Superior metabolic improvement of polycystic ovary syndrome traits after GLP1-based multi-agonist therapy.

Polycystic ovary syndrome (PCOS) is a heterogeneous condition, defined by oligo-/anovulation, hyper-androgenism and/or polycystic ovaries. Metabolic complications are common in patients suffering PCOS, including obesity, insulin resistance and type-2 diabetes, which severely compromise the clinical course of affected women. Yet, therapeutic options remain mostly symptomatic and of limited efficacy for the metabolic and reproductive alterations of PCOS. We report here the hormonal, metabolic and gonadal responses to the glucagon-like peptide-1 (GLP1)-based multi-agonists, GLP1/Estrogen (GLP1/E), GLP1/gastric inhibitory peptide (GLP1/GIP) and GLP1/GIP/Glucagon, in two mouse PCOS models, with variable penetrance of metabolic and reproductive traits, and their comparison with metformin. Our data illustrate the superior efficacy of GLP1/E vs. other multi-agonists and metformin in the management of metabolic complications of PCOS; GLP1/E ameliorates also ovarian cyclicity in an ovulatory model of PCOS, without direct estrogenic uterotrophic effects. In keeping with GLP1-mediated brain targeting, quantitative proteomics reveals changes in common and distinct hypothalamic pathways in response to GLP1/E between the two PCOS models, as basis for differential efficiency. Altogether, our data set the basis for the use of GLP1-based multi-agonists, and particularly GLP1/E, in the personalized management of PCOS.

Kisspeptins centrally modulate food intake and locomotor activity in mice independently of gonadal steroids in a sexually dimorphic manner.

Kisspeptins are essential regulators of the reproductive axis, with capacity to potently activate gonadotropin-releasing hormone neurons, acting also as central conduits for the metabolic regulation of fertility. Recent evidence suggests that kisspeptins per se may also modulate several metabolic parameters, including body weight, food intake or energy expenditure, but their actual roles and site(s) of action remain unclear. We present herein a series of studies addressing the metabolic effects of central and peripheral administration of kisspeptin-10 (Kp-10; 1 nmol and 3 nmol daily, respectively) for 11 days in mice of both sexes. To assess direct metabolic actions of Kp-10 versus those derived indirectly from its capacity to modulate gonadal hormone secretion, kisspeptin effects were tested in adult male and female mice gonadectomized and supplemented with fixed, physiological doses of testosterone or 17ß-estradiol, respectively. Central administration of Kp-10 decreased food intake in male mice, especially during the dark phase (~50%), which was accompanied by a reduction in total and nocturnal energy expenditure (~16%) and locomotor activity (~70%). In contrast, opposite patterns were detected in female mice, with an increase in total and nocturnal locomotor activity (>65%), despite no changes in food intake or energy expenditure. These changes were independent of body weight, as no differences were detected in mice of both sexes at the end of Kp-10 treatments. Peripheral administration of Kp-10 failed to alter any of the metabolic parameters analyzed, except for a decrease in locomotor activity in male mice and a subtle increase in 24 h food intake in female mice, denoting a predominant central role of kisspeptins in the control of energy metabolism. Finally, glucose tolerance and insulin sensitivity were not significantly affected by central or peripheral treatment with Kp-10. In conclusion, our data reveal a potential role of kisspeptins in the control of key metabolic parameters, including food intake, energy expenditure and locomotor activity, with a preferential action at central level, which is sex steroid-independent but sexually dimorphic.

Metabolic and Low-Grade Inflammation Risk in Young Adults with a History of Extrauterine Growth Restriction.

Children with a history of extrauterine growth restriction (EUGR), later at prepubertal age, exhibit an increased metabolic risk including risen insulin resistance and low-grade inflammation. However, the progression of such metabolic changes after puberty and the lasting health implications have not yet been investigated. The objective of this study was to ascertain whether young adults with a history of EUGR faced increased vulnerability to metabolic disorders. A study was conducted comparing a group of adults with a history of EUGR with a healthy reference group. A total of 110 young adults (36 from the EUGR group and 74 from the control group) were included. Anthropometric variables, blood pressure (BP), general biochemical parameters, plasma inflammatory biomarkers, and adipokines were assessed. Compared to the reference group, the EUGR group had a shorter height and body weight with higher lean mass and waist circumference, as well as a greater percentage of individuals with high BP. In addition, EUGR patients had higher values of insulin, HOMA-IR, nerve growth factor, and leptin, and lower levels of adiponectin and resistin. The present study suggests that young adults with a history of EUGR present increased metabolic risk factors therefore, clinical follow-up should be considered.

