Dehydroepiandrosterone induced alterations in rat ...

Carcinogenesis vol.9 no. 11 pp.2039-2043, 1988

Dehydroepiandrosterone induced alterations in rat liver carbohydrate metabolism

Doris Mayer, Edgar Weber, Malcolm A.Moore1, Iris Letsch, Erika Filsinger and Peter Bannasch Institut fur Experimentelle Pathologie, Deutsches Krebsforschungszentram, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG and 'Carcinogenesis Research Unit, School of Pathology, University of NSW, Australia

Introduction Considerable attention has been focused recently on the naturally occurring adrenal hormone dehydroepiandrosterone (DHEA; 3/3-hydroxy-5-androsten-17-one*) which has been shown capable of inhibiting the development of tumours in different organs of rats and mice, such as lung, colon, mammary gland, liver and skin (for references see (1,2)). Furthermore, it has been observed that women with subnormal urinary levels of androsterone and etiocholanolone, the primary excretory products of DHEA, have an enhanced risk for developing breast cancer (2,3). DHEA has also been observed to exhibit beneficial effects in genetically obese rats and mice, where lipogenesis and weight gain were considerably reduced without lowering food intake (4,5), and in genetic and streptozotocin-induced diabetes plasma insulin levels were improved and pancreatic beta cells protected against destruction (6). The mechanism by which DHEA protects against cancer, obesity and diabetes remains unclear. Earlier work has shown that DHEA is a non-competitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH) (7,8). Reduction of G6PDH-activity has also been observed in vivo in livers of genetically obese rats fed DHEA (9,10). It has therefore been speculated that a decreased cellular NADPH pool resulting from G6PDH-inhibition was the limiting factor for lipogenesis and biosynthesis of ribonucleotides and deoxyribonucleotides (4,5). However, more recently it became evident from a number of observations (see ref. 2) that DHEA-effects cannot be simply •Abbreviations: DHEA, dehydroepiandrosterone; G6PDH, glucose-6-phosphate dehydrogenase; G6Pase, glucose-6-phosphatase; PK, pyruvate kinase; FBPase, fructose-1,6-bisphosphatase; HK, hexokinase; GK, glucokinase; SYN, glycogen synthase; PHO, glycogen phosphorylase; PAS, periodic acid Schiff s reaction. © IRL Press Limited, Oxford, England

Materials and methods Animals and treatment Male Sprague-Dawley strain rats (purchased from Hannover, Zentralinstitut fur Versuchstierzucht, FRG) weighing - 180 g at the start of the investigation were maintained under constant conditions as described earlier (17). The hormone DHEA was mixed into the pellet diet (Altromin®, Lage/Lippe) to give a final concentration of 0.25% by weight. The rats were divided into two groups: animals fed regular diet served as controls, the DHEA group received hormonesupplemented diet for 27 weeks. At sacrifice the livers were sliced and frozen in liquid isopentane at 140°C, pre-cooled by liquid nitrogen. Frozen tissue was stored in tightly closed vessels at -80°C until used. Biochemical assays The livers from 10 DHEA-treated and 10 control animals were homogenized (5% homogenates) in buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaF, 4 mM EDTA and 0.1 mM dithiothreitol) using a glass/teflon homogenizer. Homogenates were filtered through four layers of surgical gauze and used directly for measuring glycogen content and the activities of glycogen synthase (SYN) (EC 2.4.1.11), glycogen phosphorylase (PHO) (EC 2.4.1.1), glucose-6-phosphatase (G6Pase) (EC 3.1.3.9), glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) and a-glucosidase (EC 3.2.1.20). Parts of the homogenates were used for preparing 105 g supernatants which were run for 30 min at 4°C in a 50Ti rotor. The supernatants were used for measuring the activities of hexokinase (HK) and glucokinase (GK) (EC 2.7.1.1), pyruvate kinase (PK) (EC 2.7.1.40) and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11). PHO was measured according to Wang and Esmann (18). PHO a was measured in the absence of AMP, total PHO in the presence of 1 mM AMP. SYN was measured by the method of Thomas et al. (19). The assay contained no G6P for determination of SYN I and 6 mM G6P for total SYN activity. G6Pase was measured according to Baginski et al. (20), G6PDH by the method of Delbriick et al. (21), HK and GK according to Weber et al. (22), pyruvate kinase according to Bergmeyer (23), however using 200 jil of protein solution instead of 20 pi, and FBPase by the method of Weber and Camera (24). a-glucosidase was measured by a fluorometric assay, modified according to Salafsky and Nadler (25) using 4-methylumbelliferyl-a-D-glucopyranoside as substrate. Fluorescence was determined in a Farrand MK2 fluorometer with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. 4-methylumbelliferone (0.01-0.5 iM) was used as standard. Glycogen was measured according to Roehrig and Allred (26) and protein according to Heinzel (27). All NAD(P)H forming and consuming enzymes were measured in a LKB Ultrospec 4050 equipped with thermostatted cuvettes and

