- Email: [email protected]
Lactate Ratios
Gluconeogenesis in Isolated Chicken Hepatocytes: Effect of Fatty Acids, j3-Hydroxybutrate, Ethanol, and Various Pyruvate/Lactate Ratios P. SCHULTZ and S. P. MISTRY Laboratory of Nutritional Biochemistry, Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication February 11, 1980)
1981 Poultry Science 6 0 : 6 5 3 - 6 5 8 INTRODUCTION
An important consideration in the use of isolated parenchymal cells for studies on gluconeogenesis is the control of the intracellular oxidation-reduction states. The importance of the redox state is in its determination of the direction of certain common reactions for glycolysis and gluconeogenesis. Lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase catalyze reversible equilibrium reactions common to both glycolysis and gluconeogenesis; therefore, the influence of the oxidation-reduction potential of NAD + -couple is important in gluconeogenesis. Furthermore, the oxidation-reduction state of NAD + -couple in mitochondria can play a significant role in determining the relative productions of oxalacetate and malate. Consequently, malate and aspartate transport into cytosol during gluconeogenesis could be influenced by the mitochondrial oxidation-reduction state. Hydrogen resulting from mitochondrial oxidation could be made available in the cytosol, by the malate-aspartate shuttle, for the reduction of 1,3-diphosphoglycerate to glyceraldehyde-3phosphate. In a preceding paper (Schultz and Mistry, 1981), we described a technique for the isolation of chicken liver cells and reported certain aspects of gluconeogenesis. In the present study we have examined the effect of fatty acids, pMiydroxybutyrate, ethanol, and various ratios
of pyruvate/lactate on glucose synthesis in chicken hepatocytes. MATERIALS AND METHODS
Animals. Crossbred chicks (Gallus domesticus: New Hampshire male X Columbian female) were placed in raised wire-floor cages with adequate water and heating and fed ad libitum a corn-based diet (Shen and Mistry, 1977) until the day of sacrifice. The chicks were used when they were about 50 days old. Chemicals and Enzymes. The chemicals, enzymes, and hormones used in this study were: sodium pentobarbital from Holmes Serum Company, Inc.; acetoacetate, bovine serum albumin, glucose oxidase reagent, glucagon, (3-hydroxybutyrate, oleate, L-lactate, and pyruvate from Sigma Chemical Company; collagenase Type II from Worthington Biochemical Corporation; and Longdwell catheter (teflon) from Becton, Dickinson, and Company. Isolation of Liver Parenchymal Cells, Incubation Medium, and Assays. These are described in detail in our preceding paper (Schultz and Mistry, 1981). RESULTS AND DISCUSSION The Effect of Pyruvate/Lactate Ratios. Figure 1 shows the effect of various pyruvate/lactate ratios on the rate of glucose syn-
653
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
ABSTRACT The effect of fatty acids, 0-hydroxybutyrate, ethanol, and different pyruvate/lactate ratios on gluconeogenesis in isolated chicken hepatocytes was investigated. Gluconeogenesis was significantly affected by a change in the oxidation-reduction (pyruvate/lactate) ratio, and this effect was greater than could be accounted for by the additive effects of these substrates. Substituting lactate with nongluconeogenic substrates, such as/3-hydroxybutyrate or ethanol, increased the formation of glucose by 80 and 200 %, respectively, demonstrating the beneficial effect of the increased reducing equivalents in the hepatocytes. Oleic acid per se had no effect but when added, complexed with albumin, it had a negative effect on gluconeogenesis. (Key words: chicken hepatocytes, gluconeogenesis, fatty acids, pyruvate/lactate ratios)
SCHULTZ AND MISTRY
654
3 £? to to
005
\n
8
v 01
LL
.2
X
.9
o u
.6
~i
CD to
to
Pyruvate 5 5 5 5 Lactate - 1 2 4
o Q Qdl - - mM 1 2 4 mM
FIG. 1. The effect of various pyruvate/lactate ratios on gluconeogenesis. Data are expressed as the means of four experiments + SEM (vertical lines). Significance of difference (P) from the (-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
c
(UU/
thesis by isolated chicken hepatocytes; the indicated substrate concentrations are those at the beginning of the incubations. It can be seen that the rate of gluconeogenesis was affected by varying the ratio of the oxidized/reduced substrate. Furthermore, the increase in glucose synthesis was greater in the presence of both lactate and pyruvate than with the individual substrates at the same concentration. This effect has not been observed in the perfused rat liver (Ross et al, 1967a; Exton and Park, 1969) or in isolated rat hepatocytes (Garrison and Haynes, 1973). When as little as 1 mM lactate was included in the medium containing pyruvate, a significant (P<.005) increase in gluconeogenesis was observed. The metabolism of lactate provides NADH in the cytosol (Krebs et al, 1967), and the reduced pyridine nucleotide is used for gluconeogenesis at the glyceraldehyde-3 -phosphate dehydrogenase step. Thus, the stimulatory effect of lactate on gluconeogenesis from pyruvate could be due to the reducing equivalents supplied by lactate. The Effect of (5-Hydroxybutyrate and Ethanol. Since the metabolism of nongluconeogenic substrates such as /3-hydroxybutyrate and ethanol also provide mitochondrial
I/)
5
LO
$
1.2 -
-P Q)
~ 9
£
I/)
^ ^
,n
8?-?8
o^a.^ .L
±
^
1
o
CJ
<5
6
• i
en O
-J
E
