Phosphoenolpyruvate carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism
© Hakimi et al; licensee BioMed Central Ltd. 2005
Received: 21 October 2005
Accepted: 21 November 2005
Published: 21 November 2005
The metabolic function of PEPCK-C is not fully understood; deletion of the gene for the enzyme in mice provides an opportunity to fully assess its function.
The gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 22.214.171.124) (PEPCK-C) was deleted in mice by homologous recombination (PEPCK-C-/- mice) and the metabolic consequences assessed.
PEPCK-C-/- mice became severely hypoglycemic by day two after birth and then died with profound hypoglycemia (12 mg/dl). The mice had milk in their stomachs at day two after birth and the administration of glucose raised the concentration of blood glucose in the mice but did not result in an increased survival. PEPCK-C-/- mice have two to three times the hepatic triglyceride content as control littermates on the second day after birth. These mice also had an elevation of lactate (2.5 times), β-hydroxybutyrate (3 times) and triglyceride (50%) in their blood, as compared to control animals. On day two after birth, alanine, glycine, glutamine, glutamate, aspartate and asparagine were elevated in the blood of the PEPCK-C-/- mice and the blood urea nitrogen concentration was increased by 2-fold. The rate of oxidation of [2-14C]-acetate, and [5-14C]-glutamate to 14CO2 by liver slices from PEPCK-C-/- mice at two days of age was greatly reduced, as was the rate of fatty acid synthesis from acetate and glucose. As predicted by the lack of PEPCK-C, the concentration of malate in the livers of the PEPCK-C-/- mice was 10 times that of controls.
We conclude that PEPCK-C is required not only for gluconeogenesis and glyceroneogenesis but also for cataplerosis (i.e. the removal of citric acid cycle anions) and that the failure of this process in the livers of PEPCK-C-/- mice results in a marked reduction in citric acid cycle flux and the shunting of hepatic lipid into triglyceride, resulting in a fatty liver.
The factors that regulate the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 126.96.36.199) (PEPCK-C) have been studied in great detail over the years . However, its metabolic role in the various tissues in which the gene is expressed has received far less attention. Discussion of the function of PEPCK-C in mammals has largely been confined to its role in gluconeogenesis, with only a brief mention of alternative possibilities. PEPCK-C has become an important marker for hepatic gluconeogenesis; studies of the mechanisms involved in diabetes and related diseases, often include an analysis of the alterations in the mRNA for PEPCK-C. What is seldom mentioned, however, is the fact that PEPCK-C activity is present in a number of tissues that do not make glucose, including white and brown adipose tissue, mammary gland during lactation, small intestine, brain, lung, muscle and a number of others (see  for a review of this subject). Besides its role in gluconeogenesis, PEPCK-C is involved in glyceroneogenesis in adipose tissue  and liver [4, 5]. This pathway is an abbreviated version of gluconeogenesis and results in the synthesis of glyceride-glycerol from precursors other than glucose or glycerol [6, 7]. PEPCK-C can also catalyze the formation of oxalacetate, a citric acid cycle intermediate; it has been suggested that the enzyme "refills" the cycle during periods of biosynthesis (a process known as anaplerosis). However, the major biosynthetic tissues such as the liver and adipose tissue have considerable activity of pyruvate carboxylase, which itself generates oxalacetate. PEPCK-C is also the major cataplerotic enzyme, (cataplerotic enzymes remove citric acid cycle anions that are formed by the entry of the carbon skeletons of amino acids into the cycle). Both gluconeogenesis and glyceroneogenesis are cataplerotic processes, which use intermediates of the citric acid cycle for their specific biosynthetic process. As we will show in this paper, cataplerosis is a critical metabolic function. Finally, Hahn and Nowak  proposed that in brown adipose tissue a cycle operates between the mitochondria and the cytosol in which PEPCK-C converts GTP to GDP, to insure a continued citric acid cycle flux (GDP is a substrate for succinyl CoA synthase).
