Hexokinase isozyme pattern in CCl4-injured rat liver

77

Clinica Chimica Acta, 60 (1975) 77-84 0 Elsevier Scientific Publishing Company,

Amsterdam

~ Printed

in The Netherlands

CCA 6959

HEXOKINASE

MASATOSHI

ISOZYME PATTERN

UEDA,

KAZUHISA

TAKETA

IN Ccl,-INJURED

and KIYOWO

First Department of Internal Medicine, Okayama 5-l 2-Chome, Shikata-cho, Okayama 700 (Japan) (Received

November

University

RAT LIVER

KOSAKA Medical

School,

12, 1974)

Summary Activities of hexokinase isozymes in carbon tetrachloride (Ccl, )-injured rat liver were determined quantitatively by DEAE-cellulose column chromatography and compared with those of regenerating liver, fetal liver and ascites hepatoma cells (AH 130). The Ccl4 -injured liver revealed an isozyme distribution with predominant Types I, II and III (3.2, 8.8 and 6.8 times higher than the control values, respectively) and with undetectable activity of Type IV hexokinase (glucokinase). Although the isozyme pattern generally resembled that of fetal liver or hepatoma cells, the relatively high activity of hexokinase Type III in Ccl4 t,reatment characterizes the pattern of hexokinase isozyme in acue liver damage. -

Introduction Two groups of glucose-phosphorylating enzymes have been demonstrated in livers of rat [1,2], man [3,4] and other animal species [5]. One of them, glucokinase, is relatively specific to glucose with a high K, toward this substrate and its level in the tissue is dependent upon the nutritional state of the animal and the presence of insulin, hence this enzyme appears to play an important role in regulating glucose utilization by the liver. The other, low-K, hexokinase, is less specific to the substrate with a relatively low K, for glucose and its tissue level is stable under dietary and hormonal changes. Low-K, hexokinase is known to consist of three isozymes separable from each other and from glucokinase not only by electrophoresis on starch gel [6,7] or cellulose acetate membrane [8,9] but also by chromatography on DEAE cellulose

[6,7,101. The activity of glucokinase has been reported to be greater than that of low-K, hexokinase in livers of well nourished man [ 31 and rat [1,2] . Our previous communication revealed that hepatic low-K, hexokinase increased significantly in patients with advanced liver diseases (hepatitis, liver cirrhosis

78

and hepatoma) with a concomitant decrease in the glucokinase level, as compared with control individuals [ll] . Isozyme studies on Cellogel membrane suggested that Type III hexokinase was dominant in the acute stage of hepatitis, while Type I hexokinase increased markedly in hepatic cirrhosis and primary hepatoma [9]. Similarly, animal studies in our laboratory have also indicated that low-K, hexokinase activity was much greater than the glucokinase level in rat livers injured experimentally by Ccl,. The isozyme pattern on cellulose acetate strip resembled that of fetal liver or transplantable hepatoma [ 121 . These interesting clinical and experimental observations led us to investigate a quantitative aspect of the alteration in hepatic hexokinase isozymes in acute liver damage by comparing the isozyme distribution of Ccl, -injured rat liver with those of fetal liver, regenerating liver and hepatoma cells. DEAEcellulose column chromatography was employed in the present study to obtain each isozyme activity quantitatively. Materials

and Methods

Unless otherwise stated, 150 to 250 g male Sprague-Dawley rats were purchased from a local supplier and maintained in separate cages with free access to Oriental Laboratory Chow MF for several days prior to each experiment. Ccl, -injured livers were obtained from 5 animals according to the following schedule: The animals which had been starved overnight were given a single dose of 20% Ccl, solution in liquid paraffin (0.5 ml/100 g body weight) through a gastric tube and placed in each cage without feeding for 48 hours. Weight-matched controls were studied with the animal being given an equal volume of liquid paraffin in place of the CCL solution. Regenerating livers were prepared from 5 fed rats 48 h after partial hepatectomy was performed by the method of Higgins and Anderson [13]. Fetal livers were collected from 8 pregnant rats of the same strain 2 to 4 days prior to the expected parturition. Cells of Yoshida ascites hepatoma (AH 130) were obtained from ascites of the same strain rats 9 days after intraperitoneal inoculation. Animals were sacrificed by decapitation and exsanguination. Pooled rat livers in each experimental condition were homogenized in an equal volume of a buffer solution composed of 0.01 M Tris/HCl, 1 mM EDTA and 4 mM acetylcysteine (pH 7.5) using a high speed Waring blender followed by centrifugation at 105 000 X g for 1 h at 0°C. Ascites tumor cells were washed twice by centrifugation at room temperature for 5 min at 1000 rpm using 0.154 M KC1 containing 0.32 mM KHC03 and 4 mM EDTA (pH 7.5), and then treated as described for rat livers. The supernatants thus prepared served as enzyme sources. Activities of low-K, and high-K, hexokinases were determined within an hour with 0.5 and 100 mM glucose as substrates, respectively, by the spectrophotometric method of Viiiuela et al. [l] . The method of Lowry et al. [14] was employed for the measurement of protein concentration. The electrophoresis of hexokinase isozymes on Cellogel membrane was detailed in our previous communication [ 91 . DEAE-cellulose column chromatography was carried out essentially according to the method of Shatton et al. [15] with

