(Received for publication, August 28, 1995; and in revised form, October 30, 1995)
From the
The effect of zinc ions on carbohydrate metabolism and
intracellular Zn was studied in hepatocytes from fed
rats. The addition of ZnCl
to the medium led to an almost
3-fold increase in lactate production and an increase in net glucose
production of about 50%. Half-maximal rates occurred at about 40
µM ZnCl
. These effects were not seen with
Mn
, Co
, or Ni
up
to 80 µM, whereas Cu
at 80 µM and Cd
or Pb
at 8 µM exhibited similar effects as 80 µM ZnCl
.
Changes in intracellular Zn
were followed by single
cell epifluorescence using zinquin as a specific probe. Intracellular
free Zn
in isolated hepatocytes was 1.26 ±
0.27 µM, and the addition of ZnCl
led to a
concentration-dependent increase in epifluorescence. CdCl
or PbCl
at 8 µM was as potent as
ZnCl
at 20-80 µM, whereas NiCl
at 80 µM was without effect. ZnCl
completely abolished the inhibition of glycolysis by glucagon
(cAMP). Glucagon led to a pronounced drop in cytosolic
Zn
. Both glucagon and zinc stimulated glycogenolysis
by increasing the phosphorylation of glycogen phosphorylase but acted
oppositely on glycolysis. Zinc overcame the inactivation of pyruvate
kinase by glucagon without changing the hormone-induced protein
phosphorylation. The antagonistic action of zinc and cAMP on glycolysis
together with the rapid and marked decrease in free zinc concentration
induced by glucagon (cAMP) may indicate an as yet unknown role of zinc
as an important mediator of regulation of carbohydrate metabolism.
Glycolysis in the liver is generally assumed to be regulated at
the steps catalyzed by hexokinase/glucokinase, phosphofructokinase-1,
and pyruvate kinase. We have shown that in vitro a 11.5-kDa
Zn-binding protein (ZnBP), (
)which is
identical with parathymosin, interacts in a zinc-specific manner with
key enzymes of carbohydrate metabolism, including the liver enzymes
aldolase, phosphofructokinase-1, hexokinase, glucose-6-phosphate
dehydrogenase, glycerol-3-phosphate dehydrogenase,
glyceraldehyde-3-phosphate dehydrogenase, and
fructose-1,6-bisphosphatase(1) . ZnBP is present in the liver
at high concentrations of about 20 µM(2) , yet its
physiological function is unknown, though ZnBP was discovered by its
property to inactivate phosphofructokinase-1 in a reversible,
zinc-dependent manner(3) .
So far only few data are available on the effects of zinc on carbohydrate metabolism. With cytosol from muscle, zinc at half-maximal concentration of about 0.1 mM stimulated lactate production from glucose 6-phosphate(4) . Rognstad (5) reported that zinc markedly inhibited glycogen synthesis in hepatocytes from starved rats; moreover, in hepatocytes from fed rats no effect could be observed on the glycogenolysis. In rat adipocytes zinc stimulated glucose transport (6, 7) , incorporation of glucose into lipids(8) , and glucose oxidation by both pathways, glycolysis and hexose monophosphate shunt(9) .
Zinc is known to play a
pivotal role in the regulation of DNA binding and activation of
transcription factors or in the regulation of apoptosis. As for
Ca intracellular zinc levels have been suggested to
be under homeostatic control. The latter may be essential to maintain
the diversity of biochemical functions in the intermediary metabolism,
which are dependent on zinc proteins and enzymes(10) . However,
little is known about the level of Zn
in intact cells
and to what extent changes in intracellular distribution may influence
cellular events. Hitherto the rather high affinity constants of
metalloenzymes for zinc have inferred the free zinc concentration in
cells to be in the order of 10
M or even
lower(11) . Recently, Zalewski et al.(12) have introduced the fluorescent indicator zinquin
(ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy) acetate
as an intracellular probe for Zn
. Using Jurkat
lymphoid cells pretreated with pyrithione and zinc, they estimated the
free or ``labile'' intracellular zinc as detected by zinquin
to be in the order of 2 nmol/10
cells(12) , which
would correspond to an intracellular concentration of
10
-10
M, assuming
at least 10
cells/g of wet weight. The concentration of
free zinc detected by these authors in cultured rat hepatocytes or in
liver slices resembled the total zinc concentration in liver, and
therefore, zinquin should be used with caution as a probe to measure
Zn
in living cells(13) .
