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Inhibition of glycogen synthesis in rat hepatocytes by medium Zn2+
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 726-733
Vol. 122, No. 2, 1984 July 31, 1984
INHIBITION OF GLYCOGEN SYNTHESIS IN RAT HEPATOCYTES
BY MEDIUM Zn 2+
Robert Rognstad Cedars-Sinai
Medical Center, Los Angeles,
CA 90048
Received June 21, 1984
SUMMARY. In hepatocytes from fasted rats, Zn 2+ in the range from 0 to 500uM has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH 3. In hepatocytes from fed rats, Zn 2+ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination (Katz et al. (1976) Proc. Natl. Acad. Sci.USA 73,3433-3437) of glucose, lactate and glutamine (all 10mM), Zn 2÷ markedly i n h i ~ t s glycogen synthesis, with total inhibition at 500uM, and a half maximal effect in the range from 50 to 100uM. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn 2+ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate.
The control of glycogen synthesis but some matters remain unresolved, "pull" mechanisms
in the liver has been extensively
studied,
e.g. the relative contribution of T~pushT~ or
to the stimulatory
effect of glucose
(1-3).
Another facet
which remains unclear is the mechanism of the pronounced stimulation of glycogen synthesis,
in hepatocytes,
caused by amino acids
significance to control in vivo.
(4-6), and whether this has
In probing possible mechanisms
of the amino
acid effect, we noted a pronounced and rather specific inhibition of liver glycogen synthesis by ZnCI 2 added to the incubation medium.
METHODS. Hepatocytes were prepared essentially as described in (7). 24 hr fasted rats were used except where otherwise noted. The cells were incubated in 25mi Erlenmeyer flasks in 5ml of a medium containing 5mM NaHC03, 50mM Hepes and salts as in (~8), with a pH of 7.4 and with 100% 02 in the gas phase. Incubations were stopped by addition of 0.5ml of 20% HCI04, and the cells plus medium made to 10ml and centrifuged. Glycogen was determined as in (4), Glucose was measure£ enzymatically (9). Incorporation of 14C from NaHI4C03 into glucose plus glycogen was used in some cases as a relative index of lactate gluconeogenesis; NaHI4c0~ was injected through a serum stopper after the flasks had been gassed with 021 and at the end of the incubation 14C02 was collected in a hanging well into which 0.3ml of 4N NaOH was injected, In these cases 8ml of the final acidified medium was put through a icm x 8cm Dowex i (acetate) column on top of a
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726
Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
icm x 10cm Dowex 50 (H +) column, and the columns washed with water to 50ml total in this glucose plus glycogen fraction. The size of the Dowex 50 column is longer than previously used, since Hepes apparently affects the retention of (14C)urea on Dowex 50. Dipicolinate and n-butylmalonate were obtained from Aldrich (Milwaukee, WI). Mercaptopicolinate was a generous gift from Dr. H.Saunders, Smith, Kline and French Laboratories (Philadelphia,PA). RESULTS AND DISCUSSION.
Several years ago, Horecker and coworkers
(i0,ii), on the
basis that Zn 2+ was a very potent inhibitor of fructose diphosphatase, that intracellular genesis.
Zn 2+ levels might play a role in the regulation of gluconeo-
At that time, we looked
medium of hepatocytes
More recently,
(briefly) at effects of adding Zn 2. to the
incubated with dihydroxyacetone,
tion of gluconeogenesis
neogenesis
occurred,
(Table I).
only at 500uM Zn 2+.
results not shown).
from xylitol.
that from lactate inhibitory
Gluconeogenesis
Gluconeogenesis
from L-lactate was
Somewhat larger inhibitory effects (only slight inhibition of
No effect of Zn 2+ was seen on glucofrom L-glutamine was inhibited more than
(in spite of the fact that glutamine tends to moderate the
effects of Zn 2+ on other substrates,
With hepatocytes significant
up to imM (Table I).
that external Zn 2+ did markedly affect
were found when NH 3 was added together with lactate ureagenesis
inhibi-
(see below), we looked at the effect of Zn 2. on gluco-
from other substrates
inhibited significantly
No significant
was found at ZnCI 2 concentrations
following observations
liver glycogen synthesis neogenesis
speculated
from ad libitum fed rats, Zn 2+ (0-500uM) caused no
changes in glycogenolysis
Table I.
see below).
(results not shown).
Effect of medium Zn 2+ on gluconeogenesis substrates,
This result suggests
from various
Rates of Glueoneogenesis from Substrates: ZnCI 2 Concentration ~M 0 i00 500 i000
Dihydroxyacetone 102 102 104 i01
(±4) (±4) (±2) (±4)
NH4CI L-Lactate L-Lactate Xylitol ~moles glucose/gm wet wt,hr 52 49 43 42
(±2) (i3) (±5) (±2)
63 62 54 42
(!2) (±6) (±3) (~3)
78 77 77 76
(±2) (±i) (±4) (±4)
L-Glutamine 41 34 26 20
(~2) (±3) (±i) (±3)
Hepatocytes from fasted rats were incubated as described in the Methods section for 45 min. with the snbstrates shown, all at 10mM. Values are the averages of at least 3 experiments, with standard errors of the mean in parentheses.
