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Genetic obesity in rats. I. The effects of food restriction on body composition and hypothalamic function
Genetic Obesity in Rats. I. The Effects of Food Restriction on Body Composition and Hypothalamic Function By George A. Bray, David A. York. and Ronald S. Swerloff The effect of controlled food intake was compared in genetically obese rats of the “Fatty” (Zucker) strain and lean animals. One group of six Fatty rats were fed ad lib., another six were pair fed to lean controls, and a third group were fed two-thirds the intake of lean animals. All rats were trained to eat in 6 hr each day. Weight gain was abolished in the Fatties fed a restricted diet, but these animals did not reduce their body fat. Fatty rats fed ad lib. or pair fed were more efficient than lean rats in storing calories as fat. Pituitary, ovary, and uterus were significantly smaller in the Fatty rats. Limiting food intake did not restore these organ weights toward normal.
Among the three groups of Fatty rats, body weight was positively correlated with the weight of the adrenal, pituitary, ovary, uterus, and liver. Urine volume was higher in the ad-lib. and pair-fed Fatties than in the lean animals but restriction of food intake reduced urine output below controls. The uptake of 1251 by the thyroid was significantly reduced in all groups of Fatties, but thyroid weights did not differ. Serum FSH was higher in the restricted Fatties but LH was not. These studies suggest that previously observed differences in hypothalamic endocrine function do not result from hyperphagia.
P
REVIOUS STUDIES from thislv8 and other laboratoriesg-l2 have shown a number of abnormalities in the genetically obese rat called Fatty. These animals are characterized by deposition of adipose tissue at all sites and have a smaller skeleton and less lean body mass than lean littermates.g The increased food intake3z10 poydipsia ,’ decreased thyroid activity,8 sterility, and irregular estrus cycles2 have all suggested that the hypothalamus might be the locus for the genetic abnormality in these rats.1*2 Studies on two other forms of experimental obesity have indicated, however, that some or all of the abnormalities might be the result of hyperphagia and that the primary defect might be enhanced food intake.13*r4 Thus, the sand rat (Psnmmomys obesus) does not develop obesity until its diet is changed from green vegetables to laboratory chow. Even with a chow diet, obesity can still be prevented by restricting food intake. r3 Similar findings have been reported From the Departments of Medicine, Harbor General Hospital, Torrance, Calif., and UCLA School of Medicine, Los Angeles, Calif. Received for pubIication August 29, 1972. Supported in part by NIH Grants RR 425 and AM 15165. George A. Bray, M.D.: Associate Chief, Endocrinology Division, Department of Medicine, Harbor GeneraI Hospital, Torrance, California; and Associate Professor of Medicine, UCLA School of Medicine, Los Angeles, Calif. David A. York, Ph.D.: Lecturer, Department of Physiology and Biochemistry, Soutkampfon University, Soutkampfon, England. Ronald S. Swerdloff, M.D.: Associafe Chief, Endocrinology Division, Departmenf of Medicine, Harbor General Hospital, Torrance, California; and Assisfanf Professor of Medicine, UCLA School of Medicine, Los Angeles, Calif. Reprint requests should be addressed to George 1000 West Carson Sfreet, Torrance, Calif. 90509. @ 1973 by Grune 6 Stratton, inc.
Metabolism. Vol. 22, No. 3 (March),
1973
A. Bray, M.D.,
Harbor
General
Hospital,
435
436
BRAY, YORK, AND SWERLOFF
for the C3HfI hybrid (Wellesley mouse) after caloric restriction.14 The present experiments were designed to clarify the relation between food intake and various endocrine and metabolic abnormalities in the Fatty rat. MATERIALS
AND METHODS
Experimental Protocol Nine lean and 21 genetically obese Fatty female rats were purchased from Dr. Zucker.* Their weight upon arrival in the laboratory averaged 128 -C 6 for lean animals and ranged between 138-262 g for Fatties. These Fatty rats were divided into two groups; 16 rats weighing 128-194 g and 5 rats weighing 238-262 g. Three lean and three Fatty rats were killed within 2 days of arrival for analysis of body composition. The remaining 6 lean and 18 Fatty rats were housed individually in stainless-steel metabolism cages and fed powdered food (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO.) 6 hr/day (9 a.m.-3 p.m.). Water was available ad lib. The 18 Fatty rats were divided into three groups of six rats each. One group was fed ad lib. (ad-lib. Fatties). Six Fatties (pair-fed Fatties) were individually pair fed to six corersponding lean rats and the third group of six Fatties (restricted Fatties) was restricted to two-thirds of the food eaten by the lean control animals. All food actually eaten was weighed. After SO days, the animals were killed in groups of four (one lean plus its’ three respective Fatties). One wk prior to sacrifice, the daily urine volume and water intake were measured for 4 consecutive days.7
Thyroid Function Seven days prior to sacrifice, animals were placed on a low iodine diet15 while still maintaining the 6-hr pair-feeding schedules. Twenty-four hr prior to sacrifice, 2OcLCi1251 were iniected intraperitoneally into each rat and thyroidal uptake assessed as described previously.8
General Procedure at Sacrifice Rats were fasted from the end of their previous feeding period until dealth 18-24 hr later. Blood was removed from the abdominal aorta under ether anesthesia and was transferred promptly to chilled tubes containing EDTA. The serum was separated and frozen for further analysis. The left perimetrial fat pad was removed, weighed, and used for metabolic studies. The pituitary ‘gland was removed, weighed, and homogenized in O&ml saline and frozen until diluted for determination of FSH and LH.16 The heart, liver, left kidney, left uterus, and left ovary were removed, weighed, and replaced in the carcass, which was frozen.