Ablating the gene for PEPCK-C in mice provides an opportunity to better assess the metabolic role of the enzyme in mammalian tissues. The gene for PEPCK-C has been deleted specifically in the livers of mice by She et al , who noted that the mice had a greatly reduced rate of hepatic gluconeogenesis from a variety of precursors. However, the animals did not develop hypoglycemia, even after as much as 48 hrs of fasting and could be induced to develop diabetes . The absence of PEPCK-C in the liver also resulted in a decrease in the level of glycogen in both the liver and muscle; there was also diminished whole-body glucose turnover. Mice in which the gene for PEPCK-C has been totally deleted in all tissues die in the first two days after birth with profound hypoglycemia. These findings indicate that the kidney (the second gluconeogenic organ) can make sufficient glucose to provide for the needs of the animal during fasting and that it responds to the hormonal alterations characteristic of diabetes by increasing renal glucose output.
A surprising finding from the studies of She et al  was the observation that the ablation of the gene for PEPCK-C caused the mice to develop a fatty liver. As we will report here, the fatty liver appears as early as the end of the first day after birth and is accompanied by a 5-fold elevation in the concentration of triglyceride in the blood. While not expected by conventional metabolic logic, the accumulation of triglyceride in the liver could be the result of a failure of the development of glyceroneogenesis in adipose tissue or to a loss of gluconeogenesis (the major cataplerotic pathway in the liver) in the absence of PEPCK-C. In this paper we assess the impact of a loss of PEPCK-C in the perinatal period and evaluate the various functions suggested for this enzyme in energy metabolism.
Restriction enzymes, Taq DNA polymerase and proteinase K, NAD, NADH, lactate dehydrogenase, and malate dehydrogenase were purchased from Roche Applied Science (Indianapolis, IN). QuickPrep total RNA kit, [5-14C]-sodium glutamate, [U-14C]-glucose, and [2-14C]-sodium acetate was purchased from Amersham Biosciences Corp. (Piscataway, NJ). Triglyceride (GPO) Liquid Reagent Set was purchased from Pointe Scientific, Inc. (Lincoln Park, MI). The NEFA kit was from Wako Chemicals USA, Inc. (Richmond, VA). The embryonic stem cells used in this study were a generous gift from Janet Rossant, University of Toronto.
Generation and maintenance of PEPCK-C-/- mice
PEPCK-C-/- mice and control littermates at one to three days of age were killed by decapitation. Their livers and blood were collected. Livers slices (5 to 10 mg) were prepared and incubated in a 10-ml Erlenmeyer flask, containing 1 ml of reaction buffer, which was composed of Krebs Ringer bicarbonate buffer at pH 7.4, 1 % defatted bovine serum albumin, and one of following substrates: 1 μCi [U-14C] glucose (5 mM), 1 μCi [2-14C] sodium acetate (2 mM), or 1 μCi [U-14C] sodium glutamate (1 mM). The flask had a thin rubber stopper that served both as a cap to close the flask and to hold a plastic bucket. The incubation was carried out at 37°C for 2 h in an atmosphere of O2/CO2 (95%/5%), in a shaking water bath. At the end of the incubation period, 200 μl of hyamine hydroxide was injected through the rubber stopper into the hanging bucket and 0.5 ml of 10% perchloric acid was injected into the incubation medium, to insure the complete liberation of CO2. After shaking for an addition 30 min, the tissue was removed, rinsed in saline and transferred to 2 ml of chloroform-methanol (2:1 v/v) for the extraction of total lipids. The lipid was extracted and its radioactivity determined. The hanging buckets containing the hyamine hydroxide were dissolved in liquid scintillation fluid and the radioactivity measured using a liquid scintillation spectrometer.
Mice were killed at two days of age and carcass analysis performed as described by Leischner et al.
Histology of the Liver
Segments of livers from 2-day-old mice were fixed in 10% formalin (Sigma, St. Louis, MO) at 4°C. The tissues were embedded in paraffin and stained with hematoxylin and eosin by the Pathology Core Facility (Case Western Reserve University, Cleveland, OH). The photographs were taken at 400× magnification.
DNA was isolated from the tails of the mice by lysis overnight at 55°C in a buffer containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 0.1% gelatin, 0.45% Nonidet P-40, 0.45% Tween20, and 24 mg/ml of proteinase K. The DNA was digested with Hind III, and the resulting fragments were separated by electrophoresis on 1% agarose gel, transferred to Gene Screen Plus®, and hybridized to a cDNA probe for PEPCK-C.
