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DATE DUE DATE DUE DATE DUE 6/01 c:/CIFtC/DateDue.p65-p. 15 ABSTRACT CARBOHYDRATE METABOLISM IN RATS FED ORAL CONTRACEPTIVE STEROIDS BY Angela Kung-Mei Young The effeCt of oral contraceptive steroids on the metabolism of different sugars (glucose, galactose, fructose and ribose) was studied in ll—week old female rats. One group of rats were fed ad libitum a basal grain diet containing the contraceptive steroids norethynodrel, a progestin and mestranol, an estrogen. A second group of rats, control, were pair-fed with the experimental rats re- ceiving only the basal grain diet. Oral sugar tolerance tests were performed by gavage of sugars and collecting blood samples one week and four weeks of steroid treatment 0, 20, 40 and 120 min. after administering the sugar solu— tion to the animals. Steroid treatment did not have any significant effect on fasting blood glucose level after one or four weeks treatment. After one week of steroid treat- ment, no effect of steroid on blood glucose level was found. However, after four weeks of treatment, those rats adminis- tered glucose (p < 0.01) or ribose (p < 0.05) had higher blood glucose levels, than the control group. Angela Kung—Mei Young Urine samples were collected after two weeks of steroid treatment to determine the quantity of urinary sugars after sugar loading. Urine was collected succes- sively for 6 and 18 hours after administering the sugar solution to the animals. No difference in urinary sugar levels was detected at 6 hours, however, at 18 hours, there was a higher urinary glucose level in the treated than the control animals when glucose (p < 0.05) or ribose (p < 0.01) was administered. CARBOHYDRATE METABOLISM IN RATS FED ORAL CONTRACEPTIVE STEROIDS BY Angela Kung—Mei Young A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Human Nutrition and Foods 1970 TO MY PARENTS THIS IS MORE THEIRS THAN MINE ii ACKNOWLEDGMENTS The author wishes to express her sincere thanks and gratitude to Dr. Modesto G. Yang for his encouragement, guidance and advice, and to Drs. Harold D. Hafs, Dorice M. Narins and William W. Wells for their valuable suggestions. The writer extends a special acknowledgment to Mr. Peng Ang for his technical assistance, and also to Dr. John Gill and Mr. Bill Allard for programming the data for computer analysis. The advice of the faculty members in the Department of Human Nutrition and Foods, Drs. Olaf Mickelsen and Satoshi Innami, was sincerely appreciated. The assistance of the graduate students in the Department of Human Nutri- tion and Foods, especially Mr. Yoram Malevski, Mrs. Jenny T. Johnson are gratefully acknowledged. iii TABLE OF CONTENTS Page INTRODUCTION 0 O O O O O O O O O O O I O 1 LITERATURE REVIEW . . . . . . . . . . . . 3 I. Structural Requirement for Activity . . . 3 II. Biological Effect of Progesterone and Estrogen on Glucose Metabolism . . . . 7 A. Progesterone . . . . . . . . . 7 B. Estrogen . . . . . . . . . . 11 C. Progesterone and Estrogen . . . . 14 III. Effect of Progesterone and Estrogen on Insulin Secretion . . . . . . 17 IV. Hormonal and Metabolic Changes which Affect Carbohydrate Metabolism During Oral Contraceptive Therapy . . . . . 20 A. Cortisol . . . . . . . . . . 22 B I Pyruvate O O O 0 O O O O O O 2 4 C. Growth Hormone . . . . . . . . 26 MATERIALS AND METHODS . . . . . . . . . . . 29 RESULTS AND DISCUSSION . . . . . . . . . . 33 I. Blood Glucose . . . . . . . . . . 33 II. Blood Galactose . . . . . . . . . . 42 III. Blood Fructose . . . . . . . . . . 44 IV. Blood Ribose . . . . . . . . . . 47 V. Urinary Glucose, Galactose, Fructose, and Ribose . . . . . . . . . . . 50 SUMMARY 0 O O O O O O O O O O O O O I 5 4 LITERATURE CITED . . . . . . . . . . . . 55 iv APPENDICES . I. II. III. IV. Composition of Basal Grain Diet (in Experimental Design Figures Tables Page . . . 67 %) . . 67 . . . 68 . . . 69 LIST OF TABLES Table Page 1. Summary of insulinogenic and insulin antagonistic properties of gonadal and contraceptive steroids in Rhesus monkeys . . . . . . . . . . . . 6 2. Fasting blood glucose levels after one or four weeks steroid treatment . . . . . 34 3. Blood glucose levels after force feeding glucose, galactose, fructose and ribose, one week or four weeks after steroid treatment . . . . . . . . 37 4. Blood galactose, fructose, and ribose levels after force feeding galactose, fructose, and ribose, respectively, one week or four weeks after steroid treatment . . . . . . . . . . . 45 5. Total urinary glucose excretion after glucose, galactose, fructose and ribose force feeding, after two weeks of steroid treatment . . . . . . . . 51 6. Total urinary galactose, fructose and ribose excretion after force feeding galactose, fructose or ribose respec- tively, after two weeks of steroid treatment . . . . . . . . . . . 53 A-l. Blood glucose levels after force feeding glucose, one week after steroid treat- ment . . . . . . . . . . . . . 71 A-2. Blood glucose levels after force feeding glucose, four weeks after steroid treatment . . . . . . . . . . . 72 A-3. Blood glucose levels after force feeding galactose, one week after steroid treatment . . . . . . . . . . . 73 vi Table A-4. A-ll. Blood glucose levels after force feeding galactose, four weeks after steroid treatment . . . . . . . . . . Blood glucose levels after force feeding fructose, one week after steroid treatment . . . . . . . . . . Blood glucose levels after force feeding fructose, four weeks after steroid treatment . . . . . . . . . . Blood glucose levels after force feeding ribose, one week after steroid treatment . . . . . . . . . . Blood glucose levels after force feeding ribose, four weeks after steroid treatment . . . . . . . . . . Blood galactose levels after force feeding galactose, one week after steroid treatment . . . . . . . . . . Blood galactose levels after force feeding galactose, four weeks after steroid treatment . . . . . . . . . . Blood fructose levels after force feeding fructose, one week after steroid treatment . . . . . . . . . . Blood fructose levels after force feeding fructose, four weeks after treatment . Total urinary glucose excretion after glucose force-feeding, following two weeks of steroid treatment . . . . Total urinary glucose excretion after galactose force-feeding, following two weeks of steroid treatment . . . Total urinary glucose excretion after fructose force-feeding, following two weeks of steroid treatment . . . Total urinary glucose excretion after ribose force-feeding, following two weeks of steroid treatment . . . . vii Page 74 75 76 77 78 79 80 81 82 83 84 85 86 Table A-l7. A-18. A-ZO. Total urinary galactose excretion after galactose force-feeding, following two weeks of steroid treatment Total urinary fructose excretion after fructose force-feeding, following two weeks of steroid treatment Total urinary ribose excretion after ribose force-feeding, following two weeks of steroid treatment . Total urinary glucose excretion after two weeks of steroid treatment viii Page 87 88 89 90 LIST OF FIGURES Figure Page 1. Structural features of pregnene or nor- testosterone (I) and estrogen (II) steroids which are essential for insulinogenic activity . . . . . . . . 5 2. Structures of two of the progestins and the estrogens commonly used in oral contraceptives . . . . . . . . . . 8 3. Blood glucose concentration after force- feeding (a) glucose, (b) galactose, (c) fructose, and (d) ribose after one week of steroid treatment . . . . . 35 4. Blood glucose concentration after force- feeding (a) glucose, (b) galactose, (c) fructose, and (d) ribose after four weeks of steroid treatment . . . . . 36 5. Blood galactose concentration after galactose force-feeding, after one or four weeks of steroid treatment . . . . . 48 6. Blood fructose concentration after fructose force-feeding, after one or four weeks of steroid treatment . . . . . 49 A-l. Body weight records of the control and the treated rats . . . . . . . . . . 69 A-2. Daily food consumption of the treated rats . . 70 ix INTRODUCTION The availability of various synthetic estrogenic compounds enables a wide use in recent years for different treatment such as, contraception, estrogen deficiency symptoms, osteoporosis, and anovulatory menses. The physi- ological actions of the oral contraceptives are due to two hormones which are the synthetic counterparts of natural estrogen and progesterone. There are many studies on the effect of oral contraceptive pills and their action on pituitary and adrenocortical secretions and metabolisms of various nutrients. The changes in carbohydrate, fat, protein, mineral, and various other metabolisms associated with their hormonal action have also been studied. In spite of the many studies, the effect of oral contraceptive drugs on carbohydrate metabolism has not been clarified. Some investigators have reported that the estrogenic part of the oral contraceptive drugs has a diabetogenic effect in both man and experimental animals as indicated by impaired glucose tolerance tests. Several studies have shown that fructose utilization is normal in diabetic man. The rate of fructose disappear- ance following intravenous infusion is only slightly prolonged in diabetic human subjects, and changes in blood lactic, pyruvic, and a-ketoglutaric acid are of the same magnitude as in normal persons. Insulin fails to influence fructose utilization. Thus, the general purpose of this study is to gather information which might help in under- standing the utilization of other monosaccharides besides glucose in oral contraceptive steroid treated subjects. Specifically, this work was undertaken to find out whether: (1) contraceptive steroids have a diabetogenic effect on the metabolism of six carbon sugars such as glucose, fructose and galactose, and a five carbon sugar, ribose, in rats by measuring blood glucose and the respec- tive sugar that was gavaged, and (2) urinary excretion rates of these sugars were affected by steroid treatment. LITERATURE REVI EW I. Structural Requirement for Activity The idea of birth control with progesterone was initiated by Beard in 1897 (6), who postulated that the corpus luteum of ovary secretes progesterone, which is responsible for the inhibition of ovulation during preg- nancy. In 1937, Makepeace gt_al. (59) demonstrated that progesterone will inhibit ovulation in rabbits. Later, Pincus and Chang (76) extended these studies and obtained similar antiovulatory activity with a series of synthetic progestins. In the early 1950's the orally active steroids with the biological prOperties of progesterone was developed by Searle Laboratories and Syntex Laboratories (28). Nor- ethynodrel, the first synthetic oral progestin, was prepared by Frank B. Coltone at the Searle Laboratories in 1952. Later, estrogen was added to the progestin in order to in- crease the effectiveness of contraception, since estrogen can reduce the incidence of Spotting and improve endometrial development (34). In addition to norethynodrel, several