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Effect of chronic caloric restriction on physiological variables related to energy metabolism in the male Fischer 344 rat
(1989) 117--133
J C R E S T R I C T I O N ON P H Y S I ( ERGY METABOLISM IN THE
FEUERS, J U L I A N A. L E A K E D. N A K A M U R A , A N G E L O T U R T U R R O and R RO? ONALD W ent o f Health and H u m a n Services, Public Health Service, F o o d and at, Drug A d, Center f o r Toxicological Research, Jefferson, Arkansas 72079 (U.S.A.) (U. J September 23rd, 1988)
tRY
behavioral m~ le present study, a number o f physiological and beha are to metabolism were continuously monitored in 19-month-ol 19-~ her :s that were fed ad libitum or fed a caloric restricted cted diet. Ca ted ,' fewer meals but consumed more food during each m meal and s me per meal than did rats fed ad iibitum. Therefore, the I, l l ~ , , timing I.lllllll~ a. I I U I d U I t l L I U I ! t of Q UI Is well as the total number o f calories consumed ma'. ly be associated with life on. Avera Average body temperature per day was signifiantl y lower in restricted rats extension. :ly temperature range per day and m o t o r activity but bod, :y were higher in restricted rats. Dramatic .tic changes in respiratory quotient, indicating rapid rapi changes in metabolic pathway and lower temperature, occurred in caloric restricted rats when carboh'ydrate reserves were depleted. Lower body temperatt aerature and metabolism during this time interval m a y result in less D N A damage, thereb thereby increasing the survival Lal o f restricted rats. Nighttime feeding was found to synchronize physiologipotential n d libitum l ibitum i h l t t n m and •nrl ,'.aln.;,', raetr;r.~oA rats , cal p e r f•enrman,'.,a o r m a n c e k~atu,~=~n between ad caloric restricted better than daytime feeding, thereby allowing investig~ stigators to distinguish the effects of caloric restriction f r o m those related solely to the; time-o time-of-day e-of-da, o f feeding.
Key words: Caloric; Restrictionn; Physiological; Metabolism; Male; Rat INTRODUCTION Caloric restriction is the onl' dy known experimental paradigm that uniformly and proportionately increases maxamum ~imum achievable life span (MAL) o f placental m a m Address all correspondence to: Peter H. Duffy, HFT-I, Building 62, National Center for Toxicological Research, Jefferson, Arkansas 72079, U.S.A.
Printed and Published in Ireland
he rate of the occurrence of most ve demonstrated this phenomenoJ and continued throughout life [ le onset of adulthood [6--8]. Cal, ve in reducing the incidence and ally induced tumors in rats and mi by which caloric restriction incre e are not clearly understood, seve e been postulated to explain the, t4] and Sacher [1] proposed that the longevity of a sp~ )ecies is in, restri o the metabolic rate per gram body mass and that caloric cah )y reducing the metabolic rate. However, McCarter et al. [15 tabolic rate per kilogram lean body mass was not no1 signific this n ad libitum fed and caloric restricted animals. To resolve re,. ph,. red qualitative as well as quantitative changes in several se if cal )ral variables that are related to metabolism to determine detern he homeostatic regulation o f these variables.
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[ALS AND M E T H O D S
t husbandry and feeding regimens male Fischer 344 rats used in this study were raised at the National Center for 10XlCOlO logical Research (NCTR) in a specific pathogen fre, free (SPF) environment at
23°C, and were entrained to a 12/12, light/dark cycle with lights on from 0600 to 1800 h daily. This regimen was maintained from the time the tl animals were weaned until the' ~ey were entered into the experiment. The test animals anin~ were divided into a restric group that received control• group that was fed ad libitum, and a caloric restricted 60070 off the ad libitum consumption starting at 14 weeks of age and continuing for the duration ation of the experiment. A d libitum rats were given more rr food than they conat all times. All rats were sumed in a daily interval so that food was available to them fed a standard NIH-31 diet at 1100 h daily and were housed singly in plastic cages with metal tops. The experimental ntal protocol was initiated when the test animals were ae the subjects were transferred from the SPF barrier 17 months of age, at which time to the testing area, where they were maintained under similar environmental conditions.
Physiological and behavioral testin esting procedures The equipment and testing procedures used in this study were similar to those used previously [16]. Briefly, food and water consumption, as well as feeding and tinuously )usl monitored with a system that measured the total motor activity, were continuous force applied to a load cell. Drinking activity was monitored by a digital pulsecounting device triggered whenn the rat made contact with the drinking tube. Deep ed by a thermistor-transmitter capsule that was surgibody temperature was measured eal cavity of the rat 60 days before the animals were cally implanted in the peritoneal
~nt an dioxide production, and respir es=ircuit flow system equipped with~ :ed ~ressure regulating device. Anima ral metabolism cages and sample flo ~ dyplied Electrochemistry model S3A del ared carbon dioxide analyzer. An of ~ed to provide process control and as data collection and analysis. of ten caloric restricted animals 18ior "e placed individually in test cages (11 in. × 7 in. X 7.5 7 in.) fo of nitiation o f the experiment. Both test groups were fed f~ 5 h aft environment~ e re it phase of the photoperiod cycle and all other envi ~l to those described previously. Data were collected for all p~ :32h ervals over a I 0-day testing period. The photoperiod was then va$ ," animals were allowed to phase shift for a 30-day rperiod so iod ed to restricted rats 5 h after the onset of the dark p]~hase of tt his Food consumption in the ad libitum group was at its it~ maximt day. All physiological and behavioral variables except exc oxyg ~on ; metabolism and RQ were subsequently measured continuou: c~ her period. This additional procedure was utilized to sym ,nchronize t egthat control and restricted rats were eating at the sOsame ~ L l X l ~ , time LX|aI~ VI ~,t~ff. ~ J O l ling t~ light-fed and dark9cedure, the same set of restricted rats were tested under u~ fed conditions. Conventional ventional statistical methods (means and standard errors; Student's t-test) and the.~cosinor statistical method [17] were used to a n a l y,ze z e the data. Using the least and the confidence limits squares method, the values for acrophase and amplitude a~ for these se functions were calculated for individual animals q(single cosinor) and test groups (population-mean cosinor) [17]. RESULTS Physiological and behavioral al data for ad libitum rats, restricted rats fed during the light period (LF), and restricted :ricted rats fed during the dark period (DF) are compared in Table I. Significant differen~ tifferences ences are observed in several of the experimental variables analyzed, including: (1) Food consumption and the total feeding time per day were higher in ad libitum animals than in LF and DF restricted animals, but water consumption and the total tal time spent drinking per day were not significantly different a m o n g the three groups; :~ups; (2) the average food consumption per feeding episode and water consumption ~n per gram of lean body mass were higher in LF and DF restricted rats than in ad libitum rats, but food consumption per gram of lean body mass and the average water ter consumption per drinking episode were not significantly different; (3) the number er o f feeding episodes per day was 3.5 times greater in ad libitum rats than in LF restricted stricted rats and 2.0 times greater than DF restricted rats, but the number of drinkin king episodes was not significantly different; (4) the
Average time active/feeding episode (rain) Average time active/drinking episode (rain)
Number drinking episodes/day
Number feeding episodes/day
Average water consumption/episode (g)
Average food consumption/episode (g)
Water consumption (g-~ LBM) (g)
Total water consumption/day (g)
Food consumption (g-~ LBM) (g)
Total food consumption/day (g)
1.693 ± 0.236 11,221 ± 0.631 11.649 ± 1.476 8.636 ± 0.384 4.896 ± 0.201
16.324 ± 0.745 0.048 ± 0.002 16.156 ± 0.910 0.047 __. 0.003 1.503 _+ 0.089
,4 d libitum group Mean +_ S.E.
1.564 ± 0.122 3.197 -+ 0.526 9.880 ± 0.893 25.993 ± 1.570 4.518 +-- 0.164
10.554 _+ 0.064 0.047 ± 0.000 14.170 ± 0.712 0.063 ± 0.003 3.900 +- 0.269
L F reste~ groupfe during li period Mean ±
- RESULTS OF T-TEST ANALYSIS - -
92.28
300.98
84.81
8.49
2.38
9.48
4.04
7.71
~7.92
~4.65
t lib.
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1.433 ± 0.069 5.650 - 0.321 10.992 ± 0.585 15.967 -+ 0.854 5.786 ± 0.697
10.165 +_ 0.046 0.045 ± 0.0002 14.796 - 0.246 0.066 ± 0.001 2.216 ± 0.291
D F restricted group f e d during dark period Mean ± S.E.
) STANDARD ERRORS FOR
(o. 172)
C = LF restricted x DF restricted comparison (significant aificant effe effect) *** = P < 0.001. LBM = Lean body mass. NA = Not applicable.
