metabolite concentrations in the liver of the adult and developing guinea pig and the control of...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 176, 171-180 (1976)

Metabolite Concentrations in the Liver of the Adult and Developing Guinea Pig and the Control of Glycolysis in Vivo

ANNE FAULKNER AND COLIN T. JONES

Nuffield Znstitute for Medical Research, University of Oxford, Headley Way, Headington, Oxford OX3 9DS, England

Received February 18, 1976

A range of metabolite concentrations have been determined in the liver of the adult and fetal guinea pig during the latter half of gestation. Adenine nucleotides showed little change during development of the fetal liver and the only major difference from the adult was a low ADP concentration. The hexose phosphates, particularly fructose 1,6- diphosphate, were higher and the triose phosphates in the glycolytic pathway after glyceraldehyde 3-phosphate were lower in the fetal liver. Cytosolic NAD+/NADH ratios were comparable in both adult and fetal livers as were cytosolic NADP+/NADPH ratios for the last 15-20 days of gestation. The metabolite concentrations have been used to indicate that glycolysis in the fetal guinea pig liver is controlled largely by hexokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate kinase.

During development of the liver large alterations in its enzyme activities, compo- sition, and the rates of its biosynthetic pathways occur (l-9). These are related to the changing metabolic demands upon the fetal liver which are different from those in adult life. Thus the factors controlling the metabolism of the fetal liver are likely to change during the course of development and to differ from those of the adult liver. The limited information which is available on the metabolite concentrations in the fetal liver has been used to indicate the points of control of glycolysis and, in particular, the role of hexokinase (10).

The present paper describes a range of concentrations of metabolites in the adult and fetal guinea pig liver during the latter half of gestation and indicates some sites of control of glucose metabolism in both the fetal and adult liver.

EXPERIMENTAL

Animals. Guinea pigs of the Dunkin Hartley strain were mated and gestational age was estimated as described by Elvidge (11). Their diet con- sisted of Dixon’s Diet 18 (Ware, Herts) plus hay ad Zibitum; drinking water was supplemented with ascorbic acid. They were anesthetized with pento-

171

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

barbitone (ca. 30 mg/kg) given via an ear vein. After approximately 3-5 min the fetuses were exposed through a longitudinal cut in the maternal body wall and uterus. The left lobes of the fetal liver were freeze-clamped (12) while the umbilical circulation was intact. The time between maternal injection and freeze-clamping the fetal liver was 5-7 min. For samples from adult male guinea pigs, animals were stunned by a blow to the back of the neck and the livers were rapidly exposed and freeze-clamped within 12-15 s. Hepatic tissue (0.5-l g) from adult guinea pigs or from one to three fetuses was pow- dered under liquid nitrogen with a ceramic pestle and mortar and then deproteinized with 1 ml of 30% HCIO, together with 5 ml of H,O. The extract was adjusted to pH 3-4 with 20% KOH. Blood samples from the heart of the adult guinea pigs and the umbilical vein of the fetal guinea pigs were collected into plastic syringes containing about 10 units of heparin. Metabolite and ion concentrations were determined in the plasma after spinning the blood at 15OOg and 4°C for 20 min.

Metabolite determinations. Inorganic phosphate was determined calorimetrically (13) and ribulose 5- phosphate was determined by a radiochemical car- boxylation assay (C. T. Jones, unpublished work). All other metabolites were assayed by standard methods (14). Hexose phosphates, pyruvate, phosphoenolpyruvate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, glycerate a-phosphate, and isocitrate were determined in an Aminco-Bow- man Spectrofluorimeter (Silver Spring, Maryland).

172 FAULKNER

The remaining metabolites were assayed spectro- photometrically. Where possible, more than one determination was performed in a single cuvette. Gly- cogen was determined as glucose after enzymic digestion with amyloglucosidase.

The [CO,] used in the nucleotide ratio calcula- tions (Table III) was calculated from pC0, in the maternal inferior vena cava blood and fetal umbilical vein blood; values were 40.7 + 4.2 (8) and 52.6 2 6.8 (14) mm Hg for the maternal and fetal blood, respectively.

