Vol. 74, No. 4, 1977
BIOCHEMICAL
Glucose
Synthesis
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by Isolated
Guinea-Pig
Hepatocytes
EFFECT OF CYCLIC AMP AND DIBUTYRYL CYCLIC AMP Ifeanyi
J. Arinze
Department of Biochemistry and Nutrition, Meharry College, Nashville, Tennessee 37208, U.S.A.
Received
January
Medical
lo,1977
Summary: In isolated guinea-pig hepatocytes, dibutyryl cyclic gluconeogenesis from 2 mM galactose by 25 and 40% respectively. of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose lactate and galactose by 26 and 34% respectively. Glucagon perfused
rat
of studies hepatic
liver
with
2) as well
the perfused
from guinea
information only
et al.
on the available
(6)
tive pig
(1,
AMP are
gluconeogenesis
livers
the
and cyclic
pigs
both
type
of glucagon,
of interest
from there
and dibutyryl
of glucose, cyclic
via
the influence
of regulation that
cyclic
these
that
liver,
cyclic of these
In Sbling
ineffec-
isolated
glycogenolysis,
some
of
species.
AMP were
recently
of
of
seen with
guinea-pig
AMP or dibutyryl
A number
has been a paucity in
the perfused
We showed
to examine
by guinea-pig
different
by the
(3-5).
the pattern
However,
with
glucagon
the output
hepatocytes
of cjluconeogenesis
of gluconeogenesis. increase
gluconeogenesis
(6-8).
of gluconeogenesis
rat
shown that
is markedly
regulation
of this
that
concentrations
was therefore
have
and rabbits
study
as stimulators
of low
liver
hormonal
activators
as by isolated
in the rat
indicated
hepatocytes
effective
AMP stimulated In the presence synthesis from
guinea-
upon addition AMP (9).
It
agents
on
hepatocytes.
Experimental: Male guinea pigs (200-250 g) of the Hartley strain were purchased from Williams Kentucky Cavies, Fern Creek, KY., and fed ad libitum a pelleted Purina laboratory chow supplemented with fresh lettuce. Theanimals had free access to water at all times. Food was withdrawn for 45-50 h before all experiments. Adenosine 3' :5'-cyclic monophosphate and 6-N, 2-0-dibutyryl-adenosine 3':5'-cyclic monophosphate were purchased from the Sigma Chemical Co., St. Louis, MO. The sources of all other chemicals'have been described (9). Hepatocytes were prepared by collagenase digestion of the perfused liver as described previously (9). Glucose was measured enzymatically by the method of Slein (10). Results
and Discussion:
glucose
synthesis
I) -
However,
from
At 0.1 mM in 2 mM lactate
in the presence
Copyright 0 1977 by Academic Press, Inc. AN rights of reproduction in any form reserved.
the incubation
or
2 mM galactose
of theophylline,
1656
cyclic
medium cyclic by only
about
AMP stimulated
AMP increased lo-12%
(Table
gluconeoISSN
0006-291
X
cyclic
Dibutyryl
AMP + theophylline
AMP 250 ? 25
263 +_ 34
concentrations Cells were incubated for 40 min at 37OC. Final cyclic AMP, 0.1 mM: dibutyryl cyclic AMP, 0.1 mM; theophylline SEM for 5 or 6 experiments.
cyclic
AMP + theophylline
Cyclic
Dibutyryl
232 + 25
AMP
Cyclic 229 f. 23
235 + 24
None per
of
Galactose
2 mM; galactose, 2 mM: expressed as means +
860 k 93
940 + 38
752 2 38
738 f 48
675 f 58
659 f 35
g wet wt.
(2 mM)
added were:lactate 0.5 mM. Pates are
642 f 48
684 2 25
689 + 38
585 + 29
583 f 41
545 -+ 31
n moles/min
Lactate
Substrate
in guinea-pig liver cells in the presence cyclic AMP and dibutyryl cyclic AMP
Theophylline
Gluconeogenesis theophylline,
231 + 17
I.
