INTRODUCTION

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OB PROTEIN (LEPTIN), the protein product of the ob gene, is a cytokine that reduces food intake after peripheral or central administration in a variety of species, including rats (3, 6, 10, 15, 26). These studies have used the common method of measuring total amount of food eaten during a specific time period after OB protein administration. Although total measurement of food intake provides information on the effectiveness of OB protein, this method does not allow characterization of the mode of action of OB protein to reduce food intake.

Meal pattern analysis, on the other hand, allows moment-to-moment study of ad libitum eating, including characterization of meal parameters such as meal size, meal duration, meal frequency, interval between meals (intermeal interval), derived analysis (e.g., feeding rate or meal size/meal duration and satiety ratio or intermeal intervals/meal size), and the temporal distribution during the nighttime and daytime periods. Thus meal pattern analysis provides a detailed description of the elements of eating, revealing the mode of action of an experimental manipulation that decreases food intake. For example, our previous studies showed that various cytokines and immunomodulators decrease food intake, including interleukin-1 (IL-1), IL-8, interferon (IFN), and ₂-microglobulin. However, these endogenous substances reduce food intake through different modes of action at doses that yield estimated pathophysiological concentrations in the cerebrospinal fluid: IL-1 (17) and IFN (16) reduce meal size and meal duration, whereas IL-8 (21) and ₂-microglobulin (22) reduce meal size exclusively. Suprapathophysiological concentrations of IL-1 also decrease meal frequency and prolong the intermeal intervals (18).

In the present study, we examined the effects of the intracerebroventricular (into the 3rd ventricle) administration of OB protein on ad libitum food consumption and meal parameters. Total intake, meal frequency, meal size, meal duration, and intermeal intervals were recorded using a computerized behavioral monitoring system. The results show that intracerebroventricularly administered OB protein decreases food intake exclusively by reducing meal size.

	MATERIALS AND METHODS

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Subjects and maintenance. Male Wistar (virus antibody free) rats weighing between 250 and 275 g at the beginning of the experiments were used. Rats were randomly assigned to groups and placed in individual cages. They were maintained ad libitum on powdered rat food (Labdiet, PMI Feeds, St. Louis, MO) or on pelleted rat chow (4.0 × 0.3 mm, 45 mg precision food pellets formula P; Noyes) and tap water as previously described (22). Lights were on from 0600 to 1800, and room temperature was kept at 21 ± 2°C. All rats were handled daily. After rats were adapted to their home cage for several days, cannulas were implanted into the third ventricle, as described below. Test solutions were administered after a postsurgical recovery period of at least 10 days.

Powdered food consumption. The measurement of powdered food intake was the same as in previous studies (21). In all cases food and water intakes were measured to within 0.1 g. Before and after the cannula implantation, the rats were fed ad libitum daily, except between 1630 and 1800, when food and water were removed to be measured and replaced and each rat's body weight was measured. Premeasured food and water were presented at 1800. Food and water intakes were measured at 2200 (4-h consumption), 0600 (nighttime consumption from 1800 to 0600), and 1630 (daytime consumption from 0600 to 1630). Total daily consumption was calculated from 1800 to 1630.

Meal parameter analyses. Meal parameters were recorded by a computerized behavioral monitoring system before and after intracerebroventricular microinfusions. Rats were housed in special test chambers built of Plexiglas and aluminum and equipped with electromechanical pellet dispensers that delivered a single pellet when photobeams detected the removal of the previous pellet. Spillage is <0.5% of the total daily intake in this system. Pellets and water were available ad libitum throughout the experiment. Individual and cumulative changes in meal parameters before and after intracerebroventricular microinfusions were analyzed. Data acquisition was through a central processing unit and a behavioral programming language that uses a Turbopascal compiler to run several experimental stations simultaneously. The data from each input channel were summed and recorded at 10-min intervals, 24 h/day. A meal was defined as the acquisition of at least five pellets preceded and followed by at least 20 min of no feeding (intermeal interval) (16, 17, 21, 22). Meal size was defined as the number of pellets eaten during a meal. Feeding rate was calculated from meal size divided by meal duration (pellets/min). Postprandial intermeal intervals were taken as the time from the last pellet in one meal to the first five pellets in the next. Satiety ratio was calculated from intermeal intervals divided by meal size.

