AMERICAN
JOURNAL
OF PHYSIOLOGY
Vol 229, No. 6, December
Acute
Prinfed in U.S.A.
1975.
suppression
of hepatic
by glucose in the intact HUEY
G. MCDANIEL
Veterans
Administration
Birmingham,
MCDANIEL,
by glucose
HUEY
in the intact
Hospital
Alabama
gluconeogenesis
animal
and Department
of Medicine,
G. Acute suppression of hepatic gluconeogenesis animal. Am. J. Physiol. 229(6) : 1569-l 575.
1975.-Within 2 h after glucose administration to fasting rats the incorporation of radioactive lactate into blood glucose and liver glycogen is decreased. Using tryptophan, which facilitates the study of gluconeogenesis prior to the phosphoenolpyruvate (PEP) carboxykinase step by increasing the level of certain hepatic metabolites,
we have
found
that
in animals
fasted
for 24 h glucose
markedly decreased hepatic malate and aspartate concentrations without a corresponding fall in that of pyruvate, suggesting a decrease in pyruvate carboxylase activity. An inhibitor of fatty acid oxidation, 4-pentenoic acid, similarly decreased the accumulation
of
these
intermediates,
and
octanoic
acid
significantly
lessened the fall in malate and aspartate with glucose. The changes in tissue metabolite levels were consistent with inhibition of the liver pyruvate carboxylase reaction by glucose treatment, and with abolition of this inhibition by octanoate administration. Alanine and glutamate levels in the liver of tryptophan-treated animals were decreased 90 and 32%, respectively, by glucose. Thus, glucose administration in the whole animal acutely decreases gluconeogenesis by apparently inhibiting the pyruvate carboxylase step and decreasing alanine levels in the liver. tryptophan;
4-pentenoic
pyruvate
carboxylase;
acid, alanine;
University
of Alabama
School
of Medicine,
3.5206
acetyl-CoA;
octanoic
acid;
malate; crossover plot
IS A COMPLEX PROCESS that is regulated by hormonal changes which alter enzyme activities and vary substrate levels. A good deal has been learned about its regulation from studies with perfused liver preparations and isolated liver cells. However, these systems have the disadvantage that they may remove some of the controls normally operating in the whole animal. For this reason we have looked at the effect of glucose on gluconeogenesis in the intact animal. In some of these studies of hepatic gluconeogenesis we have employed tryptophan to facilitate the measurement of certain metabolites by blocking gluconeogenesis at the site of conversion of oxalacetate to phosphoenolpyruvate (PEP) in the liver (27). This occurs because the tryptophan rnetabolite quinolinic acid is a competitive inhibitor of PEP carboxykinase (GTP: oxaloacetate carboxy-lyase, transphosphorylating, EC 4.1.1.32) especially when combined with a divalent ion such as ferrous iron (2 1). The inhibition of PEP carboxykinase results in a marked buildup in the level of oxalacetate and its precursors in the liver GLUCONEOGENESIS
(27). This provides a good model system for a study of the effect of variables on the accumulation of these gluconeogenie precursors in the intact animal. When glucose is given with tryptophan, there is a significant decrease in the accumulation of these intermediates (26). Lardy (15) suggested this occurs because glucose blocks the generation of dicarboxylic intermediates necessary for gluconeogenesis, possibly at the pyruvate carboxylask step. In the light of recent advances in knowledge concerning the role of certain amino acid substrates and fatty acids in gluconeogenesis, we have investigated the nature of the inhibitory effect of glucose on the accumulation of these intermediates in the liver, in order to determine how glucose turns off their accumulation, and to see if this correlates with the effect of glucose on the overall rate of gluconeogenesis in the whole animal. METHODS
