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The stimulatory effect of alloxan diabetes on citrulline formation in rabbit liver mitochondria
90
Biochimica et Biophysica Acta 839 (1985) 90 95
Elsevier BBA 22010
The stimulatory effect of alloxan diabetes on citrulline formation in rabbit liver mitochondria
Jadwiga Bryla and Maciej Garstka Institute of Biochemistry, University of Warsaw, AI. ~wirki i Wigury 93, 02-089 Warsaw (Poland)
(Received September 21st, 1984) (Revised manuscript received December 10th, 1984)
Key words: Alloxan; Diabetes: Citrulline synthesis; (Rabbit liver mitochondria) The effect of alloxan diabetes on citrulline formation from NH4CI and bicarbonate was studied in rabbit liver mitochondria incubated with glutamate or succinate as respiratory substrate, as well as with exogenous ATP in the presence of uncoupler and oligomycin. In contrast to ornithine transcarbamoylase, the activity of carbamoyl-phosphate synthetase (ammonia) was higher in mitochondria from diabetic animals than in those from normal ones. In diabetic rabbits the rates of citrulline synthesis were stimulated under all conditions studied. In contrast, levels of N-acetylglutamate, an activator of carbamoyl-phosphate synthetase (ammonia), were significantly increased only in the presence of glutamate, while the highest rates of citrulline formation occurred in uncoupled mitochondria incubated with exogenous ATP as energy source. Treatment of animals with alloxan resulted in an increase of both the intramitochondiral A T P level and the rate of adenine nucleotide translocation across the mitochondrial membrane. The results indicate that the stimulation of citrulline formation in liver mitochondria of diabetic rabbits is mainly due to an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of content of intramitochondrial ATP, a substrate of this enzyme.
Introduction
The increased urea excretion in diabetic rats can be correlated with the enhancement of the urea synthesis by liver slices [1] and isolated hepatocytes [2,3] and also with the increased citrulline synthesis by isolated mitochondria [2,3]. Levels of the main urea synthesis enzymes are also elevated in both alloxan-diabetic [1,4] and streptozotocin-diabetic rats [2]. Since carbamoyl-phosphate synthetase (ammonia) is the first step in ureagenesis, this enzyme is a likely site for regulatory control [5]. The enzyme is not dependent only upon the substrates
Abbreviation: FCCP, trifluoromethoxyphenylhydrazone.
concentrations but has an absolute requirement for N-acetylglutamate as allosteric activator [6]. Numerous studies, including those in vivo [7-9], with isolated hepatocytes [10,11] and isolated mitochondria [3,12 14] have demonstrated that rates of synthesis of urea or citrulline are mainly determined by N-acetylglutamate concentrations. The levels of this metabolite were reported to increase in response to nitrogen load [7,8,15-17], glucagon treatment [10,18-20] and diabetes [3]. Much of our knowledge about the effect of diabetes on hepatic ureagenesis has been derived from studies with rats. Therefore, the purpose of this investigation was to search for changes induced by diabetes in other mammalian species, for example, rabbit. In a previous communication from this laboratory we have reported that urea produc-
0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
91 tion from amino acids or NH4C1 as ammonia donor is stimulated in hepatocytes isolated from alloxan-diabetic rabbits [21]. In this paper we present some of factors responsible for the stimulatory effect of alloxan diabetes on citrulline synthesis in rabbit liver mitochondria. Materials and Methods
Animals. Young male white rabbtis (Popielno strain), weighing 5 0 0 - 1 0 0 0 g, were used throughout. Alloxan diabetes was introduced as described previously [21]. The mean value for the blood glucose concentration of rabbits considered as diabetic was 390 _+ 12 m g / 1 0 0 ml, as calculated for eleven animals. Liver mitochondria were isolated on day 4 after the alloxan treatment. Isolation and incubation of mitochondria. Liver mitochondria were isolated as described by Harris et al. [22], but the final wash and suspension of mitochondria were made with 0.3 M mannitol replacing the sucrose. Rabbit liver mitochondria (about 4-5 mg prot e i n / m l ) were incubated at 30°C in a basic medium containing 15 mM KC1, 10 mM NH4C1, 30 mM K H C O 3, 75 mM Tris-HC1 (pH 7.4), 1 mM EGTA, 5 mM potassium-phosphate buffer (pH 7.1), 0.2 mM aminooxyacetate 10 mM ornithine, 15 mM mannitol deriving from the mitochondrial suspension and either 10 mM glutamate, 10 mM succinate ( + 