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An Age-Related Decline in Melatonin Secretion Is Not Altered by Food Restriction

^a Department of Immunology, Concord Hospital, University of Sydney, New South Wales, 2139, Australia
^b Centre for Education and Research on Aging (CERA), Concord Hospital, University of Sydney, New South Wales, 2139, Australia

Mary F. MacGibbon, MacKillop Campus, Australian Catholic University, P.O. Box 968, North Sydney, NSW, Australia, 2059 E-mail: m.macgibbon{at}mackillop.acu.edu.au.

Decision Editor: John A. Faulkner, PhD

	Abstract

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Melatonin has been found to exhibit youth-maintaining and disease-preventing properties. The current study examined whether the age-retarding regimen of chronic food restriction (FR) slowed the decline in melatonin secretion 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 adulthood (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 approximately double those of the AL ( p = .06). The possibility that this result may reflect unusually high melatonin peaks in the FR tissues is discussed.

THE serum levels and total nocturnal secretion of melatonin in normal rats and humans have been found to decline with age ^{(1)(2)(3)(4)(5)(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)(14)(15), maintains youthful total nocturnal melatonin secretion throughout adulthood. 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 specific 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 investigations have associated disease with alteration in melatonin measures ^(18)(19)(20). An aging study ⁽²¹⁾ similar to that discussed above ⁽¹⁶⁾ also reported higher serum melatonin due to the FR regimen, but only one age was assessed, measurements were made at only one time during the night, and no editing of data in relation to pathology was reported. The current investigation created a separate category (path) for animals both fed AL and exhibiting gross pathological change.

Radioimmunoassay (RIA) of total nocturnal urinary excretion of the primary melatonin metabolite, 6 sulphatoxymelatonin (6-S-OH-MLT), was employed as a noninvasive and accurate of method of assessing melatonin secretion ^(22)(23).

	Methods

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Animals
Male outbred Wistar albino rats were purchased at 4 weeks of age from the specific pathogen-free (SpF) Combined 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 protocol was less rigorous in pathogen control than that for SpF conditions. However, there has been no indication of infectious disease outbreak in the animals during or since the study period. Sentinel Wistar AL rats, living in the same environment as those of the current study, have been tested throughout this period for Mycoplasma pulmonis, cilia-associated respiratory bacillus, Theiler murine encephalomyelitis virus, Sendai virus, and Tyzerr's (Clostridium piliformis). All serology tests, performed using enzyme-linked immunosorbent assay (ELISA), were negative. General postmortems, including histopathological examinations, of three outbred 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 ± 1°C), 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.

Criteria for Exclusion Due to Pathology
The data of any animals found before or after death to exhibit 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 premortem changes resulting in exclusion of data from the normal groups included apparent lethargy, substantial weight loss, oliguria, polyuria, and diarrhea. The postmortem examination 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, pituitary, and neoplastic disorders to which such rats are prone. The only obvious pathological change that did not result in exclusion of an animal from the healthy groupings was hydronephrosis if two thirds of the renal tissue remained and appeared healthy.

Diet
All animals were fed normal pelleted rat chow containing 22% protein, 3.3% fat, and 3.6% fiber, which was purchased 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.

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 between the first and second measures of urine volume and 6-S-OH-MLT, at p < .001, were 0.81 and 0.46, respectively.) The urine excreted by each animal between 5 PM and 8 AM was collected into a parafilm-covered beaker containing 5 mg boric acid. The volume was recorded and the urine filtered through muslin. Samples were stored in vials at –20°C until sent, in containers of dry ice, interstate for assay. Assessment at two ages was made on 12 of the 50 AL animals and 9 of the 14 FR animals, with the intervening periods averaging 100 days in both groups. All other animals, including those in the path category, were assessed only once.

Assay
The 6-S-OH-MLT concentration of each sample was determined by RIA using a technique ⁽²⁴⁾ involving competition 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.

Data Analysis
General factorial analyses of covariance (ANCOVA) were made using STATA (Stata Corp, College Station, TX) software that enabled the clustering of data and the use of robust standard errors to cater to the double set of measurements made on some animals. Regression analyses and correlations were made using SPSS-6 (SPSS Inc, Chicago, IL) software.

	Results

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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 significant linear decline in total nocturnal 6-S-OH-MLT excretion was found through adulthood in both the AL and FR groups (Fig. 1). The AL group showed an average decline 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 = .9), nor in the change in this difference with age ( p = .8).

View larger version (29K): [in this window] [in a new window]   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 ... ).

