Interference of chemicals with glycogen metabolism in isolated hepatocytes

Interference of chemicals with glycogen metabolism in isolated hepatocytes

Toxicology, 18 (1980) 213--223 © Elsevier/North-Holland Scientific Publishers Ltd. INTERFERENCE OF CHEMICALS WITH GLYCOGEN METABOLISM IN ISOLATED HEP...

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Toxicology, 18 (1980) 213--223 © Elsevier/North-Holland Scientific Publishers Ltd.

INTERFERENCE OF CHEMICALS WITH GLYCOGEN METABOLISM IN ISOLATED HEPATOCYTES

G. KRACK, F. GOETHALS*, D. DEBOYSER and M. ROBERFROID

Laboratory of Biotoxicology, Universit~ Catholique de Louvain, U.C.L.-73.69, B-1200 Brussels (Belgium)

SUMMARY

Freshly isolated hepatocytes in suspension were used to evaluate the possible effects of certain chemicals. Conditions including the choice of the incubation medium have been defined for maintaining the cells competent for a sufficient length of time. Using paracetamol alone or in combination with diethylmaleate, we have been able to show that these chemicals markedly alter the metabolic state of the cells, as indicated by an inhibition of glycogen synthesis and even by an enhancement of glycogen degradation, without modifying membrane integrity. These effects are dose-~lependent and probably mediated through modification of glycogen phosphorylase activity.

INTRODUCTION

Methods for the isolation of viable hepatocytes from adult rat liver have now been developed to the stage where this cellular model has been applied to the study of biochemical parameters. ~loreover, freshly isolated liver cells in suspension represent an "in vitro" system that may be suitable for evaluating the direct effects of chemicals [1--5]. One of the main advantages of this model is that isolated parenchymal cells may be exposed to a well defined chemical environment. It also allows to study a dose-response relationship and to compare the effects of various drugs on the same cell population. In order to assess the direct effect of the chemicals on these isolated hepatocytes, they have to remain viable and metabolically competent for a sufficient length of time. A thorough analysis of the experimental condi*Goethals, F. is Aspirant of the Fonds National de la Recherche Scientifique. Abbreviations: CCI,, carbon tetrachloride; DEM, diethylmaleate; GSH, reduced glutathione; LDH, lactate dehydrogenase.

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tions was thus carried o u t in order to ensure an o p t i m u m viability of these cells for at least 4 h. For this purpose, among the various criteria suitable for assessing cellular integrity and metabolic activity, evaluation of cytosolic lactate dehydrogenase (LDH) leakage, dye exclusion an2 glycogen synthesis were chosen. This last criterion reflects a specific function of the liver which has been shown on isolated hepatocytes to be particularly sensitive to the presence o f various exogenous factors such as glucose, amino acids and serum [6--10]. The ability of this model (hepatocytes in suspension) to detect any very early effects of chemicals was tested using carbon tetrachlorirle (CC14), paracetamol alone or in combination with diethylmaleate (DE!'1).Those chemicals have already been studied for their effects on hepatocytes [2,5,11--13]. MATERIALS AND METHODS Collagenase, 13-NADH, glycogen and glucose 1-phosphate were obtained from Sigma Chemical Co. ['4C]glucose 1-phosphate (150 mCi/mmol) was supplied by the Radiochemical Center, Amersham, U.K. Culture media and sera were provided by Flow Laboratories. All the other chemicals were of analytical grade and obtained from Herck.

Preparation of isolated hepatocytes Male Wistar rats (260--280 g), malntaine.t under a stan-lard illumination schedule, were allowed food and water ad libitum until the liver perfusion carried out under pentobarbital (6 mg/kg) anaesthesia. Hepatocytes were isolated according to the procedure developed by Berry and Frien ~ [14] and slightly modified as reported elsewhere [15]. After leaving the cells to se:lim e n t by gravity alone for 10 min, the supernatant was carefully sucked off. The yield of hepatocytes, as estimated by counting in a Bikrker cell under a phase contrast microscope, was usually in the range of 250 X 106--300 X 106 cells/liver. Isolated cells were then suspende2 in the selected me !ia at a final concentration of 5 X 10 s cells/ml in an incubator vessel (Fig. 1) designe~ according to the model o f Jeejeebhoy [16]. This vessel was kept in a thermostatic chamber at 37°C.

