Vol. 67, No. 1, 1975
BIOCHEMICAL
FUTILE
AND BIOPHYSICAL
CYCLES IN ISOLATED PERFUSED RAT LIVER AND IN ISOLATED RAT LIVER
Dallas
RESEARCH COMMUNICATIONS
Clark,
Donald
Department
Lee,
of Clinical Medical
PARENCHYMAL CELLS
Robert
Rognstad"
Biochemistry,
School,
and Joseph
University
Dunedin,
Katz*
of Otago
New Zealand
and
Received
*Cedars-Sinai
Medical
Research
Center
Los Angeles,
California
90029,
U.S.A.
September
5,197s
livers from fed rats were perfused with a medium containing SUMMARY: Isolated glucose labeled uniformly with 14C and specifically with 3H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of After 2 hours the 3H/l4C ratios in perfusate about half of the 14C in glucose. glucose decreased by 55-60% with (Z-3H, U-14C), 40-50% with (5-3H, U-14C), 2530% with (3-3H or 4-3H, U-14C) and by lo-15% with (6-38, U-14C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results Superimposed upon demonstrate recycling of carbon between glucose and pyruvate. There this there is an extensive futile cycle between glucose and glucose 6-P. is also futile cycling between fructose 6-P and fructose 1,6 P2 and to a small extent between phosphoenol pyruvate and pyruvate. Introduction Experiments of a futile
with
cycle
between
have
indicated
another
rat
hepatocytes
(1,
the
hydrolysis
6-P:
fructose
These
authors
tiated
(2-3H)
14C and 3H labeled glucose
cycle
P2 futile
cycle
that
and (5-3H)
Liver
of ATP and Mg however
there
results
tritium
the
formation
that of
suggested
that
exchange
between
6-P but
(3).
questioned
not
(3-3H)
L,6 P2 and its
glyceraldehyde
3-P is
moiety 212
position
cleavage
Labeled
glucose
from
from
(5-3H)
the
(3-3H) 5 of
cause fructose (4).
ATP and Mg, detri6-P.
by aldolase.
in position of
cycles of
2) and
P2 in both
by Hue and Hers with
presence
(1,
1,6
These
The occurrence
of tritium
can be lost
glyceraldehyde
liver
the
hepatocytes
unsupplemented
fructose
the
rat
has been
was loss
demonstrated
6-P and fructose
dissipation.
extracts,
glucose
have
6-P in rat
fructose
2) and in the -L_ in vivo
showed
indicate
and glucose
between
of ATP and energy 1,6
glucoses
In
glucose fructose
the 6-P.
presence These
6-P without
Hue and Hers
(4)
2 by transaldolase fructose
6-P
(carbons
4,
Vol. 67, No. 1, 1975
BIOCHEMICAL
5 and 6) and glyceraldehyde with
protons
sequence
by the
of
Hers
(4)
cates
the
and 6-3H,
triose-P
of
present
U-14C)
the
paper
in rat
without
fructose the
hepatic
energy
exchange
tissue
has been
exchanged
dissipation. liver
The detri-
extracts
of Hue and
This
futile
of the
then
of 38 by this
reaction.
phosphate
occurrence
is
The loss
supplemented
to this
RESEARCH COMMUNICATIONS
in triose-P
isomerase.
6-P in the
be attributed
occurrence
The tritium
an exchange
glucose
cannot
cycle
of
is
(3-3H)
In the futile
action
of reactions
tiation
3-P.
AND BIOPHYSICAL
cycle
fructose
results
indi-
in liver.
6-P:
reinvestigated
fructose
by using
1,6 (3,
P2
4, 5
glucose. Methods
Materials: Isotopes were purchased from the Radiochemical Centre, Amersham, England; Mono filament nylon mesh filters were from Tuta Laboratories (Australia) Pty. Limited, Lane Cove, N.S.W., Australia; Dacron filters were a kind gift from Pioneer Filters Inc. Beaverton, Oregon, U.S.A. Perfusion of Rat Liver. Male rats of the Wistar strain, 140-19Og, had free access to a standard diet of rat nellets and water or were starved for 48 hours, Rats were anesthetized wi;h nembutal and the isolated liver perfused essentially by the method of Hems et al. (5). Livers of fed rats were perfused with 40 ml Krebs-Ringer bicarbonate buffer containing 25% washed bovine erythrocytes, 3% bovine serum albumin and 2.8 mM glucose. Livers of fasted rats were perfused with 40 ml whole fresh rat blood. Before use both media were filtered through 4 g of tightly packed Dacron wool (6). The perfusate, before entering and after leaving the liver, was filtered through nylon blood transfusion filters packed with 0.3 g Dacron wool. Samples (0.5 ml) of the perfusion medium were deproteinized with 2.5 ml 95% v/v ethanol and assayed for glucose. The supernatant was passed through three columns: Amberlite CG120 (@), Dowex 1 (acetate) and Dowex 1 (borate). The borate column retained glucose which was eluted with 6M formic acid and assayed for 14C and tritium. Hepatocytes. previously
The preparation described (1).
