Interconversion of Pyruvate Dehydrogenase in the Isolated Perfused Rat Liver

Size: px

Start display at page:

Download "Interconversion of Pyruvate Dehydrogenase in the Isolated Perfused Rat Liver"

Gwendoline Bond
5 years ago
Views:

Eur. J. Biochem. 33, 117-122 (1973) Interconversion of Pyruvate Dehydrogenase in the Isolated Perfused Rat Liver Christoph PATZELT, Georg LOFFLER, and Otto H.

1 Eur. J. Biochem. 33, (1973) Interconversion of Pyruvate Dehydrogenase in the Isolated Perfused Rat Liver Christoph PATZELT, Georg LOFFLER, and Otto H. WIELAND Forschergruppe Diabetes und Klinisch-Chemisches Institut, Stidtisches Krankenhans Miinchen-Schwabing (Received September 12/November 14, 1972) Active form and total activity of pyruvate dehydrogenase were measured in homogenates prepared from tissue samples obtained from the isolated rat liver during perfusion. In the absence of substrate, the active portion accounted for about ZOO/, of total activity in livers from fed rats, whereas only about loo/, were found to be active in livers from starved rats. These activities remained rather constant during a perfusion period of 120 min. Addition of 10 mm pyruvate to the perfusion medium resulted in an immediate, three-fold increase of the active pyruvate dehydrogenase. A similar effect was exerted by 20 mm fructose, which is known to be rapidly converted to pyruvate in liver. I0 mm lactate led to a considerably smaller increase of pyruvate dehydrogenase activity as compared to the effects obtained with pyruvate or fructose. Total activities of the enzyme did not change significantly during perfusion. If the activities of active pyruvate dehydrogenase from livers perfused with and without substrate were plotted against medium pyruvate concentrations an exponential correlation could be observed. Addition of 2 mm oleate abolished the effects of fructose or pyruvate on the formation of active pyruvate dehydrogenase. The simultaneous addition of D-( )-decanoylcarnitine inhibited the action of oleate, indicating that fatty acid oxidation is required for the inactivation of pyruvate dehydrogenase by interconversion of the active to the inactive form. The possible physiological significance of pyruvate and fatty acids in the regulation of pyruvate dehydrogenase interconversion is discussed. In homogenates prepared from quick-frozen livers of normal fed rats less than ZOO/, of the pyruvate dehydrogenase complex is present in the active form [l]. This proportion is changed depending on the nutritional and hormonal state of the animals. In general, situations accompanied by a rise of plasmafree fatty acids lead to a lowering of active pyruvate dehydrogenase, whereas an elevation was observed at decreased plasma free fatty acids [I]. These and other studies [2] suggested that the relative supply of fatty acids as substrate of energy metabolism may represent a determinant of the state of the interconvertible pyruvate dehydrogenase system. In order to investigate these interrelationships in more detail we have chosen the model of the isolated rat liver, which allows perfusion under well-defined conditions as well as measurement of pyruvate dehydrogenase activities in samples taken repeatedly in the course of a single experiment. The data presented here demonstrate that the state of the pyruvate dehydrogenase system in the isolated perfused rat liver depends on the nature of the substrate offered, and that free fatty acids are capable of antagonizing the effects of substrates, which themselves lead to increased formation of active pyruvate dehydrogenase. A preliminary report has been given elsewhere [3]. MATERIALS AND METHODS The technique of perfusions and methods for medium analysis as well as the source of materials have been described elsewhere in detail [4]. During the perfusion liver samples weighing mg were cut off the various lobes in intervals of 20 min each and frozen immediately in liquid nitrogen. The sequence of the lobes was varied to exclude lobular differences. The frozen liver samples were then pulverized in a mortar and homogenized with three volumes of 20 mm potassium-phosphate buffer ph 7.0 at 0 "C. Protein concentrations in the homogenates were about 40 mg/ml. Measurements of pyruvate dehydrogenase were performed as described previously [I].