79

minor modifications. Five to eight ml of the supernatant solutions were applied directly to a column (1.7 cm X 16 cm) containing DEAE cellulose (Whatman DE52, W and R Balston Ltd, England), equilibrated with the same Tris buffer solution prepared as a homogenizing medium, and then fractionated into 80 tubes (8 ml each) with a linear KC1 gradient at 3” C and with a flow rate of 1.5 ml/min. The enzyme activities were measured at 37°C with 50- to 200+1 effluents in a final volume of 0.5 ml. In order to minimize the loss of glucokinase activity, enzyme assays on effluents at 100 mM glucose concentration commenced within 12 h after the decapitation of animals and those at 0.5 mM glucose were carried out the next day. Fractions of peak activities were concentrated separately by Collodion bags (Sartorius Membranfilter GmbH, Germany) and submitted to Cellogel electrophoresis for identification of the isozymes. The linearity of the KC1 gradient was confirmed by measuring the potassium concentrations. A pure preparation of hexokinase-free glucose-6-phosphate dehydrogenase was obtained from Boehringer Mannheim GmbH. ATP and NAD’ were purchased from Sigma Chem. Co. Results Table I summarizes the data on enzyme extracts which were applied to the DEAE-cellulose column. The usefulness of the present chromatographic method for the quantitation of hexokinase isozymes was tested with a liver extract from normal, well-fed rats (Fig. la). Four peaks designated as I, II, III and IV were separated by the linear KC1 gradient, each corresponding to potassium concentrations of 60, 150, 200 and 220 mM, respectively. Upon electrophoresis of the peak fractions, each of these four hexokinase isozymes revealed a single main band having a mobility identical to that of the original isozyme band resolved by direct application of the extract for electrophoresis, although there existed some cross contamination among Peaks II, III and IV (Fig. 2). Isozyme IV was detected only when a high concentration of glucose was used as a substrate, hence it was characterized as glucokinase. The total recovery in

TABLE

I

HEXOKINASE

Conditions

ACTIVITIES

OF

EXTRACTS

of animal

and tissue

APPLIED

TO

THE

Number

Total

of livers

applied

pooled

DEAE-column (mg)

DEAE-CELLULOSE

protein to the

Hexokinase (units/g Low

K,

COLUMN

activities

supernatant High

protein) Km

Total

Liver Normal.

fed

Normal.

48-h

CClq-treated. Partially

48-h

fasted

hepatectomized,

Fetal, 17-19 Tumor cells Ascites

fasted 48-h

hepatoma

fed 115

days (AH

130)

3

347

4.73

5

345

4.41

3

344

5

342 275 142

57.8

21.9

26.63

6.03

10.44

0

18.6

6.6

8.6

15.2

8.1

0.2

8.3

18.6

46.9

104.7

Fraction

0 08_

Number

CCIL-Treated q

b 5

R

_: x 4a

0.06.

;

004.

2 E

002.

4

: .,: 200 i? 5. -100

t-4 :

OC 0

10

20

30

40 Fraction

50 60 Number

IV

70%

012

n

Hepatectomized

JL

Fractron

Number

70

.

80

10

z

81

0.08r

II 023

AH 1.

130 fL

Fraction

Number

Fig. 1. Elution patterns of rat liver hexokinase from collected and assayed as described in the text. 0-d. ‘)_ _ _ _ _ _c, values at low glucose concentration; ---, CClq-treated. 48-h fasted; c. partially hepatectomized. ascites hepatoma (AH 130).