(
1.005--1
279.91 4- 6.87 1.373 + 0.037
x LF restricted comparison (significant effec(:)* = P <
417.65 _+ 25.98 1.354 ± 0.064 0.911--0.883 (0.028)
B = A d libitum X DF restricted comparison (signfiicant effec(0"* = P <
A = Adlibitum
Average CO 2 production/day (per rat) (ml h-') Average oxygen c o n s u m p t i o n / d a y (g-~ LBM) (ml g-J h -t) Average respiratory quotient/h (24-h range)
Average oxygen c o n s u m p t i o n / d a y (per rat) (ml h-9
(24-h range) (°C) Average total activity/day (my s -j)
36.58 4- 0.19 37.25-(2.26) 37.38--: (1.56) 0.1959 4- 0.001 308.83 4- 8.31
37.40 _+. 0.12 37.65--37.00 (0.65) 38.02--36.81 (1.21) 0.1705 4- 0.0053 463.34 4- 21.84
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6.888 + 0.311 46.931 4- 4.28.~ 3.312 4- 0.711 74.481 +-. 0.38[
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6.161 4- 0.144 60.883 .4- 7.817 3.768 4- 0.438 101.040 4- 0.827 36.485 + 0.24 37.17--36.22 (0.95) 37.74--35.92 (1.81) 0.2059 +_ 0.0068
)7.81
t4.28
)1.40
~7.04
~6.65
[ 1.92
~.8.92
17.69
13.71
~7.90
66.494 + 6.297 4.490 + 0.550 68.701 + 1.252
7%O8
ode was higher in LF and DF rest time active per drinking episode spent more total time feeding tha t difference between acl libitum daily food consumption to total d in ad libitum rats than in LF anc output per day was significantly hi 1 rats but no significant difference restricted rats; (8) the average bo~ • was significantly lower in LF and DF restricted rats rats than in re was no significant difference in average body temp)erature b xicted rats; (9) the average (whole body) oxygen consumptio cot rage (whole body) carbon dioxide production per day da3 was sigr restricted rats than in ad libitum rats. However, when a tption per day was normalized to metabolic output per p gram [ the difference between the ad libitum and LF restricted restri~ grou
lar]
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~mparison was made between LF restricted rats and DF rest md (1) t onant differences were observed in the following variables: varial 9n and the average food consumption per episode were weJ signific r in dift )od ricted rats than in DF restricted rats. This extremely small : more food than LF rats: ~ption indicated that DF rats spilled and wasted m (2) the number of feeding episodes per day, the average Ume tim spent feeding per epi;nificantly greater in DI. md the total time spent feeding per day were sign sode, and restricted rats than LF restricted rats; (3) the total time spent drinking was antly g~ greater in DF restricted rats than LF restricted rats but no differences significantly nd in any other drinking-related variables. are found consumption, respiratory aaal circadian (24 h) profiles for activity, oxygen cc Normal illustrated tt, food consumption, and temperature are illustrate for ad libitum rats in quotient, Fig. 1 a,b and for LF restricted[ rats in Fig. 2 a,b. In the adlibitum group there was a high degree of synchronization for any given parameter between animals and among internal synchronization). Respiratory quotient (RQ), parameters in a single animal (internal tant (0.883--0.911) across the circadian interval with however, remained fairly constant .m daytime and nighttime components. The circadian no significant variation between '.d groups were significantly different from the ad libiwaveforms for the LF restricted turn group. Metabolic output in LF restricted rats was expended in a bimodal man.+ ed to the 1100 h feeding that occurred 5 h after lights ner, with one peak synchronized on (HALO) and another peak: synchronized to the dark phase of the photoperiod ld LF restricted rats were fed at the same time, LF cycle. Although ad libitum and 1me their ration during the light period, immediately restricted rats started to consume reas ad libitum rats and DF restricted rats consumed after food was presented, whereas nearly all of their food durin g the dark phase. It is important to note that body
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-1; -4 -2 0 2 4 6 8 10 12 14 16 Time In Hours After Lights On (HALO) and body temperature a Fig. 1. (a)) Twenty-four-hour patterns for oxygen metabolism, total motor activity, 344 rats. (b) Twenty-fourFisct expressedI as hourly mean values ± standard error in ad libitum male Fischer ~ressed as hourly mean values _+ terns for respiratory quotient (RQ) and feeding activity express~ hour patterns feedil activity units are converted standard error in ad libitum male Fischer 344 rats. Total activity and feeding rages of restricted maximum hourly values. Oxygen consumption iis expressed in percent of daily to percentag :eding time is denoted by ( ~ Feed). Bars above the graphs denote pperiods of light and dark. range. Feeding
piratory quotient reached their ature, activity, oxygen consumption, and respirator3 temperature, daily minimum values in LF restricted rats during the transition phase between dark and light. In LF restricted rats, body temperature, activity and oxygen metabolism started to rise approximate oximately mtel 3 h before feeding (Fig. 2a), demonstrating an entrained anticipatory behavior tvior, whereas RQ started to rise only after food was consumed (Fig. 2 b). Unlike thee a d filibitum animals that showed little circadian variation in RQ, LF restricted rats expressed a dramatic change in RQ, from a minimum of 0.83 just prior to feeding, to a maximum of 1.01, approximately 2 h after feeding (Fig. 3 a). Circadian body temperaturee profiles expressed in hours after lights on (HALO) are shown in Figure 3 b for a d~l libitum rats, for LF restricted rats fed during their inactive phase (0500 HALO), and for the DF restricted rats after they were phase shifted so that restricted and a~d d libitum rats were feeding at the same time of day (1700 HALO). Circadian boddy temperature and total activity profiles for DF
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Time In Hours After Lights On (HALO) act Fig. 2. (a)) T w e n t y - f o u r - h o u r patterns f o r o x y g e n m e t a b o l i s m , m o t o r activity, and body temperature e x p r e s s e d as h o u r l y mean values _4- standard error in caloric restricted male male Fischer 344 rats that were fed during the; light phase (LF). The time at which temperature, activity, and oxygen ox3 metabolism show anticipatory ressponse to the food cue are expressed with arrows and letters T, A, and O, respectively. (b) Twent~:y-four-hour patterns for respiratory quotient (RQ) and feeding activity expressed as hourly m e a n values les + standard error in LF caloric restricted male Fischer 344 rats. rat The time at which feeding activity and ld RQ show anticipatory response to the food cue are expressed )ressed with wit arrows and letters F and R, respectively ly. Total activity and feeding activity units are converted to percentages percer of restricted maximum hourly values. Oxygen consumption is expressed in percent o f daily range. Feeding time is denoted by ( ~ Feed). Bars above the graphs denote periods of light and dark.
restricted rats are illustrated ini Fig. 4 a and feeding and drinking activity for DF restricted rats are illustrated in Fig. 4 b. A d libitum rats maintained a fairly constant 1 - - 3 7 . 1 5 o c ) during the inactive (light) phase even basal body temperature (36.81--37.1 ~ormly low during this period. However, LF and DF though activity levels were uniforml restricted rats both had daily periods ~eriods of hypothermia (temperature < 36.5 °C) and ion phase between dark (active) and light (inactive) low activity during the transition eforms for DF restricted rats were more similar to ad periods. The physiological waveforms Lcted rats because the bimodal activity and body temlibitum rats than were LF restricted perature resulting from feedingg during the light period were eliminated when food
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Time In Hours After Lights On (HALO) Fig. 3. (a),) Twent~ Twenty-four-hour patterns for respiratory quotient (RQ) expres,, ~ressed as hourly means ± standPatterns for body temperature ard error in LF caloric restricted rats and ad iibitum Fischer 344 rats. (B) 1: expressed! as hourly means ± standard error in ad libitum, LF caloric restricted rcstri and DF caloric restricted rats. Relative Jtive feeding times for restricted groups are marked with ( ~ Feed).
