Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification.

Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol. For complete details on the use and execution of this protocol, please refer to Han et al. (2020).

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Tyrosine Hydroxylase Neurons Regulate Growth Hormone Secretion via Short-Loop Negative Feedback.

Classical studies suggest that growth hormone (GH) secretion is controlled by negative-feedback loops mediated by GH-releasing hormone (GHRH)- or somatostatin-expressing neurons. Catecholamines are known to alter GH secretion and neurons expressing TH are located in several brain areas containing GH-responsive cells. However, whether TH-expressing neurons are required to regulate GH secretion via negative-feedback mechanisms is unknown. In the present study, we showed that between 50% and 90% of TH-expressing neurons in the periventricular, paraventricular, and arcuate hypothalamic nuclei and locus ceruleus of mice exhibited STAT5 phosphorylation (pSTAT5) after an acute GH injection. Ablation of GH receptor (GHR) from TH cells or in the entire brain markedly increased GH pulse secretion and body growth in both male and female mice. In contrast, GHR ablation in cells that express the dopamine transporter (DAT) or dopamine ß-hydroxylase (DBH; marker of noradrenergic/adrenergic cells) did not affect body growth. Nevertheless, less than 50% of TH-expressing neurons in the hypothalamus were found to express DAT. Ablation of GHR in TH cells increased the hypothalamic expression of Ghrh mRNA, although very few GHRH neurons were found to coexpress TH- and GH-induced pSTAT5. In summary, TH neurons that do not express DAT or DBH are required for the autoregulation of GH secretion via a negative-feedback loop. Our findings revealed a critical and previously unidentified group of catecholaminergic interneurons that are apt to sense changes in GH levels and regulate the somatotropic axis in mice.SIGNIFICANCE STATEMENT Textbooks indicate until now that the pulsatile pattern of growth hormone (GH) secretion is primarily controlled by GH-releasing hormone and somatostatin neurons. The regulation of GH secretion relies on the ability of these cells to sense changes in circulating GH levels to adjust pituitary GH secretion within a narrow physiological range. However, our study identifies a specific population of tyrosine hydroxylase-expressing neurons that is critical to autoregulate GH secretion via a negative-feedback loop. The lack of this mechanism in transgenic mice results in aberrant GH secretion and body growth. Since GH plays a key role in cell proliferation, body growth, and metabolism, our findings provide a major advance to understand how the brain regulates the somatotropic axis.

Insulin signaling in LepR cells modulates fat and glucose homeostasis independent of leptin.

Hypothalamic neurons detect changes in circulating hormones such as leptin and insulin and put forward outputs to sustain energy and glucose homeostasis. Because leptin and insulin receptors colocalize in ~40-60% of neurons in the hypothalamus, we characterized the metabolic phenotype of mice with selective deletion of the insulin receptor (InsR) in LepR cells. LRΔInsR mice presented no difference in body weight and insulin levels but increased fat mass. In the light phase, LRΔInsR mice exhibited increased food intake, locomotor activity, carbon dioxide production, and respiratory exchange rate. These mice showed reduced fat oxidation and reduced expression of cluster of differentiation 36 and AMP-activated protein kinase-α1 in the liver, increased glucose oxidation in the light phase, and overall reduced basal glucose levels. To verify the impact of InsR deletion in LepR cells in obesity, we generated ob/ ob InsRfl, ob/ ob LRcre, and ob/ ob LRΔInsR mice. The ob/ ob LRΔInsR mice had higher body weight, fat mass, and expression of genes related to fat metabolism in the liver. No difference in food intake despite increased neuropeptide Y and agouti-related peptide expression, and no difference in energy expenditure, fat, or glucose oxidation was found in ob/ ob LRΔInsR compared with LRcre or LRΔInsR controls. Remarkably, basal glucose levels were reduced, and the expression of genes associated with glucose metabolism in the liver was higher. Insulin signaling in LepR cells is required for the proper fat and glucose oxidation. These effects are independent of leptin given that the leptin-deficient ob/ ob LRΔInsR mice also presented reduced glycemia and higher adiposity. The mechanisms underlying these responses remain to be unveiled.

Obesity and High-Fat Diet Induce Distinct Changes in Placental Gene Expression and Pregnancy Outcome.