n Pyruvat :e 5 5 5 5 5 mM /5-OH-Buty - 1 2 3 10 mM FIG. 2. The effect of (3-hydroxybutyrate on gluconeogenesis. Data are expressed as the means of four experiments ± SEM (vertical lines). Significance of difference (P) from the t-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
.3
lO
(Zahlten et al, 1973; Arinze et al., 1973) and cytosolic (Williamson et al, 1969) NADH, respectively, the effect of these metabolites on glucose synthesis from pyruvate was tested. Figures 2 and 3 show the effect of /3-hydroxybutyrate and ethanol, respectively, on gluconeogenesis from pyruvate as the substrate. Assuming that the rate of gluconeogenesis from pyruvate was limited by a general deficiency of reducing equivalents in the mitochondria and the cytosol, then indeed (3-hydroxybutyrate and ethanol proved to be quite effective; gluconeogenesis was stimulated by about 80 and 200%, respectively. Increased glucose production from pyruvate by /3-hydroxybutyrate and ethanol could reflect increased availability of NADH for the reduction of 1,3-diphosphoglycerate. Our results are in agreement with those obtained with isolated rat hepatocytes (Zahlten et al., 1973; Garrison and Haynes, 1973), with the perfused rat (Arinze et al., 1973; Kaden et al., 1969), and guinea pig liver (Arinze et al., 1973), but not with the data secured with rat kidney cortex slices, mouse liver slices, and pigeon liver hemogenates (Krebs
GLUCONEOGENESIS IN ISOLATED CHICKEN HEPATOCYTES
^ 1.9
Q
8 oo
£ 1.2-
Q- Q_
O)
0CO .9 .6
o .3 £ Pyruvate Ethanol
5 5 5 mM - 1 3 mM
FIG. 3. The effect of ethanol on gluconeogenesis. Data are expressed as the means of four experiments + SEM (vertical lines). Significance of difference (P) from the t-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
et al, 1967), where ethanol had no effect on gluconeogenesis from pyruvate. In perfused rat liver (Williamson et al, 1969) and in the intact rat (Stubbs et al, 1972), supplying ethanol leads to a rapid decrease in NAD+/NADH ratios in cytosol and mitochondria. A decrease in this ratio should inhibit the conversion of a NAD + -linked substrate such as lactate to glucose. This has been shown to occur in the perfused guinea pig liver (Arinze et al, 1973), but the opposite was true in the case of the perfused rat liver. This difference in response has been explained on the basis of the intracellular location of hepatic phosphoenolpyruvate carboxykinase (PEPCK) in the two species. Since chicken liver also has substantial amounts of PEPCK in the mitochondria, it is possible that gluconeogenesis from lactate would be inhibited in the presence of ethanol as was the case in the guinea pig liver (Arinze et al, 1973). However, with substrates at the same
oxidation level as glucose or with substrates having a net requirement for NADH during gluconeogenesis, ethanol should stimulate glucose production. This was the case in the perfused rat liver (Arinze et al, 1973; Kaden et al, 1969) in the perfused guinea pig liver (Arinze et al, 1973), in isolated rat hepatocytes (Zahlten et al, 1973), and also in the present study with isolated chicken hepatocytes. Fatty Acids and Glucagon in Gluconeogenesis. Table 1 shows the effect of free and bound fatty acids on gluconeogenesis. Glucagon stimulated glucose production. Under our experimental conditions, free fatty acids (FFA) had no effect on gluconeogensis, but when added complexed with albumin (OBA), they inhibited glucose production. Such a decrease in gluconeogenesis has been observed in the perfused guinea pig liver (Soling et al, 1970), but the opposite was true in the case of the perfused rat liver (Ross etal, 1967b; Williamson et al, 1966; Williamson, 1967). This inhibition by OBA of gluconeogensis in chicken hepatocytes was also observed in the presence of glucagon (Table 1). It is possible that the inhibition was due to an unspecific effect of OBA. Hence, we measured the oxygen uptake of the cells at the beginning and end of the 30 min incubation period. As seen from Table 2, OBA in the presence or absence of glucagon did not inhibit oxygen uptake but did inhibit glucose production from pyruvate (Table 1). In fact, with increasing levels of OBA there was a progressive increase in oxygen consumption even after 30 min of incubation (Table 2), which suggests that OBA was metabolized. However, FFA did not seem to be utilized, since the oxygen consumption after 30 min was consistently lower than with pyruvate alone; very likely this was because FFA were supplied in an unphysiological state. Therefore, the decrease in gluconeogenesis observed in the presence of OBA was not due to an unspecific effect of the fatty acids on cell metabolism but very likely was the result of the removal of oxalacetate as maltate because of the increased generation of NADH during j3-oxidation. This effectively depletes oxalacetate, the substrate for PEP synthesis essentially taking place in liver mitochondria of this species. Struck et al (1965) have suggested that the stimulatory effect of glucagon on gluconeogenesis was the result of increased fatty acid oxidation due to the activation of the
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
o u Z5
655
656
SCHULTZ AND MISTRY TABLE 1. The effect of free and bound fatty acids on gluconeogenesis
Treatment
mM
Glucose produced (jumole/g wet cells/min)
Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate
4 10~ 5 .4 1.0 .4 1.0 .4 1.0 .4 1.0
.36+ . 0 1 ' .40 ± .03 .36 ± .02 .32 ± .02 .28 ± .01 .21 ± .01 .35 + .03 .34 ± .02 .34 ± .03 .25 + .01
+ FFA + FFA + OBA + OBA
(a)* (b) (a) (a,c) (c)
(d) (a,b) (a) (a) (d)
Each value represents the mean ± SEM of six experiments.
