Perinatal development of gluconeogenesis in guinea-pig liver

PERINATAL

DEVELOPMENT OF GLUCONEOGENESIS IN GUINEA-PIG LIVER RENC;ACIIARI RAC~IIUXATHAS ANI) IFFASYI J. ARI~ZE

Department

of Biochemistry

and Nutrition,

.Mcharry

.Mcdical College,

(Keceiccd I8 March

Nashville.

TN 37208. U.S.A.

IY77)

Abstract-

I. At mid-gestation the activities of guinea-pig liver pyruvate carboxylase, glucose-6-phosphatase, and cytosolic phosphoenolpyruvate carboxykinase were less than 0.4 unit:g, whereas the activity of fructose-1,6_diphosphatase was 1.6 unit/g. Throughout gestation the activity of cytosolic phosphoenolpyruvate carboxykinase remained unchanged while all other enzymes exhibited a 2- to 3-fold increase over the levels at mid-gestation, beginning at 45-50 days. The actrvttics of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in the foetus were largely mitochondrial. 2. No increases in enzyme activities were evident in the first 2-3 hr following birth. Subsequently, increases were noted for all enzymes examined. with an overshoot occurring between 7 and I4 days post purtum for pyruvate carboxylase. glucose-6-phosphatasc. and fructose- ,6_diphosphatase.

in blood

5: appreciable 6. The birth.

taken from show

level I after birth foetuscs and of glucose lactate and that canacitv . - for

IUTRODUCTION Current understanding of the regulation of gluconcogenesis in the immediate neonatal period in mammals has been largely based on many studies using the rat as a model (Hanson c( al., 1975). In this animal the capacity for gluconeogenesis is virtually absent during foetal lift (Ballard & Oliver, 1963 ; & Hanson. Yeung & 1967; Philippidis Ballard, 1969) is acquired after birth parallcl with postnatal induction phosphocnol pyruvate boxykinasc (GTP) 4.1. 1.32) & Hanson, The initial of this during deappears to part of complex mechanthat triggers initiation of gluconeogenesis the rat birth (Greengard, Ballard, 1971 Hanson et 1975). In liver, phosphocnolcarboxykinase is cytosolic (Nordhc Lardy, 1963) very little activity is during foetal (Ballard & 1967). It becoming increasingly however, that presence of phosphoenolpyruvate carboxykinase the liver a large of mammals determine to large extent pattern of of hcpatic genesis (Soling al., 1970; Arinzc et al., Ray, 1976). part of study of development of genesis in other than rat we showed that near-term foctal from the can synthesize from pyruvate the virtual of cytosolic carboxykinase (Arinze, In perfused from adult and guinea-pigs, mitochondrial form this enzyme been shown make significant to overall production from substrates (Arinzc nl., 1973; et ul.,

reduced

to

gluconeogencsis

than 20% after Caesar&n in

The purpose the present pattern dcvelopmcnt of gluconeogcnesis during pig. MATERIALS

2hr. synthesized is acquired

was to potential for in the

the

METHODS

NaH’4C03 Aquasol were from New Nuclear Core.. MA. U.S.A. analvtical and cofactors purchased from Mannhcim Corp.. U.S.A. All chemicals were reagent grade were obtained readily available

Animals Pregnant of the strain were chased at days gestation Williams Kentucky Fern Creek, U.S.A.. and ad lihitum pelleted laboratory chow with fresh tuce. The had fret to water all times, intervals during pregnancy, foetuses removed by section under ether anaesthesia. was collected drainage after Litter mates used immedtatcly Caesarean delivery placed in Humidicrib at until used experiments. The litter size 3-4 and in this of anivaried between days. Gestational was determined the date mating. Enzyme

Animals were by decapitation a blow the head the livers quickly removed, in 0.25 sucrose solution 1 mM tctraacetic acid at 4-C. for enzyme were prepared homogenizing the with the EDTA solution 1 min. homogenate (IO%, was centrifuged 100,000 g 1 hr a Beckman ultracentrifugc. The fraction was for the

Liter perfusion

assay of fructose-l. 6-diphosphatase (EC X1.3.1 I) and cytosolic phosphoenolpyruvatc carboxykinase. The particulate fraction was sonificd for four 1%~ bursts using a Branson cell disrupter, Model W185. at a setting of 5. Mitochondrial phosphocnolpyruvate carboxykinase and pyruvatc carboxylase (EC 6.4.1.1) were measured in the sonilied particulate fraction. In some experiments the liver homogenale was fractionated into subcellular fractions by differential centrifugation (Schneider & Hogcboom, 1950). The fractions without further washing were sonified as before and used as such for cn7ymc measurements.

