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. 2017 May 29;158(9):2826–2836. doi: 10.1210/en.2017-00200

Effects of Endogenous Oxytocin Receptor Signaling in Nucleus Tractus Solitarius on Satiation-Mediated Feeding and Thermogenic Control in Male Rats

Zhi Yi Ong 1,✉, Diana M Bongiorno 2, Mary Ann Hernando 3, Harvey J Grill 1
PMCID: PMC5659667  PMID: 28575174

Abstract

Central oxytocin receptor (OT-R) signaling reduces food intake and increases energy expenditure, but the central sites and mechanisms mediating these effects are unresolved. We showed previously that pharmacological activation of OT-R in hindbrain/nucleus tractus solitarius (NTS) amplifies the intake-inhibitory effects of gastrointestinal (GI) satiation signals. Unexplored were the energetic effects of hindbrain OT-R agonism and the physiological relevance of NTS OT-R signaling on food intake and energy expenditure control. Using a virally mediated OT-R knockdown (KD) strategy and a range of behavioral paradigms, this study examined the role of endogenous NTS OT-R signaling on satiation-mediated food intake inhibition and thermogenic control. Results showed that, compared with controls, NTS OT-R KD rats consumed larger meals, were less responsive to the intake-inhibitory effects of a self-ingested preload, and consumed more chow following a 24-hour fast. These data indicate that NTS OT-R signaling is necessary for normal satiation control. Whereas both control and NTS OT-R KD rats increased core temperature following high-fat diet maintenance (relative to chow maintenance), the percent increase in core temperature was greater in control compared with NTS OT-R KD rats during the light cycle. Hindbrain oxytocin agonist delivery increased core temperature in both control and NTS OT-R KD rats and the percent increase relative to vehicle treatment was not significantly different between groups. Together, data reveal a critical role for endogenous NTS OT-R signaling in mediating the intake-inhibitory effects of endogenous GI satiation signals and in diet-induced thermogenesis.

Using genetic knockdown and pharmacobehavioral strategies, we showed that NTS OT-R signaling is required for GI signal-mediated feeding inhibition and light phase diet-induced thermogenesis.

Oxytocin (OT), a nonapeptide well established for its roles in lactation (1), anxiety (2), and social behaviors (3), is also implicated in energy balance control. Accumulating evidence from rodent and nonhuman primate studies show that peripheral or central OT administration reduces food intake, body weight, and fat mass and also increases energy expenditure (4). More recently, studies in humans report that intranasal OT delivery reduces palatable food intake in lean (5, 6) and obese men (7), and promotes body weight loss (8). The complementary findings observed across species and energy states provide support for the potential of OT as an antiobesity treatment (9).

Whereas OT holds promise as a weight loss treatment, the central sites of action and mechanisms by which OT receptor (OT-R) signaling contributes to energy balance are unresolved. OT-synthesizing neurons are located in the supraoptic nucleus and paraventricular nucleus of the hypothalamus (PVH). PVH OT neurons project to a variety of brain sites that express OT-R and activation of OT-R in some of these sites including the nucleus accumbens core (10), arcuate nucleus of the hypothalamus (11, 12), ventral tegmental area (13), or nucleus tractus solitarius (NTS) (14) reduces food intake. Further, current evidence suggests that OT-R signaling interacts with gastrointestinal (GI) satiation signal processing to mediate the intake-inhibitory effects of OT-R signaling (15–17). In support of this view, we demonstrated that NTS OT delivery amplifies the intake-inhibitory effects of endogenous GI signals (stimulated by a self-ingested preload), a result that is consistent with electrophysiological data showing that OT increases the amplitude of solitary tract stimulation-evoked excitatory postsynaptic potentials on NTS neurons (18). We also showed that self-ingestion of a liquid diet (Ensure) elevated endogenous OT content in the dorsal vagal complex (which comprises the NTS) thereby providing an endogenous source of OT that could facilitate the interaction between NTS OT-R and vagal afferent signal transmission to reduce food intake (14). The physiological role of NTS OT-R signaling on food intake control was not directly assessed and is one of the subjects of this study.

Central OT-R signaling also plays a role in energy expenditure control and neurons in the hypothalamus (19, 20), rostral medullary raphe (21) and thoracic spinal cord (22) are implicated in mediating these energetic effects. Given the presence of sympathetic premotor neurons in the hindbrain including the NTS (23), it is conceivable that NTS OT-R signaling may also contribute to the energetic effects of central OT-R signaling. Adequate evaluative data on the effects of NTS OT-R signaling on energy expenditure are however, still lacking.