2 118 Pyruvate Dehydrogenase Interconversion in Perfused Rat Liver Eur. J. Biochem Time of perfusion (min) Fig. 1. Active pyrucate dehydrogenase and total activity in livers of fed and 24-hour-starved rats during perfusion in the absence o/ substrate. Vertical bars represent S.E.M. In the brackets the numbers of experiments are given. (-) Fed; (----) starved; (0) active form; (0) total activity For the determination of the active portion, liver homogenates were assayed without further pretreatment. In order to obtain total pyruvate dehydrogenase activity, the inactive form present in the homogenate has to be converted into the active form prior to the assay. As described earlier [I], the endogenous pyruvate dehydrogenase phosphatase in concentrated homogenates is sufficient to complete activation, if homogenates are incubated with 20 mm Mg2 for 60 min. D-()-Carnitine was purchased from Otsuka Pharmaceutical Factory (Naruto, Tokushima, Japan) D-( f )-Decanoylcarnitine was preparcd by a slight modification of the procedure described by Bremer [5]. RESULTS Active Pyruuate-Dehydrogenase and l'otal Pyruvate-Dehydrogenase Activities in Livers Perfused without Substrate The state of the pyruvate dehydrogenase system in livers from fed or 24-hour-starved rats is illustrated Time of perfusion (min) Fig.2. Influence of fructose and pyruvate on the state of interconvertible pgruvate dehydrogenase in perfused liver of fed rats. 400 mg fructose as a single injection followed by an infusion of 200 nig/h or 1 mmol pyruvate as pulse injection followed by an infusion of 2 mmol/h were added to the perfusion medium (arrow) after 40 min preperfusion. The number of experiments is given in brackets. (-) Active form; (----) total activity; (0) controls without substrate; (0) fructose; (f) pyruvate in Fig. 1. The active form amounted to about ZOO/, of total activity, whereas in livers from starved rats only 100/, were found to be in the active state. Total activities of both groups did not differ significantly. During a perfusion period of 120 min in the absence of any added substrate the activities of both remained rather constant. Influence of Glucose, Fructose, Pyruvate, and Lactate on the State of Pyruvate Dehydrogenase in Perfused Livers from Zed Rats Addition of glucose to the medium in concentrations up to 40 mm resulted in a slight but not significant increase of active pyruvate dehydrogenase in livers from fed rats. However, if fructose instead of glucose was added by a pulse injection followed by constant infusion leading to a final concentration of about 20mM, considerable increase of the active form occurred (Fig.2). After 80min about 600/, of

3 Vol.33, No. 1, 1973 C. PATZELT, G. L~FFLER, and 0. H. WIELAND 119 Table 1. Active pyruvate dehydrogenase in the isolated perfused rat liver and medium substrate concentrations at the end of the perfusion period Pyruvate dehydrogenase activity is expressed as mu/mg protein, substrate concentrations as pmol/ml. Mean values & S.D. are given Additions to the perfusion mediurri Active pyrnvate dehydrogcnasc Pyruvatc Lactstc Ketone bodies (ncetoacetatc 0- hvdrosvlriitvra t,n) inu/mg protcin &mol/ml R7it1iout substrate Livers from fed rats f & f 0.08 Livers from 24-h-fasted rats f ;t f & 1.03 ~ ~ Fed rats Glucose (40 mm) & & f & 0.07 Fructose (20 mm) f & & & 0.39 Pyruvate (10 mm) & & & f 0.42 Pyruvate (4 mm) & & & 0.38 Lactate (10 mbl) f & f & 0.64 ~- Fed rats Fructose (20 mm) oleate& & & & 0.43 Pyruvate (10 mm) oleate& f & f 0.53 Pyruvate (4 mm) oleateb & & & & 0.30 Pyruvate (4 mm) oleateb f f D-()-decanoylcarnitine Pyruvate (4 mm) acetatec f & a 200 Vmol oleate were added as a single iniection (total volume 100 ml). 150 VmoI oleate were added as a pulse injection followed by a infusion of GOO wniol/h. 2 mmol sodium acetate were added as a pulsc iniectlon followed by a infusion of 4 mmol/h. total enzyme activity appeared as active pyruvate dehydrogenase, whereas total activity was not significantly altered. Similar effects were observed if pyruvate was added to the medium after 40min preperfusion. As illustrated in Fig. 2, the active portion of the enzyme rose rapidly to about 70 /, of total activity after pyruvate addition, whereas total activity was not influenced. In these studies pyruvate concentration in the medium was kept at 10 mm throughout perfusion. The reversibility of the increase in the active pyruvate dehydrogenase activity due to pyruvate could be demonstrated in experiments, where 30 min after the addition of pyruvate the perfusion medium was exchanged against a medium containing no pyruvate. Immediately after the exchange the values of the active portion dropped nearly to the level of the preperfusion period. Using 10 mm lactate instead of pyruvate only a slight increase of the active portion of the enzyme could be observed (Fig.5, Table 1). Effect of Fatty Acids on the State of Pyruvate Dehydrogenase in Livers from Fed Rats Fig. 3 illustrates the effect of fatty acids on active pyruvate dehydrogenase activity. After 40 min of preperfusion, pyruvate (10 mm) was added in order to elevate the active form. 40 min later 200 pmol oleate emulsified in an albumin solution according to Wieland and Frohlich [I41 were added to t,he perfusion medium. As may be seen there occurred an immediate drop of the active form, whereas again total activity remained essentially unchanged. Acetate in quantities equimolar to the number of carbon atoms of oleate remained without effect on pyruvate dehydrogenase. As may be seen from Table 1 the depressing effect of oleate is also visible when high levels of the active portion are initially installed by fructose. Oleate abolished the increase of the level of the active form and brought it back to the control values. To get further insight in the mechanism, by which fatty acids might act on pyruvate dehydrogenase interconversion, the effect of D-( )-decanoylcarnitine, a competitive inhibitor of the acylcarnitine transferase [el, was investigated. As shown in Fig.4 the decrease of active pyruvate dehydrogenase due to oleate perfusion was completely abolished in the presence of 1 mm D-( )-decanoylcarnitine. Table 1 summarizes the data of active liver pyruvate dehydrogenase as well as medium levels of pyruvate, lactate and ketone bodies at the end of perfusion periods. DISCUSSION From the studies described herein it is clear that the state of the pyruvate dehydrogenase system in the isolated perfused rat liver can be varied depending on the kind of substrate offered to the liver. A

