Metabolic control of puberty: 60 years in the footsteps of Kennedy and Mitra's seminal work.

An individual's nutritional status has a powerful effect on sexual maturation. Puberty onset is delayed in response to chronic energy insufficiency and is advanced under energy abundance. The consequences of altered pubertal timing for human health are profound. Late puberty increases the chances of cardiometabolic, musculoskeletal and neurocognitive disorders, whereas early puberty is associated with increased risks of adult obesity, type 2 diabetes mellitus, cardiovascular diseases and various cancers, such as breast, endometrial and prostate cancer. Kennedy and Mitra's trailblazing studies, published in 1963 and using experimental models, were the first to demonstrate that nutrition is a key factor in puberty onset. Building on this work, the field has advanced substantially in the past decade, which is largely due to the impressive development of molecular tools for experimentation and population genetics. In this Review, we discuss the latest advances in basic and translational sciences underlying the nutritional and metabolic control of pubertal development, with a focus on perspectives and future directions.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determine the role of glutamatergic signaling from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces LH release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LepRb-Cre mice. We collected blood sequentially before and for 1h after iv. clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of cFos immunoreactive neurons in the PMv. Next, females with deletion of Vglut2 in LepRb neurons (LepR∆VGlut2) showed delayed age of puberty, disrupted estrous cycles, increased GnRH concentration in the axon terminals and disrupted LH responses, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LepRloxTB) with concomitant deletion of Vglut2 (Vglut2-floxed) mice. Rescue of Lepr and deletion of Vglut2 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LepRloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation and became pregnant, while LepRloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic signaling from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions. This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Deletion of Androgen Receptor in LepRb Cells Improves Estrous Cycles in Prenatally Androgenized Mice.

Androgens are steroid hormones crucial for sexual differentiation of the brain and reproductive function. In excess, however, androgens may decrease fertility as observed in polycystic ovary syndrome, a common endocrine disorder characterized by oligo/anovulation and/or polycystic ovaries. Hyperandrogenism may also disrupt energy homeostasis, inducing higher central adiposity, insulin resistance, and glucose intolerance, which may exacerbate reproductive dysfunction. Androgens bind to androgen receptors (ARs), which are expressed in many reproductive and metabolic tissues, including brain sites that regulate the hypothalamo-pituitary-gonadal axis and energy homeostasis. The neuronal populations affected by androgen excess, however, have not been defined. We and others have shown that, in mice, AR is highly expressed in leptin receptor (LepRb) neurons, particularly in the arcuate (ARH) and the ventral premammillary nuclei (PMv). Here, we assessed if LepRb neurons, which are critical in the central regulation of energy homeostasis and exert permissive actions on puberty and fertility, have a role in the pathogenesis of female hyperandrogenism. Prenatally androgenized (PNA) mice lacking AR in LepRb cells (LepRbΔAR) show no changes in body mass, body composition, glucose homeostasis, or sexual maturation. They do show, however, a remarkable improvement of estrous cycles combined with normalization of ovary morphology compared to PNA controls. Our findings indicate that the prenatal androgenization effects on adult reproductive physiology (ie, anestrus and anovulation) are mediated by a subpopulation of LepRb neurons directly sensitive to androgens. They also suggest that the effects of hyperandrogenism on sexual maturation and reproductive function in adult females are controlled by distinct neural circuits.

Ablation of Growth Hormone Receptor in GABAergic Neurons Leads to Increased Pulsatile Growth Hormone Secretion.

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH.

Effects of the Isolated and Combined Ablation of Growth Hormone and IGF-1 Receptors in Somatostatin Neurons.

Hypophysiotropic somatostatin (SST) neurons in the periventricular hypothalamic area express growth hormone (GH) receptor (GHR) and are frequently considered as the key neuronal population that mediates the negative feedback loop controlling the hypothalamic-GH axis. Additionally, insulin-like growth factor-1 (IGF-1) may also act at the hypothalamic level to control pituitary GH secretion via long-loop negative feedback. However, to the best of our knowledge, no study so far has tested whether GHR or IGF-1 receptor (IGF1R) signaling specifically in SST neurons is required for the homeostatic control of GH secretion. Here we show that GHR ablation in SST neurons did not impact the negative feedback mechanisms that control pulsatile GH secretion or body growth in male and female mice. The sex difference in hepatic gene expression profile was only mildly affected by GHR ablation in SST neurons. Similarly, IGF1R ablation in SST neurons did not affect pulsatile GH secretion, body growth, or hepatic gene expression. In contrast, simultaneous ablation of both GHR and IGF1R in SST-expressing cells increased mean GH levels and pulse amplitude in male and female mice, and partially disrupted the sex differences in hepatic gene expression. Despite the increased GH secretion in double knockout mice, no alterations in body growth and serum or liver IGF-1 levels were observed. In summary, GHR and IGF1R signaling in SST neurons play a redundant role in the control of GH secretion. Furthermore, our results reveal the importance of GH/IGF-1 negative feedback mechanisms on SST neurons for the establishment of sex differences in hepatic gene expression profile.

