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Effects of altered hormonal states and fasting on rat-liver mitochondrial phosphoenolpyruvate carboxykinase levels
214
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 172
EFFECTS OF A L T E R E D HORMONAL STATES AND FASTING ON R A T - L I V E R M I T O C H O N D R I A L 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 LEVELS ROBERT C. NORDLIE, FREDERICK E. VARRICCHIO AND DAROLD D. HOLTEN Guy and Bertha Ireland Research Laboratory, Department of Biochemistry, University of North Dakota Medical School, Grand Forks, N.D. (U.S.A.)
(Received May I9th, 1964)
SUMMARY I. Rat-liver phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxylyase (transphosphorylating), EC 4. I. 1.32) levels have been measured following various hormonal treatments and fasting, and in control animals. 2. Adrenalectomy and hydrocortisone administration were without significant effect on mitochondrial carboxykinase levels, although amounts of this enzyme in soluble fraction of these same livers were nearly doubled b y administration of the glucocorticoid. 3- Allaxan diabetes, insulin administration, and fasting also were without statistically significant effect on levels of mitochondrial carboxykinase expressed per mg protein; increases of 55 % and 51%, respectively, in mitochondrial activity per g liver or per IOO g body weight were noted in diabetic animals. 4. It is concluded that any concerted participation of rat-liver pyruvate carboxylase (pyruvate:CO z ligase (ADP), EC 6.4.1.1) plus phosphoenolpyruvate carboxykinase in glucogenesis must involve principally the extramitochondrial carboxykinase.
INTRODUCTION NORDLIE AND LARDY1 previously have demonstrated a marked difference in subcellular distribution pattern of liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.I.I.3 2) among various m a m malian species. While guinea-pig enzyme activity, for example, was present in mitochondrial and soluble fractions in an approx. 2 : I ratio, about 9 ° % of total rat-liver activity was in soluble fraction. Carboxykinase of guinea-pig liver soluble fraction has been reported ~ to rise due to starvation or mannoheptulose administration while this activity in mitochondria of these animals was unaffected. Much more extensive studies 2 indicated that the carboxykinase of soluble fraction of rat liver responds markedly to a considerable number of hormonal and metabolic alterations. It has been suggested ~-4 that the carboxykinase plus pyruvate carboxylase (pyruvate: CO s ligase (ADP), EC 6.4.1.1) 4-6 systems may function together as an energetically favorable pathway for the conversion of pyruvate to phosphoenolpyruvate in the overall process of glucogenesis from lactate, pyruvate, or certain amino acids. The rat-liver carboxylase appears to be confined to mitochondria 3& The simplest concerted mechaBiochim. Biophys. Acta, 97 (1965) 214-221
MITOCHONDRIAL CARBOXYKINASE
215
nism for these two enzymes logically would involve mitochondrial carboxykinase, since a shuttling of intramitochondrially-produced oxaloacetate and, perhaps, GTP, through the mitochondrial membrane prior to reaction in the presence of solublefraction carboxykinase would be unnecessary. Since it has been reported that in normal rats, less than io % of total liver carboxykinase is found in mitochondria 1, we believed it possible that this mitochondrial enzyme activity might be markedly elevated by certain hormonal or metabolic stimuli which cause significant increases in blood glucose and liver glycogen levels; the intramitochondrial carboxykinase, normally only slightly active, would then function as part of a specialized mechanism responding to certain stressful conditions. For these reasons, rat-liver preparations were selected for the present investigations. Results of studies of the effects of adrenalectomy and hydrocortisone administration on both soluble fraction and mitochondrial phosphoenolpyruvate carboxykinase levels, and of alloxan diabetes, insulin administration, and fasting on the latter activity are described in this paper. MATERIALS AND METHODS
Young, adult male rats of Sprague-Dawley origin, weighing between 15o and 21o g, obtained through Simonsen Laboratories, Inc., White Bear Lake, Minn., were employed in these experiments. Adrenalectomies were performed by the supplier. All animals, unless otherwise noted, were maintained on Purina Lab Chow and water (or 1% NaC1 solution in the case of adrenalectomized animals), ad libitum. Animals were acclimated for at least 4 days after receipt from the supplier. Preparation of reagents, fractionation of livers, protein determinations, and phosphoenolpyruvate assays (Series I, Table I ; and Series III and IV, Table II) were as described previously1, 2. For the assays of enzyme from animals described in Series II, Table I, the mercury methodS, 9 was employed for phosphopyruvate determinations. Identical values were obtained with this method and with the LOHMANNM E Y E R H O F F 8'1° KI3 procedure in a supplementary, comparative experiment. Hydrocortisone, obtained from Sigma Chemical Co, was suspended in 1 % NaC1 solution and injected subcutaneously once (Series I, Table I) or twice (Series II, Table I) per day for a period of 4 days; total daily dosage was IO mg per animal. Diabetes (Series III, Table II) was produced by intraperitoneal injection of alloxan (200 mg per kg body weight). Those animals having blood sugar levels (determined by the SOMOGYI-NELSON11method) of between 285 and 47 ° mg per IOOml, were sacrificed I week after injection. A second group of animals in this series received daily subcutaneous injections of 20 units of protamine zinc insulin (Lilly) daily for 3 days, and were sacrificed 4 h after the last injection. Insulin-treated animals had blood sugar levels of 17-45 mg per IOO ml, while a third, untreated control group had levels between 71 and 98 mg glucose per IOO ml blood. Another group of animals (Series IV, Table II) was fasted for 48 h while controls for this group received Purina Lab Chow ad libitum, as before. Phosphoenolpyruvate carboxykinase activity in soluble fraction of liver was assayed as described previously1,2. Supplementary experiments were conducted to determine conditions under which initial reaction velocities for the relatively inactive mitochondrial enzyme also could be determined. In general, assay conditions employed with the soluble-fraction enzyme also were found satisfactory for mitochonBiochim. Biophys. Acta, 97 (1965) 214-221
R . C . NORDLIE et al.
