INTRODUCTION

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MANY INVESTIGATORS HAVE SUGGESTED that opioids are involved in the "rewarding" aspects of feeding (10, 17, 32). This hypothesis is based on evidence that opioid agonists increase (9, 20), whereas opioid antagonists decrease (11, 15), intake of preferred foods more robustly than nonpreferred foods. For example, in food-deprived rats that choose between high-fat and high-carbohydrate diets, doses as low as 0.01 mg/kg of the opiate antagonist naloxone decrease intake of the preferred food, whereas doses as high as 3 mg/kg do not alter consumption of the nonpreferred diet (18). Several investigators have examined the role of opioids in the acquisition and expression of flavor preferences conditioned by either sweet tastes or intragastrically infused sucrose (3, 43). Peripheral administration of the opioid antagonist naltrexone decreased imbibition of the flavored solutions but failed to affect either acquisition or expression of the conditioned flavor preferences. On the other hand, opioids do seem to be involved in the expression, but not the acquisition, of sucrose-reinforced conditioned place preference (13).

Naloxone's anorectic effects on preferred food consumption are even more powerful under conditions of chronic energy restriction (34, 42). For example, when naloxone is administered to separate groups of rats provided with either a preferred sucrose diet or a less preferred cornstarch diet, its anorectic effect is modulated by the energy state of the subjects. In nocturnally fed, food-restricted rats, naloxone decreased intake of food in the high sucrose-fed rats but not in cornstarch-fed rats. However, in non-food-deprived rats naloxone suppressed intake in both sucrose- and starch-fed groups. These results indicate that opioids are especially effective under conditions that involve both energy state and presentation of preferred foods.

The heightened anorectic actions of naloxone on preferred food intake may, however, be influenced by different rates of food consumption among foods that contrast in "palatability" (34, 42). For example, food-restricted rats fed the sucrose diet consumed almost twice as much food compared with the cornstarch-fed rats during a 30-min period; that is, they appeared to have eaten at a much faster rate. However, in this study we did not measure the actual rate of food intake (local rate), which reflects the amount of food ingested divided by the time spent eating (excluding pauses). Without accurately measuring the time spent eating by using an automated feeding system or lickometer device, one cannot evaluate whether naloxone's anorectic effect is impacted by eating rate.

Energy restriction may not only affect the rate of food consumption but also the total amount of food consumed. Thus another possible explanation for nalaxone's relatively powerful anorectic effect on preferred foods under conditions of food restriction may be related to the total amount of food eaten. Previous data from our laboratory indicate that naloxone decreases intake of freely imbibed sucrose solutions much more effectively than intake of small amounts of the same sucrose solutions given in an operant chamber (8). Perhaps animals must consume a minimum amount of food before opioids are released and only after this release could naloxone block the effects of the opioids. While naloxone blocks sham feeding, it does not affect intake early in the session, but rather only after a significant amount of nutrients are consumed (28-30). The latter finding suggests some interaction between the orosensory/gastrointestinal tract and the brain is necessary for naloxone to decrease food intake.

The evidence thus reviewed suggests the possibility that naloxone may preferentially affect consumption of the high-sucrose diet for reasons other than "reward"; its effect may be "rate dependent" or dependent on total food intake. To further evaluate the potent anorectic effect of naloxone on consumption of a palatable diet, naloxone's actions on meal microstructure were analyzed using an automated weighing system in food-restricted rats eating either a high-sucrose diet or a high-cornstarch diet. Also, we tested the effect of a preload on naloxone's anorectic effects to evaluate the importance of total food intake.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Eighteen male Sprague-Dawley rats (225-250 g Harlan, Madison, WI) were housed individually in a controlled temperature (21-23°C) vivarium with a 12:12-h light-dark (light on at 0700) cycle. Rats were given standard Teklad Rodent chow and water ad libitum for a 2-wk period. Rats were then food restricted to reduce body weight to 85% of beginning weight. Laboratory chow was put into the cage late in the lights-on cycle (1400-1600). After reaching stable body weights, rats were placed daily (1400) into an automatic weighing system (25) that contained ground Teklad diet in food cups for a 2-h period. Animals received a subcutaneous injection of saline 15 min before placement into cages. No additional food was given to rats while in their home cages, but water was available ad libitum. After this 5-day acclimatization period, rats were divided into two groups: one receiving a high-sucrose diet and one receiving a high-cornstarch diet (Table 1). After rats received this diet and were injected with saline for a 10-day period, the experiment was initiated. Rats were injected subcutaneously with naloxone (0.1, 0.3, 1, or 3 mg/kg) or saline at 1400, held in plastic boxes for 15 min, and placed into the weighing system cages for 2 h. Each rat received all doses of naloxone or vehicle in a counterbalanced design every other day. Rats received saline injections at 1400 on the day between experiments.