Kisspeptin signaling in astrocytes modulates the reproductive axis.

Reproduction is safeguarded by multiple, often cooperative, regulatory networks. Kisspeptin signaling, via KISS1R, plays a fundamental role in reproductive control, primarily by regulation of hypothalamic GnRH neurons. We disclose herein a pathway for direct kisspeptin actions in astrocytes that contributes to central reproductive modulation. Protein-protein interaction and ontology analyses of hypothalamic proteomic profiles after kisspeptin stimulation revealed that glial/astrocyte markers are regulated by kisspeptin in mice. This glial-kisspeptin pathway was validated by the demonstrated expression of Kiss1r in mouse astrocytes in vivo and astrocyte cultures from humans, rats, and mice, where kisspeptin activated canonical intracellular signaling-pathways. Cellular coexpression of Kiss1r with the astrocyte markers GFAP and S100-ß occurred in different brain regions, with higher percentage in Kiss1- and GnRH-enriched areas. Conditional ablation of Kiss1r in GFAP-positive cells in the G-KiR-KO mouse altered gene expression of key factors in PGE2 synthesis in astrocytes and perturbed astrocyte-GnRH neuronal appositions, as well as LH responses to kisspeptin and LH pulsatility, as surrogate marker of GnRH secretion. G-KiR-KO mice also displayed changes in reproductive responses to metabolic stress induced by high-fat diet, affecting female pubertal onset, estrous cyclicity, and LH-secretory profiles. Our data unveil a nonneuronal pathway for kisspeptin actions in astrocytes, which cooperates in fine-tuning the reproductive axis and its responses to metabolic stress.

Central androgen action reverses hypothalamic astrogliosis and atherogenic risk factors induced by orchiectomy and high-fat diet feeding in male mice.

Hypogonadism in males confers elevated cardiovascular disease (CVD) risk by unknown mechanisms. Recent radiological evidence suggests that low testosterone (T) is associated with mediobasal hypothalamic (MBH) gliosis, a central nervous system (CNS) cellular response linked to metabolic dysfunction. To address mechanisms linking CNS androgen action to CVD risk, we generated a hypogonadal, hyperlipidemic mouse model with orchiectomy (ORX) combined with hepatic PCSK9 overexpression. After 4 wk of high-fat, high-sucrose diet (HFHS) consumption, despite equal body weights and glucose tolerance, androgen-deficient ORX mice had a more atherogenic lipid profile and increased liver and leukocyte inflammatory signaling compared with sham-operated control mice. Along with these early CVD risk indicators, ORX markedly amplified HFHS-induced astrogliosis in the MBH. Transcriptomic analysis further revealed that ORX and high-fat diet feeding induced upregulation of inflammatory pathways and downregulation of metabolic pathways in hypothalamic astrocytes. To interrogate the role of sex steroid signaling in the CNS in cardiometabolic risk and MBH inflammation, central infusion of T and dihydrotestosterone (DHT) was performed on ORX mice. Central DHT prevented MBH astrogliosis and reduced the liver inflammatory signaling and monocytosis induced by HFHS and ORX; T had a partial protective effect. Finally, a cross-sectional study in 41 adult men demonstrated a positive correlation between radiological evidence of MBH gliosis and plasma lipids. These findings demonstrate that T deficiency in combination with a Western-style diet promotes hypothalamic gliosis concomitant with increased atherogenic risk factors and provide supportive evidence for regulation of lipid metabolism and cardiometabolic risk determinants by the CNS action of sex steroids.NEW & NOTEWORTHY This study provides evidence that hypothalamic gliosis is a key early event through which androgen deficiency in combination with a Western-style diet might lead to cardiometabolic dysregulation in males. Furthermore, this work provides the first evidence in humans of a positive association between hypothalamic gliosis and LDL-cholesterol, advancing our knowledge of CNS influences on CVD risk progression.

Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility.

BACKGROUND: Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets and kisspeptins originating from them to the control of reproduction and eventually other bodily functions remains to be fully determined. METHODS: To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. To this end, mice with Tac2 cell-specific Kiss1 KO (TaKKO) were generated and subjected to extensive reproductive and metabolic characterization. RESULTS: TaKKO mice displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis, especially in males, and locomotor activity, specifically in females. CONCLUSIONS: Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as for adult metabolic homeostasis. SIGNIFICANCE STATEMENT: Neurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females, and adult metabolic homeostasis. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders.

Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice.

Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.

Selective loss of kisspeptin signaling in oocytes causes progressive premature ovulatory failure.

STUDY QUESTION: Does direct kisspeptin signaling in the oocyte have a role in the control of follicular dynamics and ovulation? SUMMARY ANSWER: Kisspeptin signaling in the oocyte plays a relevant physiological role in the direct control of ovulation; oocyte-specific ablation of kisspeptin receptor, Gpr54, induces a state of premature ovulatory failure in mice that recapitulates some features of premature ovarian insufficiency (POI). WHAT IS KNOWN ALREADY: Kisspeptins, encoded by the Kiss1 gene, are essential for the control of ovulation and fertility, acting primarily on hypothalamic GnRH neurons to stimulate gonadotropin secretion. However, kisspeptins and their receptor, Gpr54, are also expressed in the ovary of different mammalian species, including humans, where their physiological roles remain contentious and poorly characterized. STUDY DESIGN, SIZE, DURATION: A novel mouse line with conditional ablation of Gpr54 in oocytes, named OoGpr54-/-, was generated and studied in terms of follicular and ovulatory dynamics at different age-points of postnatal maturation. A total of 59 OoGpr54-/- mice and 47 corresponding controls were analyzed. In addition, direct RNA sequencing was applied to ovarian samples from 8 OoGpr54-/- and 7 control mice at 6 months of age, and gonadotropin priming for ovulatory induction was conducted in mice (N = 7) from both genotypes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte-selective ablation of Gpr54 in the oocyte was achieved in vivo by crossing a Gdf9-driven Cre-expressing transgenic mouse line with a Gpr54 LoxP mouse line. The resulting OoGpr54-/- mouse line was subjected to phenotypic, histological, hormonal and molecular analyses at different age-points of postnatal maturation (Day 45, and 2, 4, 6 and 10-11 months of age), in order to characterize the timing of puberty, ovarian follicular dynamics and ovulation, with particular attention to identification of features reminiscent of POI. The molecular signature of ovaries from OoGpr54-/- mice was defined by direct RNA sequencing. Ovulatory responses to gonadotropin priming were also assessed in OoGpr54-/- mice. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific ablation of Gpr54 caused premature ovulatory failure, with some POI-like features. OoGpr54-/- mice had preserved puberty onset, without signs of hypogonadism. However, already at 2 months of age, 40% of OoGpr54-/- females showed histological features reminiscent of ovarian failure and anovulation. Penetrance of the phenotype progressed with age, with >80% and 100% of OoGpr54-/- females displaying complete ovulatory failure by 6- and 10 months, respectively. This occurred despite unaltered hypothalamic Gpr54 expression and gonadotropin levels. Yet, OoGpr54-/- mice had decreased sex steroid levels. While the RNA signature of OoGpr54-/- ovaries was dominated by the anovulatory state, oocyte-specific ablation of Gpr54 significantly up- or downregulated of a set of 21 genes, including those encoding pituitary adenylate cyclase-activating polypeptide, Wnt-10B, matrix-metalloprotease-12, vitamin A-related factors and calcium-activated chloride channel-2, which might contribute to the POI-like state. Notably, the anovulatory state of young OoGpr54-/- mice could be rescued by gonadotropin priming. LARGE SCALE DATA: N/A. . LIMITATIONS, REASONS FOR CAUTION: Conditional ablation of Gpr54 in oocytes unambiguously caused premature ovulatory failure in mice; yet, the ultimate molecular mechanisms for such state of POI can be only inferred on the basis of RNAseq data and need further elucidation, since some of the molecular changes observed in OoGpr54-/- ovaries were secondary to the anovulatory state. Direct translation of mouse findings to human disease should be made with caution since, despite the conserved expression of Kiss1/kisspeptin and Gpr54 in rodents and humans, our mouse model does not recapitulate all features of common forms of POI. WIDER IMPLICATIONS OF THE FINDINGS: Deregulation of kisspeptin signaling in the oocyte might be an underlying, and previously unnoticed, cause for some forms of POI in women. STUDY FUNDING/COMPETING INTEREST(S): This work was primarily supported by a grant to M.P. and M.T.-S. from the FiDiPro (Finnish Distinguished Professor) Program of the Academy of Finland. Additional financial support came from grant BFU2017-83934-P (M.T.-S.; Ministerio de Economía y Competitividad, Spain; co-funded with EU funds/FEDER Program), research funds from the IVIRMA International Award in Reproductive Medicine (M.T.-S.), and EFSD Albert Renold Fellowship Programme (S.T.R.). The authors have no conflicts of interest to declare in relation to the contents of this work. TRIAL REGISTRATION NUMBER: N/A.

