The stimulatory effect of alloxan-diabetes on the gluconeogenesis from alanine and glutamine in rabbit hepatocytes

0020.711X/81/0+&713-08SO2.00010 Copynghr 6 1981 Pergamon Press Ltd

Inr. J. Biochem. Vol. 13, pp. 713 to 720. 1981 Printed in Great Britain. All rights reserved

THE STIMULATORY EFFECT OF ALLOXAN-DIABETES ON THE GLUCONEOGENESIS FROM ALANINE AND GLUTAMINE IN RABBIT HEPATOCYTES JAN ZALESKI. KRZYSZTOF ZABLOCKIand JADWIGA BRYLA* Institute

of Biochemistry.

University of Warsaw, Al. Zwirki 02-089 Warszawa Poland

(Receioed

14 Nocrmber

i Wigury

93,

1980)

Abstract-l. The stimulatory effect of alloxan-diabetes on the rate of gluconeogenesis from alanine and glutamine in rabbit hepatocytes was accompanied by both an increase in oxygen uptake and a decrease of the 3-hydroxybutyrate/acetoacetate ratio in hepatocyte suspension. 2. Diabetes produced an acceleration of the transport of amino acids into hepatocytes as well as an elevation of their accumulation in the intracellular space. 3. Activities of alanine aminotransferase, aspartate aminotransferase and glutaminase II were significantly increased in livers of diabetic animals. while those of both glutamate dehydrogenase and glutaminase I remained unaltered. 4. Diabetes produced changes in the levels of gluconeogenic intermediates in hepatocytes incubated with alanine which were consistent with facilitation of reactions located in the gluconeogenic pathway between pyruvate and phosphoenolpyruvate and between fructose 1.6-bisphosphate and glucose. Increases in levels of first metabolites of gluconeogenic sequence were also observed in hepatocytes of diabetic animals incubated with glutamine. 5. A decreased rate of lactate production from fructose in hepatocytes of diabetic rabbits was accompanied by a lower pyruvate kinase activity and an increased K,,, value of the enzyme for phosphoenolpyruvate in comparison with those determined in livers of fed animals. 6. The data indicate that the stimulation of gluconeogenesis from alanine and glutamine in hepatocytes of alloxan-diabetic rabbits may result from: (i) an accelerated rate of transport of alanine and glutamine into cells; (ii) enhanced activities of aminotransferases: and (iii) a facilitation of reactions of first steps of gluconeogenic sequence and of those catalyzing the conversion of fructose l,6-bisphosphate to glucose

INTRODUCTION It is known that there are differences in the metabolic response of various mammalian species to diabetes. Humans with diabetes and alloxan-diabetic rats show enhancement of gluconeogenesis from lactate, pyruvate, alanine or glutamate in uioo (Wagle & Ashmore, 1961; DeMuetter & Shreeve, 1963; Friedman et al.. 1965). Perfused livers as well as isolated hepatocytes from diabetic rats produce glucose from lactate, pyruvate, alanine and fructose at much higher rates than normal (Exton et al., 1972, 1973; Ingebretsen et al.. 1972: Pilkis et a/., 1974). An enhancement of gluconeogenesis from pyruvate was also observed in liver slices of diabetic Chinese hamsters (Chang & Schneider, 1970). In contrast, cells isolated from livers of streptozotocin-diabetic guinea-pigs and alloxandiabetic rabbits show no increase in the gluconeogenie rate from either lactate, pyruvate, propionate or fructose as substrate (Elliott & Pogson. 1977a; Zaleski & Bryla, 1978). However, glucose synthesis with either alanine or glutamine as gluconeogenic precursor is markedly enhanced in hepatocytes of diabetic rabbits in comparison with that in fed animals (Zaleski & Bryla, 1978). The present report is concerned with an analysis of the reasons for the stimula-

* To whom

all correspondence

should

effect of alloxan-diabetes on the gluconeogenesis from alanine and glutamine in rabbit hepatocytes.

