Evolution of pyruvate carboxylase and other biotin ... - Springer Link

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Manuel Vargas. Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Granada. Campus de Cartuja s/n, Granada, Spain.
Molecular and Cellular Biochemistry 200: 111–117, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney Rafael Salto, María Dolores Girón, María del Mar Sola and Alberto Manuel Vargas Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Granada. Campus de Cartuja s/n, Granada, Spain Received 20 August 1998; accepted 12 March 1999

Abstract The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period. (Mol Cell Biochem 200: 111–117, 1999) Key words: pyruvate carboxylase, perinatal development, rat, biotin, gluconeogenesis, mitochondria

Introduction Pyruvate carboxylase (EC 6.4.1.1) is an enzyme encoded in the nuclear genome and exclusively located in mitochondria. Pyruvate carboxylase is a biotin-enzyme that catalyzes the ATPdependent carboxylation of pyruvate rendering oxalacetate [1, 2], a crucial reaction in the anaplerosis of the Krebs’s cycle, in the lipogenesis and in the gluconeogenesis [3–5]. In rat hepatocytes, under some metabolic conditions, pyruvate carboxylase has the highest flux-control coefficient in the gluconeogenic pathway from lactate and pyruvate [6].

The in vivo regulation of pyruvate carboxylase activity relies on the concentration of substrates and allosteric effectors as well as on the concentration of the enzyme [1, 2]. The regulation of the enzyme levels constitutes a long term mechanism and it is mediated by the control of the synthesis and degradation of the enzyme. It has been widely documented the action of glucocorticoids and thyroid hormones on the induction of pyruvate carboxylase [7, 8]. Its important metabolic role together with its nuclear gene localization makes of pyruvate carboxylase a useful instrument to follow mitochondrial differentiation during perinatal

Address for offprints: A.M. Vargas, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n., 18071 Granada, Spain

112 development, a subject of particular relevance due to the central role of mitochondria in the oxidative metabolism. Birth in mammals represents a change from a situation in which the fetus has a constant supply of nutrient (a high-carbohydrate and low-fat supply) to starvation until suckling is established (a high-fat and low-carbohydrate diet) [9]. The fetus metabolizes glucose anaerobically as the main nutrient due to the absence of mature mitochondria and consequently of functional oxidative phosphorylation [10–11]. At birth, the newborn relies on his own reserves. Therefore, due to the high glycolitic metabolism, a severe hypoglycemia is produced reverting several hours later by the induction of liver gluconeogenesis and by the decrease in the glucose utilization as the main metabolic fuel [12]. As liver and kidney are the main gluconeogenic organs in vertebrates and the mitochondrial carboxylation of pyruvate is an obligatory step to produce glucose from the most abundant three carbons’ precursors, we have studied the evolution of this enzyme in liver and kidney during the perinatal development.

Materials and methods Animals Wistar rats (200–210 g, University of Granada breeding colony) were housed in a temperature (22°C) and light controlled (12-h cycle) animal facility and were fed on a standard diet. Animals were studied in compliance with our institution’s guidelines for animal research. Females were caged overnight with males and conception was assumed next morning by the presence of spermatozoa in the vagina. In this strain, parturition occurs on day 22 of gestation. Accordingly, fetuses were delivered on days 20 and 21 of gestation from cervically dislocated pregnant rats. These fetuses as well as 6 hours to 21 day-old pups were killed by cervical dislocation. The newborns were maintained suckling until they were killed. Their livers and kidneys were quickly removed and placed in ice cold PBS. For kidneys removed between day 5 and adult age, the medulla was discarded and only the cortex was used. Preparation of extracts Liver and kidney samples were homogenized in 10 vols of 50 mm Tris HCl pH 7.4 containing 250 mM sucrose and 1 mm Na4EDTA and were centrifuged at 14,000 × g for 15 min. The pellets were freeze-dried and extracted with 50 mM Tris HCl pH 7.6 containing 150 mM KCl, 10 mM Mg2Cl and 10% (v/v) glycerol. After centrifugation at 15,000 × g for 15 min, the pyruvate carboxylase activity and amount of the enzyme were

assayed in the supernatant. All buffers contained 4 µM PMSF and 15 kallikrein-inactivating units of aprotinin/ml as proteases’ inhibitors.