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Long-term dietary administration of the adrenal hormone dehydroepiandrosterone (DHEA) to male Sprague-Dawley rats induced significant alterations in the activities of enzymes involved in liver carbohydrate metabolism. Although glycogen synthase activity was increased and phosphorylase decreased, glycogen stores were reduced. This was presumably related to lysosomal glycogen degradation, since a-glucosidase was increased. All rate-limiting enzymes of glucose metabolism which were studied (glucose-6-phosphate dehydrogenase, total hexokinases, pyruvate kinase, fructose-l,6-bisphosphatase) revealed markedly reduced activity, only glucose-6-phosphatase activity was increased. These enzymatic changes point to a far-reaching metabolic shift towards energy loss via decreased glucose consumption and increased glucose output. The enzyme pattern induced by DHEA is in many respects opposite to that induced in preneoplastic and neoplastic liver lesions by chemical hepatocarcinogens.

due to G6PDH-inhibition, Thus, after long-term administration of the hormone, G6PDH-activity is often normal (11,12) and the activity of malic enzyme which also produces NADPH is markedly increased (10,12). More detailed enzymatic studies on fatty acid metabolism in rats lead to the proposal of an enhanced energy expenditure via net dephosphorylation of ATP caused by DHEA (10). A number of studies have shown that the development of epithelial and mesenchymal tumours in the liver of carcinogentreated rats can be retarded by DHEA (13,14) or modulated in a way that tumours develop which may be less malignant (15,16). Since the development of hepatocellular tumours is usually associated with marked alterations in key enzyme pattern of carbohydrate metabolism (15-17) including a G6PDH-increase, it was of interest to study the long-term effect of DHEA administration alone on the activity of such enzymes in normal rat liver. Conditions of DHEA-treatment were chosen under which significant retarding and modulating effects on tumour development had been observed earlier (15,16).

D.Mayer et al.

Table I. Glycogen content and activities of some enzymes of carbohydrate metabolism in DHEA-treated and control rats Glycogen content (mg/mg protein) and enzyme activities (nmol/mg protein/min)

Control rats (10) mean ± SD

DHEA-rats (10) mean ± SD

Wilcoxon test

Glycogen Glycogen synthase / Glycogen synthase I + D Glycogen phosphorylase a Glycogen phosphorylase a + b Hexokinase Glucokinase Pyruvate kinase Glucose-6-phosphatase Fructose-1,6-bisphosphatase Glucose-6-phosphate dehydrogenase a-Glucosidase

0.45 3.5 23.2 166.1 159.3 11.1 44.8 13.1 61.8 57.8

0.37 4.1 24.9 119.4 114.5 6.4 21.8 9.6 75.0 39.2

P < NSa NSa P < P < P < P < P < P < P
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-*A Fig. 2. Enzyme histochemical demonstration of G6PDH activity in the livers of a control (a) and a DHEA-treated (b) rat. Magnification x54. Bar represents 100 pm.

