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Induction of mitochondrial phosphoenolpyruvate carboxykinase in cultured human fibroblasts
792
Biochimica et Biophysica Acta, 521 (1978) 792--804 © Elsevier/North-Holland Biomedical Press
BBA 99315
INDUCTION OF MITOCHONDRIAL PHO~PHOENOLPYRUVATE CARBOXYKINASE IN CULTURED HUMAN FIBROBLASTS
IFEANYI J. ARINZE, R E N G A C H A R I
RAGHUNATHAN
and J A M E S D. R U S S E L L
Department of Biochemistry and Nutrition and the Division of Genetics and Molecular Medicine, Meharry Medical College, Nashville, Tenn. 37208 (U.S.A.) (Received April 24th, 1978)
Summary Mitochondria of cultured normal human fibroblast cells were found to contain the enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme in these cells is increased 2- to 3-fold by addition of 5 . 1 0 - 4 M dibutyryl cyclic AMP, or 1.5- to 2-fold by the addition of dexamethasone ( 2 - 1 0 -7 M) or hydrocortisone (1.38" 10 -6 M). These increases in enzyme activity were inhibited by cycloheximide and actinomycin D, suggesting they are dependent upon de novo protein synthesis. Cultured human fibroblasts may thus provide a useful system for studying the regulation of mitochondrial phosphoenolpyruvate carboxykinase.
Introduction
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is one of the key regulatory enzymes in gluconegenesis [1]. Two immunologically distinct forms, mitochondrial and cytosolic, of this enzyme exist in tissues of a variety of mammalian species [2,3]. In many species including the human, both forms of the enzyme are present in liver in about equal proportions whereas in others the predominating form is either the mitochondrial or the cytosolic form [3,4]. The activity of the cytosolic enzyme can be altered by dietary and hormonal manipulations [4--8] and the control of the activity of the enzyme during these manipulations appears to involve regulation of enzyme synthesis [9--11 ]. The mitochondrial form of phosphoenolpyruvate carboxykinase has generally been regarded as a non-inducible enzyme since its activity is not altered by conditions which lead to changes in the overall rate of gluconeogenesis [2,12--14]. However, Elliot and Pogson [15] have recently suggested that the mitochondrial form of the enzyme in guinea-pig liver might in fact be 'induced' under starvation conditions. In species which have both forms of the enzyme, the
793 mitochondrial form has been shown to contribute up to 50% of the carbon flow to glucose during gluconeogenesis from lactate in the perfused liver
[16--19]. As a part of our program to develop a suitable system to study genetically determined metabolic abnormalities using cultured cells, we showed recently that fibroblast cultures derived from human skin contain measurable activities of phosphoenolpyruvate carboxykinase [20]. The present paper shows that the activity of this enzyme in these fibroblasts is associated only with mitochondria and cochromatographs with the mitochondrial phosphoenolpyruvate carboxykinase from guinea-pig liver. In these cells the activity of the enzyme is elevated by dexamethasone and dibutyryl cyclic AMP in a manner suggesting enhanced rates of enzyme synthesis. Materials and Methods
Chemicals. NaH14CO3, Aquasol, [5-3H]uridine, and I~[5-3H]proline were purchased from N e w England Nuclear Corporation. Culture medium F-10, fetal bovine serum, and trypsin were obtained from Grand Island Biological Company. Actinomycin D, cycloheximide, theophylline, dibutyryl adenosine cyclic 3',5'-monophosphate (dibutyryl cyclic AMP), hydrocortisone, and dexarnethasone (A1'2,9a-fluoro,16a-methylprednisolone) were obtained from the Sigma Chemical Company. Insulin was a gift from Dr. William W. Bromer, Eli Lilly and Company, Indianapolis. Tissue culture. Fibroblast cultures were initiated as described [20]. Except for the experiments reported in Table Ill,all experiments were performed using fibroblast strain 130. The general characteristics of this strain have been described [20]. Solutions of hormones or inhibitors were made with the appropriate culture medium before addition to the cultures as indicated in the legends to tables and figures. Enzyme assays. Cell monolayers were washed twice with 0.85% NaCl solution and scraped from the dishes with a rubber policeman. The cells from 8 60-ram dishes were then suspended in 2 ml of 0.25 M sucrose and sonified for 4 15-s intervals in a Branson cell disrupter, Model W185, at a setting of 5. The sonified extract was used for enzyme assays. In some experiments the cells were released from monolayer by treatment with 0.025% solution of trypsin in medium F-10. Phosphoenolpyruvate carboxykinase activity was measured at 37°C by a modification [14] of the 14C bicarbonate fixation method of Chang and Lane [21]. Lactate dehydrogenase (EC 1.1.1.27) and citrate synthase (EC 4.1.3.7) activities were measured as described [22]. All enzyme assays were conducted in duplicate under conditions where enzyme activitieswere proportional to both time and enzyme concentration. Protein was determined by the method of Lowry et al. [23]. Measurement of radioactivityin RATA and protein. For the incorporation of radioactivity into total R N A and protein, the cells were pulsed for I h with [3H]uridine (1.5 /zCi/ml of medium F-10 containing 10% fetal bovine serum) and [3H]proline (7 ;zCi/ml of the same medium), respectively. At the end of the labeling period, the medium was removed, 2 ml/dish of cold trichloroacetic acid solution (5%, w/v) were added and after incubation for 20 rain at 4°C,
794
the dishes were rinsed twice with 2 ml of the trichloroacetic acid solution. They were further rinsed twice with 2 ml of H:O. 1 ml of a 0.25% solution of trypsin (in medium F-10) was then added and the dishes incubated for 20 min at 37°C. At the end of this period the whole volume (1 ml) was counted for radioactivity after adding 10 ml of Aquasol.
Separation of phosphoenolpyruvate carboxykinase activities by hydroxyapatite chromatography. Cell or tissue extracts were applied to the hydroxyapatite (Biorad HPT) column (1 × 14 cm) and phosphoenolpyruvate carboxykinase was eluted with a linear gradient of 10--250 mM potassium phosphate buffer, pH 7.0, at 2°C [24]. Results
Response of phosphoenolpyruvate carboxykinase activity to addition of various hormones to the culture medium. At the concentrations tested, hydrocortisone and dexamethasone increased the activity of phosphoenolpyruvate carboxykinase by 1.6- and 1.8-fold, respectively, in 6 h after an initial 2-h lag period (Table I). This lag was clearly discernible in the case of hydrocortisone, and has been observed in a similar response of this enzyme to steroid hormones in adrenalectomized rats [6] and in Reuber H-35 hepatoma cells [ 2 5 ] . Insulin, at I • 10 -s M, had no effect on the activity of the enzyme at 6 h b u t when added with dexamethasone it suppressed the dexamethasone effect b y a b o u t 30%. Addition of dibutyryl cyclic AMP at a final concentration of 5 • 10 -4 M (in the presence of 1 mM theophyUine) led to a 2.5-fold elevation in the activity o f the enzyme within 6 h. Unlike the steroid-induced increases in
TABLE I EFFECT OF HYDROCORTISONE, DEXAMETHASONE, INSULIN, AND DIBUTYRYL CYCLIC AMP O N PHOSPHOENOLPYRUVATE C A R B O X Y K I N A S E A C T I V I T Y I N H U M A N F I B R O B L A S T S All a d d i t i o n s w e r e m a d e a t z e r o t i m e to c u l t u r e s in s t a t i o n a r y p h a s e . Cells w e r e c o l l e c t e d a f t e r 2 a n d 6 h and processed for e n z y m e d e t e r m i n a t i o n . Values are m e a n s ~ S.E.M. for three e x p e r i m e n t s . The activ/ty o f the e n z y m e at zero t/me was 2.21 ± 0.07 n m o l / m g protein per rain. Addition to culture medium
Final conc. (M)
Phosphoenolpyruvate
carboxykinase activity ( n m o l / m g p r o t e i n p e r r a i n ) at 2h
None Hydrocortisone Dexamethasone Insulin De x a m e t h a s o n e + insulin D i b u t y r y l cyclic AMP + theophylline Theophylline D i b u t y r y l cyclic AMP + theophylllne + dexamethasone
1.38 • 10-6 2 . 0 • 10-7 1.0 10-8 2.0 10-~ 10-8 1.0 10-4 5.0 10-3 1.0 10-3 1.0 10-4 5.0 10-3 1.0 10-7 2.0
2.19 2.13 2.55 1.47
6h ± 0.10 ± 0.08 ± 0.07 ± 0.10
2.22 3.44 3.95 2.32
± + ± ~
0.07 0.12 0.22 0.10
2.06 ± 0.12
3.43 ± 0.11
3.71 ± 0 . 2 3 2.22 ± 0.05
5.43 ± 0 . 2 0 2 . 4 5 +_ 0 . 0 8
3 . 5 2 +- 0 . 2 4
5.41 ± 0 . 2 6
795
enzyme activity, no lag was associated with the increase caused by dibutyryl cyclic AMP. Theophylline alone had a negligible effect and in cells to which dexamethasone, dibutyryl cyclic AMP, and theophylline were added together, the increase in enzyme activity was equal to that produced by dibutyryl cyclic A M P and theophylline, suggesting that no synergistic interaction between the cyclic nucleotide and the steroid had occurred. Effect of serum. Many previous studies on enzyme induction in cultured cells have been performed in serum-free media. These studies have usually involved cells of neoplastic origin [25--28]. The fibroblasts used in our studies were derived from normal dermis, display no evidence of transformation, and cannot be grown or maintained for an appreciable length of time in the absence of serum. Therefore, the effect of serum on the induction of phosphoetlo/pyruvate carboxykinase by dexamethasone was investigated. Fig. 1 A shows that in the serum-containing medium, there was no appreciable change in the basal activity of the enzyme in control cultures during the dexarnethasoneinduced increases in enzyme activity. However, when the medium was changed at the time of addition of hormone to one lacking serum, the basal activity of the enzyme could not be maintained and actually was reduced by about 3 0 % in 4 h before gradually rising to initial basal levels (Fig. 1B). Although it was still possible to demonstrate increases in enzyme activity due to hormone addition in the serum-free medium, the hormone appeared to merely sustain the original basal activity for the first 6 h. In cultures starved of serum for 18 h prior to addition o f hormone, only 50% of the basal activity of phosphoenol. pyruvate carboxykinase could be detected (data n o t shown). The enzyme activity was still elevated u p o n the addition of steroid to these cultures b u t the basal activity of the enzyme in the serum-free cultures was progressively changing
40
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TIME, HOUR Fig. 1. The e f f e c t o f 0el~um and t h e time cou-,~ o f t h e i n d u c t i o n o f p h o $ p h o e n o l p y ~ v a t e elurboxy]rJuaMe b y dexlmaethBlone. DeY=metbaaone (@) (2 • 10 -7 M) we= a d d e d at zero time to I t a t i n n a r y phale c u l t u l ~ l i n IerIH1n-con~Ilnln.I m e d i u m A a n d i n N r u m - f r e e m e d / u m B. T h e e n z y m e a c t i v i t y w a I m c u u . ~ d a t v a r i o u I times thereafter. Dexamethuone waI not added to control (o) cultures. The values plotted in A are m e a n s ~ S.E.I~L f o r t h r e e e x p e r i m e n t s . T h e v a l u e s i n B a r e m e a n s f o r t w o e x p e r i m e n t s .
