PfliJgersArchiv
Pflfigers Arch. 370, 4 5 - 4 9 (1977)
EuropeanJournal of Physio~3y 9 by Springer-Verlag 1977
On the Mechanism of Glucagon Stimulation of Hepatic Gluconeogenesis MATILDE S. AYUSO-PARRILLA, ANGELES MARTIN-REQUERO, and ROBERTO PARRILLA Department of Metabolism, Institute G. Marafi6n, C.S.I.C., Velazquez 144, Madrid-6, Spain
Summary. The addition of L-alanine as substrate to a perfused rat liver preparation produced a five-fold increase in the rate of glucose production. This enhancement of the gluconeogenic flux seems to be a consequence of a rise in the steady-state levels of pyruvate and oxaloacetate subsequent to the rise in alanine concentration. Glucagon (2x 10 -9 M) increased the gluconeogenic flux from alanine (10raM) by 50 percent, even though the concentration of the substrate in the perfusion fluid was at saturation. This effect was accompanied by a rise in the intracellular concentration of alanine. However, the steady-state concentrations of pyruvate and oxaloacetate were decreased, probably as a consequence of a more reduced state of the nicotinamide-nucleotide system. In vivo, the intraperitoneal administration of glucagon to starved rats was accompanied by a decrease in the hepatic alanine and pyruvate concentrations despite the striking effects raising the plasma glucose levels. These observations seem to indicate that the effect of the hormone increasing the hepatic glucose output must be mediated through some other mechanism(s) independent of the intracellular variations in the hepatic amino acids levels.
enous fuel mobilization and enhanced glucose production, like fasting or diabetes, display elevated plasma levels of glucagon (Unger et al., 1970; Marliss et al., 1970). Nevertheless, in spite of the physiological importance of glucagon in the glucose homeostasis, very little is known about its mechanism of action in the control of hepatic gluconeogenesis. The control of gluconeogenesis from amino acJLds is particularly important since amino acids seem to be the most important substrates for gluconeogenesis in vivo. Glucagon is known to decrease plasma amino acid levels in vivo (Bromer and Chance, 1969; Martin et al., 1976) and also to stimulate the hepatic uptake of some amino acids in vivo as well as in the perfused isolated rat liver (Mallette et al., 1969 b). On this basis, it has been suggested that glucagon might stimulate gluconeogenesis by accelerating the entry of amino acids into the hepatic cell and the attainment of a steady-state level of gluconeogenic substrates (Mallette et al., 1969b). The experiments reported here seem to indicate that the effects of glucose increasing amino acid transport and stimulating glucose synthesis are fully independent from each other.
Key words: Glucagon action - Hepatic gluconeogenesis - Perfused liver.
INTRODUCTION The pancreatic hormone glucagon is known to have gluconeogenic effects in vitro (Miller, 1960; Exton et al., 1966; Garcia et al., 1966; Ross et al., 1967; Mallette et al., 1969 a). It is also known that most of the in vivo situations characterized by an increased endogSend offprint requests to Ayuso-Parrilla at the above address
MATERIALS AND METHODS Male Wistar rats of 190 - 2 1 0 g body weight were used. The animals were purchased when approximately 120 g in weight and kept in our animal room under standard conditions until they weighed 200 g. They were starved for 2 4 - 36 h prior to their experimental use. The rats were anesthetized by an intraperitoneal injection of Nembutal (40 mg/kg body weight). The portal vein and inferior vena cava were cannulated in situ and then infused with a hemoglobin-free Krebs-Henseleit bicarbonate buffer (Krebs and Henseleit, 1932) containing 3 ~ fraction V bovine albumin. The albumin was previously dissolved in a small volume of bicarbonate buffer and dialyzed against two or three changes of the same buffer. The pH of the medium after equilibration with 95 ~ Oz: 5 ~ CO2 in the gaseous phase was 7.4. Prior to its use the medium
46 was filtered through Mill• membranes having 0.45 gm pore size. Each liver was perfused with 100 ml of recirculating medium. The perfusion apparatus was equipped with a disc oxygenator (Scholz, 1968; Kerstens, 1969), and the liver was in contact w~th oxygen (Clark type) and pH electrodes. The rate of circulation of the perfusion medium was 28 ml/min (Goodman et al., 1973). In all the experiments the livers were allowed to equilibrate by perfusing them for 30 min in the absence of any substrate; then, when indicated, alan• (10 raM) or L-alanine (10 raM) plus glucagon (2.1 x 10 9 M) were added, and the perfusion was continued for an additional period of 60 min. At this time the livers were immediatly frozen using aluminum clamps (Wollenberger et al., 1960) which had been precooled in liquid nitrogen. Representative portions of the livers were freeze-dried and extracted with 35 volumes (w/v) of 8 ~ perchloric acid. Acid extracts were brought to pH 6.0 with potassium carbonate and immediatly used for the fluorimetric measurement of oxaloacetate (Williamson and Corkey, 1968). Perfusate samples (0.5 ml) were taken every 10 rain, deproteinized in cold 6 ~ (w/v) perchloric acid and brought to pH 6.0 with potassium carbonate for the determination of glucose and alanine. Metabolic rates were calculated over the 6 0 - 9 0 min interval. Glucose, pyruvate, glutamate, aspartate and e-ketoglutarate were measured spectrophotometrically according to methods described previously (Bergmeyer, 1965). Atanine was determined with alanine dehydrogenase and NAD + in Tris-hydrazine buffer. In the in vivo experiments rats were anesthetized, and glucagon was infused intraperitoneally at a rate of 26 gg/min for the first 5 rain and of 10 gg/min thereafter. At 20 rain the abdomen was opened and liver biopsies were taken and processed as described above. Control animals were infused with the same volume of saline. Most of the reagents were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). Enzymes were purchased from Boehringer (Mannheim, Germany).
