Eur. J. Biochem. 76, 567-571 (1977)
Gluconeogenesis in the Guinea Pig Effect of Glucagon on Gluconeogenesis from Lactate by Isolated Perfused Guinea-pig Liver David E COOK Department of Biochemistry, University of Nebraska Medical Center, Omaha (Received December 31, 1976)
Gluconeogenesis was stimulated by glucagon in fed but not fasted isolated perfused guinea pig livers. Both the amount and the rate of incorporation of radioactivity into glucose from ~-[U-'~C]lactatewere inci-eased in fed livers by the addition of glucagon to the perfusate. The glucagon-stimulated increase in gluconeogenesis was accompanied by an increase in oxygen consumption, an increase in the amount of lactate carbon converted to glucose and a decrease in the amount of lactate carbon converted to COz. The results are interpreted to indicate that glucagon affects gluconeogenesis from lactate in fed livers by redirecting the fate of substrate from other products toward glucose.
Much of our knowledge concerning the hormonal control of hepatic gluconeogenesis, especially by glucagon, has been derived from studies with rats, perfused rat livers or isolated hepatocytes prepared from rat livers. The general applicability of such knowledge, however, remains uncertain. For example, Soling et al. [l] have demonstrated that, unlike its positive effect on gluconeogenesis in the rat, glucagon does not stimulate gluconeogenesis in fasted isolated perfused guinea pig liver. In addition, the rat and a few other mammalian species have liver phosphoenolpyruvate carboxykinase activity located almost entirely in the cytosol. Most mammalian species including man and guinea pig, however, have an equal distribution of distinct mitochondria1 and cytosolic phosphoPnolpyruvate carboxykinase activity (cf. [2]). Consequently, the importance of studying the control of hepatic gluconeogenesis in an animal model such as the guinea pig has often been emphasized [I, 3 - 51. These and other considerations led to the present investigations on the effects of glucagon on hepatic gluconeogenesis in the guinea pig. MATERIALS AND METHODS Male English short-hair guinea pigs (CAMM Research Institute, Wayne, N.J.) weighing 229 to 349 g Enzyme. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1. 1.32).
were allowed to feed ad lib. or fasted 48 h prior to perfusion. The isolated livers were perfused at 37 "C with a Krebs-Ringer bicarbonate medium containing 3 % fatty-acid-free bovine serum albumin (Miles Laboratories, Elkhart, Ind.) and 20 % washed bovine erythrocytes in a recirculating system as previously described [6]. In this system the livers were perfused for 60 min prior to the addition of substrate. The substrate was 10 mM L-lactate (Schwartz-Mann, (AmerOrangeburg, N.J.) containing ~-[U-'~C]lactate sham-Searle, Arlington Heights, Ill.) to give a specific activitity of 1600 dis. min-' pmol-'. Glucagon (a generous gift from Dr William Bromer, Eli Lilly and Co., Indianapolis, Ind.) was prepared in 3 % bovine serum albumin and kept frozen until used. When used, 5 pg of glucacon was added to the perfusate at 30 min and 60 min after the start of the perfusion [6]. Perfusate samples (2 ml) were taken at the indicated times, deproteinized, and assayed for glucose and other metabolities as previously described [6,7]. All of the radioactivity in the neutral fraction obtained by this method was found to represent glucose. In earlier evaluations of this procedure, it was determined that glucose accounted for more than 90 % of the radioactivity in the neutral fraction [7] and lactate for more than 70 % of the radioactivity in the anionic or 'lactate' fraction [6]. The radioactive anionic or 'lactate' fraction will be referred to as [14C]lactate. I4CO2 produced during the perfusion was trapped by aspirating effluent from the oxygenator through KOH and
568
Effect of Glucagon on Gluconeogenesis in the Guinea-pig 200
,
I200
oxygen tension of the samples with an IL 213 pH blood gas analyzer (Instrumentation Laboratories, Inc., Lexington, Mass.). These values, together with the flow rate of perfusate through the liver, were used to calculate the rate of oxygen consumption as previously described [8]. Scintillation counting was done in either Bray's solution [9] or Aquasol (New England Nuclear, Boston, Mass.) with a Unilux I1 scintillation counter (Nuclear Chicago) and the channels ratio method was used to correct for quenching. The significance of differences between means was established by Student's t-test from t values determined with the Monroe Alpha 325 Significance Pak program number 9212V, t-test for independent means, as supplied by Monroe, The Calculator Company. Significance is shown when P < 0.05. RESULTS
