Angela K. Odle

The Somatotrope as a Metabolic Sensor: Deletion of Leptin Receptors Causes Obesity

Leptin, the product of the Lep gene, reports levels of adiposity to the hypothalamus and other regulatory cells, including pituitary somatotropes, which secrete GH. Leptin deficiency is associated with a decline in somatotrope numbers and function, suggesting that leptin may be important in their maintenance. This hypothesis was tested in a new animal model in which exon 17 of the leptin receptor (Lepr) protein was selectively deleted in somatotropes by Cre-loxP technology. Organ genotyping confirmed the recombination of the floxed LepR allele only in the pituitary. Deletion mutant mice showed a 72% reduction in pituitary cells bearing leptin receptor (LEPR)-b, a 43% reduction in LEPR proteins and a 60% reduction in percentages of immunopositive GH cells, which correlated with reduced serum GH. In mutants, LEPR expression by other pituitary cells was like that of normal animals. Leptin stimulated phosphorylated Signal transducer and activator of transcription 3 expression in somatotropes from normal animals but not from mutants. Pituitary weights, cell numbers, IGF-I, and the timing of puberty were not different from control values. Growth curves were normal during the first 3 months. Deletion mutant mice became approximately 30-46% heavier than controls with age, which was attributed to an increase in fat mass. Serum leptin levels were either normal in younger animals or reflected the level of obesity in older animals. The specific ablation of the Lepr exon 17 gene in somatotropes resulted in GH deficiency with a consequential reduction in lipolytic activity normally maintained by GH and increased adiposity.

Selective deletion of leptin receptors in gonadotropes reveals activin and GnRH-binding sites as leptin targets in support of fertility.

The adipokine, leptin (LEP), is a hormonal gateway, signaling energy stores to appetite-regulatory neurons, permitting reproduction when stores are sufficient. Dual-labeling for LEP receptors (LEPRs) and gonadotropins or GH revealed a 2-fold increase in LEPR during proestrus, some of which was seen in LH gonadotropes. We therefore investigated LEPR functions in gonadotropes with Cre-LoxP technology, deleting the signaling domain of the LEPR (Lepr-exon 17) with Cre-recombinase driven by the rat LH-β promoter (Lhβ-cre). Selectivity of the deletion was validated by organ genotyping and lack of LEPR and responses to LEP by mutant gonadotropes. The mutation had no impact on growth, body weight, the timing of puberty, or pregnancy. Mutant females took 36% longer to produce their first litter and had 50% fewer pups/litter. When the broad impact of the loss of gonadotrope LEPR on all pituitary hormones was studied, mutant diestrous females had reduced serum levels of LH (40%), FSH (70%), and GH (54%) and mRNA levels of Fshβ (59%) and inhibin/activin β A and β B (25%). Mutant males had reduced serum levels of GH (74%), TSH (31%), and prolactin (69%) and mRNA levels of Gh (31%), Ghrhr (30%), Fshβ (22%), and glycoprotein α-subunit (Cga) (22%). Serum levels of LEP and ACTH and mRNA levels of Gnrhr were unchanged. However, binding to GnRH receptors was reduced in LEPR-null LH or FSH gonadotropes by 82% or 89%, respectively, in females (P < .0001) and 27% or 53%, respectively, in males (P < .03). This correlated with reductions in GnRH receptor protein immunolabeling, suggesting that LEPs actions may be posttranscriptional. Collectively, these studies highlight the importance of LEP to gonadotropes with GnRH-binding sites and activin as potential targets. LEP may modulate population growth, adjusting the number of offspring to the availability of food supplies.

Ghrelin restoration of function in vitro in somatotropes from male mice lacking the Janus kinase (JAK)-binding site of the leptin receptor.

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptins postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P < .002) and the secretion of GH 1.8-3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7-23%). When added with 1-3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.

