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0013-7227/79/1042-0350$02.

00
Endocrinology Vol. 104, No. 2
Copyright © 1979 by The Endocrine Society Printed in U.S.A.

Daily Rhythms in Adrenal Responsiveness to


Adrenocorticotropin Are Determined Primarily by the
Time of Feeding in the Rat*
CHARLES W. WILKINSON,! JEANETTE SHINSAKO, AND MARY F. DALLMAN^
Department of Physiology and Metabolic Research Unit, University of California, San Francisco,
California 94143

ABSTRACT. These experiments were done to determine: 1) In none of these experiments was an ACTH rhythm demon-
whether feeding-related shifts in daily corticosterone rhythms strable by analysis of variance. Neither ACTH levels nor adrenal
are dependent upon changes in ACTH rhythms, 2) whether and plasma corticosterone levels were higher in animals fed 2
restricted feeding of rats results in abnormally high ACTH and h/day than in rats eating ad libitum. Peak corticosterone levels
corticosterone levels (i.e. stress), and 3) whether changes in occurred just before feeding, and the restricted feeding paradigm
either insulin or glucose levels might be the concomitants of appeared to sharpen the daily rhythms. However, there was also
feeding that change adrenal responsiveness to ACTH. Young an effect of the light-dark cycle on corticosterone measures.
male rats (80-90 g) on a 12-h light, 12-h dark cycle were allowed Dietary fat content was directly related to increases in body
access to one of three diets for 2 h/day beginning either at lights weight and mean insulin levels and inversely related to adrenal
off or lights on. The diets contained 3%, 4.5%, or 11% fat. A group responsiveness to ACTH. The data show that: 1) the time of
of rats had ad libitum access to the food containing 4.5% fat. On feeding determines the timing of the corticosterone rhythm in
day 20 of this regimen, rats were killed at 2- to 4-h intervals the absence of a rhythm in ACTH, 2) restricted feeding is not a
during the next 24 h, and plasma ACTH, corticosterone, insulin, stress, and 3) neither insulin nor glucose has a substantial
and glucose were measured. Adrenal weight and corticosterone influence on adrenal responsiveness to ACTH. (Endocrinology
content were also determined. 104: 350, 1979)

C IRCADIAN rhythms in plasma corticosteroid con-


centrations of many mammals are characterized by
the occurrence of maximal levels at approximately the
costeroids is driven by that in ACTH. However, in
ACTH-implanted, hypophysectomized rats, in which cir-
cadian ACTH cyclicity was presumed to have been abol-
beginning of daily activity (1-5). In nocturnally active ished, plasma corticosterone rhythms are maintained
murids, the circadian peak occurs at, or shortly before, (12). Furthermore, in intact rats under 12-h light, 12-h
the onset of darkness (4, 5). It has been reported repeat- dark (12:12 LD) conditions, the rhythms in plasma
edly that restriction of rats or mice to a brief period of ACTH are of much smaller amplitude than those of
eating or drinking early in the light phase of the cycle plasma corticosterone, and often do not reach statistical
results in a shift of the daily plasma corticosterone peak significance (13).
to the time immediately before eating (6-9). However, it We have recently obtained evidence in rats that adre-
is debatable whether the shift in corticosterone repre- nal responsiveness to both endogenous and exogenous
sents a shift in the diurnal rhythm or the stress of fasting ACTH is approximately 2.5 times greater at the peak
which is relieved by food presentation. Circadian (lights off) of the daily corticosterone rhythm than at its
rhythms of circulating ACTH in rats and man have been nadir (lights on) and that the ACTH and adrenal
reported to be in phase with those of corticosteroids (10, rhythms are independent (13). Evidence for time of day
11), and it has been assumed that the rhythm in corti- dependent changes in responsiveness of the human ad-
renal to ACTH has also been reported (14). In light of
Received July 3, 1978. these findings and in the absence of measurements of
Address requests for reprints to: Dr. Charles W. Wilkinson, Depart- plasma ACTH or adrenal corticosterone concentration in
ment of Physiology, University of California, School of Medicine, San
Francisco, California 94143. previous investigations of restricted feeding, we have
* This work was supported in part by a NASA-University Consor- attempted to determine the relationships among feeding-
tium Interchange Agreement NCA2-OR665-602 and by USPHS Grant induced reversals of plasma corticosterone rhythms and
AM-06704.
f Recipient of Postdoctoral Fellowship AM-05681. rhythms in ACTH and adrenal responsiveness to ACTH.
| Recipient of Research Career Development Award NS-00072. Adrenal and plasma corticosterone concentrations and
350
FOOD AND ADRENAL RESPONSIVENESS TO ACTH 351