Obese women are at high risk of pregnancy complications, including preeclampsia, miscarriage, preterm birth, stillbirth, and neonatal death. In the current study, we aimed to determine the effects of obesity on pregnancy outcome and placental gene expression in preclinical mouse models of genetic and nutritional obesity. The leptin receptor (LepR) null-reactivatable (LepRloxTB), LepR-deficient (Leprdb/+), and high-fat diet (HFD)-fed mice were assessed for fertility, pregnancy outcome, placental morphology, and placental transcriptome using standard quantitative polymerase chain reaction (qPCR) and qPCR arrays. The restoration of fertility of LepRloxTB was performed by stereotaxic delivery of adeno-associated virus-Cre into the hypothalamic ventral premammillary nucleus. Fertile LepRloxTB females were morbidly obese, whereas the wild-type mice-fed HFD showed only a mild increase in body weight. Approximately 80% of the LepRloxTB females had embryo resorptions (∼40% of the embryos). In HFD mice, the number of resorptions was not different from controls fed a regular diet. Placentas of resorbed embryos from obese mice displayed necrosis and inflammatory infiltrate in the labyrinth and changes in the expression of genes associated with angiogenesis and inflammation (e.g., Vegfa, Hif1a, Nfkbia, Tlr3, Tlr4). In contrast, placentas from embryos of females on HFD showed changes in a different set of genes, mostly associated with cellular growth and response to stress (e.g., Plg, Ang, Igf1, Igfbp1, Fgf2, Tgfb2, Serpinf1). Sexual dimorphism in gene expression was only apparent in placentas from obese LepRloxTB mice. Our findings indicate that an obese environment and HFD have distinct effects on pregnancy outcome and the placental transcriptome.

Hypothalamic growth hormone receptor (GHR) controls hepatic glucose production in nutrient-sensing leptin receptor (LepRb) expressing neurons.

OBJECTIVE: The GH/IGF-1 axis has important roles in growth and metabolism. GH and GH receptor (GHR) are active in the central nervous system (CNS) and are crucial in regulating several aspects of metabolism. In the hypothalamus, there is a high abundance of GH-responsive cells, but the role of GH signaling in hypothalamic neurons is unknown. Previous work has demonstrated that the Ghr gene is highly expressed in LepRb neurons. Given that leptin is a key regulator of energy balance by acting on leptin receptor (LepRb)-expressing neurons, we tested the hypothesis that LepRb neurons represent an important site for GHR signaling to control body homeostasis. METHODS: To determine the importance of GHR signaling in LepRb neurons, we utilized Cre/loxP technology to ablate GHR expression in LepRb neurons (LeprEYFPΔGHR). The mice were generated by crossing the Leprcre on the cre-inducible ROSA26-EYFP mice to GHRL/L mice. Parameters of body composition and glucose homeostasis were evaluated. RESULTS: Our results demonstrate that the sites with GHR and LepRb co-expression include ARH, DMH, and LHA neurons. Leptin action was not altered in LeprEYFPΔGHR mice; however, GH-induced pStat5-IR in LepRb neurons was significantly reduced in these mice. Serum IGF-1 and GH levels were unaltered, and we found no evidence that GHR signaling regulates food intake and body weight in LepRb neurons. In contrast, diminished GHR signaling in LepRb neurons impaired hepatic insulin sensitivity and peripheral lipid metabolism. This was paralleled with a failure to suppress expression of the gluconeogenic genes and impaired hepatic insulin signaling in LeprEYFPΔGHR mice. CONCLUSION: These findings suggest the existence of GHR-leptin neurocircuitry that plays an important role in the GHR-mediated regulation of glucose metabolism irrespective of feeding.

AMPKα2 in Kiss1 Neurons Is Required for Reproductive Adaptations to Acute Metabolic Challenges in Adult Female Mice.

