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ob mice and hyperphagia in lean mice
Physiology& Behavior,Vol. 32, pp. 935-939. Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/84 $3.00 + .00
Food Intake During Tumor Growth: Anorexia in Genetically Obese OB/OB MICE and Hyperphagia in Lean Mice C A R L I. T H O M P S O N
Deparment o f Psychology, Wabash College, Crawfordsville, I N 47933 JOHN W. KREIDER
Departments o f Pathology and Microbiology, The Milton S. Hershey Medical Center o f The Pennsylvania State University, Hershey, PA 17033 AND DAVID L. MARGULES
Department o f Psychology, Temple University, Philadelphia, PA 19122 R e c e i v e d 29 A u g u s t 1983 THOMPSON, C. I., J. W. KREIDER AND D. L. MARGULES. Food intake during tumor growth:Anorexia in genetically obese ob/ob mice and hyperphagia in lean mice. PHYSIOL BEHAV 32(6) 935-939, 1984.--Recent f'mdings indicate that obese (ob/ob) mice suffer a low incidence of lung metastasis and survive longer than lean (+/?) littermates following injection with BI6 melanoma cells [34]. The present study examined the food intake of obese and lean mice during the growth of this tumor. Mice from both groups increased their food intake by small and approximately equal amounts during the first three quarters of the survival period following injection with 106 cells, and body weights remained fairly stable. During the final quarter, however, obese mice became anorexic whereas lean mice became intensely hyperphagic; body weights changed accordingly. Thus, food intake is differentially affected by tumor growth in this form of genetic obesity. Food intake
Body weight
Cancer
B 16 melanoma
Obesity
ob/obMouse
Metastasis
Survival
reverse is true, with a negative association between body build and breast cancer risk [4,42]. In both males and females, controlled prospective studies indicate that people who weigh most at original screening are least likely to develop subsequent malignancy [35]. In a recent paper [34] we presented evidence that the ob/ob mouse, whose obesity is caused by a single recessive gene [14], may provide an animal model for studying increased resistance to cancer in obese humans. After injection with B16 melanoma at 10-11 months of age the primary tumor grew to a smaller terminal size in obese mice than in lean littermates, and incidence of lung metastasis at autopsy was greatly reduced. When injection occurred at 4-7 months the primary tumor grew to the same size in obese and lean animals, but obese mice again exhibited a reduced incidence of lung metastasis. Overall, obese mice survived 10.1% longer than lean mice following injection, despite the fact that under standard laboratory conditions the lean mouse lives more than a year longer than its obese counterpart [15]. During the first of the above studies [34] (mice 10-11 months old), we observed that the body weights of obese mice decreased during the terminal phase of survival
C A N C E R and nutrition are closely interconnected. At least three aspects of this relationship have been identified [39]. First, diet plays a role in the etiology o f cancer; second, nutritional status is profoundly affected by cancer; and third, dietary manipulations could play a part in cancer prevention and treatment. Because of their altered nutritional status, obese individuals might be expected to differ from normals in cancer incidence and mortality. Indeed, such differences have been reported. Early investigators suggested that cancer risk was elevated in overweight people [30], but recent findings indicate that the opposite relationship is more common, with obese individuals typically exhibiting a decreased incidence of cancer. Thus, malignancies occur less often in morbidly obese men than in the general population [10], and data from the Framingham study indicate that cancer death rates decrease steadily with increases in body build for men aged 40-69 years [29]. Of the many types of cancer only two, cancer of the endometrium and cancer of the kidney in women, appear positively linked to obesity [40,41]. Obese postmenopausal women may also exhibit an increased incidence of breast cancer [7,19], but in younger women the
1Supported in part by Grant BNS 8216104 from the National Science Foundation to D. L. M.
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THOMPSON, K R E I D E R A N D M A R G U L E S
whereas lean mice exhibited dramatic weight gains. To examine this phenomenon more closely, food intake and body weight both were measured during the second study (mice 4-7 months old). The present report documents the body weight and food intake of obese and lean mice during these investigations. We report here that obese mice became anorexic during the later stages of tumor growth, whereas lean mice became intensely hyperphagic. METHOD
Animals Fifty-eight pairs of genetically obese (C57BL/6J ob/ob) and littermate control (+/?) mice were used. All animals were female and were obtained from the breeding colony of the Psychology Department at Temple University. Sixteen pairs were injected with melanoma cells at 10-11 months of age (Experiment I), and 42 pairs were injected at 4-7 months of age (Experiment 2).
Procedure Ten days prior to injection the mice were housed individually in stainless steel shoebox cages (394 cm 2 floor area) covered with Easi Litter (Westminster Scientific Co.). Water and food (Charles River Rat, Mouse and Hamster Formula) were available ad lib. Food pellets (average weight about 5 grams each) were placed directly on the cage floor in order to maximize food accessibility. Every four days all uneaten pieces were removed and weighed on a Mettler balance, litter was changed, and fresh food was weighed and placed into the cage. Every effort was made to maximize the precision of food intake measurement. All detectable food remnants were retrieved for weighing, and we found no reason to question the assumption that the variability of any unweighed residue was equal for obese and lean animals. On the day that mice were to be injected (Day 0), a B16C3 stock tumor was removed from a freshly sacrificed C57BL/6J mouse. The B16C3 is a thrice-cloned line of B16 cells selected at each cloning for stability and intensity of melanogenesis [16]. Tumor fragments were dissociated by washing in a 0.04% Versene solution for 30 minutes and were separated from the Versene by centrifugation (1000 rpm for 15 minutes). They were resuspended in phosphate-buffered saline, counted in a hemacytometer, and adjusted to 1× l0" cells per 0.1 cc solution. Mice were injected subcutaneously with 10~ cells in the dorsal right posterior quadrant. Tumor diameters were measured with a Ve,::ier caliper at two-day intervals, beginning 12 days after ,.njection and co,atinuing until death. Measurements on any given day were calculated as the geometric n. ~n of two diameters (GMD), the first being the longest tumor diameter and the second taken perpendicular to the first. The day of initial primary tumor appearance was arbitrarily designated as the first day that the GMD was t>5 mm. Body weights were obtained in both Experiment I and 2, beginning six days prior to injection and continuing every four days thereafter until death. Food consumption was measured in Experiment 2. Food intake was calculated as 4-day totals, beginning six days prior to injection and continuing until death.