plasma ACTH levels were measured at 2- to 4-h intervals human plasma as the source of transcortin. Insulin was mea-
throughout 24 h in rats maintained for 20 days on feeding sured by RIA using kits from Amersham-Searle and standards
periods of 2 h/day at either lights on or lights off. of human insulin. Glucose was determined by a glucose oxidase
Food ingestion is a potent stimulus to insulin secretion, technique using a Beckman Autoanalyzer. For each hormone
which in turn may be related to adrenal responsiveness assay, samples from individual groups within an experiment
were measured in each run of an assay so that interassay
to ACTH. Human diabetics and alloxan-diabetic rats
variation was incorporated into individual group means and
have been reported to exhibit increased adrenocortical variances. Wet weight of the adrenals was determined, and the
responsiveness to stress or ACTH administration as well adrenals were homogenized (1:500, wt/vol) in a 20% ethanol-
as a flattening of circadian rhythms in plasma corticoste- isotonic saline solution for determination of adrenal corticos-
roids (15-17). Insulin treatment of alloxan-diabetic rats terone by competitive protein binding. In each of the three
returned adrenal responsiveness and circadian rhyth- experiments, a different diet (differing primarily in fat content,
micity to normal (16). There is also evidence that circa- as stated by the manufacturers), was fed to the rats. In Exp 1,
dian rhythms in plasma insulin levels are 180° out of rats were fed Berkeley diet (Feedstuffs Processing, San Fran-
phase with ACTH or corticosteroid rhythms in man (18) cisco, CA) containing 3% fat; in Exp 2, they were fed Purina
and rats (7), although there are contradictory data for laboratory chow containing 4.5% fat; and in Exp 3, they were
given Purina mouse chow, containing 11% fat. In chronological
the rat (19, 20). Therefore, we also measured plasma
sequence Exp 3 followed Exp 1 and the change in diet was an
glucose and insulin levels in these animals in a prelimi- independent manipulation on the part of the Animal Care
nary attempt to determine the specific concomitants of Facility that was not discovered until the experiment was well
eating and/or drinking that are capable of altering adre- in progress. An intermediate diet was then selected by the
nocortical rhythms. In each of three separate experi- experimenters for use in Exp 2, the final experiment.
ments, a different diet was provided to the animals in Data were processed for Bartlett's test for homogeneity of
order to investigate the effect of dietary fat content (or variance, one- or two-way analysis of variance, Tukey's HSD
caloric intake) on insulin concentrations and on the multiple range test, and linear correlation on an IBM 370
rhythm of adrenal responsiveness to ACTH. computer (24). One-way analyses were used to determine time
of day effects (i.e. presence or absence of daily rhythms) and
effects of dietary fat content. Paired comparisons between
Materials and Methods groups receiving different diets were made using Tukey's HSD
Male Sprague-Dawley rats, weighing 80-90 g (Simonsen, test. Two-way ( 2 x 2 factorial) analyses were used to determine
Gilroy, CA), were received 2-3 days before the beginning of effects and interactions of time of feeding and phase of the light
each experiment and were housed two per cage in hanging wire cycle. Only data obtained from animals (480 rats out of a total
baskets in animal rooms maintained at 21-23 C. Rats were of 500) for which all measurements were made successfully
exposed to a 12:12 LD schedule with lights on between were included in statistical analyses or in figures.
0900-2100 h (with the exception of the dark-fed animals in Exp
3, which were housed in a separate room with a reversed Results
lighting schedule). In each of three experiments, groups of 64-76
Levels of hormones, adrenal responsiveness to ACTH,
rats were given pellets of food inside their cages at lights on,
and equal numbers of animals were given food similarly at and glucose during 24 h in rats fed ad libitum
lights off. All remaining food was removed from the cages 2 h The data obtained from rats fed ad libitum are shown
after its presentation. In one experiment (no. 2), a third group in Fig. 1. In addition to plasma ACTH, insulin, glucose,
of 76 rats fed ad libitum was included. All rats were given free
and plasma and adrenal corticosterone concentrations
access to water throughout all experiments. Animals were
weighed at the beginning of the experiment and on alternate plotted against time of day, adrenal responsiveness (ad-
days throughout. After 20 days on the 2-h feeding schedule, renal corticosterone/log ACTH) is also shown in the
groups of six to eight rats from each of the feeding conditions figure. The use of this measure is justified by reports that
were sacrificed at 8 or 12 times during a 24-h day. Groups were adrenal synthesis and secretion of corticosterone are
killed 10 min before feeding, 10 min before food removal, and directly proportional to the logarithm of the ACTH
at multiples of 2-h intervals from these times. Both animals in concentration under conditions of bioassay for ACTH
a cage were killed by decapitation within 15 sec of opening the (25,26). Because the responsiveness measure is a function
cage, and trunk blood was collected in heparinized plastic tubes of adrenal rather than plasma corticosterone concentra-
and immediately chilled in ice. Left adrenals were removed and
tion, it is not directly affected by changes in the rate of
kept moist in petri dishes on filter paper saturated with saline,
and terminal body weights were recorded. The blood was clearance of corticosterone from the circulation. Al-
centrifuged at 4 C shortly after collection and plasma was though ACTH levels were highest just before lights out
separated and frozen in separate aliquots for 1) ACTH and and were significantly lower in the sample obtained just
corticosterone, and 2) for insulin and glucose determinations. before lights on, one-way analysis of variance did not
ACTH was measured by RIA (21, 22). Corticosterone was reveal a significant rhythm in ACTH. By contrast, sig-
measured by a competitive protein-binding technique (23) using nificant time of day effects were found for adrenal re-
352 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1979
Vol 104 • No 2

