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Possible role of pyruvate kinase in the hormonal control of dihydroxyacetone gluconeogenesis in isolated hepatocytes
Possible Role of Pyruvate Kinase in the Hormonal Control of Dihydroxyacetone Gluconeogenesis in Isolated Hepatocytes S. J. Pilkis, T. H. Claus, J. P. Riou, and C. R. Park
H E SITE of action of glucagon on the gluconeogenic pathway remains ununknown. Evidence from the laboratory of Exton and Park pointed to a site somewhere between pyruvate and phosphoenolpyruvate. 1 More recently, attention has been focused on the nonmitochondrial portion of the pathway. Glucagon has been shown to stimulate gluconeogenesis from dihydroxyacetone, fructose, and galactose and evidence suggested that the phosphofructokinase-fructose diphosphatase enzymes may be the site of action of the hormone. a3 Another possible site of glucagon action, which has not been carefully studied, is the pyruvate kinase step. Inhibition of this enzyme by glucagon would reduce the amount of phosphoenolpyruvate that is converted to pyruvate and would favor the formation of glucose. We have chosen dihydroxyacetone as the substrate with which to study the effects of glucagon on both glucose synthesis and flux through pyruvate kinase in isolated hepatocytes. Changes in flux through pyruvate kinase were evaluated by measuring changes in the amount of lactate produced. We have also investigated the effect of glucagon on the enzyme activity of hepatocyte pyruvate kinase.
T
METHODS
Isolated hepatocytes from fed or starved rats were prepared by a modification of the method of Berry and Friend 4 that has been described in detail. 5 9 Glucose synthesis was determined by measuring the conversion of [U-14C] dihydroxyacetone to labeled glucose. 5,6 Lactate production was measured either chemically or by separation of labeled lactate by ion exchange chromatography? Chemical determinations of various intermediary metabolites were performed as previously described 6 9 by published procedures. L~ Pyruvate kinase activity was assayed by the method of Llorente et al. II RESULTS AND DISCUSSION
Glucagon stimulated the conversion of lmM[U-~4C] dihydroxyacetone to glucose by twofold in hepatocytes from fed rats (Table 1). However, glucagon had no significant effect on the uptake of dihydroxyacetone. These data suggest that glucagon alters the distribution of dihydroxyacetone carbon to allow greater conversion to glucose. The effect of glucagon concentration on the metabolism of 1.5 m M [U-~4C] dihydroxyacetone in hepatocytes from fed rats is shown in Fig. l. Essentially From the Department of Physiology, Vanderbilt University Medical School, Nashville, Tenn. Reprint requests should be addressed to Dr. Simon Pilkis, Department of Physiology, Vanderbilt University, School of Medicine, Nashville, Tenn. 37232. 9 1976 by Grune & Stratton, Inc. Metabolism, Vol. 25, No. 11, Suppl. 1 (November), 1976
1355
1356
PILKIS ET AL.
Table 1. Effect of Glucagon and Ethanol on Dihydroxyacetone Metabolism in Hepatocytes From Fed and Starved Rats Production of 14C.Glucos e 14C_Lactate #moles [U-14C]-substrate converted to product/15 min/rng DNA
Source of Cells and Incubation Conditions
Dihydroxyacetone Utilized #moles/15 min/mg DNA
Fed rats 5.9 4- 0.4
7.0 • 0.4
13.3 • 1.9
Glucagon Ethanol
Basal
11.1 • 0.5 8.6 ~- 0.3
3.4 • 0.2 3.1 • 0.2
14.7 • 2.4 I
Ethanol & Glucagon
10.1 • 0.9
2.4 i 0.02
--
8.8 • .7 10.1 • .9
1.7 4- .2 1.2 4:.9
Starved 96 hr Basal Glucose
10.0 4_ 3.0 11.4 -~ 2.4
Hepatocytes from fed and 96-hr starved rats were incubated far 15 min with 1 mM [U-14C]-dihydroxyace tone with and without 10 nM glucagon or 8 mM ethanol, 14C-glucose and 14C-lactate as well as chemical dihydroxyacetone were determined in neutralized extracts as described in methods. The data represent the mean ~k S.E.M. from four cell preparations.
