Development of hepatic enzymes for the phosphorylation of glucose and fructose

DEVELOPMENT OF HEPATIC ENZYMES FOR THE PHOSPHORYLATION OF GLUCOSE AND FRUCTOSE D. G. WALKER Department of Biochemistry,Universityof Birmingham, Edgbaston, Birmingham,England INTRODUCTION THE unique role of the liver as an organ capable of reversibly converting glucose into a readily-available storage form, glycogen, and as the site of a homeostatic mechanism for the control of the blood glucose level and hence of carbohydrate metabolism in the whole mammalian organism has been recognized for over a century. Claude Bernard's classical researches were elaborated by Soskin and coworkers(1) some thirty years ago, but it is only in the last decade that the full enzymic basis of the regulatory mechanisms possessed by the liver has become clear. Glucose-6-phosphatase is present and is specially suited to the role of the liver in the production of glucose from glucose-6-phosphate (glucose-6-P), which may be formed from both glycogen or gluconeogenic sources, and is under complex dietary and hormonal control.(2) It is of pertinent interest that this enzyme develops late in the gestational period in many species(8, 4) and is hence present when the newborn animal has to rely upon its own mechanisms for metabolic control. These facts concerning glucose-6-phosphatase development represent only one aspect of the maturation of carbohydrate metabolism control mechanisms around birth and during the neonatal period. During the gestational period the fetus obtains its nutrients via the placenta. This transitory organ is able to transfer most monosaccharides (fructose being an exception) by a rapid process be it free permeability or a facilitated transport mechanism.(5) The placenta contains glycogen but the levels do not change rapidly(6) as does liver glycogen in response to changes in blood glucose. The placenta appears to contain a typical non-specific hexokinase having a low K,,, value for glucose(~) and suggestions that it contains glucose-6-phosphatase(a) have been questioned,t9) so that it almost certainly cannot fulfil the role of a temporary liver as originally suggested by Bernard.(to) The maternal liver must therefore play a major role in the control of carbohydrate metabolism within the conceptus until the fetus is able to provide its own means of control. This 163

164

D.G. WALKER

argument ignores probable additional regulatory mechanisms involving hormones and changes thereof during development. The adult liver takes up more glucose and synthesizes glycogen faster as the blood glucose level rises.(n) Enzymic expression of such physiological observations has become available in the past four years following the development of a greatly improved system for the assay of glucose phosphorylation using high initial glucose concentrations.(12) Subsequent use of this method and its application to both dialysed liver-supernatant preparations(la) and to ammonium sulphate fractions of such preparations(14) lead to the demonstration of the presence of two enzymes in liver tissue which can catalyze the phosphorylation of glucose, one of which is an adaptive enzyme.(15-m This enzyme, provisionally designated glucokinase,tt3, 14) is absent from the fetal liver of rats, guinea-pigs and rabbits and develops after birth.( 7, 13) The development of this hepatic enzyme therefore represents another feature of hepatic maturation. The ability to phosphorylate fructose by the specialized fructokinase(20, 21) also appears in the neonatal period(z2) and it is the development of glucokinase and fructokinase, two enzymes unique to the liver, which form the subjects of this article. MATERIALS AND METHODS

The methods used for experimental work to be reviewed in this article have been described elsewhere.( in, 17, 22, 2s) Wistar strain rats and Pirbright strain albino guinea-pigs were used. RESULTS AND DISCUSSION

Many features concerning hepatic glucokinase have been described in the last Symposium.its, 19) It is essential that the trivial names commonly used and the nature of the enzymes being discussed should be clarified. The following is a brief summary of the enzymes present in liver which can catalyze the phosphorylation of glucose and fructose by normal adult liver tissue. Some of the relevant facts concerning the adaptive nature of the enzymes in adult animals is also recorded to serve as a basis for discussion of the problems of the induction and control of the development of the enzymes.

Glucose Phosphorylation by Adult Liver Two enzymes are present as has been demonstrated kineticallytla) (see Fig. 1) and by their separation.tl4, 24, 25) (a) Hexokinase appears to be a typical animal hexoldnaset26) in that it can phosphorylate a number of hexoses. Its apparent aifmity for glucose (K~ is 1 - - 3 × 10-5 M) {13, 14) and other substrates( ts, 25) is such that it will always be saturated with substrate at physiological glucose concentrations so that

165

DEVELOPMENT OF HEPATIC ENZYMES

other sugars will not be phosphorylated when glucose is present. This hexokinase occurs in the non-parenchymal cells of liver tissue( is, 19) but may also be present in parenchymal cells and be able to function under some conditions(~7) in spite of it being inhibited by glucose-6-P.(2e)

25

2O

lS

10

I

| 5

0 60

J 10

10"1 (G LUCO6E] (M-I)

3G

10!

I

I

I

O

1

I

I

I

i

e

e

$

4

5

6

'~

10-4

F[o. 1

Reciprocal plots of reaction rate (measured as N A D P H production by the glucose6-P formed in the presence of glucose-6-P dehydrogenase) versus glucose concentration for a dialysed supernatant preparation from adult rat liver, the animal having been starved for 48 hr. This has the effect of lowering the amount of the high-Kin enzyme present. The points in the top graph are the same as the first six in the bottom graph. For kinetic analysis of these graphs, see Walker.(18~

(b) Giucokinase is the recently-discovered( 13, 14, ~s) unique enzyme which is almost certainly confined to hepatic parenchymal cells(19) and having a relatively high K,~ for glucose (about 10 mM)(is, 14, 29) which makes it of physiological significance. It is changes in the levels of this enzyme under various dietary and hormonal conditions(~SqT) which account for the changes in the total glucose-phosphorylating activities of liver preparations.~ ~, 3o-~) Glucokinase activity falls to low levels during starvation("-17) and the rate at which the recovery of activity on refeeding occurs seems to depend partly upon how low the activity reaches during the starvation period.(is, 16) The reappearance of glucokinase on refeeding represents net enzyme

166

D.G. WALKER

synthesis.(x6-19) Glucokinase activity also falls to low levels in alloxandiabetes(15-xv) (see also Fig. 2) and de novo resynthesis of the enzyme occurs following insulin administration to the diabetic animals.(15-17) The use of the term glucokinase for this enzyme is perfectly satisfactory from the physiological point of view and dearly distinguishes it from a hexokinase (see previous discussion by Sols(19)) but enzymically the name is less satisfactory because of its action on other substrates.( 19, 24, 25)

3O

GLUCOglNASE I0 I

--

I

I

j//°

.1~ 25

I

I

I

•

10 1015

~

1¢)

I °

J

1

I

I

,

I

.

|

.