216
drial-enzyme assay. In addition, however, 4 Fg oligomycin A plus B plus C (a gift of Dr. H. A. LARDY) per 1. 5 ml reaction mixture was included in the mitochondrialenzyme assay system, since it was found partially to suppress the rather active ITPase present in frozen-thawed mitochondria (see Fig. i). Oligomycin was found to be without effect on carboxykinase activity itself as long as ITP concentration did not become limiting, but prolonged the incubation period under which linear incubation time v s . activity plots could be obtained. Data in Fig. 2 illustrate the linearity of phosphoenolpyruvate production as a function of protein concentration obtained ,sdth the reaction mixtures (described in the legend to this Figure) employed in the assays of mitochondrial and soluble-fraction carboxykinase activities. Data in Series II, Table I, were calculated from slopes of lines obtained by determining phosphopyruvate formation as a function of protein concentration, while in all other experiments activities were assayed in quadruplicate with one concentration of mitochondrial protein within the limits of linearity of protein concentration and incubation time. All mitochondrial preparations were frozen (--15 °) and thawed (o °) between five and seven times before assay, since previous studies 1 had indicated maximal activation by this treatment; soluble-fraction preparations were maintained frozen ( - - i 5 °) until assayed. 1.2
3"00 l
-
1.50[ ~o
o
0
"I
~. 0.75
::L f
0
EL
5
/lg
6 Olicjomycin
112
0
2
4
6
8
I0
12
14
mg Protein
Fig. i. Effect of oligomycin A plus B plus C on five times frozen a n d t h a w e d rat-liver mitochondrial I T P a s e activity. Reaction m i x t u r e s contained, in a volume of 1. 5 ml, 1.6/*moles GSH, 9.0/*moles ITP, 22. 4/*moles MgSO4, 138/*moles sucrose, 20/*moles NaF, ioo/*moles T r i s - H C l buffer, 3.5 mg mitochondrial protein, a n d indicated a m o u n t s of oligomycin (introduced as a solution in 0.06 ml 50 % ethanol). Reaction m i x t u r e p H was 8.0. E t h a n o l b y itself as above was w i t h o u t effect on enzymic activity. I n c u b a t i o n s were carried out at 3 °0 with shaking. PI was m e a s u r e d colorimetrically TM. Activity is expressed as d/*moles Pt per 1. 5 ml reaction m i x t u r e per 15 min incubation. Fig. 2. Effects of protein concentration on rat-liver soluble-fraction (squares) a n d mitochondrial (circles) p h o s p h o e n o l p y r u v a t e carboxykinase activities. Activities designated b y closed s y m b o l s were obtained with p r e p a r a t i o n s from hydrocortisone-treated animals while those indicated b y open s y m b o l s were observed with p r e p a r a t i o n s from normal, control animals receiving vehicle, only. Assay m i x t u r e s for the soluble-fraction activity contained, in a volume of 1.5 ml, 6. 7 / , m o l e s oxaloacetate, 1.6 /*moles GSH, 9.0 /*moles ITP, 22. 4 /*moles MgSO4, 138 /*moles sucrose, 20/*moles NaF, and 146/*moles T r i s - H C 1 buffer. Reaction m i x t u r e p H 8.0. Assay m i x t u r e s for mitochondrial enzyme were as for soluble-fraction activity, b u t were s u p p l e m e n t e d with 4/*g oligomycin. I n c u b a t i o n s (3 o°) for soluble-fraction assays were for 5 m i n while those for mitochondrial enzyme were for 15 min (linearity of activity with time could be obtained up to 30 min with the lowest mitochondrial protein concentration indicated). All activities are expressed as z] /*moles p h o s p h o e n o l p y r u v a t e (PEP) per 1. 5 ml per min × io.
Biochim. Biophys. Acta, 97 (1965) 214 221
MITOCI-IONDRIAL CARBOXYKINASE
217
RESULTS AND DISCUSSION
Phosphoenolpyruvate carboxykinase activity of soluble fraction of liver was found to be decreased slightly in adrenalectomized as compared with normal animals, and to be increased significantly in hydrocortisone-treated animals, as previously reported ~. The effect was somewhat greater in those animals receiving two rather than one injection daily (compare Series I (one daily injection), and Series II (two daily injections), Table I). However, neither adrenalectomy nor hydrocortisone treatment caused a significant change in activity per mg protein or per g liver in mitochondria prepared from the same livers from which the soluble fraction was obtained. The 53 % increase in mitochondrial carboxykinase activity expressed per IOO g body weight (Table I, Series I) is partially compensated for by a depression of rate of growth due to hormonal administration. Hydrocortisone-treated animals weighed an average of 32 % less at sacrifice than did controls in this series. Since hydrocortisone administration caused no change in mitochondrial carboxykinase levels, activity of this enzyme in these particles also was measured following various other treatments which previously 2 had been found to cause marked alterations in levels of the soluble-fraction enzyme of rat liver. Neither insulin injection nor alloxan diabetes produced a significant change in the mitochondriat-enzyme levels calculated per mg protein, although blood sugar concentrations responded significantly to these treatments (see Table II, Series III) as before 2. Statistically significant increases of 55 % in