This study therefore aimed to examine the physiological relevance of endogenous NTS OT-R signaling on energy intake and energy expenditure. To assess the impact of endogenous NTS OT-R signaling on energy balance control, we developed an adeno-associated virus (AAV) with a short hairpin RNA (shRNA) that targets OT-R (or scrambled sequences as controls) and injected it bilaterally into the caudomedial NTS to knockdown (KD) NTS OT-R. Using this NTS OT-R KD rodent model together with complementary behavioral paradigms, we provide novel evidence that NTS OT-Rs are required for the intake-inhibitory effects of GI signals and diet-induced thermogenesis.

Materials and Methods

Animals

Male Sprague Dawley rats (250 to 265 g on arrival; Charles River Laboratories, Wilmington, MA) were housed in metal hanging steel cages under a 12-hour light/12-hour dark cycle. Rats had ad libitum access to pelleted chow (Purina 5001; Purina, St. Louis, MO) and water, unless otherwise stated. Behavioral experiments were conducted in a within-subjects, counterbalanced design, unless otherwise specified. All procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Development of AAV-shRNA

AAV shRNA production was conducted as previously described (24). Briefly, OT-R plasmid constructs were commercially obtained (TG709488; OriGene Technologies, Rockland, MD) and screened in vitro for KD efficacy using a rat immortalized cell line (R-8; Cedarland Laboratories, Burlington, NC). The shRNA targeting the OT-R sequence that achieved the most robust KD (NM_012871; 5′ ACA GGT CAC CTC TTC CAC GAA CTC GTG CA 3′) was used and packaged into an AAV serotype 1 (AAV1), driven by a U6 promoter and expressing enhanced green fluorescent protein (GFP) for visualization of injection site (AAV1 shOT-R). An AAV shRNA with scrambled sequences (5′ GAC ACC TGG ACT TAG TCA CCT ACG CTC AC 3′) was used as a control [AAV1 shRNA control (shCtrl)]. Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to determine OT-R and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeper) messenger RNA (mRNA) expression and %KD between groups (see methods for qRT-PCR later).

Cannula implantation surgery and viral delivery

Rats were anesthetized with intramuscular ketamine (90 mg/kg; Butler Animal Health Supply, Dublin, OH), xylazine (2.7 mg/kg; Anased, Shenandoah, IA), and acepromazine (0.64 mg/kg; Butler Animal Health Supply) followed by subcutaneous analgesia (2.0 mg/kg Loxicom; Midwest Veterinary Supply, Norristown, PA) postsurgery. Infusions of either the AAV shRNA designed to target and KD OT-R (1.1 × 1012 genome copies/mL; KD group), scrambled sequences (9.1 × 1011 genome copies/mL; control group), or phosphate-buffered saline (PBS; nonvirus injected controls) were made bilaterally (200 nL/side) to the caudomedial NTS at the coordinates: 15° angle (anterior to posterior direction), at midline, +1.9 mm from occipital suture, –6.8 mm from skull. A subset of KD and control rats were implanted with a guide cannula (26 gauge; Plastics One, Inc., Roanoke, VA) targeting the fourth ventricle (4V; coordinates: at midline, +2.5 mm from occipital suture, –5.2 mm from skull). Injection tips that extend 2 mm below the cannula were used. Placement of the 4V cannula was verified by sympathoadrenal-mediated glycemic response to 5-thio-d-glucose (210 µg in 2 µL artificial cerebrospinal fluid) (25). An increase in blood glucose level of 100% or greater from baseline was required for subject inclusion. Viral injection site was examined post mortem by visualizing GFP on hindbrain sections under a fluorescence microscope. All behavioral experiments were conducted 2 weeks after viral delivery.

Telemetric transponder surgery

HRC 4000 and G2 telemetric transponders (VitalView, STARR Life Sciences, Holliston, MA) were used to measure core temperature. In rats with 4V cannula (Experiment 3), abdominal implantation of HRC 4000 telemetric transponders was performed as previously described (26). Viral-injected rats (KD and control; Experiments 4 and 5) on the other hand, were implanted with the G2 telemetric transponders. Briefly, a small incision was made ∼1 cm below the sternum. Each G2 was secured to the abdominal wall with surgical absorbable sutures and the wound was closed up with sutures and wound clips. To ensure that temperature measurements of these two transponders are comparable, we compared average 2-hour light cycle core temperature measurements in rats implanted with either HRC 4000 or G2 and found similar results (HRC 4000: 37.2 ± 0.1°C; G2: 37.3 ± 0.1°C).

Experiment 1: Effect of NTS OT-R KD on body weight, food intake, and meal patterns

Rats (n = 9 KD, n = 9 control, n = 2 PBS) were matched for body weight and food intake prior to viral delivery. Postviral delivery, daily body weight, and food intake (accounting for spillage) were measured for 23 days. Growth trajectory and chow intake between PBS-injected controls and viral injected rats revealed no differences between groups, indicating no negative impact of viral injections on growth rate and food intake (Supplemental Fig. 1 (71.2KB, docx) ). These rats were used subsequently in Experiments 2a and 2b.