In hepatocytes the bulk of cellular zinc is thought to be bound as metallothioneins (MT). Accordingly, a rise in total cellular zinc has been observed following increased de novo synthesis of MT in the presence of glucocorticoids or glucagon(14) . However, little is known about short term effects on the distribution of zinc ions within the cell.
The present study was designed to look for the effects of zinc
ions on carbohydrate metabolism of hepatocytes as an indirect
indication of an involvement of ZnBP in the regulation of glycolysis
and gluconeogenesis. If the zinc-dependent interaction of ZnBP with key
enzymes of the glycolytic and gluconeogenic pathways were of any
importance in vivo, a rise in intracellular Zn should result in marked changes of flux rates. Further, it is
unknown if and by which mechanism intracellular zinc can be mobilized
to increase the cytosolic concentration of free zinc.
Here we report
that incubation of hepatocytes from fed rats with ZnCl leads to a 2-3-fold increase in the rate of lactate
production and to an enhanced glucose production, due to an enhanced
glycogenolysis and an increased flux from glucose-6-phosphate through
the glycolytic pathway. We show further that zinc counteracts the
inhibition of glycolysis by glucagon or cAMP.
In parallel, we
measured zinquin-dependent epifluorescence of single hepatocytes. The
effect of various metal ions and glucagon or cAMP on the rapid
exchangeable cytosolic zinc concentration was investigated. The
similarity of increase in glycolysis seen with ZnCl as
compared with CdCl
or PbCl
can be explained by
their ability to mobilize intracellular zinc. Moreover, by single cell
measurement we can show for the first time that glucagon (or cAMP)
rapidly decreases the level of intracellular Zn
to
less than 20% of that of control.
Cell
integrity was measured by trypan blue exclusion and usually resulted in
90-95% of the cells appearing viable. Because of a possible cell
toxicity of ZnCl or other heavy metal ions used, the
activity of lactate dehydrogenase in the incubation medium was measured
over the length of experiments.
The cells were incubated at 30 °C for up to 30 min
under conditions as specified in the text. For fluorescence
measurements hepatocytes (1-2 mg of wet weight) were withdrawn at
time intervals as indicated, suspended in 2 ml of oxygenated HMS medium
in a Petri dish (Falcon 3001, with a centered 10-mm glass window), and
immediately placed in the light path of the microscope. The
fluorescence of single hepatocytes at 380-390 nm excitation was
monitored at 480-540 nm (see below). Zn was
determined from the increase in fluorescence. Usually the
epifluorescence of 20-25 single hepatocytes under each condition
was averaged. To correct for autofluorescence, control incubation(s)
using hepatocytes ``preloaded'' in the presence of solvent
but otherwise treated identically were always run in parallel. The
average in epifluorescence of again 20-25 single hepatocytes was
subtracted from the corresponding values measured in the presence of
the dye. The concentration of the solvent(s) in the incubation never
exceeded 1% (v/v). The same amount of solvent was added to control
incubations.
Fluorescence measurements were performed using a Fura-2 data acquisition system (Luigs & Neumann GmbH, Ratingen, FRG) mounted to an inverted microscope (Zeiss IM 35). The sampling rate was 2 measurements/s. For a more detailed description and evaluation of the equipment see Neher(16) .
The concentration of
Zn was calculated according to Grynkiewicz et
al.(17) . Maximal fluorescence was determined in the
presence of 40 µM Zn
plus maltol (0.1
mM), and minimal fluorescence was determined with N,N,N`,N`-tetrakis
(2-pyridylmethyl)ethylenediamine (50 µM) plus maltol (0.1
mM). The stability constant of the
Zn
-zinquin complex was determined according to
Hagenmuller (18) using a medium resembling the intracellular
environment (130 mM KCl, 15 mM NaCl, 0.5 mM MgCl
, 50 mM Mops, pH 7.05, and 0.01% bovine
serum albumin). The dissociation constant for the 1:1 complex present
under our condition was 1.14 ± 0.16
10
mol/liter. This value compares favorably with a value of 0.37
10
mol/liter given by Zalewski et
al.(12) .