2
Different letters in parentheses indicate valid statistical differences (P<.01 from the t-test) between treat:nts. 3 4
FFA = free fatty acids (Oleic acid, approximately 99% pure). OBA = Oleic acid bound to albumin; prepared as described by Ross et al. (1967b).
hormone-sensitive lipase. Glucagon and cyclic AMP activate lipolysis in adipose tissue, and Claycomb and Kilsheimer (1969) and others have shown an effect of glucagon on lipolysis also in liver preparations. Glucagon was ketogenic in liver slices and in the perfused liver (Menahan et al., 1968; Regen and Terrell, 1968), and this was ascribed to a stimulation of endogenous lipid catabolism. Addition of fatty
acids to rat liver and kidney preparations did stimulate gluconeogenesis under certain conditions (Williamson et al, 1966; Williamson, 1967). Despite the attractiveness of this hypothesis, the following observations indicate that the stimulatory effect of glucagon on gluconeogenesis was not secondary to lipolysis. First, our data show that in the chicken, glucagon stimulated gluconeogenesis whereas
TABLE 2. The effect of free and bound fatty acids on oxygen uptake Oxygen uptake umole
o2/g wet
cells/min
% of Control
Treatment
mM
0 min
30 min
0 min
30 min
Control (cells alone) Pyruvate (substrate) Pyruvate + glucagon Pyruvate + FFA* Pyruvate + FFA Pyruvate + FFA Pyruvate + OBA a Pyruvate + OBA Pyruvate + OBA Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon +
4 10~ s .4 1.0 2.0 .4 1.0 2.0 .4 1.0 2.0 .4 1.0
.79 1.42 1.40 1.54 1.68 1.15 1.44 1.75 2.02 1.50 1.27 1.56 1.48 1.99
.79 1.34 1.55 .91 .91 .92 1.33 1.67 1.79 1.67 1.61 1.60 1.95 1.80
100 179 176 193 212 145 181 220 254 189 159 197 186 251
100 170 197 115 115 116 168 212 227 212 203 203 247 228
FFA FFA FFA OBA OBA
Abbreviations are given under Table 1.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
1
(substrate) + glucagon + FFA 3 + FFA + OBA4 + OBA + glucagon + glucagon + glucagon + glucagon
GLUCONEOGENESIS IN ISOLATED CHICKEN HEPATOCYTES
Fourth, the change in the level of gluconeogenic intermediates induced by glucagon in the perfused rat liver (Exton and Park, 1969; Exton et al, 1969) differed markedly from that produced by unbound fatty acids (Williamson et al, 1966; Williamson, 1967). The stimulatory effect of unbound fatty acids on gluconeogenesis is probably an artifact, since it was not observed when the fatty acids were added bound to albumin or when added in physiological concentrations (Exton et al, 1970). Fifth, as shown by Ross et al (1967b) in the perfused rat liver, the gluconeogenic effect of unbound oleate was additive with that of glucagon. This indicates separate mechanisms of action of glucagon and unbound fatty acids and that the hormone does not act by increasing the supply of fatty acids for oxidation. REFERENCES Arinze, I. J., A. J. Garber, and R. W. Hanson, 1973. The regulation of gluconeogenesis in mammalian liver. J. Biol. Chem. 248:2266-2274. Claycomb, W. C , and G. S. Kilsheimer, 1969. Effect of glucagon, adenosine 3',5' monophosphate and theophylline on free fatty acid release by rat liver slices and on tissue levels of coenzyme A esters. Endocrinology 84:1179-1183. Exton, J. H., and J. G. Corbin, and C. R. Park, 1969. Control of gluconeogenesis in liver. IV. Differ-
