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 in- duced 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 Downloaded by guest on September 27, 2021 VOL. 53, 1965 BIOCHEMISTRY: LARDY, PAETKA U, AND WALTER 1411 mannoheptulose, or pancreatectomy), and following the administration of glucocorti- coids.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 mnito- chondria 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 Source of intramitochondrial OAA Extramitochondrial Rate of product Expt. no. and/or aspartate OAA-trapping system accumulation* 1 7 mM Pyr, 10 mM bicarbonate None 0.013 2 7 mM Pyr, 10 mM bicarbonate, Cr-Crk None 0.003 3 7 mM Pyr, 10 mM bicarbonate, 5 mM Glu None 0.015 4 7 mM Pyr, 10 mM bicarbonate, 5 mM Glu, None 0.021 Cr-Crk 5 5 mM malate, 5 mM Glu, Cr-Crk MDH, 0.5 mM DPNH (1 0.11 mM aKG present) 6 5 mM malate, 5 mM Glu, Cr-Crk MDH, 0.5 mM DPNH, 0.99 GOT, 1 mM aKG 7 5 mM malate, 5 mMI Glu MDH, 0.5 mM DPNH, 0.54 GOT, 1 mM aKG 8 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu None 0.00 9 7 mM Pyr, 10 mM bicarbonate 1.8 U PEP-CK 0.04 10 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 2.5 U PEP-CK 0.04 11 7 mM Pyr, 10 m1\I bicarbonate, 3 mMI Glu 1.25 U PEP-CK, GOT 0.13 12 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 1.8 U PEP-CK, GOT 0.29 13 7 mMI Pyr, 10 mM bicarbonate, 3 mM Glu 2.5 U PEP-CK, GOT 0.29 14 7 mM Pyr, 10 mMI bicarbonate 0.75 U PEP-CK 0.00 15 7 mM Pyr, 10 mlI bicarbonate 1.50 U PEP-CK 0.00 16 7 mM Pyr, 10 mMl bicarbonate, 3 mM Glu 0.75 U PEP-CK 0.09 17 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 1.50 U PEP-CK 0.17 18 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 0.75 U PEP-CK, GOT 0.23 19 7 mM Pyr, 10 mMI bicarbonate, 3 mM Glu 1.50 U PEP-CK, GOT 0.43 20 7 mM Pyr, 10 mM bicarbonate, 3 mM Glu 0.75 U PEP-CK, GOT 0.31 (2 mM malonate present) 21 7 mM Pyr, 10 m.M bicarbonate, 3 mM Glu 1.50 U PEP-CK, GOT 0.43 (2 mM malonate present) *jmoles/min/gm liver. OAA = oxalacetate; Glu = glutamate; Asp = aspartate; aKG = a-ketoglutarate; Pyr = pyruvate; Cr- CrK = 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 = 370; Pi = 6 mM; triethanolamine (Cl-) buffer = 7 mM; MgS4 = 6 mM; oxalacetate was determined by the 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. Downloaded by guest on September 27, 2021 1412 BIOCHEMISTRY: LARDY, PAETKAU, AND WALTER PROC. N. A. S. per cent of the rate required for normal gluconeogenesis. The addition of glutamic- oxalacetic 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 indi- cate that malate was oxidized to oxalacetate in the mitochondria and transaminated there to form aspartate which diffused from the mitochondria. The added gluta- mate-oxalacetate transaminase and a-ketoglutarate convert aspartate to oxalacetate, and the latter oxidizes extramitochondrial DPNH in the presence of malate dehy- drogenase. 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 liber- ated 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 reason- able. TABLE 2 FORMATION OF ORGANIC ACIDS* FROM PYRUVATE, BICARBONATE, AND GLUTAMATE BY RAT LIVER MITOCHONDRIA ,-System without Glutamate--- -System with Glutamate-- Acids 0-5 min 0-10 min 0-5 min 0-10 min Pyruvate used 3.47 3.70 3.80 3.64 Glutamate used - 1.30 1.13 Malate formed 0.87 0.85 1.09 1.05 C"-Malate formedt 0.75 0.68 0.84 0.77 Citrate formed 0.68 0.72 0.52 0.56 C14-Citrate formedt 0.56 0.56 0.35 0.42 Aspartate formed 0.85 0.90 C14-Aspartate formed t 0.70 0.62 Alanine formed 0.40 0.28 a-Ketoglutarate formed 0.11 0.06 0.56 0.40 Total C14 productst 1.31 1.24 1.89 1.81 * pmoles/min/gm liver.