Pyruvate carboxylase determinations Enzyme activity was assayed by a 14CO2-fixation method [4]. The oxalacetate produced was determined after its conversion into citrate by citrate synthase using in situ synthesized acetyl-coenzyme A from acetyl phosphate and coenzyme A. One unit of enzyme activity represents the synthesis of 1 µmol of oxalacetate/min. The amount of pyruvate carboxylase in the different samples was measured by a competitive ELISA. For this purpose, rabbit polyclonal antibodies against purified rat kidney pyruvate carboxylase were prepared and the IgG fraction of the serum was used for the quantification [13]. Standard calibration curves were constructed using purified rat liver and kidney cortex pyruvate carboxylase [14]. The ELISA results were reassessed by western-blot using the same antibodies. The presence of biotin containing enzymes in the samples have been detected by western-blot using a streptavidin-peroxidase complex (EstrAvidin-peroxidase from Sigma Chemical Co.) at a 1:250 working dilution.

RNA isolation and reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNA has been isolated from tissue by a guanidinium isothiocyanate water saturated phenol extraction method [15]. First strand cDNA synthesis was performed on 5 µg total RNA using a NotI-oligodT primer and Moloney murine leukemia virus reverse transcriptase in a final volume of 15 µl as described by the manufacturer (Pharmacia First Strand Synthesis kit). PCR amplification of a 287 bp fragment corresponding to the carboxyl end of the pyruvate carboxylase was accomplished by using a set of oligonucleotide primers. Reaction containing no reverse transcripted samples were run to demonstrate absence of genomic DNA contamination. Oligonucleotide primers used for amplification were: PC-forward 5′-GACCTTGCACATCAAAGCCC-3′; PCreverse: 5′-CTCCATGGGCGAAGTCACC-3′. Reactions were performed in a DNA thermal cycler (Ericomp EasyCycler) in a 50 µl final volume of reaction buffer (50 mm KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl pH 9.0) containing 2.5 µl of transcripted RNA, 100 pmol of each primer and 0.2 µm concentration of dNTPs. Cycling conditions were: An initial denaturation at 94°C for 5 min, denaturation at 91°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min. PCR reaction was repeated 25 cycles. PCR products (5 µl) were electrophoresed in 2% agarose gels and stained with ethidium

113 bromide. The amount of mRNA in each sample was quantified by densitometry. Optimization of reverse transcriptase PCR conditions is shown in Fig. 1. All the samples analyzed were within the lineal range of the optimization curve. Pyruvate carboxylase mRNA levels in the samples have been normalized by using a set of primers that amplify a fragment of the glyceraldehide 3-phosphate dehydrogenase as a control gene (Data not shown).

Results Pyruvate carboxylase during perinatal development in liver and kidney The activity and the amount of enzyme in both organs has been studied from the day 20 after pregnancy, considered two days before parturition, to weaning. These results were compared with those obtained in 6 month old rats, considered as adult animals. Pyruvate carboxylase activity was assayed in the mitochondrial fractions by a 14CO2 fixation method while the amount of enzyme was quantified in the same samples by an indirect ELISA (Table 1). In liver, pyruvate carboxylase activity shows a remarkable profile with three major peaks. On day –2 no pyruvate carboxylase was detected, but one day before parturition there is an increase in enzyme activity that turns undetectable at birth. Six h after delivery there is a new rise in the activity that

Fig. 1. Optimization of RT-PCR conditions. A photograph of PCR products amplified with increasing numbers of PCR cycles is shown at top of the panel. PCR products were separated by electrophoresis in 2% agarose gels and stained with ethidium bromide. The amount of mRNA amplified in each sample was quantified by densitometry and plotted against the cycle number. In all analyses of samples, PCR products were collected before the reaction began to reach the plateau phase.