About l-3 days prior to normal delivery. foetuscs were removed by Cacscrean section. Livers wcrc pcrfuscd immediately after d&very using the non-recirculating system described elsewhere (Arinzc c/ (II.. 1973). Lirtcr mates nor perfused immediately after delivery were placed in a Humidicrih at 37°C. and used 24 hr later. Pcrfunatc llow was maintained at 8-15 mljmin and carefully controlled to

avoid disruption of the liver by hydrostatic pressure. Glucose in the pcrfusatc was measured as described previously (:irinxc

Blood glucose was measured by the method of Hugert and Xixon (1957). Glycogen was dctcrmincd hy the

All enzyme assrfys wcrc conducted under conditions where enLymc actlvltics wcrc proportional to time and cnzymc concentration. Pyruvate carboxylasc was measured at 37,,C by the [“‘Clbicarbonatc fixation assay described by Utter and Keech (1963) except that acctyl-CoA was

method

was assayed at 25 C,

pH 7.5. by the coupled enzyme system dcscrihed by Crisp and Pogson (1972) except that 6-phosphogluconate dchydrogenasc was omitted. The reaclion was started by the addition of fructose-1. 6-diphosphate. Glutamate dchgdrogenase (FC 1.4. I .2) activily was mcasurcd in the presence of ADP. at 25’C, in the direction

of NADH oxidation (Schmidt, 1965). Lactate dchydrogcnase (EC 1.1.1.27) activity was determined at 25°C by the rate of oxidation of NADH at 340 nm (Bcrgmeycr & Bernt, assayed with according to

All enzyme activities arc expressed in unit/g wet tissue. One unit of enyymc activity is defined as the formation of one pmolc of the assay. Table

of product

I. Intracellular

per min

distribution

under

Pyruvate carboxylase

Foetal Whole homogenate Nuclear Mitochondrial Microsomal Cystol

the conditions

of pyruvatc

2.99 + 0.27 0.94 f 0.12 1.68 f 0.19 0.01 If: O.ooO6 0.18 + 0.04

(1965).

Since in foetal rat liver the activities of pyruvate carboxylasc and phosphoenolpyruvatc carboxykinase arc negligible (Ballard & Hanson, 1967). we initialI> examined the activities of these two important enzymes in the near-term foetal guinea-pig liver. The intracellular distribution of pyruvate carboxylasc. phosphocnolpyruvatc carboxykinase. and marker enzymes in livers from 55-60 days foetal and adult animals are shown in Table 1. In both the foetal and adult liver. approx 56% of the pyruvate carboxylase activity was in the mitochondrial fraction: less than 1% was detected in either the 100,0(X) q supcmatant or the microsomal fraction. In contrast to the adult liver, cytosolic phosphoenolpyruvatc carboxykinase was negligible in the foetus (< 1%) where about 45’:h of the total activity was associated with the mitochondrial fraction. If these activities are corrcctcd for mitochondrial damage using citrate synthasc and glutamate dchydrogenase as marker enzymes for mitochondria (Table l), it is evident that pyruvatc carboxylase and phosphoenolpyruvate carboxykinase are virtually absent in the cytosol of the foetal liver. In these experiments the recovery of lactate dchydrogcnase in the cytosol fraction was 93’%. The relatively high nuclear activities are probably the result of in-

assays the reaction was stopped by adding 0.5 ml of a 10% (w/v) solution of trichloroacctic acid. The unreacted [“%]bicarhonate was Rushed out of the protein-free solution by gassing for 10 min with Oz. Aliquots were then counted for radioactivity in a Beckman Model 355 liquid scintillation spectrommctcr after adding IO ml of Aquasol. Glucose-6-phosphatase (EC 3.1.3.9) in the crude homogcnate was measured at 37’C as dcscribcd by Harper

1974). Citrate synthase (E.C. 4.1.3.7) was 5-5’-dithiobis-(2-nitrobenzoic acid) at 25’C Shepherd and Garland (lY69).

of Pflcidercr

RESULTS

generated in situ as described by Ilcnning and Seubert (1964). Phosphoenolpyruvatc carboxykinasc activity was detcrmincd at 37’C by a modification (Ballard & Hanson, 1967) of the method of Chang and Lane (1966). For both

