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. 2010 Aug 4;151(10):4736–4744. doi: 10.1210/en.2010-0151

Molecular, Immunohistochemical, and Pharmacological Evidence of Oxytocin’s Role as Inhibitor of Carbohydrate But Not Fat Intake

Pawel K Olszewski 1, Anica Klockars 1, Agnieszka M Olszewska 1, Robert Fredriksson 1, Helgi B Schiöth 1, Allen S Levine 1
PMCID: PMC2946140  PMID: 20685878

Abstract

Oxytocin (OT) facilitates feeding termination stemming from high osmolality, stomach distention, and malaise. Recent knockout (KO) studies suggested a crucial function for OT in carbohydrate intake: OT−/− mice had increased preference for carbohydrates, including sucrose, but not fat (Intralipid). In striking contrast, sugar appetite was unaffected in the OT receptor KO mouse; data from wild-type animals have been insufficient. Therefore, we examined the involvement of OT in the regulation of sucrose vs. fat intake in C57BL/6 mice that served as a background KO strain. We exposed mice to a meal of sucrose or Intralipid and determined that the percentage of c-Fos-immunoreactive paraventricular hypothalamic OT neurons was elevated at termination of intake of either of the tastants, but this increase was 2-fold higher in sucrose-fed mice. A 48-h exposure to sucrose compared with Intralipid caused up-regulation of OT mRNA, whereas inherent individual preferences for sucrose vs. fat were not associated with differences in baseline OT expression as established with quantitative PCR. We found that L-368,899, an OT receptor antagonist, increased sugar intake when sucrose was presented alone or concurrently with Intralipid; it had no effect on Intralipid or total calorie consumption. L-368,899 affected Fos immunoreactivity in the paraventricular hypothalamus, arcuate nucleus, amygdala, and nucleus of the solitary tract, areas involved in aversion, satiety, and reward. This pattern serves as neuroanatomical basis of OT’s complex role in food intake, including sucrose intake. The current findings expand our knowledge on OT and suggest that it acts as a carbohydrate-specific inhibitor of feeding.

The role of oxytocin in termination of feeding is macronutrient-specific.

Oxytocin (OT), a nonapeptide primarily synthesized in the hypothalamus, released within the brain or (via the posterior pituitary) to the general circulation, has been implicated in a variety of processes, including social bonding (1), sexual behavior (2), pain perception (3), lactation (4), and parturition (5). Importantly, OT has been linked with inhibition of consummatory behavior. Initial experiments showed that intraventricular infusion of OT and OT receptor agonists produced a dose-dependent decrease in food consumption in schedule-fed rats as well as in rats refed after food deprivation of moderate length (6,7,8). The anorexigenic effect was similar in males and females (7). Although some authors observed mild hypophagia upon peripheral administration of OT, it could be achieved only with very high doses (7,8). Therefore, a consensus has been reached that the central rather than peripheral pool of OT regulates food intake. It was later discerned that OT’s anorexigenic effect stems from the involvement of this peptide in many mechanisms, such as gastric motility (9,10) and control of gastric distention (11,12), responses to increased plasma osmolality that often accompanies food intake (13,14), as well as with the role of OT in termination of feeding upon consumption of aversive tastants and avoidance of such tastants upon subsequent presentations (15,16).

Recent studies using OT knockout (KO) mice have provided exciting data that suggest OT’s anorexigenic action may be nutrient specific, namely, by limiting consumption of carbohydrates, including sugar. In the primary report, Amico and colleagues (17) found that the OT gene deletion in mice is associated with enhanced intake of sucrose solutions upon both initial and chronic exposure. Sclafani et al. (18) showed that absence of OT resulted in an increased daily intake of palatable sweet and nonsweet solutions of carbohydrate, including sucrose, Polycose, and starch, but it did not affect consumption of fat (a soybean oil emulsion, Intralipid). Miedlar and co-workers (19) confirmed the link between carbohydrate intake and the OT status with the KO model by testing a range of sucrose and Intralipid concentrations; the effect of genotype was observed only in relation to sucrose intake.

