THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 247, No. 11, Issue of June 10, pp. 3606-3617, 1972

Printed in U.S.A.

Influence of Cortisol on Glycogen Synthesis and Gluconeogenesis in Fetal Rat Liver in Organ Culture*

(Received for publication, December 16, 1971)

CARL V~ONDER$ AND ALENA COUFALIK

From the Research Institute New York 10055

of the Hospital for Joint Diseases, Mount Sinai School of Medicine, New York,

SUMMARY

Cortisol stimulated the synthesis of glycogen in fetal rat liver explants. The increase was proportional to the amount of glucose present in the incubation medium and occurred slowly over a 44-hour period. The ability of cortisol to stimulate glycogen synthesis was accelerated after prior in- cubation of explants for 42 hours with no steroid. Glucose alone led to the synthesis of very little glycogen. [UJ4C]- glucose added to the incubation medium was incorporated into glycogen. The specific activities of [U-14C]glucose in the medium and in glycogen synthesized over a 44hour period were approximately the same whether or not cortisol was present, indicating that glycogen was derived mainly from glucose. Fetal livers synthesized glucose and glycogen from L-[UJ4C]alanine to a limited degree. The incorpora- tion of label ranged from 0.2% to 1.6% of the dose; the ex- tent was determined by the conditions of incubation. Corti- sol did not affect the over-all gluconeogenic process, but it increased the proportion of label incorporated into glycogen and decreased the incorporation into glucose. Gluconeo- genesis from L-[UJ4C]alanine did not take place in the ab- sence of glucose. Cortisol did not induce any changes which could be correlated with glycogen synthesis in the activities of phosphoenolpyruvate carboxylrinase, glycogen phosphorylase, glucose 6-phosphatase, fructose 1,6-diphos- phatase, pyruvate kinase, alanine aminotransferase, or as- partate aminotransferase. Accumulation of glycogen under all conditions studied was directly proportional to changes in glycogen synthetase b. The a form of the enzyme was not affected by cortisol. It is concluded that (a) the major source of glycogen in fetal rat liver is glucose; (b) synthesis of glycogen from glucose is stimulated by cortisol; (c) gluco- neogenesis takes place in fetal liver to a measureable extent; (d) cortisol does not influence gluconeogenesis; (e) cortlsol directs a common six-carbon intermediate towards glycogen synthesis and away from glucose formation; (f) the effect of cortisol on glycogen synthesis depends on its ability to in- crease glycogen synthetase b activity.

* This work was supported by Grant AM09006 from the Na- tional Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service.

$ Recipient of United States Public Health Service Career De- velopment Award AM06841.

In the mammalian fetus, liver glycogen normally increases in situ as term approaches to levels higher than in adult liver (1). When the animal is born, this glycogen is rapidly depleted in order to support the life processes of the newly independent organism. The events which lead to accelerated glycogen storage just prior to birth are highly complex and involve an interplay of hormonal, endogenous, and nutritional stimuli. The relative importance of these components has been the sub- ject of intensive study. There is much evidence that hormones play a major role in fetal glycogenesis. Anencephalic fetuses do not synthesize the enzymes of glycogenesis and gluconeogenesis (a), while the ability to make glycogen is restored by glucocor- ticoids (3). The injection of insulin, glucagon, cyclic adenosine 3’, 5’-monophosphate, or corticosteroids at appropriate states of gestation leads to the premature induction or suppression of enzymes of glucose metabolism (I, 4). The role of each indi- vidual component cannot be readily established in the intact fetus, because of the complexity of interactions which occur within it and between the fetus and its mother.

In the unborn rat, glucose is the main contributor to liver glycogen, and gluconeogenesis has been found to contribute little (5) or not at all (6) to glycogenesis before birth. Among the hormones which induce the accumulation of glycogen in fetal livers are the glucocorticoids. It is not clear whether corti- costeroids alone may lead directly to the formation of glycogen in prenatal liver (7), whether additional hormones are also required (8, 9), or whether the action of corticosteroids is sec- ondary to their effect on extrahepatic metabolism (10, 11).

In fetal rat liver in situ accumulation of glycogen has been found to correlate well with the increase in glycogen synthetase (2), but whether the relationship is causal is uncertain (12, 13). Similarly, the relationships between gluco- or glycogenesis and the activities of glucose 6-phosphatase (3, 14-16), glycogen phosphorylase (17), phosphoenolpyruvate carboxykinase (6, IS), and a variety of aminotransferases (19, 20) are not firmly established.’

1 The enzymes described in this paper are referred to by the International Union of Biochemistry (21) as follows: glycogen synthetase, UDP glucose:glycogen cr4-glucosyltransferase (EC 2.4.1.11) ; glucose 6-phosphatase, n-glucose 6-phosphate phospho- hydrolase (3.1.3.9) ; glycogen phosphorylase, a-1,4-glucan: ortho- phosphate glucosyltransferase (2.4.1.1) ; phosphoenolpyruvate carboxykinase, GTP:oxaloacetate carboxylyase (transphospho- rylating (4.1.1.32)) ; fructose 1,6-diphosphatase, n-fructose 1,6-

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Issue of June 10, 1972 C. Monder and A. Coufalik

In studying these areas of uncertainty, fetal liver in organ culture (18, 19) presents certain advantages. Under these conditions the influence of a hormone on metabolism of the isolated organ may be studied untrammeled by compensating interactions of other hormones or by secondary effects, as must inevitably occur in vivo. The effect of hormones on several interdependent parameters can therefore be investigated with reasonable assurance that the hormone itself is directly respon- sible for any observed changes.

In this study, fetal rat liver explants have been shown to convert glucose and n-alanine into glycogen in the presence of cortisol. Changes in levels of certain enzymes in response to steroid have been examined in order to determine whether these can be correlated with the steroid-induced increase in glycogen.