F1o. 2

Reciprocal plots similar to those in Fig. 1 for (a) an alloxan-diabetic adult rat (showing only a trace of the high-Kinglucokinase), Co) fetal-rat liver (gestational age 19 days) and (c) rat placenta.

Fructose Phosphorylation by Adult Liver Theoretically there are three known enzymes which may phosphorylate fructose. (a) The non-specific hexokinase mentioned above can catalyze the phosphorylation of fructose but, because its phosphorylation coefficient(u) is very low (0.01-0.02 compared to 1 for glucose--calculated from previous resultsOa, st)) it is unlikely to act under physiological conditions.

DEVELOPMENT OF HEPATIC ENZYMES

167

(b) The unique glucokinase can phosphorylate fructoset14, 19. 25~ but the / ~ for fructose is extremely high ~25>so again it is not of physiological signio ficance. (c) Fructokinase (adenosine triphosphate-D-fructose 1-phosphotransferase, EC 2.7.1.3) phosphorylates fructose to form fructose-l-phosphate. This enzyme has a high affinity for fructose but is strongly inhibited by ADP and when the concentration of ATP is greater than that of Mg ++ ions.t21~ Such properties may represent important intraceUular control mechanisms but its role in fructose metabolism is accepted~SS~ and there has been one report of it being an adaptive enzyme.<~)

The Post-natal Development of Hepatic Glucokinase Glucokinase is absent from supernatant preparation of rivers from fetal rats, rabbits and guinea-pigs.~ 7. is> This is apparent from the fact that the rate of glucose phosphorylation at both 100 mM and 0.5 mM glucose (the latter concentration being sufficient to give maximum rates with hexokinase but a negligible rate with glucokinase) are virtually identical. This is further illustrated by the comparisons of plots of 1/v against 1Is (Figs. 1 and 2). The plot for fetal fiver is similar to that for a tissue possessing only a nonspecific hexokinase (placenta). The plot for alloxan-diabetic rat fiver shows only a trace of glucokinase activity. Further kinetic analysis of these plots has been given.¢is. 2g) There are complications in the plots obtained with guinea-pig liver supernatant preparations which make interpretation ditiicult.tls> However, the indication is that glucokinase develops in the first week to ten days after birth in this species and this correlates with recent metabolic studies.~ST~ The development of glucokinase in the neonatal rat does not begin until about 16 days after birth~~a, 24> (Fig. 3), and adult levels are reached some 10-12 days later. The maximum rate of increase occurs between 21 and 26 days after birth. Two further points concerning the natural development of glucokiuase should be made. First, analysis of individual results revealed that there were no sex differences in developmental behavior. Secondly, there was go correlation between the weight of the animal and either the time at which glucoldnase first appeared or age at which half adult levels were attained. There was, in fact, a very wide range of activities for random animals of any given age in the developmental period (see S.E.M. values in Fig. 3) and even for fitter-mates of identical age. For the first two weeks of extrauterine life the neonatal rat presents a unique situation in that it is without glucokinase and yet is not diabetic (i.e. the blood glucose levels are normal). This is reasonable because Lazarow and coworkers¢ss~ have shown by microdissection techniques that the rat pancreas contains insulin for the last few days before birth and thereafter

168

D.G. WALKER

normal adult levels are present. That insulin is both available and effective in the neonatal rat is implied by the normal blood glucose levels and by observations that (a) injections of insulin result in the usual fall in the blood sugar level and (b) administration of chlorpropamide, a hypoglycaemic

1.81z 0

i

|

t

*

•

t

•

i

I SO

'

.

.

n

'

.

i

•

.

.

.

n

I IS

*

I

I

'

~ 1.o

I O.e

0 is

AGE

IN

I //I 10W A

DAYII

F:o. 3

The normal post-natal development of hepatic glucokinase in the rat. Each point represents the mean -4- S.E.M. of at least 4 (and up to 21) individual animals taken at random from a breeding colony. drug o f the sulphonyl urea group which is believed to act primarily as an insullnogenic agent,(39) both orally and intraperitoneally to both fed and starved neonatal rats results in a decrease of the blood glucose level.(2a) The slowness of the rate of glucokinase development immediately suggests that enzyme synthesis is involved, for any type of activation process is likely to be much quicker. A whole series o f experiments was performed which

169

DEVELOPMENT OF HEPATIC ENZYMES

should detect the presence of activators or inhibitors. Both undialyzed and dialyzed preparations of supernatant fractions of livers from animals of many ages in the range from the late gestational stage to 6 weeks old were combined in a variety of ways but in all tests the resultant total glucokinase 1.0

I

0.8 D

(2)

I

O)

(4) 0.4 I

(2)

2

O) C AGE (Days)

21

24

24

21

24

24

|4

TREATMENT

FIG. 4 The effect of inhibitors of protein synthesis upon the normal development o f hepatic giucokinase in the rat. The results are for separate experiments on 2 fitters of rats, and each result represents the mean :i: S.D. for the number of determinations on individual animals given in parentheses. The treated animals were given one intraperitoneal injection on each of days 21-23 of saline solutions of one or more of the following as indicated: L-ethionine, 50 rag/100 g body wt.; L-methionine, 100 mg/100 g body wt.; vL-p-fluorophenylalanine, 50 mg/100 g body wt.; L-~-phenylalanine, 100 mg/100 g body wt.