The KO data showing differential effects of OT on sucrose vs. Intralipid intake allowed an attractive hypothesis to be formulated that OT mediates satiety specific to carbohydrates, including sucrose. However, this hypothesis needs to be corroborated by evidence obtained in studies on mice with the intact genes encoding components of the OT system. This is particularly important as mice lacking the OT receptor were reported not to show any change in preference for sucrose, which is in stark contrast to the findings obtained in OT−/− animals (20).

The aim of this set of experiments was to examine the involvement of OT in the regulation of carbohydrate vs. fat intake in C57BL/6 mice routinely used in murine feeding studies that also served as a background strain for the OT KO model. Therefore, we exposed mice to sucrose or Intralipid and determined 1) the percentage of OT-immunoreactive (IR) neurons displaying a marker of neuronal activity, c-Fos, and 2) relative expression of the OT gene in the hypothalamus using real-time PCR. Real-time PCR was also used to determine whether inherent differences in preference for sugar vs. fat are associated with different OT mRNA levels. We evaluated the effect of L-368,899, a unique nonpeptide OT receptor antagonist capable of crossing the blood-brain barrier (21,22), on the intake of sucrose and Intralipid solutions presented independently or concurrently. This compound’s utility and specificity in rodents has been established using peripheral tissue assays (23,24). To our knowledge, no other feeding or feeding-related brain activity studies employing this antagonist exist. Hence, we also examined its effect on the intake of regular chow and determined whether peripheral injection of this compound changes c-Fos IR in central sites known to regulate food intake.

Materials and Methods

Animals

Male C57BL/6J mice (Scanbur BK AB, Sollentuna, Sweden) were housed individually or, wherever indicated, in groups of two in a temperature- (21–23 C) and humidity-controlled facility with a 12-h light, 12-h dark cycle (lights on at 0700 h). They weighed approximately 28 g at the beginning of the experiment. Tap water and standard chow (Lactamin, Lidköping, Sweden) were available ad libitum unless specified otherwise.

All procedures described herein received a prior approval from the Uppsala animal welfare committee and followed the guidelines on animal experimentation imposed by the Swedish (Animal Welfare Act SFS1998:56) and European Union (Convention ETS123 and Directive 86/609/EEC) laws.

c-Fos IR of OT neurons in the paraventricular nucleus of the hypothalamus (PVN) after the intake of Intralipid vs. sucrose solution

Mice exposed previously to Intralipid and sucrose as part of the feeding studies (see below) were used in this study (therefore, they were not naive to either sucrose or Intralipid); 8–10 d elapsed between the end of the earlier experiments and the immunohistochemical study to allow the animals to minimize any potential effects of the previous experimental manipulations. The animals were deprived of food overnight, whereas water was available ad libitum. On the experimental day at 0900 h, water was removed from the cages and a bottle containing either 10% sucrose or 4.1% Intralipid was presented. The animals were allowed to drink the fluid for 1 h (animals consumed between 2.1 and 2.6 ml of either sucrose or Intralipid; two mice that ingested less than 2 ml and one mouse that drank more than 3 ml were not included in the study). One hour after the beginning of consumption or 1 h after the end of consumption, animals were deeply anesthetized with pentobarbital and perfused through the aorta with 5 ml of 0.9% followed by 50 ml of 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The perfusion schedule was aimed at visualizing c-Fos IR of OT neurons that coincided with initiation or termination of consumption. It was based on previous reports that maximum c-Fos IR is observed about 60–90 min after the actual onset of neuronal activation (25). Brains were dissected out and postfixed overnight in the same fixative at 4 C. Each experimental group consisted of five to six animals.

Sectioning and staining

Coronal 50-μm Vibratome sections were processed for single and double immunohistochemistry. Every fourth brain section was used in the single c-Fos staining, and every fourth one containing the PVN was stained for c-Fos and OT. There were equal distances between sections used in the immunohistochemistry experiments.

Sections were treated for 10 min in 3% H2O2 and 10% methanol [diluted in Tris-buffered saline (TBS), pH 7.2] and incubated for 48 h at 4 C in the primary goat anti-Fos antibody (1:1100; Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, tissue was incubated for 1 h in the rabbit antigoat antibody (1:400; Vector Laboratories, Burlingame, CA). After the 1-h incubation in the avidin-biotin complex (ABC, 1:800; Vector), peroxidase was visualized with 0.05% 3,3′-diaminobenzidine tetrahydrochloride (Sigma Diagnostics, St. Louis, MO), 0.01% H2O2, and 0.3% nickel sulfate. All incubations in antibodies were performed in the TBS-based mixture of 0.5% Triton X-100 (Sigma Diagnostics) and 0.25% gelatin (Sigma Diagnostics). Intermediate rinsing steps were done in TBS.