796 and resembled the pattern shown in Fig. lB. Since the data in Fig. 1 demonstrate that the activity of phosphoenolpyruvate carboxykinase can be elevated by dexamethasone either in the presence or absence of serum, and because the basal enzyme activity could not be maintained relatively constant in the absence of serum, all subsequent experiments were performed in cultures maintained in serum-containing media. The effect o f serum on er#zyme induction by dibutyryl cyclic AMP was not investigated. Time course of the effect of dexamethasone. The maximum increase (70%) in phosphoenolpyruvate carboxykinase activity produced by the addition of dexamethasone at a final concentration of 2 - 10 -7 M occurred at 6 h (Fig. 1A). Subsequently ~the enzyme activity rapidly decreased to only 27% of the control by 24 h. When t h e cultures were re-fed at two 6-h intervals beginning at 6 h after the intial treatment with the hormone, the enzyme activity was slightly higher (<10%) than indicated in Fig. 1A but the decline in enzyme activity which begins at 6 h was still apparent, indicating that the decline was not due to depletion of nutrients in the medium. Dose-response relationships. These are shown in Fig. 2. The optimal concentration of dexamethasone found (2" 10 -7 M) is clearly in the physiological range since the circulating levels of glucocorticoids in humans are about 3 • 10 -7 M [29]. The optimal dose (5 • 10 -4 M) of dibutyryl cyclic AMP noted in Fig. 2 is identical to the concentration of this nucleotide which has been shown to maximally induce phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 cells [26].
Effect of dexamethasone and dibutyryl cyclic AMP on phosphoenolpyruvate carboxykinase activity during cell growth. We have previously shown that the specific activity of phosphoenolpyruvate carboxykinase in human fibroblasts increases during logarithmic growth and attains a constant level during stationary phase [20]. The growth pattern of strain 130 used in the present study is
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R i ~ i A ! 10-7 10-5 10-3 CONCENTRATION, M FiE. 2. R e s p o n s e o f phosphoenolpyruvate earboxykiname a c t i v i t y t o varying c o n c e n t r a t i o n s o f d e x a m e t h a s o n e and d i b u t y r y l c y c l i c AMP. D e w n m e t h a s o n e ( o ) and d i b u t y r y l c y c l i c AMP (o) w e r e a d d e d t o fibroblast c u l t u r e s in s t a t i o n a r y plmse. T h e o p h y U i n e (1 r a M ) w a s a d d e d at t h e s a m e t i m e t o all a d d i t i o n s o f d l b u t y r y l c y c l / c A M P and t h e e n z y m e a c t i v i t y w a s m e a s u r e d 6 h a f t e r addition. T h e ~Peeific a c t i v i t y o f phosphoenoipyruvate c a r b o x y k i n a s e in c o n t r o l c u l t u r e s w a s 1.99 n m o l / m g p r o t e i n per rain. ~
2.0
i 10-9
i
797 similar to those previously reported. It was therefore of interest to re-examine the activity of phosphoenolpyruvate carboxykinase throughout the culture cycle in cells grown in the presence of dexamethasone. In the presence of an optimal inducing dose of the steroid (2 • 10 -7 M) in the culture medium, the specific activity of the enzyme was a b o u t 2-fold higher than in control cultures b u t the pattern of increase throughout the growth cycle was identical in both cases (Fig. 3A). This pattern of increase is consistent with the observation that the specific activities of many enzymes vary in parallel fashion with increased cell density [30]. Increased levels of cyclic AMP have been reported with increased growth of cultured cells [31] although it is n o t clear whether the increased levels of this nucleotide cause the high levels of enzyme activity seen at confluency. Fig. 3B shows that when dibutyryl cyclic AMP was added (in the presence of 1 mM theophylline) to growing cells the specific activity of phosphoenolpyruvate carboxykinase, measured at 6 h after the addition, increased 2.6-fold in cells which were in stationary phase. The effect was much greater in cells in log phase b u t the maximum attainable specific activity was not observed in cells in early log phase. It was significant, however, that the maximum induced activity was reached 3--4 days before the cells entered confluency and much earlier than in cells treated with dexamethasone.
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DAYS IN CULTURE F i g . 3. C h a n g e s i n t h e s p e c i f i c a c t i v i t y o f phosphoenolpyruvate c a r b o x y k i n a s e d u r i n g c e l l g r o w t h . I n A, c e l l s w e r e p l a n t e d ( 5 • 1 0 --4 c e l l s / d i s h ) i n 3 5 m m plastic p e t r l d i s h e s a n d f e d d a i l y w i t h n u t r i e n t m e d i u m c o n t a i n i n g ( e ) o r l a c k i n g ( o ) d e x a m e t h a s o n e ( 2 . 1 0 - 7 M). T h e e n z y m e a c t i v i t y w a s m e a s u r e d o n t h e m o r n i n g o f t h e d a y s i n d i c a t e d . I n B, t h e c e l l s w e r e g ~ o w n as i n A w i t h o u t t h e i n c l u s i o n o f d e x a m e t h a s o n e i n t h e m e d i u m . O n the d a y s i n d i c a t e d , t h e cells w e r e r e f e d w i t h f r e s h m e d i u m c o n t a i n i n g e i t h e r
d e x a m e t h a s o n e ( 2 • 1 0 - 7 M) ( e ) o r d i b u t y r y l c y c l i c A M P ( 5 • 1 0 - 4 M) p l u s 1 m M t h e o p h y l l J n e (m). T h e enzyme activity was measured 6 h later.
798
Effect of actinomycin D and cycloheximide on hormone-induced increases in phosphoenolpyruvate carboxykinase activity. In order to ascertain whether the increases in phosphoenolpyruvate carboxykinase activity in human fibroblasts involved alterations in enzyme levels or activation of a pre-existing enzyme species, we studied the effects of dexamethasone and dibutyryl cyclic AMP in the presence of inhibitors of RNA and protein synthesis. Neither actinomycin D nor cycloheximide affected the basal activity of phosphoenolpyruvate carboxykinase for up to 6 h in uninduced cultures (Table II). In cells treated for 6 h with dexamethasone after 30 min prior exposure to actinomycin D, the dexamethasone-induced increase in enzyme activity was abolished. The activity of the enzyme was still elevated by dibutyryl cyclic AMP; the increase being only about 56% of that produced by the nucleotide in the absence of the transcriptional inhibitor. The concentration of actinomycin D (! gg/ml) used was sufficient to block total RNA synthesis by 94% 1 h after the addition of the inhibitor (Table II). When cycloheximide was added at a concentration of 5 #g/ml which blocks the synthesis of cell-retained proteins b.Y 93%, both the dexamethasone- and dibutyryl cyclic AMP-induced increases in enzyme activity were suppressed. These findings strongly suggest that de novo synthesis of the enzyme is increased by both dibutyryl cyclic AMP and dexamethasone. However, more direct evidence must await further studies involving direct quantitation of phosphoenolpyruvate carboxykinase protein in these cells. T A B L E lI EFFECT OF INHIBITORS OF RNA AND PROTEIN CARBOXYKINASE ACTIVITY
SYNTHESIS
ON PHOSPHOENOLPYRUVATE
A c t i n o m y c i n D (1 p g / m l ) and c y c l o h e x i m i d e (5 ~ g / m l ) w e r e a d d e d to stationary phase cultures 3 0 min prior to the a d d i t i o n o f d e x a m e t h a s o n e (2 • 1 0 - 7 M) o r d i b u t y r y l cyclic A M P ( 5 . 1 0 --4 M). T h e o p h y l l i n e , 1 r a M , w a s a d d e d t o g e t h e r w i t h d i b u t y r y l c y c l i c A M P . T h e ce il- w e r e pulse-labelled w i t h [3H]ttrldine and [ ~ H ] p r o l i n e for t w o 1-h p e r i o d s beginning at the time o f a d d i t i o n o f h o r m o n e (zero time), and again at the 5th h after the a d d i t i o n , i n c o r p o r a t i o n o f radioactivity i n t o trichloroacetie acid-preciplteble m a t e r i a l w a s m e a s u r e d . T h e e n z y m e activity w a s m e a s u r e d in the appropriate cultures 0 and 6 h after the a d d i t i o n o f d e x a m e t h a s o n e o r d i b u t y r y l cyclic AMP. T h e results g/yen is rapresenteUve o f three e x p e r i m e n t s . Inhibitor
Treatment
Time:
Control
Control
D i b u t y r y l cyclic AMP + theophylline
Dexamethuone
Oh
6h
6h
6h
None AeClnomycin D Cyclohextmlde
Phosphoenoipyruvate carboxykinue, nmol/mg protein per min 2.09 2.19 5.44 3.53 1.08 1.95 3.06 1.69 2.95 2.03 2.41 2.52
None Act/nomycin D Cyclochexlmide
Radioact/vlty incorporated into total 7 688 8 063 7 478 194 7 237 6 265 5
Nome Aetinomycin D Cyelohexim/de
Rad/oactivity incorporated into protein, epm/dlah 34 278 24 68$ 22 58a 34 973 14 746 14 8 9 5 2 524 1 706 1 667
RNA, epm/dish 106 184 412
7 453 180 6 223 23 7 1 4 14 6 6 8 1 783
799
Effect of dexamethasone and dibutyryl cyclic AMP on phosphoenolpyruvate carboxykinase activity in different fibroblast strains. A previous study from our laboratories had demonstrated a wide variation in the activities of phosphoenolpyruvate carboxykinase in human fibroblast strains [20]. This variation was n o t unexpected since the strains represented cultures from unrelated individuals. In fact, in the population studied two strains completely lacked the activity o f the enzyme [20]. A comparison of our previous data [20] and the present study shows that the basal levels of phosphoenolpyruvate carboxykinase in some o f the strains previously studied were, in fact, almost as high as the induced levels demonstrated for strain 130 in the present study. The inducing effects of b o t h dexamethasone and dibutyryl cyclic AMP in nine different strains selected from our previously studied population which exhibit widely different basal activities of phosphoenolpyruvate carboxykinase are presented in Table III. In one strain (strain no. 18) there was no detectable activity of the enzyme either in the presence or absence of dexamethasone or dibutyryl cyclic AMP. In all other strains examined, the addition of dexamethasone increased the activity of the enzyme by about 1.5- to 1.8-fold while the increase produced b y addition of dibutyryl cyclic AMP was 2.5- to 3-fold. These data indicate that the inducibility of phosphoenolpyruvate carboxykinase is a c o m m o n phenomenon occurring in human fibroblasts and is n o t unique to a single cell line.