Total RNA was extracted from the livers of one and two-day old mice using a QuickPrep total RNA kit (Amersham Pharmacia Biotech) by a modified acid-phenol/guanidine thiocynate procedure  and Northern blotting was performed as described in detail previously . Briefly, 20 μg of total RNA was separated by electrophoresis on an agarose gel, transferred to a Gene Screen Plus membrane and hybridized with a 1.0kb Sma I fragment of the 3'end of the PEPCK-C cDNA. The DNA probe was labeled by using [α-32P]-dCTP.
The following metabolites were measured in the blood of mice killed up to two days after birth. Blood glucose was determined using an Encore® Glucometer. The concentrations of triglyceride, β-hydroxybutyrate, and urea nitrogen in the blood were determined by Veterinary Diagnostic Services in Marshfield Laboratories (Marshfield, WI), using standard clinical procedures. The concentrations of amino acids in plasma were measured with a high-pressure liquid spectrophotometer equipped with a fluorescent detector, using a o-phthaldehyde derivative and pre-column derivatization . Livers of mice were used to determine the content of glycogen  or were freeze-clamped and metabolites extracted as described previously . Spectrophotometric methods were used to determine the concentrations of malate , lactate , pyruvate  and ammonia  in the liver extracts or in the blood. The concentration of hepatic triglyceride was determined using the triglyceride reagent set from Pointe Scientific, Inc. (Lincoln Park MI). Briefly, segments of liver were saponified in ethanol-KOH, the sample diluted 1:5 and one ml of triglyceride reagent added to 10 μl of diluted sample and incubated at 37°C for 5 min. The reaction product was analyzed using spectrophotometer (A500) and the concentration determined by linear regression using standards treated with one ml of triglyceride reagent. The activity of PEPCK-C in the liver cytosol of two-day-old PEPCK-C-/- mice and control littermates was determined as described by Ballard and Hanson  and the activity expressed as units/g tissue, where one unit equals one μmole of substrate converted to product per min.
All data are reported as means ± the SE. The statistical analysis was performed using SigmaPlot from Systat Software, Inc. (Point Richmond, CA).
Metabolic alterations in PEPCK-C-/- mice at two days of age.
80.3 ± 0.30
79.26 ± 0.22
3.48 ± 0.36
1.85 ± 0.57
% Lean Mass
16.42 ± 0.63
18.88 ± 0.78
Metabolites in liver
4.17 ± 1.00
9.58 ± 1.75
0.19 ± 0.02
0.37 ± 0.09
0.24 ± 0.05
2.84 ± 1.58
Metabolites in plasma
23.7 ± 6.3
36.3 ± 21.5
0.14 ± 0.02
0.45 ± 0.28
29.0 ± 7.0
65.5 ± 28.5
0.28 ± 0.13
0.72 ± 0.47
5.83 ± 0.21
17.95 ± 0.70
Since the deletion of the gene for PEPCK-C should alter the levels of intermediates in related metabolic pathways, we determined the level of malate and lactate in the liver by freeze-clamping the livers of PEPCK-C-/- mice and controls at two days after birth. Intermediates were extracted from the frozen livers and their concentrations measured (Table 1). The concentration of malate was increased by 10-fold in the livers of PEPCK-C-/- mice and the lactate concentration was elevated 2.5-fold. We did not distinguish between the malate in the cytosol and the mitochondria, but it is likely that there is an increase in the concentration of this citric acid cycle intermediate in both compartments of the hepatocyte. This finding of an elevated concentration of malate in the liver suggests that in absence of PEPCK-C the rate of oxalacetate conversion to PEP is limited and this intermediate is converted instead to malate, which accumulates. A similar increase in the concentration of malate in the livers of PEPCK-C-/- mice was reported by She et al. .
The conversion of glucose, acetate and glutamate to CO2 and lipid by liver slices from PEPCK-C-/- and control mice.