Sex Differences in Somatotrope Dependency on Leptin Receptors in Young Mice: Ablation of LEPR Causes Severe Growth Hormone Deficiency and Abdominal Obesity in Males

Leptin receptor (LEPR) signaling controls appetite and energy expenditure. Somatotrope-specific deletion of the LEPRb signaling isoform causes GH deficiency and obesity. The present study selectively ablated Lepr exon 1 in somatotropes, which removes the signal peptide, causing the loss of all isoforms of LEPR. Excision of Lepr exon 1 was restricted to the pituitary, and mutant somatotropes failed to respond to leptin. Young (2-3 mo) males showed a severe 84% reduction in serum GH levels and more than 60% reduction in immunolabeled GH cells compared with 41%-42% reductions in GH and GH cells in mutant females. Mutant males (35 d) and females (45 d) weighed less than controls and males had lower lean body mass. Image analysis of adipose tissue by magnetic resonance imaging showed that young males had a 2-fold increase in abdominal fat mass and increased adipose tissue density. Young females had only an overall increase in adipose tissue. Both males and females showed lower energy expenditure and higher respiratory quotient, indicating preferential carbohydrate burning. Young mutant males slept less and were more restless during the dark phase, whereas the opposite was true of females. The effects of a Cre-bearing sire on his non-Cre-recombinase bearing progeny are seen by increased respiratory quotient and reduced litter sizes. These studies elucidate clear sex differences in the extent to which somatotropes are dependent on all isoforms of LEPR. These results, which were not seen with the ablation of Lepr exon 17, highlight the severe consequences of ablation of LEPR in male somatotropes.

Adipocyte Versus Pituitary Leptin in the Regulation of Pituitary Hormones: Somatotropes Develop Normally in the Absence of Circulating Leptin

Leptin is a cytokine produced by white fat cells, skeletal muscle, the placenta, and the pituitary gland among other tissues. Best known for its role in regulating appetite and energy expenditure, leptin is produced largely by and in proportion to white fat cells. Leptin is also important to the maintenance and function of the GH cells of the pituitary. This was shown when the deletion of leptin receptors on somatotropes caused decreased numbers of GH cells, decreased circulating GH, and adult-onset obesity. To determine the source of leptin most vital to GH cells and other pituitary cell types, we compared two different leptin knockout models with Cre-lox technology. The global Lep-null model is like the ob/ob mouse, whereby only the entire exon 3 is deleted. The selective adipocyte-Lep-null model lacks adipocyte leptin but retains pituitary leptin, allowing us to investigate the pituitary as a potential source of circulating leptin. Male and female mice lacking adipocyte leptin (Adipocyte-lep-null) did not produce any detectable circulating leptin and were infertile, suggesting that the pituitary does not contribute to serum levels. In the presence of only pituitary leptin, however, these same mutants were able to maintain somatotrope numbers and GH mRNA levels. Serum GH trended low, but values were not significant. However, hypothalamic GHRH mRNA was significantly reduced in these animals. Other serum hormone and pituitary mRNA differences were observed, some of which varied from previous results reported in ob/ob animals. Whereas pituitary leptin is capable of maintaining somatotrope numbers and GH mRNA production, the decreased hypothalamic GHRH mRNA and low (but not significant) serum GH levels indicate an important role for adipocyte leptin in the regulation of GH secretion in the mouse. Thus, normal GH secretion may require the coordinated actions of both adipocyte and pituitary leptin.

Arousal from slices to humans

Most psychiatric and neurological disorders exhibit sleep disorders, and in some cases presage the disease. Study of the control of sleep and waking has the potential for making a major impact on a number of disorders, making translational neuroscience research on this area critical. One element of the reticular activating system (RAS) is the pedunculopontine nucleus (PPN), which is the cholinergic arm of the RAS, and projects to the thalamus to trigger thalamocortical rhythms and to the brainstem to modulate muscle tone and locomotion. We developed a research program using brainstem slices containing the PPN to tell us about the cellular and molecular organization of this region. In addition, we developed the P13 midlatency auditory evoked potential, which is generated by PPN outputs, preparation in freely moving rats. This allows the study of PPN cellular and molecular mechanisms at the level of the whole animal. We also study the P50 midlatency auditory evoked potential, which is the human equivalent of the rodent P13 potential, allowing us to study processes detected in vitro, confirmed in the whole animal, and tested in humans. This translational research program led to the discovery of a novel mechanism of sleep-wake control, pointing the way to a number of new clinical applications in the development of novel stimulants and anesthetics.

