!:or r :.CT 0: FOOD RESTRICTION

ON

SERUM .~rID PINE.IJ. INDOLES

By

COlISTAHCE LU-YEE CHIK, B.SC., M.D •

.~ Thesis

Submitted to the School of Graduate Studies

in Partial Fulfilment of the Requirements

for the Degree

Doctor of Philosophy

McMaster University

March, 1986 <S)

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FOOD RESTRICTION ON SERU11 AND PINEAL lllDOLES

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OOC70R OF PHILOSOPh""! (1985)

(MEDICAL SCIENCES)

:lcl-\ASTER UllIVERSITY

Hamilton, Ontario

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TITLE: Effect of food restriction on serum and ?ineal indoles

AUT.~OR: Constance Lai-Yee Chik, B.Sc. (University of Toronto)

n.D. (University of Toronto)

SUPERVISOR: Professor G.n. Bro.;n

lru:1SER of ?AGES: ·x, 134

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ABSTRACT

Food restriction has profound effects on various endocrine

axes and on a.lline metabolism. In the present study, the effect of

'reduced food availability on pineal and serum indole was determined

in adult male \.listar rats. Under a lighting regimen of'14 h li&~t

and 10 h dark, 3 weeks of 50~ food restrictio~ to a reduction in"~

24 h ~€an seru~ ~ryptophan and serum s€rotonin levels but an increase

in seru~ ~latonin levels. The duration of ~ ni&~;-timemelatonin" "

-rise ..-as increased secondary to an earlier rise of both pineal and

sen..," melatonin. Such changes in circulating melatonin rr.ay account

for the gonadal" regression observed in und~rfed ani~ls. This

pineal-gonadal interaction was further investigated after animals

were subjected to shortened photoperiod or after plnealectomy.

Shortened photoperiod failed to influence either the serum melatonin"~

profile or the undernutrition-related gonadal regression.

Pinealectorny, however, was able to reverse though incompletely -the

gonadal regression in underfed animals. When the pinea~'

responsiveness to beta-adrenergic stimulation was determined in food

restricted animals, both the time course and the dose responses were

altered. The changes in pineal and serum melatonin

post-stimulation, however, were atypical of either a sub- or

supersensitive pineal gland.

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Basec on the present study, food availability proves to be

another factor that can influence pineal activity. Its effect on the. '- ..

pineal, h9lever, depends on the duration of food restriction and the

~ enVironmental light/dark cycle.

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ACKNOWLElXiEMENTS

I thank my family for their .constant support and,.

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encouragement. The expert technical assistance given by my husband

Tony and my sister Florence is greatly appreciated.

The research work presented in this thesis is made possible

by a felloWship from the Medical Research Council of canada. I am

indebted to my supervisor, Dr. G.M. Brown for his guidance throughout

I also thank other members of the supervisory

E. Werstiuk, Dr. L.J, Grota, Dr. J. Laidlaw'and Dr.

L.P. Niles for their helpful advice. The assistance so freely given

by Mrs. G.E.K.· Johansson, Mr. M.G. Joshi and Ms. L. Campbell, work

amVotherwise, is greatly appreciated. I am also most grateful to

M'~ A. Murray for her patience and excellent secretarial skills.

Thanks are also extended to the other members of the

examining committee, Dr. G. Evans, Dr. B.J. Galef, Dr. W.J. Leigh and

especially to Dr. H.J. Lynch for acting as the external examiner.

TABLE OF CONTEllTS

ABSTR.ltCTACKNOWLEDG2-!ENTS

. TABLE OF COtlTENTSLIST OF TABLES .LIST OF FIGURES

Cf'.APTER OllEINTRODUCTIOU

1.1 Perspectives of the present study1.2 The pineal gland and melatonin1.3 Biochemistry of melatonin synthesis

'1.4 Regulation of melatonin synthesis1.5 Factors that influence ~neal N-acetyltransferase

rhyt!'.'ll1.6 Horoonal regulation of melatonin synthesis1.7 ~~thods in studying pineal function1.8 Pineal effects on the reproductive system1.9 Mechanism of action of melatonin1.10 Potentiating factors

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C:iAPTER THO•

E.F t ECT OF FOOD RESTRICTIOIl ON THE 24 H RHYTHM OF SERtJ:·'AND PINE.4.L HELATONI:1

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AbstractIntroductionMaterial and methodsResultsDiscussion

3031323543

CHAPTER THREEEFFECT OF SHORTEIlED PHOTOPERIOD .~ PINEALECTOMY ONUNDERFED RATS

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3.13.23·33.43.5

AbstractIntroductionMaterial and methodsResultsDiscussion

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5052535564

CH.OJ>TER :-OURALTERED PnlEAL BETA-ADRENERGIC RESPONSIVENESS T0ISOPROTERENOL

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4'.14.2·4.34.44.5

AbstractIntroductionMaterial and methodsResultsDiscussion

6869707281

CHAPTER FIVEcl'r ECT OF FOOD RESTRICTION ON SERUH TRYPTOPftl\H ANDSEROTONIH

5.1 Abstract 875.2 Introduction 885.3 Material and methods 885.4 Results 925.5 Discussion 95

CHAPTER SIX .GEHER.l\L DISCUSSION P'> 100 '.

