[CANCER RESEARCH 53. 42(14-4211. September 15, 1993]

Reduction in the Expression of Glucose Transporter Protein GLUT 2 inPreneoplastic and Neoplastic Hepatic Lesions and Reexpression ofGLUT 1 in Late Stages of Hepatocarcinogenesis1

Rainer Grobholz, Hans JörgHacker, Bernard Thorens, and Peter Bannasch2

Abteilung fürCytopalhologie (0310), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany /R. G., H. J. H., P. B.], and Institut dePharmacologie, Universitéde Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland ¡B.T.]

ABSTRACT

Expression of two important glucose transporter proteins, GLUT 2(which is the typical glucose transporter in hepatocytes of adult liver) andthe erythroid/brain type glucose transporter GLUT 1 (representing thetypical glucose transporter in fetal liver parenchyma), was studied iniimi-

nocytochemically during hepatocarcinogenesis in rats at different timepoints between 7 and 65 wk after cessation of 7-wk administration of 12mg/kg of body weight of \-nitrosomorpholine p.o. (stop model). Foci of

altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci(MCF) composed of both glycogenotic and glycogen-poor cells were pre

sent at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes.Morphometrical evaluation of the focal lesions revealed that the numberand volume fraction of GSF increased steadily until Wk 65. MCF wererare at 7 wk, increased slightly in number and size until Wk 37, butshowed a pronounced elevation in their number and volume fraction fromWk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreasedor partially absent at all time points. Consequently, foci of decreasedGLUT 2 expression showed a steady increase in number and volumefraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurredin some MCF from Wk 50 onward. The liver type glucose transporterGLUT 2 was decreased in all adenomas and hepatocellular carcinomas(HCC). In three of seven adenomas and 10 of 12 carcinomas, expression ofGLUT 1 was increased compared with normal liver parenchyma. In twocases of adenoid HCC, cells of ductular formations coexpressed GLUT 2and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, andcystic cholangiomas expressed only GLUT 1. Seven of 12 HCC containedmany microvessels intensely stained for GLUT 1, a phenomenon neverobserved in normal liver. Whenever adenoid tumor formations occurred,GLUT 1-positive microvessels were located in the immediate vicinity of

these formations. Only in one HCC were such microvessels found in theabsence of adenoid formations. Our studies indicate that a reduction ofGLUT 2 expression occurs already in early preneoplastic hepatic foci andis maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a fewMCF and in the majority of adenomas and carcinomas.

INTRODUCTION

Glycogen storage and glucose release of hepatocytes play a pivotalrole in the regulation of blood glucose homeostasis. In liver, glucoseis transported into parenchymal cells mainly via a facilitative transportmechanism. Within the family of facilitative glucose transporters thathave been detected so far (1), the low-affinity liver type glucose

transporter GLUT 2, which occurs in all hepatocytes of adult animals,is responsible for a symmetrical transport of glucose and, thus, part ofthe blood glucose level regulating system in hepatocytes (2, 3). Inaddition, a second glucose transporter, namely, the erythroid/braintype transporter GLUT 1, is found in liver, especially in the layer ofhepatocytes surrounding the terminal hepatic vein (4). During rat liver

Received 3/25/93; accepted 7/13/93.The cosls of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate Ihis fact.

' Dedicated to Rolf Preussmann on the occasion of his 65th birthday.2 To whom requests for reprints should be addressed.

development a shift in the expression of glucose transporters fromGLUT 1 present in high amounts in embryonic liver to GLUT 2 occurs2 days after birth (5, 6).

GLUT 1 has a high affinity for glucose and shows an asymmetricaltransport, i.e., in direction of glucose uptake (3, 7). Fasting causes a 3-to 4-fold GLUT 1 protein expression involving additional perivenular

hepatocytes, whereas GLUT 2 protein expression remains unchanged.During refeeding, however, GLUT 2 increases, whereas GLUT 1 fallsto basal level (8). These observations suggest that GLUT 1 is important for the glucose supply of hepatocytes under glucose-deficient

conditions. In addition to these physiological changes in GLUT expression, recent studies have shown that human liver tumors knownfor their increased glucose uptake express high amounts of GLUT 1,GLUT 2, and GLUT 3 mRNA (9, 10), suggesting shifts in glucosetransporter gene expression during neoplastic transformation.

Investigations in various species revealed that characteristicchanges in the morphological and biochemical phenotype of hepatocytes including fundamental alterations in the different pathways ofcarbohydrate metabolism emerge in focal hepatic lesions long beforehepatocellular adenomas and carcinomas develop (11). These changeshave been particularly well studied in the rat (11-13). On the basis of

correlative cytomorphological, cytochemical, and morphometric studies in this species, a predominant sequence of cellular changes hasbeen inferred, leading from clear and acidophilic cell foci excessivelystoring glycogen (GSF)3 over MCF and HCA composed of glycog

enotic and glycogen-poor cells to HCC in which glycogen-poor, ba-

sophilic cells rich in ribosomes prevail (14). This sequence of cellularchanges is associated with alterations in the activities of importantenzymes involved in glycogen degradation, gluconeogenesis, glycoly-

sis, and the oxydative pentose phosphate pathway (11, 15, 16). Sincethese different metabolic pathways depend on a sufficient supply ofglucose, it was of interest to study the expression of glucose transporter proteins in preneoplastic and neoplastic liver lesions. Here, wereport that the expression of the liver type glucose transporter GLUT2 is markedly reduced in preneoplastic hepatic foci and hepatocellularneoplasms, whereas GLUT 1 is often reexpressed in hepatocellularadenomas and carcinomas.