Data Analysis Body weights were analyzed separately for Experiments
1 and 2, with comparisons made at five points in time. A baseline weight was obtained six days prior to tumor cell injection, and the four subsequent weights were those last obtained (at the routine 4-day weighing periods) during each of the four quarters of the survival period, as individually determined for each animal. Standard analysis of variance techniques were utilized, with Body Size (obese, lean) as a between-group measure and Time (baseline, survival quarters 1--4) as a repeated measure. Food intakes (Experiment 2) also were compared across 5 time periods. A baseline intake was calculated as the average daily intake during the 4-day period from six to two days prior to tumor cell injection, and the remaining four measurements were the average daily intakes throughout each quarter of the survival period. For purposes of analysis, the first quarter began 2 days prior to cell injection. Only data from complete 4-day periods were used; the end of each quarter was designated as the last measure obtained during each fourth of the survival period, as calculated individually for each animal. A two-way analysis of variance again utilized Body Size as a between-group measure and Time as a repeated measure. F-ratios occurring with a probability o f p <0.05 were considered statistically significant. Comparisons subsequent to significant F-ratios were made using the Newman-Keuls procedure. RESULTS
Experiment I Body weight. Body weights of lean mice increased during the later stages of tumor growth whereas obese mice lost weight (Fig. 1), and the interaction between Body Size and Time was statistically significant, F(4,120)= 18,42, p<0.001. F o r obese mice, body weights at the end of the 4th quarter (72.9+2.8 gm) were 12.8% below preinjection baseline weights (83.6_+2.2 gm, p<0.01), whereas for lean mice body weights at the end of the 4th quarter (38.4_+ 1.5 gm) were 19% above baseline (32.1 _+1.0 gm, p<0.01). Tumor, metastasis and survival. Other data from Experiment 1 were reported earlier [34] and can be summarized as follows:' The tumor first appeared (~5 mm) after approximately equal post-injection intervals in the obese mice (17.4-+ 1.2 days) and lean mice (16.0_+ 1.0 days). Obese mice lived an average of 61.6_+5.0 days after tumor injection. whereas lean mice survived 53.9_+ 1.6 days. Diameter of the primary tumor at death averaged 18.4-+1.5 mm for obese mice and 29.0+0.8 mm for lean mice. At autopsy, visible lung metastases were present in 25.0% of the obese mice and in 93.8% of the lean mice. Experiment 2 Body weight. Obese mice gained a small amount of body weight during Experiment 2. As in Experiment 1, however, the weight gains of lean mice were considerably greater than those of obese mice (Fig. I), and the interaction between Body Size and Time was statistically significant, F(4,328)=43.91, p <0.001. Obese mice weighed an average of 68.9+ l.l gm at baseline and 71.3_+1.4 gm at the end of the fourth quarter, an increase of 3.5% (p<0.01). Lean mice weighed 25.8_+0.3 gm at baseline; their weights increased 16% by the end of the third quarter to 29.9_+0.5 gm (p<0.01), and 53% by the end of the fourth quarter to 39.5___0.8 gm (p<0.01).
FOOD I N T A K E D U R I N G T U M O R G R O W T H
EXPERIMENT
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EXPERIMENT
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2
3
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LEAN
QUARTER
FIG. 1. Changes in body weight, relative to a pre-injection baseline, at the end of each quarter of the survival period for female mice injected with BI6 melanoma. Sixteen pairs of genetically obese (ob/ob) and lean (+/?) mice were injected at 10-11 months of age in Experiment l, and 42 pairs were injected at 4-7 months in Experiment 2. At baseline, mean weights (-+SE) for obese mice in Experiments 1 and 2 were 83.6_+2.2 grams and 68.9-+ 1.1 grams, respectively; weights for lean mice were 32.1-+1.1 grams and 25.8-+0.3 grams.