If not otherwise stated, the values given are
the means ± S.E. of 20-25 single cells/dish under each
condition. The experiments were repeated at least three times with
independent cell preparations. Total zinc and calcium were measured by
atomic absorption after acid extraction and appropriate dilution of the
sample in 0.1 M HCl with 0.3 g of LaCl/100 ml.
For cross-over studies, at least 6 ml of
suspension were used from each incubation. Protein was precipitated by
the addition of 0.1 vol of 35% HClO and removed by
centrifugation. An aliquot of the supernatant was neutralized with 5 M KOH and centrifuged. The pellet was washed twice with 0.1 M KCl. The combined supernatants were treated with Florisil
until they became colorless and were lyophilized. The samples were
dissolved in 1-1.5 ml of H
O. Determinations of
metabolites and adenine nucleotides were performed essentially as
described by Bergmeyer(19) , but in addition, all assays
contained 1 mM diethylenetriaminepentaacetic acid.
Linearity of
metabolic rates was assured by measuring lactate and glucose production
in 10-min intervals up to 40 min. Metabolic rates were constant for at
least 30 min under all conditions used (minus or plus ZnCl,
glucagon, or Bt
-cAMP). Therefore, metabolic rates were
regularly determined from the 10 and 20 min values; metabolites were
determined at 20 min or at an earlier time point.
A preincubation of
10 min proved to be sufficient for hepatocytes to equilibrate in terms
of temperature and oxygen. This time was also sufficient to reach a
constant zinc level in the medium. This is shown in Fig. 1,
where the uptake of zinc is depicted from experiments with 0.05 and
0.10 mM ZnCl added to the medium. With hepatocytes
from starved rats, the time course of zinc uptake was comparable with
that seen with hepatocytes from fed rats. Interestingly the endogenous
zinc concentration was always higher in hepatocytes from rats starved
overnight than from fed rats. The mean value ± S.E. was 415
± 15 nmoles of zinc/g of wet weight (n = 9) in
hepatocytes from starved rats, as compared with 264 ± 11
nmoles/g of wet weight (n = 14) in hepatocytes from fed
rats. Interestingly, Coyle et al.(23) have found the
MT concentration in hepatocytes from fasted rats were double those from
fed rats.
Figure 1:
Time course of zinc uptake. Freshly
prepared hepatocytes (102 mg of wet weight/ml) from fed rats were
incubated in the presence of 0.1 mM ZnCl (open
symbols) or 0.05 mM ZnCl
(closed
symbols) in the medium and under conditions as given under
``Experimental Procedures.'' At the given time points,
samples of 0.5 ml were removed. Cells were separated from the medium by
centrifugation in an Eppendorf centrifuge for 15 s, and the
supernatants were removed immediately. Both fractions were treated with
HClO
to a final concentration of 3.5%, denaturated protein
was sedimented by centrifugation, and the content of zinc was
determined by atomic absorption spectroscopy after appropriate
dilution. The dashed lines give the amount of zinc in the cell
fraction, and solid lines show the amount of zinc in the
medium. The values are calculated for 1 ml of cell
suspension.
Apart from following the exclusion of trypan blue, the
leakage of lactate dehydrogenase was followed over the time of
experiments as an additional indicator of cell membrane integrity. In
general, the activity of lactate dehydrogenase found in the medium was
about 5% of the total activity, and after 30 min of incubation this
amount increased to about 10%. Intracellular calcium was measured as an
additional indicator of cell viability. During the incubation period of
20 min, the total cellular calcium remained constant in the control
incubations and did not change in the presence of 0.1 mM ZnCl. The total calcium concentration was 2.5
µmol/g of wet weight, which is about twice that found in the intact
liver (24) .
Figure 2:
Effect of zinc on the rates of lactate and
glucose production. Hepatocytes from fed rats were incubated without
and with increasing ZnCl concentrations for 20 min. The
changes of lactate and glucose concentrations were determined from each
incubation. Further conditions of incubation are given under
``Experimental Procedures.'' The values are the means
± S.E. of the numbers of experiments from independent cell
preparations given in the bars.
