Non-seasonal breeders include spontaneous ovulating species, such as humans, rats, mice, and naked mole rats, as well as induced ovulating species, such as the domestic cat. Unlike short-day and long-day breeders, the literature on non-seasonal breeders is more consistent, and GnIH and its receptor were shown to be present in all three levels of the HPG axis (13, 15, 30, 35, 105). Interestingly, puberty in humans has been hypothesized to be better anticipated by measuring the accumulation of fat rather than using age or environmental stimuli as a predictor (106). Therefore, it is not surprising that a direct role of GnIH on the hypothalamic control of reproduction appears to be substantially less critical than in seasonal breeders, as demonstrated by the reduced number of projections of GnIH neurons to GnRH neurons in the mouse (14) and GnIH projections to the median eminence (ME) in the rat and mouse (70, 77, 107). However, IV administration of GnIH still resulted in an inhibition of the GnRH-induced LH production in ovariectomized rats (107, 108). Although a decline in the expression of both GnIH and GnIH-R were reported around sexual maturation in male rats, while in females, the expression of GnIH increased and the expression of GnIH-R significantly declined between 28 and 49 days of age (36), again indicating a possible sex-related difference in the regulation of and sensitivity to GnIH. In naked mole rats, only dominant females can undergo spontaneous ovulation and breeding females display elevated numbers of kisspeptin cells while non-breeders presented an elevated number of GnIH cells (105). Beyond puberty and ovulation, GnIH levels in rats significantly declined immediately following parturition (109), likely playing a role in facilitating postpartum estrus in this species (110, 111). Thus, although the role of GnIH may be more discrete in non-seasonal breeders, the inhibitory impact on reproduction is still conserved.

Since its initial discovery as a novel gene in humans (112), kisspeptin, along with its receptor (GPR54/Kiss-1r; 113), was shown to control puberty and reproduction in mammals via direct stimulation of GnRH neurons (114–119). In fact, along with neurokinin B and dynorphin, kisspeptin is part of an intricate neuronal circuitry responsible for the pulsatile secretion of GnRH, referred to as the KNDy neurons (for review: 120). Interestingly, GnIH-Rs are expressed in 9-16% of RP3V kisspeptin neurons in rats (24), as well as in 5-10% of the anteroventral periventricular nucleus (AVPV) and 25% of ARC Kiss1 neurons in mice (121). Additionally, Kiss1 neurons in the ARC are in close proximity to GnIH fibres (121), suggesting that GnIH may directly inhibit a subset of kisspeptin neurons and thus inhibit reproduction (122), although a reciprocal effect was not identified (121). In mice, GnIH-R knockout (KO) resulted in a weaker disruption of LH secretion (123) compared to GPR54 KO (124). However, Kiss1 mRNA was found to increase in GnIH-R KO mice (123), and with 33% of GnRH neurons also expressing GnIH-R in rats, GnIH likely acts at multiple levels (GnRH and kisspeptin neurons) to inhibit GnRH synthesis and release (24). This is further supported by the similarities in expression patterns between GnIH-R and GPR54 as previously reported (15, 125).

In long-day breeders, shorter day lengths result in increased expression of GnIH due to higher melatonin (86), while the number of kisspeptin-positive cells in the ARC and Kiss1 mRNA levels decline (126–129). Interestingly, when hamsters maintained under a short-day length were treated with Kp10, maturation of the reproductive tract was observed, with organ weights comparable to that under long days (96), indicating that exogenous kisspeptin is able to override the need for photostimulation. As discussed further in the following sections, beyond photoperiod, switching the activity of the HPG axis from inhibitory to stimulatory may intimately be linked to metabolic status and the hypothalamic control of energy reserves and nutrient partitioning (130, 131). This is further supported as a relationship between kisspeptin, photoperiod, and food availability has previously been established in seasonal mammals (132).

Furthermore, as avian species require a significant shift in energy partitioning towards egg production, metabolic status may be the primary cue controlling the activation of the HPG axis (133). Thus, the absence of a Kp gene in several avian species (134, 135) may allow GnIH to take a central role in balancing energy status and reproduction.

