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THE JOURNAL 0% BIOLOGICAL CIIEMI~TRV Vol. Z-18, No. 7, Issue of April 10, pp. ZZGG-2271, 1973 Printed in C S.A. The Regulation of Gluconeogenesis in Mammalian Liver THE ROLE OF lLlITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASE* (Received for publication, August 18, 1972) IFEANYI J. ARINZE, ALAN J. GARBER,~ AND RICHARD W. HANSONS From the Fels Research Institute and Department oj Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 SUMMARY The significance of mitochondrial phosphoenolpyruvate formation for gluconeogenesis was evaluated in isolated, perfused livers from rats, guinea pigs, and rabbits. In guinea pig liver, 0.2 mu octanoate decreased by 40 and 30% the rate of gluconeogenesis from lactate and alanine, re- spectively, but not from pyruvate. In rat liver, gluconeogene- sis from all three substrates was increased. The inhibition of gluconeogenesis in guinea pig liver was associated with an increased level of reducing equivalents in the mitochondria as indicated by the NAD+:NADH ratios in freeze-clamped livers before and during infusion of octanoate. Infusion of /3-hydroxybutyrate also increased gluconeogenesis from pyruvate and lactate in rat liver but inhibited glucose forma- tion from lactate in guinea pig liver. Low concentrations of the artificial electron acceptor, phenazine methosulfate, reversed the inhibitory effects of excess mitochondrial reducing equivalents. Aminooxyacetate, which inhibits aminotransferases, totally abolished glucose formation from lactate (but not from pyruvate) in perfused rat liver but reduced gluconeogenesis by only 50 to 60% in guinea pig liver. This inhibition was further increased by /3-hydroxy- butyrate infusion. In livers from fed rabbits, aminooxy- acetate had no effect, but in livers from fasted rabbits in which the activity of cytosolic P-enolpyruvate carboxykinase is induced, the inhibition by aminooxyacetate was quantita- tively similar to that in the guinea pig liver. The data sho.w that in the guinea pig and rabbit liver both the cytosolic and mitochondrial forms of P-enolpyruvate carboxykinase actively function during gluconeogenesis from lactate and alanine. The gluconeogenic flux via the mito- chondrial enzyme accounts for at least one-half of the over- all rate of glucose formation from lactate. It is concluded that the regulation of gluconeogenesis in species containing a mitochondrial activity of P-enolpyruvate carboxykinase is significantly different from that operating in rat liver and reflect to a large extent the compartmentation of P-enol- pyruvate carboxykinase. * This work was supported by Grants AM-11279, AM16009. CA-10916, and CA-12227 from the National Institutes of Health. 1 Present address, Division of Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO. 63110. 5 Recipient of Career Development Award K4-AM-15365 from the United States Public Health Service. The intracellular distribution of phosphoenolpyruvate car- boxykinase (EC 4.1.1.32), a key enzyme in gluconeogenesis, varies from species to species. In rat, mouse, or hamster liver (1, 2), more than 90% of the total activity is cytosolic, whereas in rabbit (1) or avian (3-5) liver, the enzyme is essentially mito- chondrial. In a variety of other species, such as humans, cattle, sheep, guinea pigs, and pigs (I, 6-13), the enzyme is distributed nearly equally in both compartments. The mitochondrial en zyme appears to be immunochemically (14) and physicochem- ically (15) distinct from the cytosolic form. In all species so far examined, the activity of the cytosolic enzyme increases with increased gluconeogenesis, such as during starvation, diabetes, glucagon administration (9, 16-19), and immediately after birth (2). Under these conditions, the mitochondrial activity of P-enolpyruvate carboxykinase remains unchanged. In the rat, the proposed mechanism of hepatic or renal glu- coneogenesis from alanine, lactate, or pyruvate involves the translocation of either malatc or aspartate or both, across the mitochondrial membrane into t,he cytosol where oxalacetate may be regenerated from these intermediates to form P-enol- pyruvate (20-22). However, it is not certain that this mech- anism is universally applicable to other species which possess a significant capacity for mitochondrial P-enolpyruvate formation and in which more complex mitochondrial-cytosolic interactions may exist. In this paper, the hypothesis that the mitochondrial formation of P-enolpyruvate is an integral part of gluconcogenesis has been evaluated in isolated perfused livers from rats, guinelr pigs, and rabbits. It is demonstrated that in the guinea pig and rabbit liver, both the cytosolic and the mitochondrial forms of P-enolpyruvate carboxykinase, actively function during glu- coneogenesis from alanine and lactate. Preliminary accounts of parts of this work have been presented previously (23, 24). MATERIALS AND METHODS Chemicals-Enzymes and coenzymes were purchased from Boehringer Mannheim Corp. L( +)-Lactic acid, octanoic acid, aminooxyacetic acid, and phenazine methosulfatc were ob- tained from Sigma Chemical Co., St. Louis, MO.; pyruvate and @-hydroxybutyrate from Calbiochem, and Fermcozyme 952 I>11 from G. D. Searle and Co., Chicago, Ill. Animals-Male Sprague-Dawley rats (190 to 220 g) were fed Wayne Lab Blox (Allied Mills, Chicago, Ill.) ad Zibitum and fasted for 22 to 26 hours prior to use. Male guinea pigs (250 to 300 g) of the Hartley strain were maintained on a pelleted 2266 by guest on July 5, 2018 http://www.jbc.org/ Downloaded from

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THE JOURNAL 0% BIOLOGICAL CIIEMI~TRV Vol. Z-18, No. 7, Issue of April 10, pp. ZZGG-2271, 1973

Printed in C S.A.

