Obesity Alters POMC and Kisspeptin Neuron Cross Talk Leading to Reduced Luteinizing Hormone in Male Mice.

Obesity is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms. Herein, we used mice fed chronic high-fat diet, which mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone (LH), which then regulates testosterone. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. LH pulse frequency is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that the LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with anorexigenic POMC neurons, which are affected by obesity, we examined their cross talk, and determined that the LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. Together, our studies determined that obesity leads to the downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism.

Single-Cell Transcriptomics Identifies Pituitary Gland Changes in Diet-Induced Obesity in Male Mice.

Obesity is a chronic disease with increasing prevalence worldwide. Obesity leads to an increased risk of heart disease, stroke, and diabetes, as well as endocrine alterations, reproductive disorders, changes in basal metabolism, and stress hormone production, all of which are regulated by the pituitary. In this study, we performed single-cell RNA sequencing of pituitary glands from male mice fed control and high-fat diet (HFD) to determine obesity-mediated changes in pituitary cell populations and gene expression. We determined that HFD exposure is associated with dramatic changes in somatotrope and lactotrope populations, by increasing the proportion of somatotropes and decreasing the proportion of lactotropes. Fractions of other hormone-producing cell populations remained unaffected. Gene expression changes demonstrated that in HFD, somatotropes became more metabolically active, with increased expression of genes associated with cellular respiration, and downregulation of genes and pathways associated with cholesterol biosynthesis. Despite a lack of changes in gonadotrope fraction, genes important in the regulation of gonadotropin hormone production were significantly downregulated. Corticotropes and thyrotropes were the least affected in HFD, while melanotropes exhibited reduced proportion. Lastly, we determined that changes in plasticity and gene expression were associated with changes in hormone levels. Serum prolactin was decreased corresponding to reduced lactotrope fraction, while lower luteinizing hormone and follicle-stimulating hormone in the serum corresponded to a decrease in transcription and translation. Taken together, our study highlights diet-mediated changes in pituitary gland populations and gene expression that play a role in altered hormone levels in obesity.

Increased body weight in mice with fragile X messenger ribonucleoprotein 1 (Fmr1) gene mutation is associated with hypothalamic dysfunction.

Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene are linked to Fragile X Syndrome, the most common monogenic cause of intellectual disability and autism. People affected with mutations in FMR1 have higher incidence of obesity, but the mechanisms are largely unknown. In the current study, we determined that male Fmr1 knockout mice (KO, Fmr1-/y), but not female Fmr1-/-, exhibit increased weight when compared to wild-type controls, similarly to humans with FMR1 mutations. No differences in food or water intake were found between groups; however, male Fmr1-/y display lower locomotor activity, especially during their active phase. Moreover, Fmr1-/y have olfactory dysfunction determined by buried food test, although they exhibit increased compulsive behavior, determined by marble burying test. Since olfactory brain regions communicate with hypothalamic regions that regulate food intake, including POMC neurons that also regulate locomotion, we examined POMC neuron innervation and numbers in Fmr1-/y mice. POMC neurons express Fmrp, and POMC neurons in Fmr1-/y have higher inhibitory GABAergic synaptic inputs. Consistent with increased inhibitory innervation, POMC neurons in the Fmr1-/y mice exhibit lower activity, based on cFOS expression. Notably, Fmr1-/y mice have fewer POMC neurons than controls, specifically in the rostral arcuate nucleus, which could contribute to decreased locomotion and increased body weight. These results suggest a role for Fmr1 in the regulation of POMC neuron function and the etiology of Fmr1-linked obesity.

Altered GnRH neuron and ovarian innervation characterize reproductive dysfunction linked to the Fragile X messenger ribonucleoprotein (Fmr1) gene mutation.

Introduction: Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene cause Fragile X Syndrome, the most common monogenic cause of intellectual disability. Mutations of FMR1 are also associated with reproductive disorders, such as early cessation of reproductive function in females. While progress has been made in understanding the mechanisms of mental impairment, the causes of reproductive disorders are not clear. FMR1-associated reproductive disorders were studied exclusively from the endocrine perspective, while the FMR1 role in neurons that control reproduction was not addressed. Results: Here, we demonstrate that similar to women with FMR1 mutations, female Fmr1 null mice stop reproducing early. However, young null females display larger litters, more corpora lutea in the ovaries, increased inhibin, progesterone, testosterone, and gonadotropin hormones in the circulation. Ovariectomy reveals both hypothalamic and ovarian contribution to elevated gonadotropins. Altered mRNA and protein levels of several synaptic molecules in the hypothalamus are identified, indicating reasons for hypothalamic dysregulation. Increased vascularization of corpora lutea, higher sympathetic innervation of growing follicles in the ovaries of Fmr1 nulls, and higher numbers of synaptic GABAA receptors in GnRH neurons, which are excitatory for GnRH neurons, contribute to increased FSH and LH, respectively. Unmodified and ovariectomized Fmr1 nulls have increased LH pulse frequency, suggesting that Fmr1 nulls exhibit hyperactive GnRH neurons, regardless of the ovarian feedback. Conclusion: These results reveal Fmr1 function in the regulation of GnRH neuron secretion, and point to the role of GnRH neurons, in addition to the ovarian innervation, in the etiology of Fmr1-mediated reproductive disorders.

Sexual dimorphism in obesity is governed by RELMα regulation of adipose macrophages and eosinophils.

Obesity incidence is increasing worldwide with the urgent need to identify new therapeutics. Sex differences in immune cell activation drive obesity-mediated pathologies where males are more susceptible to obesity co-morbidities and exacerbated inflammation. Here, we demonstrate that the macrophage-secreted protein RELMα critically protects females against high fat diet-induced obesity. Compared to male mice, RELMα levels were elevated in both control and high fat dietfed females and correlated with adipose macrophages and eosinophils. RELMα-deficient females gained more weight and had pro-inflammatory macrophage accumulation and eosinophil loss, while both RELMα treatment and eosinophil transfer rescued this phenotype. Single cell RNA-sequencing of the adipose stromal vascular fraction was performed and identified sex and RELMα-dependent changes. Genes involved in oxygen sensing and iron homeostasis, including hemoglobin and lncRNA Gm47283, correlated with increased obesity, while eosinophil chemotaxis and response to amyloid-beta were protective. Monocyte-to-macrophage transition was also dysregulated in RELMα-deficient animals. Collectively, these studies implicate a RELMα-macrophage-eosinophil axis in sex-specific protection against obesity and uncover new therapeutic targets for obesity.

Anti-metastatic outcome of isoform-specific prolactin receptor targeting in breast cancer.

Controversy exists concerning the role of the long prolactin receptor (PRLR) in the progression of breast cancer. By targeting pre-mRNA splicing, we succeeded in knocking down only the long PRLR in vivo, leaving the short forms unaffected. Using two orthotopic and highly-metastatic models of breast cancer, one of which was syngeneic (mouse 4T1) to allow assessment of tumor-immune interactions and one of which was endocrinologically humanized (human BT-474) to activate human PRLRs, we examined the effect of long PRLR knockdown on disease progression. In both models, knockdown dramatically inhibited metastatic spread to the lungs and liver and resulted in increased central death in the primary tumor. In the syngeneic model, immune infiltrates in metastatic sites were changed from innate inflammatory cells to lymphocytes, with an increase in the incidence of tumor-specific cytotoxic T cells. Long PRLR knockdown in three-dimensional culture induced apoptosis of tumor-initiating/cancer stem cells (death of 95% of cells displaying stem cell markers in 15 days). We conclude that the long PRLR plays an important role in breast cancer metastasis.