mitochondrial enzymic activity expressed per g liver and 51% in activity per IOO g body weight were noted in diabetics compared with control animals. However, these changes are small compared, for example, with the IO.I-fold increase in soluble-fraction carboxykinase activity per IOO g body weight, and 5.7-fold increase in this activity per mg protein in diabetic animals ~. Significant also is the relatively low level of mitochondrial as compared with soluble-fraction carboxykinase activity in control animals. Even if mitochondrial carboxykinase activity were doubled in diabetic animals, the absolute amount of enzymic activity, as well as the ratio of mitochondrial to soluble-fraction activities, still would be relatively small. A 48-h fast, which previously 2 was demonstrated to cause an approx. 2.6-fold increase in carboxykinase activity per mg soluble-fraction protein, also was without effect on the level of mitochondrial enzyme activity (Table II, Series IV). It is concluded from these studies that mitochondrial phosphoenolpyruvate carboxykinase is unaffected, or relatively but slightly elevated, by certain factors which cause dramatic alterations in levels of this enzymic activity present in soluble portion of rat-liver cells. The participation of rat-liver mitochondrial phosphoenolpyruvate carboxykinase in a physiologically important metabolic pathway involved in glucogenesis appears unlikely, since the originally relatively very low levels of this activity either are unaffected or but slightly elevated by glucocorticoid administration or alloxan diabetes, both of which bring about marked elevation of blood glucose levels12, TM. This lack of response of mitochondrial carboxykinase could be observed either (a) if the mitochondrial enzyme, as opposed to the soluble-fraction enzyme, simply were not responsive to altered hormonal states or fasting, or (b) if mitochondrial permeability were a factor. Relative to the latter possibility, LITWACK et al. '4 have demonstrated that a proportionately small amount of injected [14C]hydrocortisone Biochim. Biophys. Acta, 97 (1965) 214-221
4
ADMINISTRATION
AND ADRENALECTOMY
ON RAT-LIVER
SOLUBLE
FRACTION
AND MITOCHONDRIAL
PHOSPHOENOLPYRUVATE
57-4 ~ lO.6 lO9.O ± 18.6
lI
Series
8.0
± 1A
6.08 ~ 2.14 6 . I 6 L 1.5
4.8
A d r e n a l e c t o m i z e d (8)
:!:: 1.r ~ 1.2
( x zoO)
P e r mg of protein
4.4 4.9
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
o.1--o.2
~O.OI
P
± 0.7 T 0.7 ± i.i
2.4z +_ 0.54 5.3 ± 0.76
3.3
5"3
3.7
Per g of liver
~o.5
0.3-0. 4
0. 3 0. 4
P
± 0. 3
~ 0. 3 ± 0. 3
2.44 ~ 0.58 1.89 _-: 0.76
1. 4
1. 3 1. 3
( x zo)
Per g of liver
Phosphoenolpyruvale carboxykinase activity
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
Experimental animal
10.2
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
~
42.1 ± 14.6
81.2
50,3 ±
( x ro~)
Per mg of protein
Phosphoenolpyruvate carboxykinase activity
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8) A d r e n a l e c t o m i z e d (8)
Experimental animal
Mitochondrial enzyme
II
Series
Soluble-fraction enzyme
0.4-0. 5
0.2 0. 3
~-0. 5
P
-~o.oi 0.3-0. 4
P
~ 1. 4 i 2.9 ± 2.2
9.8 ± 2 . 0 13.6 ± 3.0
4.7 ~ I.O
4.3 ~ 0.8 6.6 ± 0. 9
Per zoo g of body weight ( × zo)
9.76 L- 1.86 38.2 • 5.9
12.8 28,0 io.6
Per zoo g of body weight
O.l-O.2
0.3-0. 4
~o.oI
P
~o.oi
~O.Ol ~o.o1
P
A s s a y m i x t u r e s w e r e as d e s c r i b e d i n t h e l e g e n d t o F i g . 2 ; o t h e r d e t a i l s of t h e e n z y m i c a s s a y s a r e g i v e n i n t h e t e x t . V a l u e s g i v e t h e e n z y m e a c t i v i t y i n u n i t s ± S.D. i U n i t of e n z y m e a c t i v i t y is t h a t a m o u n t c a t a l y z i n g t h e p r o d u c t i o n of i t, m o l e p h o s p h o e n o l p y r u v a t e p e r m i n p e r 1. 5 m l r e a c t i o n m i x t u r e . T h e n u m b e r of a n i m a l s i n e a c h e x p e r i m e n t a l g r o u p is g i v e n i n p a r e n t h e s e s . P is t h e p r o b a b i l i t y of c h a n c e of o c c u r r e n c e of d i f f e r e n c e b e t w e e n m e a n s of n o r m a l , c o n t r o l g r o u p a n d i n d i c a t e d e x p e r i m e n t a l g r o u p .
ACTIVITIES
OF HYDROCORTISONE
EFFECTS
CARBOXYKINASE
I
TABLE
©
1",5
t~
4~
t~
¢
4
tZ
OF
EFFECTS
INSULIN
ADMINISTRATION,
ALLOXAN
DIABETES,
AND
FASTING
ON
RAT-LIVER
MITOCHONDRIAL
PHONPHOENOLPYRUVATE-CARBOXYKINANE
IV
III
Series
N o r m a l (6) F a s t e d (IO)
N o r m a l (7) I n s u l i n t r e a t e d (5) A l l o x a n d i a b e t i c (6)
Experimental animal
5-5 -k 1.7 5.7 ± 0.9
7.1 ± 1. 3 6. 4 :j_ 1.6 8.2 i 0.8
> 0. 5
0.3 - 0 . 4 o.o5-o.i 1.6 ± 0. 5 2.1 ~= o. 5
2.0 4- 0.3 1.8 i 0.3 3.1 ± 0. 7
o.o5-o.lo
0.2 - 0 . 3
P
5.7 ± 2.3 6.o ± o.8
6.5 :ix 1.4 6. 4 ~ I.O 9.8 Jz 2.8
( × xo)
Per zoo g body
> o. 5
) 0. 5 o.o2 o.o5
P
82 29 336
71-98 I7-45 285-47o
Range
Average
( x zo)
Per g of liver
Per mg of protein ( X z o 3)
P
Blood sugar levels (rag glucose per IOO ml)
Phosphoenolpyruvate earboxykinase activity
A s s a y m i x t u r e s w e r e a s d e s c r i b e d i n t h e l e g e n d s t o F i g s . i a n d 2; o t h e r d e t a i l s of t h e e n z y m i c a s s a y s a r e g i v e n i n t h e t e x t . F o r d e t a i l s of n o t a t i o n see l e g e n d t o T a b l e I.