120 Pyruvate Dehydrogenase Interconversion in Perfused R)at Liver Eur. J. Biochem. 1 J / i /h- -I4 0 20 40 60 80 100 120 Time of perfusion (min) Fig.3.

4 120 Pyruvate Dehydrogenase Interconversion in Perfused R)at Liver Eur. J. Biochem. 1 J / i /h- -I Time of perfusion (min) Fig.3. Influence of oleate and pyruuate on the leuel of active pyruuate dehydrogenase in perfused livers of fed rats. For added pyruvate quantities see legend to Fig.2. After a perfusion period of 80 min, 200 pmol oleate were given into the medium (arrow). Significance against control group: f, P < 0.001; f, P < 0.02 (----) Pyruvate; (-) pyruvate oleate; (0) active form; (0) total activity three-fold increase of the active form of the enzyme occurred within 20 min after addition of pyruvate and, less rapidly, after fructose (Fig.2). Lactate produced smaller changes whereas glucose, even at 40 mm concentrations, remained ineffectual (Table 1). Also alanine added to the medium in a concentration of about 10 mm did not significantly alter the state of pyruvate dehydrogenase interconversion. Simultaneously medium pyruvate levels were not changed. These different responses may be related to the rates at which pyruvate is formed from the respective substrates as indicated by the pyruvate concentrations of the perfusate. When pyruvate concentrations are plotted against the levels of active pyruvate dehydrogenase there is an exponential correlation. From Fig. 5 the pyruvate concentration leading to half-maximal increase ofthe active form of the enzyme can roughly be estimated to lie in the range of 1 mm. Incubation of isolated rat liver mitochondria also results in a dose dependent formation of active 0 1 I I I I I Time of perfusion (rnin) Fig.4. Irc,fluence of D-() -decanoylcurnitine on the effect of oleate on pyruuate dehydrogenase interconversion in perfused livers of fed rats. 0.5 mmol pyruvate as a single injection followed by an infusion of 1.8 mmol/h were given at the beginning of tho perfusion. After 30 min, 160 pmol oleate followed by an infusion of 600 pmol/h (-) and oleate 1 mm D-()-decanoylcarnitine (----) were given (arrow). Significance of the differences: f, P< 0.01; ff, P< 0,005. The full shaded column represents the mean value of perfusions without substrate pyruvate dehydrogenase, the half-maximal effect being obtained at about 0.3 mm pyruvate [7]. Studies in vitro on purified pyruvate dehydrogenase preparations have shown that pyruvate protects the enzyme from ATP-dependent inactivation [S, 19,201 which would result in a relative increase of the active portion. It seems likcly that in the perfused liver the same mechanism is responsible for the shift from the inactive to the active form induced by pyruvate. An increase of active pyruvate dehydrogenase in the livers from rats after intravenous injection of fructose has been reported by Soling et al. [9] and was ascribed to a fall in liver ATP due to fructose. Since fructose leads to an increase in pyruvate formation, this could also explain the fructose effect. It seems possible that physiologically both factors act together in the regulation of the pyruvatc dehydrogenase interconversion. In extending earlier observations on intact rats the perfusion experiments with oleate more directly demonstrate the effect of fatty acids on pyruvate dehydrogenase interconversion. From these studies it is clear that addition of oleate absolished the effect of fructose or pyruvate on the active portion of the enzyme and this occurs at undiminished or even increased pyruvate concentrations (Table 1). From the fact that in the presence of D-()-decanoylcarnitine the oleate effect was no longer visible