Body Composition The frozen carcass was thawed; the intestinal tract from pylorus to sigmoid was stripped from the mesentry and removed. The remaining carcass was reweighed and minced with two weights of anhydrous sodium sulfate. After three grindings and a final blending in a commercial Waring Blender, the resulting uniform mixture was reweighed and duplicate aliauots removed for the determination of water content bv drying to constant weight at 100°C. Duplicate aliquots were extracted 3 times with chloroform:methanol (2:1, vol/vol), filtered, and the phases separated by addition of water. The dried chloroform extract was considered to be total lipid. Estimates of the initial body composition was made by taking the percentage compositions from the lean or fatty rats of nearest weight killed at the beginning and multiplying these percentages by the body weight of the rat in the experimental group.
Statistical Analysis The data from the four groups *Dr. Lois Zucker,
Stow, Mass.
of animals
have been compared
with students
t test for
GENETIC paired
OBESITY
differences
IN RATS.
437
I
and by two-way
analysis
of variance
using
a desk calculator.
RESULTS
The Effect
of Food
intake
on Body Weight
The changes in mean body weight for the four groups of rats used in this experiment are shown in Table 1. The two groups receiving food ad lib. gained weight steadily. The pair-fed Fatties gained weight at a somewhat slower rate than the lean controls and significantly less rapidly than the ad lib. Fatties. The “restricted Fatties” showed almost no change in body weight. Food Intake
and Caloric
Storage
Table I shows the effect of dietary intake on body weight and body composition in the four groups. Initial body fat of lean rats was between 4.3% and 4.6%. During the course of the experiment, the lipid content of lean rats increased to more than 10% of the total body weight. In the Fatties, lipid initially accounted for nearly one-third total body weight. There was essentially no increase in lipid content of the restricted Fatties. However, the content of body fat in pair-fed and ad-lib. groups of Fatties rose to over 50%. The water content of the lean animals rose by SOO/O and the fat-free dry mass by 100%. In the pair-fed and restricted Fatties, water content remained unchanged, but in the ad lib. Fatties it rose 30%. Although caloric intake of lean and pair-fed Fatty rats was essentially identical, the Fatty rats stored .soO,& more calories than the lean controls. Over 90% of these calories were deposited in lipid stores by the pair-fed Table 1. Body Composition
of Caloric Content of Lean and Obese Rats Lean
Diet
Body wt (9)
Initial Final
Body fat (9)
n initial Final
Body water (9)
n Initial Final
Fat-free dry wt (9)
n Initial Final n
Food intake Caloric intake* Stored calories: (kcal) Stored calories (O/Oof intake)
Fatty
Ad Lib.
totalt lipid non-lipid
128 2 223 r 94 -t 5.3-t
6 7 7 0.27
27.6_’ 2.5 22.3? 2.2 86.15 4.1 128.42 5.3 42.3r 6.0 36.7% 1.9 66.7% 3.8 30.0? 3.6 745 ? 36 2831 -r-136.8 318.8% 16.8 201.62 21 117.2-t 13 11.22 0.4
Restricted
176 & 18 187 ” 10 11 -e 8 51.3% 7.3 57.2% 4.2 5.9-c 3.5 87.0-c 6.9 91.9% 9.7 4.9, 6.5 38.0% 4.0 38.5? 8.7 0.5% 6.3 47.9* 17 1829 -+-646 53.12 45 50.4t 32 -2.7% 32 2.9& 1.0
Pair Fed
187
2
246 ‘-t 59 I+ 54.8 100.2&
12
Ad Lib.
184
2
16
5 8 5.4 3.4
303 -t 21 118 I? 19 54.61 7.t 126.41 11.0
45.4I+ 5.0 91.42 4.5 95.02 5.2 3.6? 2.9 41.oi 2.9 51.0* 2.0 10.0% 1.7 710 1?129 2698 k-1 10.2 448.6& 43 408.62 45 40 2 6.4 16.7t 1.4
71.8f 10.6 90.1-t 5.6 121.4-c 13.1 31.3& 13.1 39.3-1- 3.6 54.21: 10.7 14.91 10.2 943 & 69 3583 1262.2 708 i 78 648 17 96 60 -5 27 19.8? 1.4 -
Caloric intake = food intake x 3.8. tStored calories = (A fat x 9) + (A protein x 4).
438
BRAY, YORK, AND SWERLOFF
Table 2. Organ Weights of Lean and Fatty Rats
Group Lean Ad Lib.
Body weight (g) Adrenal (mg) Pituitary (mg) Thyroid (mg) Ovary (mg) Uterus (mg) Liver(g) Kidney (g)
Fatty Pair Fed
Restricted
187 +lo 223 -c 7’ 26.6 I? 8.0 40.7 f 4.3 4.03& 0.79 8.432 0.63 16.75a 2.47 12.05-c 2.59 23.2 & 8.6 59.7 a14.3 29.2a-e13.92 136.2 1b31.3 5.422 0.47 7.17& 0.95 0.66% 0.10 0.75’ 0.06
246 -r-5 35.7 24.2 4.5820.32 ll.98*5.30 26.6 +6.5 30.28&7.19 6.5121.39 0.7l-cO.06
Ad Lib.
Analysis of Variance Between Groups P
303 +21 42.2 -+ 9.2 5.67+ 1.71 14.3 * 4.1 43.0 113.2 62.2 238.3 8.37-c 1.56 0.87* 0.15
=O.Ol NS <0.025 NS <0.025
*Mean ? SEM. Fatties but only 60% of the added calories were stored as lipid in lean animals. The ad-lib. Fatties stored twice as many calories as the lean animals and over 90% of this storage was in the form of lipid.