90
60
120
150
180
Time (min)
[14C]Glucose Production
B
Fed \\ \
0 60
90
120 150 Time (rnin)
180
Fig. 1. The time course (f[14C]glucose production (-) and ['4C]lactate uptake (-----) during the perfusion of guinea pig t e 100 ml qf recirculating perlivers with 10 mM ~ - [ U - ' ~ C ] l a c t ain fusate in either the absence ( 0 )or presence (Aj of added glucagon. Substrate was added at 60 min and glucagon (5 kg) at 30 and 60 min after the start of the perfusion. The curves in (A) were obtained with 48-h-fasted livers and those in (B) with fed livers. Each point represents the mean and the vertical bars f SE of 5-9 perfusions. Points identified with an asterisk are significantly different from the corresponding control (absence of glucagon) points (P ranges from < 0.05 to < 0.01)
In order to eliminate consideration of the contribution of endogenous substances to glucose production in either the absence or presence of glucagon, radioactive substrate, ~-[U-'~C]lactate, was used throughout the study. In 48-h-fasted guinea pig livers, glucagon had no significant effect on either the total amount of ['4C]glucose produced (Fig.1A) or on the rate of [14C]glucose production during the first hour after substrate addition (Table l), a period of relatively rapid ['4C]glucose production (Fig. 1 A). These observations are in agreement with the previous report that glucagon does not affect gluconeogenesis in fasted guinea pig liver [l].The time course for the production of [14C]glucosein fed livers is shown in Fig. 1 B. It can be seen that the total amount of ['4C]glucose produced by fed guinea pig livers (Fig. 1 B) is about onehalf of that produced in fasted livers (Fig. 1A). Also in contrast to fasted livers, addition of glucagon to fed livers resulted in a significant increase in the total amount of [14C]glucoseproduced (Fig. 1B) as well as an increase in the rate of ['4C]glucose production (Table 1). This glucagon-stimulated rate was more than 70 % greater in the first 30 min and 35 greater in the second 30 min following substrate addition than the corresponding rates observed in the absence of glucagon (Table 1). ['4C]Lactate Uptake
subsequently transferring the trapped 14C02 to hyamine hydroxide for scintillation counting as previously described [6]. The rate of oxygen con'sumption was determined by simultaneously sampling the perfusate just prior to entry into and just subsequent to exit from the liver and immediately determining the
Glucagon did not significantly affect the amount or rate of [14C]lactateuptake in fasted livers (Fig. 1A and Table 2, respectively). Its effect on lactate utilization by fed livers, however, was variable. The total amount of ['4C]lactate utilized was not significantly greater in glucagon-treated livers compared to control
D. E. Cook
569
Table 1. Effect of glucagon and fasting on the rate of ['4C]glucose production in isolated perfused guinea pig livers The rates of glucose production were calculated from the amount of ['4C]glucose that appeared in the perfusate during the first 30 min (60-90 min) and second 30 rnin (90- 120 min) of perfusion after the addition of substrate (~-[U-'~C]lactate). The perfusion condiwas tions are described in Fig. 1. Substrate (10 mM ~-[U-'~C]lactate) added to the perfusate at 60 min and glucagon (5 pg) at 30 and 60 min after the start of the perfusion. The numbers given represent the mean i- SE for the indicated number (n) of perfusions Liver
Addition
(n)
Table 3. Effect ofglucagon andfasting on the ratio of the rate of uptake of radioactivity,from [I4C]lactate to the rate of incorporation of radioactivity into ['4C/glucose The ratios were calculated individually for each of the perfusions represented in Tables 1 and 2 as follows: (rate of [I4C]lactale uptake)/(6/5) (rate of [14C]glucoseproduction). The numbers given represent the mean f SE Liver
Addition
['4C]Lactate ~ptake/['~C]glucose produced
['4C]Glucose production
60 - 90 min
90 - 120 min
60 - 90 min
90
~
120 rnin
Fasted
None Glucagon
2.16 i 0.11 1.80 & 0.13
1.71 0.14 1.71 2 0.13
Fed
None Glucagon
4.19 2 0.31 3.06 f 0.12"
2.72 & 0.14 2.21 2 0 . 1 I b
dis. min-' (g liver)-' min-' Fasted
None Glucagon
(6) (7)
Fed
None Glucagon
(8) (9)
1150 i 140 1492 2 153
1098 2 147 952 -I- 95 a
a
352 -I- 33 613 -I- 52"
447 2 28 605 & 57b
P < 0.01 versuscontrol. P < 0.05 versus control.