. . . . ~_-II J_._-'____ ~IL._ - I _ _ 1 . . . . .'~--II TIt . . . . . . . . . L - - .,l. . . . However, ~the duration of response in DF was presented during the darkk period. ariables restricted mice for all four variables ~les was compressed into a short time interval eas physiological response in ad libitum rats was disaround the feeding time, whereas dark period. In DF restricted rats, as was seen in LF persed evenly throughout the dark ure and activity started to rise several hours before restricted rats, body temperature ined antici anticipatory behavior. feeding time, showing an entrained an1 Population-mean cosinor anal lalysis ~ was used to determine the time-of-day at which lbles reach their maximum levels (acrophase) and to the various physiological variables ween maximum and minimum values (amplitude). determine the difference between A comparison o f the timing off acrophase for various measures is illustrated for ad ted rats in Fig. 5. Acrophase and amplitude values for liibitum and LF and DF restricted these test groups and variables are given in Table II. Significant differences in acrophase for drinking activity, feeding ;ding activity, water consumption, body temperature,
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Time In Hours After Lights On (HALO) Fig. 4. (a)) Twent Twenty-four-hour patterns for body temperature and total mot( motor activity expressed as hourly means :t: standard error in DF caloric restricted male Fischer 344 rats. The T] time at which temperature and activit, ity show anticipatory response to the food cue are expressed with aarrows and the letters T and A, respectively. (b) Patterns for feeding activity and drinking activity expresse¢ .'ly. (b) )ressed as hourly means _+ standard errors in D F caloric restricted male Fischer 344 rats. Feeding time is denoted denote~ by ( ~ Feed). Bars above the graphs denote :note periodic periods of light and dark.
oxygen consumption, carbon dioxide production, and motor activity were found between the LF restricted and ad libitum rats (P < 0.05) and between DF restricted and LF restricted rats Is (P < 0.05). A comparison was not possible for RQ hythm was found in the acl libitum group. The timing since no significant circadian rh' raters monitored in the ad libitum group and the DF of acrophase for all the parameters group, with the exception of R(Q, was almost simultaneous (1624-- 1804 HALO and 1718--1900 HALO, respectively) ly) and occurred in the middle of the dark cycle. The • ious measures varied significantly in the LF restricted timing of acrophase for the various ad occurred approximately four hours after feeding group (0624--1128 HALO) and (approx. 3 h before lights off).
+ DF Feeding Time
DF Restricted A
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Drinking Activity
} t
t I Feeding Activity t t Water Consumption Food ConsurnptJor Temperature ~ I Activity
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Drinking Activity Activity Water Consumption Food Consumption
I I I Drinking Activity I ~ I Feeding Activity ~ 1 ~ 4 Water Consumption @ F o o d Consumption I---~-I Tempetat ur e
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e time o f day at which various physiological measures reach their daily maxir )5% confidence intervals) are given for LF and DF restricted rats and ad libitz :ant cosine r h y t h m and ( ~ Feed) denotes feeding time. Bars above abovl graphs re] lark.
rotes of
DISCUSSION
The results •esults o f this study show that caloric restricted LF and DF rats ate fewer meals, consumed onsumed more food at each meal, and spend more time l eating during each an did ad libitum rats. However, the total time spe meal than )ent feeding was significantly lower )wer in LF restricted rats but not in DF restricted rats. r~ The fact that water J J ztly reduced in LF and DF caloric restricted rats and consumption was not significantly the ratio o f food consumption to water consumption was significantly reduced in both restricted groups, suggestt that water consumption is important not only in maintaining a proper osmotic balance but in partially filling the stomachs of restricted rats, thereby relieving;their hunger and compensating for the lack of food. The interaction between the timing of food consumption, meal patterns, and caloric restriction is not clearly¢ understood. Nelson and Halberg [18] reported no significant difference in life span m for restricted rats fed one to six meals at different times o f the day. However, the te caloric restriction was only 24.1 °70 in the Nelson study compared to a 40°?o restriction iction level in another study [4] that closely emulated the experimental conditions usedd in the present study. The greater level of restriction in the Yu study [4] produced a two-fol fold increase in the percentage of life extension two-fold .r
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)xygen
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respiratory quotient (RQ)
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Acrophase and amplitude in ad libitum rats is significantly different dil from DF restricted rats for t all measurements (~ osine fit (best estimate); not used for group mean determination. C2.1. = 95°70 confidence interval for acrophase. cosine
16:24 (16:04, 16:40)
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0.886 ± 0.264 pulse/s 0.0031 ± 0.0004 m v / s 0.039 ± 0.004 m g / s 0.023 ± 0.002 m g / s 0.644 + 0.048 oC
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;ULTS OF P O P U L A T I O N M E A N COSIb
the Therefore, a synergistic interactic severity of caloric restriction in th~ tdy nic tted to life extension and the on sly. als ere the presentation of food to res~ the active phase of the circadian c 35e lronizer (lights-on or lights-off), il LF Its-on during the middle of the inat md fter lights-off during the middle ive The bimodal distribution of activity, core tempen ~erature, an mparameters indicates that LF restricted animals were w~ synch the : synchronizer as well as to the photoperiod cycle. The activity 'ith ; seemed to produce a "masking effect" that partially the The the eriod rhythm that appeared to remain unaltered [19,20]. [19 • analysis supported this conclusion. The average acrophase acrc fo ~erotal motor activity, RQ, as well as oxygen and carbo~)n dioxide t in :ricted animals was 1015 H A L O . Acrophase for these the parann red )). o the time o f lights-off (1200 HALO) than to the feeding feed: time ( versely, the average acrophase for food and water cc consumpti~ ing inking activity in LF restricted rats occurred only 67 6 min af~ ing anizer. Therefore, activity involved with food and water col vas anized to the food cue, while non-absorptive, sponta ~ontaneous activity remained synchronized to the photoperiod cycle. Unlike the ad libitum animals where acrophase ase for feeding, core temperature, total activity and an, metabolic parameters occurred .~d at approximately the same time, in LF restricted rats : acrophase for food consumaption occurred 3.5 h earlier than acrophase for total atal activity, core temperature, and ad metabolic variables (P < 0.01). Therefore, a condition cone of internal desynchronization ,ation existed in restricted rats entrained to dayti ~ime feeding, caused by shifting the feeding synchronizer out of phase with the photo photo period synchronizer. It btfnl t h a t i n t o r n u l c l ~ . ~ v n e h r n n i T n t l c m a l t P r ~ l l f o c n ~ n since ~i is doubtful that internal desynchroniz nization alters life span chronization Finger [21] reported that internal desynchronization n caused by shifting of the photoperiod cycle did not significantly alter the mean survival vival time o f rodents. The results o f this study indicated licated significant differences in physiological performance between LF restricted rats ats and DF restricted rats. DF restricted rats expressed a greater number o f feeding episodes fisodes per day, an increase in total time spent feeding per day, a decrease in the avera 'age food consumption per episode, and a decrease in the average time active per feedin ding episode, compared to LF restricted rats, thereby more closely emulating feedingg behavior in ad libtium rats. Also, the bimodal waveform and internal desynchronization ization, characteristic of metabolic, feeding, activity, and temperature variables in LF restricted rats, was completely eliminated by switching to nighttime feeding,, causing acrophase for these variables to resynchronize by shifting into the dark Iphase. Therefore, these results indicate that feeding during the dark phase promotes more normal behavior, thereby allow-