The relationship between reproduction and body weight has been previously described in mammalian and avian species (133, 136–139), with a minimum body weight threshold required to initiate the activation of the HPG axis (140, 141). Prior to sexual maturation, animals undergo a rapid weight gain and growth phase while the HPG axis remains suppressed, possibly via elevated levels of GnIH. For example, moderate to high intraperitoneal doses of GnIH in mice were reported to evoke an increase in body mass (142), and chronic GnIH ICV injection elevated both body weight and feed intake in male mice (143). Initially, it was proposed that body weight was under the dual control of both GnIH and its stimulatory counterpart, as GnRH agonist treatment also resulted in a dose-dependent body mass gain in rats (144). However, the impact GnRH had on body weight was likely the outcome of a GnRH-stimulated increase in the expression of neuropeptide Y (NPY), as this peptide not only stimulates feed intake but has additionally been implicated in the preovulatory surge (145, 146). Furthermore, as described in the following section, more recent evidence suggests that beyond the central nervous system, GnIH can also influence body weight via direct control of adiposity in male mice, acting independently of reproductive steroids or the melanocortin system (96).

In view of the promiscuity between ligand and receptors from the RFamide peptide family, it is reasonable to question whether the impact of GnIH on body weight is mediated through its own receptor. This could partly be answered using GnIH-R KO, and in mice, inactivation of the GnIH-R resulted in significantly heavier females, yet it did not impact the weight of males (39), suggesting that the impact of GnIH on body weight is sex-dependent and most likely related to reproduction. Since in mammals, the GnIH-R was shown to couple to both Gα_i3 and Gα_s (64), the observed differences between males and females may point to a differential receptor activation and signalling between sexes (147). In fact, when GnIH-KO and control mice were fed high-fat diets (HFD) and low-fat diets (LFD), males displayed declined locomotor activity in both KO groups, while female mice in both KO groups demonstrated elevated fat mass over the control (39). However, regardless of sex, obese (ob/ob) mice displayed lower GnIH mRNA levels in the dorsal-medial nucleus than their wild-type counterparts (148). Taken together, these studies suggest that as proposed by Cázarez-Márquez et al. (96), the impact of GnIH and its receptor on body weight may be linked to adiposity, which is also intimately associated with reproduction. Back in 1974, Frisch and McArthur (106) postulated the ‘critical weight hypothesis,’ which states that the accumulation of body fat stores may be a better indicator of the timing of sexual maturation rather than age or body weight itself. This has further been confirmed in rats, as well as chickens, with insufficient fat stores resulting in pubertal delay (136, 149, 150) and diet-induced obesity resulting in declined reproductive capacity (151, 152). In a recent study, GnIH injections resulted in the elevation of serum total triglycerides and cholesterol (153), leading to increased uptake of triglycerides by the adipose tissue (142). Thus, it could be hypothesized that GnIH acts to control fat deposition in adipose tissue not only in immature animals but also in adults. This is partially supported by the association between GnIH-induced increased body weight and increased brown adipose tissue (BAT) mass and liver mass (143). However, although GnIH appears to stimulate fat deposition, male GnIH-R KO mice were not protected from body weight gain on a high-fat diet (39), and a lack of GnIH-R signalling did not prevent obesity. Additionally, with GnIH-stimulated feed intake reported to trigger dyslipidemia (153) and abnormal glucose metabolism (143, 154), GnIH may play a larger peripheral role in the control of metabolism than previously thought (discussed in a following section).

It is well-established that the melanocortin system controls feed intake through the orexigenic peptides, neuropeptide Y (NPY) and agouti-related peptide (AgRP), and the anorexigenic peptides, pro-opiomelanocortin (POMC) and cocaine-and-amphetamine regulated transcript (CART) (155–160). Neurons from the melanocortin system have been localized throughout many regions of the hypothalamus (24, 161) and shown to be in close contact with GnIH neurons in the DMH, with GnIH-containing fibres projecting to the ARC and PVN in mice (77, 162). In fact, the orexigenic effect of GnIH is mediated through the modulation of POMC and NPY neuronal activity (24, 161, 163, 164), with POMC downregulated in the presence of GnIH (161, 164, 165). In GnIH-R KO male mice, POMC mRNA levels in the hypothalamus are elevated compared to wild type (39), further suggesting a direct role for GnIH and its receptor in the inhibition of the anorexigenic response. However, the direct role of GnIH on NPY appears to be more ambiguous (161, 164). In mice, GnIH has been reported to inhibit the neuronal activation of NPY (161), yet in chicks, sheep, and rats, GnIH was shown to stimulate NPY, prompting an increase in feed intake (165, 166). Nonetheless, whether acting on both branches of the melanocortin system or not, ICV administration of GnIH increases feeding duration (167) and overall feed intake in chickens and rats (70, 108, 165, 168). However, the injection of GnIH into the amygdala of rats also resulted in an opposite effect with the suppression of food intake (37). Interestingly, despite mediating increases in feed intake in sheep, GnIH administration did not result in a reduction in energy expenditure, as measured by calorimetry (166). This strongly suggests that the effect of GnIH on feed intake is not intended to achieve or restore energy homeostasis but may rather coordinate the partitioning of energy away from reproduction (169). However, chronic ICV injection of GnIH in mice did decrease energy expenditure and increased feed intake, contributing to a rapid decline in brown adipose tissues (BAT) activity prior to an accumulation in lipid droplets, thus elevating BAT deposition overall (143). This effect also resulted in a GnIH-induced decrease in core body temperature during short exposure to the dark phase, suggesting for the first time that GnIH contributes to the conservation of energy (143).