and hexokinase activities were always the arithmetic sum of the activities in the individual samples. This type of test has been shown to give a false picture in one instance,(4°) but in the case of glucokinase there is no known cofactor analogous to that demonstrated for tryptophan pyrrolase. Further evidence that the development is true de n o v o enzyme synthesis has come from the use of inhibitors of protein synthesis on the normal

170

D . G . WALKER

course of development. Figure 4 shows that, during the period of maximum increase in glucokinase activity, daily injections for 3 days of either ethionine(41) or p-fluorophenylalanine(4z) resulted in lower glucokinase activities at the end of the 3-day period and that simultaneous injections of methionine or phenylalanine, respectively, largely overcomes this inhibition and permits net enzyme synthesis. Similar results have been obtained using several different litters of rats. (Because of the wide variation of activities in the developmental period, all these studies have been performed using only one set of litter-mates for each individual experiment rather than pool the results of many more animals having very wide ranges of activity under any set of experimental conditions.) The effects of puromycin and actomycin upon development were investigated but both agents have proved to be too toxic for use in neonatal animals over a sufficient period of time to permit statistical assessment of an effect upon glucokinase levels.

Factors Affecting the lnitial Appearance of Glucokinase Attempts have been made to determine the reason for the absence of glucokinase in the rat before the third week of extrauterine life. The work on adult animals already summarized and other work(4a, 44) suggests the glucokinase is induced by its substrate glucose in the presence of insulin. The presence of insulin in the pancreas since before birth and its effectiveness upon other criteria of insulin action already mentioned together with the fact that the neonatal rat has plenty of carbohydrate in its normal diet suggests that there must be some reason other than a lack of glucose and insulin which accounts for the non-appearance of glucokinase before a definite stage of development. Experiments have been performed in an attempt to advance the age at which glucokinase first appears. These fall into several groups as follows: (a) Litters of young rats were divided into 2 groups by marking but left with the mother. One group was given daily intraperitoneal injections of glucose (at the rate of 2 mmoles of glucose per 100 g body wt in the form of an 18 per cent (w/v) aqueous solution of glucose, 2 ml per I00 g body wt) and the other group received a similar volume of saline. One each of the glucoseand saline-treated animals was withdrawn from the cage and Jused for glucokinase assay on each of several days. The results of this type of experiment may be thus summarized. (i) The effect of glucose treatment beginning at age 6 days or any other time up to 14 days old was to advance the appearance of glucokinase by not more than two days from the usual time of 16 days. (ii) Glucose-treated animals having injections beginning before 14 days old usually resulted in slightly higher glucokinase values compared to the control animals in the region of 14--18 days old. The results for 2 typical litters are shown in Fig. 5. However, because of the considerable range of glucokinase

DEVELOPMENT OF HEPATIC ENZYMES

171

values, statistical analysis of the results for several litters (Fig. 6) gave signitic.ant differences between treated and control animals at 14 days of age only. Thus, glucose infusion had only a marginal effect upon glucokinase induction.

o.[-

,i,]il:i t 1|

15 AGE

18 DAYS

IN

Zl

FiG. 5 The effect of glucose infusions on the development of hepatic glucokinase in the neonatal rat. The treated animals were given daily injections of 2 • m o l e s of glucose/100 g body wt. intraperitoneaUy from when 6 days old. C) C), litter A, control animals; I-'l V], litter A, glucose-treated animals; • 0, litter B, control animals; • M, litter B, gluome-treated animals. iI i

0.0

i

(4)

(S)

0

AGE (1~15) TRE~TMKNT WITHGLUCO6E 81[GNIFICANC! O F DIFFERENCE

14 + p(0.01

II

1|

+ 0.1(p40.2

+ 0,1
FIG. 5 T h e e f f e c t o f g l u c o s e i n f u s i o n o n the d e v e l o p m e n t o f h c l ~ t i c g l u c o k i n s ~ in t h e n e o n a t a l r a t . T h e s e results a r e b a s e d u p o n n o r m a l m i d s l u c o s ~ mlim~s

frmn 4 fitters, treated as in Fig. 5. Each result is the mean 4- S.D. with the number of animals in parentheses. Treatment belpm when 10 days old and other details are as in Fig. 5.

t72

D.G. WALKER

(b) Similar types of experiments were performed in which again a litter was divided into 2 or 3 groups by marking and each group was treated for a period of 3-10 days (i.e. starting when about 11 or 4 days old, respectively) in attempts to advance glucokinase development compared with control litter-mates. Groups of litter-mates were treated in typical experiments with (i) glucose (as above) alone or together with insulin (protamine zinc insulin, intravenously, at rate of 0.5, 1 or 2 units per 100 g body wt per day), (ii) insulin alone or with glucose (same doses as before), (iii) glucose alone or together with chlorpropamide (given intraperitoneally at the rate of 0.5 ml per 100 g body wt of a suspension in saline containing 10 mg/ml, or intragastrically by stomach tube using an aqueous suspension of chlorpropamide at the same rate) and (iv) chlorpropamide alone (intraperitoneaUy or intragastrically) or with glucose. Altogether some 35 litters were used in these experiments and in every case treated animals were compared with normal controls. In addition to its role as substrate, an injection of glucose should produce an insulinogenic response. Chlorpropamide should also result in insulin release from the pancreas and this was substantiated by determinations of the blood glucose levels at the time of death which showed that the chlorpropamide-treated animals had significantly lower glucose concentrations in the blood than control animals. However, none of these treatments resulted in a significant advancement of the time at which glucokinase was first detectable. Small differences such as those recorded in Fig. 5 were sometimes observed but statistical significance was not obtained. It thus appears that the non-appearance of glucokinase in the rat liver before about 16 days after birth is not due to lack of substrate or of insulin, both of which are necessary for glucokinase synthesis in the adult animal. However, it was essential to show that both exogenous glucose and insulin are necessary for glucokinase induction to occur.