Food intake. F o o d intake increased dramatically during the final stages of tumor growth in lean mice, whereas the intake of obese mice dropped to below baseline levels (Fig. 2). The interaction between Body Size and Time was statistically significant, F(4,328)= 129.69, p<0.001. Lean mice consumed significantly less food than obese mice during the baseline period and during the first three quarters of the survival period (all ps<0.01). During the 4th quarter, however, lean mice consumed more food than did the obese mice; in fact, the average daily consumption of lean mice during this quarter was greater than that of obese mice during any of the five measurement periods (all ps<0.01). F o r lean animals only, daily food intake fin'st rose significantly above baseline during the 3rd quarter (32.6% above baseline), with a further increase occurring during the 4th quarter (83.7% above baseline; ps<0.01). Obese mice exhibited an intake that was greater than baseline during each of the first three quarters of survival (18.2%, 25.8% and 22.7%, respectively), but their intake dropped to 9.1% below baseline during the 4th quarter (all ps<0.01). F o r both lean and obese animals, temporal changes in food intake were less apparent when food intake was expressed as a percentage of body weight. During each day of the baseline period, lean mice consumed an amount of food equal to 19.0% of their body weight (as measured 6 days prior to injection), and percentages during the four quarters of survival (calculated on the basis of body weight at the end of each period) were 18.5%, 19.0%, 21.7% and 22.8%, respectively. For obese mice, the amount of food consumed daily was equivalent to 9.6% of their body weight during the baseline period, and percentages during the subsequent four quarters of the survival period were 11.6%, 12.2%, 11.7% and 8.4%, respectively. Tumor, metastasis and survival. These data were reported earlier [34] and can be summarized as follows: The tumor first appeared (~5 mm) after similar post-injection intervals in the obese mice (14.6-+0.7 days) and lean mice (13.9-+1.1 days). Obese mice lived an average of 48.6-+1.5 days after tumor injection, and lean mice survived 44.9-+ 1. l
,
I
I
I
I
I
B
l
2
3
4
SURVIVAL
QUARTER
FIG. 2. Average daily food intake for 42 pairs of genetically obese (ob/ob) and lean (+/?) female mice during a 4-day baseline period (B) and during each quarter of the survival period following injection with B16 melanoma. Vertical bars represent standard errors of the mean.
days. Diameter of the primary tumor at death averaged 29.1_+0.8 mm for obese mice and 28.8_+0.6 mm for lean mice. At autopsy, visible lung metastases were present in 38.1% of the obese mice and 85.7% of the lean mice. DISCUSSION Obese and lean mice increased their food intake by approximately equal amounts during the In'st three quarters of the survival period. In the third quarter of Experiment 2, for example, daily intake exceeded baseline values by 1.5 grams in obese mice and 1.6 grams in lean mice. By the final quarter of survival, however, sizeable group differences appeared. Obese mice became anorexic, with average daily intake dropping 2.1 grams below the third quarter figure (and 0.6 gm below baseline), whereas lean mice became increasingly hyperphagic, with daily intake averaging 2.5 grams more than during the third quarter. The extent of this hyperphagia is evident from the fact that the 9.0 gram average daily intake of lean mice during the fourth quarter was significantly higher than that of obese mice at any point in the study. Body weights of obese and lean mice remained fairly stable throughout the first three quarters of survival. During the final quarter, however, lean mice gained considerable weight whereas obese mice either lost weight (Experiment l) or exhibited minimal gains (Experiment 2). The greater weight loss in Experiment l, where animals were older than those in Experiment 2, does not reflect a natural tendency for obese mice to lose weight with advancing age; unpublished data from our laboratory indicate that the weights of non-cancerous obese females exhibit a linear increase between 2 and 14 months of age. Tumor masses were not measured at autopsy, but it seems clear that the greater weight gains of lean mice relative to their obese littermates cannot be attributed entirely to a greater tumor mass. This is especially true in Experiment 2, where lean mice gained an average of 11.3 grams more than obese mice despite the fact that final tumor diameters were virtually identical for the two groups.
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THOMPSON, KREIDER AND MARGULES
Decreases in food intake and body weight comparable to those seen in the obese mice have frequently been observed in tumor-bearing organisms. Anorexia and subsequent weight loss are part of a cachexic syndrome that occurs in one-third to two-thirds of human patients with advanced cancer [33], and in experimental animals an initial weight gain following tumor cell injection typically gives way to a progressively increasing anorexia, hyperlipemia, and weight loss [18,23]. Many mechanisms have been proposed to account for cancer cachexia [1, 3, 5, 8, 13, 17, 43], but the cause is not completely understood. Elucidation of the mechanism is complicated by the fact that cachexia does not always develop, a point illustrated by the progressive hyperphagia and weight gain of lean animals in this study. Whatever its cause, weight loss has generally been associated with a sharp reduction in survival. In one large clinical study, weight loss was a stronger predictor of death than stage of tumor, histologic type, performance index or type of chemotherapy [6], and in a recent study of patients with a spectrum of tumor types the median survival of patients who experienced weight loss before chemotherapy was about half that of patients who had not lost weight [9]. The present data, however, raise a question concerning whether weight loss is detrimental to survival in obese animals. Obese mice became anorexic and lost weight during the last few days of life, but they nevertheless lived longer than lean mice, who ate avidly and gained weight. At least two interpretations of these data are possible. The first is that anorexia and weight loss are detrimental, regardless of body size. This interpretation suggests (a) that the relatively short life span of lean animals was nevertheless longer than it would have been had cachexia developed, and (b) that the death of the obese mice, although initially postponed by some unspecified factor, was ultimately accelerated by loss of appetite and body weight. An alternative interpretation is that anorexia is part of a protective homeostatic response during advanced cancer, possibly serving as a means of inhibiting tumor growth. Although continued weight loss eventually would be incompatible with life, obese animals presumably could survive this loss longer than non-obese organisms. This interpretation suggests that the fourth-quarter anorexia in obese mice actually contributed to their extended life span. Further data clearly are needed. However, the idea that weight loss might be beneficial is supported by recent studies