Under all conditions,
the addition of the membrane-impermeable chelating agent
diethylenetriaminepentaacetic acid to the medium at concentrations
equimolar to those of ZnCl abolished the effects of zinc
(data not shown, but see Fig. 3). Moreover, no difference in the
oxygen consumption of hepatocytes could be detected in the presence of
maltol or maltol plus 0.1 mM ZnCl
as compared with
controls (results not shown).
Figure 3:
Mobilization of Zn by
various cations. Hepatocytes were preloaded with zinquin as given under
``Experimental Procedures.'' Cells (25-30 mg of wet
weight/ml) were thermoequilibrated in HMS medium in the presence of 15
mM glucose at 37 °C under oxygenation in a shaking water
bath. After 5 min, chloride salts of the various cations were added at
the concentrations indicated, and the incubation was continued for 30
min. An aliquot of 50 µl was withdrawn and mixed with 2 ml of HMS
buffer in a Falcon Petri dish, and epifluorescence of single
hepatocytes was determined as given under ``Experimental
Procedures.'' A reference incubation of cells preloaded with
solvent instead of zinquin but otherwise treated identically was
analyzed in parallel (autofluorescence, open bars). The
average (mean ± S.E.) in epifluorescence at 390 nm excitation of
20-30 cells/dish is plotted. DPTA, diethylenetriaminepentaacetic
acid.
Because we had to use a medium without
phosphate or bicarbonate, the zinc effect was compared in parallel
incubations using HMS medium and KRB buffer with 1.2 mM CaCl. Because zinc is completely insoluble in KRB
buffer alone, maltol had to be added at much higher concentrations
(0.42 mM) to maintain a Zn
concentration of
0.1 mM. This titration was verified by atomic absorption
spectroscopy. Under these conditions we could show that the zinc
effects in both media were comparable. Glycolysis was doubled in both
media, the glycogenolysis (lactate plus glucose formation) in terms of
glucose equivalents increased by 44% ± 13 (3) in KRB
buffer and 55% ± 16 (3) in HMS medium. In both media the
complete inhibition of glycolysis by 0.05 mM Bt
-cAMP (see below) was abolished by 0.1 mM zinc.
CdCl and PbCl
were
cell toxic at concentrations above 40 µM. At 8
µM, however, both cations were as effective stimulating
the rates of lactate and glucose production as ZnCl
at 80
µM.
Glucose formation was increased by glucagon due to
enhanced glycogenolysis. ZnCl had no additional effect at
maximally effective concentrations of glucagon, whereas at submaximal
rates of glucose formation, it had an additive effect (not shown).
Whereas stimulation of glycogenolysis by glucagon is caused by
increased levels of cAMP, vasopressin acts by increasing the cytosolic
concentration of free calcium via inositol 1,4,5-trisphosphate.
Vasopressin (0.5-5 10
M)
itself had no effect on the net lactate production, although the cells
were hormone-sensitive as indicated by the enhanced glucose formation
(
143%). The addition of ZnCl
increased the rate of
lactate formation in the presence of vasopressin (see Table 1) to
the rate found with ZnCl
alone. It is therefore unlikely
that the effect of zinc on glycolysis is due to a mobilization of
calcium. This conclusion is strengthened by our observation that in the
presence of ZnCl
the O
consumption of
hepatocytes was not different from control incubations (results not
shown).
In order to exclude the possibility of an interaction of
zinc with the glucagon receptor, experiments were performed with
Bt-cAMP. As shown in Table 1(part C), 25, 50, and
100 µM Bt
-cAMP strongly inhibited lactate
formation. The addition of 0.1 mM ZnCl
to the
medium overcame this inhibition and led to an enhanced glycolysis. With
increasing concentration of Bt
-cAMP, the zinc-induced
stimulation of glycolysis was reduced. Glucose output stimulated by
Bt
-cAMP was not altered by the addition of
ZnCl
.