The Effect of Starvation and of Diabetes on Glucokmase Induction in the Neonatal Rat Production of alloxan-diabetes in the neonatal rat has proved to be a difficult problem mainly because of the very wide responses to intrapedtoneal injections of alloxan. The number of animals achieving the required chronic diabetic state was less than 20 per cent of the total injected. However, enzyme assays on animals made diabetic between 10 and 14 days old and examined between 16 and 24 days old all showed negligible levels of glucokinase activity. If diabetic animals were subsequently treated with insulin (2 units/day/100 g body wt) glucokinase activity reappeared. A typical experiment is shown in Table 1. The diabetic-insulin treated animals more than recovered the glucokinase activity of their litter-mates after 3 days of insulin treatment. Hence whereas a deficiency of insulin is not the reason for the absence of gluco-

173

DEVELOPMENTOF HEPATIC EI~ZYMES

kinase in the newborn rat, insulin is necessary for normal glucokinase development, a conclusion to be expected f r o m the work with adult animals. Rats at the stage o f development where glucokinase activity increases most rapidly were starved for up to 3 days (water being permitted ad libitum). This resulted in a lowering o f the hepatic glucokinase activities (Fig. 7) so TABLE 1 The Role of Insulin in the Development of Hepatic Glucokinase Age when assayed (days) 24 24 24

Previous treatment

Blood glucose (mg %)

Glucokinase activity ~moles of glucose phosphorylated/ min/g of liver

None, normal animals Diabetic, injected with alloxan when 17 days old Diabetic, as above, then treated with insulin daily from when 21 days old

118, 111 370, 348

0.83, 0.81 0.03, 0.03 1.04, 1.06, 1.37

131, 194, 192

Data from Walker and Holland. (~s)

"~1

0.6

o~ ~mlO.4 rjo

~

0.~

I

I

I

I

21

22

23

24

AGE IN Fro. 7

DAYS

The effect of starvation on hepatic glucokinase during the developmental period. Three litters of rats were used and each point represents the mean 4- S.E.M. of 3 determinations on individual animals. 0 O, normal fed animals; [] [] animals starved from when 21 days old.

174

D.G. WALKER

that, after 3 days, the activities of the starved animals were significantly lower (p < 0.01) than for the normal fed animals. Hence dietary glucose (and not glucose formed by gluconeogenic mechanisms) is essential for glucokinase development in the neonatal animal. Again this is as to be expected from the adult-animal studies.

An Effect of Hydrocortisone on Development of Glucokinase Previous experiments on adult animals showed that cortisone-treated rats (5 mg twice daily for 7 days) resulted in only a small lowering of hepatic glucokinas¢ levels. (17) Weinhouse and coworkers~ xs) noted a slower rate of

[

1.~

--

D.e

D.| 0 Acz c~y.~ tl

it

t'~

t'.

t~

tt

FIG. 8 The effect of hydrocortisone on the development of hepatic glucokinase in the neonatal rat. The daily hydrocortison¢ treatment commenced when the animals were 8 days old (hydrocortisoneacetate as suspension in saline, 5 mg/100 g body wt.). Each point represents the mean 4- S.E.M. of 3 or 4 determinations on individual animals. recovery of normal glucokinase activity upon refeeding starved-adrenalectomized rats compared to normal starved-refed animals, but the adrenocorticosteroid control of glucokinase levels has in general shown little if any effect.(45~ It is of interest, therefore, that treatment of neonatal rats with hydrocortisone (hydrocortisone acetate suspension in saline, 2 mg per rnl, 0.5 ml per 100 g body wt., intraperitoneally) resulted in a slight advancement of glucokinase induction (Fig. 8). The similar rates of glucokinase appearance in both the hydrocortisone-treated and saline-injected control animals imply that the

DEVELOPMENT OF HEPATIC ENZYMES

175

effect of the hydrocortisone is upon the initiation of enzyme synthesis rather than on the rate of enzyme formation once initiated. Commencement of hydrocortisone treatment at earlier ages (3 experiments starting at 2, 6 and 8 days old, respectively, were performed) did not affect the initiation of glucokinase development any more than that shown in Fig. 8. This conclusion suggests that adrenocorticosteroids may play some role in producing the conditions necessary for glucokinase synthesis to commence. The nature of this process is unknown but it is of interest that there is evidence that the pituitary-adrenocortical system is not fully functional in the rat until several days after birth and, in particular, that the marked increase of corticosterone in the blood of the rat brought about by stress, which is typical of the adult animal, is very low in the early postnatal period and shows a large increase towards adult response during the third week of life.(4e) However, there is no indication that hydrocortisone affects glucokinase formation as it does the synthesis of certain transaminases( 47, 4s) and various enzymes of gluconeogenesis(49) in adult animals. Further, the period of glucokinase development is very similar to that shown by tryptophan pyrrolase(50) and the development of this latter enzyme is not affected by adrenalectomy.(51) The Synthesis of Glucokinase on Refeeding Starved Neonatal Rats It has so far been shown that starvation severely limits the synthesis of glucokinase in the period (21-24 days old) when its normal development is proceeding at the maximum rate. Because the measured amount of an enzyme at any one time must represent the net balance between the rates of synthesis and breakdown, it is reasonable to presume (in the absence of any evidence that there are any changes in the rate of enzyme degradation-an assumption which may need revision in the light of the recent observation by Schimke and coworkersO2) concerning tryptophan pyrrolase degradation) that the rate of glucokinase synthesis is continuously increasing during the period of development. It follows that the rate of enzyme synthesis in the developmental period, say at 21 or 24 days of age, is lower than in the adult animal. Neonatal rats were starved for 3 days and then given a glucose infusion and free access to normal diet for 16 hr. Hepatic glucokinase activities were then determined and compared with the activities of normal litter-mates who had received no treatment. Figure 9 shows that the starved-refed animals were able to regain and sometimes exceed the activities of the normal animals when the starvation period was either 19-22 days or 21-24 days of age. Hence, although the glucokinase activity has only been gradually increasing in the normal animals, the ability to synthesis glucokinase upo n refeeding has been developing even during the period of starvation and perfifits a rapid recovery of the normal glucokinase level within 16 hr after refeeding.