727
Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that external Zn 2+ does not materially affect either cyclic AMP or cytosolic Ca 2+ levels, both of which are known to accelerate glycogenolysis. effect of Zn
2+
The lack of
on xylitol metabolism is also in accord with unchanged Ca 2+ (12).
While gluconeogenesis, ureagenesis and glycogenolysis all can readily be produced in isolated hepatocytes at rates fully comparable to those which occur in vivo, comparable rates of glycogen synthesis had been more difficult to produce.
However~ in 1976 Katz et al. (4) showed that addition of fairly high levels
of certain amino acids, to a combination of glucose and a gluconeogenic substrate, produced active rates of glycogen synthesis in isolated hepatocytes
Using this
combination of substrates~ we found (Table II) that addition of ZnCI 2 to the medium caused a pronounced inhibition of glycogen synthesis.
Half maximal
inhibition generally occurred between 50 and 100uM Zn 2+, with inhibition becoming complete at 200uH to 500uM.
When NaH
14
CO 3 was included in the medium~ no
significant inhibition of gluconeogenesis from lactate (plus glutamine) was found under the conditions used, even at 500u~{ Zn 2+. ureagenesis was found, at the high Zn 2+ levels
A moderate inhibition of
(not shown).
A similar inhibi-
tion by Zn 2+ of glycogen synthesis was found when dihydroxyacetone replaced L-lactate.
The exact site of action of Zn 2+ is not yet known, but glycogen
synthetase or its regulatory enzymes would be suspected~ since this is the major rate controlling step in glycogen synthesis.
Table II.
ZnCI 2 Concentration uM 0 so I00 2oo SO0
Effect of medium Zn 2+ on glycogen synthesis
Rate of Glycogen Synthesis umoles/gm wet wt'hr 24 14 9 2 0
(i2) (t3) (t4) (fl) (tO)
Relative 14C Yield in Glucose (from NaHI4c03 ) i00 i02 zoo 103 ZOO
(±2) (/3) (ti) (±3)
Hepatocytes from fasted rats were incubated with glucose (10mM), L-lactate (10mM) and Lglutamine (10mM) for 60 min. 14C yields in glucose are expressed relative to control at ZnCI 2 = 0 set equal to I00. Values are the averages of 5 experiments with standard errors of the mean in parentheses.
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Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
We have looked briefly at the effects of some other divalent cations on livem carbohydrate metabolism.
Cu
glycogen synthesis as does Zn2+0
2+
causes fairly similar inhibition of liver
Cu 2+ at the same concentration
a much greater inhibition of lactate gluconeogenesis0 tends to prevent this inhibition~ effects on glycogen synthesis.
as Zn 2+ causes
However, L-glutamine
Co 2+ and Ni 2+ have markedly lower inhibitory
Mn 2+ is unusual in that it is more inhibitory
to glycogen synthesis at 50uH than at 500uM.
At present~
in view of its speci-
ficity of action~ we regard Zn 2+ as the best candidate for a possible physiological regulator,
but the studies on other cations have been only sketchy~
Since the concentrations considerably lugm/ml
of Zn 2+ used in these in vitro experiments
are
higher than normal plasma levels of Zn 2+ (which are of the order of
(13))~ our initial interpretation was that these results probably had
no physiological
significance to regulation in vivo,
However D since much of the
Zn 2+ in the liver cytosol is apparently bound to various proteins, metallothionein
(14), ferritin
e0g~
(15), or to other lower molecular weight compounds
C16) s it is possible that changes in intmacellular free Zn 2+ may be greatly damped relative to large changes in extracellular interesting
if rather tenuous extrapolation
Zn 2+ levels,
Thus a more
of our results was suggested
endogenous Zn 2+ might control s by inhibition~
that
the rate of liver glycogen 2+
synthesis.
It is known that amino acids bind Zn
(17). The stimulation of glycogen synthesis
with considerable affinity
caused by amino acids
(4) might
3ossibly be related to a lowering of free Zn 2+ caused by the chelating action of :hese amino acids, and/or various products formed from them. We examined whether known zinc chelators might also stimulate glycogen ;ynthesis in hepatocytes.
Most chelating agents do not possess great specificity
n regard to the metal ion binding, ,ere too toxic +o be useful. rthophenanthrolene,
The toxicity to lymphoblasts
was reversed by Zn 2+ (18).
as too generally inhibitory, pplicable,
?[any of the potential Zn 2+ chelating agents of the chelator~
However~ orthophenanthrolene
in the mM range~ to liver cell metabolism to be
5m}{ orthophenanthrolene
tom L-!actate s and of ureagenesis
caused >95% inhibition of glueoneogenesis
from L-glutamine.
