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The adipocyte-derived hormone leptin is required for normal pubertal maturation in mice and humans and, therefore, leptin has been recognized as a crucial metabolic cue linking energy stores and the onset of puberty. Several lines of evidence have suggested that leptin acts via kisspeptin expressing neurons of the arcuate nucleus to exert its effects. Using conditional knockout mice, we have previously demonstrated that deletion of leptin receptors (LepR) from kisspeptin cells cause no puberty or fertility deficits. However, developmental adaptations and system redundancies may have obscured the physiologic relevance of direct leptin signaling in kisspeptin neurons. To overcome these putative effects, we re-expressed endogenous LepR selectively in kisspeptin cells of mice otherwise null for LepR, using the Cre-loxP system. Kiss1-Cre LepR null mice showed no pubertal development and no improvement of the metabolic phenotype, remaining obese, diabetic and infertile. These mice displayed decreased numbers of neurons expressing Kiss1 gene, similar to prepubertal control mice, and an unexpected lack of re-expression of functional LepR. To further assess the temporal coexpression of Kiss1 and Lepr genes, we generated mice with the human renilla green fluorescent protein (hrGFP) driven by Kiss1 regulatory elements and crossed them with mice that express Cre recombinase from the Lepr locus and the R26-tdTomato reporter gene. No coexpression of Kiss1 and LepR was observed in prepubertal mice. Our findings unequivocally demonstrate that kisspeptin neurons are not the direct target of leptin in the onset of puberty. Leptin signaling in kisspeptin neurons arises only after completion of sexual maturation.

Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the Pgr gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the Pgr deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.

The transcription factor steroidogenic factor 1 (SF-1) is exclusively expressed in the brain in the ventral medial hypothalamic nucleus (VMH) and is required for the development of this nucleus. However, the physiological importance of transcriptional programs regulated by SF-1 in the VMH is not well defined. To delineate the functional significance of SF-1 itself in the brain, we generated pre- and postnatal VMH-specific SF-1 KO mice. Both models of VMH-specific SF-1 KO were susceptible to high fat diet-induced obesity and displayed impaired thermogenesis after acute exposure to high fat diet. Furthermore, VMH-specific SF-1 KO mice showed significantly decreased LepR expression specifically in the VMH, leading to leptin resistance. Collectively, these results indicate that SF-1 directs transcriptional programs in the hypothalamus relevant to coordinated control of energy homeostasis, especially after excess caloric intake.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in Lepr Cre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine- N -oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons ( Lepr ΔVGlut2 ) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, 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 ( Lepr loxTB ) with concomitant deletion of Slc17a6 (Vglut2 flox ) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control Lepr loxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while Lepr loxTB ;Vglut2 flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

A critical amount of energy reserve is necessary for puberty initiation, for normal sexual maturation and maintenance of cyclicity and fertility in females of most species. Therefore, the existence of circulating metabolic cues which directly modulate the hypothalamus-pituitary-gonad axis is predictable. The adipocyte-derived hormone leptin is one of these cues having been studied extensively in the context of regulating the reproductive physiology. Humans and mice lacking leptin (ob/ob) or leptin receptor (LepR, db/db) are infertile. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility. LepR is expressed in brain, pituitary gland and gonads, but studies using genetically engineered mouse models determined that the brain plays a major role. Recently, it has been made clear that leptin acts indirectly on gonadotropin-releasing hormone (GnRH)-secreting cells via actions on interneurons. However, the exact site(s) of leptin action has been difficult to determine. In this review, we discuss the recent advances in the field focused on the identification of potential site(s) or specific neuronal populations involved in leptin’s effects in the neuroendocrine reproductive axis.

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepRKiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IRKiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.