(1965). I-‘ructose-l,6-diphosphatase

et al., 1973).

and phosphocnolpyruvate guinea-pig liver

carboxylase

Phosphenol pyruvate carboxykinase

carhoxykinasc in foctal and adult

Glutamate Citrate dehydrogenase synthasc (I*mole:‘min per g of liver)

2.68 + 0.14 0.96 5 0.12

5.1 * 0.73 1.68 k 0.37

143.8 f 29.2 49.5 2 22.5

I.19 + 0.13 0.01 + 0.007 0.18 f 0.03

3.x5 + 0.65 0.1’) + 0.03 0.25 + 0.03

64.5 & 17.5 3.14 7 0.86 1.22 f 0.58

Lactate dehydrogcnasc

105.4 f 15.7 16.2 -t 3.0 3.25 f 0.5 4.63 + 0.X 97.73 5 Il.9

Adult Whole homogenate Nuclear Mitochondrial Microsomal Cytosol

2.34 0.59 1.44 0.02 0. I2

k * + *

0.16 6.58 + 0.38 9.33 * 1.07 159.7 & 13.3 72.2 i 0.5 0.10 0.73 I 0.08 2.05 rf: 0.26 54. I + I I .7 5.6 f 1.3 0.14 3.69 f 0.18 5.55 * 0.73 79.3 = 3.8 2.9 + 0.9 0.006 0.04 * 0.01 0.24 + 0.06 4.1 ‘- 0.08 I.!, i_ 0.5 + 0.03 1.68 +_ 0.28 0.74 & 0.14 I.9 + 0.10 63.7 + 2.0 .__-._ poetuses were obtained from 55.-60 days pregnant guinea-pigs. Livers were homogenized and fractionated according to Schneider and Hogcboom (1950). Enzyme activities were determined as described in Methods. The values given are means + S.E.M. of 4-5 animals.

Development of gluconeogenesis homogenization or contamination by mitochondria rather than true localization as judged by the activities of the mitochondrial marker enzymes in this fraction. Similar levels have been noted for other enzymes (Nordlic & Lardy, 1963; Garbcr & Hanson, 1971; Johnson et ul., 1973). complete

in guinea-pig

139

liver

Valuable information on the early development of the potential for gluconeogenesis may be obtained by studying the pattern of devclopmcnt of key enzymes involved in glucose synthesis. The changes in the activitics of four important gluconeogcnic cnzymcs during the foetal to neonatal transition in guinea-pig liver

I

I I 35-40

45-50 55-M FOETAL

I

T

I I 1 L_..L I6 B 24 DAYS AFTER BIRTH

A

b!

(b)

g-l.-L.. 35.4C

45-50 FOETAL

.I, 55-W

-2-2

8 DAYS

T

_

74 “IHTH

16 AFTFH

A

~_-

T

J u

1

I I I I I I I I I I I I I I I I

liI

s‘.’

2.0

I I 30-40

45-50 FOETAL

55-60

I

T

, B DAYS

(4

,

, 16 AFTER

, 24 BIRTH

,

,

A

M

35-40

45-W ‘OETAL

55-60

T

B DAYS

I”‘!’ 16 AFTER

24 BIRTH

(4

Fig. 1. Activities of gluconeogenic enzymes in foetal. neonatal and adult guinea-pig liver. Foetuses wcrc removed after Caesarean delivery under light anaesthesia of the mothers. Livers were homogenized and glucose-6-phosphatasc activity was determined in the whole homogenate. The homogenate was separated into particulate and supernatant fractions by centrifugation at 100,000 9 for I hr. Mitochondrial phosphocnolpyruvate carboxykinase and pyruvate carboxylase were assayed in the particulate fraction, while the supcrnatant fraction was used for the assay of cytosolic phosphoenolpyruvatc carboxykinasc: and fructose-1.6~diphosphatasc as described in the Methods section. Results are expressed as means f S.E.M. of five to eight diffcrcnt animals except for the 35-40 day foctal livers. which were pooled. Each point represents results from 34 different litters. The letters A and M reprcscnt adult and maternal livers. respectively. The symbols used arc: (a) phosphoenolpyruvate carboxykinasc; particulate (0); cytosolic (0); (b) pyruvate carboxylase: (c) fructose-1.6~diphosphatase; (d) glucose6-phosphatase.

