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Control of gluconeogenesis in chick (Gallus domesticus) isolated hepatocytes: Effect of redox state and phosphoenolpyruvate carboxykinase (EC 4.1.1.32) location
lr,r J Bw~licnt, Vol 13, pp 437 ,u 444. ,9X, ,n Great Br,la,n All r,gh,s rcscrved
0020-71 Ix;x1/040437-0xs02.00~0 CopyrIght 0 1981 Pergamon Press Ltd
Prlntcd
CONTROL OF GLUCONEOGENESIS IN CHICK (GALLUS DOMESTICUS) ISOLATED HEPATOCYTES: EFFECT OF REDOX STATE AND PHOSPHOENOLPYRUVATE CARBOXYKINASE (EC 4.1.1.32) LOCATION DONALD W. BANNISTERand IRIS E. O’NEILL Agricultural Research Council’s Poultry Research Centre, Roslin, Midlothian, EH25 9PS, Scotland (Received 26 August
1980)
Abstract--l. Hepatocytes isolated from 3- to S-week old chicks synthesized glucose from a variety of precursors. Lactate and fructose proved the most effective with pyruvate less so and glycerol and alanine relatively poor. 2. Oleate inhibited gluconeogenesis from lactate, pyruvate and glycerol. 3. Ethanol inhibited gluconeogenesis from lactate, glycerol, glyceraldehyde and fructose but stimulated gluconeogenesis from pyruvate. All these effects were abolished by pyrazole. 4. Phosphoenolpyruvate carboxykinase was located almost exclusively within the mitochondria. Quinolinate produced inhibition of gluconeogenesis by hepatocytes at high concentration (2G-50 mM) whereas the enzyme was inhibited at much lower concentration (24 mM). 5. The aminotransferase inhibitor, aminooxyacetate, inhibited gluconeogenesis from lactate, glycerol and to a small extent from fructose but not from pyruvate.
INTRODUCTION Most theories concerning metabolic regulation in animals derive from studies with rats as the experimental model. However, there is increasing evidence of substantial differences between both various species of mammal and other vertebrates. In this context our understanding of the regulation of gluconeogenesis in birds is poor and further information might help illuminate these differences. It is well established that birds have higher (12-14 mM) plasma glucose concentrations than mammals. A contributory factor may well be greater hepatic gluconeogenic capacity in avian species, indeed certain in vitro evidence supports this view. Perfused pigeon liver (Sling et al., 1973) and isolated chick hepatocytes (Ochs & Harris, 1978a) were both shown to be more active at synthesizing glucose than corresponding mammalian preparations. The difference, however, is not only quantitative since there are substantial variations in the ease with which different precursors can be used. Perfused pigeon liver utilized lactate readily but was not able to synthesize glucose from pyruvate (Sling et al., 1973), on the other hand chick hepatocytes could use pyruvate to a limited extent but glycerol and alanine were poor precursors (Brady et al., 1979; Dickson & Langslow, 1978). Undoubtedly differences such as these are consequences of differing regulatory mechanisms, arising for example, from the fact that phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is located in the mitochondria in pigeon liver and in the cytosol in rat liver (Sijling et al., 1973). With regard to the domestic fowl, however, the position is less clear. Evidence on location of the enzyme is conflicting. Some reports suggest that it is essentially mitochondrial (Felicioli et al., 431
1967; Utter, 1959) while others indicate that it is present to a significant extent in the cytoplasm (Jo et al., 1974). Another area in which different regulatory mechanisms between the fowl and the rat is indicated is that of hepatic redox state. It is well known that, upon starvation, the redox potential of rat liver becomes more reduced. In the fowl, to judge from data on blood lactate/pyruvate, and 3-hydroxy butyratelacetoacetate levels (Belo et al., 1976; Davison & Langslow, 1975), starvation causes the cytoplasmic redox state to become more oxidized and the mitochondria more reduced. More information is desirable in an attempt to clarify these questions. Accordingly we have chosen the technique of isolated hepatocytes because it has gained wide acceptance in studying metabolic regulation since its introduction (Berry 8~ Friend, 1969; Howard et al., 1967).
MATERIALS AND
METHODS
Birds Female H & N “Nick Chicks” (supplied by Pfizer. H & N Inc., Dunbar, East Lothian, G.-K.) or- the Poultry Research Centre’s S-line chicks were used in all exoeriments. Both are strains of small-bodied bird selected for egg-laying capacity. They were housed in a 4-tier heated brooder and allowed unrestricted access to water and to a commercial-type chick-mash. They were used when between 3- and 5-weeks old. When preparations from starved birds were required, food was withheld overnight (18-20 hr). A portion of the cell preparations was added to 0.1% trypan blue solution and counted on a Fuchs Rosenthal Haemacytometer; only preparations which showed > 90%
DONALD W. BANNISTERand IRIS E. O’NEILL
438
intact cells were used. The mean percentage of damaged cells in the experiments included in this work was 5.70 + 0.29 (73). Preparation
of hepatocytrs
Isolated liver parenchymal cells were prepared essentially by the method of Cramb (1980). After cervical dislocation, the rib cage was opened and the hepatic portal vein cannulated. The liver was perfused immediately for 2. 3 min with Ca’+-free Krebs-Ringer bicarbonate buffer (Krebs & Henseleit, 1932) containing 0.66”; defatted bovine serum albumin at 40°C which was gassed continuously with O,-CO, mixture (95:s). The initial 20ml of perfusate flowing from the aorta contained mainly red blood cells and was therefore discarded. At this point the perfusion was stopped and the liver carefully dissected out and transferred to an organ chamber at 40°C where the perfusion was resumed. Collagenase (EC 3.4.4.19) (IS mg) in Ca”-free Krebs-Ringer bicarbonate contaming 0.66”,, defatted bovine serum albumin was added to the reservoir and the final volume adjusted to 50 ml. The perfusion was continued for B-1Omin with recirculation of the medium which was gassed continuously with 0,&O, mixture (95: 5). When collagenase digestion was complete, the liver was removed from the chamber, minced thoroughly and then transferred to 20 ml of oxygenated medium at 40°C. The suspension was shaken gently for 2 min (with gassing) before being passed through a single layer of nylon mesh, This procedure was repeated except that the hepatocytes were filtered through one layer of nylon bolting cloth. The filtrate was centrifuged at 350~ for 5 min and the pellet resuspended in oxygenated medium at 40°C and recentrifuged at 6Og for Zmin. The cell pellet was suspended finally in the same medium supplemented with Ca*’ (final concentration 0.55 mM). Incubation
Incubations were performed in quadruplicate in plastic pots. Each contained Ca’*-supplemented medium, gluconeogenic precursor (all precursors were at an initial concentration of 10 mM) and. where required. effecters of gluconeogenesis at the concentrations given in the tables and figures, plus cells in a total volume of 1.5 ml. After addition of cells, each pot was gassed with O,-CO, mixture (95:5) for approx 20 sec. sealed and transferred to a shaking water bath at 40 C for 30min. Reactions were stopped by rapid centrifugation and glucose was measured in portions of cell supernatant by a modification of the method of Hugget & Nixon (1957). Portions of cell suspension and cell-free medium were dried to constant weight to determine the dry weight of each preparation. Pots were also incubated without precursor to determine endogenous glucose production which was used to determine net glucose synthesis. Data are expressed as pmol glucose (net)/g dry wt/hr. In experiments where various substances were tested for their effects on the rate of gluconeogenesis data were normalized by expressing values as a percentage of those given by untreated cells from the same preparation; significance w;ts assessed using a paired r-test.