Distribution of androgen receptor mRNA in the prepubertal male and female mouse brain.

Androgens are steroid hormones that play a critical role in brain development and sexual maturation by acting upon both androgen receptors (AR) and estrogen receptors (ERα/ß) after aromatization. The contribution of estrogens from aromatized androgens in brain development and the central regulation of metabolism, reproduction, and behavior is well defined, but the role of androgens acting on AR has been unappreciated. Here, we map the sex specific expression of Ar in the adult and developing mouse brain. Postnatal days (PND) 12 and 21 were used to target a critical window of prepubertal development. Consistent with previous literature in adults, sex-specific differences in Ar expression were most profound in the bed nucleus of the stria terminalis (BST), medial amygdala (MEA) and medial preoptic area (MPO). Ar expression was also high in these areas at PND 12 and 21 in both sexes. In addition, we describe extra-hypothalamic and extra-limbic areas that show moderate, consistent and similar Ar expression in both sexes at both prepubertal time points. Briefly, Ar expression was observed in olfactory areas of the cerebral cortex, the hippocampus, several thalamic nuclei, and cranial nerve nuclei involved in autonomic sensory and motor function. To further characterize forebrain populations of Ar expressing neurons and determine whether they also coexpress estrogen receptors, we examined expression of Ar, Esr1 and Esr2 in prepubertal mice in selected nuclei. We found populations of neurons in the BST, MEA and MPO that coexpress Ar, but not Esr1 or Esr2, whereas others express a combination of the three receptors. Our findings indicate that various brain areas express Ar during prepubertal development and may play an important role in female neuronal development and physiology.

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.

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice.

Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Dissociated Pmch and Cre Expression in Lactating Pmch-Cre BAC Transgenic Mice.

The melanin-concentrating hormone (MCH) system plays a role in many physiological processes including reproduction and lactation. However, research regarding the function of MCH on different aspects of the reproductive function lags, due in part to a lack of validated genetic models with which to interrogate the system. This is particularly true in the case of female reproduction, as the anatomy and function of the MCH system is not well-characterized in the female mouse. We set out to determine whether the commercially available Pmch-Cre transgenic mouse line is a viable model to study the role of MCH neurons in distinct female reproductive states. We found that Pmch is transiently expressed in several nuclei of the rostral forebrain at the end of lactation. This includes the medial subdivision of the medial preoptic nucleus, the paraventricular nucleus of the hypothalamus, the ventral subdivision of the lateral septum, the anterodorsal preoptic nucleus and the anterodorsal nucleus of the thalamus. The Pmch expression in these sites, however, does not reliably induce Cre expression in the Pmch-Cre (BAC) transgenic mouse, making this line an inadequate model with which to study the role of MCH in behavioral and/or neuroendocrine adaptations of lactation. We also contribute to the general knowledge of the anatomy of the murine MCH system by showing that lactation-induced Pmch expression in the rostral forebrain is mostly observed in GABAergic (VGAT) neurons, in contrast to the typical MCH neurons of the tuberal and posterior hypothalamus which are glutamatergic (VGLUT2).

Lack of AR in LepRb Cells Disrupts Ambulatory Activity and Neuroendocrine Axes in a Sex-Specific Manner in Mice.