4 ~tM rotenone) or 5 mM ATP ( + 4 / ~ M rotenone + oligomycin (1 / ~ g / m g protein) + 0.1-0.2 ~M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)), as indicated in the tables. The incubations were started by the addition of either mitochondrial suspension or uncoupler to the reaction medium. The incubation mixture was continuously gassed with O 2 / C O 2 (95% : 5%). At 2, 4, 6, 8 and 10 min of incubation 1-ml samples were withdrawn from the suspensions. Samples were deproteinized with 0.1 volume of 40% HC104 (v/v) and centrifuged. Supernatants were collected and used for citrulline measurements. The rate of citrulline synthesis was linear with time up to 20 min of incubation. After 15 min of incubation a 7-ml sample was withdrawn from the reaction medium. Mitochondria were separated from the suspension by rapid centrifugation and after careful removal of the
supernatant used for determination of N-acetylglutamate level, as described by Meijer and Van Woerkom [13]. Intramitochondrial ATP content was determined in samples taken after 8 min of incubation, using an atractylate stopping method [23]. When aminooxyacetate was omitted from the reaction medium, the mitochondrial ATP content was higher only about 10-15% in comparison with that estimated in the presence of aminooxyacetate, while the rate of citrulline production was practically not affected by aminooxyacetate (cf. also Ref. 24). ADP uptake. Translocation of [~4C]ADP into mitochondria (0.5-0.8 m g / m l protein) was measured at 2°C as described by Bryla et al. [25] using an atractylate stopping method [23]. [14C]ADP (0.4 ~tCi/ml) was used at 50 ~tM concentration. Enzyme assay. Carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine transcarbamoylase (EC 2.1.3.3) were assayed in the presence of 0.1% (v/v) Triton X-100 under conditions as described by McGivan et al. [26]. Protein. Mitochondrial protein was measured by biuret reaction, as described by Cleland and Slater [27], with bovine serum albumin as standard. Determination of metabolites. Citrulline was measured according to Archibald [28], as modified by Bryl~a and Harris [29]. N-Acetylglutamate levels in mitochondria were determined according to Meijer and Van Woerkom [13]. ATP was assayed according to Williamson and Corkey [30]. Chemicals. Oligomycin, N-acetylglutamate, aminooxyacetate, EGTA and atractyloside were obtained from Sigma (St. Louis, MO, U.S.A.). Triton X-100 and Tris were obtained from Serva (Heidelberg, F.R.G.). L-Ornithine and L-citrulline were from Fluka (Switzerland). FCCP was a gift from Dr. P. Heytler. [~4C]ADP was obtained from the Institute for Research Production and Uses of Radioisotopes (Prague, Czechoslovakia). Scintillator Unisolve 1 was from Koch-Light Laboratories (Colnbrook, U.K.). Other chemicals were of analytical grade from Polish Chemicals (Gliwice, Poland). Expression of results. The rates of citrulline synthesis were calculated using the least-square curve-fitting technique and expressed as means _+
92 S.E. from the n u m b e r of experiments shown in parentheses. The statistical significance of results ( P ) was calculated by the Student's t-test.
Citrulline synthesis and levels of intramitochondrial A TP and N-acetylglutamate Since rabbit liver m i t o c h o n d r i a exhibit phos-
phoenolpyruvate carboxykinase activity [31], the Results
Carbamoyl-phosphate synthetase (ammonia) and ornithine transcarbamoylase activities Since citrulline synthesis is the result of the sequential action of c a r b a m o y l - p h o s p h a t e synthetase ( a m m o n i a ) a n d o r n i t h i n e t r a n s c a r b a m o y lase, we have d e t e r m i n e d activities of these two enzymes in T r i t o n X-100-treated mitochondria. The values are similar to those reported for rat liver m i t o c h o n d r i a [26]. I n agreement with studies of Jorda et al. [3] o n diabetic rat, c a r b a m o y l - p h o s phate synthetase ( a m m o n i a ) activity was higher in m i t o c h o n d r i a from diabetic rabbits than in those from n o r m a l ones (56.0_+ 2.5 a n d 44.5 _+ 2.3 n m o l / m i n per mg protein, respectively, as calculated for 4 a n i m a l s of each group). In contrast, o r n i t h i n e t r a n s c a r b a m o y l a s e activity r e m a i n e d the same in m i t o c h o n d r i a isolated from livers of both groups of a n i m a l s (740.4 _+ 45.5 a n d 691.2 _+ 30.4 n m o l / m i n per mg protein, as calculated for m i t o c h o n d r i a isolated from livers of 5 n o r m a l a n d 6 diabetic animals, respectively). The latter observation is in agreement with that reported by Sochor et al. [4] for liver of diabetic rat.