RESULTS
Effect of Alan• and Alanine plus Glucagon on Glucose Output and Steady-State Levels of Hepatic Ketoacids. Addition of L-alanine (10 mM) to a perfused isolated rat liver preparation produced an increase in the glucose output of more than five-fold (Table 1). This increase in glucose production was accompanied by a rise in the steady-state concentration of pyruvate and oxaloacetate and a decrease in e-ketoglutarate (Table 1). Since pyruvate carboxylase (E.C. 6.4.1.1) and phosphoenolpyruvate-carboxykinase (E.C. 4.1.1.32) respond readily to variations in the concentration of pyruvate and oxaloacetate within the physiological range, the increased glucose production from L-alanine should ultimately be interpreted as a result of the rise in the steady-state levels of pyruvate and oxaloacetate. Glucagon stimulated glucose production from 67 to 95 gmoles/100 g body weight/h; however, in contrast to the effect of L-alanine alone, pyruvate and oxaloacetate steady-state levels decreased.
Hepatic Steady-State Levels of Transaminase Reactants. The hepatic steady-state levels of the reactants of alan• (E.C. 2.6.1.2) and arpartate (E.C. 2.6.1.1) amino transferases in control and glucagon treated rats are shown in Tables 2 and 3. The equilibrium expres-
Pfltigers Arch. 370 (1977) Table 1. Effect of glucagon on the hepatic steady-state concentration of some ketoacids during gluconeogenesis from L-alan• Livers were perfused for 30 min without any substrate and then Lalanine and glucagon were added. The perfusion was continued for 60 min at the end of which the livers were freeze-clamped with aluminium tongs precooled in liquid nitrogen. Metabolic rates were calculated over the last 30-rain period of perfusion Additions to the perfusate .....
None
10 mM L-alanine
10 mM L-alanine + 2 x 10 .9 M glucagon
788 • 85 529 • 30 18 • 1.7
445 _+ 48 460 • 35 t3 _+ 0.8
67_+ 4
95 _+ 3
nmoles/g dry wt Pyruvate 106 • 12 e-ketoglutarate 1150 • 160 Oxaloacetate 13 • 3a Glucose production gmoles/100 g bodywt/h 12_+ 0.9
Calculated from the aspartate aminotransferase equilibrium reaction. Values are means of at least eight experiments _+ standard error
sions for both reactions remained virtually unchanged in both instances in spite of significant changes in the concentrations of their reactants. This observation indicates once more (Williamson et al., 1967; Parrilla and Goodman, 1974) that the hepatic transaminating reactions are near equilibrium and then the variations in the concentration of ketoacids are largely dependent of the variations in the amino acid supply. However, factors other than the steady-state concentration of amino acids must be playing an important role since glucagon, in spite of increasing concentration of alanine (Table 2), was unable to raise the concentration of pyruvate. The equilibrium was maintained in this situation by a rise in the glutamate levels.
Intracellular Levels of L-Alanine in Control and Glucagon-Treated Livers. As shown in Table 4 the rat liver is able to accumulate alan• against a gradient as its intracellular concentration was found to be more than twice that of the perfusion fluid. Glucagon raised the intracellular concentration of alanine by more than 20 ~ and the ratio of intra- to extracellular concentrations was increased from 2.4 to 3.2. This finding prompts the question whether such an increase in the intracellular concentrations of amino acids was responsible for the increased gluconeogenic flux. In order to elucidate this point the relationship between alan• supply and hepatic metabolic activity was studied. As may be seen in Figure 1 the metabolic activity of the liver, as reflected by the rates of oxygen utilization, displayed saturation type kinetics, being saturated at alanine concentrations over 2 mM. The apparent "Kin" was found to be 1.1 mM. Similar results were obtained by plotting alanine concentration
M. S. Ayuso-Parrilta et al. : Glucagon Stimulation of Hepatic Gluconeogenesis
47
Table 2. Effect of glucagon on the hepatic concentration of the alanine aminotransferase reactants.The experimental details were the same as described in Table 1. Each value is the mean of at least eight experiments _+ standard error Additions to the perfusate
L-Alanine
c~-Ketoglutarate
Pyruvate
Glutamate
[Pyruvate] [Glutamate] [L-Alanine] [c~-Ketoglutarate]
nmoles/g dry wt Alanine (10 mM) Alanine (10 raM) + Glucagon (2x 10 -9 M)
45955 _+ 4083
529 _+ 30
788 + 85
10172 _+ 960
3.03
54047 _+ 4059
460 _+ 35
445 _+ 48
12090 _+ 895
4.62
Table 3. Effect of glucagon on the hepatic concentration of the aspartate aminotransferase reactants. The experimental details were the same as described in Table 1. Each figure represents the average value of at least eight experiments + standard error Additions to the perfusate
c~-Ketoglutarate
Aspartate
Glutamate
[ct-Ketoglutarate] [Aspartate]
Oxaloacetate
[Glutamate] [Oxaloacetate]
nmotes/g dry wt L-Alanine (10 raM) L-Alanine (10 raM) + Glucagon (2 x 10 -9 M)
529 + 30
13075 _+ 1290
10172 _+ 960
18 + 1.7
37.6
460 + 35
14682 _+ 2223
12090 + 895
13 + 0.8
42.4
16
Table 4. Effect of glucagon on the intracellular concentration of L-alanine in isolated rat livers perfused with L-alanine (10 raM) as a substrate. The experiments were carried out as described in Table I. For the calculation of the hepatic intracellular concentration of L-alanine, the extracellular water content was taken to be 0.2 ml/g wet weight (Mallette et al., 1969a), and the ratio of dry to wet liver weights 4.94 _+ 0.06, the mean of 87 determinations + standard error Additions to the perfusate
L-Alanine concentration Intracellular
Ratio IN/OUT
Da" 0~ z
Perfusion fluid
I
o !
