BY HENRY A. LARDY, VERNER PAETKAU, AND PAUL WALTER. DEPARTMENT OF ..... Foster, E. Shrago, and P. D. Ray, Advan. Enzyme Reg., 2, 39 (1964).
PATHS OF CARBON IN GLUCONEOGENESIS AND LIPOGENESIS: THE ROLE OF MITOCHONDRIA IN SUPPLYING PRECURSORS OF PHOSPHOENOLPYR UVA TE* BY HENRY A. LARDY, VERNER PAETKAU, AND PAUL WALTER DEPARTMENT OF BIOCHEMISTRY AND INSTITUTE FOR ENZYME RESEARCH, UNIVERSITY OF WISCONSIN, MADISON
Communicated April 2, 1965
Gluconeogenesis-the synthesis of glucose and glucose-containing polysaccharide from compounds other than hexoses-is a process of considerable magnitude in normal animals and one that is subject to grave alterations in certain disease states. In mammals gluconeogenesis occurs mainly, if not exclusively, in liver and kidney.2 Carbohydrate is synthesized by these tissues from lactate and pyruvate during periods of heavy muscular work, and the glucose formed is returned to muscle to serve as a glycolytic energy source (Cori cycle). During long intervals between meals, and especially during fasting, amino acids from tissue proteins serve as a source of carbon for gluconeogenesis. In the absence of adrenal corticosteroids, protein reserves are not converted to carbohydrate sufficiently rapidly to maintain normal blood sugar levels3 while in the diabetic this conversion is so rapid as to elevate blood sugar above the renal threshold.4 The main pathway of carbon in gluconeogenesis differs from the reverse of the glycolytic sequence at 3 steps5 and consequently gluconeogenesis is subject to some controls that are without effect on carbohydrate degradation. One of these steps-the formation of phosphoenolpyruvate from pyruvate-seemed likely to be a site at which metabolic control of gluconeogenesis would be effected6 since it is at the point where pyruvate from either lactate or amino acid residues enters the route to hexose formation. Pyruvate enters the gluconeogenic route by being carboxylated to a dicarboxylic acid.7 This is accomplished by the pyruvate carboxylase (reaction 1) of Utter and Keech which is located predominantly in the mitochondria of liver cells:8-11 acetyl CoA
Pyruvate + CO2 + ATP -
Oxalacetate + ADP + Pi. (1) Pyruvate carboxylase increases in amount in the livers of fasted rats,"3 those treated with hydrocortisone,"1 and diabetic rats'1 12 in keeping with its proposed role in gluconeogenesis. Another possible means of carboxylating pyruvate-via malic enzymel4-was suggested by Wagle and Ashmore" to be involved in the enhanced gluconeogenesis of the diabetic rat. However, this enzyme is not sufficiently active to account for the pyruvate converted to carbohydrate in normal liver, is not induced by fasting or hydrocortisone, and is in fact greatly diminished in diabetic rats' livers.6 Instead, malic enzyme participates in lipogenesis by converting oxalacetate, via malate, to pyruvate and generating TPNH. 17,17 Phosphoenolpyruvate carboxykinase, the enzyme that converts oxalacetate to phosphoenolpyruvate (reaction 2), was discovered by Utter and Kurahashi18 and was found to be located in the soluble fraction of rat, mouse, and hamster liver.'9 Oxalacetate + GTP (or ITP) 2 Phosphoenolpyruvate + GDP (or IDP) (2) Its activity in liver is greatly enhanced by fasting, by diabetes (induced by alloxan, 1410
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mannoheptulose, or pancreatectomy), and following the administration of glucocorticoids.6 20 It is sufficiently active to account for the rate of gluconeogenesis in normal rat liver and in the metabolic alterations mentioned immediately above.6 These findings indicated that pyruvate must be carboxylated to oxalacetate in the mitochondria, whereas conversion of oxalacetate to phosphoenolpyruvate and the succeeding reactions of gluconeogenesis occur in the extramitochondrial portion of the cell. Experiments designed to verify this scheme led to the finding that virtually no oxalacetate accumulated in media containing pyruvate, bicarbonate, and rat liver mnitochondria under a variety of incubation conditions (Table 1 and experiments to be presented elsewhere). The addition of creatine and crystalline creatine kinase22 as a phosphate acceptor system to enhance pyruvate oxidation23 did not enhance oxalacetate production. Therefore, the production of other 4-carbon compounds by mitochondria was studied. When malate and glutamate are both supplied to mitochondria (expts. 5-7), oxalacetate is produced and liberated to the soluble phase of the system at a rate of only 0.11 Atmole per minute by mitochondria from 1 gm of liver. This is about 10 TABLE 1 AN EXAMINATION OF THE METABOLIC PATHWAY FROM PYRUVATE TO PHOSPHOENOLPYRUVATE Expt. no.