None
Addition
Table
Vol. 74, No. 4, 1977
genesis
from
penetrates from
lactate
cells
lactate
by 27%.
in Table
glycogenolysis of these cytes
In our
mation
from the
(l-2
liver
hormonal
coneogenesis
species
however,
of basic
enzyme, whether
coneogenesis. on perfused
significant
livers
hormonal
similar
AMP tested
gluconeogenesis increase this
(0.1
shown
to the
concentration rat
in gluconeogenesis
nucleotide
in rat
mM) had no effect
in
to enhance
in isolated
to the report
by the perfused
guinea
that
It
hepatonoted
hepatocytes
on glucose
by Sibling
pig
liver
is possible
in
that
for-
effect
on gluconeogenesis
maximally
elevated in the
regulation
to the intracellular
variation
carboxykinase also
exist
on perfused
of normal
mice
(14)
initially
on gluconeogenesis 1658
by regard
in isolat-
12).
rates
Also, of glu-
of starvation.
of gluconeogenesis
among
compartmentation (13).
It
in the hormonal
guinea-pig
influ-
at the very
(4,
the basal
as a result
(6)
concentrations
increased
in which
not
In this
at low pyruvate is
et al.
experiments
demonstrable.
concentration some livers
is
easily
demonstrated
substrate
influence
not
the hormonal
is readily
the work
AMP.
are
phosphoenolpyruvate
Like
(9) and is
cyclic
difference
species
cyclic
contrast
effects
seem to be attributable
regulatory
substantially
20 mM, used in the perfusion
may be masked
are already
A number
out
lactate
as the
effects
was not
has been previously
for
in sharp
hormonal
which
which
butyrate
of dibutyryl
mM) diminishes
of theophylline.
AMP or dibutyryl
reported
of lactate,
cells
of
tested.
has been shown recently
ed rat
AMP, which gluconeogenesis
upon addition
AMP-induced
sodium
from
(6),
cyclic
even in the absence
formation
to stimulate
I are
infusion
--et al.
glucose
cells
cyclic
substrates
concentrations
SBling
liver
same range
gluconeogenesis
high
dibutyryl AMP, stimulated
was noted
to that
reported
in Table
enced by the
cyclic
m+l) of cyclic
identical
experiments
The data
it
is
in the
(11) -
that
(0.1
The dibutyryl I is
hand,
additions.
nucleotides
in Table
change
in guinea-pig
(4).
than
endogenous
The concentration experiments
other
by 25 and 40%, respectively,
I,
by the various
these
On the
No appreciable
As indicated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
much more readily
and galactose
theophylline.
altered
BIOCHEMICAL
liver
from lactate
not
clear,
regulation
(6),
failed
is
of a key
of glu-
experiments
to indicate
carried any
or pyruvate,
prompt
Vol. 74, No. 4, 1977
ing
BIOCHEMICAL
the suggestion
that
mouse or the guinea the
authors
(15).
(17),
that
(19).
the experimental species
be unique
is apparent
with
which
from Table
I that
synthesis
been noted explants
in
to hormonal
in guinea
lamb liver (18),
of response the
pig
cells but
and isolated
AMP or its rabbit
chicken
strictly results
carboxykinase
the
also
(16),perfused
same, these
of hepatic
however,
liver
and cyclic
cannot
the
and cyclic
from lactate
cells
phosphoenolpyruvate regulation
cells,
by glucagon
by glucagon
are not always
in either
mouse liver
not only
in culture
the magnitude
have mitochondrial
respect
work with
of gluconeogenesis
conditions
gluconeogenesis
of gluconeogenesis
glucose
liver
Although
regulate
In subsequent
has also
human fetal
may not
stimulation
Enhancement
derivative
hepatocytes since
(14).
stimulate
from galactose.
liver
It
nucleotides
dibutyryl
pig
have reported
nucleotides cyclic
hormones
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be compared suggest may not
gluconeogenesis.
