The physiological role of leptin is thought to be a driving force to reduce food intake and increase energy expenditure. However, leptin therapies in the clinic have failed to effectively treat obesity, predominantly due to a phenomenon referred to as leptin resistance. The mechanisms linking obesity and the associated leptin resistance remain largely unclear. With various mouse models and a leptin neutralizing antibody, we demonstrated that hyperleptinemia is a driving force for metabolic disorders. A partial reduction of plasma leptin levels in the context of obesity restores hypothalamic leptin sensitivity and effectively reduces weight gain and enhances insulin sensitivity. These results highlight that a partial reduction in plasma leptin levels leads to improved leptin sensitivity, while pointing to a new avenue for therapeutic interventions in the treatment of obesity and its associated comorbidities.

The perforated‐patch‐clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration. I K(IR) was blocked in a voltage‐dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4‐aminopyridine (15% inhibition) and insensitive to 10 mM tetra‐ethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 μM La3+. In physiological saline, 100 μM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 ± 0.7 mV from a mean resting potential of −59.6 ± 0.8 mV. Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 ± 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects. The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately −50 mV revealed that spike frequency in the presence of CRH (109 ± 7 spikes (5 min)−1) was greater than in control (60 ± 5 spikes (5 min)−1) or Ba2+‐treated (77 ± 15 spikes (5 min)−1) corticotropes. The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.

A healthy nutritional state is required for all aspects of reproduction and signaled by the adipokine leptin. Leptin acts in a relatively narrow concentration range; too much or too little will compromise fertility. The leptin signal timing is important to prepubertal development in both sexes. In the brain, leptin acts on ventral premammillary (PMV) neurons which signal kisspeptin (Kiss1) neurons to stimulate gonadotropin releasing hormone neurons (GnRH). Suppression of Kiss1 neurons occurs when AgRP neurons are activated by reduced leptin, because leptin normally suppresses these orexigenic neurons. In the pituitary, leptin stimulates production of GnRH receptors (GnRHR) and follicle-stimulating hormone (FSH) at midcycle, by activating pathways that de-repress actions of the mRNA translational regulatory protein Musashi. In females, rising estrogen stimulate a rise in serum leptin, which peaks at midcycle, synchronizing with nocturnal Luteinizing hormone (LH) pulses. The normal range of serum leptin levels (10-20 ng/ml) along with gonadotropins and growth factors, promote ovarian granulosa and theca cell functions and oocyte maturation. In males, the prepubertal rise in leptin promotes testicular development. However, a decline in leptin levels in prepubertal boys reflects inhibition of leptin secretion by rising androgens. In adult males, leptin levels are 10-50% of those in females, and high leptin inhibits testicular function. The obesity epidemic has elucidated leptin resistance pathways with too much leptin in either sex leading to infertility. Under conditions of balanced nutrition however, the secretion of leptin is timed and regulated within a narrow level range that optimizes its trophic effects.

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.

Leptin, the ob protein, regulates food intake and satiety and can be found in the anterior pituitary. Leptin antigens and mRNA were studied in the anterior pituitary (AP) cells of male and female rats to learn more about its regulation. Leptin antigens were found in over 40% of cells in diestrous or proestrous female rats and in male rats. Lower percentages of AP cells were seen in the estrous population (21 +/- 7%). During peak expression of antigens, co-expression of leptin and growth hormone (GH) was found in 27 +/- 4% of AP cells. Affinity cytochemistry studies detected 24 +/- 3% of AP cells with leptin proteins and growth hormone releasing hormone (GHRH) receptors. These data suggested that somatotropes were a significant source of leptin. To test regulatory factors, estrous and diestrous AP populations were treated with estrogen (100 pM) and/or GHRH (2 nM) to learn if either would increase leptin expression in GH cells. To rule out the possibility that the immunoreactive leptin was bound to receptors in somatotropes, leptin mRNA was also detected by non-radioactive in situ hybridization in this group of cells. In estrous female rats, 39 +/- 0.9% of AP cells expressed leptin mRNA, indicating that the potential for leptin production was greater than predicted from the immunolabeling. Estrogen and GHRH together (but not alone) increased percentages of cells with leptin protein (41 +/- 9%) or mRNA (57 +/- 5%). Estrogen and GHRH also increased the percentages of AP cells that co-express leptin mRNA and GH antigens from 20 +/- 2% of AP cells to 37 +/- 5%. Although the significance of leptin in GH cells is not understood, it is clearly increased after stimulation with GHRH and estrogen. Because GH cells also have leptin receptors, this AP leptin may be an autocrine or paracrine regulator of pituitary cell function.

