EHTERACfiQRS BETWEEN i‘HfiififitfiéE, CORTESORE, ALLQ‘XAE‘!‘ ARE“) EWLEN OH CARBOHYDFUAE Aim PROTEM METABOQSM éé’é RATS Thais fix the W 3“ Pix. 3. (WWW SFA'E'E COLLEGE" Afbfifl'fi fienmém Wééwméa W55 [H 5313 LIB R A R Y Michigan State University This is to certify that the has been accepted towards fulfillment of the requirements for Maegan: in l . M 0-169 INTERACTIONS BETWEEN THISMINE, CORTISONE, ALLOXAN AND INSULIN - ON CARBOHYDRATE AND PROTEIN METABOLISM IN RATS By Alberto Monteiro Wilwerth A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 1955 /- 7" ‘3"? 6- 2'1 :3"! ACKNOWLEDGMENT The author wishes to express his sincere gratitude to Dr.J. Meites, Professor in the Department of Physiology and Pharmacology, for his patience, guidance and advice during the course of this research and preparation of the manuscript. He also wishes to express his appreci- ation to Dr. B. V. Alfredson, head of the Department of Physiology and Pharmacology, for providing facilities and laboratory Space to carry on this work. Special acknowledgments are due Dr. L, Michaud of Merck and Co., Rahway, New Jersey, for supplying cortisone acetate; and to Dr. R. R. Chen of Eli Lilly and 00., Indianapolis, Indiana, for supplying zinc insulin (Illetin). Thanks are due Mr. John Monroe for his help in the care of the animals used in this study. The author is very much indebted to the Rockefeller Foundation and the Escola de Veterinaria da U. R. E. M. G., Belo Horizonte, Minas Gerais, Brazil, and wishes to express his sincere appreciation to them for making possible his stay in the United States. Finally, the writer is indebted to the Michigan Agricultural Experiment Station and the United States Public Health Service for pro- viding financial support to the project under which this work was carried out, aaaaaaa*** aaaaaaaa aaaaaa *aaa %* * ii DEDICATION To Odette F. Wilwerth iii INTERACTIONS BEThEEE-J THIDJINE, COhiTISOI-JB, .LLLOXAN AID II‘iSULIN ON CARBOHYDREE AND PROTEIN E'ILI‘ILLDOLIS-i IN ILATS By Alberto Monteiro Wilwerth AN ABSThACT Submitted to the School of Graduate Studies of Mickfigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology Year 1955 Approved ABSTRACT I. When young rats were maintained on a thiamine-free diet, symptoms of thiamine deficiency developed within 15 to 20 days. Supple- mentation of the diet with 2 mg. of thiamine per kilo of diet increased appetite and body weight gains, slightly increased blood sugar and greatly increased glucose tolerance. 2. When h mg. of cortisone acetate daily were injected into thiamine- deficient rats, there was a slight increase in the excretion of urinary nitrogen, a slight or no increase in blood glucose, decreased glucose tolerance, reduction in body weight gains and reduced appetite. When 2 mg. of thiamine per kilo of diet (or higher levels of thiamine) were fed to cortisone-treated rats and they were allowed to eat ag_libitum, urinary nitrogen increased greatly, blood glucose increased moderately, glucose tolerance was partially improved and body weight was maintained at the same initial level or was slightly increased. 3, Thiamine at high levels, fed to rats on a limited-food intake, largely prevented the develOpment of thiamine-deficiency symptoms but was unable to increase the blood glucose of cortisone-treated rats. It slightly increased urinary nitrogen excretion. It is concluded that large doses of thiamine, greater than normal requirements for growing rats, can partially counteract the protein catabolic action of cortisone by increasing food consumption and increasing the availability and utilization of carbohydrate by'tie organism. iv h. Cortisone partially interferred with the favorable action of large doses of thiamine on the efficiency of food utilization for body growth. Hyperglycemia, glucosuria, increased nitrogen excretion and increased insulin resistance were noted, and therefore less carbohydrate was available to exert a "sparing action" on protein for transformation into body weight gains. 5. (a) When young rats were fed a thiamine-free diet, the weight of the kidneys, heart and adrenals were increased and the weight of the thymus and seminal vesicles were greatly decreased. When 1 mg. daily of cortisone acetate was injected into thiamine-deficient rats, a still greater increase in the weight of the kidneys and heart was noted, and a slight increase was found in the weight of the testes and adrenals. The low thymus weight was not decreased further by cortisone treatment, while the seminal vesicles weighed twice as much as those of thiamine- deficient rats. (b) When thiamine was fed to cortisone-injected rats, the kidneys, heart and testes showed a slight increase in weight. The thymus showed less involution, the adrenals were reduced in weight and the seminal vesicles were slightly but not significantly increased in size. The increases in thymus and seminal vesicles weights apparently were not due to thiamine pggvgg_but to the concomittant increase in food intake. 6. Alloxan-diabetes did not further reduce the efficiency of food utilization of thiamine-deficient rats or rats on a limited-food intake, but slightly reduced the efficiency of food utilization of thiamine- adequate rats. In the latter there was a consistent increase in blood glucose and urinary nitrogen, while in the thiamine-deficient rats there was neither an increase in blood glucose nor of urinary nitrogen. Rats on a limited food intake but fed thiamine showed a consistent increase in both blood glucose and urinary nitrogen which decreased progressively as chronic inanition deve10ped. ‘When the treatment of the thiamine-deficient rats was reversed, by administering large amounts of thiamine, there was a pronounced hyperglycemia and an increase in urinary nitrOgen excretion. It is concluded that thiamine, by increas- ing food intake, permits hyperglycemia to develop in alloxan-diabetes. 7. When guinea pigs were maintained on a thiamine-free diet, symptoms of thiamine-deficiency deve10ped within 25 days. Supplemen- tation of their diet with 16 mg. of thiamine per kilo of diet increased appetite and body weight gains. Injections of S or 10 mg. of cortisone acetate daily did not appear to reduce body weight significantly in thiamine-deficient guinea pigs in contrast to rats. When cortisone was injected into thiamine-deficient guinea pigs there was no increase in urinary nitrogen or blood glucose, as in rats. When 16 mg. of thiamine or more per kilo of diet were fed to cortisone-treated guinea pigs, only a slight increase in blood glucose was observed with 5 mg. of cortisone injected daily and a consistent increase with 10 mg. of cortisone daily. Cortisone did not increase blood glucose of thiamine-deficient guinea pigs at any level. 8. Insulin was much more effective in reducing blood glucose in normal and alloxanized rats than in cortisone-treated rats maintained vi on either a thiamine-deficient or adequate diet. A thiamine-deficient diet reduced the hypoglycemic action of insulin, indicating that thiamine is essential for the maximum action of insulin. The greatest resistance to insulin was found in cortisone-treated rats, confirming the observa- tion that cortisone increases insulin resistance. 9. It is suggested that the over-all effect of large doses of cortisone in young rats, by virtue of its ability to interfere with carbohydrate utilization but at the same time increase the secretion of insulin, is to increase the need for thiamine. The beneficial action of a“ large intake of thiamine in cortisone-treated rats is believed to be brought about by its ability to increase carbohydrate intake and utilize- tion in the presence of hyperinsulinism. TABLE OF CONThNTS IKTRODUCTION..................................................... l KWHEDBEREHEL.H.....u.H . . .. ...... . ............... h Introduction................................... . ..... ..... .. h Thiamine........ ......... . ...... . . .... ..... . .. ...... .. h a) Requirements for thiamine under different conditions.... h b) Effects of thiamine on carbohydrate fat and protein metacolism. ....... .................................... 6 0) Effects of thiamine on organ weights... ......... ........ lO Adrenal cortical hormoneS..................................... 13 a) Carbohydrate metabolism................................. 13 b) Protein metabolism and body growth...................... lh C) Hair and thymus growth ..... ............................. 17 d) Organ weightS........................................... 17 .Alloxan-diabetes and Insulin.................................. 20 Effects of alloxan..... ......... ................ ........... 20 affects of insulin on carbohydrate metabolism.............. 22 Effects of insulin on fat metabolism....................... 2h Effects of insulin on protein metabolism................... 25 Mechanism of insulin action................................ 27 The hexekinase theory of insulin action.......... ..... .. 27 The permeability theory of insulin action............... 28 Control of insulin secretion.... ......... .................. 29 Relation of insulin to nutrition........................... 29 EXPERIMENTAL..................................................... 32 Experiment I - Effects of cortisone on thiamine requirements of young ratS......................... ..... .. 32 Experiment II - Effects of cortisone on thiamine requirements of young ratS. ................ . .. . ..... h2 Experiment III - Effects of thiamine and cortisone on body weight, carbohydrate and protein metabolism of guinea pigS............................... 51 Experiment IV - Effects of thiamine, cortisone and alloxan on body growth, OlOOd glucose and urinary nitrogen in ratS............................. 61 viii TASLE OF CONTENTS - Continued Page Experiment V - Effects of thiamine, cortisone, alloxan and insulin on body growth, blood glucose and urinary nitrogen in ratS..................... 71 Experiment VI and VII - Glucose utilization in normal, alloxan-diabetic and cortisone-treated rats as influenced by thiamine............ ....... . 82 DI$USSIOI\IOO00.00.000.000...00.0.0000000000000000.0.0000000000000 89 SUL‘IE‘IARY.’OOOOOOOOOOOOOOOOOOOO00.000.000.000... ........... 00...... 98 BIBLlOflPifl-OOOOOOOOOOOOOOOOOOOOOOOOOO 000000 000.000.000.000000000102 APPE‘EDIXO000000.00000000-0000a...0000.00.00.00...0000900000000... 123 ix TABLE II III IV VI VII VIII XII XIII XIV LIST OF TABLES Page Effects of thiamine and cortisone on body weight, food intake and efficiency of food utilization... ..... .......... 39 Effects of thiamine and cortisone on organ weights......... hl Effects of thiamine and cortisone on body weight, food intake and efficiency of food utilization.. ..... ........... hb Effects of thiamine and cortisone on organ weights......... 50 Effects of thiamine and cortisone on body weight gains of ng-nea pigsooooooooooooooo00.00.00.000...coo.00000000000000 57 Effects of thiamine and cortisone on blood glucose and urinary nitrogen in guinea pigS............................ 59 Effects of thiamine deficiency on blood glucose before and after cortisone administration. ...... . ....... .............. 60 Effects of thiamine, cortisone and alloxan on body weight, food intake and efficiency of food utilization............. 68 Effects of thiamine, cortisone and alloxan on blood glucose and urinary nitrogen in rate................... .......... .. 70 Effects of thiamine, cortisone and alloxan on body weight, food intake an efficiency of food utilization.............. 78 Effects of thiamine, cortisone and alloxan on blood glucose and urinary nitrogen in ratS... ..... ....................... 80 Effects of insulin on blood glucose after pretreatment with thiamine, cortisone or alloxan............ ..... ............ 81 Effects of thiamine, cortisone and alloxan on glucose telerance teStOOOOOO ..... IOOOOOOOOOOOOOOOOOCOOOO...0.00.... 65 Effects of thiamine, cortisone and alloxan on glucose tOlerance teSt.00......000.00.000.00.0.0.0000...O... ..... O. 67 INTRODUCTION Large doses of cortisone has been shown to stimulate pancreatic islet function in the rat (Baker E£,El~ 1952) and guinea pigs (Hausberger 33 _a_1_l_. 1953), but at the same time to'interfere with the action of insulin on carbohydrate utilization (Ingle 3: El. l9h5). Thiamine has been demonstrated to be necessary for the full effects of insulin on carbohydrate metabolism (Samuels, l9h8). Long (l9Sh) sug- gested that the influence of insulin on carbohydrate metabolism is accomplished through three main metabolic pathways:~ (a) an increase in the amount of glucose or glycogen which is oxidized to CO2 and water (b) polymerization of glucose into glycogen, both in the liver and muscle and (c) conversion of glucose to fatty acids, both in the liver and adipose tissues. This suggests that thiamine, cortisone and inSulin may be interdependent insofar as their actions on carbohydrate and protein metabolism are concerned. In previous reports from this laboratory, Meites (1951, 1952, 1953) observed that large doses of vitamin B12 partially counteracted the inhibitory effects of large doses of cortisone on body, hair and thymus growth in young rats. He demonstrated that large doses of vitamin 812 exerted a beneficial effect by increasing food intake and permitting a greater utilization of carbohydrate. Feng (l95h) reported that in- jections of 2 to h mg. daily of cortisone increased the urinary excretion of radioactive vitamin B12, in rats fed a vitamin BIZ-deficient diet. She also reported that on a diet meeting just the normal requirements for vitamin Big (20 mcg. per kilo of diet), cortisone did not increase urinary vitamin Blg excretion unless at least h mg. of cortisone were injected daily. It was concluded.tlat the amount of vitamin 312 ex- creted in the urine bore a direct relationship to the dose of cortisone given and to the amount of vitamin Blg fed in the diet. The increased excretion of vitamin 812 was explained on the basis of the ability of cortisone to interfere with insulin action and carbohydrate utilization, and hence to reduce the body need for vitamins concerned with carbo- hydrate metabolism. The beneficial effects of large doses of vitamin B13 was explained on the basis of its ability to enhance insulin action and thus counteract the inhibitory effects of cortisone on carbohydrate utilization. It was the purpose of this thesis to determine the nature of and the possible relationships between the actions of thiamine, cortisone and insulin on protein and carbohydrate metabolism and on body growth. Specifically, this thesis will deal with the following questions: 1. What are the effects of large doses of cortisone on thiamine- deficient, thiamine-adequate and thiamine-abundant rats and guinea pigs? Do large doses of cortisone increase or decrease the requirements for thiamine? 2. Through what means does a large intake of thiamine partially counteract some of the catabolic actions of large doses of cortisone? 3. Why do not cortisone-treated rats and guinea pigs fed a thiamine-deficient diet develOp hyperglycemia? Is this due to reduced food intake or to lack of thiamine? h. To what extent does alloxan-diabetes or insulin alter the body need for thiamine? 5. To what extent is thiamine essential for glucose utilization in normal, alloxanized and cortisone-treated rats? LITERATURE REVIEW Introduction Since this thesis deals with the interactions between thiamine, cortisone and insulin as related to carbohydrate and protein metabolism it is pertinent to briefly review some of the salient actions of the former on the latter. The writer felt it necessary to choose from a vast literature, and for the most part the articles reviewed here were selected because of their direct bearing on the thesis problem. T HIMQEE Requirements for thiamine under different conditions. The requirement for thiamine by the rat is a function of several factors. The composition of the diet is very important. It was shown by'Wainio (19h2) that rats receiving a diet containing 62 percent sucrose and 20 percent casein required 33 mcg. of thiamine per day, whereas only 20 mcg. per day was required in a diet containing 6h per- cent casein and 19.6 percent sucrose. Dann (l9h5) was able to maintain rats for more than a year on a thiamine-free synthetic diet containing 80 percent purified casein. Increasing the carbohydrate intake resulted in diminution of urinary output of thiamine (Reinhold 21°. 3;. 