Individual and combined effects of 5-year exposure to hyperandrogenemia and Western-style diet on metabolism and reproduction in female rhesus macaques.

STUDY QUESTION: What is the impact of prolonged exposure to hyperandrogenemia (T), Western-style diet (WSD) and the combination on metabolic and reproductive function in female rhesus macaques, particularly in the post-partum period? SUMMARY ANSWER: Combined T + WSD worsened measures of insulin sensitivity and parameters of cyclicity following prolonged (5 years) exposure, but there was no effect on post-partum metabolic function. WHAT IS KNOWN ALREADY: Women with hyperandrogenemia due to polycystic ovary syndrome are at higher risk for gestational diabetes and Type 2 diabetes post-partum, but it is unknown if this is related to hyperandrogenemia. Hyperandrogenemia in the presence of a WSD worsens metabolic function in female nonhuman primates. STUDY DESIGN, SIZE, DURATION: Female rhesus macaques began treatment near menarche (roughly 2.5 years of age) consisting of either cholesterol (control; C) or testosterone (T) implants (average serum levels 1.4 ng/ml) and exposure to standard monkey chow or a WSD (15 vs 36% of calories from fat, respectively). The four groups were maintained on treatment for 3 years, underwent a fertility trial in Year 4 and continued with treatments through Year 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolic measurements (glucose tolerance tests and double X-ray absorptiometry scans) were performed yearly, and results from 5 years of treatment are reported for all animals. Animals were bled daily for 30 days at 5 years to capture changes in ovarian cycle hormones, and ultrasound measurements were performed during the early follicular and luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: After 5 years of treatment, WSD exposure moderately increased body weight and body fat, although control animals also had a high body mass index due to ad libitum feeding. Animals in the T + WSD group had increased fasting insulin and insulin secretion during an intravenous glucose tolerance test. WSD exposure also altered ovarian cycles, delaying the time to the E2 surge, decreasing progesterone and anti-Müllerian hormone levels and increasing the number of antral follicles present by ultrasound. Longitudinal assessment of metabolic function for only those animals that became pregnant in Year 4 of treatment revealed no differences in post-partum metabolism between groups, although WSD resulted in overall elevated weights, body fat and measures of insulin resistance. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The small sample size and heterogeneity in metabolic effects observed in the T + WSD group are limitations of the current study, with only a subset of animals in this group showing impaired insulin resistance relative to controls. In addition, obesity in the C group prevented comparisons to lean animals. WIDER IMPLICATIONS OF THE FINDINGS: Hyperandrogenemia combined with WSD had a greater impact on insulin sensitivity and ovarian function than either treatment alone. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH grant P50 HD071836 to C.T.R., J.H. and C.T. and P51 OD011092 for support of the Oregon National Primate Research Center. All authors declare no competing interests.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

STUDY QUESTION: Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? SUMMARY ANSWER: Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. STUDY DESIGN, SIZE, DURATION: Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. WIDER IMPLICATIONS OF THE FINDINGS: Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.

Maternal and postnatal high-fat diet consumption programs energy balance and hypothalamic melanocortin signaling in nonhuman primate offspring.

Maternal high-fat-diet (HFD) consumption during pregnancy decreased fetal body weight and impacted development of hypothalamic melanocortin neural circuitry in nonhuman primate offspring. We investigated whether these impairments during gestation persisted in juvenile offspring and examined the interaction between maternal and early postnatal HFD consumption. Adult dams consumed either a control diet (CTR; 15% calories from fat) or a high-saturated-fat diet (HFD; 37% calories from fat) during pregnancy. Offspring were weaned onto a CTR or HFD at ~8 mo of age. Offspring from HFD-fed dams displayed early catch-up growth and elevated body weight at 6 and 13 mo of age. Maternal and postnatal HFD exposure reduced the amount of agouti-related peptide fibers in the paraventricular nucleus of the hypothalamus. Postnatal HFD consumption also decreased the amount of agouti-related peptide fibers in the arcuate nucleus of the hypothalamus. Postnatal HFD was associated with decreased food intake and increased activity. These results support and extend our previous findings of maternal diet effects on fetal development and reveal, for the first time in a nonhuman primate model, that maternal HFD-induced disturbances in offspring body weight regulation extended past gestation into the juvenile period. Maternal HFD consumption increases the risk for offspring developing obesity, with the developmental timing of HFD exposure differentially impacting the melanocortin system and energy balance regulation. The present findings provide translational insight into human clinical populations, suggesting that profound health consequences may await individuals later in life following intrauterine and postnatal HFD exposure.

Sex Differences in Androgen Regulation of Metabolism in Nonhuman Primates.