ly distinguish between caloric res ~cts ~ith the time-of-day of feeding. d after feeding in LF restricted rat Lad icted animals make rapid change ~lic lbility (Fig. 3 a). The average RQ i ted prior to feeding which indicated gen ~e animals were metabolizing prote ure e. The rapid rise in RQ to a ma: .01 ndicated that restricted rats shifte ein :metabolism to carbohydrate metabolism. Unlike the th ad libit hat ined a constant RQ throughout the day, indicating a constar rorid, and carbohydrate metabolism, calorically restricted restrict anima be to store glycogen reserves which resulted in a g radual d as ydrates were depleted. The fact that a period of hypol )othermia o ing iod of low RQ, may indicate that the depletion of glycogen ;igLmetabolic protection reaction resulting in lower m, metabolisrr md ~mperature (torpor). Lis study, as was reported in an earlier study [18], caloric calc restric SOvith a significant reduction in average body temperatu )erature per da if iiuction in body temperature (torpor) for a short inteT interval at a s of L ~ I ¢111~.1 1.11" I ~ 3 L I I k , L C U I ~ats LL~ 5ng the transition stage between dark and light in ILF LI and DF b). These results support a previous study [22] that showed sh a high correlation betweena torpor and photoperiod and suggest that hypothern ~othermia is involved in energy conservation 'ation and metabolic recovery. Restricted animals were we unable to maintain a constantLt body temperature during the inactive period without wit expending motor activity,, suggesting that increased level of activity in restricte :ted rats may be necessary to comIpensate for reduced thermoregulatory capability. Therefore, Tt metabolic and temperature ature regulation by the central nervous system appear~ ~ears to be altered by caloric restricti,Lon a n d / o r the reduced lean body mass and almost cc complete depletion of fat ; ~ rrestriction a e t r ; t - t ; r ~ n animals anlmqlc m ~ay r ~ l t ~ r ttheir h ~ ; r tthermal h~rnn~l ~ t~netllot in caloric ma, alter conductance, thereby accelerating the rate of heat transfer andd lowering body temperature during periods of basal metabolism and low activity outl ttput. Another possibility is that the low body temperature in restricted rats m a y be.~ a result of the rapid depletion of specific essential amino acids that are required for the biosynthesis of the neurotransmitters )f me1 metabolism and thermoregulation. associated with the regulation of The caloric restriction related ed changes in temperature and metabolism reported here support the results of other ~r studies run concurrently in our laboratory to determine the effect of age and caloric ric restr restriction striction on the levels of specific hepatic enzymes related to intermediary metabolism )lism and drug metabolism [23--25]. The activities of enzymes involved with glycol3tsis and lipid syntheses in rats maintained on caloric restricted diets were found to be ~e decreased and enzymes supporting gluconeogenesis were maintained or increased compared to control animals fed ad libitium. There-
ort alter hepatic metabolism for the g ion tions o f limited and efficient eneJ led supported by the high RQ values ( igh ats at the same 2 h after feeding in crease role for insulin and an incJ for I is also suggested. Preliminary fi est in n may be to retard the age-associa and to selectively induce other hep; talon changes indicate that the plasma restriction [24,; ; of several hormones may be altered by caloric restric pid interesting to speculate that the reduced body ternLperature ; of specific metabolic pathways towards low glycogen gly~ an ge, d for restricted rats, may significantly alter events at a the mol by tg DNA damage or by increasing DNA repair capat aabilities th, ing .~of aging [26--28]. Kayser [29] and Duffy et ai. [16] suggeste )rig m o f hibernating animals is related to the effects of o hypoth :ral life ;ators have suggested that a low core temperature sit;nificantl z slowing the age-related loss o f immunological ;ical response respc [30,3 tits study clearly indicate that in vitro caloric restriction restrictio studies )ne roper techniques to insure that tissues are prepared and speci ers nitored at the broad range of temperatures expressed [pressed . . l l [ ; ~ ; I d by U y restri( l [ ; ~ l . l l l , . L l ~ l d ll~ll.b 111 Lhis lllb properly emulate the actual in vivo conditions. McCarter 2arter et aL [15] and Masoro [32] reported me )orted that the metabolic output per gram lean b o,dy d mass is not significantly lowered by caloric restri(;tion. The results of the present study stud~ support this conclusion. However, restricted aanimals consumed fewer calories; but were able to maintain a higher level o f activity than tt ad libitum rats, with no incre; ease in metabolism. These results may indicate an Increase in~ in metabolic efficiency in restricted rats, since more work was done at the tt same rate of energy expenditure. iture. A possible reason that the caloric restriction-r~ restriction-related increase in motor , p n ~ r t ~ c l here h ~ r ~ was , u ~ f l c not n n t rreported F.nr~rt~cl m ; n previous n r a . v ; ~ n e studies e t n r l ; , ~ e is that motion detecting activity rreported equipment that measures animal al movement based on changes in position (photoelectric cells, infrared beams, etc.)I are not necessarily accurate indicators of total work done, and therefore data gathered ~ered in these systems can not be directly related to oxygen consumption (energy output) utput) resulting from motor activity. The system used in this study monitored parameters aeters such as force and velocity that can be directly related to energy output. Therefq ffore, it could accurately estimate the energy partition associated with gross motor activity. The significant changes in body temperature, motor activity and RQ reported here indicate that qualitative ch :hanges in metabolism, resulting from caloric restriction-induced alterations in metabolic abolic pathways, may modulate the aging process and the onset of chronic diseases bby triggering the onset of torpor. The results of this study also indicate that the physiolo wsiological variables that regulate energy output are
tored at frequent ric restriction
10
11
12 13 14 15 16
17 18 19
intervals througl
I to
a n d a g i n g . S i n g l e tit
er-
r a daily interval may be deceptiv¢
ing
a effects
ian
may
occur
only
at spe
and life prolongation. In C. Finch and t s.), Idbook o f the Biology o f Aging, Van Nostrand Reinhold, New York Yor (1977). [. McCay, M.F. Crowell and L.A. Maynard, The effect of retardq trded growth t i of ~pan and upon the ultimate body size. J. Nutr., 10 (1935) 63--79. • Yu, E.J. Masoro, I. Murata, H . A . Bertrand and F.T. Lynd, Life Li span stu~ her male rats fed ad libitum or restricted diets: longevity, growth, lea lean body ma • J+ ontol., 37(1982) 130--141. :l. • Yu, E.J. Masoro and C.A. McMahan, Nutritional influences or on aging of F sical, metabolic, and longevity characteristics. J. Gerontol., 40 (1985) 091 657--67 daeda, C.A. Gleiser, E.J. Masoro, l. Murata, C.A. M c M a h o n an and B.P. Yu, flu+ 1985) ,,s on aging of Fischer 344 rats. lI. Pathology. J. GerontoL, 40 (198 ~, 671--688 I. Ross, Length of life and caloric intake. Am. J. Clin. Nutr., 25 (l ~1972) 834--4 ~/eindruch and R.L. Walford, Dietary restriction in mice beginnin~ ,,tuning at one yea on ;pan and spontaneous cancer incidence. Science, 215 (1982) 1415-;--1418. • Goodrick, D.K. Ingrain, M.A. Reynolds, J.R. Freeman and N.L. Cider, Dif sol" ?mittent feeding and voluntary exercise on body weight and life sp~ ~an in adult ol., t983) 36--45. i. Sarkar, G. Fernandes, N.T. Telang, l.A. Kourides and R.A. Good, Go Low-calorie diet prevents the develo :levelopment of m a m m a r y tumors in C3H mice and reduces circulating circu prnlactin level, murine m a mamary m a r tumor virus expression, and proliferation of m a m m a r y alveolar alw cells• Proc. NatL Acad. Sci., USA, 79 (1982) 7758--7762. B.A. Ruggeri, D.M. Klurfeld and D. Kritchevsky, Biochemical alterations in 7,12-dimethylbenz,[a]anthracene-induced m a m m a r y tumors from rats subjected to caloric restriction. Biochim. Biophys. Acta, 929 (1987)239--246. D. Kritchevsk~ ~'ritchevsky, M.M. Weber and D.M. Klurfeld, Dietary fat versus; caloric ca content in initiation and promotion notion of 7,12-dimethylbenz[a]anthracene-induced m a m m a r y tumorigenesis tt in rats. Cancer Res., 44 (1984) 3174--3177. aollard and P P.bl. Luckert_ Tumori Tumorioenic indJ M. Pollard . H . Luckert, amorigenic effects of direct- and indirect-acting chemical carcinogens in rats on a restricted diet. J. Natl. Cancer Inst., 74 (1985) 1347-- 1349. B.D. Roebuck, D.D. Yager, D.S. Longnecker and S.A. Wilpone, Promotion by unsaturated fat of inogenesis in the rat. CancerRes., 41 (1981) 3961--3966. azeserine-induced pancreatic carcim R. Pearl, The Rate o f Living, Alfred red Knopf, New York (1928) 1--185. LP. Yu, Does food restriction retard aging by reducing the metaR. McCarter, E.J. Masoro and B.P. bolic rate? Am. J. Physiol., 248 (1985) l 985) E488--E490. 1. Hart, Effect of age and torpor on the circadian rhythms of body P.H. Duffy, R.J. Feuers and R.W. temperature, activity, and body weight in the mouse (Peromyscus leucopus). In J. Pauly and L. Scheving (eds.), Advances in Chronobiology, Part B., Alan R. 1Ass, Inc., New York (1987) 111--120. ~4elson, W. Runge and R.B. Sothern, Autorhythmometry proceF. Halberg, E.A. Johnson, W. Nelson lrements and their analysis. Physiol. Teacher, I (1972) l - - 1 l• dures for physiological self-measuremen! d-timing, circadian rhythms and life span of mice. J. Nutr., 116 W. Nelson and F. Halberg, Meal-timin (1986) 2244--2253. J. Aschoff, Exogenous and endoogenous components in circadian rhythms. Cold Spring Harbor Sympos. QuaL Biol., 24 (1960) 11--28.
a of
Ferman, Feeding schedules and the circadi 1 (1980) 39--65. tion shorten life? Chronobiologia, 10 (198." and R. Alosia, Relationships of light inten sernating ground squirrel, Citellus lateralis y, A. Turturro, R.A. Mittelstaedt and RA nzymes of intermediary metabolism in thq -189. J. Bazare, P . H . Duffy, R.H. Feuers and F atic drug metabolizing enzymes in the Fisc genase system. Mech. Ageing Dev., 48 (191 • Leakey, H•C. C u n n y , P.J. Webb, J. Bazare, P.H. Duffy, R . L Feuers F and F lging and caloric restriction on hepatic drug metabolizing enzyn 'mes in the F ects on conjugating enzymes• Mech• Ageing andDev., 48 (1989) 157--166. 15 Koizumi, R. Weindruch and R.L. Walford, Influences of dietar y restrictio~ yrne activities and liver peroxidation in mice• J. Nutr., 117 (1978) 361--367. 3, ,. Walford, S.B. Harris and R. Weindruch, Dietary restriction and aging: :hanisms and current directions. J. Nutr., Nutr. 117 (i 987) 1650-- 1654. Andahl and B. Nyberg, Rate o f depurination of native deoxyribo~ tribonucleic acid ~2) 3610--3618. Kayser, M a m m a l i a n hibernation• I. Hibernation versus hypothern ~othermia. Bull. A "yard University, 124 (1960) 1--29. ,. Walford, The Immunologic Theory o f Aging, Munksgaard, Cop Copenhagen (1 ~. Liu and R.L. Walford, The effect o f lowered body temperature on life spa -immune processes. Gerontologia, 18 (1972) 363--388. • Masoro, Mini-review: Food restriction in rodents: An ~evaluation evaluati . ¥ t ~ l . l U a l . l L l l l Iof d l ll.~ its lrcu l l . lg. J. Gerontol., 43 (1988) B59--64.