3.5
2.4
3.2
3.2
~
/
/. 4 I
o,//
/
27 r
o
versus glucose output. The oxygen uptake, nevertheless, offers the advantage of comprising the energy expenditure not only for glucose synthesis but also for other biosynthetic processes enhanced by the substrate, including the urea production.
o?
0
mM L-Alanine (10 mM) 8.5 L-Alanine (10 raM) + Glucagon (2 x 10-9 M) 10.3
9~
14
o
; I/[ALANINE]
,'o
', [ALANINE] m M
Fig. 1. Rates of oxygen uptake by the isolated rat liver perfused with different concentrations of L-alanine. The experiment was carried out with a non-recirculating perfusion system. L-alanine was added to the perfusion medium so as to reach the indicated concentrations. Oxygen concentration in the liver outflow was determined with a Clark-type oxygen electrode and continuously recorded. The insert shows a double reciprocal plot of the data
Effects of Glucagon Administration in vivo. In order to find out whether the aforementioned effects of glucagon were an artifact due to the high substrate levels used (10 mM), the effect of glucagon was studied in vivo. As shown in Table 5, glucagon administration was accompanied by a rise in plasma glucose levels
and a decrease in the hepatic concentration of ketoacids; however, contrary to what has been observed (Table 2) in the isolated liver perfused with high substrate levels, the hepatic alanine concentration was found to decrease.
48
Pfltigers Arch. 370 (1977)
Table 5. Effect of glucagon administration in vivo to starved rats on the hepatic concentrations of alanine, pyruvate and oxaloacetate and plasma glucose levels. For experimental details see methods. Results are mean values of eight observations _+ SEM Control
Glucagon
nmoles/g dry wt Alanine Pyruvate Oxaloacetate Plasma glucose (raM)
3320 287 14 8.5
+_ 285 _+ 47 _+ 1.8 _+ 0.2
2150 159 8 11.3
+_ 300* +_ 28* _+ 1" • 0.4**
By t test * P < 0.05, ** P < 0.001
DISCUSSION The rise in the hepatic steady-state concentration of alanine brings about an increase in pyruvate and oxaloacetate and a decrease in c~-ketoglutarate. These results are in agreement with the fact that e-ketoglutarate is consumed during alanine transamination, and its initial rate of utilization probably exceeds its rate of formation from intramitochondrial glutamate deamination or its rate of transport across the mitochondrial membrane (Parrilla and Ayuso-Parrilla, 1976). The rise in the steady-state concentration of pyruvate and oxaloacetate must be the cause for the observed increase in the gluconeogenic flux, since the two gluconeogenic enzymic steps using these ketoacids, pyruvate carboxylase and phosphoenolpyruvate-carboxykinase, are characterized by having K m values for these substrates above their normal hepatic concentration. The effect of glucagon increasing glucose production in the perfused isolated rat liver was accompanied by an increase in the hepatic content of alanine and a rise in the intra-to extracellular ratio of the amino acid concentrations (Tables 2 and 4). However, this increase in alanine concentration was not followed by a further increase in the concentrations of pyruvate and oxaloacetate which, on the contrary, were found to decrease. This observation suggests that the effect of glucagon increasing glucose output could not possibly be simply the result of an accelerated rate of alanine transport into the hepatocyte, since, as stated above, the only way by which a rise in hepatic alanine content may increase glucose production is by raising the steady-state level of pyruvate and glucagon was found to decrease the concentration of this ketoacid (Table 2). The effect of glucagon decreasing the steady-state level of pyruvate is probably the result of a shift to a more reduced state of the nicotinamide nucleotide system brought about by the hormone (Williamson et al., 1969; Parrilla et al., 1975).
