Acta pharmacol. cif toxicol. 1974, 34, 232-240.

From the lnstitutc of Physiology, University of Aarhus, DK-8000 Aarhus C , Denmark

Effects of Phenformin and Phenformin-Ethanol Synergism on the Perfused Rat Liver

BY

Christian Olsen and Niels Krarup

(Received November 14, 1973; Accepted December 19, 1973)

AbJtrac?: The metabolic eKects of phenformin and phenformin-ethanol were studied in isolated pcrfused liver preparations from fed rats. Whether phenformin was given alone or together with ethanol, the hepatic oxygen consumption decreased and thc lactate production increased. Phenformin caused a decrease in ethanol elimination. The mitochondrial redox level was reduced by phenformin in all types of cxperiments, but a significant reduction of the cytoplasmic redox levcl was seen only when phenformin and ethanol were given together. The findings indicate that phen- formin, in addition to an inhibitory effect on oxidative phosphorylation, impairs the transport of reducing equivalents across the mitochondrial membrane. This effect may be secondary to a lack of energy in the transport system.

Key-words: Ethanol metabolism - transport of reducing equivalents - lactate - pyruvate - kctone bodies.

The inhibitory effect of phenethylbiguanide (phenformin) on oxidative phos- phorylation leads to an increased blood lactate concentration in man (KREIS- BLKG ct al. 1972). This seems mainly to be due to increased lactate production in muscles as evidenced from the increase in Cori cycle activity after phen- formin (SEARLE ct al. 1969). Ethanol, which also increases the concentration of lactate in the blood, acts primarily on the liver, which after ethanol ingestion becomes less able to clear the lactate liberated in the periphery (KREISBERG et al. 1972). It is therefore not surprising that consumption of ethanol by sub- jects treated with phenformin can increase the lactate concentration to toxic levels (LACHER & LASAGNA 1966), and that lethal cases of lactic acidosis under such conditions have been described (SCHAFFALITZKY DE MUCKADELL et al. 1973). In man it was demonstrated that phenformin and ethanol act syner- gistically on the lactate concentration in peripheral blood (KREISBERG et al. 1972).

The mechanism of phenformin action on the liver is still disputed. Except for the effect on oxidative phosphorylation (DAVIDOFF 1968; SCHAFER 1969)

PHENFORMIN AND ETHANOL ON PERFUSED R A T LIVER 233

an effect on the transport of reducing equivalents across the mitochondrial membrane has been suggested (TOEWS et af. 1971).

In the present experiments the mechanism of the phenformin effect and a possible phenformin-ethanol synergism were studied on the isolated perfused rat liver. An effect on the oxidative phosphorylation was assessed from the oxygen consumption and a possible effect on the transport of reducing equiv- alents across the mitochondrial membrane was estimated from the redox levels on each side of the mitochondrial membrane. These parameters were followed partly under conditions where the flux of reducing equivalents was from mito- chondria to cytosol (KREBS et al. 1967) (non ethanol metabolizing livers) and partly where the reducing equivalents were transported in the opposite direc- tion (ethanol metabolizing livers). Furthermore, lactate and ketone body pro- duction was followed up both before and after the addition of phenformin.

Methods

Atrimals. Male Wistar rats weighing about 180 g maintained on Altromin pellets nd libitum were used.

Operative technique and perfusion were essentially as described elsewhere (HEMS et a/. 1966). The animals were anaesthetized by an intraperitoneal injection of nembutal'B (mebu- malum NFN) (0.1 ml of 6 % solution per 100 g body wt.). 100 i.u. of heparin in 100 p1 physiological saline was then administered intraperitoneally. The perfusion was started immediately after the portal vein was cannulated. Thus the liver was never without blood supply, and the time with impaired portal flow was about 30 sec. After cannulation of the inferior caval vein the first 5-10 ml samples of venous blood were discarded. The liver was left in situ, and the rat transferred to the perfusion apparatus, where recirculation of the perfusion medium was started. The perfusion medium entered the portal vein at a pressure of about 15 cm of water and left the liver at a negative pressure of about 5 cm of water. The flow was recorded continuously by an electronic drop counter which also served as an air bubble trap. The flow rate was about 0.7 ml/g liver (wet wt.)/min. The temperature of the cabinet and perfusion medium was controlled. The temperature of the liver was kept close to 38" and recorded by a thermistor placed in contact with the right liver lobe.