For meal patterns analyses, a separate group of naïve rats (n = 8 KD, n = 15 control) were habituated to modified hanging wire cages equipped with an automated food intake measuring system (DiaLog instruments). Each cage had access to a food cup (filled with powdered chow; Purina 5001) that rested on a load cell connected to specialized software (LabVIEW, National Instruments). Powdered chow is required in this system to allow precise measurements of chow consumed. The software recorded change in food cup weight every 10 seconds, which allowed detailed measurement of cumulative food intake as well as other meal parameters including meal size and meal number. Each meal was defined as >0.25 g ingested with an intermeal interval of at least 10 minutes (27). Two weeks postsurgery, food intake was measured for 24 hours and averaged across 2 days.

Experiment 2: Effect of NTS OT-R KD on sensitivity to the intake-inhibitory effects of gastrointestinal satiation signals using (a) preload and (b) 24-hour fast refeed

Experiment 2a

It is well-established that consumption of a nutritionally complete liquid diet results in gastric distension and increases GI hormone release and vagal afferent activity (28–31); therefore, rats (n = 8 KD, n = 9 control) were trained to consume a fixed volume of preload (Ensure; 1.42 kcal/mL) as a strategy to physiologically activate a range of GI signals. As described previously (14), chow was removed 2 hours before dark onset and rats were given 12 mL of Ensure to consume to entirety within 10 minutes of dark onset. During test day, rats had access to 0 mL, 7 mL, or 12 mL of Ensure for 10 minutes, after which chow was returned and chow intake (accounting for spillage) measured at 0.5 hour, 1 hour, and 1.5 hours.

Experiment 2b

Rats (n = 7 KD, n = 9 control) were food deprived for 24 hours. The next day at dark onset, chow was returned and chow intake (accounting for spillage) was measured at 0.5 hour, 1 hour, and 1.5 hours.

Experiment 3: Effect of hindbrain OT-R signaling on core temperature

Naïve rats (n = 10) implanted with a 4V cannula and HRC 4000 transponder received 4V delivery of vehicle (Veh; water) or OT (1 µg, 5 µg, 10 µg) in the early light cycle and core temperature was recorded throughout the light cycle. Experiments were conducted in the light cycle and chow was removed prior to injections to prevent a diet-induced thermogenic effect that might confound the measurement of hindbrain OT-R signaling on core temperature. Chow was subsequently returned 30 minutes before dark onset.

Experiment 4: Effect of NTS OT-R KD on high-fat diet–induced thermogenesis

Naïve rats (n = 8 KD, n = 8 control) were implanted with G2 telemetric transponders. Two weeks after viral injections, core temperature measurements were recorded and averaged across 2 days in chow-maintained KD or control rats. To examine whether NTS OT-R are required for high-fat diet–mediated increases in energy expenditure, chow was replaced with HFD (32% kcal/fat, Research Diets, New Brunswick, NJ) and rats were given ad libitum access to HFD for 3 days. After one day of HFD access to eliminate novelty, core temperature was measured and averaged over 2 days. HFD was subsequently replaced with chow and rats were maintained on chow for the rest of the experimental period. In a subset of rats (control n = 5, KD n = 5), food intake was also measured at the end of the light cycle and dark cycle.

Experiment 5: Effect of NTS OT-R KD on hindbrain OT-induced thermogenesis

The experiment was conducted during the light cycle and food was removed prior to injections. Rats from Experiment 4 (n = 8 KD, n = 8 control, with G2 and 4V cannula) were injected with Veh or 3 µg OT to the 4V. Core temperature was measured throughout the light cycle and food replaced 30 minutes prior to dark cycle onset.

Brain tissue collection for viral injection placement and qRT-PCR analyses

At 5 weeks after viral injections, rats were lightly anesthetized and killed by rapid decapitation. Brains were immediately removed and flash frozen in cold isopentane. Isolated brains were stored at –80°C before tissue processing. Serial coronal sections (Bregma –13.5 mm to Bregma –14.4 mm) were obtained using a cryostat at –20°C. Using a number 11 scalpel blade, NTS was isolated from 12 coronal sections that are 80 µm thick each, carefully avoiding the underlying dorsal motor nucleus of the vagus (DMX), and used for qRT-PCR (described later). For every fourth section, slices were cut at 30 µm, mounted on slides and viewed under a fluorescent microscope for GFP visualization and verification of the injection site. Representative images of the injection site labeled with GFP are shown for AAV1 shCtrl- and AAV1 shOT-R-injected rats in Fig. 1(a) and 1(b).

Figure 1.

Figure 1.