showing that acute periods of starvation can enhance immune effector systems in both humans [24,37] and rodents [36,38]. It has long been known that chronic underfeeding reduces the incidence of various tumors in mice [31,32] and rats [22, 27, 28]. It also is well known that food intake is exquisitely sensitive to changes in the internal environment, and that organisms adjust their dietary intakes to maximize longevity under a multitude of adverse conditions [26]. Survival times were longer in Experiment l, for both obese and lean mice, than they were in Experiment 2 (57.8 days and 46.7 days, respectively, p<0.001). Moreover, the primary tumors of obese mice grew more slowly in Experiment l, and reached a smaller terminal size, than they did in Experiment 2. The reasons underlying this increased resistance for mice in Experiment 1 are not clear, but two points might be noted. First, obese animals lost more weight, and lean mice gained less, during Experiment I than they did during Experiment 2; this indirectly supports the hypothesis that weight loss may have a positive effect upon survival in tumor-bearing animals. Second, the mice in Experiment 1 were about five months older than those in Experiment 2 when testea. Although the present studies were not specifically designed to evaluate age differences, it may be that resistance to B 16 melanoma increases in older animals. Numerous factors may have contributed to the reduced metastasis and extended longevity of the ob/ob mouse. For example, we recently reported that obese mice exhibit enhanced immunocompetence relative to lean mice [2]. This in turn may be related to elevated B-endorphin levels in obese mice [1 l, 20, 25], since a growing body of evidence indicates that neuropeptides such as B-endorphin enhance mitogeninducing lymphocyte proliferative responses [ 12] and natural cytotoxicity of tumor cells [21]. Another possibility is that a reduction in available metabolic energy is related to the enhanced resistance to cancer of obese animals. The ob/ob mouse, like many obese organisms, is hyperinsulinemic [11]. This condition perpetuates the absorptive state of metabolism and inhibits the utilization of stored fat as an energy source. These two possibilities are by no means exhaustive. In conclusion, obese ob/ob mice reduce their food intake during the final stages of BI6 melanoma tumor growth, whereas lean _+/? mice become hyperphagic. Obese animals concurrently exhibit a lower incidence of metastasis and an increase in survival time. Additional work is needed to determine if a causal relationship exists.
REFERENCES
1. Bernstein, I. L. and R. A. Sigmundi. Tumor anorexia: A learned food aversion? Science 209: 416--418, 1980. 2. Black, P. L., M. Holly, C. I. Thompson and D. L. Margules. Enhanced tumor resistance and immunocompetence in obese (ob/ob) mice. Life Sci 33: Suppl. 1,715-718, 1983. 3. Bozzetti, F., A. M. Pagnoni and M. Del Vecchio. Excessive caloric expenditure as a cause of malnutrition in patients with cancer. Surg Gynecol Obstet 150: 229-234, 1980. 4. Choi, N. W., G. R. Howe, A. B. Miller, V. Matthews, R. W. Morgan, L. Munan, J. D. Burch, J. Feather, M. Jain and A. Kelly. Am J Epidemiol 107: 510-521, 1978. 5. Costa, G. Cachexia, the metabolic component of neoplastic diseases. Cancer Res 37: 2327-2335, 1977. 6. Costa, G., R. Vincent, M. Aragon, P. Tracy and P. Homan. Weight loss and cachexia in lung cancer. Proc Am Assoc" Cancer Res 20: 387, 1979.
7. DeWaard, F. and E. A. B. Halweijn. A prospective study in general practice of breast cancer risk in post-menopausal women, lnt J Cancer 14: 153-160, 1974. 8. DeWys, W. D. Anorexia in cancer patients. Cancer Res 37: 2354-2358, 1977. 9. DeWys, W. D. Nutritional care of the cancer patient. J Am Med Assoc 244: 374-376, 1980. 10. Drenick, E. J., G. S. Bale, F. Seltzer and D. G. Johnson. Excessive mortality and causes of death in morbidly obese men. J Am Med Assoc 243: 443-445, 1980. 11. Garthwaite, T. L., D. R. Martinson, L. F. Tseng, T. C. Hagen and L. A. Menahan. A longitudinal hormonal profile of the genetically obese mouse. Endocrinology 107: 671-676, 1980. 12. Gilman, S. C., J. M. Schwartz, R. J. Milner. F. E. Bloom and J. D. Feldman. B-Endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sei USA 79: 4226--4230, 1982.
FOOD INTAKE DURING TUMOR GROWTH 13. Holland, J. C. B., J. Rowland and M. Plum. Psychological aspects of anorexia in cancer patients. Cancer Res 37: 2425-2428, 1977. 14. Ingallis, A. M., M. M. Dickie and G. D. Snell. Obese, a new mutation in the house mouse. J Hered 41: 317-318, 1950. 15. Jax Notes, No. 405, November, 1970. 16. Kreider, J. W., M. Rosenthal and N. Lengle. Cyclic adenosine 3',5'-monophosphate in the control of melanoma cell replication and differentiation. J Natl Cancer Inst 50: 555-558, 1973. 17. Liebelt, R. A., C. Bordelon and A. G. Liebelt. Adipose tissue system and food intake. Prog Physiol Psychol 5:211-252, 1973. 18. Liebelt, R. A., G. Gehring, L. Delmonte, G. Schuster and A. G. Liebelt. Paraneoplastic syndromes in experimental animal model systems. Ann N Y Acad Sci 230: 547-564, 1974. 19. Lin, T. M., K. P. Chen and B. MacMahon. Epidemiologic characteristics of cancer of the breast in Taiwan. Cancer 27: 14971504, 1971. 20. Margules, D. L., B. Moisset, M. J. Lewis, H. Shibuya and C. Pert: Beta-endorphin is associated with overeating in genetically obese mice (ob/ob) and rats OCa/fa). Science 202: 988-991, 1978. 21. Matthews, P. M., C. J. Froelich, W. L. Sibbitt, Jr. and A. D. Bankhurst. Enhancement of natural beta-endorphin cytotoxicity. J lmmunol 130: 1658-1662, 1983. 22. McCay, C. M., M. F. Crowell and L. A. Maynard. The effect of retarded growth upon the length of life span and upon the ultimate body size. J Nutr 10: 63-79, 1935. 23. McEuen, C. S. and D. L. Thomson. Effect of hypohysectomy on the growth of the Walker rat. Br J Exp Pathol 14: 384-391, 1933. 24. Murray, J. and A. Murray. Suppression of infection by famine and its activation by refeeding--a paradox? Perspect Biol Med 20: 471-483, 1977. 25. Recant, L., N. R. Voyles, M. Luciano and C. B. Pert. Naltrexone reduces weight gain, alters B-endorphin, and reduces insulin output from pancreatic islets of genetically obese mice. Peptides 1: 309-313, 1980. 26. Richter, C. P. Total self regulatory functions in animals and human beings. Harvey Lect Set 38: 63-103, 1942-1943. 27. Ross, M. H. and G. Bras. Influence of protein under- and overnutrition on spontaneous tumor prevalence in the rat. J Nutr 103: 944-963, 1973.