729
One Zn
2+
chelator which
Vol. 122, No. 2, 1 9 8 4 .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
did stimulate glycogen synthesis, to comparable levels as equal concentrations of L-glutamine,
was dipicolinate
(pyridine 2,6 dicarboxylate).
has been used to remove Zn 2+ from carbonic anhydrase rate )103 faster than did orthophenanthrolene,
Dipicolinate
(19), which it did at a
in spite of the fact that the
stability constants of each of these compounds for Zn 2+ are similar Table III shows the effect of 10mM dipicolinate synthesis
in rat hepatocytes.
source.
because dipicolinate causes considerable L-lactate.
on activation of glycogen
A mixture of dihydroxyacetone
(10mM) was used as the gluconeogenic
inhibitors,
quinolinate
(10mM) and L-lactate
L-lactate alone could not be used
inhibition of gluconeogenesis
The site of inhibition is not known,
kinase would be suspected,
(19~20)
Phosphoenolpyruvate
because of the similarity of dipicolinate
(21) and mercaptopicolinate
to McDonald and Lardy (23)~ dipicolinate Dipicolinate caused no appreciable
(22)$ however~
carboxyto known
according
does not inhibit the isolated enzyme.
inhibition of ureagenesis
(not shown).
The fact that dipicolinate also inhibits lactate gluconeogenesis to considerable ambiguity in the interpretation of the results° Katz (24) have previously synthesis~
shown that mercaptopicolinate
from dihydroxyacetone
plus glucose,
carboxykinase,
accelerates the synthesis of an activating
Okajima and
in rat hepatocytes.
producing an accumulation factor.
might be attributed to either a "metal chelation '~ or
leads
stimulates glycogen
attributed the stimulation to the fact that mercaptopicolinate enolpyruvate
from
They
inhibits phospho-
of some metabolite which
Thus, dipicolinate
effects
"metabolite accumulation ~
Table III, Effect of dipicolinate and n-butylmalonate on glycogen synthesis
Addition
Concentration mM
None L-Glutamine Dipicolinate n-Butylmalonate
i0 i0 i0
Rate of Glycogen Synthesis umoles/ gm wet wt.hr 4 24 28 27
(±i) (!2) (~2) (±2)
Hepatocytes from fasted rats were incubated with glucose (10mM), L-lactate (10mM) and dihydroxyacetone (lOmM), plus additions as shown~ for 60 min. Values are the averages of 3 experiments with standard errors of the mean in parentheses.
730
Vol. 122, No. 2, 1984
hypothesis.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Under the conditions of Table III, mercaptopicolinate
(imM) was
only about half as effective in the stimulation of glycogen synthesis as was dipicolinate.
However, another compound which was about equally as effective as
dipicolinate was n-butylmalonate* (Table III).
Since n-butylmalonate has been
reported to inhibit gluconeogenesis from L-lactate (25), the metabolite accumulation hypothesis might be favored here, although n-butylmalonate would also be expected to have some ability to chelate Zn2+°
We tested the effect of dipicolinate and n-butylmalonate on the inhibition of glycogen synthesis produced by exogenous Zn 2+ (under conditions as in the legend to Table III, with the addition of 10mM dihydroxyacetone), Dipicolinate completely prevented the inhibitory effect of medium Zn 2+ even at 500uM Zn 2+ (results not shown).
However, n-butylmalonate did not.
At low Zn 2+ concentrations (to 50uM)
n-butylmalonate in some instances actually potentiated the Zn 2+ inhibition, Dipicolinate presumably decreases the Zn 2+ inhibition mainly by forming an extracellular Zn(dipicolinate) 2
2-
complex (which form predominates at high dipicolinate
zoncentrations (~9)).
This large and doubly charged species probably would not
~nter the hepatocyte.
However, n-butylmalonate should form an uncharged (i:I)
~n 2+ complex°
There is some evidence that intestinal absorption of Zn 2+ can be
Lncreased by certain Zn 2+ complexing agents (16,26).
Conceivably the effects of
~-butylmalonate on exogenous Zn 2+ could include increased transfer of Zn 2+ Lnto the cell.
Admittedly, it is more difficult at present to fit these n-butylmalonate ,esults into a Zn 2+ chelation hypothesis, than the effects of dipicolinate, an stablished Zn 2+ chelator.
On the other hand, in recent attempts to gain
vidence for the metabolite accumulation hypothesis, only the dipicolinate data ppeared to be in some accord with this mechanism.
Thus (under the conditions
f Table III), 10m~ dipicolinate inhibited lactate gluconeogenesis (14C incornration from NaHI4CO 3 into glucose plus glycogen) by about 50%, and increased
The original observations on stimulation of glycogen synthesis by n-butylmalonate ere made by Drs. Peter Zuumendonk and Joseph Katz.
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Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the malate concentration about 2 fold~
However, 10mM n-butylmalonate decreased
lactate gluconeogenesis by only 20%, and no increase in malate was found, While dipicolinate is not a known product of hepatic metabolism~ it might be noted that various other hepatic compounds exist, with a somewhat similar chelation site, i.e. containing the grouping
NN~O00 -
, where the nitrogen
is part of a ring structure with different degrees of unsaturation,
Thus, L-proline
L-pyroglutamate~ orotate, and the cyclic forms of glutamic semialdehyde and a-ketoglutaramate contain groupings fairly similar to the picolinate chelating site, and might be pursued as potential natural Zn 2+ chelators.
Okajima and
Katz [24) found that the amino acid stimolation of glycogen synthesis was decreased By transaminase inhibito~s.