The EfFect of Food intake on Organ Weights Table 2 shows the organ weights of lean and Fatty rats. Analysis of variance revealed highly significant differences between body weight and pituitary, ovarian, and uterine weights. However, there was no significant difference in the weights of the adrenals, thyroid, liver, or kidney. Organ weights corrected by dividing by the gross body weight or by (body weight)0.7 did not abolish the differences between the lean and fat animals. In the three groups of Fatty rats organ weights were related to body weight and/or food intake. A regression analysis between body weight and organ weight of the Fatty rats showed a significant correlation with r ranging between 0.81 and 0.92. Food restriction, however, did not restore these organs to the weight of comparable organs in the lean animals. The EfJects of Food Restriction
on Thyroid Function
Thyroid weight was similar in all four groups of animals (Table 3, but the Table 3. Uptake of 1251 by Thyroid
of Lean and Genetically
Thyroid Weight (mg)
Obese Rats*
125f Uptake (% Dose)
Lean
16.8 f
2.5t
12.0 + 1.0
Fatty Restricted Pair fed Ad lib.
12.1 & 2.6 12.0 % 5.3 14.3 f 4.1
a.5 2 0.9 a.2 + 0.6 a.1 * 0.7
p
*Animals were fed a low iodine diet for 7 days prior to sacrifice. Twenty PCi 1251 was injected i.p. in 0.2 ml saline 24 hr prior to sacrifice. Thyroids were dissected free and counted in a well-type counter. tMean & SEM.
GENETIC
OBESITY
IN RATS. I
Fig. 1. Urine volume of lean and Fatty rats. The mean -C SEM for each group is plotted over a 4-day period. Analysis of variance showed a significant day-to-day variation and a significant difference between groups.
i
DAYS
i
i
uptake of iz51 by the thyroid was significantly greater in the lean animals than in the three groups of Fatty rats. Restriction of food intake and the corresponding reduction of iodine intake in the Fatties did not alter the differences in uptake of 125I between the fat and lean rats. Urinary Volume During Food Restriction Analysis of variance revealed a significant day-to-day variation in urine volume as well as a significant difference in urine output between the four groups of animals (Fig. 1). Both ad-lib. and pair-fed Fatties had higher urine volumes and water intakes (measured but not recorded here) than the lean rats. Only when caloric intake was restricted to two-thirds of lean intake was the urine volume reduced below control values for lean rats. Gonadotropins Serum FSH increased with food restriction and was significantly higher in the restricted Fatties (Table 4). In contrast, serum LH was unaffected by food intake or obesity, even though ovarian and uterine weights were significantly smaller in the Fatties (Table 2). The quantity of FSH in the pituitary of Fatty rats appeared to rise with food restriction as did the serum FSH. The content of FSH, whether expressed per animal or per mg of pituitary, was significantly higher in the group with restricted or pair-fed diets than in the ad lib.-fed Fatties. The total quantity of LH in the pituitary was lower in the Fatties than in the lean animals. When expressed per mg of pituitary, the restricted and pair-fed Fatties had significantly higher concentrations of LH than lean controls. Pituitary extracts from both lean and obese rats gave curves in the immunoassay that were parallel to standard preparations of LH and FSH suggesting that there were no immunologic differences in the gonadotropins of these animals.
BRAY, YORK, AND SWERLOFF
440
Table 4. Serum and Pituitary
Gonadotropins
in Lean and Obese Rats” Fatty
Lean Restricted
Pair Fed
Ad Lib.
Serum LH ng/ml FSH ng/ml
61.1 2 6.0 197 -t-19
52.1 & 4.5 266 &20t
53.0 i- 5.0 212 k-22
68.5 k10.0 196 -c 8.5
Pituitary LH pg/pituitary aglmg FSH pg/pituitary
191 k18.5 16.5 k 2.2 15.6 2 1.2
140.5 & 8.4t 34.9 k 2.1* 22.9 & 1.9*
145 413.5 31.7 -c2.95* 21.7 k 1.6$
139 k11.5 24.5 ?I 2.02 18.4 e 1.0
1.85% 0.14
yglmg
*LH and FSH were determined
5.69*
4.75k
0.47*
using a radioimmunoassay
Distribution Program for Rat Pituitary tp <0.05 compared to lean. Sp
Hormones
0.35*
with reagents
3.242 provided
0.18t by the
of the NIAMD.14
DISCUSSION
Although restriction of caloric intake of Fatty rats limited their rate of growth, it did not restore body composition to normal’r Indeed, lean rats gain more weight than the pair-fed Fatties, but the Fatty rats stored a significantly greater fraction of their caloric intake as fat. Less than 10% of the stored calories were added to nonlipid depots in Fatty rats. In the lean animals, on the contrary, protein and carbohydrate account for 40% of the caloric storage. Thus not only do Fatty rats appear to retain an increased fraction of ingested calories, but they also have a preferential storage of calories in lipid. This increased deposition of energy, however, is not unique to the Fatty rat,12 since similar observations have been made in forms of experimental obesity.17-lg The genetically obese rats initially had 10 times as much fat as in the lean animals, yet none of this fat was utilized for growth by the Fatty rats fed the restricted diet. This could have occurred because the diet was deficient in total calories or because the diet was deficient in some other substance which prevented use of their fat stores. To provide a partial answer to this question, three groups of female albino rats received food for 6 hr each day for 42 days. One group of six animals was allowed to eat ground chow ad lib., a second group was pair fed two-thirds of the food intake eaten by the ad-lib, group, and a third group received the same quantity of chow supplemented by enough fat to produce an isocaloric diet with that of the ad lib. fed rats.* Over the course of the experiment, the rats fed ad lib, gained weight steadily, but the animals fed a restricted diet gained only a little weight. The animals receiving the corn oil supplement gained weight at a rate identical to that of the group fed ad lib. Thus, the nutrients available in two-thirds daily intake of chow did not restrict albino rats from utilizing dietary fats for growth, We might thus conclude that in the experiments on Fatty rats, nutrients oher than calories were no deficient in the group receiving a restricted intake. The failure Ysocaloric
diet
prepared
by blending
5000
g ground
chow
with
1100 g corn
oil.