P < 0.01 versus control. P < 0.02 versus control
Table 4. Eflect of glucagon and ,fasting on I4Co2 production and consumption The I4CO2 produced represents the total amount of 14C02 produced as subduring 180 min of perfusion with 10 mM ~-[U-'~C]lactate strate. The rate of oxygen consumption was calculated from the difference between the pre-liver and post-liver perfusatc oxygen tensions and the flow rate at 90 min of perfusion as described in Methods and Materials. The perfusion conditions are the same as those described in Fig. 1. Substrate was added to the perfusate at 60 min and glucagon ( 5 pg) at 30 and 60 min after the start of the perfusion. The numbers given represent the mean -I- SE for the indicated number (n) of perfusions 0 2
Table 2. Effect ofglucagon andfasting on the rate of ['4C]lactate uptake in isolated perfused guinea pig livers The rates of lactate uptake were calculated from the amount of [14C]lactate(see Methods and Materials) that disappeared from the perfusate during the indicated times. The values were obtained under the conditions described in Table 1. The numbers given represent the mean & SE for the indicated number (n) of perfusions Liver
Addition
(n)
[l4C]Lactate uptake
60 - 90 rnin
90 - 120 min
Liver
Addition
(n)
I4CO2 produced
0 2
consumption
dis. min-' (mg liver)
pmol min-' (g liver)-'
1.74 -I- 0.17 1.94 & 0.15
dis. min-' (g liver)-' min-' Fasted
None Glucagon
(6) (7)
2895 i 212 3108 i- 129
2142 5 153 1924 k 188
Fed
None Glucagon
(8) (9)
1707 f 94 2206 i- 136"
1427 2 119 1564 119
a
Fasted
None Glucagon
(6) (6)
32 i 2 29 k 2
Fed
None Glucagon
(8) (9)
33 2 27 2 5"
P < 0.05 versuscontrol a
livers except at 120 and 150 rnin of perfusion (Fig. 1B). Likewise, the rate was variably affected. In the first 30 min after substrate addition, glucagon caused a significant increase in the rate of [14C]lactate uptake. This increase was not evident, however, in the second 30 rnin following substrate addition (Table 2). Relationshk of the Rates of (14C]Lactate Uptake to ('4C]Glucose Production
One measure of the efficiency of gluconeogenesis from a particular substrate would be a measure of that portion of the substrate that is converted to glucose with respect to time. In the present case, this
*
0.94 i- 0.07 1.26 2 0.08
P < 0.05 versus control. P < 0.02 versuscontrol.
relationship may be described by the ratio of the rate of [14C]lactate uptake to the rate of [14C]glucose production. When this ratio was determined for fasted livers, the value was found to be approximately 2 in the absence or presence of glucagon (Table 3 ) . By contrast, in fed livers without added glucagon, this ratio was almost twice as high as that observed in the fasted livers. Furthermore, addition of glucagon significantly increased the efficiency of gluconeogenesis from lactate in the fed livers as indicated by the decreased ratios observed under these conditions (Table 3 ) .
570
I4CO2 Production, 0 2 Utilization and Oxidation-Reduction States
Since the above data indicate that in fed livers glucagon increased the efficiency of conversion of lactate to glucose under conditions where gluconeogenesis was stimulated (Fig. 1 B, Table 2), it is reasonable to assume that the conversion of lactate to other products should be decreased under these same conditions. The 18% decrease in the amount of 14C02 produced under these conditions compared to control conditions (Table 4) is in agreement with such an assumption. In a similar manner, I4CO2 production was found to be depressed during glucagonstimulated gluconeogenesis from [U-'4C]xylitol in fasted perfused rat liver [6]. The approximate doubling of the rate of gluconeogenesis that resulted from fasting (Table 1) was accompanied by a correspondingly almost doubled rate of 0 2 consumption (Table 4). In parallel fashion, the increased rate of glucagon-stimulated gluconeogenesis observed in fed livers (Table 1) was accompanied by a 34% increase in the rate of oxygen consumption (Table 4). Alterations of the hepatic oxidation-reduction state, especially the intramitochondrial oxidation-reduction state. can affect gluconeogenesis [lo]. Glucagon, however, did not significantly alter the oxidationreduction state in either the cytosol or mitochondria of fed or fasted guinea pig livers as indicated by unaltered lactate : pyruvate and 3-hydroxybutyrate : acetoacetate ratios, respectively (unpublished observations). Although these ratios were determined from the concentrations of the components of the redox couples found in the perfusate after 90 min of perfusion, they were assumed to reflect the ratios that would be present in the liver [ l l ] . These results can be interpreted to indicate that the effect of glucagon on gluconeogenesis in the guinea pig is not mediated through a change in the hepatic redox state. DISCUSSION It has previously been shown, in vivo, that when guinea pigs are fasted, an approximately 3-fold increase occurs in the incorporation of radioactivity from lactate into glucose [l]. The present results also show that the rate of glucogenesis in fasted isolated perfused livers is 2 - 3-fold greater than the corresponding rate in fed livers (Table 1). This higher gluconeogenic activity in the fasted compared to the fed state, coupled with the observations that glucagon does not further stimulate gluconeogenesis in fasted guinea pig livers, can be interpreted to indicate that fasting effects a maximum increase in this process in the guinea pig that cannot be further stimulated by glucagon. Fasted guinea pig livers are responsive to glucagon,