ACTIVITY
II
TABLE
t~
t~
o~
~0 to © N
> t*
o
o
R. C. NORDLIE
220
appears recently
in mitochondria as compared with soluble portion of rat liver. pyruvate carboxylase4-6, a mitochondrial enzyme3,7, has been
et d.
However, shown to
increase in activity following administration of cortisol (a 3-fold increase in activity per mg protein was noted15), as a result of alloxan diabetes (an approx. 3-fold increase in activity per g wet liver was observed’), and during fasting15; these observations strongly suggest that mitochondrial impermeability is not in itself responsible for the lack of response of mitochondrial carboxykinase to the various hormonal and metabolic conditions studied. While mitochondrial
carboxykinase
levels responded
slightly
or not at all to
the various experimental treatments employed in these studies, a significant variation in average mitochondrial carboxykinase levels among control groups for the various series of experiments performed was noted. While animals (males of “Sprague-Dawley origin”) of approximately the same size, obtained through the same supplier, were employed in all experiments, those animals in Series I (the series which had the lowest average control mitochondrial carboxykinase specific activity) were shipped from the supplier’s California branch while all other animals were supplied from their Minnesota colony. In addition, the various series of experiments were performed with separate groups of rats, one series at a time, over a period of approx. 6 months. These observations suggest that undefined factors other than the variables specifically studied in the present investigation may have some effect on levels of the mitochondrial enzyme. The results obtained in the present study with rat-liver preparations are consistent with the previous findings that guinea-pig liver mitochondrial carboxykinase was unresponsive to starvation or mannoheptulose administration which enhanced this activity in soluble fraction 2. Studies currently in progress in our laboratory16 indicate that carboxykinases fractions of livers of this animal
partially purified from soluble and mitochondrial differ subtly in certain catalytic properties. The
variation in response to hormonal alterations and fasting by enzymes of the two rat-liver fractions is consistent with the idea that carboxykinases from mitochondria and soluble fraction are indeed different enzymes. The results of these experiments also confirm our original fmdingsr of variation in sub-cellular distribution pattern of carboxykinase in livers of various mammals. The differences in response of mitochondrial and soluble-fraction enzymes to alterations in hormonal states and fasting rule out the possibility that observed variation in carboxykinase distribution patterns in different species could have been due to varying fragility of mitochondria with the accompanying leakage of the enzyme into the soluble fraction during sub-cellular fractionation. If the enzyme in soluble and mitochondrial fractions had originated from a single source in the unfractionated liver, activities found in both fractions following isolation would have been expected to show the same responses to the various treatments to which the animals were subjected. (Further ruling against the possibility of leakage of carboxykinase from mitochondria during fractionation are results of recent studieP which indicate that for the guinea pig (the liver of which originally was foundi to have this enzymic activity more nearly evenly distributed between mitochondrial and soluble fractions than was the case with rat liver), the ratio, mitochondrial carboxykinase activity: soluble fraction carboxykinase activity, remained constant whether fractionation was carried out at o0 or at room temperature (approx. 23’). The latter treatment has Biochim.
Bioph>fs.
Acta,
97 (1965)
r14-221
MITOCHONDRIAL CARBOXYKINASE
221
been shown to solubilize mitochondrial pyruvate carboxylase during fractionationL Finally, it is concluded from these studies that if, indeed, liver pyruvate carboxylase plus phosphoenolpyruvate carboxykinase do play a significant role in glucogenesis (recent studies 1~ indicate a glucogenic response to glucocorticoid administration even in the presence of actinomycin D which prevents protein synthesis at the soluble-RNA level), the physiologically significant process involves intramitochondrial carboxylase and extramitochondrial carboxykinase in the rat. ACKNOWLEDGEMENTS
Our thanks to Dr. W. SCHMID for valuable advice regarding the statistical analyses, and to Dr. F. A. JACOBS for helpful suggestions. F.E.V. was supported by a training grant from National Institutes of Health, while D.D.H. holds a U.S. Public Health Service Pre-doctoral Fellowship. This work was supported in part by grants from the Hill Family Foundation and the National Institutes of Health, U.S. Public Health Service (Grant AM 07141-Ol). REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18
R. C. •ORDLIE AND H. A. LARDY, J. Biol. Chem., 238 (1963) 2259. E. SHRAGO, H. A. LARDY, R. C. NORDLIE AND D. O. FOSTER, J. Biol. Chem., 238 (1963) 3188. M. F. UTTER, Iowa State f . Sci., 38 (1963) 97. D. n. KEECH AND M. F. UTTER, J. Biol. Chem., 238 (1963) 2609. M. F. UTTER AND D. B. KEECH, J. Biol. Chem., 235 (196o) PCI 7. M. F. UTTER AND D. B. KEECH, f . Biol. Chem., 238 (1963) 26o3. S. R. WAGLE, Biochem. Biophys. Res. Commun., 14 (1964) 533K. LOHMANN AND O. MEYERHOF, Biochem. Z., 273 (1934) 60. G. H. MUDGE, H. W. NEUBERG AND S. W. STANBURY, J. Biol. Chem., 21o (1954) 965. R. S. BANDURSKI AND F. LIPMANN, J. Biol. Chem., 219 (1956) 741. M. J. SOMOGYI, J . Biol. Chem., 195 (1952) 19. J. ASHMORE, A. B. HASTINGS, F. B. NESBETT AND A. E. RENOLD, J . Biol. Chem., 218 (1956) 77. G. WEBER AND A. CANTERO, Endocrinology, 61 (1957) 7Ol. G. LITWACK, IV[. L. SEARS AND T. 1. DIAMONDSTONE,J. Biol. Chem., 238 (1963) 3o2. H-V. HENNING, I. SEIFFERT AND W. SEUBERT, Biochim. Biophys. Acta, 77 (1963) 345R. C. NORDLIE AND D. D. HOLTEN, unpublished observations. P. D. RAY, D. O. FOSTER AND H. A. LARDY, Federation Proc., 23 (1964) 482. C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375-
Biochim. Biophys. Acta, 97 (1965) 214-221
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 172
EFFECTS OF A L T E R E D HORMONAL STATES AND FASTING ON R A T - L I V E R M I T O C H O N D R I A L 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 LEVELS ROBERT C. NORDLIE, FREDERICK E. VARRICCHIO AND DAROLD D. HOLTEN Guy and Bertha Ireland Research Laboratory, Department of Biochemistry, University of North Dakota Medical School, Grand Forks, N.D. (U.S.A.)