other progestational compounds have been used in the oral contraceptive pills. These include: norethindrone, norethindrone acetate, methroxyprogesterone acetate, ethy- nodiol diacetate and chlormadinone acetate. Numerous reports have shown structure-activity re- lationships for pregnene and estrogen derivatives with regard to glucose and insulin metabolism. The structural features of pregnene or nortestosterone and estrogen, i.e., steroids are shown in Fig. l. The presence or absence of the C19 methyl group or of hydroxyl, ethinyl, acetyl, of acetoxy substitution at the C 7 position do not appear to l alter glucose or insulin metabolism. On the other hand, the presence of a partial positive charge at the C5 carbon is common to mestranol and all of the pregnene and nortes- tosterone derivatives which are associated with increased insulin production following glucose stimulation. A double bond at the C6 position appears to be a convenient way of separating the gluconeogenic or hyperglycemic effects of the steroid nucleus from its ability to stimulate the islet cells of the pancreas or sensitize them to glucose stimu- lation. In monkeys, it has been shown that steroid compounds with a relatively positive charge at C5 possess insulino- genic as well as insulin-resistance activities (Table l) (7) which generally neutralize each other when measured in terms of the disposal of intravenously administered glucose. These two activities may be separable by the introduction of unsaturation at the C6 position of the B ring of these steroid compounds. Fig. 1. Structural features of pregnene or nortestosterone (I) and estrogen (II) steroids which are essential for insulinogenic activity. + denotes a partial positive charge; e', electrons. Progesterone, and norethindrone are 4-dehydro-, 3-ketopregnene steroids; norethynodrel is a 5(10) dehydro- 3-keto- steroid; chlormadinine, 4-dehydro—, 6-dehydro-, 6 chloro-, 3-keto-steroid. Mestranol is a 3-methy- ether estrogen (7). pmnsmmma uoc mmcmno on mmnos no commmuomp Um>OHQEH Ho ommmmuocw ll +—+<:I o + o Hoflamuumm Handgun o + o Howuumm o I o Hoflpmuumm mcwmmuumm o I o mumpoommo a0m©0c>num m>Hum>HumU wcoumumoummuuoz oacmmocaasmcflIaoz .U o + + Hocmnummz m>fium>wum© :mmouumm o I + mcoupcflnumuoz o + + Hmuoocxnumuoz mm>flum>flump mconmumoummuuoz o + + mcoumumwmoum m>Hum>flHmU mcmqmon oaumflcomMDGMIcHHSmcw .OAGOmocwHSmcH .m + I + mcocflmeHoHno m>Hum>flHmw mamaqum oaumficommuaMIcoc .oficowocwHSmcH .m mocmumaoa :HadmcH on mmaommmm mmOODHw mocmpmwmwm :AHSmcH .Ahv mmmxcoa mummsm :H mowonmum m>flummomuucoo paw Hmpmcom mo mmfluummonm owumacommucm awasmcfl paw owcmmocwasmcw mo ammEEdm .H mqmde The chemical structure of two commonly used pro- gestins in oral contraceptives are shown in Fig. 2. Nor- ethynodrel, a proqestin which is used in Enovid,1 has a double bond at the 5'th position. This double bond at the 5'th position is biologically significant. In addition to being progestational, it makes norethynodrel estrogenic and devoid of androgenic effects in both animals and men (27, 90). The two estrogens presently used in all oral contra- ceptive preparations are either ethynylestradiol 3-methyl ether, mestranol, or ethynylestradiol (Fig. 2). It has been observed that mestranol behaves in a significantly different way with respect to carbohydrate metabolism than do other estrogens. The administration of mestranol pro- duced a significant increase in the plasma insulin response to glucose and resistance to the hypoglycemic action of exogenous insulin, even though there was no significant alteration in the rate of glucose disposal. II. Biological Effect of Progesterone and Estrogen on Glucose Metabolism A. Progesterone Although the effect of progesterone on the deposi- tion of glycogen and glucose in the endometrial tissue has been established (105), very little is known about the lEnovid- norethynodrel 5.0 mg and mestranol (ethy— nylestradiol 3-methy1 ether) 0.075 mg. PROGESTINS OH OH C‘CH --CECH ' O O Norethynodrel Norethindrone ESTROGENS OH 0H --CECH '“'C5CH HO H3CO Mestranol Ethynylestradiol Fig. 2. Structures of two of the progestins and the estro- gens commonly used in oral contraceptives. systemic effects of progesterone on carbohydrate metabolism (72). Human studies provide good evidence that the pro- gestin components of at least some oral contraceptive agents may alter the glycometabolic effect of synthetic estrogens. Nevertheless, analysis of the data for individual birth control pills shows that not all contraceptive progestins affect carbohydrate tolerance adversely, and the effect may depend on the quantity and chemical structure of the pro- gestin administered and the type of glucose tolerance test employed. A few studies suggested that the 6-dehydro- derivatives of progesterone may actually improve carbohy- drate tolerance by counteracting the diabetogenic effect of the estrogen present in the preparation. Studies by Beck, O'Haver and Bestley (8), found that neither subclinical nor non-diabetic individuals showed consistent changes in oral glucose tolerance over a 2 1/2 month period of treatment with 0.5 mg chlormadinone (6 a-chloro, 6-dehydro, 17-d- acetoxyprogesterone) daily. However, the mean integrated plasma insulin response to glucose was elevated in the non- diabetic subjects, but not in the diabetic subjects, after 2 l/2 months of treatment. Recently, Benjamin and Casper (9) found that the carbohydrate tolerance improved in women with endometrial hyperplasia or carcinoma after treatment with l7-hydroxy- progesterone caproate. They also found that there was a 10 depression in plasma inorganic phosphate level in every case following the glucose administration. They suggested that the hormone may in some way favorably influence total body glucose utilization and metabolism, as reflected by an im- provement glucose tolerance curve. Probably, the improve- ment was not mediated through the adrenal cortex since the urinary excretion of l7-hydroxycorticosteroids and 17- ketosteroids was unchanged by the hormone. In contrast, Schreibman (92) has reported that glucose tolerances deteri- orated in several diabetic subjects treated with the same compound. In Beck's recent work (7), it has been shown that there were no consistent changes in either the mean fasting serum glucose or insulin concentrations in rhesus monkeys afterthree weeks of treatment with progesterone. Although he found the mean serum progesterone level (17 mug/m1) 16 hours after the first progesterone injection was 12 times greater than the control value. However, progesterone treatment enhanced the mean plasma insulin response in monkeys without significant alterations in the glucose dis- appearance rates following Intravenous (I.V.) glucose ad- ministration. Beck (7) also found that progesterone reduced the sensitivity of these animals to the hypoglycemic action of insulin. Thus, it was shown clearly that progesterone enhanced insulin release following glucose stimulation in the rhesus monkey and produce a mild but significant per- ipheral resistance to the hypoglycemic action of insulin. 11 And he suggested that the insulin antagonistic effect of progesterone did not appear to be mediated by growth hormone since fasting serum growth hormone concentrations were not altered significantly by progesterone treatment. Wynn and Doar (126) reported that there was a higher incidence of abnormal oral glucose tolerances in women who received ethynodiol diacetate plus mestranol than mestranol alone. Thus it was indicated that ethynodiol diacetate could enhance the diabetogenic effect of mestranol. B. Estrogen It has been suggested that the estrogen component of the contraceptive drugs is apparently responsible for the changes in carbohydrate metabolism, because of the fact that treatment with a progestational agent alone does not alter glucose tolerance (39, 80). In 1954, Houssay and co-workers (48) clearly de- scribed the effect of estrogenic substances on carbohydrate tolerances. They found that during the first month of estrogen treatment the severity of diabetic symptoms was greater in the estrogen-treated animals than in the control animals. Subsequently, the hyperglycemia and glucosuria were attenuated or suppressed completely in the estrogen- treated animals due to the enhancement of peripheral glucose utilization by estrogens. Nevertheless, treatment with estrogens has not always resulted in improvement in glucose metabolism. In some depancreatized diabetic animals, 12 estrogens either did not affect or increase the severity of diabetes. Javier and associates (49) were probably first to show that glucose tolerance might deteriorate in human sub- jects treated with mestranol alone as well as with mestranol in combination with norethynodrel (35). Beck (7) indicated that subclinical diabetic individuals were much more sub- jected to deterioration of glucose tolerance than non- diabetic individuals following exposure to mestranol. It seems that mestranol produces deterioration of glucose tolerance only if the increased peripheral resistance to the hypoglycemic action of insulin is not compensated by an additional elaboration of insulin (7). Consequently, only those subjects with borderline or limited pancreatic islet insulin reserve will show deterioration of carbohydrate tolerance following exposure to mestranol. This is the reason why the subclinical diabetic individuals are more subjected to deterioration of glucose tolerance following exposure to oral contraceptive agents than non-diabetic individuals (7). Diddle 22 El’ (18) investigated 525 patients who received norethindrone and norethindrone acetate with or without ethinyl estradiol during 24-77 cycles. They found that norethindrone and norethindrone acetate with or without ethinyl estradiol did not produce deleterious metabolic effects when administered continuously for as long as 6 13 years. In contrast, Pyorala and Lampinen (82) reported a significant slowing of glucose disappearance rate in 5 sub- jects treated for 20 days with ethinyl estradiol. They indicated that this estrogen may be an insulin antagonist in man as well as in monkey. However, they also reported that ethinyl estradiol did not appear to produce hyper- glycemia as readily as mestranol in man. Currently, mestranol is extensively employed in estrogen therapy in oral contraceptive because of its marked inhibitory effect on FSH secretion, and its capacity to stimulate the genital tract. DiPaola and co-workers (19) found that only those patients who received mestranol had highly significant percentage of abnormal prednisone glucose tolerance tests (PGTT).l The percentage of diabetic type PGTT was higher in women with diabetic family histories and early menarche (before the twelfth year) than non-diabetic and later menarche. Since it is known that estrogen can cause elevations in plasma protein-bound iodine and plasma cortisol levels without necessarily increasing their functional levels (1, 47, 56, 69), it is possible that these plasma protein ele- vations may have a short-term effect in decreasing glucose tolerance in both animals and humans. Over a longer time l O I I I I I Prednisone administration causes an increase in blood glucose levels and in normal subjects an increase in insulin secretion. 