Glutamate dehydrogenase (EC 1.4.1.2) was assayed by the method of Solomon (1959) at 25°C in the direction of glutamate formation. Protein was measured by the method of Lowry rt cri.. (195 I) with bovine serum albumin as standard. Chemicals
Precursors, coenzymes, enzymes and certain inhibitors were purchased from Boehringer Corp. (London) Ltd. Lewes, East Sussex, U.K., or Sigma (London) Chemical Co. Ltd. Poole Dorset, U.K. ~-Cyano-4-hydroxy-cjnnamic acid was obtained from Aldrich Chemical Co. Ltd. Gillingham, Dorset, U.K. AI! other chemicals were of Analar grade (or the best grade available) and were obtained from BDH Chemicals Ltd, Poole, Dorset, U.K. or Koch-Light Laboratories Ltd, Colnbrook, Bucks. U.K. except for bovine serum albumin (Fraction V) which was purchased from Armour Pharmaceutical Co.. Eastbourne. Sussex, U.K. Gelatin. which was used in certain experiments. was ex-
tensively dialysed against distilled water and freeze-dried before use. Bovine serum method of Chen (1967).
albumin
was
defatted
RESULTS Effect
qf protein
type
on yluconeoyenesis
A comparison of the inclusion of either gelatin or bovine serum albumin at various concentrations is given in Fig. I. It should be noted that protein was omitted from the medium during preparation of hepatocytes. Whereas the rates of gluconeogenesis remained fairly constant with increasing concentration of bovine serum albumin, the results obtained with gelatin were erratic and gluconeogenesis from glycerol declined between 0.33 and 2.0% (w/v) protein concentration (the data bordered on statistical significance 0.05 < P < 0.10). In general the results with A r
t3 r
Prufein concentration,
Portions of liver were chopped with scissors and gently homogenized in ice-cold 250 mM sucrose-10 mM triethanolamine buffer. pH 7.5 and fractionated according to the method of De Duve (1963,‘1964). Enzyme assays
Phosphoenolpyruvate carboxykinase was assayed by the method of Seubert & Huth (1965) at 25°C in the direction of phosphoenolpyruvate formation. The product formed was measured by the method of Czok & Lamprecht (1974).
by the
% fw/vl
Fig. 1. Effect of protein on glu~oneogenesis by chick hepatocytes. Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr) except that no protein was included in the perfusion medium. Bovine serum albumin (A) or gelatin (B) were included in the incubation medium at the concentrations shown. Gluconeogenesis was measured from: A = glycerol; M = fructose and l = lactate with each precursor at an initial concn. of 10mM. Results are given as the mean. with verticai bars representing SEM, for 3 observations.
Gluconeogenesis
439
in chick hepatocytes
was observed. In contrast, the addition of NH.&1 was accompanied by progressive inhibition, reaching 47% at 1OmM. Gluconeogenesis in fed and starved stales Net glucose synthesis by hepatocytes from fed and starved chicks using a variety of potential precursors is given in Table 1. The order of effectiveness in fed chicks was: lactate > fructose > oxaloacetate > pyruvate > glycerol. Synthesis from the other precursors was small and from the amino acids was negligible. Starvation elicited significant increases in the rate of gluconeogenesis from lactate, pyruvate, glyceraldehyde and alanine. The order of effectiveness now became lactate > fructose > glyceraldehyde > pyruvate > oxaloacetate > glycerol > alanine. Oxaloacetate remained a good precursor despite there being no increase in its rate of conversion to glucose on starving. Of the amino acids, only alanine was a significant precursor. I
Effect offatty acids on gluconeogenesis
I
0
5
IO
In contrast to the rat, in which gluconeogenesis is stimulated by fatty acid oxidation (Saling et al., 1968; Williamson et al., 1969), we observed a significant decrease in gluconeogenesis from glycerol, lactate and pyruvate (the decrease from pyruvate, in presence of octanoate fell just short of statistical significance). Gluconeogenesis from glyceraldehyde and fructose was unaffected (Table 2). Acetate produced a slight reduction in gluconeogenesis from lactate and also from pyruvate, in the latter case, however, it was not statistically significant. The effect is opposite to that seen in rat, for which a specific stimulation of gluconeogenesis from lactate was observed (Whitton et al., 1979).
Concentmtion of additive, mM
Fig. 2. Effect of lysine or NH,CI on gluconeogenesis by chick hepatocytes. Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). The precursor was lactate and was included at an initial concentration of 10 mM. Results are given as the mean, with vertical bars representing SEM, for 7 observations. 0 = lysine; n = NH,CI.
gelatin tended albumin.
to be lower than with bovine
serum
EfSect of lysine and NHICl on gluconeogenesis from lactate
Effect of redox state on gluconeogenesis
The effect of including lysine or NH&l in the incubation medium is given in Fig. 2. Lysine caused a small (approx 476) inhibition at 1 mM concentration but thereafter no further reduction in gluconeogenesis
The effects of including substances known to alter intracellular redox state are given in Table 3. Butan-1,3-diol and ethanol were added to decrease the cytoplasmic free NAD+-free NADH ratio. Gluco-
Table 1. Gluconeogenesis from a variety of precursors by hepatocytes from fed or starved
Net glucose. Fed
Precursor Propionate Lactate Pyruvate Oxaloacetate Malate Glycerol D-Glyceraldehyde Fructose Glycine Serine Alanine Glutamate Aspartate
chicks
44.0 288.8 70.5 105.9 36.7 67.0 42.4 193.4 4.1 7.0 3.9 9.2 6.5
++ f + f f + * * i + f f
11.9 (5) 10.3(5) 9.9 (5) 22.9 (5) 10.5(5) 10.3 (5) 5.5 (5) 20.1 (5) 3.5 (5) 3.3 (5) 3.9 (5) 3.6(5) 2.6 (5)
pmol/g
dry wt/hr Fasted 30.7 563.6 108.1 76.7 19.5 73.0 188.4 416.5 9.3 I 1.4 60.1 7.7 10.2
+ i i k k F k f f f + + *
5.9 (6) 35.8 (16)$ 8.4 (16)* 16.5 (6) 2.5 (5) 5.2 (16) 37.7 (6)t 33.9 (16)t 3.1 (5) 3.0 (6) 8.2 (6)f 1.8 (5) 3.4 (5)
Hepatocytes were prepared as described under Materials and Methods from fed chicks or from ones starved overnight (18-20 hr). All precursors were included at an initial concentration of 1OmM and incubations performed for 30min. Data are presented as the mean f SEM for the number of observations in parentheses. * P < 0.05; t P < 0.01; $ P < 0.001 compared to fed birds.
440
DONALDW. BANNISTER and IRIS E. O’NEILL Table 2. Effect of fatty acids on gluconeogenesis by chick hepatocytes Percentage of control value Octanoate
Acetate
Precursor Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
91.4 85.6 102.8 94.5 99.4
+ f + + *
76.3 90.6 46.9 95.2 97.6
3.0(5)? 7.4(5) 8.3 (8) 4.9 (6) 5.9 (5)
k + k k &
Oleate 76. I 78.1 52.0 94.2 102.9
2.1 (5)$ 4.6 (7) 12.4(5)* 6.1 (8) 7.4(5)
i * i f k
2.4 3.1 9.6 6.2 9.7
(5)k (5): (5)* (7) (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). All precursors were included at an initial concentration of 10 mM and fatty acids at 2 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean f SEM for the number of observations in parentheses. Significance of difference was calculated between control and experimental treatments using a paired ‘I’-test. *P<0.05:tP=0.05; ~P
neogenesis from pyruvate was stimulated whereas there was a small but significant reduction from lactate. Gluconeogenesis from glycerol was strongly inhibited. Ethanol proved more effective than butan-1,3-diol in producing these effects and also inhibited gluconeogenesis from glyceraldehyde and fructose. When 0.1 M-pyrazole (an inhibitor of alcohol dehydrogenase (EC i.l.l.l), Theorell & Yonetani, 1963) was present, the effects of ethanol were overcome in agreement with findings of Krebs rt al. (1969) using perfused rat liver. Inclusion of 3_hydroxybutyrate, to decrease the mitochondrial free NAD+-free NADH ratio, reduced gluconeogenesis from glycerol but was otherwise without effect. Effect qf inhibitors on gluconeogenesis Data on the effects of inhibitors are given in Table 4. Aminooxyacetate. an inhibitor of aminotransferases (Rognstad & Katz, 1970), strongly inhibited gluconeogenesis from lactate and glycerol. There was a little
inhibition from fructose but none from glyceraldehyde or pyruvate. In this respect the chick differs from the pigeon in which there is no inhibition of gluconeogenesis from lactate (Siiling et al., 1973). m-Cyano-4-hydroxycinnamate is established as an inhibitor of mitochondrial pyruvate transport (Halestrap & Denton, 1974) and, as anticipated, inhibited gluconeogenesis from lactate and pyruvate strongly but also caused some inhibition from other precursors. Quinolinate was chosen as an inhibitor of phosphoenolpyruvate carboxykinase (Veneziale et al., 1967) and, at a concentration of 5 mM inhibited gluconeogenesis from lactate and pyruvate by l&12% (Table 4). Gluconeogenesis from other precursors was not affected. SGling et al. (1970), using perfused rat and guinea-pig livers have demonstrated the strong inhibitory effect of quinolinate on gluconeogenesis. Addition of 2.4 mM-quinolinate resulted in a decrease in glucose synthesis of 90 and 55% respectively. Perfused pigeon liver was not affected by quinolinate even at concentrations up to 28 mM and this was attributed to mitochondrial impermeability.