Distribution of phosphoenolpyruvate carboxykinase in control and dibutyryl cyclic AMP-treated human fibroblast 130 cells. The standard technique of cell organelle fractionation by differential centrifugation was used. Fibroblasts were initially trypsinized to make them more rounded and presumably easier to homogenize. The whole homogenate was then fractionated b y the technique previously used for mammalian liver [22]. The activity of phosphoenolpyruvate carboxykinase in these fractions as well as that of marker enzymes, citrate synthase and lactic dehydrogenase, were determined. As evident in Table IV a b o u t 80% of the total activity of phosphoenolpyruvate carboxyT A B L E III EFFECT OF DEXAMETHASONE AND DIBUTYRYL CYCLIC AMP ON PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN DIFFERENT STRAINS OF HUMAN FIBROBLASTS E n z y m e a c t / v i t l e s w e r e d e t e r m i n e d i n cells f r o m s t a t i o n a r y p h a s e c u l t u r e s . D e x a m e t h a s o n e (2 • 1 0 - ' / M) o r d / b u t y r y l c y c l i c A M P (5 • 1 0 - 4 M) a n d t h e o p h y l l i n e ( 1 0 - 3 M) w e r e a d d e d t o t h e c u l t u r e s 6 h b e f o r e t h e c e l l s w e r e u s e d f o r e n z y m e assay. T h e v a l u e s g i v e n are t h e average o f t w o e x p e r i m e n t s .
Strain number
21 24 30 52 130 18 38 44 50
Treatment (nmol/mg protein per min)
Control
Dexamethasone
Dibutyryl cyclic AMP + theophyliine
1.28 1.53 0.96 1.65 2.14 0 0.61 1.96 3.21
2.07 2.22 2.18 2.82 3.82 0 1.08 3.43 5.07
3.36 3.66 3.24 3.67 5.44 0 1.83 4.98 7,84
800 T A B L E IV
I N T R A C E L L U L A R D I S T R I B U T I O N OF P H O S P H O E N O L P Y R U V A T E C A R B O X Y K I N A S E A C T I V I T I E S IN C O N T R O L A N D D I B U T Y R Y L C Y C L I C A M P - T R E A T E D H U M A N F I B R O B L A S T S E n z y m e activities w e r e m e a s u r e d in cells h a r v e s t e d f r o m s t a t i o n a r y p h a s e c u l t u r e s o f strain 130. In d i b u tYlTI cyclic A M P - t r e a t e d c u l t u r e s , cells w e r e h a r v e s t e d for e n z y m e assays 8 h a f t e r a d d i t i o n of d i b u t y r y l cyclic A M P (5 • 10 -4 M) a n d t h e o p h y l l l n e (10 -3 M). T h e cells w e r e s u s p e n d e d using 0 . 0 2 5 % s o l u t i o n of t r y p s i n in F-10. F e t a l b o v i n e s e r u m was a d d e d to the s u s p e n s i o n (10% v / v ) t o i n h i b i t f u r t h e r p r o t e o l y t i c a c t i v i t y a n d t h e cells w e r e c o l l e c t e d b y c e n t r i f u g a t i o n a n d w a s h e d w i t h saline. T h e cells w e r e t h e n h o m o g e n i z e d a n d f r a c t i o n a t e d as d e s c r i b e d [ 2 2 ] . T r y p s i n i z a t i o n h a d n o e f f e c t on t h e t o t a l a c t i v i t y o f p h o s p h o enolpyruvate c a r b o x y k l n a s e i n t h e c u l t u r e s . T h e v a l u e s are given in n m o l / d i s h p e r m i n a n d are t h e average of three experiments.
Phosphoenolpyruvate
Citrate
carboxykinase
synthase
Lactate dehydrogenasc
Control Whole h o m o g e n a t e Nuclear pellet Mitochondrial pellet Postomitochondrial supernatant
1.87 0.13 1.50 0
29.8 3.8 21.3 0
389 57 4 288
D i b u t y r y l cyclic AMP Whole h o m o g e n a t e Nuclear pellet Mitoehondrial pellet Post-mitochondrial supernatant
4.38 0.24 3.99 0
40.4 6.1 31.9 0
585 72 13 483
kinase was associated with the washed mitochondria. No detectable activity was found in the post-mitochondrial supernatant. Less than 1% of the activity of the mitochondrial marker enzyme, citrate synthase was found in the postmitochondrial supernatant, while 72% of its total activity was associated with the mitochondria. The recovery of lactate dehydrogenase, an enzyme marker for cytosol, in the post-mitochondrial supernatant was about 74%. Contamination of the mitochondrial pellet by the cytosol fraction was judged to be less than 2% on the basis of the activity of lactate dehydrogenase activity found in the mitochondrial fraction. It can also be seen from Table IV t h a t in dibutyryl cyclic AMP-induced cells, a 2.7-fold increase in phosphoenolpyruvate carboxykinase activity was observed in the mitochondrial fraction which is in general agreement with the effect of this nucleotide in whole cells (Table I, see also Fig. 2 and 3); again, no detectable activity could be seen in the post-mitochondrial supernatant even after dibutyryl cyclic AMP induction. The data in Table IV is consistent with the conclusion that within the limits of our assay only the mitochondrial form of phosphoenolpyruvate carboxykinase is contained in these fibroblasts. Additional evidence is provided by the data in Fig. 4 where this activity has been examined after chromatography on h y d r o x y a p a t i t e columns. It has been demonstrated by Ballard [24] that a mixture of the cytosolic and mitochondrial forms of phosphoenolpyruvate carboxykinase can be completely separated at 2°C on hydroxyapatite columns by applying a linear gradient of 10--250 mM potassium phosphate at neutral pH. The technique can be applied to impure enzyme preparations [24]. Fig. 4A shows that this technique completely resolved the two activities of this enzyme normally present in guinea-pig liver, the mitochondrial activity was eluted first at low concentrations of phosphate, while the cytosolic enzyme was eluted at
801
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:IE nr
E c I0 20 FRACTION NUMBER
Fig. 4. C h r o m a t o g r a p h y o f p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e o n h y d r o x y a p a t i t e c o l u m n s . H y d r o x y a p a t i t e ( 4 g) w a s p r e p a r e d in I 0 m M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 0 , a n d p o u r e d i n t o a c o l u m n 1 c m in d i a m e t e r . T h e c o l u m n w a s e q u i l i b r a t e d w i t h t h e s a m e b u f f e r . 2 . 5 m l o f s o n i f i e d g u i n e a - p i g liver t i s s u e o r f i b r o b l a s t e x t r a c t s w e r e a p p l i e d t o t h e c o l u m n a n d t h e e n z y m e w a s e l u t e d at 2°C w i t h a linear g r a d i e n t of 10 to 250 mM potassium phosphate buffer, pH 7.0. 2.5-mi fractions were collected and the activity o f phosphoenolpyruvate c a r b o x y k i n a s e in e a c h f r a c t i o n w a s d e t e r m i n e d u s i n g 1 0 0 /~I a l i q u o t s o f e a c h f r a c t i o n for assay. T h e c o l u m n s w e r e r e u s e d o n l y o n c e . A, g u i n e a - p i g liver w h o l e h o m o g e n a t e ( 1 0 % w/v)" B, t h r i c e w a s h e d g u l n e a - p i g liver m i t o c h o n d r i a l s u s p e n s i o n ; C, c y t o s o l ( 1 0 0 0 0 0 X g s u p e r n a t a n t ) o f g u i n e a - p i g liver; D , f i b r o b l a s t e x t r a c t o f strain 1 3 0 at c o n f l u e n c y .