Adipocyte Versus Somatotrope Leptin: Regulation of Metabolic Functions in the Mouse.

Leptin regulates food intake and energy expenditure (EE) and is produced in adipocytes, the pituitary, and several other tissues. Animals that are leptin or leptin receptor deficient have major metabolic complications, including obesity. This study tests the hypothesis that the pituitary somatotrope may contribute a source of leptin that maintains some of these metabolic functions. We created 2 different tissue-specific leptin knockout animals: a Somatotrope-Lep-null model and an Adipocyte-Lep-null model. Metabolic analysis of both models, along with a global deletion model, was performed. The Somatotrope-Lep-null animals had fewer somatotropes, and females had a 76% decrease in serum prolactin. During the dark (feeding) phase, females had a 35% increase in ambulation coupled with a 4% increase in EE. Mutants showed no change in food intake or weight gain and EE was unchanged in males. During the light (sleep) phase, Somatotrope-Lep-null mutant males had lower EE and females continued to have higher EE. The respiratory quotients (RQs) of mutants and littermate controls were decreased in males and increased in females; all were within the range that indicates predominant carbohydrate burning. The massively obese Adipocyte-Lep-null animals, however, had significant increases in food intake, sleep, and increased EE, with decreased activity. Changes in RQ were sexually dimorphic, with female mutants having higher RQ and males having decreased RQ. We conclude that both adipocyte and somatotrope leptin contribute to the metabolic homeostasis of the mouse, and that extraadipocyte sources of leptin cannot overcome the major metabolic challenges seen in these animals.

Giant Mice Reveal New Roles for GH in Regulating the Adipose Immune Microenvironment