Congenital ablation of Tacr2 reveals overlapping and redundant roles of NK2R signaling in the control of reproductive axis.

Tachykinin (TAC) signaling is an important element in the central control of reproduction. TAC family is mainly composed of substance P (SP), neurokinin A (NKA), and NKB, which bind preferentially to NK1, NK2, and NK3 receptors, respectively. While most studies have focused on the reproductive functions of NKB/NK3R, and to a lesser extent SP/NK1R, the relevance of NK2R, encoded by Tacr2, remains poorly characterized. Here, we address the physiological roles of NK2R in regulating the reproductive axis by characterizing a novel mouse line with congenital ablation of Tacr2. Activation of NK2R evoked acute luteinizing hormone (LH) responses in control mice, similar to those of agonists of NK1R and NK3R. Despite the absence of NK2R, Tacr2-/- mice displayed only partially reduced LH responses to an NK2R agonist, which, nonetheless, were abrogated after blockade of NK3R in Tacr2-/- males. While Tacr2-/- mice displayed normal pubertal timing, LH pulsatility was partially altered in Tacr2-/- females in adulthood, with suppression of basal LH levels, but no changes in the number of LH pulses. In addition, trends for increase in breeding intervals were detected in Tacr2-/- mice. However, null animals of both sexes were fertile, with no changes in estrous cyclicity or sex preference in social behavioral tests. In conclusion, stimulation of NK2R elicited LH responses in mice, while congenital ablation of Tacr2 partially suppressed basal and stimulated LH secretion, with moderate reproductive impact. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.NEW & NOTEWORTHY We have explored here the impact of congenital ablation of the gene (Tacr2) encoding the tachykinin receptor, NK2R, in terms of neuroendocrine control of the reproductive axis, using a novel Tacr2 KO mouse line. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.

Central Ceramide Signaling Mediates Obesity-Induced Precocious Puberty.

Childhood obesity, especially in girls, is frequently bound to earlier puberty, which is linked to higher disease burden later in life. The mechanisms underlying this association remain elusive. Here we show that brain ceramides participate in the control of female puberty and contribute to its alteration in early-onset obesity in rats. Postnatal overweight caused earlier puberty and increased hypothalamic ceramide content, while pharmacological activation of ceramide synthesis mimicked the pubertal advancement caused by obesity, specifically in females. Conversely, central blockade of de novo ceramide synthesis delayed puberty and prevented the effects of the puberty-activating signal, kisspeptin. This phenomenon seemingly involves a circuit encompassing the paraventricular nucleus (PVN) and ovarian sympathetic innervation. Early-onset obesity enhanced PVN expression of SPTLC1, a key enzyme for ceramide synthesis, and advanced the maturation of the ovarian noradrenergic system. In turn, obesity-induced pubertal precocity was reversed by virogenetic suppression of SPTLC1 in the PVN. Our data unveil a pathway, linking kisspeptin, PVN ceramides, and sympathetic ovarian innervation, as key for obesity-induced pubertal precocity.