As mentioned the activity of pyruvate carboxylase responds readily to variations in pyruvate concentration within the physiological range. That is, the normal pyruvate concentration is close to the Km of pyruvate carboxylase for this intermediate. On this basis a decrease in hepatic pyruvate content in conjunction with an increase in flux through this enzyme step after glucagon stimulation (Table 1) can possibly be explained only if the enzyme was activated by the hormone. When pyruvate carboxylase was not activated a decrease in pyruvate levels should have resulted in a decrease rather than the observed increase in flux through this step. Consequently, the effect of glucagon increasing gluconeogenic flux does not seem to be related to a possible acceleration of the alanine entry into the hepatocyte. On the other hand the intracellular concentration of alanine in livers perfused with 10 mM L-alanine as substrate was found to be 8.5 mM, indicating that the system, according to the kinetics of saturation shown in Figure 1, was already oversaturated with substrate. Thus, although glucagon increased the intracellular concentration of alanine, this effect should not be expected to have any repercussion on the rate of glucose production. The experiments in vivo clearly seem to support the point of view expressed above. In this situation gluconeogenesis was significantly increased as judged by the rise in plasma glucose (Table 5); however, the hepatic steady-state levels of alanine decreased. Once again glucagon stimulation as in the isolated liver preparation was accompanied by a significant decrease in the pyruvate concentration. In summary, it seems reasonable to conclude that the metabolic effects of glucagon accelerating alanine transport and enhancing glucose synthesis are metabolic events apparently unrelated to each other. Acknowledgements. This work has been supported by grants from Comisi6n Asesora para el Desarrollo de la Investigaci6n Cientffica, Lilly Indiana de Espafia, S. A. and Essex Espafia. A.M.R. is a recipient of a fellowship from the Spanish Secretary of Science and Education. The authors wish to thank Mr. TomS~s Fontela and Mr. Fernando Martfn for skillful and devoted technical assistance.
REFERENCES Bergmeyer, H. U.: Methods of enzymatic analysis. New York: Academic Press 1965 Bromer, W. W., Chance, R. H. : Zinc glucagon depression of blood amino acids in rabbits. Diabetes 18, 748-754 (1969) Exton, J. H., Jefferson, L. S., Butcher, K. W., Park, C. R. : Gluconeogenesis in the perfused liver. The effects of fasting, alloxan diabetes, glucagon, epinephrine, adenosine 3'-5' monophosphate and insulin. Am. J. Med. 40, 709-715 (1966) Garcia, A., Williamson, J. R., Cahill, G. F., Jr.: Studies on the perfused rat liver. II. Effect of glucagon on gluconeogenesis. Diabetes 15, 188-193 (1966)
M. S. Ayuso-Parrilla et al. : Glucagon Stimulation of Hepatic Gluconeogenesis Goodman, M. N., Parrilla, R., Toews, C. J. : Influence of fluorocarbon emulsions on hepatic metabolism in perfused rat liver. Am. J. Physiol. 225, 1384-1388 (1973) Kerstens, P. J. : La perfusi6n du foie isol6e, pp. 26-38. Bruxelles: Edditions Arscia 1969 Krebs, H. A., Henseleit, K.: Untersuchungen fiber die Harnstoffbildung im Tierk6rper. Hoppe-Seylers Z. Physiol. Chem. 210, 33 - 6 6 (1932) Marliss, E. B., Aoki, T. T., Unger, R. H., Soeldner, J. S., Cahill, G. F., Jr.: Glucagon levels and metabolic effects in fasting man. J. Clin. Invest. 49, 2256-2270 (1970) Mallette, L. E., Exton, J. H., Park, C. R.: Control of gluconeogenesis from amino acids in the perfused rat liver. J. Biol. Chem.. 244, 5713 - 5723 (1969 a) Mallette, L. E., Exton, J. H., Park, C. R. : Effects of glucagon on amino acids transport and utilization in the perfused rat liver. J. Biol. Chem. 244, 5724-5728 (1969b) Miller, L.L.: Glucagon: A protein catabolic hormone in the isolated perfused rat liver. Nature 185, 248 (1960) Parrilla, R., Ayuso-Parrilla, M. S. : Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver. Pflfigers Arch. 362, 4 9 - 5 4 (1976) Parrilla, R., Goodman, M. N. : Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis. Biochem. J. 138, 341-348 (1974) Parrilla, R., Jim6nez, M. I., Ayuso-Parrilla, M. S. : Glucagon and insulin control of gluconeogenesis in the perfused isolated rat
49
liver. Effects on cellular metabolite distribution. Eur. J. Biochem. 56, 375-383 (1975) Ross, B. D., Hems, R., Krens, H. A. : The rate of gluconeogenesis from various precursors in the perfused rat liver. Biochem. J. 102, 942--951 (1967) Scholz, R.: In: Stoffwechsel der isoliert perfundierten Leber, pp. 25 - 31, Berlin-Heidelberg-New York: Springer 1969 Unger, R.H., Aguilar-Parada, E., Mtiller, W.A., Eisenstraut, A. M. : Studies of pancreatic alpha cell function in normal and diabetic subjects. J. Clin. Invest. 49, 837-848 (1970,) Williamson, D. H., Lopes-Vieira, D., Walker, B. : Concentrations of free gluconeogenic amino acids in livers of rats subjected to various metabolic stresses. Biochem. J. 104,497-502 (1967) Williamson, J. R., Browning, E. T., Thurman, R. G., Scholz, R. : Inhibition of glucagon effects in perfused rat liver by (+) decanoylcarnitine. J. Biol. Chem. 244, 50055- 50064 (1969) Williamson, J. R., Corkey, B. E. : Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. In: Methos of enzymology, vol. 13, pp. 434 (J. M. Lowenstein, ed.). New York: Academic Press 1968 Wollenberger, A., Ristau, O., Schoffa, G.: Eine einfache Technik der extrem schnellen Abktihlung gr613erer Gewebestiicke. Pfl/igers Arch. ges. Physiol. 270, 399-412 (1970)
Received March 22, 1977
Pflfigers Arch. 370, 4 5 - 4 9 (1977)
EuropeanJournal of Physio~3y 9 by Springer-Verlag 1977
On the Mechanism of Glucagon Stimulation of Hepatic Gluconeogenesis MATILDE S. AYUSO-PARRILLA, ANGELES MARTIN-REQUERO, and ROBERTO PARRILLA Department of Metabolism, Institute G. Marafi6n, C.S.I.C., Velazquez 144, Madrid-6, Spain
Summary. The addition of L-alanine as substrate to a perfused rat liver preparation produced a five-fold increase in the rate of glucose production. This enhancement of the gluconeogenic flux seems to be a consequence of a rise in the steady-state levels of pyruvate and oxaloacetate subsequent to the rise in alanine concentration. Glucagon (2x 10 -9 M) increased the gluconeogenic flux from alanine (10raM) by 50 percent, even though the concentration of the substrate in the perfusion fluid was at saturation. This effect was accompanied by a rise in the intracellular concentration of alanine. However, the steady-state concentrations of pyruvate and oxaloacetate were decreased, probably as a consequence of a more reduced state of the nicotinamide-nucleotide system. In vivo, the intraperitoneal administration of glucagon to starved rats was accompanied by a decrease in the hepatic alanine and pyruvate concentrations despite the striking effects raising the plasma glucose levels. These observations seem to indicate that the effect of the hormone increasing the hepatic glucose output must be mediated through some other mechanism(s) independent of the intracellular variations in the hepatic amino acids levels.