higher phosphate concentrations. The identity of the two peaks was established in separate runs {Fig. 4B, C). When an extract of human fibroblast 130 was applied to the column and a similar phosphate gradient was applied, only one peak of enzyme activity could be detected (Fig. 4D), supporting the data in Table IV that only one form of phosphoenolpyruvate carboxykinase is present in the fibroblasts. This activity was eluted at the same position as the mitochondrial phosphoenolpyruvate carboxykinase from guinea-pig liver. When the fibrobtast extract was mixed with a 100-fold dilution of washed mitochondrial extract from guinea-pig liver the mixture chromatographed as a single peak which resembled Fig. 4D. The specific activity of phosphoenolpyruvate carboxykinase in fibroblast mitochondria was 9.03 + 0.43 nmol/mg protein per min as opposed to 202 + 8 nmol/mg protein per min for the enzyme in guineapig liver mitochondria. Discussion Cell culture provides a relatively homogeneous source of tissue which Can be utilized to investigate the metabolic processes of intact cells as well as regula-
802 tion of specific enzymes. Glucorticoids and cyclic AMP have been shown to increase the rate of phosphoenolpyruvate carboxykinases synthesis in fetal [32] as well as adult rat liver [11] in vivo, and in Reuber H-35 hepatoma cells [25--28]. The cyclic AMP-stimulated increase in the activity of the enzyme in the perfused rat liver is also dependent on new protein synthesis as evidenced b y its inhibitability b y cycloheximide [33]. Although either cyclic AMP or glucocorticoids can independently cause increases in the activity of phosphoenolpyruvate carboxykinase, studies in the perfused rat liver suggest a permissive effect of glucocorticoids on the effect of cyclic AMP [33]. The results of the present studies show that the phosphoenolpyruvate carboxykinase activity in human fibroblasts is also increased by dibutyryl cyclic AMP and glucocorticoids. The effects of these agents do not appear to be synergistic. The optimal concentration of dexamethasone needed to achieve maximal increase in phosphoenolpyruvate carboxykinase activity in fibroblasts compares favorably with the circulating levels of glucocorticoids in human plasma which have been reported to be 3 • 10 -7 M [29]. Unlike Reuber H-35 cells where the activity of the enzyme remains elevated at the induced levels even after removal of the inducing steroid for up to 6 h, the activity of the enzyme in human fibroblasts begins to decline almost immediately after maximal elevation b y dexamethasone even in the continued presence of the steroid (Fig. 1A). In the presence of dibutyryl cyclic AMP, a maximal increase in enzyme activity is achieved at a concentration (5- 10 -4 M) (Fig. 2) which has been shown to produce a 7-fold increase in the synthesis of phosphoenolpyruvate carboxykinase-specific protein in Reuber H-35 cells where the specific activity of the enzyme is increased a b o u t 1.6-fold [26]. Although the specific molecular mechanism(s) of the action of glucocorticoids and cyclic AMP on phosphoenolpyruvate carboxykinase induction is not y e t fully understood, a great deal of our current understanding of these molecular events has come from the studies in the laboratories of Wicks [25, 27,34] and Hanson [11,26,28] using Reuber H-35 hepatoma cells. Direct radioimmunochemical measurements [26,28] using these cells have demonstrated repeatedly that the increase in phosphoenolpyruvate carboxykinase activity provoked b y either glcocorticoids or cyclic AMP results from a rise in the synthesis rate of the enzyme. According to Wicks [34], glucocorticoids p r o m o t e an increase in translatable m R N A for phosphoenolpyruvate carboxykinase, while cyclic AMP enhances enzyme synthesis by stimulating the translation of pre-existing specific m R N A templates. Other studies [26], however, suggest that dibutyryl cyclic AMP may affect multiple sites in such a way as to act translationally or posttranscriptionally. In fact, the recent studies by Iynedjian and Hanson [35] provide evidence suggesting that in the rat liver the cyclic nucleotide acts transcriptionally to increase the amount of translatable phosphoenolpyruvate carboxykinase-specific mRNA. Cyclic AMP has also recently been shown to increase the synthesis of m R N A for tyrosine aminotransferase in rat liver [36]. The phosphoenolpyruvate carboxykinase in rat kidney cortex and in Reuber H-35 cells is localized predominantly in the cytosol. Purified antibodies prepared in rabbits or goats against rat liver cytosolic phosphoenolpyruvate carboxykinase precipitate the enzymes in rat kidney cortex [37] or Reuber
803 H-35 cells [25--28]. However, this antibody does not precipitate the phospho-
enolpyruvate carboxykinase in rat liver mitochondria, guinea-pig liver or kidney mitochondria, or any other phosphoenolpyruvate carboxykinase of mitochondrial origin [2]. This and other evidence [24,38--40] show that the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase have different immunochemical, kinetic, and physicochemical properties. Our present studies indicate that the phosphoenolpyruvate carboxykinase activity in human fibroblasts is associated with mitochondria. These fibroblasts completely lack the cytosolic enzyme (Table IV, Fig. 4). To our knowledge this is probably the only system investigated in which one form of this enzyme appears to be completely lacking. Cultured human fibroblasts may provide a potentially useful model system to probe the specific regulation of mitochondrial phosphoenolpyruvate carboxykinase. As seen in the present study, the activity of this enzyme in these cells can be increased by various agents in a manner consistent with new protein synthesis (Table II). The actual intracellular site of synthesis cannot be discerned from the present data. However, the sensitivity of the induction of the enzyme in these cells to cycloheximide which does not inhibit mitochondrial protein synthesis [41] suggests that the phosphoenolpyruvate carboxykinase in human fibroblasts may be synthesized on cytoplasmic ribosomes. The inability to detect any activity of the enzyme in the cytosol during induction by dibutyryl cyclic AMP (Table IV) suggests that the newly synthesized enzyme may be inactive or that it is rapidly translocated into the mitochondria soon after synthesis. In previous work in animal systems the mitochondrial phosphoenolpyruvate carboxykinase, unlike the cytosolic enzyme has generally been found to be constitutive [ 12--14], and virtually nothing is known regarding the specific regulation of its level in mitochondria. The presence in human fibroblasts of mitochondrial phosphoenolpyruvate carboxykinase the activities of which varies with different culture conditions should facilitate studies on aspects of the regulation of this enzyme at the molecular level. These cells might also prove useful in investigating the possible involvement of altered levels or regulation of phosphoenolpyruvate carboxykinase in human disease.
Acknowledgements We thank Dr. Shirley B. Russell for valuable help with some of the experiments, Mrs. Kathryn M. Trupin for excellent technical assistance, and Drs. Sidney P. Colowick, Richard W. Hanson and Mulchand S. Patel for advice during the preparation of the manuscript. This work was supported in part by grants HD 08792 and CA 17229 from the National Institutes of Health, and by a Basil O'Connor Research Starter Grant, 5-89, from the National FoundationMarch of Dimes. I.J.A. and J.D.R. are recipients of Career Development Awards, HD 00226 and CA 00122, from the National Institutes of Health. References 1 Scrutton, M.C. and Utter, M.F. (1968) Annu. Rev. Biochem. 37, 294--302 2 Ballard, F.J. and Hanson, R.W. (1969) J. Biol. Chem. 244, 5 6 2 5 - - 5 6 3 0 3 Hanson, R.W. and Garber, A.J. (1972) Am. J. Clin. Nutr. 25, 1010--1021
804
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
N o r d l i e , R.C. a n d L a r d y , H . A . ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 2 2 5 4 - - 2 2 6 3 S h r a g o , E., L a r d y , H . A . , N o r d l i e , R.C. a n d F o s t e r , D.O. ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 3 1 8 8 - - 3 1 9 2 F o s t e r , D . O . , R a y , P . D . a n d L a r d y , H . A . ( 1 9 6 6 ) B i o c h e m i s t r y 5, 5 5 5 - - 5 6 2 S o l i n g , H.-D., Willms, B., K l e i n e k e , J. a n d G e h l h o f f , M. ( 1 9 7 0 ) Eur. J. B i o c h e m . 16, 2 8 9 - - 3 0 2 Wicks, W.D., Lewis, W. a n d M c K i b b i n , J.B. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 6 4 , 1 7 7 - - 1 8 5 H o p g o o d , M . F . , B a l l a r d , F . J . , R e s h e f , L. a n d H a n s o n , R.W. ( 1 9 7 3 ) B i o c h e m . J. 1 3 4 , 4 4 5 - - 4 5 3 T i i g h m a n , S.M.. H a n s o n , R . W . , R e s h e f , L., H o p g o o d , M.F. a n d B a l l a r d , F.J. ( 1 9 7 4 ) Proc. Natl. A c a d . Sci. U.S. 4 8 . 