Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Centrally administered urocortin 2 decreases gorging on high-fat diet in genetically prone
diet-induced obese rats
Pietro Cottone, Ph.D.1,2,3*; Valentina Sabino, Ph.D.1,2,3*; Tim R. Nagy, Ph.D. 4; Donald V.
Coscina, Ph.D.5; Barry E. Levin, M.D.6, 7; Eric P. Zorrilla, Ph.D. 1,3
1
Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La
Jolla, CA, USA; 2Laboratory of Addictive Disorders, Departments of Pharmacology and Psychiatry,
Boston University School of Medicine, Boston, MA, USA; 3Harold L. Dorris Neurological Research
Institute, The Scripps Research Institute, La Jolla, CA, USA; 4Department of Nutrition Sciences,
University of Alabama at Birmingham, Birmingham, AL, USA;
5
Departments of Psychology, and
Nutrition & Food Sciences, College of Liberal Arts & Sciences, and Departments of Psychiatry &
Behavioral Neurosciences, and Internal Medicine, School of Medicine, Wayne State University, Detroit,
MI, USA;
7
6
Neurology Service (127C), VA Medical Center, East Orange, New Jersey, USA,
Department of Neurology and Neurosciences, New Jersey Medical School, UMDNJ, Newark, New
Jersey, USA
*These authors equally contributed to this work.
Correspondence and requests for materials should be addressed to:
Pietro Cottone (Email: cottone@bu.edu)
Laboratory of Addictive Disorders, Departments of Pharmacology and Psychiatry
Boston University School of Medicine
72 E Concord St, R-618
Boston, MA 02118 USA
Phone: 617-638-5662 / Fax: 617-638-5668
and
Eric P. Zorrilla (Email: ezorrill@scripps.edu)
Committee on the Neurobiology of Addictive Disorders, SP30-2400
The Scripps Research Institute
10550 N. Torrey Pines Road
La Jolla, CA 92037 USA
Phone: 858-784-7416 / Fax: 858-784-7405
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Supplementary materials and methods
Subjects
Male genetically-selected Diet-Induced Obesity (DIO) (n=10) and Diet Resistant (DR) (n=10),
offspring of rats from the original colonies of DIO and DR rats bred by Levin et al (Levin et al., 1997),
were born at The Scripps Research Institute. Rats were maintained in a 12:12 hr reverse-lighting cycle in
a humidity- (60%) and temperature-controlled (22 °C) vivarium. Rats had access to Harlan Teklad LM485 Diet 7012 chow (65.0% of kcal from carbohydrate, 13.0% from fat, and 21.0% from protein; energy
= 3.1 kcal/g; Harlan Teklad, Indianapolis, IN) and water ad libitum before the beginning of experiments.
Procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH Publication number 85-23, revised 1996) and the “Principles of laboratory animal care”
(http://www.nap.edu/readingroom/bookslabrats) and were approved by the Institutional Animal Care
and Use Committee of The Scripps Research Institute.
Drugs
Rat Urocortin 2 (Ucn 2) and angiotensin II, generously provided by Dr. Jean Rivier (The Salk
Institute, La Jolla, CA), were synthesized by solid-phase methodology and characterized as previously
described
1, 2
. Ucn 2 was freshly dissolved in sterile water and then diluted with phosphate-buffered
saline (PBS) to attain the desired concentrations in a final vehicle of 0.5X PBS (pH=7.4). Angiotensin II
was dissolved in 1X PBS (pH=7.4).
Intra-cranial surgery and injection procedures
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Cannulae were directed at the third ventricle 3 because type 2 urocortins are known to suppress
food intake at least in part via hypothalamic sites of action
3, 4
. Anesthetized (isoflurane, 2–3% in
oxygen) subjects were each stereotaxically (David Kopf Instruments, Tujunga, CA, USA) implanted
with a single stainless steel, 24-gauge guide cannula (Plastics One, Inc., Roanoke, VA, USA) above the
third ventricle (3v), terminating 3 mm above the final injection site. Each cannula was positioned 0.8
mm posterior to bregma on the midline, terminating 3.5 mm below the outer surface of the skull with the
interaural bar set at flat skull (dorsal/ventral: bregma=lamda). Validated coordinates
3, 5
were based on
the atlas of Paxinos and Watson 6. A dummy stylet (Plastics One) maintained patency.
For testing, drug solutions or vehicle (2 µl) were injected manually over 90 sec with a Hamilton
microsyringe linked by PE 20 tubing to a 31-gauge stainless steel injector projecting 3 mm beyond the
tip of the guide cannula. Injectors were left in place for 1 additional min for diffusion. Rats were
maintained in familiar holding cages during the pretreatment interval and then returned to their
enclosures, with monitoring of nose-pokes beginning immediately. Cannula placements were
functionally verified at the conclusion of all testing as a positive dipsogenic response (>5 ml water
intake within 30 min) to 3v angiotensin II (100 ng/2 µl).