glucose levels; however, the lowest point coincides with


40 the minimum in insulin concentration at 1900 h.
Plasma Levels of hormones, adrenal responsiveness to ACTH,
ACTH 20 and glucose levels during 24 h in rats fed for 2 h at
(pg ml)
lights on or lights off
0
80 Mean values of data for each of the three experiments
Adrenal are plotted as a function of the time of day in Fig. 2. One-
B 40 way analyses of variance revealed significant time of day
effects for all variables except ACTH in each feeding
condition in every experiment. A significant rhythm in
ACTH was never obtained in these studies. However,
mean ACTH levels generally appeared to be highest
during the 6 h before feeding and lowest 12 h later.
Rhythms in adrenal responsiveness and corticosterone
reached their maxima during the hours just before feed-
ing. In five of the six restricted feeding groups, the daily
minimum in adrenal responsiveness to ACTH occurred
at the first time point after lights on, as did the minima
for adrenal and plasma corticosterone concentrations in
the three dark-fed groups. Minima for light-fed rats were
less predictable, but for all variables except ACTH they
occurred during the first 8 h after lights on.
In Exp 2 (4.5% fat diet) a direct comparison can be
made between hormone levels from rats eating this diet
ad libitum and rats allowed to feed only 2 h/day. Mean
ACTH levels ranged between 14-38 pg/ml in all three
conditions (Fig. 1 vs. Fig. 2, ). Peak values of adrenal
and plasma corticosterone levels and adrenal responsive-
ness to ACTH did not differ between rats feeding ad
libitum and those fed for 2 h/day. Peak adrenal corticos-
0900 2100 0900 terone levels were 5.1 ± 0.8, 6.4 ± 0.9, and 5.4 ± 0.8
Time of day (hours) /xg/100 mg, and peak plasma corticosterone levels were
FIG. 1. Plasma ACTH, corticosterone (B), insulin, and glucose concen- 14.1 ± 3.0,19.4 ± 1.6, and 16.2 ± 2.8 /xg/100 ml in the rats
trations of rats with free access to food containing 4.5% fat. Also plotted fed ad libitum, for 2 h at lights off, and for 2 h at lights
are adrenal corticosterone concentrations and adrenal responsiveness on, respectively. No significant differences in ACTH,
to ACTH (micrograms of B per g divided by log picograms of ACTH adrenal corticosterone, or adrenal responsiveness
per ml). Each point represents the mean (±SEM) value for 5-10 rats. In
several cases, the SEM is too small to be visible beyond the dimensions rhythms could be demonstrated among the ad libitum
of the symbol. The two points connected by dotted lines at the right of and the two restricted feeding groups using two-way
each line are repetitions of the first two points at the left. Asterisks at analyses of variance.
the far right indicate significant (P < 0.0001) time of day effects by Figure 3 shows pooled data from the three experiments
analysis of variance. For ACTH, P = 0.60; for glucose, P = 0.43.
plotted as a function of time of feeding rather than time
of day. On the left side of the figure are shown data for
sponsiveness to ACTH and for the corticosterone meas- the eight times common to all three experiments. Adrenal
ures. Adrenal responsiveness and corticosterone levels corticosterone content is included as well as corticoster-
increased at 1700 h and remained elevated for the first 6 one concentration. One-way analyses of variance of the
h after lights out. Plasma corticosterone levels peaked at combined data from all three experiments revealed sig-
a level of 14 /xg/lOO ml in the sample obtained just before nificant rhythms for all measures for both light-fed and
lights out. Minima in adrenal responsiveness and the dark-fed groups, with the exception of plasma ACTH in
corticosterone measures occurred in the samples col- the dark-fed group (Table 1). Peak values of ACTH,
lected just before lights on. Plasma insulin concentrations plasma and adrenal corticosterone concentrations, adre-
rose sharply from a nadir at 1900 h to a relatively stable nal responsiveness, and adrenal corticosterone content
plateau achieved between 2100-0900 h, during the time all occurred at the onset of feeding regardless of its
when rats normally eat. There was no rhythm in plasma relationship to the light cycle. Again, in the pooled data,
FOOD AND ADRENAL RESPONSIVENESS TO ACTH 353