1 3 0 4 - - 1 3 0 8 T i l g h m a n , S.M., B a l l a r d , F.J. a n d H a n s o n , R.W. ( 1 9 7 6 ) in G l u c o n e o g e n e s i s , Its R e g u l a t i o n in M a m m a l i a n S p e c i e s ( H a n s o n , R.W. a n d M e h l m a n , M.A., eds.), pp. 4 7 - - 9 1 , J o h n Wiley, N e w Y o r k S o l i n g , H . - D . a n d K l e i n e k e , J. ( 1 9 7 6 ) in G l u c o n e o g e n e s i s , Its R e g u l a t i o n in M a m m a l i a n S p e c i e s ( H a n s o n , R.W. a n d M e h l m a n , M.A., eds.), pp. 3 6 9 - - 4 6 2 , J o h n Wiley a n d S o n s , N e w Y o r k N o r d l i e , R . C . , V a r r i c c h i o , F . E . a n d H o l t e n , D.D. ( 1 9 6 5 ) B i o e h i m . B i o p h y s . A c t a 97, 2 1 4 - - 2 2 1 B a n a r d , F . J . a n d H a n s o n , R.W. ( 1 9 6 7 ) B i o e h e m . J. 1 0 4 , 8 6 6 - - 8 7 1 Elliot, K . R . F . a n d P o g s o n , C.I. ( 1 9 7 7 ) B i o c h e m . J. 1 6 4 , 3 5 7 - - 3 6 1 A r i n z e , I.J., G a r b e r , A . J . a n d H a n s o n , R.W. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 2 2 6 6 - - 2 2 7 4 P e n g , Y.-S., B r o o k s , M., E l s o n , C. a n d S h r a g o , E. ( 1 9 7 3 ) J. N u t r . 1 0 3 , 1 4 8 9 - - 1 4 9 5 H u i h r e g t s e , C . A , R u f o , G . A . a n d R a y , P.D. ( 1 9 7 7 ) B i o e h i m . B i o p h y s . A c t a 4 9 9 , 9 9 - - 1 1 0 A r i n z e , I.J. a n d H a n s o n , R.W. ( 1 9 7 3 ) F E B S L e t t . 31, 2 8 0 - - 2 8 2 R a g h u n a t h a n , R., Russell, J.D. a n d A r i n z e , I.J. ( 1 9 7 7 ) J. Cell. P h y s i o l . 9 2 , 2 8 5 - - 2 9 2 C h a n g , H.-C. a n d L a n e , M.D. ( 1 9 6 6 ) J. Biol. C h e m . 2 4 1 , 2 4 1 3 - - 2 4 2 0 R a g h u n a t h a n , R. a n d A r i n z e , I.J. ( 1 9 7 7 ) Int. J. B i o e h e m . 8, 7 3 7 - - 7 4 3 L o w r y , O . H . , R o s e b r o u g h , N . J . , Fax'r, N.J. a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 B a l l a r d , F.J. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 4 2 , 4 7 0 - - 4 7 2 B a r n e t t , C.A. a n d Wicks, W.D. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 2 0 1 - - 7 2 0 6 G u n n , J.M., T i l g h m a n , S.M,, H a n s o n , R.W., R e s h e f , L. a n d B a l l a r d , F.J. ( 1 9 7 5 ) B i o c h e m i s t r y 14, 2350--2357 Wicks, W.D. a n d M c K i b b i n , J.B. ( 1 9 7 2 ) B i o c h e m . B i o p h y s , Res. C o m m u n . 4 8 , 2 0 5 - - 2 1 1 T i l g h m a n , S.M., G u n n , J . M . , F i s h e r , L.M., H a n s o n , R.W., R e s h e f , L. a n d B a i l a r d , F.J. ( 1 9 7 5 ) J. Biol. Chem. 250, 3322--3329 Allen, C. a n d K e n d a l l , J.W. ( 1 9 6 7 ) E n d o c r i n o l o g y 80, 9 2 6 - - 9 3 0 M e l l m a n , W.J. ( 1 9 7 1 ) A d v . H u m a n G e n e t . 2, 2"59~--306 Z a c c h e l l o , F. ( 1 9 7 2 ) B i o c h e m . J. 1 2 6 , 27 p. H a n s o n , R.W., R e s h e f , L. a n d B a l l a r d , J . F . ( 1 9 7 5 ) F e d . P r o c . 34, 1 6 6 - - 1 7 0 K r o n e , W., M a r q u a r d t , W., Seitz, H . J . a n d T r a n o w s k i , W. ( 1 9 7 6 ) B i o c h i m . B i o p h y s , A c t a 4 5 1 , 7 2 - - 8 1 Wicks, W.D. ( 1 9 7 4 ) in A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h ( G r e e n g a r d , P. a n d R o b i n s o n , G . A . , eds.), Vol. 4, p p . 3 3 5 - - 4 3 8 , R a v e n Press, N e w Y o r k I y n e d j i a n , P.B. a n d H a n s o n , R.W. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 6 5 5 - - - 6 6 2 E r n e s t , M.J. a n d F e i g e l s o n , P. ( 1 9 7 8 ) J. Biol. C h e m . 2 5 3 , 3 1 9 - - 3 2 2 I y n e d j i a n , P.B. a n d H a n s o n , R.W. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 8 3 9 8 - - 8 4 0 3 H o l t e n , D . D . a n d N o r d l t e , R.C. ( 1 9 6 5 ) B i o c h e m i s t r y 4, 7 2 3 - - 7 3 1 B a l l a r d , F . J . ( 1 9 7 0 ) B i o c h e m . J. 1 2 0 , 8 0 9 - - 8 1 4 D i e s t e r h a f t , M., S h r a g o , E. a n d S a l l a c h , H.J. ( 1 9 7 1 ) B i o c h e m . Med. 5, 2 9 7 - - 3 0 3 S c h a t z , G , a n d M a s o n , T.L. ( 1 9 7 4 ) A n n u . Rev. B i o c h e m . 4 3 , 5 1 - - 8 7
Biochimica et Biophysica Acta, 521 (1978) 792--804 © Elsevier/North-Holland Biomedical Press
BBA 99315
INDUCTION OF MITOCHONDRIAL PHO~PHOENOLPYRUVATE CARBOXYKINASE IN CULTURED HUMAN FIBROBLASTS
IFEANYI J. ARINZE, R E N G A C H A R I
RAGHUNATHAN
and J A M E S D. R U S S E L L
Department of Biochemistry and Nutrition and the Division of Genetics and Molecular Medicine, Meharry Medical College, Nashville, Tenn. 37208 (U.S.A.) (Received April 24th, 1978)
Summary Mitochondria of cultured normal human fibroblast cells were found to contain the enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme in these cells is increased 2- to 3-fold by addition of 5 . 1 0 - 4 M dibutyryl cyclic AMP, or 1.5- to 2-fold by the addition of dexamethasone ( 2 - 1 0 -7 M) or hydrocortisone (1.38" 10 -6 M). These increases in enzyme activity were inhibited by cycloheximide and actinomycin D, suggesting they are dependent upon de novo protein synthesis. Cultured human fibroblasts may thus provide a useful system for studying the regulation of mitochondrial phosphoenolpyruvate carboxykinase.
Introduction
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is one of the key regulatory enzymes in gluconegenesis [1]. Two immunologically distinct forms, mitochondrial and cytosolic, of this enzyme exist in tissues of a variety of mammalian species [2,3]. In many species including the human, both forms of the enzyme are present in liver in about equal proportions whereas in others the predominating form is either the mitochondrial or the cytosolic form [3,4]. The activity of the cytosolic enzyme can be altered by dietary and hormonal manipulations [4--8] and the control of the activity of the enzyme during these manipulations appears to involve regulation of enzyme synthesis [9--11 ]. The mitochondrial form of phosphoenolpyruvate carboxykinase has generally been regarded as a non-inducible enzyme since its activity is not altered by conditions which lead to changes in the overall rate of gluconeogenesis [2,12--14]. However, Elliot and Pogson [15] have recently suggested that the mitochondrial form of the enzyme in guinea-pig liver might in fact be 'induced' under starvation conditions. In species which have both forms of the enzyme, the
793 mitochondrial form has been shown to contribute up to 50% of the carbon flow to glucose during gluconeogenesis from lactate in the perfused liver
[16--19]. As a part of our program to develop a suitable system to study genetically determined metabolic abnormalities using cultured cells, we showed recently that fibroblast cultures derived from human skin contain measurable activities of phosphoenolpyruvate carboxykinase [20]. The present paper shows that the activity of this enzyme in these fibroblasts is associated only with mitochondria and cochromatographs with the mitochondrial phosphoenolpyruvate carboxykinase from guinea-pig liver. In these cells the activity of the enzyme is elevated by dexamethasone and dibutyryl cyclic AMP in a manner suggesting enhanced rates of enzyme synthesis. Materials and Methods
Chemicals. NaH14CO3, Aquasol, [5-3H]uridine, and I~[5-3H]proline were purchased from N e w England Nuclear Corporation. Culture medium F-10, fetal bovine serum, and trypsin were obtained from Grand Island Biological Company. Actinomycin D, cycloheximide, theophylline, dibutyryl adenosine cyclic 3',5'-monophosphate (dibutyryl cyclic AMP), hydrocortisone, and dexarnethasone (A1'2,9a-fluoro,16a-methylprednisolone) were obtained from the Sigma Chemical Company. Insulin was a gift from Dr. William W. Bromer, Eli Lilly and Company, Indianapolis. Tissue culture. Fibroblast cultures were initiated as described [20]. Except for the experiments reported in Table Ill,all experiments were performed using fibroblast strain 130. The general characteristics of this strain have been described [20]. Solutions of hormones or inhibitors were made with the appropriate culture medium before addition to the cultures as indicated in the legends to tables and figures. Enzyme assays. Cell monolayers were washed twice with 0.85% NaCl solution and scraped from the dishes with a rubber policeman. The cells from 8 60-ram dishes were then suspended in 2 ml of 0.25 M sucrose and sonified for 4 15-s intervals in a Branson cell disrupter, Model W185, at a setting of 5. The sonified extract was used for enzyme assays. In some experiments the cells were released from monolayer by treatment with 0.025% solution of trypsin in medium F-10. Phosphoenolpyruvate carboxykinase activity was measured at 37°C by a modification [14] of the 14C bicarbonate fixation method of Chang and Lane [21]. Lactate dehydrogenase (EC 1.1.1.27) and citrate synthase (EC 4.1.3.7) activities were measured as described [22]. All enzyme assays were conducted in duplicate under conditions where enzyme activitieswere proportional to both time and enzyme concentration. Protein was determined by the method of Lowry et al. [23]. Measurement of radioactivityin RATA and protein. For the incorporation of radioactivity into total R N A and protein, the cells were pulsed for I h with [3H]uridine (1.5 /zCi/ml of medium F-10 containing 10% fetal bovine serum) and [3H]proline (7 ;zCi/ml of the same medium), respectively. At the end of the labeling period, the medium was removed, 2 ml/dish of cold trichloroacetic acid solution (5%, w/v) were added and after incubation for 20 rain at 4°C,
794
the dishes were rinsed twice with 2 ml of the trichloroacetic acid solution. They were further rinsed twice with 2 ml of H:O. 1 ml of a 0.25% solution of trypsin (in medium F-10) was then added and the dishes incubated for 20 min at 37°C. At the end of this period the whole volume (1 ml) was counted for radioactivity after adding 10 ml of Aquasol.
