Archives of

Gynecology

Arch. Gynecol. 227, 349-354 (1979)

© J. F. Bergmann Verlag 1979

Investigations of Gestation-induced Metabolic Changes in the Rat Liver I. Glycogen Metabolism P. Brockerhoff, U. Nauroth, H. Koeberlin, V. Friedberg, and G. H. Rathgen Gynaecological Clinic and Department for Experimental Endocrinology,University of Mainz, Langenbeckstral3e 1, D-6500 Mainz, Federal Republic of Germany

Untersuehungen zu gestationsbedlngten Stoffwechselverlinderungen in der Rattenleber I. Glykogenabbau Zusammenfassung. Die Aktivit/iten von o~-Glukan-Phosphorylase und Phosphoglucomutase sowie der Glykogengehalt wurden in der Leber tr/ichtiger und nichttr/ichtiger Ratten bestimmt. Bei der statistischen Auswertung der Untersuchung lieBen sich keine signifikanten Aktivit/its- bzw. Konzentrationsunterschiede zwischen normalen und tr/ichtigen Tieren nachweisen. Im Verlauf der Tr/ichtigkeit ver~inderten sich die Enzymaktivit~iten nicht signifikant. Dies deutet im Zusammenhang mit anderen Untersuchungen auf einen unver~inderten Glykogenstoffwechsel in der Gravidit~it hin. Sehliisselwiirter: Gestation - Rattenleber Phosphoglucomutase - Glykogen

o~-Glukan-Phosphorylase -

Summary. The activities of oL-glucan-phosphorylase and phosphoglucomutase and the concentration of glycogen were measured in the liver of pregnant and non-pregnant rats. There were no significant differences between normal nonpregnant and pregnant animals nor was there any change of enzyme activities during pregnancy. Our results lend support to the idea that glycogen metabolism is not changed during normal pregnancy. Key words: Gestation - Rat liver - cr-glucan-phosphorylase - Phosphoglucomutase - Glycogen

During normal pregnancy there are changes in intermediary metabolism. The precise reason for these is very difficult to demonstrate (Hytten et al., 1971; Brockerhoff et al., 1977). Glucose tolerance is of particular interest from a clinical point of Offprint requests to: P. Brockerhoff, M.D. (address see above)

0170-9925/79/0227/0349/$ 01.20

350

P. Brockerhoff et al,

view. Investigations of the regulatory processes were mainly done with hepatic tissue which has a central place in carbohydrate metabolism. By measuring the activity of various enzymes and the concentration of certain products of carbohydrate metabolism in the rat liver, changes in several parameters of carbohydrate metabolism were noted particularly glycolysis (Butt et al., 1957; Burt et al., 1959; Rathgen et al., 1970). Activated cr-Glucan-phosphorylase (EC 2.4.1.1.) breaks down glycogen to glucose-l-phosphate by an irreversible reaction involving adenylcyclase, cyclic 3', 5'adenosine monophosphate and adenosine triphosphate. Subsequently glucose-lphosphate is converted by phosphoglucomutase (EC 2.7.5.1.) to glucose-6-phosphate, the starting substrate for the further degradation of glucose. Since the activity of c~-glucan-phosphorylase is particularly subject to hormonal control (Wosilait et al., 1956) we decided to investigate the activity of these enzymes in hepatic tissue during pregnancy. We also determined the glycogen content of the rat liver in pregn a n c y and in the non-pregnant state.