(Received May I9th, 1964)
SUMMARY I. Rat-liver phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxylyase (transphosphorylating), EC 4. I. 1.32) levels have been measured following various hormonal treatments and fasting, and in control animals. 2. Adrenalectomy and hydrocortisone administration were without significant effect on mitochondrial carboxykinase levels, although amounts of this enzyme in soluble fraction of these same livers were nearly doubled b y administration of the glucocorticoid. 3- Allaxan diabetes, insulin administration, and fasting also were without statistically significant effect on levels of mitochondrial carboxykinase expressed per mg protein; increases of 55 % and 51%, respectively, in mitochondrial activity per g liver or per IOO g body weight were noted in diabetic animals. 4. It is concluded that any concerted participation of rat-liver pyruvate carboxylase (pyruvate:CO z ligase (ADP), EC 6.4.1.1) plus phosphoenolpyruvate carboxykinase in glucogenesis must involve principally the extramitochondrial carboxykinase.
INTRODUCTION NORDLIE AND LARDY1 previously have demonstrated a marked difference in subcellular distribution pattern of liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.I.I.3 2) among various m a m malian species. While guinea-pig enzyme activity, for example, was present in mitochondrial and soluble fractions in an approx. 2 : I ratio, about 9 ° % of total rat-liver activity was in soluble fraction. Carboxykinase of guinea-pig liver soluble fraction has been reported ~ to rise due to starvation or mannoheptulose administration while this activity in mitochondria of these animals was unaffected. Much more extensive studies 2 indicated that the carboxykinase of soluble fraction of rat liver responds markedly to a considerable number of hormonal and metabolic alterations. It has been suggested ~-4 that the carboxykinase plus pyruvate carboxylase (pyruvate: CO s ligase (ADP), EC 6.4.1.1) 4-6 systems may function together as an energetically favorable pathway for the conversion of pyruvate to phosphoenolpyruvate in the overall process of glucogenesis from lactate, pyruvate, or certain amino acids. The rat-liver carboxylase appears to be confined to mitochondria 3& The simplest concerted mechaBiochim. Biophys. Acta, 97 (1965) 214-221
MITOCHONDRIAL CARBOXYKINASE
215
nism for these two enzymes logically would involve mitochondrial carboxykinase, since a shuttling of intramitochondrially-produced oxaloacetate and, perhaps, GTP, through the mitochondrial membrane prior to reaction in the presence of solublefraction carboxykinase would be unnecessary. Since it has been reported that in normal rats, less than io % of total liver carboxykinase is found in mitochondria 1, we believed it possible that this mitochondrial enzyme activity might be markedly elevated by certain hormonal or metabolic stimuli which cause significant increases in blood glucose and liver glycogen levels; the intramitochondrial carboxykinase, normally only slightly active, would then function as part of a specialized mechanism responding to certain stressful conditions. For these reasons, rat-liver preparations were selected for the present investigations. Results of studies of the effects of adrenalectomy and hydrocortisone administration on both soluble fraction and mitochondrial phosphoenolpyruvate carboxykinase levels, and of alloxan diabetes, insulin administration, and fasting on the latter activity are described in this paper. MATERIALS AND METHODS
Young, adult male rats of Sprague-Dawley origin, weighing between 15o and 21o g, obtained through Simonsen Laboratories, Inc., White Bear Lake, Minn., were employed in these experiments. Adrenalectomies were performed by the supplier. All animals, unless otherwise noted, were maintained on Purina Lab Chow and water (or 1% NaC1 solution in the case of adrenalectomized animals), ad libitum. Animals were acclimated for at least 4 days after receipt from the supplier. Preparation of reagents, fractionation of livers, protein determinations, and phosphoenolpyruvate assays (Series I, Table I ; and Series III and IV, Table II) were as described previously1, 2. For the assays of enzyme from animals described in Series II, Table I, the mercury methodS, 9 was employed for phosphopyruvate determinations. Identical values were obtained with this method and with the LOHMANNM E Y E R H O F F 8'1° KI3 procedure in a supplementary, comparative experiment. Hydrocortisone, obtained from Sigma Chemical Co, was suspended in 1 % NaC1 solution and injected subcutaneously once (Series I, Table I) or twice (Series II, Table I) per day for a period of 4 days; total daily dosage was IO mg per animal. Diabetes (Series III, Table II) was produced by intraperitoneal injection of alloxan (200 mg per kg body weight). Those animals having blood sugar levels (determined by the SOMOGYI-NELSON11method) of between 285 and 47 ° mg per IOOml, were sacrificed I week after injection. A second group of animals in this series received daily subcutaneous injections of 20 units of protamine zinc insulin (Lilly) daily for 3 days, and were sacrificed 4 h after the last injection. Insulin-treated animals had blood sugar levels of 17-45 mg per IOO ml, while a third, untreated control group had levels between 71 and 98 mg glucose per IOO ml blood. Another group of animals (Series IV, Table II) was fasted for 48 h while controls for this group received Purina Lab Chow ad libitum, as before. Phosphoenolpyruvate carboxykinase activity in soluble fraction of liver was assayed as described previously1,2. Supplementary experiments were conducted to determine conditions under which initial reaction velocities for the relatively inactive mitochondrial enzyme also could be determined. In general, assay conditions employed with the soluble-fraction enzyme also were found satisfactory for mitochonBiochim. Biophys. Acta, 97 (1965) 214-221
R . C . NORDLIE et al.
