Disordered eating in early childhood: DRD4 and DAT1 gene polymorphisms and quality of mother-child interaction.

PURPOSE: Eating disturbances are complex heritable conditions that can be influenced by both genetic and environmental factors but are poorly studied in early development. The aim of this research was to investigate the association of genetic polymorphisms within dopaminergic pathways with early feeding problems. METHODS: We analyzed the presence of VNTR polymorphisms of DRD4 (rs1805186) and DAT1 (rs28363170) in overeating (N = 45), undereating (N = 48) and control (N = 44) young children. We also assessed presence of externalizing, internalizing and dysregulation symptoms by the Child Behavior Checklist and quality of mother-child interactions during feeding by the Italian adaptation of the Scale for the Assessment of Feeding Interaction, respectively. RESULTS: Both polymorphisms were associated with children's eating behavior, psychological symptoms and quality of interaction with their mothers, suggesting that: (a) the DRD4 4-repeat allele behaves as a protective factor, the 2-repeats and 7-repeats alleles as risk factors, for undereating behavior, the general quality of mother-child interaction and internalizing, externalizing and dysregulated symptoms; and (b) the DAT1 9-repeats allele behaves as a protective factor, the 10-repeats allele as a risk factor, for overeating behavior, the general quality of mother-child interaction, internalizing, externalizing and dysregulated symptoms. Finally, a gene x gene interaction is suggested between the DAT1 9-repeat or 10-repeat allele and the DRD4 4-repeat allele. CONCLUSIONS: Our results suggest a role for DRD4 and DAT1 in an early susceptibility to eating disturbances. LEVEL OF EVIDENCE III: Evidence obtained from well-designed case-control analytic study.

Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries.

Administration of 1000-1500 mg/day D-Chiro-Inositol (DCIns) or a combination of Myo-Inositol (MyoIns) and DCIns in their plasma molar ratio (40:1) for three or more months are among recommended treatments for metabolic syndrome and/or Polycystic Ovary Syndrome (PCOS). We previously confirmed the efficacy of this formulation (8.2 mg/day MyoIns and 0.2 mg/day DCIns for 10 days) in a mouse PCOS model, but also observed negative effects on ovarian histology and function of formulations containing 0.4-1.6 mg/day DCIns. We therefore analyzed effects of higher doses of DCIns, 5, 10 and 20 mg/day, administered to young adult female mice for 21 days, on ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. Five mg/day DCIns (human correspondence: 1200 mg/day) altered ovarian histology, increased serum testosterone levels and reduced the amount of aromatase of negative controls, suggesting the induction of an androgenic PCOS model. In contrast, 10-20 mg/day DCIns (human correspondence: 2400-4800 mg/day) produced ovarian lesions resembling those typical of aged mice, and reduced serum testosterone levels without affecting aromatase amounts, suggesting a failure in steroidogenic gonadal activity. Notwithstanding physiological/biochemical differences between mice and humans, the observed pictures of toxicity for ovarian histology and function recommend caution when administering DCIns to PCOS patients at high doses and/or for periods spanning several ovulatory cycles.

Experts' opinion on inositols in treating polycystic ovary syndrome and non-insulin dependent diabetes mellitus: a further help for human reproduction and beyond.

Introduction: This Experts' opinion provides an updated scientific support to gynecologists, obstetricians, endocrinologists, nutritionists, neurologists and general practitioners on the use of Inositols in the therapy of Polycystic Ovary Syndrome (PCOS) and non-insulin dependent (type 2) diabetes mellitus (NIDDM).Areas covered: This paper summarizes the physiology of Myo-Inositol (MI) and D-Chiro-Inositol (DCI), two important molecules present in human organisms, and their therapeutic role, also for treating infertility. Some deep differences between the physiological functions of MI and DCI, as well as their safety and intestinal absorption are discussed. Updates include new evidence on the efficacy exerted in PCOS by the 40:1 MI/DCI ratio, and the innovative approach based on alpha-lactalbumin to overcome the decreased therapeutic efficacy of Inositols in some patients.Expert opinion: The evidence suggests that MI, alone or with DCI in the 40:1 ratio, offers a promising treatment for PCOS and NIDDM. However, additional studies need to evaluate some still unresolved issues, such as the best MI/DCI ratio for treating NIDDM, the potential cost-effectiveness of reduced gonadotropins administration in IVF due to MI treatment, or the benefit of MI supplementation in ovulation induction with clomiphene citrate in PCOS patients.