t-ffect o f Glucagon on Gluconcogenesis in the Guinea-pig
however, since other metabolic pathways such as ketogenesis and ureogenesis are stimulated by glucagon in fasted livers [l]. The use of radioactive lactate as substrate clearly indicates that the glucagon-stimulated increase in gluconeogenesis observed in fed livers (Fig. 1B, Table 1) was due to an increase in gluconeogenesis from added substrate. The increment increase caused by glucagon in the rate of radioactive glucose production in the first 30 min following substrate addition, 291 dis. min-' (g liver)-' min-' calculated from the data in Table 1, can be more than accounted for by the increment increase in the rate of radioactive lactate uptake during this same 30-min period, 580 dis. min-' (g liver)-' min - I , calculated from the data in Table 2. This suggests that glucagon may be acting to increase gluconeogenesis in fed livers by increasing the rate of lactate uptake. Such a mechanism would be analogous to the one proposed for the action of glucagon on gluconeogenesis from lactate in perfused rat liver [12]. The lack of a significant effect of glucagon on the rate of lactate uptake during the second 30 min following substrate addition (Table 2), when there is a glucagon-stimulated rate of glucose production (Table I), however, argues against such a mechanism. In this regard, the calculated ratios shown in Table 3 suggest that glucagon may be acting to increase the rate of gluconeogenesis from lactate in fed livers by redirecting the fate of this substrate from other products toward glucose. The decreased accumulation of I4CO2 that occurs (Table 4) coincident with glucagon stimulation of gluconeogenesis (Fig. 1 A, Table 1) is supportive of such a mechanism. This type of mechanism is similar to the mechanism by which glucagon appears to stimulate gluconeogenesis from xylitol is fasted perfused rat liver [6]. It is also possible to use this type of mechanism to explain the stimulation of gluconeogenesis from lactate by glucagon in fed perfused rat livers. If the data presented by Exton et al. [I21 on the incorporation of I4C into glucose from ~-[U-'~C]lactate by perfused rat livers are used to calculate the ratio ; lactate carbon atoms utilized/ 100 g body weight: carbon atoms incorporated into glucose/100 g body weight, the values obtained are 3.44,2.04 and 1.94 for fed, fed plus glucagon, and fasted livers, respectively. These values are strikingly similar to those observed with guinea pig livers under similar conditions (Table 3). It would appear, therefore, that glucagon may stimulate gluconeogenesis from lactate in fed isolated perfused livers of both these species by a dual mechanism: (a) an increase in substrate uptake and (b) a redirection of the fate of that substrate. This does not imply, however, that gluconeogenesis in both species is affected identically by glucagon since it would appear that the latter of these mechanisms may be more important in the guinea pig. Also, a difference in the magnitude of the response
571
D. E. Cook
to glucagon by fed livers exists between these species since glucagon stimulated gluconeogenesis in fed rat livers to about the same level as that found in fasted livers [12], a situation that was not observed in the present experiments with fed guinea pig livers. These investigations were supported by National Institutes of Health grant AM 17400 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. The author gratefully acknowledges the excellent technical assistance of Margery Fienhold.
REFERENCES 1. Soling, H.-D., Willms, B., Kleineke, J. & Gehloff, M. (1970) Eur. J. Biochem. 16, 289- 302. 2. Tilghman, S. M., Hanson, R. W. & Ballard, F. J. (1976) in Gluconeogenesis: Its Regulation in Mammalian Species (Hanson, R. W. & Mehlman, M. A,, eds) pp. 47-91, John Wiley & Sons, New York.
3. Hanson, R. W. & Garber, A. J. (1972) Am. J . Clin. Nutr. 25, 3010-1021. 4. Hanson, R. W. (1974) Nutr. Rev. 32, 1- 8. 5. Arinze, I. J. & Rowley, D. L. (1975) Biochem. J . 152, 393-399. 6. Blair, J. B., Cook, D. E. & Lardy, H. A. (1973) J. Bid. Chem. 248, 3601 - 3607. 7. Cook, D. E., Blair, J. B. & Lardy, H. A. (1973) J . Biol. Chem. 248, 5272- 5277. 8. Exton, J. H. & Park, C. R. (1967) J. B i d . Chern. 242, 26222632. 9. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 10. Soling, H.-D. & Kleineke, J. (1976) in Gluconeogenesis: Its Regulation in Mammalian Species (Hanson, R. W. & Mehlman, M. A,, eds) pp. 369-462, John Wiley & Sons, New York. 11. Willms, B., Kleineke, J. & Soling, H.-D. (1971) in Regulation oJ’Gluconeogenesis (Soling, H.-D. & Willms, B., eds) pp. 103113, Academic Press, New York. 12. Exton, J. H., Corbin, J. G. & Harper, S. C. (1972) J . Biol. Chem. 247,4996-5003.
