- Email: [email protected]
Alternate day fasting decreases preference for a calorically dense diet by increasing chow intake and altering meal pattern parameters
Accepted Manuscript Alternate day fasting decreases preference for a calorically dense diet by increasing chow intake and altering meal pattern parameters
Michelle Frankot, Yada Treesukosol PII: DOI: Reference:
S0031-9384(18)30732-7 https://doi.org/10.1016/j.physbeh.2018.11.039 PHB 12389
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
3 September 2018 19 October 2018 29 November 2018
Please cite this article as: Michelle Frankot, Yada Treesukosol , Alternate day fasting decreases preference for a calorically dense diet by increasing chow intake and altering meal pattern parameters. Phb (2018), https://doi.org/10.1016/j.physbeh.2018.11.039
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ACCEPTED MANUSCRIPT Alternate Day Fasting Decreases Preference for a Calorically Dense Diet by Increasing Chow Intake and Altering Meal Pattern Parameters
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Michelle Frankot and Yada Treesukosol
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Department of Psychology, California State University Long Beach, Long Beach CA
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90840, USA
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Correspondence to be sent to:
Department of Psychology
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California State University, Long Beach
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Yada Treesukosol, Ph.D.
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1250 Bellflower Blvd, Long Beach CA 90840-090
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Email: [email protected]
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Michelle Frankot’s present address: Department of Psychology, West Virginia
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University, 53 Campus Drive, Morgantown, WV 26506
ACCEPTED MANUSCRIPT Abstract Alternate day fasting (ADF) is an effective dietary strategy for weight loss in both humans and rats. However, fasting can elicit hyperphagia in rats, particularly upon access to a calorically dense, high-energy (HE) diet. To examine the effects of ADF on HE diet
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preference, male and female Sprague-Dawley rats were randomly assigned to receive
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either ad-libitum or alternate day access to both chow and HE food. Meal pattern analysis
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was conducted to provide a more detailed explanation of changes in HE preference. ADF rats had a decreased preference for the HE diet compared to controls. Both male and
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female ADF rats increased in overall intake of chow. However, for male ADF rats, the
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decrease in HE preference was driven by an increase in both size and number of chow meals; for females, it was driven only by an increase in number of chow meals. Meal size
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is controlled by both positive feedback (e.g. from the oral cavity) and negative feedback
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(e.g. from postoral inhibitory signals). Thus, for males, fasting appeared to increase orosensory stimulation and/or decrease sensitivity to inhibitory cues towards chow. For
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females, fasting appeared to decrease sensitivity to inhibitory cues towards chow. The
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decrease in HE preference observed in the current study may contribute to the
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effectiveness of ADF as a dietary strategy for weight loss.
ACCEPTED MANUSCRIPT Highlights Alternate day fasting decreased preference for palatable food in male and female rats.
Shifts in diet preference were driven by increased chow intake.
For males, fasting increased size and number of chow meals.
For females, fasting increased number of chow meals.
Alternate day fasting mitigated high fat diet-induced weight gain.
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Keywords Alternate Day Fasting
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Meal Size
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Meal Number High Fat Diet
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Diet Preference
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Weight Loss
ACCEPTED MANUSCRIPT 1. Introduction Human overconsumption of fatty and sugary food is a main contributor to obesity [1, 2]. The diet induced obesity (DIO) model provides an analogue of this behavior in animals. In the DIO model, animals presented with a calorically dense, high energy (HE)
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diet subsequently overeat and gain weight. Specifically, HE diet presentation is
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associated with increases in meal size [3-6]. Further, providing animals with a choice
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between HE and other food items, a model more comparable to the choices humans face, may amplify the hyperphagia elicited by the HE diet [7, 8].
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Among various dietary interventions that have the potential to buffer the negative
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effects of HE diets is alternate day fasting (ADF). An ADF regimen involves the repetition of a 2-day cycle beginning with a day of ad-libitum access to food followed by
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a day of zero or limited access to food. In humans, ADF leads to weight loss even when
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participants are given a high fat diet (45% fat) regimen for eight weeks [9]. An ADF schedule with humans also reduces several factors associated with heart disease, such as
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body weight, BMI, cholesterol, and triglyceride concentrations [10] and results in weight
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loss without unsafe or aversive outcomes [11] . In rodents, ADF has a number of beneficial health benefits beyond weight loss including glucose and insulin regulation
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[12, 13], lifespan prolongation [14, 15], and mitigation of cognitive deficits associated with poor physical health [16, 17]. However, the mechanisms underlying these beneficial effects of ADF remain largely unknown. Meal pattern analysis, a method of quantifying ingestive behavior as a combination of meal size, frequency, and distribution over time, may be useful in providing a more detailed understanding of dietary changes during ADF. Meal pattern
ACCEPTED MANUSCRIPT parameters (e.g., meal size, meal number) shift both as a result of HE diet presentation [3, 5, 18] as well as fasting [19]. Meal size is directly controlled by positive feedback that maintains feeding and negative feedback that terminates feeding [20]. Positive feedback includes stimulation of gustatory, olfactory, and somatosensory oral receptors (i.e.,
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orosensory stimulation), whereas negative feedback includes postoral inhibitory signaling
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from the gut to the brain [20-23]. Thus, increases in meal size can be attributed to
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increases in orosensory stimulation and/or decreased sensitivity to postoral inhibitory signaling. Given that eliminating inhibitory signaling via sham feeding decreases
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intermeal intervals [24] and that administration of gut peptides, such as gastrin-releasing
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peptide [25] and cholecystokinin [24] lengthens intermeal interval, it can be inferred that changes in meal number involve changes in postoral inhibitory signaling.
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The purpose of the current study was to examine the effect of ADF on HE
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preference. Meal pattern analysis was used to elucidate the underlying neural mechanisms of potential shifts in HE preference resulting from ADF. It was hypothesized
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that ADF would elicit an overconsumption of palatable food, thus increasing preference
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for the HE diet. In order to test this hypothesis, some rats received ad-libitum access to both HE and chow, while others were subjected to fasting. Meal size, meal number, and
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total intake of both HE and chow were recorded in order to explain potential shifts in diet preference.
2. Materials and Methods 2.1 Subjects
ACCEPTED MANUSCRIPT A total of 29 male and 35 female adult Sprague Dawley rats were used. The rats were raised at California State University, Long Beach. Rats were obtained from the litters of breeders provided by Charles River (Hollister, CA). Litters were culled to 10 pups on postnatal day 3 and kept with the dam until weaning on postnatal day 21.
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Following weaning, rats were group housed with same-sex litter mates. Rats were
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housed in a temperature-controlled room and maintained on a 12:12 light-dark cycle. All
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procedures were approved by the Institutional Animal Care and Use Committee at
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California State University, Long Beach.
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2.2 Apparatus
For testing, rats were single housed in DietMax System food intake monitoring
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cages (AccuScan Instruments, Inc., Columbus OH). The clear, polycarbonate cages were
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42 × 42 × 30 cm (length × width × height). The floor was a solid base, and a lid covered the top of the cage. There were two openings in the cages that led to food jars, which
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were placed on scales. Each opening was 6.5 × 6.5 cm, and the two openings were 11.2
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cm apart. Rats received ad libitum access to powdered chow (3.1 kcal/g, calories from protein 24%, calories from fat 18%, calories from carbohydrate 58%; PicoLab Rodent
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Diet 20, Lab Diet) and water while habituating to the food intake monitoring cages.