A temporary and reversible inhibition of the hypothalamo-pituitary-gonadal axis is adaptive when energy reserves are diminished, allowing individual survival and energy accumulation for eventual reproduction. The AMP-activated protein kinase (AMPK) works as a cellular sensor of the AMP to ATP ratio and ultimately of energy availability. Activation of AMPK suppresses ATP-consuming processes and stimulates ATP-producing pathways. The AMPK α2 catalytic subunit is expressed in multiple hypothalamic nuclei including those associated with reproductive control, ie, the anteroventral periventricular nucleus and the arcuate nucleus. Subsets of kisspeptin neurons in the anteroventral periventricular nucleus (20% in females) and arcuate nucleus (45% in males and 65% in females) coexpress AMPKα2 mRNA. Using the Cre-loxP approach, we assessed whether AMPKα2 in Kiss1 cells is required for body weight and reproductive function. The AMPKα2-deleted mice show no difference in body weight and time for sexual maturation compared with controls. Males and females are fertile and have normal litter size. The AMPKα2-deleted and control females have similar estradiol feedback responses and show no difference in Kiss1 mRNA expression after ovariectomy or ovariectomy plus estradiol replacement. In males, acute fasting decreased Kiss1 mRNA expression in both groups, but no effect was observed in females. However, after an acute fasting, control mice displayed prolonged diestrous phase, but AMPKα2-deleted females showed no disruption of estrous cycles. Our findings demonstrate that the AMPKα2 catalytic subunit in Kiss1 cells is dispensable for body weight and reproductive function in mice but is necessary for the reproductive adaptations to conditions of acute metabolic distress.

Hypothalamic action of phoenixin to control reproductive hormone secretion in females: importance of the orphan G protein-coupled receptor Gpr173.

Sexual maturation and maintenance of reproductive function are regulated by neurohormonal communication between the hypothalamus, pituitary, and gonads (referred to as the HPG axis). Phoenixin (PNX) is a newly identified, endogenous peptide abundantly produced in the hypothalamus and shown to be an important mediator of ovarian cyclicity. However, the underlying mechanisms by which phoenixin functions within the HPG axis are unknown. Previous in vitro studies demonstrated a direct action of PNX on gonadotrophs to potentiate gonadotrophin-releasing hormone (GnRH) induced luteinizing hormone (LH) secretion. Therefore, we hypothesized that centrally derived phoenixin regulates the preovulatory LH surge required for ovarian cyclicity. We observed a significant dose-related increase in the level of plasma LH in diestrous, female rats that were given an intracerebroventricular injection of PNX compared with vehicle-treated controls. While this suggests that even under low-estrogen conditions, PNX acts centrally to stimulate the HPG axis, further characterization is contingent on the elucidation of its cognate receptor. Using the "deductive ligand receptor matching strategy," we identified the orphan G protein-coupled receptor, Gpr173, as our top candidate. In cultured pituitary cells, siRNA-targeted compromise of Gpr173 abrogated PNX's action to potentiate GnRH-stimulated LH secretion. In addition, siRNA-mediated knockdown of endogenous Gpr173, which localized to several hypothalamic sites related to reproductive function, not only significantly extended the estrous cycle but also prevented the PNX-induced LH secretion in diestrous, female rats. These studies are the first to demonstrate a functional relationship between PNX and Gpr173 in reproductive physiology and identify a potential therapeutic target for ovulatory dysfunction.

Obesity-induced hypogonadism in the male: premature reproductive neuroendocrine senescence and contribution of Kiss1-mediated mechanisms.

Reproduction is sensitive to insufficient body energy reserves, especially in females. Metabolic regulation of the male reproductive axis is less obvious, and the impact of conditions of persistent energy excess has received moderate attention. Yet, the escalating prevalence of obesity and the clinical evidence of its deleterious effects on male fertility have raised considerable concerns. We report here phenotypic and mechanistic studies of the reproductive impact of postnatal nutritional manipulations (mainly overnutrition) coupled to a high-fat diet (HFD) after weaning. Metabolic and hormonal analyses in young (4 months old) and middle-aged (10 months old) animals revealed that HFD caused profound metabolic perturbations, including glucose intolerance, which were worsened by precedent postnatal overfeeding; these were detectable already in young males but aggravated in 10-month-old rats. Impairment of reproductive parameters took place progressively, and HFD alone was sufficient to explain most of these alterations, regardless of postnatal under- or overnutrition. In young males, testosterone (T) levels and steroidogenic enzyme expression were suppressed by HFD, without compensatory increases of LH levels, which were in fact partially inhibited in heavier males. In addition, obese males displayed suppressed hypothalamic Kiss1 expression despite low T, and HFD inhibited LH responses to kisspeptin. Overweight anticipated some of the neuroendocrine effects of aging, such as the suppression of hypothalamic Kiss1 expression and the decline in serum T and LH levels. Nonetheless, HFD per se caused a detectable worsening of key reproductive indices in middle-aged males, such as basal LH and FSH levels as well as LH responses to kisspeptin. Our study demonstrates that nutritional stress, especially HFD, has a profound deleterious impact on metabolic and gonadotropic function as well as on the Kiss1 system and precipitates neuroendocrine reproductive senescence in the male.