After the completion of c-Fos staining, PVN sections were further processed for visualization of OT. The procedure was similar to that described above. However, rabbit anti-OT was used as the primary antibody (1:17,000; Millipore, Temecula, CA), and nickel sulfate was not added to the 3,3′-diaminobenzidine tetrahydrochloride solution to obtain the brown instead of black color.

Sections were mounted on gelatin-coated slides, dried, dehydrated in ascending concentrations of alcohol, soaked in xylene, and embedded in DPX (Fluka, Steinheim, Germany) and analyzed using light microscopy.

In the double staining, the following estimates were assessed per section and then per PVN: the total number of OT neurons and the number of OT neurons positive for c-Fos. Cells were counted bilaterally, and the percentage of neurons containing Fos-positive nuclei was tabulated.

Statistical analysis was done using one-factor ANOVA followed by Fisher’s least significance test. Values were considered significantly different when P < 0.05.

OT gene expression in sucrose vs. fat consumption models

RT-PCR analysis of OT gene expression was performed on the hypothalamic tissue collected in the study whose partial mRNA data as well as feeding results have been previously published (26).

Hypothalamic OT expression after 48-h consumption of sucrose or Intralipid solutions

Group-housed mice (n = two per cage) were given a bottle of 10% sucrose or 4.1% Intralipid for 48 h (in addition to chow); controls (n = 8) had chow only. Sucrose and Intralipid solutions were isocaloric (0.4 kcal/g), and the energy content of chow was 3.6 kcal/g. Mice were decapitated after 48 h (between 1100 and 1200 h). OT expression in the hypothalamus was studied with real-time PCR.

Hypothalamic OT expression in mice that differ in their preference for sugar vs. fat

Mice were given 10% sucrose and 4.1% Intralipid for 7 d (in addition to chow). During the first 2 d, they were allowed to get accustomed to the solutions. The remainder of that period was used to establish a 5-d tastant preference for each animal. Following the standards applied in previously published reports (27,28), the mice were divided into the following groups based on the 5-d preference: 1) sucrose preferrers (n = 7), 57.0 ± 2.2% of sucrose plus Intralipid calories came from the sucrose solution; 2) fat preferrers (n = 7), 39.2 ± 3.0% of sucrose plus Intralipid calories came from sucrose; and 3) neutral (n = 7), sucrose was the source of 46.9 ± 1.9% of their daily of sucrose plus Intralipid calorie intake. After 3 wk, sucrose and Intralipid were removed from cages, and the animals were maintained on chow for the next 21 d (washout phase) so that gene expression levels at decapitation were not affected by the consumption of different amounts of each of the three tastants (sucrose, Intralipid, and chow). The animals were decapitated (1100–1200) and hypothalami dissected. OT expression in the hypothalamus was studied with real-time PCR. Body weights at the end of the palatable tastant availability period and at the end of the washout phase did not differ between mice belonging to different preference groups.

RNA extraction and cDNA synthesis

Hypothalami were dissected, immersed in RNAlater (Ambion, Austin, TX), kept at room temperature for about 2 h to allow the solution to infiltrate the tissue and thereafter stored at −80 C until further processing.

RNA was extracted and cDNA was synthesized as described before (29). In short, tissue samples were homogenized using a Branson sonifier (Branson Ultrasonics Corp., Danbury, CT) in TRIzol (Invitrogen, Stockholm, Sweden). RNA was extracted using chloroform, and overnight incubation in isopropanol was used to precipitate RNA. The samples were centrifuged and the pellet was washed before being air dried, and it was dissolved in the 1× deoxyribonuclease buffer. The samples were incubated at 37 C for 90 min with ribonuclease-free deoxyribonuclease I (Roche Diagnostics, Stockholm, Sweden) to remove genomic DNA. The absence of genomic DNA was established by PCR: 0.5 μl template was mixed in a final volume of 10 μl containing 1× PCR mix [1 μl MgCl2-free buffer 10×, 0.3 μl 50 mm MgCl2, 0.25 μl 1% Tween, 0.1 μl 20 mm dNTP, 1 μl primer mix (with forward and backward primers at 10 pmol/μl each), 0.1 μl Taq polymerase, 5 U/μl (Biotools, Madrid, Spain), and 6.75 μl MilliQ H2O]. As a positive control, 0.5 μl 100 ng/μl genomic DNA was used, and 0.5 μl MilliQ water was added in a negative control. The product was analyzed with electrophoresis. Total RNA concentration was measured using Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE).