MATERIALS AND METHODS

Animals and Tissue Preparation. Adult male Sprague-Dawley rats (pur

chased from the Institut fürVersuchstierzucht, Hannover, Germany) were keptunder constant conditions (room temperature, 22°C;humidity, 55%; light:dark

cycle, 12 h:12 h). The experimental animals were treated with /V-nitrosomor-

pholine which was given in the drinking water at a concentration of 120mg/liter for 7 wk (stop model) (11); control animals received tap waler. Theanimal experiment was conducted by Dr. E. Weber (17). A'-Nitrosomorpholine

was kindly provided by Dr. R. Preußmann,Abteilung fürUmweltcarcinogen-

ese, DKFZ, Heidelberg. Groups of 5 experimental and 5 control animals weresacrificed at Wk 7, 11, 15, 20, 27, 37, 50, and 65 after cessation of the

3 The abbreviations used are: GSF, glycogen storage focus; MCF, mixed cell focus;

HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; PAS, periodic acid-Schiffreaction; G6P, glucose 6-phosphate.

4204

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

GLUCOSE TRANSPORT IN HEPATOCARC1NOGENESIS

carcinogenic treatment and examined for preneoplastic and neoplastic liverlesions by conventional histology. Liver samples of 3 experimental animalsand one control animal from each time point were selected for cytochemicaldemonstration of glycogen content and glucose transporter proteins. Tumorswere gained from 15 animals that were sacrificed at Wk 50, 57, 62, 64, 65, 77,78, 79, and 80 after the end of treatment. Animals were invariably sacrificedbetween 9 and 10 a.m. to avoid variations in carbohydrate metabolism due tothe circadian rhythm (18). The livers were removed immediately and slices 5to 10 mm thick were snap-frozen in isopentane at -150°C. The tissue was

stored at -80°C until used.

Cytochemistry. Antibodies against GLUT 1 and GLUT 2 were raised inrabbits and prepared as previously described by Thorens et al. (19-21). Serial

sections of liver samples 6 jxm in thickness were cut in a cryostat (Leica,Nußloch,Germany), and the first section was stained for glycogen by PAS andcounterstained with toluidine blue in order to identify the different types ofpreneoplastic and neoplastic lesions. The following serial sections were fixedin 0.1% alcoholic periodic acid (optimal for GLUT 2) or 1% alcoholic formaldehyde (optimal for GLUT 1), and a biotin-streptavidin-amplified indirect

immunoalkaline phosphatase method was used for detection of GLUT 1 and 2proteins as described in detail earlier (22). Briefly, as a secondary antibody ahighly biotinylated goat anti-rabbit antibody (Biogenex, San Ramon, CA) anda streptavidin-conjugated intestinal alkaline phosphatase (Biogenex) were used

for demonstration of GLUT 1 and GLUT 2. Application of Fast Red TR salt(Biogenex) resulted in a red-colored precipitate at the antigenic site. Pictureswere taken with a Leitz (Leica, Wetzlar, Germany) comparison photomicro-

scope using an AGFAAPX 25 film combined with a monochromatic interference filter Amax= 553 nm for sections stained for GLUT 1 and GLUT 2, andAmax= 560 nm for sections stained by periodic acid-Schiff in order to enhance

the black and white contrast.Morphometric Evaluation. Preneoplastic and neoplastic liver lesions

were classified according to Bannasch and Zerban (23). GSF, MCF, foci ofGLUT 1 expression, and foci of decreased GLUT 2 expression were correlatedusing a comparison microscope (Leica). The number and area of the lesionswere determined with the aid of a Zeiss Ultraphot microscope converted forprojection and a digitizer (Kontron Digicad, München,Germany) combinedwith a semiautomatic image analyzing system (Compaq Deskpro 386/20); forevaluation of the number and size of focal lesions, a computer program wasused as described by Zerban et al. (24). Only altered areas of more than 0.003mm2 were counted as foci. Calculation of the volume fraction was performed

according to the method of Enzmann et al. (25).Statistical Analysis. The morphometric parameters, area (A ) of focal le

sions (mm2), total area of parenchyma A (cm2), number of focal lesions (N),density of focal lesions D = N/A (number/cm2), and volume fraction V (%)

were determined in each animal for GSF, MCF, and GLUT 2. Arithmetic meansand standard deviations were calculated as descriptive parameters. The dependency of the morphometric parameters on the time of the cessation of treatmentwas analyzed by linear regression methods (26). Morphometric parameters ofGLUT 2 were correlated linearly with the respective parameters of GSF, MCF,and the sum of MCF and GSF by linear regression methods and graphicallyillustrated by scatter plots. Variance explained by the regression model (A2)

and the P value of the statistical significance test for a positive or negativeslope of the regression line were used for the assessment of a linear relationship.