Figure 4:
Effect
of glucagon or 8-bromo-cAMP on Zn level in isolated
rat hepatocytes. Hepatocytes were preloaded with zinquin as given under
``Experimental Procedures.'' Cells (25-30 mg of wet
weight/ml) were thermoequilibrated at 37 °C in a shaking water
bath. After 5 min a sample of 50 µl was withdrawn for immediate
measurement of epifluorescence, the agonists were added, and the
incubation was continued for 20 min. At 10 and 20 min, identical
aliquots were removed for epifluorescence measurements. Other
conditions were as given in Fig. 3. The epifluorescence at 390
nm excitation was averaged for 20-30 cells/dish, and the average
in autofluorescence of again 20-30 cells/dish was subtracted. The
calculation of free Zn
was done as given under
``Experimental Procedures.'' Representative experiments are
depicted. A, glucagon, 10
M; B, 8-bromo-cAMP, 0.1 mM. Open symbols,
control; filled symbols, agonist. Inset, dose
dependence of glucagon effects on free Zn
concentration. The conditions were as given above, except that 34
mg of wet weight/ml were used. The samples were taken after 10 min of
incubation.
To assess whether the rapid decrease in
cytosolic Zn was due to an increased binding of the
cation by MT, the potential of Cd
(8 µM)
to discharge zinc from MT was investigated. The ability of
Cd
to raise cellular Zn
was similar
in the absence or the presence of cAMP, indicating that a portion of
zinc not associated with MT is reduced (not shown).
A
distinct cross-over point at pyruvate kinase indicates that the
conversion of phosphoenolpyruvate to pyruvate was increased in the
presence of zinc. Furthermore, the data show that changes in levels of
metabolites caused by glucagon are counteracted by the addition of
ZnCl.
The observed increase in glycogenolysis and
glycolysis by zinc could be due to a stimulation of phosphorylase b and of phosphofructokinase-1 by a rise in AMP. In our experiments
the ATP concentrations were between 1.5 and 2.1 µmol/g of wet
weight, and the AMP concentrations were about 34 nmol/g of wet weight.
Both parameters did not change considerably under conditions where the
rate of lactate production increased 3-fold due to the addition of
ZnCl. The ATP/ADP ratios were between 3.02 and 3.5 and also
not affected by the presence of zinc.
Figure 5:
Phosphorylation state of pyruvate kinase,
glycogen phosphorylase, and fructose-1,6-bisphosphatase extracted from
hepatocytes incubated with and without ZnCl with
Bt
-cAMP and with Bt
-cAMP plus ZnCl
.
Hepatocytes from fed rats were preincubated with
[
P]P
(1 mCi/g of wet weight) for 30
min. The medium was changed, and the cells were thereafter incubated
without (lanes 1) and with 0.1 mM ZnCl
(lanes 2), with 0.1 mM Bt
-cAMP (lanes 3), and with Bt
-cAMP plus 0.1 mM ZnCl
(lanes 4), as given under
``Experimental Procedures.'' After 20 min of incubation,
cells were sedimented and extracted with 0.2% triton X-100. From
aliquots (0.1 ml) of the supernatants pyruvate kinase, glycogen
phosphorylase, and fructose-1,6-bisphosphatase were immunoprecipitated,
separated by SDS-polyacrylamide gel electrophoresis, and
autoradiographed as described under ``Experimental
Procedures.'' F, fructose-1,6-bisphosphatase; GP, glycogen phosphorylase; PK, pyruvate
kinase.
We have shown here that incubation of hepatocytes from fed
rats with zinc in the medium strongly enhances the formation of lactate
and glycogenolysis. The effect depends on the concentration of
ZnCl added to the medium and is significant at
concentrations of 33 µM and higher. A comparable effect of
zinc was also seen with KRB buffer, provided it had been supplemented
with enough maltol. The failure of Rognstad (5) to observe this
effect was most probably due to the insolubility of zinc in a medium
containing phosphate and bicarbonate. Using his medium we were unable
to detect zinc in the solution by atomic absorption spectroscopy, even
with concentrations as high as 0.5 mM ZnCl
.
Therefore, genuine zinc effects cannot be expected under these
conditions without an appropriate membrane-permeable chelator (i.e. maltol).