tory

be addressed. 713

MATERIALS AND Animals, isolation and incubation

METHODS

ofhepatocytes

Young male rabbits weighing 50&7OOg were used throughout. The conditions of animals care. induction of alloxan-diabetes as well as procedures for isolation and incubation of hepatocytes were the same as described previously (Zaleski & Bryla, 1978). Measurement of oxygen consumption The oxygen utilization by hepatocytes was measured at 37’C in the Warburg apparatus in IOml flasks containing Krebs-Henseleit phosphate buffer, the appropriate substrate at 10 mM concentration and hepatocytes (l&l5 mg dry weight). The final volume was 1 ml. On the addition of hepatocytes vessels were saturated with oxygen for 10 min and the measurements were started. The oxygen uptake by hepatocytes was linear with time at least for 60min of incubation (results not shown). Transport

of amino acids

For the measurement of alanine and glutamine transport the hepatocytes were preincubated in Krebs-Henseleit buffer (pH 7.4) in an atmosphere of 95”; O2 + 5% CO2 in the presence of 2 mM amino-oxyacetate (Edmondson et al.. 1977; Joseph et al.. 1978). The transport was initiated by the addition of cells (l&l5 mg dry wt/ml) to the incubation medium containing 1 mM L-alanine or L-glutamine

JAN ZALESKI er al.

714

together with “C-labelled amino acid (0.05 pCi/ml). At appropriate times I-ml aliquots of hepatocyte suspension were removed and the transport was terminated by centrifuging the ceils (for 15 set at 8OGO9) through OS-ml layer of silicone oil (Wacker AR 20-AR 200 = 1:l) to 0.4ml of 12”, perchloric acid. The supernatant was decanted carefully while the pellet after extraction was discarded by centrifugation. The pellet amino acid content was determined by measurement of “‘C-radioactivity in the liquid scintillation counter. The intracellular and extracellular waler contents of the hepatocyte pellets were determined in parallel hepatocyte incubations in the medium containing unlabelled amino acids, amino-oxyacetate, -‘Hz0 (0.15 &i/ml) and “‘Clabelled inulin (0.05 &i/ml). acting as a marker of extracellular space, The intracellular water space was determined as the difference between ‘H,O and “‘C-in&in spaces. It was established that the internai volume of hepatocytes isolated from fed and alloxan-diabetic rabbits is similar (1.26 + 0.29 and 1.46 k 0.21 ml/g dry wt of cells respectively, as measured for 6 separate preparations). The uptake of amino acid was taken as the amount of labelled compound appearing in the inulin-impermeable space. As shown previously (Edmondson et al,. 1977; Joseph & McGivan, 1978) preincubation of liver cells with aminooxyacetate and cycloserine prevents the metabolism of low concentrations of alanine in transport studies. These rest?: have been confirmed in our laboratory with the use, oi amino-oxyacetate alone. Similarly. no significant loss of label from 1 mM glutamine was observed under conditions of the uptake of glutamine. This is consistent with the observation that low concentrations of glutamine are not metabolized by hepatocytes (Lund & Watford, 1976). Enzymr

assays

Determinations of activity of amino acid metabolizing enzymes were performed at 37°C. Measurements of aspartate aminotra~sferase (t-aspartate: 2-oxoglutarate aminotransferase. EC 261.1). alanine aminotransferase (t-alanine: 2-oxoglutarate aminotransferase. EC 2.6.1.2), glutamate dehydrogenase (L-glutamate: NAD oxidoreductase (deaminating), EC 1.4.1.2) and glutaminase I (L-glutamine amidohydrolase. EC 3.5.1.2) were done in the presence of O.t”, Triton X-100. For assay of the activity of aspartate aminotransferase. alanine aminotransferase and glutamate dehydrogenase 2g pieces of livers were homogenized in Potter-Elvehjem homogenizer in 9 vol of ice cold medium containing 0.25 M sucrose, 0.01 M Tris-HCl buffer (pH 7.6) and 0.005 M EDTA (Sarkar, 1977). The supernatant fractions collected after centrifugation at 800 g were used for enzyme assays. Activities of aspartate aminotransferase and alanine aminotransferase were assayed according to Hsu & Fahien (1976). Measurement of glutamate dehydrogenase activity was performed by the method of Strecker (195.5) in the direction of glutamate formation. For assay of the activity of glutaminase I mitochondria were obtained by the method of Schneider & Hogeboom (1950) from crude homogenate prepared for measurement of activity of aminotransferases. The glutaminase I activity was measured according to KovaCeviE (1974). Glutamate formed during incubation of mitochondrial suspension was determined after centrifugation and neutralization of perchlorate extracts (Williamson & Corkey, 1969). Activity of glutaminase II (L-glutamine: 2-oxo-acid aminotransfer~e. EC 2.6.1.15) was assayed according to Kupchik & Knox (1970) by measuring the rate of removal of phenylpyruvate resulting in a decrease of absorbancy at 300 nm. To obtain a crude enzyme solution. 200:, liver homogenate in 0.15 M KC1 made with ground glass homogenizer was centrifuged for 40 min at 16,OOOg and the resulting supernatant was used for measurements. lncubations were carried out in 0.3 M borate buffer (pH 8.5) con-