ential effects of fatty acids and glucagon on ketogenesis and gluconeogenesis in the perfused rat liver. J. Biol. Chem. 244:4095-4102. Exton, J. H., L. E. Mallette, L. S. Jefferson, E.H.A. Wong, N. Friedmann, T. B. Miller, and C. R. Park, 1970. The hormonal control of hepatic gluconeogenesis. Recent. Prog. Horm. Res. 26:411-461. Exton, J. H., and C. R. Park, 1969. Control of gluconeogenesis in liver. III. Effect of L-lactate, pyruvate, fructose, glucagon, epinephrine and adenosine 3', 5'-monophosphate on gluconeogenic intermediates in the perfused rat liver. J. Biol. Chem. 244:1424-1433. Garrison, J. C , and R. C. Haynes, 1973. Hormonal control of glycogenolysis and gluconeogenesis in isolated rat liver cells. J. Biol. Chem. 248:5333-5343. Kaden, M., N. W. Oakley, and J. B. Field, 1969. Effect of alcohol on gluconeogenesis using the isolated rat liver perfusion technique. Amer. J. Physiol. 216:756-763. Krebs, H. A., T. Gascoyne, and B. M. Notton, 1967. Generation of extramitochondrial reducing power in gluconeogenesis. Biochem. J. 102:275—282. Menahan, L. A., B. D. Ross, and O. Wieland, 1968. Acetyl CoA level in perfused rat liver during gluconeogenesis and ketogenesis. Biochem. Biophys. Res. Commun. 30:38—44. Regen, D. M., and E. G. Terrell, 1968. Effects of glucagon and fasting on acetate metabolism in perfused rat liver. Biochem. Biophys. Acta 170:95-111. Ross, B. D., R. Hems, R. A. Freedland, and H. A. Krebs, 1967b. Carbohydrate metabolism of the perfused rat liver. Biochem. J. 105:869—875. Ross, B. D., R. Hems, and H. A. Krebs, 1967a. The rate of gluconeogenesis from various precursors in the perfused rat liver. Biochem. J. 102:942-951. Schultz, P., and S. P. Mistry, 1981. A technique for the isolation of chicken hepatocytes and their use in a study of gluconeogenesis. Poultry Sci. 60:000.
Shen, C. S., and S. P. Mistry, 1977. Activities of pyruvate and propionyl coenzyme A carboxylase in chicken tissues during normal growth and biotin deficiency. Poultry Sci. 56:1900-1903. Soling, H. D., B. Willms, J. Kleineke, and M. Gelhoff, 1970. Regulation of gluconeogenesis in the guinea pig liver. Europ. J. Biochem. 16:289—302. Struck, E., J. Ashmore, and O. Wieland, 1965. Stimulierung der gluconeogenese durch langkettige fettsauren und glucagon. Biochem. Z. 343: 107-110. Stubbs, M., R. L. Veech, and H. A. Krebs, 1972. Control of the redox state of the nicotinamideadenine dinucleotide couple in rat liver cytoplasm. Biochem. J. 126:59-65. Williamson, J. R., 1967. Effects of fatty acids, glucagon and anti-insulin serum on the control of gluconeogenesis and ketogenesis in rat liver. Adv. Enzyme Regul. 5:229-255. Williamson, J. R., R. A. Kreisberg, and P. W. Felts, 1966. Mechanism for the stimulation of gluconeogenesis by fatty acid in perfused rat liver. Proc. Nat. Acad. Sci. 56:247-254.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
OBA had the opposite effect. This was the case also in perfused guinea pig liver (Soling et al, 1970). Second, Exton et al. (1969) have shown that glucagon frequently had no effect on ketogenesis even though it markedly stimulated glycogenolysis and gluconeogenesis. Compared with the effect of exogenous fatty acids, the action of the hormone on ketogenesis appeared to be of minor importance. Again, the correlation between the effects of the hormone on glucose synthesis and hepatic fatty acid oxidation, as reflected by tissue levels of acetyl-CoA was rather tenuous (Menahan et al., 1968; Williamson, 1967). Third, short or long-chain fatty acids were never as effective as glucagon in stimulating gluconeogenesis from lactate in the perfused rat liver (Exton et al, 1970), although they increased ketogenesis to a very much greater degree than did glucagon. Since there is no reason to suspect that exogenous fatty acids are oxidized in a different manner to those arising from endogenous lipolysis, it is difficult to reconcile these observations with the hypothesis.
657
658
SCHULTZ AND MISTRY
Williamson, J. R., R. Scholz, E. T. Browing, R. G. Thurman, and M. H. Fukami, 1969. Metabolic effect of ethanol in perfused rat liver. J. Biol. Chem. 244:5044-5054.