. REFEREtlCES 11 1

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LIST OF TABLES

Table 1

.Table 2

Table 3

Table 4

Table 5

Body weights after 1 and 3 weeks of 50% foodrestriction

Effect of ·3 weeks 50% food restriction onorgan weights under a lighting regimen of 4 hli&~t and 20 h dark

Effect of 3 weeks 50% food restriction on serum .Lq and serum testosterone levels under a }ightingregimen of 4 h light and 20 h dark

Effect of pinealectomy on ~rum LH and senL~

testosterone levels after 3 weeks of 50% foodrestriction

Effect of 4 weeks 50% food restriction onorgan weights under a lighting regimen of 14 hlight and 10 h dark

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CLIST OF FIGURES

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

, Figure 8

.Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

The biosynthetic pathway of melatonin

"Metabolism of melatonin in the liver

Pineal biochemical circadian rhythms and theirrelationship to environmental lighting

A schematic representation of the retinal-pinealaxis

Effect of 1 and 3 weeks 50% food restriction onabsolute and relative testicular weight, ventralprostate wei&~t and seminal" vesicle weight

Effect of 1 and 3"weeks 50% food restrictionon serum Lq and serum testosterone levels

Effect of 1 week 50% food restriction on 24 hserum and pineal melatonin

Effect of 3 weeks 50% food restriction on ser~~

and pineal melatonin (Preliminary study)

Effect of 3 weeks 50% food restriction on 24 h ~

serum and pineal melatonin (Extended study)

Effect of 3 weeks 50% food restriction on serumand pineal melatonin under a lighting regimen of4 h light and 20 h dark

Effec~ of pinealectomy on absolute and relativeseminal vesicle weight after 3 weeks of 50% foodrestriction

Effect of pinealectomy on absolute and relativetesticular weight after 3 weeks of 50% foodrestriction

Effect of pinealectomy on absolute and relativeventral prostate weight after 3 weeks of 50% foodrestriction

Time course response of pineal N-acetyltransferaseto 0.5 mg/Kg isoproterenol

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Figure 15 Time course response of pineal melatonin to 0.5mg/Kg isoproterenol 76

------ Figure 16 Time course response of serum 'melatonin to 0.5mg/Kg isoproterenol 77

Figure 17 Dose response of pineal N-acetyltransferase 2 hpost isoproterenol stimulation 78

Figure 18 Dose response of pineal melatonin ~ h postisoproterenol stimulation 79

"Figure 19 Dose response of serum melatonin 2 h postisoproterenol stimulation 80

Figure 20 Chromatogram of a serum sample 91 ."'--:-:'

Figure 21 Effect of 3 weeks 50% food restriction onserum tryptophan Q"

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iFigure 22 Effect of 3 weeks 50% food re~trict ion on )

se!"Um serotonin 94

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CRAPER ONE

IilTRODUCTION

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1.1 Persoectives of the oresent study

. Food availability has profound infJ!'.1ences on many metabolic

processes. - The response of the endocrine system to diet"ary

restriction is not uniform. Any step of hormone action can be

affected. Indeed, changes in hormone synthesis, degradation or

tissue responsiveness have all been described (Becker, 1983).

Dependent on the particular hormone axis in question, it may be

suppressed, remain unchanged or be hyperactive. Suc..1. diversified

responses are ~hougt1t to represent adaptive mechanisms. One axis

that is suppressed during dietary restriction is the

hypothalamic-p it.'.1i tary-gonadal axis (Howland, 1975). The suppression

is believed to be at the hypothalamic level since the pituitary gland

retains its ability to respond to gonadotropin-releasing hor~one

(Campbell et aI, 1977). However, this may be simplistic since the

regulation of the r-eproductive axis is complex. One organ that. is

affected b~ food restri~ that can influence the reproductive

axis is the pineal gland. The regulating role of the pineal on the

reproductive axis is linked to >jhotoperiodism (Goldman and Darrow,

1983) • In view of this connection, it is of interest to investigate

the effect of food restriction on the pineal gland and its interaction

with the gonadal axis under different photoperiods.