Food Food
Lights on M Lights off Lights on Lights off
50
Plasma
ACTH 25
(pg/ml)
0

8
Adrenal
corticosterone 4
(>jg/IOO mg)

24

Plasma 16
corticosterone
(>ig/IOOml) 8
0L

Adrenal
responsiveness
/ \ 3 \
/ jjg B /lOOmg \
\logpgACTH/ml/ 0
0900 2100 0 9 0 0 0900 2100 0900
Time of day (hours)
FIG. 2. ACTH and adrenal measures for rats fed for 2 h at lights off (left) or for 2 h at lights on (right). Each line represents the mean values from
a different experiment plotted as a function of time of day. , Data from rats fed the 3% fat diet; , data from rats fed the 4.5% fat diet;
• - •, data from rats fed the 11% fat diet (five to eight rats per point).

ACTH and corticosterone levels resemble those observed mean values (obtained from the data shown in Fig. 3,
in ad libitum fed rats (Fig. 1). The nadir for each of the left) do not include data from either the 0900- or the
above measures, except for plasma ACTH and corticos- 2100-h time points because these points represent dark-
terone in the light-fed group, occurred at the first time light transition times (Fig. 3, C>). Two-way analyses of
point after lights on regardless of the feeding schedule. variance (light phase by feeding time; Table 2) show
The right side of Fig. 3 illustrates pooled data from Exp significant light-dark differences for all variables except
2 and 3 in which samples were taken at 12 2-h intervals. ACTH, whereas there are no significant effects of feeding
These data follow patterns similar to those shown at the time for any variable. There are significant feeding time-
left of the figure, with minima and maxima occurring in light phase interactions for both plasma corticosterone
the same relationship to the light cycle and feeding. One- concentration and plasma to adrenal corticosterone ratio,
way analyses of variance of these data did not reveal suggesting a feeding-related component in dark-light dif-
rhythms in ACTH in either light- or dark-fed rats, ferences in metabolism, distribution, or binding of corti-
whereas rhythms in all other variables are highly signif- costerone.
icant. Plasma insulin and glucose levels (Fig. 5) were lowest
For all of the adrenal measures, levels obtained during in the samples obtained immediately before feeding. In-
the dark phase of the light cycle are consistently higher sulin levels show a steep rise with a peak at the next time
than those obtained during the light phase of the cycle, point just before the removal of food; the levels then
although the daily maxima of the rhythms are synchro- gradually fall throughout the remainder of the 24 h.
nized with feeding time. The differences between dark- Analyses of variance revealed significant time of day
ness and light are shown more clearly when all data effects for both insulin and glucose. Table 3 shows that
obtained from a given feeding group at three time points there is a low but significant positive correlation between
during the dark phase are pooled and the mean values the logarithm of plasma ACTH concentration and adre-
are compared with similarly calculated mean values ob- nal corticosterone concentration both in the rats fed ad
tained during the light phase of the cycle (Fig. 4). The libitum and those fed for only 2 h/day. There is also a
354 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1979
Vol 104 • No 2

Food Food
50
Plasma
ACTH 25
(pg/ml)
0

8
Adrenal
corticosterone 4
(>jg/IOOmg)
0

24

Plasma 16
corticosterone
(^g/100 ml) 8
\x ^4, A/
'•9'
0

6
Adrenal
responsiveness

/ jjg B/IOOmg \
\logpgACTH/ml/

720
Adrenal
corticosterone content 3 6 0
(ng/adrenal)
0

Plasma-adrenal
1.
corticosterone ratio

jig/IOOml \

yug/IOOmg/ 12 18
Time after food presentation (hours)
FIG. 3. ACTH and adrenal measures pooled across the three experiments are plotted on the left as a function of time after food presentation for
the eight sampling times common to all of the experiments (means of 17-24/group ± SEM). Right, Pooled data for each of the 12 time points
common to Exp 2 and 3 (means of 9-16/group ± SEM). , Results from rats fed during the first 2 h of light; , results from rats fed during the
first 2 h of darkness. € , Samples collected at the time just before a light-dark transition; • , samples collected during the dark period; O, those
coUected during the light. Results of analyses of variance of these data are shown in Tables 1 and 2.