14 period, the effect may act as a stimulus to the beta cells' proliferation, with increased insulin production and im- provement in glucose tolerance (2, 98). C. Progesterone and Estrogen Carbohydrate and insulin metabolisms have been studied more frequently in women taking norethynodrel with mestranol (Enovid) than any other oral contraceptive agent. Relative impairment of oral and intravenous (I.V.) glucose tolerances has been found in certain women receiving estrogen-progestagen combination (35, 79, 106, 107, 108, 126). There is no uniform agreement for the incidence of altered glucose tolerance after estrogen-progestagen treat- ment (40). Peterson, Steel and Coyne (75) found that neither the dosage of the estrogenic agent nor the type of progestin used seemed to affect the incidence of decreased glucose tolerance, when they correlated decreased glucose tolerance with the type of medication the patient received. Posner and co-workers (79) found that the effect of the oral con— traceptives does not continue to progress indefinitely in their patients treated with norethynodrel plus mestranol and this might be due to a certain amount of pancreatic reserve which was taken up by the hormonal contraceptives. Buchler and Warren (12) indicated that administra- tion of diethylstilbestrol or norethynodrel with mestranol may produce within 30 days, an abnormal glucose tolerance 15 curve that mimics that seen in diabetes mellitus. But there was no change in the intravenous (I.V.) glucose tol- erance curve. Thus, they suggested that estrogen might be responsible in delaying gastrointestinal absorption rather than exerting a diabetogenic effect. A similar lack of correlation between I.V. and oral glucose tolerance tests in women during gestation has been reported by Benjamin and Casper (10). Moreover, the work of McIntyre (63) and of Unger (115) clearly demonstrated that oral glucose adminis- tration resulted in a release of intestinal factors which might play a part in regulating the rate of insulin secre— tion. Posner et_al, (78) reported that the I.V. glucose tolerance in women treated with Enovid1 or Ovulen2 was different, when the observation was done early in the treat- ment. There was a significant tendency for the K value3 to decline in women taking Enovid, but not for those taking Ovulen. They suggested that this might be due to the pro- gestin in the drug, since the progestin in Enovid is more estrogenic than progestin in Ovulen. Similarly, Puchulu et_al. (74) have shown that 13% to 66.7% of women in their lEnovid-norethynodrel 5.0 mg and mestranol (ethynyl estradiol 3-methyl ether) 0.075 mg. 2Ovulen-ethynodiol diacetate 1.0 mg and mestranol 0.1 mg. 3K values--the index of tolerance or the percentage decrease per minute of blood glucose over 10-60 min. follow- ing the rapid I.V. injection of 25 gm. of glucose (89). stt (0. me: ace to 88‘ It ce] ca: 911 se< fol Ha] pat dia tre tol. Cont meat Chan °0ntI i100d insulj 16 study develOped an abnormal oral glucose tolerance test (O.G.T.T.) or a cortisone-sensitized O.G.T.T.1 after treat- ment with a combination of mestranol and norethisterone acetate for 20 days. The corresponding figures were 6.5% to 11.1% in women treated with a combination of ethinyl estradiol and noresthisterone acetate for the same period. It indicated that the estrogen component of the contra- ceptive drugs is apparently responsible for the changes in carbohydrate metabolism. Pyorala gt 31. (82) indicated that glucose tolerance decreased during the estrogen phase of a sequential treatment and then slightly improved during the following phase of combined estrogen-progesterone treatment. Halling, Michals, and Paulsen (42) found that there was a significant impaired carbohydrate tolerance in their patients after a long-term treatment with ethynodiol diacetate-mestranol. The fasting glucose levels during treatment were unaffected, and the impaired carbohydrate tolerance reversed to normal when the treatment was dis- continued. Later, Wynn and Doar (127) reported that the mean fasting plasma glucose level was not significantly changed by oral contraceptive therapy both in a group which contained 67 women who were tested before and again while receiving oral contraceptive therapy, and another group 1 I I I I I I Cortisone administration causes an increase in blood glucose levels and in normal subjects an increase in insulin secretion. 17 which contained 24 women who were tested during therapy and after withdrawing the drugs. There was a significant im- pairment of oral and I.V. glucose tolerance during therapy and it improved after oral contraceptive therapy was dis- continued. Starup gt_al. (112) treated 27 non-diabetic women with a daily dose of 5 mg of megestrol acetate and 0.1 mg of mestranol for 12 months in their study. They reported that there were no significant changes in the fasting blood sugar, the K-value in the I.V. glucose tolerance test, the fasting serum insulin and the response in serum insulin after an I.V. glucose stimulus, which were tested before treatment started, after 12 months of treatment and one month after withdrawal of treatment. The diabetic woman in their study showed a significant increase in fasting blood glucose and a slight decrease in the K-value during the treatment period. Her insulin level was not changed during treatment. Thus they concluded that treatment with megestrol acetate and mestranol has no effect on the carbohydrate metabolism in normal fertile women with no family history of diabetes. III. Effect of Progesterone and Estrogen on Insulin Secretion There have been few studies on plasma insulin levels during oral glucose tolerance tests in subjects receiving oral contraceptives. Most (49, 106, 108, 112), but not all 18 (107, 112), previous workers have found that the fasting plasma insulin levels are unchanged during oral contracep- tive therapy. Spellacy £3 31. (102, 106, 107, 108) ob- served higher plasma insulin levels in patients during treatment than pretherapy levels during I.V. glucose toler- ance tests. This observation was confirmed by Wynn and Doar in 1969 (127). On the other hand, Starup EE.E$3 (112) found that there was no change in insulin level after steroid treatment. Javier 33 21. (49) found higher plasma levels early in treatment but, with prolonged therapy in- sulin levels tended to be lower in some subjects as glucose tolerance and insulin levels were elevated soon after con- traceptive steroids were used. Thus when the insulin level was high enough it might have brought the blood glucose level back to its pre-treatment range after several months of use. But with prolonged usage the glucose levels in some subjects would rise again, and he suggested that this inci- dence of decompensation with the resultant abnormal glucose tolerance curve might be due to the alteration in the insulin secretion mechanism, thus delaying the insulin re- lease and perhaps alter the type of insulin that is re- leased (proinsulin) (114). Javier, Gershberg and Hulse (49) found that insulin secretion tended to increase as the blood glucose level increased early in oral contraceptive treatment. They suggested that the failure of the pancreas to response to hyperglycemia might cause the decrease of l9 insulin secretion in certain women as glucose tolerance decreased with prolonged treatment. They indicated that the estrogen in the contraceptives is the major cause of these metabolic changes, though the progestin might exert an additional effect by being converted in the body to sub- stances having estrogenic activity (73) . The pathway by which estrogen impairs glucose tolerance are not clear yet. From what is known of their actions Pyorala gt g. (82) and Kitay (54) suggested that it could impair carbohydrate utilization by stimulating growth hormone (GH) and adreno- Estrogens could also act by modi- corticotropic secretion. fying liver function (55, 68), increase uterine and liver glycogen (51, 117) and influence the enzymes involved in the disPosal of glucose (5, 64). Recently, Kalkhoff, Kim and Stoddard (52) indicated that an acquired form of subclinical diabetes mellitus was found among women receiving mestranol- containing oral contraceptive agents. It was also shown that there was an impaired prednisolone glucose tolerance in these subjects and which was associated with a defective plasma insulin response. It was also clearly indicated in their study that 8 1/2 hours of prednisolone administration had a suppressive effect on the plasma insulin response to oral glucose in these subjects. Whether, mestranol has a direct deleterious effect on pancreatic islet secretion of insulin is still unknown, although there was a normal re- sponse of insulin secretion by prednisolone administration 20 intMaMmg-treated group after the contraceptive agent was wflmMamn However, Gold and his co-workers (38) found that bkmdghmose rose significantly in overt diabetics treated IdflIOWflen as well as with ethynodiol diacetate alone for Ovulen-induced hyperglycemia, demonstrable both one month. dmfingihsting and following introduction of glucose loads, (a) increased concentrations of was not accompanied by: (b) increased resistance to ad- endogenous plasma insulin, They interpreted this to mean a loss ministered insulin. of beta-cell sensitivity of "indifference" to fasting hyperglycemia while still retaining the capacity to re- It seemed sponse to acute increments in blood glucose. that in diabetics the insulin secretory response, once ini- tiated, assumes a fixed, somewhat autonomous pattern no longer related either to the magnitude or the duration of the hyperglycemic stimulus (38). IV. Hormonal and Metabolic Changes which Affect Carbohydrate Metabolism During Oral Contraceptive Therapy IBlevated circulating levels of plasma cortisol (65, 85), GH (110) and pyruvate (128) have 81) , thyroxine (26, been noted during oral contraceptive therapy, and the estrogenic component is thought to be responsible. Some in- vestigations have shown that estrogens have a direct action (a) increasing hormones on the peripheral insulin by: antagonistic to insulin (ACTH, corticoids, or human growth 21 hormone) (17, 27, 32, 41, 56, 65, 69, 77, 87), (b) increas- hm hIplasma antagonists (synalbumine, nonesterified fatty 11), and (0) increasing the combination acids, etc.) (2, 3, 46, 57, 65). The pos- of insulin with plasma protein (30, sibility exists that one or more of these may be responsible for the observed carbohydrate metabolic changes. The ele- vated plasma levels of cortisol and thyroxine are due to increased levels of the respective binding proteins, and the free non-protein bound hormone concentrations are prob- ably unchanged. Nevertheless, the possibility exists that the protein bound hormone, in general thought to be bio- logically inactive (62), may cause diminished carbohydrate tolerance in certain subjects taking oral contraceptives. It has been demonstrated by Walass (117) in adult rats that estrogens could affect carbohydrate metabolism by elevating glycogen formation in liver and which in turn may cause the increased blood sugar level. He suggested that this effect may be due to an upset of the hormonal balance in the organism. Fry, Miller and Long (33) have indicated that the estrogenic effect on liver glycogen content is probably transmitted through the pituitary—adrenal system, since they were unable to find any glycogen formation in the liver after estrogen injection in hypophysectomized or adrenalectomized rats. Walass (117), also concluded that the: changes in carbohydrate metabolism after estrogen admin- is trations may be explained by an increased anterior pituitary and adrenocortical secretion. ._____ .....