The in-depth characterization of sex differences relevant to human physiology requires the judicious use of a variety of animal models and human clinical data. Nonhuman primates (NHPs) represent an important experimental system that bridges rodent studies and clinical investigations. NHP studies have been especially useful in understanding the role of sex hormones in development and metabolism and also allow the elucidation of the effects of pertinent dietary influences on physiology pertinent to disease states such as obesity and diabetes. This chapter summarizes the current state of our understanding of androgen effects on male and female NHP metabolism relevant to hypogonadism in human males and polycystic ovary syndrome in human females. This review will also focus on the interaction between altered androgen levels and dietary restriction and excess, in particular the Western-style diet that underlies significant human pathophysiology.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP.

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

Use of genetic models of idiopathic hypogonadotrophic hypogonadism in mice and men to understand the mechanisms of disease.

Mutations in the genes encoding the neuropeptides kisspeptin and neurokinin B, as well as their receptors, are associated with gonadotrophin-releasing hormone (GnRH) deficiency and a failure to initiate and/or progress through puberty. Although the total number of patients studied to date is small, mutations in the kisspeptin pathway appear to result in lifelong GnRH deficiency. Mice with mutations in kisspeptin and the kisspeptin receptor, Kiss1(-/-) and Kiss1r(-/-), respectively, appear to be phenocopies of the human with abnormal sexual maturation and infertility. In contrast, mutations in the neurokinin B pathway lead to a more variable adult reproductive phenotype, with a subset of hypogonadotrophic individuals demonstrating paradoxical recovery of reproductive function later in life. While 'reversal' remains poorly understood, the ability to recover reproductive function indicates that neurokinin B may play different roles in the initiation of sexual maturation compared with the maintenance of adult reproductive function. Mice with mutations in the gene encoding the neurokinin B receptor, Tacr3, have abnormal oestrous cycles and subfertility but, similar to their human counterparts, appear less severely affected than mice with kisspeptin deficiency. Further investigations into the interaction between the kisspeptin and neurokinin B pathways will reveal key insights into how GnRH neuronal modulation occurs at puberty and throughout reproductive life.

Suppression of food intake by Glp1r/Lepr-coexpressing neurons prevents obesity in mouse models.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A potentially unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-Seq of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance caused by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.

Cross-species analysis defines the conservation of anatomically segregated VMH neuron populations.

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing translating ribosome affinity purification with RNA-sequencing (TRAP-seq) data with single-nucleus RNA-sequencing (snRNA-seq) data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of six major genetically and anatomically differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

Central and Peripheral Administration of Fibroblast Growth Factor 1 Improves Pancreatic Islet Insulin Secretion in Diabetic Mouse Models.

Fibroblast growth factor 1 (FGF1) has been shown to reverse hyperglycemia in diabetic rodent models through peripheral and central administration routes. Previous studies demonstrated that insulin is required for central and peripheral FGF1 metabolic improvements; however, it is unknown if FGF1 targets insulin secretion at the islet level. Here we show for the first time that FGF1 increases islet insulin secretion in diabetic mouse models. FGF1 was administered via a single intracerebroventricular or multiple subcutaneous injections to leptin receptor-deficient (db/db), diet-induced obese, and control mice; pancreatic islets were isolated 7 days later for analysis of insulin secretion. Central and peripheral FGF1 significantly lowered blood glucose in vivo and increased ex vivo islet insulin secretion from diabetic, but not control, mice. FGF1 injections to the cisterna magna mimicked intracerebroventricular outcomes, pointing to a novel therapeutic potential. Central effects of FGF1 appeared dependent on reductions in food intake, whereas peripheral FGF1 had acute actions on islet function prior to significant changes in food intake or blood glucose. Additionally, peripheral, but not central, FGF1 increased islet ß-cell density, suggesting that peripheral FGF1 may induce long-term changes in islet structure and function that are not present with central treatment.

Synergistic Effects of Hyperandrogenemia and Obesogenic Western-style Diet on Transcription and DNA Methylation in Visceral Adipose Tissue of Nonhuman Primates.

