Regulation of Glycogenolysis in the Uterus of the Mouse during Postimplantation Pregnancy: 2. The Role of Phosphorylase Enzyme

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1 BioSMART ISSN: X Volume 3, Nomor 2 Oktober 2001 Halaman: 1-6 Regulation of Glycogenolysis in the Uterus of the Mouse during Postimplantation Pregnancy: 2. The Role of Phosphorylase Enzyme SUTARNO Jurusan Biologi FMIPA UNS Surakarta Received: July 2, Accepted: August 31, ABSTRACT The aim of this experiment was to investigate the activity of phosphorylase enzyme in uterus and liver during pregnancy in response to treatment with ethanol, epinephrine and glucagon. The intraperitoneal injection of ethanol (3.0 g/kg body weight) in day 9-pregnant mice significantly increased (P<0.05) glycogen phosphorylase a and t activities in the liver, but not in the uterus when measured 1 h after treatment. The subcutaneous administration of epinephrine to day 9-pregnant mice produced no significant increase in either the activity of phosphorylase a or total phosphorylase 1 hour after treatment, while intravenous injection of 10 ug of glucagon also show that the activities of phosphorylase a and t were not significantly altered in either the liver or uterus 1 h after treating day 9-pregnant mice. Key words: pregnancy, epinephrine, glucagon, ethanol, phosphorylase. INTRODUCTION Glycogen phosphorylase is an enzyme responsible for the breakdown of glycogen to glucose (glycogenolysis). The enzyme is known to exist in two forms, a less active (nonphosphorylated) "b" form and in an active (phosphorylated) "a" form. Activation of glycogen phosphorylase occurs via a cascade mechanism that is initiated by the stimulation of adrenergic receptors or glucagon receptors on the cell surface. Generally, stimulation of these receptors results in the activation of adenylate cyclase that in turn causes cyclic AMP formation from ATP. Increasing intracellular cyclic AMP then activates protein kinases. The cyclic AMP-dependent protein kinase then activates phosphorylase kinase which in turn then phosphorylates phosphorylase b to active phosphorylase a. From this view, ethanol, epinephrine and glucagon, which affect glycogen breakdown, should also affect glycogen phosphorylase. This reaction commonly occurred in a stress reaction, that is defined as disturbance of homeostasis that are commonly linked to enhanced activity of hypothalamo-pituitary-adrenal (Einarson, 1996; Minton, 1994) Understanding the events of this period and their application to fertility control, both in humans and commercially valuable animals have been made in recent years, however, the physiology and biochemistry of the peri-implantation stages of pregnancy are still incomplete. One of the important metabolic aspects, glycogenolysis, that occurs in the uterus during post-implantation period also remains unclear and need to be investigated more extensively. Glycogen, a polymeric form of glucose stored in animal cells, which can be degraded on demand. In most tissue, including muscle, the role of glycogen is as a glycolytic fuel that provides energy locally when glucose or oxygen becomes scarce. In the liver, where it serves as a buffer to maintain a constant level of blood glucose, glycogen is broken down to release glucose between meals. In contrast when glucose supply is abundance, the liver can convert glucose into glycogen. Muscle glycogen serves as an important source of energy for muscle contraction, although a major portion of the energy supply of muscle comes from the breakdown of fatty acids. In uterus, the importance of uterine glycogen has been suggested as an energy store for the metabolic demands of egg implantation (Walaas, 1952) and it also act as an important energy source for both embryonic development (Demers et. al., 1972) and parturition (Chew and Rinard, 1979) Jurusan Biologi FMIPA UNS Surakarta