- pr _; 22 A. Cortisol It is well known that glucocorticoids promote hyper- glycemia by augmenting hepatic glucose production (122) and impairing peripheral glucose utilization (83). And it was found that estrogens greatly potentiate this hormonal action because of the elevation of plasma free cortisol (71, 77) and an increased cortisol secretory rate (53). Transcortin, a plasma protein with high affinity for cortisol, and corticosterone, had been shown to be elevated during pregnancy and following administration of estrogens. In the same time, there was an increase of plasma cortisol (87, 97, 118). A more clear picture was given by Sandberg £5 21. (88) in 1963. They found that administration of adequate amounts of estrogens produced increased concentra- tions of transcortin within 3 to 7 days, followed by a rise in the levels of the l7-hydroxycorticosteroids (l7-HOCS). The levels declined to normal within 7 to 10 days after cessation of estrogen treatment. It has been indicated that estrogen is the only steroid which can cause the ele- vation of plasma transcortin level. Sandberg gt; a_l_._. (88) also postulated two hypotheses from indirect evidences that (a) biologically inactive, transcortin-bound cortisol is: 66, 67, 74, 119). and (b) unavailable for catabolism (15, The first hypothesis was supported by the observation that transcortin prevented cortisol-induced glycogen deposition The in adrenalectomized mice treated with cortisol (96). 23 second hypothesis was clarified by the findings that less cortisol was metabolized in the presence of plasma from pregnant women or subjects given estrogens, than was ob- served in the presence of normal plasma or no plasma at all (88). In 1965, Leach and Margulis (57) investigated the inhibition of adrenocortical responsiveness during progestin therapy. They found that there was an increased value of l7-hydroxysteroid excretion which was obtained two months following cessation of therapy. It indicated that there was a definite suppression of responsiveness to metyraponel during treatment with norethindrone acetate, 2.5 mg, and ethynyl estradiol, 0.05 mg. Since the response to adminis- tered ACTH was not affected they suggested that the changes due to the estrogenic activity appeared to be related more to inhibition of pituitary responsiveness than to altered adrenal cortical activity. In another study a reduced level of ACTH was noted in some of the Enovid users with reduced glucose tolerance. These findings suggested a direct action of steroids especially estrogen on adrenocortical secretion (116). Kitay (54) reported that spayed female rats had lower plasma steroid concentrations at rest and after stress or after ACTH injection when compared with intact controls. Also there was an impaired steroid turnover in spayed female 1A compound which can stimulate the secretion of Pituitary hormones. 24 ratsvdth prolonged steroid half-life and decreased liver activity. In his in_vitro study he demonstrated that es- tradiol stimulated steroid production by increasing adrenal steroidogenesis. He suggested that ACTH secretion is modi- fied by the gonadal hormones without mediation by the L r adrenal glands. Mills gt_al. (67) found that the half-time for disappearance of plasma hydrocortisone was considerably i prolonged by administration of estrogen. They also found i that administration of estrogen had two distinct effects on hydrocortisone metabolism. The effects are: (a) a marked rise in the level of protein-bound steroid, and (b) an increased total pool, out of proportion to the rise in the level of plasma hydrocortisone and simultaneously a decreased excretion of metabolites. They suggested that this probably represents protection from destruction by the liver by means of greater protein bindings. B. Pyruvate It has been noted that both the fasting blood pyruvate level and/or the maximum blood pyruvate increment above the fasting level can be increased in certain women receiving oral contraceptives. These changes resemble the q changes found in subjects receiving glucocorticoid drug (128). JBut it is not known whether the increased blood pyruvate levels were due to an increased rate of pyruvate production and/or impaired pyruvate removal by the estrogen and/or progestagen, although some findings indicated that 25 it is due to increased rates of production rather than impaired removal of this metabolite. It has been suggested thattimse increased rates of production were mainly due to the estrogen component of the drug which may increase amounts of glucose passing down the glycolytic pathway to pyruvate. In a cross-sectional study by Wynn and Doar in 1966 (128), it was found that there were impaired oral and I.V. glucose tolerances, elevated fasting plasma non- esterified fatty acids levels, and elevated venous blood pyruvate levels, both before and after oral or intravenous glucose administration in women taking oral contraceptives. Their recent study (127) confirmed the majority of these findings with the exception that mean plasma non-esterified fatty acids levels before and after glucose administration were not affected by oral contraceptive therapy. Most of these metabolic changes were reversed after oral contra- ceptive therapy had been discontinued. Elevation of blood pyruvate levels were found in six women treated with estro- gen alone (127). It was not determined with the progestagen treatmentl Furthermore, it is not known whether these changes result from increased levels of other circulating hormones such as cortisol, growth hormone and thyroxine. Some studies suggested that the fasting blood pyruvate level is neither increased in thyrotoxicosis (22) , nor in control subjects after L-triiodothyronine adminis- tration (111). No elevation of blood pyruvate levels could ”r "I: 26 be found above control values during oral and I.V. glucose tolerance tests in acromegalic subjects (22), nor during I.V. glucose tolerance tests performed after administration of human growth hormone to control subjects (22). The ele- vated blood pyruvate levels during oral contraceptive ther- apy resembled those found during glucocorticoid therapy (44) and also those found in obesity (24, 25). It is possible that the biological inactive protein, transcortin (62), which is thought to be involved in the elevation of plasma cortisol levels during oral contraceptive therapy may have biological activity in certain tissues, such as the liver, and thus may be responsible for the changes of glucose tolerance and blood pyruvate levels found during oral contraceptive treatment. Since the plasma cortisol levels are normal in obesity and the cortisol production rate is often increased (93), the hepatic clearance of cortisol must therefore be increased and it is possible that this may be responsible for the impaired oral glucose tolerance and elevated blood pyruvate levels commonly found in this condition. C. Growth Hormone The effect of estrogens on growth hormone secretion has not been clarified. Early work by Young (129) demon- strated GH has diabetogenic effect. Other investigations have shown that estrogens can cause a marked increase in pituitary secretion and plasma levels of growth hormone in 27 human. This effect may be due to enhanced sensitivity of the growth hormone releasing mechanisms by estrogens. A similar effect was found when the various endogenous estro- gen production associated with differences in sex, age, and time of the menstrual cycle were correlated with plasma GH levels (104). Frantz and Rabkin (32) found that the eleva- tion of plasma human growth hormone (HGH) in response to insulin was not affected by estrogen treatment, but the plasma HGH level of the fasting subject before insulin in- jection was elevated in the estrogen treated group. They indicated that increased pituitary secretion, rather than decreased peripheral degradation, was responsible for the elevated HGH levels seen after estrogen administration. In- creased pituitary secretion may be due to the possibility that estrogen in some way increases intracellular glucose requirement, not entirely reflected by the blood sugar, and that this in turn acted as the stimulus for growth hormone release. Spellacy et‘al. (102, 109) found that there was a significant elevation of the HGH values in fasting women at rest produced by the oral contraceptive. In other studies, they confirmed that both blood glucose and plasma human growth hormone levels were elevated in drug treated group after three months (110) and twelve months (103) of treat- ment. They suggested that the estrogenic action of the tab- lets caused an elevation in the circulating HGH levels. The 28 HGH can then antagonize insulin action and this results in an elevation of blood glucose level, and a resistance to an insulin-induced hypoglycemia. MATERIALS AND METHODS One hundred and four, 10 weeks old female Sprague- Dawley rats (Spartan Research Animals, Inc., Williamston, Mich.), having an average body weight of 220 grams (196 - 255) were used in this experiment. All the rats were housed individually in metabolism cages, and maintained at a con- stant temperature of 27°C with 12 hours each of light and darkness. All the animals were fed ad_libitum a grain diet (14) (Appendix 1) for one week. At this time the rats were paired according to body weight, and one of each pair was fed the grain diet plus norethynodrel and mestranol ad libitum. This diet was prepared by dissolving the two steroids in 70% ethyl alcohol and mixing the solution with the grain diet with a Hobart mixer. The diet contained 0.0275 mg mestranol and 1.83 mg norethynodrel per kg diet and provided approximately similar dosage used by women (0.1 mg norethynodrel and 0.0015 mg mestranol per kg per day). The dosage used in the study was based on several other experiments wherein feed containing the steroids had been.measured (61). The amount of diet containing steroids consumed by individual rats was measured every afternoon at 2:00 o'clock 29 30 throughout the experiment. For this study, the average food consumption was 15 g per day (Appendix Fig. 2). Since their average body weight (Appendix Fig. l) was 250 g, the average mestranol and norethynodrel consumed was 0.0017, 0.11 mg per kg body weight. Diet spillage was measured and recorded, if there were any, and subtracted from the total daily food intake. The data were then used as a basis for the individual pair-feeding of the remaining 52 control rats with the basal diet only. At the end of the first, second and fourth week of the pair feeding, all rats were weighed and then fasted from 5:00 pm to 8:00 am. The rats were then lightly anesthetized with ether and gavaged with one of four different sugars (300 mg in 1 ml of water/100 g body weight). During the first and fourth week sugar tolerance tests were obtained for four pairs of rats for each sugar at each time interval of 20, 40 and 120 minutes (Appendix II) after gavage by ob- taining blood samples with a cardiac puncture technique (13). Another four pairs of rats were bled immediately after fasting, and considered as 0 time interval in the sugar tolerance test. The other gavage at the end of the second week was done in order to measure urinary excretion rate of the four sugars by collecting urine for 6 and the subsequent 18 Jhours. Urine was collected by metabolism funnels into flasks containing toluene to prevent bacterial growth. 