RESULTS

In the liver parenchyma of control rats, glycogen was uniformlydistributed in perivenular and periportal regions of the liver lobule(18). GLUT 2 occurred as a basolateral protein in all hepatocytes. Incontrast, GLUT 1 was only found in a small rim of hepatocytessurrounding the hepatic vein, in the wall of portal veins, and in the bileduct epithelium as described in detail earlier (8, 20, 22). In livers oftreated animals, a zonal reduction of glycogen was observed in perivenular areas 7 wk after the end of the carcinogenic treatment (Fig.la). However, the glycogen content returned to normal in these zoneswithin the following 4 wk. The reduction of glycogen was accompanied by a decreased expression of GLUT 2 and an increased expression of GLUT 1 (Fig. 1, b and c). Like the perivenular changes in

i '

, •¿�, -_.

.

.

* 3•¿�.

V .*•>-'•r - ; v. ^ -r- ^

^'>; •¿�

K-•-- ' l? **,>•-fl.*?k•¿�

Fig. 1. Serial sections through rat liver 7 wk after stop of a 7-wk treatment with 120mg/liter of W-nttrosomorpholine. Bar represents 100 p.m. a, zonal reduction of glycogenin perivenular areas (PAS reaction); b, decreased expression of glucose transporter GLUT2 closely related to the zone of glycogen depletion; and c, increased expression of GLUT1 related to the same zone, h and c, indirect immunoalkaline phosphalase method.

glycogen content, this pattern of glucose transporter expression wasreversible. However, it returned to normal only 20 wk after cessationof treatment.

4205

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

GLUCOSE TRANSPORT IN HEPA I(K AKCIMKiiiNLSIS

Table 1 Number, area, and volume fraction of (!SF and MCF after 7 wk of treatment with N-nitrosomorpholmc

Wk afterIrcutmcnl7111521127375065A2No./cm28.38

±0.60"11.54

±3.5417.66±7.0028.40±8.8539.21±17.1342.6(1±19.6558.67±10.9357.29±32.860.6055GSFArea

(mm2)0.016

±0.0070.015±0.0070.017

±0.0020.038±0.0020.051±0.005(1.067±0.0040.1

28±0.0200.164±0.0310.9259Vol.

fraction(%)0.140.190.301.081.962.827.378.720.8249No./cm~1.01

±1.750.72±1.260.71±1.241.65

±0.553.30±2.982.62±0.8010.01±9.4124.57±2.520.6494MCFArea

(mrrr)0.006

±0.0110.007±0.0120.019

±0.0320.061±0.0230.054±0.0480.097±0.0110.185±0.0540.283±0.0620.8511Vol.

fraction(%)0.1130.020.040.100.28(1.251.556.960.6392"

Mean ±SD.

In contrast to the reversible perivenular reduction of glycogen, fociof altered hepatocytes, namely, GSF (which correspond to clear andacidophilic cell foci as seen in conventional hematoxylin/eosin-

stained sections) and MCF, were present at all time points studied.GSF developing preferentially in intermediate and peripheral zones ofthe liver lobule were the predominant type of focus found throughoutthe observation period. The morphometric evaluation of the focallesions revealed that the number and volume fraction of GSF increased from Wk 7 until Wk 65 (Table 1). MCF were rarely found 7wk after withdrawal of the carcinogen. They increased only slowly innumber and volume fraction until Wk 37, but showed a pronounced

elevation in these parameters from Wk 37 to Wk 65 (Table 1). Evaluation with the comparison microscope showed that GLUT 2 wasdecreased or absent in the majority of both the GSF and MCF at alltime points (Fig. 2, a and c). Consequently, foci of decreased GLUT2 expression showed a steady increase in number and volume fractionfrom Wk 7 to Wk 65 (Table 2). GLUT 1 was absent from GSF butemerged in some MCF starting at Wk 50 after the cessation of treatment, particularly in glycogen-poor, basophilic cells which appeared

in mixed cell foci with time (Fig. 2, c and d). The morphometric datafor the foci expressing GLUT 1 indicated only a slight increase in thenumber and volume fraction of these lesions (Table 2).

V-'/;. >>'•-) tf .••¿�-,- .•¿�... îl'fWV. -„'v

.•v*:>

» if - -•-

¿v•¿�- >* ,-*

**>•¿�i *~ '

^. ¡ *«

* •¿�•¿�v''

-

dFig. 2. Serial sections through a GSF (a, b) and a MCF (c, d). Bar represents 100 p.m. a and c. demonstration of glycogen by PAS reaction; b, strongly decreased expression of

GLUT 2 in cells excessively storing glycogen; note basolateral localization of GLUT 2 in surrounding hepatocytes (arrows); d, reexpression of glucose transporter GLUT 1 in the MCF.b and d, indirect immunoalkaline phosphatase reaction.