Control of cellular membrane integrity Dye exclusion was tested using 0.5 ml of the cell suspension mixed with 0.1 ml of a 0.36% aqueous solution of erythrocin B. This dye was chosen instead of Trypan blue because it has a lower affinity for serum protein [17]. The number of dead cells stained red was determined in a Bfirker cell. The activity of lactate: NADLoxidoreductase (EC 1.1.1.27) was determine l by applying the procedure of Wrobleski [18] either on the culture me~lium or on the cell pellet o0tained after centrifugation at 400 rev./min (17.4 g). Enzymatic activity was measured in the presence of 0.03% Triton X-100 which released the enzyme containe2 in the pellet w i t h o u t inhibiting its

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B

E I} Fig. 1. Incubator vessel designed to maintain isolated hepatocytes in suspension: (A) aperture through which the cells are introduced; (B) thermometer; (C) pH electrode; (D) sampling syringe; and (E) gas bubbling (95% 02 - 5% CO2).

activity. The results are expressed activity and are c o m p a r e d to the incubation time. The percentage mo r e than 15% at the end o f the was f o u n d between 5ye exclusion only one presented in the results.

as a ratio of release:l activity to the total released e n z y m e level det erm i ned at zero of dead cells and LDH leakage is never isolation procedure. Since no :liscrepancy and LDH leakage, this last test will be the

Glycogen content An aliquot (1-ml) of h e p a t o c y t e s suspension was centrifuged at low speed (17.4 g) and washed with 0.9% NaCl in order to eliminate external glucose contained in the incubation medium. The cellular glycogen was extracte:l by 1 ml KOH (1 N) at 100°C for 10 min an:! the protein precipitateJ from the e x t r a c t with an equal volume of acetic acid (1.5 N). 0.5 ml of this ext ract

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was incubated for 30 min with 0.1 mg amylo 1-4, 1-6 glucosidase (EC 3.2.1.1) and the glucose generated was quantified using the glucose oxidase m e t h o d developed b y Huggett and Nixon [ 1 9 ] . ~esults are expresse ^] as p m o l of glucose equivalent/g of protein. IntraceUular glycogen content estimated as a function of time was used as an expression of glycogen metabolic ability of the cells. Phosphorylase A activity was assayed in the direction of glycogen synthesis b y estimating the incorporation of radioactivity from ['4C]glucose 1-phosphate into glycogen. This radiochemicai assay was applied as described [20] in the presence of 0.1 g m o l of caffeine used to reduce the activity of the B form to a negligible value. One unit of phosphorylase A is the a m o u n t of enzyme that incorporates 1 pmol of ['4C]glucose 1-phosphate into glycogen per min. The a m o u n t o f protein was estimated by applying the proce:iure of L o w r y [21] using bovine serum albumin as standard. RESULTS AND DISCUSSION

Experimental conditions for cell incubation Isolated hepatocytes are usually maintained in suspension either in salt balanced buffer or in culture medium designed to reconstitute plasma composition. The cells were thus first suspended in Krebs Ringer bicarbonate (pH 7.4), but, since they did not survive more than 90 rain, this system was discarded. Among the various culture media commercially available, 4 were chosen for comparative study: Ham F,o, Leibovitz (L,s), Dulhecco and Waymouth. The first 2 media have a low carbohydrate concentration (6 mM glucose and 5 mM gaiactose, respectively) whereas the last 2 have a high glucose concentration (25 mM and 27.8 mM, respectively). When hepatocytes (5 × 10 s cells/ml) were maintained in merlin with low carbohydrate concentration (Ham Fl0 an.J Lls), they appeared rapidly damaged as judged by LDH leakage, d y e exclusion and the rapid decrease in glycogen content to approx. 40--20% of the initial value in less than 30 min (Fig. 2A). However, when those cells separated from the culture m e ] i a after a low speed centrifugation (17.4 g) were resuspenJed in Krebs Ringer solution containing 50 mM glucose, their intracellular glycogen content increased as a function of time (up to 60 min). They were therefore still able to synthesize glycogen. Ten percent newborn calf serum had a positive effect on both LDH leakage and dye exclusion b u t it failed to protect the cells against the decrease of glycogen (Fig. 2B). As compareJ with newborn calf serum, the addition of foetal bovine serum or horse serum =lid n o t further improve the incubation conditions. Adding 20% newborn calf serum to Ham F,o medium was not a further improvement (data not shown). On the other hand, when isolated cells were incubated with either Dulbecco or Waymouth culture medium w i t h o u t serum, they also appeared significantly damaged as estimated by the membrane integrity tests (Fig. 3A). The glycogen content of the surviving cells, however, remaine,2 constant and 216