Material and Method Female Sprague-Dawley rats were used for the experiments. The mean body weight of the pregnant animals was 270 g and that of the nonpregnant animals was 230 g. The mean number of fetuses ranged from 3 to 15 and the mean length of the fetuses was 20 mm. The animals received Altromin standard diet as food. They were killed by dislocation of the cervical spine subsequent decapitation and exsanguination. The livers were carefully and rapidly excised, a portion being weighed on a torsion balance and processed further immediately. For determining enzyme activities liver tissue was homogenized in a four-fold volume of ice-cold 0.01 M sodium phosphate buffer pH 7.5 with continuous cooling using an Ultra-Turrax over two 15-s periods. The homogenate was centrifugedfor 30 min at 20,000 rpm, and a constant temperature of 4° C. The clear supernatant was then carefully decanted and used for determining the activities of the two enzymes. To measure glycogen content, 20% trichloroacetic acid was added to the liver sample in the ratio of 4 : 1 and the mixture was homogenizedwith continuous cooling during two 15-s periods using an Ultra-Turrax. The homogenate was transferred to a centrifugetube, 2 ml of 30% potassium hydroxide solution was added and the mixture was then heated for 15 rain on a boiling water bath. A 3.5 ml volume of ethanol was added and the system was heated briefly again and finally cooled slowly to room temperature. The system was then centrifugedfor 10 rain at 5,000 rpm., decanted and the centrifugate was washed with 3 ml ethanol. The ethanol was evaporated on a water bath and the precipitate was taken up in 2 ml 2 N sulphuric acid and then heated for 120 min on a boiling water bath. After cooling to room temperature, the system was neutralized with 2 N sodium hydroxide solution and sufficient distilled water was added to make 10 ml. A 0.05 ml volume of this solution was used for the measurement, ce-Glucan-phosphorylasewas measured by the method described by Hiilsmann et al. (1961) with slight modifications.Phosphoglueomutaseactivity was likewise measured optically using a method reported by Bergmeyer (1971). Glycogen was measured according to the method of Klotzsch and Bergmeyer (1962). Checks on the accuracy of this method showed a 81.6%-90.6% recovery of substrate.

Test Systems 1 [Footnote see page 351] 1. o~-Gluean-phosphorylase 0.05 ml 1.50 ml 0.10 ml 0.02 ml 0.02 ml

NaC1 (1.875 x 10-4 M/ml system) sodium phosphate buffer (0.01 M, pH 7.5) glycogen (60 mg/ml) PGM (2 mg/ml) G-6-PDH (1 mg/ml)

Investigations of Gestation-induced Metabolic Changes in the Rat Liver 0.05 1.00 0.05 0.19 0.01

ml ml ml ml ml

351

MgC12 (1.5 × 10-4 M/ml system) TRIS buffer (7.5 × 10-4 M/ml system, pH 7.5) TRILON (10 g Titriplex III/100 ml distilled water neutralized with 2 N NaOH) distilled water NADP (0.355 × 10-3 M/ml system)

0.01 ml homogenate supernatant as starter (equivalent to 2.5 mg liver fresh weigh0 2. Phosphoglucomutase 0.50 0.10 0.05 0.02 2.72 0.01

ml ml ml ml ml ml

MgC1 z (1.5 × 10-4 M/ml system) G-1-P (20 mg/ml) TRILON (10 g Titriplex III/100 ml distilled water, neutralized with 2 N NaOH) G-6-PDH (1 mg/ml) TRA-buffer (0.1 M, pH 7.5) homogenate supernatant (equivalent to 2.5 mg liver fresh weight)

0.05 ml NADP as starter (10 mg/ml) The final volume for both enzyme determinations was 3 ml and the thickness of the layer of the test system was 1 cm. The reacttions were measured with an Eppendorf photometer at 366 m~ and a constant temperature of 25 ° C over a period of 60 rain for phosphorylase and over 20 rain for phosphoglucomutase. 3. Determination of Glycogen as D-Glucose The following were consecutively pipetted into a l-era cell at room temperature: 2.58 ml TRA-buffer (7.5 × 10 7 M/ml, pH 7.6) 0.04 ml PEP solution (3 × 10-2 M) 0.06 ml NADH solution (1.2 × 10-2 M) 0.10 ml KC1 solution (2 M) 0.I0 ml MgSO 4 solution (0.5 M) 0.10 ml ATP solution (3 × 10-2 M) 0.01 ml LDH solution (suspension) (10 rag/2 ml) 0.01 ml PK solution (suspension) (2 rag/1 ml) 0.05 rrd sample 0.05 ml HK solution (2 rag/1 ml) The reaction was started with 0.05 ml (2 rag/1 ml) hexokinase. The measurement was made in a l-era cell using an Eppendorf photometer at 366 rap, with a volume of 3 ml at a constant temperature of 25 ° C. In blood the activities of phosphorylase and phosphoglucomutase (Holzer et al., 1956) and the concentration of glycogen (Brummer, 1943) are so low that corrections made for the measured blood content of the liver would be smaller than the margin of error of the laboratory methods used and so these corrections were not applied. Statistical analysis of the results was done by testing mean values, standard deviations, correlation and regression coefficients using Student's t-test for non-paired data.

