The individual and combined anorexigenic effects of oxytocin and naltrindole in rodents

Abstract

Obesity is a characterized by excess body mass, and it reflects a failure to match energy intake to energy expenditure. Energy intake is regulated by a plethora of mechanisms, including the oxytocin (OT) and opioid systems, making both attractive targets for obesity pharmacotherapy. Initial studies utilizing OT and mu-opioid receptor antagonist naltrexone (NTX) suggest that combined targeting of OT and opioid signaling may enhance hypophagia. Thus far, there have no attempts to examine potential synergy between OT and delta opioid receptor blockers. The work presented in the current thesis was conceptualized to bridge this gap in knowledge by evaluating the anorexigenic potential of OT and naltrindole (NTI) when administered both individually and in combination.

The literature describing OT effects on fat intake remains inconsistent. Macronutrient-focused studies that assess the anorexigenic effect of OT on fat consumption using liquid emulsions, most commonly Intralipid, report no suppression of intake. Conversely, studies which use solid high-fat foods more often report OT-induced hypophagia, despite not being designed to systematically test the influence of fat content. Therefore, in Specific Aim 1 of this thesis I systematically determined whether OT suppresses the consumption of solid fatty foods. Adult male rats received intraperitoneal (i.p.) OT (0.1–10.0 mg/kg) and were tested in no-choice and two-choice paradigms using solid mash diets providing 62%, 79%, or 91% of energy from fat; chow diets providing 10% or 60% of energy from fat; or a matrix-matched, cornstarch-rich control chow. The effect of fatty food consumption on neuronal activity was measured using c-Fos immunohistochemistry and c-Fos-OT double staining, where sated rats were either given small meals of 10% fat chow, 60% fat chow, or no food. In the single choice tests, OT reduced the consumption of the solid diets across a range of fat contents, and in the two-choice paradigm, OT-treated rats consumed less 60% fat chow than 10% fat chow compared to control animals. Consumption of 60% fat chow, but not 10% fat chow, increased activation of OT neurons in the PVN and SON and increased c-Fos immunoreactivity in multiple feeding-related regions. These findings demonstrate that OT suppresses intake of solid high-fat foods and help reconcile the discrepancy between Intralipid-based and solid-diet paradigms. In the context of the overarching aim of this thesis, our findings also indicate that conclusions about the anorexigenic effect of OT drawn from Intralipid intake data should be interpreted as specific to Intralipid, rather than assumed to generalise to dietary fat consumption more broadly. The literature lacks a consensus on how NTI affects sucrose intake. Prior reports frequently describe mixed or null effects of NTI on sucrose consumption, but these findings are commonly derived from paradigms in which animals are not habituated to sucrose and instead receive sucrose access in acute, ad hoc test sessions. For this reason, Specific Aim 2 of this thesis was to determine the effect of NTI, and by extension the role of DOR, on sucrose intake in rats that habitually consume sucrose solution. Adult male rats were habituated to daily 1 h access to sucrose solution for three weeks. Following habituation, rats received i.p. NTI (5 or 10 mg/kg) prior to their regular sucrose access period and sucrose intake was measured. We also assessed whether NTI altered deprivation-induced water intake or produced a conditioned taste aversion (CTA). Additionally, c-Fos immunoreactivity was assessed in rats with habitual sucrose access and in sucrose-naïve controls, with and without NTI treatment. NTI at 10 mg/kg reduced sucrose solution consumption independent of changes in thirst and without evidence of a CTA. Moreover, NTI induced changes in neuronal activity across a broader set of feeding-related regions in sucrose-habituated rats than in sucrose non-naïve rats. Together, these findings demonstrate that NTI can suppress sucrose intake once consumption is habitual, and that habituation is associated with a broader recruitment of central feeding-related mechanisms following NTI treatment. Of relevance to the overarching aim of this thesis, these results indicate that null effects of NTI observed under ad hoc sucrose access conditions should not be assumed to reflect NTI efficacy once sucrose intake is predictable and habitual.

Finally, having established foundational knowledge of the individual effects of OT and NTI on intake in Specific Aims 1 and 2, we next examined their combined administration. Based on prior reports indicating that simultaneous targeting of OT and opioid signalling can produce stronger anorexigenic effects than either system alone, we tested OT and NTI in combination. We aimed to determine whether co-administration could overcome context-dependent limitations observed with each drug individually, including OT’s failure to suppress Intralipid intake and the dependence of NTI effects on habitual sucrose exposure. Specific Aim 3 of this thesis was to test whether the simultaneous targeting of OT receptors and DOR, through combined administration of OT and NTI, synergistically suppresses the consumption of liquid diets. Adult male mice received i.p. OT (0.1–3.0 mg/kg), NTI (3.0–10 mg/kg), or OT+NTI prior to 2 h access to saccharin, Intralipid, or sucrose solutions, and intake was measured. We also assessed post–water deprivation water consumption and characterised treatment-induced changes in neuronal activation in feeding-related regions using c-Fos immunohistochemistry. NTI, but not OT, reduced saccharin consumption, whereas OT+NTI produced a stronger reduction than NTI alone, consistent with a synergistic anorexigenic effect. In contrast, OT, but not NTI, reduced sucrose consumption, and neither drug alone nor the combination altered Intralipid intake. These intake outcomes occurred independent of changes in thirst. At the neuronal activation level, OT × NTI interactions were observed in the nucleus accumbens core and shell and in hypothalamic nuclei including the ventromedial and suprachiasmatic nuclei. Although combined treatment did not overcome the individual limitations observed for sucrose and Intralipid intake, the synergistic suppression of saccharin consumption is notable and extends evidence that combined targeting of OT and opioid signalling may hold promise for development of anorexigenic treatments.

In summary, this thesis provides novel insights into the context-dependent anorexigenic actions of both OT and NTI, demonstrated by the efficacy of OT as an inhibitor of solid fatty food consumption but not Intralipid, and by the dependence of NTI-induced sucrose suppression on habitual consumption. Furthermore, this thesis provides additional evidence that simultaneous targeting of OT and delta opioid signalling can produce anorexigenic effects which extend beyond those achieved with single-drug treatment, as demonstrated by the synergistic suppression of saccharin intake observed with OT and NTI co-administration.