A

C

RI.\GZCHAHI RAGHWAIIIA~

740

are shown in Fig. I. In livers obtained at 3540 days of gestation. the activities of pyruvate carboxylase. glucose-6-phosphatasc. and cytosolic phosphocnolpyruvatc carboxykinasc were less than 0.4 unit/g whcrcas the activity of fructose-l. 6-diphosphatasc was 1.6 unit:g. Throughout gestation. the activity of cytosolic phosphocnolpyruvatc carboxykinase remained unchanged; all other enzymes exhibited a 2- to 3-fold increase over the levels at mid-gestation. beginning at 45 50 days. These increased levels wcrc not appreciably altered at term. During the postnatal period. dramatic increases were noted for all enz?mcs examined, with an ovcrshoot occurring between 7 and 14 days post purtum for pyruvatc carboxylasc. glucose-&phosphatasc, and fructose-l,6-diphosphatase. The highest activities of glucose-6-phosphatase and fructose- I .6-diphosphatase wcrc observed at 14 days (9.6 F 0.2 and 7.5 f 0.2 unitig. respcctivcly). Pyruvatc carboxylasc activity increased to 5 unit!g at 7 days after birth and subscquently declined sharply to adult levels. I!nlikc the particulate phosphoenolpyruvatc carboxykinase which increased in activity only 2-fold following birth. the activity of the cytosolic form of the enzyme was elevated l5-fold in the first 2 days, and resembled the pattern of postnatal increase reported for this enzyme in neonatal rat liver (Ballard & Hanson. 1967; Ycung & Oliver, 1967). No appreciable difl’crcnces were noted between the enzyme activities in maternal versus the adult liver. The results presented in Fig. 1 show that the enzymatic capacity for gluconcogencsis in the newborn guinea-pig is acquired before birth. Consequently the degree of neonatal hypoglycaemia in this animal might bc expected not to be as pronounced as has been rcportcd for the newborn rat (Cake 01 al., 1971). It seemed important therefore to follow closely the changes in cnzymc levels in relation to blood glucose levels in the immediate postnatal period. The gestational period in guinea-pigs is quite imprecise (Sisk, 1976). Therefore in order to follow these changes in the first few hours after birth., foctuses were removed from their mothers by Caescrcan section I -3 days prior to expected normal deliver. Table 2 shows the levels of blood glucose and hcpatic glycogen in thcsc animals. One hour after delivery, the blood glucose lcvcl (54.3 mg%) was about 40% less than that at birth. At 2 hr the blood glucose had risen to over Table 2. Blood glucose and hcpatic glycogen born guinea-pigs -.

.~

Time alicr delivcrv (hr) ’ 0 0.5 1 2 4 8 12 24 48

Blood glucose

(ms?J 92.9 52.3 54.3 75.0 82.2 87.0 81.3 100.3 99.4

IT 6.3 rf- 5.3 + 2.1 f 4.7 + 5.9 k 4.4 = 7.3 f 3.8 * 4.6

The values are the means “Not determined.

levels in new-

Glycogen (m&g liver) 67.0 + 2.0 62.0 + 2.0 39.8 f 2.5 23.0 + 1.2 18.0 _+ 1.1 14.0 * 1.2 N.D.” X.1). 16.3 +_ 0.9

k S.E.M. for 7- IO animals.

ANI) IFFASYI J. AKINZI:

HOURS

AFTER DELIVERY

Fig. 2. Gluconeogcnic enzyme activities in liver during the first 12 hr after delivery. Foetuscs wcrc removed by Cacsercan section, I-3 days prior to expected normal delivery. and the levels of the four gluconeogcnic enzymes were determined at O-12 hr after delivery. Other details are given in the legend to Fig. 1. The symbols used arc (a) phosphocnolpyruvate carboxykinasc: particulate (0); cytosolic (0): (b) pyruvate carboxylasc (c) fructose- I ,&iiphosphatase and (d) glucose-Cphosphatasc.

