Full text of DOI:10.1093/gerona/56.1.B21

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 1, B21–B26
An Age-Related Decline in Melatonin Secretion Is
Not Altered by Food Restriction
Mary F. MacGibbon,1 Ronald S. Walls,1 and Arthur V. Everitt2
1Department of Immunology and 2Centre for Education and Research on Aging (CERA), Concord Hospital,
University of Sydney, New South Wales, 2139, Australia.
Melatonin has been found to exhibit youth-maintaining and disease-preventing properties. The current study ex-
amined whether the age-retarding regimen of chronic food restriction (FR) slowed the decline in melatonin secre-
tion reported to occur with age. Total nocturnal melatonin secretion was assessed by radioimmunoassay of the
primary metabolite, 6-sulphatoxymelatonin (6-S-OH-MLT), in urine. Measurements were made through adult-
hood (70 to 765 days) on male Wistar rats maintained on the FR regimen (60% of the normal intake) with the
control animals fed ad libitum (AL). The data of animals exhibiting gross pathology were excluded. Analyses of
covariance found the FR regimen had no effect on either the levels or pattern of decline observed in 6-S-OH-
MLT excretion through adulthood. However, the FR body-weight–indexed metabolite measures were approxi-
mately double those of the AL ( p ϭ .06). The possibility that this result may reflect unusually high melatonin
peaks in the FR tissues is discussed.
HE serum levels and total nocturnal secretion of mela-
tonin in normal rats and humans have been found to de-
tonin (6-S-OH-MLT), was employed as a noninvasive and
accurate of method of assessing melatonin secretion (22,23).
T
cline with age (1–6), although some studies report no age
change in these measures (7,8). There are various indications
that a decline in melatonin secretion may both reflect and be
causal in normal aging, as discussed previously in a theory
paper by MacGibbon (9). Influences of melatonin thought to
modulate aging and disease include temperature reduction
(10), antioxidation (11), and immunoenhancement (12).
The current study tested the hypothesis that the well-
established age-retarding regimen of chronic food-restriction
(FR), 60% of normal food intake (13–15), maintains youth-
ful total nocturnal melatonin secretion throughout adult-
hood. Control animals were fed ad libitum (AL).
A previous investigation (16) assessed the pineal content
and serum levels of melatonin in AL and FR rats at two ages
only (3 and 28 months) and collected the tissues at one spe-
cific time during the night (6 hours into the dark cycle). All
the older AL animals, but only one FR animal, exhibited
gross pathological changes of tumors or bilateral cataracts.
FR was associated with increased levels of melatonin both in
the serum and in the pineal gland. Melatonin measures made
at one or a few times during the night may, however, provide
erroneous data, because both aging (4) and FR (17) have been
shown to alter the pattern of melatonin secretion, including
changes in the acrophase. The study was further limited by
the presence of obvious disease processes. Various investiga-
tions have associated disease with alteration in melatonin
measures (18–20). An aging study (21) similar to that dis-
cussed above (16) also reported higher serum melatonin due
to the FR regimen, but only one age was assessed, measure-
ments were made at only one time during the night, and no
editing of data in relation to pathology was reported. The cur-
rent investigation created a separate category (path) for ani-
mals both fed AL and exhibiting gross pathological change.
Radioimmunoassay (RIA) of total nocturnal urinary excre-
tion of the primary melatonin metabolite, 6 sulphatoxymela-
METHODS
Animals
Male outbred Wistar albino rats were purchased at 4
weeks of age from the specific pathogen-free (SpF) Com-
bined Universities Laboratory Animal Supplies facility at
Little Bay, NSW, Australia. Thereafter they were maintained
at Concord Hospital, NSW, under clean conditions whereby
persons entering the animal housing area were required to
wear sterile gloves, overshoes, a mask, and a gown. The pro-
tocol was less rigorous in pathogen control than that for SpF
conditions. However, there has been no indication of infec-
tious disease outbreak in the animals during or since the
study period. Sentinel Wistar AL rats, living in the same en-
vironment as those of the current study, have been tested
throughout this period for Mycoplasma pulmonis, cilia-asso-
ciated respiratory bacillus, Theiler murine encephalomyelitis
virus, Sendai virus, and Tyzerr’s (Clostridium piliformis).
All serology tests, performed using enzyme-linked immuno-
sorbent assay (ELISA), were negative. General postmor-
tems, including histopathological examinations, of three out-
bred AL 2.5-year-old rats in the animal house at the time of
the study also found no indication of infectious disease.