D. E,. Cook, Department of Biochemistry, University of Nebraska Medical Center, 42nd Street and Dewey Avenue, Omaha, Nebraska, U.S.A. 68 105
Gluconeogenesis in the Guinea Pig Effect of Glucagon on Gluconeogenesis from Lactate by Isolated Perfused Guinea-pig Liver David E COOK Department of Biochemistry, University of Nebraska Medical Center, Omaha (Received December 31, 1976)
Gluconeogenesis was stimulated by glucagon in fed but not fasted isolated perfused guinea pig livers. Both the amount and the rate of incorporation of radioactivity into glucose from ~-[U-'~C]lactatewere inci-eased in fed livers by the addition of glucagon to the perfusate. The glucagon-stimulated increase in gluconeogenesis was accompanied by an increase in oxygen consumption, an increase in the amount of lactate carbon converted to glucose and a decrease in the amount of lactate carbon converted to COz. The results are interpreted to indicate that glucagon affects gluconeogenesis from lactate in fed livers by redirecting the fate of substrate from other products toward glucose.
Much of our knowledge concerning the hormonal control of hepatic gluconeogenesis, especially by glucagon, has been derived from studies with rats, perfused rat livers or isolated hepatocytes prepared from rat livers. The general applicability of such knowledge, however, remains uncertain. For example, Soling et al. [l] have demonstrated that, unlike its positive effect on gluconeogenesis in the rat, glucagon does not stimulate gluconeogenesis in fasted isolated perfused guinea pig liver. In addition, the rat and a few other mammalian species have liver phosphoenolpyruvate carboxykinase activity located almost entirely in the cytosol. Most mammalian species including man and guinea pig, however, have an equal distribution of distinct mitochondria1 and cytosolic phosphoPnolpyruvate carboxykinase activity (cf. [2]). Consequently, the importance of studying the control of hepatic gluconeogenesis in an animal model such as the guinea pig has often been emphasized [I, 3 - 51. These and other considerations led to the present investigations on the effects of glucagon on hepatic gluconeogenesis in the guinea pig. MATERIALS AND METHODS Male English short-hair guinea pigs (CAMM Research Institute, Wayne, N.J.) weighing 229 to 349 g Enzyme. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1. 1.32).
were allowed to feed ad lib. or fasted 48 h prior to perfusion. The isolated livers were perfused at 37 "C with a Krebs-Ringer bicarbonate medium containing 3 % fatty-acid-free bovine serum albumin (Miles Laboratories, Elkhart, Ind.) and 20 % washed bovine erythrocytes in a recirculating system as previously described [6]. In this system the livers were perfused for 60 min prior to the addition of substrate. The substrate was 10 mM L-lactate (Schwartz-Mann, (AmerOrangeburg, N.J.) containing ~-[U-'~C]lactate sham-Searle, Arlington Heights, Ill.) to give a specific activitity of 1600 dis. min-' pmol-'. Glucagon (a generous gift from Dr William Bromer, Eli Lilly and Co., Indianapolis, Ind.) was prepared in 3 % bovine serum albumin and kept frozen until used. When used, 5 pg of glucacon was added to the perfusate at 30 min and 60 min after the start of the perfusion [6]. Perfusate samples (2 ml) were taken at the indicated times, deproteinized, and assayed for glucose and other metabolities as previously described [6,7]. All of the radioactivity in the neutral fraction obtained by this method was found to represent glucose. In earlier evaluations of this procedure, it was determined that glucose accounted for more than 90 % of the radioactivity in the neutral fraction [7] and lactate for more than 70 % of the radioactivity in the anionic or 'lactate' fraction [6]. The radioactive anionic or 'lactate' fraction will be referred to as [14C]lactate. I4CO2 produced during the perfusion was trapped by aspirating effluent from the oxygenator through KOH and
568
Effect of Glucagon on Gluconeogenesis in the Guinea-pig 200
,
I200
oxygen tension of the samples with an IL 213 pH blood gas analyzer (Instrumentation Laboratories, Inc., Lexington, Mass.). These values, together with the flow rate of perfusate through the liver, were used to calculate the rate of oxygen consumption as previously described [8]. Scintillation counting was done in either Bray's solution [9] or Aquasol (New England Nuclear, Boston, Mass.) with a Unilux I1 scintillation counter (Nuclear Chicago) and the channels ratio method was used to correct for quenching. The significance of differences between means was established by Student's t-test from t values determined with the Monroe Alpha 325 Significance Pak program number 9212V, t-test for independent means, as supplied by Monroe, The Calculator Company. Significance is shown when P < 0.05. RESULTS