Tetrahydrocannabinolic acid A (THCA-A) reduces adiposity and prevents metabolic disease caused by diet-induced obesity.

Medicinal cannabis has remarkable therapeutic potential, but its clinical use is limited by the psychotropic activity of Δ9-tetrahydrocannabinol (Δ9-THC). However, the biological profile of the carboxylated, non-narcotic native precursor of Δ9-THC, the Δ9-THC acid A (Δ9-THCA-A), remains largely unexplored. Here we present evidence that Δ9-THCA-A is a partial and selective PPARγ modulator, endowed with lower adipogenic activity than the full PPARγ agonist rosiglitazone (RGZ) and enhanced osteoblastogenic effects in hMSC. Docking and in vitro functional assays indicated that Δ9-THCA-A binds to and activates PPARγ by acting at both the canonical and the alternative sites of the ligand-binding domain. Transcriptomic signatures in iWAT from mice treated with Δ9-THCA-A confirmed its mode of action through PPARγ. Administration of Δ9-THCA-A in a mouse model of HFD-induced obesity significantly reduced fat mass and body weight gain, markedly ameliorating glucose intolerance and insulin resistance, and largely preventing liver steatosis, adipogenesis and macrophage infiltration in fat tissues. Additionally, immunohistochemistry, transcriptomic, and plasma biomarker analyses showed that treatment with Δ9-THCA-A caused browning of iWAT and displayed potent anti-inflammatory actions in HFD mice. Our data validate the potential of Δ9-THCA-A as a low adipogenic PPARγ agonist, capable of substantially improving the symptoms of obesity-associated metabolic syndrome and inflammation.

Gonadal hormone-dependent vs. -independent effects of kisspeptin signaling in the control of body weight and metabolic homeostasis.

BACKGROUND: Kisspeptins, encoded by Kiss1, have emerged as essential regulators of puberty and reproduction by primarily acting on GnRH neurons, via their canonical receptor, Gpr54. Mounting, as yet fragmentary, evidence strongly suggests that kisspeptin signaling may also participate in the control of key aspects of body energy and metabolic homeostasis. However, characterization of such metabolic dimension of kisspeptins remains uncomplete, without an unambiguous discrimination between the primary metabolic actions of kisspeptins vs. those derived from their ability to stimulate the secretion of gonadal hormones, which have distinct metabolic actions on their own. In this work, we aimed to tease apart primary vs. secondary effects of kisspeptins in the control of key aspects of metabolic homeostasis using genetic models of impaired kisspeptin signaling and/or gonadal hormone status. METHODS: Body weight (BW) gain and composition, food intake and key metabolic parameters, including glucose tolerance, were comparatively analyzed, in lean and obesogenic conditions, in mice lacking kisspeptin signaling due to global inactivation of Gpr54 (displaying profound hypogonadism; Gpr54-/-) vs. Gpr54 null mice with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54-/-Tg), where kisspeptin signaling elsewhere than in GnRH neurons is ablated but gonadal function is preserved. RESULTS: In male mice, global elimination of kisspeptin signaling resulted in decreased BW, feeding suppression and increased adiposity, without overt changes in glucose tolerance, whereas Gpr54-/- female mice displayed enhanced BW gain at adulthood, increased adiposity and perturbed glucose tolerance, despite reduced food intake. Gpr54-/-Tg rescued mice showed altered postnatal BW gain in males and mildly perturbed glucose tolerance in females, with intermediate phenotypes between control and global KO animals. Yet, body composition and leptin levels were similar to controls in gonadal-rescued mice. Exposure to obesogenic insults, such as high fat diet (HFD), resulted in exaggerated BW gain and adiposity in global Gpr54-/- mice of both sexes, and worsening of glucose tolerance, especially in females. Yet, while rescued Gpr54-/-Tg males displayed intermediate BW gain and feeding profiles and impaired glucose tolerance, rescued Gpr54-/-Tg females behaved as controls, except for a modest deterioration of glucose tolerance after ovariectomy. CONCLUSION: Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. SUMMARY OF TRANSLATIONAL RELEVANCE: Kisspeptins, master regulators of reproduction, may also participate in the control of key aspects of body energy and metabolic homeostasis; yet, the nature of such metabolic actions remains debatable, due in part to the fact that kisspeptins modulate gonadal hormones, which have metabolic actions on their own. By comparing the metabolic profiles of two mouse models with genetic inactivation of kisspeptin signaling but different gonadal status (hypogonadal vs. preserved gonadal function), we provide herein a systematic dissection of gonadal-dependent vs. -independent metabolic actions of kisspeptins. Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. These data pave the way for future analyses addressing the eventual contribution of altered kisspeptin signaling in the development of metabolic alterations, especially in conditions linked to reproductive dysfunction.

