IIIHIIHIH I I _| THE ROLE OF HEPATIC GLUCOSE-b-PHOSPHATASE IN ADAPTATION T0 EXERCISE AND ELECTRICAL STRESS IN ADULT MALE ALBINO RATS Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY David R. Lamb 1965 THESIS 3‘ LIBRARY t,- ” Michigan State 3 Universit I Y r This is to certify that the thesis entitled The Role of G1ucose-6—Phosphatase in Adaptation to Exercise and Electrical Stress in Adult Male Albino Rats presented by David R. Lamb has been accepted towards fulfillment of the requirements for ML. degree in W Major professor 0-169 ABSTRACT THE ROLE OF HEPATIC GLUCOSE—6—PHOSPHATASE IN ADAPTATION TO EXERCISE AND ELECTRICAL STRESS IN ADULT MALE ALBINO RATS by David R. Lamb The purpose of this study was to assess the role of hepatic glucose-6-phosphatase in adaptation to exercise and electrical stress in adult male albino rats. Two possible stimuli for induced enzyme activity were hypothesized: decreased blood glucose as a result of in- creased glucose utilization by exercising musCles, and increased hormonal secretions as the result of a general stress response. Five groups of 18 adult male rats (Sprague-Dawley strain) were assigned randomly to experimental regimens: (l) sedentary housing with no other stress, (2) voluntary activity housing with no other stress, (3) sedentary housing with electrical stress, (4) voluntary activity‘ housing with electrical stress, and (5) sedentary housing with electrical stress and forced-exercise stress. The experiment lasted for 70 days. The forced exercise consisted of a daily one-half hour swim with a weight equivalent to two per cent of the rat's body weight taped to the base of its tail. David R. Lamb The electrical stress, designed to elicit a general stress response of a hormonal nature, consisted of electrical shocks (60 volts and 15 milliamperes) administered once every 15 seconds for one-half hour daily through a stainless steel grid, which served as flooring for the stress compartments. The sedentary animals lived in standard 10 x 8 x 7 inch small~animal cages. The voluntary activity animals were housed in similar cages with attached five—inch spontaneouséactivity drums which were 14 inches in diameter. The rats were sacrificed by ether anesthesia at the end of the 70-day period. The livers were removed, weighed and frozen at -20 degrees Centigrade for five months prior to enzyme assay. Enzyme activity was measured as the amount of inor— ganic phosphate released by the entire liver per 100 grams of body weight during 15 minutes of incubation with a 0.08 molar substrate solution at a temperature of 37 degrees Centigrade and a pH of 6.5. Mean values and their estimated standard errors for each group were: (1) 68.2 : 3.M, (2) 59.9 i 3.5, (3) 7u.5 .i 3.8, (4) 66.1 i 2.8, and (5) 69.9 i 3.7. A one—way fixed—effects model of the analysis of variance detected no significant (p < .05) differences between groups. The power of the F-test was calculated to be .81 with group mean differences of the magnitude observed in this study. David R. Lamb The results indicate that neither the forced exercise nor the electrical stress utilized in this experiment has any effect on hepatic glucose-6-phosphatase activity in adult male albino rats. Hepatic glucose-6-phOSphatase apparently plays no significant role in adaptation to forced exercise or electrical stress. THE ROLE OF HEPATIC GLUCOSE-é-PHOSPHATASE IN ADAPTATION IO EXERCISE AND ELECTRICAL SIRESS IN ADULT MALE AIBINO RATS (X .32“ David B? Lamb Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health, Physical Education and Recreation 1965 ACKNOWLEDGMENTS The assistance of Dr. Michael Maksud, without whose diligent efforts this study could not have been completed, is gratefully acknowledged. Credit is given to my wife, Cozette, who performed the vital task of typing and editing the manuscript. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS. . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . V LIST OF EIGI; RES. . . . . . . . . . . . . . vi LIST OF APPENDICES. . . . . . . . . . . . . Vii Chapter I. INTRODUCTION . . . . . . . . 1 Statement of the Problem . . 2 Limitations of the Study . . . 3 Definitions of Terms. . . . . 4 II. RELATED LITERATURE 5 Exercise and Glucose-é-Phosphatase Activity 5 Stress and Glucose-6—Phosphatase Activity. 5 Hormones and Glucose-6-Phosphatase Activity 6 Adrenal Hormones and Adrenalectomy . . 6 Pituitary Hormones and Hypophysectomy . . 9 Pancreatic Hormones and Diabetes . . . . 9 Thyroid Hormones . . . . . . . . . 10 Blood Glucose and Glucose-6~Phosphatase Activity . . . . . . . . . . . . 10 III. RESEARCH METHODS. . . . . . . . . . , 13 Experimental Design . . . . . . . . . 13 Forced Exercise Regimen. . . . . . . . 14 Electrical Stress Regimen . . . . . . . 14 General Animal Care . . . . . . . . . 15 Enzyme Assay . . . . . . . 15 Statistical Treatment of Data. . . . . . 17 IV. RESULTS AND DISCUSSION. . . . . . . . . 18 Results . . . . . . . . . . . 18 Discussion of Results . . . . . . . . 18 iii lapter Page Absence of Enzyme Induction by lowered Blood Glucose. . . . . . . . . Absence of Enzyme Induction in Response to Stress Hormones . , . . . . . . ;_.J I\_) n) m U.) (I) V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS. Summary . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . Recommendations . . . . . . . . . . I\) I\) ID (ID (I) 00 LIIERAICPE CIIED . . . . . . . . . . . . . DOM 01%) APPENDICES . . , . . . . . , . . . . . . iv LIST OF TABLES Table Page 1. Results of Analysis of Variance. . . . . . . 19 V LIST OF FIGURES Figure Page 1. Glucose-E-PhOSphatase Activity . . . . . . . 20 vi LIST OF APPENDICES Appendix Page A. Test-Retest Reliability of Assay Procedure . . 36 s. Assay of Glucose-6-Phosphatase Activity . . . 