Determination of intracellular Na+ and K+ concentrations. The inulin spaces of adult and fetal liver were determined by injection of [3H] inulin into the umbilical vein (0.5 &i; 0.1 ml) of fetal guinea pigs or into the ear vein (2 &i; 0.4 ml) of adult guinea pigs. After 2 min a sample of liver and blood was taken and the radioactivity in each was determined.

For ion determinations, samples of fetal and adult liver were obtained as described above and digested in 0.1 M HN03. After dilution with 0.1 M

HN03, Na+ and K+ concentrations were determined with a Pye-Unicam SP90 Atomic Absorption Spec- trophotometer. Intracellular ion concentrations were calculated as: (liver content per gram - inulin space X plasma concentration)/(l - inulin space).

Expression of results. All results are expressed as means + SD with the number of observations in parentheses. Significance was estimated using Stu- dent’s t test.

Chemicals. NAD+, NADH, NADP+, NADPH, ATP, phosphoenolpyruvate, citrate lyase (EC 4.1.3.6.1, phosphoribulokinase (EC 2.7.1.19.1, n-ribulose-1, 5-diphosphate carboxylase (EC 4.1.1.39.), and triethanolamine hydrochloride were obtained from Sigma (London) Chemical Company Ltd., Kingston-upon-Thames, Surrey. All other enzymes and 2-ketoglutarate were obtained from Boehringer (London) Corp., Lewes, East Sussex. NaH’%O, and [3Hlinulin were obtained from the Radiochemical Centre, Amersham, Bucks. Pentobarbitone (Nem-

AND JONES

butal) was obtained from Abbott Laboratories Ltd. (Queenborough, Kent). Heparin was obtained from Evans Medical Ltd. (Speke, Liverpool). All other chemicals were obtained from British Drug Houses, Poole, Dorset.

RESULTS

The metabolite and ion concentrations in livers of fetal and adult male guinea pigs (400-600 g) are given in Tables I and II. The metabolite concentrations for livers obtained from nembutal anesthetised adult guinea pigs were similar to those observed in the livers of stunned animals except for citrate and aspartate, which were 407 2 52 (6) and 567 k 91 (6) nmol/g, respectively.

Nat and K+

The inulin spaces in the adult and fetal livers were 0.304 + 0.075 (8) and 0.28 + 0.086 (16) ml/g of liver, respectively. There was no change in the value for the fetal liver between 50 days and term. During development there was a rise in the intracellular [K+l and a fall in the intracellular [Na+] of the fetal liver. Plasma [K+l and [Nat] did not change during fetal life; the [K+] was slightly lower (P ;: 0.001) in the fetal compared with maternal plasma (Ta- ble I).

Adenine Nucleotides

The ATP concentration in the fetal liver at 40-51 days was not significantly lower than that in the adult. After 50 days it fell so that between 58 days and term the con-

TABLE I HEPATIC AND PLASMA Na+ AND KC CONCENTRATIONS IN ADULT AND FETAL GUINEA PIGS”

Ion concentrations of plasma or intracellular water (~moliml)

Adult Fetal

40-49 days 50-59 days 60 days-term

Liver Plasma

[Na+l W+l [Na+l [KC1 20.9 k 12 128 t 25 143 t 1.8 6.7 t 0.05

41.3 f 3.7**b 71.3 -t 6.2** - 33.8 + 1.1 ** 84.8 T 7.0** 142.2 ? 2.6’ 6.3 + 0.13’ 30.3 2 7.3 99.1 ? 8.4* -

cz Ion concentrations were measured in tissue extracts from adult and 50-day fetal guinea pigs as described in the Experimental seciton. The values are means ? SD of 4-10 determinations.

b The P values for comparison of fetal with adult male are: * c Mean value for 45- to 65-day fetuses.