31 Similar collection of urine were made for the four pairs of rats used for the zero time blood collection. These rats were not force-fed any sugar. Serum and urine samples were kept in the freezer until analysis. Before analyzing for the sugars in the serum 1.8% Ba(OH)2 and 2% ZnSO4 were used to deproteinize and neutralize the serum respectively (70, 99, 100). The ratio for the serum, the two reagents, and water for de- proteinization was l:5:5:9. This mixture was centrifuged and the supernatant used for sugar determinations. Urine was treated by a modified method of Saloman and Janson (86) before analysis. Amberlite (Rohn and Jass Co.) Ion Exchange resins, IR-120 and IR-45 were washed with distilled water, dried in air at room temperature and com- bined in a 1-1 mixture. Three ml of urine were added to 3.0 g of resin in a small beaker. After standing for 30 min., the moist resin mass was transferred to a glass funnel and the deionized urine was removed by suction into a test tube within a filter flask. The deproteinized sera and urine eluates were analyzed for glucose in addition to the appropriate sugar. Glucose was determined by the method of Washka 33 'al, (120) using "GLUCOSTAT." Galactose was determined by a similar enzymatic method "GALACTOSTAT" ( 4). Fructose *was determined by using the method of Roe 33 21. (84). And ribose was determined according to the method of Dische and Borenfreund (20). 32 All data were analyzed by analysis of variance (21). Since a few rats died during blood collection due to hemorrhage caused by uncontrollable accidental puncturing of major blood vessels around the heart, and in order to have the same number of observations needed for the com- puter analysis, only half the original number was used for statistical analysis. Data used for statistical analysis were chosen randomly and analysis of variance (21) performed by using the University computer. Dr. John Gill (Dairy Department) and Mr. Bill Allard (Computer Center) helped in programming the data for the statistical analysis. RESULTS AND DISCUSSION I. Blood Glucose Fasting or 0-time blood glucose levels were not significantly higher in the experimental group than in the control group either after one or four weeks of steroid treatment (Table 2). This finding has been reported by many other workers (42, 78, 79, 108, 112, 127) for women receiving oral contraceptives. Other reports however indi- cated an opposite effect in that patients receiving oral contraceptive steroids had increased fasting blood glucose levels (35, 50). The overall blood glucose level was sig— nificantly higher in the treated group than in the control group, taking into consideration all sugars that were force fed and the two time intervals used for the tolerance tests (p < 0.02) (Figs. 3, 4). However, after one week of treat- ment, the statistical analysis showed that there was no significant difference in blood glucose levels between the control group and the experimental group regardless of *which sugar solution was force fed to the animals (Table 3). IAlso there was no significant difference between these two (groups for the individual blood glucose level after differ- ent.sugar tolerance test at this time. After four weeks of 33 34 .cooHn mo cofluomaaoo cu Howum meson ma Umummm mums mameflcm e.m m.n m.ma m.na .o.m m.moa m.mm m.mm s.sm new: m.moa H.mm v.wm m.mm e s.Hm «.moa m.om ~.sm m N.Hoa m.mm m.HmH H.moa m m.maa m.voa o.sm s.mm a mumm .Hudxm mumm Houucoo mumm .Hudxm mumm Houucoo scamm>mwmno ucmEummHB paoumum mxmmz Mach ucwEumeB Ufloumum xmwz wco E Assumm HE ooa\mev .ucwfiuomuu cfloumum mxmmz Meow no wco Hmumm mam>ma omoosam UOOHQ Hmceummm .m mummy mg/100 ml serum mg/100 ml serum 35 225'r a. 225T b. Control A ------ Exptl. 205» " 2051 I \ I \ 185‘? I, \\ 1850 l \ I 5 1654- H155. : ,/\\ 145., ,' 'g 145. r \ \ \ o ‘ O 125. ~q 125. U‘ e 105. 1050 i / 85« 85. o 20 40 126 0 2'0 4‘0 131' time (min) time (min) 2251' c. 225? d. 205 « 2054* 185 4» 185(- 165.. 5,165 Q) ~ I!) 1451 I, "E" 145. O O 1sz 1125 105l> 105‘ / 85. 85« I’ 4 i —i T t <1 1 0 20 40 120 0 20 40 120 time (min) time (min) Fig. 3. Blood glucose concentration after force-feeding (a) glucose, (b) galactose, 1c) fructose, and (d) ribose (after one week of steroid treatment). 36 Control Exptl. I 2401- a, I‘ it ' I 220.. I ‘ 220 I. I I I 200-» , I 2001- , i 180-» ’ \ 180 I \ 5 5 I: 8 1600- m160 ‘ m H H E E 140.. c,140 .. O O 2 a E. 120.»I s 100 so ' T : : I T o 20 40 120 0 time (min) C. 200.» 200 - mg/100 ml serum Fig. 120 (min) g ‘I 40 time (min) 120 4. (b) galactose, weeks of steroid treatment). 120 time (min) Blood glucose concentration after force-feeding (a) glucose, (c) fructose, and (d) ribose (after four 37 .Amo.o v mo Hmucmeflummxm How can» MoBOH advancemecmflm we Houucoo MOM :mmz .AHo.o v my Hmucmeflummxm coco Hm3oH mHuCMOHMflcmHm we Homecoo How cmmzm .ucmfimB moon 0 ooa\c0Hp5H0m nouns no He H cw swoon comm m0 m8 comm H.m + 0.0m“ m.omH s.HNH H.5NH s.mau m.voa «.mw m.nom v.mn m.em N.m~H mmmonflm o.H~H ~.mmH m.mmH m.~mn. m.sHH s.HmH m.nma m.mHH o.mHH m.mm m.mmH m.mmH mmouosnm xmmz m.emn_ H.e H m.HmH o.mmH H.sHH m.emu r m.mHH m.nm 0.0m H.mm m.moa m.mHH omouomamo mbom m.m H. «.mmu m.msHH m.omn o.RNH «.msu m.oma e.hm H.mvm m.mm m.noa m.mna momoosao o.HNH ~.mmu s.o~H s.smH s.o~H o.mmH w.HHH m.ama m.ova «.moa m.nma m.vma mmonem ~.m H s.m~+ o.m~H H.mHH m.s~H m.o~H v.mmH m.mwa N.oma o.oma o.wha m.mva emouosum xmmz m.H~H o.eou «.mau «.mmu «.mmH. m.mmu v mzo m.om «.mma H.mea m.aoa m.mma m.moa omOpomHmo H.5HH s.sHH ~.mmu «.mmn m.o~H m.smn n.mma m.mma m.mam m.mma e.mea n.0na omoosao oma ow om omH ow om . mcflpommImouom mcflpmmm mouom ummsm Hmumm Caz Hmmsw mumm .Hudxm mumm Houucoo AGOAumfl>mo pumpcmum H_mmmm Ium>m .Esuom He ooa\mec .ucoEummuu pfloumum Hmumm memos “now no x003 moo .mmonflu one mmouosum .mmOpomamm .mmoosam mascomm wouow umumm maw>wa mmoooam cooam .m flames H 38 steroid treatment, the blood glucose levels in those ani- mals which were force fed glucose (p < 0.01) or ribose (p < 0.05) were significantly higher in the treated group than in the control group. There was also a significantly higher overall blood glucose level in the treated group than the control group after four weeks of treatment (p < 0.05). In the present study, therefore, the effect of steroids on glucose and ribose metabolism can only be found after four weeks of steroid treatment (Table 3). The impaired glucose tolerance in rats after four weeks of steroid treatment supports many previous studies using human subjects (35, 79, 106, 107, 108, 121). Results of this study showed that oral contraceptive steroids fed for one or four weeks did not have a significant effect on galactose and fructose metabolisms, in terms of blood glucose level (Fig. 3b,c and Fig. 4b,c). The effect of steroids on ribose metabolism, in terms of blood glucose level, was obvious only after four weeks of steroid treat- ment (Fig. 4d), since there was a significantly higher blood glucose level (p < 0.05) in the experimental group compared to the control group. The blood glucose level of those animals treated with steroids for one week and force fed fructose was lower than that found in control rats (Fig. 3ek But after four weeks of steroid treatment, the condition ‘was reversed (Fig. 4c). The blood glucose level of those Ianimals treated for one week with steroids and force fed 39 galactose solution was higher than that of control (Fig. 3b). After four weeks of steroid treatment, the blood glucose level rose even higher in the experimental group (Fig. 4b); the mean difference was larger at this time compared to the mean difference after one week steroid treatment (Table 3). Thus it is clear that four weeks of steroid treatment has a significant effect on glucose and ribose metabolism by raising the blood glucose level after the administration of these sugars (Fig. 4a,d). But it can be concluded that contraceptive steroids might have an effect on galactose and fructose metabolism if treatment were extended. This pos- sibility will be discussed later. Elevated circulating levels of plasma cortisol, thyroxine, growth hormone, pyruvate or hormones antagonistic to insulin, increased in plasma antagonists and insulin binding protein (2, 3, ll, 17, 27, 30, 32, 41, 46, 56, 57, 65, 69, 77, 81, 85, 87, 110, 128) have been suggested as possible mechanisms impairing glucose tolerances after con- traceptive steroid treatment. That estrogens may cause a decrease in the response of the beta-cell of the pancreas to glucose loads by the formation of an insulin—binding pro- tein, transcortin, has been suggested by Slaunwhite, Sand- berg and Wallace (87, 88, 89, 96, 97, 118, 119), as the most likely mechanism of all those proposed for the impaired tolerance. 