38 vii CHAPTER I INTRODUCTION With recent advances in instrumentation, physiologists have paid increasing attention to the effects of exercise and training at the molecular level. (4, 6, 9, l6, 17) The experiments and hypotheses of Jacob and Monod (24) on genetic mechanisms of enzyme repression and induc- tion seem to have wide application to the study of bio— chemical adaptations to exerzise. Since physical activity is essentially an intricate series of chemical reactions, a partial explaration for the adaptation which occurs in response to repeated strenuous exercise is that repression of some enzymes and induction of others cause the chemical reactions to occur at rates more favorable to efficient bodily function durin the exercise. One of the factors which may influence adaptive enzyme repression and induction under conditions of physical training is the action of the hormones released during muscular exercise. Hormones of the pituitary-adrenal axis have been recognized as vital factors in adaptation to many types of stress, including the stress of strenuous physical activity. (32, 27, 35) 1 Other possibly important non—hormonal factors in adaptive enzyme repression and induction are the levels of substrates, co—factors, co—enzymes and end-products in various rate-limiting reactions, (39) The relationships between adaptive enzyme changes which are brought about by hormonal and non-hormonal factors during training would seem to be a fruitful area of in— vestigation for exercise physiologists interested in elucidating at the molecular level the mechanisms of functional changes, Statement of the Problem The purpose of this study was to assess the role of hepatic glucose-6-phosphatase in adaptation to exercise and electrical stress in adult male albino rats. Hepatic glucose—6-phosphatase was investigated because its role in liberating free glucose to the blood for use by the peripheral tissues is well known (2, 10), and that role seemed to be an important link in the process of adaptation to strenuous activity. Since carbohydrate is generally accepted to be the primary fuel for strenuous muscular exercise (6, 11), and since the carbohydrate stored in the muscles as muscle glycogen is rapidly depleted during severe exercise (9, 37), it was hypothesized that an increased synthesiscyfhepatic glucose-6-phosphatase might occur as a training adaptation which would more 'efficiently supply needed fuel to the muscles, TWO likely mechanisms for such an adaptation were postulated. First, since several experiments (2, 22, 42) have demonstrated a possible relationship between blood glucose levels and glucose—6-phosphatase activity, it was though that an increase in activity might be caused by release of end-product (glucose) repression of enzyme synthesis. Second, since there have been many reports of a high positive correlation between pituitary-adrenal hormone levels and glucose—ouphOSphatase activity (1, la, 21, 39, MO, Ml, M2), it was thought that an increase in enzyme activity might occur as a result of enzyme induction by a hormonal mechanism associated with Selye's General Adaptation Syndrome. (33) Limitations of the Study l. The results of this study are applicable only to adult male rats of the Sprague-Dawiey strain under similar conditions of exercise and electrical stress. 2. The analysis of enzyme activity was carried out in an artificial situation with disruption of cell membranes, spatial disorientation of intracellular molecules, and incubation at a non—physiologic pH and substrate concentration. The in vivo activity of the enzyme may be greatly different than that observed in a test tube, 3, so :ontrols were utilized to test the effect or r.+ enzyme activity of the ether anesthesia used Q.) Definition of Terms :ne following terms are used in this S‘ C dy: Glucose-E-prosphatase activityo-the capacity of the liver enzyme to catalyze the reaction: glucose-E-phosphate;.a glucose + inorganic phosphate, 'ln this study the activity is expressed as milligrams of inorganic phosphate released by the entire liver per 100 grams of body weight during 15 minutes of incubation with a 0,08 molar substrate solution at a temperature of 37 degrees Centigrade and a pa of 6,5, Enzyme irdu:tion or repression--increased or decreased activity of an enzyme brought about by genetic or non- genetic mechanisms, In this study genetic mechanisms stimulated by chronic stress were of most interest, CHAPTER II RELATED LITERATURE The purpose of this study was to assess the role of hepatic glucose-é-phophatase in adaptation to exercise and electrical stress in adult male albino rats. The results of other experiments on the relationships between glucose-6-phosphatase activity and four other variables (exercise, other stresses, hormones and blood glucose) were reviewed. Exercise and Glucose—é—Phosphatase Activity No previous investigations of the relationship between exercise and glucose-o-phosphatase activity were discovered. '03 tre s and Glucose-h-PhoSphatase Activity U) Yudaev and Lebedeva (M2) found a 28 per cent increase in the hepatic glucose-o-phosphatase activity of rats l8 and 48 hours after the animals had been subjected to a sham operation under ether anesthesia, Since it is known that ether and surgery produce elevated levels of corticosteroids in the blood, and since they found similar increases in enzyme activity with injections of cortisone at a dosage of one milligram per 100 grams of body weight; 5 they concluded that the increased enzyme activity after sham operation was due to the hypersecretion of adrenal steroids. Ashmore and Weber (2) also reported increases in glucose-o-phosphatase activity, when expressed per average cell, from one to eight days after sham operations. Beaton (3) found a 26 per cent increase in activity of the enzyme after subjecting experimental rats to tempera- tures of two to three degrees Centigrade for seven days; moreover, Vaughan, Hannon and Vaughan (36) increased the enzyme activity with 28 days of exposure to temperatures of four degrees Centigrade. One report (8) of insignificant effects on glucose- 6-phosphatase activity due to x-irradiation of 750 or 1000 roentgens was contradicted by another (30) which demonstrated decrements in activity with 600 roentgens. Hormones and Glucose-é—Phosphatase Activity Adrenal Hormones and Adrenalectomy.