SO. ME PHYSIOLOGICAL EFFECTS ASSOCIATED WITH CHRONIC CALORIC RESTRICTION MADIEOX, WiscoNsIN tumors is well established (1, 2), but litte is known concerning the mecha- nism by which this occurs. “As early as xor4 Rous (3) observed that the development of mammary tumor transplants and metastases in the mouse was de- layed by food restriction and he suggested that this effect was due to a delay in the vascularization and in the development of a supporting stroma upon which the tumor is dependent. ‘The inhibition of the formation of spontaneous mammary cancer in mice on a reduced food intake has been explained on the basis of a pituitary insufi- |. clency producing decreased ovarian secretion (4). ‘Thus, one ofthe factors essential | for the éccurrence of this type of tumor is deficient when caloric intake is restricted. | However, the mechaitism of the inhibiting effect of simple caloric restriction on the development of other types of neoplasms remains obscure. In order to obtain further information on this problem, a preliminary survey of certain orgen weights (pituitary, thyroid, adrenal, thymus, ovary, uterus, heatt, liver and kidney) was made on groups of 12 to 16mice kept on ad libitum or restricted diets + for one week, one month and seven months. ‘The results confitmed the conclusion ‘that a reduction in caloric intake decreased ovariai function and in addition suggested that it increased adrenal cortical activity. In order to obtain further information concerning a possible adrenal stimulation, several criteria that indicate the activity of this organ were investigated. These included data on the weight and ascorbic acid content of the adrenals, the content of glycogen in the liver under different conditions and the activity of the lymphatic system as measured by the weight of the thymus| and by lymphocyte counts. T= Inhibiting effect of caloric restriction on the formation of experimental srETHODS Male and female mice of the ABC and Rockland strains, 2 to 3 months of age, were used. Mice of only one strain and one sex were used in any particular experi. ment. Groups of 6to 18 mice were keptin screen bottom cages and fed from a special typeof feeder (5) either at ad libitum: (generally x0 to x2 cal/mouse/day) or at re- stricted levels (6 to 7.2 cal/mouse/day). The diet was weighed into the feeders daily and the mice were fed at about g¢0 aat. ‘The composition of the artificial type diets is shown in table r. Water was available to both groups at all times ‘Received for publication July 22, 1948. "This investigation was supported hy a grant from the American Cancer Society on recom- rendation of the Committee on Growth ofthe National Research Council and by «grant from the Wisconsin Division of the Amerean Cancer Society.318 RK. DOUTWELL, M. K, BRUSH AND HP, RUSCH Volume 15 Samples for organ weight, liver glycogen, blood sugar and adrenal ascorbic acid determinations were taken from mice as soon as the comeal reflex disappeared follow- ing the injection of sodium pentobarbital. Blood was withdrawn from the right ‘ventricle of the exposed heart with a syringe moistened with a solution of the sodium alt of heparin (xo mg/cc). The weights of organs under 50 mg. were quickly deter- mined to 0.02 mg. on a torsion balance. “Liver samples were dropped without delay’ nto a tared tube containing 2.0 cc. of 3o per cent alkali and glycogen digests were made according to the method of Good, Kramer and Somogyi (6). Glucose deter- inations on the digests and on deproteinized blood filtrates were made by the fodo- tnetric copper method of Somogyi (7). The liver glycogen values were expressed in terms of the glucose equivalent. ‘The adrenal ascorbic acid determinations were ‘ade by the method of Roe and Kuether (8). Tail blood was used forthe totalwhite call counts. ‘The estrus eycle was followed by the usual vaginal smear technique. i ‘Taste 1. Costostmox oF iets Cereose . 