Thus, a-ketoglutaramate formation by
transamination from L-glutamine (27), might be involved in the formation of a natural chelator. We would stress that the effectiveness of a Zn 2+ chelator in this process would not necessarily depend solely upon relative stability constants~ but could depend also upon kinetic or specific structural factors~ as in the case of Zn 2+ removal from carbonic anhydrase (19).
Quinolinate (pyridine 2~3 dicarboxylate)
which is quite similar to dipicolinate (pyridine 2~6 dicarboxylate) produces only a rather small stimulation of glycogen synthesis under the same conditions (results not shown). To examine the physiological significance of the Zn 2+ effect more rigorously, it would be helpful to develop a method to estimate free cytosolic Zn 2+ levels~ e.g. perhaps by a technique analogous to the Ouin-2 method for estimating cytosolic free Ca 2+ (28).
Endogenous Zn 2+ chelators (if such compounds with
this specific function exist) would lower the level of cytosolic free Zn2+~ another related possibility is that they directly remove Zn 2+ from a Zn 2+ activated
inhibitor protein.
The possible regulatory role of an endogenous
Zn 2+ chelator presumably would be a permissive one, upon which the more established regulatory mechanisms of liver glycogen metabolism (I) are superimposed° Whether or not Zn 2+ is ultimately shown to have any regulatory role in liver glycogen metabolism in vivo, we feel that, because of the relative specificity
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Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the inhibition, Zn 2+ could prove to be a useful tool in elucidating the mechanism of the amino acid stimulation of liver glycogen synthesis (4).
ACKNOWLEDGEMENT
Supported in part by USPHS Grants No, AM 20417 and AM 21437.
REFERENCE S i. 2. 3. 4, 5. 6. 7, 8o 9. i0, ii. 12. 13. 14. 15o 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Hers, H.G. (1976) Ann.Rev. Biochem. 45, 167-189, Nordlie~ R.C., Sukalski, K.A. and Alvares~ F.L. (1980) J.Biol,Chem. 255, 1834-1838. E1 Refai, ~, and Bergman, R.N. (1976) Am.J.Fhysiol. 23], 1608-1619 Katz~ J.~ Golden, S. and Wals, P.A. (1976) Proc.Natl.Acad.Sci.USA 73,34339437. Katz~ J., Golden, S, and Wals~ P.A. (1979) Biochem.J, 180, 389-402 Golden~ S., Wals, P,A., Okajima, F. and Katz, J. (1979) Biochem J.182~727-734. Katz, J,, Wals, P.A. and Rognstad, R. (1978) J.Biol.Chem. 253, 4530-4536~ Krebs~ H.A. and Henseleit, K. (1932) HoppeSeyler'sZ.Physiol.Chem. 210~ 33-66. Rognstad, R0 (1981) Biochim.Biophys.Acta 676, 373-378. Tejwani, G.A., Pedrosa, F.O., Pontremoli, S. and Horecker, B.L. (1977) Proc.Natl.Acad.Sci.USA 73, 2692-2695. Pontremoli, 8,, Melloni, E., Salamino, F., Spartone, B. and Horecker~ B.L. (1978) Arch.Biochem. Biophys. 188, 90-97. Kneer, N.M., Wagner, M.J. and Lardy, H.A. (1979) J.Biol,Chem. 254 12160-12168. McCormick~ C.C, Menard, M.P. and Cousins, R.J. (1981) A m , J , P h y ~ l . 240, Kagi, J.H. and Nordberg, M, (1979) ~etallothionein, Birkhauser, Basel Price, D. and Joshi, J.G. (1982) Proc.Natl.Acad.Sci.USA 79, 3116-3119. DiSilvestro, R.A. and Cousins, R.J. (1983) Ann,Rev.Nutr. ~, 261-288. Prasad, A.S. and Oberleas, D. (1970) J.Lab.Clin.Med. 76, 416-425. Falchuk, K.H. and Krisham, A. (1977) CancerRes. 37, 2050-2056. Pocket, Y. and Fong, C.T.O. (1980) Biochemistry 19, 2045-2050. Sillen, L.G. and Martell, A.E. (1971) Stability Constants of Metal Ion Complexes, Special Publication No~ 25, The Chemistry Soclety, London Veneziale, C.M., Walter, P., Kneer,N. and Lardy, H.A. (1967) Biochemistry ~, 2129-2138, Kostos~ V., DiTullio, N.W,, Rush, J.~Cieslinski, L. and Saunders~ H.L. (1975) Arch.Biochem. Biophys. 171, 459-465. HacDonald, M.J. and Lardy, H.A. (1978) Bioorg. Chem. 7, 251-262 Okajima, F. and Katz D J. (1979) Biochem. Biophys~Res.~omm. 87, 155-162. Williamson, J.R., Anderson, J. and Browning, B.T. (1970) J.Biol. Chem. 245, 1717-1726. Suso, F.A. and Edwards, H.M. (1971) Proc.Soc°Exp,Biol.Med. 138, 157-162. Cooper, A.J.L. and Meister, A. (1981) Comp.Biochem.Physiol. 69B, 137-145 Tsien, R.Y., Pozzan, T. and Rink; T.J. (1982) J.Cell,Biol. 94, 325-334.