176

D.G. WALKER

These rapid increases in activity u p o n refeeding previously starved neonatal rats are almost certainly due to d e h o v e synthesis o f enzyme because the increases are inhibited (Fig. 10) by ethionine, p-fluorophenylalanine and also 1.4

1.2

1.0

F

m

0.8

lo,

lo., 0" A G E (Days)

22

23 2 3

22

TREATMENT

a.

b

a

a = Starved for 3 days

23 23 -

b

21 24 25 25 -

a

-

b

21 2 4 25 25 a

-

b

b = starved for 3 days, then refed 16 hours

FIo. 9 The effect of hepatic glucokinase of refeeding neonatal animals after starvation for 3 days. Each group of results represents a single litter and each separate result is the activity of a pooled preparation of two livers from similar animals. The first two litters were starved when 19 days old and refed when 22 days old; the third and fourth litters were starved when 21 days old, and refed when 24 days old. Refed animals were given a single intraperitoneal injection of glucose (as in Fig. 5) and permitted access to normal diet and killed 16 hr later. Results for normal untreated litter-mates are given for comparison.

actinomycin D, an inhibitor o f R N A synthesis.(53) The inhibition by the first two agents is largely overcome by simultaneous injections o f methionine and phenylalanine, respectively. These results add strong support to the above conclusion concerning the continued development o f a potential ability to synthesize glucokinase, when refed, during the starvation period.

DEVELOPMENT

OF HEPATIC

177

ENZYMES

The Control of Glucokinase Induction and Synthesis in the Neonatal Animal Glucokinase develops at a very definite stage in the growth o f the animal and, in the case o f the rat, the point is well after parturition. Prior to this glucokinase apparently cannot be synthesized in spite o f the presence o f dietary carbohydrate and adequate insulin and the obvious ability to synthesize very m a n y other proteins. The attempts to hasten glucokinase appearance 1°0

m

m

0.8 D

0

0.4

--

o

÷

0 AGE(I~.ys)

2 4 25 25 25 2 5

NORMAL STARVED

STARVED THEN REFED

23 2 4 2 4 2 4 24

+

24 25:15

+

+

÷

+

+

÷

25

+ +

+

+

+

+

+

Fio. 10 The effects of inhibitors of protein synthesis upon the increase in hepatic glucokinase when neonatal animals were starved for 3 days and then refed for 16 hr. Each group of results represents a single litter and each separate result is the activity of a pooled preparation of two livers from similar animals. The first and third litters were starved when 21 days old and refed when 24 days old; the second litter was starved when 20 days old and refed when 23 days old. Other details as in Figs. 4 and 9; the single dose of actinomycin D was 0.07 mg/100 g body wt. as a solution in saline. Inhibitors were injected intraperitoneally at the same time as the glucose.

and its rate o f development have given marginal effects only. The small effect o f hydrocortisone m a y be brought a b o u t via a complex series o f interactions. One is forced to conclude that some factor is missing f r o m the liver until a certain developmental stage. The nature o f the inability to synthesize glucokinase prior to this must

178

D.G. WALKER

remain a matter for speculation until more information becomes available. There would appear to be two main problems. First, very little is known concerning the environment of the hepatic cell with respect in particular to hormones at this period of animal growth. That both qualitative and quantitative changes in hormonal activity will be occurring is to be expected and it is conceivable that there could be either a simple or very complex series of alterations which result in an altered rate of glucokinase synthesis. That fetal hormones are involved in the control of biochemical differentiation has been recently demonstrated by the work on enzymes of glycogen metabolism and the effects of decapitation of fetal rats by Jacquot and Kretchmer.(54) Further work on glucokinase activities in various hormonal conditions in the adult animal may provide an indication of possible regions for further investigation in the neonatal period. Secondly, because this is essentially a problem in the control of protein synthesis, the role of insulin in the control of enzyme synthesis in the liver of the adult animal bears examination. Two opposing effects of insulin on hepatic enzyme synthesis have been reported within the past two years. Weber and coworkers(49) have demonstrated that insulin represses the formation of several of the key enzymes involved in the process of gluconeogenesis. On the other hand glucokinase synthesis is dependent upon the presence of insulin, as has already been described, and Steiner(55) has recently shown that insulin affects the rate of synthesis of glycogen synthetase (UDPG-glyeogen glucosyl transferase). This latter effect is detectable after a shorter delay than that of 6 hr which is required to detect an increase of glucokinase(xS) following the administration of insulin to a severely-diabetic rat. Thus we have to explain how the formation of at least certain hepatic enzymes may be either enhanced or repressed by insulin. Glucose, the substrate of glucokinase, is also a repressor of the induction of certain mammalian enzymes.t4a) Hence insulin may itself act as both an inducer and repressor of specific enzyme synthesis at the gene level or, alternatively, such effects may be mediated via an initial action affecting some other agent or agents which in turn may act in one of such possible ways upon the mechanisms regulating protein synthesis.tSe) If a site of insulin action is indeed at the level of the transcription of the genetic message into messenger RNA, and this is probable because of the observed effects of actinomycin D on all three cases mentioned, it would be interesting to know whether there is any subtle connection between the known instances of direct insulin action in the liver. The results presented in Figs. 9 and 10 suggest that something continues to develop during the period in which the neonatal animals were starved. This may be the mechanism by which insulin affects transcription, but there is the possibility that this may be in some way related to the unknown events occurring in the diabetic animal during the lag period following treatment with insulin before glucokinase synthesis begins. A possible connection, which is