90
60
120
150
180
Time (min)
[14C]Glucose Production
B
Fed \\ \
0 60
90
120 150 Time (rnin)
180
Fig. 1. The time course (f[14C]glucose production (-) and ['4C]lactate uptake (-----) during the perfusion of guinea pig t e 100 ml qf recirculating perlivers with 10 mM ~ - [ U - ' ~ C ] l a c t ain fusate in either the absence ( 0 )or presence (Aj of added glucagon. Substrate was added at 60 min and glucagon (5 kg) at 30 and 60 min after the start of the perfusion. The curves in (A) were obtained with 48-h-fasted livers and those in (B) with fed livers. Each point represents the mean and the vertical bars f SE of 5-9 perfusions. Points identified with an asterisk are significantly different from the corresponding control (absence of glucagon) points (P ranges from < 0.05 to < 0.01)
In order to eliminate consideration of the contribution of endogenous substances to glucose production in either the absence or presence of glucagon, radioactive substrate, ~-[U-'~C]lactate, was used throughout the study. In 48-h-fasted guinea pig livers, glucagon had no significant effect on either the total amount of ['4C]glucose produced (Fig.1A) or on the rate of [14C]glucose production during the first hour after substrate addition (Table l), a period of relatively rapid ['4C]glucose production (Fig. 1 A). These observations are in agreement with the previous report that glucagon does not affect gluconeogenesis in fasted guinea pig liver [l].The time course for the production of [14C]glucosein fed livers is shown in Fig. 1 B. It can be seen that the total amount of ['4C]glucose produced by fed guinea pig livers (Fig. 1 B) is about onehalf of that produced in fasted livers (Fig. 1A). Also in contrast to fasted livers, addition of glucagon to fed livers resulted in a significant increase in the total amount of [14C]glucoseproduced (Fig. 1B) as well as an increase in the rate of ['4C]glucose production (Table 1). This glucagon-stimulated rate was more than 70 % greater in the first 30 min and 35 greater in the second 30 min following substrate addition than the corresponding rates observed in the absence of glucagon (Table 1). ['4C]Lactate Uptake
subsequently transferring the trapped 14C02 to hyamine hydroxide for scintillation counting as previously described [6]. The rate of oxygen con'sumption was determined by simultaneously sampling the perfusate just prior to entry into and just subsequent to exit from the liver and immediately determining the
Glucagon did not significantly affect the amount or rate of [14C]lactateuptake in fasted livers (Fig. 1A and Table 2, respectively). Its effect on lactate utilization by fed livers, however, was variable. The total amount of ['4C]lactate utilized was not significantly greater in glucagon-treated livers compared to control
D. E. Cook
569
Table 1. Effect of glucagon and fasting on the rate of ['4C]glucose production in isolated perfused guinea pig livers The rates of glucose production were calculated from the amount of ['4C]glucose that appeared in the perfusate during the first 30 min (60-90 min) and second 30 rnin (90- 120 min) of perfusion after the addition of substrate (~-[U-'~C]lactate). The perfusion condiwas tions are described in Fig. 1. Substrate (10 mM ~-[U-'~C]lactate) added to the perfusate at 60 min and glucagon (5 pg) at 30 and 60 min after the start of the perfusion. The numbers given represent the mean i- SE for the indicated number (n) of perfusions Liver
Addition
(n)
Table 3. Effect ofglucagon andfasting on the ratio of the rate of uptake of radioactivity,from [I4C]lactate to the rate of incorporation of radioactivity into ['4C/glucose The ratios were calculated individually for each of the perfusions represented in Tables 1 and 2 as follows: (rate of [I4C]lactale uptake)/(6/5) (rate of [14C]glucoseproduction). The numbers given represent the mean f SE Liver
Addition
['4C]Lactate ~ptake/['~C]glucose produced
['4C]Glucose production
60 - 90 min
90 - 120 min
60 - 90 min
90
~
120 rnin
Fasted
None Glucagon
2.16 i 0.11 1.80 & 0.13
1.71 0.14 1.71 2 0.13
Fed
None Glucagon
4.19 2 0.31 3.06 f 0.12"
2.72 & 0.14 2.21 2 0 . 1 I b
dis. min-' (g liver)-' min-' Fasted
None Glucagon
(6) (7)
Fed
None Glucagon
(8) (9)
1150 i 140 1492 2 153
1098 2 147 952 -I- 95 a
a
352 -I- 33 613 -I- 52"
447 2 28 605 & 57b
P < 0.01 versuscontrol. P < 0.05 versus control.
