Effects of cortisol on the levels of metabolites and enzymes controlling glucose production from pyruvate

EFFECTS

OF

METABOLITES GLUCOSE

CORTISOL AND

ON THE

ENZYMES

PRODUCTION

FROM

LEVELS

OF

CONTROLLING PYRUVATE*

W. SEUBERT, H. V. HENNING, W. SCHONER and M. L'AGE I nstitut fiir vegetative Physiologic,Chemisch-physiologisches lnstitut der Universit~t, Frankfurt/Main, Germany

INTRODUCTION

Fog many years it has been generally assumed, as an important principle in biochemistry, that synthetic and degradative processes occur via reversible reaction sequences. Substantial support for this concept has been obtained by studies on the reversal of fatty acid oxidation(l), glycolysis(2,3), glycogenolysis(4,5) and other metabolic processes. By the use of purified enzyme preparations, a simple reversal of the above processes could be demonstrated in vitro. The physiological significance of these findings, however, seemed doubtful in view of new metabolic pathways elucidated during the last decade(6,7). On the basis of these results, the new concept of complete or at least partially separate and distinct systems for synthetic and degradative processes was developed. This principle is also valid for glucose synthesis and degradation. Both processes diverge at the levels pyruvate/phosphoenolpyruvate (PEP), fructose-6-phosphate/fructose- 1,6-diphosphate, and glucose-6-phosphate/ glucose (Fig. l). The reactions involved in glucose degradation at these levels represent energy barriers for the synthetic process. They are bypassed by new exergonic reactions(8-16, 108): fructose-6-phosphate and glucose are formed by simple hydrolysis of esterphosphate bonds. PEP is synthesized by coupling two nucleoside triphosphate cleavage reactions (Fig. 1). The equilibria of these steps are thus shifted in favor of synthesis. This principle is not only verified for carbohydrate metabolism but also for the synthesis and degradation of fatty acids, triglycerides, complex lipids, terpenes and protein. In many cases, "one way" steps have been characterized as rate limiting steps of a complex metabolic process. Acceleration or retardation *Supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany. 153

154

W. SEUBERT, H. V. H E N N I N G , W. S C H O N E R A N D M. L ' A G E

~

Lactate

DPN+ DPNH

AcetyI-CoA/Oiolocetot e ADP" ~

PhosphOenole_

ADP/

pyruvate

2- phosphog'lyceric acid

II

.3- phosphoglyceric

acid

I ~ A TP ~'~-A DP I,3-diphosphoglycericacid

~

Phosphate

'-DPNH

~"[~DP

N+

Glyceraldehyde-3-phosphate1, Dihydroxyocetone phosphate I

lr

ADP ~rFructose-1.6-diphosphate ~ k ATP ~" ~ AD~ ATP

Fructose-G- phosphate----""Phosphate •

x

Glucose-G-phosphate Glucose

Phosphate

FIG. 1

Synthesis and degradation of glucose.

of such steps is thus an ideal way to shift the metabolic flux in a given direction. The mechanisms involved are as follows: 1. Induction and repression of the synthesis of the enzymes involved in these steps. 2. Activation and inhibition of these enzymes. 3. Control of enzyme inactivation. All the above mechanisms may be controlled by hormones and metabolites. In the following presentation, early (3-6 hr) effects of glucocorticoids

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 155 on the levels of the enzymes and metabolites which control the glucogenic capacity of kidney and liver are explored.

MATERIALS

AND METHODS

Treatment of the animals, extraction of tissues, assays of enzymes and metabolites have been described, and are referred to by citing pertinent studies published elsewhere (13-17,20). Experimental conditions not yet published are described in conjunction with the presentation of results.

RESULTS

AND DISCUSSION

Relation between Glucogenic Capacity of Kidney and Liver and the .4 ctivities of Pyruvate Carboxylase and PEP Carboxykinase Besides its role as an energy source in brain~ muscle, and erythrocytes, glucose has also an important physiological function as precursor of many essential components of life. Under normal conditions enough glucose is supplied for these requirements from exogeneous sources. Under metabolic situations characterized by insufficient exogeneous glucose, it is synthesized by the kidney and the liver from protein and lactate. The enzyme system responsible for gluconeogenesis in these organs can adapt itself to the individual needs for glucose in the body. Its glucogenic capacity is controlled by humoral and other factors. This is evident from the increased incorporation of radioactive COs, alanine or pyruvate into glucose and glycogen which has been demonstrated after treatment with cortisoi or after starvation(18,20-22). An increase in the rate of conversion of pyruvate or oxaloacetate, respectively, into PEP by an induction of pyruvate carboxylase and PEP carboxykinase, dependent on a hormone or starvation, was considered as one of the causes of this increased capacity of the kidney and liver to synthesize glucose. Experimental support for this assumption was presented by Lardy's group(23,24) and by Henning et al. (13, 16) (Table 1): 6 hr after a single dose of cortisol and after prolonged starvation the activities of the soluble PEP carboxykinase and pyruvate carboxylase are approximately doubled. The activities of the mitochondrial pyruvate carboxylase respond only after prolonged starvation(16,25) or after hormone treatment over a period of several days (26). Both enzymes from kidney behave in a similar fashion (16). As one of the primary effects of cortisol, the increase in pyruvate carboxylase and PEP carboxykinase in kidney and liver should also contribute to the increased glucogenic capacity of both organs. In order to exclude the control of gluconeogenesis by fatty acids (see p. 166),

Normal Normal, starved (24 hr)

Normal Normal, cortisol (5 days)

Wagle(26)

Adrenalectomized Adrenalectomized + cortjsol (6 hr) Normal, starved (72 hr)

Adrenalectomized Adrenalectomized + cortisol (6 hr) Adrenalectomized starved (12 hr) Normal, starved (24 hr)

Pretreatment of animals

Freedman and Kohn (25)

Henning et al. (13) Henning e t al. (16)

Lardy e t al. (23) Foster et al. (24)

Authors

m

1.28

3.08

m

B

2.44

1.41

6.9 3.1

6.3

1.9 2.0

1.3 2.3

m

Pyruvate carboxylase Soluble Insoluble /~moles/min/g liver /~moles/min/g liver

5.7

2.6

/.~moles/min/g liver

96.2

97.4

78

48

PEP carboxykinase mp.moles/min/mg protein

TABLE 1 Effects of Cortisol and Starvation on PEP Carboxykinase and P' ,ruvate Carboxylase in Rat Liver

> Z

¢3 ¢) Z

Z

z

r~ z

.<

m

rn C rn

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 157 mobilized under the influence of the hormone in the periphery, the glucogenic capacity was assayed in kidney cortex slices after various treatments of the animals. Succinate and pyruvate were selected as glucogenic substrates, since both compounds are converted to glucose via the steps catalyzed by PEP carboxykinase and pyruvate carboxylase. In addition they easily permeate the cell membrane of the kidney cortex. The result of these investigations is summarized in Fig. 2: with an excess of substrate, Pyruvate (20mM) 80--

80--

Succinate (20mM)

!

70--

6O

60--

.c ~0-o~= 4 o - - !

40--

u~ 3 0 - a~

30--

:k 2o--

2oI_ 10

IO--

0 Adrenalect

i

Adrenalect Starved for +cortisol 7'2 hr 6hr

Adrena~ect Adrenalect +COrIiSOI 6hr

Starved foe 72 hr

F1G. 2 Effect of cortisol and starvation on the glucogenic capacity of kidney cortex (Henning et al. (I 6)).

gluconeogenesis in kidney cortex slices is significantly increased 6 hr after treatment of the animals with a single dose of cortisol and after starvation. The increase of gluconeogenesis observed is of the same order of magnitude as the increment in activities of PEP carboxykinase and pyruvate carboxylase (170-210% of controls). 'The other two key enzymes of gluconeogenesis, fructose-l,6-diphosphatase and glucose-6-phosphatase, do not respond within 6 hr to the doses applied in kidney(27) and liver(22,28,29). Conversion of pyruvate and oxaloacetate, respectively, to phosphoenolpyruvate was therefore considered to be the rate-limiting step for gluconeogenesis under the experimental conditions described (excess of substrate)(16). Similar conclusions have been drawn by Exton and Park (30, 31) from perfusio'n studies in the liver, by Krebs et al. (32) from an investigation of the effects of acetate and short chain fatty acids on gluconeogenesis, and by Seubert and Huth (15) from experiments with inhibitors of pyruvate carboxylase.