——A comprehensive review by Ashmore and Weber (2) of the relationships between hormones and glucose-S—phosphatase activity noted increased enzyme activity with injections of cortisone (25 milligrams per 100 grams body weight and 1.5 milligrams per 100 grams body weight), as well as with hydrocortisone, in intact or hypophysectomized rats, and decreased activity following adrenalectomy of normal rats. Weber §t_al, (40) found statistically significant enzyme increases with injections of cortisone (2.5 milligrams per 100 grams of body weight), hydrocortisone, medrol, and triamcinolone. In the case of cortisone, they demonstrated optimum induction with a dose of ten milligrams per 100 grams of body weight. Weber (39) reviewed studies on inhibition of cortisone- induced enzyme activity by ethionine and puromycin and concluded that enzyme induction in response to cortisone was due to increased genetic synthesis of the enzyme as opposed to substrate-level activation. Later work (40) with actinomycin supported his conclusion. Willmer (Al) found that adrenalectomy caused a 25 to 30 per cent decrease in enzyme activity in rats. Restoration of activity to normal values was affected by treatment with cortisone or corticosterone. Fed rats also regained normal enzyme activity with hydrocortisone and progesterone, but fasted rats did not respond to these two compounds. Freedland and Taylor (14) injected cortisone or hydrocortisone into normal rats and produced a nearly twofold increase in glucose-o—phOSphatase activity. They also observed a marked decrease in enzyme activity with adrenalectomy. Yudaev and Lebedeva (A2) administered cortisone to rats in doses of 0.3, 0.5, 0.8, 1.0, 1.5, 5.0 and 10.0 milligrams per 100 grams of body weight and found increases in enzyme activity in all cases, with increases of 25 to 225 per cent occurring with doses of 1.0 milligrams and above in a progressive manner. In another part of their experiment, Yudaev and Lebedeva found a 35 per cent decrease in activity three days after adrenalectomy in rats fed a normal diet. This decreased activity was raised to 90 per cent of normal with injections of 0.15 to 0.20 milligrams of cortisone per 100 grams of body weight. However, when adrenalectomized animals were fed a diet containing 80 per cent sucrose, the enzyme activity was raised to slightly above normal. Injections of cortisone (0.15 to 0.20 milligrams per 100 grams of body weight) to these rats caused a further increase in activity to 180 per cent of normal. This was slightly greater than values given for intact, non-injected animals on the sucrose diet. A similar pattern of responses was found by the same authors when a dose of 0.5 milligrams of cortisone per 100 grams of body weight was injected, but a dose of 1.0 milligrams per 100 grams of body weight, likewise administered to adrenalectomized animals, caused a greater increase in activity than did the sucrose diet administered to intact animals. 0ka (28) found increased activity of glucose-6- phosphatase in rats after injections of adrenaline. Pituitary Hormones and Hypophysectomy.--In the review by Ashmore and Weber (2) no conclusive evidence of a rela— tionship between growth hormone and activity of glucose-6- phosphatase was reported, but an activity effect due to corticotropin (ACTH) secretion by ACTH-secreting tumors was noted. Ashmore and Weber also reviewed studies on rat hypophysectomy, which produced marked decreases in glucose-é—phosphatase activity that were corrected neither by growth hormone, hydrocortisone, nor thyroxine injected separately, but were corrected by combined injections of both hydrocortisone and thyroxine. Hypophysectomy in rats also caused a marked decline in enzyme activity in the experiments of Freedland and Taylor (14) and Harper and Young. (21) Since apparent activity changes may be due to changes in liver composition or body weight, Harper and Young recommended that the enzyme activity be expressed as activity of the entire liver per 100 grams of body weight. Pancreatic Hormones and Diabetes.-—Hawkins et. a1. (22) injected six units of protamine zinc insulin into rats and produced a 30 per cent decrease in glucose—6— phosphatase activity. Chronic insulin treatment, however, stimulated an increased enzyme activity. Inhibiting effects of insulin were also reported by Weber et a1, (40), Harper (20), and Ashmore et a1. (1). 10 On the basis of experiments utilizing inhibitors of protein synthesis, Weber et. a1. hypothesized that insulin acts as a repressor of genetic enzyme synthesis, and that the corticosteroids act on the insulin molecule to release it from the genes, allowing the genes to direct the synthesis of more enzyme protein. Ashmore and Weber (2) found consistent reports of increased glucose-o-phosphatase activity in alloxan diabetic rats. The activity was returned to normal by insulin injections or adrenalectomy. Two studies were reviewed which showed increases in enzyme activity after injection of glucagon. .Thyroid Hormones.