Separation of phosphoenolpyruvate carboxykinase activities by hydroxyapatite chromatography. Cell or tissue extracts were applied to the hydroxyapatite (Biorad HPT) column (1 × 14 cm) and phosphoenolpyruvate carboxykinase was eluted with a linear gradient of 10--250 mM potassium phosphate buffer, pH 7.0, at 2°C [24]. Results
Response of phosphoenolpyruvate carboxykinase activity to addition of various hormones to the culture medium. At the concentrations tested, hydrocortisone and dexamethasone increased the activity of phosphoenolpyruvate carboxykinase by 1.6- and 1.8-fold, respectively, in 6 h after an initial 2-h lag period (Table I). This lag was clearly discernible in the case of hydrocortisone, and has been observed in a similar response of this enzyme to steroid hormones in adrenalectomized rats [6] and in Reuber H-35 hepatoma cells [ 2 5 ] . Insulin, at I • 10 -s M, had no effect on the activity of the enzyme at 6 h b u t when added with dexamethasone it suppressed the dexamethasone effect b y a b o u t 30%. Addition of dibutyryl cyclic AMP at a final concentration of 5 • 10 -4 M (in the presence of 1 mM theophyUine) led to a 2.5-fold elevation in the activity o f the enzyme within 6 h. Unlike the steroid-induced increases in
TABLE I EFFECT OF HYDROCORTISONE, DEXAMETHASONE, INSULIN, AND DIBUTYRYL CYCLIC AMP O N PHOSPHOENOLPYRUVATE C A R B O X Y K I N A S E A C T I V I T Y I N H U M A N F I B R O B L A S T S All a d d i t i o n s w e r e m a d e a t z e r o t i m e to c u l t u r e s in s t a t i o n a r y p h a s e . Cells w e r e c o l l e c t e d a f t e r 2 a n d 6 h and processed for e n z y m e d e t e r m i n a t i o n . Values are m e a n s ~ S.E.M. for three e x p e r i m e n t s . The activ/ty o f the e n z y m e at zero t/me was 2.21 ± 0.07 n m o l / m g protein per rain. Addition to culture medium
Final conc. (M)
Phosphoenolpyruvate
carboxykinase activity ( n m o l / m g p r o t e i n p e r r a i n ) at 2h
None Hydrocortisone Dexamethasone Insulin De x a m e t h a s o n e + insulin D i b u t y r y l cyclic AMP + theophylline Theophylline D i b u t y r y l cyclic AMP + theophylllne + dexamethasone
1.38 • 10-6 2 . 0 • 10-7 1.0 10-8 2.0 10-~ 10-8 1.0 10-4 5.0 10-3 1.0 10-3 1.0 10-4 5.0 10-3 1.0 10-7 2.0
2.19 2.13 2.55 1.47
6h ± 0.10 ± 0.08 ± 0.07 ± 0.10
2.22 3.44 3.95 2.32
± + ± ~
0.07 0.12 0.22 0.10
2.06 ± 0.12
3.43 ± 0.11
3.71 ± 0 . 2 3 2.22 ± 0.05
5.43 ± 0 . 2 0 2 . 4 5 +_ 0 . 0 8
3 . 5 2 +- 0 . 2 4
5.41 ± 0 . 2 6
795
enzyme activity, no lag was associated with the increase caused by dibutyryl cyclic AMP. Theophylline alone had a negligible effect and in cells to which dexamethasone, dibutyryl cyclic AMP, and theophylline were added together, the increase in enzyme activity was equal to that produced by dibutyryl cyclic A M P and theophylline, suggesting that no synergistic interaction between the cyclic nucleotide and the steroid had occurred. Effect of serum. Many previous studies on enzyme induction in cultured cells have been performed in serum-free media. These studies have usually involved cells of neoplastic origin [25--28]. The fibroblasts used in our studies were derived from normal dermis, display no evidence of transformation, and cannot be grown or maintained for an appreciable length of time in the absence of serum. Therefore, the effect of serum on the induction of phosphoetlo/pyruvate carboxykinase by dexamethasone was investigated. Fig. 1 A shows that in the serum-containing medium, there was no appreciable change in the basal activity of the enzyme in control cultures during the dexarnethasoneinduced increases in enzyme activity. However, when the medium was changed at the time of addition of hormone to one lacking serum, the basal activity of the enzyme could not be maintained and actually was reduced by about 3 0 % in 4 h before gradually rising to initial basal levels (Fig. 1B). Although it was still possible to demonstrate increases in enzyme activity due to hormone addition in the serum-free medium, the hormone appeared to merely sustain the original basal activity for the first 6 h. In cultures starved of serum for 18 h prior to addition o f hormone, only 50% of the basal activity of phosphoenol. pyruvate carboxykinase could be detected (data n o t shown). The enzyme activity was still elevated u p o n the addition of steroid to these cultures b u t the basal activity of the enzyme in the serum-free cultures was progressively changing
40
~3.0
H
E E c
~ 2.0
g
g
TIME, HOUR Fig. 1. The e f f e c t o f 0el~um and t h e time cou-,~ o f t h e i n d u c t i o n o f p h o $ p h o e n o l p y ~ v a t e elurboxy]rJuaMe b y dexlmaethBlone. DeY=metbaaone (@) (2 • 10 -7 M) we= a d d e d at zero time to I t a t i n n a r y phale c u l t u l ~ l i n IerIH1n-con~Ilnln.I m e d i u m A a n d i n N r u m - f r e e m e d / u m B. T h e e n z y m e a c t i v i t y w a I m c u u . ~ d a t v a r i o u I times thereafter. Dexamethuone waI not added to control (o) cultures. The values plotted in A are m e a n s ~ S.E.I~L f o r t h r e e e x p e r i m e n t s . T h e v a l u e s i n B a r e m e a n s f o r t w o e x p e r i m e n t s .
796 and resembled the pattern shown in Fig. lB. Since the data in Fig. 1 demonstrate that the activity of phosphoenolpyruvate carboxykinase can be elevated by dexamethasone either in the presence or absence of serum, and because the basal enzyme activity could not be maintained relatively constant in the absence of serum, all subsequent experiments were performed in cultures maintained in serum-containing media. The effect o f serum on er#zyme induction by dibutyryl cyclic AMP was not investigated. Time course of the effect of dexamethasone. The maximum increase (70%) in phosphoenolpyruvate carboxykinase activity produced by the addition of dexamethasone at a final concentration of 2 - 10 -7 M occurred at 6 h (Fig. 1A). Subsequently ~the enzyme activity rapidly decreased to only 27% of the control by 24 h. When t h e cultures were re-fed at two 6-h intervals beginning at 6 h after the intial treatment with the hormone, the enzyme activity was slightly higher (<10%) than indicated in Fig. 1A but the decline in enzyme activity which begins at 6 h was still apparent, indicating that the decline was not due to depletion of nutrients in the medium. Dose-response relationships. These are shown in Fig. 2. The optimal concentration of dexamethasone found (2" 10 -7 M) is clearly in the physiological range since the circulating levels of glucocorticoids in humans are about 3 • 10 -7 M [29]. The optimal dose (5 • 10 -4 M) of dibutyryl cyclic AMP noted in Fig. 2 is identical to the concentration of this nucleotide which has been shown to maximally induce phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 cells [26].
Effect of dexamethasone and dibutyryl cyclic AMP on phosphoenolpyruvate carboxykinase activity during cell growth. We have previously shown that the specific activity of phosphoenolpyruvate carboxykinase in human fibroblasts increases during logarithmic growth and attains a constant level during stationary phase [20]. The growth pattern of strain 130 used in the present study is
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R i ~ i A ! 10-7 10-5 10-3 CONCENTRATION, M FiE. 2. R e s p o n s e o f phosphoenolpyruvate earboxykiname a c t i v i t y t o varying c o n c e n t r a t i o n s o f d e x a m e t h a s o n e and d i b u t y r y l c y c l i c AMP. D e w n m e t h a s o n e ( o ) and d i b u t y r y l c y c l i c AMP (o) w e r e a d d e d t o fibroblast c u l t u r e s in s t a t i o n a r y plmse. T h e o p h y U i n e (1 r a M ) w a s a d d e d at t h e s a m e t i m e t o all a d d i t i o n s o f d l b u t y r y l c y c l / c A M P and t h e e n z y m e a c t i v i t y w a s m e a s u r e d 6 h a f t e r addition. T h e ~Peeific a c t i v i t y o f phosphoenoipyruvate c a r b o x y k i n a s e in c o n t r o l c u l t u r e s w a s 1.99 n m o l / m g p r o t e i n per rain. ~
2.0
i 10-9
i
797 similar to those previously reported. It was therefore of interest to re-examine the activity of phosphoenolpyruvate carboxykinase throughout the culture cycle in cells grown in the presence of dexamethasone. In the presence of an optimal inducing dose of the steroid (2 • 10 -7 M) in the culture medium, the specific activity of the enzyme was a b o u t 2-fold higher than in control cultures b u t the pattern of increase throughout the growth cycle was identical in both cases (Fig. 3A). This pattern of increase is consistent with the observation that the specific activities of many enzymes vary in parallel fashion with increased cell density [30]. Increased levels of cyclic AMP have been reported with increased growth of cultured cells [31] although it is n o t clear whether the increased levels of this nucleotide cause the high levels of enzyme activity seen at confluency. Fig. 3B shows that when dibutyryl cyclic AMP was added (in the presence of 1 mM theophylline) to growing cells the specific activity of phosphoenolpyruvate carboxykinase, measured at 6 h after the addition, increased 2.6-fold in cells which were in stationary phase. The effect was much greater in cells in log phase b u t the maximum attainable specific activity was not observed in cells in early log phase. It was significant, however, that the maximum induced activity was reached 3--4 days before the cells entered confluency and much earlier than in cells treated with dexamethasone.
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DAYS IN CULTURE F i g . 3. C h a n g e s i n t h e s p e c i f i c a c t i v i t y o f phosphoenolpyruvate c a r b o x y k i n a s e d u r i n g c e l l g r o w t h . I n A, c e l l s w e r e p l a n t e d ( 5 • 1 0 --4 c e l l s / d i s h ) i n 3 5 m m plastic p e t r l d i s h e s a n d f e d d a i l y w i t h n u t r i e n t m e d i u m c o n t a i n i n g ( e ) o r l a c k i n g ( o ) d e x a m e t h a s o n e ( 2 . 1 0 - 7 M). T h e e n z y m e a c t i v i t y w a s m e a s u r e d o n t h e m o r n i n g o f t h e d a y s i n d i c a t e d . I n B, t h e c e l l s w e r e g ~ o w n as i n A w i t h o u t t h e i n c l u s i o n o f d e x a m e t h a s o n e i n t h e m e d i u m . O n the d a y s i n d i c a t e d , t h e cells w e r e r e f e d w i t h f r e s h m e d i u m c o n t a i n i n g e i t h e r
d e x a m e t h a s o n e ( 2 • 1 0 - 7 M) ( e ) o r d i b u t y r y l c y c l i c A M P ( 5 • 1 0 - 4 M) p l u s 1 m M t h e o p h y l l J n e (m). T h e enzyme activity was measured 6 h later.