158

W. SEUBERT, H. V. HENNING, W, SCHONER AND M. L'AGE

The lntracellular Location of Pyruvate Carboxylase The activity of the soluble pyruvate carboxylase may be determined optimally only by using salt-containing, isotonic solutions(16). This corresponds to about 30-50% of the total enzymatic activity of liver. Glutamic dehydrogenase, an enzyme exclusively located in mitochondria, is only solubilized by this treatment to about 3-6% of the total activity present in the liver. This different behavior of pyruvate carboxylase and glutamic dehydrogenase extracted from liver could be the result of: 1. Different solubilities of both enzymes of mitochondrial origin. 2. A possible location of pyruvate carboxylase in an extramitochondrial compartment as well as in the mitochondria. Different solubilities of mitochondrial pyruvate carboxylase and glutamic dehydrogenase could be excluded. As is evident from Table 2, extraction TABLE 2 Extraction of P, 'ruvate Carboxylase and Glutamic Dehydrogenase from Mitochondria* Pyruvate carboxylase Homogenization min

Glutamic dehydrogenase

Units/g liver

% of total

U nits/g liver

% of total

0.97 1.42 1.49 2.22

25.3 36.8 50.5 57.8

0.55

15.7 30.0 51.4 60.00

1.05 1.80

2.10

Ratio of % pyruvate carboxylase/ glutamic dehydrogenase solubilized 1.6 1.2 0.98 0.96

* Sediment between 1,000 and 12,000 x g. Extraction medium ( 1/ 10) according to H enning et al. (16). Total activity of the mitochondrial fraction was determined by extraction with

the same medium containing 0.1% Na-desoxycholate. Pyruvate carboxylase: 3.85 units/g liver; glutamic dehydrogenase (forward reaction): 3.5 units/g, liver. The amounts of deoxycholate introduced with the enzyme extracts in the assay mixtures had no inhibitory effect. One unit is defined as the amount of enzyme catalysing the conversion of I/zmole substrate/ rain at 30°C (Ohly and Seubert(33)).

of purified mitochondria with buffered isotonic solutions results in solubilization of both enzyme activities to the same degree. An extramitochondrial location of pyruvate carboxylase is evident from the different behavior of the latter enzyme and glutamic dehydrogenase on extraction of liver with isotonic solutions supplemented by various additions (Table 3): solubilization of pyruvate carboxylase is favored by monovalent cations such as Na ÷ or K ÷. Acetyl-CoA can substitute for these cations. In contrast to pyruvate carboxylase the activities of glutamic dehydrogenase in the soluble portion are not altered by this treatment. Additional experiments are necessary to locate these activities of

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 159 TABLE 3 Effects of Various Agents on the Solubility of Pyruvate Carboxylase and Glutamic Dehydrogenase from Rat Liver in lsotonic Solutions Pyruvate Carboxylase

Glutamic Dehydrogenase

Medium U nits/g liver

% of total

0.50

10.5

1.5

5.9

Sucrose (0.24 M), Glutathione (1 mM) Versene I1 mM) TriethanolamineHC1, pH 7.2 (20 mM)*

1.68

35.4

1.6

6.';

Sucrose (0.28 M), Glutathione (1 raM) Versene (1 mM), Na-acetate 50 mM)

1.76

37.2

0.3

1.2

Sucrose (0.28 M), Glutathione (I mM) Versene (1 mM), Acetyl-CoA (0.5 mM)

1.55

33.0

0.25

1.0

Sucrose (0.3 M), Glutathione ( 1 mM) Versene [1 mM)

Units/g liver

% of total

*Adjusted by the addition of KOH. Extractions (1:10) were carried out according to Herming e t al.(16). Total enzyme activities of liver were extracted with the triethanolamine-medium containing 0.1% Nadeoxycholate. Pyruvate c arboxylase: 4.75 units/g liver; glutamic dehydrogenase (back reaction): 25.4 units/g liver. Additional details, see Table 2. (Ohly and Seubert(33)).

pyruvate carboxylase. It seems likely that part of pyruvate carboxylase is bound to the external surface of mitochondria or to other subcellular particulate fractions as has been reported for other enzymes(34-37). However, it can already be concluded from the present results that carboxylation of pyruvate is not exclusively restricted to the mitochondrial compartment of rat liver as postulated by others (25,38-40).

Control of Gluconeogenesis by Glucocorticoids Independent of de novo Synthesis of Enzymes in Liver Acceleration of gluconeogenesis by glucocorticoids was originally explained exclusively in terms of induction of the enzymes in liver. As was demonstrated by Ray et al.(41) and others(42,43), actinomycin D, an inhibitor of R N A synthesis, completely suppresses the hormoneinduced elevation of enzymatic activities. Carbohydrate synthesis, however, is only partially suppressed(41,44). These findings, that gluconeogenesis is accelerated by glucocorticoids independent of de novo synthesis of enzymes in liver, suggested an additional regulatory role of these hormones in carbohydrate metabolism at the level of the meta-

160

w.

S E U B E R T , H. V. H E N N I N G ,

W . S C H O N E R A N D M. L ' A G E

bolites (feedback control). Two mechanisms had to be considered: activation, and inhibition of.enzymes involved in the unidirectional steps of gluconeogenesis and of glycolysis, respectively. Metabolite control o f glucogenic enzymes. Metabolite activation and inhibition of the key enzymes of gluconeogenesis have been reported for pyruvate carboxylase, fructose-l,6-diphosphatase and glucose-6-phosphatase(45). Physiological significance of these control mechanisms has only been demonstrated substantially for pyruvate carboxylase (45). According to Utter and Keech(9, 10), this enzyme shows an absolute requirement for catalytic amounts of acetyl-CoA, in contrast to the bacterial enzyme. The enzyme from rat liver is activated by concentrations in the range of 3 × 10-5 to 1.5 × 10-4M acetyl-CoA (Fig. 3). Changes in the levels of this metabolite in the above-mentioned range could thus control the rate of oxaloacetate synthesis and, in turn, gluconeogenesis from pyruvate. Experimental support for a regulatory role of acetyl-CoA was presented by Krebs et al. (32) who demonstrated that acceleration of gluconeogenesis by acetate and short-chain fatty acids t~Jmoles pyr uvote inc orporotea

$0.

A &

&e

4

20.

10.

J

1

Z

. '--4 8a10 M J,cety|-CoA

FIG. 3 Relation b e t w e e n incorporation of 14C - pyruvate into citrate, and the concentrations of a c e t y l - C o A in the carboxylase a s s a y according to H e n n i n g and Seubert(14). U p p e r curve: pyruvate carboxylase f r o m Pseudomonas citronellolis. L o w e r curve: rat liver pyruvate carboxylase.

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 161

in vitro is associated with a rise in intracellular acetyl-CoA. A participation of acetyl-CoA in the control of gluconeogenesis by glucocorticoids is supported by the assays of acetyl-CoA in liver which are summarized in Fig. 4. As is evident from this Figure, there is a significant rise of intracellular acetyl-CoA within 3 hr after a single dose of cortisol. This rise reaches 150% of the level in control animals within 6 hr. The fixation of CO2 into glycogen corresponds to the increased glycogen deposition in liver. Acety I- CoA I--1Control mlmCortisol treated onimols ]=

80

40

91c4

,,2,

20 3

£

Hours offer treatment p~O,05 p.~O,OI G I ycogen 8--

~

171

(~z) j l (~6)

(6) a M 3 6 Hour,= offer treatment

]¢

p ,,:0,001

p
CO z - fixot ion 2000 - - i n t o

;9)

gly~en

.=- I 0 0 0

._=

(9)0 8

6 Hours ofter treatment p
Fio. 4 Effect of cortisol on hepatic acetyi-CoA, glycogen and fixation of 14CO2 into glycogen in vivo (Schoner et ai. (18)). Experimental conditions: adrenalectomized rats, 18-hr starved. Cortisol (5 rag/100 g body weight) was applied by use of a stomach tube. Controls received 0.5 ml 0.9% NaCI solution. Liver samples were excised 3 and 6hr after treatment in phenobarbital anesthesia (150mg/100g body weight L for 20 min) within 7-15 sec after laparotomy by use of "freezingstop'-technique. For assays of 14CO2-fixation into glycogen, 6.5/zC KH14CO3 (spec. activity 0.53/~C//zmole) were injected intraperitoneally 30 rain prior to excision of liver samples. Values are means-+ S.E.M.

162

w . SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

The postulated role of acetyl-CoA in controlling gluconeogenesis after cortisol administration is further strengthened by a comparison of the tissue levels of acetyl-CoA summarized in Fig. 4 (39-68 m/zmoles/g wet wt.) with the concentration range of this metabolite which activates pyruvate carboxylase (30-150p~M) (Fig. 3). Although a calculation of the intracellular concentrations of a metabolite is not possible from determinations of the tissue contents, it seems very likely on the basis of the above data that the intracellular concentrations of acetyl-CoA are in the range which is responsible for the acetyl-CoA activation of pyruvate carboxylase. Changes in acetyl-CoA level in the reported range may thus alter the glucogenic capacity of liver. Metabolite control of glucose degradation. As well as by an activation and induction of glucogenic enzymes, the glucogenic capacity of liver may also be increased by repression and inhibition of key enzymes of glucose degradation. In starvation and diabetes both mechanisms seem to be involved(46). The effect of cortisol on glucose degradation is only manifested in terms of inhibition of the enzymes involved in this process. Possible effectors and their points of attack are summarized in Table 4. TABLE 4

Controlof Key Enzymesof Glucose Degradation Enzyme

Inhibitor

Glucokinase

Fatty acids(47)

P-fructokinase

ATP (45) Citrate(45) Fatty acids(47)

Activator

ADP, 5'-AMP 3',5'-AMP, inorganicP Fructose-6-P, Fructose-1,6diphosphate(45,48)

CoA, Acetyl-CoA(54) ATP(15) Fatty acids(47) Alanine(18) Proline(18) 3-Phosphoglycerate(18)

Pyruvate kinase

Fructose- 1,6-diphosphate (49)

i

Pyruvate dehydrogenase ' Acetyl-CoA(50) N A D H (51)

Experimental support for a participation of these effectors in the control of glucose synthesis in liver under the influence of cortisol has so far only been obtained for citrate and acetyl-CoA. As illustrated in Figs. 4 and 5, the levels of these metabolites are elevated in liver within 3 hr after a single dose of cortisol. Elevated levels of citrate in liver and kidney have been observed in many metabolic situations characterized by increased gluconeogenesis

E F F E C T S OF CORTISOL ON LEVELS OF M E T A B O L I T E 8 A N D E N Z Y M E S

163

(fluoroacetate poisoning(52,53), diabetes(52,54), starvation(54,55,57), fatty acid administration in vivo and in vitro(54,56,57), glucagon treatment(55,58)). In all cases investigated this rise of citrate was accompanied by a rise of glucose-6-phosphate. It was interpreted as a limitation of glycolysis at the fructose-6-phosphate/fructose-l,6-diphosphate step due to citrate inhibition of phosphofructokinase. As illustrated in Fig. 5,

r-"-'l 300F~

Control

Citrate

Cortisol treated animals 05)