low negative correlation between plasma insulin levels groups (r = -0.265, P < 0.001). Similar inconsistent
and both adrenal corticosterone concentration and ad- correlations were found between glucose and adrenal
renal responsiveness to ACTH which only achieves sig- responsiveness measures. The inconsistencies in the signs
nificance in the rats fed 2 h/day. Despite the statistical of the glucose correlations with adrenal measures indi-
significance of the insulin correlations with adrenal cor- cate that changes in glucose levels are not responsible for
ticosterone and responsiveness, neither r2 value exceeds diurnal variations in adrenal responsiveness to ACTH.
0.035. In other words, less than 4% of the variability in Adrenal and plasma corticosterone levels are highly cor-
adrenal corticosterone or adrenal responsiveness can be related with one another, and r2 values are 0.640 and
attributed to changes in circulating insulin levels. Glucose 0.558 for the ad libitum and 2-h feeding groups, respec-
values show a nonsignificant positive correlation with tively. Thus it appears that reversal of the plasma corti-
adrenal corticosterone levels in the ad libitum groups (r costerone rhythm by reversal of the feeding schedule is
= +0.119, P = 0.156) and a significant negative correla- determined primarily by changes in adrenal corticoster-
tion with adrenal corticosterone in the restricted feeding one secretion, but diurnal fluctuations in metabolism,
FOOD AND ADRENAL RESPONSIVENESS TO ACTH 355

TABLE 1. Results of analyses of variance of pooled data from Exp 1-3 (data shown in Fig. 3, left)

Dark fed Light fed


Effect; of time after feeding on:
df df
Plasma ACTH 1.08 (7, 153) N.S. 2.97 (7, 144) <0.01
Adrenal corticosterone concentration 11.65 (7, 153) <0.0001 8.97 (7, 144) <0.0001
Adrenal responsiveness to ACTH 8.25 (7, 153) <0.0001 5.60 (7, 144) <0.0001
Adrenal corticosterone content 12.02 (7, 153) <0.0001 8.94 (7, 144) <0.0001
Plasma corticosterone 17.04 (7, 153) <0.0001 15.72 (7, 144) <0.0001
Plasma corticosterone/adrenal corticosterone 2.57 (7, 153) <0.02 6.89 (7, 144) <0.0001

Light-fed Dark-fed Light-fed


Adrenal
responsiveness
Plasma ACTH
/ >ig B/IOOmg \
(pg/ml)
llogpgACTH/miy

Adrenal
Adrenal corticosterone corticosterone content
(.pg/IOOml) (ng/adrenal/IOOgBW)

Plasma-adrenal
corticosterone ratio
Plasma corticosterone
(>ig/IOOmg)
Q JZL
/ jjg/IOOml
\ p g/IOOmg n
I I Light ME Dark
FIG. 4. Mean values from the three sample collections during the darkness and from the three sample collections during the light period in light-
fed and dark-fed groups shown in Fig. 3. Data from samples collected just before a light-dark transition are not included. Each bar represents data
from 55-59 rats. Analysis of these data (Table 3) shows an effect of the phase of the light cycle independent of the time of feeding for all measures
except for plasma ACTH.

distribution, and/or binding also contribute to the circa- from only eight time points were compared; however, the
dian variation in plasma corticosterone levels. mean values were significantly different on both meas-
ures when all data from these experiments were included
Effect of diet on mean levels of hormones, adrenal in the analyses. .
responsiveness to ACTH, glucose, and body weight in
rats fed 2 h/day Discussion
The effects of differences in dietary composition are The results of these experiments confirm earlier obser-
illustrated in Fig. 6. The data shown are overall mean vations that daily rhythms in plasma corticosterone con-
values for the eight time points common to each of the centration can be synchronized by restricted feeding or
three experiments. Weight gained during the 20 days of watering schedules. Feeding-dependent rhythms char-
the experiment, plasma insulin levels, and plasma glucose acterized by maxima at the time points immediately
concentrations all increase with increasing dietary fat preceding food presentation are also demonstrated for
content. However, mean adrenal corticosterone concen- adrenal corticosterone concentration and content and
tration was highest in the group fed the low fat diet and adrenal responsiveness to ACTH. Significant rhythms in
lowest in the rats fed the high fat diet, despite the fact plasma ACTH concentration were found only when data
that mean plasma ACTH concentration was highest in from all three experiments were pooled and then only for
the latter group. Adrenal responsiveness to ACTH, rela- the light-fed groups. In this case also, values were highest
tive adrenal weight, and adrenal corticosterone content in samples taken just before the onset of feeding. The
were all inversely related to the dietary fat content, absence of an ACTH rhythm in these experiments may
increase in body weight, and plasma insulin levels. Dif- have resulted from the fact that animals on both feeding
ferences in adrenal responsiveness to ACTH and adrenal schedules as well as the animals fed ad libitum were all
corticosterone content between Exp 2 and 3 (4.5% and housed in the same room in Exp 1 and 2. The disturb-
11% dietary fat content) were not significant when data ances associated with food presentation and removal may
356 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1979
Vol 104 • No 2

Food Food
en
o

.001
SIS
a.

