Effects of metabolic inhibitors on ingestive behavior and physiology in preweanling rat pups

Appetite (2000) 35, 9±25 doi:10.1006/appe.2000.0330, available online at http://www.idealibrary.com on

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Effects of metabolic inhibitors on ingestive behavior and physiology in preweanling rat pups S.E. Swithers Department of Psychological Sciences, Purdue University (Received 4 January 2000, revision14 March 2000, acceptedinrevised form 3 April 2000, publishedelectronically13 June 2000)

We have previously demonstrated that administration of 2-Mercaptoacetate (MA) stimulates independent intake after 1 h in 12 and 15-day-old rat pups, but not younger pups. MA also produces decreases in -HBA levels, consistent with the development of a role of altered fatty acid oxidation in modulating independent ingestion in rat pups by 12 days of age. The present experiments extended investigations of the role of changes in energy utilization in young rats by investigating the duration of the effects of altered fatty acid oxidation and the effects of combined blockade of fatty acid oxidation and glucose utilization. Pups were tested at 9, 12 or 15 days of age 3 or 6 h following administration of a dose of 0, 11
4, 22
8, 45
6 or 91
2 mg/kg MA. In pups aged 12 and 15 days, moderate doses of MA stimulated intake 3 h, but not 6 h, following administration. Administration of the highest dose of MA produced significant decreases in -HBA levels in pups at all ages when tested after 3 h, but not after 6 h. In the second set of experiments, behavioral and physiological responses to administration of MA (0, 11
4 or 22
.8 mg/kg) combined with 2-Deoxyglucose (2-DG: 0, 100 or 200 mg/kg) were investigated in pups aged 6, 9, 12 or 15 days of age. The results demonstrated that while administration of 2-DG produced physiological responses, intake was not stimulated at any age by 2-DG alone or in combination with MA. In fact, in 12 and 15-day-old pups, administration of 2-DG blocked the stimulatory effects of administration of MA. Therefore, while altered utilization of glucose does not appear to be an effective stimulus for increased independent ingestion in pups at this age, altered fatty acid oxidation may be an early metabolic modulator of intake. # 2000 Academic Press

Introduction Changes in the utilization of energy have been routinely demonstrated to profoundly influence ingestive behavior in adult rats. For example, administration of agents that selectively decrease the availability or utilization

I thank Roberto Melendez, Becky Peters, Melissa McCurley, Terah Schamberg and Rick Davis for technical assistance and Dr Javier Morell and Alicia Doerflinger for their comments on a previous version of this manuscript. Portions of these data were presented at the 1997 Annual meetings of the Society for the Study of Ingestive Behavior and Society for Neuroscience. Supported by NIH grant DK55531. Address all correspondence to: S.E. Swithers, Department of Psychological Sciences, Purdue University, West Lafayette, IN 47907-1364, U.S.A. E-mail: [email protected] 0195±6663/00/040009+17 $35.00/0

of glucose or fatty acids produce increases in intake within minutes to hours in adult rats (e.g. Friedman & Tordoff, 1986; Friedman et al., 1986; Langhans & Scharrer, 1987; Miselis & Epstein, 1975; Ritter & Slusser, 1980; Scharrer & Langhans, 1980; Smith & Epstein, 1969). In addition, combined block of fatty acid oxidation and glucose utilization produces greater increases in intake in adult rats than either alone (Friedman & Tordoff, 1986; Friedman et al., 1986). On the other hand, a number of studies have suggested that young rat pups are insensitive to alterations in the availability or utilization of energy until approximately 25±30 days of age (Houpt & Epstein, 1973; Leshem et al., 1990; Lytle et al., 1971; Williams & Blass, 1987). However, the failure to observe effects of altered energy utilization on intake in young pups may result from testing intake when pups are suckling from the dam. Although suckling does represent the typical method # 2000 Academic Press

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S.E. Swithers

for young rats to obtain food and fluids, suckling may represent an ontogenetic adaptation, and not the developmental precursor to adult ingestion (e.g. Hall & Williams, 1983). In fact, some studies have suggested that when ingestive responsiveness is examined using tests that are independent of the dam and the suckling situation, changes in metabolic or caloric status first appear to affect intake between 6 and 15 days of age, much earlier than the 25±30 days of age for suckling intake (e.g. Phifer & Hall, 1988; Phifer et al., 1986; Swithers & Hall, 1989). Recent work from our laboratory has suggested that the metabolic signal that begins to influence independent intake in rats between 6 and 15 days of age may be related to oxidation of fatty acids. Rat pups preferentially use fat-derived ketone bodies for metabolic energy, and dam's milk is a high-fat energy source (e.g. Fernando-Warnakulasuriya et al., 1981; Lockwood & Bailey, 1970, 1971; Wells, 1985; Yeh & Sheehan, 1985; Yeung & Oliver, 1967). In one study, we demonstrated that blockade of fatty acid oxidation by administration of 2-Mercaptoacetate (MA) first stimulates independent intake in rat pups between 9 and 12 days of age (Swithers, 1997). Pups tested at 6 or 9 days of age fail to alter ingestion, while older pups increase intake of a high-fat milk diet or a non-fat glucose diet 1 h following administration of moderate doses of MA. In addition, administration of MA was accompanied by physiological changes consistent with a blockade of fatty acid oxidation; levels of the ketone body -hydroxybutyrate ( -HBA) were significantly lower in 9, 12 and 15-dayold pups 1 h following administration of MA than in control pups. The effects of MA on intake were also demonstrated to be specific to caloric diets; 12-day-old pups increased consumption of either a milk or glucose diet, but intake of water was not altered in pups at either 9 or 12 days of age (Swithers et al., 1999). The demonstration that MA begins to stimulate intake of a caloric diet between 9 and 12 days of age suggests that pups develop a mechanism that permits them to detect changes in the utilization of fats and are able to translate this into a behavioral response between 9 and 12 days of age. In addition, the time frame during which responsiveness to MA-emerges suggests that changes in the utilization of fatty acids may represent the first metabolic mechanism by which pups modulate ingestion. The present experiments examined two questions related to this emergent metabolic signal. The first set of experiments examined the duration of the behavioral and physiological effects of MA in rat pups. In the second set of experiments, we examined whether a combined blockade of both glucose and fatty acid utilization produced different effects from administration of either agent alone in rat pups.

General method Subjects Subjects were progeny of Sprague-Dawley (Harlan, Indianapolis, IN, U.S.A.) rats mated in the breeding colony in the laboratory. Animals were housed in plastic maternity cages lined with aspen shavings and allowed ad libitum access to food (Lab Diets chow #5012) and water. Temperature was maintained at 22± 25 C and the animals were on a 14 : 10 light : dark cycle. Female rats were placed in pairs with male rats for 1 week for mating, then separated from the males and housed in pairs until the week of parturition, when females were placed in individual maternity cages. Cages were checked daily for births and any litters present at 1700h were considered to be born that day, designated as Day 0. Litters were culled to 10 pups (five male, five female where possible) on the day following birth. Except for routine maintenance, litters remained undisturbed with the dam until the day of testing.