939 28. Saxton, J. A., G. A. Sperling, L. L. Barnes and C. M. McCay. The influence of nutrition upon the incidence of spontaneous tumors of the albino rat. Acta Unio Intern Contra Cancrum 6: 423-43 l, 1948. 29. Sorlie, P., T. Gordon and W. B. Kannel. Body build and mortality: The Framingham study. J Am Med Assoc 243:1828-1831, 1980. 30. Tannenbaum, A. Initiation and growth of tumors. Am J Cancer 38: 335-350, 1940. 31. Tannenbaum, A. Relationship of body weight to cancer incidence. Arch Pathol 30: 509-517, 1940. 32. Tannenbaum, A. The genesis and growth of tumors. II. Effects of caloric restriction. III. Effects of a high-fat diet. Cancer Res 2: 460-475, 1942. 33. Theologides, A. Cancer Cachexia. In: Nutrition and Cancer, edited by M. Winick. New York: Wiley, 1977, pp. 75-94. 34. Thompson, C. I., J. W. Kreider, P. L. Black, T. J. Schmidt and D. L. Margules. Genetically obese mice: Resistance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses. Science 220: 1183-I 185, 1983. 35. Wallace, R. B., C. Rost, L. F. Burmeister and P. R. Pomrehn. Cancer incidence in humans: Relationship to plasma lipids and relative weight. J Natl Cancer Inst 68: 915-918, 1982. 36. Wing, E. J., L. K. Barczynski and S. M. Boehmer. Effect of acute nutritional deprivation on immune function in mice. I. Macrophages. Immunology 48: 543-550, 1983. 37. Wing, E. J., R. T. Stanko, A. Winkelstein and S. A. Adibi. Fasting-enhanced immune effector mechanisms in obese subjects. Am J Med 75: 91-96, 1983. 38. Wing, E. J. and J. B. Young. Acute starvation protects mice against Listeria monocytogenes. Infect Immun 28: 771-776, 1980. 39. Winick, M. Nutrition and Cancer. New York: Wiley, 1977. 40. Wynder, E. L., G. C. Escher and N. Mantel. An epidemiological investigation of cancer of the endometrium. Cancer 19: 489-520, 1966. 41. Wynder, E. L., K. Mabuchi and W. F. Whitmore. Epidemiology of adenocarcinoma of the kidney. J Natl Cancer lnst 53: 1619-1634, 1974. 42. Wynder, E. L., I. D. J. Bross and T. Hirayama. A study of the epidemiology of cancer of the breast. Cancer 13: 559-601, 1960. 43. Young, V. R. Energy metabolism and requirements in the cancer patient. Cancer Res 37: 2336-2347, 1977.
0031-9384/84 $3.00 + .00
Food Intake During Tumor Growth: Anorexia in Genetically Obese OB/OB MICE and Hyperphagia in Lean Mice C A R L I. T H O M P S O N
Deparment o f Psychology, Wabash College, Crawfordsville, I N 47933 JOHN W. KREIDER
Departments o f Pathology and Microbiology, The Milton S. Hershey Medical Center o f The Pennsylvania State University, Hershey, PA 17033 AND DAVID L. MARGULES
Department o f Psychology, Temple University, Philadelphia, PA 19122 R e c e i v e d 29 A u g u s t 1983 THOMPSON, C. I., J. W. KREIDER AND D. L. MARGULES. Food intake during tumor growth:Anorexia in genetically obese ob/ob mice and hyperphagia in lean mice. PHYSIOL BEHAV 32(6) 935-939, 1984.--Recent f'mdings indicate that obese (ob/ob) mice suffer a low incidence of lung metastasis and survive longer than lean (+/?) littermates following injection with BI6 melanoma cells [34]. The present study examined the food intake of obese and lean mice during the growth of this tumor. Mice from both groups increased their food intake by small and approximately equal amounts during the first three quarters of the survival period following injection with 106 cells, and body weights remained fairly stable. During the final quarter, however, obese mice became anorexic whereas lean mice became intensely hyperphagic; body weights changed accordingly. Thus, food intake is differentially affected by tumor growth in this form of genetic obesity. Food intake
Body weight
Cancer
B 16 melanoma
Obesity
ob/obMouse
Metastasis
Survival
reverse is true, with a negative association between body build and breast cancer risk [4,42]. In both males and females, controlled prospective studies indicate that people who weigh most at original screening are least likely to develop subsequent malignancy [35]. In a recent paper [34] we presented evidence that the ob/ob mouse, whose obesity is caused by a single recessive gene [14], may provide an animal model for studying increased resistance to cancer in obese humans. After injection with B16 melanoma at 10-11 months of age the primary tumor grew to a smaller terminal size in obese mice than in lean littermates, and incidence of lung metastasis at autopsy was greatly reduced. When injection occurred at 4-7 months the primary tumor grew to the same size in obese and lean animals, but obese mice again exhibited a reduced incidence of lung metastasis. Overall, obese mice survived 10.1% longer than lean mice following injection, despite the fact that under standard laboratory conditions the lean mouse lives more than a year longer than its obese counterpart [15]. During the first of the above studies [34] (mice 10-11 months old), we observed that the body weights of obese mice decreased during the terminal phase of survival