19th), indicating a greater use for this vitamin in metabolism. When fat is the sole source of non-protein energy much less vitamin B is necessary for growth than when sucrose is the only source of such energy (Evans gt El. 1929). However, when thiamine is fed at high levels, a sucrose diet can hardly be considered disadvantageous. When rats ingest dextrinized cornstarch as the sole carbohydrate, bacterial synthesis of thiamine and riboflavin is capable of lowering the dietary requirement for these vitamins (Guerrant gt El. l93h). This contrasts with the poor synthesis induced by feeding commercial cornstarch, sucrose, glucose or lactose. Temperature is another factor which influences the requirement for thiamine. Mills gt 3}. (l9h6) showed that 80 mcg. of thiamine per 100 gm. of food was mildly inadequate in the cold and markedly so in the heat for weanling rats, and that rats 12 months old required 120 mcg. in the cold and 200 mcg. per 100 gm. of food in the heat. The requirements for thiamine ranges from 80 to 200 mcg. per 100 gm. of food (Brown §t_§l. l9h9) according to different conditions to which the rat is submitted. .An animal receiving a thiamine-deficient diet shows a loss of weight and food intake is reduced to a low level. After adding a small fraction of a milligram of thiamine to the diet however, appetite is restored and weight increases considerably (Jansen, l9h9). This led to the supposition that thiamine was involved in cell metabolism. The work of Kinnersley and Peters (1929) on polyneuritic- pigeons showed that an increased amount of lactic acid in the brain was the only abnormality found. Kinnersley and Peters (1929) also showed that thiamine plays an important role in carbohydrate metabolism, especially in pyruvic acid metabolism.'Thqrdemonstrated that the oxygen uptake by the brain of polyneuritic-pigeons was less than that of the brain of normal pigeons. The addition of a solution of thianine to minced brain of polyneuritic-pigeons suspended in pyruvic acid solution increased the oxygen uptake of this mixture. This effect was shown to be due not to thiamine itself but to a compound synthesized from the free vitamin which decarboxylates pyruvic acid to acetaldehyde. Effects of thiamine on carbohydrate, fat and protein metabolism. It has been long known that thiamine exerts a preponderant role in carbohydrate metabolism. Neuberg (1911) demonstrated that yeast can cause the decarboxylation of pyruvic acid to acetaldehyde and carbon- dioxide. He termed this enzyme carboxylase. Simola (1932) showed that tissues of rats maintained on a thiamine-deficient diet had a greatly reduced content of carboxylase. It was also shown that washed yeast cells lost the capacity to perform this reaction, and it was established that a diffusible cofactor was required for decarboxylation. Since the enzyme which catalyzes this reaction was termed carboxylase, the cofactor was named cocarboxylase. Lohmann §£_§l. (1937) succeeded in isolating this cocarboxylase, and showed it to be thiamine perphOSphate. The phOSphorylation of ‘thiamine to give cocarboxylase may be effected either chemically or enzymatically with ATP as the phosphorylating agent. Thiamine pyro- phOSphate (TPP) functions not only as the prosthetngroup of the yeast carboxylase but in other reactions representing the principal pathways of pyruvic acid metabolism (Stotz l9h5). Thus, TPP is essential for the simple decarboxylation of pyruvic acid to acetaldehyde and cog, and also for the oxidative decarboxylation of pyruvate to form acetyl-Coi. TPP likewise appears to be essential for the oxidative decarboxylation of alpha—keto glutarate to succinic acid, through succinyl-Col. Green gp‘gl. (l9hl) assumed that the enzymatic decarboxylation of keto-acids other than pyruvic acid also appears to involve cocarboxylase as a cofactor. Lipoic acid is also involved in this system. Thiamine perphosphate is conjugated to lipoic acid (amide linkage) giving rise to lipothiamide perphosphate (LTPP). This seems to be the active form of cocarboxylase. Since only catalytic amounts of LTPP are required to generate acetyl-Coi from pyruvate, the reduced LTPP produced in this transportation must be converted to oxidized LTPP for the reaction to continue over and over again. Oxidation of reduced LTPP is accomplished by DPN. It was shown that TPN does not replace DPN in this reaction (Reed, 1953). The generation of active acetate (acetyl-Col.) from pyruvate by purified pyruvate oxidase preparations from bacterial and animal sources has been postulated by Reed gt El. (1953), as shown in the following reactions: Pyruvate + DPN + CoA.-——+ Acetyl-Coa. + DPNH.+CO2 + H+ (l) Reed.gt El. (1953) studied soluble pyruvate apooxidase preparations from E. coli mutant which cannot synthesize LTPP and revealed that re- \ action (1) did not proceed in the absence of S-LTPP. This reaction comprises two steps: 3‘ ACGLyl-S~ Pyruvate + I’LTPP .___. ,LTPP + co2 (2) s as Acetyl-S\ HS. ’LTPP + Cod-Sn -—+ LTPP + Coi-S-icetyl H8 H5’ fis‘ S\ + ’LTPP + DPN -e ‘ LTPP + DPNH + H (3) as s Lactate reacts in a similar manner to produce acetyl-CoA. but it requires lactic dehydrogenase and pyruvate apooxidase for the reaction to be performed. In thiamine deficiency the amount of free thiamine and of TPP in the tissues fall markedly and there is an increase in the level of pyruvate and lactate in the blood and urine. Accumulation of lactic acid appears to be due to the fact that the action of lactic dehydrogenase is inhibited by high concentration of pyruvate (Sherman gt El- 1936). The role of coenzyme.a and DPN in the oxidative decarboxylation of pyruvate and in the transfer of the acetyl group to the Krebs cycle has been studied by several workers (Korkes gt El- 1951; Littlefield 22.2;- 1952; Sanadi gt al. 1953). They all showed that thiamine pyrophosphate is involved in the reaction. Sure and Smith (1929) showed that hypoglycemia in cases of avitaminosis-B was progressive and not just a premortal state. Since the hypoglycemia was quite evident during a period of prolonged vitamin B depletion without appreciable changes in body weight, they concluded that B vitamins had a determining influence in carbohydrate metabolism. Long before, Magne and Simonnet (1922) had stated that an injection of glucose failed to raise the R.Q. of rats with beriberi but succeeded in cases of inanition. Styron gt_§l. (19h2) showed that in diabetic rats fed a thiamine-deficient diet, glucosuria decreased toward the end of the deprivation period. They also showed that tolerance for glucose was impaired by the end of the depletion period and that the administration of h to 8 times the maintainance dosage of thiamine appeared to improve consistently the tolerance for glucose. Lowry gt El- (l9h5) found, after a depletion period of 16 days, a drOp in urinary glucose of alloxan- diabetic rats. After the injection of 50, 100 and 200 mcg. of thiamine, all rats showed an alleviation of the deficiency symptoms and the excretion of glucose in urine increased consistently. On the other hand, it was claimed that in diabetes induced by alloxan in rats, thiamine requirements were not increased (Styron.§t g1. l9h2; Lowry gt 31. 19h5). Thiamine appears not to interfere in fat metabolism. Boxer gt a}, (l9hh) could not find any difference in the deposition of newly formed fat in rats that were fed on a complete diet compared to rats on a thiamine free diet. They concluded that thiamine was necessary but not the sole condition for the formation of fats from carbohydrate. It was shown that fats exert a "sparing" action on thiamine. Evans gt El. (1929) showed that when a diet containing 50 percent lard but no anti-neuritic vitamin was fed, symptoms of deficiency were not observed. This finding was later confirmed by other workers (Lecoq, 1932; Banerji, l9h0; MacDonald.§t 31. 19h0). It seems that thiamine is not involved in protein metabolism. It was shown that diets containing a high percentage of protein decreased the requirements for thiamine (wainio, l9h2) and the survival of rats maintained on a thiamine-free diet containing 80 percent casein was very long (Dann, 19b5). Effects of thiamine on organ weights. It is well known that thiamine-deficiency induces anatomical and histological changes in certain organs. Stoerck and Zucker (l9hh) - reported that thiamine-deficient rats showed a decrease in thymus weight greater than in riboflavin or pantothenic acid deficient fed rats, but less than in pyridoxine-deficient rats. Even in partial thiamine deficiency the pattern was the same; otherwise the weight losses were not so accentuated. Severe deficiency of thiamine induces weight changes in the adrenals and thymus gland (Deane gt 31. l9h7). These changes were due to a stimu- lation of the adrenal cortex by'ACTH'resulting in increased adrenal weight and production of corticosteroid hormones which induced involution of the thymus. Deane gt_§l. (19h?) concluded that the stimulation of the adrenals occurred earlier in thiamine deficiency than during a comparable degree of inanition, and therefore thiamine deficiency was a stronger stressor than starvation alone. Goodsell (l9hl) found 11 increases in the weight and in the steroid concentration of the adrenals of dogs in acute vitamin Bl deficiency. Similar findings were reported by Skelton (1950) in rats, who found in addition to adrenal hypertrophy and thymus involution, a renal hypertrOphy which was considered to be due to the prolonged negative nitrOgen balance, and failure of deveIOpment of the sex organs due to unbalanced pro- duction of pituitary gonadotrophic hormones. During starvation or chronic undernutrition, the vital organs usually lose weight together with loss of body weight. In pair-fed rats it was observed that starvation produced similar organ/body weight ratios to 3d libitum fed controls, while thiamine-deficient rats showed enlargement of the adrenals and kidneys and little change in heart size (Pecora, 1952). It was concluded that the kidneys and the adrenals were enlarged because of lack of thiamine and not inanition, and that in thiamine-deficient rats starvation pg: g3 exerted little or no effect on organ size. Later, Pecora 23 El. (1953) reported data on the organ/ body weight ratios of the heart, kidneys, testes and pituitary in thiamine-deficient, pair-fed and positive-control rats. They showed that the values for both the thiamine-deficient and the pair-fed groups were the same, except for the kidneys, indicating that changes in organ sizes were not due to the effect of thiamine deficiency but to food restriction. 'The values for the kidney/body weight ratio were considerably greater in the thiamine-deficient group than in the pair- fed rats, due to the effect of thiamine deficiency superimposed on 12 food restriction. The absolute organ weights of thiamine-deficient and pair-fed rats showed equal weight losses, except for the kidneys of the thiamine-deficient group which showed little weight loss. Dunn gt El. (19b?) observed higher organ/body weight ratios for the heart and brain of thiamine-deficient mice, although actual enlargement of the organs was not found. 13 ADREI‘CAL CORTICAL fDRI/iONES Effects of ACTH and cortisone on carbohydrate andgprotein metabolism, and body growth. a) Carbohydrate metabolism The possible interaction between adrenal cortical hormones and carbohydrate metabolism was first demonstrated by Britten (1932) and Britten.gtfl§l. (1937), Long, Katzin and Fry (l9h0) and Ingle (l9hO) demonstrated that large doses of ACTH or cortisone produced transitory hyperglycemia and glucosuria and a negative nitrogen balance. This suggested that blood glucose might come at least in part from gluconeo- genesis from protein. Ingle (l9hl) and Ingle st 31. (l9h5, l9h6, 1951) reported that ACTH or cortisone were able to induce hyperglycemia and glucosuria in force-fed rats. Long gt El. (l9h0) showed that cortisone or other ll-oxysteroids could intensify the glucosuria of partially depancreatized rats. This finding was confirmed later by Ingle (19h0) and Ingle g3 31. (l9hl). Glucosuria induced in normal force-fed rats by the administration of large doses of cortisone acetate daily was not sustained throughout the treatment with the steroid (Lazarow'gp 31, 1950). Diabetic-rats (alloxan-treated or partially depancreatized) were more sensitive to the diabetogenic effects of ACTH or cortisone (Long gt El. 19h0; Kendall, l9h2). ACTH or cortisone can cause alteration of the beta cells in the islets of Langerhans. Kobernickret 31. (1950) observed the develOpment of a diabetic state associated with hydrOpic degeneration in the islet cells in the pancreas of rabbits given 20 mg. of cortisone daily. This observation was confirmed later by Franckson et a1. (1953) who showed a similar degeneration in the rat after prolonged treatment with cortisone. Baker gt 31. (1952) reported that in the rat ACTH induced degranulation, hypertrOphy and increase in number of the beta cells in the islet of Langerhans. Steroid diabetes has been reported to increase resistance to insulin (Ingle, 19h5; Sprague, 1951; Franckson, 1953). Swingle gt 31, (1953) reported the induction of diabetes insipidus in adrenalectomized dogs, with accentuated polyuria and polydipsia, when cortisone was given in high doses. This finding confirmed the work of Sirek et a1. (1952) who reported that a syndrome closely resembling diabetes insipidus could be produced in dogs by daily injections of cortisone in doses of 50 to 300 mg. daily. b) Protein metabolism and body growth It has been demonstrated that large doses of ACTH'and cortisone induced very striking effects on the nitrogenous constituents of the body. Cortisone has been shown to be a potent growth inhibitor in normal young rats (Ingle et 31. l9h0, l9hl; Wells, 19h0; Kuizenga 23 31. 19h}; Winter elval. 1950). A single injection of 0.25 mg. of 15 cortisone given to baby rats at 2h hours of age resulted in a failure to gain weight normally for several days (Parmes §t_al. 1951). They also found that five daily injections of 0.1 mg. of cortisone beginning at 2h hours of age resulted in marked inhibition of growth of the new- born rat and failure to regain body weight three months after the cessation of injections. Kuizenga gt El. (19h3) demonstrated that when immature adrenalectomized rats were treated with cortisone in doses of 0.25 to 1.0 mg. daily, they were able to survive and grow, but at a subnormal rate. Food intake has been shown to be reduced in cortisone treated rats (winter 93 3;. 1950, Meites 1950,- 1951, 1952), and this can account at least in part for the retardation of growth. Injection of ACTH or cortisone in relatively high doses inhibits growth due to failure in the synthesis of protein and protein catabolism. The growth inhibiting potency of ACTH and cortisone parallels the magnitude of tie negative nitrOgen balance (Ingle l9h6). It was demonstrated by Ingle (l9h1) and Ingle 33 El. (l9h5, 19h6) that the temporary diabetes in rats due to the administration of ACTH or cortisone was accompanied by loss in body weight and a pronounced increase in urinary nitrogen. It was also observed that the organism had some ability to adapt itself to the catabolic effects of these hormones. The peak of nitrOgen excretion was not sustained during the administration of ACTH or cortisone. It kad.already been demonstrated (Long gt g1. 19h0; Ingle, l9h0) that large doses of either ACTH or cortisone produced negative nitrogen 16 balance in laboratory animals. Bennet gt 31. (19h8) showed that ACTH induced nitrogen losses in rats with alloxan-induced diabetes and Bennet (19h8) also showed that ACTH increased both urinary glucose and nitrogen excretion of hypophysectomized-diabetic rats. Engel gt 31. (19h9) reported an increase in urea formation beginning three hours after the subcutaneous administration of adrenal cortical extracts (AC5) to fasted nephrectomized rats. This increase was prevented by intra- peritoneal injections of glucose three hours after ACE. He stated that the action of ACE in nitrogen metabolism was on whole protein rather than on amino acids. He suggested that the amount of glucose or of glycogen precursors available might be determining factors in whether protein catabolism would be stimulated by ACE. This view was supported by Ingle gt a1. (1950) who found that cortisone accelerated the rise of amino acids in the blood of liverless rats. It was reported by Goodman 23': a_l_. (1951) that ACTH induced proteinuria in normal rats and aggravated tie proteinuria of renin- treated rats. Clark (1953), employing isotOpic glycine, studied the effects of cortisone on protein metabolism in the rat and reported a decreased protein synthesis in cortisone-treated animals as compared to control rats. Inhibition of incorporation of isotOpic glycine into protein by adrenalectomized and by adrenalectomized-thyroidectomized- parathyroidectomized rats due to the administration of cortisone was demonstrated by Heberman (1950). These observations supported the View of Albright (l9h3) tlat the effects of cortisone-like steroids are arddanabolic rather than catabolic. 17 c) Hair and thymus growth Hair growth has been.shown to be inhibited by percutaneous appli- cation of cortisone or by injection of ACTH (Whitaker 33 El. 19h8; Baker 33 El.- 19118; Winter 33 El. 1950), probably due to induction of atrophic changes in accessory structures of the skin such are known to occur in Cushing's disease. Meites (1952) demonstrated that wlen.large doses of cortisone were injected into rats on a vitamin BIZ-deficient diet, the deficiency symptoms became aggravated as indicated by inhibi- tion of body and hair growth. This was counteracted in part by feeding 20 times the normal requirements for vitamin B12. The involution of the thymus which follows cortisone administration (Antopol, 1950; Winter 22 _a_1_. 1950) was partially prevented by vitamin 1312 (Meites, 1952) but the adrenal atrOphy was not prevented. This finding was confirmed recently by Venkatarman gt 31. (195k). d) Organ weights The adrenal glands increase in weight when an animal is submitted to any type of stress (Selye, 1937; Ingle, 1938, 1939), and it is believed that this response is an aspect of the functional adaptation of the adrenals to increased requirements for cortical hormones by the organism. Ingle (1938) showed that during work, rats Show'adrenal hypertrophy which does not appear if the animal is hypOphysectomized. This hypertophic condition of the adrenals in stressing situations has been confirmed by many authors. 0n the other hand, large doses of ACE may induce atrOphy of the adrenal cortices of rats (Ingle and 18 Kendall, 1937). Later, Ingle gt 2}. (1938) demonstrated atrOphy of the adrenal cortices when pellets of cortisone were implanted. No atrOphy was observed in the absence of the pituitary gland, showing that the mechanism which controls the size and function of the adrenal in stressing conditions lies in the pituitary gland. Sayers §t_al. (19b9) found that release of ACTH was inhibited by the administration of cortisone given prior to subjecting rats to stress. Selye and Dorne (19h2) showed adrenal atrOphy in animals submitted to any stress but treated with significantly high doses of adrenal cortical hormones. However, when rats under a stress (cold) were given ascorbic acid, lgpertrophy of the adrenals was prevented (Dugal EE.§l~ l9h9). intOpol (1950) observed the effects of administering large doses of cortisone to mice: a striking lymphOpenia, loss in body weight, atrophy of the thymus and spleen and reduction in size of the adrenals. Testes, seminal vesicles and prostate were smaller than in the controls and the ovaries also appeared smaller. These findings were confirmed later by Ingle 33 _a_._]_._. (1952). Administration of cortisone or ACTH in high doses to rats or mice causes atrophy of the thymus, spleen and lymph nodes, and a decrease in the number of lymphocytes in the blood (Sprague, 1951). Heart and kidney enlargement, adrenal atrOphy and failure of body growth were reported by Hall gt 31. (1952) following irgection of adrenal cortical hormones. These findings are in disagree- ment with winter 239 _a_l_. (1950), who did not find any effect of cortisone on the Size of the kidneys and heart. 