2.3 Diet Manipulations After approximately 3 days of habituation, rats were randomly assigned to one of three diet conditions, which involved the addition of powdered HE food (4.73 kcal/g; calories from protein 20%, calories from fat 45%, calories from carbohydrate 35%;
ACCEPTED MANUSCRIPT D12451, Research Diets). In the first condition (AD LIB), rats had ad-libitum access to both standard chow and HE food every day. In the second condition (INT), rats had adlibitum access to standard chow every day and ad-libitum access to HE food every other day. In the third condition (ADF), rats only had ad-libitum access to both standard chow
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and HE food every other day; no food was administered on the alternate days. Days
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during which all rats received access to both chow and HE will be referred to as feeding
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days, and alternate days will be referred to as fasting days (Figure 1).
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Figure 1. Outline of groups (AD LIB, INT, and ADF) and the presentation schedule of the two diets (high energy diet HE or chow)
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2.4 Behavioral Testing For 23 hours per day, rats remained in the food intake monitoring cages undisturbed. During this time, food intake was constantly recorded via weight fluctuations of the powdered food on the scales using the computer program Fusion 6.0
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DietMax. For approximately 1 hour per day, no data from the scales were recorded. All
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rats were removed from the food intake monitoring cages and weighed. Vaginal smears were performed on female rats to account for estrous cycles. Water bottles were weighed
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in order to measure daily water intake, and food was refilled or removed daily per diet condition. Preference for the HE diet was assessed on each feeding day as a ratio of HE
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food consumed to total food consumed. Meal size (kcal), meal number, and total intake
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(kcal) of the HE and chow diets were also recorded on each feeding day. All procedures
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occurred at the same time every day for 3 consecutive feeding days until termination of the experiment.
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2.5 Data analysis
A meal was defined as a bout of eating that consisted of a minimum of 0.2 g of
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food. A meal was considered terminated after 10 minutes without food intake. These
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parameters for defining a meal have been used in previous meal pattern research in order to segment feeding bouts into meaningful meals while accounting for a majority of the data [26-28].
The data obtained using Fusion 6.0 Diet Max contained some missing values due to technological malfunctions. Missing values were excluded from all analyses. Meal parameters for the groups were compared using two-way mixed ANOVAs with a between subjects factor of diet condition and a within subjects factor of day. Bonferroni
ACCEPTED MANUSCRIPT corrections were applied to all post hoc analyses. Greenhouse-Geisser adjustments were applied to the degrees of freedom for within-subjects analyses that violated Mauchly’s Test of Sphericity. Data for male and female rats were analyzed separately. In order to account for potential confounding effects of the female estrous cycle, one-way ANOVAs
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were conducted across group for each feeding day with total caloric intake as the
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dependent variable. Then, these one-way ANOVAs were repeated with the addition of
cycles contributed to changes in food intake.
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3. Results
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estrus as a dichotomous covariate (i.e., estrus vs. non-estrus) to determine if estrous
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3.1 Diet Preference
Both the female and male ADF groups had a lower preference for the HE diet
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than the INT groups, but only the female ADF group had a lower preference for the HE
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diet than the AD LIB group; for males there was no significant difference between the ADF and AD LIB group (Figure 2). For females, a two-way ANOVA (group x day)
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revealed a main effect of group (F(2, 21) = 10.39, p = .001). Post hoc analyses indicated
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that female ADF rats had a lower HE preference than both INT and AD LIB rats. There was no main effect of day (F(2, 42) = 2.52, p = .092) nor significant interaction (F(4, 42)
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= 1.045, p = .40). For males, a two-way ANOVA revealed a main effect of group (F(2, 22) = 12.084, p < .001). Post hoc analyses indicated that male ADF rats had a lower HE preference than INT but not AD LIB rats. There was no main effect of day (F(2, 44) = 3.10, p = .055) nor significant interaction (F(4, 44) = 2.14, p = .092). The effect size of the shift in preference across group was slightly larger for males (η2 = .523) than females (η2 = .497).
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Figure 2. Preference for the HE diet on three consecutive feeding days for both female
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(left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey
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symbols) groups. Scores above the dashed line at 0.5 indicate that rats consumed more
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than 50% kcal from HE compared to chow.
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3.2.1 HE intake
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3.2 Food Intake
The lower preference for the HE diet observed for male ADF rats was partly
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driven by a decrease in overall HE intake. Although for females, there were no significant group differences in HE intake, for males, the INT group had a larger HE intake than the ADF and AD LIB groups (Figure 3). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 1.79, p = .19). There was a main effect of day (F(1.48, 30.99) = 4.09, p = .037). Post hoc analyses indicated that HE intake was larger on the first feeding day than the second. There was no group by day interaction (F(2.95, 30.99) = 0.64, p = .59. For males, a two-way ANOVA revealed a main effect of group (F(2, 22) =
ACCEPTED MANUSCRIPT 10.03, p = .001). Post hoc analyses indicated that male INT rats had a larger HE intake than both ADF and AD LIB rats. There was a main effect of day (F(1.63, 35.91) = 4.53, p = .024). Post hoc analyses indicated that HE intake was larger on the first feeding day than the second. There was no group by day interaction (F(3.26, 35.91) = 0.69, p = .58.
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Figure 3. Total caloric intake from the HE diet on three consecutive feeding days for
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both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and
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INT (grey symbols) groups.
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3.2.2 Chow intake
The lower preference for the HE diet observed for both male and female ADF rats was partly driven by an increase in overall chow intake. Both the male and female ADF groups had a larger chow intake than the INT and AD LIB groups (Figure 4). For females, a two-way ANOVA revealed a main effect of group (F(2, 21) = 10.67, p = .001). Post hoc analyses indicated that female ADF rats had a larger chow intake than both INT and AD LIB rats. There was no main effect of day (F(2, 42) = 3.03, p = .06) nor
ACCEPTED MANUSCRIPT significant interaction (F(4, 42) = 0.55, p = .70). For males, a two-way ANOVA revealed a main effect of group (F(2, 22) = 16.91, p < .001). Post hoc analyses indicated that male ADF rats had a larger chow intake than both INT and AD LIB rats. There was no main effect of day (F(2, 44) = 0.67, p = .52) nor significant interaction (F(4, 44) = 1.12, p =
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.36). Chow Intake (Males) 70 60 50 Intake (kcal)
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Figure 4. Total caloric intake from the chow diet on three consecutive feeding days for
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both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and
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INT (grey symbols) groups.
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3.2.3 Total intake
Although both male and female ADF groups increased in chow intake on feeding days, when total intake from the 2-day cycles of feeding and fasting was combined, the ADF groups had a lower total intake than the INT and AD LIB groups (Figure 5). For females, a two-way ANOVA revealed a main effect of group (F(2, 19) = 19.64, p < .001). Post hoc analyses indicated that female ADF rats had a lower total intake across the 2-day cycles than both INT and AD LIB rats. There was a main effect of cycle (F(2,
ACCEPTED MANUSCRIPT 38) = 4.11, p = .026). However, when Bonferroni corrections were applied to post hoc analyses, there were no significant differences in total intake across the 2-day cycles. There was no group by cycle interaction (F(4, 38) = 0.48, p = .75). For males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 58.17, p < .001). Post hoc analyses
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nor significant interaction (F(2.91, 30.56) = 0.58, p = .63).
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INT and AD LIB rats. There was no main effect of day (F(1.46, 30.56) = 3.23, p = .067)
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Figure 5. Total caloric intake from both the HE and chow diet combined on three consecutive cycles (i.e., the summed intake from a feeding day and the following fasting
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day) for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3 Meal Parameters 3.3.1 HE meal size Changes in HE meal size did not contribute to the lower preference for the HE diet observed for ADF rats; for both male and female rats there were no significant group
ACCEPTED MANUSCRIPT differences across diet conditions in the average meal size of the HE diet (Figure 6). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 0.99, p = .39). There was a main effect of day (F(1.59, 33.28) = 4.08, p = .034). However, when Bonferroni corrections were applied to post hoc analyses, there were no significant
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differences in HE meal size across day. There was no group by day interaction (F(3.17,
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33.28) = 0.28, p = .85). For males, a two-way ANOVA revealed no main effect of group
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(F(2, 20) = 1.96, p = .17), no main effect of day (F(2, 40) = 0.51, p = .61), and no group
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by day interaction (F(4, 40) = 0.41, p = .80).