FETAL LIVER METABOLITES 173

TABLE II

METABOLITE CONCENTRATIONS IN FREEZE-CLAMPED LIVERS FROM FETAL AND ADULT MALE GUINEA PIG@

Adult male

-

Fetal (40-51 days) Adult male Fet;LEy-51

ATP

ADP

AMP

Glucose 6-phosphate

Glucose l-phosphate

Fructose 6- phosphate

Fructose 1,6-diphosphate

6-Phosphoglu- conate

Ribulose 5 phosphate

Citrate Isocitrate 2-Ketoglutarate Malate

Pi

1941 + 214

264 k 47***

147 + 59*

165 k 28***

7.2 k 4.6

39.7 + 8.3***

12.8 + 6.2***

24.6 k 20.4***

14.3 + 9.3

256 k 90 6.6 + 3.8** 130 k 26*** 265 + 65

2323 + 347

2023 ? 265

576 + 128

194 k 66

80.5 r 29.1

5.9 + 3.2

22.0 k 8.7

1.7 2 1.4

7.2 + 6.1

23.8 k 23.2

267 k 122 15 k 7.8

286 f 68 282 2 105

2905 f 870

Glycerol 3- phosphate

Glyceralde- hyde 3- phosphate’

Dihydroxyac- etone phosphate

Glycerate 3- phosphate

Glycerate 2- phosphate

Phosphoenol- pyruvate

Pyruvate

Lactate

Alanine

Glutamate Aspartate Acetoacetate 3-Hydroxy-

butyrate Ammonia Glucose

146 k 43****

0.85$

95 + 33

0.5$

18.7 t 5.5***

85 + 27***

11.7 k 3.9**

41 k 24*

119 r 57***

1758 5 703*

894 + 303***

2493 2 667** 1275 A 367***

- -

10.2~ 2.6

135 ? 39

16.4 k 5.3

56 + 16.7

66 ? 33

1207 k 605

560 t 165

3409 ? 1047 321 + 154 6.2 + 4.6

23.3 k 12

188 2 142 204 k 141 2830 + 930** 4640 t 1480

n Metabolite concentrations were determined in extracts from freeze-clamped liver of 14 adult males and of fetuses from 24 guinea pigs 40-51 days pregnant, as described in the Experimental section. The values are expressed as nanomoles per gram wet weight (means k SD).

* The P values for comparison of fetal with adult males are: *<0.05; **<O.Ol; ***<O.OOl. c Calculated from the dihydroxyacetone phosphate concentration and the equilibrium constant for triose

phosphate isomerase (15).

centration was less (P < 0.001) than that at 40-51 days (Fig. 2a). No significant changes were observed in AMP, ADP, or Pi concentrations. There was a reduction in both the mass action ratio for adenylate kinase and the measured [ATP]/[ADP] [HP042-l ratio associated with low AMP and ADP concentrations in the fetal liver (Tables II and III). The calculated cytosolic [ATPMADPI [HPO,2-I ratio was much less than its measured value and it declined during development. This was determined using a glyceraldehyde 3-phosphate concentration calculated from dihydroxyacetone phosphate (Table II). The concentration in the freeze-clamped liver was not consistently detectable (i.e., about 0.3-l nmol/g), which indicates that a triose

phosphate disequilibrium of the order observed by Veech et al. (15) was not present and could not account for the very large differences in the calculated and measured [ATPMADPI [HP042-l ratios.

Glycogen and Glucose Between 40 and 50 days the glycogen

content of the fetal liver was low, at ~0.4 mglg, between 56 days and term it rose substantially (Fig. 1). The glycogen content of the adult liver was higher than that in the 40- to 56-day fetus (Fig. 1). The glucose concentration in plasma or freeze clamped liver did not change between 50 days and term. They were lower than the concentrations in the adult (Tables II and IV). Calculated intracellular glucose con-

174

25

FAULKNER AND JONES

I i

40 50 60

Fetal age, days

Term A

FIG. 1. Glycogen content of fetal and adult guinea pig liver. The glycogen content of the freeze- clamped livers from adult male and fetal guinea pigs was determined as described in the Experimental section. The values are the means + SD of 6-11 determinations. A, adult male.

centrations were 3.27 and 2 pmol/g for the adult and fetal livers, respectively.

Hexose Phosphates

Glucose 6-phosphate concentrations in the fetal liver changed little between 44 and 57 days and then rose (P < 0.001) by about 40% at the same time as the increase in liver glycogen (Fig. 3a). Fructose 6- phosphate changed in parallel and main- tained a fructose 6-phosphate/glucose 6- phosphate ratio of about 0.25. The concentrations of both were substantially higher in the fetal than adult liver (Table II). Fructose 1,6-diphosphate concentrations in the fetal liver were high between 40 and 59 days and then fell (P < 0.001) to values approaching those in the adult liver (Fig. 3b). The 6-phosphogluconate values were also high between 40 and 50 days and then declined to adult values (Fig. 3~). There were no significant changes in ribulose 5- phosphate.