Zahlten, R. N., F. W. Stratman, and H. A. Lardy, 1973. Regulation of glucose synthesis in hormone-sensitive isolated rat hepatocytes. Proc. Nat. Acad. Sci. 70:3213-3218.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
1981 Poultry Science 6 0 : 6 5 3 - 6 5 8 INTRODUCTION
An important consideration in the use of isolated parenchymal cells for studies on gluconeogenesis is the control of the intracellular oxidation-reduction states. The importance of the redox state is in its determination of the direction of certain common reactions for glycolysis and gluconeogenesis. Lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase catalyze reversible equilibrium reactions common to both glycolysis and gluconeogenesis; therefore, the influence of the oxidation-reduction potential of NAD + -couple is important in gluconeogenesis. Furthermore, the oxidation-reduction state of NAD + -couple in mitochondria can play a significant role in determining the relative productions of oxalacetate and malate. Consequently, malate and aspartate transport into cytosol during gluconeogenesis could be influenced by the mitochondrial oxidation-reduction state. Hydrogen resulting from mitochondrial oxidation could be made available in the cytosol, by the malate-aspartate shuttle, for the reduction of 1,3-diphosphoglycerate to glyceraldehyde-3phosphate. In a preceding paper (Schultz and Mistry, 1981), we described a technique for the isolation of chicken liver cells and reported certain aspects of gluconeogenesis. In the present study we have examined the effect of fatty acids, pMiydroxybutyrate, ethanol, and various ratios
of pyruvate/lactate on glucose synthesis in chicken hepatocytes. MATERIALS AND METHODS
Animals. Crossbred chicks (Gallus domesticus: New Hampshire male X Columbian female) were placed in raised wire-floor cages with adequate water and heating and fed ad libitum a corn-based diet (Shen and Mistry, 1977) until the day of sacrifice. The chicks were used when they were about 50 days old. Chemicals and Enzymes. The chemicals, enzymes, and hormones used in this study were: sodium pentobarbital from Holmes Serum Company, Inc.; acetoacetate, bovine serum albumin, glucose oxidase reagent, glucagon, (3-hydroxybutyrate, oleate, L-lactate, and pyruvate from Sigma Chemical Company; collagenase Type II from Worthington Biochemical Corporation; and Longdwell catheter (teflon) from Becton, Dickinson, and Company. Isolation of Liver Parenchymal Cells, Incubation Medium, and Assays. These are described in detail in our preceding paper (Schultz and Mistry, 1981). RESULTS AND DISCUSSION The Effect of Pyruvate/Lactate Ratios. Figure 1 shows the effect of various pyruvate/lactate ratios on the rate of glucose syn-
653
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
ABSTRACT The effect of fatty acids, 0-hydroxybutyrate, ethanol, and different pyruvate/lactate ratios on gluconeogenesis in isolated chicken hepatocytes was investigated. Gluconeogenesis was significantly affected by a change in the oxidation-reduction (pyruvate/lactate) ratio, and this effect was greater than could be accounted for by the additive effects of these substrates. Substituting lactate with nongluconeogenic substrates, such as/3-hydroxybutyrate or ethanol, increased the formation of glucose by 80 and 200 %, respectively, demonstrating the beneficial effect of the increased reducing equivalents in the hepatocytes. Oleic acid per se had no effect but when added, complexed with albumin, it had a negative effect on gluconeogenesis. (Key words: chicken hepatocytes, gluconeogenesis, fatty acids, pyruvate/lactate ratios)
SCHULTZ AND MISTRY
654
3 £? to to
005
\n
8
v 01
LL
.2
X
.9
o u
.6
~i
CD to
to
Pyruvate 5 5 5 5 Lactate - 1 2 4
o Q Qdl - - mM 1 2 4 mM
FIG. 1. The effect of various pyruvate/lactate ratios on gluconeogenesis. Data are expressed as the means of four experiments + SEM (vertical lines). Significance of difference (P) from the (-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
c
(UU/
thesis by isolated chicken hepatocytes; the indicated substrate concentrations are those at the beginning of the incubations. It can be seen that the rate of gluconeogenesis was affected by varying the ratio of the oxidized/reduced substrate. Furthermore, the increase in glucose synthesis was greater in the presence of both lactate and pyruvate than with the individual substrates at the same concentration. This effect has not been observed in the perfused rat liver (Ross et al, 1967a; Exton and Park, 1969) or in isolated rat hepatocytes (Garrison and Haynes, 1973). When as little as 1 mM lactate was included in the medium containing pyruvate, a significant (P<.005) increase in gluconeogenesis was observed. The metabolism of lactate provides NADH in the cytosol (Krebs et al, 1967), and the reduced pyridine nucleotide is used for gluconeogenesis at the glyceraldehyde-3 -phosphate dehydrogenase step. Thus, the stimulatory effect of lactate on gluconeogenesis from pyruvate could be due to the reducing equivalents supplied by lactate. The Effect of (5-Hydroxybutyrate and Ethanol. Since the metabolism of nongluconeogenic substrates such as /3-hydroxybutyrate and ethanol also provide mitochondrial
I/)
5
LO
$
1.2 -
-P Q)
~ 9
£
I/)
^ ^
,n
8?-?8
o^a.^ .L
±
^
1
o
CJ
<5
6
• i
en O
-J
E
n Pyruvat :e 5 5 5 5 5 mM /5-OH-Buty - 1 2 3 10 mM FIG. 2. The effect of (3-hydroxybutyrate on gluconeogenesis. Data are expressed as the means of four experiments ± SEM (vertical lines). Significance of difference (P) from the t-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