Glucose-U- 14 C
0.92 ± 0.26
0.30 ± 0.04
0.153 ± 0.009
0.063 ± 0.002
Acetate-2- 14 C
12.36 ± 0.88
2.72 ± 0.20
0.097 ± 0.018
0.002 ± 0.000
Glutamate-5- 14 C
5.15 ± 0.65
1.75 ± 0.65
1.778 ± 0.091
0.983 ± 0.098
It is clear from this and other studies [9, 10] that PEPCK-C is critical for life; its absence in the mouse results in death within the first two to three days after birth. We are aware of only one reported example of PEPCK-C deficiency in humans. In 1976, Vidnes and Sovik  reported the absence of PEPCK-C in the liver of an infant, which had persistent hypoglycemia and an early death, despite the presence of PEPCK-M. The rarity of PEPCK-C deficiency in humans further supports the physiological significance of this enzyme. The developmental profile of hepatic PEPCK-C is of interest in this regard. PEPCK-C is absent in the liver of all mammals during fetal development and appears dramatically at birth . In contrast, the enzyme is present in the kidney  and may appear in other tissues before birth, but the exact pattern of development in tissues other than the kidney and liver has not been determined. As an example, the time course of development of PEPCK-C in white adipose tissue is currently unknown, despite the important role that PEPCK-C plays in this tissue [3, 7]. Considerable PEPCK-M activity is present in mammalian liver before birth  but it is the appearance of PEPCK-C at birth and the marked alteration in the hepatic redox state, which occurs during this period , that are generally considered to be the important events in the initiation of hepatic gluconeogenesis. Since the role of PEPCK-C in gluconeogenesis is well established in all species , it is reasonable to assume that one of the causes of the death of the PEPCK-C-/- mice is the profound hypoglycemia which occurs at two to three days after birth. However, providing glucose to the mice by intraperitoneal injection did not rescue the animals.
Mice with a liver-specific deletion of the gene for PEPCK-C survive the perinatal period, maintain normal glucose homeostasis during fasting and can be made diabetic . Whole body glucose turnover in these mice is only slightly decreased, when compared to control animals. This suggests that the kidney can assume the function of the liver and make enough glucose for the needs of the animal. In addition, the liver can synthesize glucose from glycerol, a process that by-passes the reactions in the pathway of gluconeogenesis that require PEPCK-C. Since only 5–10% of the total PEPCK is mitochondrial in rodent species , it is unlikely that PEPCK-M plays a significant role in hepatic gluconeogenesis in mice lacking hepatic PEPCK-C. Mice lacking hepatic PEPCK-C have lower levels of liver glycogen two days after birth. As noted in Table 1, newborn PEPCK-C-/- mice have considerably less glycogen than control littermates, presumably due to the need to mobilized glycogen to support the blood glucose levels in the absence of hepatic gluconeogenesis. However, She et al  reported that mice lacking PEPCK-C in the liver had a greatly reduced rate of glycogen synthesis in both the liver and the muscle.
The marked accumulation of fat in the livers of the PEPCK-C-/- mice strongly suggests that there is another critical metabolic role for the enzyme (other than gluconeogenesis) which, when missing, can markedly alter the metabolic capacity of the animal. The concentration of hepatic triglyceride in mice two days after birth is twice that of control animals. The biochemical mechanisms responsible for the accumulation of triglyceride in PEPCK-C-/- mice are complex. In the present paper, we present evidence that in the absence of PEPCK-C hepatic cataplerosis is greatly reduced, resulting in a reduction of the ability of the liver to appropriately oxidize acetyl CoA to CO2 in the citric acid cycle. This causes an increase in the synthesis of ketone bodies and a build up of fatty acids in the liver, with the subsequent synthesis of triglyceride. Cataplerosis describes reactions involved in the disposal of citric acid cycle intermediates generated by the entry of compounds into the cycle during the breakdown of amino acids and other metabolites (i.e. propionyl CoA), or by the carboxylation of pyruvate to oxalacetate . The biological necessity for cataplerosis resides in citric acid cycle dynamics. The cycle oxidizes acetyl CoA to CO2 but cannot fully oxidize four or five carbon intermediates. This requires a series of reactions that are capable of efficiently removing citric acid cycle anions before they accumulate in the mitochondria. It is now recognized that PEPCK-C functions in cataplerosis in many tissue, not just in the more intensively studied tissues, such as the liver and kidney. For example, the net generation of alanine from glutamine, as occurs in the small intestine, involves PEPCK-C and cataplerosis. Glutamine carbon enters the citric acid cycle as α-ketoglutarate, is converted in the cycle to malate, which leaves the mitochondria, is oxidized to oxalacetate and then decarboxylated to PEP by PEPCK-C. Pyruvate kinase then converts the PEP to pyruvate, the pyruvate is transaminated by alanine aminotransferase to alanine, which is then released into the blood and transported to the liver. It has been estimated that as much as 40% of the alanine used for gluconeogenesis by the human liver during starvation is derived from the glutamine in the small intestine . Alternatively, the pyruvate can be converted to acetyl CoA in the mitochondria for the generation of energy via the citric acid cycle. A similar role for PEPCK-C has been proposed for the conversion of other amino acids to alanine in the muscle . Deleting the gene for PEPCK-C in tissues of the mouse, where it is normally present, will have the effect of altering cataplerosis and profoundly interfering with energy metabolism.