For cDNA synthesis, 5-μg RNA samples were diluted with MilliQ H2O to 12 μl. RNA was reverse transcribed in a final volume of 20 μl containing 1× Mastermix [4 μl 5× first-strand buffer, 2 μl 0.1m dithiothreitol, 0.5 μl 20 mm dNTP, 0.5 μl N6 1/6.25 (random hexamers) and 1 μl Moloney leukemia virus reverse transcriptase]. Samples were incubated at 37 C for 60 min, followed by PCR (as described above) to confirm cDNA synthesis.

RT-PCR

Twenty-five nanograms of a cDNA template from each sample was used per primer. Each RT-PCR, with a total volume of 20 μl, contained 2 μl MgCl2-free buffer 10×, 0.2 μl 20 mm dNTP, 1.6 μl 50 mm MgCl2, 0.05 μl of each primer at 100 pmol/μl (forward and reverse), 1 μl dimethyl sulfoxide, 0.5 μl Sybr Green (1:50,000), 0.08 μl Taq polymerase (5 U/μl) (Biotools), and 9.52 μl MilliQ water. All PCR were performed in duplicates, and negative controls were included on each plate. Amplification was performed as follows: denaturation at 95 C for 3 min, 50 cycles of denaturing at 95 C for 15 sec, annealing at an appropriate temperature established for the primers for 15 sec, and extension at 72 C for 30 sec. Seven housekeeping genes were analyzed (glyceraldehyde-3-phosphate-dehydrogenase, β-tubulin, ribosomal protein 19, histone H3, cyclophilin, β-actin, and succinate dehydrogenase complex subunit B). A MyiQ thermal cycler (Bio-Rad Laboratories, Sunbyberg, Sweden) was used for RT-PCR experiments.

Data analysis and relative expression calculation

RT-PCR data were analyzed as previously reported, using MyiQ software version 1.04 (Bio-Rad Laboratories, Gothenburg, Sweden) (30). Primer efficiencies were calculated with LinRegPCR (31), and samples were corrected for differences in primer efficiencies. The GeNorm protocol by Vandesompele et al. (32) was employed to calculate normalization factors on the basis of the expression levels of the housekeeping genes. Grubb’s test was used to identify and remove outliers. Differences in gene expression between groups were analyzed with ANOVA followed by Fisher’s protected least significant difference test. Values were considered significantly different when P < 0.05.

Effect of OT receptor antagonism on consummatory behavior and c-Fos IR

Effect of the OT receptor antagonist on standard chow intake

Ad libitum-fed mice were injected ip with saline or 0.3, 1, and 3 mg/kg body weight of L-368,899 (Sigma Research Biochemical Inc., St. Louis, MO), and standard chow intake was measured 2 h after injection and corrected for spillage (n = 9 per group). The amount of ingested water was also established 2 h after injection.

Effect of the OT receptor antagonist on sucrose solution intake

Animals were accustomed to receiving the 10% sucrose solution for 2 h/d (from 1500–1700 h, between the eighth and 10th hour of the light phase of the light-dark cycle) during three sessions administered every other day. At the time of sucrose presentation, chow and water were removed. Five minutes before the beginning of the fourth exposure to sucrose, mice were injected with saline or 0.3, 1 and 3 mg/kg body weight of L-368,899 (n = 9 per group), and sucrose intake was measured 2 h after injection and corrected for spillage.