P < 0.01 versus control. P < 0.02 versus control
Table 4. Eflect of glucagon and ,fasting on I4Co2 production and consumption The I4CO2 produced represents the total amount of 14C02 produced as subduring 180 min of perfusion with 10 mM ~-[U-'~C]lactate strate. The rate of oxygen consumption was calculated from the difference between the pre-liver and post-liver perfusatc oxygen tensions and the flow rate at 90 min of perfusion as described in Methods and Materials. The perfusion conditions are the same as those described in Fig. 1. Substrate was added to the perfusate at 60 min and glucagon ( 5 pg) at 30 and 60 min after the start of the perfusion. The numbers given represent the mean -I- SE for the indicated number (n) of perfusions 0 2
Table 2. Effect ofglucagon andfasting on the rate of ['4C]lactate uptake in isolated perfused guinea pig livers The rates of lactate uptake were calculated from the amount of [14C]lactate(see Methods and Materials) that disappeared from the perfusate during the indicated times. The values were obtained under the conditions described in Table 1. The numbers given represent the mean & SE for the indicated number (n) of perfusions Liver
Addition
(n)
[l4C]Lactate uptake
60 - 90 rnin
90 - 120 min
Liver
Addition
(n)
I4CO2 produced
0 2
consumption
dis. min-' (mg liver)
pmol min-' (g liver)-'
1.74 -I- 0.17 1.94 & 0.15
dis. min-' (g liver)-' min-' Fasted
None Glucagon
(6) (7)
2895 i 212 3108 i- 129
2142 5 153 1924 k 188
Fed
None Glucagon
(8) (9)
1707 f 94 2206 i- 136"
1427 2 119 1564 119
a
Fasted
None Glucagon
(6) (6)
32 i 2 29 k 2
Fed
None Glucagon
(8) (9)
33 2 27 2 5"
P < 0.05 versuscontrol a
livers except at 120 and 150 rnin of perfusion (Fig. 1B). Likewise, the rate was variably affected. In the first 30 min after substrate addition, glucagon caused a significant increase in the rate of [14C]lactate uptake. This increase was not evident, however, in the second 30 rnin following substrate addition (Table 2). Relationshk of the Rates of (14C]Lactate Uptake to ('4C]Glucose Production
One measure of the efficiency of gluconeogenesis from a particular substrate would be a measure of that portion of the substrate that is converted to glucose with respect to time. In the present case, this
*
0.94 i- 0.07 1.26 2 0.08
P < 0.05 versus control. P < 0.02 versuscontrol.
relationship may be described by the ratio of the rate of [14C]lactate uptake to the rate of [14C]glucose production. When this ratio was determined for fasted livers, the value was found to be approximately 2 in the absence or presence of glucagon (Table 3 ) . By contrast, in fed livers without added glucagon, this ratio was almost twice as high as that observed in the fasted livers. Furthermore, addition of glucagon significantly increased the efficiency of gluconeogenesis from lactate in the fed livers as indicated by the decreased ratios observed under these conditions (Table 3 ) .
570
I4CO2 Production, 0 2 Utilization and Oxidation-Reduction States
Since the above data indicate that in fed livers glucagon increased the efficiency of conversion of lactate to glucose under conditions where gluconeogenesis was stimulated (Fig. 1 B, Table 2), it is reasonable to assume that the conversion of lactate to other products should be decreased under these same conditions. The 18% decrease in the amount of 14C02 produced under these conditions compared to control conditions (Table 4) is in agreement with such an assumption. In a similar manner, I4CO2 production was found to be depressed during glucagonstimulated gluconeogenesis from [U-'4C]xylitol in fasted perfused rat liver [6]. The approximate doubling of the rate of gluconeogenesis that resulted from fasting (Table 1) was accompanied by a correspondingly almost doubled rate of 0 2 consumption (Table 4). In parallel fashion, the increased rate of glucagon-stimulated gluconeogenesis observed in fed livers (Table 1) was accompanied by a 34% increase in the rate of oxygen consumption (Table 4). Alterations of the hepatic oxidation-reduction state, especially the intramitochondrial oxidation-reduction state. can affect gluconeogenesis [lo]. Glucagon, however, did not significantly alter the oxidationreduction state in either the cytosol or mitochondria of fed or fasted guinea pig livers as indicated by unaltered lactate : pyruvate and 3-hydroxybutyrate : acetoacetate ratios, respectively (unpublished observations). Although these ratios were determined from the concentrations of the components of the redox couples found in the perfusate after 90 min of perfusion, they were assumed to reflect the ratios that would be present in the liver [ l l ] . These results can be interpreted to indicate that the effect of glucagon on gluconeogenesis in the guinea pig is not mediated through a change in the hepatic redox state. DISCUSSION It has previously been shown, in vivo, that when guinea pigs are fasted, an approximately 3-fold increase occurs in the incorporation of radioactivity from lactate into glucose [l]. The present results also show that the rate of glucogenesis in fasted isolated perfused livers is 2 - 3-fold greater than the corresponding rate in fed livers (Table 1). This higher gluconeogenic activity in the fasted compared to the fed state, coupled with the observations that glucagon does not further stimulate gluconeogenesis in fasted guinea pig livers, can be interpreted to indicate that fasting effects a maximum increase in this process in the guinea pig that cannot be further stimulated by glucagon. Fasted guinea pig livers are responsive to glucagon,