Early metabolic programming of puberty onset: impact of changes in postnatal feeding and rearing conditions on the timing of puberty and development of the hypothalamic kisspeptin system.

Kiss1 neurons have recently emerged as a putative conduit for the metabolic gating of reproduction, with leptin being a regulator of hypothalamic Kiss1 expression. Early perturbations of the nutritional status are known to predispose to different metabolic disorders later in life and to alter the timing of puberty; however, the potential underlying mechanisms remain poorly defined. Here we report how changes in the pattern of postnatal feeding affect the onset of puberty and evaluate key hormonal and neuropeptide [Kiss1/kisspeptin (Kp)] alterations linked to these early nutritional manipulations. Female rats were raised in litters of different sizes: small (four pups per dam: overfeeding), normal (12 pups per dam), and large litters (20 pups per litter: underfeeding). Postnatal overfeeding resulted in persistently increased body weight and earlier age of vaginal opening, as an external sign of puberty, together with higher levels of leptin and hypothalamic Kiss1 mRNA. Conversely, postnatal underfeeding caused a persistent reduction in body weight, lower ovarian and uterus weights, and delayed vaginal opening, changes that were paralleled by a decrease in leptin and Kiss1 mRNA levels. Kisspeptin-52 immunoreactivity (Kp-IR) in the hypothalamus displayed similar patterns, with lower numbers of Kp-IR neurons in the arcuate nucleus of postnatally underfed animals, and a trend for increased Kp-positive fibers in the periventricular area of early overfed rats. Yet, gonadotropin responses to Kp at puberty were similar in all groups, except for enhanced responsiveness to low doses of Kp-10 in postnatally underfed rats. In conclusion, our data document that the timing of puberty is sensitive to both overfeeding and subnutrition during early (postnatal) periods and suggest that alterations in hypothalamic expression of Kiss1/kisspeptin may underlie at least part of such programming phenomenon.

The anorexigenic neuropeptide, nesfatin-1, is indispensable for normal puberty onset in the female rat.

The hypothalamic peptide, nesfatin-1, derived from the precursor NEFA/nucleobindin 2 (NUCB2), was recently identified as anorexigenic signal, acting in a leptin-independent manner. Yet its participation in the regulation of other biological functions gated by body energy status remains unexplored. We show herein that NUCB2/nesfatin-1 is involved in the control of female puberty. NUCB2/nesfatin mRNA and protein were detected at the hypothalamus of pubertal female rats, with prominent signals at lateral hypothalamus (LHA), paraventricular (PVN), and supraoptic (SON) nuclei. Hypothalamic NUCB2 expression raised along pubertal transition, with detectable elevations of its mRNA levels at LHA, PVN, and SON, and threefold increase of its total protein content between late-infantile and peripubertal periods. Conditions of negative energy balance, such as 48 h fasting or sustained subnutrition, decreased hypothalamic NUCB2 mRNA and/or protein levels in pubertal females. At this age, central administration of nesfatin-1 induced modest but significant elevations of circulating gonadotropins, whose magnitude was notably augmented in conditions of food deprivation. Continuous intracerebroventricular infusion of antisense morpholino oligonucleotides (as-MONs) against NUCB2 along pubertal maturation, which markedly reduced hypothalamic NUCB2 protein content, delayed vaginal opening and decreased ovarian weights and serum luteinizing hormone (LH) levels. In contrast, in adult female rats, intracerebroventricular injection of nesfatin did not stimulate LH or follicle-stimulating hormone secretion; neither did central as-MON infusion alter preovulatory gonadotropin surges, despite suppression of hypothalamic NUCB2. In sum, our data are the first to disclose the indispensable role of NUCB2/nesfatin-1 in the central networks driving puberty onset, a function that may contribute to its functional coupling to energy homeostasis.