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Figure 6. Average meal size of the HE diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.2 Chow meal size The lower preference for the HE diet observed for male ADF rats was partly driven by an increase in chow meal size. Although for females, there were no significant
ACCEPTED MANUSCRIPT group differences in average chow meal size, for males, the ADF group had larger chow meal sizes than the INT group (Figure 7). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 1.05, p = .37), no main effect of day (F(1.48, 30.99) = 11.10, p = .081), and no group by day interaction (F(2.95, 30.99) = 1.77, p = .18). For
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males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 7.71, p = .003).
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Post hoc analyses indicated that male ADF rats had larger chow meals than INT but not
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significant interaction (F(2.81, 29.47) = 0.78, p = .51).
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Figure 7. Average meal size of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.3 HE First Meal Size For both male and female rats there were no significant group differences across diet conditions in the first meal size of the HE diet (Figure 8). For females, a two-way
ACCEPTED MANUSCRIPT ANOVA revealed no main effect of group (F(2, 21) = 0.15, p = .86), no main effect of day (F(2, 42) = 2.18, p = .13), and no group by day interaction (F(4, 42) = 0.44, p = .78). For males, a two-way ANOVA revealed no main effect of group (F(2, 20) = 0.013, p = .98), no main effect of day (F(2, 40) = 0.22, p = .81), and no group by day interaction
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(F(4, 40) = 0.46, p = .64). These findings are consistent with the average HE meal size
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data (i.e., there were no differences in average meal size across diet conditions for male
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Figure 8. First meal size of the HE diet on three consecutive feeding days for both
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female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.4 Chow First Meal Size Although for females, there were no significant group differences in first meal size of chow, for males, the ADF group had larger first chow meals than the INT and AD LIB groups (Figure 9). For females, a two-way ANOVA revealed no main effect of group
ACCEPTED MANUSCRIPT (F(2, 21) = 1.52, p = .24), no main effect of day (F(2, 42) = 1.75, p = .19), and no group by day interaction (F(4, 42) = 1.63, p = .19). For males, a two-way ANOVA revealed a main effect of group (F(2, 19) = 6.47, p = .007). Post hoc analyses indicated that male ADF rats had larger first chow meals than INT and AD LIB rats. There was no main
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effect of day (F(2, 38) = 0.11, p = .90) nor significant interaction (F(4, 38) = 0.85, p =
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.51). These findings are consistent with the average chow meal size data (i.e., ADF males
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increased in chow meal size, but there were no differences in average meal size across
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diet conditions for females).
Chow First Meal Size (Females) AD LIB ADF INT
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Figure 9. First meal size of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.5 HE meal number Changes in HE meal number did not contribute to the lower preference for the HE diet observed for ADF rats; for both female and male rats there were no group differences
ACCEPTED MANUSCRIPT across diet conditions in the meal number of the HE diet (Figure 10). For females, a twoway ANOVA revealed no main effect of group (F(2, 21) = 0.35, p = .71), no main effect of day (F(2, 42) = 0.52, p = .60), and no group by day interaction (F(4, 42) = 0.59, p = .67). Likewise for males, a two-way ANOVA revealed no main effect of group (F(2, 20)
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= 2.34, p = .12), no main effect of day (F(2, 40) = 0.86, p = .43), and no group by day
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interaction (F(4, 40) = 0.69, p = .61).
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Figure 10. Total meal number of the HE diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT
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(grey symbols) groups.
3.3.6 Chow meal number The lower preference for the HE diet observed for female and male ADF rats was partly driven by an increase in chow meal number. Both the female and male ADF groups had a larger chow meal number than the INT groups, but only the female ADF group had a larger chow meal number than the AD LIB group; for males there was no
ACCEPTED MANUSCRIPT significant difference between the ADF and AD LIB group (Figure 11). For females, a two-way ANOVA revealed a main effect of group (F(2, 21) = 10.94, p = .001). Post hoc analyses indicated that female ADF rats had more chow meals than INT and AD LIB rats. There was a main effect of day (F(2, 42) = 7.20, p = .002). Post hoc analyses
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indicated that more chow meals were taken on the third feeding day than both the two
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previous feeding days. There was no group by day interaction (F(4, 42) = 0.59, p = .68).
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For males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 8.21, p = .002). Post hoc analyses indicated that male ADF rats had more chow meals than INT
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rats but not AD LIB rats. There was no main effect of day (F(1.46, 30.55) = 0.03, p =
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.94) nor significant interaction (F(2.91, 30.55) = 0.44, p = .72).
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Figure 11. Total meal number of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.4 Weight Gain
ACCEPTED MANUSCRIPT Both the female and male ADF groups gained less weight throughout the 6 days (3 feeding days and 3 fasting days) of testing than the AD LIB groups, but only the male ADF group gained less weight than the INT group; for females there was no significant difference between the ADF and INT group (Figure 12). For females, a one-way
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ANOVA revealed a statistically significant difference in weight gain between groups
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(F(2, 31) = 7.60, p = .002). Post hoc analyses indicated that female ADF rats gained less
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weight than AD LIB but not INT rats. For males, a one-way ANOVA revealed a statistically significant difference in weight gain between groups (F(2, 26) = 45.93, p <
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.001). Post hoc analyses indicated that male ADF rats gained less weight than both AD
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LIB and INT rats. Weight Gain (Females)
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Figure 12. Total weight gain across the duration of the study for both female (left) and male (right) AD LIB (black bars), ADF (white bars), and INT (grey bars) groups.
3.5 Water Intake For both female and male rats, there were no group differences across diet conditions in water intake throughout the 6 days of feeding and fasting (Figure 13). For
ACCEPTED MANUSCRIPT females, a two-way ANOVA revealed no main effect of group (F(2, 31) = 0.20, p = .82) and no main effect of day (F(2.45, 75.95) = 1.19, p = .32). There was a significant group by day interaction (F(4.90, 75.95) = 2.88, p = .02). For males, a two-way ANOVA revealed no main effect of group (F(2, 26) = 1.63, p = .22) and no main effect of day
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(F(1.70, 44.20) = 0.15, p = .83), and no group by day interaction (F(3.40, 44.20) = 1.08, p
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= .37).
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Figure 13. Daily water intake on six consecutive feeding and fasting days for both
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female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT
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(grey symbols) groups.
