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Spontaneous activity and adipose cellularity in the genetically obese Zucker rat (fafa)
Spontaneous Activity and Adipose Cellularity Genetically Obese Zucker Rat (fafa) Judith The
development
was
studied
Rats were
of spontaneous
in obese and lean given
access
for 3 hr/day
before
hr/day
weaning.
are
after
less
active
creased
activity
follows
the
obesity. obese
S. Stern
lean
occurs
onset
of
at
adipocytes
have than
less their
for
obese
rats.
This
total
weaning
fat
lean and
appropriate
24 rats de-
hyperphagia
At 8 wk of age exercised rats
rats.
wheels
and
Zucker
than
activity
Zucker
to activity
weaning
and
Patricia
R. Johnson
Adipose ercised until
cell
size
is decreased
lean
rats.
When
8 wk
rats
body
vated
in
pared
to control
these
and
is permanently active
lean rats.
and
effect
in
weight
formerly rats.
Exercise cell
fat
active
in ex-
exercised
confined and
Adipose
decreased
decreasing
only are
of age and then
6 mo of age,
and fewer
in the
rats
until is elecom-
cell number
only
in formerly
has no long-term number
in
obese
rats.
controls.
0
BESITY, a disease of multiple etiologies, is usually accompanied by increased caloric intake and decreased activity.lm5 While there are reports linking obesity and inactivity in both humans and animals, studies to date have not established which comes first: increased adiposity or decreased activity, although it has often been proposed that decreased activity contributes to the development of obesity. Mayer has shown that spontaneous daily activity of 2mo-old obeseehyperglycemic (ob/ob) mice is less than that of 4-mo-old lean mice of equivalent body weight and, furthermore, that with increasing age and adiposity, the obese mice become even less active.6 Forced inactivity is widely used to promote weight gain and fattening in farm animals. In laboratory rats, low levels of activity are associated with increased rather than decreased food intake.’ A change in the amount of physical activity can contribute to the onset of mild obesity in adults. Greene, for example, has reported that increased weight gain in 67.5:; of adult patients that he studied occurred simultaneously with a decrease in activity.* However, we have little understanding of whether or not inactivity plays a primary role in the development of early onset obesity. Joosten and van der Kroon measured motor activity, as determined in a brief 5-min test, in ob/ob mice.’ In 7715day-old ob/ob mice, motor activity was decreased in comparison to lean littermates before any measurable indicators of obesity were present. However, this low level of motor activity returned to normal from day 15 to day 2 1. The use of increased physical exercise as a means for weight reduction has been downplayed by those treating obesity, although exercise can be an effective means of altering adiposity. Adult rats forced to swim several hours per day
From Rockefeller Universitv, New York. N. Y. Receivedfor publication Ma-v 10, 1976. Supported by NIH Grant AM 18899-02 and a grant from the Research Corporation. Reprint requests should be addressed to Dr. Judith S. Stern, Department of Nutrition. University ofCalifornia, Davis, Calif: 9.5616. @ 1977 by Grune & Stratton, Inc. Metabolism, Vol. 26, No. 4 (April), 1977
371
372
STERN
AND JOHNSON
are leaner and have smaller fat cells than nonexercised rats. If exercise is initiated before weaning, adipose cell number is also decreased.‘” In the present study we compared the level of spontaneous activity in preweaning genetically obese and lean Zucker rats that were allowed access to running wheels in order to address the question of whether or not decreased activity preceded any other measurable indicators of the obese state in this strain of genetically obese rodents. We also assessed the immediate and longterm effects of early activity level on the development of adiposity in both obese and lean Zucker rats. MATERIALS
AND
METHODS
Obesity in the Zucker rat follows the pattern of inheritance characteristic of a single Mendelian recessive gene. Since obese female rats do not breed. heterozygotes are mated to produce mixed litters with expected phenotypic ratios of 75”,, lean and 25”,, obese. The lean and obese phenotypes are not identifiable by any visible criteria until approximately 4 wk of age. Thus. mixed litters of male pups were raised eight pups per dam and housed in a temperature-controlled room with a 12-hr light dark cycle. When the pups were 16 days of age, they were randomly assigned to a nonactive control group (I), or to one of three exercise groups (II. III, IV). Group I pups were isolated from the dam for 3 hr during the dark cycle by placing them in individual hanging cages, and at 25 days of age they were weaned and housed in the same cages. Pups in groups II. III. and IV were given 3-hr daily access to Wahmann activity wheels (LC-34) during the dark cycle. but were weaned at different ages to assess the effect of time of weaning upon development of spontaneous activity: group II at 25 days (normal), group III at I9 days (early). and group IV at 44 days (late). Solid food (Purina rat chow) and water were available ad libitum to all four groups during the 3-hr period that they were separated from the dam. In summary. rats from 16 days until weaning spent 3 hr/day in activity cages: rats from weaning until 8 wk of age spent 24 hr/day in activity cages, After weaning all groups were fed Purina chow ad libitum. Food intake, body weight, and wheel turns were determined daily during