White, rnale Sprague-Dawley rats weighing from 175 to 200 g were fasted for 24 h prior to the experimental procedures. The control animals were given normal saline The tryp tophan- treated an irnals reintraperi toneally. ceived 25 mg of L-tryptophan per 100 g of body wt as a suspension in normal saline. The glucose-treated group was given 500 rng of glucose per 100 g of body wt and 25 rng of tryptophan per 100 g in normal saline. The 4-pentenoic acid group was given 25 mg of tryptophan per 100 g 3 h before their livers were freeze-clamped. They also received 25 mg of sodium pentenoate per 100 g (pH 7.4) in normal saline in traperitoneally 30 min before their livers were freeze-clamped. Sodium octanoate (20 mg/ 100 g (pH 7.4)) was given in normal saline by nasogastric tube 45 min before the livers were freeze-clamped. Exactly 3 h after the intraperitoneal injection of tryptophan or tryptophan and glucose, the animals were immobilized by cervical fracture, the abdomen was opened, and a lobe of the liver was freeze-clamped with aluminum tongs cooled in a mixture of Dry Ice and acetone. (When the animals were anesthetized with ether prior to the freezeclamping of their livers, similar results were obtained.) The livers froze within 10 s after clamping and were weighed and homogenized in the cold with 5 vol of 6 % HClO 4. The homogenate was centrifuged at 16,000 X g for 15 min and the pellet reextracted with 3 vol of 3 % HClO4. To the combined HC104 extract was added 0.1 ml of 1 M
1569
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H. G. MCDANIEL
1570 KzHPOd, and it was then carefully titrated with 1 M KOH to pH 4. After standing in the cold for 30 min, the precipitated KC104 was removed by centrifugation and the various metabolites were measured in the supernatant. ace tyl-CoA and acetoacetate were Oxalacetate, pyruvate, measured without delay. The extracts were then frozen and the other metabolites measured at a later time. The recovery of added oxalacetate (the most labile of these metabolites) was 50 70. Citrate was measured by a chemical method (18). Malate, acetoacetate, beta-hydroxybutyrate oxalacetate, pyruvate, and ATP were determined by standard enzymatic methods (34). Aspartate was measured with glutamic-oxaloacetic transaminase and malate dehydrogenase from Calbiochem, San Diego (34). Acetyl-CoA was measured by the rate assay of Allred and Guy (1). Hexokinase, glucose-6-phosphate dehydrogenase, citrate synthetase, and phosphotransacetylase were purchased from P-L Biochemicals, Lactate dehydrogenase and beta-hydroxyMilwaukee. butyrate dehydrogenase were from Calbiochem. The cytoplasmic and mitochondrial content of malate and oxalacetate were calculated by the procedure of Williamson (32). This is done by assuming that the malate and oxalacetate in the cytoplasm are in equilibrium with lactate and pyruvate, as described by the constants of the equilibrium equation for malate dehydrogenase and lactate dehydrogenase. The mitochondrial malate and oxalacetate are assumed to be in equilibrium with acetoacetate and beta-hydroxybutyrate as described by the constants of the equilibrium equation for malate dehydrogenase and beta-hydroxybutyrate dehydrogenase : uJ/P)(~LD/KMDNW WOH/‘AcAc)
(&m/&vm)
= Mx (OT-y)
= MT-~
(4 (2)
by Keech and Utter (13). PEP carboxykinase was assayed as described by Seubert and Huth (28). In the studies of 14C-labeled lactate and glycerol conversion to glucose, 4 &i of Z-14C-labeled DL-lactate or U-14C-labeled glycerol were given intraperitoneally in 2 ml of normal saline, and 1 ml of blood was withdrawn 15, 30, and 45 min later from the inferior vena cava. This blood was added to 3 ml of 6’& HC104. (The actual amount of blood was determined by weight.) The HClO4 extract was brought to pH 6, the KC104 was removed by centrifugation, and the supernatant was passed through a Dowex l-X8 column. The glucose was washed through the column with water and the lactate was then removed with 0.1 M formic acid (2). Liver glycogen was prepared by the technique of Good et al. (10). The pH 4 WC104 extract of blood from the animals given 14C-labeled glycerol was lyophilized to reduce the volume‘ and then subjected to -thin-layer chromatography on Silica Gel-G poured in 0.02 M sodium acetate with acetone :water (90: 10) (17). The plates were scraped and counted in a liquid scintillation spectrophotometer to quantify the amounts of radioactivity in glucose and glycerol. Each value in the figures and tables is the average of three or more determin ations =t the standard deviation The data were analyzed by the Student t test for statistical significance. RESULTS
Within 1 h 45 min glucose significantly decreased the incorporation of 14C-labeled lactate into glucose in the blood (Fig. 1). The measurement of 14C-labeled liver glycogen showed that the difference was not simply due to the lactate being converted to glycogen rather than glucose (legend of Fig. 1). (B ase d on a liver weight of 8 g and blood
where L P p-OH AcAc K LD K MD
&-OH
0,
0
T-Y
Mx
M T
T-x