of hepatocytes Results
The labeled
glucose
to the
attachment
a control
for
only
fresh
with
3H/l4C hour the
the
introduction
concentration
the
liver.
blood
The metabolism rats
perfused of
the
increasing
to the
with liver from
perfusion
Samples of glucose
but
of
incubation
were
as
and Discussion
was added
metabolism
rat
ratios. fasted
of
and methods
taken
there
changes
(5-3H,
fresh
during
U-14C)
rat
was rapid
3 to 7-11
is
mM within
213
in the
shown
glucose
period There
glucose
blood
30 minutes
this
by erythrocytes.
no significant of
medium
prior
served
as
was glycolysis
perfusate
glucose
by livers
from
48
in Figure
1A.
Upon
synthesis
40 minutes.
with
its
The mean maximal
Vol. 67, No. 1, 1975
BIOCHEMICAL
AND BIOPHYSICAL
FIGURE 1A
RESEARCH COMMUNICATIONS
FIGURE 1B
Figure 1A. the Livers Perfusate the Livers fresh rat
The Metabolism of (5-3H, U-14C) Glucose and Glucose Production in of Rats Starved for 48 Hours. Figure 1B. The 3H/14C Ratios in Glucose During the Metabolism of (2, 3, 4, 5 or 6-3H) Glucose in of Rats Starved for 48 Hours. Livers were perfused with 40 ml of blood containing 20 uCi of (2, 3, 4, 5 or 6-3H) glucose and 10 uCi The results with (2-3H, u-14C) and (5-3H, u-14c) glucose ~f,(Z4ZaE1~~~~*for 3 animals. Those with (3-3H, U-14C) and (6-3H, U-14C) glucose are the mean ( SD) for 2 animals while the (4-3H, U-14C) glucose perfusion was with 1 animal.
synthesis
of
residual
glucose
glycogen
Net glucose
in these in
the
production
fasted from
content
appeared
to remain
fusions.
About
one quarter
lactate
in the
acids
within
synthesis
perfusion the
of
In part
of
products
labeled
from
fasted
amino
acids
apparent cates glucose
rats
of
this
is
at
unlikely
glucose
slightly
sources of
determined
and lipids
and pyruvate. 214
as the
during
these
arises
from
Although
per-
also
amino was
glucose The nature In hepatocytes
in C02, although
lactate,
labeled
The
of glucose
and recycling
from
there
perfusions.
mainly were
glucose.
glycogen
by erythrocytes.
the presence
and synthesis,
50% of this
14C in perfusate
in these
was recovered
in
(5).
the
was sufficient
can be derived
presumably
due to glycolysis
14C glucose
least
synthesized rest
There
however
or increase
to one half
glucose
catabolism
is
and the
glycogen of
to supply
endogenous
was not
and cellular
simultaneous
the
and from
was 240 umoles.
source
constant
14C from
utilization
this
one third
was utilized. the
livers
medium
blood
glucose
experiments
(1,
7).
synthesis
of carbon
indi-
between
Vol. 67, No. 1, 1975
The loss
of tritium
detritiation
of
to 28% of
BIOCHEMICAL
the
(2-3H)
catabolism;
nevertheless
results
are
rapid
than
shown that
4 appears
from
with
lBO 5.
rapid
than
difference
(6-3H)
glucose
rapid
of glucose
3H/14C
position
to be more
rates
ratios
were
that
rate
3 hours
the
only
22 There
relative
retention The
reproducible. position
3, but In the
significantly
The
glucose.
2 is
of tritium
position
14C.
and in 14C glucose
measure fairly
ratios.
of
circulating
from
The initial
in these
than
synthesis
which
from
RESEARCH COMMUNICATIONS
and after
The detritiation
that
was not
rapid
in the
same preparation
in Figure
was no significant ratio
the
in the
most
was present
in the
two tracers
was more
was the
activity
variability
the
glucose
glucose
original
was a large
of
from
AND BIOPHYSICAL
loss after
first
different
more from
position
2 hours hour
from
the
that
of
there 3H/l4C (3-3H)
glucose. Results perfused
with
Methods). from
with
(3-3H,
livers
a buffer
In Figure U-14C)
of
fed
containing 2A the
glucose
rats
shown
albumin
synthesis is
are
shown.
of
and bovine glucose
There
sool’
in Figure
2.