.00
90
o
cj
" © © Plasma
V V
"co I 1 insulin 45
s §
u <-
CO' CO t o
CN CN CM
(nU/ml)
•a CN CN CN 0
CO " "

CO
w- ^ w- 180
Hcon Plasma
a © to >o glucose 90
i in ©
© CN rH (mg/100 ml)
cu
0 6 12 18 12 18
CU

<0.00(
bo

<0.05
Time after food presentation (hours)

NS
ron

a,
•c aa FIG. 5. Mean levels of plasma insulin and glucose in rats fed 2 h daily
u
t o tO CD either at lights on ( ) or at lights off ( ). Left, Pooled results
CN CN CN
o CN CN CN form the 8 time points common to Exp 1-3; right, mean data from the
u
12 sampling times common to Exp 2 and 3. All values are plotted as a
•51 _
***
IO •<}•
n oi H
Hiriin
CT>
function of time after food presentation.

cu
TABLE 3. Correlations between some of the variables presented in
eron

a. t/3 g CO

CO
Figs. 1 and 2
O «->
.« C
CO CO CO Pooled 2-h
o c CN CN CN Ad libitum
CJ O CN CN CN
Feeding condition feeding (n =
„ o (n = 75)
c 405)
co co i o
2?
•a TJ; O> - H
H CN
Tf O Log ACTH:adrenal corticosterone 0.299" 0.213"
CO Insulin:adrenal corticosterone -0.183'' -0.187*
veness

0.0001

a Insulin:(adrenal corticosterone/log -0.161'' -0.177"


NS
NS

a, ACTH)
Io sh V Adrenal corticosterone:plasma corti- 0.800'' 0.747''
a t j co to to
( N CN CN
costerone
cu < ^S CN CN CN
u
o rt rt -H" " P < 0.005.
c '—' '—' " ^ h
cu
ha
m I-H --i P< 0.001.
fc<co © O
(NO© ' P = 0.058.
CN

cu
c ©
£ a. CO § CO lier findings of nycthemeral rhythms in adrenocortical
ost<
tior

V responsiveness to ACTH which are independent of


tj -*2
O ^"
CO CO tO
CN CN CN
ACTH rhythms (13).
CN CN CN
8g •a
Comparison of the data obtained from rats fed ad
con
renal

O> © CN
t ~ CO —>
libitum (Fig. 1) and those allowed to feed for only 2 h at
fc
HCOO
CN lights off (Figs. 2 and 3) suggests strongly that restricted
CO CO CO
feeding does not represent a stressful stimulus to the rat.
•2 I K a. Z 2 2 ACTH levels are similar and low in the two conditions,
h
o CO CO t O
CN CN CN
and plasma and adrenal corticosterone levels were not
CN CN CN
-a
CO
i-T —T <-T different between the rats fed at lights off and those with
s
© --1 CN continuous access to food in the sample obtained just
O t - H
© © © before lights out in Exp 2 (diet containing 4.5% fat).
?1 Adrenal responsiveness to ACTH and adrenal and
plasma corticosterone levels have decreased by 4 h after
lights out in freely eating animals. It is likely that these
c
O .bp •3
SJJ
rats ate most at the onset of dark as well, since rats fed
3 S ad libitum eat more food during the 2-h interval imme-
« O diately after lights out than during any other interval
.§ SP
CN CO during the 24-h day (27). The most prominent effect on
"s "o ti hormone levels of restricting feeding to a 2-h period each
CJ O L.
$2 cu cu a)
its it: £ day is a sharpening and narrowing of the period of peak
adrenal activity. This is particularly evident in the
have constituted an environmental stimulus of sufficient marked decrease in corticosterone that occurs within 2 h
magnitude to disrupt a rhythm in ACTH. The more of food presentation in the rats fed at lights on (Figs. 2
robust rhythms in adrenal responsiveness to ACTH and and 3). Because there are no significant differences in the
in corticosterone were not disturbed, supporting our ear- maximum levels of adrenal responsiveness to ACTH or
FOOD AND ADRENAL RESPONSIVENESS TO ACTH 357

Increase
•»
40
Plasma ACTH
in body weight
(pg/ml)
(g/2Odays)
Q
Adrenal
corticosterone Adrenal FIG. 6. The effect of dietary fat content
(.ug/IOOmg) o (caloric density) on body weight, ACTH,
responsiveness
adrenal measures, insulin, and glucose
jjgB/IOOmg \ expressed as overall 24-h means of data
Plasma logpgACTH/ml I from the eight common time points from
corticosterone Exp 1-3. For each measure, group means
Oig/I00ml)
are significantly (P < 0.05) different by
Tukey's HSD test from those with a
different number of asterisks. (*), Group
mean that is not different from either of
Plasma insulin
(.uU/ml)
Relative adrenal size

(mg/100 gBW) [D_D the other two group means. Exp 1, n =


120; Exp 2, n = 102; Exp 3, n = 91.