Table 1. Values of activity and amount of pyruvate carboxylase during perinatal development in rat liver

Time

Activity U/g tissue mU/mg protein

Amount mg/g tissue µg/mg protein

–2 days –1 day 0 6h 12 h 1 day 3 days 6 days 12 days Weaning Adult

nd 0.31 ± 0.03 nd 0.42 ± 0.06 2.04 ± 0.14 0.44 ± 0.04 0.36 ± 0.07 4.42 ± 0.21 3.54 ± 0.15 1.50 ± 0.09 4.63 ± 0.25

nd 0.06 ± 0.01 nd 0.07 ± 0.01 0.17 ± 0.02 0.06 ± 0.01 0.05 ± 0.01 0.32 ± 0.01 0.38 ± 0.03 0.24 ± 0.01 0.43 ± 0.01

nd 8.23 ± 0.40 nd 8.50 ± 1.79 59.95 ± 9.17 12.23 ± 1.05 18.51 ± 0.39 254.58 ± 16.03 161.10 ± 17.41 45.80 ± 3.74 86.53 ± 2.88

nd 1.77 ± 0.18 nd 1.76 ± 0.16 4.70 ± 0.37 1.78 ± 0.07 2.87 ± 0.22 17.72 ± 1.13 13.14 ± 1.29 7.46 ± 0.67 8.16 ± 0.19

Pyruvate carboxylase activity has been assayed by a 14 CO 2 fixation method. Pyruvate carboxylase amount has been determined by a competitive ELISA using as calibration curves purified samples of rat liver pyruvate carboxylase. Results are the mean ± S.E.M. of 6 different determinations. nd – Non detectable.

peaks at 12 h, but this increase is only sustained for a few hours. The third broad peak of activity reaches the maximum on the sixth day to drops slowly until the activity it is finally increased after weaning. There is a marked relationship between the in vitro determined activity of pyruvate carboxylase and the amount of enzyme detected by ELISA. The amount of the enzyme during perinatal development has been further confirmed by a Western-blot of the pooled samples at each time point (Fig. 2, panel A). In kidney, pyruvate carboxylase showed a totally different profile. The enzyme activity was undetectable until 6 days after

Fig. 2. Evolution of the amount of pyruvate carboxylase during rat perinatal development. A representative western-blot of liver (panel A) and kidney (panel B) is shown. For this purpose, 25 µg (liver) or 50 µg (kidney) of protein from a pool of samples obtained from the mitochondrial fractions have been electrophoresed in a 10% SDS-PAGE and blotted to nitrocellulose. Pyruvate carboxylase has been detected using as first antibody an antipyruvate carboxylase IgG fraction from rabbit. The second antibody has been an anti-rabbit IgG peroxidase linked.

114 parturition. From this time-point, low levels of activity were measured during the lactation period. After weaning, the activity was increased to reach the adult values, close to those found in liver extracts. We also found a tight relationship between activity and amount of pyruvate carboxylase in kidney as assayed by indirect ELISA. To further confirm the low, if any, levels of expression of pyruvate carboxylase during the early perinatal period, we have used a more sensitive detection system than the indirect ELISA. By using a Westernblot technique we were able to discriminate small amounts of enzyme from the background signal. It can be observed traces of positive signals for the enzyme before the sixth day in the Western-blot of the renal samples (Fig. 2, panel B) although to detect these levels, a double amount of total protein (50 µg) compared to liver samples (25 µg) had to be loaded.

Pyruvate carboxylase mRNA levels during perinatal development Based on the sequence of the rat cDNA for pyruvate carboxylase [16], it has been possible to design oligonucleotides able to be used as primers for RT-PCR quantitation of the enzyme mRNA as described in Materials and methods. Total RNA was isolated from liver and kidneys at different stages of development and cDNA was obtained by reverse transcription and PCR amplification of a 287 bp fragment corresponding to the carboxyl end of the pyruvate carboxylase was done in order to semi-quantitate the level of expression of the gene. The results are shown in Fig. 3.

Fig. 3. Liver and kidney pyruvate carboxylase mRNA levels during rat perinatal development. Pyruvate carboxylase mRNA was semiquantitated by RT-PCR as described in materials and methods from total RNA. The inset shows a photograph of ethidium bromide-stained agarose gels from liver (A) and kidney (B) amplified samples. A plot of the areas of each band is shown. Liver (¨), Kidney (¡).