The Regulation of Gluconeogenesis in Mammalian Liver

THE ROLE OF lLlITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASE*

(Received for publication, August 18, 1972)

IFEANYI J. ARINZE, ALAN J. GARBER,~ AND RICHARD W. HANSONS

From the Fels Research Institute and Department oj Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

SUMMARY

The significance of mitochondrial phosphoenolpyruvate formation for gluconeogenesis was evaluated in isolated, perfused livers from rats, guinea pigs, and rabbits. In guinea pig liver, 0.2 mu octanoate decreased by 40 and 30% the rate of gluconeogenesis from lactate and alanine, re- spectively, but not from pyruvate. In rat liver, gluconeogene- sis from all three substrates was increased. The inhibition of gluconeogenesis in guinea pig liver was associated with an increased level of reducing equivalents in the mitochondria as indicated by the NAD+:NADH ratios in freeze-clamped livers before and during infusion of octanoate. Infusion of /3-hydroxybutyrate also increased gluconeogenesis from pyruvate and lactate in rat liver but inhibited glucose forma- tion from lactate in guinea pig liver. Low concentrations of the artificial electron acceptor, phenazine methosulfate, reversed the inhibitory effects of excess mitochondrial reducing equivalents. Aminooxyacetate, which inhibits aminotransferases, totally abolished glucose formation from lactate (but not from pyruvate) in perfused rat liver but reduced gluconeogenesis by only 50 to 60% in guinea pig liver. This inhibition was further increased by /3-hydroxy- butyrate infusion. In livers from fed rabbits, aminooxy- acetate had no effect, but in livers from fasted rabbits in which the activity of cytosolic P-enolpyruvate carboxykinase is induced, the inhibition by aminooxyacetate was quantita- tively similar to that in the guinea pig liver.

The data sho.w that in the guinea pig and rabbit liver both the cytosolic and mitochondrial forms of P-enolpyruvate carboxykinase actively function during gluconeogenesis from lactate and alanine. The gluconeogenic flux via the mito- chondrial enzyme accounts for at least one-half of the over- all rate of glucose formation from lactate. It is concluded that the regulation of gluconeogenesis in species containing a mitochondrial activity of P-enolpyruvate carboxykinase is significantly different from that operating in rat liver and reflect to a large extent the compartmentation of P-enol- pyruvate carboxykinase.

* This work was supported by Grants AM-11279, AM16009. CA-10916, and CA-12227 from the National Institutes of Health.

1 Present address, Division of Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO. 63110.

5 Recipient of Career Development Award K4-AM-15365 from the United States Public Health Service.

The intracellular distribution of phosphoenolpyruvate car- boxykinase (EC 4.1.1.32), a key enzyme in gluconeogenesis, varies from species to species. In rat, mouse, or hamster liver (1, 2), more than 90% of the total activity is cytosolic, whereas in rabbit (1) or avian (3-5) liver, the enzyme is essentially mito- chondrial. In a variety of other species, such as humans, cattle, sheep, guinea pigs, and pigs (I, 6-13), the enzyme is distributed nearly equally in both compartments. The mitochondrial en zyme appears to be immunochemically (14) and physicochem- ically (15) distinct from the cytosolic form. In all species so far examined, the activity of the cytosolic enzyme increases with increased gluconeogenesis, such as during starvation, diabetes, glucagon administration (9, 16-19), and immediately after birth (2). Under these conditions, the mitochondrial activity of P-enolpyruvate carboxykinase remains unchanged.

In the rat, the proposed mechanism of hepatic or renal glu- coneogenesis from alanine, lactate, or pyruvate involves the translocation of either malatc or aspartate or both, across the mitochondrial membrane into t,he cytosol where oxalacetate may be regenerated from these intermediates to form P-enol- pyruvate (20-22). However, it is not certain that this mech- anism is universally applicable to other species which possess a significant capacity for mitochondrial P-enolpyruvate formation and in which more complex mitochondrial-cytosolic interactions may exist. In this paper, the hypothesis that the mitochondrial formation of P-enolpyruvate is an integral part of gluconcogenesis has been evaluated in isolated perfused livers from rats, guinelr pigs, and rabbits. It is demonstrated that in the guinea pig and rabbit liver, both the cytosolic and the mitochondrial forms of P-enolpyruvate carboxykinase, actively function during glu- coneogenesis from alanine and lactate. Preliminary accounts of parts of this work have been presented previously (23, 24).

MATERIALS AND METHODS

Chemicals-Enzymes and coenzymes were purchased from Boehringer Mannheim Corp. L( +)-Lactic acid, octanoic acid, aminooxyacetic acid, and phenazine methosulfatc were ob- tained from Sigma Chemical Co., St. Louis, MO.; pyruvate and @-hydroxybutyrate from Calbiochem, and Fermcozyme 952 I>11 from G. D. Searle and Co., Chicago, Ill.

Animals-Male Sprague-Dawley rats (190 to 220 g) were fed Wayne Lab Blox (Allied Mills, Chicago, Ill.) ad Zibitum and fasted for 22 to 26 hours prior to use. Male guinea pigs (250 to 300 g) of the Hartley strain were maintained on a pelleted

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guinea pig diet (Teklab Inc., Manmouth, Ill.) supplemented with fresh lettuce. Male New Zealand white weanling rabbits weighing 1.0 to 1.7 kg were similarly fed. Guinea pigs were fasted for 46 to 50 hours, and rabbits for 96 hours prior to use. All animals had free access to water.

Liver Perjusion-Animals were anesthesized by intraperitoneal injection (50 mg per kg) of sodium pentobarbital (Nembutal). Details of the cannulation of the portal vein and the inferior vena cava, and the isolation of the liver have been described (25). The entire surgical procedure was completed in 5 to 8 min, and the time elapsing between the incision of the portal vein and its cannulation was less than 5 s.