Polycystic ovary syndrome (PCOS) is a major reproductive disorder that is responsible for 80% of anovulatory infertility and that is associated with hyperandrogenemia, increased risk of obesity, and white adipose tissue (WAT) dysfunction. We have previously demonstrated that the combination of chronic testosterone (T) treatment and an obesogenic Western-style diet (WSD) exerts synergistic functional effects on WAT, leading to increased lipid accumulation in visceral adipocytes by an unknown mechanism. In this study, we examined the whole-genome transcriptional response in visceral WAT to T and WSD, alone and in combination. We observed a synergistic effect of T and WSD on gene expression, resulting in upregulation of lipid storage genes concomitant with adipocyte hypertrophy. Because DNA methylation is known to be associated with body fat distribution and the etiology of PCOS, we conducted whole-genome DNA methylation analysis of visceral WAT. While only a fraction of differentially expressed genes also exhibited differential DNA methylation, in silico analysis showed that differentially methylated regions were enriched in transcription factor binding motifs, suggesting a potential gene regulatory role for these regions. In summary, this study demonstrates that hyperandrogenemia alone does not induce global transcriptional and epigenetic response in young female macaques unless combined with an obesogenic diet.

Reelin is modulated by diet-induced obesity and has direct actions on arcuate proopiomelanocortin neurons.

OBJECTIVE: Reelin (RELN) is a large glycoprotein involved in synapse maturation and neuronal organization throughout development. Deficits in RELN signaling contribute to multiple psychological disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder. Nutritional stress alters RELN expression in brain regions associated with these disorders; however, the involvement of RELN in the neural circuits involved in energy metabolism is unknown. The RELN receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR) are involved in lipid metabolism and expressed in the hypothalamus. Here we explored the involvement of RELN in hypothalamic signaling and the impact of diet-induced obesity (DIO) on this system. METHODS: Adult male mice were fed a chow diet or maintained on a high-fat diet (HFD) for 12-16 weeks. HFD-fed DIO mice exhibited decreased ApoER2 and VLDLR expression and increased RELN protein in the hypothalamus. Electrophysiology was used to determine the mechanism by which the central fragment of RELN (CF-RELN) acts on arcuate nucleus (ARH) satiety-promoting proopiomelanocortin (POMC) neurons and the impact of DIO on this circuitry. RESULTS: CF-RELN exhibited heterogeneous presynaptic actions on inhibitory inputs onto ARH-POMC-EGFP neurons and consistent postsynaptic actions. Additionally, central administration of CF-RELN caused a significant increase in ARH c-Fos expression and an acute decrease in food intake and body weight. CONCLUSIONS: We conclude that RELN signaling is modulated by diet, that RELN is involved in synaptic signaling onto ARH-POMC neurons, and that altering central CF-RELN levels can impact food intake and body weight.

Maternal High-Fat Diet Effects on Adaptations to Metabolic Challenges in Male and Female Juvenile Nonhuman Primates.

OBJECTIVE: This study aimed to determine whether maternal high-fat diet (HFD) consumption in nonhuman primates alters the ability of offspring to adapt metabolically to nutrient and caloric challenges. METHODS: Offspring from Japanese macaque dams fed either a control (CTR) diet or HFD were weaned onto a CTR diet creating two groups: maternal HFD (mHFD, n = 18) and maternal CTR (mCTR) diet (n = 12). Male and female offspring were exposed to a 5-day 30% calorie restriction and to a 35-day HFD challenge (HFDC), at 16 and 24 months of age, respectively. Caloric intake, body weight, and energy expenditure were measured. RESULTS: Offspring from both groups showed similar body weight, food intake, and metabolic adaptations to a 5-day calorie restriction. mHFD offspring demonstrated increased food intake and early weight gain in response to a 35-day HFDC; however, group differences in weight dissipated during the challenge. Unlike mCTR animals, the mHFD group had a significant increase in fasting insulin after acute HFD exposure. CONCLUSIONS: The current findings indicate that offspring exposed to an mHFD show metabolic adaptations to calorie restriction that are largely similar to those of offspring exposed to a mCTR diet but show delayed adaptation upon exposure to an acute HFDC.

Early High-Fat Diet Exposure Causes Dysregulation of the Orexin and Dopamine Neuronal Populations in Nonhuman Primates.