lactate pyruvate be ta-hydroxybutyrate acetoacetate constant for lactate dehyequilibrium drogenase dehyequilibrium constant for malate drogenase constant for beta-hydroxyequilibrium butyrate dehydrogenase oxalacetate in the cytoplasm (total oxalacetate in the mitochondria oxalacetate minus the amount in the cytoplasm) malate in the cytoplasm malate in the mitochondria (total malate minus the amount in the cytoplasm) total content of malate or oxalacetate
The resulting two equations with two unknowns can readily be solved. The equilibrium constants used for malate dehydrogenase, lactate dehydrogenase, and betahydroxybutyrate were 2.78 X 10m5, 1.11 X 10V4, and 4.93 x 1o-2 (30). Pyruvate carboxylase activity . was assayed as described
1% 12
7L’
I
, /
f”
I ,/ / /
,A ---_-f
Control
Glucose
I LI I/ OV 0
TIME
I
I
I5
30 IN MINUTES
1 45
1. Each animal was given 4 ,&i of 2J4C-labeled lactate intraperitoneally after 24 h of fasting. Counts per minute (cpm) in glucose per 1.06 g (1 ml) blood is plotted vs. time after injection. Glucose group was given 500 mg of glucose per 100 g body wt, ip, 1.5 h before lactate. Counts per min in glycogen per gram of liver with glucose were 4,564 & 2,100 at 15 min, 1,408 k 400 at 30 min, and 902 & 504 at 45 min. In control group it was 1,236 =t 532/g liver at 30 min. Lactate values were 767 rt 312 and 735 AZ 162 pmol/l.O6 g blood in control and glucose groups. Each value represents average & SD of 3 animals. Specific activity of hepatic lactate at 15 min was 5.8 & 0.1 &i/mm01 in control group and 4.6 =t 0.1 in glucose group. FIG.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 10, 2019.
ACUTE
SUPPRESSION
OF
GLUCONEOGENESIS
IN
LIVER
volume of 16 ml, 30 min after lactate the total counts per minute in glucose and hepatic glycogen were 217,376 in the control and 87,072 in the glucose groups.) The blood lactate levels were not significantly different in the two groups, so this did not play a role in the suppression of glucose formation from lactate in the animals receiving glucose (legend of Fig. 1). The specific activity of hepatic lactate was decreased 21 70 in the glucose group, but this change was much less than the decrease in lactate incorporation into glucose. In order to be sure that the effect of glucose was occurring below the level of glyceraldehyde 3-phosphate in the gluconeogenic pathway, we measured the effect of glucose on the incorporation of 14C-labeled from Table 1 that glycerol in to glucose. It is apparent glycerol conversion to glucose is not significantly affected (if at all) by glucose at 2 h. A key enzyme in gluconeogenesis which has been shown to .be suppressed by feeding is PEP carboxykinase (29). The activity of this enzyme in 24-hfasted animals was 98 & 8 nmol/mg of protein per min and 81 + 9 nmol/mg of protein 2 h after glucose (100,000 X g liver supernatant was used in the assay). To investigate further the inhibition of gluconeogenesis by glucose, we used tryptophan to isolate the initial phase of gluconeogenesis prior to the PEP carboxykinase reaction. The amount of tryptophan used in these experiments produces approximately a fivefold increase in the level of oxalacetate and two of its immediate precursors-citrate and aspartate (Table 2). The other direct precursor, malate, is increased over 19-fold. There is a much smaller increase in the level of pyruvate and lactate. The total content of these six intermediates is increased from a control value of 1,737 + 150 nmol to 12,176 & 854 nmol/g of liver, wet wt. When glucose is given with tryptophan, there is a 44 % decrease in the total content of these intermediates (Fig. 2). The greatest fall in the tryptophan and glucose group compared to tryptophan alone occurs in malate (57 %) (Table 2). Aspartate con tent is also markedly reduced (52 %) (Table 2). Th ere is a very modest fall in lactate (P < 0.02) and an actual increase in the levels of oxaloacetate (P < 0.05) and pyruvate (92%, P < 0.01). Both betahydroxybutyrate and acetoacetate are down sharply in the glucose group (P < O.Ol), and the citrate values are essentially unchanged. However, animals given glucose and tryptophan after 12 h of fasting have a drop in citrate in addition to aspartate and malate (Table 3). The marked fall in malate, which requires a supply of NADH for its formation from oxalacetate, and the drop in ketone bodies suggest that glucose is acting by decreasing the oxidation of fatty acids. In order to test this idea we TABLE
Glycerol conversion to ghcose and glycogen
1.