In songbirds and zebra finches, food deprivation results in a decline in the number of GnIH-ir cells (170), although no differences in mRNA or peptide were observed (171, 172). As hypothesized by Fraley et al. (167), this decrease in GnIH-ir cells following feed restriction is likely the result of chronic metabolic stress. Of interest, declining GnIH-ir cell numbers have been correlated to declining body mass in female songbirds (170). When GnIH-R deficient male mice underwent 12-h fasting, despite a large decline in body mass, no change in LH secretion was detected. Conversely, in their wild-type counterparts, an immediate decline in LH secretion occurred following a 12-h fasting (123). Taken together, these studies provide an alternative pathway by which GnIH is able to mediate feed-seeking behavior via body mass fluctuations (170).

Since the leptin receptor is co-expressed with NPY and POMC, leptin is also thought to regulate feed intake (173, 174). Thus, a possible integration between GnIH and leptin has been investigated. This led to the identification of the long form of the leptin receptor (LepRb), present on 15 to 20% of GnIH neurons (148). This finding highlights a possible pathway between GnIH and the regulation of adiposity via leptin and feed intake (175). With diminished levels of GnIH observed in the leptin-deficient ob/ob mice, a direct inhibition from leptin via the small subset of LepRb-expressing neurons is possible, yet alternative indirect hypotheses have also been proposed (148). However, leptin acts through the PKC-dependent pathway to promote intracellular Ca²⁺ signalling within GnIH neurons, thereby potentially eliciting an indirect effect on appetite control and an indirect negative feedback to GnRH neurons (176), which lack the LepRb (177). Thus, elevated circulating leptin results in a decline in GnIH activity (178), leading to low LH levels in ob/ob mice, resulting in infertility (179). However, reactivation of the HPG axis is possible with leptin treatment (180, 181), illustrating a possible multipronged link between metabolism and reproductive success (182, 183), with leptin additionally proposed as a regulator in the timing of puberty (184). Furthermore, as both leptin and GnIH activate the PI3K/Akt signalling pathway (185, 186), Anjum et al. (175) hypothesized that PI3K/Akt signalling in ventromedial nucleus of the hypothalamus (VHM) neurons is responsible for the coordination of energy homeostasis (further discussed in a following section).

In addition to the melanocortin system, ghrelin, a hormone traditionally referred to as the ‘hunger hormone,’ is also involved in the control of appetite. As for GnIH, ghrelin elicits a stimulatory effect on food intake and an inhibitory effect on gonadotropin secretion through the growth hormone secretagogue receptor (GHS-R) (187) present on GnRH neurons (188). While this hormone has been primarily linked to the gastrointestinal tract, it was also identified in the brain of chickens (189), mice, and rats (190, 191), pointing to a neuroendocrine role beyond the gut. Specifically, ghrelin-containing hypothalamic neurons have been proposed to interact with NPY and AgRP-containing neurons to stimulate the secretion of orexigenic peptides in rodents (190, 191). Beyond appetite control, ghrelin can also inhibit LH (192), and testosterone production through the inhibition of steroidogenic enzymes (193), contributing to the suppression of the HPG axis during periods of insufficient energy stores (reviewed by: 194). Interestingly, when fed a HFD, GnIH-R KO mice demonstrated a complete resistance to the central administration of ghrelin, failing to prompt an increased cumulative feed intake compared to their wild-type counterparts (39). This suggests that the effect of ghrelin on appetite control may be in part mediated via GnIH and its receptor. However, the suppression of ghrelin effects in GnIH-R KO was not sustained when mice were fed a LFD (39). Since it has previously been reported that rats fed a HFD do not display ghrelin-induced hyperphagia but do maintain the increased adiposity also observed in LFD-fed rats, ghrelin may utilize separate mechanisms to act on appetite and lipid metabolism (195). In fact, Anjum et al. (175) suggested that the ghrelin receptor involved in the brain may be different and not yet identified.

