**3. Leptin regulation of gonadotropes**

Shortly after leptin was discovered, pioneering studies by Yu et al. [10] demonstrated that leptin stimulated LH and FSH release, *in vitro*, from hemi-anterior pituitaries. They reported a dose dependent increase in LH after 3 h in 10−11-10−9 M leptin. Higher concentrations, however, were not stimulatory suggesting the development of leptin resistance. It is interesting to note that the relatively narrow concentration range that stimulates gonadotropin release matches that of the normal cyclic rise in leptin [36, 39]. Yu et al. reported that leptin alone stimulated LH release, *in vitro* and it did not add to the stimulatory effect of GnRH [11]. Their studies also identified nitric oxide as a signaling pathway for leptin regulation of gonadotropes. They showed that a competitive inhibitor of nitric oxide synthase (NOS), N6-monomethyl-L-arginine (NMMA), inhibited the leptin stimulation of LH release *in vitro* [11] suggesting that leptin may use the NOS pathway to stimulate gonadotropes directly.

Our studies on the importance of leptin to gonadotropes began with the detection of leptin receptors (LEPR) in dispersed pituitary cells from male and cycling female rats and mice [1]. The expression of LEPR varied with the stage of the cycle and was seen in 40-50% of anterior pituitary cells from males and females in metestrus or diestrus. LEPR expression increased to 60-80% of AP cells in proestrous and estrous females, which coincided with the midcycle rise in serum leptin [1].

To determine if the increase reflected changes in gonadotrope receptivity, dual labeling was performed for LEPR and gonadotropins. The results showed that 90% of gonadotropes identified by the stores of luteinizing hormone (LH) or follicle stimulating hormone (FSH) expressed LEPR in males and throughout all stages of the cycle in females [1]. Some of the increase in LEPR in proestrous females was due to an increase in cells expressing LH and LEPR, which occurs just before the LH surge.

The findings showing an overall increase in LEPR early in the estrous cycle stimulated studies to determine potential regulators for this expression. We treated one day cultures of anterior pituitary cells from diestrous female mice with estradiol overnight and then treated a subset of these cultures with 10 or 100 pg./ml neuropeptide Y (NPY) for 3 h. **Figure 1** shows that estradiol or NPY alone (100 pg./ml) stimulated a

### **Figure 1.**

*Estradiol and NPY stimulate LEPR expression in 1-day cultures of anterior pituitary cells. \* = significantly higher values than all other values; ANOVA + Bonferroni's post hoc test. Note: These are original data, not published elsewhere.*

significant 2--fold increase in LEPR-bearing cells and that the effects of the two were not additive. In contrast, NPY did not stimulate LEPR expression in anterior pituitary cells from male mice (data not shown). Collectively, these data support the hypothesis that rising estradiol early in the cycle may stimulate an increase in pituitary receptivity to leptin which may serve as a gateway for leptin's permissive actions [7].

Having established the presence of the receptor population in gonadotropes, we determined if leptin acted on gonadotropes through the Janus Kinase-Signal Transducer and Activator of Transcription (JAK–STAT) pathway. Following leptin stimulation for 10-60 min *in vitro*, pituitary cells from diestrous females were dual immunolabeled for phosphorylated STAT3 and LH or FSH [1]. In 30-60 min, leptin stimulation increased the overall percentage of LH or FSH-bearing gonadotropes and the percentages of gonadotropes bearing pSTAT3 [1]. Thus, leptin acts through both NOS [11] and the JAK–STAT [1] pathways to increase LH or FSH stores in gonadotropes.