8 Casein 5 Corn el 2 Liver cone 1 : Salt mis 4 4 09) 86 ‘Vitamins added: ma/ts gm. of easin; thiamine by docbloride, 3; tofavinyeg yeoxine ydonherde, oy niatnamide, cles pantothenate, 20; nota 33 paminobenzoic ai oaoline chloe, gr pteroyilutaie aid, 0. nd biotin, ot. Halibt live il was aed BS ere il at the level of eo vitamin A uae and 18 vitamin D units per kx. (6 drops « igo aid. . "Pontus, P.H, as FB, Haas. J. Bia. Chim. 109% 6571938 EXPERIMENTAL AND RESCES “Liver Glycogen ond Blood Sugor. The duroalfuctation of liver lvcogen and i also bioed sug was followed in mice adapted to the restricted and ad tbitum diets. Sie the feeling habits ofthese mice differed, this variation must be considered in secon tothe results, Mice, restlcted in food, consumed their daily allotment in aehan one hour after the 9:00 436. feeding time. However, si hour ater their ee stake wove stil distended with ration, which was mostly in an undigested, sem Sarat. ‘Even 12 hours after feeding time considerable amounts of partially ‘Tested food were stil present, but by 24 hours the stomach was empty. The mice cee pe allowed the full ration were also fed at goo At but the major portion of apat nd wos eaten between 6:00 73. and avidnight. At no time was the stomach tistended nor was a dry mass of ct found therein. ier an equilibration period of four weeks or longer, the content of glycogen in the ther wes determined it ce taken ftom the to groups at 300 #3, 9:00 7. aa ines car the following morning (6 12 and 2g bours after feeding time, respec Bees? tng teslts are plotted in igre 1. Each point fan average of 19 20 see ane curve representing the restricted group and of 6 mice toa point inthet : i i September 1998 CHRONIC CALORIC RESTRICTION s19 ‘curve depicting the mice on the od libitum diet. The same data were replotted for the second 24-hour period to emphasize the cyclic nature of the changes. This diurnal variation in liver glycogen of restricted animals was apparent by the fourth day after caloric restriction was begun and reached the extremes similar to those shown in figure 1 by the tenth day, after which there was no change for as long as 193 days, ‘The unusually high level of liver glycogen in the restricted mice 6 hours after feeding ‘ Figs Fig. 2 Fig. x (let). Day exes "AL VantaTION inthe amount of glycogen in the liver of mice maine icted det, if), BEYEcr OF A pR0LoNCED Fast on the level of glycogen in the livers of mice ‘ibitim and restricted diets. The last fond was given 24 hours prot to the te {ime of these curves, and the zero ime ofthis Ggure corresponds tothe points aty Aston the curves in gute x. ‘The fast was continued until the death of the animals, ‘Tamer 2. AVERAGE LEVEL OF BLOOD SEGAR OF MICE ADAPTED TO RESTRICTED AND AD) UBITOME [FEEDING HABITS AT 6, 12 AND 24 WOURS AFTER FEEDING Tate. THE NOME OF ARIMALE PER me sorgee soumon 6 10r-4 (7) 114.5) a 65.46) 92.79) 4 97-48) 138.3) ‘was of the same magnitude as that reported by Long and his associates (o) for mice given injections of cortin and allowed ration ad libitum, At the same time that the mice were sacrificed for the glycogen determination, blood was obtained for the analysis of sugar. There was a daily variation with the {feeding habits of the mice in both groups, yet the well fed mice showed a consistently higher level of blood sugar (table 2). This finding is in agreement with the earlier observation of Rusch, Johnson and Kline (10). tis well known that the administration of certain adrenal cortical steroids to a fasting mouse will prevent the expected depletion of liver glycogen reserves (0). This principle was the basis for an experiment to tet the endogenous production of cortical hormones by mice on restricted and ad libitum feeding habits. The fast wasa 520 R. K, BOUTWELL, M. K. BRUSH AND H. PL RUSCH _Velune 155 ‘begun by removing the food containers from the cages of both groups of mice at 9:00 ‘AM, 24 hours after the last feeding time, and accordingly this point is represented as ‘zero time in the presentation of the data (fg. 2). It also follows that the glycogen ‘values at this time are the same as at 960 4.1. in the experiment described in the preceding paragraphs, In the case of mice on the ad libitum regimen, the level of liver slycogen rapidly fell to values below 100 mig. per cent