Intake tests On the day of testing, pups were removed from the dam, stimulated to urinate and defecate by stroking with an artist's brush wetted with warm water, and weighed. Mercaptoacetate (11
5 mg/ml in a 0
135 M NaCl vehicle) and/or 2-Deoxy-d-glucose (40 mg/ml in 0
135 M NaCl vehicle) were delivered by i.p. injection as described in individual experiments. Following injection, pups were placed together in a bedding-lined cage and housed in a warm, moist incubator maintained at 32±35 C. Following injection and the appropriate delay, all pups were removed from the incubator, stimulated to urinate and defecate by stroking with a moist artist's brush, weighed, and then given an intake test. During the intake test, pups were placed individually into plastic test containers lined with paper towels wetted with a commercial half-and-half milk diet inside an incubator warmed to 32±35 C. Pups were allowed to consume the diet for 30 min; the paper towels were rewetted with warm diet every 10 min during testing as necessary. Pups were then removed from the test containers, dried carefully and re-weighed. Because pups at these ages do not readily urinate and defecate spontaneously, particularly after being stimulated to urinate and defecate, the amount of weight gained (expressed as a percentage of the pup's predeprivation body weight) during the intake test was used as a reliable measure of intake (e.g. Hall & Bryan, 1980).

Effects of MA and 2-DG in rat pups 11

Blood collection and analysis Following injection and the appropriate delay, pups were removed from the incubator and stimulated to urinate and defecate by stroking with a warm brush. Pups were then sacrificed with an overdose of Brevital and blood samples were collected from pups in the 0, 45
6 and 91
2 mg/kg MA groups by cutting the inferior vena cava and collecting into non-heparinized microhematocrit tubes (Experiment 1B). Use of Brevital was suspected to have increased blood glucose levels in Experiment 1B, therefore in Experiment 2C, pups were sacrificed by decapitation and trunk blood was collected in heparinized microhematocrit tubes. All blood samples were centrifuged immediately, plasma was separated and samples from the same animal were combined. Samples were frozen immediately and stored at ÿ80 C until assay. -HBA levels and free fatty acid (FFA) levels were determined enzymatically ( -HBA kit, Sigma Chemicals or Stat-Site -HBA cards, GDS Diagnostics; and NEFA-C kit, Wako Chemicals, respectively) while glucose was analysed using a glucometer (Beckmann Instruments). Immediately following blood collection, stomachs were removed from pups in all groups for determination of gastric contents. To determine stomach contents, stomachs were weighed, contents were removed and stomachs were reweighed. Weight of stomach contents was used as an estimate of stomach volume.

Statistical analysis All statistics were performed using Statistica (STATISTICA `99; StatSoft, Inc., Tulsa, OK; 1999) as described in individual experiments.

Experiment 1A: duration of the effects of MA on intake In a previous study, the effects of MA on physiological and behavioral responses in rat pups were evident 1 h following administration in 12 and 15-day-old pups, while younger pups failed to alter intake in response to administration of MA. One possibility is that the time following administration of MA was insufficient to stimulate an ingestive response in young pups. Although levels of -HBA were altered 1 h after administration of MA in 9-day-old pups, perhaps the ingestive behavioral responses of young pups are somehow delayed compared to older animals. In addition, because the previous study examined a single time point following administration of MA, it is unknown how enduring the effects of MA are on behavior and physiology in young pups. To determine the duration of the effects of

MA on independent ingestion and to examine the possibility that younger pups require more time to respond behaviorally to blockade of fatty acid oxidation, in this experiment, rat pups aged 9, 12 or 15 days were tested 3 or 6 h following administration of MA. The increase in time following administration of MA also produces a difference in the metabolic state of the pups at the time of testing; 3 h following administration of MA pups are 3 h deprived, while 6 h following administration of MA, pups are 6 h deprived. To control for this confound of deprivation, additional groups of pups aged 9, 12 or 15 days of age were tested after 3 or 6 h deprivation, but only 1 h following administration of MA. Thus pups were tested at each age in one of four conditions: 3-h-deprived injected 3 h prior to testing (group 3-3); 3-h-deprived injected 1 h prior to testing (group 3-1); 6-h-deprived injected 6 h prior to testing (group 6-6); 6-h-deprived injected 1 h prior to testing (group 6-1).

Method Pups were tested once at 9, 12 or 15 days of age. On the day of testing, one group of litters (N ˆ 6 litters per age) was removed from the dam, received an i.p. injection of 0, 11.4, 22.8, 45.6 or 91.2 mg/kg MA, and was placed into a warm, humid incubator where they remained undisturbed for 3 or 6 h. A second group of litters (N ˆ 6 litters per age) was removed from the dam, placed into the incubator for 2 or 5 h and then given an i.p. injection of 0, 11.4, 22.8, 45.6 or 91.2 mg/ kg MA, and returned to the incubator for 1 h. Two pups from each of six litters were tested in each condition at each age (N ˆ 11±12 per group). Baseline intakes of the half-and-half diet are different at the ages tested, thus separate two-way (Dose MAX Group) ANOVA's were conducted at each age with a value of p < 0.05 taken as significant. Post-hoc comparisons were done using LSD tests. To control for multiple comparisons, a value of p < 0.01 taken as significant in post-hoc tests.

Results and discussion Nine-day-old ln 9-day-old pups, intake was affected by deprivation (Main effect of Group; F(3, 218) ˆ 17.25, p < 0.05; Figs. 1 and 2). In these young pups, intake was higher in the 6 h deprived groups than in the 3 h deprived groups. In addition, the delay between injection and testing influenced intake. The groups that had received an injection 3 or 6 h prior to testing consumed more than groups that had received an injection only 1 h

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S.E. Swithers 6

6

(a)

(a)

*

5

4 * 3

2

Intake (% body weight)

Intake (% body weight)

5

4

3

2 * 1

1

* 0

0

9

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12 Age (days)

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5 Intake (% body weight)

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4

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2

4

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0 9

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12 Age (days)

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Figure 1. Intake of a commercial half-and-half milk diet after administration of MA in rat pups. (a) Intake measured in 3-h-deprived pups injected 3 h prior to testing. (b) Intake in 6-h-deprived pups injected 6 h prior to testing. 0 mg/kg MA, &; 11
4 mg/kg MA, ; 22
8 mg/kg MA, ; 45
6 mg/kg MA, ; 91
2 mg/kg MA; &. *p < 0
01 compared to 0 mg/kg MA.