energy generated during respiratory chain substrate oxidation in the presence of NH4C1 a n d b i c a r b o n a t e can be utilized for both phosphoenolpyruvate a n d citrulline synthesis [24]. However, in m i t o c h o n d r i a isolated from both normal a n d diabetic rabbits the rate of phosphoenolpyruvate formation in the presence of glutamate in State 4 is very low (0.6 _+ 0.1 and 2.1 _+ 0.2 n m o l / m i n per mg protein as measured for m i t o c h o n d r i a of 3 a n i m a l s of each group), p r e s u m a b l y due to an elevated N A D H / N A D + ratio, limiting the int r a m i t o c h o n d r i a l oxaloacetate synthesis [24]. As shown in Table I, in m i t o c h o n d r i a isolated from n o r m a l rabbits the rate of citrulline formation in the presence of succinate as substrate is a b o u t 30-40% higher t h a n that d e t e r m i n e d in the presence of glutamate. The highest rate of citrulline synthesis was observed in u n c o u p l e d m i t o c h o n d r i a i n c u b a t e d in the presence of exogenous ATP, suggesting that the i n t r a m i t o c h o n d r i a l A T P may limit the citrulline p r o d u c t i o n in m i t o c h o n d r i a inc u b a t e d with the respiratory chain substrate. The f i n d i n g that i n t r a m i t o c h o n d r i a l levels of A T P with succinate as substrate are a b o u t 50% higher as c o m p a r e d to those with glutamate as substrate is
TABLE I EFFECT OF DIABETES ON CITRULLINE SYNTHESIS AND INTRAMITOCHONDRIAL N-ACETYLGLUTAMATE AND ATP LEVELS Liver mitochondria isolated from either normal or diabetic rabbits were incubated. In mitochondria isolated from normal animals citrulline formation in the presence of either succinate or ATP + FCCP+ oligomycin( + rotenone) is different from that measured with glutamate (P < 0.05 and P < 0.001, respectively), while in mitochondria obtained from diabetic animals citrulline production in the presence of glutamate is different from that determined with either succinate or ATP + FCCP + oligomycin ( + rotenone) ( P < 0.05 and P < 0.005, respectively).ATP levels with succinate are significantly different from those with glutamate in mitochondria isolated from both normal and diabetic animals (P < 0.001), while N-acetylglutamate content determined with glutamate in mitochondria of diabetic rabbits is significantly different from that measured with succinate or exogenous ATP (P < 0.05). Additions
Glutamate Succinate ( + rotenone) ATP + FCCP + oligomycin ( + rotenone)
Citrulline synthesis (nmol/min per mg protein)
N-Acetylglutamate (nmol/mg protein)
ATP (nmol/mg protein)
normal
diabetic
normal
diabetic
normal
diabetic
16.6 + 1.6(7) 22.8 _+1.8(7)
39.0 _+1.5(5) " 32.2 _+2.6(6) b
1.07 +_0.08(3) 0.80 _+0.07(4)
1.42 _+0.06(4) b 1.07 + 0.08(5) ~"
2.7 + 0.2(4) 4.3 _+0.5(4)
4.3 + 0.1(3) ~ 6.1 _+0.2(3) ~
39.3 _+2.4(8)
59.1 +_1.8(5) ~
0.99 + 0.14(3)
1.18 _+0.04(3)
-
P values versus corresponding control determined in mitochondria of normal animals: a p < 0.005; b p < 0.02; " P < 0.05.
93
in agreement with this hypothesis. Alloxan diabetes resulted in an increase of both citrulline formation and mitochondrial ATP content. However, in contrast to mitochondria isolated from normal animals, the rate of citrulline formation in the presence of glutamate in mitochondria from diabetic rabbits was about 20% higher than that measured with succinate, presumably due to an increase of intramitochondrial N-acetylglutamate content. However, the addition of 5 mM N-acetylglutamate did not increase the rate of citrulline formation from succinate (not shown), since N-acetylglutamate is probably not transported into mitochondria under these conditions (cf. Ref. 32). Similarly to mitochondria from normal rabbits, the ATP level in mitochondria from diabetic animals was about 50% higher with succinate than with glutamate. The N-acetylglutamate content of the suspension of freshly isolated mitochondria from livers of diabetic rabbits was similar to that measured for the control animals (1.51 _+ 0.04 and 1.59 _+ 0.02 n m o l / m g protein, respectively, as calculated for mitochondria from 3 animals of each group). Thus, the marked elevation of mitochondrial N-acetylglutamate level found in the presence of glutamate in mitochondria of diabetic rabbits in comparison with that measured in mitochondria of normal animals may result from an acceleration of N-
T A B L E II EFFECT OF VARIOUS CONCENTRATIONS OF A T R A C T Y L O S I D E ON C I T R U L L I N E SYNTHESIS A N D N - A C E T Y L G L U T A M A T E C O N T E N T IN RABBIT LIVER MITOCHONDRIA INCUBATED WITH EXOGENOUS ATP Isolated rabbit liver mitochondria from normal animals were incubated with ATP + F C C P + oligomycin ( + rotenone) and with indicated concentrations of atractyloside. Additions
Citrulline synthesis ( n m o l / m i n per mg protein)
N-Acetylglutamate content (nmol/mg protein)
None 10 ~ M atractyloside 30 ~ M atractyloside
43.1 _+3.4(6) 31.5 _+2.5(6) a 20.8 _+2.1(6) b
1.15 _+0.14(3) 1.17 _+0.18(3) 1.15 _+0.19(3)
P values versus control with no atractyloside: a p < 0.02; b p < 0.005.
acetylglutamate synthetase activity due to generation of substrates of the enzyme during incubation of mitochondria. An enhancement of N-acetylglutamate synthetase activity was also observed in livers of diabetic rats [3]. Although increasing concentrations of atractyloside, an inhibitor of adenine nucleotide translocase [33,34], resulted in an inhibition of citrulline formation in uncoupled mitochondria incubated with oligomycin and exogenous ATP, the intramitochondrial N-acetylglutamate content was not altered (Table II), irrespective of the presence of glutamate (not shown).