Mice with a liver-specific ablation of PEPCK-C have been used by Burgress et al  to determine the importance of this enzyme in citric acid cycle flux in the intact animal. They noted that after a 24 h fast virtually all of the newly synthesized glucose was from glycerol and the formation of glucose from citric acid cycle intermediates was negligible. Flux through the citric acid cycle was 10 to 40-fold lower than noted in the livers of control mice, which correlated with an accumulation of citric acid cycle intermediates. They concluded that in the absence of PEPCK-C there was a failure of cataplerosis, leading to a decreased citric acid cycle flux, decreased fatty acid oxidation and an accumulation of liver lipid. This finding is in accord with that described in this paper using mice in which the gene for PEPCK-C is deleted in all tissues and underscores the key role of PEPCK-C in the normal functioning of the citric acid cycle in mammals.
In addition to altering cataplerosis, the deletion of the gene for PEPCK-C will result in a decrease in glyceroneogenesis in tissues of the mouse. At birth the mouse ingests milk that is relatively high in fat (13%) and low in carbohydrate (3%) when compared with some other mammalian species (i.e. human). This dietary fat, while a good source of energy, is deposited in white adipose tissue during the early perinatal period. The relatively low level of carbohydrate in the diet suggests that glyceroneogenesis in adipose tissue could play an important role in controlling the rate of triglyceride deposition in this tissue during suckling. Studies in our laboratory, using in vivo tracer methodology, have shown that glyceroneogenesis in adipose tissue of the rat is the predominant source of glyceride-glycerol, even when the animals were fed a diet high in sucrose (Nye, Hanson and Kalhan, unpublished data). If glyceroneogenesis is playing an important role in the deposition of triglyceride in the adipose tissue of the newborn mouse, the absence of PEPCK-C in the tissue could explain the very high level of triglyceride accumulation noted in the livers of the PEPCK-C-/- mice, since fatty acids not re-esterifed to triglyceride in adipose tissue, could end up being converted to triglyceride in the liver. In support of this suggestion, the tissue-specific ablation of PEPCK-C gene expression in adipose tissue of mice resulted in the development of a fatty liver.
The studies presented here and the recent work on mice with a liver-specific deletion in the gene for PEPCK-C [9, 10, 21], emphasize the important role played by this enzyme in a number of metabolic process other than gluconeogenesis. It is likely that PEPCK-C is critical for cataplerosis, especially in those tissues in which significant levels of biosynthesis occur. The dynamic nature of the citric acid cycle and its role in both energy generation and biosynthetic process requires the coordinated activity of PEPCK-C to insure a balance between carbon input into the cycle and carbon outflow. The fact that this critical function of PEPCK-C has not been appreciated for so long, underlines the importance of genetically modified animal models as tools for better understanding the control of metabolism. As an example, the metabolic function of PEPCK-M has been virtually ignored over the years despite the fact that it represents 50% of the activity of PEPCK in humans. This neglect is ironic, since Utter and Kurahashi discovered PEPCK-M in the livers of chickens  and used it to delineate the pathway of gluconeogenesis. Our understanding of the biology of PEPCK will not be complete until we directly determine the metabolic role of PEPCK-M in mammalian species. In this regard, the major limiting factor is the fact that the animal species most easily genetically modified (the mouse) has only a marginal activity of PEPCK-M in any tissue. It should be possible, however, to introduce a transgene containing the gene PEPCK-M into tissues of PEPCK-C-/- mice. This would allow us to assess the metabolic function of PEPCK-M in the absence of PEPCK-C; these studies are in progress.
This research was supported by DK 58620 and DK 25541 (RWH) by HD11089 (SCK) and by HD37934 to RAC, from the National Institutes of Health and by a United States-Israel Binational Grant 9100268 (L R).
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