The financial, social, and physical burdens of obesity have been widely studied and cited as obesity has quickly become one of the fastest growing health epidemics in America. Worldwide obesity has more than doubled since 1980. In 2014, more than a third (39%) of adults were overweight and 13% were obese, involving more than 1.9 billion adults. (http://www.who.int/mediacentre/factsheets/fs311/en/). There is growing understanding that the negative impact of the obese condition comes from the total remodeling of the microenvironment of adipose tissue, which includes adipocytes themselves and the immune cell population. In addition, we are learning that not all fat depots have the same negative impact on the body. Historically, the existence of fat as a distinct glandular organ was actively debated at the turn of the 20th century. A review by H. Batty Shaw in 1901 discussed the various studies in which scientists were trying to understand the origin of fat cells, whether fat cells existed as distinct entities or were part of a large organ, and the fluctuations in these cells during various nutritional states (1). Adipose tissue has since been well established as a complex glandular tissue existing in distinct depots in human and animal bodies. White adipose tissue (WAT) in particular, which consists of adipocytes as well as a stromal vascular fraction (SVF), is a particularly active tissue, participating in not only metabolic regulation but also immune function and glucose regulation, to name a few [see review by Trayhurn and Beattie, 2001 (2)]. Because of the large number of factors produced by adipocytes, the influence of this organ is incredibly widespread and varied, and these primary and secondary functions are particularly well studied in disease states. In 1947, French physician Jean Vague (3) described distinct patterns of obesity and their indications for disease. He found that the masculinization of obesity (so-called android obesity of the top half of the body) was associated with a much higher incidence of diabetes and atherosclerosis, when compared with the gynoid and hypergynoid (lower trunk obesity) obese states. This association of increased abdominal fat with cardiovascular disease and metabolic syndrome has been confirmed by several groups (4,–9). The SVF is composed of preadipocytes, mesenchymal stem cells, endothelial progenitor cells, and various immune cells (macrophages, T cells, B cells, mast cells, eosinophils, and neutrophils). The makeup of the SVF changes with the nutritional status (10,–14). Obesity, which increases adipokine secretion by both adipocytes and the SVF, manifests as a low-grade inflammatory state in WAT (16). The obese condition leads to recruitment of immune cells, changes in cytokine secretion, increased angiogenesis, and a shifting of macrophages from the antiinflammatory M2 state to the inflammatory M1 state (11, 15,–20) [for a comparison of immune changes in WAT in lean/obese/diabetic states, see the 2015 review by Ip et al (21)]. One hormone in particular that is involved in both immune and adipose regulation is growth hormone (GH). GH, produced and released in a pulsatile fashion by the anterior pituitary, is known to have receptors on adipocytes, preadipocytes, and the immune cells of the SVF (22,–25). GH is known to be lipolytic and promotes the proliferation of preadipocytes (26, 27). GH receptor expression differs across depots, with the highest expression of human GH receptors found in epidydimal fat (28), and the expression of the human GH receptor is regulated by GH itself (29). Our laboratory has previously shown that mice lacking the leptin signal in somatotropes have decreased GH secretion, decreased somatotrope cell numbers, and increased body weight (30). Blunted in obesity, GH is known to be stimulated by leptin, and thus, the regulation of GH and the regulation of adipocytes are intertwined (31,–35). Our mice lacking leptin receptors on somatotropes had higher serum adiponectin and resistin levels, indicating that the GH deficiency seen in this model was affecting the release of adipokines in the preobese state (36). Mice that are GH deficient have significantly increased fat mass beginning early in life, an accumulation that is independent of a change in energy intake (37). Young individuals with GH deficiency have large adipocytes, and adipose samples from obese GH-deficient individuals have increased proinflammatory markers (38). In this issue of Endocrinology, Benencia et al (39) elegantly describe the immune changes that occur in the distinct depots of WAT in a state of GH excess. At the center of the study is a well-characterized mouse model of acromegaly [the bovine GH mouse (bGH)], which exhibits a major decrease in white adipose tissue in all depots. These mice also have a shortened life span, altered immune system, alterations in circulating cytokines, and (characteristic of the acromegalic state) increased and accelerated growth. The authors hypothesize that the GH signal is imperative to the maintenance and/or activation of the immune cells of WAT. Using flow cytometry, the authors isolated the various immune cells of the SVF from different fat depots. They found that the bGH mice had differences in the numbers and types of cells in the SVF across depots and compared with wild-type mice. For example, the authors found that the bGH mice had a greater number of SVF cells in subcutaneous and mesenteric fat depots relative to these depots in wild-type mice. In addition, they observed that SVF cell number and SVF cells per gram of tissue was higher in sc and mesenteric WAT depots than epididymal depots in bGF mice. Macrophages were also demonstrated to represent a higher percentage of the SVF in bGH sc and mesenteric depots relative to wild-type controls, and these macrophages more commonly exhibited an anti-inflammatory or M2 phenotype. T helper cells were also more abundant in the sc WAT of bGH mice relative to controls. In the future, it will be interesting to determine whether these T helper cells exhibit a more proinflammatory phenotype characteristic of Th1 and Th17 cells or an anti-inflammatory phenotype characteristic of Th2 cells. The authors further demonstrated that T-regulatory cells, which are important in resolving inflammation, were elevated in the sc and mesenteric WAT depots of bGH mice relative to controls. Additionally, the use of RNA sequencing revealed a fascinating change with regard to the expression of molecules that play significant roles in immune cell biology and migration in sc fat from bGH mice, which was distinct from the expression pattern observed in epididymal fat depots. Curiously, the anti-inflammatory properties exhibited by the WAT of these mice do not contribute to longevity because these mice die young. This ground-breaking study finds that GH is able to modulate the following: 1) depot-specific changes in the proportion of SVF cells in WAT, 2) changes in the immune cell types that are recruited to the different fat depots, and 3) GH signaling in WAT that promotes an immune cell preference for the anti-inflammatory phenotype. This study will no doubt be the first in a series of investigations that will outline the differential role of GH in different fat depots.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence

The adipokine leptin signals the body’s nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin’s regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic–pituitary–gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body’s ability to delay or slow reproduction during periods of low nutrition.