1 Abbreviations: G-l-P: Glucose-l-phosphate, G-6-P: Glucose-6-phosphate, G-6-DPH: Glucose-6phosphate dehydrogenase, G-6-P-lactone: 6-phospho-gluconolaetone, AMP: Adenosine 5'-monophosphate, PGM: Phosphoglucomutase, TRIS: Tris-hydroxymethyl-aminomethane, TRA-buffer: Triethanolamine buffer, TRILON: Neutralized EDTA = ethylenediaminetetraacetate, G-1,6-DP: Glucose1,6-diphosphate, HS: Homogenate supernatant, ATP: Adenosine triphosphate, ADP: Adenosine diphosphate, NAD: Nicotiuarnide adenine dinucleotide, NADH: Nicotinarnide adenine dinueleotide, hydrated form, PEP: Phosphoenol pyruvate, LDH: Lactate dehydrogenase, PK: Pyruvate kinase, HK: Hexokinase, NADP: Nicotinamide adenine dinucleotide phosphate

P. Brockerhoff et al.

352 Table 1. Activities of PGM and Phosphorylase (mU/mg FW)

Number of animals Mean value Standard deviation

Phosphoglucomutase

Phosphorylase

nonpregnant

pregnant

nonpregnant

pregnant

n=29

n=30

n=29

n = 30

= 11.2400

i = 12.7230

i =0.5841

i = 0.7038

s = 2.4832

s = 2.8138

s =0.2471

s =0.2590

not significant

t-test for unpaired samples

not significant

Table 2. Correlation coefficient for PGM and Phosphorylase in relation to length of fetus

Correlation coefficient

Length of fetus versus PGM

Length of fetus versus phosphorylase

r = 0.1015

r = 0.0576

Number of cases

24 Significance

not significant

Table 3. Glycogen content of liver (10 -5 mol/f FW) nonpregnant

pregnant

Number of animals

n = 32

n = 25

Mean value

:~ = 13.1482

x = 10.4420

Standard deviation

s=

s=

t-test for unpaired samples

6.1734

5.3460

not significant

Results T h e values for c r - g l u c a n - p h o s p h o r y l a s e and p h o s p h o g l u c o m u t a s e are s h o w n in T a b l e 1. T a b l e 2 s h o w s relation b e t w e e n e n z y m e activity and fetal length; the latter being a m e a s u r e o f gestational age ( H a g e m a n n and Sehmidt, 1960). T a b l e 3 gives the results o f the g l y c o g e n m e a s u r e m e n t s .

Discussion T h e r e w e r e n o significant differences in the c r - g l u c a n - p h o s p h o r y l a s e and p h o s p h o g l u c o m u t a s e activities in the liver o f p r e g n a n t and n o n - p r e g n a n t rats and there were no correlations b e t w e e n e n z y m e activity and gestational age. Burt (1962) m e a s u r e d