GENETIC
OBESITY
441
IN RATS. I
to gain weight and to increase the amount of protein suggests that the Fatty rats on a restricted diet were unable to utilize he sored calories as a source of energy. The experimens described in this paper have clearly shown that most of the abnormalities previously observed in Fatty rats1*2 are not corrected by food restriction. Although urine volume was corrected by restricting food intake to two-thirds of that of lean rats, the abnormalities in thyroid and reproductive function could not be attributed to hyperphagia per se. Our data are similar, however, to observations in other syndromes of genetic obesity, 1~18,20-22but contrast with the findings in the Wellesley mouse (GfHI hybrid) l4 and the Egyptian sand rat (Psnmmamys obestls),r3 in which caloric restriction prevented obesity and its associated abnormalities. Interpretation of the data on LH and uterine weights are difficult with the information at hand. The small uteri (Table 2) and decreased estrus cycle? in the Fatty rats would be consistent with decreased levels of estrogen. If this were the case, one would expect .on the basis of feedback control to have an increase in the concentrations of LH. Yet, values for circulating LH were similar in both groups. Studies are currently underway to resolve this problem. Levels of FSH, in contrast with those for LH, are consistent with ovarian weight. The weight of the ovaries is reduced by food intake. Food restriction is associated with a rise in FSH as would be expected from the role of this gonadotropin in controllling ovarian weight. ACKNOWLEDGMENT The invaluable acknowledged.
technical
assistance
of Mrs.
B. Dukes
and Mrs.
J. Anna
is gratefully
REFERENCES 1. Bray, G. A., and York, ally transmitted obesity Rev. 51598, 1971.. 2. -:
Metabolic
and
D. A.: Genetic-
in rodents.
Physiol.
regulatory
obesity
in rats and man. Horm. Metab. Res. Suppl. 2:175, 1970. 3. Barry, W. S., and Bray, G. A.: Plasma triglycerides in genetically obese rats. Metabolism 18:833, 1969. 4. York, D. A., Steinke, J., and Bray, G. A.: Serum insulin levels and insulin insensitivity in the genetically fat rat. Diabetes 19:405, 1970. on
5. Bray, G. A., and York, D. A.: Studies food intake of genetically obese rats.
Am. J. Physiol.
223:176,
1972.
6. -, Mothon, S., and Cohen, A. S.: Mobilization of fatty acids in genetically obese rats. J. Lipid Res. 11:517, 1970. 7. York, D. A., and Bray, G. A.: Regulation of water balance in genetically obese rats. Proc. Sot. Exp. Biol. Med. 136 :798, 1971. 8. -,
Hershman,
J.
M.,
Utiger,
R.
D.,
and Bary,
G. A.:
genetically 1972.
obese
9. Zucker, accretion and 80~6, 1963.
Thyrotropin
secretion
rats. Endocrinology
T. F., and Zucker, growth
in the
L. M.: rat.
in
90 :67, Fat
J. Nutr.
10. Zucker, L. M.: Hereditary obesity in the rat associated with hyperlipemia. Ann. N.Y. Acad. Sci. 131:447, 1965. 11. -: Some effects of caloric restriction and deprivation on the obese hyperlipemic rat. J. Nutr. 91:247, 1967. 12. Zucker, T. F., and Zucker, L. M.: Hereditary obesity in the rat associated with high serum fat and cholesterol. Exp. Biol. Med. 110 :165, 1962.
Proc.
Sot.
13. Hackel, D. B., Mikat, E., Lebovitz. H. E., Schmidt-Nielsen, K., Horton, E. S., and Kinney, T. D.: The sand rat (Psantmomys obesus) as an experimental animal in studies of diabetes mellitus. Diabetologia 3:130, 1967. 14. Cahill, G. F., Jr., Jones, E. E., Lauris, V., Steinke, J., and Soeldner, J. S.: Studies
BRAY, YORK, AND SWERLOFF
442 on experimental hybrid mouse.
diabetes in the Wellesley II. Insulin levels and re-
sponse of peripheral tissues. Diabetologia 3:171, 1967. 15. Bray, G. A.: Increased sensitivity of the thyroid in iodine-depleted rats to the goitrogenic effects of thyrotropin. J. Clin. Invest. 47:1640, 1968. 16. Swerdloff, R. S., Walsh, I’. C., Jacobs, H. S., and Odell, W. D.: Serum LH and FSH during sexual maturation in the male rat: Effect of castration and cryptorchidism. Endocrinology 88:120, 1971. 17. Dickerson, G. E., and Gowen, J. W.: Hereditary obesity and efficient food utilization in mice. Science 105 :496,1947. 18. Alonzo, L. G., and Maren, T. H.: Effect of food restriction on body composition of hereditary obese mice. Am. J.