.~ 200

(14)

(13)

-~ ~,ooF l[i] ¢'3)~i 1 O--

~p
(]2)1

6 Hours after treatment

pO,05 Actinomyci +400p~g

Glucose - 6 - p h o s p h a t e 160 --

N

120 --

~

8o--

E

zL E

(15) (14)~(15) 03)tt (13) (12)~

40--

3 6 6 p~O,OiActinpomyci <0,001+400 p
Fio. 5 Effect. of cortisol and actinomycin on hepatic citrate and giucose-6-phosphate in viva (Schoner et al.(l8)). Experimental conditions as described in Fig. 4. For studies of the effects of actinomycin D the antibioticwas injectedintraperitoneally 30 rainprior to treatment with cortisoland NaCl-solution, respectively.

glucose@phosphate and citrate are simultaneously elevated in liver under the influence of cortisol with a delay of 2-3 hr. The relationship between citrate rise and inhibition of glycolysis as discussed above is further supported by these data. A role of citratein controllingglycolysis in liver, however, has to be questioned on the basis of additional data illustrated in Fig. 5: namely, treatment of rats with 400/~g actinomycin results in an elevation of citrate levels in both controls and cortisoltreated animals. There is no correlation between this increase and the levei~ of glucose-6-phosphate. A regulation of glycolysis by citrate as dl~cu~sed by various authors therefore seems unlikely-at least in liver.

164

w.

SEUBERT, H. V. H E N N I N G , W. S C H O N E R A N D M. L ' A G E

Similar conclusions were drawn by Tarnowski and Seemann(59) and Wieland(60) from a study on the changes of effectors of glycolytic and glucogenic enzymes in rat liver under conditions characterized by increased gluconeogenesis: no correlation between the rate of gluconeogenesis and the levels of citrate could be demonstrated. The activities of PEP carboxykinase (equation (2)) and of pyruvate kinase (equation (3)) in liver are 8.0 units(61) and 55 units(62) per g wet wt., respectively. Both enzymes are located in the cytoplasmic compartment. From these data, it is evident that the activities of liver pyruvate kinase would be sufficient to cleave to pyruvate again all PEP formed by pyruvate carboxylase and PEP carboxykinase. The combined action of pyruvate carboxylase (equation (1)), PEP carboxykinase (equation (2)) and pyruvate kinase (equation(3)) would thus result in the hydrolysis of G T P (equation (4)), an energy-wasting process. Pyruvate + CO2 + A T P + H 2 0 Oxaloacetate + G T P PEP + A D P

Mg++ Acetyl-CoA

)

Oxaloacetate+ADP+P

Mr+'Mn++ ) PEP + CO2 + G D P Mg++,K+

) Pyruvate + A T P

(1) (2) (3)

Mg++,K+,Mn++

Sum:

G T P + H20

AcetyJ-Co^ ) G D P + P

(4)

As already mentioned, in contrast to diabetes and starvation, the levels of pyruvate kinase are not suppressed by cortisol treatment. Metabolite inhibition of pyruvate kinase was therefore considered to favor the conversion of PEP to glucose in this hormonal state. This assumption could be supported by assays of a 100-fold purified pyruvate kinase preparation from liver in the presence of various metabolites which are or could be under the control of cortisol(18). The following metabolites were shown to be without effect: malate, citrate, fructose-6-phosphate, succinate, AMP, cyclic AMP, crotonyl-CoA, propionyl-CoA, malonyl-i2oA, a,fldehydropalmityl-CoA, fumarate, acetoacetate, lactate and pyruvate. Among 17 amino acids investigated (serine, tryptophan, phenylalanine, leucine, tyrosine, glutamate, methionine, valine, glycine, alanine, proline, lysine, threonine, arginine, histidine, isoleucine, aspartate) only proline and alanine inhibited pyruvate kinase to a greater extent. Inhibition of pyruvate kinase could also be demonstrated in the presence of 3-phosphoglycerate and ATP (Table 4). Inhibition of the enzyme from liver by acetyl-CoA reported earlier(20) could be traced to contaminations of the preparations with acetic anhydride. In agreement with Taylor and Bailey(49) and Hess et al. (63,64), glucose-6-phosphate and fructose-1,6diphosphate activated the enzyme from liver. A comparison of the tissue contents of the above inhibitors in liver

165

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES

(30, 31,44, 76,106,107) with the concentrations necessary for inhibition of pyruvate kinase supports a physiological role for the effect of alanine only. At physiological concentrations of PEP, alanine inhibits pyruvate kinase in a concentration range such as has been found by various authors in liver(76,106,107) (Fig. 6). The type of inhibition is substrate competitive(18). The inhibitory action of alanine is restricted to part of the total activity from liver, kidney and heart(42,39, and 20% respectively at 6.6 mM alanine). As demonstrated by Betheil et al. (76) treatment of rats with cortisol results in an elevation of hepatic alanine to 150% of the 0.14

0.08

._c E

o•

0.12

o

.£

0.10

o.o6 e~

I 0.04

11.06

a~ 0.02

0.02 i

i

I

i

i

2

i

i

3 ~IO'3M Alonine

!

4

I

i

8

!

i

11~

i

i

16 alOd'M PEP

FIG. 6 Inhibition of pyruvate kinase from rat liver by alanine (Schoner et al.(I 8)). Effect of alanine concentration on enzymatic activity (left side). The reaction mixture contained in 1.5 ml: 67 mM tris-HCl pH 7.2, 0.66 mM MgSO4, 18.2 mM KCi, 0.172raM PEP, 1.16ram ADP, 0.33ram D P N H , 3.6u lactate dehydrogenase from rabbit muscle, alanine as indicated, 30/zg pyruvate kinase (specific activity 8.1 u/mg). Temperature 30°C. Effect of PEP concentration on the enzymatic activity (right side). Composition of the reaction mixture (1.5 ml): 67 mM tris-HCI pH 7.2, 0.66 mM MgSO4, 12.4mM KCI, 1.16mM ADP, 0.33 mM D P N H , phosphoenolpyruvate as indicated, 3.6 units lactate dehydrogenase from rabbit muscle, 17/~g pyruvate kinase (specific activity 9.7 units/rag). Temperature 30"C. Upper curve: without alanine; lower curve: in presence of 0.66 mM alanine. Purification of pyruvate kinase: 14 g rat liver were homogenized with 140 mi 0.1 M potassium phosphate buffer, pH 7.4. The supernatant (40,000 x g for 30 mitt) was treated with 13.8 ml 1% (w/v) protamine sulfate solution and centrifuged at 40,000 × g for 20 rain. Solid (NH4hSO4 was added to the supernatant. The precipitate between 25 and 45% saturation was taken up in 5.1 ml 0.01 M potassium phosphate buffer, pH 7.4. Inactive protein was separated by addition of 20 ml CaPO4-gel (24mg/ml). The supernatant (40,000× g for 15 rain) was chromatographed after dialysis for 3 hr (0.01 M potassium phosphate buffer pH 7.7) on DEAE-Sephadex A50 ( 10 x 2 era), equilibrated with 0.01 M potassium phosphate buffer pH 7.7. Inactive protein was eluted with 400 ml 0.05 M potassium phosphate buffer, pH 7.7, pyruvate kinase with 150ml 0.1 M potassium phosphate buffer, pH 7.2

166

w . SEUBERT, H. V. H E N N I N G , W. SCHONER AND M. L'AGE

controls. The rise of intracellular PEP observed by Tarnowski et a/.(44) in this hormonal state may thus not only be due to an increased synthesis but also to a delayed degradation of this compound. In addition, a negative control of pyruvate dehydrogenase may contribute to the increased synthesis of PEP: Walter et al. (65) observed, on incubation of mitochondria with pyruvate and radioactive CO2 in presence of caprylic acid, an increased fixation of CO2 into malate, citrate and fumarate. Pyruvate degradation was suppressed (Table 5). Lardy TABLE 5

Effect of Caprylic Acid on the Metabolism of 14CO2 and Pyruvate in Mitochondria (Walter et a1.(65)) /zmoles/min/gliver Withoutcaprylate Pyruvateconsumed 14COzincorporatedinto malate, citrate and fumarate Ratio pyruvateconsumed/14CO2fixed

caprylate(2 mM)

3.62 1.40

2.13 1.73

2.59

1.23

deduced from this result a pyruvate "sparing" effect of fatty acids which favors the carboxylation of pyruvate and in turn the synthesis of C-4dicarboxylic acids. Most likely this effect of fatty acids on oxidation of pyruvic acid is the result of an inhibition of pyruvate dehydrogenase by elevated levels of acetyl-CoA, as first reported by Garland and Randle for the enzyme from muscle (50). Relation between Fatty Acid Mobilization, Acceleration of Gluconeogenesis and Elevation of Metabolites in Liver after Cortisol Treatment A rise of acetyl-CoA in liver has been observed in many metabolic states characterized by increased levels of free fatty acids (55,58, 66, 67). Most likely, the rise of acetyl-CoA and the other metabolites (Figs. 4 and 5) in liver after cortisol treatment is also a result of fatty acid mobilization in the periphery by this hormone (for a summary see (68)). The negative and positive control, respectively, of glucose degradation and gluconeogenesis by effectors of glucogenic and glycolytic enzymes may therefore be closely related to the lipolytic .action of cortisol in adipose tissue. According to studies of Fain(69), mobilization of fatty acids from isolated fat cells by growth hormone and glucocorticoids is completely suppressed by actinomycin. This finding favors an enzyme induction in adipose tissue as the primary cause of fatty acid mobilization under the influence of cortisol. Were fatty acid mobilization, acceleration of gluco-