216
drial-enzyme assay. In addition, however, 4 Fg oligomycin A plus B plus C (a gift of Dr. H. A. LARDY) per 1. 5 ml reaction mixture was included in the mitochondrialenzyme assay system, since it was found partially to suppress the rather active ITPase present in frozen-thawed mitochondria (see Fig. i). Oligomycin was found to be without effect on carboxykinase activity itself as long as ITP concentration did not become limiting, but prolonged the incubation period under which linear incubation time v s . activity plots could be obtained. Data in Fig. 2 illustrate the linearity of phosphoenolpyruvate production as a function of protein concentration obtained ,sdth the reaction mixtures (described in the legend to this Figure) employed in the assays of mitochondrial and soluble-fraction carboxykinase activities. Data in Series II, Table I, were calculated from slopes of lines obtained by determining phosphopyruvate formation as a function of protein concentration, while in all other experiments activities were assayed in quadruplicate with one concentration of mitochondrial protein within the limits of linearity of protein concentration and incubation time. All mitochondrial preparations were frozen (--15 °) and thawed (o °) between five and seven times before assay, since previous studies 1 had indicated maximal activation by this treatment; soluble-fraction preparations were maintained frozen ( - - i 5 °) until assayed. 1.2
3"00 l
-
1.50[ ~o
o
0
"I
~. 0.75
::L f
0
EL
5
/lg
6 Olicjomycin
112
0
2
4
6
8
I0
12
14
mg Protein
Fig. i. Effect of oligomycin A plus B plus C on five times frozen a n d t h a w e d rat-liver mitochondrial I T P a s e activity. Reaction m i x t u r e s contained, in a volume of 1. 5 ml, 1.6/*moles GSH, 9.0/*moles ITP, 22. 4/*moles MgSO4, 138/*moles sucrose, 20/*moles NaF, ioo/*moles T r i s - H C l buffer, 3.5 mg mitochondrial protein, a n d indicated a m o u n t s of oligomycin (introduced as a solution in 0.06 ml 50 % ethanol). Reaction m i x t u r e p H was 8.0. E t h a n o l b y itself as above was w i t h o u t effect on enzymic activity. I n c u b a t i o n s were carried out at 3 °0 with shaking. PI was m e a s u r e d colorimetrically TM. Activity is expressed as d/*moles Pt per 1. 5 ml reaction m i x t u r e per 15 min incubation. Fig. 2. Effects of protein concentration on rat-liver soluble-fraction (squares) a n d mitochondrial (circles) p h o s p h o e n o l p y r u v a t e carboxykinase activities. Activities designated b y closed s y m b o l s were obtained with p r e p a r a t i o n s from hydrocortisone-treated animals while those indicated b y open s y m b o l s were observed with p r e p a r a t i o n s from normal, control animals receiving vehicle, only. Assay m i x t u r e s for the soluble-fraction activity contained, in a volume of 1.5 ml, 6. 7 / , m o l e s oxaloacetate, 1.6 /*moles GSH, 9.0 /*moles ITP, 22. 4 /*moles MgSO4, 138 /*moles sucrose, 20/*moles NaF, and 146/*moles T r i s - H C 1 buffer. Reaction m i x t u r e p H 8.0. Assay m i x t u r e s for mitochondrial enzyme were as for soluble-fraction activity, b u t were s u p p l e m e n t e d with 4/*g oligomycin. I n c u b a t i o n s (3 o°) for soluble-fraction assays were for 5 m i n while those for mitochondrial enzyme were for 15 min (linearity of activity with time could be obtained up to 30 min with the lowest mitochondrial protein concentration indicated). All activities are expressed as z] /*moles p h o s p h o e n o l p y r u v a t e (PEP) per 1. 5 ml per min × io.
Biochim. Biophys. Acta, 97 (1965) 214 221
MITOCI-IONDRIAL CARBOXYKINASE
217
RESULTS AND DISCUSSION
Phosphoenolpyruvate carboxykinase activity of soluble fraction of liver was found to be decreased slightly in adrenalectomized as compared with normal animals, and to be increased significantly in hydrocortisone-treated animals, as previously reported ~. The effect was somewhat greater in those animals receiving two rather than one injection daily (compare Series I (one daily injection), and Series II (two daily injections), Table I). However, neither adrenalectomy nor hydrocortisone treatment caused a significant change in activity per mg protein or per g liver in mitochondria prepared from the same livers from which the soluble fraction was obtained. The 53 % increase in mitochondrial carboxykinase activity expressed per IOO g body weight (Table I, Series I) is partially compensated for by a depression of rate of growth due to hormonal administration. Hydrocortisone-treated animals weighed an average of 32 % less at sacrifice than did controls in this series. Since hydrocortisone administration caused no change in mitochondrial carboxykinase levels, activity of this enzyme in these particles also was measured following various other treatments which previously 2 had been found to cause marked alterations in levels of the soluble-fraction enzyme of rat liver. Neither insulin injection nor alloxan diabetes produced a significant change in the mitochondriat-enzyme levels calculated per mg protein, although blood sugar concentrations responded significantly to these treatments (see Table II, Series III) as before 2. Statistically significant increases of 55 % in mitochondrial