733
Vol. 122, No. 2, 1984 July 31, 1984
INHIBITION OF GLYCOGEN SYNTHESIS IN RAT HEPATOCYTES
BY MEDIUM Zn 2+
Robert Rognstad Cedars-Sinai
Medical Center, Los Angeles,
CA 90048
Received June 21, 1984
SUMMARY. In hepatocytes from fasted rats, Zn 2+ in the range from 0 to 500uM has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH 3. In hepatocytes from fed rats, Zn 2+ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination (Katz et al. (1976) Proc. Natl. Acad. Sci.USA 73,3433-3437) of glucose, lactate and glutamine (all 10mM), Zn 2÷ markedly i n h i ~ t s glycogen synthesis, with total inhibition at 500uM, and a half maximal effect in the range from 50 to 100uM. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn 2+ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate.
The control of glycogen synthesis but some matters remain unresolved, "pull" mechanisms
in the liver has been extensively
studied,
e.g. the relative contribution of T~pushT~ or
to the stimulatory
effect of glucose
(1-3).
Another facet
which remains unclear is the mechanism of the pronounced stimulation of glycogen synthesis,
in hepatocytes,
caused by amino acids
significance to control in vivo.
(4-6), and whether this has
In probing possible mechanisms
of the amino
acid effect, we noted a pronounced and rather specific inhibition of liver glycogen synthesis by ZnCI 2 added to the incubation medium.
METHODS. Hepatocytes were prepared essentially as described in (7). 24 hr fasted rats were used except where otherwise noted. The cells were incubated in 25mi Erlenmeyer flasks in 5ml of a medium containing 5mM NaHC03, 50mM Hepes and salts as in (~8), with a pH of 7.4 and with 100% 02 in the gas phase. Incubations were stopped by addition of 0.5ml of 20% HCI04, and the cells plus medium made to 10ml and centrifuged. Glycogen was determined as in (4), Glucose was measure£ enzymatically (9). Incorporation of 14C from NaHI4C03 into glucose plus glycogen was used in some cases as a relative index of lactate gluconeogenesis; NaHI4c0~ was injected through a serum stopper after the flasks had been gassed with 021 and at the end of the incubation 14C02 was collected in a hanging well into which 0.3ml of 4N NaOH was injected, In these cases 8ml of the final acidified medium was put through a icm x 8cm Dowex i (acetate) column on top of a
0006-291X/84 $1.50 Copyright © 1984byAcademic Press, Inc. Allrightsofreproductionin anyform reserved.
726
Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
icm x 10cm Dowex 50 (H +) column, and the columns washed with water to 50ml total in this glucose plus glycogen fraction. The size of the Dowex 50 column is longer than previously used, since Hepes apparently affects the retention of (14C)urea on Dowex 50. Dipicolinate and n-butylmalonate were obtained from Aldrich (Milwaukee, WI). Mercaptopicolinate was a generous gift from Dr. H.Saunders, Smith, Kline and French Laboratories (Philadelphia,PA). RESULTS AND DISCUSSION.
Several years ago, Horecker and coworkers
(i0,ii), on the
basis that Zn 2+ was a very potent inhibitor of fructose diphosphatase, that intracellular genesis.
Zn 2+ levels might play a role in the regulation of gluconeo-
At that time, we looked
medium of hepatocytes
More recently,
(briefly) at effects of adding Zn 2. to the
incubated with dihydroxyacetone,
tion of gluconeogenesis
neogenesis
occurred,
(Table I).
only at 500uM Zn 2+.
results not shown).
from xylitol.
that from lactate inhibitory
Gluconeogenesis
Gluconeogenesis
from L-lactate was
Somewhat larger inhibitory effects (only slight inhibition of
No effect of Zn 2+ was seen on glucofrom L-glutamine was inhibited more than
(in spite of the fact that glutamine tends to moderate the
effects of Zn 2+ on other substrates,
With hepatocytes significant
up to imM (Table I).
that external Zn 2+ did markedly affect
were found when NH 3 was added together with lactate ureagenesis
inhibi-
(see below), we looked at the effect of Zn 2. on gluco-
from other substrates
inhibited significantly
No significant
was found at ZnCI 2 concentrations
following observations
liver glycogen synthesis neogenesis
speculated
from ad libitum fed rats, Zn 2+ (0-500uM) caused no
changes in glycogenolysis
Table I.
see below).
(results not shown).
Effect of medium Zn 2+ on gluconeogenesis substrates,
This result suggests
from various
Rates of Glueoneogenesis from Substrates: ZnCI 2 Concentration ~M 0 i00 500 i000
Dihydroxyacetone 102 102 104 i01
(±4) (±4) (±2) (±4)
NH4CI L-Lactate L-Lactate Xylitol ~moles glucose/gm wet wt,hr 52 49 43 42
(±2) (i3) (±5) (±2)
63 62 54 42
(!2) (±6) (±3) (~3)
78 77 77 76
(±2) (±i) (±4) (±4)
L-Glutamine 41 34 26 20
(~2) (±3) (±i) (±3)
Hepatocytes from fasted rats were incubated as described in the Methods section for 45 min. with the snbstrates shown, all at 10mM. Values are the averages of at least 3 experiments, with standard errors of the mean in parentheses.