Effect of the OT receptor antagonist on Intralipid solution intake

Animals were accustomed to receiving the 4.1% Intralipid (Fresenius Kabi, Uppsala, Sweden) solution for 2 h/d (from 1500–1700 h) during three sessions administered every other day. Intralipid is a palatable lipid emulsion of soybean oil, glycerol, and egg yolk phospholipids. At the time of Intralipid presentation, chow and water were removed. Five minutes before the beginning of the fourth exposure to Intralipid, mice were injected with saline or 0.3, 1, and 3 mg/kg body weight of L-368,899 (n = 9 per group), and Intralipid intake was measured 2 h after injection and corrected for spillage.

Effect of the OT receptor antagonist on the intake of the Intralipid and sucrose solutions presented concurrently

Animals were accustomed to receiving the 4.1% Intralipid and 10% sucrose solutions concurrently for 2 h/d (from 1500–1700 h) during three sessions administered every other day. Sucrose and Intralipid were isocaloric (0.4 kcal/g). At the time of the palatable solutions’ presentation, chow and water were removed from cages. Five minutes before the beginning of the fourth exposure, mice were injected with saline or 0.3 and 1 mg/kg body weight of L-368,899 (n = 12/d) and the solutions’ intake was measured 2 h after injection. The amounts were corrected for spillage.

Analysis of feeding data

Food intake data are expressed in grams. Intake of the sucrose and Intralipid solutions when the tastants were presented separately is reported in milliliters; when they are presented concurrently, the total amount of both liquids as well as the percentage intake is given. The weights of solutions (adjusted for the predetermined weight of bottles) before and after the consumption period were determined. Ingested volumes were calculated taking into account the weight of 1 ml of Intralipid or sucrose solutions and shown in milliliters. Data are presented as group mean values (±sem). In consumption experiments, comparisons were performed with one-way ANOVA followed by Fisher’s post hoc test. A P value <0.05 was considered significant.

Fos IR in feeding-related sites in response to the lowest orexigenic dose of the OT receptor antagonist

Mice were treated ip with saline or the lowest orexigenic dose of L-368,899 (1 mg/kg body weight; six mice per treatment) as established in the experiments in which the OT receptor antagonist increased the intake of the sucrose solution. Food and water were removed from the cages of injected animals. One hour after injection, animals were anesthetized with pentobarbital and perfused; brains were dissected out, postfixed, sectioned, and immunostained for c-Fos (as described above). Every fourth brain section was used in the single c-Fos staining. In the single staining analysis, the number of Fos-IR nuclei in each region of interest was counted bilaterally in at least five sections, and the density of Fos-positive nuclear profiles (per square millimeter of each area) was calculated. Images provided by the camera attached to the Nikon microscope were analyzed using NIH 1.51 Image software (National Institutes of Health, Bethesda, MD). Densities of Fos-positive nuclei per square millimeter were averaged per animal and experimental group. Statistical analysis was done using one-factor ANOVA followed by Fisher’s least significance test. Values were considered significantly different when P < 0.05.

Results

Double immunohistochemistry revealed a higher number of Fos-IR (thus, activated) OT neurons in the PVN in animals perfused at the time corresponding to the termination of Intralipid or sucrose intake compared with the initiation of consummatory behavior (P = 0.016 and 0.011, respectively). Noteworthy, a significantly greater percentage of OT cells in the PVN was observed in animals given sugar than fat (P = 0.037; Fig. 1). It should be noted that animals consumed similar amounts of the tastants, i.e. between 2.1 and 2.6 ml of either sucrose or Intralipid.

Figure 1.

Figure 1

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C and D, The percentage of c-Fos-positive OT neurons in the PVN at the initiation (C) and termination (D) of consumption of isocaloric 10% sucrose or 4.1% Intralipid (*, P < 0.05); A and B, photomicrographs depict coronal PVN sections of animals given Intralipid and perfused at the time corresponding to the beginning (A) and end (B) of the meal. The sections were immunostained for c-Fos and OT. Open arrows, OT neurons devoid of Fos; solid arrows, Fos-positive OT neurons. The inset depicts a higher magnification of the marked area within B.