Table 3. Effect of redox state on gluconeogenesis
Precursor Lactate Pyruvate Glycerol D-Glyceraldehyde Fructose
Percentage Butan- 1.3-diol 94.4 188.2 24.0 94.9 87.1 Ethanol
Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
93.0 103.4 90.5 104.0 101.5
f + + i i
1.3 (7)’ 14.3 (61-I 7.0 (5)t 4.4 (7) 6.9 (7)
+ pyrazole i i f f +
2.6 (5) 5.6 (5) 7.6(7) 7.7 (6) 3.2 (5)
by chick hepatocytes of control
value Ethanol 84.4 206.4 11.3 86.9 81.1
* + * k +
1.9 (IO)? Il.5 (1O)t 2.8(12)t 2.3 (6)* 3.1 (Il)t
3-hydroxybutyrate 98. I 104.0 62. I 98.7 96.9
* k I + _t
3.0 3.6 9.2 8.7 6.8
(7) (5) (5)* (8) (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (I 8-20 hr). All precursors were included at an initial concentration of IOmM. Effecters were included as follows: butan-1.3-diol. IOmM: ethanol, 10 mM; 3-hydroxybutyrate, 20 mM and pyrazole. 100 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean + SEM for the number of observations in parentheses. Significance of difference was calculated between control and experimental treatments using a paired ‘?-test. * P < 0.01; t P < 0.001.
Gluconeogenesis in chick hepatocytes
441
loo-
60.
40:: 80.
Qu&olinate c~t~ion
, mM
Fig. 3. Effect of quinolinate on gluconeogenesis by chick hepatocytes and on phosphoenolpyruvate carboxykinase activity. Hepatocytes and mitochondria (for phosphoenolpyruvate carboxykinase activity) were prepared as described under Materials and Methods from the livers of chicks starved overnight (18.-20 hr). Gluconeogenesis by hepatocytes was measured using lactate (10 mM initial concentration) as precursor and phosphoenolpyruvate carboxykinase activity was determined using the method of Seubent & Huth (1965). Results are given as the mean. with vertical bars representing SEM, for 5 observations for giuconeogenesis and the mean of 2 observations for enzyme activity. l = gluconeogenesis; 0 = enzyme activity. The effect of increasing concentrations of quinolinate on gi~coneogenesis from lactate is given in Fig. 3
together with a similar experiment on mitochondrial phosphoenolpyruvate carboxykinase activity. In these experiments glutamate dehydrogenase activity was measured in mitochondrial and supernatant fractions (to estimate mitochondrial damage). Approximately 24% of total activity was present in the latter (data not shown). Phosphoenolpyruvate carboxykinase activity in the supernatant fraction was too low to measure with accuracy but was estimated to be a similar percentage to that of glutamate dehydrogenase. DISCUSSION
Isolated chick hepatocytes have not been studied as widely as those from other species and the optimum
conditions for their preparation and use have not yet been firmly establised. We felt it desirable, therefore, to examine briefly certain properties which, although not directly related to the main aim of the work, might help establish that the method employed here was giving satisfactory results. Zahlten & Stratman (1974) using guinea-pig and Brady et al. (1979) using chick hepatocytes reported that gelatin and bovine serum albumin could be used interchangeably. Our data does not support this view since in the chick the protein caused inhibition of gluconeogenesis from glycerol and the data generally tended to be erratic. Fresh preparations of rat (CornelI et al., 1974) and guinea-pig (Arinze & Rowley, 1975) hepatocytes show a lag period in gluconeogenesis from lactate that may be overcome by inclusion of either lysine or NHLCl in
Table 4. Effects of inhibitors on giuconeogenesis by chick hepatocytes
Precursor Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
Aminooxyacetate 43.2 2 100.7 + 30.7 i: 94.5 4 89.4 4
1.4(7)f 3.9 (7) 1.6 fS)$ 6.6 (4) 2.7 (5)*
Percentage of control value z-cyano-4-hydroxycinnamate 10.4 + 2.3 (5)$ 54.1 & 6.3 (5)t 72.9 + 1.7 (5); 8 I .O + 7.3 (4) 82.9 If: 1.4 (5)f
Quinolinate 90.7 k 87.1 f 97.1 f 103.7 f 106.1 f
2.3 (16)t 3.5 (7)t 5.4 (5) 2.4 (5) 2.4 (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). A11precursors were included at an initial concentration of 10 mM and the inhibitors were as follows: aminooxyacetate, 0.5 mM; ~-cyano-4-hydroxycinnamate, 0.33 mM; quinohnate, 5 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean f SEM for the number of observations in parantheses. Significance of difference was calculated between control and experimental treatments using a paired Y-test. * P < 0.05; t P < 0.01; : P < 0.001.
442
DOUALI)
W.
BANNEST~,Kand
the incubation medium. However, neither Dickson c’t al. (1978) nor Brady cr u/. (1979) reported a similar phenomenon with chick hepatocytes. Our data on melusion of lysine also indicates that in this species a lag does not occur. It is possible. therefore. that the aspartate shuttle had not become rate-limiting due to accumulation of pyruvate during preparation of the cells. Indeed Ochs & Harris (1978a) reported that chick hepatocytes do not accumulate pyruvate or lactate. Inclusion of NH,CI in the incubation medium caused progressive inhibition of gluconeogenesis from lactate which contrasts with the behaviour of rat or guinea-pig cells. Mapes & Krebs (1978) have studied this phenomenon in greater detail than reported here and postulated that it was due to diversion of carbon units way from gluconeogenesis (oxaloacetate) towards detoxification (glutamine synthesis). The ease with which various precursors were utilized was generally similar to that found by other workers who used isolated chick hepatocytes and different from that of mammals such as rat. The most notable differences were the relatively poor utilization of pyruvate in comparison to lactate and the very poor use of glycerol, alanine and the other amino acids examined compared with lactate. It is possible of course that the rather short period of starvation (18-20 hr) was insufficient to allow gluconeogenesis from amino acids to become fully established. However, it is known that for chicks of this age liver glycogen stores are small and are depleted by 6 hr starvation (Bannister et al., 1979). Our results differ from those of Dickson & Langslow (1978) in that lactate rather than fructose was the most effective precursor and also from those of Brady et aI. (1979) in that the rates we observed were generally much higher. These differences may result from variations in experimental technique or from the use of different genetic strains of bird. We have some evidence suggesting that strain differences may occur (O’Neill & Bannister, unpublished observations). The fact that lactate was the major precursor might be of physiological significance since domestic fowl possess a substantial musculature of the white fibre type and thus could be expected to produce large quantities of this metabolite. However, it is not possible to explain the low rates of gluconeogenesis from glycerol and alanine on the same basis since both are readily utilized in Coo (Davison & Langslow, 1975). Unlike perfused pigeon liver (Siiling rt ~1.. 1973) chick hepatocytes can use pyruvate at an appreciable rate. This was further stimulated (approx. 2-fold) by either butan-1.3-diol or ethanol although not reaching the level obtained with lactate. Thus, the increasingly oxidised redox state of hepatic cytoplasm upon starvation in cico (Belo c’t cl/.. 1976: Davison & Langslow. 1975) does appear to limit gluconeogenesis from pyruvate although some other, as yet unknown factor. further limits the rate in comparison with lactate. Ethanol also inhibits gluconeogenesis from a number of precursors in perfused rat liver (Krebs ut ul., 1969) and qualitatively similar results were obtained here with chick hepatocytes. Inhibition of gluconeogenesis from glycerol. however. was more severe than observed with the rat or than seen with
ISIS E.