Maternal obesity and consumption of a high-fat diet (HFD) during pregnancy has a negative impact on offspring, including an increased risk for the development of obesity in adolescence. The mechanism for this transferred metabolic risk is unclear, but many studies have focused on the brain due to its important role in appetite and body-weight regulation. Two main pathways regulate appetite in the brain; homeostatic regulation that occurs predominantly in hypothalamic circuits and hedonic regulation of feeding that occurs via dopaminergic pathways. The current proposal examined the impact of early HFD exposure on the dopaminergic control of hedonic feeding pathways in a translational nonhuman primate model. Japanese macaque offspring from mothers consuming a control (CTR) or HFD were weaned onto control or HFD at an average 8 months of age yielding four groups: maternal and post-weaning control diet (mCTRpCTR), maternal control diet and post-weaning HFD (mCTRpHFD), maternal HFD and post-weaning control diet (mHFDpCTR) and maternal and post-weaning HFD (mHFDpHFD). Brains from 13-month-old offspring were evaluated for expression of neuropeptides that regulate dopaminergic pathways including orexin, melanin-concentrating hormone (MCH) in the lateral hypothalamus (LH), and tyrosine hydroxylase expression in the ventral tegmental area (VTA). Orexin cell numbers in the LH were significantly increased in animals exposed to a post-weaning HFD, while no difference was observed for orexin mRNA content or MCH cell numbers. Orexin fiber projections to the rostral VTA were significantly reduced in mCTRpHFD, mHFDpCTR, and mHFDpHFD groups, but these differences were not significant in the caudal VTA. There was no difference in the percentage of dopamine neurons receiving close appositions from orexin fibers in either the rostral or caudal VTA, nor was there any difference between groups in the number of orexin contacts per TH cell. In conclusion, the current study finds that prolonged early exposure to HFD during the in utero and postnatal period causes alterations at several levels in the dopaminergic circuits regulating reward.

GLP-1R Signaling Directly Activates Arcuate Nucleus Kisspeptin Action in Brain Slices but Does not Rescue Luteinizing Hormone Inhibition in Ovariectomized Mice During Negative Energy Balance.

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status, and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide 1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. Because GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased after a 48-h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin(9-39), in ad libitum-fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Neurokinin B is critical for normal timing of sexual maturation but dispensable for adult reproductive function in female mice.

Humans carrying mutations in neurokinin B (NKB) or the NKB receptor fail to undergo puberty due to decreased secretion of GnRH. Despite this pubertal delay, many of these patients go on to achieve activation of their hypothalamic-pituitary-gonadal axis in adulthood, a phenomenon termed reversal, indicating that NKB signaling may play a more critical role for the timing of pubertal development than adult reproductive function. NKB receptor-deficient mice are hypogonadotropic but have no defects in the timing of sexual maturation. The current study has performed the first phenotypic evaluation of mice bearing mutations in Tac2, the gene encoding the NKB ligand, to determine whether they have impaired sexual development similar to their human counterparts. Male Tac2-/- mice showed no difference in the timing of sexual maturation or fertility compared with wild-type littermates and were fertile. In contrast, Tac2-/- females had profound delays in sexual maturation, with time to vaginal opening and first estrus occurring significantly later than controls, and initial abnormalities in estrous cycles. However, cycling recovered in adulthood and Tac2-/- females were fertile, although they produced fewer pups per litter. Thus, female Tac2-/- mice parallel humans harboring NKB pathway mutations, with delayed sexual maturation and activation of the reproductive cascade later in life. Moreover, direct comparison of NKB ligand and receptor-deficient females confirmed that only NKB ligand-deficient animals have delayed sexual maturation, suggesting that in the absence of the NKB receptor, NKB may regulate the timing of sexual maturation through other tachykinin receptors.

The integrated hypothalamic tachykinin-kisspeptin system as a central coordinator for reproduction.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r(-/-) mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons.

Cocaine- and amphetamine-regulated transcript is a potent stimulator of GnRH and kisspeptin cells and may contribute to negative energy balance-induced reproductive inhibition in females.

Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic neuropeptide implicated in both metabolic and reproductive regulation, raising the possibility that CART plays a role in reproductive inhibition during negative metabolic conditions. The current study characterized CART's regulatory influence on GnRH and kisspeptin (Kiss1) cells and determined the sensitivity of different CART populations to negative energy balance. CART fibers made close appositions to 60% of GnRH cells, with the majority of the fibers (>80%) originating from the arcuate nucleus (ARH) CART/pro-opiomelanocortin population. Electrophysiological recordings in GnRH-green fluorescent protein rats demonstrated that CART postsynaptically depolarizes GnRH cells. CART fibers from the ARH were also observed in close contact with Kiss1 cells in the ARH and anteroventral periventricular nucleus (AVPV). Recordings in Kiss1-GFP mice demonstrated CART also postsynaptically depolarizes ARH Kiss1 cells, suggesting CART may act directly and indirectly, via Kiss1 populations, to stimulate GnRH neurons. CART protein and mRNA levels were analyzed in 2 models of negative energy balance: caloric restriction (CR) and lactation. Both CART mRNA levels and the number of CART-immunoreactive cells were suppressed in the ARH during CR but not during lactation. AVPV CART mRNA was suppressed during CR, but not during lactation when there was a dramatic increase in CART-immunoreactive cells. These data suggest differing regulatory signals of CART between the models. In conclusion, both morphological and electrophysiological methods identify CART as a novel and potent stimulator of Kiss1 and GnRH neurons and suppression of CART expression during negative metabolic conditions could contribute to inhibition of the reproductive axis.