3.6 Estrous Cycles
For the first feeding day, a one-way ANOVA revealed a main effect of group (F(2, 20) = 4.07, p = .033). Post hoc analyses indicated that female ADF rats had a larger intake than AD LIB but not INT rats. When estrus was added as a covariate, a one-way ANOVA no longer revealed a main effect of group (F(2, 19) = 3.35, p = .057). For the second feeding day, a one-way ANOVA revealed a main effect of group (F(2, 20) = 7.82,
ACCEPTED MANUSCRIPT p = .003). Post hoc analyses indicated that female ADF rats had a larger intake than AD LIB but not INT rats. When estrus was added as a covariate, a one-way ANOVA still revealed a main effect of group (F(2, 19) = 7.27, p = .005). Post hoc analyses indicated that female ADF rats still had a larger intake than AD LIB but not INT rats. For the third
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feeding day, a one-way ANOVA revealed no main effect of group (F(2, 20) = 1.89, p =
effect of group (F(2, 19) = 1.70, p = .21). 4. Discussion
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.18). When estrus was added as a covariate, a one-way ANOVA still revealed no main
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The purpose of the current study was to determine how fasting affects diet
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preference in both male and female rats. It was predicted that fasting would lead to overconsumption of palatable food, thus increasing preference for the HE diet. Contrary
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to this hypothesis, ADF decreased preference for the HE diet in males and females,
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although the effect was more pronounced in males. For males, this shift in preference was driven by changes in both HE and chow intake. Specifically, male ADF rats decreased in
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HE caloric intake and increased in chow caloric intake. Using meal pattern analysis to
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further understand this shift in preference, male ADF rats had no changes in meal size or meal number of the HE diet compared to controls. However, they increased in both chow
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meal size and meal number. Thus, the change in preference for male ADF rats appeared to be caused by decreases in HE intake and increases in chow intake that were driven by increases in chow meal size and meal number. Given that the direct controls of meal size can be categorized as positive (e.g., oral) or negative (e.g., postoral inhibitory) signals [20-23, 29], it seems that ADF increased orosensory stimulation and/or decreased sensitivity to inhibitory cues towards chow for males. Lastly, male ADF rats did not
ACCEPTED MANUSCRIPT differ from controls in first meal size of HE but increased in first meal size of chow compared to controls. However, this effect was not specific to first meal size, but rather also observed in average meal size. Conversely, for female ADF rats, the decrease in HE preference only involved
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changes in chow intake; there were no differences in HE caloric intake for females.
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Rather, there was an increase in chow intake for female ADF rats driven only by an
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increase in chow meal number. Unlike males, female ADF rats did not have a larger chow meal size than controls. The increase in chow meal number but not meal size
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reflected a shortening of intermeal intervals. This suggests a dampened sensitivity to
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postoral inhibitory signaling, a form of negative feedback that is responsible for reducing food intake [21, 22]. Similar decreases in intermeal interval are seen when the effects of
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postoral inhibitory signaling are removed in a sham-feeding paradigm [24]. Lastly,
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female ADF rats did not increase in first meal size of chow or HE compared to controls. Given that first meal size also reflected average meal size for males, ADF does not
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appear to play a specific role in first meal size in the current study. Overall, it appears
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that not only does the magnitude of the shift in preference differ between males and females, the underlying causes may differ as well.
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Under different conditions, rats also demonstrate sex differences in preference [30, 31] and motivation [32] for palatable diets. This sex difference may be of particular importance due to studies that provide evidence to suggest males are more sensitive than females to the detrimental health effects of a high fat diet in mice [33] and in rats [34, 35]. Future research is needed to identify the mechanisms underlying sex differences in preference for palatable food, particularly as it is affected by fasting. Regardless of sex
ACCEPTED MANUSCRIPT differences in magnitude, the shift in preference toward chow for both male and female rats in the current study may be indicative of another reason for the efficacy of ADF for weight loss. Although it was predicted that fasting would increase preference for the HE diet,
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recent studies regarding ghrelin, an orexigenic hormone that acts to initiate meals
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following periods of hunger [36-40], may explain the shift away from the HE diet that
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was observed. While many studies indicate that ghrelin acts to increase intake of palatable food [41-44], ghrelin also appears to play a role in shifting preference toward
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chow. When rats were given a choice between chow and a high-fat diet (HFD), vehicle-
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injected controls exclusively ate the HFD; however, rats injected centrally with ghrelin shifted to eating more chow [45]. After fasting, when levels of endogenous ghrelin are
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naturally elevated [46], rats displayed increased chow consumption, just as they did when
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centrally injected with ghrelin. This effect of fasting on chow consumption was eliminated when a ghrelin antagonist was administered peripherally [47]. These findings
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provide a possible explanation for the shift in preference towards chow seen in ADF rats
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in the current study. While the two previous studies demonstrated that shifts in preference are seen in males, the current study extends these findings to females. However, the
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current data also suggest that this effect may not be as strong for females. The involvement of ghrelin may also provide a potential explanation for the sex difference in preference in the current study. In response to ADF, Martin et al. [35] found that male and female rats had different levels of endogenous ghrelin. Specifically, after 6 months of alternate day fasting, the plasma ghrelin levels in male ADF rats were comparable to those of control rats. However, female ADF rats displayed a 20%
ACCEPTED MANUSCRIPT reduction in plasma ghrelin compared to control rats [35]. It is plausible that female rats in the current study displayed a less pronounced shift towards chow due to lower levels of endogenous ghrelin. These factors should be considered in future research, as the current study did not include any measures to account for the effects of ghrelin. These
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findings are of particular interest given that humans also demonstrate elevated levels of
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endogenous ghrelin following fasting [48, 49] as well as sex differences in ghrelin levels
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in the absence of experimental manipulations [50] and in the presence of various gastrointestinal perturbances [51, 52].
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Further, despite the presentation of the HE diet, ADF provided a buffer against
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weight gain. Both male and female ADF rats gained significantly less weight than control rats that received access to food every day. This is consistent with human literature in
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which ADF has been shown to be an effective strategy for weight loss with obese
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participants given a high fat diet (45% fat) regimen for eight weeks [53]. Further, the beneficial effects of ADF surpass weight loss given that ADF also aids in regulating
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glucose and insulin tolerance in rodents [12, 13]. The effects of ADF on glucose
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tolerance in humans are less clear and appear to be sex-dependent. Heilbronn et al. [54] found that ADF impaired the glucose response after a meal in non-obese women, yet had
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no adverse consequences for men. These findings, in conjunction with evidence that ADF can mitigate cognitive deficits [16, 17] and increase the lifespan of rodents [14, 15], demonstrate the necessity to continue the study of fasting and the ways in which it may change both behavior and physiology. Stages of the estrous cycle were also taken into account in the current study given that a bulk of fasting research has focused on male animals. It was necessary to account
ACCEPTED MANUSCRIPT for potential confounding effects of cycling because food intake tends to naturally decrease on the day of estrus [55-57]. Given that estrus as a covariate had little effect on total caloric intake across group, changes in ingestive behavior seen in females in the current study can be attributed to dietary manipulations rather than hormonal changes
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across the estrous cycle.
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In conclusion, ADF shifted preference away from the HE diet towards chow for
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both male and female rats. The magnitude of the effect, as well as the driving forces behind this change, differed between males and females. The sex differences in the
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current study reinforce the importance of including female rodents when studying fasting,
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particularly given that females tend to be understudied in neuroscience [58]. Future research is needed to determine the underlying mechanisms of these sex differences and
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to assess the role of ghrelin in preference shifts during ADF. However, ADF continues to
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show promise as a dietary technique for both weight loss and a variety of other healthrelated outcomes. Future studies should aim to clarify the mechanisms underlying
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behavioral changes following fasting, with a focus on both brain areas and peripheral
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Funding
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signals that may be involved.
This work was supported by faculty start-up funds from California State University Long Beach, Department of Psychology and College of Liberal Arts to Y.T.
Acknowledgements
ACCEPTED MANUSCRIPT We would like to thank Audrey Carrillo and Alexa Gould for technical assistance with this study. Parts of this paper were presented at the 47 th Annual Meeting for the Society
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for Neuroscience, Washington DC, USA, November 2017.
ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT Highlights Alternate day fasting decreased preference for palatable food in male and female rats.
Shifts in diet preference were driven by increased chow intake.
For males, fasting increased size and number of chow meals.
For females, fasting increased number of chow meals.
Alternate day fasting mitigated high fat diet-induced weight gain.