Microstructural analysis of ingestion
Apparatus
To study the microstructure of ingestion, rats were individually housed in Plexiglas test cages
(22×22×35 cm) equipped for this purpose
7, 8
. Rats obtained individual 45-mg pellets from a trough
replenished by an automated dispenser (Med Associates, St. Albans VT). Inside the enclosure rats were
first trained using a chow diet (45 mg precision 5TUM: carbohydrate 65.5% (kcal), fat 10.4%, protein
24.1%, 3.7 kcal g−1;Test Diet/Purina Mills, Inc., Richmond, IN, USA), but ultimately tested with a high-
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
fat diet. The microstructure high-fat diet (F56381: fat 34.9% [kcal], carbohydrate 46.4%, protein 18.7%,
4.2 kcal/g; Bioserv, Frenchtown, NJ) was a 45-mg precision-pelleted variation of the high-fat diet that
rats consumed in their home cages (D12266B). The acquisition of a pellet was detected by photobeam
interruptions (0.5 sec duration) caused by the rat opening a freely swinging door to access the pellet in
the trough. An additional pellet was not delivered until the door returned to its neutral position and a
3.25 sec timeout period elapsed, to prevent duplicate pellet deliveries
3
and allow the study of feeding
with pellet-to-pellet resolution. Water delivery (0.1 ml) into a reservoir was governed by a responsecontingent solenoid (W.W. Grainger, Lincolnshire, IL) activated by nose-poke interruption (0.5 s) of
photobeams monitoring a separate hole, with a 1-sec timeout period to prevent duplicate deliveries.
Responses were recorded by a computer with 10 msec resolution 9, 10.
Study design
Beginning from 50 days of age, DR and DIO rats were provided only a high-fat diet in their
home cages (D12266B, 32.0% fat [kcal], 51.0% carbohydrate, 17.0% protein; 4.4 kcal/g; Research
Diets, New Brunswick, NJ) unless otherwise specified. Rats learned how to obtain food and water in the
microstructure enclosures beginning from 110-120 days of age. During this period, rats lived in the
microstructure enclosures continuously except for brief periods (30 min) immediately before the dark
cycle onset, when they were removed for apparatus maintenance. The dark cycle and test sessions began
at 10:00AM. After establishing stable daily food and water intakes (12-13 sessions), rats were returned
to their home cages for chronic diet exposure. At 218 days of age, rats were implanted with the 3v guide
cannulae and allowed to recover for one week. Microstructure sessions resumed at 226 days of age.
After re-attainment of stable food intake (12-13 sessions), rats were provided high-fat diet access in the
enclosure. After high-fat diet intakes stabilized (<15% food intake variation across 3 consecutive days),
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
spontaneous baseline high-fat diet intake and meal microstructure of DIO vs. DR high-fat rats were
measured at 241 and 242 days of age. To determine the effects of acute central Ucn 2 administration on
high-fat diet intake, rats then received 0, 0.1, 0.3, 1, or 3 µg doses, 10 min before testing. Doses, based
on previous studies
3, 11, 12
, were given using a within-subject Latin square design with 1-2 intervening
treatment-free days beginning from 244 days of age. Food and water intakes were monitored as
nosepoke responses for 23.5 hr.
Meal pattern analysis
Microstructure analysis used a meal definition that recognizes the existence of prandial drinking
within meals 13. For both genotypes, this analysis revealed two distinct peak distributions of log-normal
(ln)-transformed interfeeding intervals (IFI), a much larger, faster distribution of intra-meal intervals and
a slower distribution of between-meal intervals. A value that lay between both distributions for both
genotypes (300 sec between feeding or drinking events) was defined as the threshold intermeal interval
(IMI) 3. Meals were defined to contain a minimum of 0.09 g of food (2 pellets), and descriptive statistics
of average nocturnal and diurnal meal structure were calculated separately. Parameters included the
number of meals, the average size and duration of meals, and the average IMI. Overall meal duration
was calculated as the total interval from the first to last response of a meal, and duration of eating (or
drinking) within the meal was calculated as the sum of the durations of bursts of eating (or drinking)
where the duration of each burst was defined as the interval from the first to the last consecutive
response for food (or water). Thus, transitions between eating and drinking were included in total meal
duration, but not in the duration of eating or drinking. Meal sizes for eating and drinking were calculated
separately as the average number of food- or water-directed responses during meals. The IMI was
defined as the interval from the last response of a meal to the first response of the next meal.