Protein kinase Akt2/PKBß is involved in blastomere proliferation of preimplantation mouse embryos.

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.

The interplay between TGF-ß-stimulated TSC22 domain family proteins regulates cell-cycle dynamics in medulloblastoma cells.

Proteins belonging to the TGFß-stimulated clone 22 domain (TSC22D) family display a repertoire of activities, regulating cell proliferation and differentiation. The tumor suppressor activity of the first identified member of the family, TSC22D1 (formerly named TSC-22), has been extensively studied, but afterward a longer isoform encoded by the same gene turned out to play an opposite role. We have previously characterized the role of TSC22D1 and TSC22D4 in cell differentiation using granule neurons (GNs) isolated from the mouse cerebellum. However, the possibility to study the role of these factors in cell proliferation was limited by the fact that GNs readily exit from the cell-cycle and differentiate upon isolation and in vitro culture. To overcome this limitation, we have now exploited DAOY medulloblastoma cells, which are ontogenetically similar to cerebellar GNs and can be efficiently transfected with interfering RNA for gene knockdown purposes. Our findings indicate that TSC22D4-TSC22D1 short isoform heterodimers are involved in the escape from cell proliferation and exit from the cell-cycle, whereas, the TSC22D1 long isoform is required for cell proliferation, acting independently from TSC22D4. We also show that the silencing of specific expression of TSC22D4 or TSC22D1 isoforms affects the cell-cycle progression. These findings add a novel insight on the function of TSC22D proteins, with particular reference to the tumor suppressor activity of the TSC22D1 short isoform, which is re-framed within the context of a functional interplay with TSC22D4 and the mutually exclusive expression with the TSC22D1 long isoform.

Myo-inositol and D-chiro-inositol (40:1) reverse histological and functional features of polycystic ovary syndrome in a mouse model.

Mice exposed to continuous light undergo functional and histological changes that mimic those of human Polycystic Ovary Syndrome (PCOS). We herein induced the syndrome by exposing 30-day-old females to 10 weeks of permanent light. Ovarian morphology and histology, as well as reproductive parameters (time of observed pregnancy/delivery) were investigated. Ovaries of PCOS-modeled mice showed lack of tertiary follicles and corpora lutea, altered ovarian architecture, and increased thickness of the theca layer. When mice were returned to a normal light-dark regimen for 10 days, a slight, spontaneous improvement occurred, whereas a quick and almost complete recovery from PCOS signs and symptoms was obtained by treating animals with a daily supplementation of 420 mg/kg myo-inositol and D-chiro-inositol (MyoIns/DCIns) in a 40:1 molar ratio. Namely, ovaries from mice treated by this protocol recovered normal histological features and a proper ratio of theca/granulosa cell layer thickness (TGR), suggesting that the androgenic phenotype was efficiently reversed. Indeed, we identified TGR as a useful index of PCOS, as its increase in PCOS-modeled mice correlated linearly with reduced reproductive capability ( r = 0.75, p < 0.0001). Mice treated with a 40:1 formula regained low TGR values and faster recovery of their fertility, with a physiological delivery time after mating. On the other hand, a higher D-chiro-inositol treatment formula, such as MyoIns versus DCIns 5:1, was ineffective or even had a negative effect on clinical-pathological outcomes.

Broad Spectrum Anticancer Activity of Myo-Inositol and Inositol Hexakisphosphate.

Inositols (myo-inositol and inositol hexakisphosphate) exert a wide range of critical activities in both physiological and pathological settings. Deregulated inositol metabolism has been recorded in a number of diseases, including cancer, where inositol modulates different critical pathways. Inositols inhibit pRB phosphorylation, fostering the pRB/E2F complexes formation and blocking progression along the cell cycle. Inositols reduce PI3K levels, thus counteracting the activation of the PKC/RAS/ERK pathway downstream of PI3K activation. Upstream of that pathway, inositols disrupt the ligand interaction between FGF and its receptor as well as with the EGF-transduction processes involving IGF-II receptor and AP-1 complexes. Additionally, Akt activation is severely impaired upon inositol addition. Downregulation of both Akt and ERK leads consequently to NF-kB inhibition and reduced expression of inflammatory markers (COX-2 and PGE2). Remarkably, inositol-induced downregulation of presenilin-1 interferes with the epithelial-mesenchymal transition and reduces Wnt-activation, ß-catenin translocation, Notch-1, N-cadherin, and SNAI1 release. Inositols interfere also with the cytoskeleton by upregulating Focal Adhesion Kinase and E-cadherin and decreasing Fascin and Cofilin, two main components of pseudopodia, leading hence to invasiveness impairment. This effect is reinforced by the inositol-induced inhibition on metalloproteinases and ROCK1/2 release. Overall, these effects enable inositols to remodel the cytoskeleton architecture.