4206

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

OI.UCOSE TRANSPORT IN HHPATOCARC INCKil NI SIS

Table 2 Number, area, and volume fraction of foci of altered glucose transporter expression after 7 wk of treatment with N-nilrosomnrphnlme

Wk aftertreatment711152027375065R2Foci

of decreased GLUT 2expressionNo./cm29.311(1.0717.5429.8141.5442.7967.6686.S8±

2.73°±4.004.206.6822.3618.1012.41±

36.660.7771Area

(mm2)0.016

±0.0090.021±0.0170.0190.0420.0560.0590.137(UX)80.0340.0510.0070.1420.139

0.1130.8397Vol.

fraction(%)0.140.240.331.232.292.567.9311.560.754Foci

of GLUT 1expressionNo./cm2

Area (mm2) Vol. fraction(%)3.85

±2.81 0.156 ±0.0380.585.65±4.79 0.1 70 ±0.0200.950.0384

0.64220.0943"Mean ±SD.

Table 3 Gluco.se transporter expression in ht'patocellufar neoplasms

Microvessels,Glycogen GLUT 2 GLUT I GLUT 1

Tumor Histopathology content expression expression positive

HCA1HCA2HCA3HCA4HCA5HCA6HCA7HCC

1HCC2HCC3HCC4HCC5HCC6HCC7HCC8HCC9HCC10HCC11HCC

12Solid

i"Solid

1iSolid1Solid1SolidiSolidiSolid

iiTrabecular

iTrabecular1jTrabecular/adenoid1[Solid/adenoid||Solid11Solid1iSolid1ÕSolid/trabecular¡iAdenoid/solidJiAdenoid/solid

4Adenoid/solid/itrabecularAdenoid

j 1i

1111Jiiii

iiiii

iiiiiiiJI1i11i11i

itî————ttî

îîîîî——îrtt

î———————T

îTî————î

îîîTtî

î" Key: | , decrease; i |, strong decrease;

îî, strong increase.—¿�,not detectable; t . increase;

In HCA and particularly in HCC, glycogen content was decreasedin comparison to the normal liver tissue. In all HCA and HCC studied,the expression of GLUT 2 was reduced (Table 3; Fig. 3, a to d). Bycontrast, in 3 of 7 HCA and 10 of 12 HCC, expression of GLUT 1 wasincreased compared to control liver (Fig. 3, e and /). In the cells ofthese tumors GLUT 1 was localized preferentially at segments of theplasma membrane corresponding to the sinusoidal pole of normalhepatocytes. However, not all portions of the tumor tissue showed aGLUT 1 expression. In 4 HCA and 2 HCC, tumor cells did not expressGLUT 1 (Table 3). In 4 of 6 adenoid HCC, cells of the adenoidformations expressed only GLUT 1, whereas in 2 adenoid HCC, thesecells coexpressed GLUT 1 and GLUT 2 (Fig. 4, a to c). Cells ofnormal bile ducts, bile duct proliferations, and cystic cholangiomas,however, expressed only GLUT 1. Interestingly, microvessels, intensely stained for GLUT 1, were observed in 7 HCC (Table 3; Fig.4, d and e). These newly formed blood vessels usually occurred inclose vicinity to adenoid tumor formations. Only one solid HCCdisplayed areas with GLUT 1-positive microvessels in the absence of

adenoid tumor formations. Such microvessels were never found in thelivers of control animals, preneoplastic hepatic foci, or HCA.

DISCUSSION

Two basically different reaction patterns were observed in the liverparenchyma after carcinogenic treatment: (a) reversible changes related to glycogen reduction in perivenular hepatocytes and (h) persistent alterations associated with the development of preneoplasticand neoplastic lesions.

The reduction of glycogen in perivenular hepatocytes was accompanied by an increased expression of GLUT 1 and a decreased expression of GLUT 2. These changes reversed to normal 11 (glycogenreduction) and 20 wk (GLUT 1 and 2 expression), respectively, afterwithdrawal of the carcinogen. Since it is well known that the reversible perivenular loss of glycogen is a frequent consequence of anonspecific toxic effect of hepatocarcinogens (27), this effect is mostprobably also the reason for the increased expression of GLUT 1 inthe same lobular area. In this context it should be mentioned thatcultured baby hamster kidney cells likewise responded with an elevated expression of GLUT 1 to "stress" induced by arsenite (28).

Regarding the toxic lesion in perivenular hepatocytes, a transient shiftin glucose transporter expression from GLUT 2 (bidirectional glucosetransport) to GLUT 1 (preferred unidirectional transport into hepatocytes) might be necessary to meet an increased intracellular demandfor glucose.

Persistent changes in the expression of GLUT 2 were encounteredin GSF which characterize early stages of hepatocarcinogenesis. Numerous studies revealed that GSF express also many other biochemical "markers" of preneoplastic cell populations regularly preceding

the development of liver neoplasms (11-14). The early decrease or

lack of GLUT 2 in GSF suggests that there is a disturbance in glucosetransport of the hepatocytes in a way that only small amounts or noglucose can be transported in or out of the cell via this transporter. Itis difficult to understand how the excessive storage of glycogen can beachieved under these conditions. Whether preneoplastic hepatic focimight express other types of glucose transporter, such as GLUT 3found in some liver tumors (10), or use other precursors for glycogensynthesis, like láclateor amino acids (29), awaits further investigations. Previous enzyme histochemical and microbiochemical studieshave shown that the excessive storage of glycogen in GSF is usuallyassociated with a normal activity of glycogen synthase (16). In contrast, there is a disturbance in phosphorolytic glycogen breakdown,which is not due to a loss of the phosphorylasc protein (30) but to areduction in the activity of this enzyme (16). The previous enzymehistochemical finding of a reduced activity of adenylate cyclase inGSF (31 ) suggests that the reduced activity of glycogen phosphorylascmay result from alterations in superordinate regulatory mechanismsimplicating signal transduction at the plasma membrane. Moreover,many GSF show a reduced activity of the microsomal enzyme glu-cose-6-phosphatase (15) and of the a-glucosidase which is responsible

