to CoCl2.
Because PEPCK activity closely parallels PEPCK mRNA content (13), we
measured the effect of CoCl2
treatment on the concentration of PEPCK mRNA in the liver. Two
experimental protocols were employed. In the first, the relative abundance of PEPCK mRNA was measured in livers of nonfasted normal rats, diabetic rats, and diabetic rats treated with
CoCl2 (Fig. 4A).
Rats were not fasted because food deprivation increases PEPCK expression in normal rats. The content of PEPCK mRNA in the liver of
diabetic rats was increased to 3.1-fold over that found in the liver of
nontreated rats (P < 0.001).
Treatment of diabetic rats with
CoCl2 resulted in a significant
reduction in the content of PEPCK mRNA in the liver to levels similar
to those found in livers of normal rats not treated with
CoCl2
(P > 0.4). In a separate set of experiments, we found that treatment of diabetic rats with CoCl2 under nonfasting conditions
also decreased liver PEPCK mRNA content by approximately threefold
(P < 0.05; data not shown). In the
second protocol, the effect of
CoCl2 on the level of PEPCK mRNA
in the liver of nondiabetic animals was determined. In this experiment,
normal and CoCl2-treated normal
rats were deprived of food for 24 h to elicit an upregulation of PEPCK
gene expression before study (Fig.
4B). The relative abundance of
hepatic PEPCK mRNA was decreased by 33% in normal rats treated with
CoCl2 compared with the control
group (P < 0.05). The
effect of CoCl2 was also determined in nonfasted normal and
CoCl2-treated normal rats. PEPCK
mRNA content was present at low levels in liver of fasted normal rats
and decreased to below detectable levels in
CoCl2-treated rats (data not
shown).
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Fig. 4.
Effect of CoCl2 on relative
abundance of phosphoenolpyruvate carboxykinase (PEPCK) mRNA in
livers of diabetic (Diab) and normal (N) rats. Diabetic or normal rats
were treated or not treated with
CoCl2 for 12 days before assay.
Rats were killed, and livers were rapidly removed, frozen in dry ice,
and assayed within 2 wk. A:
animals were not fasted. B:
animals were deprived of food for 24 h before experiment. In each
group, results were normalized against average value in normal rats.
Results are expressed as means ± SE;
n = 5-6 rats in each group.
** P < 0.001, * P < 0.05 compared with
respective CoCl2-untreated
group.
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Effect of CoCl2 on the levels of cAMP in
the livers of diabetic and normal rats.
It is well established that the expression of the gene for the
cytosolic form of PEPCK in liver is highly regulated by several hormones, including glucagon, insulin, growth hormone, and cortisol, and by the nutritional status of the animal (10). Hence the reduction
in the levels of PEPCK mRNA in livers of
CoCl2-treated diabetic and normal
animals summarized above may well be mediated by alterations in one or
a combination of the above regulators. Because of the dominant and
opposing roles of glucagon (acting through cAMP) and insulin on the
regulation of PEPCK expression (9, 17) and because of our finding that
the concentration of serum insulin is not changed as a result of
treatment with CoCl2 in either
diabetic or normal rats (Table 1), we examined the possibility that the
concentration of hepatic cAMP is reduced in
CoCl2-treated rats (Fig.
5). Under nonfasting conditions, the concentration of cAMP is slightly (but not significantly) lower in the
livers of diabetic compared with normal rats (Fig.
5A) and decreases slightly as a
result of CoCl2 treatment.
Similarly, there was no significant change in cAMP levels in 24-h
food-deprived normal rats treated with
CoCl2 (Fig.
5B). Food deprivation in normal
rats, however, was associated with a significant increase in hepatic
cAMP levels from 625 ± 77 to 823 ± 40 pmol/g wet wt (P < 0.05).
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Fig. 5.
Effect of CoCl2 on concentration
of cAMP in livers of diabetic and normal rats. Experimental animals
were same as those in Fig. 4. Animals were either not fasted
(A) or deprived of food for 24 h
(B) before experiment. Results are
expressed as means ± SE; n = 5-6 rats in each group. None of changes induced by
CoCl2 are significant.
Concentration of cAMP in liver of food-deprived normal rats is
significantly higher than that in nonfasted normal rats
(P < 0.05).