Table 2. Values of activity and amount of pyruvate carboxylase during perinatal development in rat kidney cortex Activity Time

U/g tissue

Amount mU/mg protein

mg/g tissue

µg/mg protein

6 days 12 days Weaning Adult

0.28 ± 0.01 0.21 ± 0.01 0.78 ± 0.08 3.90 ± 0.27

12.88 ± 0.30 11.92 ± 0.70 23.76 ± 2.27 110.72 ± 5.03

0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.01 0.35 ± 0.01

1.06 ± 0.05 1.20 ± 0.03 1.27 ± 0.08 8.95 ± 0.59

Pyruvate carboxylase activity has been assayed by a 14 CO 2 fixation method. Pyruvate carboxylase amount has been determined by a competitive ELISA using as calibration curves purified samples of rat kidney pyruvate carboxylase. Results are the mean ± S.E.M. of 6 different determinations. Activity and amount of enzyme were under the detection limit until six days after parturition.

During the first few days of the study, pyruvate carboxylase is preferentially expressed in liver compared to kidney. In liver, the evolution of the amount of amplified mRNA showed peaks that precede those of protein amount and activity. As an example, the concentration of mRNA is maximum at 6 h (0.25 days) while the protein concentration and activity are maximum at 12 h, showing a delay between the transcription and the translation and import into the liver mitochondria. At certain time points, i.e. parturition and 12 h after delivery, pyruvate carboxylase mRNA is detected with no parallel detection of enzyme activity or protein inside the mitochondria, suggesting a translational regulation or a control of the import of the protein into the mitochondria during these stages of development.

Fig. 4. Evolution of biotin containing enzymes during rat perinatal development. A representative western-blot of liver (panel A) and kidney (panel B) is shown. For this purpose, 25 µg of a pool of samples from liver and 50 µg of kidney mitochondrial fractions have been electrophoresed in a 10% SDS-PAGE and blotted to nitrocellulose. Carboxylases have been detected using a streptavidin-biotin peroxidase complex. PC – Pyruvate carboxylase; Pr – Propionyl-CoA carboxylase; 3MC – 3-Methyl crotonyl-CoA carboxylase.

115 Other biotin enzymes in the mitochondria By detecting the presence of biotin, it is possible to identify other carboxylases in the mitochondrial fractions [17, 18]. For this purpose we have used a streptavidin-conjugated peroxidase detection system. Using purified rat liver and kidney pyruvate carboxylase, we have optimized the detection system to have a similar sensitivity to a Western-blot using the pyruvate carboxylase antibodies (data not shown). A photograph of liver and kidney carboxylases until the day sixth after parturition is shown in Fig. 4. In our hands, according with the described molecular weights of the subunits, pyruvate carboxylase (125 kDa), propionyl-CoAcarboxylase (75 kDa) and 3-methyl-crotonyl-CoA carboxylase (72 kDa) are clearly detected in liver but there is no signs of propionyl-CoA carboxylase in the kidney samples. Taking into account the differences in concentration among the various carboxylases, the pattern of detection is quite similar, with peaks of concentration at times –1, 0.5 and 6 days in the liver samples and a sustained low-level in the kidney ones. This result implies a similar pattern of induction of these enzymes that also may share import mechanisms into the mitochondria. Furthermore, the amount of pyruvate carboxylase detected either using specific antibodies or the streptavidin-peroxidase system is similar showing that the biotinylation system is well developed before birth.

Discussion Adaptation to extra uterine life is a crucial step in the development of mammals. Metabolism after birth has to be shifted to use a totally different diet, playing the mitochondrial maturation and proliferation an essential role. Probably, there are common mechanisms in order to establish the bioenergetic pathways of respiration and oxidative phosphorylation in the different tissues [19]. However, other mitochondrial metabolic pathways may have been specifically regulated according to the roles of each particular organ or tissue in the metabolism. Pyruvate carboxylase is one example of such phenomenon, being an enzyme with several metabolic roles that have to be developed in a tissuespecific manner. Since liver and kidney are the main, if not the only, gluconeogenic tissues contributing to the glucose homeostasis of the organism and that there are differences in the substrate selectivity of both tissues [20] we decided to study the evolution of the pyruvate carboxylase during perinatal development in both organs. We carried out determinations at close time points (6 h) after parturition to try to correlate the evolution of the enzyme with the previously described crucial steps of mitochondrial maturation [10, 21, 22].