taining 0.4 mM phenylpyruvate, 60 mM L-glutamine and 2 ml of enzyme solution in a total volume of t0 ml. After 0, 5, 10 and 20 min of incubation 2 ml aliquots were removed and the reaction was terminated by the addition of 0.8 ml of 2@, metaphosphoric acid. After 1Omin the solutions were centrifuged for 15 min at 16,OCOg and then the absorbancy was recorded. The molar absorptivity of phenylpyruvate-borate complex 3570 was used IVT calculations. For assay of pyruvate kinase activity the Sephadextreated liver homogenate was prepared by slightly modified method of Pilkis ef nf. (1978). Pieces of liver (3 gf were homogenized in 10 vol of 20 mM potassium phosphate (pH 7.2) containing 30% elvcerol. 0.1 mM EDTA. 10 mM NaF and 0.1 mM d:thiothreitol. The homogenate was then centrifuged at 12,OOOg for 45 min. Five-millilitre aliquots of supernatant were subjected to gel filtration on a Sephadex G-25 column (1.5 x 90cm) that was equilibrated with the homogenizing buffer. The pyruvate kinase fraction emerged in the void volume and was assayed at 25 C by the method of Tietz & Ochoa (1958). Protein concentration in homogenate and mitochondria was determined by a biuret method (Gornall et (II.. 1949) while that in the pyruvate kinase fraction was measured by the method of Lowry et ul. (1951). Drrerminafion

of mefaholites

Metabolites were assayed in supernatants after centrifugation of the neutralized HC104 hepatocyte extracts as desctikd previously (Zaleski & Bryla. 1977). Glucose was measured with glucose oxidase and peroxidase (Huggett & Nixon, 1957). Ketone bodies. malate, citrate and 2-oxoglutarate were assayed ~uorimetrjcally according to Williamson & Corkey (1969) while other metabolic intermediates were determined by standard enzymatic procedures (Bergmeyer, 1965) either fluorimetrically or spectrophotometritally.

Collagenase, alloxan and all substrates were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Other enzymes were purchased from either Sigma or Boehringer (Mannheim, Germany). Amino-oxyacetate was from Eastman Kodak Co. (Rochester. NY, U.S.A.). Radiochemicals were from the Radiochemical Centre (Amersham, Bucks. U.K.). Other chemicals were of analytical grade.

Expression oj resulis All results are expressed as means rf: SEM. The statistical significance of data was calculated by Student’s t-test.

RESULTS Glucose

production,

oxygen uptake und ketone bodies

formation In contrast to rat (Berry et uI., 1974: Crow et ol., 1978) and guinea-pig (Arinze & Rowley, 1975; Elliott & Pogson, 1977b) the rate of glucose production from pyruvate in hepatocytes from fed and alloxan-diabetic rabbits was much lower than that measured from lactate. Both alanine and glutamine were poor gluconeogenie substrates in hepatocytes from fed rabbits (Table l), confirming our previous findings (Zaleski & Bryia, 1978, 1979). Alloxan-diabetes resulted in a marked stimulation of rates of glucose production only from alanine or glutamine as gluconeogenic substrate. Surprisingly, the rate of oxygen uptake by hepatocytes isolated from diabetic animals was enhanced by 3(r50°,; with all substrates studied. An increase in the rate of respiration could be due to a decrease in the mito~hondrial ~A~H,~A~’ ratio.