In seasonal breeding species such as hamsters and sheep, the

importance of the pineal gland on the reproductive axis has been well

defined (Reiter, 1980; Lincoln and Short, 1980). By contr;3st, the

rat reproductive axis

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I •is relati vely unrespons 1 ve to the inhibitory

action of the pineal and its hormone melatonin err). This axis,

however, can be sensitized t~ the inhibitory action of the pineal by

manipulations such as underfeeding, olfactory bulbectomy or neonatal

steroid administration (Reiter, 1974). The focus of many previous

studies has been on changes in sensitivity to the action of the pineal

or ~IT in food restricted states. Few studies have determined the

-effect of food restriction on the activity of the pineal gland. When

adult rats are subjected to chronic food restriction, pineal activity

'increased as determined by oxygen consumption and morphologic criteria

(Walker et aI, 1978). ~nen MT is determined, short term starvation

has no effect on urinary ,IT excretion (Lynch et aI, 1975). On the

other. hand, when prepubertal rats are subjected to 5 weeks of orotein. .calorie malnutrition, determination of pineal MT content reveals lower

daytime and night-time levels (Herbert and Reiter, 1981). Even

though ~ has been accepted as the pineal hormone, the effect of food

restriction on circulating MT levels has never been determined. The

present study, therefore, investigated the effect of varying duration

of dietary restriction on the circadian rhyth.'1l of circulating MT."

Changes in pineal activity were correlated with changes in gonadal

parameters. This pineal-gonadal interaction was further investigated

by subjecting the animals to shortened photoperiod and pinealectomy.

Food restriction also has a profound influence on

neurotransmitters' which are key regulators of many hormonal axes.

For instance, many aspects of the adrenergic systems a~e influenced by

food availability. Changes in norepinephrine turnover, adrenoceptor

density and tissue responsiveness have all been described (Katovich

and Barney, 1983; Landsberg and Young, 1978; Stone, 1983).

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Since

pineal activity is inti:nately li:1ked to sympathetic activity (Zatz,,

1978), the effect of dietary restriction on pineal responsiveness to a

beta-adrenergic agonist was determined.

Food availability also leads to changes in indole metabolism.

Animals subjected to acute food deprivation have increased synthesis

and turnover of cerebral serotonin (Kantak, 1977, 1978a, 1978b,

1978c). The effect of chronic· food restriction, however, -lias not

been defined. In the last section of the present study, the effect

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of food' restriction on two cirCUlating indoles, tryptophan and

serotonin, was determined.

Since the major part of the present study is on the effect of•

food restriction on pineal-gonadal interaction, the subsequent section·

is an overview of aspects of pineal physiology.

1.2 The pineal gland and melatonin

The history of the pineal gland dates back to 325-280 Be when

Herophilos of Alexandria suggested that the pineal might function as a

valve controlling the "stream of thoughts" from the lateral ventricle

of the brain (reviewed by Kappers, 1965; Kitay and Altschute, 1954).

By contrast, Galen of Pergamon (130-200 AD) believed that the pineal

organ was merely a lymph gland. In the 17th century, the renowned

French philosopher Rene Descartes designated the pineal as the "seat

of the soul". He also suggested that the pineal receives photic

information from the eyes and thereby exercises an influence on the

body which proves to be prophetic. The first endocrine effect of the

pineal was described in 1898 when Heubner reported the association of,

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precocious puberty with a pinealoma in a four year old boy. This was

followed by Mar~rg's hypothesis that the pineal secretes a substance

that regulates the onset of pUberty. This is of particular interest

to the present study since dietary restriction can delay the onset of

puberty. In 1918, Holmgren observed similarities between the sensory

type cells in the pineal region and the cone cells of the retina in

a~hibia and fish. Taken together with the observation that

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calcification of the human pineal occurs with advancing age, this

evidence led to the hypothesis that the mammalian pineal is just a

vestigial remnant left behind by evolutionary progress. Of interest,

in 1917, McCord and Allen found that the bovine pineal glands produced

a substance that li&~tened the skin of frogs. The responsible

compound was eventually isolated and identified a'S

N-acetyl-5-;nethoxytryptamine by Lerner et al (1958, 1959) • It was

given the name "melatonin c;·iTl" because of its indole nature and its

ability to li&~ten pigment cells.•

Today, the pineal gland is recognized as an actively

functioning neuroendocrine organ that responds primarily to photic

stinuli. It exhibits circadian rhythms and influences the metabolic

activity of a host of endocrine glands. The possible mediator is the

pineal hormone melatonin.

1.3 Biochemistry of melatonin synthesis

By the use of enzyme assays (Heissbach et al, 1960; Axelrod

and Weissbach, 1960) and pineal cell culture (Shein et al, 1967; Klein

et al, 1970), the biosynthetic pathway of MT (Fig. 1) and its

regulation in the pineal has been established.

