Circadian feeding and drinking patterns of genetically obese mice fed solid chow diet

Phystology & Behawor, Vol 43, pp 651-656 Copyright©Pergamon Press plc, 1988 Printed m the U S A

0031-9384/88 $3 00 + 00

Circadian Feeding and Drinking Patterns of Genetically Obese Mice Fed Solid Chow Diet ANN HO AND ADRIENNE

CHIN

The R o c k e f e l l e r Untverstty, N e w Yor~, N Y 10021 R e c e i v e d 7 M a r c h 1988 HO, A AND A CHIN Ctrcadtan feedmg and drml.ng patterns of genett~allv obese mt, e fed sohd cho~* dtet PHYSIOL BEHAV 43(5) 651-656, 1988 --Feeding and drinking were recorded in male ob/ob mice and lean mice fed pelleted Punna Lab Chow No 5001 w~th water to dnnk The clrcadmn patterns of eating and drinking of obese m~ce d~ffered from those of lean mine, m both the proportional temporal d~strtbut~ons of feeding and of dnnkmg behawor across the 24-hour day and m the absolute amounts consumed hourly The pattern of increased food consumption by the obese m~ce was different than that underlying increased water consumption When meal parameters were analyzed m terms of 'complete meals' of both feeding and dnnkmg (the end of a meal defined as at least 12 consecutwe minutes w~th no ingestion), obese and lean m~ce had the same number of meals and their periodicity was s~mdar, but meal size was much greater m the obese m~ce In the dark, both obese and lean m~ce showed strong postprandial correlations of meal s~ze w~th time from the start of a meal to start of the next meal C57BL/6J ob/ob mice

Meal patterns

Sohd food momtor

First, there is evidence that although usually coincident, and probably regulated by a common physiological mechanism [20], the expression of circadian rhythms of feeding and drinking may be dissociated [21] Also, while the use of liquid diets affords a reliable way to measure Intake, or to change palatability, " liquid diets are unnatural" [22] For these reasons, circadian feeding and dnnklng patterns were examined in C57BL/6J oh~oh mice and in lean littermates fed a standard solid chow diet and given water to dnnk

T H E pathophysiology of the genetically obese mouse C57BL/6J ob/ob has been extensively studied This animal model of spontaneous, hereditary obesity is of particular interest because the recessive autosomal gene underlying the disorder is maintained on a background strain which is one of the best studied of inbred mice, the C57BL/6J One particularly active area of research on the C57BL strains concerns the development and expression of behavioral circadian rhythms [5-11, 15-21, 29, 30] An early study found that the 24-hour cycling in food intake typical of normal-weight mice was " either absent or barely discernible" in the C57BL/6J oh~oh mouse [1] A later study reported that ob/ob mice (on a mixed C57BL/6J-9 background) showed " no significant diurnal variations in food consumption" [2] However, subsequent studies of the free-feeding of obese mice fed hquld diets found significant diurnal differences in food intake [23,27] In the latter very thorough study of free-feeding 24-hour meal patterns, oh~oh and normal C57BL/6J mice were found to consume 72-75% of their liquid diet in the 12-hour dark phase of the hght-dark cycle [27] A detailed analysis of meal patterns is important for understanding the factors that initiate a meal, determine its size, and determine the length of intermeal intervals The possible role that each of these factors plays in the hypero phagia of the oh~oh mouse has stimulated much research [ 14, 24-26, 28] Studies that examine satiety factors are best understood in light of free-feeding, spontaneous meal taking and for such normative data in the oh~oh mouse, there is only the study of Strohmayer and Smith [27] But there are comphcations in studying Ineal patterns of mice fed liquid diets

METHOD

Animals Eight male C57BL/6J ob/ob and eight lean male littermates (9/+), eight weeks old on arrival from the Jackson Laboratory, were studied Animals were given two weeks' acclimation to individual caging in temperature-controlled rooms (22-+2°C) with a 12-12 hour light-dark cycle and free access to Purina Rodent Lab Chow No 5001 and acidulated water (6 drops HCl/hter) Mice were weighed, their cages and hardwood litter changed, their food weighed, and water volumes recorded weekly in the middle of the dally light period On arrival the obese mice weighed 43 5-+1 1 g and lean mice weighed 25 4-+0 6 g During the third week on this regimen, when a detailed examination of food and water intake was performed, obese mice weighed 49 4-+1 1 g and lean mice weighed 26 2-+0 5 g, indicating that both groups were showing the weight gain to be expected in this period of dynamic growth