80% of the value at birth. Subsequently. no significant hypoglycaemia could bc detected, the blood glucose at 4-8 hr being comparable to levels seen in l- to 2-day old newborns. No appreciable change in hcpatic glycogen content occurred in the first 0.5 hr; after that the hcpatic glycogcn levels fell rapidly. It is pcrtinent that during the initial 2 hr: gluconcogenic enzyme activities remained relatively unchalJ!cd (Fig. 2). However, the enzymes increased in actlvlty after that, reaching peak values at about 8 hr. It is clear from Table 2 that the increases in enzyme activities were not accompanied by any dramatic changes in blood glucose. The increases in enzyme acti\;ities wcrc blocked by inhibitors of protein synthesis, actinomytin D (Table 3) or cordycepin (results not shown), indicating that these increases represent de wco enzyme synthesis. Treatment with actinomycin D (100 /@/IO0 g body wt) did not cause hypoglycacmia in these animals. In fact there appeared to be a slight

Devclopmcnt Table

3. Efi’ect of acdnomycin

Time after delivery (hr)

D on changes

Glucose-6phosphatasc

Treatment

of gluconeogenesis

in guinea-pig

741

liver enzymes in liver of newborn

in the levels of

Fructose-l ,6diphosphatase

guinea-pigs

Phosphocnolpyruvate carboxykinase Cyrosol Mitochondrial (pmoleimin per g of liver)

Pyruvate carboxylase -.

0 4 X

Control Control Actinomycin Control Actinomycin

D D

1.0X 1.55 0.99 2.65 0.97

f f f + k

0.06 0.13 0.08 0.16 0.07

2.67 3.24 2.00 3.99 2.12

f + + f f

0.12 0.17 0.21 0.37 0.20

2.58 2.69 2.39 4.06 2.50

+ 1 + k f

0.13 0. I3 0.12 0.22 0.06

3.04 2.84 2.14 3.98 3.25

z I f * .f

0.33 0.05 0.48 0.24 0.24

0.46 0.74 0.45 1.43 0.33

+ 5 1 * +

0.05 0.05 0.04 0.20 0.03

Actinomycin D (100 &IO0 g of body wt) was injected intraperitoneally immediately after delivery and at 2 hr intervals thereafter. The drug was dissolved in 0.9% solution of SaCI and control animals received an equivalent volume of saline. The animals were killed at the intervals indicated and cnLyme activities were determined as described in the legend to Fig. I. The values arc the means + S.E.M for 4 animals.

elevation of the blood glucose levels (97.7 mg% in treated animals vs 82.2 mg% at 4 hr) in animals treated with this drug. Evidence that the capacity for gluconeogcncsis is acquired before birth can also be seen from the data in Table 4. When foctal guinea-pig livers were pcrfused immediately after delivery of the foetuscs, in the presence of physiological concentrations (2 mM) of lactate or glycerol they synthesized glucose at 0.284 and 0.350 jlmolejmin per g, respectively. In livers taken from 24-hr old newborns the rate of gluconeogenesis from lactate was increased (0.568 polejmin per g). These rates arc quite comparable to the rates of gluconeogencsis reported for perfused livers from fed and fasted adult guinea-pigs (Arinze er al.: 1973; Ogata et al., 1974; Jomain-Baum & Hanson, 1975). The increase in gluconeogcnesis in the 24-hr old newborn noted in Table 4 may reflect, in part, the induction of cytosolic phosphoenolpyruvatc carboxykinase which is absent in foetal liver (Fig. I, Table 1). and the increase in gluconeogenesis usually associated with fasting. It should be pointed out that except for water, the 24-hr old newborns had no access to food and therefore were equivalent to fasted animals.

The idea begin until lished from (Ballard &

that hepatic gluconcogenesis does not a few hours after birth has been estaba large number of studies with the rat Oliver, 1963; Dawkins. 1963; Ballard & Table 4. Gluconeogenesis

by isolated

Hanson, 1967; Yeung & Oliver, 1967; Vernon & Walker, 1968; Zorzoli et al., 1969) in which the initiation of gluconeogcncsis at birth follows the postnatal appearance of phosphoenolpyruvate carboxykinase, the last enzyme in the gluconeogenic sequence to develop (Ballard & Hanson, 1967; Ycung & Oliver. 1967). Consequently the importance of this enzyme is the overall regulation of neonatal gluconeogcnesis has been stressed (Ballard. 1970; Hanson et al.. 1975) although accompanying changes in hormonal levels and in the rcdox state in the liver of newborn rats also appear to be important (Philippidis el u/., 1972; Girard et al.. 1973). It should be noted, however, that rat liver lacks significant activity of the mitochondrial form of phosphocnolpyruvatc carboxykinase even in the adult (Nordlie & Lardy. 1963). The mitochondrial form of this enzyme which is present in adult liver of a wide variety of mammalian species (Hanson & Garber: 1972) strongly influences the pattern of regulation of hepatic gluconeogcncsis (Ogata et LJ/., 1974). A preliminary study from our laboratory (Arinze, 1975) indicated the presence of this form of the enzyme in foetal guinea-pig liver. In the present work a systematic study of the devclopment of all four important gluconeogenic enzymes, pyruvate carboxylase, both forms of phosphoenolpyruvate carboxykinasc, glucose-6-phosphatase and fructose-l&diphosphatase reveals that all of these enzymes except cytosolic phosphoenolpyruvate carboxykinasc are present in foetal liver from at least mid-gestation (Fig. I), the least active enzyme at this