Giuconeogenesis Table

1. Glucose

production

Glucose Fed

Substrate None L-Lactate Pyruvate L-Alanine L-Glutamine

3.0 44.4 30.0 17.5 5.5

+ * k + +

from alanine

and glutamine

in diabetic

and oxygen uptake by hepatocytes alloxan-diabetic rabbits production Alloxan-diabetic

1.0(7) 2.2 (7) 5.2 (6) 2.2 (6) 2.6(5)

4.4 59.2 37.0 41.1 33.3

* + + + *

rabbits

isolated

Oxygen

I.1 (7) 3.7 (IZ)* 4.8 (6) 3.3 (6)t 3.0(9)$

45.0 73.6 75.5 62.2 66.6

715

from

fed and

Fed

uptake Alloxan-diabetic

+ f + f *

45.5 95.4 99.5 91.2 101.6

4.2 (6) 4.0(5) 7.0(5) 8.0(6) 5.1 (4)

+ + + f f

X0(5) 2.5 (6): 9.9 (4); 3.2(6)t 7.2(6):

Rabbit hepatocytes were isolated and incubated for 30 min as described in the Material and Methods section. Pyruvate was used at 5 mM and all other substrates were added at 10 mM concentrations. Values for glucose production and oxygen uptake + SEM (in pmol/30min/g dry wt of cells) are shown for the number observations given in parentheses. * Significantly different from the corresponding fed controls. P < 0.02. t Significantly different from the corresponding fed controls. P < 0.01. 1 Significantly different from the corresponding fed controls, P < 0.001. Other differences are not significant.

as concluded from a decline of the 3-hydroxybutyrate/ acetoacetate ratio in the presence of pyruvate. lactate (Zaleski & Bryla, 1978) and alanine as substrate (Table 2). Thus, an increased pyruvate oxidation may be responsible for the lack of a significant stimulatory effect of alloxan-diabetes on gluconeogenesis from both pyruvate and lactate. In order to search for factors causing an enhanced gluconeogenesis from either alanine or glutamine in hepatocytes of diabetic rabbits, despite their increased respiratory capabilities, we have investigated the effect of alloxan-diabetes on: (i) amino acid transport into hepatocytes; (ii) the activity of enzymes involved in metabolism of alanine and glutamine: (iii) levels of

Table

metabolic intermediates of gluconeogenic and (iv) the pyruvate kinase activity. Transport

of amino acids into hepatocytes

on the time course of the transport of alanine and glutamine into rabbit hepatocytes is shown in Fig. 1, when these amino acids were added at 1 mM concentrations. In agreement with results of Joseph et al. (1978) the uptake of these amino acids was linear with time over a period of 2 min. The initial rates of transport for both alanine and glutamine were similar (15 f 2 and 18 k 2 nmol/min/mg dry wt of cells for the transport of alanine and glutamine. respectively, as calculated The effect of alloxan-diabetes

2. [3-Hydroxybutyrate]/[acetoacetate] ratio in the suspension cytes isolated from fed and alloxan-diabetic rabbits

Animals

3-Hydroxybutyrate

Fed Alloxan-diabetic

3.7 * 1.5 I.1 f 0.1*

Acetoacetate 11.1 f 2.6 10.0 * I.1

of hepato-

3-Hydroxybutyrate Acetoacetate

ratio

0.33 f 0.08 0.12 f 0.01*

Hcpatocytes were incubated for 30 min with 10 mM alanine. Values shown pmol/g dry wt of cells) are means f SEM for 4 separate experiments. * Significantly different from the corresponding fed controls, P < 0.01. Table 3. Activities of glutamate dehydrogenase. aspartate aminotransferase, alanine aminotransferase and glutaminases I and II in livers of fed and alloxan diabetic rabbits Enzyme Enzyme Glutamate dehydrogenase Aspartate aminotransferase Alanine aminotransferase Glutaminase I Glutaminase II

pathway;

Fed 244 707 86 13.0 1.1

+ & + * f

23 (8) 32 (8) 9 (7) 1.3 (7) 0.1 (6)

activity Alloxan-diabetic 228 1220 145 13.0 2.3

* + ) + +

13(8) 78 (6)* 10(7)* 1.0(7) 0.1 (6)*

Enzyme activities were assayed under conditions described in the Material and Methods section. The values (in nmol/min/mg of protein) are means + SEM for the number of observations given in parentheses. * Significantly different from the corresponding fed controls, P < 0.001.