651

652

HO A N D C H I N

Appat atu~

CIRCADIAN FEEDING PATTERN I

Drinking behavior was continuously monitored electronically in the mouse's home cage as described earher [7] Two graduated 100 ml water bottles with stainless steel dnnklng tubes fitted with stainless steel ball beanngs to reduce drlppmg were presented above a raised steel ground bar on which the mouse had to place its paw(s) m order to hck at a bottle, closing the electronic clrcmt for hck detection Each mouse was given two water bottles to facdltate calculation of the regression of volume consumed against hcks on a dally basis providing a check on the measurement of volumes consumed and the integrity of the electronic monltonng system [7] Feeding behavior was continuously monitored m the home cage as the number of seconds of actwe gnawmg detected by an electronically-fitted food hopper that sensed movement of pelleted chow To avoid vibration artifacts, a nonretrlggerable one-second pulse was the signal produced by movement of the food pellets as the mouse gnawed at them The food hopper was placed in the standard steel cage top so that the mouse could reach the pellets through the cage-top bars in the usual manner Food hopper counts were recorded and stored In the same way as hck counts [7] An earher study had shown a slgmficant difference between mice In seconds spent gnawing with no significant difference m sensitivity between individual food hoppers Thus, m comparing ammals, it is important to take total dally food retake by weight into account as described below

Data Analysts Licks of water (at both bottles) and seconds of active gnawing were cumulated and polled every six minutes The data of one 24-hour period in the third week in the laboratory for each mouse were selected for detailed analysis The day of cage service was excluded and only a day in which feeding and drinking data were representative of the mouse's usual behavior was used Each mouse's feeding and dnnklng behavior were analyzed by three measures number of hcks of water or counts of feeding in each six-minute Interval, the percent of the day's total that occurred in each interval, and the amount of water drunk (ml) or food eaten (g) in each interval calculated by multiplying the percent in each six-minute epoch by the total dally intake of the mouse Weight of chow consumed was uncorrected, measured fluid volumes were corrected for dripping and evaporation by subtracting 0 5 ml from each bottle's dally change An analysis of feeding patterns was made for each mouse using the criterion of at least one six-minute recording epoch without eating to define the end of a bout of eating An analogous examlnaUon of dnnkmg patterns was made using at least one epoch without drinking to define the end of a dnnklng bout

Criteria j o r a 'Complete Meal' and the Postprandial Correlation Episodic feeding terminated by resting or sleeping has been used as a behavioral definition of a meal in the mouse [24,27], but the behavioral observation necessary for this criterion is too costly for routine use In the study reported here, the utdlty of another definition was explored a 'complete meal' was defined as any sequence of eating or dnnking temporally separated from any other ingestive behavior by at least twelve minutes (two six-minute epochs with neither

i= •-~ 08

A

• OBESE

o LEAN

n,,,. o

:= 06

Z

,~ o4 o 02 i

14

CIRCADIAN DRINKING PATTERN

08

0

500pm 500om TIME OF DAY

FIG 1 Circadian retake patterns ot eight obese (0) and eight lean (©) mice The 12 hours of darkness (shaded) are shown in the middle of the 24-hour recording day The circadian dmtnbutlon of feeding (g chow/hour) is shown m A and that of drinking (ml water/hour) m B While the hourly consumption of chow and of water did not differ in the first few hght hours (after 5 00 a m ), food retake of obese mice is greater m later hours of the hght penod and much greater m the early and late part of the dark period Water drinking by obese mice was greater than that of leans chiefly m the dark

