Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Experimental

Cell Research

161 (1985) 551-557

Ploidy Class-dependent Metabolic Changes after Partial Hepatectomy CORNELIS J. F. VAN NOORDEN,* ILSE GERARD FRONIK, JOOP M. HOUTKOOPER

in Rat Hepatocytes

M. C. VOGEL& and JAN JAMES

Laboratory of Histology and Cell Biology, Uniwrsity of Amsterdam, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands

Glucose&phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of diierent ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: I. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.

@I 1985 Academic

Press, Inc.

The rapid compensatory growth of liver after two-thirds partial hepatectomy is a useful in vivo model for the study of mechanisms underlying controlled cellular proliferation and has also been proposed as an experimental model for the study of neoplasms [l]. Under normal conditions, the hepatocyte is long-lived and the mitotic index of liver parenchyma is very low (one mitosis in 10000-20000 hepatocytes) but partial hepatectomy triggers a rapid proliferation [2]. The organ doubles in volume by 48 h after operation and approaches its original weight by 7 days [2, 31. Many morphological and metabolic changes take place during this period of expansive growth. The number of lysosomes per cell and autophagic activity are increased in the first 10 h after hepatectomy causing early losses of endoplasmatic reticulum and mitochondria [2, 4, 51, whereas the glycogen content is completely depleted within 4 h [2, 5-91. This initial increase of lysosomal activity is followed by a decrease and protein degradation becomes diminished [4, 10, 111, whereas protein synthesis becomes elevated after 12 h with a maximum at 36 h [3]. RNA synthesis starts to increase at 6 h after operation reaching a * To whom offprint requests should be sent. Copyright @ 1985 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/85 SO3.00

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maximum at 12 h [3, 121and DNA synthesis begins at 12 h, with a maximum at 18-24 h after operation [3, 13, 14, 151.Mitosis follows DNA synthesis f&8 h later and the mitotic index (MI) rises sharply up to 5% at 32 h after partial hepatectomy 112, 13, 141.During the first mitotic wave some 30-50% of the hepatocytes are involved in mitosis [12, 131. Recently, it has been found by quantitative cytochemical means that the stem cells of liver parenchyma, the mononuclear diploid (MD) cells differ metabolically from the hepatocytes of higher ploidy classes [16, 17, 18, 191. Proportional values per genome were found for glucose-6-phosphate dehydrogenase (G6PDH) activity and single-stranded RNA (ssRNA) content in the cells of higher ploidy classes but not in the hepatic stem cell population. These mononuclear diploid (MD) cells exhibit a significantly higher G6PDH activity and ssRNA content than would be expected from the number of genome copies in comparison with hepatocytes from other ploidy classes. On the other hand, succinate dehydrogenase (SDH) activity appeared to be proportional in cells of all ploidy classes [18]. These findings lead to the hypothesis that the high relative amount of GBPDH activity and ssRNA content in MD cells is related to their function as stem cells of the liver parenchyma [16, 17, 18, 191. The present study has been performed in order to evaluate the role of the ploidy class-dependent metabolic heterogeneity of hepatocytes during the regeneration process. MATERIALS Animals

AND METHODS

and Cell Preparations

Thirty male Wistar rats, TN0 substrain (TNO, Zeist, The Netherlands), were maintained for at least two weeks under constant environmental conditions with food and water freely available. The environmental temperature was kept at 21-22°C and the relative humidity at 60%. The rats were exposed to a 12-h light-dark cycle (light, 07.0&19.00 h) throughout the acclimatization period. Partial hepatectomy was performed according to the method of Higgins Br Anderson [20]. Ligation and excision of the median and left lateral lobes of the livers resulted in two-third hepatectomy. Before operation, the animals (9ll-weeks-old) were weighed (mean+SD, 240?21 g) and anesthetized with ether (approx. 2 mitt) followed by Nembutal(40 mgIkg body wt, i.p.). At 0, 2, 10, 16, 24,48,72, 120, 168, and 336 h atIer partial hepatectomy, the animals were re-anesthetized and suspensions of isolated liver cells were obtained by a re-cycling collagenase-perfusion of the liver according to [21]. All perfusions were performed between 9.00-l 1.OO h in order to avoid diurnal variations which could affect the measurements [17, 181.