ADP uptake Since the rate of citrulline synthesis in the presence of exogenous ATP, despite the similar Nacetylglutamate levels, is 2-fold higher in mitochondira isolated from diabetic rabbits than in those from normal animals (cf. Table I) it seems likely that the activity of adenine nucleotide translocase is accelerated in mitochondria from diabetic rabbits. In order to check this possibility we have studied the effect of diabetes on the rate of [t4C]ADP uptake into rabbit liver mitochondria. We have found that in mitochondria isolated from diabetic animals the ADP.uptake rates were 3-fold higher than those determined in mitochondria from normal rabbits (1.93 _+ 0.05 and 0.66 _+ 0.09 n m o l / min per mg protein, respectively, as calculated for mitochondria of 4 diabetic animals and of 3 normal ones). This result is similar to that found following glucagon or cAMP treatment of the rat [25]. Discussion
The importance of N-acetylglutamate, an activator of carbamoyl-phosphate synthetase (ammonia) [6], in the control of urea cycle activity has been demonstrated by several authors [10,12,15, 16,18,20,35]. Jorda et al. [3] reported that changes produced by streptozotocin-diabetes on the N-acetylglutamate concentration and on N-acetylglutamate synthetase and carbamoylphosphate synthetase (ammonia) activities, directly affect the capacity of rat liver mitochondria to synthesize citrulline and the ability of hepatocytes to synthesize urea. In agreement with the findings of
94 Jorda et al. [3] the experiments described in this paper show a significant increase in carbamoylphosphate synthetase (ammonia) activity in mitochondria isolated from livers of alloxan-diabetic rabbits in comparison with that in normal animals. However, in our hands citrulline production was in a poor correlation with N-acetylglutamate levels, since the highest rates of citrulline synthesis occurred in uncoupled mitochondria incubated with exogenous ATP as energy source, i.e., under conditions when intramitochondrial Nacetylglutamate content was not increased (cf. Table I). This poor correlation between mitochondrial levels of N-acetylglutamate and rates of citrulline formation in both control and alloxan-diabetic rabbits is in agreement with that reported by Titheradge and Haynes [36] and H a m m a n and Haynes [37] for liver mitochondria isolated from glucagon-treated rats. The intramitochondrial Nacetylglutamate levels presented above (cf. Table I) are similar to those reported by several other authors [10,13,18,32] for rat liver mitochondria. McGivan et al. [12] have postulated, however, that part of the N-acetylglutamate measured is not available for the activation of carbamoyl-phosphate synthetase (ammonia), so that available Nacetylglutamate could limit the enzyme activity. Recently, the function of this metabolite as a short-term regulator of urea synthesis was questioned by Lund and Wiggins [38]. In view of our observations it seems that both an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of intramitochondrial level of ATP, one of the substrates of the enzyme, are responsible for the stimulation of citrulline production in liver mitochondria isolated from alloxan-diabetic rabbits. In vitro, the rate of citrulline synthesis varies as a function of the matrix ATP concentration [35,36,39-42]. Moreover, it has been proposed that the stimulation by glucagon of citrulline formation in rat liver mitochondria [25,43,44] is due to an elevated concentration of intramitochondrial ATP [25,36,45]. Since the glucagon concentration has been shown to be high in the blood of diabetic humans, dogs and rats [46,47], an increase of intramitochondrial ATP levels in livers of diabetic animals might be responsible for the stimulation of citrulline synthesis under these conditions.
In vivo, the mitochondrial adenine nucleotide content has been shown to increase postnatally [48-50] and in response to hormones in adults [10,51,52]. An increase of exchangeable adenine nucleotides in rat liver mitochondria has been shown to accelerate the rate of A T P - A D P translocase reactions and the activity of several mitochondrial functions when less than normal a~nounts of nucleotides were present [48,49,53]. Thus, an increase of intramitochondrial ATP level (Table I) could result in an enhancement of adenine n u c l e o t i d e t r a n s l o c a t i o n activity in liver mitochondria of diabetic rabbits. Contrary to these findings an increased content of adenine nucleotides in mitochondria of glucagon-treated rats did not greatly accelerate the rate of adenine nucleotide translocation [54]. Content of ATP in rabbit liver mitochondria (Table I) has been found to be lower than that reported for rat liver mitochondria [10,42]. According to Goldstein and Aprille [42], the matrix ATP content at which half-maximal ATP rates of citrulline synthesis were observed in intact rat liver mitochondria was about 5 n m o l / m g protein. Comparing this value with those measured for rabbit liver mitochondria it seems likely that the matrix content of ATP might limit the rate of carbamoyl phosphate formation and thus the velocity of citrulline production in rabbit liver mitochondria. It is reasonable to suppose, however, that both ATP and N-acetylglutamate may be regulatory in vivo. According to Wanders et al. [41], if the matrix ATP exceed 6 - 7 n m o l / m g protein, the effect of N-acetylglutamate may predominate by altering the magnitude of the maximum rate associated with ATP saturation.
Acknowledgements We are grateful to Mr. K. Zablocki for ATP measurements. This investigation was supported in part by the grant of the Polish Academy of Sciences (MR II.1.1.7).