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Fig. 2. (A) Isolated hepatocytes incubated in low carbohydrate media Ham F10 (o) and L~s (×): percentage of LDH leakage, glycogen content ( - - - - - - ) and glycogen synthesis ability in the presence of 50 m_~/Iglucose (~ --) estimated as a function of time. (B) Effect of 10% newborn calf serum on LDH leakage, glycogen content ( - - - - - - ) and glycogen synthesis ability in the presence of 50 n~4 glucose (~ --) estimated as a function of time.

even increased slightly during the entire incubation period. The addition of 10% newborn calf serum appreciably improved the viability of the cells throughout the 4 h of incubation (Fig. 3B). As far as glycogen metabolism is concerned, no significant improvement was however observed. In any case, serum seems to affect mainly the integrity of the plasma mem-

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Fig. 3. Isolated hepatocytes incubated in high carbohydrate media Dulbecco (e) and W a y m o u t h (×): percentage of LDH leakage, glycogen content ( ) estimated as a function of time. (B) Effect of 10% newborn calf serum on both Lq_I-~ leakage and glycogen content ( ) estimated as a function of time.

brane as shown by LDH leakage and dye exclusion but not the glycogen synthesis capacity. That physiological function of the liver has been shown in isolated hepatocytes "in vitro" to require the presence of various components in the incubation medium. Wagle [6] first reported the positive effect of an amino acid mixture on glycogen deposition and Katz [9] emphasized the role of either glutamine, asparagine or alanine as equally effective in enhancing this process. Hue et al. [8] demonstrated that glycogen Jeposition is also dependent on the potassium concentration. As expected in media with low carbohydrate content, the hepatocytes progressively loosed their glycogen content, even though they retained their capacity for glycogen synthesis, when resuspended in the presence of 50 m.~~, glucose. In media with higher glucose concentration, glycogen content remained constant or increased as a function of time. This effect is not only due to glucose [8] but also to the enrichment of the medium with amino acid, especially gluta218

mine, which was present at the concentration of 4 mM (Dulbecco) or 2.4 mM (Waymouth). In contradiction to what has been reported by Walker [ 1 0 ] , the addition of 10% serum did not significantly affect the glycogen content o f the hepatocytes. However, in that study, cells were cultured in monolayers and the adde:i serum (foetal bovine) contained 5 m~I fructose, a carbohydrate known to act synergistically with glucose on glycogen synthesis [ 1 0 , 2 2 , 2 3 ] . The newborn calf serum u s e d i n our trials contained only 0.16 mM fructose as quantified by the method o f Pooe [ 2 4 ] . It is also worth noting that under the experimental conditions used here, hepatocytes isolated from fed rats maintained glycogen synthesis throughout the incubation period, synthesis occurred at a rate o f 140 ~mol of glucose/g prot/h, a rate comparable and even greater than that previously found in isolated cells prepared from fed rats [ 8 ] , starved rats [7] or fed rats pretreated to deplete glycogen [ 2 3 ] . After 3 h of incubation, however, glycogen deposition reached a probable GLYCOGEN CONTENT

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Fig. 4. Effect of CCI4 on LDH leakage and the glycogen content of isolated hepatocytes (5 × l 0 s celis/ml) maintained in suspension in Waymouth m e d i u m supplemented with 10% newborn calf serum. Control (*), 1 raM CCi, ( × ) and 10 m.~t CCI, (r~) treated hepatocytes are isolated from the same liver and incubated together. The graphs represent 1 typical experiment of 3 determinations.

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steady state which could not be explained b y a lack of glucose since washing the cells and resuspending then in fresh medium did not induce more glycogen deposition. Our cell population also showed a large heterogeneity in the initial a m o u n t o f glycogen, apparently related to the nutritional state o f the animal. Such a physiological state is sensitive to a diurnal r h y t h m [ 2 5 ] . Evenmore, this initial glycogen content seemed to affect the new glycogen deposition ability of the hepatocytes, a point which will be discussed later [Krack, G. et al., unpublished]. Because of this variability in both the initial glycogen content and its subsequent synthesis rate, each experiment has been performed with its own control on the same cell batch.