EFFECTS

OF CORTISOL

ON LEVELS

OF METABOLITES

AND

167

ENZYMES

neogenesis and elevation of the various metabolites after cortisol treatment related to each other, these actinomycin should also suppress all these effects of the hormone. Experimental support for this assumption is presented in Fig. 7: treatment of animals with 400/zg of actinomycin Acetyl CoA i.--1 Control Cortisot treated C02 fixation after car tisoI treatment 8c --

I00 - - ~ ~

60 _

~] (12)

Citrate

• 4oaf "~ 3oo1__

/

ill)

~

-~ o 4o E ~ 2c

100

(~21.,t,('5> (11)

I3)

0 200 400 Actinomycin /xg pO,I

0 200 400 Actinomycin /.~.g pO,05

Glucose-6-phosphete 120 _

.?

fixation into (9) glycogen

16C

iCI3)

6

(12)

2000

(n)

.c I000 .~-

_~ 8o F ::k 4c E

::L

e o

200 400 Actinomycin p.g

p< 0,001 p
o

/(6),,d]¥,i '9){~ii](7(~(8) 0

200

400

g 3

Actinomycin H-g

p': O,O5

p
FIG. 7

Effect of actinomycin in viva on the cortisol-dependent rises of hepatic acetylCoA, citrate, glucose-6-phosphate, glycogen and the fixation of CO2 into glycogen 6 hr after treatment (Schoner et a/.(18)). Experimental conditions, see Figs. 4 and 5.

together with cortisol suppresses the elevation of acetyl-CoA and citrate. Simultaneously glucose-6-phosphate, glycogen and the CO2-fixation into glycogen respond only slightly to the hormone when compared with control animals which had not received the antibiotic. In agreement with the results of Ray et al.(41), a dose of 200/zg of actinomycin, which completely suppresses enzyme induction in the liver, only lowers glycogen deposition by 30%. Simultaneously, however, also the hormonemediated increase of acetyl-CoA, citrate and glucose-6-phosphate is suppressed partially only. Apparently a dose of 200/~g actinomycin is not sufficient to block mobilization of fatty acids under cortisol. Most likely this is due to the different blood supply of liver and adipose tissue. The data illustrated in Fig. 7 lend additional support to the statement of Fain, according to which fatty acid mobilization from adipose tissue under cortisol depends on de n o v a enzyme synthesis. The statement of Ray

168

w. SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

a/.(41) that "enzyme synthesis d e n o v o is not necessary for the (optimal glucogenic) effect of hydrocortisone" must therefore be restricted to the enzymes of liver. Increased supply of reducing equivalents, regenerated via the fl-oxidation of fatty acids mobilized by the hormone, also favors the synthesis of glucose at the level of triose phosphate dehydrogenase by shifting the intracellular redox potential of the DPN+-system to more negative values(40,70). Additional hydrogen for gluconeogenesis from amino acids is produced via the regeneration of aspartic acid from fumarate coupled with urea synthesis(65). Hydrogen liberated within the mitochondrion is transported with malate into the extramitochondrial compartment. The mechanisms postulated by various authors for the intracellular regeneration and transport of hydrogen are summarized in Fig. 8 (38-40, et

Acetyt - CoA

~ ~ ~ ~.

/ /./

GluCose

t" "" \

\

/

Loctote

/ pyruvate

¢..---.,s..,._

L~

Oxoloacetate

CO;t,NH 30xaloacetate

F~m,=o

DPN*e-~

/ ~ °P"'~j

~

Malate

PEP

Malnte

f

\

/ N

/ /

~Gtty acids

/

PyruvQte

J

I

"-.

Glucose

J I~lfQ~l~fldfiQl J

mitochondrio! J FIG. 8

Extra-andintramitochondrialregenerationof hydrogenfor gluconeogenesis. 65,71 ). This scheme differs from those published in one respect, namely, an additional possible carboxylation of pyruvate in the extramitochondrial compartment. This assumption seems justified on the basis of our studies on the intracellular location of pyruvate carboxylase (Table 3). Furthermore, earlier isotope experiments cannot be reconciled with an exclusively mitochondrial carboxylation of pyruvate: aspartic acid and malic acid which have been discussed as transportable forms of oxaloacetic

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES A N D ENZYMES 1 6 9

acid passing the mitochondrial membrane equilibrate completely with the fumaric acid, before conversion to glucose due to the symmetry of the former. The radioactivity of a 3-14C-aspartic acid or malic acid is therefore found to be distributed equally between the ! ,2- and 5,6-position of glucose (72,73). Equilibration of the corresponding C-atom of pyruvate (74) or lactic acid(15,75), however, is incomplete. From this different behavior it is also evident that the formation of oxaloacetate from lactate in the cytoplasmic compartment must not necessarily be coupled with an intramitochondrial conversion of pyruvate to aspartate, malate or fumarate and their transport into the cytoplasmic compartment. An extramitochondrial carboxylation of pyruvate seems also meaningful in view of the DPN-regenerating systems located in the cytoplasm which could supply the hydrogen necessary for glucose synthesis from C3-units: thus the hydrogen necessary for gluconeogenesis from lactate is regenerated completely via lactate dehydrogenase, and from the amino acids alanine, serine, cysteine and glycine, largely via urea synthesis (Fig. 8). The Physiological Significance o f Hepatic Amino Acids Mobilized by G lucocorticoids The various effects of cortisol on hepatic enzyme and metabolite levels so far discussed only alter the glucogenic capacity of liver and kidney. Increased glucose synthesis in vivo, however, necessitates not only an increased glucogenic capacity of liver and kidney, but also a sufficient supply of substrates. This need for substrates is covered by the glucogenic amino acids which are mobilized in the periphery within several hours under the influence of glucocorticoids. This is evident from assays of individual amino acids in liver, thymus, muscle and plasma carried out cortisol. Among the amino acids assayed all representatives were found to be elevated in muscle and thymus (Table 6). The chief amino acids by Betheil et al.(76) 4 hr after treatment of rats with a single dose of whose level rose in liver and plasma were glutamic acid, aspartic acid, and alanine. According to Weber et al. (77), significant increases in amino acids in liver were found as early as 2 hr after steroid injection. The high activities of glutamate-pyruvate and glutamate-oxaloacetate transaminase in liver allow for a rapid formation of alanine, aspartate and glutamate. Several authors(78-80) relate therefore amino acid mobilization in the periphery and the rise of the above amino acids in liver to the induction of tyrosine-~t-ketoglutarate transaminase (equation 5) and tryptophan pyrrolase (equation 6). Tyrosine + t~-ketoglutarate ~ hydroxylphenylpyruvate + glutamate Tryptophan + 02 --->formylkinurenine

(5) (6)

371

~--

Gly, His, Ileu, Leu, Lys, Phe, Pro, Ser, Thre, Tyv, Val

Control

Glu, Asp, Ala

Amino acids

--

483

Cortisone (4 hr)

Liver

--

38

Control

--

46

Cortisone (4 hr)

1017

265

Control

1125

328

Cortisone. (4 hr)

~moles/100 g tissue or 100 ml plasma Plasma Muscle

TABLE 6 Effect of Cortisone on the Levels of Amino Acids in Various Tissues of Rat (Betheil e t a/.(76))

434

721

Control

474

824

Cortisone (4 hr)

Thymus

Z

© Z

Z z Z

<

rn -]

E F F E C T S OF CORTISOL O N LEVELS OF M E T A B O L I T E S A N D E N Z Y M E S

171

These enzymes involved in amino acid degradation are elevated 3-10fold within a few hours after cortisol treatment. Similar results were obtained by Schole(81) by a study of the activities of L-amino acid oxidase under the influence of cortisol. Some of the authors mentioned above(78-80) assume that by reason of an increased degradation of tyrosine, tryptophan and phenylalanine after cortisol treatment, the relative concentrations of free amino acids are changed in favor of glutamic acid. This imbalance of free amino acids should retard protein synthesis in the periphery and in turn favor liberation of amino acids. The following findings are in accord with this assumption: 1. The oxidation of 14C-tryptophan is accelerated 7-fold(82) after induction of tryptophan pyrrolase in vivo. Oxidation of tryptophan is thus limited by the activities of tryptophan pyrrolase in liver. Since the intracellular contents of tryptophan are rather low, a fall in tryptophan levels may limit protein synthesis. 2. The rise of the amino acid-N in liver after cortisol treatment may be suppressed by actinomycin(83). This finding also supports an enzyme induction as the cause of amino acid mobilization. 3. The weight losses of thymus and lymph nodes as well as lymphocytolysis also contribute to the elevated amino acid levels in the periphery(78). This effect of cortisol is almost completely abolished in eviscerated animals. Hofert and White(84) conclude from these results that a factor formed in liver may be the cause of the increased lymphocytolysis under the influence of cortisol. This assumption was supported by Feigelson and Feigelson(78): many of the effects of cortisol could be imitated by administration of glutamic acid to the rat. An hepatic control of amino acid mobilization in the periphery is questioned by Smith and Long(105) on the basis of an unchanged rate of increase of plasma amino nitrogen following cortisol administration to the eviscerated rat. In contrast to the authors mentioned above, they concluded that the effects of glucocorticoids on plasma amino acid levels cannot be due solely to an effect on the liver itself but are to a large extent a consequence of their ability to increase the rate of release of amino acids from the extrahepatic tissues.