The technique of liver perfusion used was the hemoglobin-free perfusion system described by Scholz and co-workers (26-28) and employed a rotating disc oxygenator (28). The perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) which was freshly made each day and oxygenated with a continuous stream of 02 and COS (95:5). The apparatus was used as a flow-through system in which the venous effluent was not re- cycled back to the oxygenator. Oxygen consumption was con- tinuously monitored by an in-line Clark-type oxygen electrode which measured the oxygen tension in the venous effluent. Since the perfusion fluid passed through the liver only once, substrate concentrations could be maintained at low and pre- sumably near physiological levels throughout the perfusion. Unless otherwise indicated the substrate concentration was 2 mm. The nonrecycling system was found to be ideally suited for kinetic studies, because it allowed unlimited and rapid sampling of the perfusate. Perfusate flow through the liver was adjusted to ensure adequate oxygen uptake by each liver. Be- cause of the large volumes of perfusion fluid used in this system, albumin was not routinely included in the medium. This omis- sion did not affect rates of oxygen consumption, substrate utili- zation, and glucose production in either rat or guinea pig livers perfused with or without fatty acids (25). Furthermore, with this system, rates of gluconeogenesis with high (10 mM) concen- trations of lactate were found to be comparable to values pub- lished for recycling experiments in which 3$& albumin was used (9), namely, 1.0 to 1.5 pmoles of glucose per min per g wet weight of livers from 4%hour fasted guinea pigs.

Determinations of lVetabolites-Two- to five-milliliter aliquots of the perfusate were removed at frequent intervals and used directly for the analysis of glucose and ketone bodies. Glucose was determined in a Technicon autoanalyzer with the glucose oxidase-peroxidase method (29). In some experiments, livers were rapidly frozen during perfusion with aluminum clamps cooled in liquid Nz (30). The frozen tissue was pulverized in a percussion mortar and deproteinized with ice-cold 6% perchloric acid (4 ml per g of tissue). The extract was centrifuged at 20,000 x g (20 min). The supernatant solution was adjusted to pH 6 with 2 N KHC03. Five milliliters of this solution were shaken with 60- to loo-mesh Florisil (0.1 mg per ml) to remove flavins (31). After removing the Florisil by centrifugation, the resulting clear supernatant solution was used for the assay of lactate (32), pyruvate (33), fl-hydroxybutyrate (34), glutamate (35), and ammonia (36). ATP (37), ADP, and AMP (38) were determined in the supernatant solution without Florisil treat- ment. Pyruvate, oc-ketoglutarate, and acetoacetate were meas- ured in the same cuvette by sequential addition of lactate de- hydrogenase (EC 1.1.1.27)) glutamate dehydrogenase (EC 1.4.1.3)) and P-hydroxybutyrate dehydrogenase (EC 1.1.1.30). P-Hydroxybutyrate was determined after preincubating the sample with excess malate dehydrogenase (EC 1.1.1.37).

.a -

.7 -

.6-

I pyruvate

50 60 70 80 90 100 IIf

minutes of Derfusian

FIG. 1. Effect of octanoate on glucose production from lactate, alanine, and pyruvate in perfused rat liver. Substrates (2 mu) were added after 30 min of perfusion with Krebs-Ringer bicnrbon- ate buffer alone. Octanoate was introduced from an infnsion pump to deliver the indicated concentrations at the arterial input. The vertical bars represent the S.E. for four to six livers from fasted rats.

RESULTS

E$ect of Octanoate-Because the oxidation-reduction state of the NAD+-NADH system in the mitochondria of liver from fasted guinea pigs is relatively oxidized (9, 10) in contrast to the reduced state in liver of fasted rats (39), we compared the effect of an increased level of reducing tquivalents generated in the mitochondria on the rates of gluconeogenesis from a variety of substrates in isolated, perfused livers from both species. Fig. 1 shows the stimulatory effect of increased fatty acid oxidation on gluconeogenesis from pyruvate, alanine, and lactate in rat liver. Maximal enhancement of gluconeogenesis from all three sub- strates was reached at an octanoate concentration of 0.2 nlM in the perfusion medium. No further increase in gluconcogenesis was seen when the octanoate concentration was increased to 0.4 IrIM. In fact, 0.4 mM octanoate appeared to be inhibitory. Upon termination of the octanoate infusion, rates of gluconeo- genesis usually returned to control levels. When guinea pig livers were perfused under similar conditions (Fig. 2), fatty acid oxidation stimulated gluconeogenesis only from pyruvate, al- though the increase was not as marked as noted with rat liver. In sharp contrast to the result obtained with rat liver, glucose synthesis from alanine and lactate was progressively inhibited in guinea pig liver by increasing concentrations of fatty acid.

Fig. 3 depicts the course of ketone body formation and oxygen

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pyruvate

1

40 50 60 70 60 90 IC

minutes of perfusion

FIG. 2. Effect of octanoate on glucose production from lactate, alanine, and pyruvate in perfused guinea pig liver. Livers were perfused as indicated in Fig. 1, except that octanoate infusion was started after 20 min of perfusion with substrate. The vertical bars represent the S.E. for five or six livers from fasted guinea pigs.

uptake by guinea pig liver metabolizing lactate throughout the entire period of perfusion. The addition of lactate increased respiration from endogenous substrates 2- to a-fold. This in- crease was somewhat less with alanine and pyruvate as sub- strates. Increasing concentrations of octanoate enhanced oxygen consumption, ketone body production, and the P-hydroxy- butyrate to acetoacetate ratio in the perfusate, indicating an in- creased generatibn of mitochondrial reducing equivalents. Be- cause of the large quantities of acetoacetate produced relative to /3-hydroxybutyrate, the increase in perfusate /3-hydroxybutyrate to acetoacetate ratio was not impressive (0.197 f 0.029 versus 0.265 & 0.019 at 0.2 mM octanoate). However, octanoate in- fusion was associated with a progressive inhibition of gluconeo- genesis from lactate and alanine in guinea pig liver but not in rat liver (compare Figs. 1 and 2). In these experiments, each liver served as its own control, and the return of glucose production to control levels after the withdrawal of the fatty acid infusion demonstrates that linear rates of gluconeogenesis were main- tained by the nonrecyclingsystem during the entire period of per- fusion.

