**5. Leptin regulates target genes through different pathways**

After we characterized the deletion mutants lacking LEPR in gonadotropes, we hypothesized that rising leptin early in the cycle may have a permissive effect on the rise in pituitary GnRHR levels [7], which could serve as a gateway that permitted full receptivity to GnRH and facilitates the LH surge. We treated pituitary cells from normal diestrous female mice with 10 nM leptin and showed a significant increase in GnRHR proteins [7]. We also detected leptin-stimulated increases in pituitary activin (but not inhibin) mRNA (*Actßa* and *Actßb*) in the same sets of experiments. However, leptin did not stimulate increases in *Gnrhr* mRNA levels [7], which correlated well with the lack of change in mRNA levels evident in the LEPR-null gonadotropes. Thus, we identified three targets of leptin in our animal model, and proposed that leptin may activate these by different pathways.

## **5.1 Transcriptional regulation of FSH and activin by leptin**

We have demonstrated that expression of *Fshb* and activin transcripts are dependent on a normal leptin signal [1, 7]. Other workers have shown that activin and FSH may be dependent on the timing of this leptin signal during postnatal development, which is characterized by a rapid rise in serum leptin. Wen et al. [42] studied the link between the postnatal rise in leptin and FSH and reported that full co-expression of GnRHR and FSH is seen by postnatal day 7, which coincides with the peak leptin surge. A parallel rise in *Fshb* and *Actβa* and *Actβb* mRNA levels during the postnatal leptin rise has also been reported [43–46]. Researchers investigating the impact of altering the neonatal leptin surge on the reproductive system reported that blockade or alteration of the leptin surge decreased testicular or ovarian growth, delayed puberty, and reduced FSH in rat pups [47]. In addition, females showed reduced numbers of ovarian primordial follicles [48].

Another link between leptin and FSH was reported by studies that restored LEPR in gonadotropes from mice that were genetically engineered to be globally deficient in LEPR [49]. As expected, fertility was not restored, because the mice were morbidly obese, and kisspeptin and GnRH neuronal function was still deficient. However, they did report elevated FSH levels in these mice. It was not determined whether restoration of the leptin signal influenced GnRHR expression.

The reduced *Fshb* mRNA detected in our gonadotrope LEPR-null mutants correlates well with the reduced activin (*Actßa* and *Actßb*) mRNA [1], which is a critical regulator of *Fshb* transcription [50, 51]. Our studies show that leptin stimulates activin mRNA [7], which could thus serve as a pathway for FSH stimulation. Leptin regulation of FSH also agrees with studies of rats [10, 11] and non-human primates [25] in which leptin directly stimulated FSH secretion, *in vitro*. Collectively, these findings suggest that leptin may be an important transcriptional regulator of FSH production both postnatally and early in the cycle, either directly or indirectly. Additional studies are needed to determine if this pathway is mediated through JAK– STAT activation or NOS [10, 11].

**Figure 2** illustrates how the ovary and adipocytes may partner in the remodeling of gonadotropes to support the development of the follicles with key cellular regulatory pathways and outputs indicated. This cartoon focuses mainly on leptin, FSH and estrogen. We propose that normal levels of leptin permit a rise in FSH early in the cycle regulating FSH directly or through activin. This could be an important checkpoint if leptin levels drop due to fasting, for example [23] as this may signal poor nutrition and reduce FSH production. The cartoon then shows that FSH stimulates

### **Figure 2.**

*Gonadotropes are remodeled early in the cycle by estrogens, GnRH, and leptin to support the ovary. We postulate that normal levels of leptin permit FSH release directly or through activin. FSH stimulates the growing population of follicles, which produce more estrogen. This rise in estrogen may stimulate leptin release from adipocytes and the expression of LEPR in gonadotropes. Estrogen also exerts positive feedback on the neuronal circuit that regulates GnRH, which produce more rapid GnRH pulses, which also stimulate* Gnrhr *mRNA. As*  Gnrhr *mRNA rises, leptin works post-transcriptionally to permit translation of GnRHR proteins by de-repressing the actions of the translational regulator Musashi (MSI). Leptin also causes a reduction in expression of MSI. This is an original figure drawn by the corresponding author and not published elsewhere.*

### *Leptin: A Metabolic Signal for the Differentiation of Pituitary Cells DOI: http://dx.doi.org/10.5772/intechopen.100922*

ovarian follicles to produce and secrete estrogen, which stimulates a rise in serum leptin. The growth in ovarian follicles and subsequent rise in estrogen also has positive feedback actions on GnRH neurons (shown in ref. [5]) and the gonadotropes. Estrogen may also stimulate a rise in pituitary LEPR (**Figure 1**), which renders the gonadotropes more responsive to leptin.