Trioses

There were no significant changes with age in any of the trioses measured in the fetal liver. However the ratio of phosphoenolpyruvate/pyruvate, which was main- tained at 0.344 + 0.237 (52) from 45 days to term, was higher (P < 0.01) in the earlier fetal liver (40-43 days) when the ratio was 0.636 k 0.226 (9). A higher ratio (P <

0.0011 of 1.03 k 0.57 (14) was also observed in the adult liver. The concentrations of the triose phosphates before the glyceraldehyde 3-phosphate dehydrogenase reac- tion were higher, and those after were lower in the fetal than in the adult liver (Table II).

/ I / I

I 50 60 Term A

Fetal age, days

Ibl

z I

/ 1 I I

50 60

Fetal age, days

Term A

I I I

60 Term A

Fetal age, days

FIG. 2. The ATP, aspartate, and malate concentrations in the fetal and adult male guinea pig liver. Metabolites were determined in extracts of freeze- clamped livers as described in the Experimental section. (a) ATP, (b) aspartate, and (c) malate. The values are the means ? SD of 8-14 determinations. A, adult male.


TABLE III

MEASURED AND CALCULATED VALVES FOR THE PHOSPHORYLATION STATE OF THE ADENINE NUCLEOTIDES IN FETAL AND ADULT MALE GUINEA PIG LIVERY

Fetal age (days) [ATP] [AMP]/[ADP12 [ATPl/[ADPl [HPO,*-1 /.LM-’ x lo5

Measured cytosol Calculated cytosol

40-49 3.77 k 1.48 (14)*** 541 ” 105 (18)*** 41.4 k 32.4 (18)** 50-59 4.08 k 2.39 (23)*** 485 k 131 (23) 36.2 2 21.1 (23) 60-term 1.86 2 0.69 (22) 389 + 146 (22) 19.9 + 16.8 (19) Adult 1.29 + 0.56 (14)** 201 + 82 c141*** 10.9 ? 4.2 (13)*

a Details and metabolite values as for Table II. The concentrations of HPO,*- at pH 7.0 were taken as 60% of the total P,. The phosphorylation states were calculated as described by Veech et al. (18). Glyceraldehyde 3-phosphate concentrations were calculated for each liver as described in Table II. The values are expressed as means + SD. TheP values for comparison with the go-day term fetuses are *<0.05, **<0.02; ***<O.OOl.

TABLE IV

PLASMA METABOLITE CONCENTRATIONS IN FETAL AND ADULT GUINEA PIG@

Plasma metabolite concentrations, (nmol/ml)

Fetal Adult

50 days 60 days-term

Glucose 4970 f 860*** 5015 f 975*** 7780 2 410 Pyruvate 117 2 43 74.9 zt 26.4 115 ? 62 Lactate 4196 f 1241* 2733 k 575 2751 + 720 Citrate 150 -c 34 162 f 18 191 ? 62 Malate 11.6 it 3.2 - 13.9 k 5.7 Alanine 399 f 65*** 490 k 9a*** 207 2 57 Glutamate 42.3 + 26 - 58 k 25.3 Aspartate 16.6 2 8.9** - 38.8 2 9.5 NH,+ <40 - 75.8 k 61

(2 Plasma metabolites were determined in umbilical vein blood from the fetuses and in cardiac blood from adult male guinea pigs as described in the Experimental section. The results are the means ? SD of 5-22 determinations. The P values for comparison of fetal and adult male are: *<0.02, **<0.002, ***<O.OOl.

The plasma pyruvate and lactate concentrations are given in Table IV. Because these values are relatively high, a significant proportion of the measured tissue pyruvate and lactate is located in the extracellular space of the tissue. Correcting for this gives pyruvate concentrations of 45, 120, and 132 nmol/g and lactate concentrations of 545, 810, and 2064 nmol/g in the intracellular water of the livers from adult, 50-day fetal, and go-day to term fetal guinea pigs.