Implantation of brain cannulas. Under intraperitoneal ketamine (100 mg/kg) plus xylazine (5 mg/kg) anesthesia, a 23-gauge stainless steel guide cannula was implanted into the third ventricle at stereotaxic coordinates 2.1 anteroposterior and 0.0 lateral to bregma and 7.5-8.0 dorsoventral from the brain surface, as previously described (16). An incision was made through the dura mater with a dural hook. The superior sagittal sinus was carefully pulled to one side while gently lowering the guide cannula. Once the cannula was in position, the retraction of the sinus was released, and the cannula was anchored with dental acrylic. A sterile 29-gauge stainless steel obturator was used to ensure that the cannula remained patent.

Intracerebroventricular administration. Microinfusions were made into the third ventricle because of the importance of hypothalamic regions in the regulation of feeding. Intracerebroventricular microinfusions (10 µl/rat into unrestrained, undisturbed animals) were at the rate of 1 µl/min using a Harvard infusion pump (Harvard Apparatus, South Natick, MA). Each animal was infused between 1700 and 1800, i.e., before the nighttime period.

Each rat was randomly assigned to a group that received either 0.56, 1.4, or 3.5 µg/rat recombinant murine OB protein (leptin, PreproTech, Rocky Hill, NJ) or 3.5 µg/rat heat-inactivated OB protein. These doses were selected based on previous studies that used the intracerebroventricular administration of OB protein (3, 6, 26). The same OB protein stock solution (lot number 076763) was used in all experiments. OB protein stock solution was dissolved in sterile physiological saline (0.15 M NaCl) to its final concentration. OB protein inactivation was accomplished by heat treatment (90°C for 3 h); heat-treated OB protein was filtered through a sterile 0.1-µm filter (Millipore) before administration. To avoid nonspecific adsorption of the compounds on the experimental tools, all such materials were siliconized. Verification of the position of the cannula tip was as in previous studies, including the free outflow of cerebrospinal fluid through the guide cannula (17).

Data analyses. All results are expressed as means ± SE. Statistical analyses compared 1) the preinfusion levels (values for the day before infusion) to those obtained after infusion of test solutions and 2) the values among different groups after test solutions. Analyses were performed with the paired and two-sample Student's t-tests when data passed the normality (Kolmogorov-Smirnov) and equal variance (Levene Median) tests; otherwise, the data were analyzed with the Mann-Whitney or Wilcoxon tests. Power of performed tests was >0.8 in all cases. Data also were analyzed using ANOVA, with treatment and actual numbers or percentage changes as sources of variation and, where appropriate (i.e., after a significant main effect), followed by post hoc tests for pairwise multiple comparisons (Student-Newman-Keuls test). Kruskal-Wallis test was applied when data did not pass the normality test. Statistical significance was defined as P < 0.05.

	RESULTS

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Powdered food consumption. Figure 1 shows the effect of OB protein on 4-h food intake. ANOVA demonstrated that treatments differed significantly [F(3,33) = 12.3, P < 0.0001, power of performed test (ppt) = 1.0]. All three OB protein-treated groups showed significant decreases in food intake from similar baseline levels (0.56 µg/rat, from 11.6 ± 0.8 to 9.7 ± 0.6 g; 1.4 µg/rat, from 12 ± 0.6 to 8.9 ± 0.5 g; and 3.5 µg, from 11.7 ± 0.5 to 7.0 ± 0.4 g). Food intake was also decreased during the nighttime [F(3,33) = 10.3, P < 0.0001, ppt = 0.99] and total daily (H₃ degrees of freedom = 20.4, P = 0.0001) period after the intracerebroventricular administration of OB protein. Heat-treated OB protein had no significant effect in any period. Once we established the effectiveness and dose-response of this OB protein preparation, we studied the effects of intracerebroventricularly administered OB protein on meal parameters.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of intracerebroventricular administration of OB protein on 4-h (1800-2200) food intake. Bars represent means ± SE percentage differences from the preinfusion levels. Number of rats are shown in parentheses. HT, heat treated. * P < 0.05 from preinfusion level; P < 0.0001 between HT and intact 3.5 µg OB protein.