enous fuel mobilization and enhanced glucose production, like fasting or diabetes, display elevated plasma levels of glucagon (Unger et al., 1970; Marliss et al., 1970). Nevertheless, in spite of the physiological importance of glucagon in the glucose homeostasis, very little is known about its mechanism of action in the control of hepatic gluconeogenesis. The control of gluconeogenesis from amino acJLds is particularly important since amino acids seem to be the most important substrates for gluconeogenesis in vivo. Glucagon is known to decrease plasma amino acid levels in vivo (Bromer and Chance, 1969; Martin et al., 1976) and also to stimulate the hepatic uptake of some amino acids in vivo as well as in the perfused isolated rat liver (Mallette et al., 1969 b). On this basis, it has been suggested that glucagon might stimulate gluconeogenesis by accelerating the entry of amino acids into the hepatic cell and the attainment of a steady-state level of gluconeogenic substrates (Mallette et al., 1969b). The experiments reported here seem to indicate that the effects of glucose increasing amino acid transport and stimulating glucose synthesis are fully independent from each other.
Key words: Glucagon action - Hepatic gluconeogenesis - Perfused liver.
INTRODUCTION The pancreatic hormone glucagon is known to have gluconeogenic effects in vitro (Miller, 1960; Exton et al., 1966; Garcia et al., 1966; Ross et al., 1967; Mallette et al., 1969 a). It is also known that most of the in vivo situations characterized by an increased endogSend offprint requests to Ayuso-Parrilla at the above address
MATERIALS AND METHODS Male Wistar rats of 190 - 2 1 0 g body weight were used. The animals were purchased when approximately 120 g in weight and kept in our animal room under standard conditions until they weighed 200 g. They were starved for 2 4 - 36 h prior to their experimental use. The rats were anesthetized by an intraperitoneal injection of Nembutal (40 mg/kg body weight). The portal vein and inferior vena cava were cannulated in situ and then infused with a hemoglobin-free Krebs-Henseleit bicarbonate buffer (Krebs and Henseleit, 1932) containing 3 ~ fraction V bovine albumin. The albumin was previously dissolved in a small volume of bicarbonate buffer and dialyzed against two or three changes of the same buffer. The pH of the medium after equilibration with 95 ~ Oz: 5 ~ CO2 in the gaseous phase was 7.4. Prior to its use the medium
46 was filtered through Mill• membranes having 0.45 gm pore size. Each liver was perfused with 100 ml of recirculating medium. The perfusion apparatus was equipped with a disc oxygenator (Scholz, 1968; Kerstens, 1969), and the liver was in contact w~th oxygen (Clark type) and pH electrodes. The rate of circulation of the perfusion medium was 28 ml/min (Goodman et al., 1973). In all the experiments the livers were allowed to equilibrate by perfusing them for 30 min in the absence of any substrate; then, when indicated, alan• (10 raM) or L-alanine (10 raM) plus glucagon (2.1 x 10 9 M) were added, and the perfusion was continued for an additional period of 60 min. At this time the livers were immediatly frozen using aluminum clamps (Wollenberger et al., 1960) which had been precooled in liquid nitrogen. Representative portions of the livers were freeze-dried and extracted with 35 volumes (w/v) of 8 ~ perchloric acid. Acid extracts were brought to pH 6.0 with potassium carbonate and immediatly used for the fluorimetric measurement of oxaloacetate (Williamson and Corkey, 1968). Perfusate samples (0.5 ml) were taken every 10 rain, deproteinized in cold 6 ~ (w/v) perchloric acid and brought to pH 6.0 with potassium carbonate for the determination of glucose and alanine. Metabolic rates were calculated over the 6 0 - 9 0 min interval. Glucose, pyruvate, glutamate, aspartate and e-ketoglutarate were measured spectrophotometrically according to methods described previously (Bergmeyer, 1965). Atanine was determined with alanine dehydrogenase and NAD + in Tris-hydrazine buffer. In the in vivo experiments rats were anesthetized, and glucagon was infused intraperitoneally at a rate of 26 gg/min for the first 5 rain and of 10 gg/min thereafter. At 20 rain the abdomen was opened and liver biopsies were taken and processed as described above. Control animals were infused with the same volume of saline. Most of the reagents were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). Enzymes were purchased from Boehringer (Mannheim, Germany).