The change in intracellular free zinc is much lower than the changes of total zinc found in the medium after the equilibration period (Fig. 1). In liver cytosol various proteins (e.g. MT) and low molecular weight compounds will act as buffers by binding large amounts of the added zinc, depending on their stability constants, which in general are higher than that of the maltol-Zn complex, which is 5.62 (26) .
We have good
evidence that the effects of zinc on glycogenolysis and glycolysis are
specific for zinc ions. Ca, Mg
,
Mn
, Fe
, Ni
, and
Co
were ineffective. CuCl
, however, at
comparable concentration and CdCl
or PbCl
at
10-fold lower concentration showed the same effects as
ZnCl
. Those cations showing the zinc-like effect on
metabolism did indeed raise intracellular free zinc (Fig. 3). It
seems feasible to conclude that these metal ions mobilize
Zn
from Zn-MTs. This conclusion is supported by in vitro displacement studies (26) that show that
Co
and Ni
could not displace zinc
from Zn-thionein, whereas Cd
, Pb
,
and Cu
could. The rank of affinities in this study
was: Cd > Pb > Cu > Zn > Ni > Co. The metabolic effects
observed here as well as the data on intracellular Zn
mobilization fit exactly into this order of affinities.
Furthermore, using the isolated perfused liver, Kingsley and Frazier (28) have shown that exposure to cadmium increases both zinc
secretion into the perfusate and biliary excretion of zinc.
Here we
have determined the concentration of free Zn by
monitoring single cell epifluorescence using zinquin as an
intracellular probe. The advantage of this technique as compared with
bulk measurements of zinquin fluorescence in cell
suspension(12, 13) is that specific hepatocytes of
uniform size and round shape and lacking signs of cell damage can be
examined for zinquin-dependent fluorescence. Recently, Zalewski et
al.(12) reported that apoptotic HL-60 cells revealed
bright zinquin fluorescence, indicating an increased zinc
concentration, membrane blebbing, and often a decrease in cellular
volume. Conversely, zinquin seems to escape from necrotic hepatocytes,
these cells showing therefore no or a markedly reduced fluorescence.
Coyle et al.(13) , using zinquin to follow changes in
labile zinc in hepatocytes in primary culture or in liver slices
treated with dexamethasone and interleukine-6 to induce metallothionein
synthesis, have advised not to compare samples with different
concentrations of MT. All effects reported here are short term effects
(
30 min), where only negligible changes, if any, in MT levels
should occur. In freshly isolated rat hepatocytes, induction of MT by
Zn
was detectable after 2 h. Glucagon was less
effective increasing MT synthesis only by 28-35% after 5
h(23) .
The concentration of free Zn reported here (about 10
M) is
considerably higher than would be predicted from measurements with
enzymes, such as alkaline phosphatase and alcohol dehydrogenase, both
being metalloproteins with extremely high affinities for
Zn
(11) . Although we cannot exclude that some
of the zinc we are detecting could have been derived from Zn-MT, the
observed changes in Zn
-dependent fluorescence
mediated by glucagon or Cd
or Pb
do
not support a major interference.
Both zinc and glucagon stimulated
glycogenolysis due to an enhanced phosphorylation of glycogen
phosphorylase. The mechanism by which intracellular Zn activates phosphorylase kinase has to be further investigated.
With respect to the fate of glucose-6-phosphate, zinc and glucagon
(or cAMP) acted in completely different ways. Glucagon shifted
glucose-6-phosphate mainly in the direction of glucose formation and
inhibited the glycolytic pathway, whereas zinc, in addition to the
enhancement of glycogenolysis, also markedly stimulated glycolysis. In
combination, Zn acted antagonistically to the hormone
and was able to overcome the inhibition of glycolysis by glucagon or by
Bt
-cAMP.
The rapid decrease in intracellular
Zn concentration in the presence of glucagon (Fig. 4) is a new and unexpected observation and offers an
explanation for the antagonistic effects of glucagon and zinc on
glycolysis. The rapid decrease in intracellular free zinc was also seen
with 8-bromo-cAMP (Fig. 4), indicating that the action of
glucagon on intracellular Zn
is mediated via cAMP and
most probably catalyzed by protein kinase A. This could lead to a
sequestration of the cation.