MATERIALS AND METHODS

Pregnant CFE rats (Carworth Farms, Inc., New City, New York) were received at approximately 18 days after conception. When the fetuses were 20 f 1 days old, they were removed under sterile conditions after killing the mother with ether. The ages of the fetuses were determined from their crown-rump length and weight, utilizing published tables (22). Livers were re- moved and established in culture as described by Wicks (19). In experiments in which glucose content of the incubations was varied, the initial medium was specially formulated by the manufacturer (Microbiological Associates, Inc., Bethesda, Md.) to contain no glucose. Cortisol when added was dissolved in ethanol and introduced into the incubation medium in a volume of 0.1 ml to give a final concentration of 2 X 10m6 M steroid. An equal volume of ethanol was added to control dishes.

Glycogen was determined as described by Hassid and Abraham (23) after removing interfering substances with perchloric acid (24). In order to measure incorporation of radioactive pre- cursors into glycogen, the fetal livers were treated as suggested by Good et al. (25). The glycogen thus isolated was dissolved in water; perchloric acid was added to a final concentration of 0.3 M, and the precipitate formed was removed by centrifugation. Glycogen was obtained from the supernatant solution by adding 2 volumes of ethanol containing 0.1% lithium chloride. After 60 min at 49”, the suspension was cooled to room temperature. The precipitate was then collected by centrifugation, washed with ethanol, and dissolved in 2 ml of water.

Incorporation of radioactivity from L-[U-“Clalanine into glucose and glycogen in the incubation system was used as a measure of gluconeogenesis. The technique used to isolate the glucose is modified from the one described by Friedmann et al. (26). The original method led to incomplete recovery of glucose, as was also noted by DeWolf et al. (27). In the modified pro- cedure, 1 ml of deproteinized medium was diluted to a final volume of 5 ml. Three milliliters of supernatant (pH 6.8) were passed through a column of AG-2 x 8 (bicarbonate form, 10 cm x 0.6 cm) superimposed on a column of AG-50W x 8 (hy- drogen form, 10 cm x 0.6 cm). The column was washed with water, and the eflluent and washings were combined in a final

diphosphate 1-phosphohydrolase (3.1.3.11) ; pyruvate kinase, ATP:pyruvate phosphotransferase (2.7.1.46) ; aspartate amino- transferase, n-aspartate:2-oxoglutarate aminotransferase (2.6. 1.1) ; alanine aminotransferase, n-alanine:2-oxoglutarate amino- transferase (2.6.1.2) ; lactate dehydrogenase, n-1actate:NAD oxidoreductase (1.1.1.27).

volume of 25 ml. Twelve milliliters of the combined eluate (pH 5.0 to 6.0) were mixed with 1 mg of glucose oxidase and 1 mg of unlabeled glucose and incubated overnight at room tempera- ture. Sodium hydroxide (10 mu) was added to a phenolphthal- ein end point, and the entire solution was passed through a col- umn (6 cm x 0.6 cm) of AG-1 x 8 (chloride form). The column was washed with 12 ml of water and the gluconic acid was eluted with 0.1 N sodium chloride. Radioactivity in the gluconic acid of the final eluate represents the labeled glucose originally pres- ent. Recovery with known standards exceeded 90%.

The percentage of incorporation of radioactivity from L-

[U-‘*C]alanine into total hexoses was calculated using the for- mula :

y. Incorporation

= Total dpm in glycogen of explant + total dpm in glucose Total dpm of n-lU-14Clalanine added to dish

x 100 Radioactivity of aliquots was measured in Aquafluor (New

England Nuclear Corp., Boston, Mass.) using a Packard 3380 scintillation counter. All counts were converted to disinte- grations per min after making the appropriate corrections for quenching and efficiency of counting. [U-14C]Glucose (5.4 mCi per mmole) and n-[UJ*C]alanine (123 mCi per mmole) were purchased from the New England Nuclear Corp.

Phosphoenolpyruvate carboxykinase was measured as de- scribed by Sillero et al. (28), but in 0.05 M imidazole buffer, pH 6.6 (6). Residual radioactive carbon dioxide was removed by bubbling a mixture of air and carbon dioxide (98: 2) through the incubation mixture after terminating the reaction with trichlo- roacetic acid.

Glycogen synthetase was determined by the method of Leloir and Goldemberg (29). Total synthetase was measured after adding 5 mM glucose 6-phosphate to the mixture. The a form of the enzyme (30) was determined in the absence of added glucose 6-phosphate.

Glycogen phosphorylase was estimated by the method of Sutherland (31), fructose 1,6-diphosphatase by the method of Pogell and McGilvery (32), and glucose 6-phosphatase by the method described by Sillero et al. (28). Pyruvate kinase was determined as described by Sillero et al. (28), but at pH 7.5 and with 20 units of lactate dehydrogenase. Aspartate and alanine aminotransferases were determined spectrophotometrically by coupling the reactions to appropriate DPN+-dependent dehydro- genases (33, 34).

RESULTS

Xtimulation of Glycogen Synthesis by Co&sol-We have con- firmed the observations of many investigators who reported that the glycogen content of fetal rat liver increases rapidly several days before birth and drops quickly thereafter (3, 14). A positive correlation was found between the age of the fetus (determined by crown-rump length or weight) and the glycogen content of the livers. Livers of 19-day-old fetuses contained an average of 10 mg of glycogen per g wet weight of liver. Most of the experiments reported were performed with livers from fetuses 20 to 22 days old, containing 35 to 56 mg of glycogen per g wet weight.

In order to evaluate the stability of fetal liver glycogen, liver

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3610 Cortisol and Glycogenesis in Fetal Rat Liver Vol. 247, No. 11

t T (7)T

(20)

(20)

TT

b.