Investigations of Gestation-induced Metabolic Changes in the Rat Liver

353

liver phosphorylase levels in starving rats and obtained results which were similar to ours. Both enzymes play an essential role in carbohydrate metabolism. Hepatic glycogen helps to maintain a constant blood sugar level. The liver and kidney are the only organs which possess specific glucose-6-phosphatases (Swanson, 1950), which convert glucose-6-phosphate released by glycogen metabolisms into free glucose which can enter the blood stream. Its activity is markedly reduced during pregnancy (Rathgen et al, 1970). In rats, glucose-6-phosphate and glucose concentrations were found to be markedly reduced throughout pregnancy (Rathgen et al., 1970). According to Larner (1966) large quantities of glycogen stimulate the activation of ce-glucan-phosphorylase by way of activation of phosphorylase-6-kinase. However, like others (Harding et al., 1966; Butt et al., 1957) we found no significant difference between the glycogen content of the livers of pregnant and non-pregnant rats. Hence, the gross regulation of phosphorylase does not appear to be changed in pregnancy. In this connection, the unchanged activities of cr-glucan-phosphorylase and phosphoglucomutase during pregnancy suggest that glycogen breakdown remains unaltered so that the organism can still rapidly meet increased requirements for sugar. The reduction in glucose-6-phosphate activity in the liver of experimental animals and humans does not contradict this assumption. In our experiment we used normally fed animals so that there was no increase in peripheral requirements for carbohydrates which would have to have been met by the breakdown of glycogen.

References Bergmeyer, H. U.: Methoden der enzymatischen Analyse. 2. Aufl., Bd. I., S. 764. Weinheim: Verlag Chemie 1971 Brockerhoff, P., Kurtenbach, I., Netter, P., Schicketanz, K. H., Stark, W., Rathgen, G. H.: Clinical biochemical monitoring of normal and hypertensive pregnancies. Proc. VIIth As. Congr. Obstet. Gynec., p. 723. Bangkok 1977 Brummer, P.: Blood glycogen. Acta reed. Seand. 114, 373 (1943) Burt, R. L., Kimel, C. A., Winston-Salem, N. C.: Carbohydrate metabolism in pregnancy. Hepatic glycogen synthesis in the rat. Am. J. Obstet. Gynecol. 74, 295 (1957) Butt, R. L., Winston-Salem, N. C., Julian, N.: Carbohydrat metabolism in pregnancy. Observations on glucagon (hyperglycemic-glycogenolyticfactor) in normal pregnancy and the puerperium. Am. J. Obstet. Gynecol. 74, 551 (1957) Butt, R. L., Winston-Salem, N. C., Julian, N.: Liver glucose-6-phosphatase activity in pregnancy. A study utilizing albino rats. Am. J. Obstet. Gynecol. 77, 6 (1959) Burr, R. L., Pulliam, R. P.: Liver phosphorylase activity in pregnancy. Am. J. Obstet. Gynecol. g4, 764 (1962) Hagemann, E., Schmidt, G.: Ratte und Maus. Berlin: W. de Gruyter 1960 Harding, H. R., Rosen, F., Nichol, C. A.: Effects of pregnancy on several cortisol responsive enzyms in rat liver. Am. J. Physiol. 211, 1361 (1966) Holzer, H., Sedlmayer, G., Kiese, M.: Bestimmungen des Blutgehaltes von Leberproben zur Korrektur biochemischer Analysen. Biochem. Z. 328, 176 (1956) H/ilsmann, W. C., Oei, T. L., Creveld, S.: Phosphorylase activity in leucocytes from patients with glycogen storage disease. Lancet II, 581 (1961) Hytten, F. E., Leitch, I.: The physiology of human pregnancy, II. Ed. Oxford: Blackwell 1971

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Klotzsch, H., Bergmeyer, M.: Methoden der enzymatischen Analyse. Weinheim: Verlag Chemie 1962 Lamer, J.: Hormonal and nonhormonal control of glycogen metabolism. Trans. N.Y. Acad. Sci. 29, 192 (1966) Rathgen, G. H., Baumann, W., Schfimichen, C., Friedberg, V.: Untersuchungen zum Kohlenhydratstoffwechsel in der Leber wghrend der Gestation. Arch. Gyn/ik. 208, 353 (1970) Swanson, M. A.: Phosphatase of liver: Glucose-6-phosphatase. J. Biol. Chem. 184, 647 (1950) Wosilalt, W. D., Sutherland, E. W.: The relationship of epinephrine and glucagon to liver phosphorylase. II. Enzymatic inactivation of liver phosphorylase. J. Biol. Chem. 218, 469 (1956) Received November 2, 1978