.3
lO
(Zahlten et al, 1973; Arinze et al., 1973) and cytosolic (Williamson et al, 1969) NADH, respectively, the effect of these metabolites on glucose synthesis from pyruvate was tested. Figures 2 and 3 show the effect of /3-hydroxybutyrate and ethanol, respectively, on gluconeogenesis from pyruvate as the substrate. Assuming that the rate of gluconeogenesis from pyruvate was limited by a general deficiency of reducing equivalents in the mitochondria and the cytosol, then indeed (3-hydroxybutyrate and ethanol proved to be quite effective; gluconeogenesis was stimulated by about 80 and 200%, respectively. Increased glucose production from pyruvate by /3-hydroxybutyrate and ethanol could reflect increased availability of NADH for the reduction of 1,3-diphosphoglycerate. Our results are in agreement with those obtained with isolated rat hepatocytes (Zahlten et al., 1973; Garrison and Haynes, 1973), with the perfused rat (Arinze et al., 1973; Kaden et al., 1969), and guinea pig liver (Arinze et al., 1973), but not with the data secured with rat kidney cortex slices, mouse liver slices, and pigeon liver hemogenates (Krebs
GLUCONEOGENESIS IN ISOLATED CHICKEN HEPATOCYTES
^ 1.9
Q
8 oo
£ 1.2-
Q- Q_
O)
0CO .9 .6
o .3 £ Pyruvate Ethanol
5 5 5 mM - 1 3 mM
FIG. 3. The effect of ethanol on gluconeogenesis. Data are expressed as the means of four experiments + SEM (vertical lines). Significance of difference (P) from the t-test was calculated by testing the mean of each treatment against the control (pyruvate alone).
et al, 1967), where ethanol had no effect on gluconeogenesis from pyruvate. In perfused rat liver (Williamson et al, 1969) and in the intact rat (Stubbs et al, 1972), supplying ethanol leads to a rapid decrease in NAD+/NADH ratios in cytosol and mitochondria. A decrease in this ratio should inhibit the conversion of a NAD + -linked substrate such as lactate to glucose. This has been shown to occur in the perfused guinea pig liver (Arinze et al, 1973), but the opposite was true in the case of the perfused rat liver. This difference in response has been explained on the basis of the intracellular location of hepatic phosphoenolpyruvate carboxykinase (PEPCK) in the two species. Since chicken liver also has substantial amounts of PEPCK in the mitochondria, it is possible that gluconeogenesis from lactate would be inhibited in the presence of ethanol as was the case in the guinea pig liver (Arinze et al, 1973). However, with substrates at the same
oxidation level as glucose or with substrates having a net requirement for NADH during gluconeogenesis, ethanol should stimulate glucose production. This was the case in the perfused rat liver (Arinze et al, 1973; Kaden et al, 1969) in the perfused guinea pig liver (Arinze et al, 1973), in isolated rat hepatocytes (Zahlten et al, 1973), and also in the present study with isolated chicken hepatocytes. Fatty Acids and Glucagon in Gluconeogenesis. Table 1 shows the effect of free and bound fatty acids on gluconeogenesis. Glucagon stimulated glucose production. Under our experimental conditions, free fatty acids (FFA) had no effect on gluconeogensis, but when added complexed with albumin (OBA), they inhibited glucose production. Such a decrease in gluconeogenesis has been observed in the perfused guinea pig liver (Soling et al, 1970), but the opposite was true in the case of the perfused rat liver (Ross etal, 1967b; Williamson et al, 1966; Williamson, 1967). This inhibition by OBA of gluconeogensis in chicken hepatocytes was also observed in the presence of glucagon (Table 1). It is possible that the inhibition was due to an unspecific effect of OBA. Hence, we measured the oxygen uptake of the cells at the beginning and end of the 30 min incubation period. As seen from Table 2, OBA in the presence or absence of glucagon did not inhibit oxygen uptake but did inhibit glucose production from pyruvate (Table 1). In fact, with increasing levels of OBA there was a progressive increase in oxygen consumption even after 30 min of incubation (Table 2), which suggests that OBA was metabolized. However, FFA did not seem to be utilized, since the oxygen consumption after 30 min was consistently lower than with pyruvate alone; very likely this was because FFA were supplied in an unphysiological state. Therefore, the decrease in gluconeogenesis observed in the presence of OBA was not due to an unspecific effect of the fatty acids on cell metabolism but very likely was the result of the removal of oxalacetate as maltate because of the increased generation of NADH during j3-oxidation. This effectively depletes oxalacetate, the substrate for PEP synthesis essentially taking place in liver mitochondria of this species. Struck et al (1965) have suggested that the stimulatory effect of glucagon on gluconeogenesis was the result of increased fatty acid oxidation due to the activation of the
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
o u Z5
655
656
SCHULTZ AND MISTRY TABLE 1. The effect of free and bound fatty acids on gluconeogenesis
Treatment
mM
Glucose produced (jumole/g wet cells/min)
Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate
4 10~ 5 .4 1.0 .4 1.0 .4 1.0 .4 1.0
.36+ . 0 1 ' .40 ± .03 .36 ± .02 .32 ± .02 .28 ± .01 .21 ± .01 .35 + .03 .34 ± .02 .34 ± .03 .25 + .01
+ FFA + FFA + OBA + OBA
(a)* (b) (a) (a,c) (c)
(d) (a,b) (a) (a) (d)
Each value represents the mean ± SEM of six experiments.