a 6-wk period. Even at 8 wk of age the obese rats could enter and leave the activity wheel without ditiiculty. To assess the immediate effects of early exercise on the development of adiposity. approximately half the obese and lean rats in the nonactive control group (I) and in the exercise group (II) were killed at 8 wk of age. To assess the long-term effects of early exercise on adult ad)posity. the remaining rats in these groups (I and II) were housed in individual hanging cages until 6 mo of age, at which time they were killed by decapitation. Adipose cellularity was determined in three sites: the epididymal. retroperitoneal. and subcutaneous fat depots using the osmium fixation technique of Hirsch and Gallian.” Total cell number and total fat are summations of the values obtained for the three depots: mean cell size is the weighted average.” Plasma glucose was determined by a Technicon AutoAnalyzer13 and plasma immunoreactive insulin was assayed by the method of Rosselin et al.” Data were analyzed by analysis of variance and Student‘s t test. On measures of body weight. food intake, and activity from 16-25 days of age. active groups II and IV were combined in one analysis. Group 111 was omitted since data were collected only up until day I9 for this group. RESULTS
All obese rats, independent of the day of weaning, become less active than lean rats (Fig. 1). The time of separation of activity levels in the obese and lean animals varied for each group. In group II (weaned at 25 days) a striking decrease in activity was observed on the second day after weaning (day 27). In group III (weaned at 19 days) obese were less active than lean rats 2 days after weaning. However, the obese rats in this group did not actually decrease their activity. but simply maintained a low level, while lean rats were increasing their activity level. With delayed weaning (group IV), separation of activity levels of obese and lean rats occurred by day 30, at which time the dam appeared to
GENETICALLYOBESE RAT
373
GROUP II (wsonsd. day 251
GROUP,,, Cwmsd, day 19,
6000
L
GROUP IS? hv.oned. day 44)
Fig. 1. Comparison of daily activity by ages of weaning. Doily wheel turns in lean (Fa/-) and obese (fa/fa) male rat pups weaned at day 25 [group II: n = 15 (Fa/-); n = 9 (fa/fa)], day 19 [group Ill: n = 5 (fa/-); n = 4 (fa/fa)], and day 44 [group IV: n = 13 (Fa/-); n = 7 (fa/fa)]. Activity was monitored for 3 hr/day until the day of weaning, and thereafter, for all 24 hr.
32 Age
40
48
(days1
have weaned the pups, although actual separation from the dam did not occur until day 44. Independent of the day of separation from the dam, in the 21 hr immediately following the separation, wheel running increased precipitously in all obese and lean pups, perhaps reflecting the increased time of access to the wheel (3 versus 24 hr). Twenty-four hours later, the activity of obese rats declined to preseparation levels or less, while that of the lean rats was maintained or continued to increase. By 8 wk of age total wheel running in obese rats was 50”:, less than that of the lean animals (Table 1). Cumulative overall running was similar by day 52 in all three groups of lean rats, independent of the time of weaning. Cumulative running of obese rats weaned at day 19 was less than that of obese rats weaned at day 25 or day 44. In contrast to postweaning activity patterns, activity levels prior to weaning were comparable in obese and lean rats (groups II and IV) from day 16 to day 22, and from day 16 to day 19 in group III (Figs. 1 and 2). On days 23-25, activity levels of obese rats plateaued, while activity levels of lean rats continued to increase through day 24 (Fig. 2). At day 25 activity decreased in the lean rats to levels comparable to those seen in the obese rats (Fig. 2). Thus there was no significant difference in activity of obese and lean rats from day 16 to day 22
374
STERN
Table
1.
Cumulative
Activity
and
Cumulative
Food
Intake
in E-wk-Old
(X
JOHNSON
Zucker
Total Revolutions N
AND
Rats
Total Food Intake
103)
IS)
I (nonactive)
Group
Obese
(fa/fa)
Leon (Fo/-) Group
II (weaned
Lean Ill (weaned
674.9
+ 18.8
498.6
+ 10.1
day 25)
Obese
Group
6 15
9
28.1
f
2.5
760.0
i
22.1
13
57.6
f
5.5
525.1
i
18.0
day 19)
Obese
7
10.5 f
1.4
770.7
f
22.2
Lean
5
67.1
13.9
599.5
i
27.5
11.9
Group
IV (weaned
i
day 44)
Obese
7
29.0
f
3.8
397.0
*
Lean
8
71.5
f
9.6
329.9
zt 12.9
Table
2.
Results
of Analyses
(Obese
and
Lean
of Variance Rats
Weaned
for
Groups
at Day
II and
IV
25) F Valuer
Parameter
df’
Days
Genotype
x Days
Days 16-22 Body weight
l/336
Daily food intake
l/336
33.04t
68.247
Cumulative
l/336
49.277
110.80t
5.631
l/336
0.02
110.37t
0.28
0.25
food intake
Daily activity
7.187
55.09t
0.13 1.75
Days 16-25 Body weight
l/480
12.337
115.2Bt
Daily food intake
i 1480
35.7gt
64.70.t
5.257
Cumulative
l/480
59.96t
134.707
7.45t
1 12.49t
1.48t
food intake
Daily activity
l/480
4.01 t
F Values
Autopsy Data
df*
Genotype
Activity
Genotype
x Activity
At8wk Body weight
l/27
33.88t
0.02
Carcass
weight
l/27
3.41
0.68
3.23
Adrenal
weight
l/27
33.33t
45.747
0.36
Total fat
l/27
320.44t
Total cell number
l/27
1.20
32.57t
13.48t
Average
cell size
6.307
2.84
1.21
l/27
497.85t
0.02
2.89
Plasma
glucose
l/24
0.75
0.39
6.29t
Plasma
insulin
l/27
15.327
0.32
2.36
l/235
34.207
8.46t
15.99t
Cumulative
food intake
At6mo Body weight
l/26
177.55t
0.58
1.60
Total fat
l/26
424.33t
14.12t
4.60t
Total cell number
l/26
37.76t
0.14
4.54t
Average
l/26
22 1.40t
3.91
1.73
*df: degrees tValues
cell size of freedom.
that are significant
at p < 0.05.