(in

JAN ZALESKIet al.

rat liver hepatocytes. Alloxan-diabetes resulted in an increase of both the initial rate of amino acid transport (to 28 F 4 and 38 + 3 nmol/min/mg of dry wt of cells for the transport of alanine and glutamine. respectively, as calculated for 6 experiments) as well as the content of amino acids in hepatocytes. Activities of enzymes aianine and ylutamine

Incubation

0

20

40

tlme,min

Fig. 1. Time course of the transport of alanine and glutamine in hepatocytes isolated from fed and alloxan-diabetic rabbits. Rabbit hepatocytes were isolated and incubated as described in the Material and Methods section. The results obtained are expressed as means + SEM for 6 separate experiments. for 6 separate experiments). The internal and external concentrations of these two amino acids become equal within approx 1 min of initiation of transport and the subsequent uptake represents transport of the amino acids against a con~ntration gradient. These data confirm those reported by Joseph et al. (1978) for

Levels of metabolic intermediates in cell suspension Variations in the levels of intermediary metabolites in the suspensions of hepatocytes isolated from fed rabbits after incubation with either lactate, pyruvate or alanine are shown in Table 4. In the presence of either pyruvate or lactate concentrations of intermediates in hepatocyte suspensions from fed animals were similar to those measured in liver cells from fasted rabbits (Zaleski & Bryla, 1977). When alanine was used as substrate the concentrations of pyruvate, malate, 2-oxoglutarate and citrate in hepatocyte suspensions of fed rabbits were much lower than those

Table 4. Levels of intermediary metabolites in suspensions of hepatocytes isolated from fed rabbits following the incubation with either lactate. pyruvate or alanine + Lactate

Metabolite Lactate Pyruvate Malate Phosphoenolpyruvate 3-Phosphogly~rate 1,3-Diphosphoglycerate

566.4 27.7 3.37 0.27 )

qf

As shown in Table 3 the total aspartate aminotransferase activity in livers of fed rabbits was almost an order of magnitude higher than the total alanine aminotransferase activity. In agreement with earlier reports by Errera & Greenstein (1949) the activity of glutaminase I in rabbit liver mitochondria is rather low, but higher than that of glutaminase It. Similarly to the mitochondrial phosphoenolpyruvate carboxykinase of rabbit liver (Usateqko. 1970: Huibregtse et al., 1976) alloxan-diabetes did not result in a significant change of activity of glutamate dehydrogenase and glutaminase I, both located in the mitochondrial compartment (Brdiczka er al., 1968; Kalra & Brosnan, 1973). In contrast, the total hepatic activity of aspartate and alanine aminotransferases, distributed between the mitochondria and the cytosol (Sarkar, 1977) as well as that of glutaminase II. located in the cytosolic compartment (Errera, 1949). was about twice higher in livers of alloxan-diabetic rabbits in comparison with that in fed animals.

tlmr,min

30

lncubotron

involvrtl in the metubolism

k + + +

Content of metabolites + Pyruvate

24.7 (6) 4.3 (7) 0.15 (6) 0.08 (4)

77.1 f 4.5 (6) 146.3 & 14.0(4) 5.13 f 0.31 (6) 1.68 & 0.35 (4)

+ Alanine 16.8 f 1.03 f 0.53 + 0.71 *

1.9(4) 0.39 (4) 0.07 (5) 0.06 (4)

0.74 + 0.05 (6)

4.24 2 0.59 (7)

Glyceraldehyde Dihydroxyacetone3-phosphate phosphate >

0.21 4 0.02 (6)