for hydrolytic glycogen degradation in lysosomes (32). All theseobservations support the concept that the focal glycogenosis is causedby an inhibition of glycogen breakdown rather than by an increasedglycogen synthesis. Our observation of the reduced expression ofGLUT 2 in GSF suggests that there is also a diminished glucoserelease which might contribute to the accumulation of glycogen inhepatocytes. This is in accordance with the finding that GSF retain thispolysaccharide longer than the surrounding liver tissue after starvationor administration of glucagon (15, 33). In addition, reduced expres-

4207

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

•¿�•¿�\•¿�'. / <

:Fig. 3. Glucose transporter expression in hepatocellular neoplasms. Bar represents 100 /Am.a to c, serial sections of a HCA; d to /, serial sections of a HCC. a and d, demonstration

of glycogen by PAS reaction; b, decreased expression of GLUT 2 in contrast to the surrounding tissue (arrows); c, reexpression of GLUT 1 within tumor cells; e, reduced expressionof GLUT 2;/, strong reexpression of GLUT 1. b, c, e, and/, indirect immunoalkaline phosphatase reaction.

sion of GLUT 2 in hepatic GSF indicates that excessive storage of ing duct system and show a strongly reduced expression of its typicalglycogen is most probably not the consequence of increased uptake of glucose transporter GLUT 1 (34).glucose from the blood but results from a disturbance in intracellular There is convincing evidence that an increased level of G6P playsflux of metabolites. A similar conclusion has recently been drawn a central role in the long-term regulation of glycogen synthesis in afrom findings in preneoplastic and neoplastic rat renal clear cell le- glycogen-storing liver cell line which shares also many other meta-sions excessively storing glycogen, which originate from the collect- bolic features with cells of hepatic GSF in situ (35). Elevated G6P

4208

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

GLUCOSE TRANSPORT IN HEPATOCARt INIMilM SIS

b

eFig. 4. Serial sections through two different adenoid HC'C. Rar represents l(K) ¡im.a, cells lining adenoid formations (PAS reaction); b, strong expression of GLUT 2 within these

cells; r, slight expression of GLUT I within the same tumor cells; d. adenoid tumor formation (ttf) with microvessels (/m1) (PAS reaction); i\ strong expression of GLUT 1 in themicrovessels. Note also localization of GLUT 1 in tumor cells lining the adenoid formation (arrow), b. c. and t*. indirect immunoalkaline phosphatase reaction.

levels have likewise been measured in rat liver homogenates at the endof a 7-wk p.o. administration of ¿V-nitrosomorpholine (36) and inspecimens of glycogen-storing human renal clear cell carcinomas (37,

38). It is thus conceivable that G6P might also be increased in GSFand trigger both glycogen synthesis and the pentose phosphate pathway, the key enzyme of which, glucose-6-phosphate dehydrogenase,

usually shows a markedly increased activity in these lesions (15, 16).Baba ci al. (39) found a close relationship in preneoplastic liverlesions between elevated glucose-6-phosphate dehydrogenase activity

and an increased proliferation in these foci.MCF, which contain less glycogen than GSF, show the same re

duction in the expression of GLUT 2 and in the activities of glycogenphosphorylase and glucose-6-phosphatase as the GSF. Glycogen syn

thesis, however, is drastically diminished as indicated by a very lowactivity of glycogen synthase (16). In addition, there is an increasedlysosomal glycogen breakdown as demonstrated by an elevated activity of the a-glucosidase (33) and, perhaps, a more pronouncedchanneling of G6P into the pentose phosphate pathway, since glucose-6-phosphate dehydrogenase activity shows a further increase com

pared to GSF (16, 40). Because glycogen synthesis is reduced and theactivity of glyceraldehyde-3-phosphate dehydrogenase is usually increased in addition to that of glucose-6-phosphate dehydrogenase in

MCF (16), it is conceivable that metabolites are not only channeled

into the pentose phosphate pathway but also into glycolysis. Thesepathways may provide the energy and precursors of nucleic acidsynthesis to sustain the increased growth rate of MCF, which has beendemonstrated by proliferation kinetic studies using pulse labeling with[3H]thymidine (41). These considerations are in line with the meta

bolic congestion-cascade concept of neoplastic cell conversion (42),

implicating persistent, genetically or epigenetically fixed primary molecular lesions which result in an early congestion of carbohydratemetabolism, e.g., by an accumulation of G6P, initially leading to ahepatocellular glycogenosis but then increasingly channelled via acascade of intermediate steps to alternative pathways of carbohydratemetabolism. This metabolic shift might be indispensible for cell survival but leads to uncontrolled growth at the expense of differentiatedcell functions and integration of the cell in physiological cell-cell

interactions. This hypothesis receives additional support from theobservation that patients suffering from inborn hepatic glycogenosis,mostly that of the von Gierke type which is due to a genetically fixeddefect of glucose-6-phosphatase, have an exceedingly high risk of

developing hepatocellular neoplasms when they pass through adolescence (15, 42). Interestingly, common regulatory mechanisms of theglycogen metabolism, the expression of the nuclear oncogcnes c-myband c-jun, and certain aspects of development in Drosophila have

recently been discussed (43).