O’NEILL
other precursors in these experiments. Exactly why gluconeogenesis from glycerol is particularly susceptible to change in redox state is not understood. However. a parallel observation was made by Bannister & Cleland (1977) in chicks suffering from fatty liver and kidney syndrome. In this condition gluconeogenesis is blocked by the almost total absence of pyruvate carboxylase activity accompanying which there is an elevated lactate concentration and a decrease in the calculated cytoplasmic free NAD+ -free NADH ratio. Liver slices from affected chicks are also unable to use glycerol for gluconeogenesis. The authors suggested that inhibition might occur at the level of glycerol phosphate dehydrogenase (EC I. I. I .8) because of the unfavourable NAD’-NADH ratio. Another possibility is that ATP concentration could be reduced via equilibrium at the glyceraldehyde phosphate dehydrogenase (EC I .2.1.12) system, thereby reducing the ability of glycerol kinase (EC 2.7.1.30) to phosphorylate its substrate. In fatty liver and kidney syndrome, ability of glycerol kinase (EC 2.7.1.30) to phosphorylate its substrate. In fatty liver and kidney syndrome, however. freeze-clamped liver contained normal levels of ATP (Bannister & Cleland, 1977). The effects of ethanol derive from its interaction with alcohol dehydrogenase since they were abolished by pyrazole. The possiblity that 3-hydroxybutyrate might inhibit glyconeogenesis from lactate and pyruvate was tested because this has been seen with guinea-pig and rabbit liver (Arinze et (II., 1973: Garbcr & Hanson, 1971a.b) in which phosphoenolpyruvate carboxykinase also has an intramitochondrial location. We observed no effect on either precursor and attribute this to the low activity of 3-hydroxybutyrate dehydrogenase (EC 1. I. I .30) activity reportedly present in avian liver (Bailey & Horne, 1972; Brady et i/l.. 1978). Gluconeogenesis from glycerol was depressed in the presence of 3-hydroxybutyrate. for which it is difficult to offer an explanation unless one assumes that those reducing equivalents generated by 3-hydroxybutyrate are transferred rapidly to the cytoplasm. Oxidation of medium- and long-chain fatty acids caused slight inhibition of gluconeogenesis from lactate and to a lesser extent from pyruvate. This is opposite to the well known effect in rat liver (Williamson cr (I/.. 1969) but in agreement with guinea-pig (Sibling et al., 1970) and pigeon liver (Siiling rt al., 1973) and probably derives from the fact that in all these species oxaloacetate is generated intramitochondially and is reduced to malate rather than being converted to phosphoenolpyruvatc for export to the cytoplasm and thence glucose synthesis. Gluconeogenesis from glycerol was again adversely affected. The effect of acetate was examined because it is present in avian blood (Annison et cd., 1968) and because it stimulates gluconeogenesis from lactate in rat hepatocytes (Whitton c’t (II.. 1979). The effect on avian cells was to produce slight though significant inhibition of gluconeogenesis from lactate but not the other precursors. The mechanism is unclear; Whitton ef al. (1979) suggest that conversion to acetaldehyde is a possibility. with a consequent increase in cytoplasmic free NAD’-NADH ratio. In view of the evidence given earlier, inhibition of gluconeogenesis from pyruvate would bc anticipated. and although our values
G~~coneog~nesis in chick hepatocytes
were lower in these conditions the difference was not statistically significant. Siiling et al. (1973) demonstrated that aminooxyacetate did not inhibit gluconeogenesis from lactate in perfused pigeon liver, in contrast to rat liver. This was due, it was suggested, to the fact that phosphoenolpyruvate was generated intramitochondrially and therefore the transfer of carbon units to the cytopiasm as aspartate was unnecessary. Our finding that aminooxyacetate inhibited gluconeogenesis from lactate but not pyruvate might, therefore, appear unexpected. However, Ochs & Harris (1978b) made a similar observation with chick hepatocytes. They showed that inhibition was relieved by oleate and suggested that, in the absence of fatty acid, lactate was serving as an energy source for gluconeogenesis as well as a precursor. Aminoox~cetate blocked this function by causing build-up of reducing equivalents in the cytoplasm that would otherwise be transferred to the mitochondria. The fact that gluconeogenesis from glycerol was strongly inhibited (approx 70”,) supports this hypothesis. r-Cyano-4-hydroxycinnamate had the anticipated effect on inhibiting gluconeogenesis from lactate and pyruvate strongiy but also reduced gluconeogenesis from other precursors to a lesser degree, the reason for this is not apparent. We were unable to detect significant phosphoenolpyruvate carboxykinase activity in cytoplasm of chick liver and conclude, in agreement with Felicioli et al. (1967) and Utter (1959), that the enzyme is essentially mitochondrial in the fowl. However, the mitochondria of chick hepatocytes appear to be permeable to high concentrations of quinolinate, in contrast to pigeon liver (Si%ng rt al., 1973) since it was possible to inhibit gluconeogenesis by about 73% at 50mM. That this was due to permeability is indicated by complete inhibition of enzyme activity at about one-tenth the inhibitor concentration. Acknowludyemmt-We are grateful for the skilled technical assistance of Miss June R. Thomson.
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BELO P. S., ROMSOS D. R. & LEVEILLE G. A. (1976) Blood metabolites and glucose metabolism in the fed and fasted chicken. J. Nutr. 106, 1135-l 143. BERRY M. N. & FRIEND D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Ceil B&. 43, 506.520. BRADY L. J., ROMSOS D. R.. BRADY P. S., BLRC;FN W. G. & LEVEILLE G. A. (1978) The effects of fasting on body composition, glucose turnover. enzymes and mctabolites in the chicken. J. Nutr. 108, 648-657. BRADY L. J.. ROMSOS D. R. & LEVEILLEG. A. (1979) Gluconeogenesis in isolated chicken (Gal/us tlomrstic~u.s) liver cells. Camp. Biochem. Physiol. 638, 193%198. CHEN R. F. (1967) Removal of Fatty acids from serum albumin by charcoal treatment. J. hioi. Chrnl. 242. 173-181. CORNELLN. W., LUND P. &. KRERSH. A. (1974) The effect of lysine on gluconeogenesis from lactate in rat hepatocytes. Biochem. J. 142, 327-331. CRAMR G. (1980) The actions of the pancreatic hormones on isolated chicken hepatocytes. Ph.D. Thesis. Edinburgh University. _ CZOK R. & LAMPRE~HT W. (1974) Pvruvate. .ohosnhocnol. pyruvate and D-glycerate:2-phosphate. In ,Cfd&s of ~t~q’tm~tic Ana/y~is.Vol. 3 (Edited by B~RGMI:Y~:K H. t;.i. pp. 1446-1451. Academic Press, New York, DAVISONT. F. & LANGSLOW D. R. (1975) Changes in plasma glucose and liver glycogen following the administration of gluconeogenic precursors to the starving fov\ I. Camp. Biochrm. Phq’siol. 52A. 645-649. D~:Duvr C. (1963,‘64) The separation and characterization of subcellular particles. NUTI*O~L
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DONALD W. BANNISTER and IRIS E. ONEILL
die HarnstofTbiIdung im Tierkorper. Hoppe-Seyler’s Z. physiol. Chem. 210, 33-66. KREBS H. A., FREEDLAND R. A., HEMS R. & STUBBS M. (1969) Inhibition of hepatic gluconeogenesis by ethanol. Biochem. J. 112, 117-124. LOWRY 0. H., ROSEBROUGH N. .I., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. bid. Chem. 193, 265-275. MAPES J. P. & KREBS H. A. (1978) Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver. Eiothem. J. 172, 193-203. OCHS R. S. & HARRIS R. A. (1978a) Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis and pyruvate kinase activity of rat and chicken hepatocytes. Archs Biochem. Eiophys. 190, 193-201. OCHS R. S. & HARRIS R. A. (1978b) Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes. Fe&t Proc. 37, 1480. ROGNSTAD R. & KATZ J. (1970) Glugoneogenesis in the kidney cortex : effects of D-malate and aminooxyacetate. Biochem. J. 116, 483493. SEUBERTW. & HUTH W. (1965) On the mechanism of gluconeogenesis and its regulation II. The mechanism of gluconeogenesis from pyruvate and fumarate. Eiochem. Z. 343. 176-191. SBL~NG H. D., WILLMS B., FR~EDRICHS D. & KLEINEKE J. (1968) Regulation of gluconeogenesis by fatty acid oxidation in isolated perfused livers of non-starved rats. Eur. J. Biochem.