Figure 2. Intake of a commercial half-and-half milk diet after administration of MA in rat pups. (a) Intake measured in 3-h-deprived pups injected 1 h prior to testing. Bottom: Intake in 6-h-deprived pups injected 1 h prior to testing. 0 mg/kg MA, &; 11
4 mg/kg MA, ; 22
8 mg/kg MA, ; 45
6 mg/kg MA, ; 91
2 mg/kg MA; &. *p < 0
01 compared to 0 mg/kg MA.

prior to testing (e.g. compare overall intake in 9-dayold pups in top of Fig. 1 with top of Fig. 2). However, in 9-day-olds, there were no effects of administration of any dose of MA in any group. The lack of effect of MA on intake in 9-day-old pups when tested after 3 or 6 h is similar to the pattern previously described 1 h after administration (Swithers & Hall, 1989). Thus, it appears unlikely that the failure of 9-day-old pups to respond to administration of MA compared to 12-dayold pups results from a delayed behavioral response to blockade of fatty acid oxidation in the younger animals.

the group of the pups (main effect of dose MA; F(4, 220) ˆ 21
49, p < 0
05; main effect of group; F(3, 220) ˆ 29
78, p < 0
05; dose MA  group interaction; F(12, 220) ˆ 2
22, p < 0
05). In 3-h-deprived pups, a moderate dose (22
8 mg/kg) of MA increased intake (Figs. 1a and 2a), while the highest dose tested, 91
2 mg/kg, significantly suppressed intake when administered 1 h prior to testing (Fig. 2). In 6-hdeprived pups, administration of MA did not enhance intake (Figs. 1b and 2b), but the highest dose of 91
2 mg/kg MA did suppress intake when administered 1 h prior to testing (Fig. 2). In addition, pups tested 1 h following injection consumed less than pups 3 or 6 h after injection. These results suggest that moderate doses of MA stimulate intake in 3-h-deprived pups whether delivered 1 or 3 h prior to testing. MA does not stimulate intake in 6-h-deprived pups whether delivered 1 or 6 h prior to testing. In addition, the

Twelve-day-olds In contrast to the effects seen in 9-day-olds, administration of MA did alter intake in 12-day-old pups. Intake was affected by the dose administered and by

Effects of MA and 2-DG in rat pups 13

intake-suppressing effects of the high dose of MA are shorter lived than the intake-stimulating effects of more moderate doses since intake suppression was seen only 1, but not 3 or 6 h following administration of MA.

Fifteen-day-olds In 15-day-old pups, intake was affected by the group and the dose administered (main effect of group; F(3, 217) ˆ 21
348, p < 0
05; main effect of dose MA; F(12, 217) ˆ 7
799, p < 0
05), but there were no interactions. In 3-h-deprived pups, moderate doses of MA enhanced intake when delivered 3 h prior to testing (Fig. 1a). However, when moderate doses of MA were administered 1 h prior to testing in 3-h-deprived pups, only a trend towards enhanced intake was noted; no significant effects on intake were demonstrated (Fig. 2a). In 6-h-deprived 15-day-old pups, administration of MA did not enhance intake whether delivered 1 or 6 h prior to testing. In addition, the highest dose tested significantly suppressed intake in both 3 and 6-h-deprived pups when delivered 1 h, but not 3 or 6 h, prior to testing (Fig. 2). These data demonstrate that pups younger than 12 days of age do not increase independent ingestion after administration of MA even after delays of up to 6 h. Thus, a delay in behavioral responding to signals generated by blockade of fatty acid oxidation is an unlikely explanation for the failure of 9-day-old pups to respond to administration of MA. In addition, these data suggest that the stimulatory effects of moderate doses of MA in 12 and 15-day-old pups on independent intake endure for 3 h. An exception to this conclusion is the lack of stimulation of intake in the 3-h-deprived 15-day-old pups when MA was administered 1 h prior to testing. While MA appears to stimulate intake for up to 3 h, no increases in intake are observed 6 h following administration. Ingestive responding may no longer be affected 6 h after administration of MA because it is degraded within this time frame. That is, if MA is no longer active, then no signal will be generated for pups to increase intake. However, results from pups tested after 6 h of food deprivation, but only 1 h following administration of MA, suggest an alternative explanation. The 6-h-deprived pups failed to increase intake even 1 h following administration of MA. During the 6 h deprivation period, endogenous signals related to the alterations in metabolic state were likely generated by food deprivation. For example, utilization of fatty acids may have decreased due to exhaustion of available supplies, producing a floor effect. Thus, in this group of pups, saline-injected animals may already be generating a metabolic signal similar to the one generated by

administration of MA. Administration of MA may fail to stimulate intake because a signal related to a change in fat utilization has already been produced by the deprivation period. Consistent with this hypothesis, intakes in control pups are generally higher after 6 h deprivation than after 3 h deprivation; these differences are significant in 9 and 15-day-old pups. An additional method of discriminating between the effects of increased deprivation and decreased effectiveness of MA is to examine how changes in circulating levels of chemicals related to energy metabolism are altered by deprivation and administration of MA.

Experiment 1B: duration of biochemical effects of MA The results of Experiment 1A suggest that administration of MA produces increased ingestive responding in mildly deprived pups for up to 3 h following administration. These results are consistent with a previous demonstration that MA significantly affects intake within 1 h of administration in pups 12 days of age and older (Swithers, 1997), and thus reinforce the possibility that altered fatty acid utilization may be the earliest metabolic signal modifying independent ingestion in rats. Administration of MA produces biochemical changes within 1 h that are consistent with a role of altered fatty acid oxidation; circulating levels of -HBA are decreased within 1 h of administration of MA. In the present experiment, circulating levels of -HBA and other energy-related signals (free fatty acids and glucose) were measured 3 and 6 h following administration of MA to examine the time course of the physiological effects of MA in pups.

Method Pups were tested once at 9, 12 or 15 days of age. Pups received an i.p. injection of 0, 11
4, 22
8, 45
6 or 91
2 mg/kg MA and were placed into a warm humid incubator for 3 or 6 h prior to collection of blood samples. Blood samples were obtained from pups in the 0, 45
6 and 91
2 mg/kg groups (analysis of blood samples from the lower doses were conducted in Experiment 2C, below); stomachs were removed from all groups. Two pups from each of five or six litters were tested in each condition at each age (Ns ˆ 10±12 per group). To control for differences in baseline levels at each age separate 2-Way ANOVA's (delay  dose) were run on stomach contents, free fatty acid, -HBA, and glucose levels. A value of p < 0
05 was

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S.E. Swithers

taken as significant. Post-hoc comparisons were done using LSD tests. To control for multiple comparisons, a p < 0
01 was taken as significant in post-hoc comparisons. In addition, Mann-Whitney U tests were used to examine differences in stomach contents, free fatty acid, -HBA, and glucose levels in control pups across ages, with a p < 0
05 taken as significant.