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Biochimica et Biophysica Acta 839 (1985) 90 95
Elsevier BBA 22010
The stimulatory effect of alloxan diabetes on citrulline formation in rabbit liver mitochondria
Jadwiga Bryla and Maciej Garstka Institute of Biochemistry, University of Warsaw, AI. ~wirki i Wigury 93, 02-089 Warsaw (Poland)
(Received September 21st, 1984) (Revised manuscript received December 10th, 1984)
Key words: Alloxan; Diabetes: Citrulline synthesis; (Rabbit liver mitochondria) The effect of alloxan diabetes on citrulline formation from NH4CI and bicarbonate was studied in rabbit liver mitochondria incubated with glutamate or succinate as respiratory substrate, as well as with exogenous ATP in the presence of uncoupler and oligomycin. In contrast to ornithine transcarbamoylase, the activity of carbamoyl-phosphate synthetase (ammonia) was higher in mitochondria from diabetic animals than in those from normal ones. In diabetic rabbits the rates of citrulline synthesis were stimulated under all conditions studied. In contrast, levels of N-acetylglutamate, an activator of carbamoyl-phosphate synthetase (ammonia), were significantly increased only in the presence of glutamate, while the highest rates of citrulline formation occurred in uncoupled mitochondria incubated with exogenous ATP as energy source. Treatment of animals with alloxan resulted in an increase of both the intramitochondiral A T P level and the rate of adenine nucleotide translocation across the mitochondrial membrane. The results indicate that the stimulation of citrulline formation in liver mitochondria of diabetic rabbits is mainly due to an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of content of intramitochondrial ATP, a substrate of this enzyme.
Introduction
The increased urea excretion in diabetic rats can be correlated with the enhancement of the urea synthesis by liver slices [1] and isolated hepatocytes [2,3] and also with the increased citrulline synthesis by isolated mitochondria [2,3]. Levels of the main urea synthesis enzymes are also elevated in both alloxan-diabetic [1,4] and streptozotocin-diabetic rats [2]. Since carbamoyl-phosphate synthetase (ammonia) is the first step in ureagenesis, this enzyme is a likely site for regulatory control [5]. The enzyme is not dependent only upon the substrates
Abbreviation: FCCP, trifluoromethoxyphenylhydrazone.
concentrations but has an absolute requirement for N-acetylglutamate as allosteric activator [6]. Numerous studies, including those in vivo [7-9], with isolated hepatocytes [10,11] and isolated mitochondria [3,12 14] have demonstrated that rates of synthesis of urea or citrulline are mainly determined by N-acetylglutamate concentrations. The levels of this metabolite were reported to increase in response to nitrogen load [7,8,15-17], glucagon treatment [10,18-20] and diabetes [3]. Much of our knowledge about the effect of diabetes on hepatic ureagenesis has been derived from studies with rats. Therefore, the purpose of this investigation was to search for changes induced by diabetes in other mammalian species, for example, rabbit. In a previous communication from this laboratory we have reported that urea produc-
0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
91 tion from amino acids or NH4C1 as ammonia donor is stimulated in hepatocytes isolated from alloxan-diabetic rabbits [21]. In this paper we present some of factors responsible for the stimulatory effect of alloxan diabetes on citrulline synthesis in rabbit liver mitochondria. Materials and Methods
Animals. Young male white rabbtis (Popielno strain), weighing 5 0 0 - 1 0 0 0 g, were used throughout. Alloxan diabetes was introduced as described previously [21]. The mean value for the blood glucose concentration of rabbits considered as diabetic was 390 _+ 12 m g / 1 0 0 ml, as calculated for eleven animals. Liver mitochondria were isolated on day 4 after the alloxan treatment. Isolation and incubation of mitochondria. Liver mitochondria were isolated as described by Harris et al. [22], but the final wash and suspension of mitochondria were made with 0.3 M mannitol replacing the sucrose. Rabbit liver mitochondria (about 4-5 mg prot e i n / m l ) were incubated at 30°C in a basic medium containing 15 mM KC1, 10 mM NH4C1, 30 mM K H C O 3, 75 mM Tris-HC1 (pH 7.4), 1 mM EGTA, 5 mM potassium-phosphate buffer (pH 7.1), 0.2 mM aminooxyacetate 10 mM ornithine, 15 mM mannitol deriving from the mitochondrial suspension and either 10 mM glutamate, 10 mM succinate ( + 4 ~tM rotenone) or 5 mM ATP ( + 4 / ~ M rotenone + oligomycin (1 / ~ g / m g protein) + 0.1-0.2 ~M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)), as indicated in the tables. The incubations were started by the addition of either mitochondrial suspension or uncoupler to the reaction medium. The incubation mixture was continuously gassed with O 2 / C O 2 (95% : 5%). At 2, 4, 6, 8 and 10 min of incubation 1-ml samples were withdrawn from the suspensions. Samples were deproteinized with 0.1 volume of 40% HC104 (v/v) and centrifuged. Supernatants were collected and used for citrulline measurements. The rate of citrulline synthesis was linear with time up to 20 min of incubation. After 15 min of incubation a 7-ml sample was withdrawn from the reaction medium. Mitochondria were separated from the suspension by rapid centrifugation and after careful removal of the
supernatant used for determination of N-acetylglutamate level, as described by Meijer and Van Woerkom [13]. Intramitochondrial ATP content was determined in samples taken after 8 min of incubation, using an atractylate stopping method [23]. When aminooxyacetate was omitted from the reaction medium, the mitochondrial ATP content was higher only about 10-15% in comparison with that estimated in the presence of aminooxyacetate, while the rate of citrulline production was practically not affected by aminooxyacetate (cf. also Ref. 24). ADP uptake. Translocation of [~4C]ADP into mitochondria (0.5-0.8 m g / m l protein) was measured at 2°C as described by Bryla et al. [25] using an atractylate stopping method [23]. [14C]ADP (0.4 ~tCi/ml) was used at 50 ~tM concentration. Enzyme assay. Carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine transcarbamoylase (EC 2.1.3.3) were assayed in the presence of 0.1% (v/v) Triton X-100 under conditions as described by McGivan et al. [26]. Protein. Mitochondrial protein was measured by biuret reaction, as described by Cleland and Slater [27], with bovine serum albumin as standard. Determination of metabolites. Citrulline was measured according to Archibald [28], as modified by Bryl~a and Harris [29]. N-Acetylglutamate levels in mitochondria were determined according to Meijer and Van Woerkom [13]. ATP was assayed according to Williamson and Corkey [30]. Chemicals. Oligomycin, N-acetylglutamate, aminooxyacetate, EGTA and atractyloside were obtained from Sigma (St. Louis, MO, U.S.A.). Triton X-100 and Tris were obtained from Serva (Heidelberg, F.R.G.). L-Ornithine and L-citrulline were from Fluka (Switzerland). FCCP was a gift from Dr. P. Heytler. [~4C]ADP was obtained from the Institute for Research Production and Uses of Radioisotopes (Prague, Czechoslovakia). Scintillator Unisolve 1 was from Koch-Light Laboratories (Colnbrook, U.K.). Other chemicals were of analytical grade from Polish Chemicals (Gliwice, Poland). Expression of results. The rates of citrulline synthesis were calculated using the least-square curve-fitting technique and expressed as means _+
92 S.E. from the n u m b e r of experiments shown in parentheses. The statistical significance of results ( P ) was calculated by the Student's t-test.
Citrulline synthesis and levels of intramitochondrial A TP and N-acetylglutamate Since rabbit liver m i t o c h o n d r i a exhibit phos-
phoenolpyruvate carboxykinase activity [31], the Results
Carbamoyl-phosphate synthetase (ammonia) and ornithine transcarbamoylase activities Since citrulline synthesis is the result of the sequential action of c a r b a m o y l - p h o s p h a t e synthetase ( a m m o n i a ) a n d o r n i t h i n e t r a n s c a r b a m o y lase, we have d e t e r m i n e d activities of these two enzymes in T r i t o n X-100-treated mitochondria. The values are similar to those reported for rat liver m i t o c h o n d r i a [26]. I n agreement with studies of Jorda et al. [3] o n diabetic rat, c a r b a m o y l - p h o s phate synthetase ( a m m o n i a ) activity was higher in m i t o c h o n d r i a from diabetic rabbits than in those from n o r m a l ones (56.0_+ 2.5 a n d 44.5 _+ 2.3 n m o l / m i n per mg protein, respectively, as calculated for 4 a n i m a l s of each group). In contrast, o r n i t h i n e t r a n s c a r b a m o y l a s e activity r e m a i n e d the same in m i t o c h o n d r i a isolated from livers of both groups of a n i m a l s (740.4 _+ 45.5 a n d 691.2 _+ 30.4 n m o l / m i n per mg protein, as calculated for m i t o c h o n d r i a isolated from livers of 5 n o r m a l a n d 6 diabetic animals, respectively). The latter observation is in agreement with that reported by Sochor et al. [4] for liver of diabetic rat.