Control of Enzyme Synthesis by G lucocorticoids in vitro The increase of enzyme activities observed after treatment of animals with glucocorticoids in vivo, and the suppression of this increase by inhibitors of protein synthesis as well as immunological studies (for a summary see(79, 41-43)) are in accord with a control of enzyme synthesis by these hormones. No conclusion about aprimary effect of the hormones can be drawn, however, from this observation. Also altered metabolic states induced by the hormones could be responsible for the effects observed.

172

W. SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

Several teams have therefore attempted to demonstrate effects of the hormone in the isolated perfused liver or in liver slices. These attempts were successful: increased incorporation of radioactive CO2(85,93), alanine (85-93) and pyruvate (85,92) into glucose could be demonstrated in the presence of glucocorticoids in the perfused liver and in liver slices. Increased enzyme activities after hormone treatment in vitro, however, were only evident in the perfused liver(94,95) and not in liver slices(96). The latter finding was confirmed in our laboratory. No increase of pyruvate carboxylase on incubation of liver slices with cortisol or triamcinolone could be demonstrated. In contrast, a rapid decrease in pyruvate carboxylase activities was found on prolonged incubation of liver slices due to a damage of the tissue(97). As recommended by Krebs et a/.(98), kidney cortex slices proved to be a much better system for studies in vitro on the action of glucocorticoids(99). This tissue is more resistant than liver to an incubation of several hours at high shaking rate (150/min). In Table 7 the results of experiments with kidney cortex slices TABLE 7 Effect of Various Steroids and Puromycin in vitro on the Fixation of Radioactive CO2 by Kidney Cortex Slices 14CO2-Incorporated Experimental group

Control 2 × 10-5 M cortisol 2 × 10-5 M dexamethasone 2 × 10-5 M cortisol, Puromycin (30 p.g/ml) 2 × 10-5 M dexamethasone, Puromycin (30/~g/ml) 2 x 10-5 M deoxycorticosterone 2 × 10-5 M tetrahydrocortisol

c.p.m. per g of tissue 18 13 9 14

6340± 90 7030±130 7490± 190 6330_+150

<0.001 <0.005 >0.100

6510__+120

>0.100

6660± 170 6280 ± 460

>0.100 >0.100

Composition of the reaction mixtures: 8 ml phosphate buffer according to Krebs (104), 200 mg kidney cortex slices from adrenalectomized, starved rats (12-15 hr), 0.6 mg peptone/ ml, additional components as indicated. The slices were preincubated for 2 hr at 37°C under shaking (150/min), and subsequent 2 hr after addition of pyruvate (20 mM), glucose (5 mM), and NaHI4COa (10 mM, 0.1/~C//zmole). Gas phase: 100% 02. After incubation, the slices were separated by centrifugation and the remaining radioactivity after gassing with CO2(20 min) and N2(10 min) determined. Values are means ±_S.E.M. (L'age et al.(19)).

are summarized(19): after 2 hr preincubation of slices in the presence of cortisol and a subsequent incubation for 2 hr with pyruvate (20 mM) and radioactive bicarbonate (10 rnM), the fixation of radioactive CO2 is significantly elevated. The effect of the hormone is evident at concentrations found in liver(29). Cortisol may be substituted by dexamethasone. 11-

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 173 D e o x y c o r t i c o s t e r o n e and tetrahydrocortisol which have been shown to be ineffective in vivo were also unable to stimulate C O : f i x a t i o n in vitro. Increased C O : f i x a t i o n was suppressed in the presence of puromycin. This finding was in agreement with the supposition that an e n z y m e induction in vitro was the cause of the increased CO2-fixation. This assumption could be confirmed by assays of pyruvate carboxylase (Table 8): after 2 hr incubation, the activities of this enzyme were shown to be TABLE 8 Effects of Cortisol and Puromycin in vitro on the Activities of Pyruvate Carboxylase in Kidne,, Cortex Slices of Adrenalectomized Rats

Pyruvate Carboxylase after Incubation for 2 Hr Experimental Group n Control Puromyein (30 tzg/ml) Cortisol (2 x 10-5 M) Cortisol + puromycin

26 7 19 8

units/g wet wt.

P

2.98 ___0.09 3.04-----0.19 >0.1 3.49_+0.005 <0.005 2.92___0.18 >0.1

% of control

100 102 117 (123)* 98

*Additional collective. Composition of the incubation mixtures: 8 ml Krebs-Henseleit-butfer; 400 mg kidney cortex slices from adrenalectomized, starved 12-15 hr rats; 5 x 10-aM pyruvate; 0.6rag peptone/ml; additional components as indicated. The sfices were incubated for 2 hr with shaking (150/rain). Gas phase: 95% 02, 5% CO2. For assays of pyruvate carboxylase, the slices were,centrifuged down at 17,300x g for 10 rain and the "soluble" part extracted as reported earlier(16). One unit is the amount of enzyme catalyzingthe conversion of I/zmole pyruvate to oxaloacetate per rain at 30°C. The values are mean values ---S.E.M. (L'age et al.(19)). elevated in kidney cortex slices in the presence of cortisol. In agreement with the results obtained in studies on CO2-fixation, the effect of the hormone on the e n z y m e activity was abolished by puromycin (Table 8). The effects of cortisol and dexamethasone observed in vitro on the rate of CO2-fixation and pyruvate carboxylase activities are around 1 2 0 - 1 2 5 % of the controls. The extent of the hormone effect in vitro is thus lower than the effect observed in vivo. In spite of this disadvantage, kidney cortex proved to be a valuable tissue for a study of the mechanism of action of cortisol. In order to obtain optimal and reproducible results in vitro, the addition of an ~imino acid mixture proved to be necessary (Table 9). In the absence of pyruvate, in the latter system no effect of the hormone is visible(20). The amino acid mixture apparently cannot substitute for the substrate. In agreement with results of other investig a t o r s ( 6 8 , 9 5 , 1 0 0 - 1 0 3 ) , this finding points to a "permissive" role of cortisol in the control of e n z y m e synthesis. Additional studies are necessary to explore the role of both the amino acids and glucocorticoids in this process.

174

w . SEUBERT, H. V. H E N N I N G , W. SCHONER AND M. L'AGE SUMMARY

Starvation and cortisol treatment of rats result in an almost 2-fold increase in the glucogenic capacity of kidney cortex slices when supplemented with an excess of pyruvate or succinate. A rise of both pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase was considered to be the cause of the increased glucogenic capacity observed. This assumption could be supported by assays of pyruvate carboxylase and PEP carboxykinase under the influence of cortisol and after starvation: the activities of both enzymes were found to be elevated in the liver 6 hr after treatment of rats with a single dose of cortisol and in starved animals. The activities of both enzymes from kidney behaved in a similar fashion. TABLE 9 Role of Amino Acids in Glucocorticoid Action in vitro

14CO2 Incorporated Experimental group

Control 2 x 10-5 M dexamethasone Control, peptone (0.6 mg/l) 2 x 10-5 M dexamethasone, Peptone (0.6 mg/ml)

n

c.p.m, per g of tissue

10 10 5 3

3850 --- 200 3860 ± 290 3800± 190 4500 _ 300

p

>0.1

Composition of reaction mixture: ml phosphate buffer according to Krebs (104), 200 mg slices from adrenalectomized, starved rats, additional components as indicated. Experimental conditions, see Table 7 (L'age et al.(19)).

Studies on the intracellular location of pyruvate carboxylase revealed that 30-50% of the total activity present in liver and kidney may be solubilized by isotonic solutions supplemented with sodium or potassium ions. Acetyl-CoA can substitute for these cations. Glutamic dehydrogenase, an enzyme exclusively located in the mitochondria, is solubilized to 3% only by these treatments. This behavior of pyruvate carboxylase favors an extramitochondrial location of part of this enzyme which is apparently bound to the external surface of the mitochondrion or other subcellular particulate fractions. Assays of the activities of the soluble and the mitochondrial pyruvate carboxylase after administration of cortisol support a regulation of gluconeogenesis by control of the extrarnitochondrial enzyme levels. The stimulation of gluconeogenesis by glucocorticoids independent of de n o v o synthesis of enzymes in liver was studied. Two possible mechanisms were investigated: activation of glucogenic enzymes, and

EFFECTS OF CORTISOL ON LEVELSOF METABOLITESAND ENZYMES 175 inhibition of enzymes involved in glucose degradation by metabolites. Activation of pyruvate carboxylase, a key enzyme of gluconeogenesis, could be supported by assays of acetyl-CoA in livers of rats with a sensitive isotope assay. Within 3-6 hr after treatment of adrenalectomized rats with a single dose of cortisol, the levels of acetyl-CoA, an activator of pyruvate carboxylase, were significantly elevated when compared with untreated animals. A negative control of glycolysis under the influence of cortisol was supported by assays of glucose-6-phosphate in liver. Concomitant with acetyl-CoA, glucose-6-phosphate levels were also elevated. Inhibition of phosphofructokinase by citrate as the cause of the glucose6-phosphate rise could be excluded. The mechanism involved has still to be elucidated. An additional negative control of glucose degradation under the influence of glucocorticoids could be supported by a study of the effects of various metabolites on the activity of a 100-fold purified pyruvate kinase from liver. Among 32 metabolites investigated, alanine proved to be a potent inhibitor of the enzyme from liver. The inhibitory action of alanine was evident in a concentration range such as has been found in liver. Amino acid mobilization under cortisol may thus also control the glucogenic capacity of liver. Glycogenesis, fixation of CO2 into glycogen, and the rise of the various metabolites after cortisol treatment could be blocked by actinomycin D. This finding is in agreement with an enzyme induction in adipose tissue as the cause of the cortisol-dependent mobilization of fatty acids and the concomitant rise of the metabolites investigated. The dose of actinomycin necessary for the suppression of the latter effects of the hormone, however, was found to be twice (400/.~g) the dose which completely blocks de novo synthesis of enzymes in liver (150-200/~g). In order to obtain experimental evidence for a primary action in the control of enzyme synthesis, the effect of glucocorticoids on COs-fixation and on the activities of pyruvate carboxylase was studied in vitro. Since kidney cortex slices, in contrast to liver slices, did not show leakage of pyruvate carboxylase activities on prolonged incubation, this tissue was chosen for studies in vitro. Incubation of kidney cortex slices from adrenalectomized rats with cortisol or dexamethasone (2× 10-SM) resulted in a significant increase in both the activity of pyruvate carboxylase and COs-fixation into glucose and into di- and tricarboxylic acids. Puromycin completely abolished this effect in vitro. Reproducibility of the effects in vitro depended on the presence of an amino acid mixture. This finding is in accord with a "permissive" role of glucocorticoids in the control of enzyme synthesis, as suggested by others.