, -Fi~u;-e 1 The biosynthetic path'lay of melatonin

~C~.C"'''''.'COO''

~N)J_

TRYPTOPIolA""

~ T ....OIOO"'- ....d.o...'n~

HO~CIol.ClollN""lcoo"

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tH0f!rT...... ,C.........,~!'O/

15·", d·a • .,t· .. o, ..""·".'

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?i~ealocytes possess all ~~e enzymes that a~e ~equi~ed fo~ MT

synthesis. The indole amino acid t~yptopha."l is the COllI!lon precursor

of the pineal a."ld brain i~dolea'llines. lJ;:ltake of tr"yptophan from the

blood st~ean by pinealocytes is follow-ed by h:Yd~xylation at the 5-

pcsi tion by tryptopha."l hydroqlase to 5-hydroxytryptophan (5HT?)

(Lovenbe~g et al. 1967). The 5HT? thus formed is deca~boxylated to

5-hydroxytryptamine (se~otonin• ':XiT) by a~omatic-L-arnino-acid

deca~boxylase. Compa~ed to othe~ b~ai~ a~eas. the pineal has one of

the highest concentrations and turnover ~ates fo~ 5HT (Falck et· al.

1966). Se~otonin in the pineal has a complex fate: (a) OXidative

dea'llination by monoamine oxidase to 5-hyd~oxyindoleacetic acid o~

5-hydroxyt~yptophol (Hakanson a."ld 0.ma~. 1965.1966); (b) ~elease to

the ext~acellul~ space and uptake by sympathetic ne~ve terminals

(Owman. 1965; Hakanson and Owman. 1966); o~ (c) N-acetylation to

N-acetylse~otonin (NAS) by serotonin :'-acetylt~ansfe~ase (NATase) with

acetylcoenzyme-A se~ving as the acetyl d:mo~ (Io.eissbach et al. 1960).

NAS is then O-methylated by hyd~cxyindole-D-methyl t~ansfe~ase (HIO:-rr)

to form MT with S-adenosylmethionine p~oviding the methyl group (Shein

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et al. 1967). In addition to ~lAS. HIO:-rr can also D-,methylate, ,5-hyd~oxyindoleacetic acid and 5-hyd~oxytryptopho~to fo~m

5-methoxyindoleacetic acid and ·5-methoxyt~yptophol ~espectively. beth

of which have been identified in the pineal (Lerne~ et al. 1960;

McIssac .et al, 1965). Al though NATase is widely dist~ibuted in

various tissues. in the pineal it is the key enzyme in the control of

the ci~cadian rhythn of MT synthesis (Ellison et" al, 1970; Deguchi,

1975) • In cont~ast to the widesp~ead distribution of NATase, HIO:-rr

is almost completely localized in the pL"leal gland.

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Outside of the

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pineal, HI01Thas only been identified in the retina and the harderian

gland CCardinali and Wurtman, 1972) • Using immunohistoc~emical

•techniques, HT has been localized in the same tissues, retina and

harderian glands as well as the intestine (Bubenik et aI, 1977, 1978).

Nevertheless, the extrapineal contribution to the circulating pool of

MT is· small since pinealectomy in rats result in undetectable MT

levels by gas chromatography mass spectrometry CLewy et aI, 1980). MT

synthesized in the pineal appears to be secreted into the blood stream

by sL~le diffusion.· \ihether i·IT is primarily secreted into the bleed

stream or the cerebrospinal fluid remains controversial. In the rat,

the blood compartment is likely the pri~3ry site of secretion since MT

concentration in the plasma from the confluence sinuur.l· is about 8

times higher than that of trunk blood CWithyachu~narnkul and Knigge,

1980). In any case, HT crosses the blood brain barrier with ease

CAnton-Tay and Wurtman, 1969).

The ~3jor metabolic pathway of circulating ~IT is conversion to

6-hYd~xymelatonin in the liver by microsomal enzymes CKopin et aI,"-

1960) (Fig. 2). This is followed by conjugation with sulphate or

glucuronic acid and excretion mainly in the urine CKveder and ~cIsaac,

1961). Another route of metabolism is via a brain enzyme,

indolea~ine 2,3 dioxygenase, which cleaves the pyrrole ring of various

indoleamines (Fujiwara et aI, 1978). The plasma half life of HT has

been estimated to be between 15-20 minutes in rats CGibbs and Vriend,

1983). Possible~hanges in the production rate or metabolic clearance

of MT in food restricted animals have never been determined.

Fi5u~e· 2 :·iet.abolis:n of :nelator:ir: i:1 t.he liver

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Liver enzyme NAOPH andOxygen

R = Sulphate andGlucuronide

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