Effect o f various chemicals on glycogen synthesis in isolated hepatocytes Carbon tetrachloride (CC14) the hepatotoxic effects of which have been extensively studied both in vivo [26] and in vitro [3,11,12] was incubated in the presence of isolated hepatocytes (Fig. 4). At low CC14 concentration (approx. 1 mM), no cell damage was observed whereas at higher concentration (approx. 10 mM) toxic responses were obtained as shown both on LDH leakage and the glycogen content. At that concentration, CC14 produced not only a necrotic effect b u t it also impaired the metabolic activity of the surviving cells as judged by measuring the glycogen content of those cells, isolated from the medium by gentle centrifugation (17.4 g). The interpretation of such a modification of glycogen synthesis is, however, difficult in this situation because the cell membrane is probably not intact. Krebs [27] has recently suggested that, under conditions where cell membrane integrity can be preserved, evaluating the impairment of metabolic activity can be used as a means of assessing the functional integrity of the cell w i t h o u t involving cell death. To test such an hypothesis, paracetamol, which is widely used as an analgesic and known to be hepatotoxic [28] was incubated in the presence of isolated hepatocytes at the concentration of 2.5 raM. At this concentration, it did not affect cellular membrane integrity as already reported [13]. However, in our conditions, it clearly impaired the ability of these cells to synthesize glycogen after 2 h of incubation, although the phosphorylase A level remains as low as in the control incubation (Fig. 5A). Paracetamol is converted by a cytochrome P-450 dependent mixed function oxidase to a reactive species accountable for its toxic effect and intracellular reduced glutathione (GSH) traps this reactive species thus protecting the cell [ 2 9 ] . It was therefore of interest to test the effect of paracetamol on ceils incubated in the presence of DE~ ~. which reduces the GSH content [30]. Before testing the effect of the association DEM + paracetamol, preliminary experiments were, however, performed with D E r | alone. Isolated hepatocytes at 5 × 10 s cells/ml incubated in the presence of 0.9 mM DE_M showed no cell damage b u t glycogen metabolism was clearly affected (Fig. 5B). After 1-h incubation, glycogen was broken down at a rate of 3.1 ~mol glucose equivalent- g prot -1 • min-', an effect which could be 220

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Fig. 5. Effect of various chemicals on glycogen content and phosphorylase A activity in isolated hepatocytes maintained in suspension in a Waymouth medium supplemented with 10% newborn calf serum: (A) control ( , ) and 2.5 mM paracetamol ( x ) treated hepatocytes; (B) control (e) and DEM 0.23 mM ( x ) or 0.9 mM ( , ) treated hepatocytes; and (C) control (e) and DEM + paracetamol 0.23 mM. + 2.5 mM ( x ) or 0.9 raM. + 2.5 raM. ( , ) respectively treated hepatocytes. The graphs represent 1 typical experiment of at least 3 determinations.

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catalyzed by phosphorylase A activity which appeared t o be reactivated. At a lower c o n c e n t r a t i o n of DEM (0.23 mI{), even t hough intracellular GSH was similarly depleted (80% vs. 88% of cont rol value} (F. Goethals, pers. co mm.) , glycogen synthesis remained as high as in c o n t r o l cells (Fig. 5B). Finally, isolated h e p a t o c y t e s were incubated in t he presence of either 0.9 mM or 0.23 mM DEM in c o m b i n a t i o n with 2.5 m}{ paracetamol. In the presence of 0.9 mM DEM + 2.5 mM paracetamol, intracellular glycogen was b r o k e n dow n as e x p e c t e d f r om the preceding figure (Fig. 5B,C). However, the phosphorylase A activity was n o t reduced {25.2 U/g prot.) as m uch as when 0.9 mM DEM {12.5 U/g prot.) or 2.5 m}i paracetamol (14.5 U/g prot.) were used alone indicating an e n h a n c e m e n t of t he e f f e c t of paracetamol. Such a synergistic e f f e c t was even more clearly demonstrate,~ when th e cells were incubated in the presence of 0.23 m[{ DE:'~ + 2.5 m:'l paracetamo l (Fig. 5C). T h e br e a kdow n o f glycogen occurred after 90 rain incubatio n at a rate of 1.5 um ol glucose equivalent g prot.-' • min- ' and was correlated with a simultaneous reactivation o f phosphorylase A. CONCLUSION When isolated h e p a t o e y t e s are incubated in W a y m o u t h medium supplem e n t e d with 10% n e w b o r n calf serum, t h e y remain viable and metabolically c o m p e t e n t f or at least 4 h, as estimated by their ability t o synthesize glycogen. However, the m e a s u r e m e n t of glycogen c o n t e n t as a funct i on o f time allows the det ect i on of a metabolic i m p a i r m e n t which does n o t run in parallel with cell viability as estimated by the application o f m e m b r a n e integrity tests. This was confirmed when t he cells were incubated in the presence o f 2.5 mL,I paracetamol alone or in c o m b i n a t i o n with DE?L Our data illustrate the i m p o r t a n c e o f controlling n o t only the membrane integrity o f isolated h e p a t o c y t e s but also their metabolic c o m p e t e n c e . Th e d e t e c t i o n of early t oxi c effects of chemicals requires this t y p e of careful biochemical approach. REFERENCES

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