Amino Acids

Of the amino acids measured, only aspartate showed significant changes in the fetal liver, with lower (P < 0.001) concentrations at 50-57 days than earlier or later in gestation (Fig. 2b). Alanine and aspartate concentrations were higher and gluta-

mate concentrations were lower in the fetal than in the adult liver. Only alanine had a relatively high plasma concentration but correction of the freeze-clamped liver values for this had only a small ef- fect.

Krebs Cycle Intermediates

The concentrations of malate in the 40- to 60-day fetal liver were similar to those in the adult. After 60 days there was a significant (P < 0.001) rise to about twice the adult values (Fig. 2~). No changes were seen during development in the concentrations of citrate, isocitrate, or 2-ketoglutarate in the fetal liver. In the adult liver the isocitrate and 2-ketoglutarate concentrations were higher than in the fetal liver (Table II).

176 FAULKNER AND JONES

4m

1 T

Y 4 I

01 40

1 I , 50 60 Term A

Fetal ache, days

(bl

5 F .F 10 -

-2 c i

5

0 %, I ;

40 50 60 Term A

Fetal qe, days

60 1 /

40

20

50 60 Term A

Fetal age, days

FIG. 3. The concentrations of some hexose phosphates in the liver of the fetal and adult male guinea pig. Hexose phosphate concentrations were measured in extracts of the freeze-clamped livers as described in the Experimental section. (a) glucose 6- phosphate, (b) fructose 1,6-diphosphate, (c) 6-phosphogluconate. The values are the means 2 SD of 8- 14 determinations. A, adult male.

NAD+INADH and NADP+INADPH Ra- tios

The cytosolic NAD+/NADH ratios calculated from the total liver pyruvate/lactate ratios were approximately 500 for both the adult and fetal liver (Table V). When cor- rections were made for the extracellular pyruvate and lactate the NAD+/NADH ratios became 743, 1335, and 576, respectively for the adult, 50-day fetal, and 60- day to term fetal guinea pig liver. These were now similar to the ratios obtained using dihydroxyacetone phosphate/glycerol 3-phosphate, which also fell between 40 days and term in the fetal liver. Adult liver ratios were not significantly different from any of those seen in the fetal liver (Table V).

Willms et al. (16) have indicated that in the fed guinea pig both P-hydroxybutyrate dehydrogenase and glutamate dehydrogenase are at equilibrium and mitochondrial NAD+/NADH ratios calculated on those assumptions are similar. We also observed similar but much smaller calculated ratios which were 5.05 ? 3.8 (14) and 5.9 2 3.95 (8) for the glutamate dehydrogenase and fi-hydroxybutyrate dehydrogenase reactions, respectively. Little con- version of /3-hydroxybutyrate to acetoacetate occurs in the fetal guinea pig liver at about 50 days. Glutamate dehydrogenase activity in the fetal liver is about 80 units/ g at 37°C (C. T. Jones, unpublished work) but whether that is sufficient to maintain equilibrium is not clear (17). The mitochondrial NAD+/NADH ratio calculated using this enzyme was 2.26 +- 1.72 (60) for the fetal liver.

The NADP+/NADPH ratios calculated using 6-phosphogluconate dehydrogenase were similar in the adult and fetal liver between 50 days and term. Lower values were obtained using the malic enzyme substrates for calculating the ratio. In the 40- to 49-day fetal liver the high 6-phosphogluconate concentration is associated with a low calculated NADP+/NADPH ratio (Table V).

Pregnant Guinea Pigs

There was no difference in the metabolite concentrations in livers of guinea pigs

TABL

E V

THE

CAL

CU

LATE

D

NAD

+/N

ADH

AN

D N

ADP+

/NAD

PH

Rat

ios

for

the

Live

rs

of

the

Feta

l an

d Ad

ult

Mal

e G

uine

a Pi

p 2

Feta

l ag

e (d

ay&

NAD

+/N

ADH

, ca

lcul

ated

fro

m

NAD

P+/N

ADPH

, ca

lcul

ated

fro

m

3 E Py

ruva

te/la

ctat

e D

ihyd

roxy

acet

one

hos-

R

R

ibul

ose

5-ph

osph

ate/

6-

Pyru

vate

imal

ate

phat

elgl

ycer

ol

3-p

os-

phos

phog

luco

nate

2-

Keto

glut

$r..t

eiis

ocit-

r

phat

e 2

40-4

9 67

9 2

342

(18)

16

22 z

k 57

2 (1

7)**

* 0.