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Within-meal microstructure analysis
To identify differences between high-fat diet-fed DR and DIO rats in the rate and regularity of
sustained eating within meals, analysis of the ln-transformed duration of consecutive (uninterrupted by
drinking) within-meal IFIs was performed
3, 8
. The mean, standard deviation, kurtosis, skewness and
histogram entropy of the ln-transformed duration of each subject’s consecutive IFIs was individually
determined and then averaged across subjects. Histograms were constructed from ln-transformed IFI
durations that fell from e1 to e3 sec (~2.7-20.1 sec), with a bin width of e0.1. Significant increases in the
standard deviation (a measure of continuous variability, reflected in the horizontal spread of the
histogram) or the histogram entropy (a measure of categorical variability, reflected in an increasing
number of populated histogram bins, each with more similar event frequencies) would indicate a
decreased regularity of intake. Conversely, a decrease in the kurtosis of the inter-pellet interval
distribution (a measure of the distribution's 'peakedness’, reflected in a flatter top and taller tails of the
distribution) would be consistent with a decreased regularity of pellet-to-pellet feeding within meals 3.
Finally a significant increase in the positive skewness (a measure of the distribution’s right-tailed
asymmetry) would be consistent with an increase in the relative frequency of fast pellet-to-pellet intake.
Fat pad and body composition analysis
Two days after completing the Ucn 2 study, animals were euthanized (262 days of age), and their
frozen carcasses were shipped to the University of Alabama at Birmingham for chemical analysis of
body composition. Carcasses were thawed (room temperature) and weighed to determine freezingrelated water loss. Gastrointestinal tracts were removed to determine eviscerated weight. Visceral
(gonadal, retroperitoneal, mesenteric) and non-visceral (inguinal, subcutaneous) white fat pads and
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
brown adipose tissue (BAT) were dissected, weighed, and returned to the carcass for composition
analysis. Total body water, fat mass, and fat-free dry mass (FFDM) were determined 14.
Statistical analysis
To compare the time courses of ingestion by DR and DIO rats, split-plot analyses of variance
(ANOVAs) were performed on the incremental average nocturnal and diurnal intake of food during 1-hr
time bins from the two baseline days, with Genotype as a between-subjects factor and Time as a withinsubject factor. Student’s t-tests were used to identify genotype differences in the meal microstructure of
food intake and inter-pellet interval frequency histogram measures within the dark and light cycles.
To compare the time course of Ucn 2 anorexia between high-fat diet fed DIO and DR rats, a
three-way mixed ANOVA was performed on the incremental intake of food during 1-hr time bins, with
Dose and Time as within-subject factors and Genotype as a between-subjects factor. Meal pattern and
inter-pellet interval frequency histogram measures were analyzed by split-plot ANOVA with Dose as a
within-subject factor, and Genotype as a between-subjects factor. Linear contrasts were performed to
determine whether Ucn 2 treatment exerted a log-linear, dose-response effect on measures of ingestion.
In all analyses, intermeal interval durations were transformed to logarithmic values to account for the lnnormal time scale of post-meal intervals 3, 13, 15.
To compare the degree to which fat pad and whole carcass fat weights were disproportionately
increased (after controlling for differences in lean body mass), analysis of covariance (ANCOVA) was
used
16
, with the fat measure as the dependent measure, genotype as the between-subjects factor and
non-fat mass as a covariate. To allow comparison with other work, fat pad mass and whole carcass fat
also were expressed as a percentage of body weight and analyzed by Student’s t test.
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
For interpretation of main effects of repeated measures with more than two levels (e.g., Dose),
post hoc pair-wise comparisons were performed within the general linear model. Student’s unpaired ttests were used for between-subject factors having only two levels. The software packages used were
Systat 11.0 (SPSS, Chicago, IL, USA), Excel 2003 (Microsoft, Redmond, WA, USA), SigmaPlot 11.0
(Systat Software, Inc., Point Richmond, CA, USA), and InStat 3.0 (GraphPad, San Diego, CA, USA).
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Supplementary Table 1
Table 1. Differences in baseline prandial intake of high-fat diet-fed genetically
selected DR and DIO rats
Parameter
Dark Phase
Light Phase
DR
DIO
DR
DIO
Feeding
Intake (g)
22.9±1.1
22.3±1.3
6.8±0.5
6.6±0.8
Duration (min)
73.0±5.1
57.8±5.9
22.8±1.7
17.4±1.6 *
Drinking
Intake (ml)
15.0±2.2
4.8±0.6 ***
4.0±0.6
1.8±0.3 **
Duration (min)
32.0±4.1
15.9±2.7 **
10.8±1.8
6.0±1.4
Prandial feeding and drinking of genetically-selected diet-induced obesity-resistant (DR) and
susceptible (DIO) rats (n=10/genotype) fed a high-fat diet. Data represent the M±SEM quantity or
duration of food and water intake within meals of male DR and DIO rats during the dark cycle and the
light cycle and were calculated from the average of two consecutive sampling periods measured at 241242 days of age. Symbols denote significant differences: * p<0.05 compared to DR rats, ** p<0.01, ***
p<0.001, (unpaired Student’s t-test).