22 44 66 5 12 22

HOURS INCUBATION

1

FIU. 1. Effect of cortisol on the level of glycogen in fetal rat liver in organ culture. a, liver fragments were incubated in the standard system in the presence of 2 X 1W6 M cortisol ( q il) or 0.1 ml of ethanol vehicle, (0) for the designated time intervals. b, liver fragments were incubated for 42 hours in standard medium, then incubated for the indicated additional time interval in the presence of cortisol (@A) or vehicle (Cl ). Numbers in purenfheses represent number of individual observations. Limit lines repre- sent standard error of the mean for number of observations shown in parentheses.

4

cr W >

i 3 (4)

0.0 M O.llM 0.22 M

GLUCOSE CONTENT OF MEDIUM

FIG. 2. Effect of glucose and cortisol on the synthesis of glyco- gen by fetal rat liver. Liver fragments were previously incubated in standard medium containing 0.11 M glucose for 42 hours. They were then washed in 0.14 M potassium chloride and transferred for an additional 22 hours into medium containing the designated amounts of glucose and 2 X l(re M cortisol (a) or vehicle (0). Limit lines indicate standard error of the means; number of in- dividual observations is in parentheses. Dashed horizontal line indicates glycogen level after 42 hours of preliminary incubation.

explants were incubated in organ culture for various periods of time (Fig. 1). In 1 hour, no decrease in glycogen content was detected. Adult liver under the same conditions lost more than 50% of its glycogen in this time. In 5 hours, the decrease in glycogen averaged less than 20%. After 44 hours, only 0.4% of the initial glycogen remained. These results demonstrate

that the fetal livers were capable of glycolysis but that this ability was poorly developed.

Fig. 1 shows that liver incubated with 2 X 10d6 M cortisol contained more glycogen after 44 hours than the controls with no steroid. No significant differences were seen between incu- bations performed with and without steroid after 5 and 22 hours. Cortisol was either stimulating an increase in glycogen synthesis or slowing the rate of glycogen breakdown. To distinguish between these possibilities, liver explants were incubated for 42 hours without steroid to deplete them of glycogen. Subsequent addition of cortisol led to an increase in the glycogen content of the livers after 22 hours of incubation. Cortisol had therefore stimulated the net synthesis of glycogen.

It was not clear from these results whether the glycogen formed was derived entirely from glucose or whether the other compo- nents of the medium, under the influence of cortisol, were contrib- uting significantly to its synthesis. Liver fragments were incubated in standard medium containing 0.11 M glucose for 42 hours. After this preliminary incubation period, the glycogen content of the livers was usually below the limit of sensitivity of the assay method (0.075 mg per g of liver). The livers were then washed in cold 0.15 M potassium chloride and transferred to fresh medium containing either no glucose, 0.11 M glucose, or 0.22 M glucose. The fragments, which contained an average of 0.12 mg of glycogen per g of liver, were cultured for an additional 22 hours. Fig. 2 shows that in the absence of glucose, no addi- tional glycogen accumulated. Addition of 0.11 M glucose to the medium resulted in a slight increase in glycogen. The ability of livers to synthesize glycogen from glucose was severely limited, since further glucose had no additional beneficial effect. When cortisol was added to the medium, considerable glycogen was made in amounts directly proportional to the glucose level.

Comparison of Figs. lb and 2 shows that the ability of cortisol to stimulate glycogen synthesis increased when the cultures were transferred to fresh medium after a prior 42-hour period of incu- bation with no added steroid. After the preliminary incubation period, the livers had lost the capacity to synthesize significant quantities of glycogen in the absence of cortisol in the original medium. When transferred to fresh medium, some of this capacity was regained. It is possible that the medium in which the liver explants were originally suspended had accumulated an inhibitor of glycogen synthesis during preliminary incubation. The livers themselves had not deteriorated in this time, as established by their increased responsiveness to cortisol when they were transferred to fresh medium.

Synthesis of Glycogen from [U-r4C]Glucose-The ability of cortisol to stimulate glycogen synthesis was examined with radioactive precursors, in order to evaluate the relative con- tribution of glycogenesis and glyconeogenesis. Fetal rat liver has been reported to be incapable of gluconeogenesis (20,35) but can synthesize glycogen from glucose, though at a rate about three times slower than livers of adult rats (36). Table I demon- strates that cortisol greatly enhanced the net synthesis of glyco- gen in organ culture. The effect was more dramatic after 44 hours than after 22 hours. Over the 44-hour time interval, the increase ranged from about 5 to 10 times the values in the con- trols. Tissues incubated without cortisol incorporated radio- active glucose into glycogen; maximum total incorporation was achieved by 22 hours. Longer incubation periods (44 hours) without cortisol usually led to severe depletion of glycogen and a decrease in the total radioactivity incorporated into glycogen.

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Issue of June 10, 1972 C. Monder and A. Coufalik 3611

However, even without added cortisol there was further increase used by the explants to synthesize glycogen. The primary after 22 hours of the specific activity of UJ4C-glucosyl in glyco- effect of cortisol was to increase the rate of glycogen synthesis. gen. In the experiment shown in Table I, the specific activity of Synthesis of Glycogen from L-[U-14C]Alanine-The extent of the glycogen in the presence of cortisol increased over the 44-hour incorporation of n-[UJ4C]alanine of high specific activity into incubation period. The specific activity of glucose in each dish glycogen is shown in Table II. These fetal livers were capable of at the end of the experiment and the specific activity of the limited gluconeogenesis from n-[U-i4C]alanine since radioactivity glycogen after 44 hours were not significantly different, so that was detected in the minute amount of glycogen present after 22 almost all of the glycogen in the liver which had been stimulated and 44 hours of incubation in the absence of cortisol. However, by cortisol was newly formed from the gIucose in the medium. the ability to synthesize glycogen from alanine, although present, A similar conclusion can be drawn about the incorporation of was of a very low order of magnitude. Consequently, alanine [U-i4C]glucose in the absence of cortisol. Therefore, regardless contributed little to the net accumulation of glycogen. The of whether cortisol was added to the system or not, glucose was specific activity of glycogen derived from L[U-‘Qalanine was