perera.
t of 344 ects The 'ects II. iver ses,
•
11
~ol.,
and Ill
Idl y of
I . l l C bl. U l d y
J C R E S T R I C T I O N ON P H Y S I ( ERGY METABOLISM IN THE
FEUERS, J U L I A N A. L E A K E D. N A K A M U R A , A N G E L O T U R T U R R O and R RO? ONALD W ent o f Health and H u m a n Services, Public Health Service, F o o d and at, Drug A d, Center f o r Toxicological Research, Jefferson, Arkansas 72079 (U.S.A.) (U. J September 23rd, 1988)
tRY
behavioral m~ le present study, a number o f physiological and beha are to metabolism were continuously monitored in 19-month-ol 19-~ her :s that were fed ad libitum or fed a caloric restricted cted diet. Ca ted ,' fewer meals but consumed more food during each m meal and s me per meal than did rats fed ad iibitum. Therefore, the I, l l ~ , , timing I.lllllll~ a. I I U I d U I t l L I U I ! t of Q UI Is well as the total number o f calories consumed ma'. ly be associated with life on. Avera Average body temperature per day was signifiantl y lower in restricted rats extension. :ly temperature range per day and m o t o r activity but bod, :y were higher in restricted rats. Dramatic .tic changes in respiratory quotient, indicating rapid rapi changes in metabolic pathway and lower temperature, occurred in caloric restricted rats when carboh'ydrate reserves were depleted. Lower body temperatt aerature and metabolism during this time interval m a y result in less D N A damage, thereb thereby increasing the survival Lal o f restricted rats. Nighttime feeding was found to synchronize physiologipotential n d libitum l ibitum i h l t t n m and •nrl ,'.aln.;,', raetr;r.~oA rats , cal p e r f•enrman,'.,a o r m a n c e k~atu,~=~n between ad caloric restricted better than daytime feeding, thereby allowing investig~ stigators to distinguish the effects of caloric restriction f r o m those related solely to the; time-o time-of-day e-of-da, o f feeding.
Key words: Caloric; Restrictionn; Physiological; Metabolism; Male; Rat INTRODUCTION Caloric restriction is the onl' dy known experimental paradigm that uniformly and proportionately increases maxamum ~imum achievable life span (MAL) o f placental m a m Address all correspondence to: Peter H. Duffy, HFT-I, Building 62, National Center for Toxicological Research, Jefferson, Arkansas 72079, U.S.A.
Printed and Published in Ireland
he rate of the occurrence of most ve demonstrated this phenomenoJ and continued throughout life [ le onset of adulthood [6--8]. Cal, ve in reducing the incidence and ally induced tumors in rats and mi by which caloric restriction incre e are not clearly understood, seve e been postulated to explain the, t4] and Sacher [1] proposed that the longevity of a sp~ )ecies is in, restri o the metabolic rate per gram body mass and that caloric cah )y reducing the metabolic rate. However, McCarter et al. [15 tabolic rate per kilogram lean body mass was not no1 signific this n ad libitum fed and caloric restricted animals. To resolve re,. ph,. red qualitative as well as quantitative changes in several se if cal )ral variables that are related to metabolism to determine detern he homeostatic regulation o f these variables.
led ~ric len
ion O|
md ses na, or-
Lses ha~ ent ave md ion
[ALS AND M E T H O D S
t husbandry and feeding regimens male Fischer 344 rats used in this study were raised at the National Center for 10XlCOlO logical Research (NCTR) in a specific pathogen fre, free (SPF) environment at
23°C, and were entrained to a 12/12, light/dark cycle with lights on from 0600 to 1800 h daily. This regimen was maintained from the time the tl animals were weaned until the' ~ey were entered into the experiment. The test animals anin~ were divided into a restric group that received control• group that was fed ad libitum, and a caloric restricted 60070 off the ad libitum consumption starting at 14 weeks of age and continuing for the duration ation of the experiment. A d libitum rats were given more rr food than they conat all times. All rats were sumed in a daily interval so that food was available to them fed a standard NIH-31 diet at 1100 h daily and were housed singly in plastic cages with metal tops. The experimental ntal protocol was initiated when the test animals were ae the subjects were transferred from the SPF barrier 17 months of age, at which time to the testing area, where they were maintained under similar environmental conditions.
Physiological and behavioral testin esting procedures The equipment and testing procedures used in this study were similar to those used previously [16]. Briefly, food and water consumption, as well as feeding and tinuously )usl monitored with a system that measured the total motor activity, were continuous force applied to a load cell. Drinking activity was monitored by a digital pulsecounting device triggered whenn the rat made contact with the drinking tube. Deep ed by a thermistor-transmitter capsule that was surgibody temperature was measured eal cavity of the rat 60 days before the animals were cally implanted in the peritoneal
~nt an dioxide production, and respir es=ircuit flow system equipped with~ :ed ~ressure regulating device. Anima ral metabolism cages and sample flo ~ dyplied Electrochemistry model S3A del ared carbon dioxide analyzer. An of ~ed to provide process control and as data collection and analysis. of ten caloric restricted animals 18ior "e placed individually in test cages (11 in. × 7 in. X 7.5 7 in.) fo of nitiation o f the experiment. Both test groups were fed f~ 5 h aft environment~ e re it phase of the photoperiod cycle and all other envi ~l to those described previously. Data were collected for all p~ :32h ervals over a I 0-day testing period. The photoperiod was then va$ ," animals were allowed to phase shift for a 30-day rperiod so iod ed to restricted rats 5 h after the onset of the dark p]~hase of tt his Food consumption in the ad libitum group was at its it~ maximt day. All physiological and behavioral variables except exc oxyg ~on ; metabolism and RQ were subsequently measured continuou: c~ her period. This additional procedure was utilized to sym ,nchronize t egthat control and restricted rats were eating at the sOsame ~ L l X l ~ , time LX|aI~ VI ~,t~ff. ~ J O l ling t~ light-fed and dark9cedure, the same set of restricted rats were tested under u~ fed conditions. Conventional ventional statistical methods (means and standard errors; Student's t-test) and the.~cosinor statistical method [17] were used to a n a l y,ze z e the data. Using the least and the confidence limits squares method, the values for acrophase and amplitude a~ for these se functions were calculated for individual animals q(single cosinor) and test groups (population-mean cosinor) [17]. RESULTS Physiological and behavioral al data for ad libitum rats, restricted rats fed during the light period (LF), and restricted :ricted rats fed during the dark period (DF) are compared in Table I. Significant differen~ tifferences ences are observed in several of the experimental variables analyzed, including: (1) Food consumption and the total feeding time per day were higher in ad libitum animals than in LF and DF restricted animals, but water consumption and the total tal time spent drinking per day were not significantly different a m o n g the three groups; :~ups; (2) the average food consumption per feeding episode and water consumption ~n per gram of lean body mass were higher in LF and DF restricted rats than in ad libitum rats, but food consumption per gram of lean body mass and the average water ter consumption per drinking episode were not significantly different; (3) the number er o f feeding episodes per day was 3.5 times greater in ad libitum rats than in LF restricted stricted rats and 2.0 times greater than DF restricted rats, but the number of drinkin king episodes was not significantly different; (4) the
Average time active/feeding episode (rain) Average time active/drinking episode (rain)
Number drinking episodes/day
Number feeding episodes/day
Average water consumption/episode (g)
Average food consumption/episode (g)
Water consumption (g-~ LBM) (g)
Total water consumption/day (g)
Food consumption (g-~ LBM) (g)
Total food consumption/day (g)
1.693 ± 0.236 11,221 ± 0.631 11.649 ± 1.476 8.636 ± 0.384 4.896 ± 0.201
16.324 ± 0.745 0.048 ± 0.002 16.156 ± 0.910 0.047 __. 0.003 1.503 _+ 0.089
,4 d libitum group Mean +_ S.E.