40 Ribose administration or infusion, as well as glu- cose caused secretions of insulin according to Goetz 23 31. (37, 43) and Steinberg et 31. (103). It is possible that estrogen may have the same effect on ribose as on glucose by decreasing insulin availability or activity. Mechanisms for the increased insulin secretion after administration or infusion of glucose were suggested to be a direct stimu- lation of the beta-cell, Specified gastrointestinal factor, and through the mediation of the liver (11, 37, 60). How- ever, for ribose only the latter mechanism could be in- volved (37). On the other hand, some workers have suggested that estrogen can modify liver function (55, 68). This may help explain why there was a higher glucose level in steroid treated group after ribose loading. It has been reported (37) that galactose infusion can cause insulin secretion as glucose does. Thus, it is possible that oral contraceptive steroids may have the same effect on galactose metabolism as on ribose metabolism after a longer period of steroid treatment. That fructose infusion can not cause insulin secre- tion as galactose, glucose and ribose has also been reported by Goetz et a1. (37). This might apply to the finding in this study since there was a lower blood glucose level in treated group than in control group after fructose loading, and an Opposite result when the other sugars were used. 41 The rise in blood glucose level following oral ad- ministration of fructose, galactose or ribose indicated that part of the sugars was converted to glucose (Fig. 3 and 4). Blood glucose levels are significantly different among the four sugars tested (p < 0.01). This might be due to the different metabolic conversion rates of these sugars to glucose. Fructose is easily converted to glucose both in intestinal mucosa and liver through the reversed glycolytic pathway (123). The conversion of galactose to glucose is through the pathway of uridine diphosphate galactose to uridine diphosphate glucose (124). The conversion of ribose to glucose is through the reversed phosphogluconate oxida- tive pathway (125). It can be interpreted that the oral contraceptive steroids may have the same effect on galac- tose, fructose and ribose metabolism as on glucose metabo- lism in terms of the effect on blood glucose level, since these three sugars can be converted to glucose through the above mentioned metabolic pathways. From Figs. 3 and 4, it can be seen that the highest blood glucose level when glucose or ribose was force fed to the animals was 20 min. after administration. This occurred both in control and experimental groups, after one or four weeks of steroid treatment (Figs. 3a,d; 4a,d). When galac- tose or fructose was force fed to the animals, the highest blood glucose level was after 40 min (Fig. 3b,e), both in control and experimental groups but only after one week of 42 steroid treatment. After four weeks of steroid treatment, the highest blood glucose level was after 20 min. in the control group and 120 min. in the experimental group (Fig. 4b,c). Based on blood glucose peak time, it seems that oral contraceptive steroids have an effect on the conversion rate of galactose or fructose to glucose. Weinstein and Roe (121) have reported that there was a maximum of 47% rise in the total blood sugar at the end of fructose infusion. They pointed out that there was a rapid metabolic conversion of fructose to glucose. In the present experiment, the blood glucose level was higher when measured 120 min. after fructose force feeding than after force feeding galactose or ribose (Figs. 3 and 4). It seems that after a prolonged steroid treatment the effect of this drug on fructose metabolism in terms of raising blood glucose level may be due to decreasing conversion rate from fructose to glucose. The possible mechanism in- volved might be the binding of estrogen with enzymes which are involved in the conversion reaction of fructose to glucose. This assumption might also apply to galactose, since a similar blood sugar picture has been found in those animals force fed galactose. II. Blood Galactose Although galactosemia has been observed only in man, analogous physiologic changes can be induced in ani- mals by feeding diet high in galactose. It has been shown 43 that when rats are placed on 30% galactose diets, cataracts can be developed within a period of 14 to 21 days (94). This is due to the accumulation of galactose-l-P, which inhibits phosphoglucomutase and causes a decrease in glu- cose-1,6-P2 (95, 36). The physiologic significance of this interference is uncertain but it may be a factor contrib— uting to the hypoglycemia which occurs in galactosemic patients when they ingest galactose or lactose. The experi- mental observations of Foa (34) suggested that the hypo- glycemia in the galactosemic patients when they ingest galactose may be due to the stimulation of insulin release from the pancreas by the elevated blood galactose. In his experiment, 1.75 g galactose/kg body weight given orally to the galactosemic patient produced on an average 225 mg galactose/100 ml blood two hours after the dosing, at this time, the blood glucose concentration was 48 mg/100 ml blood. In the present experiment, the amount of galactose given orally to the animals was almost two times that used by Foe (31), but the average blood galactose level was 260 mg and the blood glucose level was almost near fasting level two hours after dosing compare to his values. This may indicate that the animals used in the present work were able to utilize galactose efficiently whether or not the possible effect of steroid treatment was excluded. From the present data, there appear to be no im- paired galactose tolerance with steroid treated. The 44 experimental group has a lower blood galactose level, but a higher blood glucose level, than in the control group, after one or four weeks of steroid treatment (Table 4). This suggested that the steroids may not have an effect on galactose metabolism directly. But it has the effect on blood glucose level since galactose can be converted to glucose. Present results further indicated that after four weeks of steroid treatment, there was probably a decreased conversion rate of galactose to glucose (Fig. 5), since there was an increased blood galactose level, and a de— creased blood glucose level (Tables 3 and 4). It can not be determined from the present experiment how long a period of steroid treatment is required before galactose tolerance is impaired. It may be that a longer period of steroid treatment is required before galactose tolerance is af- fected than is required before glucose tolerance is impaired. III. Blood Fructose The disturbances of fructose utilization have been observed. The possible causes may be a deficiency of aldolase in the liver (45) and the benign or idiopathic fructosuria, which involves deficiency of fructokinase in the liver (91). Two pathways are recognized by which fructose can be converted to glucose. One begins with fructose-l-P, formed by the specific fructokinase and pro- ceeds through the triose phosphates to fructose-1,6-P2, 45 .usmHOB upon 0 ooa\coHuSHom Hmum3 mo HE H ca ummdm comm no 05 com H o o o o o o o o amonam m.mH ~.dH m.nH m.¢H H.1H m.nH mews H.mH m.mH m.mH o m.ma m.~H s.oa o amouosum W. I I. I. I. I. I moon m.mma+ m.moa+ s.woa+ m.H~H+ s.mm~+ «.maa+ e.mem m.sfie m.Hm~ o H.mo~ o.som 0.0mm o 0mou0mHmu o o o o o o o o o amonfim G.HH N.NH_ o.HH m.~H o.~H m.nH gems m.sa m.~a ~.HH o m.oa H.ea m.ma o amouosumn W I I I. .I I. I mzo m.vm+ o.om+ H.RHH+ o.mma+ m.ms+ o.am+ m.ss~ m.ms~ m.aH~ o s.mam m.mme o.na~ o mmouomamo.r ems as om massage own as om assumes mcflomwm mouom Hmmsm kum< .cwz mama .Humxm weapomh couch ummsm mu.mm HOHHGOU AcofiumH>mo photomum_H mommuo>m .Eduom He ooa\mec paoumum umumm mxmms boom Ho x003 moo .>Hm>auowmmmu H.0mooflu one .mmouosnm .mmouomamm mcwommm monom umumm mam>mfl mmooau can .mmouooum .mmouomamm ooon .ucmEommHu .v mqmfifi 46 fructose-6-P and glucose-6-P. The other begins with fructose-G-P formed directly by hexokinase. After giving a large amount of fructose to rats it was shown that fructose- l-P aldolase activity was inadequate to keep up with fruc- tokinase and may represent a limitation to the first path- way (58). The abnormal high level of fructose-l-P was found and which may lead to a profound decrease in ATP and UTP, and thus may indirectly influence other physiological reactions (58). In the study of Lowry et 21. (58), an accumulation of 100~fold of fructose-l-P was found 60 min. after giving intraperitonealy 40 umoles/kg of fructose to male rats averaging 100 g in body weight. The amount of fructose force fed to the animals in the present work was equal to 17 umoles/kg of body weight, which was less than half of the amount used in Lowry's work. Thus, the level of fruc— tose-l-P formed from fructose may not be high enough to cause an impaired fructose tolerance. From present results, it seems that the steroids did not affect fructose metabolism appreciably. There was no significant difference in blood fructose level between the control and the experimental groups after one or four weeks of steroid treatment (Table 4). However, blood glucose level after force feeding fructose was lower in the experimental group than the control group after one week of steroid treatment (Fig. 3c). There was an increase in mean 47 difference of blood glucose level of these two groups after four weeks of steroid treatment (Table 3). And there seems to be a delay in the conversion of fructose to glucose in the experimental group after four weeks of steroid treatment (Fig. 4c). This indicates that the contraceptive steroids may affect fructose metabolism, in terms of blood glucose level after a prolonged treatment. This effect might be due to the fact that estrogen may bind enzymes which are in- volved in the conversion reactions of fructose to glucose which might lead to an increased blood glucose level and a decreased blood fructose level in the steroid treated animals. IV. Blood Ribose No ribose was found in any blood sample. This indicates that all the ribose force fed was converted to c>t1‘1er metabolites within 20 min. A significant mean dif- ference of blood glucose level was found between the control and the experimental groups after four weeks of steroid treatment, but not after one week of steroid treatment (Tables 3 and 4) . It is clear then that contraceptive Steroids did not have any effect on ribose metabolism. But an indirect effect has been shown by a significant rise in blood glucose level in the experimental group. CHHE} WHEEEK '11 O C.‘ 11 WEEK I? 419?- mg/100 ml serum mg/100 ml serum 5. 600 500 400 300 200 100 600 500 i 400 300 200 x 100 i 48 ———-——Control ------ Exptl. 20 40 120 time (min) L 1 I I 20 40 , , 120 time (min) Blood galactose concentration after galactose force-feeding, after one or four weeks of steroid treatment. 49 Control 18.. ------ Exptl. 