The livers
erythrocytes
and the
uptake
was considerable
were
(see of
variation
isotope between
o-
0 243 2040w 90 120 1501y ma T I hme I menI
0 203m 4060 90 120150lap 200 1 timeimlnl 1 FIGURE 2B
FIGURE2A
Figure 2A. The Metabolism of (2-3H, U-14C) Glucose and Glucose Production in Figure 2B. The 3H/l4C Ratios in Perfusate the Livers of Rats Fed Ad Libitum. in the Livers Glucose During the Metabolism of (2, 3, 4, 5 or 6-3H) Glucose Livers were perfused with 40 ml of Krebs Ringer of Rats Fed Ad Libitum. bicarbonate buffer containing 25% washed bovine erythrocytes, 3% bovine serum albumin and 2.8 mM glucose labeled with 20 pCi (2, 3, 4, 5 or 6-3H) glucose Each point is the mean (fSD) of 4 animals. and 10 $i (U-14C) glucose.
215
Vol. 67, No. 1, 1975
Table
BIOCHEMICAL
1.
The Utilization from
(18 mg cell experiment
protein
with
Incubation Period Hours
Diet
in experiment
doubly
Glucose
3
Uptake
of cell
of
in the
ratio
amount
Final 3H/l4C Ratio in Glucose*k
% Dose u-14c
-
8.4
l-3H
13
0.97
2-3~
27
0.78
3-38
16
0.96
4-3H
17
0.95
5-3H
20
0.93
6-3H
14
0.97
u-14c
19
l-3H 2-3~
30
0.93
54
0.66
3-3H
37
0.80
4-38
39
0.82
5-38
45
0.77
6-3H
32
0.93
quite
synthesis
for
the
both
from
1.00.
of glucose
were
derived
14C
protein
is
14c glucose
rapid
with
The isotope uptake from 14C glucose represents the 14C recovered in amino acids and glycogen. Water constituted between C02, lactate, 90-95% of the utilized tritium activity from (2, 3, 4 and 5-3H) glucose and from 80-90% from (1 and 6-3H) glucose.
>k>W The initial
animals
uniformly
mg*/hr
-7.2
3~ Milligrams 2s;
labeled
bicarbonate
Label in Isotope Glucose Uptak&
-4.0
Fed
1 and 30 mg in
tritium.
nmoles/lOO Fasted
by Hepatocytes
in 2 ml Krebs-Henseleit
10 mM in glucose,
and specifically
RESEARCH COMMUNICATIONS
of Labeled Glucose Fasted and Fed Rats.
2) incubated
buffer,
AND BIOPHYSICAL
glycogen
synthesized
similar. first
but
The synthesis
40 to 60 minutes. and from
lactate
216
plus
the
average
of glucose The glucose amino
acids.
rates
of
catabolism
was biphasic
with
is presumably Although
the
Vol. 67, No, 1, 1975
increase than
BIOCHEMICAL
in concentration
that
At the
with
livers
end of the
the
initial
The pattern case
there
(3-3H,
from
3 hour
was much depleted, of
of
a much greater and
(6-3H,
1 results labeled
with
In these
experiments
a small
fraction
of the
3H/l4C
ratios
as in perfused
The detritiation
greater
than
of other
order:
2> 5> 3 4> 116. The occurrence for
the
by Katz
the
of glucose
can not
be applied.
results
however
there also of
Only
triose-P
from
were from position
state
out. are
cycle
between
solely (3-3H)
through and
5 exceeds
upon glucose
fructose the (5-3H) that
aldolase glucose position
217
the
position
decline
tritium
same
2 is much is
in
the
and the methods in a forthcoming in these
estimates experiments
and steady
state
approximations
are possible. of
detritiation
this
overall
should 3,
of
P2.
it
4) There If
fructose
be about
equal.
is possible
between
of carbon
6-P.
1,6
cleavage
of carbon
recycling
and glucose
Some in hepatocytes
is recycling
6-P and fructose
from
are
tritium
present
2) There
between
with
In the
1) The pattern
3) Superimposed
with
is much the
discussed
interpretations
similar.
in this
of
reason
difficulties
was non-uniform
but
detritiation
metabolism
are are
of
conditions.
qualitative
liver
cycle
from
in glucose
There
(8).
synthesis
futile
of glucose
of recycling
are brought
is a rapid
pattern
loss
rats
2B.
was 10 mM and only
this
the
and the
cycles
steady
and pyruvate.
detritiation loss
under
perfused
a futile
futile
and Rognstad
systems
glucose
of
positions,
quantitation
in ideal
Still
small.
in Figure
obtained
of glucose For
18 to 22%
shown
ratios
positions
was utilized.
glucose
only
fasted
on the
six
greater.
medium
are
with
concentration
activity
was rather
liver. that
the
in glucose
hepatocytes
14C and in all
of the
the
greater
14C was also
containing
between
obtained
shown.
glucose
experiments
glucose.
of
content
ratios
U-14C)
with
uptake
tritium
difference
RESEARCH COMMUNICATIONS
medium was considerably
the
(5-3H)
same in the
is
and in the
and
was much the
doubly
rate
the
(2-3H)
glucose
review
rats,
The 3H/l4C
In Table
proposed
fasted
in the
activity.