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SOLING H. D., WILLMS B., KLEINEKE J. & GEHLHOFF M. (1970) Regulation of gluconeogenesis in the guinea-pig liver. Eur. J. Biochem. 16, 289-302.
SOLING H. D.. KLEINEKE J., WILLMS B.. JANSON G. & KUHN
A. (1973) Relationship between intracellular distribution of phosphoenolpyruvate carboxykinase, regulation of gluconeogenesis, and energy cost of glucose formation. Eur. J. Biochem.
37, 233-243.
SOLOMON J. B. (1959) Changes in the distribution of glutamic, lactic, and malic dehydrogenases in liver cell fractions during development of the chick embryo. I)& Bird. I, 182-198.
THEORELL H. & YONETANI T. (1963) Liver alcohol dehydrogenase-DPN-pyrazole complex: a model of a ternary intermediate in the enzyme reaction. Biochem. Z. 338. 537-553. UTTFR M. F. (1959) The role of CO: fixation in carbohydrate utilization and synthesis. .4,1r1. N. y .4ctrd. Sc,i. 72, 451-461. VENEZIALE C. M., WALTER P., KUEER N. & LARDY H. A. (1967) Influence of L-tryptophan and its metabolites on gluconeogenesis in the isolated, perfused liver. Biochemistry
6, 2129- 213X.
WHITTON P. D., RODRIGL~S L. M. & HELMSD. A. (1979) Stimulation by acetate of gluconeogenesis in hepatocyte suspensions. FEBS Lrtr. 98, 85 87. WILLIAMS~V J. R.. BROWNING E. T. & SCHOLZ R. (1969) Control mechanisms of gluconeogcnesis and ketogenesis. I. Effects of oleate on gluconeogenesis in perfused rat liver. J. hiol. Chem. 244, 46074616. ZAHL.TF\ R. N. & STRAT~~AN F. W. (1974) The isolation of hormone-sensitive rat hepatocytcs by a moditied enzymatic technique. ilrc,/l.s Hi~j<~/~c,m. B;~,[)/I~~.s.163. 600 608.
0020-71 Ix;x1/040437-0xs02.00~0 CopyrIght 0 1981 Pergamon Press Ltd
Prlntcd
CONTROL OF GLUCONEOGENESIS IN CHICK (GALLUS DOMESTICUS) ISOLATED HEPATOCYTES: EFFECT OF REDOX STATE AND PHOSPHOENOLPYRUVATE CARBOXYKINASE (EC 4.1.1.32) LOCATION DONALD W. BANNISTERand IRIS E. O’NEILL Agricultural Research Council’s Poultry Research Centre, Roslin, Midlothian, EH25 9PS, Scotland (Received 26 August
1980)
Abstract--l. Hepatocytes isolated from 3- to S-week old chicks synthesized glucose from a variety of precursors. Lactate and fructose proved the most effective with pyruvate less so and glycerol and alanine relatively poor. 2. Oleate inhibited gluconeogenesis from lactate, pyruvate and glycerol. 3. Ethanol inhibited gluconeogenesis from lactate, glycerol, glyceraldehyde and fructose but stimulated gluconeogenesis from pyruvate. All these effects were abolished by pyrazole. 4. Phosphoenolpyruvate carboxykinase was located almost exclusively within the mitochondria. Quinolinate produced inhibition of gluconeogenesis by hepatocytes at high concentration (2G-50 mM) whereas the enzyme was inhibited at much lower concentration (24 mM). 5. The aminotransferase inhibitor, aminooxyacetate, inhibited gluconeogenesis from lactate, glycerol and to a small extent from fructose but not from pyruvate.
INTRODUCTION Most theories concerning metabolic regulation in animals derive from studies with rats as the experimental model. However, there is increasing evidence of substantial differences between both various species of mammal and other vertebrates. In this context our understanding of the regulation of gluconeogenesis in birds is poor and further information might help illuminate these differences. It is well established that birds have higher (12-14 mM) plasma glucose concentrations than mammals. A contributory factor may well be greater hepatic gluconeogenic capacity in avian species, indeed certain in vitro evidence supports this view. Perfused pigeon liver (Sling et al., 1973) and isolated chick hepatocytes (Ochs & Harris, 1978a) were both shown to be more active at synthesizing glucose than corresponding mammalian preparations. The difference, however, is not only quantitative since there are substantial variations in the ease with which different precursors can be used. Perfused pigeon liver utilized lactate readily but was not able to synthesize glucose from pyruvate (Sling et al., 1973), on the other hand chick hepatocytes could use pyruvate to a limited extent but glycerol and alanine were poor precursors (Brady et al., 1979; Dickson & Langslow, 1978). Undoubtedly differences such as these are consequences of differing regulatory mechanisms, arising for example, from the fact that phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is located in the mitochondria in pigeon liver and in the cytosol in rat liver (Sijling et al., 1973). With regard to the domestic fowl, however, the position is less clear. Evidence on location of the enzyme is conflicting. Some reports suggest that it is essentially mitochondrial (Felicioli et al., 431
1967; Utter, 1959) while others indicate that it is present to a significant extent in the cytoplasm (Jo et al., 1974). Another area in which different regulatory mechanisms between the fowl and the rat is indicated is that of hepatic redox state. It is well known that, upon starvation, the redox potential of rat liver becomes more reduced. In the fowl, to judge from data on blood lactate/pyruvate, and 3-hydroxy butyratelacetoacetate levels (Belo et al., 1976; Davison & Langslow, 1975), starvation causes the cytoplasmic redox state to become more oxidized and the mitochondria more reduced. More information is desirable in an attempt to clarify these questions. Accordingly we have chosen the technique of isolated hepatocytes because it has gained wide acceptance in studying metabolic regulation since its introduction (Berry 8~ Friend, 1969; Howard et al., 1967).