Beyond Leptin: Emerging Candidates for the Integration of Metabolic and Reproductive Function during Negative Energy Balance.

Reproductive status is tightly coupled to metabolic state in females, and ovarian cycling in mammals is halted when energy output exceeds energy input, a metabolic condition known as negative energy balance. This inhibition of reproductive function during negative energy balance occurs due to suppression of gonadotropin-releasing hormone (GnRH) release in the hypothalamus. The GnRH secretagogue kisspeptin is also inhibited during negative energy balance, indicating that inhibition of reproductive neuroendocrine circuits may occur upstream of GnRH itself. Understanding the metabolic signals responsible for the inhibition of reproductive pathways has been a compelling research focus for many years. A predominant theory in the field is that the status of energy balance is conveyed to reproductive neuroendocrine circuits via the adipocyte hormone leptin. Leptin is stimulatory for GnRH release and lower levels of leptin during negative energy balance are believed to result in decreased stimulatory drive for GnRH cells. However, recent evidence found that restoring leptin to physiological levels did not restore GnRH function in three different models of negative energy balance. This suggests that although leptin may be an important permissive signal for reproductive function as indicated by many years of research, factors other than leptin must critically contribute to negative energy balance-induced reproductive inhibition. This review will focus on emerging candidates for the integration of metabolic status and reproductive function during negative energy balance.

Characterisation of arcuate nucleus kisspeptin/neurokinin B neuronal projections and regulation during lactation in the rat.

Lactation results in negative energy balance in the rat leading to decreased gonadotrophin-releasing hormone (GnRH) release and anoestrus. Inhibited GnRH release may be a result of decreased stimulatory tone from neuropeptides critical for GnRH neuronal activity, such as kisspeptin (Kiss1) and neurokinin B (NKB). The present study aimed to identify neuronal projections from the colocalised population of Kiss1/NKB cells in the arcuate nucleus (ARH) using double-label immunohistochemistry to determine where this population may directly regulate GnRH neuronal activity. Additionally, the present study further examined lactation-induced changes in the Kiss1 system that could play a role in decreased GnRH release. The colocalised ARH Kiss1/NKB fibres projected primarily to the internal zone of the median eminence (ME) where they were in close proximity to GnRH fibres; however, few Kiss1/NKB fibres from the ARH were seen at the level of GnRH neurones in the preoptic area (POA). Arcuate Kiss1/NKB peptide levels were decreased during lactation consistent with previous mRNA data. Surprisingly, anteroventral periventricular (AVPV) Kiss1 peptide levels were increased, whereas Kiss1 mRNA levels were decreased during lactation, suggesting active inhibition of peptide release. These findings indicate ARH Kiss1/NKB and AVPV Kiss1 appear to be inhibited during lactation, which may contribute to decreased GnRH release and subsequent reproductive dysfunction. Furthermore, the absence of a strong ARH Kiss1/NKB projection to the POA suggests regulation of GnRH by this population occurs primarily at the ME level via local projections.

The neuroendocrine basis of lactation-induced suppression of GnRH: role of kisspeptin and leptin.

Lactation is an important physiological model of the integration of energy balance and reproduction, as it involves activation of potent appetitive neuropeptide systems coupled to a profound inhibition of pulsatile GnRH/LH secretion. There are multiple systems that contribute to the chronic hyperphagia of lactation: 1) suppression of the metabolic hormones, leptin and insulin, 2) activation of hypothalamic orexigenic neuropeptide systems NPY, AGRP, orexin (OX) and melanin concentrating hormone (MCH), 3) special induction of NPY expression in the dorsomedial hypothalamus, and 4) suppression of anorexigenic systems POMC and CART. These changes ensure adequate energy intake to meet the metabolic needs of milk production. There is significant overlap in all of the systems that regulate food intake with the regulation of GnRH, suggesting there could be several redundant factors acting to suppress GnRH/LH during lactation. In addition to an overall increase in inhibitory tone acting directly on GnRH cell bodies that is brought about by increases in orexigenic systems, there are also effects at the ARH to disrupt Kiss1/neurokinin B/dynorphin neuronal function through inhibition of Kiss1 and NKB. These changes could lead to an increase in inhibitory auto-regulation of the Kiss1 neurons and a possible disruption of pulsatile GnRH release. While the low levels of leptin and insulin contribute to the changes in ARH appetitive systems, they do not appear to contribute to the suppression of ARH Kiss1 or NKB. The inhibition of Kiss1 may be the key factor in the suppression of GnRH during lactation, although the mechanisms responsible for its inhibition are unknown.