RESULTS
Effect of Alan• and Alanine plus Glucagon on Glucose Output and Steady-State Levels of Hepatic Ketoacids. Addition of L-alanine (10 mM) to a perfused isolated rat liver preparation produced an increase in the glucose output of more than five-fold (Table 1). This increase in glucose production was accompanied by a rise in the steady-state concentration of pyruvate and oxaloacetate and a decrease in e-ketoglutarate (Table 1). Since pyruvate carboxylase (E.C. 6.4.1.1) and phosphoenolpyruvate-carboxykinase (E.C. 4.1.1.32) respond readily to variations in the concentration of pyruvate and oxaloacetate within the physiological range, the increased glucose production from L-alanine should ultimately be interpreted as a result of the rise in the steady-state levels of pyruvate and oxaloacetate. Glucagon stimulated glucose production from 67 to 95 gmoles/100 g body weight/h; however, in contrast to the effect of L-alanine alone, pyruvate and oxaloacetate steady-state levels decreased.
Hepatic Steady-State Levels of Transaminase Reactants. The hepatic steady-state levels of the reactants of alan• (E.C. 2.6.1.2) and arpartate (E.C. 2.6.1.1) amino transferases in control and glucagon treated rats are shown in Tables 2 and 3. The equilibrium expres-
Pfltigers Arch. 370 (1977) Table 1. Effect of glucagon on the hepatic steady-state concentration of some ketoacids during gluconeogenesis from L-alan• Livers were perfused for 30 min without any substrate and then Lalanine and glucagon were added. The perfusion was continued for 60 min at the end of which the livers were freeze-clamped with aluminium tongs precooled in liquid nitrogen. Metabolic rates were calculated over the last 30-rain period of perfusion Additions to the perfusate .....
None
10 mM L-alanine
10 mM L-alanine + 2 x 10 .9 M glucagon
788 • 85 529 • 30 18 • 1.7
445 _+ 48 460 • 35 t3 _+ 0.8
67_+ 4
95 _+ 3
nmoles/g dry wt Pyruvate 106 • 12 e-ketoglutarate 1150 • 160 Oxaloacetate 13 • 3a Glucose production gmoles/100 g bodywt/h 12_+ 0.9
Calculated from the aspartate aminotransferase equilibrium reaction. Values are means of at least eight experiments _+ standard error
sions for both reactions remained virtually unchanged in both instances in spite of significant changes in the concentrations of their reactants. This observation indicates once more (Williamson et al., 1967; Parrilla and Goodman, 1974) that the hepatic transaminating reactions are near equilibrium and then the variations in the concentration of ketoacids are largely dependent of the variations in the amino acid supply. However, factors other than the steady-state concentration of amino acids must be playing an important role since glucagon, in spite of increasing concentration of alanine (Table 2), was unable to raise the concentration of pyruvate. The equilibrium was maintained in this situation by a rise in the glutamate levels.
Intracellular Levels of L-Alanine in Control and Glucagon-Treated Livers. As shown in Table 4 the rat liver is able to accumulate alan• against a gradient as its intracellular concentration was found to be more than twice that of the perfusion fluid. Glucagon raised the intracellular concentration of alanine by more than 20 ~ and the ratio of intra- to extracellular concentrations was increased from 2.4 to 3.2. This finding prompts the question whether such an increase in the intracellular concentrations of amino acids was responsible for the increased gluconeogenic flux. In order to elucidate this point the relationship between alan• supply and hepatic metabolic activity was studied. As may be seen in Figure 1 the metabolic activity of the liver, as reflected by the rates of oxygen utilization, displayed saturation type kinetics, being saturated at alanine concentrations over 2 mM. The apparent "Kin" was found to be 1.1 mM. Similar results were obtained by plotting alanine concentration
M. S. Ayuso-Parrilta et al. : Glucagon Stimulation of Hepatic Gluconeogenesis
47
Table 2. Effect of glucagon on the hepatic concentration of the alanine aminotransferase reactants.The experimental details were the same as described in Table 1. Each value is the mean of at least eight experiments _+ standard error Additions to the perfusate
L-Alanine
c~-Ketoglutarate
Pyruvate
Glutamate
[Pyruvate] [Glutamate] [L-Alanine] [c~-Ketoglutarate]
nmoles/g dry wt Alanine (10 mM) Alanine (10 raM) + Glucagon (2x 10 -9 M)
45955 _+ 4083
529 _+ 30
788 + 85
10172 _+ 960
3.03
54047 _+ 4059
460 _+ 35
445 _+ 48
12090 _+ 895
4.62
Table 3. Effect of glucagon on the hepatic concentration of the aspartate aminotransferase reactants. The experimental details were the same as described in Table 1. Each figure represents the average value of at least eight experiments + standard error Additions to the perfusate
c~-Ketoglutarate
Aspartate
Glutamate
[ct-Ketoglutarate] [Aspartate]
Oxaloacetate
[Glutamate] [Oxaloacetate]
nmotes/g dry wt L-Alanine (10 raM) L-Alanine (10 raM) + Glucagon (2 x 10 -9 M)
529 + 30
13075 _+ 1290
10172 _+ 960
18 + 1.7
37.6
460 + 35
14682 _+ 2223
12090 + 895
13 + 0.8
42.4
16
Table 4. Effect of glucagon on the intracellular concentration of L-alanine in isolated rat livers perfused with L-alanine (10 raM) as a substrate. The experiments were carried out as described in Table I. For the calculation of the hepatic intracellular concentration of L-alanine, the extracellular water content was taken to be 0.2 ml/g wet weight (Mallette et al., 1969a), and the ratio of dry to wet liver weights 4.94 _+ 0.06, the mean of 87 determinations + standard error Additions to the perfusate
L-Alanine concentration Intracellular
Ratio IN/OUT
Da" 0~ z
Perfusion fluid
I
o !