176

w . SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE ACKNOWLEDGMENTS

The authors gratefully acknowledge the co-operation of the colleagues Dr. Walter Prinz, Walter Huth and Bruno Stumpf and the technical help from Miss Brigitte Ohly, Mrs. Ursula Haag, Miss Helga Brod and Mrs. Ilse Seiffert.

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178

w . SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

44. W. TARNOWSKI, Die Regulation der Gluconeogenese, Habilitations thesis, University Hamburg, Germany (1966). 45. E. R. STADTMAN,Allosteric regulation of enzyme activity, Advances in Enzymology 28, 41-I 54 (1966). 46. G. WEBER, R. L. SINGHAL, N. I . STAMM, M. A. LEA and E. A. FISHER, Synchronous behavior pattern of key glycolytic enzymes: glucokinase, phosphofructokinase, and pyruvate kinase, Advances in Enzyme Regulation 4, 59-85 (1966). 47. G. WEBER, H. J. H. CONVERY, M. A. LEA and N. B. STAMM, Feedback inhibition of key glycolytic enzymes in liver: action of free fatty acids, Science 154, 1357-1360 (1966). 48. A. H. UNDERWOOD and E. A. NEWSHOLME, Properties of phosphofructokinase from liver and their relation to the control of glycolysis and gluconeogenesis, Biochem. J. 95,868-875 (1965). 49. C. B. TAYLOR and E. BAILEY, Activation of liver pyruvate kinase by fructose-l, 6-diphosphate, Biochem. J. 102, 32c-33c (1967). 50. P. B. GARLAND and P. J. RANDLE, Control of pyruvate dehydrogenase in the perfused rat heart by the intracellular concentrations of acetyl-CoA, Biochem. J. 91, 6c-7c (1964). 51. R. G. HANSEN and U. HENNING, Regulation of pyruvate dehydrogenase activity in Escherichia coli K 12, Biochim. Biophys. A cta 122, 355-358 (1966). 52. A. F. SPENCER and J. M. LOWENSTEIN, Citrate content of liver and kidney of rat in various metabolic states and in fluoroacetate poisoning, Biochem. J. 103, 342-348 (1967). 53. R. A. PETERS, Mechanism of the toxicity of the active constituent of Dichapetalum cymosam and related compounds, Advances in Enzymology 18, 113-159 (1957). 54. A. PARMEGGIANI and R. H. BOWMAN, Regulation of phosphofructokinase activity by citrate in normal and diabetic muscle, Biochem. Biophys. Res. Communs. 12, 268-273 (1963). 55. J. B. WILLIAMSON, B. HERCZEG, n . COLES and R. DAWISH, Studies on the ketogenic effect of glucagon in intact rat liver, Biochem. Biophys. Res. Communs. 24, 437-446 (1966). 56. E. A. NEWSHOLME and A. H. UNDERWOOD, The control of glycolysis and gluconeogenesis in kidney cortex, Biochem. J. 99, 24c-26c (1966). 57. A. H. UNDERWOOD and E. A. NEWSHOLME, Control of glycolysis and gluconeogenesis in rat kidney cortex slices, Biochem. J. 104, 300-305 (1967). 58. J. R. WILLIAMSON,Mechanism for the stimulation in vivo of hepatic gluconeogenesis by glucagon, Biochem. J. 101, 11 c - 14c (1966). 59. W. TARNOWSKI and M. SEEMANN, Konzentrations~inderungen yon Effektoren gluconeogenetischer Schliisselenzyme in der Rattenleber unter gluconeogenetischen Bedingungen, Hoppe-Seyler's Z. Physiol. Chem. 348, 1- l 0 (1967). 60. O. WIELAND, Personal communication. 61. H. A. LARDY, Gluconeogenesis: pathways and hormonal regulation, pp. 261-278 in The Harvey Lectures, Series 60, Academic Press, New York and London (1966). 62. R. YON FELLENBERG, n . EPPENBERGER, R. RICHTERiCH and H. AEm, Das glykolytische Enzymmuster yon Leber, Niere, Skelettmuskel, Herzmuskel und GroBhirn bei Ratte und Maus, Biochem. Z. 336,334-350 (1962). 63. B. HESS, R. HAECKEL and K. BRAND, FDP-activation of yeast pyruvate kinase, Biochem. Biophys. Res. Communs. 24, 824-831 (1966). 64. B. HESS and R. HAECKEL, Interaction between potassium-, ammonium- and fructose1,6-diphosphate activation of yeast pyruvate kinase, Nature 214, 848-849 (1967). 65. P. WALTER, V. PAETKAU and H. A. LARDY, Paths of carbon in gluconeogenesis and lipogenesis Iii. The role and regulation of mitochondrial processes involved in supplying precursors of phosphoenolpyruvate, J. Biol. Chem. 241, 2523-2532 (1966). 66. O. WIELAND and L. WEISS, Increase in liver acetyl-CoA during ketosis, Biochem. Biophys. Res. Communs. 10, 333-339 (1963). 67. W. BORTZ and F. LYNEN, Elevation of long chain acyl-CoA derivatives in livers of fasted rats, Biochem. Z. 339, 77-82 (1963).

EFFECTS OF CORTISOL ON LEVELS OF M.ETABOLITES AND ENZYMES 179 68. D. STEINBERG and M. VAUGHAN,Release of free fatty acids from adipose tissue in vitro in relation to rats of triglyceride synthesis and degradation, pp. 335-347 in Handbook of Physiology, Section 5:,4dipose Tissue (A. E. RENOLD and G. F. CAH1LL, JR., eds.), American Physiological Society, Washington, D.C. (1965). 69. J. N. FAIN, V. P. KOVACEV and R. O. SCOW, Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat, J. Biol. Chem. 240, 3522-3529 (1965). 70. J. R. WlLLIAMSON,R. A. K~ISBERG and P. W. FELTS, Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver, Proc. NatL ,4cad. Sci., U.S. 56,247-254 (1966). 71. H. A. Kmms, T. GASCOYNE and B. M. NOTTON, Generation of extramitochondrial reducing power in gluconeogenesis, Biochem. J. 102,275-282 (1967). 72. H. D. HOBERMAN and A. F. D'ADAMO, JR., Synthesis of liver glycogen from 3-C ~4DL-malate, evidence for complete isotopic equilibration of C4-dicarboxylic acids, J. Biol. Chem. 235,934-936 (1960). 73. B. BLOOM and D. W. FOSTER, Source of phosphoenolpyruvate for hexose synthesis in liver and muscle as studied with aspartate-3-C ~4, J. Biol. Chem. 237, 2744-2746 (1962). 74. H. D. HOBERMAN and A. F. D'ADAMO, JR., Evidence for the formation of phosphoenolpyruvate in liver by a mechanism not involving equilibration with fumarate, J. Biol. Chem. 235, 2185-2187 (1960). 75. M. F. UTTER, Pathways of phosphoenoipyruvate synthesis in glycogenesis, Iowa StateJ. Science 38, 97-113 (1963). 76. J. P. BETHEIL, M. FEIGELSON and P. FEIGELSON, The differential effects of glucocorticoids on tissue and plasma amino acid levels, Biochim. Biophys. ,4cta 104, 92-97 (1965). 77. G. WEBER,R. L. SINGHALand S. K. SRIVASTAVA,Action of glucocorticoid as inducer and insulin as suppressor of biosynthesis of hepatic gluconeogenic enzymes, Advances in Enzyme Regulation 3, 43-75 (1965). 78. M. FEIGELSON and P. FEIGELSON, Relationships between hepatic enzyme induction, glutamate formation, and purine nucleotide biosynthesis in glucocorticoid action, J. Biol. Chem. 241, 5819-5826 (1966). 79. W. E. KNOX and O. GREENGARD, The regulation of some enzymes of nitrogen metabolism-an introduction to enzyme physiology, Advances in Enzyme Regulation 3,247-313 (1965). 80. I. SANKOFF and T. L. SOURKES, The weight depressing action of a-methyi-D,Ltryptophan in rat, Canad. J. Biochem. Physiol. 40, 739-747 (1962). 81. J. SCHOLE, Direkte oder indirekte Enzyminduktion durch Cortisol, Autorenreferate, Herbsttagung der Gesellschaft f'tir Biologische Chemic, pp. 96-97, Marburg (1966). 82. J. F. MORAN and T. L. SOURKES, Induction of tryptophan pyrrolase by a-methyitryptophan and its metabolic significance in vivo, J. Biol. Chem. 238, 3006-3008 (1963). 83. G. WEBER, S. K. SRIVASTAVAand R. L. SINGHAL, Role of enzymes in homeostasis VII. Early effects of corticosteroid hormones on hepatic gluconeogenic enzymes, ribonucleic acid metabolism, and amino acid level, J. Biol. Chem. 240, 750-756 (1965). 84. J. F. HOFERT and A. WHITE, Inhibition of the lymphocytolytic activity of cortisol by total hepatectomy, Endocrinology 77,574-581 (1965). 85. T. UETE and J. ASHMORE,Effects of triamcinolone on carbohydrate synthesis in rat liver slices, J. Biol. Chem. 238, 2906-2911 (1963). 86. A. B. EISENSTEIN, Adrenocortical hormones and carbohydrate synthesis in liver, .4 dvances in Enzyme Regulation 3, 121 - 135 (1965). 87. R. C. HAYNES, Studies of an in vitro effect of glucocorticoids on gluconeogenesis, Endocrinology 71, 399--406 (1962). 88. G. OKUNO, Med..I. Osaka Univ. Dent. Soc. 10, 483 (1966), cited by UETE and ASHMOREin (85). 89. A. B. EISENSTEIN,E. BERG, D. GOLDENBERGand B. JENSEN,In vitroeffect of adrenal steroids on carbohydrate synthesis in liver, Endocrinology 74, 123-127 (1964).