In order to follow more closely the changes in intracellular oxidation-reduction states, livers were rapidly freeze-clamped before and during the infusion of octanoate, and appropriate NAD+-linked metabolites determined by standard methods. With all three substrates (alanine, lactate, and pyruvate), the infusion of fatty acid increased the tissue concentration of ketone

2.5.

2.0.

1.5 -

1.0 -

30 40 50 60 70 00 90

MINUTES OF PERFUSION

FIG. 3. Oxygen consumption and ketone body production by perfused guinea pig liver metabolizing lactate (2 mM) in the presence and absence of octanoate. The concentrations of aceto- acetate (AcAc) and p-hydroxybutyrate (p-OHB) were measured in the perfusate. The vertical bars represent the S.E. for six livers. These data were obtained in the same experiment indi- cated in Fig. 2.

bodies 2-to 5-fold. With lactate as substrate, the mitochondrial NAD+:NADH ratio calculated from the equilibrium constant (39) and the measured concentrations of the substrates of the glutamate dehydrogenase reaction decreased from 29 before fatty acid infusion to 17 after the 6-min pulse (Table I). When pyruvate and alanine were perfused, the actual mitochondrial oxidation-reduction state could not be reliably obtained from the glutamate dehydrogenase system because of the nonequilib- rium changes in the intracellular concentrations of glutamate and a-ketoglutarate caused by the action of alanine aminotrans- ferase. The mitochondrial NAD+:NADH ratio calculated from the P-hydroxybutyrate dehydrogenase couplet indicates that the mitochondrial compartment was reduced upon fatty acid infusion (Table I). Although the values are not identical with those calculated from the glutamate dehydrogenase system, other conditions have also been found (40, 41) in which values of the oxidation-reduction states calculated from both systems do not agree. It is significant, however, that with both systems this ratio shifted in the same direction. With either pyruvate or lactate as substrate, the cytosolic NAD+:NADH ratio cannot be satisfactorily calculated from the pyruvate to lactate ratio, because a considerable portion of the whole liver concentration of these intermediates is likely to be extracellular. But it may be presumed that the cytosol was also reduced by fatty acid in- fusion, since the NAD+:NADH ratio with alanine as substrate was lowered from 1175 to 506. The infusion of octanoate (0.2 mM) did not alter the adenine nucleotide balance as measured by total tissue ATP, ADP, and AMP levels or by the ratio of ATP to ADP (Table II). As in previous experiments (see Fig. 2),

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TABLE 1

Changes in concentrations of NAD-linked metabolites in freeze-clamped guinea pig livers perfused with gluconeogenic substrates in the presence or absence of octanoate

Livers perfused without substrate were freeze-clamped after drial NAD+:NADH ratio was calculated from the concentra- 30 min of perfusion with Krebs-Ringer bicarbonate buffer (pH tions of glutamate, ammonia, and a-ketoglutarate, and a Keg =

7.4) alone. Livers perfused with substrate (2 mM) in the absence 3.87 X 1O-3 (41) for the glutamate dehydrogenase reaction (A), of octanoate were freeze-clamped after 16 min of perfusion with and from the couplets of the P-hydroxybutyrate dehydrogenase substrate; those perfused with substrate plus octanoate (0.2 mM) reaction (B) using a K,, = 4.93 X 1OP (41). The cytosol NAD+: were freeze-clamped 6 min after the start of the infusion of octa- NADH ratio was calculated from the substrate components of

noate following a prior perfusion period of 16 min with substrate. the lactate dehydrogenase system using a Kes = 1.11 X 10-d (41). The metabolites were determined (see “Materials and Methods”) Metabolite concentrations are expressed as micromoles per g wet enzymatically on the neutralized tissue extracts. The mitochon- liver f S.E. for the number of livers indicated in I )arentheses.

Substrate ( Ictanoate 1 Glutamate aii.logbtamfr 1 Ammonia Pyruvate Lactate &Hydroxy- butyrate Acetoacetate Mito-

chondria -lc :ytoso1

A B I-

ZrnM

None (6) Lactate (5)

Lactate (5) Alanine (6) Alanine (5)

0.2 T?hM

- 0.418f0.0750.074~0.0090.196+0.C - 0.837zk0.0930.397+0.0520.236fO.(

+ 1.137+0.2670.323f0.0560.237fO.C - 2.350+0.6710.054f0.0120.319f0.C

+ 4.971*1.3730.058+0.0090.209*0.(

137 0.025fO.OlC 1420.034fO.OOE

159 0.021+0.00E 148 0.030*0.00: )120.021*0.00:

.193+0.0650.054f0.00C ;a 1.088f0.01: 9 33 0.034*0.00; 70 ~.071&O.OlC 29 42 0.181+0.03; 76 1.315f0.06: 17 35

.230f0.0330.042f0.00! IO 1.106&0.02> 2 51

.374*0.0770.085f0.01t 30 1.178f0.06C 0.6 43

1167

i

1175 506

TABLE II

Oxygen consumption, glucose production, and adexine nucleotide levels in guirlea pig liver perfused with gluconeogenic substrates in the

presence or absence of octanoate

NAD+:NADH

Values are means f SE. for the number of livers in parentheses. Livers are the same as in Table I. Rates and metabolite concen-

trations are expressed per g wet liver. T

I

-

ATP

2.334 f 0.124 2.091 f 0.106 2.102 f 0.117

2.300 f 0.128 2.050 f 0.091

ADP

pnoles/g

0.452 f 0.027 0.596 f 0.036

0.594 f 0.059 0.696 f 0.044

0.549 zt 0.026

Glucose production Oxygen uptake

plzoles/min/g

0.068 f 0.006 0.86 f 0.05 0.495 f 0.049 2.08 f 0.12 0.303 f 0.044 2.31 f 0.24

0.226 f 0.019 1.53 f 0.05 0.170 f 0.039 1.75 f 0.09

_.