19 Selye (1952) showed that the inhibitory effect of cortisone on body growth could be counteracted by growth hormone (STH), and that the two hormones are also antagonistic with regard to their effects upon a variety of other target organs: the involution of the thymus and adrenal cortices were counteracted by simultaneous STH-cortisone treatment. Testosterone also has been shown to counteract the catabolic effects of cortisone. Albright (19b3) reported that testosterone prOpionate induced anabolic response in patients with Cushing's syndrome and that this therapy produced some improvement in these patients. Methyl testosterone also, when administered simultaneously with ACTH prevented the development of a negative nitrOgen balance (Bartter gt gt. 19h9). These effects of cortisone and_ACTH on body organs are transitory and reversible and generally disappear within 10 to 17 days after discontinuation of injections (Winter gt gt. 1950). 20 ALLOXAN-DIABETES AND INSULIN Effects of alloxan Jacobs (1937) found that the intravenous injection of alloxan produced a transient period of hyperglycemia in rabbits, followed by a severe and fatal hypoglycemia with convulsions and death in 7-10 hours. Intravenous or intraperitoneal injection of glucose could save these animals. Later, Dunn gt gt. (l9h3) found that tie pancreas of alloxan- treated rabbits showed selective necrosis of the islets of Langerhans. This fact led to the discovery that alloxan could be used to induce permanent diabetes in animals. Bailey gt 3}. (l9h3), Goldner 23 El. (19h3) and Dunn gt El. (19h3) demonstrated the possibility of inducing diabetes mellitus in the rabbit, dog and rat, by injecting alloxan. They could protect the animals' life by injecting glucose during the hypoglycemic phase which usually followed alloxan administration. This discovery was very important for the study of experimental diabetes, since it offered a technique for destroying the insulin-producing tissue of the pancreas without requiring pancreatectomy, a technique not feasible in some Species of animals. With the exception of the guinea pig which appears to be resistant to the diabetogenic effect of alloxan, other laboratory animals are susceptible to it. The required dose of alloxan varies with the species and the mode of administration (Bailey, l9h9), the rate of injection 21 (Pincus gt g__1_. 1951;), the pH of the solution (Klebanoff gt 3;. 19511) and sex (Beach.gt_gl. 1951). The diabetogenic action of alloxan is due to its selective necrotic destruction of the beta cells in the islets of Langerhans of the pan- creas. The pancreatic islet lesions produced by a single injection of alloxan show degenerative changes of all or nearly all of the beta cells within 2b hours. Duff _e_t_ 31, (191;?) found hydrOpic degeneration of the islet of Langerhans in alloxan-treated rabbits. This confirmed the findings of Bailey gt gt. (19hh), who also found hydropic degenera- tion of the beta cells in rabbits two months after the deve10pment of alloxan diabetes. Goldner gt g_1_. (19in), Dragsted gt if. (19h3) and Thorogood gt g1. (19h5) reported a high insulin tolerance by alloxan-diabetic dogs. 0n the other hand, Thorogood gt gt. (l9h5) demonstrated that pancreatectomy of alloxan-diabetic dogs resulted in a significant reduction of their insulin requirement. The withdrawal of insulin after pancreatectomy led the animals rapidly into ketosis and coma, while alloxan-diabetic dogs could survive for a long period of time without insulin. This confirms the findings of Young (1939) who found that dogs made permanently diabetic by injections of anterior pituitary extracts required more insulin than depancreatized animals on the same diet. Dragsted (19h3) demonstrated that dogs with partial pancreatectomy required more insulin than after complete pancreatectomy. 22 Effects of insulin on carbohydrate metabolism Long (195h) has proposed tlat tie influence of insulin on the rate of glucose utilization is through three major metabolic pathways: (1) increase in the amount of glucose or glycogen which is oxidized to carbon dioxide and water; (2) polymerization of glucose to glycogen both in the liver and muscle; and (3) conversion to fatty acids, both in the liver and adipose tissue. Experiments with excised diaphragm muscle of rats have shown that extremely small amounts of insulin caused a measurable increase in the uptake of glucose by this tissue. This stimulation of glucose uptake was accompanied by an increased rate of glycogenesis (Stadie gt_gl. l9h7). Insulin also stimulated glucose consumption and glycogen synthesis in the muscle. This was demonstrated by Bouckaert gt gt. (19h?) and Wick gt gt. (1951) in the intact and eviscerated animal, and they concluded that the primary physiological effect of insulin in lowering blood glucose was its increase in the utilization of glucose in the organs and tissues of the body and decreasing the net production of glucose by the liver. Bouckaert and DeDuve (19h?) measured quantitatively the amount of glucose which disappeared in the liver and in the peripheral tissues under the action of insulin. This was done by determining the amount of glucose needed to maintain the blood sugar at a constant concentration after insulin. By comparing normal and hepatectomized animals, it was found that the liver accounted for a large fraction of total glucose 23 utilization. Insulin promotes the net uptake of glucose by the liver, since hepatectomy greatly reduced the amount of glucose necessary to maintain the blood sugar level after a large dose of insulin. Marks gt_gt. (1939) showed that upon the administration of glucose to fasted dogs the R.Q. was promptly increased, denoting an increased utilization of carbohydrate. Hewever, in the fasted diabetic animal the R.Q. was not raised. More direct evidence for an impairment of glucose oxidation in diabetics has been provided by isotOpic experi- ments in which C14-labeled glucose was administered to depancreatized dogs (Feller gt gt. 1951) or to alloxan-diabetic rats (Stetten gt gt. 1951). In each instance a markedly decreased capacity to convert the administered glucose to 01402 was observed. In depancreatized dogs given insulin the rate of glucose oxidation returned to the values ob- served for normal animals. These observations are in accordance with those of Nick gt at. (1951) who demonstrated that the oxidation of radioactive glucose to C02 in the eviscerated rabbit was increased by insulin. Villee and Hasting (19h9) who studied the effect of insulin on carbohydrate metabolism of isolated tissues found that in the rat diaphragm, insulin increased the utilization of glucose and the formation of glycogen and carbon dioxide from labeled glucose. These reactions proceeded at lower rate in the diaphragm from alloxan-diabetic rats. The energy provided from these oxidative reactions is not released in the form of heat, but is used to form substances possessing very 2L high energy which can subsequently be liberated on their breakdown. This energy is in.tle form of phosphate bonds and the important known "high energy phOSphate bond substances" are ATP and creatine phosphate. The former is probably the immediate source for mechanical energy such as is required for the performance of work in muscle contraction, growth and reproduction. The latter is perhaps only a storage form of phOSphate bond energy. Effects of insulin on fat metabolism In addition to the effects of insulin in promoting glucose oxida- tion and glycogenesis, it also induces lipogenesis from carbohydrate. This was demonstrated by Chernick gt gt. (1950) with the use of isotopes, by comparing fat synthesis by liver slices from normal and alloxan— diabetic rats, with or without insulin. Brady gt gt. (1950) also demonstrated that the synthesis of higher fatty acids from acetate tg‘ztttg was accelerated by insulin. The process of fat synthesis from two-carbon fragments requires much energy and must be coupled to energy— yielding reactions. It appears likely that any appreciable decrease in the normal rate of glucose utilization will lead to a corresponding decrease in the energy available for lipogenesis. This view was sup- ported by the observations of Baker gt gt. (1952) who noted that feeding of fructose to diabetic rats induced formation of fat from acetate. In diabetic animals the conversion of glucose to fatty acids is impaired, possibly due to a deficiency in the energy required for fat synthesis from two-carbon fragments. Consequently the tissues largely 25 oxidize fatty acids and lead to the formation of ketone bodies and ketonuria, and acidosis deve10ps. The decreased ability of liver slices of diabetic rats to synthesize fat from acetate is a secondary effect related to impairment of glucose metabolism (Stadie gt gt. l9h0). Bloch gt §_1_. (19148) and Brady 5% gt. (1950, 1951) measured the in- corporation of labeled acetate into fatty acids in surviving rat liver slices. They found that insulin produced a significant increase in the synthesis of fat from acetate by normal liver slices, while liver slices from alloxan-diabetic rats or depancreatized cats almost lost their ability to synthesize long chain fatty acids. Chernick E£.E£- (1950) also demonstrated a decreased formation of fat from labeled glucose as well as a decreased oxidation of glucose to CO2 in liver slices in alloxan—diabetic rats. Pretreatment of the diabetic rats with insulin repaired this inability to utilize carbohydrate for lipogenesis. Stetten and Boxer (19kb) claimed that the major metabolic defect in.tle diabetic organism is its inability to synthesize fat from carbohydrate. Effects of insulin on protein metabolism A further consequence of the decreased utilization of carbohydrate by diabetic animal is an accelerated rate of breakdown of tissue protein. in increased excretion of urinary nitrogen, leading the animal to a negative nitrogen balance, is observed in diabetes. It appears possible that protein formation is favored by insulin in the same way as for lipogenesis. Since the fasted diabetic animal continues to excrete 26 glucose, it appears that gluconeogenesis still proceeds in the absence of insulin. In diabetes large amounts of nitrogen are excreted in the urine (Duncan, 19h2). Bach _e_t gt. (1937) showed that insulin inhibited the deamination of amino acids by liver slices and concluded that insulin inhibited protein catabolism and consequently gluconeogenesis. This nitrogen Sparing action of insulin was further demonstrated by Gaebler gt gt. (1914.2). They showed that whereas anterior pituitary extracts administered to normal animals resulted in nitrogen retention, the same treatment in diabetic animals induced increased nitrogen excretion. Lotspeich (l9h9) observed that insulin accelerated the dis- appearance of amino acids from the blood stream and its appearance in muscle protein, and he claimed.tlat insulin could synthesize protein Further evidence for the anabolic action of insulin on protein was . given by Milman gt gt. (1951). They found that administering growth hormone to hypOphysectomized-depancreatized cats caused no retention of nitrogen but increased glucosuria. In depancreatized cats given a constant supply of food and insulin, the administration of growth hormone resulted in nitrogen storage. This storage is correlated to the amount of insulin given. The authors concluded that insulin was essential for the protein-anabolic effect of growth hormone, and probably an increased secretion of insulin follows the administration of growth hormone to normal animals. More recently Best (1952) showed that it was possible to induce the hyp0physectomized rat to grow by treatment with 1-6 units of insulin per day. 27 Mechanism of action of insulin l) The hexokinase theory of insulin action The most Specific action which has been given to insulin is in the hexokinase reaction suggested by Cori (l9h5-b6) and Price gt a}. (l9h5). The first step in the utilization of glucose for degradation or for glycogen synthesis is the formation of glucose-é-phosphate. The enzyme involved in this reaction is hexokinase (glucokinase) and phoSphate is supplied by ATP: Glucose + ITP Hexokinase Glucose-6 phosphate + ADP Price gt El. (l9h5) showed that tissue from alloxanized rats or rats which received anterior pituitary extracts had subnormal hexokinase activity; In 31332 hexokinase reaction is retarded when anterior pituitary extract is added to the constituents of the reaction. Addition of insulin was shown to abolish this inhibition (Price 21.2l. 19h5). .Adrenal cortical extract also was shown to depress hexokinase activity of muscle preparation taken from alloxan-diabetic animals. This inhibi- tion of adrenal certical extracts also could be released by addition of insulin (Colowick et al. l9b7). It is well known that the anterior pituitary and the adrenal cortex act Opposite to insulin with respect to blood sugar, and the work of Price et 31- (l9h5) helps to explain at least in part, the Hbussay animal and Long's double Operated cats. This assumption of Price 23.3l- (l9h5) of the action of insulin was not confirmed by Smith (l9h9) and Stadie 22.2lo (1950). Nevertheless, the idea that insulin acts in the initial phosphoryla- tion of glucose seems to have considerable merit. The increased glucose uptake of the rat diaphragm by insulin demonstrated by Stadie gt El° (l9h7), can only be explained by assuming that the rate of hexokinase reaction was increased. Further support for the hexokinase theory was supplied by Baker at El- (1952) who showed that fructose, but not glucose feeding improved the ability of liver slices of diabetic rats to synthe- size fat from acetate. 2) The permeability theory of insulin action The rate of transfer of metabolites such as glucose across cell membrane is thought to be influenced by insulin. Levine et_al. (l9h9) attempted to show that insulin accelerated the entrance of a metabolite into the cell. It was found that if galactose were administered to the eviscerated, nephrectomized dog the blood level decreased rapidly and finally became stationary. however, if insulin were added the final concentration was much lower than in the absence of insulin, and it was assumed that insulin caused a transfer of galactose from the blood to the tissues. They concluded that insulin acts upon the cell membranes of certain tissues in such a manner that the transfer of hexoses and perhaps other substances from the extracellular fluid into the cell is facilitated. On the other hand, Wick and Drury (1951) studied the action of insulin on the permeability of cells to sorbitol by determining the distribution of 014-labeled sorbitol in the body fluids of nephrectomized, 29 eviscerated rabbits. They observed that the distribution was not in- creased with insulin. They concluded that the entrance of sorbitol into the cell is dependent on an enzyme mechanism and not a physical one like permeability. The Levine theory still requires confirmation. Control of insulin secretion The elaboration of insulin by the pancreas appears to depend on the blood sugar level. Zunz et al. (1927) found in cross-circulation experiments (in which the pancreatic-duodenal vein of a dog was connected to the jugular vein of a second dog), that when the blood glucose of the donor dog was elevated, blood glucose concentration fell in the recipient animal. This was interpreted to be the result of increased insulin secretion in response to the stimulus of hyperglycemia. More recently Anderson et _l. (19h?) demonstrated that the secre- tion of insulin by the isolated rat pancreas was increased during perfusion for one hour with hyperglycemic fluid. Administration of large amount of glucose for a long period can also induce diabetes in some Species. Dohan gt 3;. (l9h8) reported hydropic degeneration of the beta cells and a diabetic state following glucose administration for long periods of time. Lukens (l9bh) showed that this occurred when large doses of anterior pituitary diabetogenic extracts were given to partially depancreatized cats. Relation of insulin to nutrition The work of Best, haist and Ridout (1939) established the importance of the diet in regulating the production of insulin by the pancreas. 