GENETICALLYOBESE RAT
375
4-
DAILY
FOOD
INTAKE i ;; ;I,/
3. (
0 2000
DAlLY
ACTIVITY
1500-
Fig. 2. Body weight, daily food intake, and daily wheel turns in lean (Fa/-) and obese (fa/fa) male rat pups from day 16 to day 25 (group II). Weaning occurred at day 25 or later. Activity was monitored for 3 hr during the dark cycle. Each point represents the mean & SEM for 16 obese and 34 lean PUPS.
0
16
1
16
,
~
20 Age
(doysl
22
*
24
1
(p > 0.05; Table 2). During this 16-22-day period, body weights were significantly increased in obese pups compared to lean (p < 0.05; Fig. 2, Table 2). During this period, daily solid food intake and cumulative food intake were significantly increased in the obese rats (Fig. 2, Tables 1 and 2). Cumulative food intake for the 37-day period was significantly increased in obese compared to lean rats, and was higher in active than in inactive rats. Table 3 presents the autopsy data for animals killed at 8 wk (groups I and II) and Table 2 presents the statistical analyses. In those animals killed at 8 wk of
376
STERN
AND JOHNSON
GENETICALLY
OBESE
377
RAT
Table 4.
Autopsy Data in Lean and Obese Zucker Rats at 6 mo of Age Body
Genotype
Obese (fa/fa) Exercised
N
2 12
5
Number
Mean Cell Sire
(9)
(9)
(9)
x 106
(rg lipid/cell)
12.2
179.1 f
11.3
446.0 f
Nonactive
Fat
705.0 *
Lean (Fa/-) Exercised
Total Cell
wt.
CClrCaSS
728.5 f
Nonactive
Total
wt.
11
22.0
204.969
161.667
f
f
165.165
f
f
f
3.8
7.4 11.6
8.097
1.050
f
11.240
1.984
1.3040 i
4.090
f
5.078
.0642
0.4483 f
84.415 f
.0202 1.2933
60.910 f
17.970 f
3.559
131.987
30.086 f
204.7 f
7.273
179.2
198.4
10.5 432.0
f
13.8
.0403 0.2164
f
.0209
age, there were no significant differences in body weight and carcass weight in either active lean or obese rats compared to their inactive controls. Total fat, adipose cell size, and number were greater in obese than in lean rats. The immediate effect of access to activity wheels was to decrease total fat and adipose cell number in both obese and lean rats. Adipose cell size was decreased in active lean rats, but unchanged in active obese rats. Plasma glucose levels were unchanged. Plasma immunoreactive insulin levels were significantly higher in the obese rats compared to lean rats. Activity decreased insulin levels only in lean rats. Adrenal weights in both active groups were elevated. Table 4 presents the autopsy data for animals killed at 6 mo in order to assess the long-term effects of exercise in the remaining rats from groups I and II. Results of statistical analyses are presented in Table 2. Early exposure to activity wheels was associated with increased body weight in both groups, which was reflected in an increased total fat and adipose cell size. Adipose cell number was decreased in the active lean rats compared to nonactive lean controls. Activity had no long-term effect in decreasing cell number in obese rats; in fact, formerly active obese rats had higher total cell number than nonactive obese controls. DISCUSSION
We have demonstrated that the Zucker obese rat is less spontaneously active than its lean littermate when given access to a running wheel. The decrease in spontaneous activity follows both the onset of hyperphagia and elevations in body weight that were detected before weaning. Perhaps at day 16, and certainly by day 17, the obese pups are eating more solid food than their lean littermates during the 3-hr access period. We may speculate that it was the noncompetitive free feeding situation that allowed the hyperphagia to be expressed at this early stage. Whether or not this hyperphagic behavior is expressed in the home cage when the pups are suckling the dam has not been established. Although there are indications that Zucker obese rats are significantly fatter as early as 15 days of age,15 no other study establishes significant differences in body weight between lean and obese Zucker rats before weaning. The event of weaning itself appears to play an important role in the establishment of decreased activity in the obese rat. When the time of weaning is
378
STERN
AND JOHNSON
varied, the onset of decreased activity is also changed. It is evident that obese Zucker rats are not born less active than their lean littermates, but rather the major shift to inactivity is coincident with the weaning process. There are numerous obvious changes that occur at weaning, such as change in composition of the diet, separation from the dam, and, often, separation from littermates and social isolation. Social isolation and physical separation are probably not precipitating factors, since obese pups removed from the dam at day 44 (group IV) decreased their activity around day 30 when the dam spontaneously weaned the pups. Joosten and van der Kroor? have recently reported that, in a 5-min test, locomotor activity of ob/ob mice is less than that of nonobese littermates as early as the second week of life, but during the third week of life (from day 15 to day 20) obese and nonobese mice show similar locomotor activity. From approximately day 23 on, the obese mice are less active as measured by lines crossed in an open field. These results in another obese rodent strain, and with another measure of activity, are similar to ours from 16 days. We were not able to measure spontaneous wheel running before day 16, so comparisons prior to that age are not possible. Unfortunately, Joosten and van der Kroon do not give the time of weaning in their study, but it is tempting to speculate that the decrease in activity which they report in the third week in ob/ob mice could be associated with the weaning process. In this study exercise had no effect in decreasing the hyperinsulinemia observed in Zucker obese rats. Perhaps the exercise was not severe enough to effect a change. In lean rats exercise did decrease plasma insulin values. however, these were twice as active as the exercised obese rats. Exercise resulted in increased adrenal weights; these findings are in keeping with those previously reported by Leshner.