AMP ATP:ADP ratio

0.165 f 0.051 5.3 f 0.4

0.186 f 0.021 3.5 f 0.2 0.197 f 0.050 3.7 f 0.5 0.222 f 0.062 3.3 f 0.1

0.172 f 0.038 3.7 f 0.1

octanoate

0.2 ?lLM

-

-

+

+

Substrate

2 i?ul

None (6) Lactate (5)

Lactate (5) Alanine (6) Alanine (5)

gluconeogenesis from lactate and alanine was depressed by 40

and 30%, respectively.

Effect of P-Hydroxybutyrate-Perfusion studies were carried out with P-hydroxybutyrate, rather than with octanoate, as a source of reducing equivalents. Since the metabolism of p- hydroxybutyrate by the liver involves only its oxidation to acetoacetate thereby generating mitochondrial NADH (42)) the potential effects of acetyl-CoA formation by fatty acid metabolism may be eliminated. Glucose synthesis from lactate was enhanced by P-hydroxybutyrate in the perfused rat liver but was inhibited in the guinea pig liver (Fig. 4). In both species, P-hydroxybutyrate increased gluconeogenesis from pyruvate, but this increase was more pronounced in rat than in guinea pig liver. The infusion of P-hydroxybutyrate generally increased oxygen consumption by about 5% in most experiments. These

results are identical with the octanoate experiments and are best explained by the alterations in the NAD+:NADH ratio within the mitochondria.

During gluconeogenesis from lactate, a net generation of cytosolic NADH occurs, part of which is reoxidized in the mito- chondria. Since the infusion of fi-hydroxybutyrate into the intact liver results in a decreased mitochondrial NAD+:NADH ratio, removal of the excess reducing equivalents might be ex-

petted to relieve the inhibition of gluconeogenesis induced by ,& hydroxybutyrate. Fig. 5 shows that low concentrations (4 to 8 PM) of the artificial electron acceptor, phenazine methosulfate, relieved the fl-hydroxybutyrate-induced inhibition of gluconeo- genesis in guinea pig liver with a concomitant increase in oxygen consumption. Although phenazine methosulfate can also act as an electron acceptor in the cytosol (43, 44), it is apparent from Fig. 5 that 8 PM phenazine methosulfate had a negligible effect on gluconeogenesis in livers perfused with lactate alone (i.e. in the absence of fi-hydroxybutyrate) .

E;trect of Aminooxyacetic Acid-Previous work (20, 21) has suggested that in rat liver lactate conversion to glucose involves the transamination of mitochondrial oxalacetate to aspartate followed by aspartate transfer to the cytosol and subsequent reconversion to oxalacetate. This pathway supplies mitochon- drially generated aspartate without contributing to the NADH levels in the cytosol. With pyruvate as substrate, malate efflux from the mitochondria and its conversion in the cytosol to ox- alacetate provides both the carbon and the NADH necessary for glucose formation. Rognstad and Katz (43) have shown that AOA,l an inhibitor of pyridoxal phosphate enzymes (45),

1 The abbreviation used is: AOA, aminooxyacetic acid.

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1 PYRUVATE

I I I I I I 1

, LACTATE

/3-hydroxybutyrote (mM)

8

50 60 70 80 90 100 II

minutes of perfusion

FIG. 4. Effect of P-hydroxybutyrate infusion on gluconeogene- sis from pyruvate and lactate in rat and guinea pig livers. Livers were perfused as indicated in Fig. 1. The concentration of p- hydroxybutyrate indicated in the figure represents the concentra- tions of o-p-hydroxybutyrate at the arterial input. The bars represent t,he S.E. of four to six livers from fasted animals.

.5 :

.4 * Ioctot;

.3 L

PMS@M)

.2

.I

l!!Y.lI 30 40 . 50 60 70 80 30 40 50 60 70 80

MINUTES OF PERFUSION

FIG. 5. Reversal of P-hydroxybutyrate-induced inhibition of glueoneogenesis in guinea pig liver by phenazine methosulfate (PM&‘). Lactate (2 mM) infusion was begun at 30 min. P- Hydroxybutyrate and PMS were introduced (right) as indicated. PMS (8 hi) had no effect on glucose production in livers perfused with lact.ate alone (left). The points were plotted from the average of four experiments.

blocked gluconeogenesis from lactate in rat kidney cortex slices without affecting glucose formation from pyruvate. These re- sults have been confirmed using the perfused rat liver (46) and support the hypothesis that aspartate is the major anion leaving the mitochondria when lactate is the substrate.

Importance of Mitochondrial P-enolpyruvate Formation in Gluconeogenesis-Guinea pig and rat livers were perfused with lactate plus AOA in the presence and absence of P-hydroxybu- tyrate. The infusion of AOA should inhibit P-enolpyruvate formation in the cytosol indirectly via aspartate aminotransferase, whereas the infusion of P-hydroxybutyrate should inhibit mito- chondrial P-enolpyruvate synthesis due to a shift in the mito- chondrial oxidationreduction state toward reduction. In an- other type of experiment, we evaluated the effect of AOA (0.2 mM) in perfused livers from fed and fasted rabbits. This experi- mental design permits a comparison of gluconeogenic rates in the presence and absence of a cytosolic P-enolpyruvate carboxy- kinase, since fed rabbits have negligible activity of this enzyme in liver cytosol (1, 16, 17).