DEVELOPMENT OF HEPATIC ENZYMES

179

put forward here as a basis for future investigation, is that insulin may also assert an effect upon the cytoplasmic machinery for protein synthesis, i.e. upon the translation of the genetic information. In order that such an effect could occur at all it would have to enter the liver cell, which is something unproven but likely.(55) Current ideas concerning the mechanism of protein synthesis~ 57, 58) require the assembly of the synthesizing units, ribosomes associated with rough endoplasmic reticulum, into polysomes which are organized in a specific manner upon the messenger RNA templates. These templates require a certain stability which could be influenced by factors such as hormones or substrates.(59) Free ribosomes from rat liver, on the other hand, are comparatively inactive for protein synthesis.(6°) Now fetal and neonatal rat-liver parenchymal cells contain high levels of such free ribosomes until about 5 days after birth.~ 6x) The numbers then gradually fall towards adult figures. In the developing chick liver the number of rough membranes increases as the number of free ribosomes decreases.(ee) Thus a part of the process of liver maturation involves changes in the cytoplasmic protein-synthetic apparatus of the hepatic parenchymal cell. It may be that insulin is unable to exert its effects on protein synthesis until, in the case of the developing animal, a certain maturity of organization is available and that certain enzymes must await an advanced stage of development for synthesis to be possible. Is it this organization which is affected in the diabetic animal and which must first be put right on administration of insulin? Such a suggestion is reminiscent of and a little more specific than Krahl's generalized hypothesis for insulin action3 eS) Insulin may itself be necessary for certain final stages of the structural organization to be achieved.

The Post-natal Development of Fructokinase Work on this enzyme has been confined so far to a demonstration, by direct enzyme assays, of the appearance of the enzyme in the post-natal period in three species (see Table 2). The study of this enzyme was, in part, stimulated by the fact that certain animal species, especially the ungulates, contain large quantities of the ketohexose fructose in their allantoic and amniotic fluids and also in the fetal blood. Immediately after birth the blood fructose quickly falls to very low levels.(6) Liver perfusion studies(64) on newborn lambs showed that the ability to utilize fructose developed after birth, thus supporting an earlier observation(65) that fructokinase was absent in the fetal sheep. While direct estimations on the development of fructokinase activity have not been made on a fructogenic species, all the evidence available(z2) suggests that the appearance of this enzyme after birth is a general phenomenon applicable to both fructogenie and non-fructogenic species. This rules out the possibility that the presence or absence of fructose in the

180

D.

G. WALKER TABLE 2

Summary of Results on Post-natal Development of Hepatic Fructokinase

Species

Rat Rabbit Guinea-pig

Activity in fetal liver just before birth

Period of development

Fructokinase activity of adult animals (~moles of fructose utilized/lnin/g wet wt. of tissue)

Birth---7 days Birth--10 days Birth---7 days

2.684-0.26 (3) 1. 164-0.14 (5) O. 364-0.09 (5)

The results are based upon data by WalkerC2~)and the figures represent means -4- S.D. with the number of determinations in parentheses. fetal blood depends upon the ability of the fetal liver to utilize this sugar. The newborn human baby has a very poor tolerance to fructose and this may be due to the absence of sufficient hepatic fructokinase during the first phase of extrauterine life.t66) Unlike the unique glucokinase, fructokinase activity is not decreased in diabetes and normal fructose utilization can therefore occur in insulindeficiency. The development of this enzyme appears to occur in all species directly or indirectly examined in response to the changes occurring at birth and hormonal factors may well be responsible for initiation of enzyme development.tz2, eT) Two other possible factors can be eliminated. First, considerable concentrations of fructose have been present throughout gestation in the fructogenic species and lower concentrations are also present in the nonfructogenic species so that it is not the sudden appearance of the substrate fructose which induces formation of the enzyme. Secondly, the presence of either activators or inhibitors in the liver supernatant fractions used for fructokinase assay has been eliminated by showing that the activities of combinations of fetal and adult liver preparations were always strictly additive.t 2~) Like many other enzymes which appear at birth, therefore, the elucidation of the mechanism of fructokinase induction awaits further information concerning the changes in the internal environment of the newborn animal resulting from the dramatic change in its external surroundings. SUMMARY 1. The control of carbohydrate metabolism in the developing mammal and the lack of ~ r t a i n hepatic control mechanisms prior to maturation were briefly reviewed.

DEVELOPMENTOF HEPATICENZYMES

181

2. The enzymes available in the liver for the phosphorylation of glucose and fructose were summarized and the unique roles of glucokinase and fructokinase indicated. 3. Glucokinase is absent from fetal liver and develops immediately after birth in the guinea-pig but first appears about 16 days post-parturition in the rat and reaches adult levels 10-12 days later. 4. This development of glucokinase is not due to an activation process but is almost certainly d e n o v o synthesis of new enzyme. 5. Attempts to induce glucokinase development at an earlier stage had marginal effects only, and its absence from rat liver prior to the 16th day is not due to lack of insulin. 6. Hydrocortisone had a small effect 6n advancing initiation of glucokinase appearance but did not affect the rate of apparent synthesis. 7. Glucokinase synthesis was impaired when neonatal rats were starved during the developmental period. When these starved animals were refed they were able to synthesize glucokinase enabling the activities of their untreated litter-mates to be achieved quickly. Some form of development of the cell apparatus for the regulation of enzyme synthesiswas continuing during the period of starvation, and possible mechanisms have been discussed. 8. Fructokinase also develops after birth in both fructogenic and nonfructogenic species and this may represent another example of development of an enzyme in response to hormonal changes occurring as a result of parturition.