2
Different letters in parentheses indicate valid statistical differences (P<.01 from the t-test) between treat:nts. 3 4
FFA = free fatty acids (Oleic acid, approximately 99% pure). OBA = Oleic acid bound to albumin; prepared as described by Ross et al. (1967b).
hormone-sensitive lipase. Glucagon and cyclic AMP activate lipolysis in adipose tissue, and Claycomb and Kilsheimer (1969) and others have shown an effect of glucagon on lipolysis also in liver preparations. Glucagon was ketogenic in liver slices and in the perfused liver (Menahan et al., 1968; Regen and Terrell, 1968), and this was ascribed to a stimulation of endogenous lipid catabolism. Addition of fatty
acids to rat liver and kidney preparations did stimulate gluconeogenesis under certain conditions (Williamson et al, 1966; Williamson, 1967). Despite the attractiveness of this hypothesis, the following observations indicate that the stimulatory effect of glucagon on gluconeogenesis was not secondary to lipolysis. First, our data show that in the chicken, glucagon stimulated gluconeogenesis whereas
TABLE 2. The effect of free and bound fatty acids on oxygen uptake Oxygen uptake umole
o2/g wet
cells/min
% of Control
Treatment
mM
0 min
30 min
0 min
30 min
Control (cells alone) Pyruvate (substrate) Pyruvate + glucagon Pyruvate + FFA* Pyruvate + FFA Pyruvate + FFA Pyruvate + OBA a Pyruvate + OBA Pyruvate + OBA Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon + Pyruvate + glucagon +
4 10~ s .4 1.0 2.0 .4 1.0 2.0 .4 1.0 2.0 .4 1.0
.79 1.42 1.40 1.54 1.68 1.15 1.44 1.75 2.02 1.50 1.27 1.56 1.48 1.99
.79 1.34 1.55 .91 .91 .92 1.33 1.67 1.79 1.67 1.61 1.60 1.95 1.80
100 179 176 193 212 145 181 220 254 189 159 197 186 251
100 170 197 115 115 116 168 212 227 212 203 203 247 228
FFA FFA FFA OBA OBA
Abbreviations are given under Table 1.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
1
(substrate) + glucagon + FFA 3 + FFA + OBA4 + OBA + glucagon + glucagon + glucagon + glucagon
GLUCONEOGENESIS IN ISOLATED CHICKEN HEPATOCYTES
Fourth, the change in the level of gluconeogenic intermediates induced by glucagon in the perfused rat liver (Exton and Park, 1969; Exton et al, 1969) differed markedly from that produced by unbound fatty acids (Williamson et al, 1966; Williamson, 1967). The stimulatory effect of unbound fatty acids on gluconeogenesis is probably an artifact, since it was not observed when the fatty acids were added bound to albumin or when added in physiological concentrations (Exton et al, 1970). Fifth, as shown by Ross et al (1967b) in the perfused rat liver, the gluconeogenic effect of unbound oleate was additive with that of glucagon. This indicates separate mechanisms of action of glucagon and unbound fatty acids and that the hormone does not act by increasing the supply of fatty acids for oxidation. REFERENCES Arinze, I. J., A. J. Garber, and R. W. Hanson, 1973. The regulation of gluconeogenesis in mammalian liver. J. Biol. Chem. 248:2266-2274. Claycomb, W. C , and G. S. Kilsheimer, 1969. Effect of glucagon, adenosine 3',5' monophosphate and theophylline on free fatty acid release by rat liver slices and on tissue levels of coenzyme A esters. Endocrinology 84:1179-1183. Exton, J. H., and J. G. Corbin, and C. R. Park, 1969. Control of gluconeogenesis in liver. IV. Differ-