Analyses of nighttime, daytime, and total daily meal parameters. Parameters analyzed for the nighttime (period of largest OB protein effect after the acute intracerebroventricular administration), daytime, and total daily periods were total pellet intake, meal frequency, meal size, meal duration, feeding rate (meal size/meal duration), intermeal intervals, and satiety ratio (intermeal intervals/meal size). Vehicle alone or heat-treated OB protein had no effect on any meal parameter. The data comparing the profiles induced by heat-treated and active OB protein are summarized in Figs. 2-8 and Tables 1 and 2. Preinfusion values were similar for both groups.

Total intake of pellets. The intracerebroventricular administration of heat-treated OB protein had no effect on nighttime, total daily, or daytime intake of pellets. Intact OB protein decreased nighttime pellet intake 45% from a mean of 447 to 246 pellets (Fig. 2). Total daily and daytime intake of pellets decreased by 40% (Table 1) and 28% (Table 2), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of intracerebroventricular administration of heat-treated or intact OB protein (3.5 µg/rat, n = 10 each group) on nighttime (1800-0600) pellet intake. Bars represent means ± SE preinfusion day (open bars) and infusion day (filled bars).

The cumulative intake of pellets in 1-h intervals for the nighttime period is shown in Fig. 3. The significant decrease in nighttime pellet intake is clearly apparent beginning 2 h after intracerebroventricular administration of the OB protein preparation. Two-way ANOVA showed significant main effect for treatment [F(1,216) = 411.2, P < 0.0001, ppt = 1.0] and time [F(11,216) = 95.1, P < 0.0001, ppt = 1.0]; there was also a significant treatment × time interaction [F(11,216) = 10.5, P < 0.0001, ppt = 1.0].

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of intracerebroventricular administration of heat-treated () or intact () OB protein (3.5 µg/rat, n = 10 each group) on nighttime cumulative pellet intake in 1-h intervals after intracerebroventricular administration. Each point represents means ± SE. ANOVA revealed that groups differed significantly (see RESULTS).

Meal frequency. The intracerebroventricular administration of heat-treated or intact OB protein did not significantly affect the nighttime, total daily, or daytime meal frequency (Fig. 4 and Tables 1 and 2).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of intracerebroventricular administration of heat-treated or intact OB protein on nighttime meal frequency. Other explanations are as for Fig. 2.

Meal size. Nighttime and total daily meal size were significantly different between the heat-treated and intact OB protein-treated groups (Fig. 5 and Table 1). In the latter group, meal size decreased by 42 and 36% for the nighttime and total daily periods, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of intracerebroventricular administration of heat-treated or intact OB protein on nighttime meal size. Other explanations are as for Fig. 2.

Analysis of meal size of individual meals was also performed. OB protein decreased meal size beginning from the second meal after the intracerebroventricular administration: the first meal for the OB protein-treated group was 38 ± 6 (preinfusion day) vs. 40 ± 6 (infusion day) pellets; for the second meal, values were 72 ± 6 vs. 28 ± 5 pellets, respectively (P < 0.003); and for the third meal 54 ± 7 vs. 26 ± 4 pellets, respectively (P < 0.003). The intermeal interval between the first and second meals was identical, that is, 59 ± 7 and 59 ± 12 min for the preinfusion and infusion days, respectively.

Meal duration. There were no significant differences in nighttime, total daily, or daytime meal duration (Fig. 6 and Tables 1 and 2).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effects of intracerebroventricular administration of heat-treated or intact OB protein on nighttime meal duration. Other explanations are as for Fig. 2.