energy generated during respiratory chain substrate oxidation in the presence of NH4C1 a n d b i c a r b o n a t e can be utilized for both phosphoenolpyruvate a n d citrulline synthesis [24]. However, in m i t o c h o n d r i a isolated from both normal a n d diabetic rabbits the rate of phosphoenolpyruvate formation in the presence of glutamate in State 4 is very low (0.6 _+ 0.1 and 2.1 _+ 0.2 n m o l / m i n per mg protein as measured for m i t o c h o n d r i a of 3 a n i m a l s of each group), p r e s u m a b l y due to an elevated N A D H / N A D + ratio, limiting the int r a m i t o c h o n d r i a l oxaloacetate synthesis [24]. As shown in Table I, in m i t o c h o n d r i a isolated from n o r m a l rabbits the rate of citrulline formation in the presence of succinate as substrate is a b o u t 30-40% higher t h a n that d e t e r m i n e d in the presence of glutamate. The highest rate of citrulline synthesis was observed in u n c o u p l e d m i t o c h o n d r i a i n c u b a t e d in the presence of exogenous ATP, suggesting that the i n t r a m i t o c h o n d r i a l A T P may limit the citrulline p r o d u c t i o n in m i t o c h o n d r i a inc u b a t e d with the respiratory chain substrate. The f i n d i n g that i n t r a m i t o c h o n d r i a l levels of A T P with succinate as substrate are a b o u t 50% higher as c o m p a r e d to those with glutamate as substrate is
TABLE I EFFECT OF DIABETES ON CITRULLINE SYNTHESIS AND INTRAMITOCHONDRIAL N-ACETYLGLUTAMATE AND ATP LEVELS Liver mitochondria isolated from either normal or diabetic rabbits were incubated. In mitochondria isolated from normal animals citrulline formation in the presence of either succinate or ATP + FCCP+ oligomycin( + rotenone) is different from that measured with glutamate (P < 0.05 and P < 0.001, respectively), while in mitochondria obtained from diabetic animals citrulline production in the presence of glutamate is different from that determined with either succinate or ATP + FCCP + oligomycin ( + rotenone) ( P < 0.05 and P < 0.005, respectively).ATP levels with succinate are significantly different from those with glutamate in mitochondria isolated from both normal and diabetic animals (P < 0.001), while N-acetylglutamate content determined with glutamate in mitochondria of diabetic rabbits is significantly different from that measured with succinate or exogenous ATP (P < 0.05). Additions
Glutamate Succinate ( + rotenone) ATP + FCCP + oligomycin ( + rotenone)
Citrulline synthesis (nmol/min per mg protein)
N-Acetylglutamate (nmol/mg protein)
ATP (nmol/mg protein)
normal
diabetic
normal
diabetic
normal
diabetic
16.6 + 1.6(7) 22.8 _+1.8(7)
39.0 _+1.5(5) " 32.2 _+2.6(6) b
1.07 +_0.08(3) 0.80 _+0.07(4)
1.42 _+0.06(4) b 1.07 + 0.08(5) ~"
2.7 + 0.2(4) 4.3 _+0.5(4)
4.3 + 0.1(3) ~ 6.1 _+0.2(3) ~
39.3 _+2.4(8)
59.1 +_1.8(5) ~
0.99 + 0.14(3)
1.18 _+0.04(3)
-
P values versus corresponding control determined in mitochondria of normal animals: a p < 0.005; b p < 0.02; " P < 0.05.
93
in agreement with this hypothesis. Alloxan diabetes resulted in an increase of both citrulline formation and mitochondrial ATP content. However, in contrast to mitochondria isolated from normal animals, the rate of citrulline formation in the presence of glutamate in mitochondria from diabetic rabbits was about 20% higher than that measured with succinate, presumably due to an increase of intramitochondrial N-acetylglutamate content. However, the addition of 5 mM N-acetylglutamate did not increase the rate of citrulline formation from succinate (not shown), since N-acetylglutamate is probably not transported into mitochondria under these conditions (cf. Ref. 32). Similarly to mitochondria from normal rabbits, the ATP level in mitochondria from diabetic animals was about 50% higher with succinate than with glutamate. The N-acetylglutamate content of the suspension of freshly isolated mitochondria from livers of diabetic rabbits was similar to that measured for the control animals (1.51 _+ 0.04 and 1.59 _+ 0.02 n m o l / m g protein, respectively, as calculated for mitochondria from 3 animals of each group). Thus, the marked elevation of mitochondrial N-acetylglutamate level found in the presence of glutamate in mitochondria of diabetic rabbits in comparison with that measured in mitochondria of normal animals may result from an acceleration of N-
T A B L E II EFFECT OF VARIOUS CONCENTRATIONS OF A T R A C T Y L O S I D E ON C I T R U L L I N E SYNTHESIS A N D N - A C E T Y L G L U T A M A T E C O N T E N T IN RABBIT LIVER MITOCHONDRIA INCUBATED WITH EXOGENOUS ATP Isolated rabbit liver mitochondria from normal animals were incubated with ATP + F C C P + oligomycin ( + rotenone) and with indicated concentrations of atractyloside. Additions
Citrulline synthesis ( n m o l / m i n per mg protein)
N-Acetylglutamate content (nmol/mg protein)
None 10 ~ M atractyloside 30 ~ M atractyloside
43.1 _+3.4(6) 31.5 _+2.5(6) a 20.8 _+2.1(6) b
1.15 _+0.14(3) 1.17 _+0.18(3) 1.15 _+0.19(3)
P values versus control with no atractyloside: a p < 0.02; b p < 0.005.
acetylglutamate synthetase activity due to generation of substrates of the enzyme during incubation of mitochondria. An enhancement of N-acetylglutamate synthetase activity was also observed in livers of diabetic rats [3]. Although increasing concentrations of atractyloside, an inhibitor of adenine nucleotide translocase [33,34], resulted in an inhibition of citrulline formation in uncoupled mitochondria incubated with oligomycin and exogenous ATP, the intramitochondrial N-acetylglutamate content was not altered (Table II), irrespective of the presence of glutamate (not shown).