The environment was also controlled for temperature (21 Ϯ
1ЊC), light (on and off at 6 am and 6 pm, respectively), and
changes in other conditions such as handling and noise,
which were kept to a minimum. The racks holding the cages
were designed to minimize differences of light exposure to
each cage. The AL animals were generally housed in pairs
and the FR animals singly.
The 77 animals involved in the study were aged from
70 to 585 days, with four aged between 640 and 765 days.
All animals were weighed fortnightly and checked twice
weekly for any indication of ill health.
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B22
MACGIBBON ET AL.
Criteria for Exclusion Due to Pathology
software that enabled the clustering of data and the use of
robust standard errors to cater to the double set of measure-
ments made on some animals. Regression analyses and cor-
relations were made using SPSS-6 (SPSS Inc, Chicago, IL)
software.
The data of any animals found before or after death to ex-
hibit gross pathology, other than minor hydronephrotic
change, were, with advice from a veterinary pathologist
specializing in laboratory animals, excluded from the main
analyses. The 12 AL animals that exhibited such evidence
of disease were placed in the separate “path” category. For
this and other studies by the authors, examples of premor-
tem changes resulting in exclusion of data from the normal
groups included apparent lethargy, substantial weight loss,
oliguria, polyuria, and diarrhea. The postmortem examina-
tion included a visual inspection of the brain and pituitary
gland and of the organs of the thoracic and abdomino-pelvic
cavities, with dissection of the lungs and kidneys. Attention
was directed at overt indicators of the respiratory, renal, pi-
tuitary, and neoplastic disorders to which such rats are
prone. The only obvious pathological change that did not re-
sult in exclusion of an animal from the healthy groupings
was hydronephrosis if two thirds of the renal tissue re-
mained and appeared healthy.
RESULTS
When the data of the animals with obvious pathology
were excluded (12 AL and 1 FR), those of 50 AL and 14 FR
animals remained.
In animals without apparent gross pathology, a signifi-
cant linear decline in total nocturnal 6-S-OH-MLT excre-
tion was found through adulthood in both the AL and FR
groups (Figure 1A). The AL group showed an average de-
cline of 40.4%, from 915 to 545 pmol, and the FR group,
over the same ages, showed an average 45.9% decline, from
935 to 505 pmol. The age-adjusted means were 748.8 pmol
and 737.8 pmol, for the AL and FR animals, respectively.
No statistical differences were found in levels of excretion
of 6-S-OH-MLT ( p ϭ .9), nor in the change in this differ-
ence with age ( p ϭ .8).
Diet
Regression curves of best fit (the unit of 6-S-OH-MLT is
pmol; age is measured in days):
All animals were fed normal pelleted rat chow containing
22% protein, 3.3% fat, and 3.6% fiber, which was pur-
chased from Doust and Rabbidge pty. Ltd., Sydney, NSW,
Australia. The FR animals were supplied, at noon each day,
60% of the average weight of the intake of the AL animals,
for which food was available at all times.
AL 6-S-OH-MLT = 980 – 0.676 age (p < .001)
FR 6-S-OH-MLT = 1010 – 0.790 age (p < .05) .
Figure 1B shows the total nocturnal 6-S-OH-MLT excre-
tion per unit of body weight (6-S-OH-MLT/bw) of the two
groups. As expected, given the age-associated weight
changes (Figure 1C), both groups exhibited a significant de-
cline with age. An initially rapid then slower decline was
seen in the AL group, with an exponential best-fitting re-
gression curve; although, as indicated below, a linear de-
cline fits nearly as well. The FR measures showed a steady,
linear, and shallower decline with age. Comparison of the
two groups found a nearly statistical difference in metabo-
lite excretion/body weight (p ϭ .06) and that this difference
did not change significantly through the ages studied (p ϭ
1.0). Age-adjusted means were 1.232 pmol/g and 2.383
pmol/g in the AL and FR groups, respectively.
Urine Collection
Animals were placed singly into metabolism chambers
for 4 consecutive days, the first 2 days for acclimatization
and the second 2 days for data collection. The final results
were based on an average of these two measures. (A previous
rat study by the authors indicated that a baseline of 6-S-OH-
MLT excretion was reached after 2 nights in these chambers.
In the current investigation, the correlation coefficients be-
tween the first and second measures of urine volume and
6-S-OH-MLT, at p Ͻ .001, were 0.81 and 0.46, respec-
tively.) The urine excreted by each animal between 5 pm
and 8 am was collected into a parafilm-covered beaker con-
taining 5 mg boric acid. The volume was recorded and the
urine filtered through muslin. Samples were stored in vials
at –20ЊC until sent, in containers of dry ice, interstate for as-
say. Assessment at two ages was made on 12 of the 50 AL
animals and 9 of the 14 FR animals, with the intervening peri-
ods averaging 100 days in both groups. All other animals, in-
cluding those in the path category, were assessed only once.