30 .A high fat diet, fasting or insulin administration lowers the insulin content of the pancreas below that on a high carbohydrate diet alone. On the other hand, high protein diets give intermediate values. It appears probable that the carbohydrate in the diet determines the pro- duction of insulin. If insulin production is stimulated at high levels, due either to too high intake of carbohydrate or administration of diabetogenics hormones such as adrenocorticotrophic, growth or adrenocortic hormones (Lukens, l9hh), the islet cells may be exhausted and permanent diabetes may develOp. The relation between the amount of insulin and the carbohydrate which can be utilized appears to be tie principal problem in controll- ing diabetic patients. The decreased utilization of carbohydrate in the insulin-deficient individual is followed by a decreased need for the accessory factors involved in carbohydrate metabolism. Thiamine, niacin and perhaps riboflavin and pantothenic acid are known to be in- volved in carbohydrate oxidation systems as coenzymes. The need for these vitamins is reduced in diabetes, according to Samuels (l9h8), and is also reduced when animals are kept on a high fat, low carbohydrate diet (Evans gt 2.1- 1929). Styron at El- (l9h2) found no significant difference in the length of time required by diabetic and non-diabetic rats on a thiamine-free diet to develOp signs of thiamine deficiency. The urinary output of thiamine has been demonstrated to decrease when the intake of carbo- hydrate increases (Reinhold 33.3l- l9hh). The requirements of B vitamins 31 increase following the administration of insulin, due to the increased utilization of carbohydrate. The effectiveness of insulin appears to depend an the presence of these vitamins. Martin (1937) found that depancreatized dogs on a vitamin-B-deficient diet became resistant to insulin. Insulin resistance and poor glucose tolerance tests were also shown by Burke gt El. (1938) and Lepkovsky §t_al. (1930) in vitamin B- deficient animals. A progressive decrease in the response to insulin in a woman as a result of a deficiency of B-vitamins was reported by Elsom et_al. (l9h0). Upon administration of thiamine and riboflavin the subject became abnormally sensitive to insulin. Vitamin therapy was found by Biskind (l9h5) to be effective in decreasing tie hormone requirement of insulin-resistant diabetes. Feng (l95h) found that vitamin 813 was essential for maximum insulin action. Single injections of insulin, 2.0 units per rat, were more effective in reducing blood glucose in normal, alloxan-diabetic and cortisone-treated rats on a vitamin B12- adequate than on a vitamin Blz-deficient diet. 32 FDCPERII-iFNTAL Experiment I - The Effects of Cortisone on Thiamine- Requirements of Young Rats Purpose Large doses of cortisone may increase requirements for vitamin 812 in the young rat (Meites 1951, 19S2a,b) and the baby pig (Wahlstrom gt §l~ 1951) and for other B-vitamins such as pantothenic acid (Schultz gt a}, 1952), pyridoxine (Kiel, 1953) and riboflavin (Wilwerth El ail. 1953). It was the purpose of the present study to determine the effects of large doses of cortisone on the requirements for thiamine in the young rat. Methods Fifty-three male Carworth rats weighing 65 gm. were fed a semi- synthetic diet from which thiamine was omitted for a period of 10 days, when body growth stOpped and began to decrease. The composition of this semi-synthetic diet is presented in detail in the appendix. After the depletion period the rats were divided into five uniform groups on the basis of body weight, and were kept in metal cages with raised screen bottom at a mean room temperature of 760110 F. Artificial light was supplied from 7.00 a.m. to 6.00 p.m. daily; water and food were avail- able at all times. At the end of this period, the following treatment was inaugurated: Group 1 - positive controls, 2 mg. of thiamine per kilo of diet Group 2 - negative controls, no thiamine Group 3 - no thiamine, 1 mg. of cortisone daily Group h - 2 mg. of thiamine per kilo of diet, 1 mg. cortisone daily Group 5 - 10 mg. of thiamine per kilo of diet, 1 mg. cortisone daily Body weight and food consumption were measured every two days. Cortisone acetate (Cortone, Merck) was given subcutaneously in daily injections. it the end of 18 days the rats were killed and the organs were removed and weighed on a holler-Smith balance to the nearest milligram. In this and in all subsequent experiments, the standard error of the mean was determined by the following formula: 3.13. = d3 n (n-l) Significant differences between groups were determined as follows: S.D. 2 m1 - m2 Vai+h§ Results 1. Effects on body weight (Table I and Figure l). At the beginning of the depletion period the rats averaged 65.0 gm. mn. 0 each and at the end of the depletion period the rats averaged 103.2 each in body weight, at which time the diets were changed according to tie schedule already outlined. At the end of the experiment, the rats which were fed 2 mg. of thiamine per kilo of diet (group 1) showed an . + increase in average body weight of from 101.7:2.3 gm. to 151.3- h.l gm., or a gain of b9.6 gm. The group of rats maintained on the thiamine— free diet (group 2) lost an average of 25.0 gm. in body weight and was far below the average weight of the positive controls (group 1), showing a final average body weight of 80.8:3.l gm. (Figure 1). An even greater loss in body weight occurred in the thiamine- deficient rats which received 1 mg. of cortisone daily (group 3). This group which started at an average of 105.2:2.3 gm. in body weight at the beginning of the experiment finished with an average weight of 6h.8:2.9 gm., showing a loss of bh.b gm. by the end of the 18 days of cortisone treatment. 0n the other hand, when thiamine (2 mg. per kilo of diet) was added to the ration and cortisone was given daily (group b), body weight was maintained about.tle same throughout the experiment. These rats started at an average body weight of 102.0:2.7 gm. and by the end of the experiment the average weight was 110.8:S.l gm., representing a gain of 8.8 gm. 'When the amount of thiamine in the diet was increased five-fold (group 5), these rats were able to almost completely counteract the growth- inhibiting effect of cortisone and the growth curve of this group followed quite closely that of the positive controls (group 1). The initial average body weight of these rats (group S) was 101.8+2.8 gm. and at the end of the experiment was 137.6:h.9 gm., representing an average body weight gain of 35.8 gm. The daily average gain was much greater in the first five days following the addition of thiamine to the diet, as compared to the gain of the positive controls (group 1) during the same period. 35 In addition to the body weight loss and extreme emaciation the thiamine—deficient rats exhibited typical symptoms of deficiency such as hunched posture, paralysis and spinning when picked up by the tail (groups 2 and 3). These symptoms were more pronounced in the thiamine- deficient, cortisone-injected rats (group 3). Eight of ten rats in this group showed pronounced priapism, a phenomenon not seen in any of the other rats. The addition of 2 mg. of thiamine per kilo of diet enabled cortisone-treated rats (group h) to overcome partially the depression of body growth and eliminated all the other gross symptoms of thiamine deficiency. 2. Effects on food intake (Table I and Figure 1). Food intake was affected primarily by the deficiency of thiamine in the diet and later by the administration of cortisone. During the 10-day depletion period all rats showed a progressive decrease in appe- tite, drOpping from a daily average food intake of 9,2 gm. at the begin- ning to 6.1 gm. by the end of the depletion period. When thiamine was added to the diets of groups 1, h and 5, appetite was restored. The rats in group 1 ate an average of 153.0 gm. each and group h consumed 135.0 gm. each, while the rats in group 5 with a higher level of thia- mine consumed an average of 170.0 gm. each, as shown in Table I. The groups which were maintained on the thiamine-free diet (groups 2 and 3) ate less every day. By the end of the experiment the total food intake was about 65.0 gm. for the thiamine-deficient controls (group 2) and 62.0 gm. for the thiamine—deficient, cortisone-treated rats (group 3). 36 3. Effects on organ weights (Table II) Since a deficiency of thiamine has been shown to influence the weights of certain vital organs (Skelton, 1950; Pecora, 1952; Pecora gt 31. 1953), and on the other hand cortisone has been demonstrated to reduce the size of the thymus and adrenals and increase the weight of the kidneys (Ingle, 1938, 1939; Ingle £339. §_1_. 1952; Selye, 1937, 1952; AntOpol, 1950; Meites, 1951, 1952), it was of interest to determine the effects of both thiamine-deficiency and cortisone administration on the weights of the adrenals, thymus, kidneys, seminal vesicles, testes and heart. The results are shown in Table II. Both the actual weights of tde organs and weight of organ per 100 gm. of body weight are presented. The adrenals were greatly increased in size in the rats which were main- tained on the thiamine-deficient diet (group 2) or on the thiamine- deficient diet and cortisone (group 3). Adrenal weights averaged 32 and 26 mg. per 100 gm. of body weight, respectively. When 2 or 10 mg. of thiamine per kilo of diet were fed to the cortisone—treated rats (groups h and 5) the weight of the adrenals averaged the same on a body weight basis as in the positive controls (group 1). The average weight of the kidneys are also increased in both the thiamine-deficient rats (group 2) and cortisone-treated, thiamine- deficient rats (group 3). These two groups showed an average kidney weight of 1550 mg. per 100 gm. of body weight when compared to the positive control rats (group 1), which averaged only 9h0 mg. per 100 gm. 37 of body weight. The cortisone-treated rats maintained on an adequate diet (group h) and on 10 mg. of thiamine per kilo of diet (group 5), had an average kidney weight of 1219 and 1090 mg. per 100 gm. of body weight, respectively. The thymus gland showed a very accentuated involution on the thiamine-deficient diet (group 2), while the rats treated with cortisone on the thiamine-deficient diet (group 3), showed an average of 33 mg. per 100 gm. of body weight. The thymus of the cortisone-treated rats fed 2 mg. (group b) or 10 mg. of thiamine per kilo of diet (group 5) weighed 2 to 3 times as much as in group 3, although the average weights were far below that of the positive controls (group 1). The seminal vesicles were very much decreased in the thiamine- deficient rats (group 2) and in the cortisone-treated, thiamine- deficient rats (group 3), averaging 3b and 73 mg. per 100 gm. of body weight, respectively. The average weight of the seminal vesicles of the cortisone-treated rats maintained on an adequate diet (group b) and on 10 mg. of thiamine per kilo of diet (group 5) were above that of the positive control rats (group 1). Group D showed an average of 202 mg. and group 5 had 211 mg. per 100 gm. of body weight, while the positive controls (group 1) averaged 171 mg. per 100 gm. of body weight. The heart appeared to be slightly larger in both the thiamine- deficient and cortisone-treated rats. The most striking increase was shown by the cortisone-treated, thiamine-deficient rats (group 3) which had an average weight of 600 mg. per 100 gm. of body weight. No con- sistent difference was observed in.tle other rats (groups 2, h and 5) 38 which averaged about 500 mg. per 100 gm. of body weight, although this average was above that of the positive controls (group 1) which showed only h26 mg. per 100 gm. of body weight. The weight of the testes was not consistently altered in this experiment. with tie exception of the cortisone-treated, thiamine- deficient rats (group 3), which showed an average testes weight of 233h mg. per 100 gm. of body weight, the other groups averaged about 1600 mg. per 100 gm. of body weight. Conclusions l. Thiamine-deficiency in young rats induced a decrease in food intake and body weight, as demonstrated in previous experiments by other workers. Cortisone also decreased appetite and caused loss of body weight, and aggravated the symptoms of thiamine deficiency when given to thiamine-deficient rats. It was concluded that normal, and particu- larly excessive amounts of thiamine (10 mg. per kilo of diet) partially counteracted the depressant effects of cortisone on appetite and body weight gains, but the catabolic effects were not completely overcome. 2. Insofar as organ weights are concerned, it was concluded that thiamine deficiency induced atrOphy of the thymus and seminal vesicles and enlargement of the adrenals, kidney and heart, and cortisone- treatment depressed.tle thymus and adrenals and slightly increased the size of the kidney, heart and seminal vesicles. TABLE I 39 EFFECTS OF THIAMINE AND CORTISONE ON BODY WEIGET, FOOD INT} E AND EFFICIENCY OF FOOD UTILIZATION Group and Initial“ Final Avg. Food Intake Number Treatment Body Weight Body Weight Total Per gm. of Rats Gain Body Weight an]. Em. E0 m. 1 (13) 2 mg. thiamine loin-T2,?" 151.5%.1‘” 153 3.0 2 (10) No thiamine 105,612.07 80.8:3.1 o5 - 3 (10) No thiamine+ 105.212. 3 oh.6i2.9 62 - cortisone u (10) 2 mg. thiamine+ 102.0327 110.8311 135 15.3 cortisone 5 (lo) 10 mg. thiamine+ 101.8i2.8 137.61h.9 170 11.7 cortisone * Represents average body weight after thiamine-depletion period. ** Standard error of the mean. (from Wilwerth gt El. 1953) he I - 2 mg. thiamine II - no thiamine . III - no thiamine, cortisone IV - 2 mg. thiamine,.cortisone V - 10 mg. thiamine, cortisone r Daily food intake (em) . 10 150 130 110 90 70 in thiamine ‘\‘-“\ andfior cortisone ‘\-III 50 l l ' I1 L l 1 I I ‘ 1. e 12 16 20 2h 28 D A Y S Fig. 1 - Effects of thiamine and cortisone on food intake and body weight. hl .omce cap mo soaps pumpempm ** .Eoppoo co psmflmz Avon mo .Ew OOH pod vzwflms pew mop :o quMflms compo Hmfiuoq * o.ooflaaoa e.oanmas o.a new m.HHoH m.HHHHHm m.kuammH odomaonoo w.mouooma m.omnbmo a.mauoma m.mumm m.efiuoam m.mmnmmHN o.amH .ooasoeso .we oH m H.mmnaamfi m.afineam e.anao H.HH~H H.mm-NoN w.mo hwmwfi cocoaoooc s.mmueema a.maumom a.au®e m.HuoN b.mmuomm N.mmflmoamfl w.oHH .ooasdaso .mg m a ®.amwoama m.awuooo m.muom «.mwbm H.muma a.wansmmm osoneoooo m.amuaaoH a.wauowm c.NuHN m.HfleH m.eflwo m.owuoome m.eo .osesseoo oz m w.amummmH m.ofims©s a.mumm a.Humm H.musm m.eamamafi n.moflmmma c.mauoam w.m~am n.Huom 5.4uwm w.aaunmoa n.0w osaeowao oz N m.mmmama m.m Home w.mauaaa n.o-®H H.sHHHaH m.mmmmmmH s.amummsa s.aeubso w.aauawm H.Hnem H.Hmummm m.aeuaawm m.HmH reassess .ms N H ** .mE .we .we .mE .mE .mE .Ew mhmcpfix ppmmm mdezxe mamecmpw mmaofimo> menace pzmflmz HmcfiEmm heom Hagen pamEpMmpe mocha «HmOHmz,zeUmO zo mzomHHmoo Q24 MZszHmB mo meoflmmm HH mqmee L2 Experiment II - The effects of cortisone on thiamine- requirements of young rats. ose In this experiment an attempt was made to confirm the findings of Experiment I and in addition to find out whether thiamine doses larger than five times the normal requirement for growing rats would produce an increased counteraction of the depressing effects of cortisone on appetite and body growth. The effects of large amounts of thiamine on cortisone-treated rats whose food intake was limited were also observed. Methods Seventy-five male Carworth rats were divided into eight groups by body weight and fed a thiamine-free ration for 18 days. The initial body weight of all rats averaged h6.710.8 gm. and at the end of the depletion period averaged 73.8i1.6 gm. As in the previous experiment, the rats were housed in metal cages at a mean temperature of 76°ilOF and had artificial light from 7:00 a.m. to 6:00 p.m., daily. With the exception of group b whose food intake was limited, all other groups were permitted unlimited food at all times. After the depletion period of 18 days, the following treatments were begun: Group 1 - positive controls, 2 mg. of thiamine per kilo of diet Group 2 - negative controls, no thiamine Group 3 - no thiamine, 1 mg. of cortisone daily L3 Group h - limited food intake to group 3, hO mg. of thiamine per kilo of diet, 1 mg. of cortisone daily Group 5 - 2 mg. of thiamine per kilo of diet, 1 mg. of cortisone daily Group 6 - 10 mg. of thiamine per kilo of diet, 1 mg. of cortisone daily Group 7 - 20 mg. of thiamine per kilo of diet, 1 mg. of cortisone daily Group 8 - LO mg. of thiamine per kilo of diet, 1 mg. of cortisone daily. Body weight and food intake were measured every two days. Cortisone was given daily in subcutaneous injections of 1 mg. At the end of the experiment, after killing the rats, the organs were removed and weighed. Results 1. Effects on body weight (Table III and Figure 2). The findings are summarized in Table III. At the end of the depletion period all rats averaged 73.8 gm. When thiamine was added in the amount of 2 mg. per kilo of diet (group 1), there was an average body weight gain of 30.0 gm. by the end of 10 days. The rats maintained on the thiamine-free diet (group 2) showed a loss in body weight of 12.8 gm. and were h5.3 gm. below the average body weight of group 1 at the end of the lO-day experiment. Greater loss of body weight was observed in the thiamine-free, cortisone-treated rats (group 3). This group showed a very pronounced cannibalism which resulted in the death of 8 out of 9 rats by the end of the 10 day experimental period. It was because of this group that the experiment was terminated at the end of 10 days. bh Group b, whose food intake was limited to that of group 3 but received 20 times the normal requirement for thiamine, together with cortisone, showed an average body weight loss of 10.8 gm. but all rats survived and showed no gross symptoms of thiamine deficiency. 0n the contrary, they all were in very good condition deSpite the loss of body weight. When cortisone was given to the rats on the diet containing 2 mg. of thiamine per kilo of food (group 5), body weight was maintained at the same level throughout the experiment. 0n the other hand, when higher levels of thiamine were added to the diet (S, 10 and 20 times the normal requirement, groups 6, 7 and 8), there was a body weight gain in all these groups of about 2h.0 gm. per rat. However, body weight did not reach the same level as in the positive control rats given 2 mg. of thiamine per kilo of food without cortisone (group 1). 2. Effects on food intake (Table III and Figure 2). During the depletion period food intake drOpped from 9.0 to h.0 gm. by the end of the 18-day period. When thiamine was fed to groups 1, S, 6, 7 and 8, recovery of appetite and increased food consumption was observed (Figure 2). The rats which were maintained on an adequate- thiamine diet (group 1) had an average total food intake of 82.0 gm. per rat, while in group S on a similar thiamine intake but also given cortisone, the total food intake averaged 61.0 gm. per rat. The groups receiving higher levels of thiamine in their diets (S, 10 and 20 times the normal requirement, groups 6, 7 and 8) showed hS a total food intake above that of group 1, with the greatest value in the group given the highest level of thiamine. In the rats maintained on a thiamine-deficient diet (group 2), total food intake was very low, averaging only about 30.0 gm. per rat during the experiment. When cortisone was given to the thiamine-deficient rats (group 3), appetite was very much depressed and a severe canibalism developed, resulting in the death of 8 rats in this group. The food intake in this group could not be measured. 