VCE-004.8, A Multitarget Cannabinoquinone, Attenuates Adipogenesis and Prevents Diet-Induced Obesity.

Over the past few years, the endocannabinoid system (ECs) has emerged as a crucial player for the regulation of food intake and energy metabolism, and its pharmacological manipulation represents a novel strategy for the management of metabolic diseases. The discovery that VCE-004.8, a dual PPARγ and CB2 receptor agonist, also inhibits prolyl-hydroxylases (PHDs) and activates the HIF pathway provided a rationale to investigate its effect in in vitro models of adipogenesis and in a murine model of metabolic syndrome, all processes critically regulated by these targets of VCE-004.8. In accordance with its different binding mode to PPARγ compared to rosiglitazone (RGZ), VCE-004.8 neither induced adipogenic differentiation, nor affected osteoblastogenesis. Daily administration of VCE-004.8 (20 mg/kg) to HFD mice for 3-wks induced a significant reduction in body weight gain, total fat mass, adipocyte volume and plasma triglycerides levels. VCE-004.8 could also significantly ameliorate glucose tolerance, reduce leptin levels (a marker of adiposity) and increase adiponectin and incretins (GLP-1 and GIP) levels. Remarkably, VCE-004.8 increased the FGF21 mRNA expression in white and brown adipose, as well as in a BAT cell line, qualifying cannabinoaminoquinones as a class of novel therapeutic candidates for the management of obesity and its common metabolic co-morbidities.

Sex-Biased Physiological Roles of NPFF1R, the Canonical Receptor of RFRP-3, in Food Intake and Metabolic Homeostasis Revealed by its Congenital Ablation in mice.