In addition, we evaluated the effect of a diet preload on naloxone's anorectic effects. Rats were allowed to eat 4 g of either the starch diet or the sucrose diet before naloxone injection (1, 0.1, 0.01 mg/kg). Fifteen minutes after naloxone administration they were placed into the weighing system cages, and food intake during the first meal was evaluated. We also measured the minutes to the first eating event and the minutes to the termination of the first meal.

The eating microstructure was analyzed using data obtained from our automated weighing system. Latency to eat, meal length (end of meal = 10 min without intake), time of meal termination, overall rate of eating (g intake/meal), local rate of eating (g intake/min actual eating), and eating episodes (end of episode = 8 s without intake) were calculated based on data derived from the automated weighing system. Food intake for the first meal was measured by subtracting spillage from total gram intake.

In previous studies of naloxone's effect on food intake, intake was measured at selected time points. Our measurement of meal parameters did not measure intake at selected time points, but rather during the complete meal. That is, intake was quantified at the time at which the meal ended. To compare these data to other studies, we also estimated intake at 30 min. In some cases, animals were actively eating at the 30-min time point. To estimate 30-min intake we used the amount of food ingested closest to the 30-min time point, divided by the number of minutes at that time, and multiplied by 30 min.

All data are presented as means ± SE. Data were subjected to two-factor analysis of variance [diet type × naloxone dose (repeated measure)], and means were compared using Fisher's least-significant difference test. When only two groups were compared, a Student's t-test was used to compare means.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sucrose-fed rats exhibited a higher overall eating rate (g intake/meal) during their first meal compared with cornstarch-fed rats [0.34 ± 0.04 vs. 0.20 ± 0.02 g/min, respectively, P = 0.005] (Table 2). Collapsed across all treatments there was a main effect of diet type [F(1,16) = 11.4, P = 0.0038] on overall eating rate with no main effect of naloxone [F(4,64) = 1.49, P = 0.22] on overall rate, but a significant diet type × naloxone interaction [F(4,64) = 5.79, P = 0.0005] (Table 2). While naloxone failed to significantly decrease the overall rate of consumption of the diets, there appeared to be a trend for a reduction in the overall rate of sucrose diet intake. This is supported by the significant diet type × naloxone interaction, suggesting that naloxone affected the overall rate of diet intake differently in the starch diet than the sucrose diet.

Sucrose-fed rats did not exhibit a higher local eating rate (food intake/min spent eating during meal, pauses excluded) during their first meal compared with cornstarch-fed rats [0.78 ± 0.09 vs. 0.76 ± 0.10 g/min] (Table 2). Naloxone seemed to increase the local rate of the starch diet intake while decreasing the local rate of intake of the sucrose diet. However, collapsed across all treatments there was no main effect of naloxone on local eating rate [F(4,64) = 0.24, P = 0.92], but there was a main effect of diet type [F(1,16) = 12.11, P = 0.003] on local eating rate. There was no diet type × naloxone interaction [F(4,64) = 2.11, P = 0.09] (Table 2).

There was a main effect of diet type [F(1,16) = 6.33, P = 0.023] on latency to the first eating episode, but neither a significant main effect of naloxone [F(4,64)= 0.978, P = 0.426] nor a diet type × naloxone interaction [F(4,64)= 0.978, P = 0.964]. Rats presented with the sucrose diet began eating more quickly than rats presented with the cornstarch diet (Table 2). However, naloxone had no effect on the latency to begin eating in either the sucrose or cornstarch groups.