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Verification of NTS OT-R KD. Representative images of viral injection sites in the NTS of (a) control and (b) KD rats. OT-R mRNA expression was significantly reduced in (c) embryonic hypothalamic cells transfected with shOT-R and in (d) NTS of rats that received AAV-shOT-R. Expression of (e) V1a, (f) V1b, and (g) V2 mRNA in the NTS was not different between NTS OT-R KD and control rats, indicating a specific KD of OT-R. *P < 0.05.

qRT-PCR

RNA was isolated from NTS-enriched tissues using TRIzol (Invitrogen, Life Technologies, Grant Island, NY) and the RNeasy kit (QIAGEN, Germantown, MD). A high-capacity complementary DNA reverse transcription kit (Applied Biosystems, Life Technologies, Grant Island, NY) was used to synthesize complementary DNA from the RNA. mRNA expression of OT-R, vasopressin receptor subtypes (V1a, V1b, V2) and GAPDH (internal control) was determined using TaqMan gene expression kits and PCR reagents (OT-R: Rn00563503_m1; V1a: Rn00583910_m1; V1b: Rn01490541_m1; V2: Rn00569508_g1; GAPDH: Rn01775763_g1; Applied Biosystems). Relative mRNA levels were analyzed using the comparative cycle threshold (ΔΔCt) method, as previously described (32).

Statistical analyses

Results are expressed as mean ± standard error of the mean. Meal patterns and deprivation refeed data were analyzed using a mixed-design analysis of variance (ANOVA), with time as the repeated measure variable and group (control vs KD) as the between-subject variable. When a significant group effect or interaction is found, data are further analyzed using Student’s unpaired t test (between subjects) to determine group differences at specific time points. Two-way repeated-measures ANOVA were performed for the preload experiment and one-way repeated-measures ANOVA was performed for the 4V OT thermogenic dose response experiment, followed by pairwise comparisons with Bonferroni correction. Student’s paired or unpaired t tests were performed for the OT-R KD validation and OT-R KD thermogenic experiments. All statistical analyses were conducted using Statistica software (Statsoft) and statistical significance was defined as P < 0.05.

Results

OT-R KD validation

Real time PCR analyses of OT-R expression (relative to the housekeeper, GAPDH) in (1) rat immortalized hypothalamic cells transiently transfected by shOT-R and (2) NTS-enriched sections of rats injected with AAV1 shOT-R to the NTS revealed significant reduction in OT-R expression, when compared with control transfections [P < 0.05; Fig. 1(c) and 1(d)]. To ensure that the KD was specific to OT-R, expression of vasopressin receptor subtypes (V1a, V1b, V2) in the NTS were also analyzed. No differences in the expression of V1a, V1b or V2 were detected between NTS OT-R KD and control rats [Fig. 1(e–g)].

NTS OT-R KD increased average meal size

To assess the meal patterns of control and KD rats, meal parameters including meal size and meal number were examined 2 weeks after viral injections and averaged across 2 days. Mixed-design ANOVA analyses revealed an overall effect of group (F1,21 = 4.43, P < 0.05), time (F10,120 = 28.43, P < 0.001) and group × time interaction (F10,120 = 2.47, P < 0.01) on meal size. Subsequent comparisons between groups showed that meal sizes at 3 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dark cycle onset were greater in KD rats compared with controls [P < 0.05; Fig. 2(a)]. For meal number, there were significant effects on time (F10,120 = 399.29, P < 0.001) and group × time interaction (F10,120 = 5.12, P < 0.001). Here, KD rats consumed fewer meals at 8 hours and 24 hours after dark cycle onset when compared with controls [P < 0.05; Fig. 2(b)]. The combination of larger meal size and fewer meals taken resulted in the absence of an effect on cumulative chow intake between KD and control rats [F1,21 = 1.74, NS; Fig. 2(c)].

Figure 2.

Figure 2.

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Meal patterns of NTS OT-R KD and control rats. (a) Average meal size in the mid-late dark cycle was greater in the NTS OT-R KD rats than controls. (b) NTS OT-R KD rats consumed less meals compared with controls, with effects only observed in the late dark cycle. (c) The increase in cumulative intake arising from the increase in meal size was compensated by a decrease in meal number in NTS OT-R KD rats; this resulted in no difference in cumulative chow intake between the NTS OT-R KD and control rats. *P < 0.05; ^P = 0.05.