Physiol. 183:284, 1955. 19. Brooks, C. M., and Lambert, E. F.: A study of the effect of limitations of food intake and the method of feeding on the rate of weight gain during hypothalamic obesity in the albino rat. Am. J. Physiol. 147~695, 1946. 20. Seidman, I., Horland, A. A., and Teebor, G. W.: Glycolytic and gluconeogenie enzyme activities in the hereditary obese-hyperglycemic syndrome and in acquired obesity. Diabetologia 6313, 1970. 21. Chang, A. Y., and Schneider, D. I.: Abnormalities in hepatic enzyme activities during development of diabetes in db mice. Diabetologia 6:274, 1970. 22. Dulin, W. E., and Wyse, B. M.: Diabetes in the KK mouse. Diabetologia 6 :317, 1970.
Among the three groups of Fatty rats, body weight was positively correlated with the weight of the adrenal, pituitary, ovary, uterus, and liver. Urine volume was higher in the ad-lib. and pair-fed Fatties than in the lean animals but restriction of food intake reduced urine output below controls. The uptake of 1251 by the thyroid was significantly reduced in all groups of Fatties, but thyroid weights did not differ. Serum FSH was higher in the restricted Fatties but LH was not. These studies suggest that previously observed differences in hypothalamic endocrine function do not result from hyperphagia.
P
REVIOUS STUDIES from thislv8 and other laboratoriesg-l2 have shown a number of abnormalities in the genetically obese rat called Fatty. These animals are characterized by deposition of adipose tissue at all sites and have a smaller skeleton and less lean body mass than lean littermates.g The increased food intake3z10 poydipsia ,’ decreased thyroid activity,8 sterility, and irregular estrus cycles2 have all suggested that the hypothalamus might be the locus for the genetic abnormality in these rats.1*2 Studies on two other forms of experimental obesity have indicated, however, that some or all of the abnormalities might be the result of hyperphagia and that the primary defect might be enhanced food intake.13*r4 Thus, the sand rat (Psnmmomys obesus) does not develop obesity until its diet is changed from green vegetables to laboratory chow. Even with a chow diet, obesity can still be prevented by restricting food intake. r3 Similar findings have been reported From the Departments of Medicine, Harbor General Hospital, Torrance, Calif., and UCLA School of Medicine, Los Angeles, Calif. Received for pubIication August 29, 1972. Supported in part by NIH Grants RR 425 and AM 15165. George A. Bray, M.D.: Associate Chief, Endocrinology Division, Department of Medicine, Harbor GeneraI Hospital, Torrance, California; and Associate Professor of Medicine, UCLA School of Medicine, Los Angeles, Calif. David A. York, Ph.D.: Lecturer, Department of Physiology and Biochemistry, Soutkampfon University, Soutkampfon, England. Ronald S. Swerdloff, M.D.: Associafe Chief, Endocrinology Division, Departmenf of Medicine, Harbor General Hospital, Torrance, California; and Assisfanf Professor of Medicine, UCLA School of Medicine, Los Angeles, Calif. Reprint requests should be addressed to George 1000 West Carson Sfreet, Torrance, Calif. 90509. @ 1973 by Grune 6 Stratton, inc.
Metabolism. Vol. 22, No. 3 (March),
1973
A. Bray, M.D.,
Harbor
General
Hospital,
435
436
BRAY, YORK, AND SWERLOFF
for the C3HfI hybrid (Wellesley mouse) after caloric restriction.14 The present experiments were designed to clarify the relation between food intake and various endocrine and metabolic abnormalities in the Fatty rat. MATERIALS
AND METHODS
Experimental Protocol Nine lean and 21 genetically obese Fatty female rats were purchased from Dr. Zucker.* Their weight upon arrival in the laboratory averaged 128 -C 6 for lean animals and ranged between 138-262 g for Fatties. These Fatty rats were divided into two groups; 16 rats weighing 128-194 g and 5 rats weighing 238-262 g. Three lean and three Fatty rats were killed within 2 days of arrival for analysis of body composition. The remaining 6 lean and 18 Fatty rats were housed individually in stainless-steel metabolism cages and fed powdered food (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO.) 6 hr/day (9 a.m.-3 p.m.). Water was available ad lib. The 18 Fatty rats were divided into three groups of six rats each. One group was fed ad lib. (ad-lib. Fatties). Six Fatties (pair-fed Fatties) were individually pair fed to six corersponding lean rats and the third group of six Fatties (restricted Fatties) was restricted to two-thirds of the food eaten by the lean control animals. All food actually eaten was weighed. After SO days, the animals were killed in groups of four (one lean plus its’ three respective Fatties). One wk prior to sacrifice, the daily urine volume and water intake were measured for 4 consecutive days.7
Thyroid Function Seven days prior to sacrifice, animals were placed on a low iodine diet15 while still maintaining the 6-hr pair-feeding schedules. Twenty-four hr prior to sacrifice, 2OcLCi1251 were iniected intraperitoneally into each rat and thyroidal uptake assessed as described previously.8
General Procedure at Sacrifice Rats were fasted from the end of their previous feeding period until dealth 18-24 hr later. Blood was removed from the abdominal aorta under ether anesthesia and was transferred promptly to chilled tubes containing EDTA. The serum was separated and frozen for further analysis. The left perimetrial fat pad was removed, weighed, and used for metabolic studies. The pituitary ‘gland was removed, weighed, and homogenized in O&ml saline and frozen until diluted for determination of FSH and LH.16 The heart, liver, left kidney, left uterus, and left ovary were removed, weighed, and replaced in the carcass, which was frozen.
Body Composition The frozen carcass was thawed; the intestinal tract from pylorus to sigmoid was stripped from the mesentry and removed. The remaining carcass was reweighed and minced with two weights of anhydrous sodium sulfate. After three grindings and a final blending in a commercial Waring Blender, the resulting uniform mixture was reweighed and duplicate aliauots removed for the determination of water content bv drying to constant weight at 100°C. Duplicate aliquots were extracted 3 times with chloroform:methanol (2:1, vol/vol), filtered, and the phases separated by addition of water. The dried chloroform extract was considered to be total lipid. Estimates of the initial body composition was made by taking the percentage compositions from the lean or fatty rats of nearest weight killed at the beginning and multiplying these percentages by the body weight of the rat in the experimental group.