The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing 2.4 % (w/v) bovine serum albumin (SIGMA), washed, aged human erythrocytes yielding a hae- matocrit value of about 30 % and of glucose up to a concentration of 5.5 mM. The medium was equilibrated with a gas mixture containing 75 % N2, 20% Of, and 5 D/O C 0 2 .

Analyses. Samples of the medium were taken from the tubes carrying the medium to and from the liver and immediately pipetted into ice cold 5.1 '/n perchloric acid. Pyruvate (P), lactate (L), acetoacetate (Ac), and 0-hydroxybutyrate (HB) were determined by an en- zymatic fluorometric micromethod (OLSEN 1971). For the determination of ethanol 50 pI of the supernatant was incubated into 1000 pl hydrazine buffer (1.1 M, pH 9.0) containing 0.33 mg N A D + (BOEHRINGER Mannheim, 15298) and 15pl alcohol dehydrogenase (BOEH- RINGER 15418). After a 60 min. incubation the assay material was diluted with 10 ml 0.07 M-HCI, and the resulting N A D H was determined by its native fluorescence. In the range of the standards used (0.3-3.3 niM) the recovery was 99.9-103.9%, and the coefficient

234 CHRISTIAN OLSEN A N D NIELS KRARUP

of variation was 0.9-2.7 YO. Thc hacmoglobin concentration and oxygen saturation were determined as previously described (KRARUP 1973).

Types of expeririients, statistics, rind calculations. Four types of experiments were carried out:

A. Now ethanol rnetuholizing h e r s : I . Perfusion without addition of phenformin (n=6). 2. Phenformin hydrochloride (PHARMACIA) was added to a concentration of 40 mg/100

nil in the medium after 55 min. pcrfusion (n=3). B. Ethanol metnbolizing liuers: 3. Perfusion without addition of phenformin. Ethanol was added to a concentration of

about 5 niM in the medium after 30 min. perfusion. This concentration was nearly maintained by continuous infusion (n = 5) .

4. Phenformin was added to a concentration of 40 mg/100 ml in the medium after 75 min. of perfusion. Ethanol as above (n=6). To ensure an equilibrium period with ethanol, phenformin was added later than in type A2.

Samples were withdrawn at 10 min. intervals for at least 2 hours in all experiments. P-values wcre calculatcd by t-test for the difference between the means of thc above series. The uptake or output of the metabolites by the liver was calculated by multiplying the concentration differences across the liver by the flow. The L/P and HB/Ac ratios in the effluent medium were used as estimates of the redox levels in cytosol and mitochondria rcspectively (SCHOLZ 1968).

Results

The oxygen uptake in tlie different types of experiments is shown in fig. 1. There is a slight spontaneous fall in the oxygen consumption of the controls. Ethanol does not influence the oxygen consumption whereas a significant de- crease is seen after the addition of phenformin. Steady state conditions were not obtained, but the changes with time of the parameters followed were al- most linear except immediately after the addition of phenformin. In the follow- ing the mean values of the parameters immediately before the time of phen- formin addition and 50 minutes thereafter will be presented.

