From an Empty Stomach to Anxiolysis: Molecular and Behavioral Assessment of Sex Differences in the Ghrelin Axis of Rats

Abstract

Ghrelin, a stomach-produced hormone, is well-recognized for its role in promoting feeding, controlling energy homeostasis, and glucoregulation. Ghrelin’s function to ensure survival extends beyond that: its release parallels that of corticosterone, and ghrelin administration and fasting have an anxiolytic and antidepressant effect. This clearly suggests a role in stress and anxiety. However, most studies of ghrelin’s effects on anxiety have been conducted exclusively on male rodents. Here, we hypothesize that female rats are wired for higher ghrelin sensitivity compared to males. To test this, we systematically compared components of the ghrelin axis between male and female Sprague Dawley rats. Next, we evaluated whether anxiety-like behavior and feeding response to endogenous or exogenous ghrelin are sex divergent. In line with our hypothesis, we show that female rats have higher serum levels of ghrelin and lower levels of the endogenous antagonist LEAP-2, compared to males. Furthermore, circulating ghrelin levels were partly dependent on estradiol; ovariectomy drastically reduced circulating ghrelin levels, which were partly restored by estradiol replacement. In contrast, orchiectomy did not affect circulating plasma ghrelin. Additionally, females expressed higher levels of the endogenous ghrelin receptor GHSR1A in brain areas involved in feeding and anxiety: the lateral hypothalamus, hippocampus, and amygdala. Moreover, overnight fasting increased GHSR1A expression in the amygdala of females, but not males. To evaluate the behavioral consequences of these molecular differences, male and female rats were tested in the elevated plus maze (EPM), open field (OF), and acoustic startle response (ASR) after three complementary ghrelin manipulations: increased endogenous ghrelin levels through overnight fasting, systemic administration of ghrelin, or blockade of fasting-induced ghrelin signaling with a GHSR1A antagonist. Here, females exhibited a stronger anxiolytic response to fasting and ghrelin in the ASR, in line with our findings of sex differences in the ghrelin axis. Most importantly, after GHSR1A antagonist treatment, females but not males displayed an anxiogenic response in the ASR, and a more pronounced anxiogenesis in the EPM and OF compared to males. Collectively, female rats are wired for higher sensitivity to fasting-induced anxiolytic ghrelin signaling. Further, the sex differences in the ghrelin axis are modulated, at least partly, by gonadal steroids, specifically estradiol. Overall, ghrelin plays a more prominent role in the regulation of anxiety-like behavior of female rats.

Ghrelin and the Control of Energy Balance in Females

“…Böchers et al recently confirmed that female rats have higher circulating ghrelin levels than males, and that ovariectomy resulted in reduced acyl-ghrelin concentrations using a well characterized acyl-ghrelin assay. These authors also and also showed that females have lower plasma concentrations of the liver anitimicrobial peptide-2 (LEAP-2), an endogenous antagonist to the GHSR (63,64).…”

“…Using quantitative polymerase chain reaction (qPCR), a recent study compared GHSR mRNA expression in the ARC and amygdala of male and female ad lib fed mice and mice that were fasted overnight. Female mice showed higher overall GHSR expression in both of these regions, especially in the amygdala (63). In another study, the issue of ghrelin sensitivity was examined indirectly by measuring Fos immunoreactivity (Fos-ir) in response to icv ghrelin infusions in female rats at different phases of the cycle.…”

Blockade of ghrelin receptor signaling enhances conditioned passive avoidance and context-associated neural circuit activation in fasted male rats

“…Future research also should explore sex differences in passive avoidance behavior and the role of ghrelin signaling (and other interoceptive feedback signals) in that behavior, given our previous report that food deprivation suppresses conditioned passive avoidance in male, but not female, rats [7]. In this regard, recent evidence indicates that female rats have significantly higher plasma levels of acylghrelin compared to male rats under both fed and fasted conditions, and that females may be more sensitive to the anxiolytic effects of fasting-induced ghrelin signaling [25]. We reported that compared to male rats, female rats display markedly less conditioned passive avoidance behavior regardless of metabolic state at testing, consistent with other reports that female rodents show less passive and more active responses to threatening stimuli [7].…”

“…Food deprivation increases circulating levels of acyl-ghrelin [23–25], and acyl-ghrelin reduces tonic firing and stimulus-induced responsiveness of vagal sensory neurons [26,27] and postsynaptic A2 neurons in the cNTS [28]. We recently demonstrated that systemic administration of a ghrelin receptor antagonist in fasted rats partially restores the ability of vagal sensory stimulation to activate PrRP+ A2 neurons and suppress food intake [20].…”

Cerebellar irradiation does not cause hyperactivity, fear, and anxiety-related disorders in the juvenile rat brain

“…Whole brain irradiation in young rats have shown different impact on male and female [ 23 , 32 ]. Furthermore, animal behaviour in females is much more influenced by the sexual cycle [ 33 ]. To avoid the potential influence of sex hormone on the behavioural test, we used male rat pups only in this study.…”