‘(’ The cellularity data reported here confirm and extend previous findings of Johnson et al.” on the effects of early environmental manipulations on cell number of adipose depots in the Zucker rat. The early activity of the lean rats effected a permanent decrease in the total number of adipocytes. While exercised obese rats had a decreased total fat cell number at 8 wk of age, the decrease was not permanent. At 6 mo, the formerly active rats, if anything, had more fat cells than their nonactive controls. These data support the previously proposed hypothesis that the Zucker obese rat is unable to shut down the mechanism of adipocyte proliferation, unlike its lean littermate, in which a preweaning environmental stimulus may render a permanent change in adipose tissue cellularity. While the lean formerly active rat had fewer fat cells than its nonactive control, it also had larger fat cells. Since active lean rats ate more chow than did nonactive controls during the 8-wk period of wheel running, it appears possible that a continued increased caloric consumption with decreased caloric output may have contributed to the increased deposition of body fat seen in these animals. We may only speculate that such a mechanism can explain the increased lipid deposition in the inactive period that followed a period of activity, since we did not measure food intake during the inactive period. This phenomenon may, however, be a condition that parallels the familiar gain in body fat often observed in the aging retired athlete.
GENETICALLY
OBESE
379
RAT
We conclude from these studies that hyperphagia is the first consistently measurable difference between obese and lean Zucker rats. It is closely followed by elevated body weight. The decreased activity is secondary to the development of adiposity. Joosten and van der Kroon’ proposed that hyperphagia is a congenital defect in the ob/ob mouse, although their earliest measure of increased food intake in this species was at 5 wk of age. Bray et al. have proposed that the primary disturbance in the genetically obese Zucker rat is a hypothalamic lesion or malfunction, since a number of the abnormalities that have been described may be related to hypothalamic control mechanisms: e.g., thyroid function, water balance, reproductive function.‘7m2” Hyperphagia could also result from an aberrant hypothalamic control of feeding behavior. Thus, studies now in progress are designed to determine how the pattern of feeding and running evolves in the obese and lean animals. Alternatively, hyperphagia may yet prove to be secondary to a morphological or metabolic derangement which is the primary expression of the fa gene. The primary defect in this animal could be due to an inability to synthesize muscle protein at the normal rate with unused calories being diverted into fat stores.2’,22 The resulting nutrient imbalance could provide a stimulus to signal feeding behavior. In any case, increased proliferation of adipocytes followed by a requirement for lipid filling, or decreased utilization of calories by muscle resulting in excess substrate for lipid filling of preexistent fat cells, could provide metabolic signals to the central regulatory system to produce hyperphagia. Decreased activity may, however, become a contributing factor in the subsequent expression and maintenance of the obese state. ACKNOWLEDGMENT We thank Dr. Jules Hirsch and Dr. George Collier for their Noeldechen and Nuton Stewart for their technical assistance.
continued
support
and
Patricia
REFERENCES I. Chirico A, Stunkard AJ: Physical activity and human obesity. N Engl Med 263:935-940, 1960 2. Bullen FA, Reed RB, Mayer J: Physical activity of obese and nonobese adolescent girls appraised by motion picture sampling. Am J Clin Nutr 14:21 I-223, 1964 3. Yen TTT, Acton JM: Locomotor activity of various types of genetically obese mice. Proc Sot Exp Biol Med 140647-650, 1972 4. Stern JS, Johnson PR: Spontaneous activity in the Zucker obese rat (fafa). Fed Proc 33:677, 1974 (Abstract) 5. Buskirk ER: Obesity: A brief overview with emphasis on exercise. Fed Proc 33:19481951.1974 6. Mayer J: Decreased activity and energy balance in the hereditary obesity-diabetes syndrome of mice. Science 117:5044505, 1953 7. Mayer J, Marshall NB, Vitale JJ, Christen-
sen J, Mashayekki MB, Stare FJ: Exercise, food intake and body weight in normal rats and genetically obese mice. Am J Physiol 177:544-548. 1954 8. Greene JA: Clinical study of the etiology ofobesity. Ann Intern Med 12:1797-1803, 1939 9. Joosten HFP. van der Kroon PHW: Growth pattern and behavioral traits associated with the development of the obese-hyperglycemic syndrome in mice (ob/ob). Metabolism 26:1141~1147. 1974 IO. Oscai LB, Spirakis CN, Wolff CA, Beck RJ: Effects of exercise and food restriction on adipose tissue cellularity. J Lipid Res 13:588592, 1972 11. Hirsch J, Gallian E: Methods for the determination of adipose cell size in man and animals. J Lipid Res 9:110-l 19. 1968 12. Johnson PR, Stern JS. Greenwood MRC. Zucker LM. Hirsch J: Effect of early nutrition
380
on adipose cellularity and pancreatic insulin release in the Zucker rat. J Nutr 103:738-743. 1973 13. Hoffman WS: A rapid photoelectric method for the determination of glucose in blood and urine. J Biol Chem 120:51-55, 1937 14. Rosselin CR, Assan RS. Yalow RS, Berson SA: Separation of antibody-bound and unboundpeptide hormones labelled with iodine131 by talcum powder and precipitated silica. Nature 2123555358. 1966 15. Bell G, Stern JS: Development of obesity and hyperinsulinemia in the Zucker obese rat (fafa). Fed Proc 35:657. 1976 (Abstract) 16. Leshner AI: The adrenals and the regulatory nature of running wheel activity. Physiol Behav 6:551 -558. 1971
STERN
AND JOHNSON
17. Bray GA, York DA: Thyroid function ol genetically obese rats. Endocrinology 88: 1095 1099. 1971 IX. York DA, Bray GA: Regulation of water balance in genetically obese rats. Proc Sot Exp Biol Med 136:798 X01. 1971 19. York DA, Hershman JM. Utiger RD. Bray GA: Thyrotropin secretion In genetically obese rats. Endocrinology 90:67 72. 1972 20. Saiduddin S. Bray GA. York DA. Swerdloff RS: Reproductive function in the genetically obese “fatty” rat. Endocrinology 93: 1251 1256. 1973 21. Zucker L: Unpublished observations 22. Martin RJ: Genetic influence on nutrittonal aspects of metabolic regulation. 1. Fed Proc 35:2291 2294. 1976
development
was
studied
Rats were
of spontaneous
in obese and lean given
access
for 3 hr/day
before
hr/day
weaning.