equal amounts of dihydroxyacetone are converted to glucose and lactate in the absence of hormone. The stimulation by glucagon of labeled glucose synthesis could in large part be accounted for by diminished labeled lactate production. The concentration of glucagon that gave a half-maximally effective inhibition of labeled lactate production was about 0.2 nM. The same concentration gave a half-maximal stimulation of glucose synthesis. The large decrease in lactate proI
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GLUCAGON(M) Fig. 1. Effect of glucagon on the conversion of 14C-dihydroxyacetone, 14C-lactate, and 14Cglucose in hepatocytes from fed rats. Cells (50 pg DNA/ml) were incubated with 1.5 mM [U-14C] dihydroxyacetone as described in Table 1. 14C-lactate production was determined by ion-exchange chromatography of the neutralized extract as described in the Materials and Methods section. The data represent # moles of labeled dihydroxyacetone converted to either 14C-glucose or 14C-lactate. The values represent the mean • SE. of three observations from a single cell preparation.
PYRUVATE KINASE
1357
duced from dihydroxyacetone in cells from fed rats, upon addition of glucagon, suggested that the hormone may affect flux of carbon through pyruvate kinase. The glucagon stimulation of dihydroxyacetone gluconeogenesis does not require the resynthesis of phosphoenolpyruvate from pyruvate, 12,13 since several inhibitors of lactate and pyruvate metabolism did not prevent the action of the hormone (Table 2). If glucagon affects flux through pyruvate kinase, one might expect a diminished effect of glucagon in situations where flux through the enzyme is low. It has been reported that starvation for 48-96 hr greatly depresses the level of hepatic pyruvate kinase. ~4Starvation for 96 hr led to a doubling of glucose production and to a decrease in lactate production. Glucagon had no significant effect on glucose or lactate production or dihydroxyacetone uptake (Table 1). Addition of 8 mM ethanol stimulated dihydroxyacetone gluconeogenesis and suppressed labeled lactate production (Table 1). Glucagon, in the~ presence of ethanol, had only minimal effects on glucose synthesis and lactate production. These results are probably due to ethanol providing reducing equivalents to the triose phosphate dehydrogenase and thus preventing conversion of dihydroxyacetone to lactate. Instead the substrate is directed toward glucose synthesis. The effects of glucagon and ethanol are not additive, suggesting that both agents act to stimulate glucose synthesis from dihydroxyacetone by preventing lactate production. The effect of 10 nM glucagon on the levels of glycolytic intermediates in cells from fed rats incubated with 2 mM dihydroxyacetone is shown in Fig. 2. The increase in phosphoenol pyruvate and the decrease in pyruvate, along with the decreased flux to lactate, suggest that glucagon inhibited pyruvate kinase. There was also a decrease in fructose diphosphate and an increase in fructose-6phosphate. This, along with the increased flux to glucose suggests that the hormone also stimulated fructose diphosphatase and/or inhibited phosphofructokinase. Similar changes have been reported by Lardy's group. 12 It is known that insulin suppresses glucagon stimulated lactate and alanine gluconeogenesis in isolated hepatocytes.9 Table 3 shows the effect of insulin Table 2. Effect of Inhlbitors of LQctate and Pyruvate Metabolism on the Ability of Glucagon to Stimulate Dihydroxyacetone Gluconeogenesis DihydroxyacetoneGluconeogenesis # moles[U~14C] dihydroxyacetoneconverted to g!ucose/15 min/mg DNA Inhibitor None (5) Hydrazine (2 mM) (4) o~-Cyanocinnamate (0,125 mM) (1) *Tryptophan (2 mM) (4) Aminoxyacetate (0.25 mM) (2)
Basal 3.4 3.2 2.6 3.7 3.2
~ ~ ~: :k :~
0.1 0.3 0.4 0.2 0.2
Glucagon 7.5 6.0 7.5 7.4 7.4
• i • • •
0.5 0.5 0.7 0.5 0.4
*Cells were preincubated with tryptophan for 30 rain. Hepatocytes from fed rats were incubated with 1 mM IU-14C] dihydroxyacetone as described in Table 1. Conversion of labeled dihydroxyacetone to labeled glucose was determined as described in the Methods section. The number in parenthesis indicates the number of cell preparations tested with each inhibitor. Glucagon concentration was 10 nM. All the inhibitors tested were found to inhibit glucogenesis from 2 mM lactate by 80% or more. No lactate glucogenesis was detectable with 2 mM hydrozine.