C A N C E R and nutrition are closely interconnected. At least three aspects of this relationship have been identified [39]. First, diet plays a role in the etiology o f cancer; second, nutritional status is profoundly affected by cancer; and third, dietary manipulations could play a part in cancer prevention and treatment. Because of their altered nutritional status, obese individuals might be expected to differ from normals in cancer incidence and mortality. Indeed, such differences have been reported. Early investigators suggested that cancer risk was elevated in overweight people [30], but recent findings indicate that the opposite relationship is more common, with obese individuals typically exhibiting a decreased incidence of cancer. Thus, malignancies occur less often in morbidly obese men than in the general population [10], and data from the Framingham study indicate that cancer death rates decrease steadily with increases in body build for men aged 40-69 years [29]. Of the many types of cancer only two, cancer of the endometrium and cancer of the kidney in women, appear positively linked to obesity [40,41]. Obese postmenopausal women may also exhibit an increased incidence of breast cancer [7,19], but in younger women the
1Supported in part by Grant BNS 8216104 from the National Science Foundation to D. L. M.
935
936
THOMPSON, K R E I D E R A N D M A R G U L E S
whereas lean mice exhibited dramatic weight gains. To examine this phenomenon more closely, food intake and body weight both were measured during the second study (mice 4-7 months old). The present report documents the body weight and food intake of obese and lean mice during these investigations. We report here that obese mice became anorexic during the later stages of tumor growth, whereas lean mice became intensely hyperphagic. METHOD
Animals Fifty-eight pairs of genetically obese (C57BL/6J ob/ob) and littermate control (+/?) mice were used. All animals were female and were obtained from the breeding colony of the Psychology Department at Temple University. Sixteen pairs were injected with melanoma cells at 10-11 months of age (Experiment I), and 42 pairs were injected at 4-7 months of age (Experiment 2).
Procedure Ten days prior to injection the mice were housed individually in stainless steel shoebox cages (394 cm 2 floor area) covered with Easi Litter (Westminster Scientific Co.). Water and food (Charles River Rat, Mouse and Hamster Formula) were available ad lib. Food pellets (average weight about 5 grams each) were placed directly on the cage floor in order to maximize food accessibility. Every four days all uneaten pieces were removed and weighed on a Mettler balance, litter was changed, and fresh food was weighed and placed into the cage. Every effort was made to maximize the precision of food intake measurement. All detectable food remnants were retrieved for weighing, and we found no reason to question the assumption that the variability of any unweighed residue was equal for obese and lean animals. On the day that mice were to be injected (Day 0), a B16C3 stock tumor was removed from a freshly sacrificed C57BL/6J mouse. The B16C3 is a thrice-cloned line of B16 cells selected at each cloning for stability and intensity of melanogenesis [16]. Tumor fragments were dissociated by washing in a 0.04% Versene solution for 30 minutes and were separated from the Versene by centrifugation (1000 rpm for 15 minutes). They were resuspended in phosphate-buffered saline, counted in a hemacytometer, and adjusted to 1× l0" cells per 0.1 cc solution. Mice were injected subcutaneously with 10~ cells in the dorsal right posterior quadrant. Tumor diameters were measured with a Ve,::ier caliper at two-day intervals, beginning 12 days after ,.njection and co,atinuing until death. Measurements on any given day were calculated as the geometric n. ~n of two diameters (GMD), the first being the longest tumor diameter and the second taken perpendicular to the first. The day of initial primary tumor appearance was arbitrarily designated as the first day that the GMD was t>5 mm. Body weights were obtained in both Experiment I and 2, beginning six days prior to injection and continuing every four days thereafter until death. Food consumption was measured in Experiment 2. Food intake was calculated as 4-day totals, beginning six days prior to injection and continuing until death.