3. Effects on organ weight (Table IV) The effects of thiamine-deficiency and of cortisone were observed on the testes, seminal vesicles, adrenals, thymus, heart and kidneys. The results are shown in Table IV, as the absolute and relative weights. Data for group 3 could not be obtained because of the death of the animals. The adrenals of tie rats maintained on a thiamine-free diet (group 2) were significantly increased showing an average weight of 38.0 mg. per 100 gm. of body weight, while tie positive control rats (group 1) averaged 25.0 mg. The rats which were partially starved and were treated with cortisone had an average adrenal weight of 2h.0 mg. per 100 gm. of body weight. The cortisone-treated rats fed on adequate diet (group 5) and the other cortisone-treated rats (groups 6, 7 and 8) maintained on higher levels of thiamine showed a very consistent atrophy of the adrenals. D6 The weight of the thymus was very much reduced in the group main- tained on the thiamine-free diet (group 2) and in the cortisone-treated, limited food intake rats (group h) which had an average of 58.0 mg. per 100 gm. of body weight, while the positive control group (group 1) showed an average weight of 25h.0 mg. The other cortisone-treated groups which received thiamine in different amounts (groups 5, 6, 7 and 8) showed significantly greater thymus weights than in groups 2 and h. The weight of the kidneys was increased in the thiamine-deficient rats (group 2) and in the limited food, cortisone-treated animals (group b), as compared to the kidneys of the positive control rats (group 1). The cortisone-treated rats maintained on an adequate- thiamine diet (group 5) and those fed greater amounts of thiamine (groups 6, 7 and 8) showed progressively smaller kidney weights. With the exception of the thiamine-deficient rats (group 2), which showed a significant decrease in the weight of the testes, all other groups had testes of about the same weight when compared on a body weight basis. The seminal vesicles were very much decreased in the thiamine- deficient rats (group 2), weighing only 21.0 mg., while the limited food, cortisone—treated rats (group b) averaged h9.0 mg. Group 5, which was cortisone-treated and fed an adequate diet showed an average seminal vesicles weight of 77.0 mg. while the other cortisone-treated rats fed higher levels of thiamine (groups 6, 7 and 8) showed significant gains in seminal vesicles weight above that of the positive control rats (group 1). L7 Conclusions 1. The findings in this experiment, insofar as food intake, body weight and food efficiency are concerned, confirm the results of the first experiment. 2. The effects of thiamine-deficiency and cortisone on organ weight also followed much the same pattern as in Experiment I. A very pronounced decrease in the size of the thymus and seminal vesicles of the thiamine-deficient rats was observed, and also in the thymus and adrenals of the cortisone-treated rats. in increase in the size of the kidneys was found both in the thiamine-deficient and cortisone-treated rats. This confirms other reports of the effects of thiamine-deficiency on kidney weight (Pecora, 1952). The small increase in kidney weight effected by cortisone can perhaps be attributed to the salt and water retaining action of this hormone. 3. The increase in size of the seminal vesicles appears to be due mainly to increased thiamine and food intake, since cortisone did not increase the weight of the seminal vesicles in the rats given just an adequate thiamine intake (group 5). The increase in the weight of the thymus of the cortisone-treated rats given thiamine is believed to be primarily due to greater food intake. 1L8 TABLE III EFFECTS OF THIAMINE AND CORTISONE ON BODY WEIGHT, FOOD INTAKE AND EFFICIENCY OF FOOD UTILIZATION $1- Group and Initial Final Avg. Food Intake Number Treatment Body Weight Body Weight Total Per gm. of Rats _ Gain Body Weight gm . am . gm . gm . w y”. 1 (9) 2 mg. thiamine 77.311.1 107.333.8‘y 82 2.73 2 (9) No thiamine 7b.8i1.2 62,032.h 3o - 3 (10) No thiamine 73 .011 .2 - - - cortisone h (10) ho mg. thiamine, 73,811.? o3,oil.h 3o - limited food in- take to 3,corti- some 5 (10) 2 mg. thiamine, 72.03152 71.2i'1.6 61. - cortisone o (9) 10 mg. thiamine, 75.5*1.o 99.0io,3 9o h.1 cortisone 7 (9) 20 mg. thiamine, 73.1i2.03 9t.oi3.6 93 h.h cortisone 8 (9) to mg. thiamine, 71.311.S 97.7i2.1 105 h.0 cortisone * Represents the average body weight after thiamine-depletion period. . ** Standard error of the mean. 10 110 90 70 50 h? I - 2 mg. thiafllihe II - no thiamine III - no thiamine, cortisone IV - ho mg. thiamine, limited- -food, cortisone V - 2 mg. thiamine, cortisone VI - 10 mg. thiamine, cortisone VII - 20 mg. thiamine, cortisone VIII - ho mg. thiamine, cortiSone Daily food intake (gm) _' I Body weight VI .VIII -/v'vn ‘ ‘ 0.. y ‘. 0v \ . ....\...... \ ./ \ ...o \\ ‘2‘? K o .‘o...-...\ .-oo \III 1 J 1 l J 2 h 6 8 10 DAYS ON EXPERINWIm Fig. 2 - Effects of thiamine and cortisone on food intake and body weight. 50 .CMoE map mo nopno ppmpcmom ** .Eoppoo :o pnmwoz hpoo mo .ew OOH pea pzwwms one new so mpzwfimz compo depod * m.amnmaaa a.manaam N.OHHoHH H.Hflaa m.emfimea a.HanmaeH oaonaeeoo e.emaaeoa a.mmflwem n.a HmHH H.H-aa o.omueea a.manmmea e.aa .oeeeeaee .me on m w.maumaaa a.manomm 4.0HHOQH m.o|HH m.©HHmmH m.©OHflgm©H ocomfipaoo a.mauaaoa n.0mhaam m.m “mma $.0HHH w.omueNH a.0manwwmfi 0.4a _ .oeeeeeee .me cm a a.emamamma a.amuaam a.omnaaa m.fiuma o.omuaefl w.amamaeea . ozoneeeoo a.monmama a.mmnaem H.wfiuoma H.HHNH n.3flnaefi o.aanoHoH o.aa .oeaeeaee .me 0H 0 m.maaammfl m.oaueam m.afinmoa ®.Hnwa m.anae c.0eauaooa oaonaeaoo a.mmuoww N.Henmma w.ouea m.amma w.euem e.aoaueama ~.Ha .oeaeeaee .me N m ocomfipaoo am m.omunHmH a.oNnmoo a.muam a.ouem m.anaa a.moanmaaa oe eoomueoeaaea N.oauoan a.manwwm ®.Hnnm o.oumH a.ewNm . a.mmanamma o.mo oeaeeaen .mg on a mcomwpaoo . i u n u u . .oaHEdeae oz m a.mgummsa o.~anm~4 H.m Mom a.mnpm m.muam o.wmamwaaa a.0mH0ma m.oHuo0m d.H Ham e.Huam m.HuaH o.afiau0me o.mo oeaeeeee oz N a.mmnomoa 4.0HHng 4.0muamm m.OHmw m.wnmw m.~m Hempa m.eauHmHH a.mmHONm m.emmnam o.Hnam H.anaa maH.HOHnam~H m.eOH oeaeeaee .me m H .mE .ME «ME .ms .ms mime .aw whocpflx annex mdsaze mausoppq moaofimo> mmpmme pzmfioz pcmspwmpy moopu Heeanm aeom Heeaa *mamonz,24omo zo mzomHBmoo Q24 m2H24HmB mo mauflmmm >H mqmda 51 Experiment III - Effects of thiamine and cortisone on body weight, carbohydrate and protein metabolism of guinea pigs. Purpose Since it had been demonstrated that large doses of cortisone in- duced transitory diabetes in guinea pigs (hausberger 23 El- 1953) and slightly increased urinary nitrogen output, it was of interest to determine the effects of cortisone when given to normal and thiamine- deficient guinea pigs on body weight, urinary nitrogen and blood glucose. Methods Forty-three male guinea pigs of an average body weight of 35hil.22 gm. each were placed on a thiamine—depletion ration for a period of 30 days. At the end of this period they were divided into five groups, and housed in elevated, wide-meshed, wire-bottom galvanized iron cages, at a mean room temperature of 76OilOF. They were fed a standard semi- synthetic diet ad libitum and water was available at all times. The composition of the semi-synthetic diet (Reid gt al. 1953) is given in the appendix. Artificial light was supplied from 7:30 a.m. to 9:30 p.m. daily. The five groups were treated as follows: Group 1 - positive controls, 16 mg. of thiamine per kilo of diet Group 2 - negative controls, no thiamine Group 3 - no thiamine, cortisone daily 52 Group h - 16 mg. of thiamine per kilo of diet, cortisone daily Group 5 - 80.0 mg. of thiamine per kilo of diet, cortisone daily. Groups 3, h and 5 received subcutaneous injections of cortisone at a daily dose level of 5 mg. for 10 days, after which the cortisone dose was increased to 10 mg. daily for 10 more days. This increase in dosage was established because the 5 mg. dose showed little effect in raising either blood glucose or urinary nitrogen. Body weight was measured at h to 5 day intervals throughout the experiment. Food intake could not be measured because of excess scattering of food by the guinea pigs. Blood samples were collected after 10 days on the 5 mg. cortisone level and after 5 and 10 days on the 10 mg. level of cortisone treatment. Blood was collected by direct puncture of the heart with a 22 gauge needle and blood glucose was determined with 0.2 ml. of blood by the Hartmann, Shaffer and Somogy micro-method (Hawk gt El. 1951). Twenty- four hour urine samples were also collected at the same time intervals as the blood collections. For urine collections, three guinea pigs from each group were placed in a single metabolism cage in which water was available at all times. To feed the guinea pigs, they were returned to their regular cages for a period of two hours, after being in the metabolism cages for 12—lh hours. After feeding, the guinea pigs were returned to the metabolism cages until the completion of a 2h hour period. Urine was collected in flasks containing citric acid as a 53 preservative. The samples were filtered and aliquots of each were placed under refrigeration. Nitrogen was determined by a standard micro- Kjeldahl method (hawk gt 31. 1951). Both methods are described in the appendix. Results 1. Effects on body weight (Table v and Figure 3) The results obtained in this experiment on body weight are summarized in Table V and are slown more clearly in Figure 3. it the beginning of the experiment body weight averaged 35h.0 gm. in all groups. The animals receiving 16.0 mg. of thiamine per kilo of diet (group 1) showed a normal growth curve and at the end of the experiment had an average body weight gain of lhb.0 gm. Group 2, maintained on a thiamine-free diet, showed a body weight loss of 3h.0 gm. and a final difference in average body weight of 181.0 gm. when compared to group 1. The cortisone- treated animals maintained on a thiamine-free dict (group 3) showed about the same loss in body weight as group 2. When cortisone was given to guinea pigs maintained on a diet containing 16.0 mg. or 80.0 mg. of thiamine per kilo of diet (groups h and 5), body weight was not signifi- cantly depressed and was about the same as the positive controls (group 1). 2. Effects on blood sugar (Table VI) Blood glucose at the end of the depletion period averaged 9h.h mg. per 100 ml. of blood and did not show any significant variation for the thiamine-adequate and thiamine-deficient guinea pigs (groups 1 and 2) during the eXperimental period. In group 3, which was treated with cortisone and maintained on a thiamine-free diet, blood glucose in- creased slightly from 92.0 to 112.0 mg. percent after daily injections of 5 mg. of cortisone for 10 days, and was slightly reduced to 106.0 and 8h.0 mg. of glucose per 100 ml. of blood after daily injections of 10 mg. of cortisone for 5 and 10 days, respectively. Thus, no signifi- cant increase in blood glucose occurred in these cortisone-treated guinea pigs maintained on a thiamine-free diet. Blood glucose rose from 10h.0 to l2h.0 mg. percent when 5 mg. of cortisone were given for 10 days to the guinea pigs fed 16.0 mg. of thiamine, and a still greater rise in blood glucose was observed after the dose of cortisone was increased to 10 mg. per day. Blood glucose increased to lh5.0 mg. percent and after 10 days to 193.0 mg. percent. In the group maintained on the higher level of thiamine (group 5) the increase in blood glucose was greater after 5 days on 10 mg. of cortisone but did not increase further after 10 days on this dose of cortisone. 3. Effects on urinagy nitroggn (Table VI) The urinary nitrogen excretion per 100 gm. of body weight per 2h hours, after the end of the depletion period, averaged 7h.0 me. for the control guinea pigs (group 1). After the addition of thiamine to the diet the amount of nitrogen in urine varied from 72.0 to 78.0 mg. In the guinea pigs maintained on the thiamine-free diet (group 2) the nitrogen per 100 gm. of body weight was slightly but probably not significantly higher than in the thiamine-fed guinea pigs (group 1). The guinea pigs treated with cortisone (groups 3, h and 5) tended to show slight increases in nitrogen per unit of body weight but these were of doubtful significance. Conclusions l. Thiamine-deficiency in guinea pigs, as in rats, produced a decrease in food intake and body growth. The guinea pigs were not as young as the rats and therefore the symptoms of thiamine-deficiency, such as anorexia, lassitude and loss of weight did not appear until after 25 days of thiamine deprivation and were not uniform in all guinea pigs. Some guinea pigs showed considerable resistance to thiamine— deficiency while others died. In general however, thiamine-deficiency was characterized by a slowing of body growth or loss of weight. 2. Cortisone did not appear to significantly affect body weight in the guinea pigs in contrast to the rats. The hormone also did not reduce body weight in the thiamine-deficient guinea pigs. This is in agreement with the work of Hausberger at al. (1953) who showed that large doses of cortisone did not depress body weight in guinea pigs. 3. In the thiamine-fed animals, blood glucose was not significantly elevated when 5 mg. of cortisone daily was given. However, when this dose was increased to 10 mg., a significant increase in blood glucose was observed at the end of 5 and 10 days of treatment. Neither dose of cortisone affected the blood glucose level of the guinea pigs maintained on the thiamine-free diet. This latter appears to be associated with 56 reduced food intake. In an attempt to test this idea, group 5 at the end of the experiment was put on a thiamine-free diet for a period of 35 days, and then 10 mg. of cortisone were injected daily for a period of 10 days. At the end of this time, blood sugar was determined and the results are shown in Table VII. These results show that 5 out of 9 guinea pigs, which had previously shown high blood glucose levels on the diet containing 80.0 mg. of thiamine per kilo of food, did not show any significant increase in blood glucose after being depleted of thiamine for 35 days. The other h guinea pigs were hyperglycemic and perhaps this can be accounted for by animal variability and the longer period of time required for an adult animal to exhibit symptoms of thiamine-deficiency. h. Urinary nitrogen did not show any pronounced alteration by any of the treatments. This is believed to reflect a greater resistance by guinea pigs to cortisone-induced gluconeogenesis from protein. It is probable that the increased blood glucose observed with the higher levels of cortisone was due to the well established ability of the hormone to increase insulin resistance and thus decrease glucose utilization (Ingle, l9bS). TABLE v EFFECTS OF THIQHINE AND CORTISONE ON BODY WEIGHT GAINS 0F GUINEA PIGS Initial Final 'Weight Group Treatment Body Weight Body weight Change gm. 8m. 55“. l 16 mg. thiamine 357'5x 501.0, + lh3.5 ‘ + 21.5 3 90.3“ 2 No thiamine 35h.0 320.0 - 3h.0 : 13.05 i so 02 3 No thiamine, 352.0 300.0 - 52.0 cortisone 1 19.5 I hh.6 h 16 mg. thiamine, 355.5 h7h.0 + 119.5 cortisone 1 13.05 3 25.5 5 80 mg. thiamine, 351.0 h8l.5 + 130.5 cortisone illh.l : 25.7 * Standard error of the mean. 510 hSO 390 330 270 58 16 mg. thiamine no thiamine no thiamine, cortisone 16 mg. thiamine, cortisone 80 mg. thiamine, cortisone H H H lllll Body weight I (an) '- \’ \ .\ 'fi".—-"’-.’II ' ./III \_._. l I l l l 3 6 9 12 15 1 21 DAYS OF EXPERIMENT Fig. 3 - Effects of thiamine and cortisone on body weight. _> .._.—.i¢,._. .cwms one we popes pampcmom a .eooHn no .Hs OOH nod nsnemHHHHa n .powama cowpofimmp ego mo Ono one 94 m .mhdo: 4m mom semoppfic prop pcmmoamoa mosam> comoapfia Han H m.ma H.Ow m.ea m.wa O.Oa ..en OOH\z osonHeeoo a.mHe O.aHe O.Omm 0.0em m.sam mHo noeHse\z .me OH so m.OHHm.OON m.anm.maH m.Oum.aO O.aue.HOH O.euo.ma semen eoon name OH N.ae m.we m.mm H.OO m.me .ew OOH\: oeonHeeoo N.nmm a.msm a.mmm O.Osm H.me MHa eoeHsa\z .ns 0H so O.dHO.OOm m.OHHo.mnH m.OH4.OOH a.muH.OOH n.4um.ea nemsn eoon name m m.a~ m.ee s.OO m.wa a.ms .sm OOH\z enonHenoo m.mHm O.Hmm m.OOm m.mmm O.Oem mHa meaHsa\z .mg m so m.muO.an O.aHO.smH a.muO.tHH H.nuO.Od O.mHO.eOH semen eooHn mane OH O.ws 0.00 O.mm O.4O O.ee .sm OOH\z H.mmm O.sOm o.wOm a.aam m.eOm mHa nonesn\z O.NHO.ea H.mHO.sOH m.suO.ma a.muO.sO ew.mHO.Oa seamen eoon nnHoteeoO .MflE .ms .WE .ME .ME moomfipwoo ocomflonoo ocomfipnoo .oeHeeHae .eeHeeHae .oeHeeHaa oeHeeHea oeHeana mo .oE ow mo .wE 0H oz 02 mo .mE 0H m gnome a macho m macaw m dsono H macho mOHa.amzHOO 2H HZMOoaeHz mawaHeO O24 OoOOOHO OOOHm 2O ezomHanO can aZHaeHme aO mauaaae H> aqmme TABLE VII EFFECTS OF THIIMINE DEFICIENCY 0N BLOOD SUGAR BEFORE AND AFTER CORTISONE ADMINISTRATION Guinea Pig Before Cortisone lifter Cortisone Number mg. % mg. % i 8h.o 116.0 2 92.0 30h.0 3 8h.o 8h.o h 8h.o 88.0 S 92.0 9b.0 6 lhh.0 260.0 7 10h.0 172.0 8 8h.0 10b.o th.O 220.0 61 Experiment IV - The effects of thiamine, cortisone and alloxan on body growth, blood sugar and urinary nitro- gen in rats. ose Since it had been demonstrated that the administration of cortisone failed to induce hyperglycemia in most thiamine-deficient guinea pigs, even when given in doses of 5 and 10 mg. this experiment was undertaken to determine if this was due to deprivation of thiamine per g2 or merely to reduced food intake. Alloxan was also given to see whether thiamine would influence tie action of this substance in producing diabetes. Methods Eighty male, adult Carworth rats were fed a semi-synthetic diet from which thiamine was omitted for a period of 20 days. At the end of this time the first symptoms of thiamine deficiency appeared, as indi- cated by anorexia, lassitude and body weight loss. water and food were available at all times. Blood samples were collected every 5 days from the tip of the tail, and blood glucose was determined by the micro- method of Folin and Malmros (Hawk _e_3_t_ 31:. 1951), using 0.1 ml. of blood. Twenty-four hour urine samples were collected every 5 days and the total urinary nitrogen was determined by the standard micro-Kjeldahl method (Hawk _e_t _a_1_. 1951). After the 20-day depletion period the rats were divided into eight groups on the basis of body weight, and were treated as follows for an additional period of 28 days: 62 Group 1 - positive controls, 2 mg. of thiamine per kilo of diet Group 2 - negative controls, no thiamine Group 3 - 2 mg. of thiamine per kilo of diet, alloxan Group 11 - no thiamine, alloxan Group 5 - 20 mg. of thiamine per kilo of diet, but limited in food intake to group h, alloxan Group 6 - 2 mg. of thiamine per kilo of diet, h mg. of cortisone daily Group 7 - no thiamine, h mg, of cortisone daily Group 8 - 20 mg. of thiamine per kilo of diet, but limited in food intake to group 7, h mg. of cortisone daily. The limited-food groups (5 and 8) were fed a ration containing 20 mg. of thiamine per kilo of diet but their food intake was limited to that eaten by the thiamine-deficient rats. After a h8—hour fasting period, alloxan was injected subcutaneously in the rats of groups 3, h and 5 at a level of 16.0 mg. per 100 gm. of body weight from a 3 per- cent freshly prepared solution. Cortisone was given to groups 6, 7 and 8 in daily subcutaneous injections of h mg. per rat during the experimental period. Body weight and food intake were measured every two days, and urinary nitrogen and blood glucose were determined every five days. For urine collection five rats were placed in a single metabolism cage in which food was present in non-scattering metal feeders and water was available at all times. The rats remained in the cages for 2h hours and urine was collected in flasks containing 1 gm. of citric acid as a preservative. 