U-14C)
in the
glucose
period,
with
AND BIOPHYSICAL
is
the
labeling
1,6
P2 the Since
that
the
the
Vol. 67, No. 1, 1975
BIOCHEMICAL
excess
tritium
loss
(6-3H)
glucose
is
The change injection
has
is only
(10)
but the
been
and more glucose
in rabbits
In mice
(4)
rates
glucose the
the
rate
and rabbits
(10)
to body
water.
to study
the
be pointed
of liver
from
hepatic
and triose-P
out
however
between
of that
of
14C (12)
the
periphery
and in hepatocytes
the
that
of
methods
is hepatic
since recycling
glucose. (5-3H)
*
In all
species
10% of 3HOH formed
recycling
(9).
is
the
contribution tracer
as shown here
of It
those
to some extent
(4)
catalyzed
synthesis.
including
in
The fraction
In mice
possibly
glucose
to glucose
(3-3H)
in the
make a significant
overestimate and liver,
(11).
glucose,
(6-3H)
cycle
(2-3H)/
is much less.
might
that
from
same
due to extrahepatic
only
(5-3H)
the
difference
in sheep
position
available will
was little
3HOH is
In rabbits
and (6-3H)
order
there
5 or 6-3H)
tritium
(5-3H)
in the
(9)
is nearly
of
of
that
(3,
glucose
The loss
by hepatic
Cori
all
there
(5-3H)
estimated
suggest
contribution
glucose.
isomerase,
Our results
In rat
(2-3H)
liberated
detritiation
species.
with
glucose
other
intravenous
that
3 or 6-3H)
the
after
with
and
of
glucose.
ratios
there
derived
(U-14C)
3H/l4C
decreases
is
of
the
between
is
5) The utilization
glucose
Conversely
it
(4).
that
glucose.
of the
glucose
liver
randomization carbon
(2,
In rat
in
by transaldolase
the
(4).
fraction
(2-3H)
of 3HOH formed
(6-3H)
RESEARCH COMMUNICATIONS
in several
(2-3H)
of detritiation
of
greater
from
than
was intermediate
catabolism.
body
between
of
3H) glucose
the
--in vivo
of
that
than
in circulating
was slower
of detritiation
by far
ratios
of detritiation than
exchange
greater
investigated
decay
rapid
(3-3H)..:(6-
slightly
difference
the
rate
transaldolase
in 3H/l4C
was no significant glucose
via
AND BIOPHYSICAL
the
choice
should
employing cycling
in perfused
of carbon.
ACKNOWLEDGEMENT The authors assistance. This Medical Research AM 12604 for the National Science
are indebted to Mrs E. Joyce for her valuable technical work was supported in part by a grant from the New Zealand Distribution Committee (1A 200/12133) and in part by grant National Institute of Health and grant BMS 7422815 for the Foundation.
218
Vol. 67, No. 1, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Clark, D.G., Rognstad, R., and Katz, J. (1973) Biochem. Biophys. Res. Comm., 54, 1141-1148. Rognstad, R., Clark, D.G., and Katz, J. (1973) Biochem. Biophys. Res. Comm., 54, 1149-1156. D.P., Holland, P.C., and Lardy, H.A. Clark, M.G., Bloxham, (1974) J. Biol. Chem. 249, 279-290. Biophys. Res. Comn., 58, Hue, L., and Hers, H.G. (1974) Biochem. 532- 539. Hems, R., Ross, B.D., Berry, M.N., and Krebs, H.A. (1966) Biochem. J. 101, 284-292. Prichard, K.H. (1972) J. Thorac. Cardiovas. Surg. 63, 240-247. R. (1975) "Current Trends z Cellular Regulation" Katz, J., and Rognstad, In Press. Golden, S. and Rognstad, R. (1975) Eur. J. Bioch. Katz, J., Wals, P.A., In Press. Katz, J., Golden, S., Dunn, A., and Chenoweth, M.A. Submitted for Publication. Dunn, A., Chenoweth, M.A,, Golden, S., and Katz, J. Submitted for Publication. and Leng, R.A. (1972) Aust. J. Biol. SC. 24, 1313-1332. Judson, C.J., Reichard, G.A., Moury, N.F., Hochella, A.L., Patternson, A.L., and S. (1963) J. Biol. Chem. 238, 495-501. Weinhouse,
219