We have found large differences in the concentration of pyruvate carboxylase in liver and kidney during perinatal development that we ascribe mainly to the gluconeogenic role of the enzyme. There is a complex profile of expression of mitochondrial pyruvate carboxylase in liver. The import of this protein into the mitochondria with this pattern implies a programmed biochemical differentiation of the organelle. It has been described that at birth there is a marked increase in respiration and oxidative phosphorylation [21] being pyruvate the main energy substrate oxidized [22]. Taking together the transient decline in pyruvate carboxylase at birth (this paper) and the induction of the pyruvate dehydrogenase complex at the same time [23], the lack of pyruvate carboxylase switches completely the metabolism of pyruvate to its oxidation. Later, the rise in glucagon and the consequent decline in plasma insulin may be as for phosphoenolpyruvate carboxykinase [24] the determinant of pyruvate carboxylase induction. As mRNA peaks for pyruvate carboxylase always precede the peaks of activity and amount of the enzyme, it seems reasonable to propose that the differentiation of liver mitochondria to a gluconeogenic state is mediated partly by the induction of the pyruvate carboxylase gene. Besides the transcriptional control others regulatory mechanisms are also possible. At birth we have detected mRNA that does not translate into mitochondrial protein as assayed by ELISA and Western-blot, therefore a translational mechanism of regulation is possible as it has been described for other mitochondrial enzymes during the perinatal development [25]. Finally, the maturation and control of the import of proteins to the mitochondrial could play a role in the regulation of pyruvate carboxylase in the perinatal period. Our results confirm the limited role of renal gluconeogenesis during perinatal development [26, 27] as no detectable pyruvate carboxylase activity was found in kidney until the sixth day after birth. Moreover, the activity remains at a minimum and only after weaning reaches the adult levels. Many renal mitochondrial activities develop at the late stages of gestation and early after birth, as NaK-ATPase, oxygen consumption and ATP synthase. They increase in the rat kidney before the first day of life by mechanisms depending on protein synthesis de novo and on changes in the concentration of adenine nucleotides [28]. However, the biggest increase in respiration occurs later in the development [29]. It has been well documented that changes in mitochondrial biogenesis and maturation occurring at the final period of lactation in kidney are triggered by glucocorticoids and thyroid hormones [30–32]. These results are in agreement with the finding that pyruvate carboxylase is not induced in kidney by glucocorticoids at the late stages of gestation [27]. Later, the pyruvate carboxylase gene is induced by glucocorticoids [7] and then high enzyme levels appears in kidney at the end of the studied period.

116 It has been demonstrated that in rat adult liver most if not all the pyruvate carboxylase detected is fully biotinylated [33]. It has also been demonstrated that in 3T3-L adipocytes, pyruvate carboxylase is fully biotinylated before its import into the mitochondria independently of the presence of the prosthetic group of the enzyme [34]. Our studies extended these findings to the perinatal period in the rat. In liver and kidney, pyruvate carboxylase is fully biotinylated as early as 2 days before parturition and there is a close relationship between the amount of enzyme detected by antibodies and the amount of enzyme detected using streptavidin-peroxidase complexes. Therefore, the cytosol located holocarboxylase synthetase [35] is able to catalyze the incorporation of biotin during the whole perinatal studied period. The use of streptavidin-peroxidase complexes for the detection of the biotinylation of pyruvate carboxylase allowed us the study of the evolution of other mitochondrial biotin-enzymes. These enzymes, propionyl-CoA carboxylase and 3-methyl crotonyl-CoA carboxylase, have been identified based in their molecular weight upon separation in SDS-PAGE. We have assigned a molecular weight of 75 kDa to propionylCoA carboxylase and 72 kDa to 3-methyl crotonyl-CoA carboxylase, in agreement with the molecular weight previously described for these biotin-enzymes [17]. Using this technique, we have studied the evolution of these two carboxylases from 2 days before parturition until 6 days after the onset. In liver mitochondria, the most abundant biotin-enzyme is the 3-methyl crotonyl-CoA carboxylase, while the propionyl-CoA carboxylase showed the lowest levels. All the carboxylases showed a similar profile of induction, with 3 peaks of amount of enzyme, as described for the pyruvate carboxylase. In kidney, all carboxylases also showed a similar behavior. The pyruvate carboxylase and 3-methyl crotonyl-CoA carboxylase showed low levels during the perinatal development and the propionyl-CoA carboxylase was not detected during this period. The similar evolution of the three carboxylases in both organs probably could be explained by common tissuespecific mechanisms of induction and post-transcriptional regulation.

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