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Michelle Frankot, Yada Treesukosol PII: DOI: Reference:
S0031-9384(18)30732-7 https://doi.org/10.1016/j.physbeh.2018.11.039 PHB 12389
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
3 September 2018 19 October 2018 29 November 2018
Please cite this article as: Michelle Frankot, Yada Treesukosol , Alternate day fasting decreases preference for a calorically dense diet by increasing chow intake and altering meal pattern parameters. Phb (2018), https://doi.org/10.1016/j.physbeh.2018.11.039
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ACCEPTED MANUSCRIPT Alternate Day Fasting Decreases Preference for a Calorically Dense Diet by Increasing Chow Intake and Altering Meal Pattern Parameters
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Michelle Frankot and Yada Treesukosol
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Department of Psychology, California State University Long Beach, Long Beach CA
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90840, USA
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Correspondence to be sent to:
Department of Psychology
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California State University, Long Beach
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Yada Treesukosol, Ph.D.
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1250 Bellflower Blvd, Long Beach CA 90840-090
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Email: [email protected]
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Michelle Frankot’s present address: Department of Psychology, West Virginia
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University, 53 Campus Drive, Morgantown, WV 26506
ACCEPTED MANUSCRIPT Abstract Alternate day fasting (ADF) is an effective dietary strategy for weight loss in both humans and rats. However, fasting can elicit hyperphagia in rats, particularly upon access to a calorically dense, high-energy (HE) diet. To examine the effects of ADF on HE diet
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preference, male and female Sprague-Dawley rats were randomly assigned to receive
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either ad-libitum or alternate day access to both chow and HE food. Meal pattern analysis
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was conducted to provide a more detailed explanation of changes in HE preference. ADF rats had a decreased preference for the HE diet compared to controls. Both male and
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female ADF rats increased in overall intake of chow. However, for male ADF rats, the
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decrease in HE preference was driven by an increase in both size and number of chow meals; for females, it was driven only by an increase in number of chow meals. Meal size
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is controlled by both positive feedback (e.g. from the oral cavity) and negative feedback
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(e.g. from postoral inhibitory signals). Thus, for males, fasting appeared to increase orosensory stimulation and/or decrease sensitivity to inhibitory cues towards chow. For
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females, fasting appeared to decrease sensitivity to inhibitory cues towards chow. The
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decrease in HE preference observed in the current study may contribute to the
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effectiveness of ADF as a dietary strategy for weight loss.
ACCEPTED MANUSCRIPT Highlights Alternate day fasting decreased preference for palatable food in male and female rats.
Shifts in diet preference were driven by increased chow intake.
For males, fasting increased size and number of chow meals.
For females, fasting increased number of chow meals.
Alternate day fasting mitigated high fat diet-induced weight gain.
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Keywords Alternate Day Fasting
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Meal Size
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Meal Number High Fat Diet
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Diet Preference
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Weight Loss
ACCEPTED MANUSCRIPT 1. Introduction Human overconsumption of fatty and sugary food is a main contributor to obesity [1, 2]. The diet induced obesity (DIO) model provides an analogue of this behavior in animals. In the DIO model, animals presented with a calorically dense, high energy (HE)
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diet subsequently overeat and gain weight. Specifically, HE diet presentation is
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associated with increases in meal size [3-6]. Further, providing animals with a choice
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between HE and other food items, a model more comparable to the choices humans face, may amplify the hyperphagia elicited by the HE diet [7, 8].
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Among various dietary interventions that have the potential to buffer the negative
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effects of HE diets is alternate day fasting (ADF). An ADF regimen involves the repetition of a 2-day cycle beginning with a day of ad-libitum access to food followed by
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a day of zero or limited access to food. In humans, ADF leads to weight loss even when
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participants are given a high fat diet (45% fat) regimen for eight weeks [9]. An ADF schedule with humans also reduces several factors associated with heart disease, such as
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body weight, BMI, cholesterol, and triglyceride concentrations [10] and results in weight
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loss without unsafe or aversive outcomes [11] . In rodents, ADF has a number of beneficial health benefits beyond weight loss including glucose and insulin regulation
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[12, 13], lifespan prolongation [14, 15], and mitigation of cognitive deficits associated with poor physical health [16, 17]. However, the mechanisms underlying these beneficial effects of ADF remain largely unknown. Meal pattern analysis, a method of quantifying ingestive behavior as a combination of meal size, frequency, and distribution over time, may be useful in providing a more detailed understanding of dietary changes during ADF. Meal pattern
ACCEPTED MANUSCRIPT parameters (e.g., meal size, meal number) shift both as a result of HE diet presentation [3, 5, 18] as well as fasting [19]. Meal size is directly controlled by positive feedback that maintains feeding and negative feedback that terminates feeding [20]. Positive feedback includes stimulation of gustatory, olfactory, and somatosensory oral receptors (i.e.,
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orosensory stimulation), whereas negative feedback includes postoral inhibitory signaling
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from the gut to the brain [20-23]. Thus, increases in meal size can be attributed to
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increases in orosensory stimulation and/or decreased sensitivity to postoral inhibitory signaling. Given that eliminating inhibitory signaling via sham feeding decreases
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intermeal intervals [24] and that administration of gut peptides, such as gastrin-releasing
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peptide [25] and cholecystokinin [24] lengthens intermeal interval, it can be inferred that changes in meal number involve changes in postoral inhibitory signaling.
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The purpose of the current study was to examine the effect of ADF on HE
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preference. Meal pattern analysis was used to elucidate the underlying neural mechanisms of potential shifts in HE preference resulting from ADF. It was hypothesized
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that ADF would elicit an overconsumption of palatable food, thus increasing preference
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for the HE diet. In order to test this hypothesis, some rats received ad-libitum access to both HE and chow, while others were subjected to fasting. Meal size, meal number, and
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total intake of both HE and chow were recorded in order to explain potential shifts in diet preference.
2. Materials and Methods 2.1 Subjects
ACCEPTED MANUSCRIPT A total of 29 male and 35 female adult Sprague Dawley rats were used. The rats were raised at California State University, Long Beach. Rats were obtained from the litters of breeders provided by Charles River (Hollister, CA). Litters were culled to 10 pups on postnatal day 3 and kept with the dam until weaning on postnatal day 21.
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Following weaning, rats were group housed with same-sex litter mates. Rats were
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housed in a temperature-controlled room and maintained on a 12:12 light-dark cycle. All
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procedures were approved by the Institutional Animal Care and Use Committee at
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California State University, Long Beach.
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2.2 Apparatus
For testing, rats were single housed in DietMax System food intake monitoring
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cages (AccuScan Instruments, Inc., Columbus OH). The clear, polycarbonate cages were
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42 × 42 × 30 cm (length × width × height). The floor was a solid base, and a lid covered the top of the cage. There were two openings in the cages that led to food jars, which
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were placed on scales. Each opening was 6.5 × 6.5 cm, and the two openings were 11.2
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cm apart. Rats received ad libitum access to powdered chow (3.1 kcal/g, calories from protein 24%, calories from fat 18%, calories from carbohydrate 58%; PicoLab Rodent
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Diet 20, Lab Diet) and water while habituating to the food intake monitoring cages.
2.3 Diet Manipulations After approximately 3 days of habituation, rats were randomly assigned to one of three diet conditions, which involved the addition of powdered HE food (4.73 kcal/g; calories from protein 20%, calories from fat 45%, calories from carbohydrate 35%;
ACCEPTED MANUSCRIPT D12451, Research Diets). In the first condition (AD LIB), rats had ad-libitum access to both standard chow and HE food every day. In the second condition (INT), rats had adlibitum access to standard chow every day and ad-libitum access to HE food every other day. In the third condition (ADF), rats only had ad-libitum access to both standard chow
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and HE food every other day; no food was administered on the alternate days. Days
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during which all rats received access to both chow and HE will be referred to as feeding
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days, and alternate days will be referred to as fasting days (Figure 1).