Cytochemical Staining Procedures for G6PDH and SDH Activity ABer harvesting, the isolated cells used for the studies on G6PDH and SDH activity were washed and incorporated in a matrix of polyacrylamide gel as described previously [16, 19, 221. Small circular punches of the cell-containing polyacrylamide films were incubated for 30 min at 37°C in the dark for the demonstration of G6PDH activity in a phosphate-buffered medium (100 mM; pH 7.45) containing 0.67 mM glucose-6-phosphate (Serva, Heidelberg, FRG), 0.48 mM NADP+ (Boehringer, Mannheim, FRG), 4 mM MgCl,, 5 mM sodium azide, 0.1 mM l-methoxyPMS (Dojmdo Chem. Co. Kumamoto, Japan) and 1 mM tetranitro BT (Serva) [22, 231. For the cytochemical determination of SDH activity, punches with cells were incubated for 30 min at 37’C in the dark in a medium containing 100 mM phosphate buffer (pH 7.45) 50 mM succinate (sodium salt; Merck, Darmstadt, FRG), 5 mM EDTA Exp Cell Res 161 (1985)

Ploidy-dependent

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Fig. 1. G6PDH activity in isolated rat hepatocytes of different ploidy classes after partial hepatectomy. The activity is expressed as nU per set of genome copies (2n) as calculated from cytophotometric readings of 30 cells (3 rats; n= 10 per animal). m, MD cells; W, BD cells; A-A, MT cells; A-A, BT cells. (hl

(Merck), 5 mM sodium azide, 0.1 mM PMS and 1 mM tetranitro BT [24]. Control incubations were performed in the absence of substrate for the determination of non-specific background absorbance. The cytochemical determination of G6PDH and SDH activity are both valid for quantitative purposes [18, 19, 23, 24, 251.

Cuprolinic

Blue Staining

Procedure for Single-stranded

RNA

Cytocentrifuge preparations were made from the isolated hepatocyte suspensions, air-dried and fixed in ethanol : chloroform : acetic acid, 60 : 30 : 5 by volume for 10 mitt at room temperature [21,26]. The liver cell preparations were stained for 60 min at room temperature with 0.1% (w/v) Cuprohnic Blue (BDH, Poole, Dorset, England) dissolved in 25 mM acetate buffer (pH 5.6) in the presence of 1 M MgCl*. After staining, the slides were rinsed in several changes of 25 mM acetate buffer (pH 5.6) with 1 M MgClr [27]. Cuprolinic Blue is a highly specific dye for ssRNA in the presence of 1 M MgCI, and can be used for quantitative purposes [28]. The cells were counterstained in a solution of Nuclear Fast Red (0.1%; w/v; C. I. 60760) in distilled water for 1 mitt at room temperature [18]. The cell preparations were rinsed in distilled water, dehydrated in a graded series of ethanol, cleared in xylene and mounted in Euparal.

Cytophotometry The integrated absorbance of ten hepatocytes of each rat per ploidy class, incubated for both G6PDH and SDH activity either in the presence or in the absence of substrate, was measured at 534 nm with a Barr & Stroud GNS densitometer. The integrated absorbance per cell was converted into units X10e9 (nU) of G6PDH or SDH activity (1 U is 1 pm01 of substrate oxidized/min) per cell as outlined previously [23] after correction for the mean background absorption value as determined in ten hepatocytes incubated in the absence of substrate. The integrated absorbance of hepatocytes stained with Cuprolinic Blue was measured at 626 nm and expressed in arbitrary (machine) units (a.u.). The ploidy classes considered were: mononuclear diploid (MD) cells, binuclear diploid (BD) cells, mononuclear tetraploid (MT) cells, and binuclear tetraploid (BT) cells. Statistical analysis of the data was performed by one-way analysis of variance on the various histochemical measures, with respect to their variation with time. The differences between cell types were also analysed using analysis of variance. The MT cell type, which has the highest incidence in adult rats, was used as a reference, to which the cell types of other ploidy classes in the same animal were compared, taking into account the proportionality with genome multiplicity. A 5 % level of significance was applied in all tests. Exp Cell

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35 30 25 20 c ;

15 a

g 2 0

10

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Fig. 2. SDH activity in isolated rat hepatocytes of different ploidy classes after partial hepatectomy. See fig. 1 for further details and symbols.