are
after
less
active
creased
activity
follows
the
obesity. obese
S. Stern
lean
occurs
onset
of
at
adipocytes
have than
less their
for
obese
rats.
This
total
weaning
fat
lean and
appropriate
24 rats de-
hyperphagia
At 8 wk of age exercised rats
rats.
wheels
and
Zucker
than
activity
Zucker
to activity
weaning
and
Patricia
R. Johnson
Adipose ercised until
cell
size
is decreased
lean
rats.
When
8 wk
rats
body
vated
in
pared
to control
these
and
is permanently active
lean rats.
and
effect
in
weight
formerly rats.
Exercise cell
fat
active
in ex-
exercised
confined and
Adipose
decreased
decreasing
only are
of age and then
6 mo of age,
and fewer
in the
rats
until is elecom-
cell number
only
in formerly
has no long-term number
in
obese
rats.
controls.
0
BESITY, a disease of multiple etiologies, is usually accompanied by increased caloric intake and decreased activity.lm5 While there are reports linking obesity and inactivity in both humans and animals, studies to date have not established which comes first: increased adiposity or decreased activity, although it has often been proposed that decreased activity contributes to the development of obesity. Mayer has shown that spontaneous daily activity of 2mo-old obeseehyperglycemic (ob/ob) mice is less than that of 4-mo-old lean mice of equivalent body weight and, furthermore, that with increasing age and adiposity, the obese mice become even less active.6 Forced inactivity is widely used to promote weight gain and fattening in farm animals. In laboratory rats, low levels of activity are associated with increased rather than decreased food intake.’ A change in the amount of physical activity can contribute to the onset of mild obesity in adults. Greene, for example, has reported that increased weight gain in 67.5:; of adult patients that he studied occurred simultaneously with a decrease in activity.* However, we have little understanding of whether or not inactivity plays a primary role in the development of early onset obesity. Joosten and van der Kroon measured motor activity, as determined in a brief 5-min test, in ob/ob mice.’ In 7715day-old ob/ob mice, motor activity was decreased in comparison to lean littermates before any measurable indicators of obesity were present. However, this low level of motor activity returned to normal from day 15 to day 2 1. The use of increased physical exercise as a means for weight reduction has been downplayed by those treating obesity, although exercise can be an effective means of altering adiposity. Adult rats forced to swim several hours per day
From Rockefeller Universitv, New York. N. Y. Receivedfor publication Ma-v 10, 1976. Supported by NIH Grant AM 18899-02 and a grant from the Research Corporation. Reprint requests should be addressed to Dr. Judith S. Stern, Department of Nutrition. University ofCalifornia, Davis, Calif: 9.5616. @ 1977 by Grune & Stratton, Inc. Metabolism, Vol. 26, No. 4 (April), 1977
371
372
STERN
AND JOHNSON
are leaner and have smaller fat cells than nonexercised rats. If exercise is initiated before weaning, adipose cell number is also decreased.‘” In the present study we compared the level of spontaneous activity in preweaning genetically obese and lean Zucker rats that were allowed access to running wheels in order to address the question of whether or not decreased activity preceded any other measurable indicators of the obese state in this strain of genetically obese rodents. We also assessed the immediate and longterm effects of early activity level on the development of adiposity in both obese and lean Zucker rats. MATERIALS
AND
METHODS
Obesity in the Zucker rat follows the pattern of inheritance characteristic of a single Mendelian recessive gene. Since obese female rats do not breed. heterozygotes are mated to produce mixed litters with expected phenotypic ratios of 75”,, lean and 25”,, obese. The lean and obese phenotypes are not identifiable by any visible criteria until approximately 4 wk of age. Thus. mixed litters of male pups were raised eight pups per dam and housed in a temperature-controlled room with a 12-hr light dark cycle. When the pups were 16 days of age, they were randomly assigned to a nonactive control group (I), or to one of three exercise groups (II. III, IV). Group I pups were isolated from the dam for 3 hr during the dark cycle by placing them in individual hanging cages, and at 25 days of age they were weaned and housed in the same cages. Pups in groups II. III. and IV were given 3-hr daily access to Wahmann activity wheels (LC-34) during the dark cycle. but were weaned at different ages to assess the effect of time of weaning upon development of spontaneous activity: group II at 25 days (normal), group III at I9 days (early). and group IV at 44 days (late). Solid food (Purina rat