1358
PILKIS ET AL
2raM DI HYDROXYACETONE
160 140
g~ ~-- 120-
AGON
u_ I00 O
Fig. 2. Effect of glucagon on the levels of glycolytic intermediates in hepatocytes from fed rats. Hepatocytes from fed rats (600 #g D N A / 6 ml) were incubated for 15 min with 2 mM dihydroxyacetone in the absence and presence of 10 nM glucagon. Metabo|ites were determined by published enzymatic procedures 13 in neutralized extracts.
806040-
LAC
PYR
PEP DHAP FI)P F6P
GIP
GI'U
(10 nM) on conversion of 1 mM[U-14C] dihydroxyacetone to either labeled glucose or lactate in isolated hepatocytes from fed rats. Insulin had no significant effect on the basal rate of glucose or lactate synthesis. However, when added with 0.4 n M glucagon, insulin suppressed the synthesis of 14C-glucose and stimulated the production of ~4C-lactate. The suppression of glucose synthesis by the hormone could be accounted for by the increased lactate production. Since the effect of glucagon to stimulate dihydroxyacetone gluconeogenesis does not involve mitochondrial steps, it seems reasonable to postulate an extramitochondrial site for the action of insulin. The ability of insulin to stimulate lactate production suggests that this site may be the pyruvate kinase step. These results suggest that alterations in flux through pyruvate kinase are important in the action of glucagon on dihydroxyacetone gluconeogenesis in isolated hepatocytes. Evidence for glucagon actually affecting pyruvate kinase activity is shown in Fig. 3. A 15,000-supernatant fraction in 20 m M phosphate40% (v/v) glycerol buffer was prepared from cells from fed rats incubated with various glucagon concentrations. The enzyme activity was measured with 0.4 m M phosphoenolpyruvate as substrate in the absence and presence of 20 # M fructose diphosphate, an allosteric activator of the enzyme. In the absence of the activator there was a dose-dependent inhibition of activity. Clear inhibitory effects were seen with l n M glucagon and maximal concentrations inTable 3. Effect of Insulin on the Production of Glucose and Lactate from Dihydroxyacetone in Hepatocytes From Fed Rats Production of
14C.Glucose Condition Basal insulin, 10 Glucagon, Glucagon, Glucagon,
nM 0.4 nM 0.4 nM and insulin, 10 nM 10 nM
14C-Lactate
# motes[ U- 14C] dihydroxyacetone converted to product/15 min/mg DNA 4.8 • .4
7.9 • .4
4.3 7.8 6.3 9.4
7.9 5.1 6.3 4.2
• • • •
.2 .4 .3 .2
• • • •
.4 .2 .06 .1
Hepatocytes from fed rats were incubated with 1 mM [U-14C] dihydroxyacetone as described in Table 1. Labeled glucose and lactate were determined as described in the Methods section.
PYRUVATE KINASE
1359
120Fig. 3. Effect of glucagon concentration on pyruvate kinase activity in isolated hepatocytes from fed rats. Hepatocytes from fed rats ( 2 0 0 # g D N A / 5 rot) w e r e incubated with various concentrations of glucagon for 10 min. The cells were then rapidly sedimented and suspended in 1 ml of ice-cold 20 mM sodium phosphate4 0 % glycerol buffer, pH 7.8. The cells were broken with an Ultraturrax Homogenizer | (Tekmar Company, Cincinnati, Ohio) and a 15,000 xg supernatant fraction prepared. Pyruvate kinase activity in the absence or presence of 20 #M fructose diphosphate was estimated by the method of Llorente et al.11
z i~
IO0-
n-
80-
1~ ~_
60-
~
40-
;~ =
20-
a.