Regression curves of best fit (the unit of 6-S-OH-MLT/
bw is pmol/g; age is measured in days):
AL 6-S-OH-MLT/bw = 2.25 + 0.002 exp age/100) –
0.003 age (p < .0001)
FR 6-S-OH-MLT/bw = 3.24 – 0.003 age (p = .04) .
Figure 1B shows the AL linear regression:
Assay
AL 6-S-OH-MLT/bw = 2.09 – 0.003 age (p < .001) .
The 6-S-OH-MLT concentration of each sample was de-
termined by RIA using a technique (24) involving competi-
tion between the sample 6-S-OH-MLT and known amounts
of radioactive iodine-labeled 6-S-OH-MLT for antibody
raised against the metabolite. The assays were performed at
the University of Adelaide, SA.
Body weight change with age in the AL group was loga-
rithmic, with a marked initial increase followed by a steady
slower rise. The FR weights showed too much variation
and/or the numbers were too few for a statistically signifi-
cant regression to be found. A significant intergroup weight
difference occurred (p Ͻ .0001). The age-adjusted means
were 643.8 g and 300.5 g for the AL and FR groups, respec-
tively. This difference changed with time. (Figure 1C shows
regression lines that fit 50% of the points.)
Data Analysis
General factorial analyses of covariance (ANCOVA)
were made using STATA (Stata Corp, College Station, TX)

MELATONIN IN FOOD-RESTRICTED AGING
B23
Figure 1. The graphs show the change with age in A, total nocturnal 6-S-OH-MLT excretion, B, weight-indexed 6-S-OH-MLT excretion, and
C, body weight in the FR and AL groups. Graph D shows a comparison between the age change in weight-indexed 6-S-OH-MLT excretion in
the path and AL groups. The individual animal measures and group regression lines are represented, respectively, as FR ( ᭿ and —), AL (ᮀ and
---), and path (᭢ and . . . ).
Regression curves of best fit (the unit of weight is g; age
is measured in days):
gies of the path group were kidney disease and pituitary tu-
mours.
˙
AL weight = 266 ln age – 0.453 age – 706 ( p < .001)
DISCUSSION
˙
FR weight = 10.4 sin age – 0.005 age + 310 ( p = .3) .
This study found that chronic FR did not change the lev-
els or the pattern of decline of total nocturnal melatonin se-
cretion observed through adulthood, as indicated by urinary
excretion of the primary melatonin metabolite, 6-S-OH-
MLT (Figure 1A). However, the FR animals exhibited a
substantially higher body-weight–indexed 6-S-OH-MLT
excretion compared with the AL control group throughout
adulthood (Figure 1B). Also the animals with gross pathol-
ogy, the path group, showed lower weight-indexed 6-S-OH-
MLT excretion than the controls (Figure 1D).
ANCOVA of the change in weight-indexed 6-S-OH-
MLT excretion with age in the AL group and in those ex-
cluded from the AL group due to pathology (path) found a
37% reduction in the latter (p ϭ .05), with age-adjusted
means of 0.896 and 1.319 pmol/g per night in the path and
AL groups, respectively. The group difference resulted both
from lower metabolite excretion and weight of the path ani-
mals, neither variable separately showing significant inter-
group difference. (Figure 1D shows regression lines fitting
50% of the data points.) The most common gross patholo-
The group differences in weight-indexed metabolite lev-

B24
MACGIBBON ET AL.
els may reflect unusually high melatonin levels in the FR
tissues and lower-than-usual melatonin concentrations in
those animals with gross pathological change. Investiga-
tions of the distribution of melatonin after its secretion by
pinealocytes indicate that, in rats and rabbits, immediately
following its synthesis melatonin is directly and readily se-
creted into the general circulation via the confluens sinuum
(25). Melatonin is a small lipophilic amine, which passes
easily in and out of most body tissues and, in normal ro-
dents, has a short half-life of approximately 20 minutes
(25). A study (2) comparing melatonin levels in the sys-
temic circulation, confluens sinuum, and pineal in rats of
different ages found that the pineal melatonin content per
unit of body weight correlated better than the nonweight-
indexed measure with serum melatonin in the systemic
circulation. Hence, in the current investigation, although
6-S-OH-MLT excretion may reflect pineal production of
melatonin, the serum levels and availability of the hormone
to body tissues, including the brain, may also be better indi-
cated by the weight-indexed measure.
The following studies suggest that the serum melatonin
peak of the age-retarded FR animal may be not only higher,
but also earlier and shorter-lived than normal. FR and youth
have each been associated with increased amplitudes in hor-
monal, temperature, and other circadian rhythms (26–28).