Adrenal
Plasma glucose 300
corticosterone content
(mg/IOOml)
(ng/adrenal/IOOg) 0
3.0 4.5 11.0 3.0 4.5 11.0

Dietary fat content (%) Dietary fat content (%)

of adrenal or plasma corticosterone between light- and that have been food deprived for 5 or more days (27).
dark-fed groups, it seems most likely that the time of Further, pharmacological and surgical manipulations
food presentation, rather than the possible stress of food that abolish adrenal rhythms also abolish feeding and
deprivation, determines the adrenal rhythms. In support drinking rhythms in animals fed ad libitum. Treatment
of this conclusion is the report that starvation of rats for with parachlorophenylalanine abolishes rhythms in food
as long as 3 days does not result in increased plasma and water intake (33) and in plasma and adrenal corti-
corticosterone levels (27). costerone but does not affect rhythms in ACTH (13, 34).
These experiments do not address the question of Lesions of the suprachiasmatic nuclei abolish circadian
whether some aspect of feeding 1) acts as a Zeitgeber to rhythms in eating, drinking, locomotor activity, and
entrain an endogenous oscillation in the adrenal, a pos- plasma corticosterone (35-37), but restricted feeding
sibility suggested by reports of adrenal rhythms obtained schedules synchronize adrenal rhythms in these animals
in culture (28), or 2) acts as an oscillating driving force to (38).
which the adrenal responds passively. Because rats on Our experiments demonstrate also that the phase of
restricted feeding schedules drink as much as 90% of the light cycle affects adrenocortical rhythms. Adrenal
their daily water intake during the feeding period (29), corticosterone concentration and content and adrenal
these experiments do not differentiate between concom- responsiveness to ACTH, but not plasma ACTH concen-
itants of eating, as opposed to drinking, as the primary tration, are higher during the dark than during the light
determinants of adrenal rhythms. Restriction of access phase of the light cycle, regardless of the time of feeding
to water has also been shown to resynchronize adrenal in food-restricted rats (Fig. 4). Thus there are two inter-
rhythms (6). acting influences on the daily variation in adrenal re-
The strength of the coupling between time of feeding sponsiveness to ACTH: the time of feeding and the light
and/or drinking and corticosterone rhythms is shown by cycle. Interactions of feeding effects with the light cycle
the fact that reversal of the light cycle results in concom- have also been reported by others (39, 40). By changing
itant reversals in ad libitum food intake and plasma adrenal responsiveness to ACTH, feeding and light cycles
corticosterone (30), whereas the resynchronization of the amplify the adrenal expression of the circadian rhythm
rhythm in ACTH follows a longer time course (31). Also, in circulating ACTH by a mechanism that may be me-
in blinded rats, the free-running phase shifts in food diated by direct neural influences on the adrenal cortex
intake and corticosterone rhythms follow the same time or by alterations in the circulating levels of other hor-
course (32), and corticosterone rhythms disappear in rats mones or metabolites.
358 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1979
Vol 104 • No 2