ential effects of fatty acids and glucagon on ketogenesis and gluconeogenesis in the perfused rat liver. J. Biol. Chem. 244:4095-4102. Exton, J. H., L. E. Mallette, L. S. Jefferson, E.H.A. Wong, N. Friedmann, T. B. Miller, and C. R. Park, 1970. The hormonal control of hepatic gluconeogenesis. Recent. Prog. Horm. Res. 26:411-461. Exton, J. H., and C. R. Park, 1969. Control of gluconeogenesis in liver. III. Effect of L-lactate, pyruvate, fructose, glucagon, epinephrine and adenosine 3', 5'-monophosphate on gluconeogenic intermediates in the perfused rat liver. J. Biol. Chem. 244:1424-1433. Garrison, J. C , and R. C. Haynes, 1973. Hormonal control of glycogenolysis and gluconeogenesis in isolated rat liver cells. J. Biol. Chem. 248:5333-5343. Kaden, M., N. W. Oakley, and J. B. Field, 1969. Effect of alcohol on gluconeogenesis using the isolated rat liver perfusion technique. Amer. J. Physiol. 216:756-763. Krebs, H. A., T. Gascoyne, and B. M. Notton, 1967. Generation of extramitochondrial reducing power in gluconeogenesis. Biochem. J. 102:275—282. Menahan, L. A., B. D. Ross, and O. Wieland, 1968. Acetyl CoA level in perfused rat liver during gluconeogenesis and ketogenesis. Biochem. Biophys. Res. Commun. 30:38—44. Regen, D. M., and E. G. Terrell, 1968. Effects of glucagon and fasting on acetate metabolism in perfused rat liver. Biochem. Biophys. Acta 170:95-111. Ross, B. D., R. Hems, R. A. Freedland, and H. A. Krebs, 1967b. Carbohydrate metabolism of the perfused rat liver. Biochem. J. 105:869—875. Ross, B. D., R. Hems, and H. A. Krebs, 1967a. The rate of gluconeogenesis from various precursors in the perfused rat liver. Biochem. J. 102:942-951. Schultz, P., and S. P. Mistry, 1981. A technique for the isolation of chicken hepatocytes and their use in a study of gluconeogenesis. Poultry Sci. 60:000.
Shen, C. S., and S. P. Mistry, 1977. Activities of pyruvate and propionyl coenzyme A carboxylase in chicken tissues during normal growth and biotin deficiency. Poultry Sci. 56:1900-1903. Soling, H. D., B. Willms, J. Kleineke, and M. Gelhoff, 1970. Regulation of gluconeogenesis in the guinea pig liver. Europ. J. Biochem. 16:289—302. Struck, E., J. Ashmore, and O. Wieland, 1965. Stimulierung der gluconeogenese durch langkettige fettsauren und glucagon. Biochem. Z. 343: 107-110. Stubbs, M., R. L. Veech, and H. A. Krebs, 1972. Control of the redox state of the nicotinamideadenine dinucleotide couple in rat liver cytoplasm. Biochem. J. 126:59-65. Williamson, J. R., 1967. Effects of fatty acids, glucagon and anti-insulin serum on the control of gluconeogenesis and ketogenesis in rat liver. Adv. Enzyme Regul. 5:229-255. Williamson, J. R., R. A. Kreisberg, and P. W. Felts, 1966. Mechanism for the stimulation of gluconeogenesis by fatty acid in perfused rat liver. Proc. Nat. Acad. Sci. 56:247-254.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015
OBA had the opposite effect. This was the case also in perfused guinea pig liver (Soling et al, 1970). Second, Exton et al. (1969) have shown that glucagon frequently had no effect on ketogenesis even though it markedly stimulated glycogenolysis and gluconeogenesis. Compared with the effect of exogenous fatty acids, the action of the hormone on ketogenesis appeared to be of minor importance. Again, the correlation between the effects of the hormone on glucose synthesis and hepatic fatty acid oxidation, as reflected by tissue levels of acetyl-CoA was rather tenuous (Menahan et al., 1968; Williamson, 1967). Third, short or long-chain fatty acids were never as effective as glucagon in stimulating gluconeogenesis from lactate in the perfused rat liver (Exton et al, 1970), although they increased ketogenesis to a very much greater degree than did glucagon. Since there is no reason to suspect that exogenous fatty acids are oxidized in a different manner to those arising from endogenous lipolysis, it is difficult to reconcile these observations with the hypothesis.
657
658
SCHULTZ AND MISTRY
Williamson, J. R., R. Scholz, E. T. Browing, R. G. Thurman, and M. H. Fukami, 1969. Metabolic effect of ethanol in perfused rat liver. J. Biol. Chem. 244:5044-5054.
Zahlten, R. N., F. W. Stratman, and H. A. Lardy, 1973. Regulation of glucose synthesis in hormone-sensitive isolated rat hepatocytes. Proc. Nat. Acad. Sci. 70:3213-3218.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on May 25, 2015