p&used pigs

livers from foctal and ncwbom

Glucose

Source

Number of experiments

of liver

Foetus Newborn:

fasted

24 hr

4 7

Lactate (pmole/min 0.284 f 0.086 0.568 + 0.094

production

guinea

from

Glycerol per g of liver) 0.350 f 0.083 N.D.”

Foetuses were removed by Caeserean section, I 3 days prior to expcctcd normal delivery and the livers were perfused in a non-circulating fashion with Krebs Ringer bicarbonate buffer, pH 7.4, at 30 32°C. After a 50min wash-out to reduce the level of cndogenous glucose release, the substrates (2mM) were introduced into the perfusion system and the perfusion was continued for an additional 30 min. The rates of gluconeogcnesis were calculated from the average rates of glucose production within this period. For the values given, the endogenous rates of glucose production have been subtracted. The values are the means + S.E.M. ’ Not determined.

742

RENGAUIAKIRAGIIUNA.THAS ASD IFI:.~,w

time being pyruvatc carboxylase. All the enzymes except cytosolic phosphocnolpyruvatc carboxykinasc show gradual increases in activity towards birth. Most of the activity of pyruvatc carboxylase and phosphoenolpyruvate carboxykinase in the foetus arc mitochondrial, resembling the situation in the adult (Table I). The activity of mitochondrial phosphoenolpyruvate carboxykinase in foctal guinea-pig liver appears suficient to cnablc the perfused liver to synthcsizc glucose from lactate or glycerol (Table 4) at rates quite comparable to rates in the fed adult (Arinze el al.. 1973; Ogata et ~1.: 1974; Jomain-Raum & Hanson. 1975). incorporation of [3-‘4C]pyruvate and [U-‘4C]lactate into glucose in liver slices from foctai guinea-pigs has been demonstrated recently by Jones and Ashton (1976) although the rates of glucose synthesis in this preparation wcrc only lOo/, of that by the perfused foetal liver (ArinTe, 1975). The low rates of incorporation is not surprising since the use of liver slices which are not stabilized by calcium ions (Roobol & Allcyne, 1973) usually results in low rates of gluconeogencsis. In any cast these results support the view that in the guinea-pig the capacity for gluconeogcnesis is acquired before birth. Whether this capacity is expressed in ~rtrro is not known. Tn a recent abstract Warnes et al. (1974) showed that foctal lambs have substantial activities of gluconeogenic enzymes including phosphoenolpyruvatc carboxykinase but do not synthesize glucose or glycogen from isotopically labcllccl lactate irl Co. They suggest that the highly oxidized redox states in foetal liver might be limiting gluconeogcncsis in ~HEIW. Presently there arc no measurements of the oxidation reduction states in foctal liver of any species other than the rat. An important feature of the present work is the apparent lack of pronounced hypoglycacmia in newborn guinea-pigs (Table 2). In newborn rats a sharp drop in glood glucose is apparent 1 hr after birth (Dawkins, 1963). In the experiments by Yeung and Oliver (1968) with newborn rats the blood glucose dropped from 71 mg:i to 14 rng’x an 80’2 drop: bv 2 hr. Normoglvcaemia was not attained until 5- 6 hi. In the prcsci; study with newborn guinea-pigs, the drop (approx 40’%,) in blood glucose was of very short duration. being noticeable only within the first hour. By 2 hr, the blood glucose had risen to over 80% of the level at birth. We attribute the resistance to hypoglycaemia in the newborn period to the fat that this species already possesses substantial activitics of all gluconeogcnic cnzymcs before birth and can carry out gluconcogencsis (Table 4). Consequently, there is no dclaycd initiation of gluconeogenesis following birth as seen in the rat. Since the ability of newborn rats to carry out gluconcogencsis follows the postnatal development of cytosolic phosphocnolpyruvate carboxykinase (Ballard & Hanson: 1967; Yeung & Oliver. 1967): the resistance to neonatal hypoglycaemia in newborn guinea-pigs in the face of rapid glycogen depletion (Table 2) must reflect the contribution of the mitochondrial phosphoenolpyruvate carboxykinasc to glucose production. One must presume then that the postnatal increase in cytosolic phosphocnolpyruvste carboxykinase in this species would make additional contribution to overall glucose production in the first few days of lift.