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DISCUSSION |
The present study was prompted from the earlier observation that
treatment of STZ-induced diabetic rats with
CoCl2 results in a significant
reduction in the serum glucose concentration (31). In principle, the
glycemia-lowering effect of CoCl2
could be secondary to decreased systemic glucose production, increased tissue glucose uptake, or a combination of the two mechanisms. The
observed induction of GLUT-1 mRNA in several tissues of
CoCl2-treated rats suggested that
enhanced glucose uptake may well play a dominant role in the above
effect, although GLUT-1 expression and glucose transport were not
measured in that study (31). The results of studies reported herein,
however, indicate that the glycemia-lowering effect of
CoCl2 is mediated by a reduction
in the Ra of glucose and possibly
gluconeogenesis. It is worth emphasizing that the results of the
present study are internally consistent, i.e., the decrease in glycemia
of CoCl2-treated diabetic rats is
associated with a significant reduction in
Ra and a decrease in liver PEPCK mRNA content. A reduction in calorie intake as the explanation for the
reduction in glucose production is excluded by the observation that, in
accordance with previous results (31), diabetic rats treated or not
treated with CoCl2 gained weight
at an equal rate during the 2-wk period of treatment.
Previous reports indicate that glucose
Ra values in non-insulin-dependent
diabetic subjects are higher than in normal controls (22), although the
higher Ra values in diabetic
subjects are documented under conditions in which the blood glucose
concentrations are significantly higher than for nondiabetic controls
(28). Ra values measured in the
present study, whether expressed per animal or per unit body weight,
were somewhat but not significantly lower in the diabetic animals. The
reasons for this apparent discrepancy are unknown but may reflect the
fact that the model employed in the present study is one of
insulin-dependent diabetes. Moreover, unlike previous protocols,
diabetic and normal rats in this study were deprived of food for
24-30 h before measurement of glucose Ra, a condition which served to
markedly reduce the serum glucose concentration of the diabetic group
to levels approximating those in food-deprived normal rats. A profound
depressing effect of a prolonged period of food deprivation (72 h) on
hepatic glucose production in the rat has been reported previously (1).
Treatment of diabetic rats with
CoCl2 resulted in ~50%
reduction in glucose Ra. If
glucose Ra is decreased by a
similar extent under nonfasting conditions, then this change alone
would be expected to result in a significant decrease in the glycemia
of diabetic rats.
There was a tendency toward lower
2-deoxy-[14C]glucose
uptake in tissues of CoCl2-treated
rats, although only in the case of kidney and eye in diabetic rats was
the decrease significant. It is possible that the marked reduction in
glycemia of diabetic rats during the period of food deprivation results
in a lower rate of tissue glucose uptake and thereby leads to a masking
of any further decrement in glucose uptake by treatment with
CoCl2. In keeping with this
premise, it has been reported that food deprivation for 72 h in normal
rats results in a decrease in sensitivity of peripheral tissues to the
actions of insulin (1). Nevertheless, the summation of tissue uptake
values, reflecting the uptake by the organism as a whole, is apt to be
reduced in CoCl2-treated animals.
It should also be noted that the decreased clearance of glucose from
the circulation of diabetic rats treated with CoCl2 might help explain the
finding that the glycemia of these rats is not lower than that of
diabetic rats not treated with CoCl2.
Because the measurements of Ra and
2-deoxy-[14C]glucose
uptake in various tissues were performed after a fasting period of
24-30 h, it is highly likely that
Ra of glucose closely reflects
gluconeogenesis rather than glycogenolysis. We therefore explored the
possibility that treatment with
CoCl2 reduces gluconeogenesis by
the liver, a process that is controlled in part by the level of PEPCK
activity (29). Because PEPCK activity closely parallels the level of its mRNA (13), we measured the relative abundance of PEPCK mRNA in
livers of diabetic and normal rats treated with
CoCl2. In accordance with previous
findings, the abundance of PEPCK mRNA was increased in livers of
diabetic rats compared with normal controls under nonfasting conditions
(7). In addition, treatment with
CoCl2 significantly reduced
hepatic PEPCK mRNA levels in diabetic rats under fasting or nonfasting
conditions. CoCl2 treatment also
significantly reduced PEPCK mRNA levels in the livers of normal rats.
However, a direct correspondence between PEPCK mRNA and hepatic glucose production may not always exist, especially under differing
experimental and nutritional conditions. It is worth emphasizing that
the decrease in PEPCK mRNA content does not represent a nonspecific or
toxic effect of the agent because diabetic rats treated with up to 4 mM
CoCl2 for 7 wk demonstrated no
reduction in weight gain compared with diabetic controls not treated
with CoCl2 (31), and the abundances of other mRNAs such as those encoding erythropoietin (8, 30)
and GLUT-1 and GLUT-2 (31) are increased in
CoCl2-treated rats.