r--c/
o
;
~b
io'o
GLUCAGON, nM
h i b i t e d as m u c h as 7 0 ~ . T h e s e effects c a n b e seen w i t h i n 2 m i n a f t e r t h e a d d i t i o n o f h o r m o n e s . In t h e p r e s e n c e o f f r u c t o s e d i p h o s p h a t e o r w i t h h i g h p h o s p h o e n o l p y r u v a t e (>_ 2 r a M ) c o n c e n t r a t i o n s , g l u c a g o n h a d little o r n o effect. A l l o f t h e p r e c e d i n g r e s u l t s s u g g e s t t h a t t h e effect o f g l u c a g o n i n v o l v e s a l t e r a t i o n o f p y r u v a t e k i n a s e a c t i v i t y w h i c h m a y be m e d i a t e d b y a fall in f r u c t o s e d i p h o s p h a t e levels ( F i g . 2; B l a i r et al. 12 a n d / o r by m o d i f i c a t i o n o f t h e e n z y m e a c t i v i t y by p h o s p h o r y l a t i o n J 7 T h e s e p o s s i b i l i t i e s a r e c u r r e n t l y u n d e r i n v e s t i g a t i o n . Inh i b i t i o n o f p y r u v a t e k i n a s e a c t i v i t y by g l u c a g o n h a s b e e n r e p o r t e d by o t h e r s , m5,~6
REFERENCES
1. Exton JH, Park CR: Control of gluconeogenesis in liver. 111. Effects of L-lactate, pyruvate, fructose, glucagon, epinephrine, and adenosine 3',5'-monophosphate on gluconeogenic intermediates in the perfused rat liver. J Biol Chem 244:1424-1432, 1969 2. Kneer NM, Bosch AL, Clark MG, Lardy HA: Glucose inhibition of epinephrine stimulation of hepatic gluconeogenesis by blockade of the a-receptor function. Proc Natl Acad Sci USA 71:4523 4527, 1974 3. Clark MG, Kneer NM, Bosch AL, Lardy HA: The fructose 1,6-diphosphatase-phosphofructokinase substrate cycle. A site of regulation of hepatic gluconeogenesis by glucagon. J Biol Chem 249:5695--5703, 1974 4. Berry MN, Friend DS: High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structure study. J Cell Bio143:506-520, 1969 5. Pilkis SJ, Claus TH: Stimulation of dihydroxyacetone and glycerol gluconeogenesis by g[ucagon in isolated hepatocytes. Fed Proc, 1976. American Society of Biological Chemists, San Francisco
6. Pilkis SH, Claus TH: Hormonal control of [14C] glucose synthesis from [U-14C] dihydroxyacetone and glycerol in isolated hepatocytes. J Biol Chem (in press) 7. Claus TH, Pitkis SJ, Park CR: Stimulation by glucagon of the incorporation of U-14Clabeled substrates into glucose by isolated hepatocytes from fed rats. Biochim Biophys Acta 404:110-123, t975 8. Pilkis SJ, Claus TH, Johnson RA, Park CR: Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats. J Biol Chem 250:6328-6336, 1975 9. Claus TH, Pilkis SJ: Regulation by insulin of gluconeogenesis in isolated rat hepatocytes. Biochim Biophys Acta 421:246-262, 1976 10. Bergmeyer HU (ed): Methods of Enzymatic Analysis. Third Edition. vol. 1-4, New York, Academic Press, 1974 I 1. Llorente P, Marco R, Sols A: Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads. Eur J Biochem 13:45 54, 1970 12. Blair JB, Cooke DE, Lardy HA: In-
1360
fluence of glucagon on the metabolism of xylitol and dihydroxyacetone in the isolated perfused rat liver. J Biol Chem 248:3601-3607, 1973 13. Veneziale CM: Gluconeogenesis from Dglyceraldehyde and dihydroxyacetone in isolated rat liver. Stimulation by glucagon. Biochemistry t1:3286-3289, 1972 14. Tanaka T, Harano Y, Sue F, Morimura H: Crystallization,:characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J Biochem (Japan) 62:71-91, 1967 15. Cimbala M, Blair JB: Modification of rat
PILKIS ET AL.
hepatic pyruvate kinase by glucagon and cyclic AMP. Fed Proc 34:618, 1975 16. Taunton OD, Stefel FB, Greene HI-, Herman RH: Rapid reciprocal changes in rat hepatic glycolytic enzyme and fructose diphosphatase activities following insulin and glucagon injection. Biochem Biophys Res Comm 48:1663-1670, 1972 17. Ekman P, Dahlqvist U, Humble E, Engstrom L: Comparative kinetic studies on the L type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3',5'-AMP stimulated protein kinase. Biochem Biophys Acta 429:374 382, 1976
H E SITE of action of glucagon on the gluconeogenic pathway remains ununknown. Evidence from the laboratory of Exton and Park pointed to a site somewhere between pyruvate and phosphoenolpyruvate. 1 More recently, attention has been focused on the nonmitochondrial portion of the pathway. Glucagon has been shown to stimulate gluconeogenesis from dihydroxyacetone, fructose, and galactose and evidence suggested that the phosphofructokinase-fructose diphosphatase enzymes may be the site of action of the hormone. a3 Another possible site of glucagon action, which has not been carefully studied, is the pyruvate kinase step. Inhibition of this enzyme by glucagon would reduce the amount of phosphoenolpyruvate that is converted to pyruvate and would favor the formation of glucose. We have chosen dihydroxyacetone as the substrate with which to study the effects of glucagon on both glucose synthesis and flux through pyruvate kinase in isolated hepatocytes. Changes in flux through pyruvate kinase were evaluated by measuring changes in the amount of lactate produced. We have also investigated the effect of glucagon on the enzyme activity of hepatocyte pyruvate kinase.