3.5
2.4
3.2
3.2
~
/
/. 4 I
o,//
/
27 r
o
versus glucose output. The oxygen uptake, nevertheless, offers the advantage of comprising the energy expenditure not only for glucose synthesis but also for other biosynthetic processes enhanced by the substrate, including the urea production.
o?
0
mM L-Alanine (10 mM) 8.5 L-Alanine (10 raM) + Glucagon (2 x 10-9 M) 10.3
9~
14
o
; I/[ALANINE]
,'o
', [ALANINE] m M
Fig. 1. Rates of oxygen uptake by the isolated rat liver perfused with different concentrations of L-alanine. The experiment was carried out with a non-recirculating perfusion system. L-alanine was added to the perfusion medium so as to reach the indicated concentrations. Oxygen concentration in the liver outflow was determined with a Clark-type oxygen electrode and continuously recorded. The insert shows a double reciprocal plot of the data
Effects of Glucagon Administration in vivo. In order to find out whether the aforementioned effects of glucagon were an artifact due to the high substrate levels used (10 mM), the effect of glucagon was studied in vivo. As shown in Table 5, glucagon administration was accompanied by a rise in plasma glucose levels
and a decrease in the hepatic concentration of ketoacids; however, contrary to what has been observed (Table 2) in the isolated liver perfused with high substrate levels, the hepatic alanine concentration was found to decrease.
48
Pfltigers Arch. 370 (1977)
Table 5. Effect of glucagon administration in vivo to starved rats on the hepatic concentrations of alanine, pyruvate and oxaloacetate and plasma glucose levels. For experimental details see methods. Results are mean values of eight observations _+ SEM Control
Glucagon
nmoles/g dry wt Alanine Pyruvate Oxaloacetate Plasma glucose (raM)
3320 287 14 8.5
+_ 285 _+ 47 _+ 1.8 _+ 0.2
2150 159 8 11.3
+_ 300* +_ 28* _+ 1" • 0.4**
By t test * P < 0.05, ** P < 0.001
DISCUSSION The rise in the hepatic steady-state concentration of alanine brings about an increase in pyruvate and oxaloacetate and a decrease in c~-ketoglutarate. These results are in agreement with the fact that e-ketoglutarate is consumed during alanine transamination, and its initial rate of utilization probably exceeds its rate of formation from intramitochondrial glutamate deamination or its rate of transport across the mitochondrial membrane (Parrilla and Ayuso-Parrilla, 1976). The rise in the steady-state concentration of pyruvate and oxaloacetate must be the cause for the observed increase in the gluconeogenic flux, since the two gluconeogenic enzymic steps using these ketoacids, pyruvate carboxylase and phosphoenolpyruvate-carboxykinase, are characterized by having K m values for these substrates above their normal hepatic concentration. The effect of glucagon increasing glucose production in the perfused isolated rat liver was accompanied by an increase in the hepatic content of alanine and a rise in the intra-to extracellular ratio of the amino acid concentrations (Tables 2 and 4). However, this increase in alanine concentration was not followed by a further increase in the concentrations of pyruvate and oxaloacetate which, on the contrary, were found to decrease. This observation suggests that the effect of glucagon increasing glucose output could not possibly be simply the result of an accelerated rate of alanine transport into the hepatocyte, since, as stated above, the only way by which a rise in hepatic alanine content may increase glucose production is by raising the steady-state level of pyruvate and glucagon was found to decrease the concentration of this ketoacid (Table 2). The effect of glucagon decreasing the steady-state level of pyruvate is probably the result of a shift to a more reduced state of the nicotinamide nucleotide system brought about by the hormone (Williamson et al., 1969; Parrilla et al., 1975).