1.564 ± 0.122 3.197 -+ 0.526 9.880 ± 0.893 25.993 ± 1.570 4.518 +-- 0.164
10.554 _+ 0.064 0.047 ± 0.000 14.170 ± 0.712 0.063 ± 0.003 3.900 +- 0.269
L F reste~ groupfe during li period Mean ±
- RESULTS OF T-TEST ANALYSIS - -
92.28
300.98
84.81
8.49
2.38
9.48
4.04
7.71
~7.92
~4.65
t lib.
,.rcent
1.433 ± 0.069 5.650 - 0.321 10.992 ± 0.585 15.967 -+ 0.854 5.786 ± 0.697
10.165 +_ 0.046 0.045 ± 0.0002 14.796 - 0.246 0.066 ± 0.001 2.216 ± 0.291
D F restricted group f e d during dark period Mean ± S.E.
) STANDARD ERRORS FOR
(o. 172)
C = LF restricted x DF restricted comparison (significant aificant effe effect) *** = P < 0.001. LBM = Lean body mass. NA = Not applicable.
(
1.005--1
279.91 4- 6.87 1.373 + 0.037
x LF restricted comparison (significant effec(:)* = P <
417.65 _+ 25.98 1.354 ± 0.064 0.911--0.883 (0.028)
B = A d libitum X DF restricted comparison (signfiicant effec(0"* = P <
A = Adlibitum
Average CO 2 production/day (per rat) (ml h-') Average oxygen c o n s u m p t i o n / d a y (g-~ LBM) (ml g-J h -t) Average respiratory quotient/h (24-h range)
Average oxygen c o n s u m p t i o n / d a y (per rat) (ml h-9
(24-h range) (°C) Average total activity/day (my s -j)
36.58 4- 0.19 37.25-(2.26) 37.38--: (1.56) 0.1959 4- 0.001 308.83 4- 8.31
37.40 _+. 0.12 37.65--37.00 (0.65) 38.02--36.81 (1.21) 0.1705 4- 0.0053 463.34 4- 21.84
S.ZYl
•
6.888 + 0.311 46.931 4- 4.28.~ 3.312 4- 0.711 74.481 +-. 0.38[
:I: 4 . 1 3 Y
6.161 4- 0.144 60.883 .4- 7.817 3.768 4- 0.438 101.040 4- 0.827 36.485 + 0.24 37.17--36.22 (0.95) 37.74--35.92 (1.81) 0.2059 +_ 0.0068
)7.81
t4.28
)1.40
~7.04
~6.65
[ 1.92
~.8.92
17.69
13.71
~7.90
66.494 + 6.297 4.490 + 0.550 68.701 + 1.252
7%O8
ode was higher in LF and DF rest time active per drinking episode spent more total time feeding tha t difference between acl libitum daily food consumption to total d in ad libitum rats than in LF anc output per day was significantly hi 1 rats but no significant difference restricted rats; (8) the average bo~ • was significantly lower in LF and DF restricted rats rats than in re was no significant difference in average body temp)erature b xicted rats; (9) the average (whole body) oxygen consumptio cot rage (whole body) carbon dioxide production per day da3 was sigr restricted rats than in ad libitum rats. However, when a tption per day was normalized to metabolic output per p gram [ the difference between the ad libitum and LF restricted restri~ grou
lar]
if ited onted md
tivure ats
md md Nel +
gen ass fig,
~mparison was made between LF restricted rats and DF rest md (1) t onant differences were observed in the following variables: varial 9n and the average food consumption per episode were weJ signific r in dift )od ricted rats than in DF restricted rats. This extremely small : more food than LF rats: ~ption indicated that DF rats spilled and wasted m (2) the number of feeding episodes per day, the average Ume tim spent feeding per epi;nificantly greater in DI. md the total time spent feeding per day were sign sode, and restricted rats than LF restricted rats; (3) the total time spent drinking was antly g~ greater in DF restricted rats than LF restricted rats but no differences significantly nd in any other drinking-related variables. are found consumption, respiratory aaal circadian (24 h) profiles for activity, oxygen cc Normal illustrated tt, food consumption, and temperature are illustrate for ad libitum rats in quotient, Fig. 1 a,b and for LF restricted[ rats in Fig. 2 a,b. In the adlibitum group there was a high degree of synchronization for any given parameter between animals and among internal synchronization). Respiratory quotient (RQ), parameters in a single animal (internal tant (0.883--0.911) across the circadian interval with however, remained fairly constant .m daytime and nighttime components. The circadian no significant variation between '.d groups were significantly different from the ad libiwaveforms for the LF restricted turn group. Metabolic output in LF restricted rats was expended in a bimodal man.+ ed to the 1100 h feeding that occurred 5 h after lights ner, with one peak synchronized on (HALO) and another peak: synchronized to the dark phase of the photoperiod ld LF restricted rats were fed at the same time, LF cycle. Although ad libitum and 1me their ration during the light period, immediately restricted rats started to consume reas ad libitum rats and DF restricted rats consumed after food was presented, whereas nearly all of their food durin g the dark phase. It is important to note that body
~-?- 400 -80
0
-7o -60
iT ~
~
~/
~
-50 ~ -9o -40 ~
-30 o~ -20 -10
--e--Feeding Activity
3.97 ~, 3.93-
3.86-
t
P '"
-35 -30
~ _/
-25 ~
"
i,, ,~-i-.j TtT'ri'
t5 2B
-1; -4 -2 0 2 4 6 8 10 12 14 16 Time In Hours After Lights On (HALO) and body temperature a Fig. 1. (a)) Twenty-four-hour patterns for oxygen metabolism, total motor activity, 344 rats. (b) Twenty-fourFisct expressedI as hourly mean values ± standard error in ad libitum male Fischer ~ressed as hourly mean values _+ terns for respiratory quotient (RQ) and feeding activity express~ hour patterns feedil activity units are converted standard error in ad libitum male Fischer 344 rats. Total activity and feeding rages of restricted maximum hourly values. Oxygen consumption iis expressed in percent of daily to percentag :eding time is denoted by ( ~ Feed). Bars above the graphs denote pperiods of light and dark. range. Feeding
piratory quotient reached their ature, activity, oxygen consumption, and respirator3 temperature, daily minimum values in LF restricted rats during the transition phase between dark and light. In LF restricted rats, body temperature, activity and oxygen metabolism started to rise approximate oximately mtel 3 h before feeding (Fig. 2a), demonstrating an entrained anticipatory behavior tvior, whereas RQ started to rise only after food was consumed (Fig. 2 b). Unlike thee a d filibitum animals that showed little circadian variation in RQ, LF restricted rats expressed a dramatic change in RQ, from a minimum of 0.83 just prior to feeding, to a maximum of 1.01, approximately 2 h after feeding (Fig. 3 a). Circadian body temperaturee profiles expressed in hours after lights on (HALO) are shown in Figure 3 b for a d~l libitum rats, for LF restricted rats fed during their inactive phase (0500 HALO), and for the DF restricted rats after they were phase shifted so that restricted and a~d d libitum rats were feeding at the same time of day (1700 HALO). Circadian boddy temperature and total activity profiles for DF
- , : , - Activity
1100
.... o2
[90
-v-Temperature
~80
z*~
+
///_ ....
O
70
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Time In Hours After Lights On (HALO) act Fig. 2. (a)) T w e n t y - f o u r - h o u r patterns f o r o x y g e n m e t a b o l i s m , m o t o r activity, and body temperature e x p r e s s e d as h o u r l y mean values _4- standard error in caloric restricted male male Fischer 344 rats that were fed during the; light phase (LF). The time at which temperature, activity, and oxygen ox3 metabolism show anticipatory ressponse to the food cue are expressed with arrows and letters T, A, and O, respectively. (b) Twent~:y-four-hour patterns for respiratory quotient (RQ) and feeding activity expressed as hourly m e a n values les + standard error in LF caloric restricted male Fischer 344 rats. rat The time at which feeding activity and ld RQ show anticipatory response to the food cue are expressed )ressed with wit arrows and letters F and R, respectively ly. Total activity and feeding activity units are converted to percentages percer of restricted maximum hourly values. Oxygen consumption is expressed in percent o f daily range. Feeding time is denoted by ( ~ Feed). Bars above the graphs denote periods of light and dark.