A . Non rtlianol metabolizing livers. From fig. 2 it appears that the spontaneous decrease in oxygen consumption

by the untreated livers is 18% during 50 minutes, whereas a 60% decrease is seen after the addition of phenformin. The lactate production remains fairly constant in the untreated livers, whereas the addition of phenformin is followed by a pronounced increase in lactate production, although the lactate concentra- tion in tlie perfusion medium is increased from 6.9 f 1 .O m M to 14.3 k I .4 mM 50 minutes after the addition of phenformin. An insignificant pyruvate output was reversed to a slight uptake after phenformin. The ketone production is not changed, either in the untreated group or in the group given phenformin. In the untreated livers the HB as well as Ac output was not changed during the

PHENFORMIN A N D ETHANOL ON PERFUSED RAT LIVER 235

w 3

W Y lu 9- 3 - S W cn

5

3.0

2.5

2.0

1.5

1.0

0.5

T

I Y L

\ I 1

-4 J

-il : 40 60 80 I00 I20 I 40

Minutes Fig. 1 . The effect of phenformin on the oxygen consumption of the perfused rat liver. Open dots indicate the control experiments (n = 6 ) . Closed dots experiments in which phenformin was added after 55 min. (n = 3). Open triangles indicate ethanol metabolizing livers (n = 5 ) , closed triangles ethanol metabolizing livers in which phenformin was given

after 75 min. (n = 6 ) . (k S.E.M. indicated by the bars).

experiments. The addition of phenformin, however, caused an increased out- put of HB and reversed the Ac output to an uptake. These findings are also reflected in the mitochondria1 redox levels as estimated from the HB/Ac ratios

236

-

2.0

1.0

-

CHRISTIAN OLSEN AND NlELS K R A R U P

O x y g e n Lactate Ketone uptake ou tpu t output

/ i Y

P NS (0.05NS (0.05 NS NS Fig. 2. The effect of phenformin on oxygen consumption, lactate, and ketone production in the non-ethanol metabolizing livers. Open dots indicate control experiments (n = 6 ) , closed dots experiments i n which phenformin was given 5 minutes after thc first sample shown (11 = 3). The time interval between the lines is 50 minutes. (k S.E.M. indicated

by the bars).

shown in fig. 4 A. After phenformin the HB/Ac ratio was markcdly increased, whereas only a slight change in the untreated livers was seen. In the untreated livers the cytoplasmic rcdox level, estimated from the L/P ratio, were not significantly changed, and phenformin had only a slight cffcct on the L/P ratio.

B. Ethanof rnetaholizirig livcrs. From fig. 3 it appears that the effects of phenformin on oxygen consumption

and luctate production arc very similar to the effects obtained in the non ethanol metabolizing livers. A very slight uptake of pyruvate was seen before as well as after the addition of phenformin. Ketone production also remains unaltered, but again plienformin increased HB output and reversed Ac output to an up- take. It also appears from fig. 3 that in the untreated group ethanol elimination

PHENFORMIN AND ETHANOL ON PERFUSED RAT LIVER 237

Oxygen Lactate Ketone Ethano l uptake output

L

outpu t uptake

P N S < O . O l N S < O . O l N S N S N S <0 .01 Fig. 3. The effect of phenformin on oxygen consumption, lactate, and ketone production and ethanol elimination in the ethanol metabolizing likers. Open dots indicate control experiments (n = S), closed dots experiments in which phenformin was given 5 niiniites after the first sample shown (n = 6). The time interval between the lines is 50 minutes.

(k S.E.M. indicated by the bars).

is spontaneously decreased by 12% during the experiments, but a 60% de- crease occurs after phenformin. As evidenced from fig. 4 B, the HB/Ac ratio in the untreated group remains fairly constant, whereas the HB/Ac ratio is markedly increased by phenformin as in the non ethanol metabolizing livers. The addition of phenformin to the ethanol metabolizing livers increases the L/P ratio much more than in the livers not metabolizing ethanol (fig. 4 B com- pared to 4A) . The effect of phenforrnin on the HB/Ac ratio, however, was relatively greater than the effect on L/P ratio.