In consequence, in order to locate the
points at which zinc acts on carbohydrate metabolism, we focused on
points where phosphorylation of enzymes is known to be of regulatory
importance. We have analyzed the activity and grade of phosphorylation
of glycogen phosphorylase and pyruvate kinase and determined the
concentration of fru-2,6-P, an indicator of the activity of
the bifunctional enzyme
fructose-2,6-bisphosphatase/phosphofructokinase-2. The proportion of
phosphorylase a increased in the presence of Zn
as well as in the presence of glucagon from 35% to about 60% of
total phosphorylase activity (Table 4). This could be verified in
intact hepatocytes by [
P]P
incorporation into phosphorylase (Fig. 5). Moreover, the
effect of zinc and Bt
-cAMP on the phosphorylation of
glycogen phosphorylase was not additive, suggesting that zinc acts
rather via an increased activity of phosphorylase kinase and not by an
inhibition of the protein phosphatases 1 or 2a. That the effects of
zinc and of Bt
-cAMP on phosphorylase activation are
comparable and not additive can also be calculated from the metabolic
rates given in Table 1by converting the rates of glucose and
lactate production into glycosyl units (not shown). However, the
mechanism by which the increase in cytosolic Zn
activates the phosphorylase kinase remains to be elucidated. A
binding of Zn
by calmodulin has been
reported(29) , but the measured affinity (K
about 100 µM) seems to be too low to be of
physiological importance.
The mechanism by which zinc activates glycolysis remains obscure. Based on the metabolite measurements reported here, zinc exerts its effect most likely at the steps catalyzed by pyruvate kinase and by phosphofructokinase-1. The substrate concentration (fructose-6-phosphate) of the rate-limiting enzyme phosphofructokinase-1 was doubled, and at the step of pyruvate kinase the inverse changes in substrate and product concentration (cross-over) indicate another control point at this step.
The
classical explanation for metabolic regulation by allosteric activators
does hold for the antagonistic effect of zinc on the inhibition of
glycolysis by glucagon. The most potent activator of
phosphofructokinase-1, namely fru-2,6-P, was reduced by
glucagon from 12 to about 6 nmoles/g of wet weight, whereas in the
presence of glucagon plus zinc the concentration of this effector was
restored to 9 nmoles/g of wet weight. This would lead to a higher flux
through phosphofructokinase-1 in the presence of glucagon plus zinc as
compared with glucagon alone. The effect of zinc on the fru-2,6-P
level in the presence of Bt
-cAMP and the failure to
get an effect in the absence of Bt
-cAMP may well be
explained by a counteraction of Zn
exclusively on the
phosphorylated and therefore less active form of phosphofructokinase-2.
We have shown that under this condition protein kinase A is as active
as in the absence of ZnCl
(see phosphorylation of pyruvate
kinase, Fig. 5), and phosphofructokinase-2 should be
phosphorylated as well, unless there exists an as yet unknown specific
and Zn
-dependent protein phosphatase.
It is also
possible that the higher concentration of fructose-6-phosphate, which
is almost doubled in the presence of ZnCl, has more effect
on the phosphorylated than on the unphosphorylated form of
phosphofructokinase-2. The mechanism of Zn
action on
this enzyme seems to be different from that of vanadate, which also
raises glycolytic flux in hepatocytes (30) but exerts also
glycogenolytic effects by activating glycogen phosphorylase and
inactivating glycogen synthase(31) . The
``insulin-like'' effect of vanadate on glycolysis has been
related to the increase in fru-2,6-P
. Rider et al.(32) have found vanadate to be an inhibitor of chicken
liver fructose-2,6-bisphosphatase, which offers a likely explanation
for the increased flux through phosphofructokinase-1 in the presence of
vanadate. However, this explanation is not valid for the pronounced
glycolytic effect of zinc on its own, because under this condition the
concentration of fru-2-6-P
did not change.