enzymic activity expressed per g liver and 51% in activity per IOO g body weight were noted in diabetics compared with control animals. However, these changes are small compared, for example, with the IO.I-fold increase in soluble-fraction carboxykinase activity per IOO g body weight, and 5.7-fold increase in this activity per mg protein in diabetic animals ~. Significant also is the relatively low level of mitochondrial as compared with soluble-fraction carboxykinase activity in control animals. Even if mitochondrial carboxykinase activity were doubled in diabetic animals, the absolute amount of enzymic activity, as well as the ratio of mitochondrial to soluble-fraction activities, still would be relatively small. A 48-h fast, which previously 2 was demonstrated to cause an approx. 2.6-fold increase in carboxykinase activity per mg soluble-fraction protein, also was without effect on the level of mitochondrial enzyme activity (Table II, Series IV). It is concluded from these studies that mitochondrial phosphoenolpyruvate carboxykinase is unaffected, or relatively but slightly elevated, by certain factors which cause dramatic alterations in levels of this enzymic activity present in soluble portion of rat-liver cells. The participation of rat-liver mitochondrial phosphoenolpyruvate carboxykinase in a physiologically important metabolic pathway involved in glucogenesis appears unlikely, since the originally relatively very low levels of this activity either are unaffected or but slightly elevated by glucocorticoid administration or alloxan diabetes, both of which bring about marked elevation of blood glucose levels12, TM. This lack of response of mitochondrial carboxykinase could be observed either (a) if the mitochondrial enzyme, as opposed to the soluble-fraction enzyme, simply were not responsive to altered hormonal states or fasting, or (b) if mitochondrial permeability were a factor. Relative to the latter possibility, LITWACK et al. '4 have demonstrated that a proportionately small amount of injected [14C]hydrocortisone Biochim. Biophys. Acta, 97 (1965) 214-221
4
ADMINISTRATION
AND ADRENALECTOMY
ON RAT-LIVER
SOLUBLE
FRACTION
AND MITOCHONDRIAL
PHOSPHOENOLPYRUVATE
57-4 ~ lO.6 lO9.O ± 18.6
lI
Series
8.0
± 1A
6.08 ~ 2.14 6 . I 6 L 1.5
4.8
A d r e n a l e c t o m i z e d (8)
:!:: 1.r ~ 1.2
( x zoO)
P e r mg of protein
4.4 4.9
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
o.1--o.2
~O.OI
P
± 0.7 T 0.7 ± i.i
2.4z +_ 0.54 5.3 ± 0.76
3.3
5"3
3.7
Per g of liver
~o.5
0.3-0. 4
0. 3 0. 4
P
± 0. 3
~ 0. 3 ± 0. 3
2.44 ~ 0.58 1.89 _-: 0.76
1. 4
1. 3 1. 3
( x zo)
Per g of liver
Phosphoenolpyruvale carboxykinase activity
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
Experimental animal
10.2
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8)
~
42.1 ± 14.6
81.2
50,3 ±
( x ro~)
Per mg of protein
Phosphoenolpyruvate carboxykinase activity
N o r m a l (8) H y d r o c o r t i s o n e t r e a t e d (8) A d r e n a l e c t o m i z e d (8)
Experimental animal
Mitochondrial enzyme
II
Series
Soluble-fraction enzyme
0.4-0. 5
0.2 0. 3
~-0. 5
P
-~o.oi 0.3-0. 4
P
~ 1. 4 i 2.9 ± 2.2
9.8 ± 2 . 0 13.6 ± 3.0
4.7 ~ I.O
4.3 ~ 0.8 6.6 ± 0. 9
Per zoo g of body weight ( × zo)
9.76 L- 1.86 38.2 • 5.9
12.8 28,0 io.6
Per zoo g of body weight
O.l-O.2
0.3-0. 4
~o.oI
P
~o.oi
~O.Ol ~o.o1
P
A s s a y m i x t u r e s w e r e as d e s c r i b e d i n t h e l e g e n d t o F i g . 2 ; o t h e r d e t a i l s of t h e e n z y m i c a s s a y s a r e g i v e n i n t h e t e x t . V a l u e s g i v e t h e e n z y m e a c t i v i t y i n u n i t s ± S.D. i U n i t of e n z y m e a c t i v i t y is t h a t a m o u n t c a t a l y z i n g t h e p r o d u c t i o n of i t, m o l e p h o s p h o e n o l p y r u v a t e p e r m i n p e r 1. 5 m l r e a c t i o n m i x t u r e . T h e n u m b e r of a n i m a l s i n e a c h e x p e r i m e n t a l g r o u p is g i v e n i n p a r e n t h e s e s . P is t h e p r o b a b i l i t y of c h a n c e of o c c u r r e n c e of d i f f e r e n c e b e t w e e n m e a n s of n o r m a l , c o n t r o l g r o u p a n d i n d i c a t e d e x p e r i m e n t a l g r o u p .
ACTIVITIES
OF HYDROCORTISONE
EFFECTS
CARBOXYKINASE
I
TABLE
©
1",5
t~
4~
t~
¢
4
tZ
OF
EFFECTS
INSULIN
ADMINISTRATION,
ALLOXAN
DIABETES,
AND
FASTING
ON
RAT-LIVER
MITOCHONDRIAL
PHONPHOENOLPYRUVATE-CARBOXYKINANE
IV
III
Series
N o r m a l (6) F a s t e d (IO)
N o r m a l (7) I n s u l i n t r e a t e d (5) A l l o x a n d i a b e t i c (6)
Experimental animal
5-5 -k 1.7 5.7 ± 0.9
7.1 ± 1. 3 6. 4 :j_ 1.6 8.2 i 0.8
> 0. 5
0.3 - 0 . 4 o.o5-o.i 1.6 ± 0. 5 2.1 ~= o. 5
2.0 4- 0.3 1.8 i 0.3 3.1 ± 0. 7
o.o5-o.lo
0.2 - 0 . 3
P
5.7 ± 2.3 6.o ± o.8
6.5 :ix 1.4 6. 4 ~ I.O 9.8 Jz 2.8
( × xo)
Per zoo g body
> o. 5
) 0. 5 o.o2 o.o5
P
82 29 336
71-98 I7-45 285-47o
Range
Average
( x zo)
Per g of liver
Per mg of protein ( X z o 3)
P
Blood sugar levels (rag glucose per IOO ml)
Phosphoenolpyruvate earboxykinase activity
A s s a y m i x t u r e s w e r e a s d e s c r i b e d i n t h e l e g e n d s t o F i g s . i a n d 2; o t h e r d e t a i l s of t h e e n z y m i c a s s a y s a r e g i v e n i n t h e t e x t . F o r d e t a i l s of n o t a t i o n see l e g e n d t o T a b l e I.