2 2 BioSMART Vol. 3, No. 2, Oktober 2001, hal The evidence supports these function that glycogen accumulation appears cyclically during estrus cycle. The aim of this experiment was therefore to investigate the activity of phosphorylase enzymes in uterus and liver during pregnancy in response to treatment with ethanol, epinephrine and glucagon. MATERIALS AND METHODS Experimental Animals The experimental animals used in all experiments were outbreeding Quackenbush (QS) strain mice aged between 6 to 10 weeks. They were housed in white plastic cages under controlled environmental conditions (constant temperature of 22 o C, with unlimited access to fresh tap eater and food). Pairing females with fertile males of the same strain brought about pregnancy. The females were examined for copulation plugs each morning, and the day of finding a plug was designated as day 1 or the first day of pregnancy. Some animals were treated with saline (as control), while other groups were treated with ethanol, epinephrine or glucagon on day 9 of pregnancy to assess the role of these agents in the regulation of liver and uterine glycogenolysis. Phosphorylase assay Samples of liver (10mg/ml) or uterine tissue (20mg/ml) were homogenized at 4 o C in glycylglycine buffer (ph 6.2) containing 0.15 M NaF using a polytron homogenizer. The homogenates were then centrifuged at 8000g for 10 minutes at 4 o C and the supernatant containing the enzyme was retained. This buffer system was selected since it is known to affect a consistent activity of phosphorylase, and the fluoride is a potent inhibitor of phosphorylase phosphatase, the enzyme which inactivates phosphorylase a by conversion to phosphorylase b (Winston and Reitz, 1984). The reaction mixture was adjusted to maximize enzyme activity and to ensure zero-order kinetics. In order to determine whether low molecular weight effect molecules existed in the homogenates to influence phosphorylase activity, several tissue preparations were subjected to gel filtration a column (2 x 25 cm) of Sephadex G-25 that had been previously equilibrated with the isolation buffer. Figure 1. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant mice with ethanol (3.0 g/kg body weight). Value represent the mean + S.E.M for N=5.

3 SUTARNO - Glycogenolysis: 2. Phosphorylase Enzyme 3 Since the activity of enzyme preparations collected in this way did not differ significantly from those not subject to column chromatography, gel filtration was not considered a necessary step in the assay procedure and the activity values reported in the present study are those recorded without the inclusion of the column chromatography. Aliquots of enzyme preparation (120ul) were added to 120 ml of assay mixture for phosphorylase a (A solution), total phosphorylase (B solution) and control (C solution). The A solution contained 32 mm of glucose-10phosphate and 2 % glycogen; the B solution contained 32mM glucose-1-phosphate, 10mM AMP and 2% glycogen; and the C solution containing 2% glycogen only. Determination of inorganic phosphate (Pi) was based on the method of Bergmeyer (1963). Phosphorylase activities were expressed as nmol of Pi released/mg of protein/min. at 25oC. The protein concentration of samples was measured by the method of Lowry et al. (1951) using standards of bovine serum albumin. Statistical analysis The significance of results was assessed by analysis of variance and students t-test. The multiple-range test (Duncan test) was used to compare levels of glycogen in the uterus of the various reproductive stages studied. RESULTS The intraperitoneal injection of ethanol (3.0 g/kg body weight) in day 9-pregnant mice significantly increased (P<0.05) glycogen phosphorylase a and t activities in the liver, but not in the uterus when measured 1 h after treatment (Figure 1). The ratio of activities of phosphorylase a to phosphorylase t varied between 0.83 and 0.93 in the liver, and between 0.74 and 0.81 in the uterus. The specific activity of the enzymes was greater in the liver than the uterus, but this difference was not of the same magnitude as the difference in glycogen levels between the two tissues. The results presented in Figure 2 that the subcutaneous administration of epinephrine to day 9-pregnant mice produced no significant increase in either the activity of phosphorylase a or total phosphorylase 1 hour after treatment. This treatment also fail to alter the ratio of activities of phosphorylase a to phosphorylase t and, again, enzyme activity was greater in the liver than in the uterus. Figure 2. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant mice with epinephrine (1mg/kg body weight). Value represent the mean + S.E.M for N=5.