and it remained at this low level {for 60 hours when it increased to about 700 mg. per cent, an observation similar to that made by Mirski and his associates (sx). This probably indicated stimulation of adrenal cortical activity after the stress of an acute starvation. Tn contrast, the content of glycogen in the liver of restricted mice never decreased below the zero time level of 700 mg. per cent and was indicative of a high level of cortical activity prior to the period of prolonged fast. The length of the experiment was determined by the survival time of the mice in each group and the mice with the greater reserves lived the longer. ‘A more refined test of endogenous cortical activity was devised by an adaptation of the adrenal cortical assay method of Venning, Kazmin and Bell (12)... ‘They found ‘Taoce 5, ESFSCE OF CALORIC INTAKE AND OF A GLUCOSE SUPPLEMENT UFON TItE DEFOSTHIOS OF {vee cEYCOGES (MOF GLYCOGEN /100 Gx. EVER) 2 62 623, 2250 3 6 888, 2385 6 a4 Br 2420 6 493 1320 2450, ana, aooav. o3tav. 237630 1 Four mice in each group. that the injection of small amounts of glucose such that there was no increase in the amount of liver glycogen in. adrenalectomized mice increased several fold the sensi- tivity of the Reinecke-Kendail (x3) method for the assay of cortin preparations. Tn the present study, the effect of small amounts of glucose on the level of liver glycogen was determined in fasted Rockland mice previously adapted to the two diets. Two intraperitoneal injections of 0.3 ec. ofa s per cent glucose solution were given 32 and '33 hours after the lat feeding time (corresponding to 8 and 9 hours after zero time in fig. 2). A total of so mg. of glucose was given to each mouse, ‘The content of gly- ‘cogen in the liver of both the glucose injected and control mice was determined three hours after the second injection and the results of 2 typical experiment are shown in table 3, The control mice did not receive glucose and the glycogen values are com- parable to the 12-hour values shown in igure 2, It isnoted that the administration Uf the glucose increased the glycogen in the liver to an average of 2376 mg. per cent in the restricted mice, but had a very minor effect on the livers of mice accustomed to adict ofabundant calories. Since the average weight of the livers is x gm. in the mice fon the restricted diets and 1.5 gm. for those on the ad libitum ration, it can be seen that about 48 per cent of the total glucose administered was converted to glycogen in the underfed mice as compared to only 8 per cent in the fully fed animals.Sepember 1948 CHRON! ‘ALORIC RESTRICTION Adrenal and Thymus Weights. In a typical experiment, x2 male mice of the ABC strain 2} months old were restricted to six calories daily for five weeks and 8 ‘mice were allowed ration ad libitum (between x0 and s1 eal, per mouse daily). The average weight of the mice at five weeks, the average weight of the glands, and the eight of the glands expressed as mg/gm. of body weight are shown in table 4. ‘There was no difference in the absolute weight ofthe adrenals, but the adrenal weight Aelative to the body weight was more than half again as large. These findings agree ‘with those reported by Quimby (14) for underfed young male rats but differ from ‘those of Mulinos and Pomerantz (13). The later investigators found an increase adrenal weight in rats on complete starvation but a decrease in the weight of the adrenal when the animals were kept on a dict restricted by so per cent. The present ‘Tapur 4, Bony wetcurs «xD WEIGHS OF ADRENAL AND T¥AUS GTANDS APTER 35 DAYS OF No. of mice... " 8 Av. body we ei 16.7 em. 26.08. ‘Av. adrenal wt 2.38me. 2.38%, We. ratio, adrenal ody, : 144 (0.023)! 0.091 (©.086)! Aw. thymus Whine : 3.86 mg. 23.2 mE. We. ti, thymus. 5 0.236 (0.155)! 2.972 (0.168) 1 stants eon = 4/, ‘Taoue 5. EFfect o” eatonic Resrnicrios OX THE WHIGHE OP TRE OVARIES AND THE UTERUS =i)" No. of mice 6 3 Aw. body we : Sons 20.7 gm 23.6 gm. ‘Av. ovarian wt 8.39 m8. 20.5 mg, Wr ratio, ovarian body. aes, ony range... (0:381-0.606) (o.452-1.06) Av. uterine we 02000 38.5 m6. 