TABLE I

EJ’ect of cortisol on incorporation of [U-14C]glucose into glycogen of fetal rat liver explants

Liver explants from 20-day-old rat fetuses were incubated steroid. Each dish contained 1 PCi of [U-Wlglucose. Total where indicated with 2 X 10-e M cortisol added to the culture molarity of glucose was 0.11 M. Values are shown with standard medium in 0.1 ml of ethanol. No preliminary incubation period error of the mean. intervened. In the controls, 0.1 ml of ethanol replaced the

Additions Replicates

None.. . 2 0 None.. . 4 22 Cortisol.. . 4 22 None.. . . . . 4 44 Cortisol . . . 4 44

lcubatiol time

hrs

-

n

_-

-

Tissue weight

m mg/g wet weight

30.0 35.4 39.4 f 1.6 4.87 f 0.02 34.1 f 2.9 5.13 f 0.28 20.7 f 0.1 0.21 f 0.02 22.7 zk 2.7 2.76 f 0.72

Liver glycogen 1°C ill glycogen

dpm/P?~~~u&lUCOSe

-c

2,845 i 420 2,680 f 160

49,600 f 7,440 41,900 f 6,800

-

P

_-

-

rota1 incor- oration into

explanta

%

- 116,600 -

2,514 84,220 f 17,400 0.10 f 0.01 2,894 62,680 f 7,540 0.12 i 0.01 1,331 51,165 zt 3,580 0.06 zk 0.01

16,204 51,500 i 2,350 0.67 zt 0.00

“C in medium Incorporationb

(I (mg of tissue) X (~g of glycogen per mg of tissue) X (dpm per pmoles of glucose residue) = Total ,4c in glycogen (162 ILR per mole glucose residue) X 1000

6 Total 14C in glycogen/total 14C added to system X 100. c Dashes indicate absence of detectable radioactivity.

TABLE II Effect of cortisol on incorporation of L-[U-YJalanine into glycogen and glucose

I I relimi Fetal Madl$z %S kpli. a* in age cater %

ncuba- C” a- tiond

tion

__

dl%YS ‘““,“I”/ hrs hrs

21 3.37 None 4 3.37 Cortisol 4

0 22 22

Liver glycogene

19 3.37 None 4 22 3.37 Cortisol 4

42 22

tag/g wet wt dMP;;;;gw=

0.58 f 0.18 1,150 f 516 1.90 f 0.06 6,000 f 173

<0.07 0 1.58 f 0.25 34,400 f 6,9OC

0.20 f 0.04 4,055 i 245 0.62 f 0.05 8,670 f 480 025 f 0.10 5,000 zk 670 2.99 f 0.19 13,980 i 730

20 0.004 None 4 22 0.004 Cortisol 4 0.004 None 6

0 22 44

0.004 Cortisol 6 44 -

0 Estimated from crown rump length and weight. b Total activity added to each dish was 1 &i.

“C in glycogen’ W in glucosee UC in glycogen’ Total incor- poration into

glucose + glycogenf

d~m/&w?wlc &ha/dish &%/dish % of dose

298 f 46 166 zk 23 4,568 f 538 0.22 f 0.02 147 f 3 2,366 f 253 2,176 f 80 0.21 f 0.02

3,100 f 5Of 0 37,400 zk 3,070 1.70 f 0.14 2,183 zb 28 9,460 f 780 24,040 f 2,500 1.54 f 0.15

293 zk 23 130 f 5 4,640 f 335 0.22 f 0.01 259 zk 22 990 f 120 3,960 f 460 0.23 f 0.02

1,227 f 25 200 f 15 16,100 f 83 0.74 i 0.00 856 f 64 5,800 f 415 9,900 f 1,600 0.72 f 0.09

c Cortisol was added to a final concentration of 2 X 10-s M in 0.1 ml of ethanol. Controls contained 0.1 ml of ethanol in place of steroid.

J Where indicated, explants were incubated for 42 hours in culture medium with 0.11 M glucose, no alanine, and no steroid. They were then washed with 0.14 M KC1 and transferred to fresh culture dishes containing alanine and cortisol as indicated.

6 Values are shown with standard error of the mean. , Total dpm in (glucose + glycogen) in culture dish

Total dpm of n-[UJ4C]alanine added to medium x 100.

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Cortisol and Glycogenesis in Fetal Rat Liver Vol. 247, No. 11

enhanced by cortisol 3- to &fold. This was accompanied by a considerable increase in net glycogen formation. The radio- activity introduced into glycogen when cortisol was present appeared to be similar in 20- and al-day-old fetuses. The introduction of large amounts of unlabeled alanine led to glycogen of lower specific activity when no steroid was present, but the stimulatory effects of cortisol remained undiminished. Cortisol led to a decrease in specific activity of free [14C]glucose in the medium. This effect has been seen preiriously (37) and suggests that cortisol may lead to the synthesis of glycogen in part at the expense of glucose formation. Gluconeogenesis, expressed in terms of glucose plus glycogen, was quite significant. Prelimi- nary incubation of the liver fragments for 42 hours increased the level of incorporation of label from I,-[U-“C]alanine from about 0.2 y0 to 1.6 y0 of the initial dose in 22 hours. Cortisol clearly did not enhance gluconeogenesis in the absence of other added hormones, since the total incorporation of label into glucose plus glycogen was the same as in the absence of steroid. The role of cortisol was therefore to redirect a common pool of precursors into glycogen rather than glucose. In order to determine whether the low level of gluconeogenesis was due to its suppression by the glucose present in the medium, an experiment was performed to see if L-[U-14C]alanine incorporation would be increased in a glucose-free medium. The amount of alanine in the system was 10 times the normal plasma concentration, since it has been shown that this level is close to optimal for gluconeogenesis (38).