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Supplementary Table 2
Table 2. Effects of i.c.v Ucn 2 on 12h meal microstructure in
high-fat diet-fed genetically-selected DR and DIO rats
Parameter
Feeding
Meal Frequency (meals) ##
0 µg
0.1 µg
0.3 µg
1 µg
3 µg
Avg Intermeal Interval (min)
0 µg
0.1 µg
0.3 µg
1 µg
3 µg
Drinking
Avg Meal Size (ml) ##
0 µg
0.1 µg
0.3 µg
1 µg
3 µg
Avg Meal Duration (min)
0 µg
0.1 µg
0.3 µg
1 µg
3 µg
Drinking Rate (µl/sec) ###
0 µg
0.1 µg
0.3 µg
1 µg
3 µg
DR
DIO
7.3±0.2
6.4±0.1
5.9±0.1
5.9±0.1
6.4±0.2
5.1±0.2
4.4±0.1
4.3±0.1
4.4±0.2
4.7±0.2
102.7±2.9
111.4±1.8
134.2±7.0
119.0±2.7
136.5±7.3
188.8±16.9
177.7±5.5
157.2±4.4
144.0±5.1
165.0±9.2
2.5±0.2
2.2±0.1
2.3±0.2
2.0±0.1
1.7±0.1
0.9±0.1
0.9±0.1
1.5±0.1
1.1±0.1
0.9±0.1
4.3±0.2
3.7±0.1
3.4±0.2
3.4±0.2
3.5±0.2
3.5±0.1
3.6±0.3
4.1±0.4
3.2±0.2
2.7±0.2
9.9±0.4
10.0±0.3
11.2±0.3
11.0±0.4
8.5±0.3
4.5±0.2
4.0±0.1
7.3±0.3
8.0±0.6
5.8±0.3
Effect of third ventricle Ucn 2 treatment on nocturnal meal microstructure of genetically-selected
diet-induced obesity-resistant (DR) and susceptible (DIO) rats (n=10/genotype) fed a high-fat diet. Data
express the M±SEM quantity or duration of food and water intake within meals of adult male DR and
DIO rats during the first 12 hr of the dark cycle following Ucn 2 pretreatment. Subjects were pretreated
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
(-10 min) with Ucn 2 in a balanced Latin square design with test sessions beginning at the onset of the
dark cycle. ## Genotype main effect p<0.01, ### p<0.001
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Supplementary Table 3
Table 3. Adiposity in high-fat diet-fed genetically-selected DR and DIO rats
Parameter
DR
DIO
43.4±3.1
11.6±0.8
8.2±0.4
9.0±0.8
7.3±0.6
7.4±0.8
0.56±0.05
107.1±15.8 ***
29.6±3.7 ***
14.3±1.7 **
26.4±5.3 **
16.9±2.2 ***
20.0±3.5 **
0.90±0.06 ***
47.1±3.2
121.6±17.6 ***
Fat Pads
White fat pad, g
Total
Inguinal
Gonadal
Retroperitoneal
Mesenteric
Subcutaneous
Brown fat pad, g
Whole Carcass Adiposity
Fat, g
Raw fat pad and whole carcass fat weights in high-fat diet fed DR and DIO rats (n=10
rats/genotype). Values are M±SEM. Symbols denote significant differences: * p<0.05 compared to DR
rats, ** p<0.01, *** p<0.001, (Student’s t-test).
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
Supplementary Table 4
Table 4. Comparison of variables related to the microstructure of feeding between
genetically-selected DIO rats relative to DR rats in pre- and post-obese states
Parameter
Pre-obese state (Low-fat diet)
Post-obese state (High-fat diet)
↔
↓
↓
↑
↓
↑
↓
↔
↑
↔
↓
↑
↑
↔
Total food intake
Meal size
Meal duration
Meal frequency
Intermeal Interval
Eating rate
Total water intake
Differences in variables related to the microstructure of food intake between genetically-selected
DR and DIO rats in a pre-obese state (fed a low-fat chow diet) and in a post-obese state (fed a high-fat
diet). Symbols denote relative increase ↑, decrease ↓, or no change ↔ compared to DR. The findings of
both the pre-obese state
3
and the post-obese state were obtained using the same validated drinking-
inclusive meal definition 17.
Cottone et al, Feeding microstructure in high-fat diet-fed DR and DIO rats
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