86.8 mg. We ratio, uterine/body. ee 0.998 3.87 Niicanesssserseenns + (0.361-1.85) (iag-s.a7) experiment also showed a striking involution of the thymus gland of restricted mice which amounted to-a factor of seven on the actual weight basis and of four on the relative basis (table 4). Lymphocyte Counts. The average total leukocyte and Iymphocyte count of 6 ABC Eontrol mice fed ad libitum were 10,300 and 8000 respectively, and of 16 mice restricted in calories, 5700 and 1900. The decreased lymphocyte count was largely responsible for the lower total counts found in restricted mice, Adrenal Ascorbic Acid, "The ascorbic acid content ofthe adrenals ofthe strain ABC mice on the two diets was also determined. There was no difference between the two groups. Data from a typical experiment showed that this value averaged 234 mg/too gm. of adrenal (64. 9.4) for 8 restricted mice and 237 mg/10o gm. (6.4. 27.6) for 8 mice on the full fed diet. Ovarian and Uterine Weights For this experiment 8 young adult female mice522 RK, BOUTWELL, M. K, BRUSH AND H. P, RUSCH” Volume 154 of the Rockland strain were fed ad libitum and 6 were restricted in calories to 60 per cent of the other group. After 53 days the ovarian and uterine weights were deter- rmined and the ratio of these weights to body weights was calculated. The data, presented in table 5, showed that average weight and the weight ratio of these two organs was reduced by 4o per cent or more in caloric restriction ‘As evidenced by the vaginal smear technic, estrus ceased in all mice restricted to six calories daily, but was normal in all mice allowed ro or more calories ofthe coatrol ration. In order to determine whether the anestrus may have resulted from a de- creased production of estrogens or to an inkbility of the tissues to respond to the hormone, the mice on the restricted diet were injected subcutaneously with 0.08 xg. of estradiol benzoate, Estrus resulted in all the mice which proved that the vaginal epithelium was still capable of responding to this stimulus. ‘This is a confirmation ‘of an earlier short-term experiment by Mulinos el. (16) in which estrus was restored in rats subjected to complete starvation following the injection of either estradiol monobenzoate or gonadotropic hormone (Follutein). DISCUSSION Direct methods for the measurement of pituitary-adrenal-cortical activity have yet to be developed for so small an animal as the mouse. However, there ate several measurable physiological criteria that indicate the state of this system. [An involu- tion of the thymus gland (17), a decrease in the lymphocyte count (18), an increase in the weight of the adrenal gland (19, 20) and a decrease in its content of ascorbic acid (21),andan increase in gluconeogenesis(o) are some of the manifestations ofan intensi fied adrenal coctical activity. The finding ofthese changes in the mice restricted in calories strongly support the conclusion of stimulated cortex in this condition. The ‘one exception was the lack of a measurable change in the ascorbic acid content of the adrenal. Apparently the decrease in ascorbic acid is manifest only after conditions of acute stress (22), since s'milar changes have not been reported during the mild chronic stress described as Type UI by Sayers e! cl. (21). "The experiments in which liver glycogen was determined in fasting mice previ- ously adapted to the two diets and given small glucose supplements are particularly Significant, The fast eliminated the cyclic eflect of food consumption on the amount of glycogen (fg. 2) and permitted a greater spread in the glycogen content folowing the administration of low levels of glucose to the two groups. By standardizing the response in liver glycogen to that obtained with known amounts of cortical steroids, this technique is being developed for the assay of the endogenous production of hormones of the adrenal cortex, ‘The high content of glycogen inthe livers of mice on a low caloric intake probably arises in part {rom non-carbohydrate sources such as aminoacidsand protein. These ‘adrenal controlled glycogen precursors can be designated as the building