Results and discussion Nine-day-olds -HBA levels were affected by the dose and the delay since administration (main effect of dose, F(2, 66) ˆ 6
73, p < 0
05; dose  delay interaction, F(2, 66) ˆ 4
04, p < 0
05; Fig. 3). The highest dose tested, 91
2 mg/kg, produced a significant decrease in -HBA levels 3 h after administration, but not 6 h after administration. There was a trend for -HBA levels to be lower in

control pups tested after 6 h compared to those tested after 3 h, but this effect was not significant according to the criteria used for post-hoc tests (LSD test, p ˆ 0
04). FFA levels were also affected by the dose administered and the delay since administration (main effect of dose, F(2, 66) ˆ 3
61, p < 0
05; main effect of delay, F(1, 66) ˆ 4
15, p < 0
05), but there were no interactions (Fig. 4). FFA levels were significantly higher 6 h after administration of the highest dose of MA compared to all other groups. Glucose levels were affected by the dose of MA, but not by the delay (main effect of dose, F(2, 66) ˆ 11
69, p < 0
05, Fig. 5). The highest dose tested, 91
2 mg/kg, produced a significant decrease in glucose levels at both 3 and 6 h after administration. Stomach contents were affected by the dose of MA and the delay since administration (main effect of dose, F(4, 110) ˆ 16
67, p < 0
05; main effect of delay, F(1, 110) ˆ 53
89, p < 0
05; Fig. 6). Stomach contents were significantly greater in pups receiving the highest dose of MA than all other doses in both 3 and 6 h

25 2.0

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Figure 3. -hydroxybutyrate levels in pups (a) 3 h or (b) 6 h after administration of MA. 0 mg/kg MA, &; 45
6 mg/kg MA, &; 91
2 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA; **0
01 < p < 0
05 compared to 0 mg/kg at 3 h; ***p < 0
01 compared to 0 mg/kg at 3 h.

0.0

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Figure 4. Free fatty acid levels in pups (a) 3 h or (b) 6 h after administration of MA. 0 mg/kg MA, &; 45
6 mg/kg MA, &; 91
2 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA.

Effects of MA and 2-DG in rat pups 15 4

250

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Figure 5. Glucose levels in pups (a) 3 h or (b) 6 h after administration of MA. 0 mg/kg MA, &; 45
6 mg/kg MA, &; 91
2 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA.

delay conditions. In addition, stomach contents were significantly smaller in control pups 6 h after administration compared to 3 h after administration.

Twelve-day-olds -HBA levels in 12-day-old pups were affected by both the delay since administration and the dose of MA administered (main effect of dose, F(2, 53) ˆ 3
27, p < 0
05; dose  delay interaction, F(2, 53) ˆ 3
38, p < 0
05; Fig. 3). After 3 h following the administration of MA, -HBA levels were significantly lower in pups receiving either dose of MA. However, by 6 h after administration, no effects on -HBA levels were seen. Similar to the pattern observed in 9-day-old pups, there was a trend for -HBA levels to be decreased in control pups following 6 h delay compared to 3 h delay, but this effect was not statistically significant ( p ˆ 0
04). FFA levels in 12-day-olds were affected by the dose and the delay as well (main effect of dose, F(2, 53) ˆ 7
07, p < 0
05; main effect of delay, F(2, 53) ˆ 8
08, p < 0
05; Fig. 4). Similar to the pattern seen in 9-day-olds, FFA

Stomach contents (% body weight)

Glucose (mg/dl)

200

0

15

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2 * *

* 1 **

** 0

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Figure 6. Stomach contents in pups (a) 3 h or (b) 6 h after administration of MA. 0 mg/kg MA, &; 11
4 mg/kg MA, ; 22
8 mg/kg MA, ; 45
6 mg/kg MA, ; 91
2 mg/kg MA; &. *p < 0
01 compared to 0 mg/kg MA; **p < 0
01 compared to 0 mg/kg at 3 h.

levels were elevated in the pups receiving the highest dose of MA after 6 h compared to all other groups. Glucose levels were also affected by the dose and delay (main effect of dose, F(2, 53) ˆ 10
08, p < 0
05; main effect of delay, F(2, 53) ˆ 5
48, p < 0
05; Fig. 5). Glucose levels were significantly suppressed by the highest dose of MA after both 3 and 6 h and there was a trend for glucose levels to be suppressed after 6 h compared to after 3 h, but this was not significant ( p ˆ 0
05). Stomach contents were altered by delay and dose (main effect of dose, F(4, 88) ˆ 21
08, p < 0
05; main effect of delay, F(1, 88) ˆ 69
48, p < 0
05, delay  dose interaction, F(4, 88) ˆ 4
05; p < 0
05; Fig. 6). Stomach contents were significantly greater in pups receiving the two highest doses of MA after 3 h and in the pups receiving the highest dose after 6 h. In addition, stomach contents were significantly lower in control pups 6 h after administration compared to 3 h.

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S.E. Swithers

Fifteen-day-olds -HBA levels in 15-day-old pups were affected by the dose and the delay (dose  delay interaction; F(2, 54) ˆ 13
82, p < 0
05; Fig. 3). After 3 h following the administration of either dose of MA, -HBA levels were significantly lower than in control pups, however no significant effects were seen after 6 h. In 15-day-old pups, control levels of -HBA were significantly lower after 6 h compared to 3 h. FFA levels were significantly affected by the dose but not the delay (main effect of dose, F(2, 54) ˆ 6
11, p < 0
05; Fig. 4). The highest dose administered significantly elevated FFA levels in 15-day-old pups. Glucose levels were not affected by either the dose of MA delivered or the delay since administration (Fig. 5). Stomach contents were altered by the dose and delay (main effect of dose, F(2, 54) ˆ 26
16, p < 0
05, main effect of delay, F(2, 54) ˆ 48
62, p < 0
05; Fig. 6). Three hours after administration of MA, the two highest doses produced significantly greater stomach contents and 6 h after administration the highest dose produced significantly greater stomach content compared to controls. There were no differences in stomach contents in control pups between 3 and 6 h.

Across age comparisons There were no significant differences in levels of HBA or FFA in control pups across ages, but glucose levels were significantly lower in 9-day-old pups than in 12 or 15-day-old pups after 3 h ( ps < 0
05) and after 6 h, glucose levels were significantly higher in 15day-old pups compared to both 9 and 12-day olds ( ps < 0
05). The results from this experiment demonstrate that the physiological effects of MA after 3 and 6 h are similar in 9-day-old pups, which do not respond behaviorally, and in 12 and 15-day-old pups, which do respond behaviorally. Further, there are no differences in either -HBA or FFA levels in control pups at the different ages. These results support the argument that the failure of 9-dayold pups is not due to a lack of physiological effects of MA in young pups, and are thus consistent with results seen 1 h after administration of MA (Swithers, 1997). Administration of MA produces decreases in -HBA levels for up to 3 h following administration, but its effects are no longer apparent after 6 h. Thus, these results provide some suggestion as to why MA fails to increase intake 6 h after administration. In pups at 15 days of age, -HBA levels were significantly lower in the 6-h-deprived control pups than in the 3-h-deprived control pups, and a trend toward decreased -HBA levels after 6 h was observed in the younger animals.