.I.

AKISX

Further work is now in progress to assess the relative contribution to neonatal gluconcogencsis by both forms of this enzyme. Ackr~o~cl&ements National Institutes

-This work was supported bq of Health grants HD 087LI2, RR

05422-14, and by a Basil O’Connor Starter Research grant So. j-8’) from the Xational Foundation ;March of Dimes. We thank Ms. Gwendolynnc Smith and Mr. Rent Darvcaux for technical assistance. Drs. Ricahrd W. Hanson. Mulchand S. Pate1 and ,Mireillc Jomain-Baum for advice during the preparation of the manuscript. and Drs. Charles W. Johnson and Edward G. High for support and cncouragement.

AKISZI; 1. J. (lY75) On the development of phosphocnolpyruvate carboxykinasc and gluconcogen&is in guinea pig liver. Biochun. bioph13. Kcs. Commm. 65. I84 189. A&% 1. J.. GARHFR A:J: & Il~z.sos R. W. (1973) The regulation of gluconeogcncsis in mammalian liver. ‘I’hc role of mitochondrial kinasc. J. hid. Chon.

phosphocnolpyruvatc

carboxy-

248, 22662274. BAI.LAKDF. J. (1970) Gluconcogencsis and the regulation of blood glucose in the neonate. I:‘\-cwptn :LleJicu Iut. Conyr. Series No. 231. 592.-600. BALLARD F. J. (1971) Regulation of gluconcogenesis during exposure of young rats to hypoxic conditions. I~iochem. J. 121, 169-178. BALLAN)F. J. & OLIVI~KI. 1‘. (1963) Cilycogcn metabolism in embryonic chick and neonatal rat liver. Nochim. hiophy.7. Acra 71, 578-588.

BALLAKDF. J. & HASSOPI‘R. W. (1967) Phosphocnolpvruvate carboxykinase and pyruvate carboxyiase in d&L ouinrz rat liver. Biochem. J. 104. X66 871. BCK&MEY~K I-I. U. & Htl
GIKARDJ. R.. GUENDL’I‘G. S.. MARLISSE. B.. K~KVKAS A., RIE~.TORTh4. & ASSAS R. (1973) Fuels. hormones and liver metabolism at term and during the early postnatal period in the rat. J. clin. Inwst. St, 3190 3100. GREESCARD0. (1970) The dcvclopmcn tal formation 01 enzymes in rat liver. In Biochernicul Acrior7.s of Her(Edited by LITWA(.K G.) pp. 53-t;7. Academic Press. New York. HAI\SOS R. W. & GARRERA. J. (1972) Phosphocnolp!ruvate carboxykinase. Its role in gluconcogenesis. AI,I. J. din. Mutr. 25. lOI& 1021. mows

Development

13.4~~0~

K.