Fig. 1 shows the total nocturnal 6-S-OH-MLT excretion per unit of body weight (6-S-OH-MLT/bw) of the two groups. As expected, given the age-associated weight changes (Fig. 1), both groups exhibited a significant decline with age. An initially rapid then slower decline was seen in the AL group, with an exponential best-fitting regression curve; although, as indicated below, a linear decline 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 metabolite excretion/body weight (p = .06) and that this difference did not change significantly through the ages studied (p = 1.0). Age-adjusted means were 1.232 pmol/g and 2.383 pmol/g in the AL and FR groups, respectively.

Regression curves of best fit (the unit of 6-S-OH-MLT/bw is pmol/g; age is measured in days):

Fig. 1 shows the AL linear regression:

Body weight change with age in the AL group was logarithmic, 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 significant regression to be found. A significant intergroup weight difference occurred (p < .0001). The age-adjusted means were 643.8 g and 300.5 g for the AL and FR groups, respectively. This difference changed with time. (Fig. 1 shows regression lines that fit 50% of the points.)

Regression curves of best fit (the unit of weight is g; age is measured in days):

ANCOVA of the change in weight-indexed 6-S-OH-MLT excretion with age in the AL group and in those excluded from the AL group due to pathology (path) found a 37% reduction in the latter (p = .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 animals, neither variable separately showing significant intergroup difference. (Fig. 1 shows regression lines fitting 50% of the data points.) The most common gross pathologies of the path group were kidney disease and pituitary tumours.

	Discussion

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This study found that chronic FR did not change the levels or the pattern of decline of total nocturnal melatonin secretion observed through adulthood, as indicated by urinary excretion of the primary melatonin metabolite, 6-S-OH-MLT (Fig. 1). However, the FR animals exhibited a substantially higher body-weight–indexed 6-S-OH-MLT excretion compared with the AL control group throughout adulthood (Fig. 1). Also the animals with gross pathology, the path group, showed lower weight-indexed 6-S-OH-MLT excretion than the controls (Fig. 1).

The group differences in weight-indexed metabolite levels may reflect unusually high melatonin levels in the FR tissues and lower-than-usual melatonin concentrations in those animals with gross pathological change. Investigations 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 secreted 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 rodents, has a short half-life of approximately 20 minutes ⁽²⁵⁾. A study ⁽²⁾ comparing melatonin levels in the systemic 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 indicated 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 hormonal, temperature, and other circadian rhythms ^(26)(27)(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 excretion decline in amount and in amplitude with age. Investigations ^(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 mentioned. 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 basis of the weight-indexed measure of metabolite is complicated, however, particularly in the present study, because of the differences both physiologically and in body composition reported in rats of different ages and under AL and FR regimens ^{(14)(29)(30)(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 different subjects ^(29)(30)(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 obviously requires regular nocturnal blood collection. The earlier 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 sampling. 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 aging and can be obtained using currently available techniques. 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 micturition; this method enables hourly urine collection, using an automated peristaltic pump to transfer the urine ⁽³⁴⁾.

The present investigation attempted to minimize the effect of disease as a confounding factor. However, as the following 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 laboratory 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 proposed 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 presence 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 retain, in the AL group of the current investigation, those animals with relatively mild hydronephrosis where a large proportion 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 melatonin 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, appear to prevent the potent age- and disease-retarding influences of the FR regimen as shown in previous rat studies ^{(15)(21)(39)(40)(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 suggest that the noon feeding of FR rats in the current study would result in unusual feeding patterns, which could intensify hypoglycamia observed with FR. Nocturnal, compared with daytime, feeding of FR animals has been shown to result in a slower rate of consumption more comparable with the grazing mode of the AL animals ⁽²⁸⁾. One study ⁽⁴²⁾ 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 melatonin 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.

Various explanations may be given for the current findings. 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 relatively 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 exceptionally high it may reach a threshold not attained with normal aging and at which a negative feedback influence, less active 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 secrete melatonin, produce measurable amounts of the hormone following pinealectomy. Alternatively, possible nocturnal hypoglycemia in the FR group as discussed above may, without preventing certain age-retarding and life-extending influences of the regimen, compromise maintenance of the youthful pineal function that would otherwise occur.

Correlations between disease and reduced melatonin levels ^(18)(19)(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 restriction 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 melatonin 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 pinealocyte function, particularly with regard to adrenergic stimuli and the responsiveness of target cells to melatonin, as discussed in an earlier paper ⁽⁹⁾. Important mammalian differences 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 physiologically significant compartments of melatonin ⁽⁵⁰⁾.

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 influence 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 regimen are not modulated or reflected by youthful total nocturnal melatonin secretion, although higher tissue concentrations 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.

	Acknowledgments

 
We thank CERA, Concord Hospital, for the use of the animals and facilities; 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.

Received February 7, 2000

Accepted June 27, 2000

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