ADP uptake Since the rate of citrulline synthesis in the presence of exogenous ATP, despite the similar Nacetylglutamate levels, is 2-fold higher in mitochondira isolated from diabetic rabbits than in those from normal animals (cf. Table I) it seems likely that the activity of adenine nucleotide translocase is accelerated in mitochondria from diabetic rabbits. In order to check this possibility we have studied the effect of diabetes on the rate of [t4C]ADP uptake into rabbit liver mitochondria. We have found that in mitochondria isolated from diabetic animals the ADP.uptake rates were 3-fold higher than those determined in mitochondria from normal rabbits (1.93 _+ 0.05 and 0.66 _+ 0.09 n m o l / min per mg protein, respectively, as calculated for mitochondria of 4 diabetic animals and of 3 normal ones). This result is similar to that found following glucagon or cAMP treatment of the rat [25]. Discussion
The importance of N-acetylglutamate, an activator of carbamoyl-phosphate synthetase (ammonia) [6], in the control of urea cycle activity has been demonstrated by several authors [10,12,15, 16,18,20,35]. Jorda et al. [3] reported that changes produced by streptozotocin-diabetes on the N-acetylglutamate concentration and on N-acetylglutamate synthetase and carbamoylphosphate synthetase (ammonia) activities, directly affect the capacity of rat liver mitochondria to synthesize citrulline and the ability of hepatocytes to synthesize urea. In agreement with the findings of
94 Jorda et al. [3] the experiments described in this paper show a significant increase in carbamoylphosphate synthetase (ammonia) activity in mitochondria isolated from livers of alloxan-diabetic rabbits in comparison with that in normal animals. However, in our hands citrulline production was in a poor correlation with N-acetylglutamate levels, since the highest rates of citrulline synthesis occurred in uncoupled mitochondria incubated with exogenous ATP as energy source, i.e., under conditions when intramitochondrial Nacetylglutamate content was not increased (cf. Table I). This poor correlation between mitochondrial levels of N-acetylglutamate and rates of citrulline formation in both control and alloxan-diabetic rabbits is in agreement with that reported by Titheradge and Haynes [36] and H a m m a n and Haynes [37] for liver mitochondria isolated from glucagon-treated rats. The intramitochondrial Nacetylglutamate levels presented above (cf. Table I) are similar to those reported by several other authors [10,13,18,32] for rat liver mitochondria. McGivan et al. [12] have postulated, however, that part of the N-acetylglutamate measured is not available for the activation of carbamoyl-phosphate synthetase (ammonia), so that available Nacetylglutamate could limit the enzyme activity. Recently, the function of this metabolite as a short-term regulator of urea synthesis was questioned by Lund and Wiggins [38]. In view of our observations it seems that both an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of intramitochondrial level of ATP, one of the substrates of the enzyme, are responsible for the stimulation of citrulline production in liver mitochondria isolated from alloxan-diabetic rabbits. In vitro, the rate of citrulline synthesis varies as a function of the matrix ATP concentration [35,36,39-42]. Moreover, it has been proposed that the stimulation by glucagon of citrulline formation in rat liver mitochondria [25,43,44] is due to an elevated concentration of intramitochondrial ATP [25,36,45]. Since the glucagon concentration has been shown to be high in the blood of diabetic humans, dogs and rats [46,47], an increase of intramitochondrial ATP levels in livers of diabetic animals might be responsible for the stimulation of citrulline synthesis under these conditions.
In vivo, the mitochondrial adenine nucleotide content has been shown to increase postnatally [48-50] and in response to hormones in adults [10,51,52]. An increase of exchangeable adenine nucleotides in rat liver mitochondria has been shown to accelerate the rate of A T P - A D P translocase reactions and the activity of several mitochondrial functions when less than normal a~nounts of nucleotides were present [48,49,53]. Thus, an increase of intramitochondrial ATP level (Table I) could result in an enhancement of adenine n u c l e o t i d e t r a n s l o c a t i o n activity in liver mitochondria of diabetic rabbits. Contrary to these findings an increased content of adenine nucleotides in mitochondria of glucagon-treated rats did not greatly accelerate the rate of adenine nucleotide translocation [54]. Content of ATP in rabbit liver mitochondria (Table I) has been found to be lower than that reported for rat liver mitochondria [10,42]. According to Goldstein and Aprille [42], the matrix ATP content at which half-maximal ATP rates of citrulline synthesis were observed in intact rat liver mitochondria was about 5 n m o l / m g protein. Comparing this value with those measured for rabbit liver mitochondria it seems likely that the matrix content of ATP might limit the rate of carbamoyl phosphate formation and thus the velocity of citrulline production in rabbit liver mitochondria. It is reasonable to suppose, however, that both ATP and N-acetylglutamate may be regulatory in vivo. According to Wanders et al. [41], if the matrix ATP exceed 6 - 7 n m o l / m g protein, the effect of N-acetylglutamate may predominate by altering the magnitude of the maximum rate associated with ATP saturation.
Acknowledgements We are grateful to Mr. K. Zablocki for ATP measurements. This investigation was supported in part by the grant of the Polish Academy of Sciences (MR II.1.1.7).
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