Thus, the deprivation itself may provide a signal to the pups that stimulates intake and no effective signal is produced by administration of MA. Finally, these results demonstrate that high doses of MA may fail to stimulate intake because they suppress gastric emptying. Pups that start the ingestive test with a fuller stomach may consume less than other groups because gastric fill has a significant influence on termination of intake in pups (e.g. Phifer & Hall, 1988).

Experiment 2A: effects of combined blockade of glucose utilization and fatty acid oxidation The results of Experiment 1 suggest that pups younger than 12 days of age do not respond to altered fatty acid oxidation with increased intake, even 6 h following administration of MA. The failure to respond is not due to a failure of the drug to alter physiological indicators of fatty acid oxidation. One explanation for the failure of young pups to respond to administration of MA may be related to differing metabolic capacity of pups younger than 12 days of age compared to pups 12 days of age or older. For example, young rat pups have significantly higher levels of both gluconeogenesis and fatty acid oxidation compared to adult rats (Lockwood & Bailey, 1970; Yeung & Oliver, 1967). These increased levels of gluconeogenesis in young pups may produce a compensatory response following administration of drugs which block fatty acid oxidation, and thus MA alone does not produce a sufficient metabolic signal in a young pup. In fact, in Experiment 1B, there was some evidence that young pups may attempt to compensate for decreased fatty acid utilization with increased glucose utilization; glucose levels were decreased by MA in 9 and 12-day-old pups, but not in 15-day-old pups. As pups age, their gluconeogenic capacity begins to decline. This decline first emerges between 10 and 15 days of age, the same age at which the first behavioral responses to MA emerge. The decreased ability of older pups to physiologically compensate for altered fatty acid oxidation may result in a true metabolic change and thus an increased ingestive response. It may be that in order to produce increases in ingestion in pups younger than 12 days of age, administration of drugs which block both fatty acid oxidation and glucose utilization is necessary. In this experiment, we therefore examined the effects of administration of both MA and 2-DG on intake in rat pups.

Effects of MA and 2-DG in rat pups 17

Method

Six-day-olds In 6-day-old pups, there were no effects of administration of MA or 2-DG on intake after any delay. However, 3 and 6 h deprivation produced increased intake compared to 1 h deprivation and 6 h deprivation produced increased intake compared to 3 h deprivation (main effect of delay; F(2, 304) ˆ 86
51, p < 0
05; Fig. 7).

Nine-day olds The pattern of responding in 9-day-old pups was similar to that seen in 6-day-old pups; there were no significant effects of MA or 2-DG at any delay, but overall intakes after 3 and 6 h deprivation were significantly different from 1 h and from each other (main effect of delay; F(2, 308) ˆ 117
93, p < 0
05; Fig. 8).

Twelve-day olds In 12-day-old pups, overall intake following a 3 h delay was increased compared to intake following a 1 h delay (main effect of delay; F(1, 201) ˆ 18
72; p < 0
05; Fig. 9). In addition, a dose of 200 mg/kg 2-DG produced decreases in intake (main effect of dose 2-DG; F(2, 201) ˆ 5
31, p < 0
05). However, there were no significant effects of MA and no interactions.

Fifteen-day-olds Overall, increases in intake were produced by increased delay in 15-day-old pups (main effect of delay;

Intake (% body weight)

4 3 2 1 0

0

100 Dose 2-DG (mg/kg)

200

0

100 Dose 2-DG (mg/kg)

200

0

100 Dose 2-DG (mg/kg)

200

5 (b) Intake (% body weight)

Results

(a)

4 3 2 1 0

5 (c) Intake (% body weight)

Pups were tested once at 6, 9, 12 or 15 days of age. Doses were chosen to produce no effect on intake in 9-day-old pups when administered alone (based on Experiment 1 for MA and pilot work for 2-DG). Pups received an i.p. injection of 0, 100 or 200 mg/kg 2-DG combined with 0, 11
4 or 22
8 mg/kg MA. The 6 and 9day-old pups were returned to the incubator for 1, 3 or 6 h and 12 and 15-day-olds pups were returned to the incubator for 1 or 3 h. Following the delay, pups received a 30 min intake test. One or two pups from each of 12 litters were tested in each condition (Ns ˆ 12±14 per group). Due to baseline differences in intake among ages, results were analyzed separately at each age. A 3-way (delay  dose MA  dose 2-DG) ANOVA was run at each age, with a p < 0.05 taken as significant. Post-hoc tests were done using LSD tests; to control for multiple comparisons, a p < 0
01 was taken as significant in post-hoc tests.

5

4 3 2 1 0

Figure 7. Intake of a commercial half-and-half milk diet (a) 1 h, (b) 3 h or (c) 6 h, after administration of MA and 2-DG in 6-day-old rat pups. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

F(1, 205) ˆ 6
57, p < 0
05; Fig. 10). In addition, administration of either dose of 2-DG significantly decreased intake (Main effect of 2-DG; F(2, 205) ˆ 34
29, p < 0
05). There were no effects of MA or interactions. Separate ANOVAs of the effects of MA alone did demonstrate significant effects of MA on intake in 12 and 15-day-old pups.

18

S.E. Swithers 5

5

(a) Intake (% body weight)

Intake (% body weight)

(a) 4 3 2 1 0

0

100 Dose 2-DG (mg/kg)

4 3 2 1 0

200

200

0

100 Dose 2-DG (mg/kg)

200

(b) Intake (% body weight)

(b) Intake (% body weight)

100 Dose 2-DG (mg/kg)

5

5 4 3 2 1 0

0

100 Dose 2-DG (mg/kg)

200

4 3 2 1 0

Figure 9. Intake of a commercial half-and-half milk diet (a) 1 h or (b) 3 h after administration of MA and 2-DG in 12-day-old rat pups. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

5 (c) Intake (% body weight)

0

4 3 2

administered with MA. Therefore, in this experiment, we examined the effects of additional doses of 2-DG.