Acknowledgements: This work was supported by National Institute of Health Grant HD 08792 and by a Basil O'Connor Starter Research Grant #5-89 from the National Foundation-March of Dimes. I thank Mr. James Tutwiler for technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Ross, B.D., Hems R, and Krebs H.A. (1967) Biochem. J. 102:942-951 Exton, J.H. and Park, C. R. (1969) J. Biol. Chem. 244:1424-1433 Johnson, M.E.M., Das, N.M., Butcher, F.R. and Fain, J.N. (1972) J. Biol. Chem. 247:3229-3235 Zahlten, R.N., Stratman, F.W. and Lardy, H-A. (1973) Proc.Natl. Acad, Sci. U.S.A. 70:3213-3218 Haynes, R.C.Jr., Garrison, J. C. and Yamazaki, R.K. (1974) Mol. Pharmacol. 10: 381-388 Selling, H.D., Willms, B., Kleineke, J. and Gehlhoff, M. (1970) Eur. J. Biochem 16:289-302 Arinze, I.J., Garber, A.J. and Hanson, R.W. (1973) J. Biol. Chem. 248:2266-2274 Johnson, D.C., Brunsvold, R.A., Ebert, K.A. and Ray, P.D. (1973) J. Biol. Chem. 248:763-770 Arinze, I.J. and Rowley, D.L. (1975) Biochem. J. 152:393-399 Slein, M.W. (1965) IN: Methods of Enzymatic Analysis (Bergmeyer, H.U., ea.), pp. 117-123, Academic Press, New York Cornell, N.W., Lund, P., Hems, R. and Krebs H.A. (1973) Biochem. J. 134:671-672 Claus, T. H., Pilkis, S.J. and Park, C.R. (1975) Biochim. Biophys. Acta 404:110-123 Hanson, R.W. (1974) Nutr. Rev. 32:1-8 Loten,E.G., Assimacopoulos-Jeannet, F., Marchand Y.L., Singh, A. and Jeanrenaud B. (1974) Adv. Enz. Regulat. 12:45-71 Muller, P., Singh, A., Orci, L. and Jeanrenaud B. (1976) Biochfm. Biophys. Acta 428:480-494 Clark, M.G., Filsell, O.H. and Jarrett, I.G. (1976) Biochem. J. 156:671-680 Huibregtse, C.A. and Ray, P.D. (1975) Federation Proc. 34:659 (Abs.) Schwartz, A.L. and Rall, T.W. (1975) Diabetes 24:650-657 Anderson, C.E., and Langslow, D.E. (1975) Biochem. Sot. Trans. 3:1037-1039
1659
BIOCHEMICAL
Glucose
Synthesis
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by Isolated
Guinea-Pig
Hepatocytes
EFFECT OF CYCLIC AMP AND DIBUTYRYL CYCLIC AMP Ifeanyi
J. Arinze
Department of Biochemistry and Nutrition, Meharry College, Nashville, Tennessee 37208, U.S.A.
Received
January
Medical
lo,1977
Summary: In isolated guinea-pig hepatocytes, dibutyryl cyclic gluconeogenesis from 2 mM galactose by 25 and 40% respectively. of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose lactate and galactose by 26 and 34% respectively. Glucagon perfused
rat
of studies hepatic
liver
with
2) as well
the perfused
from guinea
information only
et al.
on the available
(6)
tive pig
(1,
AMP are
gluconeogenesis
livers
the
and cyclic
pigs
both
type
of glucagon,
of interest
from there
and dibutyryl
of glucose, cyclic
via
the influence
of regulation that
cyclic
these
that
liver,
cyclic of these
In Sbling
ineffec-
isolated
glycogenolysis,
some
of
species.
AMP were
recently
of
of
seen with
guinea-pig
AMP or dibutyryl
A number
has been a paucity in
the perfused
We showed
to examine
by guinea-pig
different
by the
(3-5).
the pattern
However,
with
glucagon
the output
hepatocytes
of cjluconeogenesis
of gluconeogenesis. increase
gluconeogenesis
(6-8).
of gluconeogenesis
rat
shown that
is markedly
regulation
of this
that
concentrations
was therefore
have
and rabbits
study
as stimulators
of low
liver
hormonal
activators
as by isolated
in the rat
indicated
hepatocytes
effective
AMP stimulated In the presence synthesis from
guinea-
upon addition AMP (9).
It
agents
on
hepatocytes.