t-ffect o f Glucagon on Gluconcogenesis in the Guinea-pig
however, since other metabolic pathways such as ketogenesis and ureogenesis are stimulated by glucagon in fasted livers [l]. The use of radioactive lactate as substrate clearly indicates that the glucagon-stimulated increase in gluconeogenesis observed in fed livers (Fig. 1B, Table 1) was due to an increase in gluconeogenesis from added substrate. The increment increase caused by glucagon in the rate of radioactive glucose production in the first 30 min following substrate addition, 291 dis. min-' (g liver)-' min-' calculated from the data in Table 1, can be more than accounted for by the increment increase in the rate of radioactive lactate uptake during this same 30-min period, 580 dis. min-' (g liver)-' min - I , calculated from the data in Table 2. This suggests that glucagon may be acting to increase gluconeogenesis in fed livers by increasing the rate of lactate uptake. Such a mechanism would be analogous to the one proposed for the action of glucagon on gluconeogenesis from lactate in perfused rat liver [12]. The lack of a significant effect of glucagon on the rate of lactate uptake during the second 30 min following substrate addition (Table 2), when there is a glucagon-stimulated rate of glucose production (Table I), however, argues against such a mechanism. In this regard, the calculated ratios shown in Table 3 suggest that glucagon may be acting to increase the rate of gluconeogenesis from lactate in fed livers by redirecting the fate of this substrate from other products toward glucose. The decreased accumulation of I4CO2 that occurs (Table 4) coincident with glucagon stimulation of gluconeogenesis (Fig. 1 A, Table 1) is supportive of such a mechanism. This type of mechanism is similar to the mechanism by which glucagon appears to stimulate gluconeogenesis from xylitol is fasted perfused rat liver [6]. It is also possible to use this type of mechanism to explain the stimulation of gluconeogenesis from lactate by glucagon in fed perfused rat livers. If the data presented by Exton et al. [I21 on the incorporation of I4C into glucose from ~-[U-'~C]lactate by perfused rat livers are used to calculate the ratio ; lactate carbon atoms utilized/ 100 g body weight: carbon atoms incorporated into glucose/100 g body weight, the values obtained are 3.44,2.04 and 1.94 for fed, fed plus glucagon, and fasted livers, respectively. These values are strikingly similar to those observed with guinea pig livers under similar conditions (Table 3). It would appear, therefore, that glucagon may stimulate gluconeogenesis from lactate in fed isolated perfused livers of both these species by a dual mechanism: (a) an increase in substrate uptake and (b) a redirection of the fate of that substrate. This does not imply, however, that gluconeogenesis in both species is affected identically by glucagon since it would appear that the latter of these mechanisms may be more important in the guinea pig. Also, a difference in the magnitude of the response
571
D. E. Cook
to glucagon by fed livers exists between these species since glucagon stimulated gluconeogenesis in fed rat livers to about the same level as that found in fasted livers [12], a situation that was not observed in the present experiments with fed guinea pig livers. These investigations were supported by National Institutes of Health grant AM 17400 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. The author gratefully acknowledges the excellent technical assistance of Margery Fienhold.
REFERENCES 1. Soling, H.-D., Willms, B., Kleineke, J. & Gehloff, M. (1970) Eur. J. Biochem. 16, 289- 302. 2. Tilghman, S. M., Hanson, R. W. & Ballard, F. J. (1976) in Gluconeogenesis: Its Regulation in Mammalian Species (Hanson, R. W. & Mehlman, M. A,, eds) pp. 47-91, John Wiley & Sons, New York.
3. Hanson, R. W. & Garber, A. J. (1972) Am. J . Clin. Nutr. 25, 3010-1021. 4. Hanson, R. W. (1974) Nutr. Rev. 32, 1- 8. 5. Arinze, I. J. & Rowley, D. L. (1975) Biochem. J . 152, 393-399. 6. Blair, J. B., Cook, D. E. & Lardy, H. A. (1973) J. Bid. Chem. 248, 3601 - 3607. 7. Cook, D. E., Blair, J. B. & Lardy, H. A. (1973) J . Biol. Chem. 248, 5272- 5277. 8. Exton, J. H. & Park, C. R. (1967) J. B i d . Chern. 242, 26222632. 9. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 10. Soling, H.-D. & Kleineke, J. (1976) in Gluconeogenesis: Its Regulation in Mammalian Species (Hanson, R. W. & Mehlman, M. A,, eds) pp. 369-462, John Wiley & Sons, New York. 11. Willms, B., Kleineke, J. & Soling, H.-D. (1971) in Regulation oJ’Gluconeogenesis (Soling, H.-D. & Willms, B., eds) pp. 103113, Academic Press, New York. 12. Exton, J. H., Corbin, J. G. & Harper, S. C. (1972) J . Biol. Chem. 247,4996-5003.
D. E,. Cook, Department of Biochemistry, University of Nebraska Medical Center, 42nd Street and Dewey Avenue, Omaha, Nebraska, U.S.A. 68 105