-~Freedland and Taylor (1A) and Ashmore and Weber (2) presented evidence of marked increases in glucose-o—phosphatase activity after thyroxine injections in rats. ZBeaton (3) found that when 0.3 per cent of the diet was thyroid tissue, the enzyme activity did not increase in rats. But a 0.6 per cent thyroid diet caused a 26 per cent increase in glucose—é—phosphatase activity. Blood Glucose and Glucose—G—Phosphatase Activity In Ashmore and Weber's review (2) a positive correla— tion between blood glucose and hepatic glucose-o—phosphatase activity in fasting normal, cortisone-treated ll adrenalectomized and hypophysectomized rats was noted when results were expressed as activity per 100 grams of body weight, A similiar relationship was observed in obese, hyperglycemic mice. However, Harper and Young (21) failed to observe a relationship between liver glucose-6- phosphatase activity and blood sugar level in rats subjected to hypophysectomy and hormone replacement therapy. Based on physico—chemical relationships observed in tissue homogenates, Segal (31) proposed a mechanism for the in vitro inhibition of glucose-6-phosphatase by glucose. Hawkins gt_al. (22) continually infused glucose into normal rats and found a 66 per cent reduction in enzyme activity between A8 and 72 hours after initiation of glucose infusion. Such infusion did not lower enzyme activity in alloxan diabetic rats; it increased activity in rats pretreated chronically with insulin. As insulin effects gradually disappeared during infusion in the latter case, enzyme activity was depressed, just as in normal infused animals. The authors concluded that repression of glucose- 6—phophatase in response to infused glucose was a result of normal secretion of insulin. Several investigations of the relationship between diet and glucose—6-phosphatase activity lend indirect support to the hypothesis that blood glucose levels influence glucose-6-phosphatase activity. Freedland and Harper (12) fed rats a diet in which protein, fat, galactose 12 or fructose was substituted for a direct glucose source and found a marked increase in enzyme activity. They cited previous evidence which indicated that this response was due neither to blood glucose levels nor liver glycogen stores, however. In further dietary studies Freedland and Harper (13) found that the observed increases in enzyme activity with protein or fat diets were only temporary, and normal activity levels were reached after 21 days. A sucrose diet caused increased activity which was still significant after 30 days. These results suggested some mechanism of metabolic adaptation specific to the diet. Yudaev and Lebedeva (A2) administered an 80 per cent sucrose diet to normal rats and reported that glucose-6— phosphatase activity was elevated to 180 per cent of normal values. The same diet fed to adrenalectomized animals increased enzyme activity, which had been reduced by adrenalectomy, to normal values. They concluded that the diet had an effect on enzyme activity which was activated by adrenal steroids. CHAPIEB III RESEARCH METHODS The purpose of this study was to assess the role of hepatic glucose-6-phosphatase in adaptation to exercise and elect-ical stress in adult male albino rats. Two possible stimuli for increased enzyme synthesis were hypothesized: decreased blood glucose as a result of increased glucose utilization by exercising muscles, and increased hormonal secretions as the result of a general stress response. Experimental Design Ninety adult male Sprague—Dawley rats, 100 days old at the start of the experiment, were purchased from Hormone Assay Laboratories in Chicago, Illinois, and matched into five groups on the basis of serum cholesterol values.* The groups were then assigned randomly to experi- mental regimens: (l) sedentary housing with no other stress, (2) voluntary activity housing with no other stress, (3) sedentary housing with electrical stress, (A) voluntary activity housing with electrical stress, and (5) sedentary *The matching on serum cholesterol values was done to facilitate research on other parameters for the same animals, 13 lb housing with electrical stress and forced—exercise stress. The experimental program was initiated two days after receiving the animals and was continued for 70 days. It was hypothesized that the forced exercise might induce enzyme activity by lowering blood glucose levels in addition to increasing the secretion of stress hormones, and that the electrical stress would induce enzyme activity solely by hormonal means, thus serving as a general stress control. Forced Exercise Regimen The forced exercise consisted of a daily one-half hour swim with a weight equivalent to two per cent of the rat’s body weight taped to the base of its tail. The animals swam individually in one-foot square compartments of a large steel tank in water at 35 to 37 degrees Centi- grade. All animals of the forced—exercise group could swim the full one—half hour with the designated weight by the fourth day of training. During the last 20 days of the experiment many animals were unable to complete the daily swim and had to be pulled from the bottom of the swimming tank. It was during this period that two animals drowned. Electrical Stress Regimen The electrical stress, designed to elicit a general stress response of a hormonal nature, consisted of electrical shocks (60 volts and 15 milliamperes) administered once every 15 seconds for one-half hour daily through a stainless steel grid, which served as flooring for the small, plastic-walled stress compartments. General Animal Care The rats were fed a standard ground feed ad libitum and had constant access to water. They were handled daily and weighed weekly. The sedentary animals were individually housed in standard 10 x 8 x 7 inch small—animal cages. The voluntary activity animals had similar living quarters but had access to spontaneous activity drums, five inches wide and 14 inches in diameter, in which they could run at will. Counters attached to the drums recorded revolutions as the animals exercised. Room temperature varied between 26 and 29 degrees Centigrade. No attempt was made to control humidity in the animal rooms. ‘Enzyme Assay The animals were sacrificed by ether anesthesia