Condition
of Animal
[WjGlucose, whole
cpm/l.06 blood
Fasting
16,305
&
730
Glucose
13,620
z!z 5,234
g
Liver
Glycogen,
3,516 10,149
cpm/g
liver
=t 1,530 *
9,023
Each animal was given 4 &i of [UJ4C]glycerol in 2 ml saline, 1.5 h after 500 mg glucose per 100 g of body wt (both intraperitoneally). Blood and liver samples were collected as for Fig. 1. The radioactivity present in the 15-, 30-, and 45-min samples was essentially the same, so they were averaged.
1571
BY GLUCOSE
Metabolite, nmol/g liver wet wt
Saline
Tryptophan
Tryptophan + Glucose
6,409&365*
2,724&840
Tryptophan. + 4-~c$eno1c
-___M alate Oxalacetate
328zk74 3&l
Aspartate
490 zt95
Citrate
343 zt89
Pyruvate
29*9 544zkl86
Beta-hydroxybutyrate
760%120
Acetoacetate
245 A30
16&l*
1,523&203
31 zt8
2,490*751*
37+14
1,205zk119
1,910&245*
1,617zt360 197&107
1,874&310 98 xt9
51 zk7t
1,000&157*
284 zt36
864 zk40
713zt109
2,462&357
T;y,.$~~~~en + Octanoic Acid 4,937&425 7=t3
2,016&526 1,905*350 36 zt9
821 zk180
598zk197
237 zt34
336 zk2 1
ATP -All tp < Tryptophan
Tryptophon
a “:ose Octonotc
Aad
TrypPphan GlucoseTr YP. T
4-l&
II
AC.
FIG. 2. Each column shows total value for 6 metabolites (lactate, pyruvate, citrate, aspartate, malate, and oxalacetate). Value in tryptophan-treated animals (12,176 nmol/g liver) is taken to be 100%. Difference between group receiving tryptophan and glucose and group receiving tryptophan, glucose, and octanoic acid is significant (p < 0.05). (Bar above each column is 1 SD.) Tryptophan and octanoic acid give a total value of 11,409 & 602 nmol/g liver. (In this group there was a slight increase in malate and citrate and a slight fall in aspartate.)
decided to block fatty acid oxidation and see if this duplicated the effect of glucose. For this we chose 4-pentenoic acid, which has been shown to interrupt the oxidation of fatty acids by tying up coenzyme A and carnitine so that they are not available for use in fatty acid oxidation (5). When 4-pentenoic acid is given with tryptophan, there is a 50 % decline in the total content of these intermediates, similar to the effect of glucose (Fig. 2). Again there is a marked fall in malate (P < 0.01) and aspartate and a significant rise in oxalacetate and pyruvate (P < 0.01) the role of fatty acid (Table 2). I n order to investigate oxidation further, we studied the effect of giving octanoic acid along with glucose and tryptophan on the metabolite levels in the liver. Octanoic acid, a fatty acid that can only be oxidized and not esterified or elongated (14), diminishes the fall in the total content of these metabolites from 44 to 20 % (P < 0.05) (Fig. 2). When only octanoic acid and
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1572
H. G. MCDANIEL
3. Efect of glucose in U-h-fasted -~-~--
TABLE
Metabolite,
nmol/g wet wt
Substance
liver Tryptophan
animals _-
-~__--
Given
Tryptophan
P
+ Glucose
.~..-
~_~
Citrate
3,241
+
346
1,174
*
347
JOURNAL
OF PHYSIOLOGY
Vol 229, No. 6, December
Acute
Prinfed in U.S.A.