Hypothalamic levels of OT mRNA were higher (P = 0.009; Fig. 2) in mice allowed 48-h access to a diet enriched with sucrose (chow plus 10% sucrose) than to a diet containing Intralipid (chow plus Intralipid). The two solutions were palatable; each animal ingested on average 8.1 kcal of Intralipid and 7.4 kcal of sucrose per day. In both of these groups, animals derived more than 50% of calories from the palatable solutions. Controls fed only chow, which is high in carbohydrates, had OT mRNA concentration similar to that in sucrose- plus chow-consuming mice (P = 0.178) and significantly higher than animals given Intralipid plus chow (P = 0.043; Fig. 2). Total caloric intake (chow alone, chow and sucrose, or chow and Intralipid) per mouse was 10.3 kcal in the chow group, 14.1 kcal in the sucrose group, and 12.9 kcal in the Intralipid group. It should be emphasized that although controls consumed fewer calories than sucrose- and Intralipid-fed animals, their consumption of chow was elevated compared with the remaining two groups.

Figure 2.

Figure 2

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Relative expression of OT in the hypothalamus. A, In mice that were exposed for 48 h to palatable Intralipid or sucrose solutions in addition to chow (n = 8 per group); B, in mice that differed in their preference for fat (Intralipid) vs. sucrose (n = 7 per group of fat preferrers, sucrose preferrers, and neutrals) as established in the 7-d preference test when Intralipid and sucrose were offered simultaneously. *, P < 0.05.

Although intake of sucrose and fat differentially affected hypothalamic OT gene expression, animals that displayed distinct preference profiles for sugar vs. Intralipid dubbed fat preferrers (39.2 ± 3.0% cal came from sucrose), sucrose preferrers (57.0 ± 2.2% cal from sucrose), and neutrals (46.9 ± 1.9% cal from sucrose) did not differ in their baseline OT mRNA levels (Fig. 2).

Administration of L-368,899 in sated mice having access only to chow did not change the amount of food consumed in the 2-h period after the injection (Fig. 3). In the chow-only paradigm, it was also determined that antagonism of the OT receptor did not alter drinking behavior, because the control and L-368,899-treated mice drank similar amounts of water (saline, 0.3 ± 0.2 ml; 0.3 mg L-368,899, 0.5 ± 0.3 ml; 1 mg L-368,899, 0.2 ± 0.2 ml). Feeding studies presented herein show that injections of the OT receptor antagonist specifically influence sugar consumption. When 10% sucrose or 4.1% Intralipid were presented alone in the cage for 2 h, L-368,899 increased the intake of sugar (1 mg/kg body weight, P = 0.036; and 3 mg/kg body weight, P = 0.031) and had no effect on Intralipid consumption (Fig. 4). When the two tastants were offered simultaneously, 1 mg/kg body weight L-368,899 preferentially elevated the intake of sugar (P = 0.019), without affecting the total amount of ingested energy. The 0.3-mg dose that had not produced any change in sucrose intake when the sugar bottle was presented alone, also in this sucrose-Intralipid choice study, did not affect consummatory behavior (Fig. 4).

Figure 3.

Figure 3

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The effect of the OT receptor antagonist L-368,899 on chow intake in sated mice. L-368,899 was injected at 0.3, 1, and 3 mg/kg body weight; saline (sal) was used in controls. Chow intake was measured 2 h after injection.

Figure 4.

Figure 4

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The effect of the OT receptor antagonist L-368,899 on the intake of isocaloric 4.1% Intralipid and 10% sucrose presented independently (A, Intralipid; B, sucrose) or concurrently (C and D). L-368,899 was injected ip; saline (sal) was used in controls. Sucrose and Intralipid intake was assessed 2 h after injection. Preference for sucrose vs. fat (Intralipid) shown in C is expressed as the percentage of the sucrose solution consumed in the total volume of both ingested tastants. D, Total volume of consumed Intralipid and sucrose solutions. *, P < 0.05.

Finally, L-368,899 injected at the 1-mg dose effective in altering consumption profile induced c-Fos IR in a number of sites involved in the regulation of energy- and reward-driven feeding, including the PVN (P = 0.034), arcuate nucleus (P = 0.045), medial preoptic area (P = 0.029), nucleus of the solitary tract (P = 0.021), and central nucleus of the amygdala (P = 0.018; Fig. 5). The values showed a strong trend toward up-regulation of c-Fos IR as they approached significance in the area postrema and dorsal motor nucleus of the vagus (P = 0.090 and 0.082, respectively).

Figure 5.