During the first meal (eating continuously without a 10-min hiatus) there was no main effect of diet type [F(1,16) = 2.19, P = 0.16], a significant main effect of naloxone [F(4,64) = 12.80, P < 0.0001], and no diet type × naloxone interaction [F(4,64) = 1.03, P = 0.40] on food intake. Rats injected with the vehicle control ate the same amount of the sucrose diet as the cornstarch diet (9.5 ± 1.3 vs. 9.2 ± 0.9 g, respectively). All doses of naloxone significantly reduced food intake (38-60%) during the first meal in both the cornstarch and sucrose diets (Fig. 1).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effect of naloxone on food intake (g) during the first meal. A: no preload; B: 4 g preload given to rats before injection of naloxone. *P < 0.05 compared with the vehicle control.

We estimated 30-min food intake by using the food intake data closest to 30 min, determining the overall rate, and extrapolating the data to 30-min intake. An estimation of 30-min intake was necessary due to the fact that the scale can only measure intake accurately at a time when the rats are not eating and thereby applying pressure to the scale. As we have found in the past, naloxone altered intake at this time point only in the sucrose group (Fig. 2). Although there was no main effect of diet [F(1,16) = 2.62, P = 0.13], there was a significant naloxone effect [F(4,64) = 5.50, P = 0.0007] and a significant diet type × naloxone interaction [F(4,64) = 3.41, P = 0.01].

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effect of naloxone on food intake (g) during the first 30 min. *P < 0.05 compared with the vehicle control.

There was a main effect of diet type [F(1,16) = 19.82, P = 0.0004] and naloxone [F(4,64) = 4.61, P = 0.0025] on length of the first meal, but no significant diet type × naloxone interaction [F(4,64) = 0.231, P = 0.92] (Fig. 2). Sucrose-fed rats took less time to complete their first meal compared with cornstarch-fed rats (33.8 ± 4.7 vs. 52.3 ± 9.3 min, respectively). Naloxone significantly reduced the time to the end of the first meal in sucrose-fed rats across all doses, whereas naloxone did not significantly affect the termination time of the first meal in cornstarch-fed rats (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of naloxone on length of the first meal (min). A: no preload; B: 4 g preload given to rats before injection of naloxone. *P < 0.05 compared with the vehicle control.

We also evaluated the number of eating episodes (termination defined as an 8-s hiatus) during the first hour of the study. There were more episodes of eating during the first hour in the starch group than in the sucrose group [F(1,16) = 48.02, P < 0.0001], but naloxone had no effect on the number of eating episodes in either group [F(4,64) = 2.07, P = 0.10] (Table 2).

Food intake during the second meal was not affected by naloxone [F(4,64) = 1.21, P = 0.32], perhaps due to its short-term effects (Table 3). However, the rats did ingest more of the sucrose diet than the starch diet [F(1,16) = 9.38, P < 0.0007]. There was no effect of diet or naloxone on length of the second meal (data not shown). Termination of the second meal occurred at 96.2 ± 10.9 min for the starch diet and 74.6 ± 7.5 min for the sucrose diet. Most rats ingesting the cornstarch diet did not eat a third meal, whereas those ingesting sucrose did. Naloxone had no effect on intake or length of the third meal (data not shown). Termination of the third meal occurred at 106.3 ± 7.6 min for the starch diet and 94.4 ± 9.5 min for the sucrose diet. Overall and local rate were unaffected by diet or naloxone during the second and third meals (data not shown).

The above data indicate that naloxone decreased food intake by decreasing meal length but not rate of eating. Because naloxone did not impact overall or local rate, it was unlikely that naloxone affected the percentage of time spent eating or pausing. Although there was a significant effect of diet on the percent time rats spent eating or pausing [F(1,16) = 12.86, P = 0.0024], there was no effect of naloxone on these parameters [F(4,16) = 0.75, P = 0.56] (Table 2).

Thus naloxone's major effect was to decrease both the amount of food consumed and the time to the end of the first meal in the sucrose-fed rats. These results suggest that naloxone may be producing an early satiety, analogous to exposure to food. To examine this possibility, we conducted another study in which rats were given a diet preload.