Discussion Previous studies have shown that long-term administration of DHEA to rats leads to a reduction of body weight which is not due to a toxic effect of the steroid but to alterations in enzyme activities of lipid metabolism resulting in a waste of energy (4,5,10). The data presented in this paper show that long-term administration of DHEA to male Sprague—Dawley rats caused also profound alterations in enzyme activities of liver carbohydrate metabolism. Although our data do not provide information on the flux of metabolites through the various metabolic pathways, it may be assumed, and this assumption seems to be justified since we have studied rate-limiting enzymes of carbohydrate metabolism, that glycolysis and gluconeogenesis both are restrained in these livers. Furthermore it may be concluded from the reduced GK activity that the input of glucose into hepatic metabolism via phosphorylation is lowered. On the other hand glucose release via hydrolysis of G6P by G6Pase may be markedly increased. This would lead to a net output of glucose. The elevated hydrolytic cleavage of glycogen by an increased or-glucosidase activity would fit in this conception of glucose output. G6PDH activity has been described to be only weakly reduced or even normal in rat livers after long-term DHEA administration (10—12). In our model a clear reduction of G6PDH activity was found in liver homogenates of DHEA-treated rats compared to untreated controls, which from the histochemical findings could be predominantly attributed to a reduction in non-parenchymal 2042

cells (Figure 2). However, from Figure 2 it is conceivable that G6PDH activity is also reduced in parenchymal cells. Slight differences are not easily visible in sections treated by enzyme histochemistry, especially if the activity is as low as in hepatocytes of male rats. The finding that G6PDH is reduced in both parenchymal and non-parenchymal cells (Kupffer cells and endothelial cells) which per se have a much higher G6PDH activity than hepatocytes (33) may be of interest for the observation that rats given simultaneously N-nitrosomorpholine and DHEA develop fewer hemangiosarcomas than rats treated with TV-nitrosomorpholine alone (16) and for the observation that chemically induced hepatocarcinogenesis in rats which is accompanied by an increase in G6PDH activity, is also retarded and modulated by DHEA (16,39). The enzyme pattern observed in the DHEA-treated livers points to a reduction of glucose consumption and to a waste of energy due to increased glycogen and G6P hydrolysis. Cleary et al. (10) have observed similar changes in lipid metabolism in DHEAtreated rat liver, such as increase in long-chain fatty acyl-CoA hydrolase and a decrease in fatty acid synthetase. This was interpreted as a futile cycle of fatty acid metabolism leading to net ATP consumption, since liberated fatty acids need ATPdependent reattachment of CoA (34). Increased G6Pase and decreased glucokinase activity as demonstrated in Table I may also represent such a futile cycle, since liberated glucose needs rephosphorylation by ATP before entering further metabolism. It has been published previously (35-38) that in preneoplastic liver lesions induced by chemical carcinogens the glycogen


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DHEA induced alterations in rat liver

Acknowledgement This work was supported by grants from the Deutsche Forschungsgemeinschaft.