Figure 5

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The effect of the OT receptor antagonist L-368,899 on c-Fos IR in central sites involved in feeding regulation. *, P < 0.05. AP, Area postrema; ARC, arcuate nucleus; CeA, central nucleus of the amygdala; DMH, dorsomedial hypothalamic nucleus; DMNV, dorsal motor nucleus of the vagus; MPOA, medial preoptic area; NTS, nucleus of the solitary tract; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.

Discussion

The traditional approach to OT’s role in food intake regulation emphasized this peptide’s involvement in facilitating feeding termination as a protective response due to excessive stomach distention, osmotic challenge, or malaise. Subsequent studies showing responsiveness of brain stem-projecting OT neurons in the PVN to the adipocyte-derived hormone leptin (33) further substantiated the importance of OT as an energy balance regulator. Recent murine OT KO studies suggested a possible crucial function of OT in the regulation of sugar intake; OT gene deletion resulted in the increased preference for sucrose, whereas the drive to ingest Intralipid remained intact (17,18,19). This preference shift has been shown to extend beyond sucrose onto other carbohydrates, such as starch and Polycose (18). Enthusiasm regarding the outcome of the OT KO model experiments was somewhat hampered by the inability to affect sucrose vs. fat intake through the genetic deletion of the OT receptor. It is worth noting that the two types of KO models may involve different background strains. The OT KO is of a B6 background, whereas the OT receptor KO may have been expressed on a 129 background (the latter was not clear in the report). This is potentially important because B6 mice consume more sugar than 129 mice, in part, because of differences in their sweet taste receptor sensitivity (34,35). The lack of systematic pharmacological, neuropeptidergic, or molecular studies linking OT with the regulation of sugar and fat consumption in wild-type animals has nonetheless been the major obstacle in understanding the results obtained in KO mice, especially considering conflicting sugar/fat preference phenotypes.

Our immunohistochemical detection of c-Fos and OT revealed that an increase in PVN OT neuronal activity coincides with termination of ingestive behavior. Intakes of similar amounts of sucrose or Intralipid, both resulting in feeding termination, generated this effect. One should note that the OT system responds both to the liquid diet intake studied herein as well as to consumption of solid food used in previous studies (36). It supports the notion that OT acts as an inhibitor of consummatory activity irrespective of the composition, texture, flavor, and calorie density of a tastant, which parallels the basic, homeostatic role of the peptide. It is in concert with reports showing that PVN OT mediates effects of peripheral and central satiety factors, including cholecystokinin and glucagon-like peptide-1 (37,38,39).

The finding that twice as many OT neurons coexpress c-Fos in sucrose- than Intralipid-fed group at the end of the meal indicates that the OT system is much more responsive to the consumption of sugar than fat. Under circumstances unrelated to excessive stomach distention or dangerous increase in salt loading, such as during most meals, increase in activity of anorexigenic OT pathways is more pronounced when high-carbohydrate than high-fat foods are ingested. It suggests that the feeding termination response induced by OT may be also an essential part of the fine control of food intake.

This proposed sensitivity of OT neurons to carbohydrate intake was confirmed in the real-time PCR experiment. The hypothalamic preparations obtained from animals consuming sucrose had a higher OT mRNA content than the hypothalami of Intralipid-fed animals. In the Intralipid group, OT mRNA was down-regulated compared with the chow-only controls, although the significance level was not as high as between Intralipid- and sucrose-fed mice (all groups had chow available; however, the intake of bland pellets was decreased once the calories from palatable sucrose or fat solutions were available). This corroborates evidence from the OT KO studies that intake of other carbohydrates is regulated by OT, too; carbohydrates, mainly starch (∼85% of the carbohydrate content; the rest is shared by lactose, glucose, fructose, and sucrose), constitute approximately 50% of weight of standard laboratory chow. The issue of whether there exists a special link between the OT system and intake of one particular carbohydrate (or this pertains to carbohydrates in general) needs to be further explored. For example, the recent study using schedule-fed rats presented with either a high-sugar or regular chow showed up-regulation of OT mRNA in the sucrose group (40). Other high-carbohydrate diets (e.g. high Polycose) need to be studied in this context.

Noteworthy, the hypothalamic OT mRNA content does not reflect animals’ baseline preferences for sugar vs. fat, although this relationship has been shown for other feeding-related genes (26). It suggests that OT is part of the system involved in transient or fast regulation of consumption, but it does not participate in shaping innate, long-term preferences for sucrose vs. fat.

