The present study investigated whether abolishing hypoleptinaemia with a physiologically-relevant dose of leptin was capable of restoring GnRH/LH secretion during negative energy balance. The results obtained demonstrate that restoring leptin to normal basal values does not attenuate LH inhibition in a 50% CR. This dose of leptin was also incapable of maintaining LH levels during a 48-h fast. Therefore, although leptin may be a permissive signal for the suppression of cyclic reproductive function during negative energy balance, it appears unlikely that elimination of hypoleptinaemia is the critical driver of LH restoration. ARC Kiss1 is also suppressed with CR and fasting, and exogenous leptin could not restore ARC Kiss1 levels in the CR models, although there was a small effect of leptin in the fasting study.
There is a wealth of literature on the importance of leptin for normal reproductive function. Leptin is required for normal reproductive development because mutations in leptin or leptin receptors result in abnormal reproductive function (9
), and exogenous leptin administration results in early onset of pubertal development (46
). Studies across many species have also shown stimulation of LH by exogenous leptin under both normal and metabolically challenged conditions (7
) and, critically, human studies suggest that leptin may be a viable treatment for women with exercise-induced hypothalamic amenorrhea (48
Although the literature clearly points to an important role for leptin in reproductive function, recent studies have complicated the hypothesised role of leptin for negative energy balance-induced reproductive inhibition (49
) found that when food restricted ewes are refed, LH parameters were restored before any increases in circulating leptin levels, suggesting that the elimination of hypoleptinaemia is not required for restoration of LH after negative energy balance. A recent study from our laboratory found that restoring leptin to normal basal levels had no effect to restore LH levels in the lactation model of negative energy balance (36
). This dose of leptin, also used in the present study, resulted in normal basal leptin levels that are at least 50-fold lower than the levels reported with a standard pharmacological dose previously shown to attenuate LH inhibition (7
). Indeed, the vast majority of studies demonstrating leptin-induced attenuation of LH inhibition have used similarly large pharmacological doses (7
). However, it is important to acknowledge that although the administration of leptin by minipump in the present study resulted in serum levels in the normal physiological range, this administration cannot itself be termed ‘physiological’ because the diurnal leptin pattern was disrupted with continuous leptin infusion. Therefore, it remains possible that the effects of leptin in the present study were not observed as the result of a lack of diurnal rhythm.
Our previous work demonstrating a lack of effects of leptin upon negative energy balance-induced LH inhibition was performed in the lactation model (36
), and we hypothesised that the effects of leptin in this model may have been masked by other inhibitory signals specific to lactation, such as the suckling stimulus (39
). Therefore, the present study was designed to determine if leptin was ineffective during lactation as a result of the redundant inhibitory signals specific to this model or the lower dose of leptin administered. Seventy-two hours of exogenous leptin treatment, which restored leptin to normal basal levels, was incapable of restoring LH during 50% CR, suggesting the previously demonstrated lack of effects of leptin during lactation may not be a characteristic specific to this model. Additionally, although pharmacological leptin did prevent LH inhibition during a fast, consistent with previous results (7
), maintaining leptin at normal basal levels did not. Leptin levels rise modestly when animals exit negative energy balance to return to normal basal levels and do not reach the very high levels observed with pharmacological doses (7
). Therefore, although leptin appears to be required for normal reproductive development and can stimulate LH at pharmacological concentrations, hypoleptinaemia may not be the critical signal responsible for suppression of LH during negative energy balance.
In addition to LH inhibition, the present study has demonstrated that ARC Kiss1 is inhibited in both fasting and CR models of negative energy balance. Previous data describing fasting effects on ARC Kiss1 have been inconsistent, with data arguing both for and against inhibition of these cells (34
). Given previous results suggesting a role for ARC Kiss1 in negative steroid feedback of GnRH release, the current results of ARC Kiss1 inhibition in the present study are consistent with previous work demonstrating a loss of pulsatile LH during a 48-h fast (12
). AVPV Kiss1, which is considered to contribute to positive steroid feedback and the GnRH/LH surge, was only significantly inhibited in the 50% CR study, and not during fasting or the 40% CR experiments. An unexpected finding in the 40% CR study was the significant inhibition of AVPV Kiss1 in the presence of leptin. There are no obvious explanations for this result because inhibitory effects of leptin on AVPV Kiss1 have not been reported. AVPV Kiss1 is also suppressed during lactation (19
), indicating this population may only be inhibited under severe conditions of negative energy balance. Interestingly, NKB and PDYN, which are coexpressed within the ARC KNDy neurones, were not consistently inhibited with negative energy balance, although NKB levels were inhibited with the more severe 50% CR, similar to lactation (19
). More research is needed to understand how differential regulation of these three reproductive neuropeptides within the same KNDy cells may contribute to reproductive inhibition.
Similar to the LH results, restoring leptin to normal basal values was unable to attenuate inhibition of ARC Kiss1 or NKB mRNA or AVPV Kiss1 mRNA inhibition in the 50% CR experiment. Despite previous results showing leptin-receptor expression on ARC KNDy neurones in the mouse (27
), the present study found a dramatic lack of ARC Kiss1/pSTAT3 colocalisation after acute treatment with a pharmacological dose of leptin, arguing against a strong direct regulatory relationship in the rat. Taken together with recent evidence observing a lack of leptin-receptor signalling in AVPV Kiss1 neurones (52
), these findings suggest Kiss1 neurones are unlikely to be the cell population relaying leptin signalling to GnRH neurones. Pharmacological leptin treatment has been shown to stimulate Kiss1 levels (27
) and, in the present study, leptin replacement to normal basal levels appeared to partially attenuate inhibition of ARC Kiss1 in the least severe model of negative energy balance (i.e. the 48-h fast model), suggesting there may be differential regulation of ARC Kiss1 by leptin depending on the model of negative energy balance.