Statistical Analysis The data from the four groups *Dr. Lois Zucker,
Stow, Mass.
of animals
have been compared
with students
t test for
GENETIC paired
OBESITY
differences
IN RATS.
437
I
and by two-way
analysis
of variance
using
a desk calculator.
RESULTS
The Effect
of Food
intake
on Body Weight
The changes in mean body weight for the four groups of rats used in this experiment are shown in Table 1. The two groups receiving food ad lib. gained weight steadily. The pair-fed Fatties gained weight at a somewhat slower rate than the lean controls and significantly less rapidly than the ad lib. Fatties. The “restricted Fatties” showed almost no change in body weight. Food Intake
and Caloric
Storage
Table I shows the effect of dietary intake on body weight and body composition in the four groups. Initial body fat of lean rats was between 4.3% and 4.6%. During the course of the experiment, the lipid content of lean rats increased to more than 10% of the total body weight. In the Fatties, lipid initially accounted for nearly one-third total body weight. There was essentially no increase in lipid content of the restricted Fatties. However, the content of body fat in pair-fed and ad-lib. groups of Fatties rose to over 50%. The water content of the lean animals rose by SOO/O and the fat-free dry mass by 100%. In the pair-fed and restricted Fatties, water content remained unchanged, but in the ad lib. Fatties it rose 30%. Although caloric intake of lean and pair-fed Fatty rats was essentially identical, the Fatty rats stored .soO,& more calories than the lean controls. Over 90% of these calories were deposited in lipid stores by the pair-fed Table 1. Body Composition
of Caloric Content of Lean and Obese Rats Lean
Diet
Body wt (9)
Initial Final
Body fat (9)
n initial Final
Body water (9)
n Initial Final
Fat-free dry wt (9)
n Initial Final n
Food intake Caloric intake* Stored calories: (kcal) Stored calories (O/Oof intake)
Fatty
Ad Lib.
totalt lipid non-lipid
128 2 223 r 94 -t 5.3-t
6 7 7 0.27
27.6_’ 2.5 22.3? 2.2 86.15 4.1 128.42 5.3 42.3r 6.0 36.7% 1.9 66.7% 3.8 30.0? 3.6 745 ? 36 2831 -r-136.8 318.8% 16.8 201.62 21 117.2-t 13 11.22 0.4
Restricted
176 & 18 187 ” 10 11 -e 8 51.3% 7.3 57.2% 4.2 5.9-c 3.5 87.0-c 6.9 91.9% 9.7 4.9, 6.5 38.0% 4.0 38.5? 8.7 0.5% 6.3 47.9* 17 1829 -+-646 53.12 45 50.4t 32 -2.7% 32 2.9& 1.0
Pair Fed
187
2
246 ‘-t 59 I+ 54.8 100.2&
12
Ad Lib.
184
2
16
5 8 5.4 3.4
303 -t 21 118 I? 19 54.61 7.t 126.41 11.0
45.4I+ 5.0 91.42 4.5 95.02 5.2 3.6? 2.9 41.oi 2.9 51.0* 2.0 10.0% 1.7 710 1?129 2698 k-1 10.2 448.6& 43 408.62 45 40 2 6.4 16.7t 1.4
71.8f 10.6 90.1-t 5.6 121.4-c 13.1 31.3& 13.1 39.3-1- 3.6 54.21: 10.7 14.91 10.2 943 & 69 3583 1262.2 708 i 78 648 17 96 60 -5 27 19.8? 1.4 -
Caloric intake = food intake x 3.8. tStored calories = (A fat x 9) + (A protein x 4).
438
BRAY, YORK, AND SWERLOFF
Table 2. Organ Weights of Lean and Fatty Rats
Group Lean Ad Lib.
Body weight (g) Adrenal (mg) Pituitary (mg) Thyroid (mg) Ovary (mg) Uterus (mg) Liver(g) Kidney (g)
Fatty Pair Fed
Restricted
187 +lo 223 -c 7’ 26.6 I? 8.0 40.7 f 4.3 4.03& 0.79 8.432 0.63 16.75a 2.47 12.05-c 2.59 23.2 & 8.6 59.7 a14.3 29.2a-e13.92 136.2 1b31.3 5.422 0.47 7.17& 0.95 0.66% 0.10 0.75’ 0.06
246 -r-5 35.7 24.2 4.5820.32 ll.98*5.30 26.6 +6.5 30.28&7.19 6.5121.39 0.7l-cO.06
Ad Lib.
Analysis of Variance Between Groups P
303 +21 42.2 -+ 9.2 5.67+ 1.71 14.3 * 4.1 43.0 113.2 62.2 238.3 8.37-c 1.56 0.87* 0.15
=O.Ol NS <0.025 NS <0.025
*Mean ? SEM. Fatties but only 60% of the added calories were stored as lipid in lean animals. The ad-lib. Fatties stored twice as many calories as the lean animals and over 90% of this storage was in the form of lipid.