at the end of the 70-day period. The livers were removed, trimmed of excess connective tissue, weighed to the nearest milligram on a Mettler electronic balance, wrapped in plastic freezer bags, placed in small, insulated sample jars, frozen in dry ice and stored at —20 degrees Centigrade for five months prior to enzyme assay. Freezing at this 16 temperature has been shown to have no effect on glucose— 6—phOSphatase activity. (10) The assay of glucose—6—phosphatase activity was based on the capacity of the enzyme in a liver homogenate to catalyze the reaction (glucose—6—phosphate ~* glucose + inorganic phosphate) upon incubation with a solution of glucose—é—phosphate. Except for the preliminary freezing procedure, the assay was carried out in accordance with the procedure of Harper. (19) The phosphate determination was performed at a wave length of 660 millimicrons, and enzyme activity was expressed as milligrams of inorganic phosphate released by the entire liver per 100 grams of body weight during 15 minutes of incubation with a 0.08 molar substrate solution at a temperature of 37 degrees Centigrade and a pH of 6.5. The liver samples were randomly selected from various lobes of the frozen livers.* Triplicates of each sample were analyzed, and mean values were used in the final statistical treatment of data. Since two animals died during the forced exercise phase of the experiment, and five livers were destroyed in a pilot study, data were collected on 83 of the original 90 animals. *It has been shown that the concentration of glucose— 6-phosphatase activity is the same between lobes but is greater in the peripheral portions than it is in the central portion of any particular lobe. (7) 17 A test-retest reliability coefficient of .68 was calculated for this method on the basis of two independent analyses on each of 18 livers. These data are presented in Appendix A. In 14 of the 18 livers, differences between samples were no greater than differences within the same sample. Statistical Treatment of Data For statistical analysis the initial matching on the basis of serum cholesterol values was disregarded, and a completely randomized assignment of animals to the five experimental groups was assumed. There was no evidence from other studies or from these data that serum cholesterol values and glucose—6-phosphatase activity were in any way dependent on one another. A one-way fixed—effects model of the analysis of variance was used to analyze the data. The probability of making a Type I statistical error was held to the .05 level. The probability of making a Type II error was calculated to be .19 with mean differences of the magnitude obtained in this study. CHAPTER IV RESULIS AND DISCUSSION The purpose of this study was to assess the role of hepatic glucose-6—phosphatase in adaptation to exercise and electrical stress in adult male albino rats. Five groups of animals were involved: (1) sedentary housing with no other stress, (2) voluntary activity housing with no other stress, (3) sedentary housing with electrical stress, (4) voluntary activity housing with electrical stress, and (5) sedentary housing with electrical stress and forced-exercise stress. It was hypothesized that the forced exercise might induce enzyme activity by lowering blood glucose levels in addition to increasing the secretion of stress hormones, and that the electrical stress would induce activity solely by hormonal means, thus serving as a general stress control. Results The group means and estimated standard errors for hepatic glucose—o-phosphatase activity, expressed as milligrams of inorganic phosphate released by the entire liver per 100 grams of body weight in 15 minutes of incubation with a 0.08 molar substrate solution at a 18 19 temperature of 37 degrees Centigrade and a pH 6.5, are presented graphically in Figure 1. The raw data are shown in Appendix B. The analysis of variance detected no significant (p < .05) differences between groups. The results of the analysis are presented in Table 1. TABLE 1.--Results of analysis of variance Sums of Degrees Mean _ Source Squares of Freedom Squares F F,95(4,78) Among Groups 1916.47 4 479.12 2.41 2.51 Within Groups 15485.07 78 198.53 Total 17401.54 82 Discussion of Results Absence of enzyme induction by lowered blood glucose.-- If lowered blood glucose levels are associated with increased activity of g1ucose—6—phosphatase as others (2) have suggested, three explanations for the failure of the forced swimming to induce activity are apparent: (l) the exercise was not sufficient to diminish muscle glycogen stores and create a demand for hepatic glucose, (2) normal quantities of the liver enzyme were adequate to supply any needed hepatic glucose, and (3) the exercising animals conserved blood glucose by metabolizing non-carbohydrate nutrients. 804. 70 60 IVITY* (MEANS : STANDARD ERRORS) 50 40 {-4. (J <1 [1.] 33 30 H . h h z a L p p L s.© CU CU CU CU CU Q) 4—> +3 +3 4—> +9 o 1* C2 C Q E C‘. )2-4 m 3 Q) 3 G)O 'U H "C H 'Ufiq Cl) 0 (I) O Q) «n- U) > Cf) > 01+ 1 2 3 4 5 EXPERIMENTAL REGIMENS l.--Glucose—6-Phosphatase Activity P04 released by entire liver per 100 gm. body weight in %5 min. with a 0.08 M. substrate solution at 37° 0., pH ,.5. 