feeding nor drinking) This analysis better reflects the observed behavioral patterns of the mouse fed sohd chow, with its repeated switches between eating and drinking interspersed with pauses of varying lengths Indeed, from a nutritional point of view, a complete meal includes the water necessary for the metabohsm of the food eaten The pattern of 'complete meals' was used to determine if there were any differences between the obese and lean mice in number of meals, meal size in grams, periodicity of meals (measured as time elapsed from the start of one meal to the start of the next), and length of the lntermeal interval (the time between the end of a meal and the start of the next meal) Two methods were used to examine the postprandial correlation of meal size with the interval between that meal and the next meal The regression was calculated of time from meal-start to meal-start on meal size, a method analogous to that of Le Magnen and Tallon with the rat [12,13] A separate regression was calculated of the lntermeal interval (time between the end of a meal to the next meal-start) on meal size, a method analogous to that of Strohmayer and Smith [27] Meals and mtervals between them that fell across the transitions between light and dark were not included in these analyses RESULTS Obese mice both ate and drank more than lean mice Obese mice consumed 7 4_+0 4 g of chow daily compared to 4 2_+0 1 g of chow eaten by lean mice (t=7 80, p < 0 001)

E A T I N G AND D R I N K I N G PATTERNS OF OBESE MICE

653

Obese mice drank 9 4___1 0 ml of water compared to 4 4-+0 3 ml for lean mice (t=4 81, p < 0 001)

MEAL SIZE

Circadian Intake Patterns The circadian feeding pattern of each group may be seen in Fig 1A The amount of chow consumed by each mouse in each hour was summed, these sums were used to calculate hourly means for each group, and these were plotted with the active 12-hour dark period in the center of the 24-hour day The corresponding circadian drinking patterns may be seen in Fig 1B There was a strong daily rhythm charactenzed by much lower levels of eating and drinking for both groups d u n n g the light portion of the daily lighting cycle Both groups showed a three-peaked pattern of dnnking found in normal C57BL/6J mice [7,16], although the second peak occurred later in the obese group The circadian feeding pattern was three-peaked in the lean mice but was more like a " d a w n and dusk" two-peaked pattern in the obese mice Thus, obese mice ate much more food than lean mice during the early and late parts of the 12-hour dark period, but they drank more water then lean mice m the early, middle, and latter parts of the dark period, corresponding to their threepeaked drinking pattern Examination of the percent of each mouse's eating and dnnking was used to determine if the relative distribution of total intake d u n n g the light and dark portions differed between groups Obese mice ate 28 2-+3 6% of their food in the light portion compared to 16 6-+2 2% for lean mice (t=2 75, p < 0 05) There was no significant difference between groups in the distribution of dnnklng by this measure Obese mice did 16 5-+3 7% of their drinking in the hght compared to 14 4-+3 2% by lean mice Thus, obese mice show increased food intake during the light compared to lean mice, but for both groups, drinking behavior was rather tightly confined to the dark portion of the lighting cycle

Feeding Patterns For each mouse, the distribution and size of bouts of feeding were calculated (without regard to drinking) and significant differences between the obese and lean groups were found in several parameters Obese mice initiated fewer feeding bouts per day than did lean mice, 16 3--_1 0 compared to 20 9-+1 2 (t=2 95, p < 0 05), but the amount consumed in these bouts was significantly greater Bouts of feeding initiated in the 12-hour dark portion of the lighting cycle by obese mice were twice as large as those of the lean 0 6 0 - + 0 0 6 g vs 0 3 1 - + 0 0 6 g ( t = 3 4 1 , p < 0 0 1 ) Bouts of feeding begun in the light by the obese mice were three times as large 0 32-+0 04 g compared to 0 ll_+0 02 g (t=4 67, p < 0 001)

Drmkmg Patterns Water consumption of each mouse was analyzed into dnnklng bouts in an analogous manner (without regard to feeding) When the distribution of drinking bouts and the size of bouts were calculated, quite a different pattern emerged for dnnklng than had been found for feeding Obese mice initiated drinking bouts significantly more often than lean mice 26 0_ 1 5 bouts started by obese mice vs 17 6-+ 1 1 bouts started by lean mice (t=4 52, p < 0 001) Furthermore, this increased number of bouts was due to more bouts started in the dark by the obese mice, 19 3-+1 6 bouts compared to 12 3_+1 5 bouts (t=3 20, p < 0 01) The amount