The OT receptor antagonist injection studies further clarify the role of OT in the fine control of food intake. When either sugar or Intralipid was presented independently as the sole source of calories, antagonism of the OT receptor with 1 and 3 mg/kg body weight of L-368,899 led to an approximately 20% increase in sucrose consumption but had no effect on the intake of the palatable lipid emulsion. In line with our aforementioned speculation on the unique relationship between OT and intake of sucrose, doses of the antagonist that were effective in reducing sugar consumption failed to affect the consumption of carbohydrate-rich chow. When Intralipid and sucrose were presented concurrently, antagonism of the OT receptor augmented sucrose intake without affecting total calories. These data are in concert with KO mouse findings that the lack of the OT gene does not lead to obesity (18,19). They are also in agreement with reports showing that other OT receptor antagonists did not affect energy intake (6). Noteworthy, those initial publications did not dissect sucrose/carbohydrate vs. fat intake.

Our results, in conjunction with previous data, show that OT is involved in energy balance regulation. OT neuronal activity coincides with the end of any meal. OT interacts with classical molecules of the energy balance mechanism, such as leptin, α-MSH, or glucagon-like peptide 1 (33,36,38,41). OT has also a fine feeding control function, when inhibition of energy intake is in fact secondary to the reduction of carbohydrate intake.

Distribution of c-Fos expression in animals injected with the OT receptor antagonist reflects the vast array of OT’s functions in feeding. Changes in c-Fos IR in the hypothalamic PVN, arcuate nucleus, amygdala, and nucleus of the solitary tract, induced by OT receptor antagonism, have been also detected upon aversion, satiety, and reward-driven consumption (15,42). This high degree of c-Fos pattern overlap between all aspects of feeding controlled by OT and OT receptor ligand injection may be treated as a functional-neuroanatomical basis of OT’s role as a regulator of food intake. Additionally, next to the recent study showing that iv administered L-368,899 accumulates in limbic brain areas in monkeys (22), our c-Fos experiment further supports the assumption that this compound’s action occurs, at least to some extent, via the central nervous system. One should note although that a thorough evaluation of this compound’s action at known splice variants of the OT receptor in various species is needed. This seems particularly crucial in primates because, according to the most comprehensive Ensembl database (www.ensembl.org), they express several spliced transcripts of this molecule. Only one splice variant has been annotated in the mouse, which decreases the likelihood of a differential effect of L-368,899 at the central vs. peripheral murine receptor.

Although this project concerns the detailed role of anorexigenic OT in feeding, the results allow us to present perspectives that reach beyond the aspect of just one peptide. In the current literature, orexigenic factors are very well described regarding their roles in appetite for either calorie-dense or palatable foods. For example, opioids are thought to drive eating for pleasure, whereas neuropeptide Y drives feeding for energy. Anorexigenic factors, on the other hand, are predominantly studied only with respect to the relationship with satiety vs. aversive responses, whereas food composition-based targeting of their action remains neglected. Our findings indicate that involvement of anorexigenic peptides in the development of satiety may be geared toward particular components of a diet; therefore, satiety cannot be always generalized as a uniform mechanism that leads to overall abandonment of consummatory activity. The example of OT’s specific function in feeding control as an inhibitor of carbohydrate rather than fat intake illustrates that in the environment rich in easily available diets that vary in composition, it is possible to overeat while in search of reaching satiety specific to multiple nutrients.

Footnotes

This work was supported by the Swedish Research Council (VR, Medicine), AFA Insurance, Swedish Brain Research Foundation, Svenska Läkaresällskapet, Åhlens Foundation, Novo Nordisk Foundation, Göran Gustafssons Foundation, National Institute of Drug Abuse (R01DA021280), Systembolagets råd för alkoholforskning (SRA), and National Institute of Diabetes and Ingestive and Kidney Diseases (P30DK50456).

Disclosure Summary: The authors have nothing to declare.

First Published Online August 4, 2010

Abbreviations: IR, Immunoreactivity; KO, knockout; OT, oxytocin; PVN, paraventricular nucleus of the hypothalamus; TBS, Tris-buffered saline.

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