Not shown in this cartoon is GnRH, which is secreted in response to estradiol positive feedback to stimulate gonadotrope production of gonadotropins and GnRHR (pathway shown in ref. [5]). GnRH and estradiol both stimulate *Gnrhr* mRNA during this time (reviewed in [9]). Leptin's role is to de-repress Musashi's actions on *Gnrhr* mRNA and permit translation. Leptin also reduces Musashi expression [7, 8]. Thus, our studies show that, whereas leptin does not regulate *Gnrhr* mRNA directly, it works in partnership with estradiol and GnRH to permit its translation by regulating MSI. This is another checkpoint in reproductive cycles [7, 9]. Reduced leptin, due to fasting for example, may signal poor nutrition and thus reduce translation of GnRHR [7–9] and GnRH binding sites [23]. Ultimately, leptin reduction of ablation slows or prevents reproduction. Our animal models lacking LEPR in gonadotropes support this hypothesis [1, 5, 7].

### **5.2 Post-transcriptional regulation of GnRHR by leptin**

**Figure 2** also shows the pathway that regulates the third target for leptin, GnRHR. This receptor appears to be regulated post-transcriptionally by leptin, because *Gnrhr* mRNA is unchanged when diestrous female or male gonadotrope LEPR-null mutants were compared with control males or diestrous females. Additionally, stimulation of control diestrous female pituitary cultures by leptin increases GnRHR, but not *Gnrhr* mRNA levels [7, 8]. We investigated post-transcriptional mediators of leptin action and determined that a putative miRNA repressor of *Gnrhr* mRNA translation, *miR-581/669d,* was increased in LEPR-null gonadotropes [7]. The most promising regulation, however, came from the translational regulatory protein, Musashi (MSI), as we identified 3 consensus binding elements for Musashi (MBEs) in the 3' UTR of murine *Gnrhr* mRNA [8]. The evolutionarily conserved Musashi family of sequence-specific RNA binding proteins (Musashi1 and Musashi2) have long been known to be expressed in stem and progenitor cell populations, where they act to oppose differentiation and promote stem cell self-renewal [52]. Although originally identified as a repressor of target mRNA translation, Musashi was subsequently shown capable of directing translational activation of target mRNAs in a context-dependent manner [53].

Our studies of leptin stimulation of GnRHR proteins showed a dose response relationship between leptin and expression of GnRHR (detected by enzyme assays) or Biotinylated GnRH binding to living pituitary cells (detected cytochemically) [8] After we confirmed that leptin stimulated GnRHR proteins, but not mRNA, we determined by electrophoretic mobility shift assays that Musashi1 interacted directly with the *Gnrhr* 3' UTR [8] . This pituitary association was confirmed by immunoprecipitation with anti-Musashi antibody and the detection of an enrichment of the endogenous *Gnrhr* mRNA (17-fold over control immunoprecipitates). Moreover, the use of luciferase mRNA reporter assays showed that Musashi1 repressed translation of the *Gnrhr* 3' UTR. Tests of leptin actions on Musashi showed that leptin stimulation caused a reduction in Musashi protein levels in gonadotropes, suggesting that leptin may inhibit Musashi expression [8].

To summarize, our studies of leptin actions on gonadotropes have shown severe functional deficiencies in gonadotropes lacking exon 1 of LEPR. The total absence of the LEPR caused infertility in a subset of females [7]. Collectively, studies of these animal

models point to key gene products that are affected by loss of leptin signals. Leptin may be important in the transcription of *Fshb* mRNA either directly and/or through the transcription of activin. In addition, leptin's actions may serve to regulate the translation of GnRHR protein [7–9]. Our studies suggest that leptin opposes Musashidependent repression of target mRNAs and/or reduces expression of Musashi directly in gonadotropes, leading to enhanced translation of the *Gnrhr* mRNA. This may provide a pathway which permits full expression of GnRHR early in the cycle to reach peak levels in diestrus and proestrus. Estradiol may also stimulate the expression of LEPR, which peaks on proestrus (**Figure 1**). Rising leptin may then partner with estradiol to promote the production of GnRHR (**Figure 2**). We hypothesize that leptin's permissive actions on GnRHR may be to de-repress actions of the translational regulatory protein Musashi and promote full receptivity of the gonadotrope to GnRH [7–9].