1975.
suppression
of hepatic
by glucose in the intact HUEY
G. MCDANIEL
Veterans
Administration
Birmingham,
MCDANIEL,
by glucose
HUEY
in the intact
Hospital
Alabama
gluconeogenesis
animal
and Department
of Medicine,
G. Acute suppression of hepatic gluconeogenesis animal. Am. J. Physiol. 229(6) : 1569-l 575.
1975.-Within 2 h after glucose administration to fasting rats the incorporation of radioactive lactate into blood glucose and liver glycogen is decreased. Using tryptophan, which facilitates the study of gluconeogenesis prior to the phosphoenolpyruvate (PEP) carboxykinase step by increasing the level of certain hepatic metabolites,
we have
found
that
in animals
fasted
for 24 h glucose
markedly decreased hepatic malate and aspartate concentrations without a corresponding fall in that of pyruvate, suggesting a decrease in pyruvate carboxylase activity. An inhibitor of fatty acid oxidation, 4-pentenoic acid, similarly decreased the accumulation
of
these
intermediates,
and
octanoic
acid
significantly
lessened the fall in malate and aspartate with glucose. The changes in tissue metabolite levels were consistent with inhibition of the liver pyruvate carboxylase reaction by glucose treatment, and with abolition of this inhibition by octanoate administration. Alanine and glutamate levels in the liver of tryptophan-treated animals were decreased 90 and 32%, respectively, by glucose. Thus, glucose administration in the whole animal acutely decreases gluconeogenesis by apparently inhibiting the pyruvate carboxylase step and decreasing alanine levels in the liver. tryptophan;
4-pentenoic
pyruvate
carboxylase;
acid, alanine;
University
of Alabama
School
of Medicine,
3.5206
acetyl-CoA;
octanoic
acid;
malate; crossover plot
IS A COMPLEX PROCESS that is regulated by hormonal changes which alter enzyme activities and vary substrate levels. A good deal has been learned about its regulation from studies with perfused liver preparations and isolated liver cells. However, these systems have the disadvantage that they may remove some of the controls normally operating in the whole animal. For this reason we have looked at the effect of glucose on gluconeogenesis in the intact animal. In some of these studies of hepatic gluconeogenesis we have employed tryptophan to facilitate the measurement of certain metabolites by blocking gluconeogenesis at the site of conversion of oxalacetate to phosphoenolpyruvate (PEP) in the liver (27). This occurs because the tryptophan rnetabolite quinolinic acid is a competitive inhibitor of PEP carboxykinase (GTP: oxaloacetate carboxy-lyase, transphosphorylating, EC 4.1.1.32) especially when combined with a divalent ion such as ferrous iron (2 1). The inhibition of PEP carboxykinase results in a marked buildup in the level of oxalacetate and its precursors in the liver GLUCONEOGENESIS
(27). This provides a good model system for a study of the effect of variables on the accumulation of these gluconeogenie precursors in the intact animal. When glucose is given with tryptophan, there is a significant decrease in the accumulation of these intermediates (26). Lardy (15) suggested this occurs because glucose blocks the generation of dicarboxylic intermediates necessary for gluconeogenesis, possibly at the pyruvate carboxylask step. In the light of recent advances in knowledge concerning the role of certain amino acid substrates and fatty acids in gluconeogenesis, we have investigated the nature of the inhibitory effect of glucose on the accumulation of these intermediates in the liver, in order to determine how glucose turns off their accumulation, and to see if this correlates with the effect of glucose on the overall rate of gluconeogenesis in the whole animal. METHODS
White, rnale Sprague-Dawley rats weighing from 175 to 200 g were fasted for 24 h prior to the experimental procedures. The control animals were given normal saline The tryp tophan- treated an irnals reintraperi toneally. ceived 25 mg of L-tryptophan per 100 g of body wt as a suspension in normal saline. The glucose-treated group was given 500 rng of glucose per 100 g of body wt and 25 rng of tryptophan per 100 g in normal saline. The 4-pentenoic acid group was given 25 mg of tryptophan per 100 g 3 h before their livers were freeze-clamped. They also received 25 mg of sodium pentenoate per 100 g (pH 7.4) in normal saline in traperitoneally 30 min before their livers were freeze-clamped. Sodium