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

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Vol. 67, No. 1, 1975 BIOCHEMICAL FUTILE AND BIOPHYSICAL CYCLES IN ISOLATED PERFUSED RAT LIVER AND IN ISOLATED RAT LIVER Dallas RESEARCH COMMUNIC...
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