In addition to the effects of feeding and light cycles on 4. Guillemin, R., W. E. Dear, and R. A. Liebelt, Nycthemeral varia-
adrenal responsiveness to ACTH, a third factor that tions in plasma free corticosteroid levels of the rat, Proc Soc Exp
BiolMed 101: 394, 1959.
results in even greater excursions in the rhythm in plasma 5. Halberg, F., J. H. Galicich, F. Ungar, and L. A. French, Circadian
corticosterone is the time of day difference in metabo- rhythmic pituitary adrenocorticotropic activity, rectal temperature,
lism, distribution, or binding of corticosterone (see Fig. and pinnal mitosis of starving, dehydrated C mice, Proc Soc Exp
Biol Med 118: 414, 1965.
4). Plasma to adrenal corticosterone ratios during the 6. Johnson, J. T., and S. Levine, Influence of water deprivation on
darkness are significantly higher than during the light adrenocortical rhythms, Neuroendocrinology 11: 268, 1973.
phase of the cycle, indicating more rapid disappearance 7. de Gasquet, P., and E. Pequignot, Changes in adipose tissue and
heart lipoprotein lipase activities and in serum glucose, insulin and
of corticosterone from the blood during the light than corticosterone concentrations in rats adapted to a daily meal, Horm
during the darkness. This consistent finding (13) supports Metab Res 5: 440, 1973.
previous reports of nycthemeral rhythms in the disap- 8. Krieger, D. T., Food and water restriction shifts corticosterone,
temperature, activity and brain amine periodicity, Endocrinology
pearance rate of corticosterone from plasma (41). 95: 1195, 1974.
Finally, the diet consumed had a significant effect on 9. Nelson, W., L. Scheving, and F. Halberg, Circadian rhythms in
mice fed a single daily meal at different stages of lighting regimen,
mean daily levels of adrenal corticosterone, relative ad- JNutr 105: 171, 1975.
renal weight, and adrenal responsiveness to ACTH. Rats 10. Cheifetz, P., N. Gaffud, and J. F. Dingman, Effects of bilateral
were placed on the restricted feeding schedule at a time adrenalectomy and continuous light on the circadian rhythm of
corticotropin in female rats, Endocrinology 82: 1117, 1968.
of extremely rapid growth. In none of the experiments 11. Krieger, D. T., W. Allen, F. Rizzo, and H. P. Krieger, Characteri-
was growth rate normal. From observation of the stom- zation of the normal temporal pattern of plasma corticosteroid
achs of rats at the end of the feeding period, it seems levels, J Clin Endocrinol Metab 32: 266, 1971.
likely that stomach capacity limited the amount of food 12. Meier, A. H., Daily variation in concentration of plasma corticoste-
roid in hypophysectomized rats, Endocrinology 98: 1475, 1976.
consumed during the 2-h period of food availability. It is 13. Dallman, M. F., W. C. Engeland, J. C. Rose, C. W. Wilkinson, J.
likely, therefore, that the three dietary groups differed Shinsako, and F. Siedenburg, Nycthemeral rhythm in adrenal
primarily in the calories consumed, and differences in responsiveness to ACTH, Am. J Physiol, in press.
14. Forsham, P. H., V. DiRaimondo, D. Island, A. P. Rinfret, and R. H.
corticosterone levels and adrenal responsiveness may Orr, Dynamics of adrenal function in man, Ciba Found Colloq
reflect different degrees of chronic malnutrition (42). The Endocrinol 8: 279, 1955.
inverse relationship obtained between the mean insulin 15. Lentle, B. C, and J. P. Thomas, Adrenal function and the compli-
cations of diabetes mellitus, Lancet 2: 544, 1964.
levels for each dietary group and the corresponding mean 16. L'age, M., W. Fechner, J. Langholz, and H. Salzmann, Relationship
values of adrenal responsiveness to ACTH may be a between plasma corticosterone and the development of ketoacidosis
coincidental finding reflecting independent relationships in the alloxan diabetic rat, Diabetologia 10: 131, 1974.
17. L'age, M., J. Langholz, W. Fechner, and H. Salzmann, Disturbances
of insulin and adrenal responsiveness to caloric intake. of the hypothalamo-hypophysial-adrenocortical system in the al-
The low negative correlation obtained between insulin loxan-diabetic rat, Endocrinology 95: 760, 1974.
levels and adrenal responsiveness to ACTH suggests that 18. Lakatua, D. J., E. Haus, and F. Halberg, Habitual circadian timing
insulin may exert a very minor inhibitory influence on of growth hormone (STH), adrenocorticotrophic hormone (ACTH),
insulin, cortisol and glucose in human serum, In Aschoff, J., F.
the adrenal cortex but that it is not the primary deter- Ceresa, and F. Halberg (eds.), Chronobiological Aspects of Endo-
minant of adrenal responsiveness to ACTH. Results of crinology, Verlag, Stuttgart, 1974, p. 185.
preliminary experiments directly testing the acute effect 19. Jolin, T., and A. Montes, Daily rhythm of plasma glucose and
insulin levels in rats, Horm Res 4: 153, 1973.
of insulin on adrenal responsiveness to ACTH in vivo 20. Bellinger, L. L., V. E. Mendel, and G. P. Moberg, Circadian insulin,
and in vitro support this conclusion (Wilkinson, C. W., GH, prolactin, corticosterone, and glucose rhythms in fed and
and M. F. Dallman, unpublished). Neither circulating fasted rats, Horm Metab Res 7: 132,1975.
21. Dallman, M. F., D. DeManincor, and J. Shinsako, Diminishing
insulin levels nor circulating ACTH levels are good pre- corticotrope capacity to release ACTH during sustained stimula-
dictors of circadian variations in resting adrenal or tion: the twenty-four hours after bilateral adrenalectomy in the rat,
plasma corticosterone concentrations. Endocrinology 95: 65,1974.
22. Rees, L. H., D. M. Cook, J. W. Kendall, C. F. Allen, R. M. Kramer,
J. G. Ratcliffe, and R. A. Knight, A radioimmunoassay for rat
Acknowledgments plasma ACTH, Endocrinology 89: 254, 1971.
23. Murphy, B. E. P., Some studies of the protein-binding of steroids
We thank Mr. Brian Matsumura for corticosterone assays and Ms. and their application to the routine micro and ultramicro measure-
Anne Aldrich for glucose assays. ment of various steroids in body fluids by competitive protein-
binding radioassay, J Clin Endocrinol Metab 27: 973,1967.
24. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H.
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