block reser Tncontrast, the well fed mouse has a rate of cortical steroid secretion which apparent causes the conversion of only negligible amounts of this reserve to carbohydrate, = fs shown by the lower content of liver glycogen. Perhaps this regulation by the laine the inhibiting effect of calorie restriction on tumnor formation. ( In imal all available nutrients may well be required for the maintenance adrenal expSeptember 148 CHRONIC CALORIC RESTRICTION 533 oflifebutin the wellfed animal there is sufficient energy to satisty the critical require- rents and still allow a reserve of building blocks that could be readily available to respond to carcinogenic stimuli with the ultimate appearance of a tumor. These conditions apply only during the early stages of tumor development and have litle effect after the establishment of an independent blood supply to the neoplasm ‘The results of this experiment appear to be the first demonstration of an acti vated pituitary-adrenocortical mechanism in mice on prolonged caloric restriction In contrast, the decreased ovarian and uterine weights and the cessation of estrus the restricted mice reflect a lowered secretion of the gonadotropic hormones, a con- clusion also reached by Huseby and his associates (4). This variance in the elabora- tion of two of the hormones of the pituitary suggest that the ‘general adaptation syndrome’ described by Selye (23) might be operative in the mouse restricted in calories with a shift to the production of the essential adrenocorticotropin at the ex- pense ofthe les critical hormones. Accordingly, the term pseudohypophysectomy used by Mulinos and Pomerantz (24) to describe the condition in chronic or complete starvation cannot be used to accurately characterize the condition ofthe pituitary in the mouse chronically restricted in calories, Although mice on restricted diets were in anestrus, itis significant that they zespond normally to physiological amounts of estradiol. This demonstrated that the end-organ response wvas not impaired and that cell proliferation was not incompatible with caloric restriction. SuscaRY ‘The state of activity of the adrenal cortex and of the ovary and uterus was in- vestigated in two groups of young adult albino mice maintained for long periods on diets varying as to caloric value. One group was allowed a diet ad libitum while the other group was restricted in calories to 6o per cent of the other. ‘The mice on the restricted diet were in anestrus, but they responded normally to physiological amounts of estradiol. ‘Thus, cell proliferation was still compatible with caloric restriction. The study of the estrus cycle together with the determina- tion of ovarian and uterine weights indicated 2 lowered ovarian hormone production in mice restricted in calories. In contrast to this, these same mice showed evidence of an increased activity of the adrenal cortex; the involution of the thymus, the decrease in lymphocytes, the increase in the ratio of the adrenal to body weight and the increase in gluconeogenesis all favored this concept. The apparent variance in the secretion of the gonadotropic and adrenocorticatropic hormones from the pituitary suggested that the ‘general adaptation syndrome’ might be operative in mice restricted in calories, ic. a shift by the pituitary to the elaboration of the essen- tial adrenocorticotropin at the expense of other less critical hormones. ‘The relation of these findings to the inhibiting effect of caloric restriction on tumor formation is described. Weare indebted to Professor R. K. Meyer of the Department of Zoology of the University of Wisconsin for assistance with some preliminary work inthis problem, We also express out thanks to Dr. Augustus Gon of Merck and Company fora generous supply of the B vitamins and to De 1B. L, Hutchings of the Lederle Laboratories for the pterylglutamie acid used in ths study.RK, BOUTWELL, M, K, BRUSH AND HP, RUSCH —— Volume 154 REFERENCES 1. Rosen, HD. Physiol. Res. 241 177, 1944 2 -tawvespauas, A. Cancer Research §: 609, 1945. 5: Rovs, B,J. 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