08

I-I OBESE F~ LEAN

L

~06

I.LI N

_

~04 <:[ W 02

LIGHT

pARK

LIGHTING PHASE IN WHICH MEAL ENDED

FIG 2 Meal size in g (mean_+SEM) of eight obese (dotted columns) and eight lean (hatched columns) mice in the light and m the dark phases of the 12-12 hour light-dark cycle A period of at least 12 minutes without eating or drinking was used to determine a meal's end Analysis of variance showed a significant dtfference between groups, F(1,14)=10 38, p<0 01, and a significant effect of hghtlng phase, F(1,14)=31 18, p<0 001, with no significant mteractmn

consumed by obese mice in these drinking bouts, however, was not significantly greater than that of lean mice Obese mice drank 0 41_+0 05 ml of water per bout in the dark while lean mice drank 0 31-+0 03 ml water There was no significant difference between obese and lean mice in either the number of bouts started in the light or in their size Obese mice drank 6 8-+0 7 bouts in the light while lean mice drank 5 4-+1 0 bouts In the light, obese mice drank 0 25-+0 06 ml of water per bout, lean mice drank 0 14---0 03 ml of water per bout Thus both groups started about the same number of dnnking bouts in the light, and these bouts were approximately half the size of bouts drunk in the dark The greater total water intake of the obese mice was accomplished chiefly through an increase in the number of bouts of drinklng in the dark

Complete Meals An analysis of both feeding and dnnklng behavior in units of a 'complete meal' was undertaken to determine number of meals and meal size during the light and dark phases All meals were categorized according to whether they ended in the light or in the dark phase of the lighting cycle While there were no slgmficant differences in number of meals between obese and lean mice in the dark (6 4-+0.7 vs 7 3-+0 8 meals) or in the light (6 8-+0 7 vs 6 9-+0 6 meals), there was a significant difference between groups in meal size across both parts of the day as may be seen in Fig 2 A repeatedmeasures analysis of variance of mean meal size for each mouse at each time of day resulted in a Group, F(1,14)=10 38, p < 0 01, and a Light Phase, F(1,14)=31 18, p < 0 001 There was no significant Group × Light Phase interaction ( F < I 00) Thus, while both obese and lean mice initiated about the same number of meals in the light and in the dark, and both groups showed the same average differ-

654

HO A N D CHIN TABLE 1 PERIODICITY OF MEALS AND INTERMEALINTERVALS

Group Light Obese Lean Dark Obese Lean

Meal Start to Meal Start Interval*

Intermeal Intervalt (End of Meal to Start of Next)

119 _+ 12 103 _+ 8

82 _+ 10a 67 ± 6a

104 _+ 17 97 +_ 8

53 _+ 7" 41-*- 7a

TABLE 2 POSTPRANDIALCORRELATIONS Meal Size With Interval From Start of This Meal to Start of Next* Group

*Interval m minutes (number of epochs × 6) from the start of one meal to the start of the next, expressed as mean _+ SEM Method analogous to that of Le Magnen and Tallon [12,13] tlnterval in minutes from the end of one meal to the start of the next Method analogous to that of Strohmayer and Smith [27] ~The only significant difference found was between lntermeal intervals m hght vs dark phases of the light-dark cycle F(1,14)=12 69, p<0 005

Light Obese Lean Dark Obese Lean

Meal Size With Intermeal Interval (This Meal's End to Start of Next)t

r

n

I?

r

n

1;

100 518

51 52

NS 0 001

044 135

51 52

NS N S

724 713

48 52

0 001 0 001

- 085 412

48 52

NS 0 01

Pearson r of meal size (g) with mtervals m minutes, n=meals pooled across mice m each group in each phase of the light-dark cycle *Method analogous to that of Le Magnen and Tallon [12,13] CMethod analogous to that of Strohmayer and Smith [27]

DISCUSSION ence in meal size from light to dark, the meals of obese mice were slgmficantly larger than those of leans both in the light and in the dark Pertodtttt~ lntermeal lntervalv and Postprandtal ( orrelatton