798
Effect of actinomycin D and cycloheximide on hormone-induced increases in phosphoenolpyruvate carboxykinase activity. In order to ascertain whether the increases in phosphoenolpyruvate carboxykinase activity in human fibroblasts involved alterations in enzyme levels or activation of a pre-existing enzyme species, we studied the effects of dexamethasone and dibutyryl cyclic AMP in the presence of inhibitors of RNA and protein synthesis. Neither actinomycin D nor cycloheximide affected the basal activity of phosphoenolpyruvate carboxykinase for up to 6 h in uninduced cultures (Table II). In cells treated for 6 h with dexamethasone after 30 min prior exposure to actinomycin D, the dexamethasone-induced increase in enzyme activity was abolished. The activity of the enzyme was still elevated by dibutyryl cyclic AMP; the increase being only about 56% of that produced by the nucleotide in the absence of the transcriptional inhibitor. The concentration of actinomycin D (! gg/ml) used was sufficient to block total RNA synthesis by 94% 1 h after the addition of the inhibitor (Table II). When cycloheximide was added at a concentration of 5 #g/ml which blocks the synthesis of cell-retained proteins b.Y 93%, both the dexamethasone- and dibutyryl cyclic AMP-induced increases in enzyme activity were suppressed. These findings strongly suggest that de novo synthesis of the enzyme is increased by both dibutyryl cyclic AMP and dexamethasone. However, more direct evidence must await further studies involving direct quantitation of phosphoenolpyruvate carboxykinase protein in these cells. T A B L E lI EFFECT OF INHIBITORS OF RNA AND PROTEIN CARBOXYKINASE ACTIVITY
SYNTHESIS
ON PHOSPHOENOLPYRUVATE
A c t i n o m y c i n D (1 p g / m l ) and c y c l o h e x i m i d e (5 ~ g / m l ) w e r e a d d e d to stationary phase cultures 3 0 min prior to the a d d i t i o n o f d e x a m e t h a s o n e (2 • 1 0 - 7 M) o r d i b u t y r y l cyclic A M P ( 5 . 1 0 --4 M). T h e o p h y l l i n e , 1 r a M , w a s a d d e d t o g e t h e r w i t h d i b u t y r y l c y c l i c A M P . T h e ce il- w e r e pulse-labelled w i t h [3H]ttrldine and [ ~ H ] p r o l i n e for t w o 1-h p e r i o d s beginning at the time o f a d d i t i o n o f h o r m o n e (zero time), and again at the 5th h after the a d d i t i o n , i n c o r p o r a t i o n o f radioactivity i n t o trichloroacetie acid-preciplteble m a t e r i a l w a s m e a s u r e d . T h e e n z y m e activity w a s m e a s u r e d in the appropriate cultures 0 and 6 h after the a d d i t i o n o f d e x a m e t h a s o n e o r d i b u t y r y l cyclic AMP. T h e results g/yen is rapresenteUve o f three e x p e r i m e n t s . Inhibitor
Treatment
Time:
Control
Control
D i b u t y r y l cyclic AMP + theophylline
Dexamethuone
Oh
6h
6h
6h
None AeClnomycin D Cyclohextmlde
Phosphoenoipyruvate carboxykinue, nmol/mg protein per min 2.09 2.19 5.44 3.53 1.08 1.95 3.06 1.69 2.95 2.03 2.41 2.52
None Act/nomycin D Cyclochexlmide
Radioact/vlty incorporated into total 7 688 8 063 7 478 194 7 237 6 265 5
Nome Aetinomycin D Cyelohexim/de
Rad/oactivity incorporated into protein, epm/dlah 34 278 24 68$ 22 58a 34 973 14 746 14 8 9 5 2 524 1 706 1 667
RNA, epm/dish 106 184 412
7 453 180 6 223 23 7 1 4 14 6 6 8 1 783
799
Effect of dexamethasone and dibutyryl cyclic AMP on phosphoenolpyruvate carboxykinase activity in different fibroblast strains. A previous study from our laboratories had demonstrated a wide variation in the activities of phosphoenolpyruvate carboxykinase in human fibroblast strains [20]. This variation was n o t unexpected since the strains represented cultures from unrelated individuals. In fact, in the population studied two strains completely lacked the activity o f the enzyme [20]. A comparison of our previous data [20] and the present study shows that the basal levels of phosphoenolpyruvate carboxykinase in some o f the strains previously studied were, in fact, almost as high as the induced levels demonstrated for strain 130 in the present study. The inducing effects of b o t h dexamethasone and dibutyryl cyclic AMP in nine different strains selected from our previously studied population which exhibit widely different basal activities of phosphoenolpyruvate carboxykinase are presented in Table III. In one strain (strain no. 18) there was no detectable activity of the enzyme either in the presence or absence of dexamethasone or dibutyryl cyclic AMP. In all other strains examined, the addition of dexamethasone increased the activity of the enzyme by about 1.5- to 1.8-fold while the increase produced b y addition of dibutyryl cyclic AMP was 2.5- to 3-fold. These data indicate that the inducibility of phosphoenolpyruvate carboxykinase is a c o m m o n phenomenon occurring in human fibroblasts and is n o t unique to a single cell line.
Distribution of phosphoenolpyruvate carboxykinase in control and dibutyryl cyclic AMP-treated human fibroblast 130 cells. The standard technique of cell organelle fractionation by differential centrifugation was used. Fibroblasts were initially trypsinized to make them more rounded and presumably easier to homogenize. The whole homogenate was then fractionated b y the technique previously used for mammalian liver [22]. The activity of phosphoenolpyruvate carboxykinase in these fractions as well as that of marker enzymes, citrate synthase and lactic dehydrogenase, were determined. As evident in Table IV a b o u t 80% of the total activity of phosphoenolpyruvate carboxyT A B L E III EFFECT OF DEXAMETHASONE AND DIBUTYRYL CYCLIC AMP ON PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN DIFFERENT STRAINS OF HUMAN FIBROBLASTS E n z y m e a c t / v i t l e s w e r e d e t e r m i n e d i n cells f r o m s t a t i o n a r y p h a s e c u l t u r e s . D e x a m e t h a s o n e (2 • 1 0 - ' / M) o r d / b u t y r y l c y c l i c A M P (5 • 1 0 - 4 M) a n d t h e o p h y l l i n e ( 1 0 - 3 M) w e r e a d d e d t o t h e c u l t u r e s 6 h b e f o r e t h e c e l l s w e r e u s e d f o r e n z y m e assay. T h e v a l u e s g i v e n are t h e average o f t w o e x p e r i m e n t s .
Strain number
21 24 30 52 130 18 38 44 50
Treatment (nmol/mg protein per min)
Control
Dexamethasone
Dibutyryl cyclic AMP + theophyliine
1.28 1.53 0.96 1.65 2.14 0 0.61 1.96 3.21
2.07 2.22 2.18 2.82 3.82 0 1.08 3.43 5.07
3.36 3.66 3.24 3.67 5.44 0 1.83 4.98 7,84
800 T A B L E IV
I N T R A C E L L U L A R D I S T R I B U T I O N OF P H O S P H O E N O L P Y R U V A T E C A R B O X Y K I N A S E A C T I V I T I E S IN C O N T R O L A N D D I B U T Y R Y L C Y C L I C A M P - T R E A T E D H U M A N F I B R O B L A S T S E n z y m e activities w e r e m e a s u r e d in cells h a r v e s t e d f r o m s t a t i o n a r y p h a s e c u l t u r e s o f strain 130. In d i b u tYlTI cyclic A M P - t r e a t e d c u l t u r e s , cells w e r e h a r v e s t e d for e n z y m e assays 8 h a f t e r a d d i t i o n of d i b u t y r y l cyclic A M P (5 • 10 -4 M) a n d t h e o p h y l l l n e (10 -3 M). T h e cells w e r e s u s p e n d e d using 0 . 0 2 5 % s o l u t i o n of t r y p s i n in F-10. F e t a l b o v i n e s e r u m was a d d e d to the s u s p e n s i o n (10% v / v ) t o i n h i b i t f u r t h e r p r o t e o l y t i c a c t i v i t y a n d t h e cells w e r e c o l l e c t e d b y c e n t r i f u g a t i o n a n d w a s h e d w i t h saline. T h e cells w e r e t h e n h o m o g e n i z e d a n d f r a c t i o n a t e d as d e s c r i b e d [ 2 2 ] . T r y p s i n i z a t i o n h a d n o e f f e c t on t h e t o t a l a c t i v i t y o f p h o s p h o enolpyruvate c a r b o x y k l n a s e i n t h e c u l t u r e s . T h e v a l u e s are given in n m o l / d i s h p e r m i n a n d are t h e average of three experiments.