0.23 + 0.03 (7)

0.20 f 0.03 (5)

Fructose 1.6-bisphosphate Fructose 6-phosphate Glucose 6-phosphate 2-Oxoglutarate Citrate Aspartate Glutamate

0.08 f 0.01 (6) 0.15 f 0.01 (6) 0.37 & 0.03 (6) 3.81 & 0.37 (4) 2.57 5 0.17 (7) 1.03 + 0.12(6) -

0.09 0.15 0.34 8.36 6.23

0.08 * 0.01 (4) 0.15 + 0.01 (5) 0.39 + 0.01 (5) 0.65 & 0.09 (4) 0.87 f 0.1216) 4.53 i 0.64 (7) 30.4 + 3.9(6)

f O.Ol(7) f O.Ol(6) &-O.Ol(6) & 0.96 (4) & 0.64 (7)

1.12 i 0.13(6)

Hepatocytes isolated from fed rabbits were incubated for 30 min with either 10 mM lactate, 5 mM pyruvate or 10 mM alanine. Values shown are means + SEM (in nmol/g dry wt of cells) for the number of observations in parentheses.

Gluconeogenesis

from alanine

and glutamine

in diabetic

rabbits

717

150

I

LACTATE

1

::

2

e

e t

ALANINE

0;

I La,

I Fyr

I Mel

I I PEP PGI

1 TP

$3) I FOP F6P

I ‘6P

1 ctt

I KQ

1 ASP

I Gl”

Fig. 2. The effect of alloxan-diabetes on the content of metabolic intermediates in suspension of rabbit hepatocytes incubated with pyruvate. lactate or alanine as gluconeogenic precursor. The concentrations of intermediates in suspensions of hepatocytes of alloxan-diabetic rabbits are expressed as percentage of control values measured in those of fed animals. Experimental conditions as well as metabolite levels for suspension of hepatocytes isolated from fed rabbits are presented in Table 4. Points with vertical lines represent the means & SEM of the number of experiments shown in parentheses. P values (against the corresponding values of metabolites determined for suspensions of hepatocytes of fed rabbits) are shown where significant. Lact. lactate; Pyr. pyruvate: Mal, malate: PEP, phosphoenolpyruvate; PGA. 3-phosphoglycerate + 1,3-diphosphoglycerate: TP. triosephosphate: G6P. glucose 6-phosphate; FDP, fructose l.6-bisphosphate; F6P. fructose 6-phosphate; Cit. citrate; Kg. 2-oxoglutarate; Asp, aspartate: Glu, glutamate. measured

with either

lactate

or pyruvate

as gluconeo-

genie substrate. The metabolic changes induced by alloxan-diabetes in rabbit hepatocytes incubated with lactate. pyruvate or alanine’are presented in Fig. 2. The concentrations of intermediates in suspensions of hepatocytes isolated from alloxan-diabetic rabbits are expressed as a percentage of values measured in suspensions of cells obtained from fed animals. With all three substrates studied alloxan-diabetes resulted in a significant decrease in the levels of gluconeogenic intermediates in the sequence from fructose 1.6-bisphosphate to glucose 6-phosphate, suggesting a facilitation of reactions catalyzed by fructose 1.6-bisphosphatase and glucose 6-phosphatase. In contrast to both lactate and pyruvate when alanine was used as substrate alloxan-diabetes resulted in a significant increase in levels of malate, phosphoenolpyruvate, 3-phosphoglycerate + 1,3-diphosphoglycerate and aspartate. These changes could be due to: (i) an accelerated transport and accumulation of alanine in hepatocytes (cf Fig. 1); (ii) a higher pyruvate production in the reaction catalyzed by increased

Table 5. The content of glutamate, ammonia aspartate, 2-oxoglutarate and malate in hepatocyte suspension of fed and alloxan-diabetic rabbits incubated with glutamine Content Fed