180

W. SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

90. A. B. EISENSTEIN, M. RONA and B. BRUMMER, In vitro effects of puromycin aminonucleoside on liver carbohydrate synthesis, Endocrinology 76, 191 - 193 (1965). 91. A. B. EISENSTEIN,S. SPENCER,S. FLATNESS and A. BRODSKY,Carbohydrate synthesis in the isolated perfused rat liver: role of the adrenal cortex, Endocrinology 79, 182-186 (1966). 92. R. C. HAYNES, The control of gluconeogenesis by adrenal cortical hormones, Advances in Enzyme Regulation 3, I I I - I 19 (1965). 93. C. SONGSAWADE and J. ASHMORE, Effects of actinomycin D and puromycin on in vitro effects of triamcinolone on hepatic metabolism, Advances in Enzyme Regulation 3,237-244 (1965). 94. L. GOLDSTEIN, E. J. STELLA and W. E. KNOX, The effect of hydrocortisone on tyrosine-a-ketoglutarate transaminase and tryptophan pyrrolase activities in the isolated, perfused rat liver, J. Biol. Chem. 237, 1723-1726 (1962). 95. O. BARNABEI and F. SERENI, Cortisol-induced increase of tyrosine-a-ketoglutarate transaminase in the isolated perfused rat liver and its relation to ribonucleic acid synthesis, Biochim. Biophys. Acta 91,239-247 (1964). 96. M. CIVEN and W. E. KNOX, The independence of hydrocortisone and tryptophan inductions of tryptophan pyrrolase, J. Biol. Chem. 234, 1787-1790 ( 1959). 97. H . V . HENNING andW. SEUBERT, unpublished experiments. 98. H. A. KREBS, D. A. H. BENNETT, P. DE GASQUET, T. GASCOYNE and T. YOSH1DA, Renal gluconeogenesis, The effect of diet on the gluconeogenic capacity of rat-kidneycortex slices, Biochem. J. 86, 22- 27 (1963). 99. H. V. HENNING, W. HUTH and W. SEUBERT, An in vitro effect ofcortisol on pyruvate carboxylase and gluconeogenesis, Biochem. Biophys. Res. Communs. 17, 496-501 (1964). 100. C. PERAINO, C. LAMAR and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver IV. Effects of cortisone and phenobarbital, J. Biol. Chem. 241, 2944-2948 (1966). 101. H. KR6GER, J. PmLIPP and A. WICKE, Wirkungsdauer geringer Cortison-Dosen bei der Substrat-lnduktion yon Tyrosin-a-Ketoglutarat-Transaminase und TryptophanPyrrolase, Biochem. Z. 344,227-232 (1966). 102. H. KR6GER and B. GREUER, Influence of cortisone on the substrate induction of enzymes in rat liver, Nature 210,200 (1966). 103. H. S. MARVER, A. COLLINS and D. D. TSCHUDY, The "permissive" effect of hydrocortisone on the induction of c$-aminolaevulate synthetase, Biochem. J. 99, 31c-33c (1966). 104. H. A. KREBS and P. DE GASQUET, Inhibition of gluconeogenesis by a-oxo acids, Biochem. J. 90, 149-154 (1964). 105. O. K. SMITH and C. N. H. LONG, Effect of cortisol on the plasma amino nitrogen of eviscerated adrenalectomized-diabetic rats, Endocrinology 80, 561-566 (1967). 106. D. H. WILLIAMSON,O. LOPES-V|EIRA and B. WALKER,Concentrations of free glucogenic amino acids in livers of rats subjected to various metabolic stresses, Biochem. J. 104,497-502 (1967). 107. E. KIRSlEN, R. KIRSTEN, H. J. HOHORST and TH. BOCHER, Free amino acids in alloxan diabetic rat livers, Biochem. Biophys. Res. Communs. 4, 169-174 (1961). 108. E. S. SHRAGO, H. A. LARDY, R. C. NORDLIE and D. O. FOSTER, Metabolic and hormonal control of phosphoenolpyruvate carboxykinase and malic enzyme in rat liver, J. Biol. Chem. 238, 3188-3192 (1963).

DISCUSSION

Sm HANS A. KREBS: l would like to ask Dr. Seubert whether he could elaborate on the differences between his results and interpretation and those of Lardy and his group? W. SEUBERT: The path of carbon in gluconeogenesis postulated by

E F F E C T S O F C O R T I S O L ON L E V E L S OF M E T A B O L I T E S A N D E N Z Y M E S

181

Lardy is based upon studies on the intracellular distribution of pyruvate carboxylase in chicken and rat liver carried out by Keech and Utter (J. Biol. Chem. 238, 2609 (2963)), Freedman and Kohn (Science 145, 58 (1964)), Shrago and Lardy (J. Biol. Chem. 241, 663 (1966)) and Henning and Seubert (Biochem. Z. 340, 160 (1964)). The first three groups of investigators mentioned could not find pyruvate carboxylase in the cytosol obtained by extraction of liver with isotonic sucrose solution. Also Dr. Henning in my laboratory could solubilize by this treatment only 15 to 20% of the total activity present in rat liver. As already pointed out in this and earlier papers, the soluble fraction of pyruvate carboxylase could be considerably increased by extraction with isotonic solutions supplemented by K +, Na + or acetyl-CoA. Studies on the intracellular distribution of pyruvate carboxylase in Dr. Utter's laboratory have to my knowledge been restricted to the enzyme from chicken liver. As has been pointed out by Nordlie and Lardy (J. Biol. Chem. 238, 2259 (1963)) and by Haynes (Adv. Enzyme Regul. 3, l I l (1965)), there are great differences between various species concerning the intracellular location of PEP carboxykinase and the response of the glucogenic enzymes to glucocorticoids. One should therefore be cautious in drawing conclusions on a metabolic pathway in rat liver from studies carried out with chicken liver. | also would like to make a few remarks on the various assays employed by other investigators for the determination of pyruvate carboxylase in the cytosol or crude extracts. These assays are: I. The coupling of the carboxylation of pyruvate with the reduction of oxaloacetate formed to malate by DPNH and malic dehydrogenase (eq. 1-2) (Keech and Utter). The activity of pyruvate carboxylase is calculated in this assay from the rate of D P N H oxidation. In presence of lactate dehydrogenase a blank without acetyl-CoA is used as a control. 2. Coupling of pyruvate carboxylation with PEP carboxykinase (eq. 1 and 3) (Shrago and Lardy). In this assay formation of PEP was considered as a measure of the activities of pyruvate carboxylase in the cytosol. 3. The isotope assay which is based upon the fixation of radioactive bicarbonate into oxaloacetate and citrate respectively in the presence of citrate synthase (eq. l and 4) (Freedman and Kohn). 4. The "exchange" assay (eq. 5). This assay is based upon the exchange of radioactive pyruvate with cold oxaloacetate. It is independent of Mg++ and acetyl-CoA. Mg++

(l) Pyruvate + CO* + ATP acetyl-CoAoxaloacetate* + A D P + P (2) Oxaloacetate + D P N H + H +

> malate + DPN +

182

W . S E U B E R T , H. V. H E N N I N G , W . S C H O N E R A N D M. L ' A G E

(3) Oxaloacetate+ ITP

Mg~,, Mn++

(4) Oxaloacetate* ÷ acetyl-CoA

~ P E P + I D P ÷ CO2 ) citrate* ÷ C o A - S H

(5) Oxaloacetate+ Enzyme-biotin ~ pyruvate*+ Enzyme-biotin-CO2 Using the optical assay (eq. 1 and 2), we could not detect activity of pyruvate carboxylase in the cytosol of rat liver, although there was pyruvate carboxylase present. This finding, however, had to be expected because of the high activities of lactic dehydrogenase present in the cytosol which cause a rapid oxidation of DPNH. This renders impossible any assay of pyruvate carboxylase due to the insufficiency of this enzyme in the system. The same experience apparently was made by Utter and Keech. In purifying pyruvate carboxylase, they replaced "in initial extracts in which lactate dehydrogenase activity was quite high the optical a s s a y . . , by one involving fixation of 14CO2" (J. Biol. Chem. 238, 2603 (1963)). We can also confirm, using their assay system, the negative results of Shrago and Lardy concerning the extramitochondrial location of pyruvate carboxylase in rat liver: there is almost no PEP formed upon incubation of pyruvate with the cytosol under the conditions described by these investigators. Examination of a possible inhibitory action of the various components of their assay mixture in our assay, however, revealed ITP and fluoride (fluoride is added to inhibit enolase) to be inhibitors of pyruvate carboxylase. In addition, contamination of the cytosol with citrate synthase (eq. 4) may interfere with PEP carboxykinase (eq. 3) by converting part of the oxaloacetate formed to citrate. We therefore consider this assay for pyruvate carboxylase not to be a reliable one for assays in the cytosol or crude extracts. The statement of Freedman and Kohn that "all the pyruvic carboxylase activity is located in the mitochondrial fraction of rat liver" could not be confirmed by us using their assay method. As expected on the basis of studies with our assay, there is a considerable avidin-sensitive CO2-fixation catalyzed by the cytosol of rat liver. The relation between soluble and total activity of pyruvate carboxylase is the same as observed with our assay (Table 1). In fact the above authors also reported in Table 1 of their paper CO~-fixation in the 9000 g-supernatant. From our experience, optimal CO2-fixation was in some cases only observed when citrate synthase was added (Table 1). Apparently the amount of citrate synthase present is not sufficient to overcome a Mg+÷- and enzyme-dependent decarboxylation of oxaloacetate (Fig. 1) by conversion of the latter to citrate. Although the isotope assay based upon COsfixation (equ. 1 and 4) is more convenient than our assay (incorporation