007

2 0.

012

WQ

**

0.02

2 -c

0.0

12

c17j

**

0.01

3 k

0.01

7 (1

8)

E 50

-59

467

2 20

0 (2

3)

1212

+ 3

75 (

22)

0.03

3 L

0.03

3 (2

4)

0.02

1 2

0.01

1 (2

4)

0.03

6 -t

0.02

1(24

) $

60-te

rm

528

k 28

3 (2

2)

880

k 64

2 (1

9)

0.02

6 zt

0.0

2 (2

0)

0.01

2 f

0.00

9 (2

2)

0.03

3 2

0.02

2 (2

1)

z Ad

ult

550

rt_ 1

64 (

14)

1271

s

583

(10)

0.

038

f 0.

028

(14)

0.

009

2 0.

005

(14)

0.

026

2 0.

015

(14)

o D

etai

ls

and

met

abol

ite

valu

es

as fo

r Ta

ble

2. T

he

NAD

+/N

ADH

ra

tio

from

gl

ycer

ol

3-ph

osph

ate

dehy

drog

enas

e w

as

calc

ulat

ed

as t

dihy

drox

yac-

ii r

eton

e ph

osph

ate]

/[gly

cero

l 3-

phos

phat

e]/K

w

here

K

g9

x 10

d5 a

t 37

°C a

nd

pH

7.0

(32)

. Th

e ot

her

nucl

eotid

e ra

tios

wer

e ca

lcul

ated

as

des

crib

ed

by

Veec

h et

al.

(24)

. Th

e N

ADP+

INAD

PH

ratio

s w

ere

calc

ulat

ed

on t

he

follo

win

g es

timat

es

of l

iver

[C

O,],

fe

tal:

1.6

mol

ig,

mat

erna

l 1.

3 m

ol/g

. Th

e 2 cn

va

lues

ar

e m

eans

k

S.D

. Th

e P

valu

es

for

com

paris

on

with

th

e ra

tios

of t

he

60 d

ay-te

rm

feta

l liv

er

are:

*<

0.05

; **

<0.0

1;

***<

0.00

1.


35-56 days pregnant compared with adult males except for the alanine concentration in the pregnant guinea pigs, which at 278 t- 178 (9) nmol/g was significantly lower (P < 0.002) than in the adult male.

DISCUSSION

Adult Liver Metabolites

In the rat liver [ATPl[AMPl/1ADP12 values are close to the equilibrium constants for adenylate kinase and the calculated and measured [ATPI/[ADPl[HP0,2-l ratios are similar (18, 19). In the guinea pig liver the ADP and Pi content is lower (16, 20) with adenylate kinase apparently out of equilibrium and the calculated and measured [ATPI/[ADPl[HP042-l ratios differing by a factor of more than 20. This suggests that the glyceraldehyde 3-phosphate dehydrogenase-glycerate 3-phosphate kinase couple is out of equilibrium and that mitochondrial and cytosolic adenine nucleotide concentrations are substantially different.

The hexose and triose phosphates, particularly fructose 1,6-diphosphate, are substantially higher in the rat (19, 21) than in the guinea pig liver although the mass action ratios for the equilibrium enzymes between phosphoglucose isomerase and phosphoglycerate mutase are comparable in both species. The concentrations of the amino acids and Krebs cycle intermediates in the guinea pig liver are comparable with those reported for the rat (18, 21, 22).

The cytosolic NAD+/NADH ratio calculated from the pyruvate/lactate ratio is about twice the value previously reported for the guinea pig liver (16, 20) and about half those reported for the rat liver (18, 21, 23). The mitochondrial values calculated from acetoecetate/3-hydroxybutyrate and 2-ketoglutarateglutamate ratios are about one-half to one-quarter of those previously reported for the guinea pig liver (16, 20) and are comparable with the rat liver values (23).

Cytosolic NADP+/NADPH ratios calculated both from ribulose Sphosphate/6- phosphogluconate and 2-ketoglutarateliso- citrate are about four- to eightfold higher in the guinea pig than in the rat liver (21, 24). A lower ratio calculated from the py-

ruvate/malate couple is probably the re- sult of the much lower activity of malic enzymes than 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase 03).