MATERIALS AND
METHODS
Birds Female H & N “Nick Chicks” (supplied by Pfizer. H & N Inc., Dunbar, East Lothian, G.-K.) or- the Poultry Research Centre’s S-line chicks were used in all exoeriments. Both are strains of small-bodied bird selected for egg-laying capacity. They were housed in a 4-tier heated brooder and allowed unrestricted access to water and to a commercial-type chick-mash. They were used when between 3- and 5-weeks old. When preparations from starved birds were required, food was withheld overnight (18-20 hr). A portion of the cell preparations was added to 0.1% trypan blue solution and counted on a Fuchs Rosenthal Haemacytometer; only preparations which showed > 90%
DONALD W. BANNISTERand IRIS E. O’NEILL
438
intact cells were used. The mean percentage of damaged cells in the experiments included in this work was 5.70 + 0.29 (73). Preparation
of hepatocytrs
Isolated liver parenchymal cells were prepared essentially by the method of Cramb (1980). After cervical dislocation, the rib cage was opened and the hepatic portal vein cannulated. The liver was perfused immediately for 2. 3 min with Ca’+-free Krebs-Ringer bicarbonate buffer (Krebs & Henseleit, 1932) containing 0.66”; defatted bovine serum albumin at 40°C which was gassed continuously with O,-CO, mixture (95:s). The initial 20ml of perfusate flowing from the aorta contained mainly red blood cells and was therefore discarded. At this point the perfusion was stopped and the liver carefully dissected out and transferred to an organ chamber at 40°C where the perfusion was resumed. Collagenase (EC 3.4.4.19) (IS mg) in Ca”-free Krebs-Ringer bicarbonate contaming 0.66”,, defatted bovine serum albumin was added to the reservoir and the final volume adjusted to 50 ml. The perfusion was continued for B-1Omin with recirculation of the medium which was gassed continuously with 0,&O, mixture (95: 5). When collagenase digestion was complete, the liver was removed from the chamber, minced thoroughly and then transferred to 20 ml of oxygenated medium at 40°C. The suspension was shaken gently for 2 min (with gassing) before being passed through a single layer of nylon mesh, This procedure was repeated except that the hepatocytes were filtered through one layer of nylon bolting cloth. The filtrate was centrifuged at 350~ for 5 min and the pellet resuspended in oxygenated medium at 40°C and recentrifuged at 6Og for Zmin. The cell pellet was suspended finally in the same medium supplemented with Ca*’ (final concentration 0.55 mM). Incubation
Incubations were performed in quadruplicate in plastic pots. Each contained Ca’*-supplemented medium, gluconeogenic precursor (all precursors were at an initial concentration of 10 mM) and. where required. effecters of gluconeogenesis at the concentrations given in the tables and figures, plus cells in a total volume of 1.5 ml. After addition of cells, each pot was gassed with O,-CO, mixture (95:5) for approx 20 sec. sealed and transferred to a shaking water bath at 40 C for 30min. Reactions were stopped by rapid centrifugation and glucose was measured in portions of cell supernatant by a modification of the method of Hugget & Nixon (1957). Portions of cell suspension and cell-free medium were dried to constant weight to determine the dry weight of each preparation. Pots were also incubated without precursor to determine endogenous glucose production which was used to determine net glucose synthesis. Data are expressed as pmol glucose (net)/g dry wt/hr. In experiments where various substances were tested for their effects on the rate of gluconeogenesis data were normalized by expressing values as a percentage of those given by untreated cells from the same preparation; significance w;ts assessed using a paired r-test.
Glutamate dehydrogenase (EC 1.4.1.2) was assayed by the method of Solomon (1959) at 25°C in the direction of glutamate formation. Protein was measured by the method of Lowry rt cri.. (195 I) with bovine serum albumin as standard. Chemicals
Precursors, coenzymes, enzymes and certain inhibitors were purchased from Boehringer Corp. (London) Ltd. Lewes, East Sussex, U.K., or Sigma (London) Chemical Co. Ltd. Poole Dorset, U.K. ~-Cyano-4-hydroxy-cjnnamic acid was obtained from Aldrich Chemical Co. Ltd. Gillingham, Dorset, U.K. AI! other chemicals were of Analar grade (or the best grade available) and were obtained from BDH Chemicals Ltd, Poole, Dorset, U.K. or Koch-Light Laboratories Ltd, Colnbrook, Bucks. U.K. except for bovine serum albumin (Fraction V) which was purchased from Armour Pharmaceutical Co.. Eastbourne. Sussex, U.K. Gelatin. which was used in certain experiments. was ex-
tensively dialysed against distilled water and freeze-dried before use. Bovine serum method of Chen (1967).
albumin
was
defatted
RESULTS Effect
qf protein
type
on yluconeoyenesis
A comparison of the inclusion of either gelatin or bovine serum albumin at various concentrations is given in Fig. I. It should be noted that protein was omitted from the medium during preparation of hepatocytes. Whereas the rates of gluconeogenesis remained fairly constant with increasing concentration of bovine serum albumin, the results obtained with gelatin were erratic and gluconeogenesis from glycerol declined between 0.33 and 2.0% (w/v) protein concentration (the data bordered on statistical significance 0.05 < P < 0.10). In general the results with A r
t3 r
Prufein concentration,
Portions of liver were chopped with scissors and gently homogenized in ice-cold 250 mM sucrose-10 mM triethanolamine buffer. pH 7.5 and fractionated according to the method of De Duve (1963,‘1964). Enzyme assays
Phosphoenolpyruvate carboxykinase was assayed by the method of Seubert & Huth (1965) at 25°C in the direction of phosphoenolpyruvate formation. The product formed was measured by the method of Czok & Lamprecht (1974).
by the
% fw/vl
Fig. 1. Effect of protein on glu~oneogenesis by chick hepatocytes. Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr) except that no protein was included in the perfusion medium. Bovine serum albumin (A) or gelatin (B) were included in the incubation medium at the concentrations shown. Gluconeogenesis was measured from: A = glycerol; M = fructose and l = lactate with each precursor at an initial concn. of 10mM. Results are given as the mean. with verticai bars representing SEM, for 3 observations.
Gluconeogenesis
439
in chick hepatocytes
was observed. In contrast, the addition of NH.&1 was accompanied by progressive inhibition, reaching 47% at 1OmM. Gluconeogenesis in fed and starved stales Net glucose synthesis by hepatocytes from fed and starved chicks using a variety of potential precursors is given in Table 1. The order of effectiveness in fed chicks was: lactate > fructose > oxaloacetate > pyruvate > glycerol. Synthesis from the other precursors was small and from the amino acids was negligible. Starvation elicited significant increases in the rate of gluconeogenesis from lactate, pyruvate, glyceraldehyde and alanine. The order of effectiveness now became lactate > fructose > glyceraldehyde > pyruvate > oxaloacetate > glycerol > alanine. Oxaloacetate remained a good precursor despite there being no increase in its rate of conversion to glucose on starving. Of the amino acids, only alanine was a significant precursor. I
Effect offatty acids on gluconeogenesis
I
0
5
IO
In contrast to the rat, in which gluconeogenesis is stimulated by fatty acid oxidation (Saling et al., 1968; Williamson et al., 1969), we observed a significant decrease in gluconeogenesis from glycerol, lactate and pyruvate (the decrease from pyruvate, in presence of octanoate fell just short of statistical significance). Gluconeogenesis from glyceraldehyde and fructose was unaffected (Table 2). Acetate produced a slight reduction in gluconeogenesis from lactate and also from pyruvate, in the latter case, however, it was not statistically significant. The effect is opposite to that seen in rat, for which a specific stimulation of gluconeogenesis from lactate was observed (Whitton et al., 1979).
Concentmtion of additive, mM
Fig. 2. Effect of lysine or NH,CI on gluconeogenesis by chick hepatocytes. Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). The precursor was lactate and was included at an initial concentration of 10 mM. Results are given as the mean, with vertical bars representing SEM, for 7 observations. 0 = lysine; n = NH,CI.
gelatin tended albumin.
to be lower than with bovine
serum
EfSect of lysine and NHICl on gluconeogenesis from lactate
Effect of redox state on gluconeogenesis
The effect of including lysine or NH&l in the incubation medium is given in Fig. 2. Lysine caused a small (approx 476) inhibition at 1 mM concentration but thereafter no further reduction in gluconeogenesis
The effects of including substances known to alter intracellular redox state are given in Table 3. Butan-1,3-diol and ethanol were added to decrease the cytoplasmic free NAD+-free NADH ratio. Gluco-
Table 1. Gluconeogenesis from a variety of precursors by hepatocytes from fed or starved
Net glucose. Fed
Precursor Propionate Lactate Pyruvate Oxaloacetate Malate Glycerol D-Glyceraldehyde Fructose Glycine Serine Alanine Glutamate Aspartate
chicks
44.0 288.8 70.5 105.9 36.7 67.0 42.4 193.4 4.1 7.0 3.9 9.2 6.5
++ f + f f + * * i + f f
11.9 (5) 10.3(5) 9.9 (5) 22.9 (5) 10.5(5) 10.3 (5) 5.5 (5) 20.1 (5) 3.5 (5) 3.3 (5) 3.9 (5) 3.6(5) 2.6 (5)
pmol/g
dry wt/hr Fasted 30.7 563.6 108.1 76.7 19.5 73.0 188.4 416.5 9.3 I 1.4 60.1 7.7 10.2
+ i i k k F k f f f + + *
5.9 (6) 35.8 (16)$ 8.4 (16)* 16.5 (6) 2.5 (5) 5.2 (16) 37.7 (6)t 33.9 (16)t 3.1 (5) 3.0 (6) 8.2 (6)f 1.8 (5) 3.4 (5)
Hepatocytes were prepared as described under Materials and Methods from fed chicks or from ones starved overnight (18-20 hr). All precursors were included at an initial concentration of 1OmM and incubations performed for 30min. Data are presented as the mean f SEM for the number of observations in parentheses. * P < 0.05; t P < 0.01; $ P < 0.001 compared to fed birds.