Metabolite Glutamate Ammonia Aspartate 2-Oxoglutarate Malate

31.4 18.5 1.95 0.54 0.59

f + + k *

6.4(4) 2.2 (6) 0.24 (5) 0.08 (4) 0.10 (5)

of metabolites Alloxan-diabetic 71.8 78.8 3.13 0.90 0.93

+ 9.6 (5)t + 16.5(7)* +_ 0.35 (5)$ f 0.12 (6)$ + 0.10 (6)$

Hepatocytes isolated from either fed or alloxan-diabetic rabbits were incubated for 30 min with 10 mM glutamine. Values shown (in pmo)/g dry wt of cells) are means k SEM of the n:Amber of experiments indicated in parentheses. * Significantly different from the corresponding fed controls. P < 0.001. t Significantly different from the corresponding fed controls, P < 0.02. 1 Significantly different from the corresponding fed controls, P < 0.05. 3 Significantly different from the corresponding fed controls. P -c 0.1.

JAN ZALESKI et al.

718

Table 6. Pyruvate kinase activity and its Ko.s value for phosphoenolpyruvate in the Sephadex-treated liver homogenate of fed and alloxan-diabetic rabbits

Animals

Pyruvate kinase activity (nmol/min/mg of protein)

Ko.s (mM)

64.5 ) 3.1(11) 44.2 + 3.0(6)*

1.03 + 0.03(9) 1.60 + O.l5(5)t

Fed Alloxan-diabetic

Pyruvate kinase activity was assayed in the presence of 4 mM phosphoenolpyruvate by the method described in the Materials and Methods section. K,,, value was obtained by the Hill plot of L’ vs log[phosphoenolpyruvate], log __ Vma, - I’ where r and V,,, denote the initial obtained by the Lineweaver-Burk

and maximal

plot

initial

V,,, was of saturating

velocities.

in the region

phosphoenolpyruvate

concentrationsThe values shown are means ) SEM for the number of observations given in parentheses. * Significantly different from the corresponding fed controls, P < 0.01. t Significantly different from the corresponding fed controls, P < 0.05.

activity of alanine aminotransferase (cf Table 3); (iii) an enhanced pyruvate carboxylation and phosphoenolpyruvate formation; and (iv) a decline of pyruvate kinase activity caused by an inhibitory effect of alanine (Llorente et al., 1970) and an almost complete suppression of fructose 1,6_bisphosphate, an activator of the enzyme (Irving & Williams, 1973). As shown in Table 5 when glutamine was used as glucose precursor the content of first metabolites in the gluconeogenic sequence was also increased in suspension of hepatocytes isolated from diabetic rabbits. This might be due to (i) an accelerated transport of glutamine into hepatocytes (cf Fig. 1) and (ii) an enhanced activity of both glutaminase II and aspartate aminotransferase (cf Table 3). An increased production of ammonia in hepatocytes of alloxandiabetic rabbits might result in the stimulation of glutaminase I (Joseph & McGivan, 1978a.b) providing more glutamate and ammonia. An enhanced production of urea from glutamine in rabbit hepatocytes under conditions of alloxan-diabetes (Zaleski & Bryia, 1978) is consistent with this suggestion. Pgrwate

kinase actiuit)

The data of Table 6 show that the activity of pyruvate kinase is significantly lower in livers of diabetic rabbits than in those of fed animals. Although the of the enzyme K ,,5 value for phosphoenolpyruvate Table 7. Lactate of hepatocytes

Incubation period (min) 0 30

formation from fructose in the suspension isolated from fed and alloxan-diabetic rabbits Lactate formation (pmol/g dry wt of cells) Alloxan-diabetic Fed 11.5 &- 3.0(5) 57.7 + 10.4(5)

8.1 + 0.7(7) 10.0 + 2.6 (7)*

Rabbit hepatocytes were isolated and incubated for 30 min with 10 mM fructose as described in the Material and Methods section. Values are shown for the number of experiments given in parentheses. * Significantly different from the fed control. P < 0.001.

extracted from livers of fed animals is similar to that reported by Irving & Williams (1973). however. the enzyme of diabetic rabbits exhibits a lower affinity for phosphoenolpyruvate than that of fed animals. A decline of lactate production from fructose in hepatocytes of alloxan-diabetic rabbits in comparison with that in fed animals (Table 7) is consistent with the suggestion that pyruvate kinase activity is decreased under conditions of alloxan-diabetes. so the recycling in the pathway of gluconeogenesis may be reduced. DISCUSSION