Feeding rate. Significant changes occurred in the intact OB protein-treated group. Feeding rate decreased by 30% for the nighttime (Fig. 7) and 25% for the total daily period (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of intracerebroventricular administration of heat-treated or intact OB protein on nighttime feeding rate. Other explanations are as for Fig. 2.

Intermeal intervals. No significant changes were obtained in either group.

Satiety ratio. This ratio increased by 101, 102, and 115% for the nighttime (Fig. 8), total daily (Table 1), and daytime (Table 2) periods, respectively, in the OB protein-treated group.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effects of intracerebroventricular administration of heat-treated or intact OB protein on nighttime satiety ratio. Other explanations are as for Fig. 2.

	DISCUSSION

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The data presented show that the intracerebroventricular administration of OB protein decreases food intake through a specific reduction of meal size. Meal duration and meal frequency are not significantly affected. Because of the decrease in meal size, the feeding rate decreased, whereas the satiety ratio increased. The data also show that a single intracerebroventricular administration of OB protein decreases meal size during the nighttime, daytime, and total daily periods. Because OB protein half-life is short (<1 h), this long-term inhibition of meal size must depend on other long-lasting events triggered by OB protein.

OB protein is secreted from adipose tissue into the circulation, and, as an endocrine signal, it is proposed to act on the central nervous system (CNS) networks that control feeding and energy balance (2). The precise action of OB protein on meal size after the intracerebroventricular administration conforms to that seen with activation of the ventromedial hypothalamus (7, 11) and suggests that OB protein-induced feeding inhibition is not due to a generalized depression of feeding behavior or to an impairment in the rat's capacity to start a meal. Because OB protein may be a mediator of fat tissue-to-brain communication, the specific role of OB protein on spontaneous meal size supports its action as a satiety signal and as a long-term regulator of food intake.

Perspectives

The inhibitory effect of OB protein on feeding, however, exhibits significantly longer duration relative to the orexigenic effect of exogenous NPY. Thus OB protein also may interact with other endogenous signals that affect meal size. Cholecystokinin can inhibit meal size (5, 30), and a synergistic interaction between OB protein and cholecystokinin to reduce feeding has been reported (1). OB protein also interacts with glucagon-like peptide-1 neurons to suppress food intake (9). OB protein increases insulin sensitivity in normal rats (27). Insulin can act as a prandial satiety signal by reducing the size of spontaneous meals (34), and intracerebroventricular insulin potentiates the satiety effect of cholecystokinin (24). Insulin, as OB protein, is proposed to be an adiposity signal to the brain (12). OB protein also modulates proopiomelanocortin neurons (4), hypothalamic melanocortin receptor subtypes action (8), and the corticotropin-releasing factor-ACTH-glucocorticoid pathway (23).

These few examples bring into context potential endogenous interactions that may play a role in the specific reduction of meal size induced by OB protein. There are many known interactions between OB protein and other feeding-regulatory signals. These interactions often follow a cascade pattern. Based on the numerous putative satiety signals, a myriad of other interactions can be proposed and tested experimentally. The obvious critical issue is that multiple neuronal and chemical interactions may participate in the control of meal size by OB protein. Feeding behavior involves a variety of psychological factors; neuronal and humoral mechanisms; and gastrointestinal, metabolic, and nutrient factors (18). Modification in one or various of these components could potentially reduce meal size. Interactive endogenous chemical models involved in the control of meal size are consistent with the multiple interactions among neurotransmitters, neuropeptides, and gut-brain peptides that occur during the physiological regulation of a spontaneous meal. OB protein, as an adipose tissue-to-brain signal, can be considered within these multiple interactions. Cytokine cytokine (e.g., OB protein and IL-1) (19) neuropeptide (e.g., NPY, CRF) (31) neurotransmitter (e.g., serotonin, catecholamines) interactions may also participate in the inhibition of meal size during pathophysiological conditions (20). Therefore, understanding the integration among short-term (meal related) and long-term (adiposity related) regulatory factors is essential to characterize the underlying mechanisms involved in the alteration of meal size in both health and disease.