octanoate (20 mg/ 100 g (pH 7.4)) was given in normal saline by nasogastric tube 45 min before the livers were freeze-clamped. Exactly 3 h after the intraperitoneal injection of tryptophan or tryptophan and glucose, the animals were immobilized by cervical fracture, the abdomen was opened, and a lobe of the liver was freeze-clamped with aluminum tongs cooled in a mixture of Dry Ice and acetone. (When the animals were anesthetized with ether prior to the freezeclamping of their livers, similar results were obtained.) The livers froze within 10 s after clamping and were weighed and homogenized in the cold with 5 vol of 6 % HClO 4. The homogenate was centrifuged at 16,000 X g for 15 min and the pellet reextracted with 3 vol of 3 % HClO4. To the combined HC104 extract was added 0.1 ml of 1 M
1569
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H. G. MCDANIEL
1570 KzHPOd, and it was then carefully titrated with 1 M KOH to pH 4. After standing in the cold for 30 min, the precipitated KC104 was removed by centrifugation and the various metabolites were measured in the supernatant. ace tyl-CoA and acetoacetate were Oxalacetate, pyruvate, measured without delay. The extracts were then frozen and the other metabolites measured at a later time. The recovery of added oxalacetate (the most labile of these metabolites) was 50 70. Citrate was measured by a chemical method (18). Malate, acetoacetate, beta-hydroxybutyrate oxalacetate, pyruvate, and ATP were determined by standard enzymatic methods (34). Aspartate was measured with glutamic-oxaloacetic transaminase and malate dehydrogenase from Calbiochem, San Diego (34). Acetyl-CoA was measured by the rate assay of Allred and Guy (1). Hexokinase, glucose-6-phosphate dehydrogenase, citrate synthetase, and phosphotransacetylase were purchased from P-L Biochemicals, Lactate dehydrogenase and beta-hydroxyMilwaukee. butyrate dehydrogenase were from Calbiochem. The cytoplasmic and mitochondrial content of malate and oxalacetate were calculated by the procedure of Williamson (32). This is done by assuming that the malate and oxalacetate in the cytoplasm are in equilibrium with lactate and pyruvate, as described by the constants of the equilibrium equation for malate dehydrogenase and lactate dehydrogenase. The mitochondrial malate and oxalacetate are assumed to be in equilibrium with acetoacetate and beta-hydroxybutyrate as described by the constants of the equilibrium equation for malate dehydrogenase and beta-hydroxybutyrate dehydrogenase : uJ/P)(~LD/KMDNW WOH/‘AcAc)
(&m/&vm)
= Mx (OT-y)
= MT-~
(4 (2)
by Keech and Utter (13). PEP carboxykinase was assayed as described by Seubert and Huth (28). In the studies of 14C-labeled lactate and glycerol conversion to glucose, 4 &i of Z-14C-labeled DL-lactate or U-14C-labeled glycerol were given intraperitoneally in 2 ml of normal saline, and 1 ml of blood was withdrawn 15, 30, and 45 min later from the inferior vena cava. This blood was added to 3 ml of 6’& HC104. (The actual amount of blood was determined by weight.) The HClO4 extract was brought to pH 6, the KC104 was removed by centrifugation, and the supernatant was passed through a Dowex l-X8 column. The glucose was washed through the column with water and the lactate was then removed with 0.1 M formic acid (2). Liver glycogen was prepared by the technique of Good et al. (10). The pH 4 WC104 extract of blood from the animals given 14C-labeled glycerol was lyophilized to reduce the volume‘ and then subjected to -thin-layer chromatography on Silica Gel-G poured in 0.02 M sodium acetate with acetone :water (90: 10) (17). The plates were scraped and counted in a liquid scintillation spectrophotometer to quantify the amounts of radioactivity in glucose and glycerol. Each value in the figures and tables is the average of three or more determin ations =t the standard deviation The data were analyzed by the Student t test for statistical significance. RESULTS
Within 1 h 45 min glucose significantly decreased the incorporation of 14C-labeled lactate into glucose in the blood (Fig. 1). The measurement of 14C-labeled liver glycogen showed that the difference was not simply due to the lactate being converted to glycogen rather than glucose (legend of Fig. 1). (B ase d on a liver weight of 8 g and blood
where L P p-OH AcAc K LD K MD
&-OH
0,
0
T-Y
Mx
M T
T-x