restricted rats are illustrated ini Fig. 4 a and feeding and drinking activity for DF restricted rats are illustrated in Fig. 4 b. A d libitum rats maintained a fairly constant 1 - - 3 7 . 1 5 o c ) during the inactive (light) phase even basal body temperature (36.81--37.1 ~ormly low during this period. However, LF and DF though activity levels were uniforml restricted rats both had daily periods ~eriods of hypothermia (temperature < 36.5 °C) and ion phase between dark (active) and light (inactive) low activity during the transition eforms for DF restricted rats were more similar to ad periods. The physiological waveforms Lcted rats because the bimodal activity and body temlibitum rats than were LF restricted perature resulting from feedingg during the light period were eliminated when food
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. . . . ~_-II J_._-'____ ~IL._ - I _ _ 1 . . . . .'~--II TIt . . . . . . . . . L - - .,l. . . . However, ~the duration of response in DF was presented during the darkk period. ariables restricted mice for all four variables ~les was compressed into a short time interval eas physiological response in ad libitum rats was disaround the feeding time, whereas dark period. In DF restricted rats, as was seen in LF persed evenly throughout the dark ure and activity started to rise several hours before restricted rats, body temperature ined antici anticipatory behavior. feeding time, showing an entrained an1 Population-mean cosinor anal lalysis ~ was used to determine the time-of-day at which lbles reach their maximum levels (acrophase) and to the various physiological variables ween maximum and minimum values (amplitude). determine the difference between A comparison o f the timing off acrophase for various measures is illustrated for ad ted rats in Fig. 5. Acrophase and amplitude values for liibitum and LF and DF restricted these test groups and variables are given in Table II. Significant differences in acrophase for drinking activity, feeding ;ding activity, water consumption, body temperature,
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oxygen consumption, carbon dioxide production, and motor activity were found between the LF restricted and ad libitum rats (P < 0.05) and between DF restricted and LF restricted rats Is (P < 0.05). A comparison was not possible for RQ hythm was found in the acl libitum group. The timing since no significant circadian rh' raters monitored in the ad libitum group and the DF of acrophase for all the parameters group, with the exception of R(Q, was almost simultaneous (1624-- 1804 HALO and 1718--1900 HALO, respectively) ly) and occurred in the middle of the dark cycle. The • ious measures varied significantly in the LF restricted timing of acrophase for the various ad occurred approximately four hours after feeding group (0624--1128 HALO) and (approx. 3 h before lights off).
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rotes of
DISCUSSION
The results •esults o f this study show that caloric restricted LF and DF rats ate fewer meals, consumed onsumed more food at each meal, and spend more time l eating during each an did ad libitum rats. However, the total time spe meal than )ent feeding was significantly lower )wer in LF restricted rats but not in DF restricted rats. r~ The fact that water J J ztly reduced in LF and DF caloric restricted rats and consumption was not significantly the ratio o f food consumption to water consumption was significantly reduced in both restricted groups, suggestt that water consumption is important not only in maintaining a proper osmotic balance but in partially filling the stomachs of restricted rats, thereby relieving;their hunger and compensating for the lack of food. The interaction between the timing of food consumption, meal patterns, and caloric restriction is not clearly¢ understood. Nelson and Halberg [18] reported no significant difference in life span m for restricted rats fed one to six meals at different times o f the day. However, the te caloric restriction was only 24.1 °70 in the Nelson study compared to a 40°?o restriction iction level in another study [4] that closely emulated the experimental conditions usedd in the present study. The greater level of restriction in the Yu study [4] produced a two-fol fold increase in the percentage of life extension two-fold .r
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the Therefore, a synergistic interactic severity of caloric restriction in th~ tdy nic tted to life extension and the on sly. als ere the presentation of food to res~ the active phase of the circadian c 35e lronizer (lights-on or lights-off), il LF Its-on during the middle of the inat md fter lights-off during the middle ive The bimodal distribution of activity, core tempen ~erature, an mparameters indicates that LF restricted animals were w~ synch the : synchronizer as well as to the photoperiod cycle. The activity 'ith ; seemed to produce a "masking effect" that partially the The the eriod rhythm that appeared to remain unaltered [19,20]. [19 • analysis supported this conclusion. The average acrophase acrc fo ~erotal motor activity, RQ, as well as oxygen and carbo~)n dioxide t in :ricted animals was 1015 H A L O . Acrophase for these the parann red )). o the time o f lights-off (1200 HALO) than to the feeding feed: time ( versely, the average acrophase for food and water cc consumpti~ ing inking activity in LF restricted rats occurred only 67 6 min af~ ing anizer. Therefore, activity involved with food and water col vas anized to the food cue, while non-absorptive, sponta ~ontaneous activity remained synchronized to the photoperiod cycle. Unlike the ad libitum animals where acrophase ase for feeding, core temperature, total activity and an, metabolic parameters occurred .~d at approximately the same time, in LF restricted rats : acrophase for food consumaption occurred 3.5 h earlier than acrophase for total atal activity, core temperature, and ad metabolic variables (P < 0.01). Therefore, a condition cone of internal desynchronization ,ation existed in restricted rats entrained to dayti ~ime feeding, caused by shifting the feeding synchronizer out of phase with the photo photo period synchronizer. It btfnl t h a t i n t o r n u l c l ~ . ~ v n e h r n n i T n t l c m a l t P r ~ l l f o c n ~ n since ~i is doubtful that internal desynchroniz nization alters life span chronization Finger [21] reported that internal desynchronization n caused by shifting of the photoperiod cycle did not significantly alter the mean survival vival time o f rodents. The results o f this study indicated licated significant differences in physiological performance between LF restricted rats ats and DF restricted rats. DF restricted rats expressed a greater number o f feeding episodes fisodes per day, an increase in total time spent feeding per day, a decrease in the avera 'age food consumption per episode, and a decrease in the average time active per feedin ding episode, compared to LF restricted rats, thereby more closely emulating feedingg behavior in ad libtium rats. Also, the bimodal waveform and internal desynchronization ization, characteristic of metabolic, feeding, activity, and temperature variables in LF restricted rats, was completely eliminated by switching to nighttime feeding,, causing acrophase for these variables to resynchronize by shifting into the dark Iphase. Therefore, these results indicate that feeding during the dark phase promotes more normal behavior, thereby allow-
ly distinguish between caloric res ~cts ~ith the time-of-day of feeding. d after feeding in LF restricted rat Lad icted animals make rapid change ~lic lbility (Fig. 3 a). The average RQ i ted prior to feeding which indicated gen ~e animals were metabolizing prote ure e. The rapid rise in RQ to a ma: .01 ndicated that restricted rats shifte ein :metabolism to carbohydrate metabolism. Unlike the th ad libit hat ined a constant RQ throughout the day, indicating a constar rorid, and carbohydrate metabolism, calorically restricted restrict anima be to store glycogen reserves which resulted in a g radual d as ydrates were depleted. The fact that a period of hypol )othermia o ing iod of low RQ, may indicate that the depletion of glycogen ;igLmetabolic protection reaction resulting in lower m, metabolisrr md ~mperature (torpor). Lis study, as was reported in an earlier study [18], caloric calc restric SOvith a significant reduction in average body temperatu )erature per da if iiuction in body temperature (torpor) for a short inteT interval at a s of L ~ I ¢111~.1 1.11" I ~ 3 L I I k , L C U I ~ats LL~ 5ng the transition stage between dark and light in ILF LI and DF b). These results support a previous study [22] that showed sh a high correlation betweena torpor and photoperiod and suggest that hypothern ~othermia is involved in energy conservation 'ation and metabolic recovery. Restricted animals were we unable to maintain a constantLt body temperature during the inactive period without wit expending motor activity,, suggesting that increased level of activity in restricte :ted rats may be necessary to comIpensate for reduced thermoregulatory capability. Therefore, Tt metabolic and temperature ature regulation by the central nervous system appear~ ~ears to be altered by caloric restricti,Lon a n d / o r the reduced lean body mass and almost cc complete depletion of fat ; ~ rrestriction a e t r ; t - t ; r ~ n animals anlmqlc m ~ay r ~ l t ~ r ttheir h ~ ; r tthermal h~rnn~l ~ t~netllot in caloric ma, alter conductance, thereby accelerating the rate of heat transfer andd lowering body temperature during periods of basal metabolism and low activity outl ttput. Another possibility is that the low body temperature in restricted rats m a y be.~ a result of the rapid depletion of specific essential amino acids that are required for the biosynthesis of the neurotransmitters )f me1 metabolism and thermoregulation. associated with the regulation of The caloric restriction related ed changes in temperature and metabolism reported here support the results of other ~r studies run concurrently in our laboratory to determine the effect of age and caloric ric restr restriction striction on the levels of specific hepatic enzymes related to intermediary metabolism )lism and drug metabolism [23--25]. The activities of enzymes involved with glycol3tsis and lipid syntheses in rats maintained on caloric restricted diets were found to be ~e decreased and enzymes supporting gluconeogenesis were maintained or increased compared to control animals fed ad libitium. There-