The results of the experiment shown in Table III led to the conclusion that, contrary to expectations, glucose was required for the incorporation of radioactivity from [U-Wlalanine into glycogen. Although glucose in the medium stimulated glyco- genesis from L-[U-14C]alanine in the absence of steroid, accu- mulation of glycogen was especially marked in the presence of cortisol. Total incorporation into bound and free hexoses was the same with or without cortisol. Cortisol, as before, led to increased glycogenesis at the expense of gluconeogenesis. These results lead to the conclusion that, in fetal explants, the effects of cortisol are localized at the bifurcation between glucose pro- duction and glycogen synthesis. It can therefore be predicted that cortisol will have no effect on the enzymes of gluconeogene- sis and will affect only those enzymes which directly control glycogen synthesis and glucose release. To test this prediction, we examined key enzymes of sugar metabolism.

Phosphoemlpyruvate Carboxykinase-The effects of incubation conditions on phosphoenolpyruvate carboxykinase activity are shown in Table IV. The enzyme doubled in activity in cultures incubated for 44 hours in medium containing 0.11 M glucose and 2 X 10m6 M cortisol (Series I), and glycogen content increased about 5-fold. No appreciable change in phosphoenolpyruvate carboxykinase activity occurred in the absence of cortisol. When fetal liver fragments were depleted of glycogen by pre- liminary incubation for 42 hours in the absence of cortisol, subsequent incubation in the same medium with cortisol for 22

TABLE III E$ect of glucose and cortisol on incorporation of L-[U-l”C]alanine into glycogen

Explants were previously incubated for 42 hours in culture medium with 0.11 M glucose, no alanine, and no steroid. They were washed with 0.14 M KC1 and transferred to fresh culture dishes containing glucose or cortisol as indicated. Each dish contained 1 &i of L-[UJ4C]alanine and a total concentration of 3.37 mM L-alanine. Cortisol was added in 0.1 ml of ethanol. Controls with no cortisol contained 0.1 ml of ethanol. Analyses were performed after 44 additional hours of culture.

Glucose Cortisol Replicates Liver glycogen *PC in glycogen W in glucose Incorporation into total hexoses

M M mgjg tissue dfim/dish &n/dish %

0 0 6 0.20 f 0.10 0.11 0 6 0.25 f 0.09 84 f 10 22,780 f 8,822 1.04 zk 0.40

0 2 x 10-C 6 0.11 2 x 10-S 6 1.69 f 0.36 5,685 f 1,115 16,014 f 2,426 0.99 f 0.12

Series

I

II

III

- I

Effect of incubation conditions on p shosphoenolpyruvate carboxykinase and glycogen synthesis

Preliminary incubation conditions

- I Incubation

conditions

Not previously EMEc incubated EME

42 hrs in EME

42 hrs in EME Fresh medium transferd

-I-

EME EME

TABLE IV

Incubation Cortisola

hrs

44 44 0

22 22

22 22

M

0 2 x 10-6

0

0 2 x 10-6

0 2 x 10-e I -

Phosphoenolpyruvate carboxykinaseb

0.084 f 0.003 0.154 f 0.007 0.060 f 0.002

0.068 f 0.004 0.132

0.073 f 0.022 0.076 f 0.001

Glycogen

mg/g wet wight

0.30 1.58 48.1

0.37 f 0.05 0.50 f 0.09

0.51 f 0.01 1.15 f 0.14

a Added in 0.1 ml of ethanol per culture dish. 6 Nanomoles of [Wlbicarbonate incorporated per min per mg of protein with standard error of the mean for four replicates. c Eagle’s minimum essential medium with Hanks’ balanced salt solution and 0.11 M glucose. d Cultures after preliminary incubation were washed in 0.14 M potassium chloride and transferred to fresh medium.

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Issue of June 10, 1972 C. Mender and A. Coufalik 3613

TABLE V

Effect of cortisol on glycogen synthetase activity

Experimental Preliminary incubation

time”

hrs hrs

0 44 0 44 0 0

42 42

0

42 42

0

42 42 42

0

22 22

0

5 5 0

22 22

0 0

Incubation Incubation medium Cortisol’

Original Original

Original Original

Original Original

Transferredd Transferred

M

0 2 x 10-6

0

0 2 x 10-C

0

0 2 x 10-6

0

0 2 x 10-6

Glycogen synthetase”

ofb a

0.24 i 0.01 0.12 0.63 f 0.1 0.12

2.60 0.24

0.26 f 0.03 0.12 0.35 f 0.07 0.12

1.70 0.24

0.33 f 0.05 0.11 0.37 f 0.02 0.12

2.7 0.20

0.31 f 0.04 0.12 0.51 f 0.03 0.10 0.34 f 0.03 0.11

2.30 0.24

Glycogen

n&g/g wet wi 0.32 2.29

0.22 0.41

0.37 0.25

0.09 0.96

0 Cultures were maintained under standard conditions without c Standard errors of the mean are for four determinations. cortisol for the time interval indicated. d After preliminary incubation, the cultures were washed in 0.15

b Subsequent to preliminary incubation, 2 X 10-e M steroid or M KC1 and transferred to fresh medium containing cortisol or ethanol (0.1 ml) was added to the medium as indicated and incu- ethanol (0.1 ml) as indicated. bation was continued in the original medium for the designated time.

hours also led to a doubling of phosphoenolpyruvate carboxyki- 0.50

nase activity, but glycogen synthesis was only slightly greater than in the absence of cortisol (Series II). Finally, preliminary w incubation of livers for 42 hours followed by transfer to fresh

VI a .