Data Analysis Body weights were analyzed separately for Experiments
1 and 2, with comparisons made at five points in time. A baseline weight was obtained six days prior to tumor cell injection, and the four subsequent weights were those last obtained (at the routine 4-day weighing periods) during each of the four quarters of the survival period, as individually determined for each animal. Standard analysis of variance techniques were utilized, with Body Size (obese, lean) as a between-group measure and Time (baseline, survival quarters 1--4) as a repeated measure. Food intakes (Experiment 2) also were compared across 5 time periods. A baseline intake was calculated as the average daily intake during the 4-day period from six to two days prior to tumor cell injection, and the remaining four measurements were the average daily intakes throughout each quarter of the survival period. For purposes of analysis, the first quarter began 2 days prior to cell injection. Only data from complete 4-day periods were used; the end of each quarter was designated as the last measure obtained during each fourth of the survival period, as calculated individually for each animal. A two-way analysis of variance again utilized Body Size as a between-group measure and Time as a repeated measure. F-ratios occurring with a probability o f p <0.05 were considered statistically significant. Comparisons subsequent to significant F-ratios were made using the Newman-Keuls procedure. RESULTS
Experiment I Body weight. Body weights of lean mice increased during the later stages of tumor growth whereas obese mice lost weight (Fig. 1), and the interaction between Body Size and Time was statistically significant, F(4,120)= 18,42, p<0.001. F o r obese mice, body weights at the end of the 4th quarter (72.9+2.8 gm) were 12.8% below preinjection baseline weights (83.6_+2.2 gm, p<0.01), whereas for lean mice body weights at the end of the 4th quarter (38.4_+ 1.5 gm) were 19% above baseline (32.1 _+1.0 gm, p<0.01). Tumor, metastasis and survival. Other data from Experiment 1 were reported earlier [34] and can be summarized as follows:' The tumor first appeared (~5 mm) after approximately equal post-injection intervals in the obese mice (17.4-+ 1.2 days) and lean mice (16.0_+ 1.0 days). Obese mice lived an average of 61.6_+5.0 days after tumor injection. whereas lean mice survived 53.9_+ 1.6 days. Diameter of the primary tumor at death averaged 18.4-+1.5 mm for obese mice and 29.0+0.8 mm for lean mice. At autopsy, visible lung metastases were present in 25.0% of the obese mice and in 93.8% of the lean mice. Experiment 2 Body weight. Obese mice gained a small amount of body weight during Experiment 2. As in Experiment 1, however, the weight gains of lean mice were considerably greater than those of obese mice (Fig. I), and the interaction between Body Size and Time was statistically significant, F(4,328)=43.91, p <0.001. Obese mice weighed an average of 68.9+ l.l gm at baseline and 71.3_+1.4 gm at the end of the fourth quarter, an increase of 3.5% (p<0.01). Lean mice weighed 25.8_+0.3 gm at baseline; their weights increased 16% by the end of the third quarter to 29.9_+0.5 gm (p<0.01), and 53% by the end of the fourth quarter to 39.5___0.8 gm (p<0.01).
FOOD I N T A K E D U R I N G T U M O R G R O W T H
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FIG. 1. Changes in body weight, relative to a pre-injection baseline, at the end of each quarter of the survival period for female mice injected with BI6 melanoma. Sixteen pairs of genetically obese (ob/ob) and lean (+/?) mice were injected at 10-11 months of age in Experiment l, and 42 pairs were injected at 4-7 months in Experiment 2. At baseline, mean weights (-+SE) for obese mice in Experiments 1 and 2 were 83.6_+2.2 grams and 68.9-+ 1.1 grams, respectively; weights for lean mice were 32.1-+1.1 grams and 25.8-+0.3 grams.
Food intake. F o o d intake increased dramatically during the final stages of tumor growth in lean mice, whereas the intake of obese mice dropped to below baseline levels (Fig. 2). The interaction between Body Size and Time was statistically significant, F(4,328)= 129.69, p<0.001. Lean mice consumed significantly less food than obese mice during the baseline period and during the first three quarters of the survival period (all ps<0.01). During the 4th quarter, however, lean mice consumed more food than did the obese mice; in fact, the average daily consumption of lean mice during this quarter was greater than that of obese mice during any of the five measurement periods (all ps<0.01). F o r lean animals only, daily food intake fin'st rose significantly above baseline during the 3rd quarter (32.6% above baseline), with a further increase occurring during the 4th quarter (83.7% above baseline; ps<0.01). Obese mice exhibited an intake that was greater than baseline during each of the first three quarters of survival (18.2%, 25.8% and 22.7%, respectively), but their intake dropped to 9.1% below baseline during the 4th quarter (all ps<0.01). F o r both lean and obese animals, temporal changes in food intake were less apparent when food intake was expressed as a percentage of body weight. During each day of the baseline period, lean mice consumed an amount of food equal to 19.0% of their body weight (as measured 6 days prior to injection), and percentages during the four quarters of survival (calculated on the basis of body weight at the end of each period) were 18.5%, 19.0%, 21.7% and 22.8%, respectively. For obese mice, the amount of food consumed daily was equivalent to 9.6% of their body weight during the baseline period, and percentages during the subsequent four quarters of the survival period were 11.6%, 12.2%, 11.7% and 8.4%, respectively. Tumor, metastasis and survival. These data were reported earlier [34] and can be summarized as follows: The tumor first appeared (~5 mm) after similar post-injection intervals in the obese mice (14.6-+0.7 days) and lean mice (13.9-+1.1 days). Obese mice lived an average of 48.6-+1.5 days after tumor injection, and lean mice survived 44.9-+ 1. l
,
I
I
I
I
I
B
l
2
3
4
SURVIVAL
QUARTER
FIG. 2. Average daily food intake for 42 pairs of genetically obese (ob/ob) and lean (+/?) female mice during a 4-day baseline period (B) and during each quarter of the survival period following injection with B16 melanoma. Vertical bars represent standard errors of the mean.