4209

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

Ol.UrOSK TRANSPORT IN HKPATOCARC INOCil NI SIS

In our studies, 3 of 7 HCA and 10 of 12 HCC displayed an elevatedGLUT 1 expression. Since GLUT 1 has a low K„,for glucose influxand a high Km for glucose efflux, an increase of this transporterprotein (7) endows tumor cells with a high capacity for glucose uptakeeven at low blood glucose concentrations. The high glucose consumption of HCC is also reflected hy an isozyme shift from glucokinase tohexokinase (44). In a different model of hepatocarcinogenesis Fischerel al. (45) found hy microhiochemical means that this isozyme shiftoccurs already in preneoplastic hepatic foci (as defined hy a deficiency in glucose-6-phosphatase or membrane-hound adenosine-tri-

phosphatase activity). However, in our experimental model, similarmicrobiochemical studies on the different types of preneoplastic foci(as defined by cytoplasmic glycogen content and basophilia) recentlyrevealed that hexokinase activity is not significantly altered in GSFand MCF but shows a pronounced increase in all HCC (46). Thepartial reexpression of GLUT 1 in some late MCF (Table 2) mightcorrelate with the beginning of this isozyme shift in tumor development. The observation that in 2 HCC and 3 HCA neither GLUT 1 norGLUT 2 expression could be detected within the limits of our methodsdoes not preclude the presence of very low amounts of these transporter proteins or the existence of another type of glucose transporter,e.g., GLUT 3, which has been found in human hepatomas (10).Although these authors demonstrated an elevated GLUT 2 mRNAlevel in one human hepatoma, none of the 12 HCC in our study hada pronounced GLUT 2 protein expression. This might be due tospecies-related differences in glucose transporter expression, to varia

tions in glucose transporter expression pattern in hepatocellular neoplasms, or to alterations in the translation of this protein in neoplastichepatocytes.

Glucose transporter expression in adenoid HCC deserves specialattention because the cells lining adenoid formations were in 4 casespositive only for GLUT 1, but in 2 cases for GLUT 2 and GLUT 1.Fully differentiated bile duct epithelium in control liver (22), bile ductproliferations, and cystic cholangiomas, however, express only GLUT1. Previous ultrastructural and immunocytochemical studies on primary adenoid HCC by Rúan et al. (47) provided evidence for atransdifferentiation (metaplasia) from cells with a hepatocellular phe-

notype to cells resembling bile ductular epithelium in adenoid formations. Some of the adenoid formations contained cells with a transitional phenotype expressing features of both hepatocytes and bileductular epithclia. The occasional coexpression of GLUT 1 and 2 inepithelial cells of adenoid formations in these HCC appears to supportthe concept of transdifferentiation in the development of adenoidstructures of HCC. Another remarkable finding in adenoid HCC is thefrequent presence of microvessels usually located in the immediatevicinity of adenoid formations, which exhibit a strong expression ofGLUT 1. Since particularly high cell proliferation in the cells liningthese formations was found,4 the concentration of microvessels

around the adenoid formations might indicate a high demand forglucose in these rapidly growing cellular subpopulations of HCC.

ACKNOWLEDGMENTS

The authors thank Erika Filsingcr for technical assistance and JoachimHollatz for photographic work. We are also grateful to Renate Rausch and Dr.Lutz F.dler for the statistical evaluation.

REFERENCES

1. Silverman. M. Structure and function of hcxose transporters. Annu. Rev. Biochem..60: 757-794. 1991.

2. Muecklcr. M. Family of glucose-transporter genes. Diabetes. 39: 6-11. 1991).3. Thorens. B. Molecular and cellular physiology of CiLUT-2. a high-K,,, facilitated

10.

18.

19.

20.

23.

24.

25.

28.

29.

30.

31.

33.

4 Unpublished observation. 34.

4210

diffusion glucose transporter. Int. Rev. Cytol.. I37A: 209-238. 1992.Tal. M.. Schneider, D. t... Thorens, B., and l.odish. H. F. Restricted expression of thecrythroid/brain glucose transporter isoform to perivenous hepatocytes in rats. Modulation hy glucose. J. Clin. Invest.. Ä6:986-992, 199(1.

Wang. C.. and Brcnnan. W. A. Rat skeletal muscle, liver, and brain have different fetaland adult forms of glucose transporter. Biochim. Biophys. Acta. Q4fi: 11-18. 1988.Asano. T. Shibasaki. Y., Kasuga. M.. Kanazawa, Y.. Takaku. F., Akanuma. Y. andOka. Y. Cloning of a rabbit glucose transporter cDNA and alteration of glucosetransporter mRNA during tissue development. Biochem. Biophys. Res. Commun..¡54:1204-1211, 1988.