chow) and water were available ad libitum to all four groups during the 3-hr period that they were separated from the dam. In summary. rats from 16 days until weaning spent 3 hr/day in activity cages: rats from weaning until 8 wk of age spent 24 hr/day in activity cages, After weaning all groups were fed Purina chow ad libitum. Food intake, body weight, and wheel turns were determined daily during a 6-wk period. Even at 8 wk of age the obese rats could enter and leave the activity wheel without ditiiculty. To assess the immediate effects of early exercise on the development of adiposity. approximately half the obese and lean rats in the nonactive control group (I) and in the exercise group (II) were killed at 8 wk of age. To assess the long-term effects of early exercise on adult ad)posity. the remaining rats in these groups (I and II) were housed in individual hanging cages until 6 mo of age, at which time they were killed by decapitation. Adipose cellularity was determined in three sites: the epididymal. retroperitoneal. and subcutaneous fat depots using the osmium fixation technique of Hirsch and Gallian.” Total cell number and total fat are summations of the values obtained for the three depots: mean cell size is the weighted average.” Plasma glucose was determined by a Technicon AutoAnalyzer13 and plasma immunoreactive insulin was assayed by the method of Rosselin et al.” Data were analyzed by analysis of variance and Student‘s t test. On measures of body weight. food intake, and activity from 16-25 days of age. active groups II and IV were combined in one analysis. Group 111 was omitted since data were collected only up until day I9 for this group. RESULTS
All obese rats, independent of the day of weaning, become less active than lean rats (Fig. 1). The time of separation of activity levels in the obese and lean animals varied for each group. In group II (weaned at 25 days) a striking decrease in activity was observed on the second day after weaning (day 27). In group III (weaned at 19 days) obese were less active than lean rats 2 days after weaning. However, the obese rats in this group did not actually decrease their activity. but simply maintained a low level, while lean rats were increasing their activity level. With delayed weaning (group IV), separation of activity levels of obese and lean rats occurred by day 30, at which time the dam appeared to
GENETICALLYOBESE RAT
373
GROUP II (wsonsd. day 251
GROUP,,, Cwmsd, day 19,
6000
L
GROUP IS? hv.oned. day 44)
Fig. 1. Comparison of daily activity by ages of weaning. Doily wheel turns in lean (Fa/-) and obese (fa/fa) male rat pups weaned at day 25 [group II: n = 15 (Fa/-); n = 9 (fa/fa)], day 19 [group Ill: n = 5 (fa/-); n = 4 (fa/fa)], and day 44 [group IV: n = 13 (Fa/-); n = 7 (fa/fa)]. Activity was monitored for 3 hr/day until the day of weaning, and thereafter, for all 24 hr.
32 Age
40
48
(days1
have weaned the pups, although actual separation from the dam did not occur until day 44. Independent of the day of separation from the dam, in the 21 hr immediately following the separation, wheel running increased precipitously in all obese and lean pups, perhaps reflecting the increased time of access to the wheel (3 versus 24 hr). Twenty-four hours later, the activity of obese rats declined to preseparation levels or less, while that of the lean rats was maintained or continued to increase. By 8 wk of age total wheel running in obese rats was 50”:, less than that of the lean animals (Table 1). Cumulative overall running was similar by day 52 in all three groups of lean rats, independent of the time of weaning. Cumulative running of obese rats weaned at day 19 was less than that of obese rats weaned at day 25 or day 44. In contrast to postweaning activity patterns, activity levels prior to weaning were comparable in obese and lean rats (groups II and IV) from day 16 to day 22, and from day 16 to day 19 in group III (Figs. 1 and 2). On days 23-25, activity levels of obese rats plateaued, while activity levels of lean rats continued to increase through day 24 (Fig. 2). At day 25 activity decreased in the lean rats to levels comparable to those seen in the obese rats (Fig. 2). Thus there was no significant difference in activity of obese and lean rats from day 16 to day 22
374
STERN
Table
1.
Cumulative
Activity
and
Cumulative
Food
Intake
in E-wk-Old
(X
JOHNSON
Zucker
Total Revolutions N
AND
Rats
Total Food Intake
103)
IS)
I (nonactive)
Group
Obese
(fa/fa)
Leon (Fo/-) Group
II (weaned
Lean Ill (weaned
674.9
+ 18.8
498.6
+ 10.1
day 25)
Obese
Group
6 15
9
28.1
f
2.5
760.0
i
22.1
13
57.6
f
5.5
525.1
i
18.0
day 19)
Obese
7
10.5 f
1.4
770.7
f
22.2
Lean
5
67.1
13.9
599.5
i
27.5
11.9
Group
IV (weaned
i
day 44)
Obese
7
29.0
f
3.8
397.0
*
Lean
8
71.5
f
9.6
329.9
zt 12.9
Table
2.