727
Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that external Zn 2+ does not materially affect either cyclic AMP or cytosolic Ca 2+ levels, both of which are known to accelerate glycogenolysis. effect of Zn
2+
The lack of
on xylitol metabolism is also in accord with unchanged Ca 2+ (12).
While gluconeogenesis, ureagenesis and glycogenolysis all can readily be produced in isolated hepatocytes at rates fully comparable to those which occur in vivo, comparable rates of glycogen synthesis had been more difficult to produce.
However~ in 1976 Katz et al. (4) showed that addition of fairly high levels
of certain amino acids, to a combination of glucose and a gluconeogenic substrate, produced active rates of glycogen synthesis in isolated hepatocytes
Using this
combination of substrates~ we found (Table II) that addition of ZnCI 2 to the medium caused a pronounced inhibition of glycogen synthesis.
Half maximal
inhibition generally occurred between 50 and 100uM Zn 2+, with inhibition becoming complete at 200uH to 500uM.
When NaH
14
CO 3 was included in the medium~ no
significant inhibition of gluconeogenesis from lactate (plus glutamine) was found under the conditions used, even at 500u~{ Zn 2+. ureagenesis was found, at the high Zn 2+ levels
A moderate inhibition of
(not shown).
A similar inhibi-
tion by Zn 2+ of glycogen synthesis was found when dihydroxyacetone replaced L-lactate.
The exact site of action of Zn 2+ is not yet known, but glycogen
synthetase or its regulatory enzymes would be suspected~ since this is the major rate controlling step in glycogen synthesis.
Table II.
ZnCI 2 Concentration uM 0 so I00 2oo SO0
Effect of medium Zn 2+ on glycogen synthesis
Rate of Glycogen Synthesis umoles/gm wet wt'hr 24 14 9 2 0
(i2) (t3) (t4) (fl) (tO)
Relative 14C Yield in Glucose (from NaHI4c03 ) i00 i02 zoo 103 ZOO
(±2) (/3) (ti) (±3)
Hepatocytes from fasted rats were incubated with glucose (10mM), L-lactate (10mM) and Lglutamine (10mM) for 60 min. 14C yields in glucose are expressed relative to control at ZnCI 2 = 0 set equal to I00. Values are the averages of 5 experiments with standard errors of the mean in parentheses.
728
Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
We have looked briefly at the effects of some other divalent cations on livem carbohydrate metabolism.
Cu
glycogen synthesis as does Zn2+0
2+
causes fairly similar inhibition of liver
Cu 2+ at the same concentration
a much greater inhibition of lactate gluconeogenesis0 tends to prevent this inhibition~ effects on glycogen synthesis.
as Zn 2+ causes
However, L-glutamine
Co 2+ and Ni 2+ have markedly lower inhibitory
Mn 2+ is unusual in that it is more inhibitory
to glycogen synthesis at 50uH than at 500uM.
At present~
in view of its speci-
ficity of action~ we regard Zn 2+ as the best candidate for a possible physiological regulator,
but the studies on other cations have been only sketchy~
Since the concentrations considerably lugm/ml
of Zn 2+ used in these in vitro experiments
are
higher than normal plasma levels of Zn 2+ (which are of the order of
(13))~ our initial interpretation was that these results probably had
no physiological
significance to regulation in vivo,
However D since much of the
Zn 2+ in the liver cytosol is apparently bound to various proteins, metallothionein
(14), ferritin
e0g~
(15), or to other lower molecular weight compounds
C16) s it is possible that changes in intmacellular free Zn 2+ may be greatly damped relative to large changes in extracellular interesting
if rather tenuous extrapolation
Zn 2+ levels,
Thus a more
of our results was suggested
endogenous Zn 2+ might control s by inhibition~
that
the rate of liver glycogen 2+
synthesis.
It is known that amino acids bind Zn
(17). The stimulation of glycogen synthesis
with considerable affinity
caused by amino acids
(4) might
3ossibly be related to a lowering of free Zn 2+ caused by the chelating action of :hese amino acids, and/or various products formed from them. We examined whether known zinc chelators might also stimulate glycogen ;ynthesis in hepatocytes.
Most chelating agents do not possess great specificity
n regard to the metal ion binding, ,ere too toxic +o be useful. rthophenanthrolene,
The toxicity to lymphoblasts
was reversed by Zn 2+ (18).
as too generally inhibitory, pplicable,
?[any of the potential Zn 2+ chelating agents of the chelator~
However~ orthophenanthrolene
in the mM range~ to liver cell metabolism to be
5m}{ orthophenanthrolene
tom L-!actate s and of ureagenesis
caused >95% inhibition of glueoneogenesis
from L-glutamine.