T
METHODS
Isolated hepatocytes from fed or starved rats were prepared by a modification of the method of Berry and Friend 4 that has been described in detail. 5 9 Glucose synthesis was determined by measuring the conversion of [U-14C] dihydroxyacetone to labeled glucose. 5,6 Lactate production was measured either chemically or by separation of labeled lactate by ion exchange chromatography? Chemical determinations of various intermediary metabolites were performed as previously described 6 9 by published procedures. L~ Pyruvate kinase activity was assayed by the method of Llorente et al. II RESULTS AND DISCUSSION
Glucagon stimulated the conversion of lmM[U-~4C] dihydroxyacetone to glucose by twofold in hepatocytes from fed rats (Table 1). However, glucagon had no significant effect on the uptake of dihydroxyacetone. These data suggest that glucagon alters the distribution of dihydroxyacetone carbon to allow greater conversion to glucose. The effect of glucagon concentration on the metabolism of 1.5 m M [U-~4C] dihydroxyacetone in hepatocytes from fed rats is shown in Fig. l. Essentially From the Department of Physiology, Vanderbilt University Medical School, Nashville, Tenn. Reprint requests should be addressed to Dr. Simon Pilkis, Department of Physiology, Vanderbilt University, School of Medicine, Nashville, Tenn. 37232. 9 1976 by Grune & Stratton, Inc. Metabolism, Vol. 25, No. 11, Suppl. 1 (November), 1976
1355
1356
PILKIS ET AL.
Table 1. Effect of Glucagon and Ethanol on Dihydroxyacetone Metabolism in Hepatocytes From Fed and Starved Rats Production of 14C.Glucos e 14C_Lactate #moles [U-14C]-substrate converted to product/15 min/rng DNA
Source of Cells and Incubation Conditions
Dihydroxyacetone Utilized #moles/15 min/mg DNA
Fed rats 5.9 4- 0.4
7.0 • 0.4
13.3 • 1.9
Glucagon Ethanol
Basal
11.1 • 0.5 8.6 ~- 0.3
3.4 • 0.2 3.1 • 0.2
14.7 • 2.4 I
Ethanol & Glucagon
10.1 • 0.9
2.4 i 0.02
--
8.8 • .7 10.1 • .9
1.7 4- .2 1.2 4:.9
Starved 96 hr Basal Glucose
10.0 4_ 3.0 11.4 -~ 2.4
Hepatocytes from fed and 96-hr starved rats were incubated far 15 min with 1 mM [U-14C]-dihydroxyace tone with and without 10 nM glucagon or 8 mM ethanol, 14C-glucose and 14C-lactate as well as chemical dihydroxyacetone were determined in neutralized extracts as described in methods. The data represent the mean ~k S.E.M. from four cell preparations.
equal amounts of dihydroxyacetone are converted to glucose and lactate in the absence of hormone. The stimulation by glucagon of labeled glucose synthesis could in large part be accounted for by diminished labeled lactate production. The concentration of glucagon that gave a half-maximally effective inhibition of labeled lactate production was about 0.2 nM. The same concentration gave a half-maximal stimulation of glucose synthesis. The large decrease in lactate proI
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g.~
o,s
0.4
I---J
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~
5~
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/
!
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GLUCAGON(M) Fig. 1. Effect of glucagon on the conversion of 14C-dihydroxyacetone, 14C-lactate, and 14Cglucose in hepatocytes from fed rats. Cells (50 pg DNA/ml) were incubated with 1.5 mM [U-14C] dihydroxyacetone as described in Table 1. 14C-lactate production was determined by ion-exchange chromatography of the neutralized extract as described in the Materials and Methods section. The data represent # moles of labeled dihydroxyacetone converted to either 14C-glucose or 14C-lactate. The values represent the mean • SE. of three observations from a single cell preparation.