lactate pyruvate be ta-hydroxybutyrate acetoacetate constant for lactate dehyequilibrium drogenase dehyequilibrium constant for malate drogenase constant for beta-hydroxyequilibrium butyrate dehydrogenase oxalacetate in the cytoplasm (total oxalacetate in the mitochondria oxalacetate minus the amount in the cytoplasm) malate in the cytoplasm malate in the mitochondria (total malate minus the amount in the cytoplasm) total content of malate or oxalacetate
The resulting two equations with two unknowns can readily be solved. The equilibrium constants used for malate dehydrogenase, lactate dehydrogenase, and betahydroxybutyrate were 2.78 X 10m5, 1.11 X 10V4, and 4.93 x 1o-2 (30). Pyruvate carboxylase activity . was assayed as described
1% 12
7L’
I
, /
f”
I ,/ / /
,A ---_-f
Control
Glucose
I LI I/ OV 0
TIME
I
I
I5
30 IN MINUTES
1 45
1. Each animal was given 4 ,&i of 2J4C-labeled lactate intraperitoneally after 24 h of fasting. Counts per minute (cpm) in glucose per 1.06 g (1 ml) blood is plotted vs. time after injection. Glucose group was given 500 mg of glucose per 100 g body wt, ip, 1.5 h before lactate. Counts per min in glycogen per gram of liver with glucose were 4,564 & 2,100 at 15 min, 1,408 k 400 at 30 min, and 902 & 504 at 45 min. In control group it was 1,236 =t 532/g liver at 30 min. Lactate values were 767 rt 312 and 735 AZ 162 pmol/l.O6 g blood in control and glucose groups. Each value represents average & SD of 3 animals. Specific activity of hepatic lactate at 15 min was 5.8 & 0.1 &i/mm01 in control group and 4.6 =t 0.1 in glucose group. FIG.
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ACUTE
SUPPRESSION
OF
GLUCONEOGENESIS
IN
LIVER
volume of 16 ml, 30 min after lactate the total counts per minute in glucose and hepatic glycogen were 217,376 in the control and 87,072 in the glucose groups.) The blood lactate levels were not significantly different in the two groups, so this did not play a role in the suppression of glucose formation from lactate in the animals receiving glucose (legend of Fig. 1). The specific activity of hepatic lactate was decreased 21 70 in the glucose group, but this change was much less than the decrease in lactate incorporation into glucose. In order to be sure that the effect of glucose was occurring below the level of glyceraldehyde 3-phosphate in the gluconeogenic pathway, we measured the effect of glucose on the incorporation of 14C-labeled from Table 1 that glycerol in to glucose. It is apparent glycerol conversion to glucose is not significantly affected (if at all) by glucose at 2 h. A key enzyme in gluconeogenesis which has been shown to .be suppressed by feeding is PEP carboxykinase (29). The activity of this enzyme in 24-hfasted animals was 98 & 8 nmol/mg of protein per min and 81 + 9 nmol/mg of protein 2 h after glucose (100,000 X g liver supernatant was used in the assay). To investigate further the inhibition of gluconeogenesis by glucose, we used tryptophan to isolate the initial phase of gluconeogenesis prior to the PEP carboxykinase reaction. The amount of tryptophan used in these experiments produces approximately a fivefold increase in the level of oxalacetate and two of its immediate precursors-citrate and aspartate (Table 2). The other direct precursor, malate, is increased over 19-fold. There is a much smaller increase in the level of pyruvate and lactate. The total content of these six intermediates is increased from a control value of 1,737 + 150 nmol to 12,176 & 854 nmol/g of liver, wet wt. When glucose is given with tryptophan, there is a 44 % decrease in the total content of these intermediates (Fig. 2). The greatest fall in the tryptophan and glucose group compared to tryptophan alone occurs in malate (57 %) (Table 2). Aspartate con tent is also markedly reduced (52 %) (Table 2). Th ere is a very modest fall in lactate (P < 0.02) and an actual increase in the levels of oxaloacetate (P < 0.05) and pyruvate (92%, P < 0.01). Both betahydroxybutyrate and acetoacetate are down sharply in the glucose group (P < O.Ol), and the citrate values are essentially unchanged. However, animals given glucose and tryptophan after 12 h of fasting have a drop in citrate in addition to aspartate and malate (Table 3). The marked fall in malate, which requires a supply of NADH for its formation from oxalacetate, and the drop in ketone bodies suggest that glucose is acting by decreasing the oxidation of fatty acids. In order to test this idea we TABLE
Glycerol conversion to ghcose and glycogen
1.