NTS OT-R KD rats were less sensitive to the intake inhibitory effects of a self-ingested preload

To examine whether NTS OT-Rs are required for the intake inhibitory effects of GI signals, self-ingestion of Ensure was used as a proxy for GI signal activation and the impact of these signals on chow intake was examined following the preload. In control rats, two-way repeated-measures ANOVA analysis of chow intake postpreload revealed a significant main effect of time (F2,16 = 34.83, P < 0.0001) and preload (F2,16 = 19.62, P < 0.001). Control rats significantly reduced chow intake at all time points measured (0.5 hour, 1 hour, 1.5 hours) following 12 mL preload [P < 0.01; Fig. 3(a)]. By contrast, there was no overall effect on preload volume [F2,14 = 1.76, NS; Fig. 3(b)] on chow intake in KD rats but there was a significant main effect of time (F2,14 = 29.74, P < 0.001). At 24 hours, chow intake was not different between control and KD rats whether rats were given 0 mL (control: 25.4 ± 1.3 g, KD: 27.8 ± 0.9 g; NS), 7 mL (control: 27.2 ± 1.5 g, KD: 28.5 ± 0.7 g; NS), or 12 mL (control: 27.5 ± 1.1 g, KD: 29.1 ± 0.9 g; NS) preload.

Figure 3.

Figure 3.

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Sensitivity to the intake-inhibitory effects of endogenous GI satiation signals in NTS OT-R KD and control rats. (a) Control rats significantly reduced chow intake in response to 12 mL preload from 0.5 hour up to 1.5 hours. (b) NTS OT-R KD rats failed to reduce chow intake with any volumes of preload or at any other time points measured. **P < 0.01.

NTS OT-R KD rats consumed more chow following 24-hour fast

As an additional test of whether NTS OT-R KD rats are less sensitive to the satiation effects of GI signals, a deprivation refeed experiment was conducted where chow intake (motivated by a 24-hour food deprivation period) was measured in control and KD rats. Two-way repeated-measures ANOVA showed a main effect of group (F1,14 = 6.54, P < 0.05) and time (F2,28 = 28.14, P < 0.0001) on chow intake following a 24-hour fast. At 0.5 hour and 1 hour after refeed, NTS OT-R KD rats consumed significantly more chow than controls (P < 0.05; Fig. 4). Chow intake was no longer different between groups at the 1.5-hour (Fig. 4), 2-hour (control: 12.6 ± 0.7 g, KD: 14.3 ± 0.7 g; NS), or 3-hour (control: 14.9 ± 0.4 g, KD: 15.5 ± 0.9 g; NS) time point.

Figure 4.

Figure 4.

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Effects of NTS OT-R KD on chow intake during refeed after a 24-hour fast. NTS OT-R KD rats consumed more chow during 0.5 hour and 1 hour of refeed following a 24-hour fast when compared with controls. *P < 0.05.

NTS OT-R KD impaired HFD-induced thermogenesis

To determine whether NTS OT-Rs are necessary for HFD-induced thermogenesis, core temperature was measured in control and KD rats when maintained on HFD and on chow. The percent increase in core temperature in response to HFD maintenance relative to chow maintenance was determined with the formula: [(HFD – Chow)/Chow] × 100%. When examined in either light and/or dark phases of the light/dark cycle, HFD intake increased core temperature (thermic effect of food) in both control and NTS OT-R KD rats [P < 0.05; Fig. 5(a) and 5(b)]. Percent increase in core temperature was however higher in control rats compared with KD rats in the light cycle but not in the dark cycle or when light and dark cycle temperatures were combined [24 hour; P < 0.05; Fig. 5(c)].

Figure 5.

Figure 5.

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Effects of hindbrain NTS OT-R signaling on core temperature (Tc) following HFD maintenance. In (a) control and (b) NTS OT-R KD rats, Tc measured during the light, dark, or 24-hour (both light and dark) periods was higher when the rats were maintained on HFD compared with when they were maintained on chow, indicating a HFD-induced thermogenic response in both control and NTS OT-R KD rats. (c) Percent increase in Tc during HFD maintenance relative to chow maintenance was significantly higher in control rats than NTS OT-R KD rats during the light cycle, but not in the dark or throughout a 24-hour period. *P < 0.05; **P < 0.01.

To validate that this increase in core temperature was mediated via an increase in caloric intake, we measured chow and HFD intake during the light and dark cycles and showed that HFD intake was significantly higher than chow intake in both control (light cycle: chow 11.5 ± 3.2 kcal, HFD 31.1 ± 1.8 kcal; dark cycle: chow 82.9 ± 3.7 kcal, HFD 134.4 ± 5.9 kcal; P < 0.01) and KD rats (light cycle: chow 12.7 ± 1.8 kcal, HFD 27.3.1 ± 3.8 kcal, t = 11.62, df = 4; dark cycle: chow 75.9 ± 3.8 kcal, HFD 115.6 ± 9.6 kcal; P < 0.01). HFD intake measured during the light (control: 31.1 ± 1.8 kcal, KD: 27.3.1 ± 3.8 kcal) and dark cycles (control: 134.4 ± 5.9 kcal, KD: 115.6 ± 9.6 kcal) revealed no differences in food intake between control and KD rats.