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by dehydroepiandrosterone. Carcinogenesis, 9, 1191-1195. 17. Hacker,H.J., Moore,M.A., Mayer.D. and Bannasch.P. (1982) Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver. Carcinogenesis, 3, 1265-1272. 18. Wang,P. and Esmann.V. (1972) A new assay of phosphorylase based on the filter paper technique. Anal. Biochem., 47, 495-500. 19. Thomas.J.A., Schlender.K.K. and LarnerJ. (1968) A rapid filter paper assay for UDP glucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-l4C-glucose. Anal. Biochem., 25, 486-499. 20. Baginski.E.S., Foa,P.P. and Zak,B. (1974) Glucose-6-phosphatase. In Bergmeyer.H.U. (ed.), Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim/Bergstrasse, pp. 909-913. 21.Delbriick,A., Zebe,E. and Bucher,T. (1959) Uber Verteilungsmuster von Enzymen des Energie liefernden Stoffwechsels im Flugmuskel, Sprungmuskel and Fettkorper von Locusta migratoria und ihre cytologische Zuordnung. Biochem. Z , 331, 273-296. 22. Weber.G., Singhal,R.L., Stamm,N.B., Lea,M.A. and Fisher.E.A. (1966) Synchronous behavior pattern of key glycolytic enzymes: glucokinase, phosphofructokinase and pyruvate kinase. Adv. Enzyme Regul., 4, 59—81. 23. Bergmeyer.H.U. (1974) Pyruvat-Kinase. In Bergmeyer.H.U. (ed.), Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim/Bergstrasse, pp. 544-545. 24. Weber,G. and Cantero,A. (1959) Fructose-1,6-diphosphatase and lactic dehydrogenase activity in hepatoma and in control human and animal tissues. Cancer Res., 19, 763-768. 25. Salafsky,J.S. and Nadler,H.L. (1973) A fluorometric assay of alphaglucosidase and its application in the study of Pompe's disease. J. Lab. Clin. Med., 81, 450-454. 26. Roehrig,K.L. and Allred,J.B. (1974) Direct enzymatic procedure for the determination of liver glycogen. Anal. Biochem., 58, 414—421. 27. Heinzel.W. (1960) Ein neues Verfahren zur quantitativen Eiwei/3bestimmung. Verh. Dtsch. Ges. Inn. Med., 66, 636-637. 28. Pearse,A.G.E. (1972) Histochemistry, Theoretical and Applied. Churchill Livingstone, Edinburgh. 29. Hacker.H.J. (1978) Histochemische Darstellung der Glykogenphosphorylase (EC 2.4.1.1) mit Htife semipermeabler Membranen. Histochemistry, 58, 289-296. 30. Benner.U., Hacker.H.J. and Bannasch.P. (1979) Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes. Histochemistry, 65, 41-47. 31. Meijer.A.E.F.H. and de Vries.G.P. (1974) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. IV. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating). Histochemistry, 40, 349—359. 32. Bannasch.P., Mayer.D. and Hacker.H.J. (1980) Hepatocellular glycogenosis and hepatocarcinogenesis. Biochim. Biophys. Acta, 605, 217-245. 33.Knook,D.L., Sleyster.E.C. and Teutsch.H.F. (1980) High activity of glucose-6-phosphate dehydrogenase in Kupffer cells isolated from rat liver. Histochemistry, 69, 211-216. 34. Shepherd,A. and Cleary.M.P. (1984) Metabolic alterations following dehydroepiandrosterone treatment in Zucker rats. Am. J. Physiol., 246, E123-128. 35. Klimek.F., Mayer.D. and Bannasch.P. (1984) Biochemical microanalysis of glycogen content and glucose-6-phosphate dehydrogenase activity in focal lesions of the rat liver induced by /V-nitrosomorpholine. Carcinogenesis, 5, 265-268. 36. Bannasch.P., Hacker,H.J., Klimek.F. and Mayer.D. (1984) Hepatocellular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis. Adv. Enzyme Regul., 22, 97-121. 37. Fischer,G., Ruschenburg,J., Eigenbrodt.E. and Katz.N. (1987) Decrease in glucokinase and glucose-6-phosphatase and increase in hexokinase in putative preneoplastic lesions of rat liver. J. Cancer Res. Clin. Oncol., 113,430—436. 38. Mayer.D., Klimek.F., Hacker.H.J., Seelmann-Eggebert.G. and Bannasch.P. (1989) Carbohydrate metabolism in hepatic preneoplasia. In Bannasch.P., Keppler,D. and Weber.G. (eds), Liver Cell Carcinoma, MTP Press, Lancaster, in press. 39. Moore.M.A., Weber.E. and Bannasch.P. (1988) Modulating influence of dehydroepiandrosterone administration on the morphology and enzyme phenotype of dimethylaminoazobenzene-induced hepatocellular foci and nodules. Virchows Arch. B Cell Pathol., in press. Received on April 25, 1988; revised on July 11, 1988; accepted on July 28, 1988

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content was increased 2-fold, G6PDH activity 3- to 6-fold, PK up to 2-fold and HK 2.5-fold. G6Pase activity was reduced to 46% of that observed in normal tissue. The striking opposition in enzyme alterations caused by chemical carcinogens and the 'anti-carcinogen' DHEA needs further investigation. Several effects of DHEA on preneoplastic liver lesions preceding the development of hepatocellular carcinomas have been described. DHEA treatment leads not only to reduction of the total number of preneoplastic foci (39) but also to a modulation of the morphological phenotype of the foci (15,16,39) in the way that lesions which lead to less malignant tumours are prevailing. Furthermore the altered enzyme pattern which is typically observed in preneoplastic liver foci is modulated by DHEA in the same manner as it is observed in normal liver (15,39). It is not clear so far whether the anti-carcinogenic effect of DHEA is due to abolishment of carcinogen-induced enzyme alterations or to more profound alterations of cellular metabolism on the base of transcription or translation or posttranslational modification of the enzymes.