Allowing rats to consume a preload altered naloxone's effects on food intake and termination time of the first meal but no other aspects of meal architecture. During the first meal after the preload (eating continuously without a 10-min hiatus), there was a main effect of diet type [F(1,76) = 7.68, P = 0.007] and naloxone [F(3,76) = 22.52, P = 0.0001] on food intake but no diet type × naloxone interaction [F(3,76) = 1.34, P = 0.267]. Rats injected with the vehicle control ate more of the sucrose diet than the cornstarch diet (10.2 ± 0.5 vs. 6.9 ± 0.5 g, respectively). The 1, 0.1, and 0.01 mg/kg doses of naloxone significantly decreased food intake in the sucrose group, whereas only the 1 mg/kg dose decreased intake in the cornstarch group (Table 4).

After the preload, there was a main effect of diet type [F(1,76) = 6.54, P = 0.012] and naloxone on termination time of the first meal [F(3,76) = 8.65, P = 0.0001] but no significant diet type × naloxone interaction [F(3,76) = 0.18, P = 0.905]. The amount of time to the termination of the first meal was not different in the sucrose and the starch-diet groups (38.4 ± 4.4 vs. 31.7 ± 2.6 min, respectively). Naloxone significantly reduced the time to the end of the first meal in sucrose-fed rats (33-67% reduction) across all doses, whereas naloxone did not affect the termination time of the first meal in cornstarch-fed rats (Table 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It has been previously shown that naloxone's anorectic effect is enhanced in food-restricted rats consuming high-sucrose foods (34, 42). Such findings are consistent with suggestions that opioids are importantly involved in the rewarding aspects of food consumption (10, 17). However, food-restricted rats fed a high-sucrose diet eat much more of the high-sucrose diet during a 30-min period (42); thus it is also possible that naloxone preferentially decreases food ingested at a high rate. Alternatively, naloxone's anorectic effect may be influenced by the total amount of food eaten. These possibilities were investigated by analyzing the meal patterns of food-restricted rats fed either a high-sucrose or a high-cornstarch diet.

Rats ate at a faster overall rate (food intake/length of meal) when fed a sucrose-based diet compared with a cornstarch-based diet. Although not statistically significant, there was a tendency for naloxone to decrease overall eating rate of the sucrose diet. Also, naloxone failed to significantly alter the local eating rate (food intake/time of eating) in both groups. These results suggest that if naloxone had an effect on overall or local rate of food intake, it was not robust. Instead, naloxone's major effect was to decrease the amount of food consumed during the first meal in the sucrose-fed rats. Thus the present results indicate that naloxone reduces the amount of sucrose or cornstarch diet consumed irrespective of how fast it is eaten. Although naloxone decreased food intake of both diets, it did seem to have greater effects on intake of the sucrose diet, particularly if one used a 30-min time point. In agreement with our previous finding (18), the present results suggest that both the magnitude and potency of naloxone's anorectic effect seemed to be heightened in sucrose-fed rats, relative to cornstarch-fed rats. However, naloxone's heightened effectiveness in the sucrose-fed rats was less notable if one examined the entire meal, rather than a fixed time point (e.g., 30 min). It should be noted that the rats eating the cornstarch diet took longer to end their meal than the sucrose diet. Because naloxone is a relatively short-acting compound, it is possible that naloxone's anorectic effect diminished by the end of the starch meal.

Naloxone's stronger anorectic effects in sucrose-fed rats may be interpreted solely in terms of the "sweet" or "rewarding" properties of this diet. However, within the fixed time periods during which food intake is typically measured in feeding studies, sucrose-fed rats also consume more food than starch-fed rats. For example, over 30 min, sucrose-fed rats consume as much as 30% more diet than starch-fed rats. Thus these results might have been due to differences in the amount of food ingested among the two groups. This possibility was examined by preloading animals before testing with naloxone. It was found that preloading starch-fed rats did alter the magnitude of naloxone's effects on food intake. For example, under vehicle, unloaded and preloaded starch-fed rats had similar amounts of food in the gut, but at the 1.0 mg/kg dose naloxone had a stronger effect in the preloaded animals (61% when preload excluded from food intake; 75% when 4 g preload included in food intake) compared with unloaded group (35%). The preload did not have a major affect on the percent reduction in the sucrose group (64% when preload was excluded from food intake; 74% when preload was included in food intake) compared with the percent reduction observed in the unloaded intake (59%). Thus it seems unlikely that equating food intake in sucrose- or starch-fed rats by preloading can alone account for the present results.