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Figure 1. Outline of groups (AD LIB, INT, and ADF) and the presentation schedule of the two diets (high energy diet HE or chow)
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2.4 Behavioral Testing For 23 hours per day, rats remained in the food intake monitoring cages undisturbed. During this time, food intake was constantly recorded via weight fluctuations of the powdered food on the scales using the computer program Fusion 6.0
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DietMax. For approximately 1 hour per day, no data from the scales were recorded. All
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rats were removed from the food intake monitoring cages and weighed. Vaginal smears were performed on female rats to account for estrous cycles. Water bottles were weighed
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in order to measure daily water intake, and food was refilled or removed daily per diet condition. Preference for the HE diet was assessed on each feeding day as a ratio of HE
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food consumed to total food consumed. Meal size (kcal), meal number, and total intake
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(kcal) of the HE and chow diets were also recorded on each feeding day. All procedures
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occurred at the same time every day for 3 consecutive feeding days until termination of the experiment.
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2.5 Data analysis
A meal was defined as a bout of eating that consisted of a minimum of 0.2 g of
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food. A meal was considered terminated after 10 minutes without food intake. These
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parameters for defining a meal have been used in previous meal pattern research in order to segment feeding bouts into meaningful meals while accounting for a majority of the data [26-28].
The data obtained using Fusion 6.0 Diet Max contained some missing values due to technological malfunctions. Missing values were excluded from all analyses. Meal parameters for the groups were compared using two-way mixed ANOVAs with a between subjects factor of diet condition and a within subjects factor of day. Bonferroni
ACCEPTED MANUSCRIPT corrections were applied to all post hoc analyses. Greenhouse-Geisser adjustments were applied to the degrees of freedom for within-subjects analyses that violated Mauchly’s Test of Sphericity. Data for male and female rats were analyzed separately. In order to account for potential confounding effects of the female estrous cycle, one-way ANOVAs
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were conducted across group for each feeding day with total caloric intake as the
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dependent variable. Then, these one-way ANOVAs were repeated with the addition of
cycles contributed to changes in food intake.
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3. Results
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estrus as a dichotomous covariate (i.e., estrus vs. non-estrus) to determine if estrous
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3.1 Diet Preference
Both the female and male ADF groups had a lower preference for the HE diet
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than the INT groups, but only the female ADF group had a lower preference for the HE
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diet than the AD LIB group; for males there was no significant difference between the ADF and AD LIB group (Figure 2). For females, a two-way ANOVA (group x day)
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revealed a main effect of group (F(2, 21) = 10.39, p = .001). Post hoc analyses indicated
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that female ADF rats had a lower HE preference than both INT and AD LIB rats. There was no main effect of day (F(2, 42) = 2.52, p = .092) nor significant interaction (F(4, 42)
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= 1.045, p = .40). For males, a two-way ANOVA revealed a main effect of group (F(2, 22) = 12.084, p < .001). Post hoc analyses indicated that male ADF rats had a lower HE preference than INT but not AD LIB rats. There was no main effect of day (F(2, 44) = 3.10, p = .055) nor significant interaction (F(4, 44) = 2.14, p = .092). The effect size of the shift in preference across group was slightly larger for males (η2 = .523) than females (η2 = .497).
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Figure 2. Preference for the HE diet on three consecutive feeding days for both female
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(left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey
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symbols) groups. Scores above the dashed line at 0.5 indicate that rats consumed more
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than 50% kcal from HE compared to chow.
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3.2.1 HE intake
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3.2 Food Intake
The lower preference for the HE diet observed for male ADF rats was partly
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driven by a decrease in overall HE intake. Although for females, there were no significant group differences in HE intake, for males, the INT group had a larger HE intake than the ADF and AD LIB groups (Figure 3). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 1.79, p = .19). There was a main effect of day (F(1.48, 30.99) = 4.09, p = .037). Post hoc analyses indicated that HE intake was larger on the first feeding day than the second. There was no group by day interaction (F(2.95, 30.99) = 0.64, p = .59. For males, a two-way ANOVA revealed a main effect of group (F(2, 22) =
ACCEPTED MANUSCRIPT 10.03, p = .001). Post hoc analyses indicated that male INT rats had a larger HE intake than both ADF and AD LIB rats. There was a main effect of day (F(1.63, 35.91) = 4.53, p = .024). Post hoc analyses indicated that HE intake was larger on the first feeding day than the second. There was no group by day interaction (F(3.26, 35.91) = 0.69, p = .58.
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HE Intake (Males) 140
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Figure 3. Total caloric intake from the HE diet on three consecutive feeding days for
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both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and
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INT (grey symbols) groups.
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3.2.2 Chow intake
The lower preference for the HE diet observed for both male and female ADF rats was partly driven by an increase in overall chow intake. Both the male and female ADF groups had a larger chow intake than the INT and AD LIB groups (Figure 4). For females, a two-way ANOVA revealed a main effect of group (F(2, 21) = 10.67, p = .001). Post hoc analyses indicated that female ADF rats had a larger chow intake than both INT and AD LIB rats. There was no main effect of day (F(2, 42) = 3.03, p = .06) nor
ACCEPTED MANUSCRIPT significant interaction (F(4, 42) = 0.55, p = .70). For males, a two-way ANOVA revealed a main effect of group (F(2, 22) = 16.91, p < .001). Post hoc analyses indicated that male ADF rats had a larger chow intake than both INT and AD LIB rats. There was no main effect of day (F(2, 44) = 0.67, p = .52) nor significant interaction (F(4, 44) = 1.12, p =
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.36). Chow Intake (Males) 70 60 50 Intake (kcal)
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Figure 4. Total caloric intake from the chow diet on three consecutive feeding days for
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both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and
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INT (grey symbols) groups.
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3.2.3 Total intake
Although both male and female ADF groups increased in chow intake on feeding days, when total intake from the 2-day cycles of feeding and fasting was combined, the ADF groups had a lower total intake than the INT and AD LIB groups (Figure 5). For females, a two-way ANOVA revealed a main effect of group (F(2, 19) = 19.64, p < .001). Post hoc analyses indicated that female ADF rats had a lower total intake across the 2-day cycles than both INT and AD LIB rats. There was a main effect of cycle (F(2,
ACCEPTED MANUSCRIPT 38) = 4.11, p = .026). However, when Bonferroni corrections were applied to post hoc analyses, there were no significant differences in total intake across the 2-day cycles. There was no group by cycle interaction (F(4, 38) = 0.48, p = .75). For males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 58.17, p < .001). Post hoc analyses
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indicated that male ADF rats had a lower total intake across the 2-day cycles than both
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nor significant interaction (F(2.91, 30.56) = 0.58, p = .63).
Total Intake (Males)
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Total Intake (Females)
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INT and AD LIB rats. There was no main effect of day (F(1.46, 30.56) = 3.23, p = .067)
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Figure 5. Total caloric intake from both the HE and chow diet combined on three consecutive cycles (i.e., the summed intake from a feeding day and the following fasting
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day) for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3 Meal Parameters 3.3.1 HE meal size Changes in HE meal size did not contribute to the lower preference for the HE diet observed for ADF rats; for both male and female rats there were no significant group
ACCEPTED MANUSCRIPT differences across diet conditions in the average meal size of the HE diet (Figure 6). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 0.99, p = .39). There was a main effect of day (F(1.59, 33.28) = 4.08, p = .034). However, when Bonferroni corrections were applied to post hoc analyses, there were no significant
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differences in HE meal size across day. There was no group by day interaction (F(3.17,
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33.28) = 0.28, p = .85). For males, a two-way ANOVA revealed no main effect of group
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(F(2, 20) = 1.96, p = .17), no main effect of day (F(2, 40) = 0.51, p = .61), and no group
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by day interaction (F(4, 40) = 0.41, p = .80).