Our initial experiments with AOA in rat livers perfused with In rat liver, AOA (0.2 mM) reduced gluconeogenesis from either lactate or pyruvate produced results which in general lactate by more than 90%, and the addition of P-hydroxybutyr- agreed with those of Rognstad and Katz (43). It is conceivable ate (8 mM) did not reverse or enhance this inhibition (Fig. 7). that aminooxyacetate inhibits the mitochondrial aspartate When a physiological mixture of lactate and pyruvate (ratio = aminotransferase as well as the cytosolic enzyme. In these ex- 10 : 1 (39, 47)) was used as substrate, a slight reversal of this in-

.4 -

.3-

.2-

.5-<

.4 -

.3

.2-

.I -

5

pyruvote

0.4

0.1

Aminooxyacetote (mM)

10 60 70 80 90 100

minutes of perfusion

FIG. 6. Effect of AOA on gluconeogenesis from pyruvate and lactate in rat (O- - -0) and guinea pig (0-0) livers.

periments, the metabolic consequences would be the same re- gardless of whether one or both isozymes were inhibited. A comparison of the effects of increasing concentrations of AOA on gluconeogenesis in perfused rat and guinea pig liver is shown in Fig. 6. Gluconeogenesis from pyruvate was not appreciably affected by AOA in either species. With lactate as substrate, the inhibition by AOA was much less marked in guinea pig than in rat liver, suggesting that in guinea pig liver a significant amount of glucose production proceeded by a pathway which did not involve aspartate formation. The effect of AOA was about equal at both 0.2 and 0.4 mM, indicating that saturating levels of the inhibitor were attained. Since glucose formation from the P-enolpyruvate generated in the mitochondria would not be sensitive to inhibition by AOA, these data imply that mitochon- drial P-enolpyruvate formation and efflux into the cytosol must play a significant role during gluconeogenesis in the guinea pig liver.

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% L I C c 60- : 6p

$ 0 guinea pig liver

5 . .

- j..-..,.

g 4o I :

s 0

z+ :

20 b\\ i-,

90. 0. 0 rat liver AOA --~~0.0-0.0-00- - _ -” -o--- 0.2mM

L

c R-OHB

O-

h $0 7’0 sb 9’0 100 minutes of perfusion

FIG. 7. Effect of p-hydroxybutyrate infusion on gluconeogene- sis from lactate in rat and guinea pig livers in the presence of aminooxyacetate. Livers from fasted animals were perfused as indicated in Fig. 1. At 60 min AOA was introduced and main- tained at 0.2 rnrvr by constant infusion throughout the experiment. fi-Hydroxybutyrate (8 mM) was infused as indicated in the figure. The initial rate represents the mean rate of glucose production in the 20 min preceding the infusion of AOA and was similar to rates indicated in Figs. 1 to 4.

hibition was observed (Fig. S), which is in accord with the stim- ulation of gluconeogenesis in rat liver by fi-hydroxybutyrate (Fig. 4). ,In contrast, BOA lowered gluconeogenesis in guinea pig liver by 50 to 55oj,, and the addition of P-hydroxybutyrate further increased this inhibition (Fig. 7) to about 70%. Upon termination of the fl-hydroxybutyrate infusion, glucose produc- tion returned to the same level noted before its addition. This pattern of sequential inhibition in the guinea pig liver was also observed when a physiological mixture of lactate and pyruvate (ratio = 3O:l (9, 10)) was employed as substrate (Fig. 8). These data further support the contention that mitochondrial P-enolpyruvate formation is a significant process for gluconeo- genesis, and that the flux of carbon through mitochondrial P- enolpyruvate carboxykinase is inhibited when the mitochondrial NAD+:NADH ratio is shifted toward reduction (cf. Figs. 4 and 5). In these experiments, AOA may have secondary effects in addition to its inhibition of aspartate aminotransferase. For example, an AOA-induced inhibition of this enzyme should inter- fere with the operation of the malate-aspartate shuttle and thereby result in a decreased utilization of NADH either in the cytosol or in the mitochondria. Although phenazine metho- sulfate (8 PM) had no effect on glucose synthesis from lactate alone (Fig. 5), it significantly reversed the AOA-induced inhibi- tion of glucose formation from lactate with a concomitant in- crease in oxygen uptake (Fig. 9, see also Ref. 43). This increase in oxygen uptake could result from the reoxidation of NADH either via mitochondrial or microsomal (48) electron transport pathways. A rise in the NADH level in the guinea pig liver mitochondria may subsequently inhibit mitochondrial P-enol- pyruvate formation. It seems probable therefore that a sec- ondary inhibition of P-enolpyruvate formation, presumably mediated by the mitochondrial oxidation-reduction potential, also contributes to the inhibition of gluconeogenesis by AOA.

60-

R-OH0 8mM

0'50 4 8 60 70 00 90 100 I

minutes of perfusion

FIG. 8. Effect of fi-hydroxybutyrate infusion on gluconeogene- sis in rat and guinea pig livers in the presence of aminooxyacetate. Livers were perfused as indicated in Fig. 7, except that the sub- strate was a physiological mixture of lactate plus pyruvate as follows: for rat liver (39) a lactate to pyruvate ratio of 1O:l; for guinea pig liver (10) a lactate to pyruvate ratio of 3O:l. In each substrate mixture, the concentration of lactate was 2 mM.