ort alter hepatic metabolism for the g ion tions o f limited and efficient eneJ led supported by the high RQ values ( igh ats at the same 2 h after feeding in crease role for insulin and an incJ for I is also suggested. Preliminary fi est in n may be to retard the age-associa and to selectively induce other hep; talon changes indicate that the plasma restriction [24,; ; of several hormones may be altered by caloric restric pid interesting to speculate that the reduced body ternLperature ; of specific metabolic pathways towards low glycogen gly~ an ge, d for restricted rats, may significantly alter events at a the mol by tg DNA damage or by increasing DNA repair capat aabilities th, ing .~of aging [26--28]. Kayser [29] and Duffy et ai. [16] suggeste )rig m o f hibernating animals is related to the effects of o hypoth :ral life ;ators have suggested that a low core temperature sit;nificantl z slowing the age-related loss o f immunological ;ical response respc [30,3 tits study clearly indicate that in vitro caloric restriction restrictio studies )ne roper techniques to insure that tissues are prepared and speci ers nitored at the broad range of temperatures expressed [pressed . . l l [ ; ~ ; I d by U y restri( l [ ; ~ l . l l l , . L l ~ l d ll~ll.b 111 Lhis lllb properly emulate the actual in vivo conditions. McCarter 2arter et aL [15] and Masoro [32] reported me )orted that the metabolic output per gram lean b o,dy d mass is not significantly lowered by caloric restri(;tion. The results of the present study stud~ support this conclusion. However, restricted aanimals consumed fewer calories; but were able to maintain a higher level o f activity than tt ad libitum rats, with no incre; ease in metabolism. These results may indicate an Increase in~ in metabolic efficiency in restricted rats, since more work was done at the tt same rate of energy expenditure. iture. A possible reason that the caloric restriction-r~ restriction-related increase in motor , p n ~ r t ~ c l here h ~ r ~ was , u ~ f l c not n n t rreported F.nr~rt~cl m ; n previous n r a . v ; ~ n e studies e t n r l ; , ~ e is that motion detecting activity rreported equipment that measures animal al movement based on changes in position (photoelectric cells, infrared beams, etc.)I are not necessarily accurate indicators of total work done, and therefore data gathered ~ered in these systems can not be directly related to oxygen consumption (energy output) utput) resulting from motor activity. The system used in this study monitored parameters aeters such as force and velocity that can be directly related to energy output. Therefq ffore, it could accurately estimate the energy partition associated with gross motor activity. The significant changes in body temperature, motor activity and RQ reported here indicate that qualitative ch :hanges in metabolism, resulting from caloric restriction-induced alterations in metabolic abolic pathways, may modulate the aging process and the onset of chronic diseases bby triggering the onset of torpor. The results of this study also indicate that the physiolo wsiological variables that regulate energy output are
tored at frequent ric restriction
10
11
12 13 14 15 16
17 18 19
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er-
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and life prolongation. In C. Finch and t s.), Idbook o f the Biology o f Aging, Van Nostrand Reinhold, New York Yor (1977). [. McCay, M.F. Crowell and L.A. Maynard, The effect of retardq trded growth t i of ~pan and upon the ultimate body size. J. Nutr., 10 (1935) 63--79. • Yu, E.J. Masoro, I. Murata, H . A . Bertrand and F.T. Lynd, Life Li span stu~ her male rats fed ad libitum or restricted diets: longevity, growth, lea lean body ma • J+ ontol., 37(1982) 130--141. :l. • Yu, E.J. Masoro and C.A. McMahan, Nutritional influences or on aging of F sical, metabolic, and longevity characteristics. J. Gerontol., 40 (1985) 091 657--67 daeda, C.A. Gleiser, E.J. Masoro, l. Murata, C.A. M c M a h o n an and B.P. Yu, flu+ 1985) ,,s on aging of Fischer 344 rats. lI. Pathology. J. GerontoL, 40 (198 ~, 671--688 I. Ross, Length of life and caloric intake. Am. J. Clin. Nutr., 25 (l ~1972) 834--4 ~/eindruch and R.L. Walford, Dietary restriction in mice beginnin~ ,,tuning at one yea on ;pan and spontaneous cancer incidence. Science, 215 (1982) 1415-;--1418. • Goodrick, D.K. Ingrain, M.A. Reynolds, J.R. Freeman and N.L. Cider, Dif sol" ?mittent feeding and voluntary exercise on body weight and life sp~ ~an in adult ol., t983) 36--45. i. Sarkar, G. Fernandes, N.T. Telang, l.A. Kourides and R.A. Good, Go Low-calorie diet prevents the develo :levelopment of m a m m a r y tumors in C3H mice and reduces circulating circu prnlactin level, murine m a mamary m a r tumor virus expression, and proliferation of m a m m a r y alveolar alw cells• Proc. NatL Acad. Sci., USA, 79 (1982) 7758--7762. B.A. Ruggeri, D.M. Klurfeld and D. Kritchevsky, Biochemical alterations in 7,12-dimethylbenz,[a]anthracene-induced m a m m a r y tumors from rats subjected to caloric restriction. Biochim. Biophys. Acta, 929 (1987)239--246. D. Kritchevsk~ ~'ritchevsky, M.M. Weber and D.M. Klurfeld, Dietary fat versus; caloric ca content in initiation and promotion notion of 7,12-dimethylbenz[a]anthracene-induced m a m m a r y tumorigenesis tt in rats. Cancer Res., 44 (1984) 3174--3177. aollard and P P.bl. Luckert_ Tumori Tumorioenic indJ M. Pollard . H . Luckert, amorigenic effects of direct- and indirect-acting chemical carcinogens in rats on a restricted diet. J. Natl. Cancer Inst., 74 (1985) 1347-- 1349. B.D. Roebuck, D.D. Yager, D.S. Longnecker and S.A. Wilpone, Promotion by unsaturated fat of inogenesis in the rat. CancerRes., 41 (1981) 3961--3966. azeserine-induced pancreatic carcim R. Pearl, The Rate o f Living, Alfred red Knopf, New York (1928) 1--185. LP. Yu, Does food restriction retard aging by reducing the metaR. McCarter, E.J. Masoro and B.P. bolic rate? Am. J. Physiol., 248 (1985) l 985) E488--E490. 1. Hart, Effect of age and torpor on the circadian rhythms of body P.H. Duffy, R.J. Feuers and R.W. temperature, activity, and body weight in the mouse (Peromyscus leucopus). In J. Pauly and L. Scheving (eds.), Advances in Chronobiology, Part B., Alan R. 1Ass, Inc., New York (1987) 111--120. ~4elson, W. Runge and R.B. Sothern, Autorhythmometry proceF. Halberg, E.A. Johnson, W. Nelson lrements and their analysis. Physiol. Teacher, I (1972) l - - 1 l• dures for physiological self-measuremen! d-timing, circadian rhythms and life span of mice. J. Nutr., 116 W. Nelson and F. Halberg, Meal-timin (1986) 2244--2253. J. Aschoff, Exogenous and endoogenous components in circadian rhythms. Cold Spring Harbor Sympos. QuaL Biol., 24 (1960) 11--28.
a of
Ferman, Feeding schedules and the circadi 1 (1980) 39--65. tion shorten life? Chronobiologia, 10 (198." and R. Alosia, Relationships of light inten sernating ground squirrel, Citellus lateralis y, A. Turturro, R.A. Mittelstaedt and RA nzymes of intermediary metabolism in thq -189. J. Bazare, P . H . Duffy, R.H. Feuers and F atic drug metabolizing enzymes in the Fisc genase system. Mech. Ageing Dev., 48 (191 • Leakey, H•C. C u n n y , P.J. Webb, J. Bazare, P.H. Duffy, R . L Feuers F and F lging and caloric restriction on hepatic drug metabolizing enzyn 'mes in the F ects on conjugating enzymes• Mech• Ageing andDev., 48 (1989) 157--166. 15 Koizumi, R. Weindruch and R.L. Walford, Influences of dietar y restrictio~ yrne activities and liver peroxidation in mice• J. Nutr., 117 (1978) 361--367. 3, ,. Walford, S.B. Harris and R. Weindruch, Dietary restriction and aging: :hanisms and current directions. J. Nutr., Nutr. 117 (i 987) 1650-- 1654. Andahl and B. Nyberg, Rate o f depurination of native deoxyribo~ tribonucleic acid ~2) 3610--3618. Kayser, M a m m a l i a n hibernation• I. Hibernation versus hypothern ~othermia. Bull. A "yard University, 124 (1960) 1--29. ,. Walford, The Immunologic Theory o f Aging, Munksgaard, Cop Copenhagen (1 ~. Liu and R.L. Walford, The effect o f lowered body temperature on life spa -immune processes. Gerontologia, 18 (1972) 363--388. • Masoro, Mini-review: Food restriction in rodents: An ~evaluation evaluati . ¥ t ~ l . l U a l . l L l l l Iof d l ll.~ its lrcu l l . lg. J. Gerontol., 43 (1988) B59--64.
perera.
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