days. Diameter of the primary tumor at death averaged 29.1_+0.8 mm for obese mice and 28.8_+0.6 mm for lean mice. At autopsy, visible lung metastases were present in 38.1% of the obese mice and 85.7% of the lean mice. DISCUSSION Obese and lean mice increased their food intake by approximately equal amounts during the In'st three quarters of the survival period. In the third quarter of Experiment 2, for example, daily intake exceeded baseline values by 1.5 grams in obese mice and 1.6 grams in lean mice. By the final quarter of survival, however, sizeable group differences appeared. Obese mice became anorexic, with average daily intake dropping 2.1 grams below the third quarter figure (and 0.6 gm below baseline), whereas lean mice became increasingly hyperphagic, with daily intake averaging 2.5 grams more than during the third quarter. The extent of this hyperphagia is evident from the fact that the 9.0 gram average daily intake of lean mice during the fourth quarter was significantly higher than that of obese mice at any point in the study. Body weights of obese and lean mice remained fairly stable throughout the first three quarters of survival. During the final quarter, however, lean mice gained considerable weight whereas obese mice either lost weight (Experiment l) or exhibited minimal gains (Experiment 2). The greater weight loss in Experiment l, where animals were older than those in Experiment 2, does not reflect a natural tendency for obese mice to lose weight with advancing age; unpublished data from our laboratory indicate that the weights of non-cancerous obese females exhibit a linear increase between 2 and 14 months of age. Tumor masses were not measured at autopsy, but it seems clear that the greater weight gains of lean mice relative to their obese littermates cannot be attributed entirely to a greater tumor mass. This is especially true in Experiment 2, where lean mice gained an average of 11.3 grams more than obese mice despite the fact that final tumor diameters were virtually identical for the two groups.
938
THOMPSON, KREIDER AND MARGULES
Decreases in food intake and body weight comparable to those seen in the obese mice have frequently been observed in tumor-bearing organisms. Anorexia and subsequent weight loss are part of a cachexic syndrome that occurs in one-third to two-thirds of human patients with advanced cancer [33], and in experimental animals an initial weight gain following tumor cell injection typically gives way to a progressively increasing anorexia, hyperlipemia, and weight loss [18,23]. Many mechanisms have been proposed to account for cancer cachexia [1, 3, 5, 8, 13, 17, 43], but the cause is not completely understood. Elucidation of the mechanism is complicated by the fact that cachexia does not always develop, a point illustrated by the progressive hyperphagia and weight gain of lean animals in this study. Whatever its cause, weight loss has generally been associated with a sharp reduction in survival. In one large clinical study, weight loss was a stronger predictor of death than stage of tumor, histologic type, performance index or type of chemotherapy [6], and in a recent study of patients with a spectrum of tumor types the median survival of patients who experienced weight loss before chemotherapy was about half that of patients who had not lost weight [9]. The present data, however, raise a question concerning whether weight loss is detrimental to survival in obese animals. Obese mice became anorexic and lost weight during the last few days of life, but they nevertheless lived longer than lean mice, who ate avidly and gained weight. At least two interpretations of these data are possible. The first is that anorexia and weight loss are detrimental, regardless of body size. This interpretation suggests (a) that the relatively short life span of lean animals was nevertheless longer than it would have been had cachexia developed, and (b) that the death of the obese mice, although initially postponed by some unspecified factor, was ultimately accelerated by loss of appetite and body weight. An alternative interpretation is that anorexia is part of a protective homeostatic response during advanced cancer, possibly serving as a means of inhibiting tumor growth. Although continued weight loss eventually would be incompatible with life, obese animals presumably could survive this loss longer than non-obese organisms. This interpretation suggests that the fourth-quarter anorexia in obese mice actually contributed to their extended life span. Further data clearly are needed. However, the idea that weight loss might be beneficial is supported by recent studies
showing that acute periods of starvation can enhance immune effector systems in both humans [24,37] and rodents [36,38]. It has long been known that chronic underfeeding reduces the incidence of various tumors in mice [31,32] and rats [22, 27, 28]. It also is well known that food intake is exquisitely sensitive to changes in the internal environment, and that organisms adjust their dietary intakes to maximize longevity under a multitude of adverse conditions [26]. Survival times were longer in Experiment l, for both obese and lean mice, than they were in Experiment 2 (57.8 days and 46.7 days, respectively, p<0.001). Moreover, the primary tumors of obese mice grew more slowly in Experiment l, and reached a smaller terminal size, than they did in Experiment 2. The reasons underlying this increased resistance for mice in Experiment 1 are not clear, but two points might be noted. First, obese animals lost more weight, and lean mice gained less, during Experiment I than they did during Experiment 2; this indirectly supports the hypothesis that weight loss may have a positive effect upon survival in tumor-bearing animals. Second, the mice in Experiment 1 were about five months older than those in Experiment 2 when testea. Although the present studies were not specifically designed to evaluate age differences, it may be that resistance to B 16 melanoma increases in older animals. Numerous factors may have contributed to the reduced metastasis and extended longevity of the ob/ob mouse. For example, we recently reported that obese mice exhibit enhanced immunocompetence relative to lean mice [2]. This in turn may be related to elevated B-endorphin levels in obese mice [1 l, 20, 25], since a growing body of evidence indicates that neuropeptides such as B-endorphin enhance mitogeninducing lymphocyte proliferative responses [ 12] and natural cytotoxicity of tumor cells [21]. Another possibility is that a reduction in available metabolic energy is related to the enhanced resistance to cancer of obese animals. The ob/ob mouse, like many obese organisms, is hyperinsulinemic [11]. This condition perpetuates the absorptive state of metabolism and inhibits the utilization of stored fat as an energy source. These two possibilities are by no means exhaustive. In conclusion, obese ob/ob mice reduce their food intake during the final stages of BI6 melanoma tumor growth, whereas lean _+/? mice become hyperphagic. Obese animals concurrently exhibit a lower incidence of metastasis and an increase in survival time. Additional work is needed to determine if a causal relationship exists.
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