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One Zn
2+
chelator which
Vol. 122, No. 2, 1 9 8 4 .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
did stimulate glycogen synthesis, to comparable levels as equal concentrations of L-glutamine,
was dipicolinate
(pyridine 2,6 dicarboxylate).
has been used to remove Zn 2+ from carbonic anhydrase rate )103 faster than did orthophenanthrolene,
Dipicolinate
(19), which it did at a
in spite of the fact that the
stability constants of each of these compounds for Zn 2+ are similar Table III shows the effect of 10mM dipicolinate synthesis
in rat hepatocytes.
source.
because dipicolinate causes considerable L-lactate.
on activation of glycogen
A mixture of dihydroxyacetone
(10mM) was used as the gluconeogenic
inhibitors,
quinolinate
(10mM) and L-lactate
L-lactate alone could not be used
inhibition of gluconeogenesis
The site of inhibition is not known,
kinase would be suspected,
(19~20)
Phosphoenolpyruvate
because of the similarity of dipicolinate
(21) and mercaptopicolinate
to McDonald and Lardy (23)~ dipicolinate Dipicolinate caused no appreciable
(22)$ however~
carboxyto known
according
does not inhibit the isolated enzyme.
inhibition of ureagenesis
(not shown).
The fact that dipicolinate also inhibits lactate gluconeogenesis to considerable ambiguity in the interpretation of the results° Katz (24) have previously synthesis~
shown that mercaptopicolinate
from dihydroxyacetone
plus glucose,
carboxykinase,
accelerates the synthesis of an activating
Okajima and
in rat hepatocytes.
producing an accumulation factor.
might be attributed to either a "metal chelation '~ or
leads
stimulates glycogen
attributed the stimulation to the fact that mercaptopicolinate enolpyruvate
from
They
inhibits phospho-
of some metabolite which
Thus, dipicolinate
effects
"metabolite accumulation ~
Table III, Effect of dipicolinate and n-butylmalonate on glycogen synthesis
Addition
Concentration mM
None L-Glutamine Dipicolinate n-Butylmalonate
i0 i0 i0
Rate of Glycogen Synthesis umoles/ gm wet wt.hr 4 24 28 27
(±i) (!2) (~2) (±2)
Hepatocytes from fasted rats were incubated with glucose (10mM), L-lactate (10mM) and dihydroxyacetone (lOmM), plus additions as shown~ for 60 min. Values are the averages of 3 experiments with standard errors of the mean in parentheses.
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Vol. 122, No. 2, 1984
hypothesis.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Under the conditions of Table III, mercaptopicolinate
(imM) was
only about half as effective in the stimulation of glycogen synthesis as was dipicolinate.
However, another compound which was about equally as effective as
dipicolinate was n-butylmalonate* (Table III).
Since n-butylmalonate has been
reported to inhibit gluconeogenesis from L-lactate (25), the metabolite accumulation hypothesis might be favored here, although n-butylmalonate would also be expected to have some ability to chelate Zn2+°
We tested the effect of dipicolinate and n-butylmalonate on the inhibition of glycogen synthesis produced by exogenous Zn 2+ (under conditions as in the legend to Table III, with the addition of 10mM dihydroxyacetone), Dipicolinate completely prevented the inhibitory effect of medium Zn 2+ even at 500uM Zn 2+ (results not shown).
However, n-butylmalonate did not.
At low Zn 2+ concentrations (to 50uM)
n-butylmalonate in some instances actually potentiated the Zn 2+ inhibition, Dipicolinate presumably decreases the Zn 2+ inhibition mainly by forming an extracellular Zn(dipicolinate) 2
2-
complex (which form predominates at high dipicolinate
zoncentrations (~9)).
This large and doubly charged species probably would not
~nter the hepatocyte.
However, n-butylmalonate should form an uncharged (i:I)
~n 2+ complex°
There is some evidence that intestinal absorption of Zn 2+ can be
Lncreased by certain Zn 2+ complexing agents (16,26).
Conceivably the effects of
~-butylmalonate on exogenous Zn 2+ could include increased transfer of Zn 2+ Lnto the cell.
Admittedly, it is more difficult at present to fit these n-butylmalonate ,esults into a Zn 2+ chelation hypothesis, than the effects of dipicolinate, an stablished Zn 2+ chelator.
On the other hand, in recent attempts to gain
vidence for the metabolite accumulation hypothesis, only the dipicolinate data ppeared to be in some accord with this mechanism.
Thus (under the conditions
f Table III), 10m~ dipicolinate inhibited lactate gluconeogenesis (14C incornration from NaHI4CO 3 into glucose plus glycogen) by about 50%, and increased
The original observations on stimulation of glycogen synthesis by n-butylmalonate ere made by Drs. Peter Zuumendonk and Joseph Katz.
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Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the malate concentration about 2 fold~
However, 10mM n-butylmalonate decreased
lactate gluconeogenesis by only 20%, and no increase in malate was found, While dipicolinate is not a known product of hepatic metabolism~ it might be noted that various other hepatic compounds exist, with a somewhat similar chelation site, i.e. containing the grouping
NN~O00 -
, where the nitrogen
is part of a ring structure with different degrees of unsaturation,
Thus, L-proline
L-pyroglutamate~ orotate, and the cyclic forms of glutamic semialdehyde and a-ketoglutaramate contain groupings fairly similar to the picolinate chelating site, and might be pursued as potential natural Zn 2+ chelators.
Okajima and
Katz [24) found that the amino acid stimolation of glycogen synthesis was decreased By transaminase inhibito~s.