21 If muscle glycogen stores were not depleted by the exercise, an increased need for hepatic glycogenolysis and glucose—6—phosphatase activity would not be expected. However, an 800-fold increase in glycogenolysis of the gastrocnemius muscles of anesthetized rats occurs after a l5-second tetanic electrical stimulation, according to Cori. (9) This gives some indication that strenuous muscular contraction at least causes the rate of muscle glycogen depletion to increase markedly, but still does not solve the problem of how rapidly it is depleted by a given level of exercise. Walker, Boyd and Asimov (38) stated that the glycogen content of resting muscle (0.5 to 1.0 per cent) decreases during muscular activity and may approach total depletion when the activity is particularly intense and prolonged. Wakabayashi (37) found that liver glycogen stores of rats were almost completely depleted by four five- minute runs on a treadmill with ten minutes of rest between runs when the exercise was carried out daily for five to 16 days. However, he noted that the livers of trained rats did not show such a great depletion of glycogen and attributed the difference to increased muscle glycogen stores in the trained animals. His supposition has been experimentally confirmed in studies reviewed by Hensel and Hildebrandt (23) which utilized both electrical muscle stimulation and exercise. Thus, it is possible 22 that the training increased muscle glycogen to the extent that glucose from liver stores was not necessary to complete the exercise in the final stages of the training program. Assuming that muscle glycogen was depleted, and that hepatic glucose production was necessary for continuation of the exercise, it is possible that normal liver glucose—6—phosphatase activity was adequate to meet the demands placed on it. Since metabolic systems are usually supplied with an excess of necessary enzymes (39, p, 8), and since most writers (5, ll, 18) agree that hypoglycemia in exercising man occurs only after very severe and prolonged exertion, no increase in enzyme activity may have been necessary to supply whatever glucose was needed. Finally, even if total caloric requirements during exercise were greater than could be supplied without marked increases in hepatic glycogenolysis, there is a body of evidence (6, ll, 16, 34) which indicates that much of that caloric requirement, possibly 70 per cent or more, may have been met by lipid metabolism. The rat apparently can metabolize lipids to a greater extent than man. (29) Perhaps adaptive increases in activity of lipolytic enzymes occurred as a response to training which enabled the animals to conserve carbohydrate. An indication of 23 such a response appeared in studies (13) on glucose-6— phosphatase which showed that activation by various dietary treatments in some cases was significant during the first 21 days of the diet, but not thereafter. The possibility of induced enzyme activity in response to glucose—need during exercise should not be discounted without further investigations into the nature of carbohydrate and fat metabolism during exercise. Absence of enzyme induction in response to stress hormone§.-~The apparent failure of both the forced exercise and the electrical shock to induce enzyme activity by hormonal secretions can be interpreted as a failure of these stressors to elicit a general stress response, as a metabolic adaptation to the stressors, or as a specific response to specific stressors. Adrenal weights and histological measurements of medulla and cortex size both showed progressive increases with degree of stress, when the measures were divided by body weight. (26) This is evidence that the stresses were severe enough to stimulate the adrenals. However, a similar training regimen with younger rats (25) did not' significantly decrease circulating blood eosinophils, indicating that corticosteroid liberation was not greatly increased by the training. Most stress studies have measured stress responses a few minutes, hours or days after the stress was applied. It is possible that a metabolic adaptation to stress occurred in the present experiment as a consequence of the prolonged stress period. This adaptation may have enabled the animals to metabolize lipids, rather than carbohydrates, thus eliminating the need for increased glucose—6-phosphatase activity. The glucose—6—phosphatase response to electric shock or forced exercise might be quite different than the response to cold, sham operation, or corticosteroid administration. The induction of enzyme activity with cold is probably due to the increased secretion of thyroid hormone (3), while the induction after sham operation may be due to some effect of the anesthesia used. Increases in glucose-6—phosphatase activity with corticosteroid administration have been noted only with large doses. Geller_et_a1. (l5) calculated that 3.5 micrograms of corticosterone were in the circulation of a rat 15 minutes after having been subjected to agitation and noise stress severe enough to elevate plasma and adrenal corticosterone levels and to deplete adrenal ascorbic acid. The same workers induced enzyme activities with an injection of five milligrams of cortisol per 100 grams of body weight in normal rats but could not do so with the stress, and concluded that the enzyme induction with corticosteroids occurred only with non-physiologic doses. 25 In the writer's opinion none of these interpretations which might account for the observed lack of enzyme induction in this experiment can be discounted. The most likely explanation seems to be that the animals underwent some metabolic adaptation in response to the exercise and electrical stress which enabled the animals to conserve glucose by metabolizing non-carbohydrate nutrients. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary The purpose of this study was to assess the role of hepatic glucose-6-phosphatase in adaptation to exercise and electrical stress in adult male albino rats. Two possible stimuli for induced enzyme activity were hypothe— sized: decreased blood glucose as a result of increased glucose utilization by exercising muscles, and increased hormonal secretions as the result of a general stress response. Five groups of 18 adult male albino rats (Sprague- Dawley strain) were assigned randomly to experimental regimens: (l) sedentary housing with no other stress, (2) voluntary activity housing with no other stress, (3) sedentary housing with electrical stress, (4) voluntary activity housing with electrical stress, and (5) sedentary housing with electrical stress and forced—exercise stress. The experiment lasted for 70 days. The forced exercise consisted of a daily one-half hour swim with a weight equivalent to two per cent of the rat's body weight taped to the base of its tail. 26 27 The electrical stress, designed to elicit a general stress response of a hormonal nature, consisted of electrical shocks (60 volts and 15 milliamperes) administered once every 15 seconds for one-half hour daily through a stainless steel grid, which served as flooring for the stress com— partments. The sedentary animals lived in standard 10 x 8 x 7 inch small-animal cages. The voluntary activity animals were housed in similar cages with attached five-inch wide spontaneous activity drums which were 14 inches in diameter. The rats were sacrificed by ether anesthesia at the end of the 70—day period. The livers were removed, weighed and frozen at —20 degrees Centigrade for five months prior to enzyme assay. Enzyme activity was measured as the amount of inorganic phosphate released by the entire liver per 100 grams of body weight during 15 minutes of incubation with a 0.08 molar substrate solution at a temperature of 37 degrees Centigrade and a pH of 6.5. Mean values and their estimated standard errors for each group were: (1) 68.2_i 3.4, (2) 59.9 i 3.5, (3) 74.5 i 3.8, (1066.1 i 2.8, and (5) 69.9 i 3.7. A one- way fixed-effects model of the analysis of variance detected no significant (p < .05) differences between groups. 28 The power of the F-test was calculated to be .81 with group mean differences of the magnitude observed in this study. Conclusions The results indicate that neither the forced exercise not the electrical stress utilized in this experi— ment has any effect on hepatic glucose-6-phosphatase activity in adult male albino rats. Hepatic glucose-6- phosphatase apparently plays no significant role in adaptation to forced exercise or electrical stress. Recommendations Future research at the molecular level on the nature of adaptation to exercise should be directed along the following lines: 1. Research is needed to determine the specific nutrients metabolized during different types of exercise of various durations. 2. Research is needed to determine if adaptive changes in nutrient metabolism occur as a result of chronic physical training of various intensities. 3. Research on enzyme induction and repression with exercise and training should consider acute mechanisms as well as chronic mechanisms. LITERATURE C ITED 29 LITERATURE CITED Ashmore, J., A. B. Hastings, F. B. Nesbett, and A. E. Renold. "Studies on Carbohydrate Metabolism in Rat Liver Slices VI. Hormonal Factors Influencing G1ucose-6—Phos hatase " Journal of Biological Chemistry. 21 : 77-88, 19561 Ashmore, J. and G. Weber. "The Role of Hepatic Glucose— 6-Phosphatase in the Regulation of Carbohydrate Metabolism," in R. S. Harris, G. E. Marrian, and K. V. Thimann (Eds.), Vitamins and Hormones, Vol. 17. New York: Academic Press, 1959, pp. 914I32. Beaton, J. R. "Note on Liver Enzyme Activities in Thyroid—Fed and in Cold-Exposed Rats,” Canadian Journal of Biochemistry and Physiology. 41: 2041-2044, 1963. Bratton, R. D., S. R. Chowdhury, W. M. Fowler, Jr., G. W. Gardner, and C. M. Pearson. "Effect of Exercise on Serum Enzyme Levels in Untrained Males," Research ggarterly. 33: 182-193, 1962. Cahil, G. F., Jr. "Some Observations on Hypoglycemia in Man," in G. Weber (Ed.), Advances in Enzyme Regulation, Vol. 2. New York: Macmillan, 1964, pp. 137-148. Carlson, L. A. and B. Pernow. "Studies on Blood Lipids During Exercise I. Arterial and Venous Plasma Concentrations of Unesterified Fatty Acids,” The Journal of Laboratory and Clinical Medicine. 53: 833-841, 1959. Chiquoin, A. D. "The Distribution of Glucose—6- Phosphatase in the Liver and Kidney of the Mouse,” Journal of Histochemistry and Cytochemistry. 1: 429—435, 1953- Coniglio, J. G., J. C. Kirschman, and G. W. Hudson. "Hepatic Glycogen, Lipogenesis, and Glucose-6- Phosphatase in X-irradiated and Control Rats,” American Journal of Physiology. 191: 350-354, 1957. Cori, C. F. "Regulation of Enzyme Activity in Muscle During Work," in 0. H. Gaebler (Ed.), Enzymes: Units of Biological Structure and Function. New York: Academic Press, 1956, pp. 573-583. 30 10. 11. 12. 13. 14. 16. 17. 18. 19. 31 Cori, G. T., and C. F. Cori. ”Glucoseu6-Phosphatase of the Liver in Glycogen Storage Disease," The Journal of Biological Chemistry. 199: 661—667, 1952. Edwards, H. T., R. Margaria, and D. B. Dill. "Metabolic Rate, Blood Sugar and Utilization of Carbohydrate,”' American Journal of Physiology. 108: 203-209, 1934. Freedland, R. A. and A. E. Harper. "Metabolic Adap- tations in Higher Animals I. Dietary Effects on Liver Glucose-6-Phosphatase," The Journal of Biological Chemistry. 228: 743-751, 1957. Freedland, R. A. and A. E. Harper. "Metabolic Adapta- tions in Higher Animals II. Changes with Time in the Adaptive Response of G1ucose-6—Phosphatase," TheBJournal of Biological Chemistry. 230: 833-841, 195 . Freedland, R. A. and A. R. Taylor. "Studies on Glucose- 6-Phosphatase and Glutaminase in Rat Liver and Kidney,‘ Biochimica et Biophysica Acta. 92: 567-571, 1964. Geller, E., A. Yuwiler, and S. Schapiro. "Comparative Effects of a Stress and Cortisol Upon Some Enzymic Activities,” Biochimica et Biophysica Acta. 93: 311-315, 1964. Gordon, E. S. ”The Effect of Exercise on Lipid Metabolism," in Exercise and Fitness. Chicago: The Athletic Institute, 1960, pp. 96-109. Gould, M. D. and w. A. Rawlinson. "Biochemical Adaptation as a Response to Exercise 1. Effect of Swimming on the Levels of Lactic Dehydrogenase, Malic Dehydrogenase and Phosphorylase in Muscles of 8, 11, and 15—Week—01d Rats,” Biochemical Journal. 