1 2 3 4
Source of intramitochondrial OAA and/or aspartate
7 mM Pyr, 10 mM bicarbonate 7 mM Pyr, 10 mM bicarbonate, Cr-Crk 7 mM Pyr, 10 mM bicarbonate, 5 mM Glu 7 mM Pyr, 10 mM bicarbonate, 5 mM Glu,
5
Cr-Crk 5 mM malate, 5 mM Glu, Cr-Crk
6
5 mM malate, 5 mM Glu, Cr-Crk
7
5 mM malate, 5 mMI Glu
8 9 10 11 12 13 14 15 16 17 18 19 20
7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 7 mM Pyr, 10 mM bicarbonate 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 7 mM Pyr, 10 m1\I bicarbonate, 3 mMI Glu 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 7 mMI Pyr, 10 mM bicarbonate, 3 mM Glu 7 mM Pyr, 10 mMI bicarbonate 7 mM Pyr, 10 mlI bicarbonate 7 mM Pyr, 10 mMl bicarbonate, 3 mM Glu 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 7 mM Pyr, 10 mMI bicarbonate, 3 mM Glu 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu
(2 mM malonate present)
7 mM Pyr, 10 m.M bicarbonate, 3 mM Glu (2 mM malonate present) *jmoles/min/gm liver.
21
Extramitochondrial OAA-trapping system
Rate of product accumulation*
None None None None
0.013 0.003 0.015 0.021
MDH, 0.5 mM DPNH (1 mM aKG present) MDH, 0.5 mM DPNH, GOT, 1 mM aKG MDH, 0.5 mM DPNH, GOT, 1 mM aKG None 1.8 U PEP-CK 2.5 U PEP-CK 1.25 U PEP-CK, GOT 1.8 U PEP-CK, GOT 2.5 U PEP-CK, GOT 0.75 U PEP-CK 1.50 U PEP-CK 0.75 U PEP-CK 1.50 U PEP-CK 0.75 U PEP-CK, GOT 1.50 U PEP-CK, GOT 0.75 U PEP-CK, GOT
0.11
1.50 U PEP-CK, GOT
0.99 0.54 0.00 0.04 0.04 0.13 0.29 0.29 0.00 0.00 0.09 0.17 0.23 0.43 0.31
0.43
OAA = oxalacetate; Glu = glutamate; Asp = aspartate; aKG = a-ketoglutarate; Pyr = pyruvate; CrCrK = 13 mM creatine + 0.1 mg crystalline creatine kinase per ml; MDH = 10 units of crystalline malate dehydrogenase (Boehringer); GOT = 10 units of glutamate-oxalacetate transaminase (Boehringer) dialyzed free of ammonia; PEP = phosphoenolpyruvate; PEP-CK = PEP carboxykinase."'s24 In all experiments, ATP = 3 mM; pH = 7.4; mitochondria from 0.17 gm rat liver were included in each ml of reaction mixture, except in expts. 5-7 where mitochondria from 6.6 mg of liver were used. In expts. 1-4, T = was determined by the 370; Pi = 6 mM; triethanolamine (Cl-) buffer = 7 mM; MgS4 = 6 mM; oxalacetate highly sensitive colorimetric method of Kalnitsky and Tapley.21 In expts. 5-7, T = 210; Pi = 1 mM; triethanol= = amine (Cl-) buffer 25 mM; MgSO4 6 mM; DPNH oxidation was measured spectrophotometrically at 340 M/A. In expts. 8-21, T = 370; Pi = 3 mM; triethanolamine (Cl-) buffer = 10 mM; MgSO4 = 18 mM; ITP = 5 mM; PEP was determined chemically." A control for expts. 5-7 had a zero rate of DPNH oxidation in the absence of either malate or malate dehydrogenase.