Vol.33, No.1, 1973 :i 44.- & 3 3 - E C. PATZELT, G. LOFFLER, and 0. H. WIELAND 121 1 I I 01 1 Pyruvate concentration in the medium (pmolirnl) 10 Fig. 5.

5 Vol.33, No.1, 1973 :i 44.- & E C. PATZELT, G. LOFFLER, and 0. H. WIELAND I I 01 1 Pyruvate concentration in the medium (pmolirnl) 10 Fig. 5. Relationship between pyruvate concentration of the perfusion medium and the level of active pyruvate dehydrogenase in the perfused livers (a) 24-hour-fasted; (0) fed; () fed pyruvate; (0) fed fructose; ( X ) fed lactate (Fig. 4) one might conclude that oleate is not working unless being oxidized. Acetate though giving rise to considerable ketone formation indicating its utilization had no significant effect in lowering the levels of active pyruvate dehydrogenase in the perfused liver. The substrate-dependent interconversion of pyruvate dehydrogenase in the perfused liver as shown here may be of physiological significance for the regulation of gluconeogenesis and of lipid synthesis from carbohydrates. Fructose is incorporated into liver triglyceride fatty acids and CO, at a rate at least 3 times that of equimolar concentrations of glucose [lo,ll]. This could be explained by the present observation that fructose, in contrast to glucose, markedly (%fold) increases the portion of activc pyruvate dehydrogenase thus raising the supply of acetyl-coa as the precursor for fatty acid synthesis. This view is based on the assumption discussed previously [I] that the pyruvate dehydrogenase reaction represents the rate-limiting step in the conversion of carbohydrate to fat in the liver. The present findings also shed some light on earlier studies on gluconeogenesis using the isolated perfused rat livcr. With lactate as the precursor for glucose synthesis, the ratio of C, units used to glucose synthesized was found to be in the range from [12,13,14], whereas with pyruvate as the substrate this ratio may reach a value of 4 [13,15]. This difference could be explained by the observation described here that pyruvate much more than lactate leads to an increase of pyruvate dehydrogenase activity. Therefore with pyruvate instead of lactate as the percursor more carbon atoms could pass the pyruvate dehydrogenase step and would thus be diverted from glucose synthesis. It has been shown that in the presence of fatty acids a greater percentage of pyruvate is converted to glucose in the perfused liver [4,15,16]. This sparing effect could be explained by the present finding that oleate lowers pyruvate dehydrogenase activity by promoting interconversion from the active to the inactive form. In addition, direct inhibition of hepatic pyruvate dehydrogenase by acetyl-coa [17], a finding originally obtained with the enzyme from heart muscle [IS], may be involved. Fatty acids are also known to stimulate gluconeogenesis from lactate. However, since lactate yields glucose at nearly the theoretical ratio of 2 : 1 already in the absence of fatty acids, a sparing mechanism can be excluded. In a previous paper an increase of active pyruvate dehydrogenase in the livers of rats pretreated with insulin has been reported [I]. All attempts to demonstrate a similar action of insulin on the perfused liver have failed so far. This is also true for glucagon which could have been expected to produce an effect comparable to that of oleate. Since the isolated perfused liver does respond readily in various other respects to glucagon and insulin it remains questionable whether these hormones are immediately involved in the control of hepatic pyruvate dehydrogenase interconversion. From these and other studies on isolated mitochondria [7] it would appear more likely that the ratio of active to inactive enzyme is dependent on the metabolic situation, pyruvate on the one side and fatty acids (and perhaps other substrates) on the other playing an antagonistic role. Obviously, the effective concentrations of pyruvate in these