Gluconeogenesis in Rabbit Liver-Perfused livers from both fed and fasted rabbits, synthesized glucose from lactate at rates two to four times above the endogenous level, with almost linear rates being maintained for up to 1 hour (Fig. IO/l). In livers from fed rabbits (Fig. IOB), AOA had no effect on glucose pro- duction, which was not surprising since fed rabbits have a neg- ligible activity of cytosolic P-enolpyruvate carboxykinase. Even in livers from fasted rabbits in which the cytosolic enzyme is induced to significantly higher levels (16, 17), the inhibition by AOA did not exceed 60% and was quantitatively similar to the results noted with guinea pig liver (compare Figs. 7 and 10B). The agreement between the two situations is excellent and indi- cates different regulatory mechanisms for hepatic gluconeo- genesis in these species as opposed to the rat.

DISCUSSION

Mitochondrial Reducing Equivalents and Gluconeogenesis in Perfused Rat and Guinea Pig Liver-Fatty acid oxidation causes an increased state of reduction of the NAD+ system in the mito- chondria and also enhances gluconeogenesis from lactate and pyruvate in isolated perfused rat liver (49, 50), as well as in vivo (51). However, it has been recently reported (9) that hexano- ate and oleate inhibit gluconeogenesis from lactate in perfused guinea pig liver at concentrations which stimulate this same process in rat liver. Seyffert and Madison (52) have also noted that an acute elevation of plasma free fatty acids produces a marked decrease in hepatic glucose output in portacaval shunted dogs. The mechanism of the stimulation of gluconeo- genesis in rat liver by fatty acid oxidation has been attributed to either an acetyl-CoA-dependent activation of pyruvate car- boxylase or to an increased supply of reducing equivalents within the mitochondria, or both (53). However, no detectable differ- ences have been found in the tissue concentrations of either free CoA or acetyl-CoA in livers from either guinea pigs or rats per- fused with lactate in the presence and absence of hexanoate (9), although the distribution of these metabolites within the cell is

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ate (

I I I I 30 40 50 60 70 80 90 100 30 40 50 60 70 80 90 IO

MINUTES OF PERFUSION

10

FIG. 9. Effect of phenazine methosulfate (PMS) on gluconeogenesis in rat and guinea pig liver metabolizing lactate in the presence of AOA. The vertical bars represent the S.E. for four and five livers from fasted rats (A) and guinea pigs (B), respectively.

uncertain. From the data presented in Tables I and II and Figs. 1 to 4, it seems apparent that the differential effect of fatty acid oxidation in both species is best explained by the shift in the mitochondrial NAD+:NADH ratio associated with fatty acid osidation.

The presence of P-enolpyruvate carboxykinase in guinea pig and rabbit liver mitochondria leads to a metabolic situation entirely different from that prevailing in rat liver, since all three key gluconeogenic intermediates, aspartate, malate, and P- enolpyruvate, may be formed within this compartment. Their relative rates of production are the result of a competition for mitochondrial oxalacetate. We have shown previously (10, 17) that in isolated guinea pig and rabbit liver mitochondria, the utilization of oxalacetate is controlled, in part, by the oxidation- reduction state of the mitochondria such that more P-enolpyru- vate relative to either aspartate or malate is synthesized as the mitochondria become increasingly oxidized. Conversely, a shift toward reduction initially favors malate formation, and at more reduced levels, aspartate synthesis, with a concomitant decrease in P-enolpyruvate formation. Decreasing the pro- portion of NADf relative to NADH lowers the steady state con- centration of oxalacetate within the matrix space by the interac- tion of the NADf:NADH ratio with malate dehydrogenase (54). This would decrease P-enolpyruvate synthesis and increase the rate of carbon flow to malate. Thus, if gluconeogenesis in guinea pig liver proceeded primarily via aspartate and malate as in rat liver, the shift towards reduction in the mitochondria associated with fatty acid oxidation and P-hydroxybutyrate in- fusion would be expected to increase rather than decrease gluco- neogenesis. Because this does not occur, except when pyruvate is the substrate, it seems reasonable to hypothesize that the in- hibition of gluconeogenesis in guinea pig liver (Figs. 2 to 4) results from an oxidation-reduction-mediated inhibition of mito-

chondrial P-enolpyruvate synthesis. The differential effects of fatty acid oxidation on gluconeogenesis in rat and guinea pig livers are therefore related to the synthesis of P-enolpyruvate for gluconeogenesis in guinea pig liver mitochondria, and, in order to sustain hepatic gluconeogenesis in the guinea pig, a consider- able fraction of the P-enolpyruvate in the cytosol must be sup- plied directly by the mitochondria. The apparent stimulation of gluconeogenesis from 2 mrvr pyruvate in guinea pig liver could represent an artifact of the high pyruvate concentration in the perfusion medium which may result in a large intramitochondrial pool of oxalacetate and therefore may be of doubtful physiological significance.

Mitochondrial P-erwlpyruvate Carboxykinase and Gluconeo- genesis-A number of studies have suggested that any partici- pation of the mitochondrial form of P-enolpyruvate carboxy- kinase in guinea pig or rabbit liver gluconeogenesis must be mediated by a form of physiological uncoupling, presumably by free fatty acids. The basis for this hypothesis has been the con- sistently increased rates of P-enolpyruvate formation by isolated mitochondria after partial uncoupling with dinitrophenol or oleic acid (55-58). However, much of this effect may be due to an increase in the NAD+:NADH ratio induced by uncouplers (59, 60). In fact, under conditions when the oxidation-reduc- tion potential is held fairly constant, uncoupling produces a con- sistent decrease in the rate of P-enolpyruvate formation (61). The results of the present study show clearly that gluconeo- genesis in the guinea pig liver was progressively inhibited by in- creasing concentrations of octanoate without any alteration in the adenine nucleotide balance. This occurred despite the in- creased rates of oxygen consumption which resulted from the fatty acid infusion. It seems probable then that an uncoupling mechanism is not required for rapid rates of gluconeogenesis in