ACKNOWLEDGMENTS Mr. M. A. Lea, Mrs. S. Rao, Miss G. Holland and Mr. M. J. Parry have collaborated in various aspects of this work. I am grateful to the Wellcome Trust for a grant which enabled the work to be commenced and to the Medical Research Council for subsequent financial support.

REFERENCES 1. S. SOSKIN,H. E. ESSEX,3. P. HERRICKand F. C. MANN,The mechanism of regulation of the blood sugar by the liver, Am. J. Physiol. 124, 558-567 (1938). 2. G. W~ER, Pathology of glucose-6-phosphate metabolism. A study in enzyme pathology, Rev. Can. Biol. 18, 245-282 (1959). 3. A. M. NE~TH, Glucose-6-pbosphatase in the liver of the foetal guinea-pig, 3". Biol. Chem. 208, 773-776 (1954). 4. M.A. LEA and D. G. WALKER,The metabolism of glucose-6-phosphate in developing mammalian tissues, Biochem. J. 91,417-424 (1964). 5. W. F. WmDAS, Transport mechanisms in the foetus, Brit. Med. Bull, 17, 107-111 (1961). 6. A. ST G. HUC,GETr, Carbohydrate metabolism in the placenta and foetus, Brit. Med. Bull. 17, 122-126 (1961). 7. D. G. W ~ , The development of hepatic hexokinases after birth, Bioehem. J. 84, 118-119P (1962).

182

D . G . WALKER

8. C. A. VILLV~, Regulation of blood glucose in the human foetus, J. Appl. Physiol. 5, 437--444 (1953). 9. M. A. LEA and D. G. WALKER,Changes in glucose-6-phosphate metabolism during development, Biochem. J. 8.5, 30P (1962). 10. C. BERNARD,De la matiere glycog~ne consid~r~ comme condition"de d6veloppement de certains tissus chez le foetus avant 1'apparition de la fonction glycogenique du role, J. de la Physiol. de rHomme, Paris, 2, 326-337 (1859). 1 I. G . F . CAHILL, J. A$HMORE, A. E. RENOLD and A. B. HASTINGS,Blood glucose and the liver, Am. J. Med. 26, 264-282 (1959). 12. D. L. DIPIETROand S. WEINHOUSE,Hepatic glucokinase in the fed, fasted and allo×andiabetic rat, J. Biol. Chem. 235, 2542-2545 (1960). 13. D. G. WALKER,On the presence of two soluble glucose-phosphorylating enzymes in adult liver and the development of one of these after birth, Biochim. Biophys. Acta 77, 209-226 (1963). 14. E. VI~UELA,M. SXL~ and A. SOLS, Glucokinase and hexokinase in liver in relation to glycogen synthesis, J. Biol. Chem. 238, PCl175-1177 (1963). 15. M. SALAS,E. VISUELAand A. SOLS, Insulin-dependentsynthesis of liver glucokinase in the rat, J. Biol. Chem. 238, 3535-3538 (1963). 16. C. SHARMA,R. MANJFS~VARand S. W~OUSE, Effects of diet and insulin on glucoseadenosine triphosphate phosphotransferases of rat liver, J. Biol. Chem. 239, 38403845 (1963). 17. D. G. WALKERand S. RAO,The role of glucokinase in the phosphorylation of glucose by rat liver, Biochem. J. 90, 360-368 (1964). 18. C. SHARMA,R. MANJI~HWARand S. WEINHOUSE,Hormonal and dietary regulation of hepatic glucokinase, Advances in Enzyme Regulation 2, 189-200 (1964). 19. A. Sol.s, M. SAL~ and E. VISTULA,Induced biosynthesis of liver glucokinase, Advances in Enzyme Regulation 2, 177-188 (1964). 20. H. G. HERS, La fructokinase du foie, Biochim. Biophys. Acta g, 416--423 (1952). 21. R. E. PARKS,E. GEN-GERSHOMand H. A. LARDY,Liver fructokinase, J. Biol. Chem. 227, 231-242 (1957). 22. D. G. WALKER,The post-natal development of hepatic fructokinase, 8iochem. J. 87, 576-580 (1963). 23. D. G. WALKERand G. HOLLAND,unpublished results. 24. D. G. WALKER, Liver enzymes for the phosphorylation of monosaccharides, Prec. Sixth Internat. Congress Biochem., New York, pp. 475--476 (1964). 25. M . J . PARRY and D. G. WALKER, unpublished results. 26. R. K. CS~NE and A. SOL% Animal tissue hexokinases, pp. 277-286 in Method~ in Enzymology, Vol. l (S. P. COLOWlCKand N. O. KAPLAN, eds.), Academic Press, New York (1955). 27. D.G. WALKERand M. A. LEA,Carbohydrate metabolism in foetal and neonatal liver, Prec. Sixth Internat. Congress Biochem., New York, p. 531 (1964). 28. D.L. DIPIE~o, C. SHAme,A and S. WEXNHOUSE,Studies on glucose phosphorylation in rat liver, Biochemistry 1,455-462 (1962). 29. D. G. WALKERand S. RAO, The effect of glucose analogues on the hepatic glucosephosphorylating enzymes, Biochim. Biophys. Acta 77, 662-665 0963). 30. H. NI~EY~, L. CLARK-TURRIand E. RAaA~ILLE,Induction of glucokinase by glucose in rat liver, Nature 198, 1096-1097 (1963). 31. M.D. BLUMENTHAL,S. ABRAHAMand I. L. CHAU
DEVELOPMENT OF HEPATIC ENZYMES