Hindbrain OT delivery increased core temperature in control and NTS OT-R KD rats

To examine whether hindbrain OT-R signaling triggers a thermogenic response, core temperature was measured in intact (non-virus-injected) rats prior to and following 4V OT administration. 4V delivery of OT (Veh, 1 µg, 5 µg, 10 µg) dose-dependently increased average core temperature during the first 2 hours postinjection [F3,27 = 21.5, P < 0.001; Fig. 6(a) and 6(b)]. In a separate study using virus-injected control and NTS OT-R KD rats, 4V OT delivery (3 µg OT; examined in the absence of food) elevated core temperature in both control and NTS OT-R KD rats (P < 0.05). The percent increase in 4V OT-induced thermogenesis relative to Veh treatment was not different between control and NTS OT-R KD rats [Fig. 6(c) and 6(d)].

Figure 6.

Figure 6.

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Effects of hindbrain NTS OT-R signaling on 4V OT agonist-induced thermogenesis. (a) Light cycle core temperature (Tc) following 4V OT delivery (black arrow) in intact, nonvirus injected rats. Dotted box represent the period of which results were analyzed. (b) Analysis of average Tc between 10:00 am to 12:00 pm following 4V OT delivery show dose-dependent increases in Tc, when compared with 4V vehicle (Veh) delivery. (c) 4V delivery of OT significantly increased Tc both in control and NTS OT-R KD rats. (d) Percent increase in Tc following 4V OT delivery relative to Veh treatment was not different between control and KD rats. *P < 0.05; **P < 0.01.

Discussion

Using a novel viral-mediated OT-R KD rodent model, we investigated the role of endogenous NTS OT-R signaling on food intake control. Collectively, the data gathered show that, compared with controls, rats with reduced NTS OT-R signaling increased meal size, were less sensitive to the intake-inhibitory effects of GI signals generated by a caloric preload, and consumed more chow in the refeed period following a 24-hour fast. These results are consistent with the hypothesis that endogenous NTS OT-R signaling is required for food intake control and is an important mediator of the intake-suppressive effects of GI satiation signals.

To directly probe the interaction between NTS OT-R and GI signaling on food intake control, the response of NTS OT-R KD rats to the intake-inhibitory effects of GI satiation signals was examined. Here, we used two strategies to engage endogenous GI signals: (1) consumption of a liquid caloric preload and (2) refeeding following a 24-hour fast. We found that NTS OT-R KD rats were less sensitive to the intake-inhibitory effects of the ingested preload. Compared with controls, NTS OT-R KD rats failed to reduce chow intake in response to 7 mL or 12 mL preload. Complementary results were observed when comparing chow intake of control and OT-R KD rats to 24-hour food deprivation. Here, NTS OT-R KD rats consumed more chow than controls during the refeed period, at both 0.5-hour and 1-hour time points. Together, these novel findings indicate an impaired sensitivity to GI satiation signals in rats with reduced NTS OT-R signaling that emphasizes the necessity of NTS OT-Rs in mediating the intake-inhibitory effects of GI satiation signals. This interaction between NTS OT-R signaling and GI signals is also consistent with electrophysiological data showing that NTS OT-R signaling amplifies the excitatory response of NTS neurons following solitary tract stimulation (18) and further supports our behavior pharmacological results showing that NTS OT-R signaling amplifies the intake-suppressive effects of a preload (14).

Although the current study used an ingested preload as a strategy to trigger a variety of GI signals, previous studies examining the interaction between hindbrain OT-R signaling and GI signals probed the effects of a single GI satiation signal, cholecystokinin (CCK). Complementary to our findings, Blevins et al. (16) showed that the intake-inhibitory effects of CCK are attenuated by hindbrain OT-R antagonism. In addition, the body weight and food intake reducing effects of chronic central OT treatment in diet-induced obese rats was shown to be in part mediated through increased CCK sensitivity (33). Interestingly, transgenic mice with enhanced hypothalamic leptin receptor signaling were more sensitive to the intake effects of endogenous CCK and had elevated NTS OT levels compared with controls (34). In humans, intranasal OT delivery increases plasma CCK (5), the mechanisms of which are however still unclear. Together, our findings and those of others indicate a GI-mediated mechanism through which central OT-R signaling reduces food intake. This understanding of the mechanistic basis of OT’s effects on food intake control is of particular importance given the potential for OT as an antiobesity treatment.

Through detailed analysis of feeding patterns, we showed that NTS OT-R KD rats ate larger meals throughout the mid-late dark cycle. Given that GI signals are critical to meal size control (35, 36), this finding supports the hypothesis that NTS OT-R signaling, which interacts with GI signals to suppress food intake, is required for normal meal size control. Consistent with a wealth of evidence that daily caloric intake is a highly regulated parameter (35, 37, 38), it was observed that the cumulative effect of increased meal size in NTS OT-R KD rats was accompanied by a decrease in meal number later in the dark cycle. It is reasonable to suggest that the cumulative effect of increased meal size resulted in a compensatory reduction on meal number that in turn led to no net difference in cumulative chow intake between control and KD rats.