73: A1-AA, 1959. Guild, Warren R. ”Pre—Event Nutrition, With Some Implications for Endurance Athletes," in Exercise and Fitness. Chicago: The Athletic Institute, 1960, pp- 135-137. Harper, A. E. ”Glucose-6-Phosphatase," in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis. New York: Academic Press, 196*, pp. 788-792. 20. 21. 22. 23. 24. 26. 27. IU 00 29. 32 Harper, A. E. "Hormonal Factors Affecting Glucose-6— Phosphatase Activity II. Some Effects of Diet and of Alloxan Diabetes in the Rat," Biochemical Journal. 71: 702-705, 1959. Harper, A. E. and F. G. Young. "Hormonal Factors Affecting Glucose-6-Phosphatase Activity I. Effect of Hypophysectomy and Replacement Therapy in the Rat," Biochemical Journal. 71: 696-701, 1959. Hawkins, R. D., M. A. Ashworth, H. Schachter, and R. E. Haist. ”Effects of Continuous Glucose Infusion on the Glucose-6—Phosphatase Activity of the Liver in Rats,” New England Journal of Medicine. 216: 434-437, 1959. Hensel, H. and G. Hildebrandt. ”Or an Systems in Adaptation: The Muscular System in D. B. Dill (Ed.), Handbook of Physiology, Section 4: Adaptation to the Environment. Baltimore: Williams and Wilkins, 1964, pp. 73‘90- Jacob, F. and J. Monod. "Genetic Regulatory Mechanisms in the Synthesis of Proteins," Journal of Molecular Biology. 3: 318-356, 1961. Kertzer, R. ”Effects of Prolonged Training on Resistance to Radiation—Induced Changes in Male Albino Rats.” Unpublished Ph. D. dissertation, Michigan State University, 1965. Maksud, M., W. Van Huss, W. Heusner, E. Smith, R. Kertzer, and K. Coutts. ”The Effects of Physical Activity and Electrical Stress on Selected Physiological, Anatomical and Biochemical Parameters in Adult Male Rats,” Proceedings, First International Congress of Psychology of Sport, 1965 (in press). Nocenti, M. R. ”Adrenal Cortex," in P. Bard (Ed.), Medical Physiology. 11th ed. St. Louis: C. V. Mosby, 1961, pp. 822-848. Oka, Motoo. "Pharmacological Studies on Flavonoids. Effect of Flavonoids on Adrenaline Action in Carbohydrate Metabolism,” Nippon Yakurigaku Zasshi. 57: 493—500, 1961. Abstracted in Chemical Abstracts. 58: 2755h, 1963. Rowett, H. G. Q. The Rat as a Small Mammal. London: John Murray, 1957, p. 50. 30. 31. 32. 33. 34. 36. 37. 39. 40. 33 Sahasrabudhe, M. B., A. D. Rahalkar, M. K. Nerurkar, and D. K. Mahajan. "Influence of Total Body X— irradiation on the Acid and Alkaline Phosphatases, Glucose-6-Phosphatase, Adenosinetriphosphatase, and Phosphorylase Enzymes in Rats," Journal of Scientific and Industrial Research. 180: 344387 1959. Segal, H. L. "Some Consequences of the 'Non—Competitive' Inhibition by Glucose of Rat Liver Glucose-6- Phosphatase,” Journal of the American Chemical Society. 81: 4047-4050, 1959. Selye, H. Annual Report on Stress. Montreal: ACTA, Inc., 1951, p. 31. Selyeé H. The Stress of Life. New York: McGraw—Hill, 195 . Stadie, William c. ”The Intermediary Metabolism of Fatty Acids,H Physiological Reviews. 25: 395-441, 1945. Turner, C. D. General Endocrinology. Philadelphia: W. B. Saunders Co., 1960, p. 257. Vaughan, D. A., J. P. Hannon, and L. N. Vaughan. ”Interrelations of Diet and Cold Exposure on Selected Liver Glycolytic Enzymes," American Journal of Physiology. 201: 33-36, 1961. Wakabayashi, Y. ”Leberglykogen und Muskeltraining,” Hoppe-Seyler's Zeitschrift fur Physiologische Chemie. 179: 79—82, 1928: Walker, B. S., W. C. Boyd, and I. Asimov. Biochemistry and Human Metabolism. Baltimore: Williams and Wilkins, 1957, p. 153. Weber, G. "Study and Evaluation of Regulation of Enzyme Activity and Synthesis in Mammalian Liver," in G. Weber (Ed.), Advances in Enzyme Regulation, Vol. I. New York: Macmillan, 1963, pp. 1—35. Weber, G., R. L. Singhal, N. B. Stamm, E. A. Fisher, and M. A. Mentendick. "Regulation of Enzymes Involved in Gluconeogenesis," in G. Weber (Ed.), Advances in Enzyme Regulation, Vol. II. New York: Macmillan, 34 41. Willmer, J. S. "Changes in Hepatic Enzyme Levels After Adrenalectomy II. Glucose-6~Phosphatase, Glucose— 6-Phosphate Dehydrogenase, and 6—PhOSphog1uconate Dehydrogenase," Canadian Journal of Biochemistry and Physiology. 38: 1449-1446, 1960. 42 Yudaev, N. A. and M. B. Lebedeva. "0 Roll Kory . Nadpochechnikov v Protessakh Adaptatsii Glukozo-6- fosfatazy Pecheni u Krys," (The Role of the Adrenal Cortex in the Glucose-6-Phosphatase Adaptation Process in Rat Liver), Voprosy Meditsinkoi Khimii. 9: 267-273, 1963. APPENDICES APPENDIX A lest~Retest Reliability of Assay Procedure 36 Test #1 Test #2 Liver Replicate Activity Mean Activity Mean A 1 64.35 , 38.55 A 2 64.35 64 35 47 10 41.40 A 3 64.35 38.55 .B l 64.35 68 55 a 2 68.55 67.15 68.55 71.40 B 3 68 55 77.10 0 1 30.00 30.00 c 2 30.00 30.00 25.65 28.55 C 3 30.00 30 00 D 1 39 75 35.25 D 2 39 75 39.75 44.10 39.70 D 3 39.75 39.75 E 1 48.60 E 2 48 60 50.05 44.10 50.00 E 3 52.95 52.95 F 1 88.20 48.60 F 2 61.80 73 55 52.95 48 55 F 3 70.65 44.10 G 1 42.90 60.00 G 2 42 90 42,90 42.90 48.60 a 3 42 90 42.90 H 1 51 45 51 45 H 2 42.90 45.75 51.45 50.00 H 3 42 90 47.10 _1 1 51.45 55.65 I 2 51.45 50.00 55 65 54 25 1 3 47 10 51.45 *‘U’U‘U T711113 (0.030 1 55 65 51.45 2 55 65 57.10 51 45 52.85 3 60 00 55 65 1 51.45 5 .65 2 51 45 50,00 51 45 51.40 3 47.10 47.10 1 42.90 42.90 2 42.90 40.00 47.10 51.45 3 34 35 64.35 1 55.50 60.90 2 60.00 58.50 56.55 60.90 3 60 00 65.25 1 64.65 56.55 2 55 50 61.60 47.85 53.65 3 64.65 56.55 1 46.20 43 50 2 50.70 50.80 43.50 47.85 3 55.50 56.55 1 76.05 62.70 2 67.20 71.65 71.70 68.70 3 71.70 71 70 1 53.70 49.20 2 49.20 49.25 53.70 50.70 3 44.85 49.20 1 67.20 67.20 2 71.70 67 20 67.20 65.70 3 62 70 62.70 Test #1 Total = 969.60 Test #2 Total = 935,65 Sum (test #1)2 2 54711.17 Sum (test#2)2 2 50430.85 Sum (test #1 x test #2) = 51829.84 - .68 38 sm.so Hmo. mos osm.sa was. smm. -- spa. ma. ofi. ma. o4 m®.:@ :mo. mm: 00:.ma sz. 0mm. :: oma. ma. 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