The EfFect of Food intake on Organ Weights Table 2 shows the organ weights of lean and Fatty rats. Analysis of variance revealed highly significant differences between body weight and pituitary, ovarian, and uterine weights. However, there was no significant difference in the weights of the adrenals, thyroid, liver, or kidney. Organ weights corrected by dividing by the gross body weight or by (body weight)0.7 did not abolish the differences between the lean and fat animals. In the three groups of Fatty rats organ weights were related to body weight and/or food intake. A regression analysis between body weight and organ weight of the Fatty rats showed a significant correlation with r ranging between 0.81 and 0.92. Food restriction, however, did not restore these organs to the weight of comparable organs in the lean animals. The EfJects of Food Restriction
on Thyroid Function
Thyroid weight was similar in all four groups of animals (Table 3, but the Table 3. Uptake of 1251 by Thyroid
of Lean and Genetically
Thyroid Weight (mg)
Obese Rats*
125f Uptake (% Dose)
Lean
16.8 f
2.5t
12.0 + 1.0
Fatty Restricted Pair fed Ad lib.
12.1 & 2.6 12.0 % 5.3 14.3 f 4.1
a.5 2 0.9 a.2 + 0.6 a.1 * 0.7
p
*Animals were fed a low iodine diet for 7 days prior to sacrifice. Twenty PCi 1251 was injected i.p. in 0.2 ml saline 24 hr prior to sacrifice. Thyroids were dissected free and counted in a well-type counter. tMean & SEM.
GENETIC
OBESITY
IN RATS. I
Fig. 1. Urine volume of lean and Fatty rats. The mean -C SEM for each group is plotted over a 4-day period. Analysis of variance showed a significant day-to-day variation and a significant difference between groups.
i
DAYS
i
i
uptake of iz51 by the thyroid was significantly greater in the lean animals than in the three groups of Fatty rats. Restriction of food intake and the corresponding reduction of iodine intake in the Fatties did not alter the differences in uptake of 125I between the fat and lean rats. Urinary Volume During Food Restriction Analysis of variance revealed a significant day-to-day variation in urine volume as well as a significant difference in urine output between the four groups of animals (Fig. 1). Both ad-lib. and pair-fed Fatties had higher urine volumes and water intakes (measured but not recorded here) than the lean rats. Only when caloric intake was restricted to two-thirds of lean intake was the urine volume reduced below control values for lean rats. Gonadotropins Serum FSH increased with food restriction and was significantly higher in the restricted Fatties (Table 4). In contrast, serum LH was unaffected by food intake or obesity, even though ovarian and uterine weights were significantly smaller in the Fatties (Table 2). The quantity of FSH in the pituitary of Fatty rats appeared to rise with food restriction as did the serum FSH. The content of FSH, whether expressed per animal or per mg of pituitary, was significantly higher in the group with restricted or pair-fed diets than in the ad lib.-fed Fatties. The total quantity of LH in the pituitary was lower in the Fatties than in the lean animals. When expressed per mg of pituitary, the restricted and pair-fed Fatties had significantly higher concentrations of LH than lean controls. Pituitary extracts from both lean and obese rats gave curves in the immunoassay that were parallel to standard preparations of LH and FSH suggesting that there were no immunologic differences in the gonadotropins of these animals.
BRAY, YORK, AND SWERLOFF
440
Table 4. Serum and Pituitary
Gonadotropins
in Lean and Obese Rats” Fatty
Lean Restricted
Pair Fed
Ad Lib.
Serum LH ng/ml FSH ng/ml
61.1 2 6.0 197 -t-19
52.1 & 4.5 266 &20t
53.0 i- 5.0 212 k-22
68.5 k10.0 196 -c 8.5
Pituitary LH pg/pituitary aglmg FSH pg/pituitary
191 k18.5 16.5 k 2.2 15.6 2 1.2
140.5 & 8.4t 34.9 k 2.1* 22.9 & 1.9*
145 413.5 31.7 -c2.95* 21.7 k 1.6$
139 k11.5 24.5 ?I 2.02 18.4 e 1.0
1.85% 0.14
yglmg
*LH and FSH were determined
5.69*
4.75k
0.47*
using a radioimmunoassay
Distribution Program for Rat Pituitary tp <0.05 compared to lean. Sp
Hormones
0.35*
with reagents
3.242 provided
0.18t by the
of the NIAMD.14
DISCUSSION
Although restriction of caloric intake of Fatty rats limited their rate of growth, it did not restore body composition to normal’r Indeed, lean rats gain more weight than the pair-fed Fatties, but the Fatty rats stored a significantly greater fraction of their caloric intake as fat. Less than 10% of the stored calories were added to nonlipid depots in Fatty rats. In the lean animals, on the contrary, protein and carbohydrate account for 40% of the caloric storage. Thus not only do Fatty rats appear to retain an increased fraction of ingested calories, but they also have a preferential storage of calories in lipid. This increased deposition of energy, however, is not unique to the Fatty rat,12 since similar observations have been made in forms of experimental obesity.17-lg The genetically obese rats initially had 10 times as much fat as in the lean animals, yet none of this fat was utilized for growth by the Fatty rats fed the restricted diet. This could have occurred because the diet was deficient in total calories or because the diet was deficient in some other substance which prevented use of their fat stores. To provide a partial answer to this question, three groups of female albino rats received food for 6 hr each day for 42 days. One group of six animals was allowed to eat ground chow ad lib., a second group was pair fed two-thirds of the food intake eaten by the ad-lib, group, and a third group received the same quantity of chow supplemented by enough fat to produce an isocaloric diet with that of the ad lib. fed rats.* Over the course of the experiment, the rats fed ad lib, gained weight steadily, but the animals fed a restricted diet gained only a little weight. The animals receiving the corn oil supplement gained weight at a rate identical to that of the group fed ad lib. Thus, the nutrients available in two-thirds daily intake of chow did not restrict albino rats from utilizing dietary fats for growth, We might thus conclude that in the experiments on Fatty rats, nutrients oher than calories were no deficient in the group receiving a restricted intake. The failure Ysocaloric
diet
prepared
by blending
5000
g ground
chow
with
1100 g corn
oil.