440
DONALDW. BANNISTER and IRIS E. O’NEILL Table 2. Effect of fatty acids on gluconeogenesis by chick hepatocytes Percentage of control value Octanoate
Acetate
Precursor Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
91.4 85.6 102.8 94.5 99.4
+ f + + *
76.3 90.6 46.9 95.2 97.6
3.0(5)? 7.4(5) 8.3 (8) 4.9 (6) 5.9 (5)
k + k k &
Oleate 76. I 78.1 52.0 94.2 102.9
2.1 (5)$ 4.6 (7) 12.4(5)* 6.1 (8) 7.4(5)
i * i f k
2.4 3.1 9.6 6.2 9.7
(5)k (5): (5)* (7) (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). All precursors were included at an initial concentration of 10 mM and fatty acids at 2 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean f SEM for the number of observations in parentheses. Significance of difference was calculated between control and experimental treatments using a paired ‘I’-test. *P<0.05:tP=0.05; ~P
neogenesis from pyruvate was stimulated whereas there was a small but significant reduction from lactate. Gluconeogenesis from glycerol was strongly inhibited. Ethanol proved more effective than butan-1,3-diol in producing these effects and also inhibited gluconeogenesis from glyceraldehyde and fructose. When 0.1 M-pyrazole (an inhibitor of alcohol dehydrogenase (EC i.l.l.l), Theorell & Yonetani, 1963) was present, the effects of ethanol were overcome in agreement with findings of Krebs rt al. (1969) using perfused rat liver. Inclusion of 3_hydroxybutyrate, to decrease the mitochondrial free NAD+-free NADH ratio, reduced gluconeogenesis from glycerol but was otherwise without effect. Effect qf inhibitors on gluconeogenesis Data on the effects of inhibitors are given in Table 4. Aminooxyacetate. an inhibitor of aminotransferases (Rognstad & Katz, 1970), strongly inhibited gluconeogenesis from lactate and glycerol. There was a little
inhibition from fructose but none from glyceraldehyde or pyruvate. In this respect the chick differs from the pigeon in which there is no inhibition of gluconeogenesis from lactate (Siiling et al., 1973). m-Cyano-4-hydroxycinnamate is established as an inhibitor of mitochondrial pyruvate transport (Halestrap & Denton, 1974) and, as anticipated, inhibited gluconeogenesis from lactate and pyruvate strongly but also caused some inhibition from other precursors. Quinolinate was chosen as an inhibitor of phosphoenolpyruvate carboxykinase (Veneziale et al., 1967) and, at a concentration of 5 mM inhibited gluconeogenesis from lactate and pyruvate by l&12% (Table 4). Gluconeogenesis from other precursors was not affected. SGling et al. (1970), using perfused rat and guinea-pig livers have demonstrated the strong inhibitory effect of quinolinate on gluconeogenesis. Addition of 2.4 mM-quinolinate resulted in a decrease in glucose synthesis of 90 and 55% respectively. Perfused pigeon liver was not affected by quinolinate even at concentrations up to 28 mM and this was attributed to mitochondrial impermeability.
Table 3. Effect of redox state on gluconeogenesis
Precursor Lactate Pyruvate Glycerol D-Glyceraldehyde Fructose
Percentage Butan- 1.3-diol 94.4 188.2 24.0 94.9 87.1 Ethanol
Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
93.0 103.4 90.5 104.0 101.5
f + + i i
1.3 (7)’ 14.3 (61-I 7.0 (5)t 4.4 (7) 6.9 (7)
+ pyrazole i i f f +
2.6 (5) 5.6 (5) 7.6(7) 7.7 (6) 3.2 (5)
by chick hepatocytes of control
value Ethanol 84.4 206.4 11.3 86.9 81.1
* + * k +
1.9 (IO)? Il.5 (1O)t 2.8(12)t 2.3 (6)* 3.1 (Il)t
3-hydroxybutyrate 98. I 104.0 62. I 98.7 96.9
* k I + _t
3.0 3.6 9.2 8.7 6.8
(7) (5) (5)* (8) (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (I 8-20 hr). All precursors were included at an initial concentration of IOmM. Effecters were included as follows: butan-1.3-diol. IOmM: ethanol, 10 mM; 3-hydroxybutyrate, 20 mM and pyrazole. 100 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean + SEM for the number of observations in parentheses. Significance of difference was calculated between control and experimental treatments using a paired ‘?-test. * P < 0.01; t P < 0.001.
Gluconeogenesis in chick hepatocytes
441
loo-
60.
40:: 80.
Qu&olinate c~t~ion
, mM
Fig. 3. Effect of quinolinate on gluconeogenesis by chick hepatocytes and on phosphoenolpyruvate carboxykinase activity. Hepatocytes and mitochondria (for phosphoenolpyruvate carboxykinase activity) were prepared as described under Materials and Methods from the livers of chicks starved overnight (18.-20 hr). Gluconeogenesis by hepatocytes was measured using lactate (10 mM initial concentration) as precursor and phosphoenolpyruvate carboxykinase activity was determined using the method of Seubent & Huth (1965). Results are given as the mean. with vertical bars representing SEM, for 5 observations for giuconeogenesis and the mean of 2 observations for enzyme activity. l = gluconeogenesis; 0 = enzyme activity. The effect of increasing concentrations of quinolinate on gi~coneogenesis from lactate is given in Fig. 3
together with a similar experiment on mitochondrial phosphoenolpyruvate carboxykinase activity. In these experiments glutamate dehydrogenase activity was measured in mitochondrial and supernatant fractions (to estimate mitochondrial damage). Approximately 24% of total activity was present in the latter (data not shown). Phosphoenolpyruvate carboxykinase activity in the supernatant fraction was too low to measure with accuracy but was estimated to be a similar percentage to that of glutamate dehydrogenase. DISCUSSION
Isolated chick hepatocytes have not been studied as widely as those from other species and the optimum
conditions for their preparation and use have not yet been firmly establised. We felt it desirable, therefore, to examine briefly certain properties which, although not directly related to the main aim of the work, might help establish that the method employed here was giving satisfactory results. Zahlten & Stratman (1974) using guinea-pig and Brady et al. (1979) using chick hepatocytes reported that gelatin and bovine serum albumin could be used interchangeably. Our data does not support this view since in the chick the protein caused inhibition of gluconeogenesis from glycerol and the data generally tended to be erratic. Fresh preparations of rat (CornelI et al., 1974) and guinea-pig (Arinze & Rowley, 1975) hepatocytes show a lag period in gluconeogenesis from lactate that may be overcome by inclusion of either lysine or NHLCl in
Table 4. Effects of inhibitors on giuconeogenesis by chick hepatocytes
Precursor Lactate Pyruvate Glycerol o-Glyceraldehyde Fructose
Aminooxyacetate 43.2 2 100.7 + 30.7 i: 94.5 4 89.4 4
1.4(7)f 3.9 (7) 1.6 fS)$ 6.6 (4) 2.7 (5)*
Percentage of control value z-cyano-4-hydroxycinnamate 10.4 + 2.3 (5)$ 54.1 & 6.3 (5)t 72.9 + 1.7 (5); 8 I .O + 7.3 (4) 82.9 If: 1.4 (5)f
Quinolinate 90.7 k 87.1 f 97.1 f 103.7 f 106.1 f
2.3 (16)t 3.5 (7)t 5.4 (5) 2.4 (5) 2.4 (5)
Hepatocytes were prepared as described under Materials and Methods from the livers of chicks starved overnight (18-20 hr). A11precursors were included at an initial concentration of 10 mM and the inhibitors were as follows: aminooxyacetate, 0.5 mM; ~-cyano-4-hydroxycinnamate, 0.33 mM; quinohnate, 5 mM. Data were normalized by expressing values as a percentage of control (no treatment) and are given as the mean f SEM for the number of observations in parantheses. Significance of difference was calculated between control and experimental treatments using a paired Y-test. * P < 0.05; t P < 0.01; : P < 0.001.