of gluconcogenesis

W., Resu~b L. & BALLAKIJF. J. (1975) Hor-

(GTP) during development. pedn Proc. Fedn Am. Sots e.sg. Uiol. .34. 166.. 171. HARPER .A.E. (1965) Glucose-6-phosphatase. In &f&hods ofI:rtzymatic Analysis (Edited by HERGSIEYER H. U.) 2nd Edn. pp. 788 792. Academic Press, New York. H~.Nx.IN&-H. V. & SIXJB~X~ W. (1964) Zum mechanism der gluconeogenese und ihrer stcuerung -I. Quantitative bcstimmung der pyruvat carboxylase in rohextraktcn der rattenleber. Biochrm. %. 340, 160 -170. HLXI-1-r A. Sr. C. & NIXON P. A. (1957) Enzymic detcrmination of blood glucose. Biochem. J. 66, 12. JOI~XOS D. C.. BKUNSV~LD R. A.. EnERr K. A. & RAY P. D. (1973) Gluconeogenesis in rabbit liver -1. Pyruvate-derived dicarboxylic acids and phosphoenolpyruvate formation in rabbit liver. J. hiol. Chem. 248, 763-770. Jo~~~~-Bauu M. & HAYSON R. W. (1975) Regulation of hepatic gluconeogencsis in the guinea-pig by fatty acids and ammonia. J. hiol. Chrm. 250. 8978-8985. Jowlis C. T. & ASIITON 1. K. (1976) The appearance. propertics, and functions of gluconcogenic enzymes in the liver and kidney of the guinea pig during fetal & early neonatal development. Archs Biochem. I3iophy.s. 174. 506 522. NORDLIE R. C. & LARIIY H. A. (1963) Mammalian liver phosphoenolpyruvate carboxykinase activities. J. hiol. Chrm. 238, 2259-2263. OGATA K.. K~~LAIS-BAL.M M. & &SSOX R. W. (1974) Phcncthylbiguanidc and the inhihition of hepatic gluconcogenesis in the guinea pig. Biochem. J. 144. 49-57. P~L~ID~~R G. (1965) Glycogen. Determination as D-glucase with hexokinase, pyruvic kinase & lactic dchydrogenase. In Merho& of Enzymatic Analysis (Edited by BERGM~.YERH. U.) 2nd Edn, pp. 59-62. Academic Press, New York. PI~ILIPI’IDIS II. & BALLARD F. J. (1969) The development of gluconeogenesis in rat liver. Experiments in uiuo. Rioc/tern. J. 113, 651 -657. PIIILII~PILXSII., HAX\‘X)I\ R. W., R~XIEF L., H~PG~~D M. 6i BN.I.ARI> F. J. (1972) The initial synthesis of proteins during development. Phosphoenolpyruvate carboxylase in rat liver at birth. &o&m. J. 126, I1 27-l 134.

in guinea-pig

liver

743

RAY P. D. (1976) Hepatic Species Regulatiotl of (Xconeoyenesis (Edited by M~HLMAN M. & HANSOS R. W.) pp. 293 333. John Wiley, New York. Roono~ A. & ALL~YS~: G. A. 0. (1973) Regulation of renal gluconeogenesis by calcium ions, hormones and adenosine 3’ : 5’-cyclic monosphosphate. Uiochem. J. 134, 157-165. SCFIMIDT E. (1965) Glutamic dehydrogenase. In Methods in Enzymcltic Analysis (Edited by BERC~MEY~K H. U.) 2nd Edn, pp. 752-756. Academic Press, New York. SCHS~ID~R W. C. & Hoccnoot.1 G. H. (1950) lntraccllular distribution of enzymes-v. Further studies on the distribution of cytochrome C in rat liver homogenate. J. hiol. Chem. 183. 123-12X. S~IEPHEKD D. & CARLA&II P. B. (1969) Citrate synthase from rat liver. In Methods in Enzymology (Edited by C~L~WICK S. P. & KAPLAX N. 0.) Vol. 13, pp. 11-16. Academic Press, New York. SISK D. B. (1976) In 7he Biology of’ the Guinea Pig (Edited by WAGNER J. E. & MANSING P. J.) pp. 63-98. Academic Press, New York. %LIWG H. D.. WILLIAMS A., KLEIX~KE J. & GEHLH~FF M. (1970) Regulation of gluconeogencsis in guinea pig liver. Eur. J. Rio&em. 16, 289 032. UXER M. F. & KEECH D. B. (1963) Pyruvate carboxylasc-I. Nature of the reaction. J. hiol. Chem. 238, 2603 2608. VEKXON F. G. & WAJ.KIX D. G. (1968) Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver. Rio&em. J. 106. 321-329. WARNFS D., BAL.IARI) F. J. & S~A~IAKK R. F. (1974) Gluconcogcnesis in fetal and neonatal lambs. J. Reprod. Fert. 36. 471472. YEUSG D. & OLIVER 1. T. (1967) Development of gluconeogenesis in neonatal rat liver. Effect of prcmaturc delivery. Rio&m. J. 105, 1229- 1233. YEXTC; D. & OLIVER I. T. (1968) Factors affecting the premature induction of phosphoenolpyruvate carboxylase in neonatal rat liver. Biochem. J. 108, 325-331. Z~RZOLI A., TURKT-\I)OPF I. J. & MUEI.I.EK V. L. (1969) Gluconeogencsis in developing rat kidney cortex. Rio&m. J. 111, 181-185.