Phosphoenolpyruvate
Citrate
carboxykinase
synthase
Lactate dehydrogenasc
Control Whole h o m o g e n a t e Nuclear pellet Mitochondrial pellet Postomitochondrial supernatant
1.87 0.13 1.50 0
29.8 3.8 21.3 0
389 57 4 288
D i b u t y r y l cyclic AMP Whole h o m o g e n a t e Nuclear pellet Mitoehondrial pellet Post-mitochondrial supernatant
4.38 0.24 3.99 0
40.4 6.1 31.9 0
585 72 13 483
kinase was associated with the washed mitochondria. No detectable activity was found in the post-mitochondrial supernatant. Less than 1% of the activity of the mitochondrial marker enzyme, citrate synthase was found in the postmitochondrial supernatant, while 72% of its total activity was associated with the mitochondria. The recovery of lactate dehydrogenase, an enzyme marker for cytosol, in the post-mitochondrial supernatant was about 74%. Contamination of the mitochondrial pellet by the cytosol fraction was judged to be less than 2% on the basis of the activity of lactate dehydrogenase activity found in the mitochondrial fraction. It can also be seen from Table IV t h a t in dibutyryl cyclic AMP-induced cells, a 2.7-fold increase in phosphoenolpyruvate carboxykinase activity was observed in the mitochondrial fraction which is in general agreement with the effect of this nucleotide in whole cells (Table I, see also Fig. 2 and 3); again, no detectable activity could be seen in the post-mitochondrial supernatant even after dibutyryl cyclic AMP induction. The data in Table IV is consistent with the conclusion that within the limits of our assay only the mitochondrial form of phosphoenolpyruvate carboxykinase is contained in these fibroblasts. Additional evidence is provided by the data in Fig. 4 where this activity has been examined after chromatography on h y d r o x y a p a t i t e columns. It has been demonstrated by Ballard [24] that a mixture of the cytosolic and mitochondrial forms of phosphoenolpyruvate carboxykinase can be completely separated at 2°C on hydroxyapatite columns by applying a linear gradient of 10--250 mM potassium phosphate at neutral pH. The technique can be applied to impure enzyme preparations [24]. Fig. 4A shows that this technique completely resolved the two activities of this enzyme normally present in guinea-pig liver, the mitochondrial activity was eluted first at low concentrations of phosphate, while the cytosolic enzyme was eluted at
801
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Fig. 4. C h r o m a t o g r a p h y o f p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e o n h y d r o x y a p a t i t e c o l u m n s . H y d r o x y a p a t i t e ( 4 g) w a s p r e p a r e d in I 0 m M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 0 , a n d p o u r e d i n t o a c o l u m n 1 c m in d i a m e t e r . T h e c o l u m n w a s e q u i l i b r a t e d w i t h t h e s a m e b u f f e r . 2 . 5 m l o f s o n i f i e d g u i n e a - p i g liver t i s s u e o r f i b r o b l a s t e x t r a c t s w e r e a p p l i e d t o t h e c o l u m n a n d t h e e n z y m e w a s e l u t e d at 2°C w i t h a linear g r a d i e n t of 10 to 250 mM potassium phosphate buffer, pH 7.0. 2.5-mi fractions were collected and the activity o f phosphoenolpyruvate c a r b o x y k i n a s e in e a c h f r a c t i o n w a s d e t e r m i n e d u s i n g 1 0 0 /~I a l i q u o t s o f e a c h f r a c t i o n for assay. T h e c o l u m n s w e r e r e u s e d o n l y o n c e . A, g u i n e a - p i g liver w h o l e h o m o g e n a t e ( 1 0 % w/v)" B, t h r i c e w a s h e d g u l n e a - p i g liver m i t o c h o n d r i a l s u s p e n s i o n ; C, c y t o s o l ( 1 0 0 0 0 0 X g s u p e r n a t a n t ) o f g u i n e a - p i g liver; D , f i b r o b l a s t e x t r a c t o f strain 1 3 0 at c o n f l u e n c y .
higher phosphate concentrations. The identity of the two peaks was established in separate runs {Fig. 4B, C). When an extract of human fibroblast 130 was applied to the column and a similar phosphate gradient was applied, only one peak of enzyme activity could be detected (Fig. 4D), supporting the data in Table IV that only one form of phosphoenolpyruvate carboxykinase is present in the fibroblasts. This activity was eluted at the same position as the mitochondrial phosphoenolpyruvate carboxykinase from guinea-pig liver. When the fibrobtast extract was mixed with a 100-fold dilution of washed mitochondrial extract from guinea-pig liver the mixture chromatographed as a single peak which resembled Fig. 4D. The specific activity of phosphoenolpyruvate carboxykinase in fibroblast mitochondria was 9.03 + 0.43 nmol/mg protein per min as opposed to 202 + 8 nmol/mg protein per min for the enzyme in guineapig liver mitochondria. Discussion Cell culture provides a relatively homogeneous source of tissue which Can be utilized to investigate the metabolic processes of intact cells as well as regula-
802 tion of specific enzymes. Glucorticoids and cyclic AMP have been shown to increase the rate of phosphoenolpyruvate carboxykinases synthesis in fetal [32] as well as adult rat liver [11] in vivo, and in Reuber H-35 hepatoma cells [25--28]. The cyclic AMP-stimulated increase in the activity of the enzyme in the perfused rat liver is also dependent on new protein synthesis as evidenced b y its inhibitability b y cycloheximide [33]. Although either cyclic AMP or glucocorticoids can independently cause increases in the activity of phosphoenolpyruvate carboxykinase, studies in the perfused rat liver suggest a permissive effect of glucocorticoids on the effect of cyclic AMP [33]. The results of the present studies show that the phosphoenolpyruvate carboxykinase activity in human fibroblasts is also increased by dibutyryl cyclic AMP and glucocorticoids. The effects of these agents do not appear to be synergistic. The optimal concentration of dexamethasone needed to achieve maximal increase in phosphoenolpyruvate carboxykinase activity in fibroblasts compares favorably with the circulating levels of glucocorticoids in human plasma which have been reported to be 3 • 10 -7 M [29]. Unlike Reuber H-35 cells where the activity of the enzyme remains elevated at the induced levels even after removal of the inducing steroid for up to 6 h, the activity of the enzyme in human fibroblasts begins to decline almost immediately after maximal elevation b y dexamethasone even in the continued presence of the steroid (Fig. 1A). In the presence of dibutyryl cyclic AMP, a maximal increase in enzyme activity is achieved at a concentration (5- 10 -4 M) (Fig. 2) which has been shown to produce a 7-fold increase in the synthesis of phosphoenolpyruvate carboxykinase-specific protein in Reuber H-35 cells where the specific activity of the enzyme is increased a b o u t 1.6-fold [26]. Although the specific molecular mechanism(s) of the action of glucocorticoids and cyclic AMP on phosphoenolpyruvate carboxykinase induction is not y e t fully understood, a great deal of our current understanding of these molecular events has come from the studies in the laboratories of Wicks [25, 27,34] and Hanson [11,26,28] using Reuber H-35 hepatoma cells. Direct radioimmunochemical measurements [26,28] using these cells have demonstrated repeatedly that the increase in phosphoenolpyruvate carboxykinase activity provoked b y either glcocorticoids or cyclic AMP results from a rise in the synthesis rate of the enzyme. According to Wicks [34], glucocorticoids p r o m o t e an increase in translatable m R N A for phosphoenolpyruvate carboxykinase, while cyclic AMP enhances enzyme synthesis by stimulating the translation of pre-existing specific m R N A templates. Other studies [26], however, suggest that dibutyryl cyclic AMP may affect multiple sites in such a way as to act translationally or posttranscriptionally. In fact, the recent studies by Iynedjian and Hanson [35] provide evidence suggesting that in the rat liver the cyclic nucleotide acts transcriptionally to increase the amount of translatable phosphoenolpyruvate carboxykinase-specific mRNA. Cyclic AMP has also recently been shown to increase the synthesis of m R N A for tyrosine aminotransferase in rat liver [36]. The phosphoenolpyruvate carboxykinase in rat kidney cortex and in Reuber H-35 cells is localized predominantly in the cytosol. Purified antibodies prepared in rabbits or goats against rat liver cytosolic phosphoenolpyruvate carboxykinase precipitate the enzymes in rat kidney cortex [37] or Reuber
803 H-35 cells [25--28]. However, this antibody does not precipitate the phospho-
enolpyruvate carboxykinase in rat liver mitochondria, guinea-pig liver or kidney mitochondria, or any other phosphoenolpyruvate carboxykinase of mitochondrial origin [2]. This and other evidence [24,38--40] show that the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase have different immunochemical, kinetic, and physicochemical properties. Our present studies indicate that the phosphoenolpyruvate carboxykinase activity in human fibroblasts is associated with mitochondria. These fibroblasts completely lack the cytosolic enzyme (Table IV, Fig. 4). To our knowledge this is probably the only system investigated in which one form of this enzyme appears to be completely lacking. Cultured human fibroblasts may provide a potentially useful model system to probe the specific regulation of mitochondrial phosphoenolpyruvate carboxykinase. As seen in the present study, the activity of this enzyme in these cells can be increased by various agents in a manner consistent with new protein synthesis (Table II). The actual intracellular site of synthesis cannot be discerned from the present data. However, the sensitivity of the induction of the enzyme in these cells to cycloheximide which does not inhibit mitochondrial protein synthesis [41] suggests that the phosphoenolpyruvate carboxykinase in human fibroblasts may be synthesized on cytoplasmic ribosomes. The inability to detect any activity of the enzyme in the cytosol during induction by dibutyryl cyclic AMP (Table IV) suggests that the newly synthesized enzyme may be inactive or that it is rapidly translocated into the mitochondria soon after synthesis. In previous work in animal systems the mitochondrial phosphoenolpyruvate carboxykinase, unlike the cytosolic enzyme has generally been found to be constitutive [ 12--14], and virtually nothing is known regarding the specific regulation of its level in mitochondria. The presence in human fibroblasts of mitochondrial phosphoenolpyruvate carboxykinase the activities of which varies with different culture conditions should facilitate studies on aspects of the regulation of this enzyme at the molecular level. These cells might also prove useful in investigating the possible involvement of altered levels or regulation of phosphoenolpyruvate carboxykinase in human disease.
Acknowledgements We thank Dr. Shirley B. Russell for valuable help with some of the experiments, Mrs. Kathryn M. Trupin for excellent technical assistance, and Drs. Sidney P. Colowick, Richard W. Hanson and Mulchand S. Patel for advice during the preparation of the manuscript. This work was supported in part by grants HD 08792 and CA 17229 from the National Institutes of Health, and by a Basil O'Connor Research Starter Grant, 5-89, from the National FoundationMarch of Dimes. I.J.A. and J.D.R. are recipients of Career Development Awards, HD 00226 and CA 00122, from the National Institutes of Health. References 1 Scrutton, M.C. and Utter, M.F. (1968) Annu. Rev. Biochem. 37, 294--302 2 Ballard, F.J. and Hanson, R.W. (1969) J. Biol. Chem. 244, 5 6 2 5 - - 5 6 3 0 3 Hanson, R.W. and Garber, A.J. (1972) Am. J. Clin. Nutr. 25, 1010--1021
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4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
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