1

Method

0

0

100 Dose 2-DG (mg/kg)

200

Figure 8. Intake of a commercial half-and-half milk diet (a) 1 h, (b) 3 h or (c) 6 h after administration of MA and 2-DG in 9-day-old rat pups. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

Pups were tested once at 6 or 9 days of age. Methods were identical to Experiment 2A except that doses of 2-DG were 0, 300 and 400 mg/kg. One to two pups from each of 8±10 litters were tested at each age (Ns ˆ 8±12 per group). A separate 2-way (dose 2DG  dose MA) ANOVA was conducted at each age.

Results and discussion

Experiment 2B Although no significant effects of combined administration of 2-DG and MA were found in Experiment 2A, in 6 and 9-day-old pups, there was a trend for 200 mg/kg 2-DG to increase intake after 3 h when

Even at increased doses of 2-DG, there were no significant effects of 2-DG, MA or the combination on intake in 6 or 9-day-old pups (data not shown). The data from these experiments reinforce the suggestion that 6 and 9-day-old pups differ from 12 and 15-day-old pups. In pups 9 days and younger, administration of MA alone or in combination with 2-DG fails

Effects of MA and 2-DG in rat pups 19 5 Intake (% body weight)

(a) 4 3 2 1 0

0

100 Dose 2-DG (mg/kg)

200

5 Intake (% body weight)

(b)

while older pups attain this capacity. Alternatively, they may reflect differences in physiological effects of administration of the drugs to pups of different ages. In the present experiment, we therefore examined the effects of administration of 2-DG and MA on glucose, hematocrit levels, -HBA, and stomach contents in 9, 12 and 15-day-old pups. In addition, Experiment 1B demonstrated that high doses of MA produced altered -HBA levels for 3 h; however, the doses at which altered -HBA was demonstrated do not consistently increase intake; in fact, 91
2 mg/kg MA consistently suppresses intake in 12 and 15-day-old pups. Thus, a second goal of this experiment was to examine the effects of doses of MA which produce increased intake on glucose, hematocrit, -HBA, and stomach contents in 9, 12 and 15-day-old pups.

4

Method

3 2 1 0

0

100 Dose 2-DG (mg/kg)

200

Figure 10. Intake of a commercial half-and-half milk diet (a) 1 h or (b) 3 h after administration of MA and 2-DG in 15-day-old rat pups. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

to produce significant changes in intake; the only changes noted were trends toward increased intake when a combination of 2-DG and MA was delivered to 9-day-old pups. In contrast, in 12 and 15-day-old pups, administration of MA alone can enhance intake but this enhancement is blocked by co-administration of 2-DG. Thus, 12 and 15-day-old pups also differ from adult rats, in which co-administration of drugs which block both fatty acid oxidation and glucose utilization produces larger increases than administration of either drug alone (Friedman & Tordoff, 1986; Friedman et al., 1986).

Experiment 2C: physiological effects of 2-DG and MA The results of Experiments 2A and B could reflect agerelated differences in detecting and processing signals related to changes in energy utilization; young pups fail to respond to signals related to altered metabolism

Pups were tested once at 9, 12 or 15 days of age. Pups received an i.p. injection of 0, 11
4 or 22
8 mg/kg MA combined with 0, 100 or 200 mg/kg 2-DG, then were placed into a warm, humid incubator for 1 h (9 and 12day olds) or 3 h (9, 12 and 15-day-olds). One to two pups from each of 10 litters were tested at each age (Ns ˆ 10±12 per group). Blood and stomachs were collected from pups in all groups; in addition, hematocrit levels were determined for all pups. To test the effects of combined administration of MA and 2-DG, separate 3-way (delay  dose 2-DG  dose MA; 9 and 12-day-old pups) or 2-way (dose 2-DG  dose MA; 15-day-olds) ANOVA's were run for each measure (glucose level, stomach content, hematocrit, -HBA levels). Post hoc-comparisons were performed using LSD tests with a p < 0
01 used to control for multiple comparisons.

Results and discussion Nine-day-olds -HBA levels in 9-day-old pups were affected by delay as well as dose of 2-DG and dose of MA (main effect of delay; F(1, 186) ˆ 72
12, p < 0
05: dose 2-DG  dose MA interaction; F(4, 186) ˆ 2
76, p < 0
05; Fig. 11). -HBA levels were significantly higher in 9-day-old pups after 3 h than after 1 h. In addition, 1 h after administration, a dose of 100 mg/kg 2-DG alone significantly decreased -HBA levels. Three hours after administration of 2-DG and MA, -HBA levels were significantly lower in pups receiving 22
8 mg/kg MA combined with either 100 or 200 mg/kg 2-DG. There were no significant effects of either dose of MA alone after 1 or 3 h, although there was a trend for decreased

20

S.E. Swithers 20

250 (a) 200

15

10

** **

Glucose (mg/dl)

β-hydroxybutyrate (mg/dl)

(a)

*

5

100 50

0

0

100 Dose 2-DG (mg/kg)

0

200

20

100 Dose 2-DG (mg/kg)

200

(b)

***

200 *

* 10

5

Glucose (mg/dl)

15

0

250

(b) β-hydroxybutyrate (mg/dl)

* 150

150

*

***

*

100 50

0

0

100 Dose 2-DG (mg/kg)

200

Figure 11. -hydroxybutyrate levels in 9-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11.4 mg/kg MA, &; 22
8 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG group; **0
01 < p < 0
05 compared to 0 mg/kg MA, 0 mg/kg 2-DG group, ***p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG at 1 h delay.

-HBA 1 h after administration of either 11
4 or 22
8 mg/kg MA alone ( ps ˆ 0
03 and 0
04 respectively). Glucose levels in 9-day-old pups were not affected by administration of MA, but were significantly affected by the delay and the dose of 2-DG (main effects of delay and dose 2-DG; F(1, 186) ˆ 70
66, p < 0
05 and F(2, 186) ˆ 9
26, p < 0
05 for delay and dose 2-DG respectively; Fig. 12). Overall, glucose levels were significantly lower in pups after 3 h than after 1 h. In addition, administration of 200 mg/kg 2-DG produced significant increases in glucose levels after 1 or 3 h and administration of 100 mg/kg 2-DG produced increases in glucose levels after 3 h. Stomach contents were significantly affected by delay, but not by administration of 2-DG or MA (main effect of delay; F(1, 186) ˆ 19
07, p < 0
05; Fig. 13). Overall, pups tested after a 3 h delay had significantly smaller stomach contents than pups tested after a 1 h delay.

0

0

100 Dose 2-DG (mg/kg)

200

Figure12. Glucose levels in 9-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11.4 mg/kg MA, &; 22
8 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG; ***p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG at 1 h delay.

Hematocrit values were also affected by the delay, but not by administration of MA or 2-DG (main effect of delay; F(1, 186) ˆ 179
11, p < 0
05; data not shown). Hematocrit levels were significantly elevated in pups tested after 3 h compared to pups tested after 1 h (Means  SEM ˆ 38
93  0
23 vs. 34
26  0
25).