HE Meal Size (Females)
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Figure 6. Average meal size of the HE diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.2 Chow meal size The lower preference for the HE diet observed for male ADF rats was partly driven by an increase in chow meal size. Although for females, there were no significant
ACCEPTED MANUSCRIPT group differences in average chow meal size, for males, the ADF group had larger chow meal sizes than the INT group (Figure 7). For females, a two-way ANOVA revealed no main effect of group (F(2, 21) = 1.05, p = .37), no main effect of day (F(1.48, 30.99) = 11.10, p = .081), and no group by day interaction (F(2.95, 30.99) = 1.77, p = .18). For
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males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 7.71, p = .003).
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Post hoc analyses indicated that male ADF rats had larger chow meals than INT but not
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significant interaction (F(2.81, 29.47) = 0.78, p = .51).
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AD LIB rats. There was no main effect of day (F(1.40, 29.47) = 0.05, p = .89) nor
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Figure 7. Average meal size of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.3 HE First Meal Size For both male and female rats there were no significant group differences across diet conditions in the first meal size of the HE diet (Figure 8). For females, a two-way
ACCEPTED MANUSCRIPT ANOVA revealed no main effect of group (F(2, 21) = 0.15, p = .86), no main effect of day (F(2, 42) = 2.18, p = .13), and no group by day interaction (F(4, 42) = 0.44, p = .78). For males, a two-way ANOVA revealed no main effect of group (F(2, 20) = 0.013, p = .98), no main effect of day (F(2, 40) = 0.22, p = .81), and no group by day interaction
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(F(4, 40) = 0.46, p = .64). These findings are consistent with the average HE meal size
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data (i.e., there were no differences in average meal size across diet conditions for male
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or female rats).
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Figure 8. First meal size of the HE diet on three consecutive feeding days for both
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female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.4 Chow First Meal Size Although for females, there were no significant group differences in first meal size of chow, for males, the ADF group had larger first chow meals than the INT and AD LIB groups (Figure 9). For females, a two-way ANOVA revealed no main effect of group
ACCEPTED MANUSCRIPT (F(2, 21) = 1.52, p = .24), no main effect of day (F(2, 42) = 1.75, p = .19), and no group by day interaction (F(4, 42) = 1.63, p = .19). For males, a two-way ANOVA revealed a main effect of group (F(2, 19) = 6.47, p = .007). Post hoc analyses indicated that male ADF rats had larger first chow meals than INT and AD LIB rats. There was no main
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effect of day (F(2, 38) = 0.11, p = .90) nor significant interaction (F(4, 38) = 0.85, p =
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.51). These findings are consistent with the average chow meal size data (i.e., ADF males
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increased in chow meal size, but there were no differences in average meal size across
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diet conditions for females).
Chow First Meal Size (Females) AD LIB ADF INT
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Figure 9. First meal size of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.3.5 HE meal number Changes in HE meal number did not contribute to the lower preference for the HE diet observed for ADF rats; for both female and male rats there were no group differences
ACCEPTED MANUSCRIPT across diet conditions in the meal number of the HE diet (Figure 10). For females, a twoway ANOVA revealed no main effect of group (F(2, 21) = 0.35, p = .71), no main effect of day (F(2, 42) = 0.52, p = .60), and no group by day interaction (F(4, 42) = 0.59, p = .67). Likewise for males, a two-way ANOVA revealed no main effect of group (F(2, 20)
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= 2.34, p = .12), no main effect of day (F(2, 40) = 0.86, p = .43), and no group by day
HE Meal Number (Females)
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interaction (F(4, 40) = 0.69, p = .61).
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Figure 10. Total meal number of the HE diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT
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(grey symbols) groups.
3.3.6 Chow meal number The lower preference for the HE diet observed for female and male ADF rats was partly driven by an increase in chow meal number. Both the female and male ADF groups had a larger chow meal number than the INT groups, but only the female ADF group had a larger chow meal number than the AD LIB group; for males there was no
ACCEPTED MANUSCRIPT significant difference between the ADF and AD LIB group (Figure 11). For females, a two-way ANOVA revealed a main effect of group (F(2, 21) = 10.94, p = .001). Post hoc analyses indicated that female ADF rats had more chow meals than INT and AD LIB rats. There was a main effect of day (F(2, 42) = 7.20, p = .002). Post hoc analyses
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indicated that more chow meals were taken on the third feeding day than both the two
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previous feeding days. There was no group by day interaction (F(4, 42) = 0.59, p = .68).
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For males, a two-way ANOVA revealed a main effect of group (F(2, 21) = 8.21, p = .002). Post hoc analyses indicated that male ADF rats had more chow meals than INT
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rats but not AD LIB rats. There was no main effect of day (F(1.46, 30.55) = 0.03, p =
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.94) nor significant interaction (F(2.91, 30.55) = 0.44, p = .72).
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Figure 11. Total meal number of the chow diet on three consecutive feeding days for both female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT (grey symbols) groups.
3.4 Weight Gain
ACCEPTED MANUSCRIPT Both the female and male ADF groups gained less weight throughout the 6 days (3 feeding days and 3 fasting days) of testing than the AD LIB groups, but only the male ADF group gained less weight than the INT group; for females there was no significant difference between the ADF and INT group (Figure 12). For females, a one-way
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ANOVA revealed a statistically significant difference in weight gain between groups
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(F(2, 31) = 7.60, p = .002). Post hoc analyses indicated that female ADF rats gained less
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weight than AD LIB but not INT rats. For males, a one-way ANOVA revealed a statistically significant difference in weight gain between groups (F(2, 26) = 45.93, p <
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.001). Post hoc analyses indicated that male ADF rats gained less weight than both AD
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LIB and INT rats. Weight Gain (Females)
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Figure 12. Total weight gain across the duration of the study for both female (left) and male (right) AD LIB (black bars), ADF (white bars), and INT (grey bars) groups.
3.5 Water Intake For both female and male rats, there were no group differences across diet conditions in water intake throughout the 6 days of feeding and fasting (Figure 13). For
ACCEPTED MANUSCRIPT females, a two-way ANOVA revealed no main effect of group (F(2, 31) = 0.20, p = .82) and no main effect of day (F(2.45, 75.95) = 1.19, p = .32). There was a significant group by day interaction (F(4.90, 75.95) = 2.88, p = .02). For males, a two-way ANOVA revealed no main effect of group (F(2, 26) = 1.63, p = .22) and no main effect of day
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(F(1.70, 44.20) = 0.15, p = .83), and no group by day interaction (F(3.40, 44.20) = 1.08, p
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= .37).
Water Intake (Males) 40
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Figure 13. Daily water intake on six consecutive feeding and fasting days for both
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female (left) and male (right) AD LIB (black symbols), ADF (white symbols), and INT
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(grey symbols) groups.