63 After 15 days, the treatments for the thiamine-deficient rats of groups h and 7 were reversed, i.e., an intraperitoneal injection of 10 mg. of thiamine was given and 2 mg. of thiamine per kilo of diet was added. The daily food intake of groups 5 and 8 was limited to that consumed daily by groups h and 7, reSpectively. Results 1. Effects on food intake and body weight (Table VIII and Figure h). The results are summarized in Table VIII and Figure b. As in previous experiments during the depletion period, appetite was markedly depressed and body weight was greatly reduced. When 2 mg. per kilo of diet were fed to groups 1, 3 and 6, appetite was restored and body weight increased steadily. No great difference was observed between the positive controls (group 1) and the alloxan treated rats on 2 mg. of thiamine per kilo of diet (group 3). 0n the other hand the rats on 2 mg. of thiamine per kilo of diet and cortisone (group 6), showed less increase in food intake and body weight was maintained at the same level during most of the experiment. The rats maintained on the thiamine-free diet during the first part of the experiment (groups 2, h and 7) showed a constant decrease in body weight and their food intake became gradually reduced. The thiamine- deficient, cortisone-treated rats (group 7) showed a more pronounced loss of body weight than any other thiamine-deficient group. This harmful effect of cortisone on thiamine-deficient rats was previously demonstrated in Emperiments 1 and 2. The two limited-food groups 6h (5 and 8), which received 20 mg. of thiamine in their ration, followed groups h and 7 very closely. Once more, the limited-food group which received cortisone (group 8) showed a more pronounced decrease in body weight than the alloxan-treated rats (group 5). After an intraperitoneal injection of 10 mg. of thiamine per rat to the previously thiamine-deficient rats (groups h and 7), appetite and body weight showed consistent increases. However, the alloxan-treated rats (group b) showed a greater increase in food intake and body weight than the cortisone-treated rats (group 7). When the limited-food groups (5 and 8) received larger amounts of food to correspond with the in- creased appetite in groups h and 7, group 5 (alloxan-treated) showed an increase in body weight similar to group b, and group 8 (cortisone- treated) stowed an increase similar to group 7. 2. Effects on blood sugar (Table IX) The effects of alloxan and cortisone on blood glucose in adequate and thiamine-free diet fed rats varied according to the diet given. After the end of the deprivation period the average blood glucose values were about 93.0 mg. per 100 ml. of blood. is can be seen in Table IX, group 1, after the addition of thiamine to the ration, showed an increase in blood glucose which remained at 120.0 to 127.0 mg. percent throughout the experiment. The thiamine-deficient control rats (group 2) showed values varying from 91.0 to 96.0 mg. percent. When alloxan was given to the rats on an adequate diet (group 3), blood glucose levels rose to an average of 260.0 mg. percent and the rats showed polydipsia and polyuria. In the thiamine-deficient, alloxanized rats (group b) only a slight and probably insignificant increase in blood glucose was observed. The amount of sugar excreted in the urine could not be determined because the diet contained 62 percent of glucose (Cerelose) and some of this food contaminated the urine samples. The limited-food rats which were fed thiamine showed a very consistent increase in blood glucose 5 days after alloxan treat- ment (2bh.0 mg. %). The cortisone-treated rats (groups 6, 7 and 8) showed greater increases in blood glucose when thiamine was fed than when it was with- held. When group b (alloxan-treated) and group 7 (cortisone-treated were injected with 10 mg. of thiamine, a very consistent increase in blood glucose was shown by the alloxan-treated rats (group b) and a lesser increase occurred in the cortisone-treated rats (group 7). The limited-food groups (5 and 8) closely followed groups h and 7, respectively, as shown in Table IX. 3. Effects on urinary nitrggen (Table IX) The average daily excretion of total urinary nitrogen per 100 gm. of body weight was about 85.0 mg. by the end of the deprivation period in all groups. After the addition of thiamine to the diet of the positive control rats (group 1), daily nitrogen excretion increased slightly, while the thiamine-deficient rats (group 2) showed no change in nitrogen. When alloxan was given to rats on an adequate diet (group 3), the daily urinary nitrogen excretion rose gradually from 66 86.0 to l8h.O mg. per 100 gm. of body weight. On the other hand, no increase in nitrogen was found in the alloxan-treated rats kept on a thiamine-free diet (group b). However, the alloxan-treated rats on a limited—food intake but given thiamine (group 5) showed a very con~ sistent increase in urinary nitrOgen. In all rats receiving cortisone (groups 6, 7 and 8) nitrogen increased almost at the same rate. When thiamine was injected into group h(alloxan-treated) and group 7 (cortisone-treated) there was a pronounced increase in the excretion of urinary nitrogen. This was also true of the corresponding limited- food groups (5 and 8). Conclusions l. Deprivation of thiamine depressed food intake and body weight, and reduced the efficiency of food utilization, as demonstrated in previous experiments. Alloxan administration slightly reduced and cortisone-treatment significantly decreased the efficiency of food utilization in the thiamine-adequate rats as compared to the positive control rats. Limitation of food intake with adequate thiamine intake produced no depressing effects on either body weight or food efficiency. Otherwise the rats showed a much better appearance. 2. When alloxan and cortisone were given to thiamine-adequate rats, hyperglycemia was produced. This was not evident when alloxan was given to thiamine-deficient rats. This contrasts with the findings of Feng (l95h), who found that in vitamin BIB-deficiency, administration of alloxan or cortisone induced hyperglycemia and glucosuria. In fact 67 cortisone induced a greater hyperglycemia in the vitamin B-lZ-deficient than in the vitamin BIB-adequate rats. This may be due to the differ— ences in the actions of thiamine and vitamin 812 on carbohydrate metabolism. 3. Urinary nitrogen excretion in cortisone-treated rats was in- creased at the same rate in all groups, irreSpective of the amount of thiamine intake. Hewever, hyperglycemia was not evident in either the thiamine-deficient rats or in the rats permitted only limited food intake. Tlds suggests the possibility that the reduced food failed to provide sufficient carbolwdrate to the organism or that gluconeogenesis from protein was at a low level or both. Only the alloxan-treated rats on the thiamine—deficient diet did not show any increase in urinary nitrogen. The increase in blood glucose and urinary nitrogen in the limited—food, alloxanized rats given thiamine suggests that thiamine rather than increased caloric in- take is primarily responsible for the manifestation of the diabetogenic action of alloxan. On the other hand, reduced food intake rather than a thiamine deficiency appears to be responsible for the absence of a pronounced diabetogenic effect in cortisone-treated rats. TlBLE VIII EFFECTS OF THIAMINE, CORTISONE AND ALLOXAN ON BODY WEIGHT, FOOD INTAKE AND EFFICIENCY OF FOOD UTILIZATION Group and Body-weight Avg. Food Intake Number Treatment Initial Middle Final Total Per gn. Gain of Rats Body'Weight {97131.1 gin.2 £ng g"). g”. 1 (1o) 2 mg. thiamine 183.8 220.0 282.1 262 8 h.3 i“6.93 $7.6 :10.1 2 (10) No thiamine 185.1 1h9.1 152 0 62.3 - i9.2 10.3 2:7.9 3 (10) 2 mg. thiamine, 182.0 19u.3 229.3 239.5 h.9 alloxan 110.1 $8.2 t1h.1 h (10) No thiamine, 162.3 139.5 195.2 150.9 7.9 alloxan i7.h $8.7 -iO.9 5 (11) 20 mg. thiamine,183.2 1h6.3 200.8 150.9 6.3 limited-food to 1“6.5 :8.2 $12.3 h, alloxan 6 (11) 2 mg. thiamine, 18h.5 165.0 167.8 17h.2 - cortisone i5.9 i6.2 i9.3 7 (11) No thiamine, 185.h 1gh.h 151.8 112.5 - cortisone i5.9 -h.7 $5.5 8 (10) 20 mg. thiamine 185.8 119.0 187.5 112.5 — limited-food to ih.6 $3.2 $8.1 7, cortisone 1 Average body weight after thiamine—depletion period. ‘ 2 Average body weight at the date of thiamine injection and food treatment reversal. 3 Standard error of the mean. 20 15 250 220 190 160 130 69 |> I 7 2 mg. of thiamine II - no thiamine III 2 mg. thiamine, alloxan no thiamine, alloxan 20 mg. thiamine, limited-food, alloxan 2 mg. thiamine, cortisone no thiamine, cortisone 20 mg. thiamine, limited-food, cortisone VI VII VIII <3 lllll ‘ haily food intake (5“) IV-V Body weight (an) ‘VII . '\-—f_;’/"VIII '\. /"——r/"I‘k“—II \?s\ ‘~. .’0 [I k \ ./’-- I, .‘L ’/ ‘-/ 1 1 1 ”‘1’ J 1 I h 8 12 16 20 2 u 28 DAYS ON EXPERIMYNT Fig. h - Effects of thiamine, cortisone and alloxan on food intake and body weight. TABLE IX EFFECTS OF THIAMINE, CORTISONE AND ALLOXAN ON BLOOD GLUCOSE AND URINARY NITROGEN IN 8115 B. G. - blood glucose (mg. per 100 m1. of blood) N/rat — urinary nitrogen (mg. per rat/2b. hours) N/lOO — urinary nitrogen (mg. per 100 gm. body weight/22 hours) Daysl Group 1 Group 2 Group 3 Group h Group 5 Group 6 Group 7 Group 8 new. Notan him. NotMmm Lm.fwd mam. NetMmm Lmnrma illoxan illoxan Thiam. Cortis. Thllhh Alloxan Cortis. 0 B. G. 91.0 93.7 98. 8 98.11 811.11. 95. 92.3 98.2 i8.22 1“5.5 —5. l 18.7 -3. 8 :8.9 i8.05 i3.1 N/rat 156.8 165.7 169.8 163.8170 2 163. 0 181.6 163.0 N/lOO 80.0 85.0 86.0 86.0 83.9 88.0 88.0 83.0 5 B. G. 117.? 93.9 2605108.2 281.0 155 0 103 8 121.9 - -8.7 i3.8 ~25.5 15.7 :10. 2 iL.8 i2 .2 1“3.1 N/rat 231.1 162.9 220.2 179.1 288.8 255.7 172.: 253.8 N/lOO 127 0 90.0 121.0 99.0 157.0 139.0 96.0 137.0 10 B. 0. 120.2 92.6 262.0 117.2 190.0 167 2 109.0 105.7 —8.1 52.9 i21.1 i3.6 $8.6 i5.2 f8.0 $2.6 7 N/rat 251.5 156.9 226.5 181.1 225.8 257 2 208 2 228.7 : N/lOO 129.0 98.9 128.8 91.1 135.0 187.0 137 0 185.7 15 B. G. 127.5 96.3 285.0 118.3 162.7 175 0 11“ 129 7 +8. 92.8 :1 .5 15.8 :95 18.6 2‘9. £238 N/rat 237.5 152.5 305.9 126.3 191.7 219.3 175.1 176.2 N/lOO 111.0 101.3 161.0 . 89.3 127.0 129.6 139.0 181.7 Reverse food treatment for groups 8 and 7 20 B. G. 129.0 95.1 258.3 268.8 831.2 169.7 132.1 111.2 59 6 53.1 i21.5 i15.1 i17.t i7.3 i8.2 2“-3.9 N/rat 278.3 159.6 376. 271 8 876.8 251.1 833.2 267. N/lOO 121.6 118.8 175.8 178 0 287.0 186.5 303.0 ‘19.? 25 B- G- 135-0 21.2 225.6 283.7 837.6 170.6 129.1 121.2 -..‘3.7 -2.1 117.6 33195 $203 $6.5 $10.6 $8.2 bf/rat 262 .2 157 .1 806 .3 382 .1 562 .5 239 .9 387 .3 308 .0 V100 109.6 105.5 179.3 189.2 293.0 182.5 227.3 220.2 - 28 B- G- 127-3 93.1 231.5 279 3 833.8 172.2 127.1 120.6 , ~ 1.7.7. -3.8 116.5 2117.9 3118.8 i7.2 29.1 15,7 N/rat 263 .5 199.8 8.23.2 8.8.0.1 878.1 283 .2 328.0 381.2 N/100 108.3 105.6 188.3 219.8 229.5 152.3 207.1 223 7 1 Days after alloxan or cortisone treatment. 2 Standard error of the mean. f"!======!\ 71 Experiment V - The effects of thiamine, cortisone, alloxan and insulin on body growth, blood glucose and urinary nitrogen in rats. Purpose In this experiment an attempt was made to confirm the results obtained in Experiment IV on blood glucose and urinary nitrogen as induced by the administration of cortisone and alloxan to rats main- tained on adequate or thiamine—deficient diets. Insulin was given to determine the response of the rats previously treated with cortisone or alloxan and maintained on thiamine-adequate or deficient diets. Methods Sixty male, adult Carworth rats with an average body weight of 225.0 gm. each were placed on a semi-synthetic, thiamine-deficient diet for a period of 25 days until their average body weight decreased to 196.0 gm. The rats were then divided into six uniform groups on the basis of body weight and were housed in metal cages with raised screen bottom. Water and food were available at all times. After this period, the following treatment was established for each group: Group 1 - positive controls, 2 mg. of thiamine per kilo of diet Group 2 - negative controls, no thiamine ,Group 3 - 2 mg. of thiamine per kilo of diet; 17.0 mg. of alloxan per 100 gm. of body weight given in one single subcutaneous injection Group h - no thiamine; 17.0 mg. of alloxan per 100 gm. of body weight given in one single subcutaneous injection 72 Group 5 - 2 mg. of thiamine per kilo of diet; 8 mg. of cortisone daily Group 6 - no thiamine; 8 mg. of cortisone daily. Body weight and food intake were measured every two days during the l6-day period of the experiment. Blood samples were collected every 5 days for a 15-day period and blood analysis were made according to the Folin and Malmros micro-method (Hawk gt El- 1951). Twentyefour hour urine samples were collected at the same time intervals as the blood collections, and nitrogen was determined by the standard micro- Kjeldahl method (Hawk _e_t 3;. 1951). After the 15-day period, 2.0 units of insulin (Illetin, Lilly) were injected into the rats of groups 1, 3 and 5, and 0.2 units into the rats of the other groups. Blood samples were collected four hours later (Feng, 1958). Because of the weakness and low food intake of the rats of groups 2, h and 6 (thiamine-deficient rats), the insulin dose was less in these groups than in those on a thiamine-adequate diet. Results 1. Effects on food intake and bodyiweight (Table X and Figure 5) The results are summarized in Table X. Deprivation of thiamine induced a markedly decrease in food intake and body-weight. ‘When 2 mg. of thiamine per kilo of diet was added to the ration of groups 1, 3 and 5, appetite was promptly restored and body weight increased. When the alloxan and cortisone—treated rats (groups 3 and 5) are compared to the positive control rats (group 1), it is observed that the 73 efficiency of food utilization was greater in the alloxan than in the cortisone-treated rats. The rats maintained on a thiamine-free diet (group 2) and those on a thiamine-free diet with alloxan (group h) or cortisone (group 6) did not show any significant variation in food intake; otherwise their appetite was very poor and they showed a pro- nounced loss in body weight. 2. Effects on blood sugar (Table XI) After the end of the deprivation period blood glucose averaged 92.8 mg. per 100 m1. of blood. Two mg. of thiamine added to the diet of the positive control rats (group 1) induced a progressive increase in the average blood glucose to 125.0 mg. percent, while the rats main- tained on a thiamine-free diet averaged 93.9 mg. percent of blood glucose throughout the experiment. 'When alloxan was given blood glucose levels rose to 381.0 mg. per- cent for the rats maintained on a thiamine-adequate diet (group 3). On the thiamine-deficient alloxanized rats (group 8), only a slight and probably not significant increase in blood glucose was observed. The cortisone-treated rats fed 2 mg. of thiamine per kilo of diet (group 5) showed a consistent and progressive increase in blood glucose during the experiment, reaching 172.6 mg. percent by the end of the experimental period. When cortisone was given to thiamine deficient rats (group 6), blood glucose only rose to 121.2 mg. percent. 7b 3. Effects on urinaryflnitrogen (Table XI) The total urinary nitrogen excretion per 100 gm. of body weight per 28 hours, after the end of the depletion period averaged 86.0 mg. ‘When 2 mg. of thiamine were added to the diet of the positive control rats (group 1) urinary nitrogen slightly increased during the experi- ment, while it remained at about the same level for the thiamine- deficient control rats (group 2). Alloxan-treated rats on a thiamine-adequate diet (group 3) showed a progressive increase in urinary nitrogen which by the end of the experiment averaged 162.0 mg. per 100 gm. of body weight. 0n the other hand, alloxan-treatment of thiamine-deficient rats (group 8) did not induce any increase in the excretion of nitrogen in the urine. Corti- sone administration somewhat increased nitrogen excretion in both thiamine-adequate (group 5) and thiamine-deficient rats (group 6). The greater increase (189.0 mg./lOO gm. body weight) was shown by group 5 (thiamine-fed) while the thiamine-deficient rats (group 6) increased only to 133.0 mg. per 100 gm. body weight. 8. Effects of insulin on blood sugar (Table x11) The effects of insulin on blood sugar are summarized in Table XII. In groups 1, 3 and 5, which were fed on a thiamine-adequate diet, insulin induced the greatest percent decrease in blood glucose in the alloxan-treated rats (group 3), with an average decrease of 62.8 per- cent. The cortisone-treated rats (group 5) were the most resistent to the hypoglycemic action of insulin, showing only a 38.5 percent 75 decrease in blood glucose compared to 53.1 percent for the positive- control rats (group 1). The rats maintained on a thiamine-free diet (group 2) showed a 51.6 percent average decrease in blood glucose, while the alloxan treated, thiamine-deficient rats (group 8) showed a decrease of 35.2 percent and the cortisone-treated rats (group 6) a decrease of only 33.0 percent. Conclusions 1. A deficiency of thiamine reduced food intake and body weight, and markedly reduced the efficiency of food utilization, as observed in previous experiments. illoxan administration to rats fed a thiamine- adequate diet slightly reduced the efficiency of food utilization while cortisone decreased it markedly. N0 aggravated effects were noted in thiamine—deficient rats given alloxan, but when cortisone was given to thiamine-deficient rats an enhancement of the deficiency'symptomS'was> observed. 