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per cent of the rate required for normal gluconeogenesis. The addition of glutamicoxalacetic transaminase in the presence of 1 mM a-ketoglutarate resulted in DPNH oxidation at the rate of 1 Mmole per minute. The data of these experiments indicate that malate was oxidized to oxalacetate in the mitochondria and transaminated there to form aspartate which diffused from the mitochondria. The added glutamate-oxalacetate transaminase and a-ketoglutarate convert aspartate to oxalacetate, and the latter oxidizes extramitochondrial DPNH in the presence of malate dehydrogenase. Omission of the phosphate acceptor system (expt. 7) slowed malate oxidation in the mitochondria to the point where only half as much aspartate was formed. In the remaining experiments (8-21) of Table 1, pyruvate plus HCO - was added to produce C4 acids; enzyme systems for converting (extramitochondrially) either oxalacetate [(i), PEP-CK + ITP] or aspartate [(ii), GOT + PEP-CK + ITP] to phosphoenolpyruvate were added, and the latter compound was measured."9 With system (i), or without glutamate, only negligible amounts of phosphopyruvate accumulated. Only in system (ii) was phosphopyruvate formation significant (expts. 18-21). In these experiments no ketoglutarate or malate was added. Oxalacetate formed by carboxylation of pyruvate transaminated with glutamate; the aspartate and ketoglutarate formed diffused out of the mitochondria where the added transaminase converted them partially to oxalacetate and glutamate. In similar ezperiments (to be published elsewhere) but without an external trapping system, about one umole of aspartate and 0.73 Mmoles of a-ketoglutarate were liberated per minute by the mitochondria from 1 gm of liver. Thus the availability of a-ketoglutarate may be limiting the rate of phosphopyruvate formation under these conditions. Considering that no attempts were made to determine conditions for maximum rates of phosphopyruvate production, the yields of the latter are reasonable. TABLE 2 FORMATION
OF
PYRUVATE, BICARBONATE, RAT LIVER MITOCHONDRIA
ORGANIC ACIDS* BY
Acids
Pyruvate used Glutamate used Malate formed C"-Malate formedt Citrate formed C14-Citrate formedt Aspartate formed C14-Aspartate formed t Alanine formed a-Ketoglutarate formed Total C14 productst
FROM
,-System without Glutamate--0-10 min 0-5 min
3.47 0.87 0.75 0.68 0.56 0.11
1.31
3.70 0.85 0.68 0.72 0.56 0.06 1.24
AND
GLUTAMATE
-System with Glutamate-0-10 min 0-5 min
3.80 1.30 1.09 0.84 0.52 0.35 0.85 0.70 0.40 0.56 1.89
3.64 1.13 1.05 0.77 0.56 0.42 0.90 0.62 0.28 0.40 1.81
* pmoles/min/gm liver. t Calculated on the basis of the specific radioactivity of the KH14COs, assuming that not more than one C02 has been incorporated per molecule. The reaction mixture contained 3 mM ATP, 7.5 mM MgSO4, 6.7 mM potassium phosphate, pH 7.4, 6.7 mM triethanolamine, pH 7.4, 10 mM KH14CO, (0.6 oc/pmole), 6.7 mM sodium pyruvate, 1.0 ml mitochondrial suspension corresponding to 0.8 gm of original rat liver, and, when indicated, 3 mM potassium glutamate. All components were added as essentially isotonic solutions, and the final volume was made up to 6.0 ml with 0.25 M sucrose. The incubations were carried out in stoppered 25-ml Erlenmeyer flasks which were shaken at 370 in a water bath. The reaction was started by adding the mitochondria after an equilibration period of 2 min and stopped by adding 6 ml of 0.66 M HC104. The deproteinized samples were neutralized with KOH, and the perchlorate salt was centrifuged off. An aliquot was freed of all nonacid components by chromatography on Dowex-2-formate, and the acids were then separated by high-voltage electrophoresis at 4500 v and pH 3 on Whatman 3 MM paper strips. The radioactive peaks were located on a paper scanner, cut out, and counted in the scintillation counter. The content of the various acids was determined according to the references cited: malate,25 pyruvate,25 a-ketoglutarate,26 citrate;26 the amino acids were determined on the Spinco amino acid analyzer,
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The insensitivity of system (ii) to malonate suggests that glutamate is required as an amino group donor rather than as a carbon source for aspartate formation (expts. 20 and 21). This is confirmed by the isotopic experiments which follow. In the experiments summarized in Table 2, the production of various organic acids from pyruvate and H14COi was measured and their specific radioactivity was determined. Malate and citrate were produced in good yields, while only a small amount of a-ketoglutarate and negligible amounts of isocitrate were found. The specific radioactivity of malate and citrate indicated that at least 78-86 per cent of the oxalacetate going into these compounds was derived directly from carboxylated pyruvate. These values are minimal because they are calculated from the specific activity of the H14CO- added and do not take into account the dilution by metabolically produced CO2. Any malate or oxalacetate originating from the tricarboxylic acid cycle would not be labeled. Thus, in mitochondria under these conditions, malate arises almost entirely by reduction of oxalacetate, and not by the tricarboxylic acid cycle. In the presence of glutamate, malate formation was slightly enhanced and at least 73-77 per cent originated from oxalacetate formed by direct carboxylation of pyruvate. Aspartate was produced in amounts significant for gluconeogenesis and this compound too originated largely (at least 69-82%) from oxalacetate produced by pyruvate carboxylation. Glutamate diminished citrate formation slightly but enhanced total C14 fixed by trapping oxalacetate as aspartate. When the phosphate acceptor system hexokinase and glucose was added to the mixtures described in Table 2, total C14 fixed in the system in the absence and presence of glutamate, respectively, decreased to 3 and 6 per cent of the amount fixed without phosphate acceptor. Discussion.-The implications of these findings for gluconeogenesis are summarized in Figure 1. Both malate and aspartate are likely precursors of oxalacetate in the extramitochondrial compartment of the liver cell. The malate oxalaceINTRAMITOCHONDRIAL
EXTRAMITOCHONDRIAL
TPN
PYR +
CO2
MAL TE
MALATE
TRIOSE P
FUMARATE 1DPN ~~~~~~TDPNH UREA CYCE A4/ ~DPNHE GAASP | ,_ASPSH,-4, ATE OAA 'PEP
DPN
DPNH
FUMA
tPYR
/