No abstract

There is a 2-to 3-fold increase in luteinizing hormonebeta (LH ) or follicle-stimulating hormone-beta (FSH ) antigen-bearing gonadotropes during diestrus in preparation for the peak LH or FSH secretory activity. This coincides with an increase in cells bearing LH or FSH mRNA. Similarly, there is a 3-to 4-fold increase in the percentage of cells that bind GnRH. In 1994, we reported that this augmentation in gonadotropes may come partially from subsets of somatotropes that transitionally express LH or FSH mRNA and GnRH-binding sites. The next phase of the study focused on questions relating to the somatotropes themselves. Do these putative somatogonadotropes retain a somatotrope phenotype? As a part of ongoing studies that address this question, a biotinylated analog of GHRH was produced, separated by HPLC and characterized for its ability to elicit the release of GH as well as bind to pituitary target cells. The biotinylated analog (Bio-GHRH) was detected cytochemically by the avidin-peroxidase complex technique. It could be displaced by competition with 100-1000 nM GHRH but not corticotropin-releasing hormone or GnRH. In cells from male rats exposed to 1 nM Bio-GHRH, 28 6% (mean .) of pituitary cells exhibited label for Bio-GHRH (compared with 0·8 0·6% in the controls). There were no differences in percentages of GHRH target cells in populations from proestrous (28 5%) and estrous (25 5%) rats. Maximal percentages of labeled cells were seen following addition of 1 nM analog for 10 min. In dual-labeled fields, GHRH target cells contained all major pituitary hormones, but their expression of ACTH and TRH was very low (less than 3% of the pituitary cell population) and the expression of prolactin (PRL) and gonadotropins varied with the sex and stage of the animal. In all experimental groups, 78-80% of Bio-GHRHreactive cells contained GH (80-91% of GH cells). In male rats, 33 6% of GHRH target cells contained PRL (37 9% of PRL cells) and less than 20% of these GHRH-receptive cells contained gonadotropins (23 1% of LH and 31 9% of FSH cells). In contrast, expression of PRL and gonadotropins was found in over half of the GHRH target cells from proestrous female rats (55 10% contained PRL; 56 8% contained FSH ; and 66 1% contained LH ). This reflected GHRH binding by 71 2% PRL cells, 85 5% of LH cells and 83 9% of FSH cells. In estrous female rats, the hormonal storage patterns in GHRH target cells were similar to those in the male rat. Because the overall percentages of cells with Bio-GHRH or GH label do not vary among the three groups, the differences seen in the proestrous group reflect internal changes within a single group of somatotropes that retain their GHRH receptor phenotype. Hence, these data correlate with earlier findings that showed that somatotropes may be converted to transitional gonadotropes just before proestrus secretory activity. The LH and FSH antigen content of the GHRH target cells from proestrous rats demonstrates that the LH and FSH mRNAs are indeed translated. Furthermore, the incre...

Pituitary cells with GnRH receptors increase over 2-fold during diestrus to reach a peak during the morning of proestrus. This is followed by a rapid fall during the afternoon of proestrus to reach a nadir by estrus. The objective of this study was to learn the identity of the new target cells added during diestrus. This was particularly important in view of recent evidence showing that gonadotropes with LH beta and FSH beta mRNA have GH antigens. Pituitary cells from diestrous and proestrous rats were exposed to biotinylated GnRH (Bio-GnRH) for 10 min. Bio-GnRH was detected by avidin peroxidase, and then the cells were immunolabeled for pituitary hormones. The percentages of cells labeled for Bio-GnRH rose during diestrus from 6.6 +/- 0.8% in the morning to 11.9 +/- 0.7% by evening (mean +/- SD). By the morning of proestrus, the percentages of Bio-GnRH target cells increased further to 16 +/- 0.7%. The percentages of pituitary cells dual labeled for LH beta antigens and Bio-GnRH rose from 4.3 +/- 0.6% to 9% +/- 1% during diestrus and averaged 13 +/- 0.7% by the morning of proestrus. At this time, 90% of cells with LH antigens bound Bio-GnRH. When percentages of pituitary cells with FSH beta antigens and Bio-GnRH-binding sites were analyzed, there was an increase during diestrus from 4 +/- 0.4% to 9.7 +/- 0.7%; a peak level of 14 +/- 0.9% was reached by the morning of proestrus. Bio-GnRH binding was expressed by 86% of FSH cells during this peak. Finally, GH antigens were also detected in GnRH target cells. The percentage of cells dual labeled for Bio-GnRH and GH increased from 4 +/- 0.8% to 8 +/- 1% during diestrus and the morning of proestrus. During the diestrous and proestrous peak periods of expression, Bio-GnRH binding was seen in 32% of GH cells. None of the other pituitary cell types showed significant GnRH binding. These studies showed that most of the new GnRH-receptive cells stem from maturing gonadotropes. Half of the GnRH-receptive cells also contain GH antigens, which correlated with results from previous studies that showed GH antigens in cells with gonadotropin mRNAs. This might reflect expression of gonadotrope functions by a subset of GH cells. Alternatively, the GH antigens may be bound to GH receptors in gonadotropes. This latter possibility may signify a paracrine regulation of gonadotrope function by GH.