Wheeler. T J.. and Hinkle. P. C. The glucose transporter of mammalian cells. Annu.Rev. Physiol.. 47: 503-517, 1985.

Thorens. B.. Flier. J. S.. Lodish. H. F. and Kahn. B. B. Differential regulation of twoglucose transporters in rat liver by fasting and refeeding and by diabetes and insulintreatment. Diabetes. 39: 712-719. 1990.Su. T.-S.. Tsai. T.-F. Chi. C -W.. Han, S.-H.. and Chou, C.-K. Elevation of facilitatedglucose-transporter messenger RNA in human hepatocellular carcinoma. Hcpatology.//: 118-122, 1990.Yamamoto. T., Scino, Y. Fukumoto. H.. Koh. G.. Yano. H.. Inagaki. N.. Yamada. Y.Inoue. K-. Manabe. T.. and Imura. H. Over-expression of facultative glucose transporter genes in human cancer. Biochem. Biophys. Res. Commun., 170: 223-230,1990.Bannasch. P.. and Zerban. H. Prédictivevalue of hepatic preneoplastic lesions asindicators of carcinogenic response. In: H. Vainio. P. Magee. D. McGregor, and A. J.McMichael (cds.). Mechanisms of Carcinogencsis in Risk Identification, pp. 381-419. Lyon: International Agency for Research on Cancer, 1992.Farber. E . and Sarma. D. S. R. Hepatocarcinogenesis: a dynamic cellular perspective.I .ah. Invest., 56.' 4-22. 1987.Pilot. H. C".Altered hepatic foci: their role in murine hepatocarcinogenesis. Annu.

Rev. Pharmacol. Toxicol.. 30: 465-500, 1990.

Bannasch. P.. Mayer. D.. and Hacker. H. J. Hepatocellular glycogenosis and hepatocarcinogenesis. Biochim. Biophys. Acta. fW5: 217-245. 198(1.

Bannasch. P.. Hacker. H. J.. Klimek. F.. and Mayer. D. Hepatocellular glycogenosisand related pattern of enzymatic changes during hcpatocarcinogenesis. Advan. Enzyme Regul.. 22: 97-121. 1984.Hacker. H. J., Moore. M. A.. Mayer. D.. and Bannasch. P. Correlative histochemistryof some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesionsin the rat liver. Carcinogenesis (Lond.). 3: 1265-1272. 1982.Weber. E. Dosisabhängigkeit der Sequen/ zellularer Veränderungenhei der A'-Nitro-somorpholin-induzierten Hepatokarzinogenese in der Ratte. Technische Hochschule(Dissertation). Darmstadt: Naturwiss. 1989.Müller.O. Der cirkadiane Glykogenrhythmus der Rattenleber (Biochemie, quantitative Histochemie, elcktronenmikroskopischc Morphometric). Acta Histochem.Suppl.. 16: 139-144, 1976.

Thorens. B.. Sarkar. H. K.. Kahack, H. R., and Lodish. H. F. Cloning and functionalexpression in bacteria of a novel glucose transporter present in liver, intestine, kidney,and (3-pancrealic islet cells. Cell. 55: 281-290. 1988.

Thorens. B.. Cheng. Z. 0 . Brown, D.. and l-odish. H. F Liver glucose Iransporter: abasolateral protein in hepatocytes and intestine and kidney cells. Am. J. Physiol. CellPhysiol.. 259: C279-C285. 1990.Thorens. B.. Lodish. H. F.. and Brown. D. Differential localisation of two glucosetransporter isoforms in rat kidney. Am. J. Physiol. Cell Physiol.. 259: C286-C294.1990.Hacker, H. J., Thorens. B., and Grohholz. R. Expression of facilitalivc glucosetransporter in rat liver and choroid plexus. A histochemical study in native cryostatsections. Histochemistry, 96: 435^*39, 1991.Bannasch. P.. and Zcrhan. H. Tumours of the liver. In: V. Turusov and U. Mohr (eds.).Pathology of Tumours in Laboratory Animals. Vol. 1. pp. 199-240. Lyon: International Agency for Cancer. 1990.Zerhan. H.. Preussmann. R.. and Bannasch. P. Dose-time relationship of the development of preneoplastic liver lesions induced in rats with low doses of W-nitroso-diethanolaminc. Carcinogenesis (Lond.). 9: 607-610. 1988.

Enzmann. H.. Edler. L., and Bannasch. P. Simple elementary method for the quantification of focal liver lesions induced hy carcinogens. Carcinogenesis (I.ond.). 8:231-235. 1987.

Draper. N. R.. and Smith. H. Applied Regression Analysis. New York: Wiley. 1981.Bannasch. P. The cytoplasm of hepatocytes during Carcinogenesis. Electron and lightmicroscopical investigations of the nitrosomorpholine-intoxicatcd rat liver. RecentResults Cancer Res.. 19: 1-100. 1968.Pasternak. C. A.. Aiyathurai. J. E. J.. Makindc. V. Baldwin. S. A.. Konieczko. E. M..and Widncll, C. C. Regulation of glucose uptake by stressed cells. J. Cell. Physiol.,¡4Q;324-331, 1991.