Plain language summary Melanin-concentrating hormone (MCH) is a neuropeptide important for the regulation of metabolism, sleep, circadian rhythms, and other body functions. MCH neurons contain the excitatory neurotransmitter glutamate, which is released via vesicular glutamate transporter 2 (VGLUT2). The release of glutamate from MCH neurons is known to play a role in metabolism in males, but its action in female physiology has not been determined. Furthermore, little is known about the role of glutamate release from MCH neurons in reproductive function. We developed a new transgenic mouse model that expresses Cre recombinase driven by the MCH gene ( Pmch ) and deleted Vglut2 from the MCH neurons. We then assessed reproductive and metabolic function in both sexes. Females exhibited late-onset leanness, delayed sexual maturation, and increased latency to pregnancy. On a high-fat diet, males showed improved insulin resistance, and both sexes displayed an improved daily pattern of food intake. These findings highlight the importance of considering sex as a relevant biological variable in MCH neuronal function. Highlights Pmch ΔVglut2 female, but not male, mice showed a delay in puberty onset and completion. Pmch ΔVglut2 female mice were (HFD)-induced disruption of estrous cycles. Pmch ΔVglut2 female mice exhibited late-onset reduced total body mass whereas males showed late-onset reduced fat mass. Pmch ΔVglut2 male, but not female, mice on HFD showed reduced insulin resistance compared to controls. HFD disrupted the daily pattern of food consumption in control Vglut2 flox male and female mice. This disruption was not observed in Pmch ΔVglut2 mice. Background Melanin-concentrating hormone (MCH) neurons contribute to the regulation of a diverse array of physiological functions including glucose and energy homeostasis. MCH neurons express genes involved in the synthesis, packaging, and release of glutamate, the brain’s predominant excitatory neurotransmitter. Deletion of vesicular glutamate transporter 2 (VGLUT2, Slc17a6 gene) in MCH neurons of male mice results in a hypophagic, hyperactive, lean phenotype. However, the metabolic and reproductive effects of VGLUT2 deletion in females have not been fully addressed. Methods Previous studies have utilized Pmch -Cre mice generated using a bacterial artificial chromosome (BAC). The loci of insertion for the BAC are random and may fail to recapitulate epigenetic regulation at the native gene locus. Here, we generated a novel knock-in Pmch -iCre mouse model and investigated the effects of deleting Slc17a6 from MCH neurons in both male and female mice. Assessment of sexual maturation, fertility, glucose homeostasis and energy balance (food intake, body composition, energy expenditure, locomotion, glucose and fat oxidation) on regular chow and high fat diet (HFD) were performed in both sexes. Results Lack of glutamate neurotransmission in MCH neurons ( Pmch ΔVglut2 mice) delays puberty onset and completion in female, but not male mice. Pmch ΔVglut2 females were also protected against (HFD)-induced disruption of estrous cycles. On a regular chow diet, neither male nor female Pmch ΔVglut2 mice showed differences in body weight, food intake, or lean and fat masses compared to littermate controls until 20 weeks of age. At 24 weeks of age, Pmch ΔVglut2 females exhibited reduced total body mass, and males had lower fat mass. Pmch ΔVglut2 female mice also gained less weight on HFD than littermate controls. An attenuation of HFD-induced disruption of daily feeding pattern was observed in Pmch ΔVglut2 mice of both sexes, while only males were protected against insulin resistance on HFD. Conclusions Glutamate neurotransmission from MCH neurons has a sex-specific role in sexual maturation, fertility, typical weight gain trajectory, and HFD-induced weight gain and insulin resistance. In addition, a sex-independent role in daily feeding pattern was observed.

Leptin is an adipocyte-derived hormone involved in a myriad of physiological process, including the control of energy balance and several neuroendocrine axes. Leptin-deficient mice and humans are obese, diabetic, and display a series of neuroendocrine and autonomic abnormalities. These individuals are infertile due to a lack of appropriate pubertal development and inadequate synthesis and secretion of gonadotropins and gonadal steroids. Leptin receptors are expressed in many organs and tissues, including those related to the control of reproductive physiology (e.g., the hypothalamus, pituitary gland, and gonads). In the last decade, it has become clear that leptin receptors located in the brain are major players in most leptin actions, including reproduction. Moreover, the recent development of molecular techniques for brain mapping and the use of genetically modified mouse models have generated crucial new findings for understanding leptin physiology and the metabolic influences on reproductive health. In the present review, we will highlight the new advances in the field, discuss the apparent contradictions, and underline the relevance of this complex physiological system to human health. We will focus our review on the hypothalamic circuitry and potential signaling pathways relevant to leptin’s effects in reproductive control, which have been identified with the use of cutting-edge technologies of molecular mapping and conditional knockouts.

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 -Cre transgenic mouse line is a viable model to study the role of MCH neurons in distinct female reproductive states. We found that 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 expression in these sites, however, does not reliably induce Cre expression in the -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 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).

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 p110β in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110β in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110β 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 p110β in the regulation of glucose and energy homeostasis via VMH neurons. Metabolism: Enzymatic subunit essential to brain’s glucose responses A particular subunit of a critical signaling enzyme is needed for neurons inside the brain’s hypothalamus to properly regulate energy metabolism. Ki Woo Kim from Yonsei University College of Dentistry, Seoul, South Korea, and colleagues explored the role that the PI3K enzyme plays in neurons of the ventromedial area toward the front of the hypothalamus, a region involved in regulating hunger and metabolism. Deleting a subunit of PI3K called p110β, which is needed for enzymatic function, made mice less responsive to insulin, the hormone that keeps blood sugar levels at healthy levels. As well as having abnormal glucose metabolism, the mice converted more brown fat, which burns energy, into white fat, which stores energy. They were also more susceptible to diet-induced obesity. The findings point toward p110β as a potential drug target for treating diabetes.