PYRUVATE KINASE
1357
duced from dihydroxyacetone in cells from fed rats, upon addition of glucagon, suggested that the hormone may affect flux of carbon through pyruvate kinase. The glucagon stimulation of dihydroxyacetone gluconeogenesis does not require the resynthesis of phosphoenolpyruvate from pyruvate, 12,13 since several inhibitors of lactate and pyruvate metabolism did not prevent the action of the hormone (Table 2). If glucagon affects flux through pyruvate kinase, one might expect a diminished effect of glucagon in situations where flux through the enzyme is low. It has been reported that starvation for 48-96 hr greatly depresses the level of hepatic pyruvate kinase. ~4Starvation for 96 hr led to a doubling of glucose production and to a decrease in lactate production. Glucagon had no significant effect on glucose or lactate production or dihydroxyacetone uptake (Table 1). Addition of 8 mM ethanol stimulated dihydroxyacetone gluconeogenesis and suppressed labeled lactate production (Table 1). Glucagon, in the~ presence of ethanol, had only minimal effects on glucose synthesis and lactate production. These results are probably due to ethanol providing reducing equivalents to the triose phosphate dehydrogenase and thus preventing conversion of dihydroxyacetone to lactate. Instead the substrate is directed toward glucose synthesis. The effects of glucagon and ethanol are not additive, suggesting that both agents act to stimulate glucose synthesis from dihydroxyacetone by preventing lactate production. The effect of 10 nM glucagon on the levels of glycolytic intermediates in cells from fed rats incubated with 2 mM dihydroxyacetone is shown in Fig. 2. The increase in phosphoenol pyruvate and the decrease in pyruvate, along with the decreased flux to lactate, suggest that glucagon inhibited pyruvate kinase. There was also a decrease in fructose diphosphate and an increase in fructose-6phosphate. This, along with the increased flux to glucose suggests that the hormone also stimulated fructose diphosphatase and/or inhibited phosphofructokinase. Similar changes have been reported by Lardy's group. 12 It is known that insulin suppresses glucagon stimulated lactate and alanine gluconeogenesis in isolated hepatocytes.9 Table 3 shows the effect of insulin Table 2. Effect of Inhlbitors of LQctate and Pyruvate Metabolism on the Ability of Glucagon to Stimulate Dihydroxyacetone Gluconeogenesis DihydroxyacetoneGluconeogenesis # moles[U~14C] dihydroxyacetoneconverted to g!ucose/15 min/mg DNA Inhibitor None (5) Hydrazine (2 mM) (4) o~-Cyanocinnamate (0,125 mM) (1) *Tryptophan (2 mM) (4) Aminoxyacetate (0.25 mM) (2)
Basal 3.4 3.2 2.6 3.7 3.2
~ ~ ~: :k :~
0.1 0.3 0.4 0.2 0.2
Glucagon 7.5 6.0 7.5 7.4 7.4
• i • • •
0.5 0.5 0.7 0.5 0.4
*Cells were preincubated with tryptophan for 30 rain. Hepatocytes from fed rats were incubated with 1 mM IU-14C] dihydroxyacetone as described in Table 1. Conversion of labeled dihydroxyacetone to labeled glucose was determined as described in the Methods section. The number in parenthesis indicates the number of cell preparations tested with each inhibitor. Glucagon concentration was 10 nM. All the inhibitors tested were found to inhibit glucogenesis from 2 mM lactate by 80% or more. No lactate glucogenesis was detectable with 2 mM hydrozine.
1358
PILKIS ET AL
2raM DI HYDROXYACETONE
160 140
g~ ~-- 120-
AGON
u_ I00 O
Fig. 2. Effect of glucagon on the levels of glycolytic intermediates in hepatocytes from fed rats. Hepatocytes from fed rats (600 #g D N A / 6 ml) were incubated for 15 min with 2 mM dihydroxyacetone in the absence and presence of 10 nM glucagon. Metabo|ites were determined by published enzymatic procedures 13 in neutralized extracts.