Experimental: Male guinea pigs (200-250 g) of the Hartley strain were purchased from Williams Kentucky Cavies, Fern Creek, KY., and fed ad libitum a pelleted Purina laboratory chow supplemented with fresh lettuce. Theanimals had free access to water at all times. Food was withdrawn for 45-50 h before all experiments. Adenosine 3' :5'-cyclic monophosphate and 6-N, 2-0-dibutyryl-adenosine 3':5'-cyclic monophosphate were purchased from the Sigma Chemical Co., St. Louis, MO. The sources of all other chemicals'have been described (9). Hepatocytes were prepared by collagenase digestion of the perfused liver as described previously (9). Glucose was measured enzymatically by the method of Slein (10). Results
and Discussion:
glucose
synthesis
I) -
However,
from
At 0.1 mM in 2 mM lactate
in the presence
Copyright 0 1977 by Academic Press, Inc. AN rights of reproduction in any form reserved.
the incubation
or
2 mM galactose
of theophylline,
1656
cyclic
medium cyclic by only
about
AMP stimulated
AMP increased lo-12%
(Table
gluconeoISSN
0006-291
X
cyclic
Dibutyryl
AMP + theophylline
AMP 250 ? 25
263 +_ 34
concentrations Cells were incubated for 40 min at 37OC. Final cyclic AMP, 0.1 mM: dibutyryl cyclic AMP, 0.1 mM; theophylline SEM for 5 or 6 experiments.
cyclic
AMP + theophylline
Cyclic
Dibutyryl
232 + 25
AMP
Cyclic 229 f. 23
235 + 24
None per
of
Galactose
2 mM; galactose, 2 mM: expressed as means +
860 k 93
940 + 38
752 2 38
738 f 48
675 f 58
659 f 35
g wet wt.
(2 mM)
added were:lactate 0.5 mM. Pates are
642 f 48
684 2 25
689 + 38
585 + 29
583 f 41
545 -+ 31
n moles/min
Lactate
Substrate
in guinea-pig liver cells in the presence cyclic AMP and dibutyryl cyclic AMP
Theophylline
Gluconeogenesis theophylline,
231 + 17
I.
None
Addition
Table
Vol. 74, No. 4, 1977
genesis
from
penetrates from
lactate
cells
lactate
by 27%.
in Table
glycogenolysis of these cytes
In our
mation
from the
(l-2
liver
hormonal
coneogenesis
species
however,
of basic
enzyme, whether
coneogenesis. on perfused
significant
livers
hormonal
similar
AMP tested
gluconeogenesis increase this
(0.1
shown
to the
concentration rat
in gluconeogenesis
nucleotide
in rat
mM) had no effect
in
to enhance
in isolated
to the report
by the perfused
guinea
that
It
hepatonoted
hepatocytes
on glucose
by Sibling
pig
liver
is possible
in
that
for-
effect
on gluconeogenesis
maximally
elevated in the
regulation
to the intracellular
variation
carboxykinase also
exist
on perfused
of normal
mice
(14)
initially
on gluconeogenesis 1658
by regard
in isolat-
12).
rates
Also, of glu-
of starvation.
of gluconeogenesis
among
compartmentation (13).
It
in the hormonal
guinea-pig
influ-
at the very
(4,
the basal
as a result
(6)
concentrations
increased
in which
not
In this
at low pyruvate is
et al.
experiments
demonstrable.
concentration some livers
is
easily
demonstrated
substrate
influence
not
the hormonal
is readily
the work
AMP.
are
phosphoenolpyruvate
Like
(9) and is
cyclic
difference
species
cyclic
contrast
effects
seem to be attributable
regulatory
substantially
20 mM, used in the perfusion
may be masked
are already
A number
out
lactate
as the
effects
was not
has been previously
for
in sharp
hormonal
which
which
butyrate
of dibutyryl
mM) diminishes
of theophylline.
AMP or dibutyryl
reported
of lactate,
cells
of
tested.
has been shown recently
ed rat
AMP, which gluconeogenesis
upon addition
AMP-induced
sodium
from
(6),
cyclic
even in the absence
formation
to stimulate
I are
infusion
--et al.
glucose
cells
cyclic
substrates
concentrations
SBling
liver
same range
gluconeogenesis
high
dibutyryl AMP, stimulated
was noted
to that
reported
in Table
enced by the
cyclic
m+l) of cyclic
identical
experiments
The data
it
is
in the
(11) -
that
(0.1
The dibutyryl I is
hand,
additions.
nucleotides
in Table
change
in guinea-pig
(4).
than
endogenous
The concentration experiments
other
by 25 and 40%, respectively,
I,
by the various
these
On the
No appreciable
As indicated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
much more readily
and galactose
theophylline.
altered
BIOCHEMICAL
liver
from lactate
not
clear,
regulation
(6),
failed
is
of a key
of glu-
experiments
to indicate
carried any
or pyruvate,
prompt
Vol. 74, No. 4, 1977
ing
BIOCHEMICAL
the suggestion
that
mouse or the guinea the
authors
(15).