2. Blood glucose was increased slightly or not at all in thiamine- deficient rats given either alloxan or cortisone, as observed in previous experiments, while in thiamine-adequate rats a significant increase in blood glucose was observed. It was reported previously by Lowry gt El. (1985) that a reduction in glucosuria was observed in diabetic rats submitted to a l6-day thiamine-depletion period, and that administration of thiamine restored the glucosuria. This and the previous experiment confirms these observations. In the cortisone- treated, thiamine-deficient rats, hyperglycemia did not follow the 76 increased nitrogen excretion by these rats. It is possible that the decreased food intake hid any manifestation of gluconeogenesis from protein. 3. The increase of urinary nitrogen in alloxan-treated rats fed a thiamine-adequate diet, and in cortisone-treated rats has already been noted in previous experiments. In the former this was due to the absence or reduction in the amount of insulin available, which has an anti-catabolic effect on protein. In the latter, this was due to the well known catabolic effect of cortisone on protein metabolism. In thiamine-deficient rats, administration of alloxan did not result in any increase in urinary nitrogen. This effect is presumably due to the reduced food consumption associated with thiamine deficiency. h. Insulin was less effective in lowering blood glucose in thiamine- deficient rats than in thiamine-adequate rats, with the possible ex- ception of the thiamine-adequate controls. This suggests that thiamine is necessary for the full action of insulin, and is in agreement with the view that there is an increased resistance to insulin in B vitamin deficiencies (Samuels 1988). Burke 23 El. (1938) had previously reported a reduced effectiveness of insulin in the absence of thiamine. The cortisone-treated groups showed an increased resistance to insulin both on the thiamine-adequate and deficient diets, confirming the findings of Ingle (1985) and Feng (19Sb) who showed that cortisone increased insulin resistance. Feng (1958) reported that insulin was twice as effective in reducing blood sugar in cortisone-treated rats 7? fed a diet abundant in vitamin 312 than on a diet deficient in vitamin 812. Hewever, since the same dose of insulin was not given to both groups in this experiment, it can only be assumed that insulin was more effective in the thiamine-treated group. 78 TABLE X EFFECTS OF THIAMINE, COHTISONE AND ALLOXAN ON BODY WEIGHT, FOOD INTAKE AND EFFICIENCY OF FOOD UTILIZATION Group Treatment InitialW Final Avg. Food Intahe_ Body Weight Body Weight Total Per gm.Gain Body-Weight __Em. gm. gm. gm. 1 2 mg. thiamine 197.2:6.9** 2hh.5:7.3** l93.5 b.06 2 No thiamine 196.1:6.3 155.7:S.6 38.5 - 3 2 mg. thiamine, 196.9is.9 215.3f8.9 16h.o 8.h5 alloxan b No thiamine, 198.2:7.2 159.1:8.1 h2.3 - alloxan 5 2 mg. thiamine, 199.1:6.7 189.6i7.6 169.5 - cortisone 6 No thiamine, 198.0i7.5 139.515.8 h1.8 - cortisone * Average body weight after thiamine-depletion period. *% Standard error of the mean. 79‘ I - 2 mg. thiamine II - no thiamine III - 2 mg. thiamine, alloxan IV - no thiamine, alloxan V - 2 mg. thiamine, cortisone VI - no thiamine, cortisone 20 -' Daily food intake 15 _ 10 - Sb '~— II #_ <___ VI I Zhor Body weight (gm) 210 3 o 180 .- ‘oLf's3g3... .°-:¢c:;& . °"-. \- ". \ ‘ ‘..‘~“\.“ .‘. “a... ‘~~._\_. -... . Iv 150 - .I'0........ \.;'(~ II - . VI 120 L i l J L l l J 2 l; 6 8 10 12 1h 16 DAYS ON EXPERIMENT Fig. 5 - Effects of thiamine, cortisone and alloxan on food intake and body weight. TABLE XI EFFECTS OF T AI'IINE , COEiI‘ISOI-LE AND ALLOXAN ON BLOOD SUGih AND UhINifiY NITROGENl GO Group Treatment Initial2 S davs 10 days 15 days mg. mg. mg, mg. 1 Positive controls + 3 + Blood glucose 92.6-h.2 115.3ih.73 12o.eih.13 125.2-h.63 N/rat 17l.h 2h2.6 269.3 276.6 N/lOO gm. 88.6 121.3 115.6 113.h 2 Negative controls + + Blood glucose 9h.8-5.5 93.7i3.b 92.9:3.l 9h.l-h.5 N/rat 151.7 1L9.6 1h6,o 139.9 N/lOO em. 83.1: 87.5 927.2 90.3 3 ideq. thiamine illoxan + + + + Blood glucose 91.7-h.6 396.8-23.S 389.3-21.2 381.6-23.7 N/rat 15b.l 235.0 310.9 325.6 N/lOO gm. 85.6 129.3 153.2 162.8 b No thiamine Alloxan + + + + Blood glucose 93.9-b.7 103.9-3.6 112.0-5.7 ll6.2-S.8 N/rat 1h9.5 153.2 15h.l lhl.6 N/lOO gm. 81.3 92.3 9h.6 89.1 5 Adeq.1fldamine Cortisone + + + + Blood glucose 96.3—h.9 16S.S-<.8 170.0-S.9 172.6-7.6 N/rat 16h.b 257.0 278.8 281.7 N/lOO gm. 89.b 135.3 1L6.2 1h9.1 6 No thiamine Cortisone + + + Blood glucose 87.8-h.1 118.6-3.8 121.3-h.6 121.2-3.9 N/rat 159.6 211 o 19h.b 196.0 N/lOO gm. 86.7 121.3 129.6 133.2 1 Represents total nitrogen per 2h hours. 2 it the end of the depletion period. 3 Standard error of the mean- TABLE XII 81 EFFECTS OF INSULIN ON BLOOD GLUCOSE AFTER PRETREA‘MENT WITH THIAI‘EINE, COhTISONE Oft ALLOXAN Group Treatment Blood g1ucose,_mg._percent First Trial Second Trial 1 2 mg. thiamine % . Before insulin 117.5ih.2 119.0i3.9 .After insulin 55.235.6 5h.7ih.8 mg. decrease 62.3 6h.3 Percent decrease 52.1 5h.1 2 No thiamine + + Before insulin 9h.3-5.5 91.8-3.h After insulin h6.2i6.3 hb.Oih.8 mg. decrease b8.l h7.8 Percent decrease 51.1 52.1 3 2 mg. thiamine, alloxan Before insulin 2h6.2:20.3 2h9.h:2l.9 liter insulin 95 .033 ,o 93 .71” 9.3 mg. decrease 151.2 155.? Percent decrease 61.5 62.h b No thiamine, alloxan + Before insulin 90.2-b.7 98.1-5.l After insulin 60.6:6.3 61.3:6.5 mg. decrease 29.6 36.8 Percent decrease 32.9 37.6 5 2 mg. thiamine, cortisone Before insulin 1h7.5io.7 loh.2i7.2 After insulin 101.317.3 102.5i7.6 mg. decrease h6.2 61.8 Percent decrease 3l.h 37.7 6 No thiamine, cortisone + + Before insulin 118.5-3.8 121.2—h.2 After insulin 81.2i6.3 79.3*S.8 mg. decrease 37.3 h1.9 Percent decrease 31.5 3h.6 * Standard error of the mean. 82 Experiment VI and VII - Glucose utilization in normal, alloxan- diabetic and cortisone-treated rats as influenced by thiamine. Purpose Styron gt 31. (l9b2) demonstrated that glucose tolerance was lower in thiamine-deficient than in thiamine-adequate rats. These experiments were designed to observe the response of alloxan—diabetic and cortisone-treated rats to glucose administration, when maintained on diets either deficient or adequate in thiamine. Methods The rats from Experiment V were used in this study. After 20 days of the treatment outlined in the previous experiment, the rats were starved for 12 hours and blood samples were taken for initial glucose measurements. A total of 750.0 mg. of glucose dissolved in 5 m1. of physiological saline was injected into each rat intraperitoneally. Blood samples were obtained 1 and 2 hours later and glucose was determined as in the previous experiments. Results of Experiment V (Table XIII and Figure 6) The results are summarized in Table XIII and Figure 6. .ill thiamine- deficient rats (groups 2, h and 6) showed a lower ability to utilize the injected glucose than the correSponding groups fed on a thiamine- adequate diet (groups 1, 3 and 5). The alloxan-treated rats fed the thiamine-adequate diet (group 3) and thiamine-deficient diet (group b) showed the highest blood glucose level after the first hour and these 53 values were only slightly reduced by the second hour. Such a glucose tolerance curve is characteristic of diabetic rats. The percent in- crease in blood sugar was greater in the thiamine-deficient rats (group h), indicating that thiamine is essential even in diabetic rats for the limited carbohydrate utilization which takes place. The cortisone-treated, thiamine-deficient rats (group 6) showed as little ability to utilize glucose as the alloxanized rats, whereas the corti- sone-treated, thiamine-adequate rats showed much greater ability to utilize glucose. The positive control rats (group 1) stewed greater ability to utilize glucose than the negative controls (group 2), although even the latter rats metabolized more glucose than either of the alloxanized or cortisone-treated groups. This is to be expected since diabetes and cortisone both interfere with carbohydrate utiliza- tion. Results of Experiment VII (Table XIV and Figure 7) The results in this experiment were essentially the same as in Experiment VI and hence require no further comment. Conclusions 1. These experiments confirm the view that a thiamine deficiency interferes with the utilization of glucose. Cortisone was shown to impair glucose utilization in both thiamine-deficient and adequate rats. However, cortisone impaired glucose utilization more in the thiamine— deficient rats than in the thiamine-adequate rats. This is to be expected 8h since either cortisone or a thiamine deficiency interferes with glucose metabolism. 2. A lack of insulin, induced by administration of alloxan, pro- duced the greatest inhibition of glucose utilization observed in these experiments, particularly in the thiamine-deficient rats. It appears that thiamine is more essential for carbohydrate utilization than insulin, since some degree of glucose metabolism can proceed in the absence of insulin but not in the complete absence of thiamine. Of course, none of the rats in this study were completely lacking in thiamine. 3. From these experiments it can be concluded that in the absence of insulin or following the administration of large doses of cortisone, there is still a need for thiamine to carry on at least a minimal degree of carbohydrate metabolism. Essentially the same has been shown to be true for vitamin B12 (Feng, 195h) under similar circumstances and the same probably applies to other vitamins which assist in the metabolism and utilization of carbohydrate. More of these vitamins are needed for the maximum effectiveness of insulin and to overcome certain catabolic effects of large doses of cortisone. Apparently'more of these vitamins are also needed during adrenal cortical insufficiency. Dumm and Ralli (19h8, 1953) and Meites (1953) showed that administration of large doses of B vitamins to adrenalectomized rats greatly prolonged life and in- duced some degree of body growth. It is possible that an increased intake of B vitamins would also be beneficial in diabetes, since it would insure at least a minimal degree of carbohydrate metabolism and utiliza- tion. TABLE XIII EFFECTS OF THIiMIhE, CORTISONE AND ALLOXAN ON GLUCOSE TOLERANCE TEST Group Treatment _Blood GlucoseJ mg. Percent Initialwr 1_hour 2 Hours 1 2 mg. thiamine - 117.5:h.7** 225.5:6.8** 130.6:6.2** % increase -- 83.h 11.1 2 No thiamine 92.5i2.h 25h.3i7.2 2oh.3:6.l % increase -- 17h.1 120.8 3 2 mg. thiamine, alloxan 2hl.8i22.3 6h1.3:35.8 558.7128.9 % increase -— 165.2 lh3.h t No thiamine, alloxan 96.3136 37913.9 3ee.3ie,3 % increase —- 293.9 280.3 5 2 mg. thiamine,cortisone 157.5io.9 3t8.7il7.3 257.5il2.3 % increase -- 12l.h 63.5 6 No thiamine, cortisone 11h.513.7 295.0:7.5 255.8:7.2 % increase 157.6 123.h * After 12 hours fasting. ** Standard error of the mean. 86 I - 2 mg. thiam1ne II - no thiamine III - 2 mg. thiamine, alloxan IV - no thiamine, alloxan V - 2 mg. thiamine, cortisone VI - no thiamine, cortisone 680 F’ Blogd lucose m8 ) .P.“‘.‘ .’ ‘.“~.‘III / / / hBO - ./ / / / ‘IV / /’ fi~~ ““‘~VI . ,r / ~.‘ I / “‘ / ’I’ / \II . 1’ t” r’/ I r 1’ 80 1 j 1 2 H O U R S Fig. 6 - Glucose tolerance curves in normal, allo- xanized and cortisone-treated rats as in- fluenced by thiamine. 87 TABLE XIV EFFECTS OF TIHAMIKE, CORTISONE AND ALLOXAN ON GLUCOSE TOLERANCE TEST ” Group Treatment _Blood Glucose,_mgA_Percent Initial* 1 hour 2 Lburs ea re as l 2 mg. thiamine 119.6:h.1 21h.5:7.6 125.0:61 % increase -— 79.05 12.7 , , + . 2 No thiamine 9o.2i 258,543.63 2011:1263 % increase -- 168.5 109.3 3 2 mg. thiamine, alloxan 2h8.li23.l 6950:3119. 579.1:3o.o % increase -- 180.1 133.h t No thiamine alloxan 99.ti2.9 393.1178 3ol.ti7,e , % increase -- 295.8 263.9 5 2 mg. thiamine, cortisone 15h.h:7.l 359.1:19.3 2h2.5:18.1 % increase -- 132.5 57.1 6 No thiamine, cortisone 120.0:h.2 308.8:8.6 273.1:9.3 % increase -- 157.2 127.5 * After 12 hours fasting. *% Standard error of the mean. 88 I - 2 mg. thiamine II - no thiamine III - 2 mg. thiamine, alloxan IV - no thiamine, alloxan V - 2 mg. thiamine, cortisone VI - no thiamine, cortisone 680 Blood lucose . r- (m8 ) ./\ \'\ / ‘\. \Q / \- ‘\. / \III /\ IV ‘/l . ’/~ ..... ‘::\\. , ‘~~ 28 ./ ’1 \~--- /’ A\ ‘\.:I ” / \\ ///// ’ z” I“‘- I \ / / \\II ’ .z’ I r’ / ’I r 1’ I 80 L l 1 2 H O U R S Fig. 7 - Glucose tolerance curves in normal, allo- xanized and cortisone-treated rats as in- fluenced by thiamine. 89 DISCUSSION A large dose of cortisone injected into young rats fed a diet just adequate in thiamine completely inhibited further body growth, depressed appetite slightly and considerably reduced the efficiency of food utilization. However, when five times or more than the normal requirement for thiamine was fed, it partially counteracted the inhibi- tory effects of cortisone on body growth and efficiency of food utili- zation. It was observed that on a thiamine-deficient diet, cortisone further aggravated the symptoms of thiamine-deficiency but did not do so in rats fed hO mg. of thiamine per kilo of diet and permitted only a very limited food intake. Alloxan did not appear to markedly alter growth in either thiamine- deficient or adequate rats. After administration of alloxan to thiamine- adequate rats, body weight was decreased during the first h to 5 days but was partially recovered during the remainder of the experiment. The efficiency of food utilization was lower in these rats but appetite was not depressed. Thus the requirement for thiamine does not appear to be increased in alloxanized rats. Lowry'gt_al, (l9h5) also concluded that there was no increase in the requirements for this vitamin in alloxan-diabetic rats. Charalampous gt El. (19h8) similarly reported that the requirement for riboflavin was not increased in alloxan- diabetic rats. Feng (l95h) found that alloxan—diabetes reduced body 90 growth and the food/gain ratio of rats on a vitamin B-12 deficient but not on a vitamin Blg-supplemented diet. A deficiency of thiamine has been reported to produce enlargement of certain organs such as the adrenals, heart and kidney (Dunn gt a1. 19h7; Pecora, 1953) and atrophy of the tiymus,testes and seminal vesicles (Skelton, 1950). Cortisone also, when given in high doses has been shown to induce atrOphy of the thymus and adrenals (IntOpol, 1950; Meites, 1951), testes and seminal vesicles (Antopol, 1950). In this study it was observed that the kidney and the heart were enlarged in thiamine-deficient rats and in cortisone-treated rats fed either adequate or thiamine-deficient diets. The increase was more pronounced however in the thiamine-deficient rats. This action of cortisone may have been due in part to its limited salt and water retain- ing action. It has also been attributed to a negative nitrogen balance (Skelton, 1950). 'Walter gt El. (1939) have presented evidence that changes in the weight of the kidney and heart are determined mainly by changes in the amount of work done by these organs. with continued starvation and consequent loss of weight the burden of the heart and kidneys may be reduced, permitting loss of weight of these organs. The functional demands on the kidneys and heart however, are closely related to the rate of metabolism. Since the metabolic rate and surface area of an animal diminish proportionally less than its body weight, the functional burden of the kidneys and heart and consequently the weights of these organs may diminish prOportionally less than body weight. 91 The adrenals were greatly enlarged in the thiamine-deficient rats, whether or not they received cortisone. A thiamine-deficiency and the concomittant inanition constitute a stress, and it has been demonstrated by Selye (1937) that in any stressing situation the adrenals are stimu- lated by the adrenocorticotrOphic hormone of the anterior pituitary gland. A dose of 1 mg. of cortisone daily did not completely overcome the enlargement of the adrenals in the thiamine-deficient rats. Cortisone however, reduced the weight of the adrenals of thiamine- adequate rats. The thymus gland was reduced in both cortisone-treated and in thiamine-deficient rats, with the greater reduction occurring in the latter. A decrease in size of the thymus can be induced directly by cortisone (Meites, 1951) or indirectly by prolonged inanition (Selye, 1937). Deane gt 31. (19h?) reported that the involution of the thymus was due to stimulation of the adrenal cortex by.ACTH, resulting in increased secretion of corticosterone-like hormones. In pair-feeding experiments, it was found that the stimulation of the sona fasciculata occurred earlier in thiamine-deficiency than with a comparable degree of inanition (Deane gt El. 19h7). Meites (1951, 1952, 1953) found that a large intake of vitamin B12 counteracted the effects of large doses of cortisone on thymus involution. In this study it was found that large doses of thiamine produced no significant counteraction of the inhibitory effects of cortisone on thymus weight. The weight of the testes was not significantly affected by corti- sone at any level of thiamine intake but was reduced in size when 92 thiamine was absent from the diet. Thiamine may have exerted some beneficial effect on the size of the testes which was unrelated to food intake, since the testes of the limited-food rats given thiamine were larger than in the thiamine-deficient rats. The size of the seminal vesicles was about the same in the cortisone- treated rats fed 2 mg. of thiamine per kilo of diet and in the control rats which received the same amount of thiamine. This is in disagree- ment with the findings of Antopol (1950) who reported atrOphy of the testes and seminal vesicles in cortisone-treated mice. In the corti- sone-treated rats fed higher levels of thiamine, the seminal vesicles were increased in weight but this appeared to be due primarily to in- creased food intake and not to cortisone. A thiamine-deficiency alone induced a decrease in the weight of the seminal vesicles, confirming the observations of Skelton (1950). He considered the failure of de- veIOpment of sex organs in thiamine-deficiency to be due to decreased production of pituitary gonadotrOphic hormone. Administration of large doses of cortisone to thiamine-deficient rats and guinea pigs was unable to or only slightly increased blood glucose, but it was able to produce a pronounced