15 w 12 1+ , CHNIB ‘WHEIEK mg/100 ml serum 0 \ culi- 0 2O 40 120 time (min) 12 4)- / FOUR M % mg/100 ml serum \0 0 20 40 120 time (min) :FViSI- 6. Blood fructose concentration after fructose force- feeding, after one or four weeks of steroid treat- ment. 50 V. Urinary Glucose, Galactose, Fructose and Ribose There was no significant difference in urinary glucose content between the control and the experimental groups in the six hour urine collection regardless of which one of the four sugars was force fed. However, the eighteen hour urine of the treated animals force fed glucose (p < 0.05) or ribose (p < 0.01) had a significantly higher quantity of glucose than control (Table 5), but not for those force fed fructose or galactose (Table 5). The treated animals having a high urinary excretion of glucose, also have a high blood glucose level. This indicates that the high blood glucose, and the high urinary glucose excre— tion are probably correlated. There was no galactose, fructose or ribose found in any of the urine samples of those animals which have not been force fed these sugars. Unfortunately, because of the spillage of feed and the contamination of the urine samples with feces in this group of rats, the level of glucose in the urine were higher than the force-fed rats (Table 5). At the present time, there was no sufficient data for making further discussions concerning the effect of steroid treatment on urinary glucose excretion per se, However, indications are that an impaired blood sugar tolerance might be in existence two weeks after treatment, since a significantly higher urinary glucose was found in 51 .AHo.o v mo HmucmEHHomxm cor» HmBOH mHucMOHMHcmHm mH Houucoo new new: a .Amo.o v my HMHGOEHHmmxm cmsu HoBOH mHucMOHMHGmHm mH Houucoo now amuse .coHuomHHoo ou HOHHQ muse: mH powwow oucB mHmEHc4m .mCHpmmm wou0m kumm meson HDOMIhucmsae .mcHowmw OOHOM Houwm mason cmouanmm .UCHommm m0H0m Hmumm mason mem .ucmHoz anon m OOH\c0Hu5Hom Hmumz mo HE H CH swoon some mo 08 OOMH mg h23H mg «.0 H «.0 as s2... H as we H N6 0892 a; EHH ma ToH m.o In NJH m.m ToH To 08003.1 Tm To H m.m Em H eta. as we H m.m o.m H Em $3033 NH SGHH as ToH m5 a.m SNAH m.m H.0HH.o 0.0.0030 13 TmHmK TNHmé its m.mH m4 ~.oHH.o mmaoz 238. mass 3 0x02 «.3: o SH 2309 rams: 3 882 mm“: a 03 mcHooom COflfiOOHHOU MO fiOHHMHDQ OOHO'H Hmmsm mumm .Huaxm mumm Homecoo H AcoHHMH>mo oumocmum H mommum>m .mev .ucmfiummuu pHoumum mo mxmmz 03» umumm .mcHommm mono“ OmOQHH pom mmouosuw .mmOHOMHmm .mmOOsHm Monmm :OHumHoxm wmoosHm >HMCHHS HMHOB .m mqmde 52 the treated rats than the control at this time. Further- more, Fenichel at 31. (29) reported that normal intact female rats treated with norgestrel, ethynyl oestradiol or their combinations had a higher blood glucose than control after two weeks of treatment. Present results did not show any significant dif- ference of urinary galactose, fructose or ribose content between the control and the experimental groups (Table 6). This indicates that oral contraceptive steroids do not enhance galactose, fructose and ribose excretion in the urine. 53 .mcHommm mouow Hmumm meson unculmucm3a a .mcHommm mouom Hmuwm meson ammuanmM .mcHowmm QOHOM umpmm muson wam .ucmHos moon m OOH\coHusH0m Hmumz mo HE H CH Hmmnm comm mo 05 OOMH m.s m.nH N.wH m.m m.nH m.mH m.H m.m m.m m.m mmoon m.s m.mH s.HH o.s o.mH m.HH H.m N.H m.m m.H mmouosum s.m s.~H s.oH H.m m.oH a.HH H.m m.H m.H m.H mmouomHmo eHmuoa may: mH uxmz «.muc m umH eHmuoa may: mH uxmz mm“: m #3 coHuowHHou mo coHumuso mama .Huaxm mpmm Houucoo AcoHumH>mo oumocmum H mommuo>m .mev .ucmeummuu pHoumum mo mxmm3 03H Hmumm .mHm>HuomammH HomooHu no mmouosnm .mmOHUMHmm mcHommw mouom Hmumm coHumuoxm mmooHH can mmouosnm meHomwm couch Hmmsm .mmOHOMHmm hnmcHHs Hmuoe .m mqm¢e SUMMARY After feeding the contraceptive steroids norethy— nodrel and mestranol to 11 week old female rats, there was a significantly higher (p < 0.05) urinary glucose excretion (24 hours) in the experimental group after two weeks of steroid treatment and a significantly higher (p < 0.05) blood glucose level after four weeks of treatment in those rats administered glucose or ribose solution. No effect on blood glucose level was noticed after one week of steroid treatment. Glucose in urine collected for 6 hours after force-feeding glucose, galactose, fructose and ribose was not significantly different between the control and the experimental groups after two weeks of steroid treatment. No significant effects of steroids on galactose or fructose metabolism was found in this study. The galactose and fructose metabolism could be affected after a longer steroid treatment, since present results show this tendency. It is suggested that the oral contraceptive should be fed for a longer period in order to give more detailed information on their effect on galactose and fructose metabolism. 54 LITERATURE CITED LITERATURE CITED Alexander, R. W., Marmorston, J.: Effect of two syn- thetic estrogens on the level of serum protein— bound iodine in men and women with atherosclerotic heart disease. J. Clin. Endoc. & Metab. 21:243, 1961. Ashton, W. 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Lancet 2: 372, 1937. APPENDI CE S APPENDIX I COMPOSITION OF BASAL GRAIN DIET (in %) alfal 2..5; l..6; :sailt, APPENDIX I COMPOSITION OF BASAL GRAIN DIET (in %) Ground corn 60.7; soybean meal (50% protein), 28.0; fa meal (17% protein), 2.0; fish meal (60% protein), dried whey (67% lactose), 2.5; limestone (38% Ca), dicalcium phosphate (18.5% P, 22-25% Ca), 1.75; iodized 0.5. Supplementary minerals and vitamins were added ‘tc> provide per kg of diet: (in mg.) Mn, 121; Fe, 95; Cu, '7; Zn, 4; 12, 4; Co, 2; Choline chloride, 400; Ca panto- ‘tliennate, 6; riboflavin, 3; niacin, 33; menadione, 2; DL- methionine, 500; (in microgram) vitamin B12, 7; (in I.U.) Vfiiizaunin A, 8010; vitamin D2, 750; vitamin E, 5. 67 W'“‘" "— ng. APPENDIX II EXPERIMENTAL DESIGN 68 mam>fluommmwu .CHE >Hm>flwommmmu .cflE mam>fluommmmn .CHE >Hm>fluommmmu .GHE omH pcm ow .om omH paw ow .om ONH can ow .om oma paw ov .om uwumm moan mnw3 nmumm cman ouo3 umumm ooan muck “mums pman mumB mcfiummm wuflmm v >Hm>m muHmm v >Hm>m muwmm v wsm>o muflmm v >Hm>m Hmumm oman x2 ifi mcflpmmw mcflpmwm mcflpmmm mcflcmmm moH0m mmonflu moHOM mmouosum mUHOM omouomamm moHOM mmoosam MOM muwma NH How mHHmm NH How mnflmm NH you mnflmm NH muflmm v T _ muflmm mm mums voa szmma H- d!- .1omm tomm £ c> ‘w cu (m5) unfitam Kpos meanwmm mama omuumum #omm 1 room 70 .mumu Umpmmuu may mo coauQEdmcoo poom >HHMQ .mu4 .mam Amuse wasp mmbmmmmmvmmmmmamommamahamamavamamaaaoa m m h w m w m m H .rm LIQH .ma Pow (m6) exequI poog APPENDIX IV TABLES 71 .mflmmamcm HMOflumfiumum MOM pom: m .ummu mocmumaou Hmmsm map mowusc coco haco UonEmm mmz awn sumo umnu 0m awn ucmumMMHp m Eoum cmuomaaoo mm3 mHQEMm comm m .unmflms mpon m ooa\coflu5H0m 0mm mo HE H CH wmoosHm m0 m8 OOMH H.>H v.va m.mm «.mm m.om m.vm .Q.m n.mma w.mma m.mHN «.mma v.mwa 5.05H cmmz mm mwa mm and mm hum ma mm N «ma mm mma v mm mma mm mma mm om mv mma v «Ha mm mma m m.moa H.mva >.mmm m.mma o.vma o.hma m o.HNH H.Nma m.mmm w.ava H.Hha m.>ma H oma ov om omH ow om msflpmmm mouom mmoosaw Hmpw< .CHE Ncowum>ummno . mo .02 mumm .Humxm mumm Houucoo AEdem HE ooa\mEV .ucwaummuu ofloumum Hmumm x003 mco .mmoosam onwvmmm mOHOM Hmumm mam>ma mmoosam poon .HI¢ mqmda H 72 .AHo.o v my Hmucmfiflnmmxm cmnu HmBOH wauchAMHcmHm ma Houucoo How cmmzm v .manmu mflnu CH Umpsaocfl mum muflmm pmumHmEoo Mom mumm maco .mumu mmuflmm co momma mums mpmm mocflm .mcflamsmm mcausm Umflo mmumEIHHmm may no oc0m .ummu mocmnmaou Hmmsm on» mcfluso moco waco poamfimm mms umu sumo umnu om umu ucoquMHp m Eoum monomaaoo mm3 mamamm 50mm .mflmmamcm Hmoaumflumum MOM pom: N .usmH03 moon m ooa\coHuDH0m Hmums mo HE H CH omoosam mo 08 oomH m.m «.mm m.m>a m.om o.h~ ¢.mv .o.m m.oma w.hm H.mvm o.mm m.noa m.mna mcmmz em oma Hgm pm we nma vb ow vm «ma eo omm v we «ma vv mma vm cam vm mm an moa mm mma m m III o.mm m.oma m In: 0.0m m.mma N n.nma m III v.nma m.HHH m III m.HHH a oma om om oma ov om mafipmmm mouom mmoosao Hound .cwz mcowum>nmmno mo .02 mumm .Humxm mumm Honucou AEdem HE ooa\mEV .ucmEummuu vfloumpm Hmumm mxmms HSOM .omoosam mcflpmmm mUHOM Hmumm maw>ma mmoosHm pooam .mnfl mqmda H 73 UmHmEmm mmB umu WI“ . n HqufM‘\ 1 1 . rum. ..Jlaiiwiliym iii: . II!‘ I . .uzmHmB moon m 00H\coHu5H0m 0mm HE H CH mmouomHmm m0 m8 oom .mHmmHmcm HmoHumHumum How pom: m .ummu mocmumHou HmmSm on» mcHHso moco >Hco comm umnu 0m umu unmumMMHU m Eoum pmuomHHoo mm3 mHmEmm 30mm N H m.Hm o.mm «.mH «.mm v.om m.mm .o.m m.mm w.mmH H.m¢H m.HoH m.mmH m.mOH cmwz N NHH mm om mm va H mOH mm mm mm.vOH a m.hOH m.mmm mv.mmH v.om m.¢mH mn.mm m mm NOH mm mmH H mmH mm mm mm omH m.mmH m mm.mw ~.mwmno mums .Humxm mumm Houucoo UHoumum kumm xmmz mco Afidumm HE o0H\mEV .mmouomHmm mchmmm moH0m Hmumm mHm>mH mmoosHm H .ucmfiummnu UOOHm .MIfl mqmdfi flirnu «V'fl‘ Eqmqrfi 74 T .oHsmu mHsu sH oomsHusH oum mHHmm ooponEou now mums hHso .mumu mouHmm so momms ouoz mums ousHm .msHHmEmm msHHso ooHo moumEIHHmm osu mo osOv m .umou ousmuoHou Hmmdm osu msHuso ouso >Hso oonEmm mmB umn sumo umsu 0m umu usouomeU m Eoum pouuoHHou mm3 onEmm summ .mHmaHmsm HmUHumHumum HON mom: m .usmHos moon 0 00H\soHusHom Omm mo HE H sH omouumHmm no me OOMH m.vm H.m m.Hm o.mm H.vH m.mm .o.w o.mHH m.nm 0.0m H.~m m.moH m.mHH smoz w an: mm NOH mm mm v III mm.mHH mm ovH w o.mmH H.mm mv.m0H H.0HH m.hm mm.hHH m mm mHH mm 00H 0 Ha mm mm mm mm m.mo m mv.mm o.mm m.~m mm.mh m.mHH m.NVH H omH ow om omH ov om msHpoom ouuom omouumHmo Houmd .st msoHum>nomso mo .02 mums .Humxm mumm Houusoo Afisuom HE OOH\mEV .usoEumoHu oHououm Houmm msoo3 HSOM H.omouumHmo msHUoom ouHOM Houmm mHo>oH omOUSHm Uoon .VId mqmdfi 75 .mHthmsm HmuHumHumum Mow momDm .umou ousmMoHou Hmmsm osu msHHsp ouso mHso poHQEmm mm3 umu sumo umsu 0m umu usouomuHU m Eouw pouuoHHou mm3 onEmm summ m .uzmH63 smog m OOH\coH»sHom 0mm mo Ha H cH «monosnm mo ms oomH ~.m v.m~ o.- H.mH m.¢~ m.om .o.m «.mmH m.ooH ~.omH m.mma 0.55H m.m¢H cams mm.moH m.vsH mm.HOH m.msH m.mmH mm.msH H MH.mmH mm.omH s.mMH m.smH o.mmH m.qu m ¢.HHH v.mmH o.smH m.o¢H o.mvH m.omH m o.mvH mm.vHH mm.NmH v.~mH m.¢sH mm.meH H ONH ow. om ONH oq om msHmoom ouuom omouusum Houws .st msonm>mwmso mumm .Hudxm mumm Houucoo AEsnom HE OOH\OEV .usoEumouu pHououm Houmm xooB oso .omouusnm msHooom ouuom Houmm mHo>oH omousHm poon .m|< mqmmMmso mumm .Humxm mums Houpsou Aesuom HE 00H\mfiv .usoEumoHu oHououm Houmm mxoo3 H50m H.owouusuw msHooom ouHOm uoumm mHo>oH omousHm poon .ml< mqmdfi 77 .ggoHoz smog o OOH\gngoHom omm mo He H gH omogH.H so me can .mHmsHmsm HmuHumHumum How pom: m .pmou ousmHoHos HmmSm osu msHHsp ouso sHso oonEmm mm3 umn sumo umsu Om umu psoHoMMHo m Eoum mouuoHHou mm3 onEmm summ N H o.H~ N.mm m.om s.vm s.o~ o.mm .g.m m.HHH m.HmH m.omH m.~0H m.smH m.mmH goo: m.mmH mm.mmH m.mmH m.mOH mHGHH mm.smH v mm.mOH m.HNH m.HHH mo.sm m.mmH mm.sHH m ms.sHH m.msH m.~mH mo.mmH N.~HH s.mom m s.vm mm.Hm m.omH m.mm mm.smH m.ms H omH ow om omH ow om ogHmoom oouom omogHm uogmm .gHz mgonm>mwmgo mumm .Humxm mumm HOHusou Afisuom HE 00H\mEV .usoEumouu UHououm Houwm sooB oso H.omosHH msHooom ouuom Houwm wHo>oH omousHm UOOHm .hls MHmse 78 .Amo.ovmvm~o.ov HmusoEHHomxo How smsu uo3OH sHvsmunHsmHm mH Honusou How smoZm v .oHsmp mHsu sH poosHusH ohm mHHmm poponEou How mums sHsO .mumH ooHHmm so oomms oHoB mpmp ousHm .msHHmEmm msHHsU UoHU moumEIHHmm osu mo oGOm .umou ousmHoHou ummSm osu msHHso ouso sHso UonEmm mmB umn sumo umsu Om umH usoquMHU m Eoum popuoHHou mm3 onEmm summ .mHmsHmsm HmuHumHumum How pom: m .ggoHoz smog o OOngngoHom omm go H2 H gH omogHu Ho ms oomH H.m 0.0m m.om m.HN H.sm s.mH .s.m o.mOH «.mm m.