3.6 Estrous Cycles
For the first feeding day, a one-way ANOVA revealed a main effect of group (F(2, 20) = 4.07, p = .033). Post hoc analyses indicated that female ADF rats had a larger intake than AD LIB but not INT rats. When estrus was added as a covariate, a one-way ANOVA no longer revealed a main effect of group (F(2, 19) = 3.35, p = .057). For the second feeding day, a one-way ANOVA revealed a main effect of group (F(2, 20) = 7.82,
ACCEPTED MANUSCRIPT p = .003). Post hoc analyses indicated that female ADF rats had a larger intake than AD LIB but not INT rats. When estrus was added as a covariate, a one-way ANOVA still revealed a main effect of group (F(2, 19) = 7.27, p = .005). Post hoc analyses indicated that female ADF rats still had a larger intake than AD LIB but not INT rats. For the third
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feeding day, a one-way ANOVA revealed no main effect of group (F(2, 20) = 1.89, p =
effect of group (F(2, 19) = 1.70, p = .21). 4. Discussion
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.18). When estrus was added as a covariate, a one-way ANOVA still revealed no main
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The purpose of the current study was to determine how fasting affects diet
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preference in both male and female rats. It was predicted that fasting would lead to overconsumption of palatable food, thus increasing preference for the HE diet. Contrary
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to this hypothesis, ADF decreased preference for the HE diet in males and females,
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although the effect was more pronounced in males. For males, this shift in preference was driven by changes in both HE and chow intake. Specifically, male ADF rats decreased in
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HE caloric intake and increased in chow caloric intake. Using meal pattern analysis to
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further understand this shift in preference, male ADF rats had no changes in meal size or meal number of the HE diet compared to controls. However, they increased in both chow
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meal size and meal number. Thus, the change in preference for male ADF rats appeared to be caused by decreases in HE intake and increases in chow intake that were driven by increases in chow meal size and meal number. Given that the direct controls of meal size can be categorized as positive (e.g., oral) or negative (e.g., postoral inhibitory) signals [20-23, 29], it seems that ADF increased orosensory stimulation and/or decreased sensitivity to inhibitory cues towards chow for males. Lastly, male ADF rats did not
ACCEPTED MANUSCRIPT differ from controls in first meal size of HE but increased in first meal size of chow compared to controls. However, this effect was not specific to first meal size, but rather also observed in average meal size. Conversely, for female ADF rats, the decrease in HE preference only involved
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changes in chow intake; there were no differences in HE caloric intake for females.
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Rather, there was an increase in chow intake for female ADF rats driven only by an
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increase in chow meal number. Unlike males, female ADF rats did not have a larger chow meal size than controls. The increase in chow meal number but not meal size
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reflected a shortening of intermeal intervals. This suggests a dampened sensitivity to
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postoral inhibitory signaling, a form of negative feedback that is responsible for reducing food intake [21, 22]. Similar decreases in intermeal interval are seen when the effects of
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postoral inhibitory signaling are removed in a sham-feeding paradigm [24]. Lastly,
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female ADF rats did not increase in first meal size of chow or HE compared to controls. Given that first meal size also reflected average meal size for males, ADF does not
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appear to play a specific role in first meal size in the current study. Overall, it appears
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that not only does the magnitude of the shift in preference differ between males and females, the underlying causes may differ as well.
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Under different conditions, rats also demonstrate sex differences in preference [30, 31] and motivation [32] for palatable diets. This sex difference may be of particular importance due to studies that provide evidence to suggest males are more sensitive than females to the detrimental health effects of a high fat diet in mice [33] and in rats [34, 35]. Future research is needed to identify the mechanisms underlying sex differences in preference for palatable food, particularly as it is affected by fasting. Regardless of sex
ACCEPTED MANUSCRIPT differences in magnitude, the shift in preference toward chow for both male and female rats in the current study may be indicative of another reason for the efficacy of ADF for weight loss. Although it was predicted that fasting would increase preference for the HE diet,
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recent studies regarding ghrelin, an orexigenic hormone that acts to initiate meals
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following periods of hunger [36-40], may explain the shift away from the HE diet that
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was observed. While many studies indicate that ghrelin acts to increase intake of palatable food [41-44], ghrelin also appears to play a role in shifting preference toward
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chow. When rats were given a choice between chow and a high-fat diet (HFD), vehicle-
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injected controls exclusively ate the HFD; however, rats injected centrally with ghrelin shifted to eating more chow [45]. After fasting, when levels of endogenous ghrelin are
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naturally elevated [46], rats displayed increased chow consumption, just as they did when
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centrally injected with ghrelin. This effect of fasting on chow consumption was eliminated when a ghrelin antagonist was administered peripherally [47]. These findings
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provide a possible explanation for the shift in preference towards chow seen in ADF rats
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in the current study. While the two previous studies demonstrated that shifts in preference are seen in males, the current study extends these findings to females. However, the
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current data also suggest that this effect may not be as strong for females. The involvement of ghrelin may also provide a potential explanation for the sex difference in preference in the current study. In response to ADF, Martin et al. [35] found that male and female rats had different levels of endogenous ghrelin. Specifically, after 6 months of alternate day fasting, the plasma ghrelin levels in male ADF rats were comparable to those of control rats. However, female ADF rats displayed a 20%
ACCEPTED MANUSCRIPT reduction in plasma ghrelin compared to control rats [35]. It is plausible that female rats in the current study displayed a less pronounced shift towards chow due to lower levels of endogenous ghrelin. These factors should be considered in future research, as the current study did not include any measures to account for the effects of ghrelin. These
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findings are of particular interest given that humans also demonstrate elevated levels of
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endogenous ghrelin following fasting [48, 49] as well as sex differences in ghrelin levels
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in the absence of experimental manipulations [50] and in the presence of various gastrointestinal perturbances [51, 52].
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Further, despite the presentation of the HE diet, ADF provided a buffer against
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weight gain. Both male and female ADF rats gained significantly less weight than control rats that received access to food every day. This is consistent with human literature in
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which ADF has been shown to be an effective strategy for weight loss with obese
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participants given a high fat diet (45% fat) regimen for eight weeks [53]. Further, the beneficial effects of ADF surpass weight loss given that ADF also aids in regulating
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glucose and insulin tolerance in rodents [12, 13]. The effects of ADF on glucose
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tolerance in humans are less clear and appear to be sex-dependent. Heilbronn et al. [54] found that ADF impaired the glucose response after a meal in non-obese women, yet had
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no adverse consequences for men. These findings, in conjunction with evidence that ADF can mitigate cognitive deficits [16, 17] and increase the lifespan of rodents [14, 15], demonstrate the necessity to continue the study of fasting and the ways in which it may change both behavior and physiology. Stages of the estrous cycle were also taken into account in the current study given that a bulk of fasting research has focused on male animals. It was necessary to account
ACCEPTED MANUSCRIPT for potential confounding effects of cycling because food intake tends to naturally decrease on the day of estrus [55-57]. Given that estrus as a covariate had little effect on total caloric intake across group, changes in ingestive behavior seen in females in the current study can be attributed to dietary manipulations rather than hormonal changes
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across the estrous cycle.
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In conclusion, ADF shifted preference away from the HE diet towards chow for
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both male and female rats. The magnitude of the effect, as well as the driving forces behind this change, differed between males and females. The sex differences in the
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current study reinforce the importance of including female rodents when studying fasting,
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particularly given that females tend to be understudied in neuroscience [58]. Future research is needed to determine the underlying mechanisms of these sex differences and
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to assess the role of ghrelin in preference shifts during ADF. However, ADF continues to
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show promise as a dietary technique for both weight loss and a variety of other healthrelated outcomes. Future studies should aim to clarify the mechanisms underlying
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behavioral changes following fasting, with a focus on both brain areas and peripheral
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Funding
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signals that may be involved.
This work was supported by faculty start-up funds from California State University Long Beach, Department of Psychology and College of Liberal Arts to Y.T.
Acknowledgements
ACCEPTED MANUSCRIPT We would like to thank Audrey Carrillo and Alexa Gould for technical assistance with this study. Parts of this paper were presented at the 47 th Annual Meeting for the Society
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for Neuroscience, Washington DC, USA, November 2017.
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ACCEPTED MANUSCRIPT Highlights Alternate day fasting decreased preference for palatable food in male and female rats.
Shifts in diet preference were driven by increased chow intake.
For males, fasting increased size and number of chow meals.
For females, fasting increased number of chow meals.
Alternate day fasting mitigated high fat diet-induced weight gain.
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