Fetal Liver Metabolites

Some information is available on the metabolite content of the fetal liver (10, 25). This reports high concentrations of hexose and triose phosphates and very low calculated cytosolic NAD+/NADH ratios compared with those in the adult rat or adult and fetal guinea pig livers.

In the fetal guinea pig liver the concentrations of the hexose phosphates (particularly fructose 1,6-diphosphate and, at 40- 50 days, 6-phosphogluconate) are higher and the concentrations of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenolpyruvate are lower than in the adult liver. Calculated cytosolic NAD+/ NADH ratios are similar, while the adult mitochondrial values are about twice those in the fetal liver.

Control of Metabolism in the Fetal Liver

Hommes (10) and Vergonet (26) have suggested that, as indicated by the high hexose and triose phosphate concentrations, particularly fructose l,gdiphos- phate, glycolysis in the fetal rat liver is largely controlled by glucose phosphorylation. Fetal guinea pig liver hexokinase may also largely control glycolysis, although at the probable intracellular ATP and glucose 6-phosphate concentrations the enzyme has little activity in vitro (A. Faulkner and C. T. Jones, unpublished work). The high fructose 1 ,&diphosphate concentration in the fetal guinea pig also indicates that the control of glycolysis by phosphofructokinase is less important than in the adult liver.

Pyruvate kinase in the fetal guinea pig liver has properties similar to the rat liver enzymes (27, 28). The high fructose 1,6- diphosphate concentrations present in the fetal liver should fully activate pyruvate kinase but its rate may be limited by the low ADP and phosphoenolpyruvate concentrations. Higher pyruvate kinase activity in the fetal than in the adult liver is


suggested by the low fetal phosphoenolpyruvate/pyruvate ratio. This may also be a consequence of a low rate of phosphoenolpyruvate production by fetal liver mi- tochondria. In the liver of both the fetal rat and guinea pig, phosphoenolpyruvate carboxylase is largely mitochondrial (9, 29). However, the low NAD+/NADH ratio has indicated that there is little mitochondrial phosphoenolpyruvate production in the fetal liver of the rat. This, together with the NAD+/NADH ratio, may be responsible for the virtual absence of gluconeogenesis in the fetal rat liver (6, 25). The situation in the fetal guinea pig is less clear since relatively high NAD+/NADH values are found. Despite this, significant rates of gluconeogenesis have not been observed in the fetal guinea pig liver in vitro until after 60 days (9).

The high mass action ratios for adenylate kinase and the large difference between the calculated and measured [ATPI/ lADPl[HP0,2-] ratios in the fetal liver is surprising. It is not possible at present to say whether this is a consequence of sub- strate compartmentation or of disequilibrium of the enzymes involved. The activity of the equilibrium enzymes of glycolysis, except for aldolase, are relatively high in the fetal guinea pig liver from 40 days to term (30). The mass action ratios for phosphoglucose isomerase, phosphoglyceromu- tase, and enolase but not for aldolase are close to the equilibrium constants for the enzymes in both the fetal and adult liver. Thus it seems likely that the major sites of control of glycolysis in the fetal guinea pig liver are hexokinase, glyceraldehyde 3- phosphate dehydrogenase-glycerate 3- phosphate kinase, and pyruvate .kinase. The role of phosphofructokinase-aldolase remains to be established.

While there were differences in the [Na+l and [K+l contents of the fetal and adult livers it is unlikely that these pro- duce appreciable differences in the control of glycolytic enzyme activity. Similar changes in ion content have been reported for the developing rat liver (31).

In the 40- to 50-day fetal guinea pig, biosynthetic activity such as fatty acid synthesis (7, 8) and the pentose phosphate

pathway activity is high (C. T. Jones, unpublished work). Thus it is surprising that the 6-phosphogluconate concentration is high and the NADP+/NADPH ratio is low at this time. The higher ratio obtained using isocitrate dehydrogenase’ indicates that 6-phosphogluconate dehydrogenase might be out of equilibrium at 40-50 days.

ACKNOWLEDGMENTS

We wish to thank Professor G. S. Dawes for his interest and encouragement and Mrs. Paula Webb for expert technical assistance. The work was sup- ported by the Medical Research Council.

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metabolite concentrations in the liver of the adult and developing guinea pig and the control of...

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