(17),
that
(19).
the experimental species
be unique
is apparent
with
which
from Table
I that
synthesis
been noted explants
in
to hormonal
in guinea
lamb liver (18),
of response the
pig
cells but
and isolated
AMP or its rabbit
chicken
strictly results
carboxykinase
the
also
(16),perfused
same, these
of hepatic
however,
liver
and cyclic
cannot
the
and cyclic
from lactate
cells
phosphoenolpyruvate regulation
cells,
by glucagon
by glucagon
are not always
in either
mouse liver
not only
in culture
the magnitude
have mitochondrial
respect
work with
of gluconeogenesis
conditions
gluconeogenesis
of gluconeogenesis
glucose
liver
Although
regulate
In subsequent
has also
human fetal
may not
stimulation
Enhancement
derivative
hepatocytes since
(14).
stimulate
from galactose.
liver
It
nucleotides
dibutyryl
pig
have reported
nucleotides cyclic
hormones
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be compared suggest may not
gluconeogenesis.
Acknowledgements: This work was supported by National Institute of Health Grant HD 08792 and by a Basil O'Connor Starter Research Grant #5-89 from the National Foundation-March of Dimes. I thank Mr. James Tutwiler for technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Ross, B.D., Hems R, and Krebs H.A. (1967) Biochem. J. 102:942-951 Exton, J.H. and Park, C. R. (1969) J. Biol. Chem. 244:1424-1433 Johnson, M.E.M., Das, N.M., Butcher, F.R. and Fain, J.N. (1972) J. Biol. Chem. 247:3229-3235 Zahlten, R.N., Stratman, F.W. and Lardy, H-A. (1973) Proc.Natl. Acad, Sci. U.S.A. 70:3213-3218 Haynes, R.C.Jr., Garrison, J. C. and Yamazaki, R.K. (1974) Mol. Pharmacol. 10: 381-388 Selling, H.D., Willms, B., Kleineke, J. and Gehlhoff, M. (1970) Eur. J. Biochem 16:289-302 Arinze, I.J., Garber, A.J. and Hanson, R.W. (1973) J. Biol. Chem. 248:2266-2274 Johnson, D.C., Brunsvold, R.A., Ebert, K.A. and Ray, P.D. (1973) J. Biol. Chem. 248:763-770 Arinze, I.J. and Rowley, D.L. (1975) Biochem. J. 152:393-399 Slein, M.W. (1965) IN: Methods of Enzymatic Analysis (Bergmeyer, H.U., ea.), pp. 117-123, Academic Press, New York Cornell, N.W., Lund, P., Hems, R. and Krebs H.A. (1973) Biochem. J. 134:671-672 Claus, T. H., Pilkis, S.J. and Park, C.R. (1975) Biochim. Biophys. Acta 404:110-123 Hanson, R.W. (1974) Nutr. Rev. 32:1-8 Loten,E.G., Assimacopoulos-Jeannet, F., Marchand Y.L., Singh, A. and Jeanrenaud B. (1974) Adv. Enz. Regulat. 12:45-71 Muller, P., Singh, A., Orci, L. and Jeanrenaud B. (1976) Biochfm. Biophys. Acta 428:480-494 Clark, M.G., Filsell, O.H. and Jarrett, I.G. (1976) Biochem. J. 156:671-680 Huibregtse, C.A. and Ray, P.D. (1975) Federation Proc. 34:659 (Abs.) Schwartz, A.L. and Rall, T.W. (1975) Diabetes 24:650-657 Anderson, C.E., and Langslow, D.E. (1975) Biochem. Sot. Trans. 3:1037-1039
1659