Baggeto. L. G. Deviant energetic metabolism of glycolytic cancer cells. Biochimie(Paris). 74: 959-974. 1992.Scelmann-Eggebert. G., Mayer. D.. Mecke, D., and Bannasch. P. Expression and

regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesionsin rats. Virchows Arch. B Cell Pathol., 5.?: 44-51. 1987.Ehemann. V. Mayer. D.. Hacker. H. J.. and Bannasch. P. Ijiss of adenylate cyclaseactivity in preneoplastic and neoplastic lesions induced in rat liver hy A/-nitrosomor-pholinc. Carcinogenesis (Lond.). 7: 567-573. 1986.Klimek. F.. and Bannasch. P. Biochemical microanalysis of nr-glucosidase activity inpreneoplastic and neoplastic hepatic lesions induced in rats hy A'-nitrosomorpholine.Virchows Arch. B Cell Palhol.. 57: 245-250. 1989.Färber.E The sequential analysis of liver cancer induction. Biochim Biophys. Acia.605: 149-166. 1980.Ahn. Y. S.. Zerhan. H.. Grobholz. R.. and Bannasch. P. Sequential changes in glyco-

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

GLUCOSE TRANSPORT IN HEPATOt AK( ÕNIXil SI SIS

gen content, expression of glucose transporters and enzymic patterns during development of clcar/acidophilic cell tumors in rat kidney. Carcinogenesis (Lond.). 13:2329-2334, 1992.

35. Mayer, D., and Letsch, I. Glucose-6-phosphate plays a central role in the regulationof glycogen synthesis in a glycogen-storing liver cell line. Cell Biochem. Funct., 7:243-256, 1989.

36. Enzmann, H.. Bettler. T.. Ohlhauser. D., and Bannasch. P. Elevation of glucose-6-phosphate in early stages of hepatocarcinogencsis induced in rats by A/-nitrosomor-pholine. Horm. Metab. Res., 20: 128-129, 1988.

37. Mayer. D.. and Bannasch, P. Activity of glycogen synthase and phosphorylase andglucose-6-phosphate contení in renal clear cell carcinomas. J. Cancer Res. Clin.Oncol., 114: 369-372, 1988.

38. Steinberg, P., Störkel. S., Oesch, F.. and Thoencs, W. Carbohydrate metabolism inhuman renal clear cell carcinomas. Lab. Invest-, 67: 506-511, 1992.

39. Baba, M., Yamamoto, R., lishi. H., Tatsuta, M„and Wada, A. Role of glucose-6-phosphate dehydrogenase on enhanced proliferation of pre-neoplastic and ncoplasliccells in rat liver induced by A'-nitrosomorpholine. Int. J. Cancer, 43: 892-895, 1989.

40. Klimek, F., Mayer, D.. and Bannasch, P. Biochemical microanalysis of glycogencontent and glucose-6-phosphate dehydrogenase activity in focal lesions of the ralliver induced by /V-nitrosomorpholine. Carcinogenesis (Lond.). 5: 265-268, 1984.

41. Zerban, H., Rabes, H. M., and Bannasch, P. Sequential changes in growth kinetics andcellular phenotype during hepalocarcinogcnesis. J. Cancer Res. Clin. Oncol., 115:329-334, 1989.

42. Bannasch. P. Phenolypic cellular changes as indicators of stages during neoplasticdevelopment. In: O. H. Iverscn (ed.). Theories of Carcinogenesis, pp. 231-249.Washington: Hemisphere Publishing Corporation, 1988.

43. Woodgett. J. R. A common denominator linking glycogen metabolism, nuclear on-cogenes, and development. TIBS. lt>: 177-181, 1991.

44. Hammond, K. D., and Balinsky, D. Isoenzyme studies of several enzymes of carbohydrate metabolism in human adult and fetal tissues, tumor tissues, and cell cultures.Cancer Res., 38: 1323-1328, 1978.

45. Fischer, G.. Ruschenburg, !.. Eigenbrodt, E., and Katz, N. Decrease in glucokinaseand glucose-6-phosphatase and increase in hexokinase in putative preneoplastic lesions of rat liver. J. Cancer Res. Clin. Oncol.. 113: 430-436, 1987.

46. Klimek, F., and Bannasch, P. Isocnzyme shift from glucokinase to hexokinase is notan early but a late event in hepatocarcinogenesis. Carcinogenesis (Lond.), in press,1993.

47. Rúan,Y-, Hacker. H. J.. Zerban. H.. and Bannasch, P. Ultrastructural and immuno-cytochemical characterization of the cellular phenotype in primary adenoid livertumors of the rat. Pathol. Res. Pract., 184: 223-233, 1989.

4211

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

1993;53:4204-4211. Cancer Res   Rainer Grobholz, Hans Jörg Hacker, Bernard Thorens, et al.   HepatocarcinogenesisReexpression of GLUT 1 in Late Stages ofGLUT 2 in Preneoplastic and Neoplastic Hepatic Lesions and Reduction in the Expression of Glucose Transporter Protein

  Updated version

  http://cancerres.aacrjournals.org/content/53/18/4204

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/53/18/4204To request permission to re-use all or part of this article, use this link

Research. on January 2, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from