medium containing cortisol led to a glycogen content twice that I- w :

of the control, but the activity of phosphoenolpyruvate carboxy- I k-

kinase did not change at all (Series III). It must be concluded 2

from these observations that although cortisol can stimulate t-

phosphoenolpyruvate carboxykinase under certain conditions, * 0.25 - 2

there is no causal relationship between its level and the ability w

of the liver to form glycogen from glucose. An interesting side- ::

light derived from the data in Table IV relates to the stability Y .

of phosphoenolpyruvate carboxykinase in fetal liver cultures. 2

No decrease in activity occurred after 66 hours of culture under a

a variety of conditions. Glycogen Xynthetase-Since the fetal rat liver synthesizes

glycogen mainly by glycogenesis, and only to a small extent by glyconeogenesis, we examined the effects of cortisol on glycogen synthetase, the enzyme generally considered to be rate-limiting in the transformation of glucose B-phosphate to glycogen. It can be seen in Table V that the enzyme in fetal liver was unstable. Liver fragments that were incubated in the absence of steroids for 42 to 66 hours lost about 90% of their glycogen synthetase activity. This loss contrasts with the increase in activity in adult liver homogenates in vitro in the presence of glucose (27). Introduction of steroid after 42 hours of preliminary incubation, when enzyme loss was at its maximum, stimulated a 40 to 70% increase in total synthetase activity. In the time intervals studied, a resurgence of activity to the original levels was never achieved. Regeneration of glycogen synthetase activity was a slow process. In 5 hours, steroid did not enhance its level; in 22 hours, a significant increase was seen. In contrast with phos-

oi 2

A GLYCOGEN (mg/g wet weight)

FIG. 3. Comparison of the effects of cortisol on increase in gly- cogen and glycogen synthetase in fetal liver in organ culture. Values are the measured changes due to cortisol under the con- ditions shown in Table VI. Four additional experiments not shown on the table are included in the figure. Values designated are for glycogen synthetase a + 6.

phoenolpyruvate carboxykinase, the effects of steroids on glyco- gen synthetase were qualitatively the same regardless of whether the livers were incubated with steriod in the original medium in which they were incubated or whether they were washed and transferred to fresh medium prior to the introduction of steroid. In each case, as Fig. 3 demonstrates, the change in glycogen reflected the change in enzyme activity. An increase in enzyme was associated with an increase in glycogen; where glycogen did

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3614 Co&sol and Glycogenesis in Fetal Rat Liver Vol. 247, No. 11

not increase, the enzyme similarly did not increase. The coeffi- cient of correlation for these effects was 0.87, significant at the 0.005 level (39). During the preliminary incubation period, the extensive drop in glycogen was accompanied by an equally precipitous decrease in glycogen synthetase. The change in enzyme activity with cortisol was almost entirely due to the alteration in synthetase b activity. Cortisol had no discernible effect on synthetase CL. The a activity was very low to begin with and decreased comparatively little during the preliminary incubation period.

TABLE VI Effect of cortisol on glycogen phosphorylase in fetal liver

Incubation Glycogen phosphorylase~ Glycogenb

time - Cortisol + Cortisol - Cortisol + Cortisol

hrs pm& Pi/100 mg )rotcin/min t&g wet weight

0 14.67 f 1.49 22 8.79 f 1.32 6.70 f 0.29 0.55 1.42 44 4.88 f 0.65 4.45 f 1.24 0.13 3.54

a Enzyme levels were estimated after incubating liver frag- ments under standard conditions with 2 X 10-e M cortisol or ethanol (0.1 ml) for time intervals indicated. Means and stand- ard deviations are shown for four independent experiments.

b Glycogen levels det.ermined in one experiment.

Glycogen Phosphorylase-Over a period of 44 hours of incu- bation, the glycogen phosphorylase activity of the fetal liver cultures decreased to about 30yo of its initial activity. Addition of cortisol to the incubation medium led to no greater drop in activity, as Table VI shows. Concurrently, there was an in- crease in glycogen. It was concluded therefore, that if the rate of degradation of glycogen is altered in the presence of cortisol, this is not reflected in the levels of phosphorylase.

Some Other Enzymes of Glucose Metabolism-Two key enzymes of gluconeogenesis, glucose 6-phosphatase and fructose 1,6- diphosphatase were examined to determine if they change in response to corticosteroid stimulation. Table VII shows that incubation of fetal livers with cortisol, under conditions in which highly significant increases in glycogen occur, did not result in any significant changes in fructose 1,6-diphosphatase or glucose 6-phosphatase, compared with controls containing no cortisol. Pyruvate k&se, a key glycolytic enzyme, also did not change. The 44-hour incubation period itself led to no ap- preciable change in fructose 1,6-diphosphatase and glucose 6-phosphatase. Pyruvate kinase decreased to about one-half of its initial value, and this was not influenced by the addition of cortisol.

Aspartate and Alanine Amirwtransjerases-The fact that incor- poration of radioactivity from L-[UJ4C]alanine into glycogen was increased by cortisol suggested that corticosteroid stimulated alanine aminotransferase in order to provide more of the gly-

TABLE VII Effect of cortisol on additional enzymes of glucose metabolism

Final enzyme activitqP Glycoge~P EnzylIle Initial enzyme activity

+ Cortisol - Cortisol + Cortisol - Cortisol

mg/g wet weight

Fructose 1,6-diphosphataseb. . 0.48 0.39 f 0.02 0.43 f 0.07 4.24 0.13 Glucose 6-phosphataseb.. 1.41 1.10 f 0.02 1.14 f 0.08 2.81 0.08

Pyruvate kinasec. 11.2 4.71 f 0.09 5.00 f 0.02 1.58 0.30

5 Values determined after 44-hour incubation without change of medium. Final cortisol concentration was 2 X lo-+ M. Means and average deviations are for two experiments. Each glycogen value is a single experiment.

b Micromoles of Pi per hour per mg of protein. c Micromoles of DPNH oxidized per min per 100 mg of protein.