Results
of Analyses
(Obese
and
Lean
of Variance Rats
Weaned
for
Groups
at Day
II and
IV
25) F Valuer
Parameter
df’
Days
Genotype
x Days
Days 16-22 Body weight
l/336
Daily food intake
l/336
33.04t
68.247
Cumulative
l/336
49.277
110.80t
5.631
l/336
0.02
110.37t
0.28
0.25
food intake
Daily activity
7.187
55.09t
0.13 1.75
Days 16-25 Body weight
l/480
12.337
115.2Bt
Daily food intake
i 1480
35.7gt
64.70.t
5.257
Cumulative
l/480
59.96t
134.707
7.45t
1 12.49t
1.48t
food intake
Daily activity
l/480
4.01 t
F Values
Autopsy Data
df*
Genotype
Activity
Genotype
x Activity
At8wk Body weight
l/27
33.88t
0.02
Carcass
weight
l/27
3.41
0.68
3.23
Adrenal
weight
l/27
33.33t
45.747
0.36
Total fat
l/27
320.44t
Total cell number
l/27
1.20
32.57t
13.48t
Average
cell size
6.307
2.84
1.21
l/27
497.85t
0.02
2.89
Plasma
glucose
l/24
0.75
0.39
6.29t
Plasma
insulin
l/27
15.327
0.32
2.36
l/235
34.207
8.46t
15.99t
Cumulative
food intake
At6mo Body weight
l/26
177.55t
0.58
1.60
Total fat
l/26
424.33t
14.12t
4.60t
Total cell number
l/26
37.76t
0.14
4.54t
Average
l/26
22 1.40t
3.91
1.73
*df: degrees tValues
cell size of freedom.
that are significant
at p < 0.05.
GENETICALLYOBESE RAT
375
4-
DAILY
FOOD
INTAKE i ;; ;I,/
3. (
0 2000
DAlLY
ACTIVITY
1500-
Fig. 2. Body weight, daily food intake, and daily wheel turns in lean (Fa/-) and obese (fa/fa) male rat pups from day 16 to day 25 (group II). Weaning occurred at day 25 or later. Activity was monitored for 3 hr during the dark cycle. Each point represents the mean & SEM for 16 obese and 34 lean PUPS.
0
16
1
16
,
~
20 Age
(doysl
22
*
24
1
(p > 0.05; Table 2). During this 16-22-day period, body weights were significantly increased in obese pups compared to lean (p < 0.05; Fig. 2, Table 2). During this period, daily solid food intake and cumulative food intake were significantly increased in the obese rats (Fig. 2, Tables 1 and 2). Cumulative food intake for the 37-day period was significantly increased in obese compared to lean rats, and was higher in active than in inactive rats. Table 3 presents the autopsy data for animals killed at 8 wk (groups I and II) and Table 2 presents the statistical analyses. In those animals killed at 8 wk of
376
STERN
AND JOHNSON
GENETICALLY
OBESE
377
RAT
Table 4.
Autopsy Data in Lean and Obese Zucker Rats at 6 mo of Age Body
Genotype
Obese (fa/fa) Exercised
N
2 12
5
Number
Mean Cell Sire
(9)
(9)
(9)
x 106
(rg lipid/cell)
12.2
179.1 f
11.3
446.0 f
Nonactive
Fat
705.0 *
Lean (Fa/-) Exercised
Total Cell
wt.
CClrCaSS
728.5 f
Nonactive
Total
wt.
11
22.0
204.969
161.667
f
f
165.165
f
f
f
3.8
7.4 11.6
8.097
1.050
f
11.240
1.984
1.3040 i
4.090
f
5.078
.0642
0.4483 f
84.415 f
.0202 1.2933
60.910 f
17.970 f
3.559
131.987
30.086 f
204.7 f
7.273
179.2
198.4
10.5 432.0
f
13.8
.0403 0.2164
f
.0209
age, there were no significant differences in body weight and carcass weight in either active lean or obese rats compared to their inactive controls. Total fat, adipose cell size, and number were greater in obese than in lean rats. The immediate effect of access to activity wheels was to decrease total fat and adipose cell number in both obese and lean rats. Adipose cell size was decreased in active lean rats, but unchanged in active obese rats. Plasma glucose levels were unchanged. Plasma immunoreactive insulin levels were significantly higher in the obese rats compared to lean rats. Activity decreased insulin levels only in lean rats. Adrenal weights in both active groups were elevated. Table 4 presents the autopsy data for animals killed at 6 mo in order to assess the long-term effects of exercise in the remaining rats from groups I and II. Results of statistical analyses are presented in Table 2. Early exposure to activity wheels was associated with increased body weight in both groups, which was reflected in an increased total fat and adipose cell size. Adipose cell number was decreased in the active lean rats compared to nonactive lean controls. Activity had no long-term effect in decreasing cell number in obese rats; in fact, formerly active obese rats had higher total cell number than nonactive obese controls. DISCUSSION
We have demonstrated that the Zucker obese rat is less spontaneously active than its lean littermate when given access to a running wheel. The decrease in spontaneous activity follows both the onset of hyperphagia and elevations in body weight that were detected before weaning. Perhaps at day 16, and certainly by day 17, the obese pups are eating more solid food than their lean littermates during the 3-hr access period. We may speculate that it was the noncompetitive free feeding situation that allowed the hyperphagia to be expressed at this early stage. Whether or not this hyperphagic behavior is expressed in the home cage when the pups are suckling the dam has not been established. Although there are indications that Zucker obese rats are significantly fatter as early as 15 days of age,15 no other study establishes significant differences in body weight between lean and obese Zucker rats before weaning. The event of weaning itself appears to play an important role in the establishment of decreased activity in the obese rat. When the time of weaning is
378
STERN
AND JOHNSON