ACTIVITY
II
TABLE
t~
t~
o~
~0 to © N
> t*
o
o
R. C. NORDLIE
220
appears recently
in mitochondria as compared with soluble portion of rat liver. pyruvate carboxylase4-6, a mitochondrial enzyme3,7, has been
et d.
However, shown to
increase in activity following administration of cortisol (a 3-fold increase in activity per mg protein was noted15), as a result of alloxan diabetes (an approx. 3-fold increase in activity per g wet liver was observed’), and during fasting15; these observations strongly suggest that mitochondrial impermeability is not in itself responsible for the lack of response of mitochondrial carboxykinase to the various hormonal and metabolic conditions studied. While mitochondrial
carboxykinase
levels responded
slightly
or not at all to
the various experimental treatments employed in these studies, a significant variation in average mitochondrial carboxykinase levels among control groups for the various series of experiments performed was noted. While animals (males of “Sprague-Dawley origin”) of approximately the same size, obtained through the same supplier, were employed in all experiments, those animals in Series I (the series which had the lowest average control mitochondrial carboxykinase specific activity) were shipped from the supplier’s California branch while all other animals were supplied from their Minnesota colony. In addition, the various series of experiments were performed with separate groups of rats, one series at a time, over a period of approx. 6 months. These observations suggest that undefined factors other than the variables specifically studied in the present investigation may have some effect on levels of the mitochondrial enzyme. The results obtained in the present study with rat-liver preparations are consistent with the previous findings that guinea-pig liver mitochondrial carboxykinase was unresponsive to starvation or mannoheptulose administration which enhanced this activity in soluble fraction 2. Studies currently in progress in our laboratory16 indicate that carboxykinases fractions of livers of this animal
partially purified from soluble and mitochondrial differ subtly in certain catalytic properties. The
variation in response to hormonal alterations and fasting by enzymes of the two rat-liver fractions is consistent with the idea that carboxykinases from mitochondria and soluble fraction are indeed different enzymes. The results of these experiments also confirm our original fmdingsr of variation in sub-cellular distribution pattern of carboxykinase in livers of various mammals. The differences in response of mitochondrial and soluble-fraction enzymes to alterations in hormonal states and fasting rule out the possibility that observed variation in carboxykinase distribution patterns in different species could have been due to varying fragility of mitochondria with the accompanying leakage of the enzyme into the soluble fraction during sub-cellular fractionation. If the enzyme in soluble and mitochondrial fractions had originated from a single source in the unfractionated liver, activities found in both fractions following isolation would have been expected to show the same responses to the various treatments to which the animals were subjected. (Further ruling against the possibility of leakage of carboxykinase from mitochondria during fractionation are results of recent studieP which indicate that for the guinea pig (the liver of which originally was foundi to have this enzymic activity more nearly evenly distributed between mitochondrial and soluble fractions than was the case with rat liver), the ratio, mitochondrial carboxykinase activity: soluble fraction carboxykinase activity, remained constant whether fractionation was carried out at o0 or at room temperature (approx. 23’). The latter treatment has Biochim.
Bioph>fs.
Acta,
97 (1965)
r14-221
MITOCHONDRIAL CARBOXYKINASE
221
been shown to solubilize mitochondrial pyruvate carboxylase during fractionationL Finally, it is concluded from these studies that if, indeed, liver pyruvate carboxylase plus phosphoenolpyruvate carboxykinase do play a significant role in glucogenesis (recent studies 1~ indicate a glucogenic response to glucocorticoid administration even in the presence of actinomycin D which prevents protein synthesis at the soluble-RNA level), the physiologically significant process involves intramitochondrial carboxylase and extramitochondrial carboxykinase in the rat. ACKNOWLEDGEMENTS
Our thanks to Dr. W. SCHMID for valuable advice regarding the statistical analyses, and to Dr. F. A. JACOBS for helpful suggestions. F.E.V. was supported by a training grant from National Institutes of Health, while D.D.H. holds a U.S. Public Health Service Pre-doctoral Fellowship. This work was supported in part by grants from the Hill Family Foundation and the National Institutes of Health, U.S. Public Health Service (Grant AM 07141-Ol). REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18
R. C. •ORDLIE AND H. A. LARDY, J. Biol. Chem., 238 (1963) 2259. E. SHRAGO, H. A. LARDY, R. C. NORDLIE AND D. O. FOSTER, J. Biol. Chem., 238 (1963) 3188. M. F. UTTER, Iowa State f . Sci., 38 (1963) 97. D. n. KEECH AND M. F. UTTER, J. Biol. Chem., 238 (1963) 2609. M. F. UTTER AND D. B. KEECH, J. Biol. Chem., 235 (196o) PCI 7. M. F. UTTER AND D. B. KEECH, f . Biol. Chem., 238 (1963) 26o3. S. R. WAGLE, Biochem. Biophys. Res. Commun., 14 (1964) 533K. LOHMANN AND O. MEYERHOF, Biochem. Z., 273 (1934) 60. G. H. MUDGE, H. W. NEUBERG AND S. W. STANBURY, J. Biol. Chem., 21o (1954) 965. R. S. BANDURSKI AND F. LIPMANN, J. Biol. Chem., 219 (1956) 741. M. J. SOMOGYI, J . Biol. Chem., 195 (1952) 19. J. ASHMORE, A. B. HASTINGS, F. B. NESBETT AND A. E. RENOLD, J . Biol. Chem., 218 (1956) 77. G. WEBER AND A. CANTERO, Endocrinology, 61 (1957) 7Ol. G. LITWACK, IV[. L. SEARS AND T. 1. DIAMONDSTONE,J. Biol. Chem., 238 (1963) 3o2. H-V. HENNING, I. SEIFFERT AND W. SEUBERT, Biochim. Biophys. Acta, 77 (1963) 345R. C. NORDLIE AND D. D. HOLTEN, unpublished observations. P. D. RAY, D. O. FOSTER AND H. A. LARDY, Federation Proc., 23 (1964) 482. C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375-
Biochim. Biophys. Acta, 97 (1965) 214-221