806040-
LAC
PYR
PEP DHAP FI)P F6P
GIP
GI'U
(10 nM) on conversion of 1 mM[U-14C] dihydroxyacetone to either labeled glucose or lactate in isolated hepatocytes from fed rats. Insulin had no significant effect on the basal rate of glucose or lactate synthesis. However, when added with 0.4 n M glucagon, insulin suppressed the synthesis of 14C-glucose and stimulated the production of ~4C-lactate. The suppression of glucose synthesis by the hormone could be accounted for by the increased lactate production. Since the effect of glucagon to stimulate dihydroxyacetone gluconeogenesis does not involve mitochondrial steps, it seems reasonable to postulate an extramitochondrial site for the action of insulin. The ability of insulin to stimulate lactate production suggests that this site may be the pyruvate kinase step. These results suggest that alterations in flux through pyruvate kinase are important in the action of glucagon on dihydroxyacetone gluconeogenesis in isolated hepatocytes. Evidence for glucagon actually affecting pyruvate kinase activity is shown in Fig. 3. A 15,000-supernatant fraction in 20 m M phosphate40% (v/v) glycerol buffer was prepared from cells from fed rats incubated with various glucagon concentrations. The enzyme activity was measured with 0.4 m M phosphoenolpyruvate as substrate in the absence and presence of 20 # M fructose diphosphate, an allosteric activator of the enzyme. In the absence of the activator there was a dose-dependent inhibition of activity. Clear inhibitory effects were seen with l n M glucagon and maximal concentrations inTable 3. Effect of Insulin on the Production of Glucose and Lactate from Dihydroxyacetone in Hepatocytes From Fed Rats Production of
14C.Glucose Condition Basal insulin, 10 Glucagon, Glucagon, Glucagon,
nM 0.4 nM 0.4 nM and insulin, 10 nM 10 nM
14C-Lactate
# motes[ U- 14C] dihydroxyacetone converted to product/15 min/mg DNA 4.8 • .4
7.9 • .4
4.3 7.8 6.3 9.4
7.9 5.1 6.3 4.2
• • • •
.2 .4 .3 .2
• • • •
.4 .2 .06 .1
Hepatocytes from fed rats were incubated with 1 mM [U-14C] dihydroxyacetone as described in Table 1. Labeled glucose and lactate were determined as described in the Methods section.
PYRUVATE KINASE
1359
120Fig. 3. Effect of glucagon concentration on pyruvate kinase activity in isolated hepatocytes from fed rats. Hepatocytes from fed rats ( 2 0 0 # g D N A / 5 rot) w e r e incubated with various concentrations of glucagon for 10 min. The cells were then rapidly sedimented and suspended in 1 ml of ice-cold 20 mM sodium phosphate4 0 % glycerol buffer, pH 7.8. The cells were broken with an Ultraturrax Homogenizer | (Tekmar Company, Cincinnati, Ohio) and a 15,000 xg supernatant fraction prepared. Pyruvate kinase activity in the absence or presence of 20 #M fructose diphosphate was estimated by the method of Llorente et al.11
z i~
IO0-
n-
80-
1~ ~_
60-
~
40-
;~ =
20-
a.
r--c/
o
;
~b
io'o
GLUCAGON, nM
h i b i t e d as m u c h as 7 0 ~ . T h e s e effects c a n b e seen w i t h i n 2 m i n a f t e r t h e a d d i t i o n o f h o r m o n e s . In t h e p r e s e n c e o f f r u c t o s e d i p h o s p h a t e o r w i t h h i g h p h o s p h o e n o l p y r u v a t e (>_ 2 r a M ) c o n c e n t r a t i o n s , g l u c a g o n h a d little o r n o effect. A l l o f t h e p r e c e d i n g r e s u l t s s u g g e s t t h a t t h e effect o f g l u c a g o n i n v o l v e s a l t e r a t i o n o f p y r u v a t e k i n a s e a c t i v i t y w h i c h m a y be m e d i a t e d b y a fall in f r u c t o s e d i p h o s p h a t e levels ( F i g . 2; B l a i r et al. 12 a n d / o r by m o d i f i c a t i o n o f t h e e n z y m e a c t i v i t y by p h o s p h o r y l a t i o n J 7 T h e s e p o s s i b i l i t i e s a r e c u r r e n t l y u n d e r i n v e s t i g a t i o n . Inh i b i t i o n o f p y r u v a t e k i n a s e a c t i v i t y by g l u c a g o n h a s b e e n r e p o r t e d by o t h e r s , m5,~6
REFERENCES
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