hyperglycemia in thiamine-adequate animals. Protein catabolism was slightly increased in the former rats as shown by the small increase in urinary nitrogen excretion. Alloxan also was unable to induce any significant imper- glycemia or increase of urinary nitrOgen in thiamine—deficient rats. Similarly, Lowry gt El. (l9h5) observed that after a thiamine depletion 93 period of 16 days, glucosuria in alloxan-diabetic rats was signifi- cantly decreased. After injecting 50 to 200 mcg. of thiamine the excretion of glucose in the urine increased consistently. Charalampous gt El- (l9h8) made similar observation in alloxan-diabetic rats fed a riboflavin-deficient diet. These authors observed a marked decrease in food intake on the deficient diet, and this probably accounted to a large degree for the decrease in the excretion of urinary glucose. It appears probable therefore, that food intake played a paramount role in the diabetogenic response to cortisone and alloxan in the conditions under which these experiments were carried out. Apparently thiamine itself is partially responsible for hyper- glycemia in alloxan-diabetic rats, since on a limited-food intake in which large amounts of thiamine are fed, there is a consistent hyper- glycemia and increased urinary nitrogen excretion. This contrasts with the results noted in the cortisone-treated, limited-food rats given thiamine, since in these rats there was no pronounced hyperglycemia or increase in urinary nitrogen. Thus, reduced food consumption rather than thiamine deficiency seems to be responsible for the comparative absence of the diabetogenic reSponse to cortisone in these rats. Feng (195b) found that 1 to h mg. of cortisone acetate given daily to vitamin BIB-deficient rats induced a progressive increase in urinary nitrogen and increased blood glucose and glucosuria to greater levels than in rats fed on a diet containing 200 mcg. of vitamin B12 per kilo of diet. The cortisone-treated rats on vitamin BIZ—deficient diet ate 914 half as much food as the rats on 200 mcg. of vitamin B13 per kilo of diet, but despite this the former rats showed values for blood glucose almost double that of the latter animals. Thus the diabetogenic action of cortisone is not influenced in the same way by thiamine and vitamin B12. Feng (l95h) also found that alloxan-diabetic rats showed higher levels of blood glucose when fed a vitamin BIZ-adequate diet as com- pared to a vitamin BIZ-deficient diet. This is similar to the results observed in the present experiment with thiamine and cortisone. The hyperglycemia induced by cortisone in this study was consider- ably lower than that reported by Feng (l95h). The diets used may have accounted at least in part for these differences. In the present study a semi-synthetic diet containing 62 percent glucose and 2h percent casein was employed. Long g: 31. (l9h0) and Engle (l9h9) noted that the administration of large amounts of carbolwdrate to cortisone-treated rats prevented the protein catabolic action of cortisone, and it is therefore possible that the readily available glucose in the diet re- duced gluconeogenesis in these experiments. In the thiamine-deficient rats, glucose utilization was reduced and insulin resistance was increased. This is in agreement with the previous reports of Burke g3 31. (1938) and Styron g§_gl. (19h2) who showed that rats maintained on a thiamine-free diet had less ability to utilize injected glucose and showed increased resistance to insulin. The administration of thiardimadecreased the hypoglycemic action of insulin and increased glucose utilization, as indicated by increase in 95 body weight. Cortisone-treated rats also showed increased resistance to insulin as had been noted previously by others (Grattan g£_gl. l9h0). It was the purpose of this thesis to determine the nature of and the possible relationships between the actions of thiamine, cortisone and insulin on body growth, carbohydrate and protein metabolism. Apparently such relationships exist. Cortisone has been shown to stimulate pancreatic islet function in rats (Baker g§_gl. 1952) and guinea pigs (Hausberger 23.21- 1953), and thiamine has been demonstrated to be necessary for maximum insulin action. Cortisone increases glu- coneogenesis from protein but at the same time interferes with the utilization of glucose by insulin (Ingle 93.319 l9h5). Insulin is essential for the conversion of glucose into energy, glycogen and fat (Long, 195h). Thiamine is essential for the intermediate metabolism of glucose. Of all the factors involved in carbohydrate metabolism only the site of action of thiamine is known. Thiamine apparently can function to a limited extent even in the absence of the pancreas (Soskin gt g1. 1952), indicating that insulin and also probably corti- sone influence the,rate of the reaction rather than the reaction itself. However, the ability of cortisone and insulin to influence carbohydrate metabolism depends in part on the level of thiamine and other B vita- mins concerned with carbohydrate metabolism. Does cortisone increase the requirements for thiamine in the young rat or guinea pig? Draper and Johnson (1953) reported that neither pyridoxine nor riboflavin metabolism was influenced by cortisone in the rat. They found that cortisone did not affect the survival time of young rats or the excretion of pyridoxine in the urine on a pyridoxine-deficient diet. Likewise, no effect was observed on the excretion of riboflavin or the riboflavin content of the liver on a riboflavin-adequate diet. Dhyse 21°. 21. (1953) found no change in the excretion of pantothenic acid, biotin, riboflavin, niacin, pyridoxine or folic acid in the rat liver following either adrenalectomy or cortisone injection on a diet adequate in these vitamins. The above reports appear to cast some doubt as to whether large doses of cortisone can actually alter requirements for thiamine in the young rat, although neither group of investigators worked with thiamine. To the writer, it appears logical that large doses of cortisone should alter the metabolism of the vitamins concerned with carbohydrate metatol- ism since cortisone interferes with carbohydrate utilization (Ingle 33.31- l9h5). The vitamins essential for carbohydrate metabolism should there- fore be eliminated more rapidly through the kidneys. It will be noted that in the work of Draper g3 31. (1953) and Dhyse 23.319 (1953), the only vitamins directly concerned with carbohydrate metabolism are riboflavin, niacin and pantothenic acid and these were fed in more than adequate quantities in tleir experiments. 0n the other hand,'Wahlstrom and Johnson (1951), Chow (1958) and Feng (195h) have demonstrated that on a vitamin Blz-deficient diet, large dose or cortisone increased the urinary excretion of vitamin B12. It would have been worth—while to study the metabolism of thiamine as influenced by large doses of cortisone. 97 The results of the present study did Show however, that large doses of cortisone aggravated the condition of young rats on a thiamine- deficient diet and that this could be at least partially counteracted by feeding large amounts of thiamine. If large doses of cortisone interfere with the utilization of glucose in the body, and increase urinary losses of vitamin B12 and possibly thiamine, how can it be claimed that requirements for these vitamins are increased? Evidence has already been presented elsewhere in this thesis that large doses of cortisone increases pancreatic islet function (Baker _t 31. 1952), and that insulin increases require- ments for thiamine (Samuels, 19h8 and this thesis) and vitamin B12 (Feng, 195D). It is suggested that a condition is produced by large doses of cortisone whereby at one and the same time there is both a lesser and greater requirement for the above vitamins. Supplementation of the diet with large amounts of vitamin B12 or thiamine are bene- ficial, apparently because they increase the action of insulin. What- ever the barrier which cortisone places in the way of insulin action, large amounts of these vitamins help overcome this barrier. This is the best hypothesis which the writer can present at this time to explain the interactions between large doses of cortisone, insulin and thiamine observed in this study. 98 Eiflhhdflf 1. When young rats were maintained on a thiamine-free diet, symptoms of thiamine deficiency developed within 15 to 20 days. Supple- mentation of the diet with 2 mg. of thiamine per kilo of diet increased appetite and body weight gains, slightly increased blood sugar and greatly increased glucose tolerance. 2. When h mg. of cortisone acetate daily were injected into thiamine- deficient rats, there was a slight increase in the excretion of urinary nitrogen, a slight or no increase in blood glucose, decreased glucose tolerance, reduction in body weight gains and reduced appetite. When 2 mg. of thiamine per kilo of diet (or higher levels of thiamine) were fed to cortisone-treated rats and they were allowed to eat 33 libitum, urinary nitrogen increased greatly, blood glucose increased moderately, glucose tolerance was partially improved and body weight was maintained at the same initial level or was slightly increased. 3. Thiamine at high levels, fed to rats on a limited-food intake, largely prevented the develOpment of thiamine-deficiency symptoms but was unable to increase the blood glucose of cortisone-treated rats. It slightly increased urinary nitrogen excretion. It is concluded that large doses of thiamine, greater than normal requirements for growing rats, can partially counteract the protein catabolic action of cortisone by increasing food consumption and increasing the availability and utilization of carbohydrate by the organism. 99 h. Cortisone partially interferred with the favorable action of large doses of thiamine on the efficiency of food utilization for body growth. Hyperglycemia, glucosuria, increased nitrogen excretion and increased insulin resistance were noted, and therefore less carbohydrate was available to exert a "Sparing action" on protein for transformation into body weight gains. 5. (a) When young rats were fed a thiamine-free diet, the weights of the kidneys, heart and adrenals were increased and the weighhsof the thymus and seminal vesicles were greatly decreased. When-l mg. daily of cortisone acetate was injected into thiamine-deficient rats, a still greater increase in the weight of the kidneys and heart was noted, and a slight increase was found in the weight of the testes and adrenals. The low thymus weight was not decreased further by cortisone treatment, while the seminal vesicles weighed twice as much as those of thiamine- deficient rats. (b) When thiamine was fed to cortisone-injected rats, the kidneys, heart and testes showed a slight increase in weight. The thymus showed less involution, the adrenals were reduced in weight and the seminal vesicles were slightly but not significantly increased in size. The increases in thymus and seminal vesicles weights apparently were not due to thiamine peg‘gg but to the concomittant increase in food intake. 6. Alloxan-diabetes did not further reduce the efficiency of food utilization of thiamine-deficient rats or rats on a limited-food intake, but slightly reduced the efficiency of food utilization of thiamine- ade uate rats. In the latter there was a consistent increase in blood q lOO glucose and urinary nitrogen, while in the thiamine-deficient rats there was neither an increase in blood glucose nor of urinary nitrogen. Rats on a limited food intake but fed thiamine showed a consistent increase in both blood glucose and urinary nitrogen which decreased progressively as chronic inanition developed. When the treatment of the thiamine-deficient rats was reversed, by administering large amounts of thiamine, there was a pronounced hyperglycemia and an increase in urinary nitrOgen excretion. It is concluded that thiamine, by increas- ing food intake, permits hyperglycemia to develOp in alloxan-diabetes. 7.‘When guinea pigs were maintained on a thiamine-free diet, symptoms of thiamine-deficiency deve10ped within 25 days. Supplementa- tion of their diet with 16 mg. of thiamine per kilo of diet increased appetite and body weight gains. Injections of S or 10 mg. of cortisone acetate daily did not appear to reduce body weight significantly in thiamine-deficient guinea pigs in contrast to rats. When cortisone was injected into thiamine-deficient guinea pigs there was no increase in urinary nitrogen or blood glucose, as in rats. When 16 mg. of thiamine or more per kilo of diet were fed to cortisone—treated guinea pigs, only a slight increase in blood glucose was observed with 5 mg. of cortisone injected daily and a consistent increase with 10 mg. of corti- sone daily. Cortisone did not increase blood glucose of thiamine- deficient guinea pigs at any level. 8. 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Somogyi-Shaffer-Hartman Method (Hawk gt a1. 1951) q A volume of 0.2 ml. of blood was drawn from the tail of each rat by a Folin micropipette and was mixed into 5.8 m1. of water in a 25-ml. Erlenmeyer flask. The pipette was then rinsed several times with the lacking water. A volume of one ml. of 1.8 percent zinc sulfate and one ml. of 0.1 N of sodium hydroxide were added and mixed. After shaking, the contents were filtered through No. 1 dry filter paper. Five m1. of the Shaffer-Hartman copper reagent were measured into a 25 x 250 mm. test tube and 5 m1. of the blood filtrate was mixed into it, siaken and covered with a glass bulb, and placed in a boiling water bath for 15 minutes. It was then cooled, 1 ml. of 5 N sulfuric acid was added, and it was titrated with 0.005 N sodium thiosulfate. Starch was added as an indicator. A blank was run on 5 ml. of the c0pper reagent after boiling with an equal volume of water. In the calculation the blank titration was subtracted from the titration of the unknown, and this gave the ml. of thiosulfate required for the unknown. For the glucose equivalent, the Table (page 525) in Hawk gt El- (1951) was con- sulted. Since this table applies to the usual 1:10 dilution of blood, and in the present case, a 1:L0 dilution was used, the mg. of glucose in 100 ml. of blood given in the table were multiplied by four. b. Folin and Malmros Method (HaWk.§E.El- 1951) A volume of 0.1 ml. of blood was drawn from the tail of each rat with a Folin micrOpipette and transferred to a centrifuge tube containing 10 ml. of dilute tungstic acid. This was mixed and centri- fuged. Four m1. of the water-clear supernatant liquid were transferred to a test tube graduated at 25 m1. To this, 2 ml. of a O.h percent potassium ferrioyanide solution and 1 m1. of a cyanide-carbonate solu- tion were added. The contents were heated in a boiling water bath for 8 minutes and cooled in running tap water for 2 minutes. Then, 5 ml. of ferric iron solution were added and mixed. Two minutes later, the contents were diluted with water almost to the 25-ml. mark, two drOps of alcohol were added to prevent foaming, and water was added exactly to the 25-ml. mark, and mixed. The solution was placed in a Fisher electrOphotometer 10 minutes later and read. A green plate filter of 525 mu. wavelength was used. The photometer was initially set to zero density with water. The preparation of the standard solution of sugar, a stock solu- tion of 1 percent glucose, was made up in saturated benzoic acid. The stock solution was diluted to 0.01 mg. and then to 0.1 mg. per mP. The Optical density for each amount was read on the photometer and a standard curve was drawn from these values. The calculation of blood glucose was as follows: density of unknown 0 Oh x 19 199 m . ercent lucose = . . g p g density of standard x h.O 0,1 2. Determination of Total Urinary Nitrogen Koch and McMeekin Method (Hawk 2; 31. 1951) One ml. from a 2h-Jnur urine Specimen was diluted to 50 ml. and mixed in a volumetric flask. One ml. of the dilute urine was pipetted 125 into a micro-Kjeldahl flask, and 1 ml. of 50 percent sulfuric acid was added and mixed. The flask was heated over a gas flame for 10 minutes, after which 3 dr0ps of 30 percent hydrogen peroxide were added. The flask was heated for 6 more minutes until all the sulfuric acid fumes disappeared. It was cooled for 30 minutes and diluted to 75 ml. with water. Fifteen ml. of Nessler's reagent were added and the whole was diluted to exactly 100 ml. This was left to stand for 10 minutes and was then read on a Fisher electrOphotometer in which a green plate filter of 525 mu. wavelength was used. For a standard nitrogen preparation, O.h71h gm. of ammonium sulfate was dissolved in one liter of water together with a few drOps of con- centrated sulfuric acid as a preservative. This solution contained 1 mg. of nitrogen per 10 ml. It was used in amounts of 0.1 ml. to 1 m1. of the stock solution, and was diluted with 15 ml. of Nessler's reagent and water to 100 ml. The values were read on the photometer and a standard curve was drawn. The calculations were as follows: Reading_of standard Reading of unknown x mg. N in the standard x urine volume body weight total nitrogen expressed as mg./1OO gm. body weight/2L-hour urine Specimen 126 3. Semi-synthetic Diet for Rats Ingredients Amount Ingredients Amount (gm/kilo) (mg,/kilo) Cerelose 620.0 Thiamine HCl 2.0 Casein (vitamin free) 2ho.o Riboflavin 5.0 Corn oil 100.0 Pyridoxine 2.0 Mineral mixture No. h h0.0 * Niacin 10.0 Choline Chloride 1.0 Calcium pantothenate 28.0 2éMe-l,h-naphthoquinone 0.h Vitamin B12 0.02 Vitamin A acetate 2000.0 I.U. Vitamin D 250.0 I.U. 127 h. Semi-synthetic Diet for Guinea Pigs (Reid 33 El- 1953) Ingredients Amount Ingredients Amount (gm./kilo) (mg,/kilo) Casein (vitamin free) 3oo.o Thiamine PC]. 16.oo Corn oil 73.0 Riboflavin 16.00 Sucrose 103.0 Pyridoxine HCl 16.00 Cellophane Spangles 150.0 Calcium pantothenate h0.00 Starch (corn) 2oo.o Niacin 200,00 Cerelose 78.0 Biotin 0.60 Potassium acetate 25.0 Folic acid 10.00 Magnesium oxide 5.0 Vitamin B12 o.oh Salt Mixture No. h 60.0 Vitamin A acetate 6.00 Choline chloride 2.0 Vitamin D 0.0h Ascobic acid 2.0 Alpha toc0pherol acetate 20.00 Inositol 2.0 2eMe-1,h-naphthoquinone 2.00 Date Due :31 ‘t w" ($39“ ‘ ... ‘3 «. \x i l'\ :‘I ‘ ‘ ‘ v .‘ v . .. ‘X 123 I f‘m: U*" Demco-293 111111111 283 ill) 143 3 1293 03 VI I." u fl " I" HI"