Condition
of Animal
[WjGlucose, whole
cpm/l.06 blood
Fasting
16,305
&
730
Glucose
13,620
z!z 5,234
g
Liver
Glycogen,
3,516 10,149
cpm/g
liver
=t 1,530 *
9,023
Each animal was given 4 &i of [UJ4C]glycerol in 2 ml saline, 1.5 h after 500 mg glucose per 100 g of body wt (both intraperitoneally). Blood and liver samples were collected as for Fig. 1. The radioactivity present in the 15-, 30-, and 45-min samples was essentially the same, so they were averaged.
1571
BY GLUCOSE
Metabolite, nmol/g liver wet wt
Saline
Tryptophan
Tryptophan + Glucose
6,409&365*
2,724&840
Tryptophan. + 4-~c$eno1c
-___M alate Oxalacetate
328zk74 3&l
Aspartate
490 zt95
Citrate
343 zt89
Pyruvate
29*9 544zkl86
Beta-hydroxybutyrate
760%120
Acetoacetate
245 A30
16&l*
1,523&203
31 zt8
2,490*751*
37+14
1,205zk119
1,910&245*
1,617zt360 197&107
1,874&310 98 xt9
51 zk7t
1,000&157*
284 zt36
864 zk40
713zt109
2,462&357
T;y,.$~~~~en + Octanoic Acid 4,937&425 7=t3
2,016&526 1,905*350 36 zt9
821 zk180
598zk197
237 zt34
336 zk2 1
ATP -All tp < Tryptophan
Tryptophon
a “:ose Octonotc
Aad
TrypPphan GlucoseTr YP. T
4-l&
II
AC.
FIG. 2. Each column shows total value for 6 metabolites (lactate, pyruvate, citrate, aspartate, malate, and oxalacetate). Value in tryptophan-treated animals (12,176 nmol/g liver) is taken to be 100%. Difference between group receiving tryptophan and glucose and group receiving tryptophan, glucose, and octanoic acid is significant (p < 0.05). (Bar above each column is 1 SD.) Tryptophan and octanoic acid give a total value of 11,409 & 602 nmol/g liver. (In this group there was a slight increase in malate and citrate and a slight fall in aspartate.)
decided to block fatty acid oxidation and see if this duplicated the effect of glucose. For this we chose 4-pentenoic acid, which has been shown to interrupt the oxidation of fatty acids by tying up coenzyme A and carnitine so that they are not available for use in fatty acid oxidation (5). When 4-pentenoic acid is given with tryptophan, there is a 50 % decline in the total content of these intermediates, similar to the effect of glucose (Fig. 2). Again there is a marked fall in malate (P < 0.01) and aspartate and a significant rise in oxalacetate and pyruvate (P < 0.01) the role of fatty acid (Table 2). I n order to investigate oxidation further, we studied the effect of giving octanoic acid along with glucose and tryptophan on the metabolite levels in the liver. Octanoic acid, a fatty acid that can only be oxidized and not esterified or elongated (14), diminishes the fall in the total content of these metabolites from 44 to 20 % (P < 0.05) (Fig. 2). When only octanoic acid and
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1572
H. G. MCDANIEL
3. Efect of glucose in U-h-fasted -~-~--
TABLE
Metabolite,
nmol/g wet wt
Substance
liver Tryptophan
animals _-
-~__--
Given
Tryptophan
P
+ Glucose
.~..-
~_~
Citrate
3,241
+
346
1,174
*
347