It is known that rat liver alanine aminotransferase exhibits a high K, value (34 mM) for alanine (Segal & Matsuzawa, 1970). Similarly, K, values for glutamine of liver glutaminase I have been reported to be 28 and 5-1OOmM according to Huang & Knox (1976) and Katunuma et al. (1968), respectively. Glutaminase II has a lower K, (2 mM for this amino acid (Errera, 1949). Thus, the intracellular concentration of alanine and glutamine in hepatocytes may be of importance for the rate of gluconeogenesis from these amino acids. Since both the initial rate as well as the levels of alanine and glutamine are significantly increased by alloxan-diabetes (cf Fig. 1) it is possible to assume that under these conditions there is a higher availability of these amino acids for alanine aminotransferase and glutaminases I and II. Moreover, the glutamine degradation may be accelerated due to the stimulation of glutaminase I activity by NH: (Joseph & McGivan. 1978a,b), known to be accumulated under conditions of alloxan-diabetes (cf Table 5). An increased transport of amino acids was also observed in rat perfused liver as well as in parenchymal cells incubated with glucagon (Mallette et al., 1969; Kelley et a/., 1978; Kletzien et al.. 1976; Le Cam & Freychet. 1976) which was pointed out to be high in the blood of diabetic humans, dogs and rats (Unger et al.. 1970: Muller et al., 1971). The data suggesting a facilitation of reactions located in the gluconeogenic pathway between pyruvate and phosphoenolpyruvate and between fructose 1,6-bisphosphate and glucose in the presence of ala-

Gluconeogenesis

from alanine

nine are in agreement with the results of Huibregtse et al. (1976) showing an increase of the activities of cytosolic phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase in livers of alloxan-diabetic rabbits. The facilitation of reactions of the first steps of gluconeogenic sequence in diabetic animals might also be due to a decline of pyruvate kinase activity resulting from an increase of Kos value of this enzyme for phosphoenolpyruvate (cf Table 6) and a significant decrease of fructose 1.6-bisphosphate level (cf Fig. 2), an activator of the enzyme (Irving & Williams, 1973). This suggestion is consistent with a decline of lactate production from fructose in hepatocytes isolated from diabetic rabbits (cf Table 7). Although a decrease of pyruvate kinase activity was also observed in livers of diabetic rats (Feliu et al., 1977; Tanaka et al., 1967; Weber et al., 1965), however, the rate of lactate production from fructose in the latter species was reported not to be aitered (Exton et al., 1973). A significant increase of the Ko.s value of pyruvate kinase for phosphoenolpyruvate in livers of diabetic rabbits (cf Table 6) is in agreement with an inverse non-linear relationship between the concentration of phosphoenolpyruvate and the activity of pyruvate kinase in liver of diabetic rat (Feliu et al., 1977). An increase of the I& value of enzyme for phosphoenolpyruvate was also observed in livers of starved and glucagon-treated rats (Kohl & Cottam, 1977; Riou et ul., 1976; Van Berkel et al., 1977). Moreover, both glucagon and starvation were reported to cause a marked depression of pyruvate kinase flux from nearly one-half that of gluconeogenesis in hepatocytes of fed rats to as low as 10% of the rate of glucose production from lactate in those from fasted animals (Rognstad & Katz. 1977). Thus, in view of the presented data a decline of pyruvate kinase activity under conditions of gluconeogenesis in hepatocytes of diabetic rabbits is very likely. especially in the presence of alanine, an inhibitor of the enzyme (Irving & Williams, 1973). A~,knowledgemenrs~The authors are greatly indebted to Professors S. G. Van den Bergh and F. Muller who kindly provided alloxan and silicone oils, respectively and to Professor J. R. Williamson for making available the fluorimeter for the measurements of levels of metabolic intermediates. The participation of Mr Szulc in experiments concerning the pyruvate kinase activity is acknowledged. The studies were supported by grant No. 11.1.1.4 from the Polish Academy of Sciences.

and glutamine

in diabetic

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