>om m.ms m.mm ~.me msmoz s NHH mm mm vm mom 5 mm mo mm mm HmH m mm moH m mm on omH mm mOH m mm H.o mHH m mm mm m mm m III ms mm m MOH m In: N m --- Hm Hm m --- m In- on MOH m --- H omH om om omH om om msHooom ounom omosHm nouns .st msoHum>Homso mo .02 mumm .Humxm mumm Houusoo AEdHom HE OOH\mEV .usosumouu mHououm “ovum mxoos usom H.omosHH msHooom ouHOH Houmm mHo>oH omousHm OOOHm .mld mqmde 79 .mHmsHmsm HmuHumHumum MOM pom: m .umou ousmHoHou Hmmsm osu manss ouso sHsO oonEmm mmz umn sumo umsu Om umu usoquMHU m Eoum UouuoHHou mm3 oHQEmm summ m .ggoHoz smog o OOH\gngoHom om: go H2 H gH omogomHmm go as oomH m.mm o.om H.5HH w.mmH m.ms o.mm .Q.m m.smm m.msm m.sH~ o.o s.mHm m.mmm o.sHm o.c smoz o.mom «.mmm m.omm o.o m.mHm m.mom m.mmH o.o v mm.msm m.m~m mH.mmm o.o mo.sHm «.mmm mm.mmm 0.0 m w.~mH mo.o~m ms.mmH o.o m.HmH m~.mmv mm.mom 0.0 m mH.o~N mw.s~m m.omm o.o mm.mom mH.mmv «.mvH o.o H omH ow om ogHgmmm omH ow om ogHgmms msHUoom ouuom omosumHmo Houmd .st NGOHWM>mMmsO mgmm .Huoxm mgmm Houugoo AEdHom HE 00H\mEV .usoEumouu oHOHoum Houmm sooB oso .omouumHmm msHUoom ouuom Houmm mHo>oH omouumHmm UOOHm .mld mamme H 80 UoHHmm so momma ono3 mums ousHm .oHsmu mHsp sH popsHusH oum mHHmm UouonEou How mums mHso .mumu .msHHmEmm mandp poHp momeIuHmm osu mo oso m .mHmsHmsm HmUHumHumum How somam .umou ousmHoHou “mono osu mansp ouso sHso monEmm mmB pmu sumo pmsu Om umu usoHoMMHU m Eonm oopuoHHou mms onEmm summN .usmHos woos m o0H\soHusHOm on mo HE H sH omouumHmm no me oomH N.mmH m.mmH h.m0H m.HNH v.mmN m.mHH .o.m s.NmN m.sHm m.HMN o.o H.m0N o.som o.oNN o.o smoz v :1: H.mNm m.msN 0.0 v III m.sms m.HMN 0.0 m ms mmm m 1:: mm mmN o 0 mm mmm 1:: mm omm o o m N.NmH ms.mMH mm.mNm o.o m.HmH m.mms mm.mmH o.o N mN.mMH mm.som v.mh o.o mn.mm N.mmm m.sm o.o H ONH om om mgHgmmm omH as om ogHgmos msHooom ouuom omouumHmU nouns .st NGOHWM>mwmso mums .Humxm mumm Houusou Henson HE o0H\mEv .usoaumouu UHOHopm Houmm mxooz HSOH .omO»umHmm wsHpoom ouH0m Houwm mHo>oH omouumHmm UOOHm .OH|< mqmse H 81 .mHmsHmsm HmuHumHumum How pomsm .umou ousmHoHOu Hmmsm osu msHHDO ouso sHso UonEmm mm3 umn sumo omsw Om umH usoHoMMHU m Eoum nouuoHHou mms onEmm summ N .ggoHos smog o OOH\:ngoHom omm go H5 H gH omogooum go as oomH m.H ~.~ o.H m.m o.~ m.m .g.m m.HH m.~H N.HH o.o m.OH H.mH N.mH o.o goo: mm mH H NH mm NH o 0 mm HH m «H mm mH 0.0 H m.HH mm.mH m.HH o.o m.OH mm.mH m.s 0.0 m ms.~H H.NH mm.m o.o mm.HH H.sH m¢.mH o.o N m.mH mm.OH o.OH o.o m.s mm.vH m.OH o.o H omH om om ogHgmms omH om om ogHgmmm msHUoom ouuom omouusnm Houmm .sHE NsOHum>HomsO Ho .oz mumm .Humxm mumm Honusoo AESHom HE o0H\mEV .usoEumoHu UHOHoum Houmm sooz oso H.omouusum msHmoom oouom Houmm mHo>oH omouusum UOOHm .HHI¢ mam¢a 82 .oHsmu mHsu CH soUCHuCH on mHHmm pouonEou How mpmp sHCO .mpmu poHHmm CO oomms oCoB mums ouCHm .mCHHQEmm mCHHCU poHU moumEIHHmm osu mo oCO¢ m .umou oquHoHOu Hmmsm osu mCHuCo ouCo sHCo UonEmm mmB umu sumo umsu Om umu “CoHoHMHC m Eoum wouuoHHOu mm3 onEmm summN .mHmsHmCm HmuHumHumum COM poms .usmHo3 woos m OOH\COHuCHOm on mo HE H CH omouusum mo mE oom H m.N N.H N.m N.m H.m m.m .a.m H.mH m.mH m.mH o.o m.MH m.NH m.mH o.o Cmoz mo NH m III mm mH o 0 mm s m III mm NH O o v o.NH mm.NH m.mH o.o N.vH ms.m «.mH 0.0 m mm wH v III mm NH o 0 mm «H m III mm mH 0.0 N v.HH ms.vH v.m o.o m.hH mv.mH m.NH o.o H omH OH om ogHgmms omH ow om ogHgmmm mCHcoom ouuom omouusum “ovum .CHz NCOHum>HomsO mo .02 mums .Humxm mumm HouuCoo AECCom HE OOH\mEv .UCoEumoHu Houmm mxoo3 snow H.omouusum mCHUoom ouuom Houmm mHo>oH omouusnm UOOHm .NHId mqmma 83 TABLE A-l3. Total urinary glucose excretion after glucose force-feeding, following two weeks of steroid treatment. (mg) Con- COIIEOEIogf 4 Exptl. gingiigogf 4 8:2: lst 3 Next 18 TOtal Rats lst 6 Next 18 TOtal hrs. hrs. hrs.2 hrs. 1 0.20 2.86 3.06 1 0.09 1.60 1.60 2 __ 5 __ 5 2 __ 5 __ 5 3 0.07 2.47 2.54 3 0.21 5.04 5.25 4 o 14 3.18 3 32 4 0 36 9.15 9.51 5 0 18 4 22 4 4o 5 0.39 8.05 8 44 6 __ 5 __ 5 6 __ 5 __ 5 7 0.026 1.686 1.706 7 0.236 3.486 3.716 8 0.106 4.796 4.896 8 0 406 3.566 3.966 9 0.056 1.616 1.666 9 0.136 5.696 5.826 10 0.116 4.146 4.256 10 0.286 4.586 4.866 11 0.166 1.406 1.566 11 0.126 3.536 3.656 12 0.226 1.996 2 216 12 0.996 3 826 4.816 Mean 0.13 2.837 2.96 Mean 0.32 4.857 5.17 S.D. 0.1 3.2 5.0. 0.4 5.6 1 300 mg of glucose in 1 m1 of H20 solution/100 g body weight. 2Six hours after force—feeding. 3Eighteen hours after force-feeding. 4 5One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. Twenty—four hours after force-feeding. 6Used for statistical analysis. 7Mean for control is significantly lower than exper- imental (p < 0.05). 84 TABLE A-l4. Total urinary glucose excretion after galactose force-feeding, following two weeks of steroid treatment. (mg) Duration of Duration of Con- ;zgi 1::l6e0§::: 18 TOtal4 Eggti' lgiléecfiigt 18 T°tal4 hrs. hrs. hrs. hrs. 1 2.77 7.16 9.93 1 2.30 12.21 14.51 2 5.82 6.24 12.06 2 2.26 4.21 6.47 3 1.965 3.905 5.855 3 2.345 1.385 3.725 4 3.30 2.08 5.38 4 1.32 3.62 4.94 5 1.595 7.695 9.285 5 1.675 7.515 9.185 6 2.765 6.415 9.175 6 4.335 2.455 6.785 7 2.275 1.455 3 725 7 1.155 9.895 11.045 8 1.64 1.64 3.28 8 2.21 9.45 11.66 9 1.81 15.44 17.25 9 3.99 5.62 9.61 10 1.815 2.325 4.135 10 3.425 3.315 6.735 11 4.065 3.905 7.965 11 3.125 4.685 7.805 12 __ 6 __ 6 12 __ 6 __ 6 __ 6 Mean 2.70 5 29 7.99 Mean 2.55 5.84 8 39 S.D. 3.0 6.6 S.D. 2.7 6.7 1300 mg of galactose in 1 m1 of H20 solution/100 g body weight. 2Six hours after force-feeding. 3Eighteen hours after force-feeding. 4Twenty-four hours after force feeding. 5Used for statistical analysis. 6One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. 85 TABLE A-15. Total urinary lucose excretion after fructose force-feeding, following two weeks of steroid treatment. (mg) 90’” 331126326? 4 Exptl. 3311122320? 4 Egg: lst 3 Next 18 TOtal Rats lst 3 Next 8 TOtal hrs. hrs. hrs. hrs. 1 0.095 0.815 0.905 1 0.635 4.695 5.325 2 0.075 3.115 3.185 2 0.855 0.945 1.795 3 0.155 1.005 1.155 3 1.615 5.565 7.175 4 0 09 1.17 1.26 4 0.25 6.14 6.39 5 __ 6 __ 6 5 __ 6 __ 6 6 0.09 1.35 1.44 6 0.43 4.19 4.62 7 0.095 5.545 5.635 7 0.335 3.535 3.865 8 0.055 5.775 5.825 8 0.145 4.995 5.135 9 0.245 3.795 4.035 9 0.355 2.455 2.805 10 0.11 4.12 4.23 10 0.23 5.13 5.36 11 __ 6 __ 6 11 __ 6 __ 6 12 0.13 6.76 6.89 12 0.30 4.95 5.25 Mean 0.11 3.34 3.45 Mean 0.51 4.26 4.77 S.D. 0.1 4.2 S.D. 0.7 1.7 1 300 mg of fructose in 1 ml of H20 solution/100 g body weight. 2Six hours after force-feeding. 3Eighteen hours after force-feeding. 4Twenty-four hours after force-feeding. 5Used for statistical analysis. 6One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. 86 TABLE A-16. Total urinary lucose excretion after ribose force-feeding, following two weeks of steroid treatment. (mg) Duration of Duration of Egg; 1221;901:332 . E3528- 1:245:32 I. hrs. hrs. hrs. hrs.3 1 __ 5 __ 5 1 __ 5 __ 5 2 0.096 2.816 2.906 2 0.156 2.626 2.776 3 __ 5 __ 5 3 __ 5 __ 5 4 __ 5 __ 5 4 __ 5 __ 5 5 0.15 1.92 2.07 5 0.08 0.98 1.06 6 0.046 0.986 1.026 6 0.116 0.626 0.736 7 0.136 3.606 3.736 7 0.086 5.136 5.216 8 0.14 5.32 5.46 8 0.10 14.87 14.97 9 0.306 5.456 5.756 9 0.206 3.166 3.366 10 0.266 6.726 6.976 10 0.166 4.916 5.076 11 0.106 4.846 4.946 111 0.136 17.976 18.106 12 0.14 7.82 7.96 12 0.37 15.80 16.17 Mean 0.15 4.387 4.53 Mean 0.15 7.347 7.49 S.D. 0.2 5.2 S.D. 0.2 10.4 1300 mg of ribose in 1 m1 of H20 solution/100 g body weight. wa Six hours after force-feeding. Eighteen hours after force-feeding. Twenty-four hours after force-feeding. 5 One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. 6 7 imental (p < 0.01). Used for statistical analysis. Mean for control is significantly lower than exper- TABLE A- 17 . 87 Total urinary galactose excretion after galac- tose force4feeding, following two weeks of steroid treatment. (mg) Con- ESIIEOEIogf 4 Exptl. ggiizizgogf 4 Egg: lst 6 Next 8 TOtal Rats lst 3 Next 8 T°tal hrs. hrs. hrs. hrs. 1 1.12 3.02 4.14 1 1.11 3.61 4.72 2 1.20 1.57 2.77 2 1.37 3.00 4.37 3 1.525 1.655 3.175 3 1.155 0.005 1.155 4 1.91 1.88 3.79 4 1.53 2.39 3.92 5 1.425 2.505 3.925 5 1.375 1.975 3.345 6 1.015 1.455 2.465 6 0.925 3.015 2.985 7 0.865 1.675 2.535 7 1.305 1.685 2.985 8 1.22 1.30 2.52 8 1.15 1.50 2.65 9 1.18 2.22 3.40 9 1.78 1.77 3.55 10 1.455 1.175 2.625 10 1.225 2.045 3.265 11 1.335 1.735 3.065 11 1.355 1.715 3.065 12 __ 6 __ 6 12 __ 6 __ 6 Mean 1.29 1.83 3.12 Mean 1.30 2.06 3.36 S.D. 13.8 0.55 1.93 S.D. 0.43 2.37 2.80 1 281x hours after force-feeding. 3Eighteen hours after force-feeding. 4 5 6 Used for statistical analysis. Twenty-four hours after force-feeding. One of the pair-mates died during sampling. 300 mg of galactose in 1 m1 of H20 solution/100 g body weight. Since data were based on paired rats, only data for completed pairs are included in this table. 88 TABLE A-18. Total urinary fructose excretion after fructose force-feeding, following two weeks of steroid treatment. (mg) Duration of Duration of E32; 122129082221. Eiiiti' 1231;661:321. hrs. hrs. hrs. hrs. 1 1.145 1.195 2.335 1 2.215 2.365 4.575 2 1.245 2.065 3.305 2 1.195 1.635 2.825 3 3.485 2.335 5.855 3 0.905 5.765 6.665 4 1 52 1.55 3 07 4 0.71 4.68 5.39 5 _‘ 6 __ 6 5 __ 6 __ 6 6 0.93 1.31 2.24 6 1.40 2.29 3.69 7 2.535 1.695 4.225 7 0.835 1.665 2.495 8 0.445 6.905 7.345 8 1.255 4.245 5.495 9 1.305 2.165 3.465 9 1.785 2.135 3.915 10 1.06 3 09 4.15 10 0.59 3.17 3.76 11 __ 6 __ 6 11 __ 6 __ 6 12 1.28 3.06 4 34 12 1 30 2 79 4.09 Mean 1.49 2.53 4 02 Mean 1.22 3.07 4.29 S.D. 1.8 3.0 S.D. 1.4 3.5 1 300 mg of fructose in 1 ml of H20 solution/100 g body weight. Six hours after force-feeding. Eighteen hours after force-feeding. Twenty-four hours after force-feeding. U'IIbUN Used for statistical analysis. 6One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. 89 TABLE A-l9. Total urinary ribose excretion after ribose force-feeding, following two weeks of steroid treatment. (mg) Con- EEIIEOEIogf 4 Exptl. EEIIEOEIOSf 4 :22: lst 6 Next 18 TOtal Rats lst 6 Next 18 T°tal hrs. hrs. hrs.2 hrs.3 1 __ 5 __ 5 1 __ 5 __ 5 2 0.006 0.796 0.796 2 0.006 1.246 1.246 3 __ 5 __ 5 3 __ 5 __ 5 4 __ 5 __ 5 4 __ 5 __ 5 5 13.45 5.62 19.16 5 0.51 7.29 7.80 6 0.006 0.536 0.536 6 0.006 5.706 5.706 7 9.136 1.076 10.206 7 2.106 0.546 2.656 8 0.00 2.81 2.81 8 9.17 0.59 9.76 9 0.006 5.006 5.006 9 2.236 0.436 2.656 10 0.006 2.536 2.536 10 6.096 1.046 7.136 11 7.116 2.256 9.366 11 3.146 0.006 3.146 12 0.14 1.50 1.64 12 0.00 0.00 0.00 Mean 3.32 2.46 5.78 Mean 2.58 1.87 4.45 S.D. 6.3 3.2 S.D. 4.2 3.3 1300 mg of ribose in 1 ml of H20 solution/100 g body weight. 2 3 Six hours after force-feeding. Eighteen hours after force-feeding. 4Twenty-four hours after force-feeding. 5One of the pair-mates died during sampling. Since data were based on paired rats, only data for completed pairs are included in this table. 6 Used for statistical analysis. 90 TABLE A-20. Total urinary glucose excretion after two weeks of steroid treatment. (mg) Con- Duration of Duratio? of 4 Collection 4 Exptl. Collect on R::: lst 3 Next 8 Total Rats let 6 Next 18 Total hrs. hrs. hrs.2 hrs.3 1 0.15 1.02 1.17 1 0.14 5.90 6.04 2 0.14 2.56 2.70 2 0.12 14.93 15.05 3 0.15 3.21 3.36 3 0.07 3.55 3.62 4 0.10 10.34 10.44 4 0.03 6.83 6.86 Mean 0.13 4.28 4.41 Mean 4.28 7.80 12.08 S.D. 0.2 5.8 S.D. 2.1 9.7 1Animals were fasted 15 hours prior to collection. 2First six hours of collection. 3Next eighteen hours of collection. 4 Total urine collection. MICHIGAN STATE UHWERSWY COLLEGE OF HOME ECGE‘IECMICS EAST LANSING, MICHIGAN