? 50

100 time

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hepatectomy

(h)

RESULTS The relative G6PDH activity per set of genome copies (2n) in isolated rat hepatocytes during the first 7 days after partial hepatectomy is presented in fig. 1. Analysis of variance demonstrated a significant rise of the G6PDH activity in mononuclear diploid (MD) cells with a maximum activity of 3.5 times the control value at 48 h after operation, whereas the activity in the cells of higher ploidy classes did not change significantly. The activity per MD cell slowly returned to normal values again at 14 days after hepatectomy (6.5 nU per MD cell). The relative activity in MD cells was significantly different from the relative activity per 2n in cells of higher ploidy classes (p
Cell

Res 161 (1985)

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Fig. 3. Content of ssRNA stained with Cuprolinic Blue in isolated rat hepatocytes of different ploidy classes after partial hepatectomy. The amount of ssRNA per cell is expressed in a.u. of integrated absorbance at 626 nm (Z&J. See fig. 1 for further details and symbols. 50

100 time

after

150 partial

hepatectomy

(h)

operation, the ssRNA content per hepatocyte is similar to that in control animals. Data for the G6PDH and SDH activity per cell and for the ssRNA content per cell at the 14th postoperative day are not shown in figs 1-3, as they are at the same level as at 5 and 7 days. DISCUSSION The stem cell of the liver parenchyma, the mononuclear diploid (MD) cell, shows a metabolic behaviour different from that of hepatocytes of higher ploidy classes (figs l-3). This finding is in agreement with previous quantitative cytochemica1 observations that the metabolism of MD cells deviates from that of hepatocytes of higher ploidy classes with respect to G6PDH activity and ssRNA content under normal conditions [16, 17, 18, 191. These metabolic differences seem to be related to the function of MD cells as stem cells of liver parenchyma 116, 17, 181. Hepatocytes other than MD cells all show a similar behaviour after partial hepatectomy. Both the SDH activity and the ssRNA content in all hepatocytes show a slight decrease in the first few hours after operation (figs 2, 3) which may well be explained by the higher lysosomal and autophagic activity during that period [2,4, 51. The decrease is followed by an increase both of the SDH activity and of the ssRNA content above normal values which is, however, significant only in MD cells. The curves’ maxima at 1624 h coincide with a period of increased RNA synthesis [l, 3, 121 and with the onset of DNA synthesis preceding the mitotic wave at 32 h [l, 3, 12-151. Cells in mitosis are essentially glycolytic and this may explain the rise in SDH activity [7, 8, 291. G6PDH activity does not alter very much in hepatocytes of higher ploidy values after partial hepatectomy, although the binuclear diploid (BD) cells show some increase which is not significant. Exp Cell

Res 161 (1985)

556 Van Noorden

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The G6PDH activity in MD cells shows a different pattern after partial hepatectomy (fig. 1) starting to increase after 10 h with a maximum at 48 h followed by a slow decrease towards normal values. Our cytochemical findings are in agreement with a biochemical study of Curtin & Sell [30] performed with homogenates of regenerating liver. However, values of G6PDH activity in liver homogenates are considerably affected by the very high activity in Kupffer cells [19, 31-341 which proliferate at 2 days after partial hepatectomy [ 13, 14, 35, 361. Although MD cells represent only a small fraction of the total population of hepatocytes in mature rats (? 5 %) [21, 37, 381, the role of the MD cells seems to be a very crucial one, especially during periods of growth. G6PDH activity is high during fetal development of the liver [30] and at this stage liver parenchyma consists of 100% of MD cells [26, 391. Postnatal growth of the liver, when hepatocytes of higher ploidy classes appear, is characterized by a relatively high G6PDH activity in MD cells [16]. These facts, together with the increased G6PDH activity in MD cells during the regeneration process after partial hepatectomy, may be explained by the fact that the MD cells as stem cells keep at least some of their fetal characteristics, such as an elevated G6PDH activity and ssRNA content. A retrodifferentiation of metabolic patterns from the adult stage via weaning and neonatal stages back to fetal stages has been described for neoplastic transformations and for regeneration processes [30, 40, 411. In the regeneration process after partial hepatectomy, retrodifferentiation is a fact at 48 h after operation and enzyme patterns investigated were similar to both fetal and neoplastic livers [30]. The present study indicates that mainly the MD cell population and to some extent the BD cell population are responsible for the elevated G6PDH activity after partial hepatectomy. It is also interesting to note that it has been found in our laboratory that the accelerated growth after partial hepatectomy, as analysed histochemically by measurements of protein content of individual hepatocytes, follows a growth pattern similar to the pattern found in the fetal period [42]. The results of the present study draw very much into doubt the conclusion by various authors that G6PDH activity would be a transformation-linked discriminant for neoplastic metabolism [43-48]. Elevated G6PDH activity in premalignant lesions but not in malignant lesions [49] and the high G6PDH activity in the marginal regenerating part of cirrhotic liver nodules [50] may well be related with the high G6PDH activity in MD cells during the fetal period, postnatal development and regeneration. REFERENCES 1. 2. 3. 4. Exp

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Exp Cell

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161 (1985)