Human aging has been shown to result in a lower (5) and
later (4) serum melatonin peak. A rat study (1) using
3-hourly urine sampling has found that both 6-S-OH-MLT
excretion and body-weight–indexed 6-S-OH-MLT excre-
tion decline in amount and in amplitude with age. Investiga-
tions (16,21) similar to the current study found higher serum
melatonin in the FR animals, although the results may be
misleading due to aspects of the protocols used, as men-
tioned. The half-life of melatonin may be reduced in the FR
rat due to more efficient hepatic function. Two rat studies
have found the FR regimen retards age-related degeneration
of the liver (13,29).
Speculation regarding serum melatonin levels on the ba-
sis of the weight-indexed measure of metabolite is compli-
cated, however, particularly in the present study, because of
the differences both physiologically and in body composi-
tion reported in rats of different ages and under AL and FR
regimens (14,29–31). The lipophilic property of melatonin
would alter its distribution in lean and adipose tissues, and,
in the current study, the absolute and relative amounts of
these tissues may be expected to differ markedly in the dif-
ferent subjects (29–31). Although specific measures of body
composition were not made, gross examination revealed
much larger fat deposits in the abdominal cavity of the AL
compared with FR animals.
Accurate assessment of serum melatonin patterns obvi-
ously requires regular nocturnal blood collection. The ear-
lier studies (16,21) of the effects of FR and aging on serum
melatonin used blood that was collected at only one time of
night, when the animals were killed. The influences of light
and stress on pineal function (32) limit the use of usual
methods of repeated serum collection in rats. One such
method (33), using tail blood, with a reported low level of
trauma allows a minimum of 1-hour intervals between sam-
pling. This method would require substantial light, which
may inhibit melatonin secretion (32). To compare melatonin
rhythms in animals of different ages and food regimens,
sampling at shorter intervals may be necessary during peak
periods to cater to differing peak acrophase and duration.
A profile of metabolite excretion through the dark period
may reveal some differences due to diet regimen during ag-
ing and can be obtained using currently available tech-
niques. Urine sampling at smaller intervals than the 3 hours
of the study referred to above (1) has been made possible by
providing rats with a liquid diet that resulted in regular mic-
turition; this method enables hourly urine collection, using
an automated peristaltic pump to transfer the urine (34).
The present investigation attempted to minimize the ef-
fect of disease as a confounding factor. However, as the fol-
lowing reports indicate, the older AL animals of the study
may, with further examination, reveal histopathological
changes. A report by Everitt and colleagues (35) of the labo-
ratory rat populations at the University of Sydney found that
the SpF-derived Wistar rats used in the current study grew
more quickly, became much heavier, and demonstrated a
shorter life expectancy compared with conventional Wistar
rats, apparently as a result of multiple pathology. It was pro-
posed that these animals demonstrate accelerated aging.
Other studies also indicate that the amount and distribution
of the fat tissue in the AL animals may predispose them to
premature aging and the onset of disease (36,37). The pres-
ence of renal disease and pituitary tumors in the path group
concurs with the usual pattern of pathology associated with
aging in male Wistar rats (38). The arbitrary decision to re-
tain, in the AL group of the current investigation, those ani-
mals with relatively mild hydronephrosis where a large pro-
portion of the kidney was apparently unaffected was based
on a premise that the remaining tissue would adequately
perform renal function and so possibly not affect the mela-
tonin biology. The study of disease in Wistar rats (38) states
that all older male Wistar rats exhibit some degree of renal
disease, indicating that it may be impossible to exclude
completely the effect of disease processes in aging studies
using these animals.
Two features of the current study that may have created
unintended differences in the environmental conditions of
the two experimental groups were the noon feeding time of
the FR animals and the paired caging of some but not all
subjects. These features of the protocol do not, however, ap-
pear to prevent the potent age- and disease-retarding influ-
ences of the FR regimen as shown in previous rat studies
(15,21,39–41) using the same protocols. Some stress effect
of such caging may be reflected in a long-term study (40)
that compared single and paired caging in AL rats and
found that the former resulted in an increase in food intake,
body weight, and renal disease, although life expectancy
was unchanged. FR rats in the same study showed the usual
increase in life expectancy and reduced incidence of disease
compared with the AL groups. The following studies sug-
gest that the noon feeding of FR rats in the current study
would result in unusual feeding patterns, which could inten-
sify hypoglycamia observed with FR. Nocturnal, compared
with daytime, feeding of FR animals has been shown to re-
sult in a slower rate of consumption more comparable with
the grazing mode of the AL animals (28). One study (42)

MELATONIN IN FOOD-RESTRICTED AGING
B25
demonstrated nocturnal hypoglycamia in FR animals fed at
the end of the light phase, and low glucose availability to
the brain has been proposed as a cause of decline in melato-
nin secretion observed in a study (43) of fasting humans. An
automated feeding machine may enable nocturnal feeding
of the FR animals without exposing them to light.