Consistent with our finding on the contribution of NTS OT-R signaling to meal size control, other studies showed that global OT-R deficient mice exhibit increased meal size (39) and chronic central OT delivery reduces meal size in diet-induced obese rats (33). By antagonizing hindbrain OT-R signaling, which resulted in increased meal size and attenuation of the meal size reducing effects of hypothalamic leucine, Blouet and colleagues (40) showed that the meal size effects of OT-R signaling are mediated through hindbrain OT-Rs. Our data here support the contribution of hindbrain OT-R in meal size control and extend these findings to highlight the NTS as a novel neural substrate contributing to the meal size effects of hindbrain OT-R signaling.

Although it is clear that NTS OT-R signaling reduces food intake via interactions with GI satiation signals, the underlying mechanisms mediating this interaction requires further investigation. We previously showed that activation of GI signals via self-consumption of a liquid preload (Ensure) increases OT content in the dorsal vagal complex/NTS (14), a brain region that receives OT projections from the PVH (41). Within the NTS, OT-R signaling amplifies the excitatory potential of solitary tract stimulation via non-NMDA mechanisms (18), which suggests the contribution of non-NMDA receptors in mediating the intake inhibitory effects of NTS OT-R signaling. CCK, on the other hand, reduces food intake via NTS NMDA receptor signaling (42), thus indicating that the interaction between CCK and OT within the NTS may not involve NMDA glutamatergic signals. Nonetheless, one potential downstream signaling pathway mediating the interaction between CCK and OT is via ERK1/2 because both CCK and PVH OT-R signaling involve the phosphorylation of ERK1/2 (43, 44). Although the downstream signaling pathways of NTS OT-R signaling are unknown, it is possible that NTS OT-R and CCK (or other GI signals) signaling converge via the ERK1/2 pathway to reduce food intake. If this hypothesis were true, it would be interesting in future studies to examine whether NTS OT-R KD rats also express reduced ERK1/2 phosphorylation compared with controls.

In addition to food intake control, we also examined the effects of NTS OT-R signaling on energy expenditure control. Previous studies show that OT-R signaling participates in the neural control of energy expenditure where acute or chronic central OT agonist delivery increases energy expenditure (45, 46), and global OT-deficient or OT-R deficient rodent models, despite having limited or no deficits in food intake control, exhibit significant reductions in energy expenditure (47–50). Similarly, rodents with postnatal ablations of PVH OT neurons also display reduced energy expenditure, as evidenced by reduced oxygen consumption following HFD maintenance, suggesting impairments in diet-induced thermogenesis (49). Supporting this outcome, here, we provide novel evidence that OT-R expressing neurons in the NTS are necessary for diet-induced thermogenesis. Whereas both control and NTS OT-R KD rats increased core temperature in response to HFD intake relative to chow intake, the percent increase in core temperature following HFD when compared with chow maintenance in the light cycle was significantly lower in NTS OT-R KD rats compared with control rats, indicating reduced diet-induced thermogenic response in rats with reduced NTS OT-R signaling. Whether these thermogenic effects are mediated directly by OT-Rs that are expressed on sympathetic premotor neurons in the NTS that project to the brown adipose tissue (the fat depot that contributes to diet-induced thermogenesis) (23), or indirectly through local interaction between NTS OT-R expressing cells and other sympathetic premotor neurons are unclear. These are important questions to pursue in future studies.

We also showed that hindbrain ventricular OT delivery increased core temperature in both control and NTS-OT-R KD rats. We offer two interpretations of this outcome: (1) As the OT-R KD was only partial (32% KD) and the concentration of OT used was relatively high, it is possible that the available/residual NTS OT-R in the NTS OT-R KD rats was sufficient to mediate a comparable effect in both groups; (2) it is also possible that energetic effects produced by 4V OT delivery may not be mediated exclusively via NTS OT-Rs but may involve other OT-R expressing hindbrain sites including the rostral medullary raphe (21) and thoracic spinal cord (22), areas that express OT-R and were previously shown to contribute to OT-mediated thermogenic control.

Central OT-R signaling is also implicated in cold-induced thermogenesis. Global OT-R knockout mice show impaired cold-induced thermogenesis (48) but this thermogenic response to cold can be restored by virally expressing OT-Rs specifically at the rostral medullary raphe (21) or the hypothalamus (19). The complete reversal of the impaired thermogenic response to cold, whether it was a result of restored OT-R expression in rostral medullary raphe or hypothalamus, suggests some redundancy in the neural mechanisms contributing to cold-induced thermogenesis. A better understanding of the neural circuits and mechanisms mediating the thermogenic effects of OT-R signaling is therefore required and future research should consider whether other OT-R expressing brain regions, including the NTS, contribute to OT-mediated cold-induced thermogenesis.