Twelve-day-olds -HBA levels in 12-day-old pups were significantly affected by the delay and by administration of 2-DG and MA (main effect of delay, F(1, 160) ˆ 33
60, p < 0
05; dose MA  dose 2-DG interaction; F(4, 160) ˆ 3
73, p < 0
05; Fig. 14). Unlike 9-day-olds, there were no differences in -HBA levels in control pups between 1 and 3 h. In addition, -HBA levels were significantly suppressed 1 h after administration of either 11
4 or 22
8 mg/kg MA alone or administration of 100 or 200 mg/kg 2-DG alone. After 1 h, combined administration of either dose of 2-DG with

2.5

21

20 (a) β-hydroxybutyrate (mg/dl)

(a)

2.0 1.5 1.0 0.5 0.0

0

100 Dose 2-DG (mg/kg)

15

10

*

*

*

*

5

0

200

0

100 Dose 2-DG (mg/kg)

200

0

100 Dose 2-DG (mg/kg)

200

20 (b)

(b) β-hydroxybutyrate (mg/dl)

Stomach contents (% body weight)

Stomach contents (% body weight)

Effects of MA and 2-DG in rat pups

2.0 1.5 1.0 0.5 0.0

0

100 Dose 2-DG (mg/kg)

200

15

10

5

0

Figure 13. Stomach contents in 9-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

Figure 14. -hydroxybutyrate levels in 12-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG group.

either dose of MA produced no significant decreases in -HBA. Three hours after administration, there were no significant effects of 2-DG or MA on -HBA levels in 12-day-old pups. Glucose levels were significantly affected by the delay and by administration of 2-DG, but not MA (main effect of delay, F(1, 165) ˆ 7
62, p < 0
05; main effect of dose 2-Dg, F(2, 165) ˆ 14
47, p < 0
05; delay  dose 2-DG interaction, F(2, 165) ˆ 6
16, p < 0
05; Fig. 15). One hour after administration of 2-DG, glucose levels were significantly elevated by either dose of 2-DG, however there were no differences in glucose levels after 3 h and there were no differences in glucose levels in control pups between 1 and 3 h. Stomach contents were affected by administration of 2-DG, but not by administration of MA or the delay (main effect of 2-DG, F(2, 166) ˆ 3
10, p < 0
05; Fig. 16). Post-hoc tests revealed a trend for either dose of 2-DG to increase stomach contents, but these differences were not significant ( ps ˆ 0
04 and 0
02 for 100 and 200 mg/kg 2-DG, respectively).

Hematocrit levels were significantly affected by the delay but not by administration of MA or 2-DG (main effect of delay; F(1, 166) ˆ 98
55, p < 0
05; data not shown). Hematocrit levels were significantly higher in pups after 3 h than after 1 h (35
54  0
37 vs. 31
25  0.22).

Fifteen-day olds -HBA levels in 15-day-old pups after 3 h were not significantly altered by administration of MA or 2-DG (Fig. 17). Glucose levels were significantly affected by 2-DG, but not MA (main effect of dose 2-DG, F(2, 86) ˆ 11
20, p < 0
05; Fig. 18). Three hours after administration, a dose of 200 mg/kg 2-DG significantly elevated glucose levels compared to either control pups or pups receiving a dose of 100 mg/kg 2-DG. Neither stomach contents (Fig. 19) nor hematocrit levels (data not shown) were significantly affected by administration of MA or 2-DG in 15-day-old pups.

S.E. Swithers Stomach contents (% body weight)

250 (a)

Glucose (mg/dl)

200

* *

150 100 50 0

0

100 Dose 2-DG (mg/kg)

200

Stomach contents (% body weight)

250 (b)

Glucose (mg/dl)

200 150 100 50 0

0

100 Dose 2-DG (mg/kg)

200

Figure15. Glucose levels in 12-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &. *p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG.

Results from this experiment suggest five conclusions. First, doses of MA which stimulate intake in 12-day-old pups produce changes in -HBA levels consistent with altered fatty acid oxidation when measured 1 h after administration of MA. These results support a role for changes in fatty acid oxidation as an early metabolic signal modulating independent ingestion in rat pups. Second, administration of 2-DG results in changes in glucose utilization in 9, 12, and 15-day-old pups, but not in changes in ingestion. These results are consistent with previous demonstrations that exogenously altered glucose utilization is not an effective ingestive stimulus in rat pups at these ages (e.g. Houpt & Epstein, 1973; Leshem et al., 1990; Lytle et al., 1971). Third, unlike the effects seen in adult animals, simultaneous blockade of both fatty acid-oxidation and glucose utilization does not produce greater increases in intake than either alone (e.g. Friedman & Tordoff, 1986; Friedman et al., 1986); instead administration of 2-DG interferes with the stimulatory effects of MA in 12 and 15-day-old pups. Fourth, physiological changes are not the same in 9 and

2.5 (a) 2.0 1.5 1.0 0.5 0.0

0

100 Dose 2-DG (mg/kg)

200

0

100 Dose 2-DG (mg/kg)

200

2.5 (b) 2.0 1.5 1.0 0.5 0.0

Figure 16. Stomach contents in 12-day-old pups (a) 1 h or (b) 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &. 20 β-hydroxybutyrate (mg/dl)

22

15

10

5

0

0

100 Dose 2-DG (mg/kg)

200

Figure 17. -hydroxybutyrate levels in 15-day-old pups 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

12-day-old pups after administration of MA and 2-DG. For example, administration of 2-DG to 12-day-old pups results in increased stomach contents, but there are no effects on stomach contents in 9-day-old pups. These

Effects of MA and 2-DG in rat pups 250

Glucose (mg/dl)

200 % 150 100 50 0

0

100 Dose 2-DG (mg/kg)

200

Stomach contents (% body weight)

Figure 18. Glucose levels in 15-day-old pups 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &. %p < 0
01 compared to 0 mg/kg MA, 0 mg/kg 2-DG and 0 mg/kg MA, 100 mg/kg 2-DG.

2.5

23

signals that have been demonstrated to be produced by higher levels of the drug. Given that older pups fail to respond to these higher doses, determining the cause of differences between 9 and 12-day-olds' behavior is complicated. However, the latter explanation seems more likely given that in 9-day-old pups a combination of 2-DG and MA does produce significant decreases in -HBA levels after 3 h and does not affect stomach contents, but this combination fails to alter intake. Finally, even in pups older than 12 days of age there is no straightforward relationship between the physiological effects of administration of MA and 2-DG and the effects on ingestive behavior. For example, in 12-dayold pups, administration of DG alone can lower -HBA levels, perhaps due to increased utilization of ketones as an alternative fuel, but intake is not affected. This lack of increased intake effect may result from altered gastric emptying produced by administration of 2-DG in 12-day-olds. In addition, the effects of moderate doses of MA on behavior endure for up to 3 h, while the effects on -HBA levels endure for only 1 h. It thus remains unclear exactly what signal produced by administration of MA results in increased intake in pups 12 days of age and older.