DEAE-cellulose column. Eight-ml fractions were enzyme activities at high glucose concentration; potassium concentrations. a, normal fed; b. 48-h fed: d, fetal. 17-19 days: and e, tumor Cells.

the effluents approached 60% of the hexokinase activity present in the original supernatant applied. The ratio of glucokinase to low-K, hexokinase activity in the original liver extract was approximately the same as that of the sum activities measured at 100 mM glucose to those at 0.5 mM glucose in the effluents. About 6% of the totally recovered activity was leaked in Fractions 2 and 3 where the most protein was eluted (not shown in the figure), and the activity was identified electrophoretically as that of Isozyme I. The elution pattern for the isozymes of the control, fasted rat liver was essentially identical to that of normal, well-fed rat liver except for a moderately depressed Peak IV activity. The isozyme pattern of Ccl, -injured liver was apparently different from that of the fasted control; markedly increased activities of Isozyme I, II and III (3.2, 8.8 and 6.8 times higher than the control, respectively) and undetectable activity of Isozyme IV (Fig. lb). No discernible band of Isozyme IV was

82

Ai

z

a

Origin1al

I

II

m

Hexokinase isozymes

m

Fig. 2. Electrophoretic patterns of hexokinase isozymes in the original supernatant DEAE-cellulose column chromatography. 0 denotes the origin.

and peak fractions

on

obtained when the fractions corresponding to Peak IV were studied by Cellogel electrophoresis. Each concentrate of the split Peak I fractions was found to have an identical mobility as Isozyme I on Cellogel membrane. Regenerating liver after partial hepatectomy showed an isozyme pattern resembling that of normal, well-fed liver except for slightly increased Peak II and moderately decreased Peak IV activities (Fig. lc). The relative increases in hexokinase Types I and II in fetal liver were similar to that of Ccl,-injured liver. However, the activity of Isozyme III was minimal, resulting in a sharp contrast to the pattern of Ccl4 injury (Fig. Id). Ascites tumor cells (AH 130) had a huge peak corresponding to Isozyme II. Cellogel electrophoresis demonstrated a faint band of Isozyme III in the original supernatant as well as in the fractions where Isozyme III was to be eluted (Fig. le). Discussion It is widely accepted that glucokinase, high-K, hexokinase, of rat liver behaves like an adaptive enzyme, decreasing by fasting or in diabetes and recovering after refeeding or insulin treatment [1,16,17]. However, very little information is available concerning the physiological role of hepatic low-K, hexokinase which consists of three isozymes designated as I, II and III, since

x3

these enzyme activities change very little under altered dietary or hormonal conditions. The present results describe the evidence indicating a marked increase of low-K, hexokinase activity and a corresponding decrease in glucokinase activity in Ccl4 -intoxicated rat liver. The isozyme pattern on DEAEcellulose column chromatography disclosed conspicuous rises in all three forms of low-K, hexokinase (I, II and III), confirming the results obtained with electrophoretic techniques [12]. High levels of Isozyme III were not obtained in regenerating liver, fetal liver and ascites tumor cells of AH 130, suggesting that the predominant Type III hexokinase characterizes the isozyme pattern of the acute hepatic injury. Previous publications have also indicated a noticeable change in two kinetically separable hexokinases in transplantable hepatoma cells or in experimentally induced hepatomas of rat, as shown in the Ccl4 -treated rat liver [l&22]. The concentration of hexokinase seems to be paralleled by the degree of loss of histological differentiation of hepatoma cells [22,23]. The isozyme pattern of tumors varies among authors. The results obtained by Sato et al. [8] support our findings by their demonstration that the higher activities of Isozyme II associated with depressed Isozyme III activities were characteristic of poorly differentiated hepatomas (Yoshida ascites hepatomas) and were also evident in fetal liver. Shatton [ 151, on the other hand, indicated that II and III were predominant in fast growing hepatomas (Novikoff strain) as well as in fetal liver. Farron [24] showed significant amounts of Isozyme III in neoplasmas which were absent from fetal tissues. These discrepancies may be due to different strains of animals or tumor cells available among authors. Our unpublished observation has revealed that the relative amount of Isozyme III varies under experimental conditions even with the same AH 130 and animal strains. Even so, the hexokinase isozyme pattern of CCL, -injured liver is quite distinguishable from those of regenerating liver, fetal liver and hepatomas. The mechanisms underlying the reciprocal changes of glucokinase and hexokinase activities in Ccl, -treated liver are obscure. The hepatolow-K, toxicity of Ccl, is likely to be due to the degradation of the hepatic endoplasmic reticulum [25] and the consequent alteration of protein synthesis [ 261 . It may be plausible that the liver-specific glucokinase is degraded rapidly, or synthesis itself is impaired in the damaged hepatocytes, while the nonspecific hexokinases are induced in the same injured parenchymal cells possibly by a mechanism involving a post-transcriptional regulation [12] . This view is supported by a histochemical demonstration that increased low-K, hexokinase activity and decreased glucokinase activity are both found in the injured parenchymal liver cells [ 271. References 1

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2

D.G.

M.

Walker,

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