Although GnIH may not be produced in the adipose tissue, its receptor was shown to be present on human fat cells, suggesting a direct role for GnIH or its orthologues on adiposity (29). Interestingly, the initial focus was placed on white adipose tissue (WAT) due to its association with energy storage (233). However, it was recently reported that GnIH treatment inhibits the activity of BAT (143), which plays a critical role in energy expenditure through thermogenesis (reviewed by: 234, 235).

In terms of WAT, circulating leptin concentrations can be used as an indirect measure of fat accumulation (96, 236–238). For example, higher leptin concentrations are observed in fat or ad libitum fed Syrian hamsters compared to their respective lean or feed-restricted counterparts (239). Of interest, leptin is known to reduce feed intake (240) and is considered anorexigenic (241). This activity opposes the role of GnIH on appetite control discussed earlier and highlights a possible antagonistic effect between leptin and GnIH (178). This relationship is further emphasized in ob/ob mice, which lack a functional leptin gene (181). Without this WAT regulator, rats demonstrated an elevation in body weight (242, 243), similar to that observed in GnIH treated mice (143).

As described previously, both leptin and GnIH activate the PI3K/Akt signalling pathway in the hypothalamus, a pathway also used during insulin-mediated glucose uptake in the adipose tissue (142), thus, raising the possibility of an interaction between, GnIH, leptin, and insulin in the regulation of glucose uptake. Since GnIH-treated male hamsters had higher levels of leptin and insulin compared to controls (96), it is possible that this resulted in increased adiposity (142), leading to increased insulin resistance (reviewed by: 244). However, this effect was not observed in female hamsters (96). In fact, it appears that the effect of GnIH may be dose-dependent as 2,000 and 20 ng induced a decline and increase in glucose levels, respectively, with a concomitant elevation in insulin receptor and GLUT8 proteins under the lowest GnIH dose only (142). Thus, GnIH may regulate insulin sensitivity, at least in male mice. As the study by Cázarez-Marquez et al. (96) used significantly higher GnIH levels (105-μg), a possible GnIH threshold may exist with supraphysiological doses eliciting opposite effects.

As described in the previous section, GnIH has been hypothesized to regulate fat accumulation in adipose tissue through the mediation of increased nutrient uptake, including glucose and triglycerides (142). In fact, when injected intraperitoneally, GnIH has been found to primarily co-localize with glucagon in α-cells, while its receptor, although present in both cell types, is primarily co-localized with insulin in β-cells of pancreatic islets (153), strongly suggesting an involvement in glucose homeostasis. Essentially, GnIH supports α-cell survival and hyperplasia through activation of the GnIH-R present in these cells, which triggers the AKT and ERK1/2 pathways (186), leading to an increase in glucagon and feed intake. When glycemia is elevated, β-cells would normally release insulin to offset the imbalance, restore glucose homeostasis, and reduce feed intake (reviewed by: 245). However, recent evidence demonstrated that chronic and acute doses of GnIH increase blood glucose levels while simultaneously reducing insulin secretion. With the colocalization of GnIH and glucagon, it is likely GnIH can promote hyperglycemia in rats (153, 246). The direct blockade of insulin has been hypothesized to occur through Gα_i and the inhibition of the AC-cAMP-PKA pathway (153, 247). This hypothesis is further supported by the reduced insulin sensitivity with chronic GnIH treatment, characterized by a decline in insulin receptor and GLUT4 in the adipose tissue (142, 153). Intriguingly, since insulin is able to relay information regarding body fat status to the central nervous system (reviewed by: 248, 249), inhibition via GnIH further supports a multipronged impact through which GnIH promotes fat accumulation and an elevation in body weight, associated with increased feed intake (see previous section). Furthermore, kisspeptin has also been reported to stimulate glucose production with an increased glucose tolerance to prevent the onset of hyperglycemic disorders (250) via its receptor. As this effect is absent in GPR54-KO mice (251), it is possible that cross-activation by various members of the RFamide family, including GnIH, can occur. This is of particular interest as insulin has been reported to be a mediator between nutritional status and reproductive success (252), and significant fluctuations in glucose levels can be lethal (reviewed by: 253).