varied, the onset of decreased activity is also changed. It is evident that obese Zucker rats are not born less active than their lean littermates, but rather the major shift to inactivity is coincident with the weaning process. There are numerous obvious changes that occur at weaning, such as change in composition of the diet, separation from the dam, and, often, separation from littermates and social isolation. Social isolation and physical separation are probably not precipitating factors, since obese pups removed from the dam at day 44 (group IV) decreased their activity around day 30 when the dam spontaneously weaned the pups. Joosten and van der Kroor? have recently reported that, in a 5-min test, locomotor activity of ob/ob mice is less than that of nonobese littermates as early as the second week of life, but during the third week of life (from day 15 to day 20) obese and nonobese mice show similar locomotor activity. From approximately day 23 on, the obese mice are less active as measured by lines crossed in an open field. These results in another obese rodent strain, and with another measure of activity, are similar to ours from 16 days. We were not able to measure spontaneous wheel running before day 16, so comparisons prior to that age are not possible. Unfortunately, Joosten and van der Kroon do not give the time of weaning in their study, but it is tempting to speculate that the decrease in activity which they report in the third week in ob/ob mice could be associated with the weaning process. In this study exercise had no effect in decreasing the hyperinsulinemia observed in Zucker obese rats. Perhaps the exercise was not severe enough to effect a change. In lean rats exercise did decrease plasma insulin values. however, these were twice as active as the exercised obese rats. Exercise resulted in increased adrenal weights; these findings are in keeping with those previously reported by Leshner.‘(’ The cellularity data reported here confirm and extend previous findings of Johnson et al.” on the effects of early environmental manipulations on cell number of adipose depots in the Zucker rat. The early activity of the lean rats effected a permanent decrease in the total number of adipocytes. While exercised obese rats had a decreased total fat cell number at 8 wk of age, the decrease was not permanent. At 6 mo, the formerly active rats, if anything, had more fat cells than their nonactive controls. These data support the previously proposed hypothesis that the Zucker obese rat is unable to shut down the mechanism of adipocyte proliferation, unlike its lean littermate, in which a preweaning environmental stimulus may render a permanent change in adipose tissue cellularity. While the lean formerly active rat had fewer fat cells than its nonactive control, it also had larger fat cells. Since active lean rats ate more chow than did nonactive controls during the 8-wk period of wheel running, it appears possible that a continued increased caloric consumption with decreased caloric output may have contributed to the increased deposition of body fat seen in these animals. We may only speculate that such a mechanism can explain the increased lipid deposition in the inactive period that followed a period of activity, since we did not measure food intake during the inactive period. This phenomenon may, however, be a condition that parallels the familiar gain in body fat often observed in the aging retired athlete.
GENETICALLY
OBESE
379
RAT
We conclude from these studies that hyperphagia is the first consistently measurable difference between obese and lean Zucker rats. It is closely followed by elevated body weight. The decreased activity is secondary to the development of adiposity. Joosten and van der Kroon’ proposed that hyperphagia is a congenital defect in the ob/ob mouse, although their earliest measure of increased food intake in this species was at 5 wk of age. Bray et al. have proposed that the primary disturbance in the genetically obese Zucker rat is a hypothalamic lesion or malfunction, since a number of the abnormalities that have been described may be related to hypothalamic control mechanisms: e.g., thyroid function, water balance, reproductive function.‘7m2” Hyperphagia could also result from an aberrant hypothalamic control of feeding behavior. Thus, studies now in progress are designed to determine how the pattern of feeding and running evolves in the obese and lean animals. Alternatively, hyperphagia may yet prove to be secondary to a morphological or metabolic derangement which is the primary expression of the fa gene. The primary defect in this animal could be due to an inability to synthesize muscle protein at the normal rate with unused calories being diverted into fat stores.2’,22 The resulting nutrient imbalance could provide a stimulus to signal feeding behavior. In any case, increased proliferation of adipocytes followed by a requirement for lipid filling, or decreased utilization of calories by muscle resulting in excess substrate for lipid filling of preexistent fat cells, could provide metabolic signals to the central regulatory system to produce hyperphagia. Decreased activity may, however, become a contributing factor in the subsequent expression and maintenance of the obese state. ACKNOWLEDGMENT We thank Dr. Jules Hirsch and Dr. George Collier for their Noeldechen and Nuton Stewart for their technical assistance.
continued
support
and
Patricia
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STERN
AND JOHNSON
17. Bray GA, York DA: Thyroid function ol genetically obese rats. Endocrinology 88: 1095 1099. 1971 IX. York DA, Bray GA: Regulation of water balance in genetically obese rats. Proc Sot Exp Biol Med 136:798 X01. 1971 19. York DA, Hershman JM. Utiger RD. Bray GA: Thyrotropin secretion In genetically obese rats. Endocrinology 90:67 72. 1972 20. Saiduddin S. Bray GA. York DA. Swerdloff RS: Reproductive function in the genetically obese “fatty” rat. Endocrinology 93: 1251 1256. 1973 21. Zucker L: Unpublished observations 22. Martin RJ: Genetic influence on nutrittonal aspects of metabolic regulation. 1. Fed Proc 35:2291 2294. 1976