4 4 BioSMART Vol. 3, No. 2, Oktober 2001, hal Figure 3. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant mice with glucagon (10 ug/animal). Value represent the mean + S.E.M for N=7. The data presented in Fig 3 show that the activities of phosphorylase a and t were not significantly altered in either the liver or uterus 1 h after treating day 9-pregnant mice with an intravenous injection of 10 ug of glucagon. Again, the enzyme activity in the liver was greater than that in the uterus, but the difference was not of the same order of magnitude as the differences in glycogen concentration. Treatment with this hormone also failed to significantly alter the ratio of activities of phosphorylase a to phosphorylase t. These treatments fail to alter the ratio of activities of phosphorylase a to phosphorylase t (table 1), and, the enzyme activity was greater in the liver than in the uterus. DISCUSSION The mechanism whereby glycogenolysis is regulated in the uterus to provide glucose for the developing embryo remains uncertain. Ethanol has been reported to rapidly promote the degradation of glycogen to glucose in the liver (Winston and Reitz, 1980; Simm and Murdoch, 1990), but not in the uterus of the mouse during post implantation pregnancy (Murdoch and Simm, 1992). The results of the present study confirm these findings and show that 1 h after treating day 9-pregnant mice with ethanol resulted in an increase of glycogenolysis in the liver (almost 50% degraded), but not in the uterus (Sutarno, 2000). The Table 1. Ratios of activities of phosphorylase a to phosphorylase t (%) 1 h after treating day 9-pregnant mice with ethanol (3.0 g/kg body weight), epinephrine (1 mg/kg body weight) or glucagon (10 ug/mouse). A/t phosphorylase activities (%) Treatment LIVER UTERUS Control Treated Control Treated Ethanol, n = Epinephrine, n = Glucagon, n =

5 SUTARNO - Glycogenolysis: 2. Phosphorylase Enzyme 5 stimulation of glycogenolysis in the liver by ethanol is most likely due to the acute activation of the sympathetic nervous system, since the alcohol is known to increase the urinary excretion (Adams and Hirst, 1984) and plasma concentration of catecholamines (Eisenhofer et al., 1983). The catecholamines produced in this response may then promote glycogenolysis in the liver via receptor mediated events involving intracellular second messengers. However, uterine glycogen concentrations were not changed in response to ethanol, suggesting that catecholamines such as epinephrine released in response to the stress reaction mobilizes only liver glycogen without interfering with uterine glycogen stores. This suggests that under conditions of stress, uterine glycogen is conserved to meet physiological demands other than those required by the maternal system to cope with the factors involved in this response. The present results support the suggestion that epinephrine mediates the effects of ethanol in this respect since the administration of the catecholamine to pregnant mice also stimulated glycogen degradation in the liver without influencing the levels of the polysaccharide in the uterus. The significantly increased activities of phosphorylase a and total phosphorylase in the liver 1 h after treatment with ethanol, but not in response to epinephrine or glucagon question the proposal that the effects of alcohol on glycogen metabolism are mediated by the adrenal medullary hormone. However, Simm and Murdoch (1990) have recently shown that the QS mouse needs a time period of 6 h to clear this dose of alcohol from the maternal system during post-implantation pregnancy, while both epinephrine and glucagon are inactivated, rapidly. Thus, the ethanol may continue to promote an epinephrine-release response in the mouse for periods in excess of 1 h while it continues to exist in the blood stream in appreciable amounts. Consequently, in order to better asses the effects of these agents on the glycogen-degrading phosphorylase system, it may be necessary to study the enzyme system within minutes of exposure before the hormones have had the opportunity to be inactivated and excreted from the maternal system. Under these conditions, it would be expected that both epinephrine and glucagon will interact with liver cell receptors and stimulate the activity of phosphorylase a. However, whether a similar response is likely to occur in the uterine cells remains to be elucidated. In this context, previous studies have demonstrated that epinephrine exposed to rat uteri in vitro resulted in increased phosphorylase a