BACKGROUND: RF-amide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibiting hormone, operates as inhibitory signal for the reproductive axis. Recently, RFRP-3 has been also suggested to stimulate feeding, and therefore might contribute to the control of body weight and its alterations. Yet, characterization of the metabolic actions of RFRP-3 has been so far superficial and mostly pharmacological. Here, we aim to investigate the physiological roles of RFRP-3 signaling in the control of feeding and metabolic homeostasis using a novel mouse model of genetic ablation of its canonical receptor, NPFF1R. METHODS: Food intake, body weight gain and composition, and key metabolic parameters, including glucose tolerance and insulin sensitivity, were monitored in mice with constitutive inactivation of NPFF1R. RESULTS: Congenital elimination of NPFF1R in male mice resulted in changes in feeding patterns, with a decrease in spontaneous food intake and altered responses to leptin and ghrelin: leptin-induced feeding suppression was exaggerated in NPFF1R null mice, whereas orexigenic responses to ghrelin were partially blunted. Concordant with this pro-anorectic phenotype, hypothalamic expression of Pomc was increased in NPFF1R null mice. In contrast, spontaneous feeding and neuropeptide expression remained unaltered in NPFF1R KO female mice. Despite propensity for reduced feeding, ablation of NPFF1R signaling in male mice did not cause overt alterations in body weight (BW) gain or composition, neither it affected BW responses to high fat diet (HFD), total energy expenditure or RQ ratios. Yet, NPFF1R KO males showed a decrease in locomotor activity. Conversely, NPFF1R null female mice tended to be heavier and displayed exaggerated BW increases in response to obesogenic insults, such as HFD or ovariectomy. These were associated to increased fat mass, decreased total energy expenditure in HFD, and unaltered RQ ratios or spontaneous locomotor activity. Finally, lack of NPFF1R signaling worsened the metabolic impact of HFD on glycemic homeostasis in males, as revealed by impaired glucose tolerance and insulin sensitivity, while female mice remained unaffected. CONCLUSION: Our data support a discernible orexigenic role of NPFF1R signaling selectively in males, which might modulate the effects of leptin and ghrelin on food intake. In addition, our study is the first to disclose the sex-biased, deleterious impact of the lack of NPFF1R signaling on body weight and fat composition, energy expenditure, locomotor activity and glucose balance, which exaggerates some of the metabolic consequences of concurrent obesogenic insults, such as HFD, in a sexually dimorphic manner. SUMMARY OF TRANSLATIONAL RELEVANCE: Our data are the first to document the nature and magnitude of the regulatory actions of RFRP-3/NPFF1R signaling in the control of feeding and metabolic homeostasis in a physiological setting. Our results not only suggest an orexigenic action of endogenous RFRP-3, specifically in males, but reveal also the detrimental impact of ablation of NPFF1R signaling on body composition, energy expenditure, locomotor activity or glucose balance, especially when concurrent with other obesogenic insults, as HFD, thereby providing the first evidence for additional metabolic effects of RFRP-3, other that the mere control of feeding. Interestingly, alterations of such key metabolic parameters occurred in a sex-biased manner, with males being more sensitive to deregulation of locomotor activity and glycemic control, while females displayed clearer obesogenic responses and deregulated energy expenditure. While our study cannot discard the possibility of RFRP-3 actions via alternative pathways, such as NPFF2R, our data pave the way for future analyses addressing the eventual contribution of altered RFRP-3/NPFF1R signaling in the development of metabolic alterations (including obesity and its comorbidities), especially in conditions associated to reproductive dysfunction.

Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways.

Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes.

Discussion of the influence of CO and CH4 in CO2 transport, injection, and storage for CCS technology.

This paper discusses the influence of the noncondensable impurities CO and CH4 on Carbon Capture and Storage (CCS) technology. We calculated and drew conclusions about the impact of both impurities in the CO2 on selected transport, injection, and storage parameters (pipeline pressure drop, storage capacity, etc.), whose analysis is necessary for the safe construction and operation of CO2 pipelines and for the secure long-term geological storage of anthropogenic CO2. To calculate these parameters, it is necessary to acquire data on the volumetric properties and the vapor-liquid equilibrium of the fluid being subjected to CCS. In addition to literature data, we used new experimental data, which are presented here and were obtained for five mixtures of CO2+CO with compositions characteristic of the typical emissions of the E.U. and the U.S.A. Temperatures and pressures are based on relevant CO2 pipeline and geological storage site values. From our experimental results, Peng-Robinson, PC-SAFT, and GERG Equations of State for were validated CO2+CO under the conditions of CCS. We conclude that the concentration of both impurities strongly affects the studied parameters, with CO being the most influential and problematic. The overall result of these negative effects is an increase in the difficulties, risks, and overall costs of CCS.

Influence of methane in CO2 transport and storage for CCS technology.

CO(2) Capture and Storage (CCS) is a good strategy to mitigate levels of atmospheric greenhouse gases. The type and quantity of impurities influence the properties and behavior of the anthropogenic CO(2), and so must be considered in the design and operation of CCS technology facilities. Their study is necessary for CO(2) transport and storage, and to develop theoretical models for specific engineering applications to CCS technology. In this work we determined the influence of CH(4), an important impurity of anthropogenic CO(2), within different steps of CCS technology: transport, injection, and geological storage. For this, we obtained new pressure-density-temperature (PρT) and vapor-liquid equilibrium (VLE) experimental data for six CO(2) + CH(4) mixtures at compositions which represent emissions from the main sources in the European Union and United States. The P and T ranges studied are within those estimated for CO(2) pipelines and geological storage sites. From these data we evaluated the minimal pressures for transport, regarding the density and pipeline's capacity requirements, and values for the solubility parameter of the mixtures, a factor which governs the solubility of substances present in the reservoir before injection. We concluded that the presence of CH(4) reduces the storage capacity and increases the buoyancy of the CO(2) plume, which diminishes the efficiency of solubility and residual trapping of CO(2), and reduces the injectivity into geological formations.