The
inhibition of glycolysis by glucagon at the step of pyruvate kinase has
been explained by the increased protein phosphorylation. The
concentration of zinc, however, which overcame the glucagon-induced
inhibition of glycolysis, did not at all affect the glucagon-induced
phosphorylation of the enzyme. This makes it also unlikely that a
protein phosphatase is involved in the antagonistic effect of zinc. In
the presence of zinc alone, pyruvate kinase remains dephosphorylated (Fig. 5) and should be in its active form. Therefore, the
elevated fru-1,6-P concentration measured under this
condition (15-23 µM) should not activate pyruvate
kinase more than under control conditions, because the fru-1,6-P
concentration necessary for half-maximal stimulation is reported
to be much lower. K
values of 0.06 µM and 0.13 µM fru-1,6-P
are given for the
unphosphorylated and the phosphorylated form of the purified rat liver
pyruvate kinase, respectively(33) . Even the highest K
values we could find in the literature,
resulting from a study using crude extracts from hepatocytes (4.2 and
9.8 µM fru-1,6-P
for control and for
hepatocytes incubated with glucagon, respectively(34) ), would
not readily explain the activation of glycolysis in the presence of
zinc alone.
The stimulation of lactate formation in hepatocytes by
zinc could also result from a strong inhibition of
fructose-1,6-bisphosphatase, a regulatory enzyme in gluconeogenesis.
This enzyme is strongly inhibited by zinc in vitro at
concentrations below 1 µM(35) , but it has also
been reported that zinc at 0.5-2 µM counteracts the
inhibition of fructose-1,6-bisphosphatase by fru-2,6-P,
shifting the K
from 3 µM to values
higher than 50 µM(36) . In our experiments an
inhibition of glucose formation from pyruvate, alanine, or
dihydroxyacetone in hepatocytes from starved rats was indeed observed
but only at concentrations of 0.1 mM ZnCl
or
higher, and this inhibition never exceeded 15-20% (see Table 2). At lower concentrations of zinc, an inhibition of
gluconeogenesis was not observed, although at these concentrations a
significant stimulation of glycolysis occurred with hepatocytes from
fed rats. In hepatocytes from fed rats, the fructose-1,6-bisphosphatase
was already in the phosphorylated state. Although this enzyme is a
substrate for protein kinase A in vitro, the incubation with
Bt
-cAMP did not lead to a further incorporation of
phosphate into the enzyme (Fig. 5). This observation resembles
results from our earlier studies analyzing the phosphorylation of
phosphofructokinase-1 in hepatocytes from fed and starved rats. In
those experiments the degree of phosphorylation was clearly determined
by the metabolic state rather than by the activation of the protein
kinase A(37) .
In summary we show here for the first time
that effects on carbohydrate metabolism induced by
Zn, Cd
, or Pb
in
the medium are accompanied by an intracellular increase in free zinc
and that glucagon or cAMP drastically reduce the free zinc
concentration. Our observation that zinc counteracts the effect of cAMP
on glycolysis as catalyzed by protein kinase A is in line with the
concept that Zn
plays a regulatory role in signal
transduction. For instance, zinc has been shown to induce the
translocation of protein kinase C to membranes that will convert the
enzyme into a more active form due to binding of zinc to the
cysteine-rich domains required for the formation of the phorbol ester
binding site(38, 39) . In Chinese hamster ovary cells
overexpressing protein kinase C
, phorbol ester
activation led to an increased phosphorylation of the insulin receptor.
This serine/threonine phosphorylation by excessive protein kinase C
seems to inhibit insulin-stimulated responses(40) .
We have
shown earlier that the 11.5-kDa ZnBP binds in vitro Zn-dependently to several enzymes of the
carbohydrate metabolism, including phosphofructokinase-1 and
fructose-1,6-bisphosphatase. In liver these two regulatory enzymes are
both covalently modified by phosphorylation but without measurable
effects on the kinetic of these enzymes. It is tempting to speculate
that zinc ions could transduce certain effects of glucagon and/or
insulin, permitting a cross-talk between these, in many respects,
antagonistic hormones. However, there is evidence that insulin-like
effects of Zn
on adipocytes occur by a mechanism
unrelated to insulin(9) . But in contrast to adipocytes,
hepatocytes have the capacity for both glycolysis and gluconeogenesis
and have a high zinc content and specific types of phosphorylatable
enzymes, so that the regulation mechanisms by insulin in hepatocytes
may differ from those in adipocytes.