Thus, a-ketoglutaramate formation by
transamination from L-glutamine (27), might be involved in the formation of a natural chelator. We would stress that the effectiveness of a Zn 2+ chelator in this process would not necessarily depend solely upon relative stability constants~ but could depend also upon kinetic or specific structural factors~ as in the case of Zn 2+ removal from carbonic anhydrase (19).
Quinolinate (pyridine 2~3 dicarboxylate)
which is quite similar to dipicolinate (pyridine 2~6 dicarboxylate) produces only a rather small stimulation of glycogen synthesis under the same conditions (results not shown). To examine the physiological significance of the Zn 2+ effect more rigorously, it would be helpful to develop a method to estimate free cytosolic Zn 2+ levels~ e.g. perhaps by a technique analogous to the Ouin-2 method for estimating cytosolic free Ca 2+ (28).
Endogenous Zn 2+ chelators (if such compounds with
this specific function exist) would lower the level of cytosolic free Zn2+~ another related possibility is that they directly remove Zn 2+ from a Zn 2+ activated
inhibitor protein.
The possible regulatory role of an endogenous
Zn 2+ chelator presumably would be a permissive one, upon which the more established regulatory mechanisms of liver glycogen metabolism (I) are superimposed° Whether or not Zn 2+ is ultimately shown to have any regulatory role in liver glycogen metabolism in vivo, we feel that, because of the relative specificity
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Vol. 122, No. 2, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the inhibition, Zn 2+ could prove to be a useful tool in elucidating the mechanism of the amino acid stimulation of liver glycogen synthesis (4).
ACKNOWLEDGEMENT
Supported in part by USPHS Grants No, AM 20417 and AM 21437.
REFERENCE S i. 2. 3. 4, 5. 6. 7, 8o 9. i0, ii. 12. 13. 14. 15o 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Hers, H.G. (1976) Ann.Rev. Biochem. 45, 167-189, Nordlie~ R.C., Sukalski, K.A. and Alvares~ F.L. (1980) J.Biol,Chem. 255, 1834-1838. E1 Refai, ~, and Bergman, R.N. (1976) Am.J.Fhysiol. 23], 1608-1619 Katz~ J.~ Golden, S. and Wals, P.A. (1976) Proc.Natl.Acad.Sci.USA 73,34339437. Katz~ J., Golden, S, and Wals~ P.A. (1979) Biochem.J, 180, 389-402 Golden~ S., Wals, P,A., Okajima, F. and Katz, J. (1979) Biochem J.182~727-734. Katz, J,, Wals, P.A. and Rognstad, R. (1978) J.Biol.Chem. 253, 4530-4536~ Krebs~ H.A. and Henseleit, K. (1932) HoppeSeyler'sZ.Physiol.Chem. 210~ 33-66. Rognstad, R0 (1981) Biochim.Biophys.Acta 676, 373-378. Tejwani, G.A., Pedrosa, F.O., Pontremoli, S. and Horecker, B.L. (1977) Proc.Natl.Acad.Sci.USA 73, 2692-2695. Pontremoli, 8,, Melloni, E., Salamino, F., Spartone, B. and Horecker~ B.L. (1978) Arch.Biochem. Biophys. 188, 90-97. Kneer, N.M., Wagner, M.J. and Lardy, H.A. (1979) J.Biol,Chem. 254 12160-12168. McCormick~ C.C, Menard, M.P. and Cousins, R.J. (1981) A m , J , P h y ~ l . 240, Kagi, J.H. and Nordberg, M, (1979) ~etallothionein, Birkhauser, Basel Price, D. and Joshi, J.G. (1982) Proc.Natl.Acad.Sci.USA 79, 3116-3119. DiSilvestro, R.A. and Cousins, R.J. (1983) Ann,Rev.Nutr. ~, 261-288. Prasad, A.S. and Oberleas, D. (1970) J.Lab.Clin.Med. 76, 416-425. Falchuk, K.H. and Krisham, A. (1977) CancerRes. 37, 2050-2056. Pocket, Y. and Fong, C.T.O. (1980) Biochemistry 19, 2045-2050. Sillen, L.G. and Martell, A.E. (1971) Stability Constants of Metal Ion Complexes, Special Publication No~ 25, The Chemistry Soclety, London Veneziale, C.M., Walter, P., Kneer,N. and Lardy, H.A. (1967) Biochemistry ~, 2129-2138, Kostos~ V., DiTullio, N.W,, Rush, J.~Cieslinski, L. and Saunders~ H.L. (1975) Arch.Biochem. Biophys. 171, 459-465. HacDonald, M.J. and Lardy, H.A. (1978) Bioorg. Chem. 7, 251-262 Okajima, F. and Katz D J. (1979) Biochem. Biophys~Res.~omm. 87, 155-162. Williamson, J.R., Anderson, J. and Browning, B.T. (1970) J.Biol. Chem. 245, 1717-1726. Suso, F.A. and Edwards, H.M. (1971) Proc.Soc°Exp,Biol.Med. 138, 157-162. Cooper, A.J.L. and Meister, A. (1981) Comp.Biochem.Physiol. 69B, 137-145 Tsien, R.Y., Pozzan, T. and Rink; T.J. (1982) J.Cell,Biol. 94, 325-334.
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