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fasted

-20 -10 0 +20 t40 0 IO 20 30

time (min)

FIG. 10. Glucose production in perfused rabbit liver. A, livers from fed and fasted rabbits were perfused with Krebs-Ringer bicarbonate buffer alone until endogenous glucose production was reduced to a minimum. The zero time in the figure indicates the time of addition of substrate (2 mM lactate). Minutes to the left of zero represent time of perfusion immediately prior to infusion of lactate and not the total period of perfusion with Krebs-Ringer bicarbonate buffer. Points plotted are means f S.E. (vertical bars) for the number of livers shown in parentheses. B, inhibition of gluconeogenesis from lactate by AOA in perfused rabbit livers. Livers from fed and fasted (96 hours) rabbits were perfused with Krebs-Ringer bicarbonate buffer alone until endogenous glucose production was reduced to a minimum as shown in A. After a 20-min perfusion with substrate, AOA (0.2 mM) infusion was begun and continued for 30 min. The zero time represents time of addition of AOA. The percentage of inhibition plotted was corrected from the rate of gluconeogenesis in control livers during this period as indicated in A. The points represent the means of five and four livers from fasted and fed animals, respectively. Vertical bars, where indicated, represent the S.E.

species containing a mitochondrial form of P-enolpyruvate car- boxykinase.

It would seem certain that species such as the rabbit and bird, both of which contain an almost totally mitochondrial form of P-enolpyruvate carboxykinase, must rely on this enzyme for hepatic glucose synthesis. However, it has been suggested, based on studies with isolated rabbit liver mitochondria (16), that only the cytosolic form of the enzyme is involved in glucose formation in the starved rabbit. Isolated mitochondria from rabbit liver have the capacity to form sufficient P-enolpyruvate under conditions of favorable oxidation-reduction states (17) to account for the measured rates of gluconeogenesis shown in Fig. 10 which is about 0.3 to 0.4 pmole per min per g of liver. In fed rabbits, cytosolic $-enolpyruvate carboxykinase accounts for only 5% of the total activity of the enzyme in the liver, yet con- siderable net glucose formation from lactate of 0.23 pmole per min per g can be observed (Fig. 10). After fasting, the activity of the cytosolic enzyme increases from 0.4 to 2.4 units per g (17), but the rate of glucose formation increases to only 0.30 pmole per min per g, suggesting that over 70% of gluconeogenesis pro- ceeds via the mitochondrial enzyme. Arninooxyacetic acid, at concentrations which totally abolish gluconeogenesis in rat liver, caused a 55 to 60% inhibition of glucose synthesis from lactate in livers from fasted rabbits but had no effect when infused into livers from fed animals.

Longshaw el al. (62) have concluded that mitochondrial P- enolpyruvate formation plays an insignificant role in gluconeo- genesis in guinea pig kidney cortex slices. This was based on the finding that aminooxyacetic acid caused a 72% inhibition of glucose synthesis from lactate (but not from pyruvate). How- ever, the influence of the mitochondrial oxidation-reduction po- tential on P-enolpyruvate formation was not considered and

therefore was not controlled during the 60.min incubation. Under the conditions of their experiment, the oxidation-reduction state of the slices would probably be relatively reduced. Since it is apparent that a marked reduction in glucose formation in guinea pig liver occurs with P-hydroxybutyrate infusion, even in the presence of aminooxyacetic acid (Figs. 4, 7, and 8), it is possible that Longshaw et al. (62) were measuring the rate of gluconeogenesis under conditions comparable to those in Fig. 7, in which fl-hydroxybutyrate and aminooxyacetic acid combined to give a 70% inhibition of glucose synthesis from lactate.

An alternative approach to understanding the role of mito- chondrial P-enolpyruvate carboxykinase in gluconeogenesis was employed by Sijling et al. (9) who used quinolinate to block cytosolic P-enolpyruvate carboxykinase. These authors noted a complete inhibition of gluconeogenesis from lactate in rat liver, a partial inhibition in perfused guinea pig liver, and no effect in perfused pigeon liver, which contains an almost totally mito- chondrial form of P-enolpyruvate carboxykinase. Since quin- olinate presumably inhibits only the cytosolic form of the en- zyme (63), gluconeogenesis can still proceed via the mitochon- drial enzyme. These studies together with the results presented in this paper strongly support a mechanism of gluconeogenesis in guinea pig and rabbit liver which involves P-enolpyruvate for- mation in both the cytosol and the mitochondria. This con- clusion, which is also valid for the cat,2 has broad physiological implications, since the livers of the majority of animal species, including humans, have both forms of the enzyme (23). A num- ber of important questions arise from these and other studies of mitochondrial metabolism in guinea pig liver. It is not clear, for example, why the NADf:NADH ratio in the mitochondria in guinea pig liver (9, 10) shifts towards oxidation during fasting, whereas the same ratio in the cytosol remains unaltered but extremely reduced when compared with the rat liver cytosol (39). These divergent changes suggest major differences in the mecha- nism by which the oxidation-reduction state of the cytosol and mitochondria is regulated in guinea pig and rat liver.

Acknowledgments-The authors wish to thank Edward Good- man, Jr., Linda Grengel, and Emilia Siojo for their skillful tech- nical assistance and also to acknowledge the help of J. Harold Mohler during the initial stages of this work. We are grateful to Dr. Mireille Jomain-Baum for valuable discussion and help during the course of this study, and to Dr. Sidney Weinhouse for advice during the preparation of the manuscript.

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Ifeanyi J. Arinze, Alan J. Garber and Richard W. HansonMITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASEThe Regulation of Gluconeogenesis in Mammalian Liver: THE ROLE OF

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