TABLE VIII Effect of cortisol on aminotransferases

Preliminary Incubation incubation timea period

hrs hrs

0 45 0 45 0 0

42 42

0

42 42

0

22 22 0

22 22 0

Incubation medium

Original Original

Original Original

Freshc Fresh

Aspartate aminotransferase Alanine aminotransferase Glycogen

pmmoles kelo acid/hr/mg protein

5.17 f 0.01 1.35 f 0.15 8.30 f 0.27 1.36 f 0.14

11.86 0.83

3.69 f 0.11 0.78 f 0.01 7.04 f 0.82 0.74 f 0.16

15.70 0.90

3.06 z!z 0.31 0.95 f 0.01 5.31 f 0.38 1.61 f 0.19

9.04 0.79

0.28 1.23

0.30 1.28

0.84 1.56

0 See Table V, Footnote a. b See Table V, Footnote b. c See Table V, Footnote d.

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Issue of June 10, 1972 C. Mender and A. Coufalik 3615

coneogenic precursor, pyruvate. Therefore, the effects of cortisol on the activities of alanine and aspartate aminotrans- ferasea were examined. The results, summarized in Table VIII, show that cortisol caused a 50 to 100% increase in aspartate aminotransferase. The increase did not depend on the con- ditions of preliminary incubation. Liver preparations previously incubated for 42 hours prior to addition of steroid responded about as well as cultures in which cortisol was present from the outset. Cultures transferred to fresh medium containing cortisol after preliminary incubation also increased aspartate aminotransferase.

Alanine aminotransferase, on the other hand, was not affected by cortisol when the tissue was incubated in the original medium, regardless of whether it was previously incubated or not. When the tissue was transferred to fresh medium after 42 hours of preliminary incubation, a significant stimulation of activity was seen in preparations cultured in the presence of cortisol for a further 22 hours. During the incubations, the activity of the enzyme increased spontaneously in many cases. No explanation can be given for this phenomenon. There was no correlation between the change in alanine aminotransferase and the change in glycogen in fetal livers.

DISCUSSION

The rapid accumulation of glycogen to a level two to three times that of adult concentration in livers of fetal rats ap- proaching term requires the cooperative interaction of a number of hormones (3, 14, 40). This increase is associated with a relatively slower rate of glycolysis (41). The slow loss of glyco- gen by isolated fetal livers reported here is in accord with these observations. In 5 hours, explants lost little glycogen, in con- trast with the rapid changes that occur in adult liver. Over a period of about 20 hours, the fetal livers did eventually lose most of their glycogen. The activity of phosphorylase in our prepara- tions was initially about 15 pmoles of Pi per 100 mg of protein per min, a value comparable to that found by others (42), and somewhat lower than the activity of adult livers of 20 bmoles of Pi per 100 mg of protein per min (42, 43). It did not appear likely that the slow rate of glycolysis was caused by inadequate levels of phosphorylase. It is of considerable interest that fetal livers in organ culture lost glyzogen, even though livers during the normal course of development in utero accumulate greater concentrations of glycogen than do normal adult livers. Glyco- gen synthesis and breakdown require the imposition of external controls which are lost when the liver is separated from its natural environment and placed in culture. Corticosteroid at physiological concentrations probably participates directly in the control of glycogen synthesis in fetal liver, since cortisol was able to stimulate the synthesis of glycogen from glucose in the absence of other added hormones in accord with the observations of Glinsmann et al. (44). There is no obligatory requirement for pituitary hormones (45) or insulin (8,9,46-48)) though they may increase the net amount synthesized. Consequently, it is con- cluded that the glucocorticoids do not act exclusively by a “permissive” action (8, 9).

Corticosteroids increase the net accumulation of glycogen in postnatal liver, and the process has been correlated with in- creased levels of glycogen synthetase (49). The increase in total synthetase (a + 6) is inadequate to explain the high degree of stimulation of glycogen synthesis in mature liver (13, 50-52). Accelerated conversion of glucose to glycogen caused by glucose

or glucocorticoids may depend on the appearance of glycogen synthetase a (53, 54) and a concurrent depression in the activity of glycogen phosphorylase a (17). In fetal liver in situ, accu- mulation of glycogen is reported to correlate well with the glyco- gen synthetase activity (2). This association is supported by our in vitro experiments. Glycogen synthetase a activity was found to be initially low, representing about 10% of the total enzyme. It decreased about half during incubation and was not affected by cortisol, in contrast with adult liver. Synthetase b was far more labile, and lost more than 90% of its activity during 42 hours or more of incubation. It was this latter form of the synthetase that responded to cortisol. Summing the activities of the synthetases a and b leads to the conclusion that cortisol increased total enzyme activity, possibly as a consequence of the stimulation by cortisol of the formation of enzyme protein, or activation of a precursor (55). The differences were not due to a protective effect of steroid on residual activity, because the introduction of cortisol after the enzyme was at its minimum led to an increase of synthetase b specifically (Table VI, Experiment 4). It can be concluded that the action of cortisol does not depend on the phosphorylation of synthetase a and that conse- quently the activation of synthetase phosphatase (54) by cortisol plays no role in fetal liver under the conditions of our experi- ments. Since glucose 6-phosphate is present in high intracellular concentration in fetal liver (56), it is not a variable. Variation in glucose 6-phosphate due to the action of glucose 6-phosphatase is ruled out because the levels of this enzyme, low to begin with (2, 3, 15), did not change in the presence of steroids. The un- responsiveness of fetal glucose 6-phosphatase, in contrast with the postnatal enzyme (4), was also seen by Wicks (18). Fetal liver contains hexokinase but no glucokinase (36, 57) and is freely permeable to glucose (16), so that phosphorylation of glucose is not limiting provided adequate supplies of glucose are available. Although the increase by cortisol of the level of glycogen synthetase may be a controlling factor in the conversion of glucose to glycogen, other variables must intervene, since the total glycogen produced depended on the quantity of glucose in the medium. The mechanism may involve an activation of the enzyme by depletion of ATP, a potent inhibitor of the phos- phorylation of glucose, and concurrent enhancement of enzyme activity by the product, glucose 6-phosphate (58).