Disorders of androgen imbalance, such as hyperandrogenism in females or hypoandrogenism in males, increase risk of visceral adiposity, type 2 diabetes, and infertility. Androgens act upon androgen receptors (AR) which are expressed in many tissues. In the brain, AR are abundant in hypothalamic nuclei involved in regulation of reproduction and energy homeostasis, yet the role of androgens acting via AR in specific neuronal populations has not been fully elucidated. Leptin receptor (LepRb)-expressing neurons coexpress AR predominantly in hypothalamic arcuate and ventral premammillary nuclei (ARH and PMv, respectively), with low colocalization in other LepRb neuronal populations, and very low colocalization in the pituitary gland and gonads. Deletion of AR from LepRb-expressing cells (LepRbΔAR) has no effect on body weight, energy expenditure, and glucose homeostasis in male and female mice. However, LepRbΔAR female mice show increased body length later in life, whereas male LepRbΔAR mice show an increase in spontaneous ambulatory activity. LepRbΔAR mice display typical pubertal timing, estrous cycles, and fertility, but increased testosterone levels in males. Removal of sex steroid negative feedback action induced an exaggerated rise in luteinizing hormone in LepRbΔAR males and follicle-stimulating hormone in LepRbΔAR females. Our findings show that AR can directly affect a subset of ARH and PMv neurons in a sex-specific manner and demonstrate specific androgenic actions in the neuroendocrine hypothalamus.

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.

P110ß in the ventromedial hypothalamus regulates glucose and energy metabolism.

Phosphoinositide 3-kinase (PI3K) signaling in hypothalamic neurons integrates peripheral metabolic cues, including leptin and insulin, to coordinate systemic glucose and energy homeostasis. PI3K is composed of different subunits, each of which has several unique isoforms. However, the role of the PI3K subunits and isoforms in the ventromedial hypothalamus (VMH), a prominent site for the regulation of glucose and energy homeostasis, is unclear. Here we investigated the role of subunit p110ß in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110ß in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110ß in SF-1 neurons leads to the whitening of brown adipose tissues and increased susceptibility to diet-induced obesity due to blunted energy expenditure. These results highlight a critical role for p110ß in the regulation of glucose and energy homeostasis via VMH neurons.

Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.

CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. OBJECTIVE: To investigate the genetic etiology of idiopathic POI. PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.

PI3K signalling in leptin receptor cells: Role in growth and reproduction.

Nutrition and growth are important signals for pubertal development, although how they are perceived and integrated in brain circuits has not been well defined. Growth hormones and metabolic cues both recruit phosphatidylinositol 3-kinase (PI3K) signalling in hypothalamic sites, although whether they converge into the same neuronal population(s) is also not known. In this review, we discuss recent findings from our laboratory showing the role of PI3K subunits in cells directly responsive to the adipocyte-derived hormone leptin in the coordination of growth, pubertal development and fertility. Mice with deletion of PI3K p110α and p110ß catalytic subunits in leptin receptor cells (LRΔα+ß ) have a lean phenotype associated with increased energy expenditure, locomotor activity and thermogenesis. The LRΔα+ß mice also show deficient growth and delayed puberty. Deletion of a single subunit (ie, p110α) in LR cells (LRΔα ) causes a similar phenotype of increased energy expenditure, deficient growth and delayed pubertal development, indicating that these functions are preferably controlled by p110α. The LRΔα mice show enhanced leptin sensitivity in metabolic regulation but, remarkably, these mice are unresponsive to the effects of leptin on growth and puberty. PI3K is also recruited by insulin and a subpopulation of LR neurones is responsive to i.c.v. insulin administration. Deletion of insulin receptor in LR cells causes no changes in body weight or linear growth and induces only a mild delay in pubertal completion. Our findings demonstrate that PI3K in LR cells plays an essential role in growth and reproduction. We will also discuss the potential neural pathways underlying these effects.

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.

Neuroanatomical Framework of the Metabolic Control of Reproduction.

A minimum amount of energy is required for basic physiological processes, such as protein biosynthesis, thermoregulation, locomotion, cardiovascular function, and digestion. However, for reproductive function and survival of the species, extra energy stores are necessary. Production of sex hormones and gametes, pubertal development, pregnancy, lactation, and parental care all require energy reserves. Thus the physiological systems that control energy homeostasis and reproductive function coevolved in mammals to support both individual health and species subsistence. In this review, we aim to gather scientific knowledge produced by laboratories around the world on the role of the brain in integrating metabolism and reproduction. We describe essential neuronal networks, highlighting key nodes and potential downstream targets. Novel animal models and genetic tools have produced substantial advances, but critical gaps remain. In times of soaring worldwide obesity and metabolic dysfunction, understanding the mechanisms by which metabolic stress alters reproductive physiology has become crucial for human health.

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.