2.0

General discussion

1.5 1.0 0.5 0.0

0

100 Dose 2-DG (mg/kg)

200

Figure 19. Stomach contents in 15-day-old pups 3 h after administration of MA and 2-DG. 0 mg/kg MA, &; 11
4 mg/kg MA, &; 22
8 mg/kg MA, &.

increases may help explain why 2-DG inhibits the effects of MA on intake in 12-day-olds. As described above, pups are sensitive to gastric volume and if pups start the test with larger stomach volumes, they may consume less. In addition, low doses of MA fail to produce significant decreases in -HBA levels in 9-day-old pups, but do alter -HBA levels in 12-day-olds. This pattern of results complicates interpretation of the failure of 9-day-olds to increase intake after administration of any dose of MA. This failure may result either from an inability of lower doses to produce a physiological signal or the inability of the pup to respond to the physiological

The results from the present set of experiments expand our understanding of the physiological and behavioral effects of altered energy metabolism in young rats. Previous study has demonstrated that blockade of fatty acid oxidation in 9-day-old pups does not affect intake within 1 h (Swithers, 1997); the present data demonstrate that intake is also not affected 3 or 6 h after MA in 9-day olds. In addition, altered glucose metabolism does not affect intake in 6 or 9-day-old pups after 1, 3 or 6 h. Further, combined blockade of fatty acid oxidation and glucose oxidation fails to produce increased intake in 6 or 9-day-old pups. Taken together with previous work attempting to demonstrate effects of altered metabolism on intake in young rats, these data support the hypothesis that pups 9 days of age or younger are insensitive to signals related to changes in metabolic state. On the other hand, in pups aged 12 or 15 days, administration of MA produces increases in intake for up to 3 h, but is no longer effective by 6 h after administration. The present results suggest that the failure of MA to enhance intake results from the 6 h deprivation period which accompanies the 6 h delay between administration of MA and testing, since 12 and 15-day-old pups tested 6 h deprived, but only 1 h following administration of MA also fail to increase intake. In addition,

24

S.E. Swithers

the behavioral results suggest dissociation between the intake-enhancing effects of moderate doses of MA and the intake-suppressing effects of higher doses. The enhancing effects are seen up to 3 h following administration of MA, and are not observed in 6-h-deprived pups. However, the suppressive effects are seen only 1 h following administration, even in pups tested 6 h deprived. In addition, while previous studies suggested that the effects of high doses of MA on gastric emptying might explain suppressed intake, the results of the present study do not entirely support that conclusion. First, at several ages and delays, a dose of MA was demonstrated to suppress gastric emptying, but intake was either unaffected or actually enhanced. In addition, in the pups tested 6 h deprived, but only 1 h after administration of MA, a large portion of the gastric contents likely emptied during 5 h of deprivation preceding administration of MA. Administration of MA to pups with empty stomachs would have had little opportunity to suppress gastric emptying, but intake was still suppressed. However, it is possible that the suppressive effects result from inhibition of gastric motility rather than gastric emptying. In that case, high doses of MA might affect intake even in the absence of overt changes in gastric volume at the end of testing. Thus, determining the mechanism by which high doses of MA suppress intake will require further investigation. In 12 and 15-day-old pups, the temporal characteristics of the effects of MA depend on the doses tested. Pups show significant changes in -HBA levels 3 h after administration of high doses of MA, but more moderate doses produce effects that last for 1 but not 3 h. By 6 h after administration, no effects of even high doses of MA are observed. In 15-day-old pups, this failure to decrease -HBA levels may result from the decreased levels observed in control pupsÐthat is, increased deprivation alone decreases -HBA levels. Consistent with this hypothesis is the demonstration that 12 and 15-day-old pups deprived for 3 h and receiving doses of MA had -HBA levels that were not different from control pups deprived for 6 h. These results suggest that -HBA levels may correlate with a signal which typically stimulates ingestion following deprivation in rat pups, and the physiological and behavioral changes noted in these pups support the idea that changes in oxidation of fatty acids represent an early metabolic stimulus to ingest. On the other hand, a signal related to a change in -HBA levels alone cannot account for all behavior observed in these experiments, or for results obtained from previous experiments on the control of ingestion in rat pups. For example, Experiment 2 demonstrated clear dissociations between the level of -HBA and intake; some pups with altered -HBA levels failed to show

increases in intake. Additional signals might therefore be involved in the increased intake following more significant deprivation. One additional signal known to alter intake in pups is hydrational status (e.g. Wirth & Epstein, 1976). In Experiment 2C evidence for extracellular dehydration after 3 h deprivation was noted. This suggests that during deprivation, signals relevant to both metabolic and hydrational status may be affecting intake; it is currently unknown whether combined blockade of fatty acid oxidation and acute dehydration produce greater increases in intake in pups than either alone. Taken together, the data on changes in -HBA, FFA, glucose and hematocrit levels in pups at the different ages tested suggest that as pups mature, not only do different behavioral responses to administration of MA occur, but physiological responsiveness is also altered. In the youngest pups tested, administration of high doses of MA produced significant effects on -HBA, glucose and FFA acid levels, but the time since the administration of MA did not have a significant effect. On the other hand, lower doses failed to produce significant effects on -HBA levels at either 1 or 3 h. These results suggest that in young pups, administration of high doses of MA has profound physiological effects that are relatively enduring. However, pups at this age do not respond to these physiological changes with enhanced ingestive behavior. The pattern of changes in all of the blood measures after high doses (decreased -HBA levels and decreased glucose levels) and the failure of lower doses to affect -HBA levels is consistent with the suggestion that young pups are more capable of compensating physiologically to alterations of fatty acid oxidation. A lack of ingestive responding may reflect this enhanced physiological responding. By 15 days of age, administration of MA no longer produces effects on blood glucose levels, suggesting an absence of physiological compensation, which may be replaced by behavioral compensation. These data extend previous findings on the ontogeny of ingestive and physiological responses to blockade of fatty acid oxidation in rat pups. The behavioral results are generally consistent with the physiological effects of altered fatty acid oxidation, supporting the conclusion that changes in the utilization of fats represents the earliest metabolic signal influencing ingestion in rat pups. It is currently unclear what developmental mechanisms are responsible for the emergence of responding to changes in fatty acid oxidation. It is not clear why 9-day-old pups are insensitive to drastic changes in -HBA levels, nor why 12 and 15-day-old pups are sensitive. However, pinpointing the ontogeny of metabolic controls of ingestion has a number of implications for furthering our understanding of ingestive responding.

Effects of MA and 2-DG in rat pups

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