The activity of phosphorylase a was not decreased by cortisol in organ culture, even though glycogen synthetase was increased, and it is concluded that phosphorylase does not play as im- portant a role in fetal liver metabolism as in adult liver metabo- lism.

The major source of glycogen in fetal liver is glucose (36). The available evidence suggests that gluconeogenesis does not appear until after birth (14,35,59) because of the absence of key enzymes such as hexosediphosphatase and cytosol phospho- enolpyruvate carboxykitiase. In organ culture, however L-[U-‘~C]- alanine was incorporated into glycogen and glucose. The mean absolute incorporation into all sugars was 0.2% of the total dose in 22 hours and 0.7% in 44 hours. If the livers were previously depleted of glycogen, the incorporation increased to about 1.4% in 22 hours. When conversion of glucose to glycogen was ex- amined, incorporation ranged from about 0.1 to 0.7% of total glucose (0.11 M) per culture dish depending on whether or not cortisol was present. It appears, therefore, that the formation of glycogen from alanine was mainly from glucose 6-phosphate, and little was derived from the [14C]glucose which had left the

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liver. Gluconeogenesis was dependent on a supply of glucose, lated by corticosteroids, whereas aspartate aminotransferase was for in the presence of glucose, incorporation of radioactivity hardly affected (71). Aspartate aminotransferase activity in from n-[U-14C]alanine was found in both glucose and glycogen, rat liver near term was initially much higher than that of alanine but neither component had detectable label in the absence of aminotransferase, and, unlike alanine aminotransferase, it in- glucose. Most significantly, cortisol had no effect on the total creased rapidly to term (20). The increase may partly reflect conversion of n-[U-14C]alanine to free and bound sugars, for the stimulation by corticosteroids and may be related to the increased percentage of conversion remained the same as in the absence of capabilities of the liver for gluconeogenesis, perhaps, as Lardy et steroid. Only the distribution of label was affected. There- al. have suggested (70)) by supplying precursors of phosphoenol- fore, cortisol facilitated the incorporation of both glucose and pyruvate. However, we have inhibited the stimulation of alanine into glycogen but had a greater effect on glucose in- aspartate transaminase by the addition of decaborane to the corporation. These findings are in accord with the conclusion culture medium and have found that gluconeogenesis is un- that the actions of glucocorticoids are directed specifically to impaired (37). glycogenesis rather than to gluconeogenesis (60, 61). These studies have shown that the fetal rat liver is capable of

Fructose 1,6-diphosphatase is a limiting enzyme on the path both glycogenesis and gluconeogenesis. Only the former process from 3-carbon precursors to glucose or glycogen. It has very low is stimulated by cortisol. In either case the rate of glycogen activity in fetal rat liver (62). Cortisol did not affect its level accumulation appears to be limited by the activity of glycogen in our experiments. The inconsistency of the effect of cortisol synthetase. The level of gluconeogenesis is quite low, and on fructose 1,6-diphosphatase activity in adult liver (63, 64) probably reflects the initial stages in the development of activity and the lack of stimulation in fetal livers suggest that it may not that is to emerge after birth. Consequently, it makes only a be involved in the control of gluconeogenesis under all conditions. small contribution to the accumulation of glycogen in the prenatal

Pyruvate kinase may also influence gluconeogenesis by pro- rat liver. The extent of glycogen synthesis from glucose is also moting the conversion of phosphoenolpyruvate to pyruvate (65). small whether or not glucose is present, when compared to the It is depressed by cortisol (66), birth (40), and low carbohydrate capacity of the liver to accumulate glycogen in utero. The low diet (65), all of which accelerate gluconeogenesis. However, in degree of glycogen accumulation under the influence of cortisol our experiments cortisol did not stimulate gluconeogenesis in seems to be associated with a lag period. In organ culture, organ culture, and pyruvate kinase did not change. steroid and tissue must be incubated together for 20 hours or

Ray et al. (60) have shown that corticosteroids stimulate more before significant glycogen accumulates. Several possible glyconeogenesis and gluconeogenesis in the livers of rats whose explanations present themselves. Perhaps during this interval ability to synthesize phosphoenolpyruvate carboxykinase has the steroid is converted to a more active form. If the unaltered been blocked by actinomycin D. Although it has been reported steroid is needed, then enzymes in the tissue which inactivate the that soluble phosphoenolpyruvate carboxykinase is absent from steroid may be lost. Possibly this amount of time is needed for fetal rat liver (6, 61), its presence has been shown by Wicks (18), the induction of glycogen synthetase. Although the fetal liver who found that its activity was not increased by corticosteroids has mechanisms for the synthesis of glycogen and glucose which in 4 hours in organ culture. We have confirmed this observation. are stimulated independently by hormones such as cortisol and Increased activity could only be shown after 22 hours in our experiments under conditions similar to those used by Wicks.

metabolites such as glucose, its capacity to respond to these

No meaningful correlation could be made between the changes in components is limited. Optimal response to its environment

phosphoenolpyruvate carboxykinase levels and the ability of the undoubtedly requires the cooperative action of all of the in-

liver to synthesize glycogen. The changes in phosphoenolpyru- dividual components.

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Carl Monder and Alena CoufalikLiver in Organ Culture

Influence of Cortisol on Glycogen Synthesis and Gluconeogenesis in Fetal Rat

1972, 247:3608-3617.J. Biol. Chem. 

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