122 C. PATZELT et al. : Pyruvatc Dehydrogenase Interconversion in Perfused Rat Liver Xur. J. Biochern. studies are above the expected physiological range.

6 122 C. PATZELT et al. : Pyruvatc Dehydrogenase Interconversion in Perfused Rat Liver Xur. J. Biochern. studies are above the expected physiological range. This, however, does not seem to represent a strong argument against the possible regulatory role of pyruvate in vivo taking into consideration the model character of the isolated perfused liver. Indccd, as will be reported elsewhere, much lower concentrations of pyruvate are effective on pyruvate dehydrogenase interconversion in isolated rat liver mitochondria [7]. This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany. The conscientious technical assistance of Mrs R. Weiss and Mrs U. Zechmeister is gratefully acknowledged. REFERENCES 1. Wielmd, O., Patzelt, C. & Loffler, G. (1972) Eur. J. Biochem. 26, Wieland, O., Siess, E., Schulze-Wethmar, F. H., v. Funcke, H. J. & Winton, B., (1971) Arch. Biochem. Biophys. 143, Patzelt, C., Loffler, G. & Wieland, 0. (1972) Proc. 1st. Eur. Meet. on the Technique of Perfusion of Tsolated Liver and Its Application (Milan, 1971) Raven-Press, New York, in press. 4. Teufel, H., Menahan, L. A, Shipp, J. C., Boning, S. & Wieland, 0. (1967) Eur. J. Biochem. 2, Bremer, J. (1968) Biochem. Prep. 12, Fritz, J. B. & Marquis, N. R. (1965) PTOC. Nut. Acad. ~S ci. U. S. A. 58, Portenhauscr, R. & Wieland, 0. H. (1972) Eur. J. Biochem. 31, Linn, T. C., Pettit, F. L. & Reed, L. J. (1969) Proc. Nut. Acad. Sci. CJ. S. A. 62, Soline, H. D. & Bernhard, G. (1971) FEBS Lett. 13, Wyshak, G. H. & Chaikoff, L L. (1953) J. Biol. Chem. 200, Pereira, J. N. & Jangaard, N. 0. (1971) ivetabozism, 20, Hems, R., Ross, B. D., Berry, N. & Krebs, H. A. (1966) Biochem. J. 101, Williamson, J. R., Browning, E. T. & Scholz, R. (1969) J. Biol. Chm. 244, Frohlich, J. & Wieland, 0. (1971) Bur. J. Biochem. 19, Menahan, L. A. & Wieland, 0. (1969) Eur. J. Biochem. -0, Menahan, L. A, Ross, B. D. & Wieland, 0. (1967) 3. Iionf. Ges. Biol. Chena. Oestrick p. 142, Springer- Verlag, Berlin, Heidelberg, New York. 17. U ieland, 0. (1971) Excerpta Med. Int. Congr. Ser. 231, Garland, P. B. & Randle, P. J. (1964) Biochem. J. 91, 6C. 19. Wieland, O., & v. Jagow-Westermann, R. (1969) FEBS Lett. 3, Siess, E., Wittmann, J. & Wieland, 0. (1971) Iloppe Seyler s Z. Physiol. Chem. 352, 447. C. Patzelt, G. Loffler, and 0. H. Wieland Institut fur Diabetesforschnng D-8000 Munchen 40, Kolner I latz 1 Federal Republic of Germany

THE GLUCOSE-FATTY ACID-KETONE BODY CYCLE Role of ketone bodies as respiratory substrates and metabolic signals

Br. J. Anaesth. (1981), 53, 131 THE GLUCOSE-FATTY ACID-KETONE BODY CYCLE Role of ketone bodies as respiratory substrates and metabolic signals J. C. STANLEY In this paper, the glucose-fatty acid cycle