Discussion

A . Non ethanol metabolizing 1iivr.c. The present results agree with the finding that phenformin inhibits oxida-

tive phosphorylation (DAVIDOFF 1968). The increase in lactate production after phenformin may reflect anaerobic glycolysis from glycogen present i n the livers of the fed rats, in an attempt to compensate for the decreased oxidative phos-

23 8 CHRISTIAN OLSEN A N D NlELS KRARUP

A. -Ethanol 6. + Ethanol

200

100 L/ P -

20

10 H B l A C

20

10

Fig. 4. The effect of phenformin on the HB/Ac and L/P ratios in the effluent medium. A. From the series shown in fig. 2 (non-ethanol metabolizing livers). Open dots indicate control experiments. closed dots experiments in which phenformin was given 5 minutes

after the first sample shown, B. From the series shown in fig. 3 (ethanol metabolizing livers). Open dots indicate control experiments, closed dots experiments in which phenformin was given 5 minutes after the

first sample.

phorylation. The increase in HB/Ac ratio after phenformin reflects a reduction in mitochondria1 redox level, probably due to a decreased electron flux through the respiratory chain. The L/P ratio increased after phenformin, but to a much smaller extent than the HB/Ac ratio. This may indicate that the transport of reducing equivalents across the mitochondria1 membrane is inhibited by phen- formin.

B. Ethanol metabolizing livers. In these experiments the effects of phenformin on oxygen consumption and

lactate production also suggest an inhibition of oxidative phosphorylation and

PHENFORMIN A N D ETHANOL ON P E R F U S E D R A T LIVER 239

a compensatory stimulation of anaerobic glycolysis. These effects of phenfor- min are apparently not influenced by ethanol.

Oxidation of ethanol leads to a reduction of NAD+ to NADH in the cytosol, and is therefore dependent on a continuous reoxidation of NADH. Although this reoxidation takes place mainly in the mitochondrial compartment, the ratio NADH/NAD+ is lowest in cytosol (SCHOLZ 1968). This indicates that the transport of reducing equivalents from cytosol to mitochondria is uphill. This transport is apparently inhibited by phenformin, as the cytosol redox level is further reduced after phenformin, despite a decrease in the production of reduc- ing equivalents in cytosol from ethanol oxidation. The inhibitory effect of phen- formin on this uphill, and hence energy dependent transport, may be a con- sequence of lack of energy from the oxidative phosphorylation. The observed decrease in ethanol elimination rate may be a result of the more reduced cyto- plasmic redox level (HAWKINS & KALANT 1972). The clearance of lactate from peripheral tissues is also dependent on the cytoplasmic redox level in the liver (KREBS et al. 1969). The synergistic effect of phenformin and ethanol on the redox level in hepatic cytosol (conf. fig. 4 B) may therefore affect the hepatic clearance of lactate. This may explain the observed phenformin-ethanol syner- gism on the blood lactate concentration in man (KREISBERG el al. 1972).

It is concluded that phenformin in the isolated perfused rat liver acts by inhibiting the oxygen consumption. This leads to an increase in anaerobic glycolysis resulting in an increased lactate production. The findings further suggest an inhibitory effect of phenformin on the transport of reducing equiv- alents across the mitochondrial membrane. This effect may be a result of the decreased energy production in the respiratory chain. The reported phenformin- ethanol synergism on lactate concentration in the intact organism (KREISBFRG et a/. 1972) is probably due to the pronounced reduction in cytoplasmic redox level in the liver, as a result of an increased production of reducing equivalents in the cytosol from ethanol, and the impaired elimination of these due to the effects of phenformin. It should be stressed that the dose of phenformin used in the experiments is much higher than therapeutic doses. It has been shown, however, that biguanids are concentrated in the liver (BECKMAN 1968), and thus the possible clinical implications of this study cannot be ruled out.

Acknowledgements We are indebted to ass. Professor J. A. Larsen for helpful suggestions during

the preparation of the manuscript. Excellent technical assistance from Lissi Hansen, Anne-Marie Krogh, and Lillian B. Nielsen is thankfully acknowledged.

240 CHRISTIAN OLSEN A N D NIELS KKAKUP

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