442
DOUALI)
W.
BANNEST~,Kand
the incubation medium. However, neither Dickson c’t al. (1978) nor Brady cr u/. (1979) reported a similar phenomenon with chick hepatocytes. Our data on melusion of lysine also indicates that in this species a lag does not occur. It is possible. therefore. that the aspartate shuttle had not become rate-limiting due to accumulation of pyruvate during preparation of the cells. Indeed Ochs & Harris (1978a) reported that chick hepatocytes do not accumulate pyruvate or lactate. Inclusion of NH,CI in the incubation medium caused progressive inhibition of gluconeogenesis from lactate which contrasts with the behaviour of rat or guinea-pig cells. Mapes & Krebs (1978) have studied this phenomenon in greater detail than reported here and postulated that it was due to diversion of carbon units way from gluconeogenesis (oxaloacetate) towards detoxification (glutamine synthesis). The ease with which various precursors were utilized was generally similar to that found by other workers who used isolated chick hepatocytes and different from that of mammals such as rat. The most notable differences were the relatively poor utilization of pyruvate in comparison to lactate and the very poor use of glycerol, alanine and the other amino acids examined compared with lactate. It is possible of course that the rather short period of starvation (18-20 hr) was insufficient to allow gluconeogenesis from amino acids to become fully established. However, it is known that for chicks of this age liver glycogen stores are small and are depleted by 6 hr starvation (Bannister et al., 1979). Our results differ from those of Dickson & Langslow (1978) in that lactate rather than fructose was the most effective precursor and also from those of Brady et aI. (1979) in that the rates we observed were generally much higher. These differences may result from variations in experimental technique or from the use of different genetic strains of bird. We have some evidence suggesting that strain differences may occur (O’Neill & Bannister, unpublished observations). The fact that lactate was the major precursor might be of physiological significance since domestic fowl possess a substantial musculature of the white fibre type and thus could be expected to produce large quantities of this metabolite. However, it is not possible to explain the low rates of gluconeogenesis from glycerol and alanine on the same basis since both are readily utilized in Coo (Davison & Langslow, 1975). Unlike perfused pigeon liver (Siiling rt ~1.. 1973) chick hepatocytes can use pyruvate at an appreciable rate. This was further stimulated (approx. 2-fold) by either butan-1.3-diol or ethanol although not reaching the level obtained with lactate. Thus, the increasingly oxidised redox state of hepatic cytoplasm upon starvation in cico (Belo c’t cl/.. 1976: Davison & Langslow. 1975) does appear to limit gluconeogenesis from pyruvate although some other, as yet unknown factor. further limits the rate in comparison with lactate. Ethanol also inhibits gluconeogenesis from a number of precursors in perfused rat liver (Krebs ut ul., 1969) and qualitatively similar results were obtained here with chick hepatocytes. Inhibition of gluconeogenesis from glycerol. however. was more severe than observed with the rat or than seen with
ISIS E.
O’NEILL
other precursors in these experiments. Exactly why gluconeogenesis from glycerol is particularly susceptible to change in redox state is not understood. However. a parallel observation was made by Bannister & Cleland (1977) in chicks suffering from fatty liver and kidney syndrome. In this condition gluconeogenesis is blocked by the almost total absence of pyruvate carboxylase activity accompanying which there is an elevated lactate concentration and a decrease in the calculated cytoplasmic free NAD+ -free NADH ratio. Liver slices from affected chicks are also unable to use glycerol for gluconeogenesis. The authors suggested that inhibition might occur at the level of glycerol phosphate dehydrogenase (EC I. I. I .8) because of the unfavourable NAD’-NADH ratio. Another possibility is that ATP concentration could be reduced via equilibrium at the glyceraldehyde phosphate dehydrogenase (EC I .2.1.12) system, thereby reducing the ability of glycerol kinase (EC 2.7.1.30) to phosphorylate its substrate. In fatty liver and kidney syndrome, ability of glycerol kinase (EC 2.7.1.30) to phosphorylate its substrate. In fatty liver and kidney syndrome, however. freeze-clamped liver contained normal levels of ATP (Bannister & Cleland, 1977). The effects of ethanol derive from its interaction with alcohol dehydrogenase since they were abolished by pyrazole. The possiblity that 3-hydroxybutyrate might inhibit glyconeogenesis from lactate and pyruvate was tested because this has been seen with guinea-pig and rabbit liver (Arinze et (II., 1973: Garbcr & Hanson, 1971a.b) in which phosphoenolpyruvate carboxykinase also has an intramitochondrial location. We observed no effect on either precursor and attribute this to the low activity of 3-hydroxybutyrate dehydrogenase (EC 1. I. I .30) activity reportedly present in avian liver (Bailey & Horne, 1972; Brady et i/l.. 1978). Gluconeogenesis from glycerol was depressed in the presence of 3-hydroxybutyrate. for which it is difficult to offer an explanation unless one assumes that those reducing equivalents generated by 3-hydroxybutyrate are transferred rapidly to the cytoplasm. Oxidation of medium- and long-chain fatty acids caused slight inhibition of gluconeogenesis from lactate and to a lesser extent from pyruvate. This is opposite to the well known effect in rat liver (Williamson cr (I/.. 1969) but in agreement with guinea-pig (Sibling et al., 1970) and pigeon liver (Siiling rt al., 1973) and probably derives from the fact that in all these species oxaloacetate is generated intramitochondially and is reduced to malate rather than being converted to phosphoenolpyruvatc for export to the cytoplasm and thence glucose synthesis. Gluconeogenesis from glycerol was again adversely affected. The effect of acetate was examined because it is present in avian blood (Annison et cd., 1968) and because it stimulates gluconeogenesis from lactate in rat hepatocytes (Whitton c’t (II.. 1979). The effect on avian cells was to produce slight though significant inhibition of gluconeogenesis from lactate but not the other precursors. The mechanism is unclear; Whitton ef al. (1979) suggest that conversion to acetaldehyde is a possibility. with a consequent increase in cytoplasmic free NAD’-NADH ratio. In view of the evidence given earlier, inhibition of gluconeogenesis from pyruvate would bc anticipated. and although our values
G~~coneog~nesis in chick hepatocytes
were lower in these conditions the difference was not statistically significant. Siiling et al. (1973) demonstrated that aminooxyacetate did not inhibit gluconeogenesis from lactate in perfused pigeon liver, in contrast to rat liver. This was due, it was suggested, to the fact that phosphoenolpyruvate was generated intramitochondrially and therefore the transfer of carbon units to the cytopiasm as aspartate was unnecessary. Our finding that aminooxyacetate inhibited gluconeogenesis from lactate but not pyruvate might, therefore, appear unexpected. However, Ochs & Harris (1978b) made a similar observation with chick hepatocytes. They showed that inhibition was relieved by oleate and suggested that, in the absence of fatty acid, lactate was serving as an energy source for gluconeogenesis as well as a precursor. Aminoox~cetate blocked this function by causing build-up of reducing equivalents in the cytoplasm that would otherwise be transferred to the mitochondria. The fact that gluconeogenesis from glycerol was strongly inhibited (approx 70”,) supports this hypothesis. r-Cyano-4-hydroxycinnamate had the anticipated effect on inhibiting gluconeogenesis from lactate and pyruvate strongiy but also reduced gluconeogenesis from other precursors to a lesser degree, the reason for this is not apparent. We were unable to detect significant phosphoenolpyruvate carboxykinase activity in cytoplasm of chick liver and conclude, in agreement with Felicioli et al. (1967) and Utter (1959), that the enzyme is essentially mitochondrial in the fowl. However, the mitochondria of chick hepatocytes appear to be permeable to high concentrations of quinolinate, in contrast to pigeon liver (Si%ng rt al., 1973) since it was possible to inhibit gluconeogenesis by about 73% at 50mM. That this was due to permeability is indicated by complete inhibition of enzyme activity at about one-tenth the inhibitor concentration. Acknowludyemmt-We are grateful for the skilled technical assistance of Miss June R. Thomson.
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