No slgmficant differences in meal-to-meal perlodloty between obese and lean mice or between light and dark parts of the day were found (see Table 1) Ttus is remarkable in that there is so much less eating and dnnklng in the light phase of the lighting cycle Thus mice initiate just as frequent but much smaller meals dunng this time There was a significant &fference m lntermeal intervals between light and dark phases of the lighting cycle as shown m Table 1 Repeated measures analysis of variance showed a significant &fference between light and dark portions of the day, Light Phase, F(I,14)=1269, p < 0 0 0 5 The difference between obese and lean mice was not significant, Group, F(1,14)=3 52, p < 0 10, and there was no slgmficant Group × Light Phase interaction ( F < 1 00) Thus it may be seen that lmtlatlon of meal taking is a perlo&c behavior m both obese and lean mice, in both hght and dark phases of the hght/dark cycle Intermeal intervals were longer in the hght when meals were smaller (when less time was spent eating) Significant postprandial correlations of the Interval from the start of the meal to the start of the next meal with meal size were found in both obese and lean mice in the dark as shown m Table 2 A test of the difference between the slopes of the regression lines for obese mice (54 6) and lean mice (92 3) was significant t ( 9 6 ) = - 2 8 2 , p < 0 01 This is consistent with the fact that although the obese mine ate larger meals, the perlodloty of meal lnmatmn did not differ slgmficantly between obese and lean mice In the hght, only the lean mice had a slgnLficant postprandial correlatmn using this measure With the postprandial correlation based on mtermeal intervals defined as the time between the end of the meal and the beginning of the next, the only significant correlation found was that of mtermeal interval with meal size of the lean mine m the dark

This study has documented vigorous circadian patterns ot teedlng and of drinking in C57BL/6J o b / o b mice fed solid food, supporting the finding of robust diurnal differences m their intake of a llqmd diet reported by Strohmayer and Smith [27] It may be that Anhker and Mayer faded to find a 24-hour peno&clty in food intake dunng the dynamic phase of weight gain because the mice were required to press a bar to obtain food pellets [1] The obese mouse may not have eaten as much at night as it would have under completely free-feeding condmons due to its documented deficit m energy expenditure [4] To the extent that it 'caught up' dunng the light part of the cycle, diurnal differences m food consumptlon would be reduced This explanation, although speculative, is consistent with the authors' comment that a lower position of the lever worked better for the very obese mine, and by same authors' finding of a 24-hour p e r l o & o t y In obese mice past the dynamic growth stage when they do not eat as much on a daily basis In any case. m the study reported here, both obese and control mice exhibit strong circadian patterns of feeding and of drinking Obese mice, however, were found to differ from lean mice in tbelr circadian intake patterns in two ways They ate proportionately more in the light phase of the hght/dark cycle than did lean mice, while their drinking showed the same light/dark proportions as lean mice Also, when feeding and dnnkmg were analyzed in the smallest temporal units available in this study, 6-minutes epochs, the pattern of feeding showed larger but not more frequent bouts of feeding in obese compared to lean mine, and more frequent but not larger drinking bouts Thus, the temporal mlcrostructure underlying the greater total dally feeding differed from that underlying dnnkmg Besides further dehneatmg differences in the patterns of food retake by obese mice, these findings add to the evidence of dissociation between the regulation of feeding and dnnklng When both feeding and dnnklng were used in the analysis of 'complete meals,' meal size in terms of grams of chow eaten per meal was greater in obese than in lean mice These results support and extend the report on mice fed hquld dl-