As mentioned the activity of pyruvate carboxylase responds readily to variations in pyruvate concentration within the physiological range. That is, the normal pyruvate concentration is close to the Km of pyruvate carboxylase for this intermediate. On this basis a decrease in hepatic pyruvate content in conjunction with an increase in flux through this enzyme step after glucagon stimulation (Table 1) can possibly be explained only if the enzyme was activated by the hormone. When pyruvate carboxylase was not activated a decrease in pyruvate levels should have resulted in a decrease rather than the observed increase in flux through this step. Consequently, the effect of glucagon increasing gluconeogenic flux does not seem to be related to a possible acceleration of the alanine entry into the hepatocyte. On the other hand the intracellular concentration of alanine in livers perfused with 10 mM L-alanine as substrate was found to be 8.5 mM, indicating that the system, according to the kinetics of saturation shown in Figure 1, was already oversaturated with substrate. Thus, although glucagon increased the intracellular concentration of alanine, this effect should not be expected to have any repercussion on the rate of glucose production. The experiments in vivo clearly seem to support the point of view expressed above. In this situation gluconeogenesis was significantly increased as judged by the rise in plasma glucose (Table 5); however, the hepatic steady-state levels of alanine decreased. Once again glucagon stimulation as in the isolated liver preparation was accompanied by a significant decrease in the pyruvate concentration. In summary, it seems reasonable to conclude that the metabolic effects of glucagon accelerating alanine transport and enhancing glucose synthesis are metabolic events apparently unrelated to each other. Acknowledgements. This work has been supported by grants from Comisi6n Asesora para el Desarrollo de la Investigaci6n Cientffica, Lilly Indiana de Espafia, S. A. and Essex Espafia. A.M.R. is a recipient of a fellowship from the Spanish Secretary of Science and Education. The authors wish to thank Mr. TomS~s Fontela and Mr. Fernando Martfn for skillful and devoted technical assistance.
REFERENCES Bergmeyer, H. U.: Methods of enzymatic analysis. New York: Academic Press 1965 Bromer, W. W., Chance, R. H. : Zinc glucagon depression of blood amino acids in rabbits. Diabetes 18, 748-754 (1969) Exton, J. H., Jefferson, L. S., Butcher, K. W., Park, C. R. : Gluconeogenesis in the perfused liver. The effects of fasting, alloxan diabetes, glucagon, epinephrine, adenosine 3'-5' monophosphate and insulin. Am. J. Med. 40, 709-715 (1966) Garcia, A., Williamson, J. R., Cahill, G. F., Jr.: Studies on the perfused rat liver. II. Effect of glucagon on gluconeogenesis. Diabetes 15, 188-193 (1966)
M. S. Ayuso-Parrilla et al. : Glucagon Stimulation of Hepatic Gluconeogenesis Goodman, M. N., Parrilla, R., Toews, C. J. : Influence of fluorocarbon emulsions on hepatic metabolism in perfused rat liver. Am. J. Physiol. 225, 1384-1388 (1973) Kerstens, P. J. : La perfusi6n du foie isol6e, pp. 26-38. Bruxelles: Edditions Arscia 1969 Krebs, H. A., Henseleit, K.: Untersuchungen fiber die Harnstoffbildung im Tierk6rper. Hoppe-Seylers Z. Physiol. Chem. 210, 33 - 6 6 (1932) Marliss, E. B., Aoki, T. T., Unger, R. H., Soeldner, J. S., Cahill, G. F., Jr.: Glucagon levels and metabolic effects in fasting man. J. Clin. Invest. 49, 2256-2270 (1970) Mallette, L. E., Exton, J. H., Park, C. R.: Control of gluconeogenesis from amino acids in the perfused rat liver. J. Biol. Chem.. 244, 5713 - 5723 (1969 a) Mallette, L. E., Exton, J. H., Park, C. R. : Effects of glucagon on amino acids transport and utilization in the perfused rat liver. J. Biol. Chem. 244, 5724-5728 (1969b) Miller, L.L.: Glucagon: A protein catabolic hormone in the isolated perfused rat liver. Nature 185, 248 (1960) Parrilla, R., Ayuso-Parrilla, M. S. : Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver. Pflfigers Arch. 362, 4 9 - 5 4 (1976) Parrilla, R., Goodman, M. N. : Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis. Biochem. J. 138, 341-348 (1974) Parrilla, R., Jim6nez, M. I., Ayuso-Parrilla, M. S. : Glucagon and insulin control of gluconeogenesis in the perfused isolated rat
49
liver. Effects on cellular metabolite distribution. Eur. J. Biochem. 56, 375-383 (1975) Ross, B. D., Hems, R., Krens, H. A. : The rate of gluconeogenesis from various precursors in the perfused rat liver. Biochem. J. 102, 942--951 (1967) Scholz, R.: In: Stoffwechsel der isoliert perfundierten Leber, pp. 25 - 31, Berlin-Heidelberg-New York: Springer 1969 Unger, R.H., Aguilar-Parada, E., Mtiller, W.A., Eisenstraut, A. M. : Studies of pancreatic alpha cell function in normal and diabetic subjects. J. Clin. Invest. 49, 837-848 (1970,) Williamson, D. H., Lopes-Vieira, D., Walker, B. : Concentrations of free gluconeogenic amino acids in livers of rats subjected to various metabolic stresses. Biochem. J. 104,497-502 (1967) Williamson, J. R., Browning, E. T., Thurman, R. G., Scholz, R. : Inhibition of glucagon effects in perfused rat liver by (+) decanoylcarnitine. J. Biol. Chem. 244, 50055- 50064 (1969) Williamson, J. R., Corkey, B. E. : Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. In: Methos of enzymology, vol. 13, pp. 434 (J. M. Lowenstein, ed.). New York: Academic Press 1968 Wollenberger, A., Ristau, O., Schoffa, G.: Eine einfache Technik der extrem schnellen Abktihlung gr613erer Gewebestiicke. Pfl/igers Arch. ges. Physiol. 270, 399-412 (1970)
Received March 22, 1977