In conclusion, the present study overcame the problem of
change in peak acrophase in animals of different ages and
diet regimens by measuring the total nocturnal excretion of
6-S-OH-MLT as an indicator of melatonin secretion. The
study also attempted to control for the complicating influ-
ence of disease. The similarity found between diet groups in
age change in 6-S-OH-MLT excretion through adulthood
may indicate that the age-retarding effects of the FR regi-
men are not modulated or reflected by youthful total noctur-
nal melatonin secretion, although higher tissue concentra-
tions of melatonin in the FR groups are suggested by the
marked group differences in the weight-indexed metabolite
excretion. Because many factors complicate the relationship
of weight-indexed metabolite to serum levels of melatonin,
direct sampling of serum melatonin is necessary to establish
the influence of FR on melatonin availability to tissues. The
difficulties of making the required regular sampling of
blood from small nocturnal mammals may preclude the use
of a rat model in such studies.
Various explanations may be given for the current find-
ings. Although a difference in 6-S-OH-MLT excretion due
to diet regimen was not found, a slowing of pineal aging
may have occurred in the FR animals, as postulated in the
following scenario. Melatonin may be secreted more rapidly
by more responsive or more efficient pinealocytes in the rel-
atively youthful FR animal, with the tissue concentrations
reaching an unusually high peak due to enhanced pineal
function and reduced tissue mass. There are indications that
the FR regimen may maintain youthful upregulating ability
in pinealocytes (9,44), although a Wistar rat study suggests
that an age-related decline in ability to synthesize melatonin
is a more likely cause of reduced secretion with age (45). If
the serum melatonin in the aging FR animals is exception-
ally high it may reach a threshold not attained with normal
aging and at which a negative feedback influence, less ac-
tive in the younger animal, is induced. Feedback control is
found in all other endocrine systems (46), and an inhibitory
effect of melatonin appears to be demonstrated in a study
(47) showing that certain tissues, which do not usually se-
crete melatonin, produce measurable amounts of the hor-
mone following pinealectomy. Alternatively, possible noc-
turnal hypoglycemia in the FR group as discussed above
may, without preventing certain age-retarding and life-
extending influences of the regimen, compromise mainte-
nance of the youthful pineal function that would otherwise
occur.
Correlations between disease and reduced melatonin lev-
els (18–20) may indicate that impaired health status in some
of the current AL group masks a higher level of metabolite
excretion usual in AL animals. A higher absolute, but not
weight-indexed, secretion of melatonin in normal AL rats
compared with those undergoing food restriction was found
in a short-term study (48) in which 3 weeks of 50% food re-
striction in male rats resulted in a decrease in pineal content
of melatonin but an increase in melatonin serum levels. In
this study also, low glucose levels may have impaired pineal
function in the food-restricted group.
Monitoring of blood glucose in studies of FR and melato-
nin and investigation of the suppressive effects of melatonin
on the pineal and other tissues are indicated. Future studies
could also explore further the effects of the FR regimen and
other temperature-lowering environmental influences on pi-
nealocyte function, particularly with regard to adrenergic
stimuli and the responsiveness of target cells to melatonin, as
discussed in an earlier paper (9). Important mammalian dif-
ferences pertinent to such studies include the reported lack of
a blood–brain barrier surrounding the pineal gland in the rat,
but not the human (49), and the differing adrenergic receptors
responsible for regulating pineal melatonin secretion (32).
Also, in some species, there is evidence that the melatonin
concentrations in the third ventricle are substantially higher
than in the plasma, raising the possibility of two physiologi-
cally significant compartments of melatonin (50).
Acknowledgments
We thank CERA, Concord Hospital, for the use of the animals and facil-
ities; Alison Ryan for care of the animals; Dr. Francis Seow for technical
advice and support; and Dr. Malcolm France of the Veterinary Pathology
Department, University of Sydney, for advice on rat pathology.
Address correspondence to Dr. Mary F. MacGibbon, MacKillop Cam-
pus, Australian Catholic University, P.O. Box 968, North Sydney, NSW,
Australia, 2059. E-mail: m.macgibbon@mackillop.acu.edu.au
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Received February 7, 2000
Accepted June 27, 2000
Decision Editor: John A. Faulkner, PhD