EATING AND DRINKING PATTERNS OF OBESE MICE ets, a n s w e n n g in the a f f i r m a t i v e t h e q u e s t i o n o f S t r o h m a y e r a n d S m i t h o f w h e t h e r d~fferences in m e a l size f o u n d b e t w e e n o b e s e a n d lean mice fed l i q m d diet w o u l d also b e f o u n d w i t h s o h d f o o d [27] I n t e r m e a l i n t e r v a l s o f t h e o b e s e mice d e f i n e d as p e r i o d s o f n o i n g e s t i o n b e t w e e n ' c o m p l e t e m e a l s ' did n o t differ f r o m t h o s e o f lean m i c e , in a g r e e m e n t with w h a t w a s f o u n d w h e n a b e h a v i o r a l " s a t i e t y s e q u e n c e " w a s u s e d [27] P e r h a p s t h e s i m p l e s t w a y to c h a r a c t e r i z e the feeding beh a v i o r o f o b e s e m i c e w o u l d b e t h a t the p e r i o d i c i t y o f m e a l m i t i a t l o n (or n u m b e r o f m e a l s p e r day) is t h e s a m e as in lean m i c e b u t t h e a m o u n t e a t e n in e a c h m e a l is m u c h g r e a t e r Th~s is true w h e t h e r a 6 - m m u t e i n t e r v a l is t a k e n to e n d a b o u t of feeding ( w i t h o u t r e g a r d to d n n k i n g ) o r a l o n g e r 12-minute Interval w i t h o u t e i t h e r f e e d i n g o r d n n k i n g is t a k e n to e n d a 'complete meal ' It h a s b e e n s u g g e s t e d t h a t abd~ty to find a significant corr e l a t i o n b e t w e e n p o s t m e a l i n t e r v a l a n d m e a l size d e p e n d s u p o n the l e n g t h o f the e n d - o f - m e a l c r i t e r i a [3] o r t h e c o m p l e t i o n o f a b e h a v i o r a l l y d e f i n e d satiety s e q u e n c e [27] In the s t u d y r e p o r t e d h e r e , h o w e v e r , slgmficant p o s t p r a n d i a l correl a t i o n s w e r e f o u n d in the d a r k for b o t h o b e s e a n d lean mice u s i n g a 12-minute e n d o f m e a l c r i t e r i o n if the p o s t m e a l interval w a s d e f i n e d as time e l a p s e d from m e a l - s t a r t to n e x t m e a l - s t a r t If l n t e r m e a l i n t e r v a l ( e n d o f m e a l to n e x t mealstart) w a s used, o n l y the lean mice s h o w e d a slgmficant p o s t p r a n d i a l c o r r e l a t i o n W h a t is i m p o r t a n t here is t h a t in t h e

655 f r e e - f e e d i n g s i t u a t i o n , i n g e s t i o n is p e r i o d i c a n d t h a t this p e r i o d i c Initiation o f m e a l s ( w i t h o u t a m i n i m u m c r i t e r i o n o f w h a t c o n s t i t u t e s a meal) s e e m s to h e m o d u l a t e d b y t h e a m o u n t e a t e n in t h e last m e a l O b e s e a n d lean m i c e d o n o t differ in t h e p e r i o d i c i t y o f m e a l m l t m t i o n , a n d b o t h s h o w t h a t the a m o u n t c o n s u m e d in a g i v e n meal in t h e d a r k m o d u l a t e s the t i m i n g o f the n e x t m e a l ' s initiation T h e p r o p o s e d definition o f a c o m p l e t e m e a l as a n y e a t i n g o r d r i n k i n g s e p a r a t e d t e m p o r a l l y b y a specified p e r i o d o f n o i n g e s t i o n s h o u l d p r o v e to b e useful in studies in w h i c h fluids are i m p o r t a n t & e t a r y c o m p o n e n t s o f the m e a l S u c h s t u d i e s w o u l d include t h o s e o f a n i m a l s offered sugar, a l c o h o l o r o t h e r caloric s o l u t i o n s to d r i n k T h i s m e t h o d w o u l d p r o v i d e i n f o r m a t i o n o n the retake o f n o n c a l o r i c s o l u t i o n s in t h e patt e r n o f m e a l taking in the m o u s e a n d c o u l d b e u s e d to d e t e r m i n e if o b e s e mice r e g u l a t e caloric a n d n o n c a l o n c i n t a k e in t h e s a m e w a y as t h e i r lean c o u n t e r p a r t s

ACKNOWLEDGEMENTS We thank Alan Lipton for his painstaking development of the food hoppers that detect gnawing, the Electronics Laboratory and Electronics Shop for continuing techmcal assastance, Seth Coplan for computer programming, and Evelyn Archibald for blbhographlc assistance This work was supported by grants from NIAAA (AA06368) and from the John L and Helen Kellogg Foundation

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