Accurate values of some thermodynamic properties for carbon dioxide, ethane, propane, and some binary mixtures.

Quasicontinuous PρT data of CO(2), ethane, propane, and the [CO(2) + ethane] mixture have been determined along subcritical, critical, and supercritical regions. These data have been used to develop the optimal experimental method and to determine the precision of the results obtained when using an Anton Paar DMA HPM vibrating-tube densimeter. A comparison with data from reference EoS and other authors confirm the quality of our experimental setup, its calibration, and testing. For pure compounds, the value of the mean relative deviation is MRD(ρ) = 0.05% for the liquid phase and for the extended critical and supercritical region. For binary mixtures the mean relative deviation is MRD(ρ) = 0.70% in the range up to 20 MPa and MRD(ρ) = 0.20% in the range up to 70 MPa. The number of experimental points measured and their just quality have enable us to determine some derivated properties with satisfactory precision; isothermal compressibilities, κ(T), have been calculated for CO(2) and ethane (MRD(κ(T)) = 1.5%), isobaric expasion coefficients, α(P), and internal pressures, π(i), for CO(2) (MRD(α(P)) = 5% and MRD(π(i)) = 7%) and ethane (MRD(α(P)) = 7.5% and MRD(π(i)) = 8%). An in-depth discussion is presented on the behavior of the properties obtained along subcritical, critical, and supercritical regions. In addition, PuT values have been determined for water and compressed ethane from 273.19 to 463.26 K up to pressures of 190.0 MPa, using a device based on a 5 MHz pulsed ultrasonic system (MRD(u) = 0.1%). With these data we have calibrated the apparatus and have verified the adequacy of the operation with normal liquids as well as with some compressed gases. From density and speed of sound data of ethane, isentropic compressibilities, κ(s), have been obtained, and from these and our values for κ(T) and α(P), isobaric heat capacities, C(p), have been calculated with MRD(C(p)) = 3%, wich is within that of the EoS.

Volumetric behavior of the {CO2 (1) + C2H6 (2)} system in the subcritical (T = 293.15 K), critical, and supercritical (T = 308.15 K) regions.

The volumetric behavior for the {CO2 (1) + C2H6 (2)} system has been studied. Density measurements of {CO2 (1) + C2H6 (2)} binary mixtures at 293.15 and 308.15 K, at several pressures and compositions, and density measurements for infinitely dilute solutions at 304.21 and 308.15 K were carried out using an Anton Paar DMA 512-P vibrating-tube densimeter calibrated with the forced path mechanical calibration model. The mean relative standard deviation of density, s(rho)(r), was estimated to be better than 0.1%, and the uncertainties in temperature and pressure were estimated as +/-0.01 K and +/-0.001 MPa, respectively. In the experimental setup, an uncertainty in the mole fraction of u(x(j)) = +/-0.0015 has been achieved. Other properties related to P-rho-T-x data such as the compressibility factor, Z, excess molar volumes, V(m)(E), and partial molar volumes, V(i) and V(i)(infinity) have been calculated. The volumetric behavior has been compared with literature data and with that obtained from the PC-SAFT EoS rescaled parameters; these parameters have been obtained from our previous experimental values for the critical temperature and pressure of pure compounds. The value for the Krichevskii parameter, A(Kr), was obtained from the experimental density data for infinitely dilute solutions measured in this work, and it has been compared with that obtained from critical properties. Structural properties such as direct and total correlation function integrals and cluster size were calculated using the Krichevskii function concept.

Critical properties and high-pressure volumetric behavior of the carbon dioxide+propane system at T=308.15 k. Krichevskii function and related thermodynamic properties.

Critical properties and volumetric behavior for the {CO2(1)+C3H8(2)} system have been studied. The critical locus was measured with a flow apparatus and detected by critical opalescence. For the mixtures, repeatabilities in critical temperature and pressure are rTc