183

36. C. KUYPER,Rat liver fructokinase as an adaptive enzyme, Arch. Internat. Physiol. 62, 566 (1954). 37. M. A. LEA and D. G. WALKER,submitted for publication. 38. P . K . DIXIT, I. P. LowE, C. B. H E c ~ m ~ o and A. L^z^Row, Insulin content of microdissected fetal islets obtained from diabetic and normal rats, Diabetes 13, 71-77 (1964). 39. S. S. LAZARUSand B. W. VOLK, Physiological basis of the effectiveness of combined insulin-tolhutamide therapy in stable diabetes, Ann. N.Y. Acad. Sci. 82, 590-602 (1959). 40. O. GREENGARDand P. FEmELSON, The activation and induction of rat liver tryptophan pyrrolase in vivo by its substrate, J. Biol. Chem. 236, 158-161 (1961). 41. S. VtLLA-TREVINO,K. H. SHULL and E. FARBER,The role of adenosine triphosphate deficiency in ethionine-induced inhibition of protein synthesis, J. Biol. Chem. 238, 1757-1763 (1963). 42. S. KIT, D. R. DUBBS and L. J. PIEKARSKI,Inhibitory effects of p-fluorophenylalanine and puromycin on the induction of thymidine kinase by vaccinia-infected mouse fibroblasts, Science 138, 992 (1962). 43. N. PinEs, A. L. KEN~AN, H. C. PrroT and R. C. WOLV, Hormonal interactions in glucokinase induction, Fed. Proc. 23, 409 (1964). 44. H. C. PITOT, The regulation of enzyme synthesis in mammalian tissues, Proc. Sixth lnternat. Congress Biochem. pp. 682-683 (1964). 45. S. W~n~ousE, personal communication. 46. K. MILKOVIC and S. MILKOVIC,Functioning of the pituitary-adrenocortical axis in rats at and after birth, Endocrinology, 73, 535-539 (1963). 47. F. ROSEN, H. R. HARDI~G, R. J. Mlt.HOt~Ar,TD and C. A. NICHO~ Glucocorticoids and transaminase activity. VI. Comparison of the adaptive increases of alanine-and tyrosine--a-ketoglutarate transaminases, J. Biol. Chem. 238, 3725-3729 (1963). 48. H . L . S ~ A L a n d Y . S. KiM, Glucocorticoidstimulation of the biosynthesis of glutamicalanine transaminase, Proc. Nat. Acad. Sci. 50, 912-918 (1963). 49. G. WEBER,R. S~GHAL, N. B. STAMM,E. A. FISHERand M. A. MENTENDmK, Regulation of enzymes involved in gluconeogenesis, Advances in Enzyme Regulation 2, 1-38 (1964). 50. A. M. NEMETX~,Mechanisms controlling changes in tryptophan pyrrolase activity in developing mammalian liver, J. Biol. Chem. 234, 2921-2924 (1959). 51. O. GR~GARD, M. A. SMn', and G. Acs, Relation of cortisone and synthesis of ribonucleic acid to induced and developmental enzyme formation, J. Biol. Chem. 238, 1548-1551 (1963). 52. R. T. SCmMKE, E. W. SWEENEYand C. M. BEaus, An analysis of the kinetics of rat liver tryptophan pyrrolase induction: the significance of both enzyme synthesis and degradation, Biochem. Biophys. Res. Commun. 15, 214-219 (1964). 53. E. REICH, R. M. FRANKLIN,A. J. SHATmN and E. L. T^TUM, Effect of actinomycin D on cellular nucleic acid synthesis and virus production, Science 134, 556-557 (1961). 54. R. JACQUOTand N. KRETCHMER,Effect of fetal decapitation on enzymes of glycogen metabolism, J. Biol. Chem. 239, 1301-1304 (1964). 55. D. F. STEINERand J. KIN~, Induced synthesis of hepatic uridine diphosphate glucoseglycogen glucosyltransferase after administration of insulin to alloxan-diabetic rats, J. Biol. Chem. 239, 1292-1298 (1964). 56. F. JACOn and J. MONOD, On the regulation of gene activity, in Cold Spring Harbor Symposia on Quantitative Biology 26, 193-211 (1961). 57. T. STA~mELIN,F. O. WETTST~IN,H. O u ~ and H. NOLL, Determination of the coding ratio based on molecular weight of messenger ribonucleic acid associated with ergosomes of different aggregate size, Nature, 201,264-270 (1964). 58. T. Htm~N, Ribosomal functions related to protein synthesis, lnternat. Rev. Cytol. 16, 1-36 (1964). 59. H. C. Pn'oT, C. PERAINO,R. BLAKEand A. L. K~NNAN, Altered template stability i n rat hepatomas, Proc. Am. Ass. Cancer Research, in press.

184

D . G . WALKER

60. P. N. CAMPBELL and J. R. SARGENT,Morphological structure and protein-synthetic activity in rat liver, Bioehem. J. 91, 18-19P (1964). 61. I . T . OLIVER, W. F. C. BLUMER and I. J. WrmAM, Free ribosomes during maturation of rat liver, Comp. Biochem. Physiol. 10, 33-38 (1963). 62. C. DUCK-CHONG,J: K. POLLAK and R. J. NORTH,The relation between the intracellular ribonucleic acid distribution and amino acid incorporation in the liver of the developing chick embryo, J. Cell. Biol. 20, 25-35 (1964). 63. M. E. I~RAHL, The Action o f Insulin on Cells, Academic Press, New York (1961). 64. W. H. H. ANDREWS, H. G. BmTTON and D. A. NIXON, Fructose and lactic acid metabolism in the peffused liver of premature lambs, Nature 191, 1307-1308 (1961). 65. H. G. HERS, Presence of sorbitol in seminal vesicles and in foetal blood of the sheep, Biochem. J. 66, 30P (1957). 66. R. SCHWARZ,H. GAMSU,P. B. MULLIGAN,S. H. REISNER,S. H. WYBREGT and M. CORNBLATH,Transient intolerance to exogenous fructose in the newborn, J. Clin. Invest. 43, 333-340 (1964). 67. A. M. NEMETH,Initiation of enzyme formation by birth, Ann. N.Y. Acad. Sci. 111, 199-202 (1963).