TABLE 1

116 000 (total activity of liver) 39 400 47300 28 000

47 3OO 506OO 31 400

Without Citrate Synthase

122 000

Citrate Synthase Added

14C-Bicarbonate Incorporated (c.p.m./g liver)

*Adjusted by the addition of KOH. Extractions were carried out according to Henning et aL [16]. Composition of the reaction mixture as described by Henning and Seubert[ 14], except that pyruvate and bicarbonate were replaced by the cold and radioactive compounds respectively (spec. radioactivity of NaH14COs; 0.1/zC//zmole). The reaction mixtures were incubated for 30 min at 30°, acidified with 0.5 ml 10% trichloracetic acid. 14CO2 was removed by gassing with unlabelled CO~ for 15 min and subsequently for 10 min with N2. After adjusting the pH to 8-9, the remaining radioactivity was counted after plating in the gas flow counter.

Sucrose (0.24 M), Glutathione (I nua), Versene (! raM), Triethanolamine-HCl pH 7.2* (20 raM), Na-deoxycholate (0.1%) Sucrose (0.24 M), Glutathione (1 rnM), Versene ( 1 mM), Triethanolamine-HCl* pH 7.2 (20 raM) Sucrose (0.3 M)

Medium

Intracellular Distribution of P mvate Carboxylase Assayed by the Fixation of 14C-Bicarbonate

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184

W. SEUBERT, H. V. HENNING, W. SCHONER AND M. L'AGE

t

3 0 ~

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r,

....

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:

D

Iw

u ZO ~0

'....

0

A

:1

i

i

i

5

10

15

I

20 min

FiG. 1 Stability of oxaloacetate under conditions of the isotope and "exchange" assay for pyruvate carboxylase. The reaction mixture contained in 0.55 ml: 50 p, moles Tris-buffer, pH 7.7; 3/zmoles oxaloacetate; 0.6p, moles pyruvate, 5/xmoles MgCI2; 0.1 ml rat liver cytosol (1:10). The mixtures were incubated at 30°. Oxaloacetate and pyruvate were converted at the various time intervals indicated to the corresponding hydroxy acids by addition of KBH4. Malate was quantitated enzymatically after acidifying and neutralization (see Table 2) according to Schoner e t al.[18]. Curve A: Complete system. Curve B: MgCI2 omitted. Curve C: Cytosol omitted. Curve D: MgCI2 and cytosol omitted. Since chemical r&luction of oxaloacetate by KBH4 results in formation of both D- and L-malate, the amounts determined by the enzymatic assay[I 8] had to be multiplied by a factor of 2.

of 14C-pyruvate into citrate), we prefer the latter one because of the difficulties in avoiding dilution of radioactive bicarbonate by contamination of the solutions with cold bicarbonate. In addition, storage of bicarbonate solutions will lower the specific radioactivity by exchange with cold COz. In our opinion quantitative determination of pyruvate carboxylase necessitates therefore in each series of experiments a simultaneous determination of the specific radioactivity of the bicarbonate in presence of all solutions employed in the assay. In earlier studies on the role of CO2 in isoprenoid degradation, we did this by coupling the bacterial pyruvate carboxylase with citrate synthase (eq. I and 4). Since the bacterial enzyme catalyzes the carboxylation of pyruvate independent of acetyl-CoA, incorporation of 14CO2 into citrate may be limited by known amounts of this substrate (Biochem. Z. 341, 23 (1964). The "exchange" assay was first employed by Scrutton et al. (J. Biol. Chem. 240, 574 (1965)) and by us (Biochem. Z. 334, 401 (1961)) for assays of pure preparations of pyruvate carboxylase from chicken liver, and Pseudomonas citronellolis, respectively. Stabilization and isolation

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 185 of labelled oxaloacetate formed from pyruvate w a s achieved by Dr. Scrutton by conversion to aspartic acid followed by chromatography. We stabilized oxaloacetate by hydrogenation with KBH4. Radioactive lactate and malate formed by this treatment were separated by high voltage electrophoresis (Fig. 2). Labelling of malate was a measure of the enzyme activity. Since under the conditions of the exchange assay (no Mg ÷+) oxaloacetate is not decarboxylated (Fig. 1), the exchange assay also permits prolonged incubation of oxaloacetate with 14Cpyruvate.

FIG. 2 Electrophoretic separation of lactate and malat¢.

In our former attempts to develop an assay for mammalian pyruvate carboxylase in crude extracts, we also investigated the reliability of the exchange assay. These studies revealed that conversion of oxaloacetate to aspartate by transamination with glutamic acid introduced with crude tissue extracts may be neglected. Incorporation into oxaloacetate and malate respectively is therefore also a measure of the activity of pyruvate carboxylase in crude extracts. From this assay, there was very little activity that could be detected in the cytosol (Table 2, column 3), although our assay showed activity to be present (Table 2, column 2). The apparent discrepancies could be clarified by supplementing the exchange assay system with acetyl-CoA. Under these conditions, considerable activity could be detected in the cytosol when compared with the deoxycholate extract which contains the total pyruvate carboxylase activity from liver. I should like to stress that the results obtained with the exchange assay in the presence of acetyl-CoA are too low; because of the activities of citrate synthase present in crude extracts, part of oxaloacetate was converted to radioactive citrate. The latter does not separate from lactate

2.5 1.6 2.1 1.1 1.7

4.7 4.0

Assayed According to Henning and Seubert [ i 4]

0.80

0.25 0.31 0.13 0.27

0.04 0.07 0.03 0.03

n

0.57

0.71

Acetyl-CoA Present

0.68

Without AcetyI-CoA

Exchange Assay

pH 7.7; 0.6/zmoles 1-14C-pyruvate (spec. activity: 7 × 105 - 9 . 5 × 10s C//~mole); 3/~moles oxaloacetate, pH 7.0. Acetyl-CoA was regenerated with acetyl-POo phosphotransacetylase and CoA-SH [14]. After incubation for 30 min at 30 °, the reaction was terminated by addition of 2-3 mg KBH4. Oxaloacetate and pyruvate are immediately reduced by this treatment to the hydroxy acids. After standing for 1-2 min at 0 °, 0.2 ml of 6% perchloric acid were added to destroy excess of KBH4. The solutions were brought to pH 8 with 0.1-0.12 ml 2 s KOH(ff'). After separation of KCIO4 a 0.05 ml aliquot was transferred to electrophoresis paper as described earlier[14, 17]. Separation of radioactive lactate and malate was achieved by a 4 h electrophoresis in pyridine-acetic acid buffer (water+pyridine+glaciai acetic acid = 9 0 0 + 1 0 0 + 1) at 1600 V. Malate was detected and counted as described earlier[14]. All values are corrected by a control in which oxaloacetate was omitted. Incorporation of radioactivity into aspartate was checked according to Prinz et al.[ 17].

Assay ofpyruvate carboxylase with the exchange assay. The reaction mixtures contained in 0.55 ml: 50/z moles Tris-buffer,

*Adjusted by the addition of KOH.

Sucrose (0.24 M), Glutathione (I mM)* Versene (I rnM), Triethanolamine-HCl, pH 7,2* (20 raM) Sucrose (0.3 M)

(0.1%)

Sucrose (0.24) M), Glutathione (I raM), Versene (! mM), Triethanolamine-HCI, pH 7.2* (20 mM), Na-deoxycholate

Medium

Pyruvate Carboxylase (Units[g liver)

TABLE 2 Intracellular Distribution of Pyruvate Carboxylase in Rat Liver Assayed with the "Exchange" Assay, and According to Henning and Seubert

~n

>-

t-

¢3 m O Z rn

9

rn X Z

.<

r~

m

OO

EFFECTS OF CORTISOL ON LEVELS OF METABOLITES AND ENZYMES 187 on electrophoresis and can therefore not be quantitated (Fig. 2). The error is especially high with tissue extracts obtained by deoxycholate treatment (Fig. 2). These extracts contain all the mitochondrial enzyme activities including the total citrate synthase present in liver. Because of the difficulties created by the indispensability of acetyl:CoA in the exchange assay, we still prefer our assay for the determination of pyruvate carboxylase in crude extracts. According to studies of Scrutton et al. (J. Biol. Chem. 240, 574 (1965~), acetyl-CoA is not necessary for the transfer of CO2 from enzyme-bound CO2 to the substrate. The stimulatory effect of acetyl-CoA in the exchange assay (eq. 5) can therefore only be explained as a stabilization of the enzyme by this substrate under the conditions of the assay (30-min incubation, crude extracts). This assumption is in accord with observations of Scrutton and Utter (J. Biol. Chem. 242, 1723 (1967)) according to which pyruvate carboxylase is protected by acetyi-CoA against inactivation by various agents.