Because of the lack of effects of leptin on Kiss1 mRNA and LH levels in the present study, it was important to demonstrate that the physiologically-relevant dose of leptin administered by minipump infusion was biologically active in the brain. To definitively answer this question, pSTAT3 staining was measured in fasted animals receiving either saline or the physiologically-relevant dose of leptin via osmotic minipump. Two hours of the physiologically-relevant leptin dose significantly increased pSTAT3 staining compared to animals receiving saline, confirming the biological activity of this dose in the brain. Although it is conceivable that leptin may be degraded at body temperature with longer minipump treatments such as those employed in Experiments 1–3, this appears unlikely given the abundance of studies showing significant effects of leptin after prolonged minipump administration for up to 2 weeks (53
). In addition to pSTAT3 staining, ARC SOCS3 mRNA was also increased in the 40% CR and the 50% CR with the low leptin treatment, although this difference only reached statistical significance in the former model. Furthermore, this dose of leptin was used previously in our laboratory and shown to completely reverse the suppression of POMC in lactating rats (36
), confirming that this dose is biologically relevant in the brain.
Unexpectedly, in the present study, POMC was not significantly inhibited in either CR model. This lack of POMC regulation with CR suggests that decreases in POMC may not be as strongly regulated with negative energy balance in females as has been previously reported for males (60
), and therefore POMC inhibition is only observed in female rats with the severe hyperphagia and negative energy balance of lactation (36
). Given the lack of POMC inhibition with CR, it was not surprising that leptin administration had no affect on POMC levels. Leptin infusion was also incapable of completely attenuating the large increases in NPY and AgRP in response to CR, consistent with previous work from our laboratory finding no effect of restoring leptin to normal basal levels on NPY and AgRP levels in lactating rats (36
). Although this lack of leptin regulation on NPY and AgRP may appear controversial, it should be noted that previous effects of leptin on NPY and AgRP have been demonstrated with male mice using pharmacological doses (60
). It appears likely that differences between the present study and previously reported leptin affects on NPY and AgRP are likely a result of either gender or doses of leptin.
Consistent with earlier fasting studies, our results found a requirement of oestradiol for negative energy balance-induced LH inhibition (12
). As expected, the low dose of oestradiol administered alone did not blunt the OVX-induced LH rise (40
); however, the oestradiol levels were clearly biologically active because significant effects on body weight, uterine weight and AVPV Kiss1 were observed. Interestingly, the results of the present study demonstrate that, although oestradiol is required for inhibition of LH, it is not required for of the inhibition of ARC Kiss1. In the 40% CR studies, ARC Kiss1 was uniformly suppressed in all CR groups with or without oestradiol treatment, whereas the LH values were widely variable, with some in the normal control range. Thus, it appears that suppression of ARC Kiss1 is not always tightly coupled to the suppression of LH secretion, lending support to the notion of Kiss1-independent regulation of LH secretion (66
). The variable LH levels with a 14-day 40% CR suggest that there may be a threshold of sufficient weight loss required for LH inhibition. The uniform suppression of LH with 50% CR suggests all animals had achieved sufficient weight loss for LH inhibition. Importantly, it appears that once animals are in a severe enough state of negative energy balance, as demonstrated with the 50% CR, restoring leptin to normal basal levels has no effect to restore LH. These findings highlight the fact that many aspects of LH inhibition remain poorly understood, and further studies are needed to understand the multitude of signals contributing to LH inhibition.
The findings of the present study, coupled with our earlier studies of lactation (36
), suggest that metabolic factors other than low leptin likely contribute to inhibition of reproductive pathways in models of negative energy balance. Previously studied candidates include ghrelin, insulin, glucose and NPY, amongst others (67
). NPY and insulin do not appear be to critical players because insulin replacement during lactation did not restore reproductive function, and attenuation of elevated levels of NPY in both lactation and 50% CR were not accompanied with changes in LH (36
). Ghrelin is also an interesting candidate for linking metabolic and reproductive function because ghrelin has been shown to be inhibitory to LH (69
). However, although the elevated levels of ghrelin during fasting are consistent with a potential role in the inhibition of LH (70
), during lactation, ghrelin levels are low (71
) and exogenous ghrelin has no affect on LH (72
), suggesting that it is unlikely to contribute to the suppression of LH. Clearly, much remains to be learned about the metabolic regulation of reproduction, although the results obtained in the present study argue against a critical role of hypoleptinaemia in the suppression of LH during negative energy balance because restoration of leptin to normal basal levels does not restore LH. Taken together with previous work clearly demonstrating a strong role for leptin in reproductive regulation, it is becoming clear that this pathway is more complex than previously hypothesised. Therefore, understanding the neurocircuitry involved in the inhibition of Kiss1 and GnRH release and the potential multitude of metabolic signals that could be involved in this process still remain two critical and unresolved questions in the field.