The mechanism by which CoCl2
modifies the level of PEPCK mRNA in the livers of diabetic rats is of
interest. When CoCl2 is added to
cells in the presence of oxygen, many of its effects on gene expression
mimic the effects noted in response to lowered oxygen concentration
(8). For example, transcription of the gene coding for GLUT-1 is
stimulated in response to hypoxia by oxygen-sensing molecules that can
also be activated by cobalt (2). Cobalt is thought to alter gene
transcription by increasing the level of hypoxia-induced factor
(HIF)-1, a transcription factor that binds to a regulatory element
(CGTGCTG) in the promoter of a number of genes, most notably
erythropoietin and vascular endothelial growth factor genes (30). The
steady-state concentration of HIF-1 is induced by hypoxia or
CoCl2 by a mechanism that involves the stabilization of the protein against degradation, resulting in an
accumulation of the transcription factor (24). The gene for PEPCK has
also been shown to respond to changes in the redox state of liver cells
in culture (11). This gene is expressed in the liver in a decreasing
gradient from the periportal to the pericentral region (14), presumably
because of higher levels of oxygen and nutrients supplied to
hepatocytes in the periportal region. Previous studies by Hellkamp et
al. (11) have also demonstrated that glucagon-induced transcription
from the PEPCK promoter is inhibited by reducing the concentration of
oxygen from 16 to 8%. The hypoxia-inducible DNA element noted in the
3'-flanking region of the erythropoietin gene is also present in
the PEPCK promoter at 
129 to 121 kb (from the
transcription start site), a position that is immediately 5' to a
control region that includes the cAMP and the nuclear factor-1
regulatory elements (120 to 80 kb), which are critical
for both basal and cAMP-induced transcription of the PEPCK gene (18,
21). However, T. Kietzmann (personal communication) has implicated a
site in the PEPCK gene between 277 and 174 kb as being
required for the negative effect of redox state on transcription from
the PEPCK promoter in primary hepatocytes in culture (15). Although it
is possible that the effect of
CoCl2 noted in the present study
is due to an inhibition of transcription of the PEPCK gene in the liver
via a mechanism involving HIF-1, it is likely that there are multiple
elements in the PEPCK promoter involved in the effect of cobalt on
PEPCK gene expression.
Cytosolic PEPCK gene expression in the liver is highly controlled by a
number of hormones and physiological conditions and most importantly by
glucagon and insulin mediation of positive and negative regulation,
respectively. Results of previous studies indicate that treatment with
CoCl2 does not alter the
concentration of insulin in the blood of normal rats (3, 31) and does
not increase the extremely low levels of insulin in the blood of
STZ-induced diabetic rats (31). It has also been reported that livers
of CoCl2-treated nondiabetic rats
are relatively "insensitive" to glucagon action, since the
release of glucose by the liver in response to glucagon is decreased
both in vivo and in vitro (4); the lower release of glucose is present,
although the livers of CoCl2-treated rats contain higher
levels of glycogen (3). Because of the critical regulation of PEPCK
gene transcription by glucagon and cAMP, we elected to measure the
levels of this nucleotide in liver of diabetic and normal rats treated
or not treated with CoCl2. The
results showed no systematic change among any of the groups examined
except for the finding that food deprivation of normal rats resulted in
a significant increase in the level of cAMP in the liver. It has been
reported that CoCl2-treated rats appear to be more "sensitive" to the action of insulin in
enhancing glucose disposal from the blood after a glucose load (3). If the liver of CoCl2-treated rats is
also more sensitive to actions of insulin, then such a finding might
help explain the lower glucose Ra
in such animals. This explanation, however, cannot be extended to
diabetic rats, given the extremely low levels of insulin in these
animals. Further studies are required to gain a better understanding of
the mechanisms mediating the apparent reduction of gluconeogenesis in
CoCl2-treated diabetic and normal
rats.
| |
ACKNOWLEDGEMENTS |
This study was supported, in part, by grants from the Diabetes
Association of Greater Cleveland and the National Institutes of Health
(DK-45945 to F. Ismail-Beigi, DK-25541 to R. W. Hanson, and HD-11089 to
S. C. Kalhan). P. Leahy and F. Saker were trainees on the National
Institute of Diabetes and Digestive and Kidney Diseases Metabolism
Training Grant DK-07319.
| |
FOOTNOTES |
F. Saker and J. Ybarra contributed equally to this study.
Address for reprint requests: F. Ismail-Beigi, Clinical and Molecular
Endocrinology, Case Western Reserve Univ., Cleveland, OH 44106-4951.
Received 22 October 1997; accepted in final form 20 February 1998.
| |
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