GENETIC
OBESITY
441
IN RATS. I
to gain weight and to increase the amount of protein suggests that the Fatty rats on a restricted diet were unable to utilize he sored calories as a source of energy. The experimens described in this paper have clearly shown that most of the abnormalities previously observed in Fatty rats1*2 are not corrected by food restriction. Although urine volume was corrected by restricting food intake to two-thirds of that of lean rats, the abnormalities in thyroid and reproductive function could not be attributed to hyperphagia per se. Our data are similar, however, to observations in other syndromes of genetic obesity, 1~18,20-22but contrast with the findings in the Wellesley mouse (GfHI hybrid) l4 and the Egyptian sand rat (Psnmmamys obestls),r3 in which caloric restriction prevented obesity and its associated abnormalities. Interpretation of the data on LH and uterine weights are difficult with the information at hand. The small uteri (Table 2) and decreased estrus cycle? in the Fatty rats would be consistent with decreased levels of estrogen. If this were the case, one would expect .on the basis of feedback control to have an increase in the concentrations of LH. Yet, values for circulating LH were similar in both groups. Studies are currently underway to resolve this problem. Levels of FSH, in contrast with those for LH, are consistent with ovarian weight. The weight of the ovaries is reduced by food intake. Food restriction is associated with a rise in FSH as would be expected from the role of this gonadotropin in controllling ovarian weight. ACKNOWLEDGMENT The invaluable acknowledged.
technical
assistance
of Mrs.
B. Dukes
and Mrs.
J. Anna
is gratefully
REFERENCES 1. Bray, G. A., and York, ally transmitted obesity Rev. 51598, 1971.. 2. -:
Metabolic
and
D. A.: Genetic-
in rodents.
Physiol.
regulatory
obesity
in rats and man. Horm. Metab. Res. Suppl. 2:175, 1970. 3. Barry, W. S., and Bray, G. A.: Plasma triglycerides in genetically obese rats. Metabolism 18:833, 1969. 4. York, D. A., Steinke, J., and Bray, G. A.: Serum insulin levels and insulin insensitivity in the genetically fat rat. Diabetes 19:405, 1970. on
5. Bray, G. A., and York, D. A.: Studies food intake of genetically obese rats.
Am. J. Physiol.
223:176,
1972.
6. -, Mothon, S., and Cohen, A. S.: Mobilization of fatty acids in genetically obese rats. J. Lipid Res. 11:517, 1970. 7. York, D. A., and Bray, G. A.: Regulation of water balance in genetically obese rats. Proc. Sot. Exp. Biol. Med. 136 :798, 1971. 8. -,
Hershman,
J.
M.,
Utiger,
R.
D.,
and Bary,
G. A.:
genetically 1972.
obese
9. Zucker, accretion and 80~6, 1963.
Thyrotropin
secretion
rats. Endocrinology
T. F., and Zucker, growth
in the
L. M.: rat.
in
90 :67, Fat
J. Nutr.
10. Zucker, L. M.: Hereditary obesity in the rat associated with hyperlipemia. Ann. N.Y. Acad. Sci. 131:447, 1965. 11. -: Some effects of caloric restriction and deprivation on the obese hyperlipemic rat. J. Nutr. 91:247, 1967. 12. Zucker, T. F., and Zucker, L. M.: Hereditary obesity in the rat associated with high serum fat and cholesterol. Exp. Biol. Med. 110 :165, 1962.
Proc.
Sot.
13. Hackel, D. B., Mikat, E., Lebovitz. H. E., Schmidt-Nielsen, K., Horton, E. S., and Kinney, T. D.: The sand rat (Psantmomys obesus) as an experimental animal in studies of diabetes mellitus. Diabetologia 3:130, 1967. 14. Cahill, G. F., Jr., Jones, E. E., Lauris, V., Steinke, J., and Soeldner, J. S.: Studies
BRAY, YORK, AND SWERLOFF
442 on experimental hybrid mouse.
diabetes in the Wellesley II. Insulin levels and re-
sponse of peripheral tissues. Diabetologia 3:171, 1967. 15. Bray, G. A.: Increased sensitivity of the thyroid in iodine-depleted rats to the goitrogenic effects of thyrotropin. J. Clin. Invest. 47:1640, 1968. 16. Swerdloff, R. S., Walsh, I’. C., Jacobs, H. S., and Odell, W. D.: Serum LH and FSH during sexual maturation in the male rat: Effect of castration and cryptorchidism. Endocrinology 88:120, 1971. 17. Dickerson, G. E., and Gowen, J. W.: Hereditary obesity and efficient food utilization in mice. Science 105 :496,1947. 18. Alonzo, L. G., and Maren, T. H.: Effect of food restriction on body composition of hereditary obese mice. Am. J.
Physiol. 183:284, 1955. 19. Brooks, C. M., and Lambert, E. F.: A study of the effect of limitations of food intake and the method of feeding on the rate of weight gain during hypothalamic obesity in the albino rat. Am. J. Physiol. 147~695, 1946. 20. Seidman, I., Horland, A. A., and Teebor, G. W.: Glycolytic and gluconeogenie enzyme activities in the hereditary obese-hyperglycemic syndrome and in acquired obesity. Diabetologia 6313, 1970. 21. Chang, A. Y., and Schneider, D. I.: Abnormalities in hepatic enzyme activities during development of diabetes in db mice. Diabetologia 6:274, 1970. 22. Dulin, W. E., and Wyse, B. M.: Diabetes in the KK mouse. Diabetologia 6 :317, 1970.