Given that our shRNA construct and strategy did not completely eliminate OT-R expression in the NTS, it is possible that the food intake and diet-induced thermogenic effects would be enhanced with a greater %KD of NTS OT-R. Greater %KD could be achieved through increased viral titer; however, we found that a 10× higher titer for both AAV1 shCtrl and AAV1 shOT-R caused sickness (weight loss, porphyrin secretions) in the rat which prevented accurate evaluation of the data collected. Other considerations into increasing viral titer and thus %KD, without negative health effects include altering the serotype of AAV vector (51). Nonetheless, we note that although NTS OT-R KD was partial, it was sufficient to interfere with meal size control, satiation-mediated food intake inhibition and diet-induced thermogenesis in the light phase, which highlights the significance of NTS OT-R in the control of food intake and diet-induced thermogenesis.

Even though NTS injections and dissections were performed with great precision and care, given the close proximity of the DMX and the NTS, we cannot exclude the possibility of the spread of AAV shOT-R to the DMX or that the dissection of NTS tissue for qRT-PCR analysis included a small number of DMX neurons. Studies show that OT-R signaling in the DMX can reduce food intake via a parasympathetic efferent mechanism, intra-DMX OT administration reduces gastric motility whereas its receptor antagonist increases motility (52, 53), and these effects are vagally mediated (54). It is unclear whether the reduced sensitivity to the intake inhibitory effects of GI signals in response to NTS OT-R KD observed in the current study might be influenced in part via some reduction in DMX OT-R signaling as the interaction between DMX OT-R signaling and GI signals in food intake control, to our knowledge, is yet to be examined. It would be important in future studies to test this hypothesis.

The sequence homologies between OT and vasopressin, as well as OT-R and vasopressin receptors (55), indicate potential interactions between the OT ligand and vasopressin receptor and vice versa. To ensure that NTS OT-R KD did not affect vasopressin receptor expression, we examined gene expression of vasopressin receptors within the NTS in KD vs control rats and found no differences between groups. Given that vasopressin also has affinity to OT-R (55), it is possible that feeding effects observed in the current study may include vasopressin activity. However, in contrast to the excitatory effects of OT, electrophysiological studies show that vasopressin reduces the excitatory inputs of solitary tract to NTS neurons (56), suggesting that OT and vasopressin in the NTS have opposing functions. Hence, it is highly likely that feeding effects observed here are specific to reduced endogenous NTS OT signaling.

In conclusion, this study showed that NTS OT-R signaling is required for GI signal-mediated food intake inhibition, which complements previous electrophysiological (18) and behavioral evidence (14, 33) that demonstrate the interaction between GI signal processing and NTS OT-R signaling. We further showed for the first time that NTS OT-Rs are necessary contributors to diet-induced thermogenesis measured in the light phase. Together, these data highlight the physiological significance of OT-R signaling in the NTS on food intake control and on diet-induced thermogenesis. Given the therapeutic potential of OT as an antiobesity treatment, future studies examining the role of other OT-R bearing brain sites and the use of diet-induced obese models are warranted to enhance our understanding of the mechanisms and pathways underlying OT-mediated effects on energy balance control.

Acknowledgments

We would like to acknowledge Dr. Matthew Hayes and Derek Zimmer for their help with the development of AAV shOT-R. We also thank Dr. Amber Alhadeff, Dr. Xue Sun, Hallie Wald, Blake Mergler, Carlos Couce, Noah Kennedy-White, Nir Lavi-Romer, Ananya Chandra, and Michelle Yang for technical assistance in this study.

Current Affiliation: Z. Y. Ong's current affiliation is the School of Psychology, University of New South Wales, Sydney, New South Wales 2052, Australia.

Acknowledgments

This study was funded by National Institutes of Health Grant NIH DK-21397 (to H.J.G.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
AAV
adeno-associated virus
AAV1
adeno-associated virus serotype 1
ANOVA
analysis of variance
CCK
cholecystokinin
DMX
dorsal motor nucleus of the vagus
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
GFP
green fluorescent protein
GI
gastrointestinal
HFD
high-fat diet
KD
knockdown
mRNA
messenger RNA
NTS
nucleus tractus solitarius
OT
oxytocin
OT-R
oxytocin receptor
PBS
phosphate-buffered saline
PVH
paraventricular nucleus of the hypothalamus
qRT-PCR
real-time quantitative polymerase chain reaction
shCtrl
short-hairpin RNA control
shRNA
short hairpin RNA.

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