within one minute, reached a maximum at two minutes and declined to a level which was not statistically different from the control level at ten minutes (Leonard, 1963).It is of interest that the present study showed that the activity of phosphorylases in the uterus was high in relation to the small amounts of glycogen that the organ contained relative to that of the liver. The liver of the mouse was found to contain about 12- fold as much glycogen as the uterus at its maximal concentration, but the activity of the phosphorylases was only about 2-fold greater in the liver than the uterus. It is possible that this reflects different isozymic forms of the enzymes between the two tissues with different Km values for substrate. This possibility requires further investigation to validate the proposal. Finally the ratio of phosphorylase a and t activities in the present study was not significantly different from control values, indicating that the proportion of active to inactive enzyme failed to alter in response to the treatments, at least when they were measured 1 h after administration. The large phosphorylase a to t activities at this time may be due to non- specific activation of the enzyme, although the same phenomenon was observed when enzyme preparations were subjected to gel filtration to remove any small activator molecules such as AMP or c-amp. Thus, the reason for these high phosphorylase a to t ratios are not apparent, but similar ratios in other tissues have been reported previously (Cornblath et al., 1963). REFERENCES Adams, MA and M. Hirst Adrenal and urinary cathecolamines during and after severe ethanol intoxication in rats: A profile of changes. Pharmacol. Biochem. Behav. 21: 125. Bergmeyer, H.U Methods of enzymatic analysis. New York: Academic Press. Bergmeyer, H.U Method of Enzymatic Analysis. New York: Academic Press. Chew, C.S. and G.A. Rinard Glycogen levels in the rat myometrium at the end of pregnancy and immediately post partum. Biology of Reproduction 20: Cornblath, M., P.J. Randle, A. Parmeggrani, and H.E. Morgan Effects of glucagon and anoxia on lactate production, glycogen content, and phosphorylase activity in the perfused isolated heart. J. Biol. Chem. 238: De Feo, V.J Decidualization. In R.M. Wynn (ed.). Cellular Biology of the Uterus. New York: ACC.

6 6 BioSMART Vol. 3, No. 2, Oktober 2001, hal Demers, L.M., K. Yoshinaga, and R.O. Greep Uterine glycogen metabolism of the rat in early pregnancy. Biology of Reproduction 9: Einarsson, S., A. Madej, V. Tsuma The influence of stress on early pregnancy in the pig. Animal Reproduction Science 42: Eisenhofer, G., D.G. Lambic, and R.H Johnson Effects of ethanol on plasma cathecolamines and norepinephrine clearance. Clin. Pharmacol. Ther.34: 143. Finn, C.A. and Porter The decidual reaction. In: C.A. Finn (ed.). The Uterus. London: Elec. Science. Leonard, S.L. and M. Crandall1963. Hormonal stimulation of phosphorylase activity in the rat uterus in vitro. Endocrinol. 73: Lowry, O.H., N.J. Rosenbrough, and R.J. Randall Protein measurement with the folin reagen. J. Biol. Chem. 193: Minton, J.E Function of Hipothalamic-pituitaryadrenal axis and the symphatetic nervous system models of acute stress in domestic farm animals. Journal of Animal Science 72: Murdoch, R.N., D.J. Kay, and M. Cross Activity and cellular distribution of mouse uterine alkaline phosphatase during pregnancy and pseudopregnancy. Journal of Reproduction and Fertility 54: Murdoch, R.N. and B. Simm Impaired glucose homeostasis during post-implantation pregnancy in the mouse following acute exposure to ethanol, with particular reference to the uterus and embryo. Biochem. Med. Metab. Biol. 54: Simm, B. and R.N. Murdoch The role of acetate in alcohol-induced alterations of uterine glucose metabolism in the mouse during pregnancy. Life Science 47: Sutarno Regulation of glycogenolysis in the uterus of the mouse during post-implantation pregnancy 1: Hormonal control. BioSMART-Journal of Biological Sciences 2 (1): 1-6. Vasilenko, P., W.C., Adams, and E.H. Frieden Uterine size and glycogen content in cycling and pregnant rats, influence of relaxin. Biology of Reproduction 25: Walaas, O Effects of oestrogens on the glycogen content of the rat uterus. Acta Endocrinologica 10: Winston, G.W. and R.C. Reitz Effects of chronic ethanol ingestion on glucose homeostasis in males and females. Life Science 26: 201. Winston, G.W. and R.C. Reitz Effects of chronic ethanol ingestion on male and female rat liver clycogen phosphorylase phosphatase. Clin. Exp. Res. 8 (3):