The present findings indicate that naloxone's anorectic actions are modulated by both the type and amount of food ingested. These data may be interpreted within the context of naloxone-mediated effects on several different mechanisms, including orosensory processing, food reward, motivational actions, or gastrointestinal signaling. One mechanism by which naloxone may affect sucrose consumption is by altering sensory processes, such as the ability of rats to discriminate sucrose. However, peripheral administration of naloxone, at doses comparable to those used in the present study, failed to affect sucrose discrimination in rats trained to discriminate 10% sucrose from water in a two-lever operant chamber (37). However, it was shown that naloxone did reduce consumption of sucrose solutions when rats were allowed to drink ad libitum from sipper tubes during a brief session.

In contrast to taste discrimination, opioid antagonists appear to affect postsensory processing of sucrose. The taste reactivity test has been used to examine hedonic reactions by analyzing patterns of stereotypic orofacial responses emitted by rats in reaction to passive oral infusions of sucrose, quinine, or sucrose-quinine solutions (6, 22). Opioid antagonists reduce ingestive reactions in response to an infusion of sucrose (38). The results derived from the taste-reactivity test indicate that naloxone may influence hedonic reactions in response to oral stimulation with sapid solutions and is consistent with hypotheses indicating that opioids are involved in food reward (17).

Whereas opioid antagonists influence hedonic reactions to sucrose, they appear to have a lesser effect on motivation for sucrose. For example, naloxone is less effective in reducing responding for a 10% sucrose solution on the progressive ratio schedule (PR) than in decreasing nonoperant sucrose drinking in the home cages (8, 23). In non-food-deprived rats, naloxone failed to reduce the break point even at a dose of 10 mg/kg when rats were working for 0.1 ml of a 10% sucrose solution (8). On the other hand, naloxone robustly reduced consumption of 10% sucrose when non-food-deprived rats were given free access to it from a sipper tube. These results may reflect the fact that, under vehicle administration, rats responding on the PR consumed <1 ml of solution per session on average, whereas under free-access conditions, rats consumed ~15 ml of solution on average. These data suggest that opioid antagonists may only have an effect on food consumption after a substantial amount of sucrose solution has been consumed. A similar pattern of effects has also been observed in genetically obese Zucker rats, where naloxone was more effective in reducing free consumption of food pellets compared with responding on a PR-3 schedule of reinforcement (19). These results may reflect a relationship between opioids and the development of satiety.

This interpretation seems to be consistent with evidence that opioid antagonists affect the maintenance of food intake. For example, it has been shown that opioid antagonists affected only the maintenance but not the initiation of feeding as measured by either observational or instrumental behavioral analyses (26, 27). Additionally, when food-restricted rats are made to work for food pellets by pressing a lever 80 times (FR80, initiation phase) to obtain the first pellet and then made to earn each subsequent pellet with three lever presses (FR3, maintenance phase), naloxone had no effect on the effort required to earn the first pellet but inhibited pressing after some food had been earned (39). By measuring the effects of opioid antagonists on free-access consumption of sapid solutions over discrete time intervals, it has also been noted that opioid antagonists affect the maintenance of feeding (7).

Naloxone is a nonselective opioid receptor antagonist that binds preferentially to the µ-receptor. Others have noted that selective µ- and -opioid receptor antagonists have differential effects on ingestion of a sucrose solution and a maltose dextrin solution in sham and real feeding (4, 5, 30, 31). Also, deprivation-induced feeding is more potently reduced by µ- and µ₁-antagonists than - and -antagonists (1, 2, 33). Therefore, in our study it is possible that different subpopulations of opioid receptors are activated and blocked by the less-selective antagonist naloxone. Analysis of meal patterns with selective opioid antagonists would address this issue.

As the previous discussion indicates, the mechanisms by which naloxone affects food consumption appear to be complex. The present results together with previously reported data indicate that naloxone's anorectic actions are modulated by at least two conditions: the sensory properties of foods, particularly those high in sucrose, and the energy state of the organism. Thus the elevated anorectic potency of naloxone may reflect the actions of opioid receptor blockade on neural circuits that mediate orosensory and/or postingestive signals.

Perspectives