Pharmacodynamics and pharmacokinetics of inositol(s) in health and disease.

INTRODUCTION: Inositol and its derivatives comprise a huge field of biology. Myo-inositol is not only a prominent component of membrane-incorporated phosphatidylinositol, but participates in its free form, with its isomers or its phosphate derivatives, to a multitude of cellular processes, including ion channel permeability, metabolic homeostasis, mRNA export and translation, cytoskeleton remodeling, stress response. AREAS COVERED: Bioavailability, safety, uptake and metabolism of inositol is discussed emphasizing the complexity of interconnected pathways leading to phosphoinositides, inositol phosphates and more complex molecules, like glycosyl-phosphatidylinositols. EXPERT OPINION: Besides being a structural element, myo-inositol exerts unexpected functions, mostly unknown. However, several reports indicate that inositol plays a key role during phenotypic transitions and developmental phases. Furthermore, dysfunctions in the regulation of inositol metabolism have been implicated in several chronic diseases. Clinical trials using inositol in pharmacological doses provide amazing results in the management of gynecological diseases, respiratory stress syndrome, Alzheimer's disease, metabolic syndrome, and cancer, for which conventional treatments are disappointing. However, despite the widespread studies carried out to identify inositol-based effects, no comprehensive understanding of inositol-based mechanisms has been achieved. An integrated metabolomics-genomic study to identify the cellular fate of therapeutically administered myo-inositol and its genomic/enzymatic targets is urgently warranted.

Does myo-inositol effect on PCOS follicles involve cytoskeleton regulation?

Inositol metabolism is severely impaired in follicles obtained from cystic ovaries, leading to deregulated insulin transduction and steroid synthesis. On the contrary, inositol administration to women suffering from polycystic ovary syndrome (PCOS) has been proven to efficiently counteract most of the clinical hallmarks displayed by PCOS patients, including insulin resistance, hyperandrogenism and oligo-amenorrhea. We have recently observed that myo-inositol induces significant changes in cytoskeletal architecture of breast cancer cells, by modulating different biochemical pathways, eventually modulating the epithelial-mesenchymal transition. We hypothesize that inositol and its monophosphate derivatives, besides their effects on insulin transduction, may efficiently revert histological and functional features of cystic ovary by inducing cytoskeleton rearrangements. We propose an experimental model that could address not only whether inositol modulates cytoskeleton dynamics in both normal and cystic ovary cells, but also whether this effect may interfere with ovarian steroidogenesis. A more compelling understanding of the mechanisms of action of inositol (and its derivatives) would greatly improve its therapeutic utilization, by conferring to current treatments a well-grounded scientific rationale.

Physiological role and clinical utility of inositols in polycystic ovary syndrome.

During the last decades, a substantial body of research has focused on the role of the two major inositol (Ins) stereoisomers, myoinositol (myoIns) and D-chiroinositol (D-chiroIns), both second messengers of insulin, in insulin-dependent processes, including polycystic ovary syndrome (PCOS). MyoIns has been shown to affect different pathways at both ovarian and non-ovarian level. On the contrary, D-chiroIns alone is unable to exert significant improvements in the ovarian cell functions, as its beneficial effects are mainly limited to the non-ovarian tissue in which it may significantly inhibit the negative cellular consequences of hyperinsulinemia. However, both Ins isomers can be positively associated in the management of PCOS patients in a ratio corresponding to their physiological plasma ratio (40:1). This appears to exert a synergistic effect according to a multi-targeted design. In this respect, new fundamental insights into the biological mechanisms displayed by Ins, as well as clinical trials based on the myoIns + D-chiroIns formulations, have already provided encouraging results.

Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI).

The combined therapy myo-inositol plus D-chiro-inositol, rather than D-chiro-inositol, is able to improve IVF outcomes: results from a randomized controlled trial.

PURPOSE: The present study aims to investigate the effects of the combined therapy myo-inositol (MI) plus D-chiro-inositol (DCI) or D-chiro-inositol treatment in oocyte quality. METHODS: Polycystic ovary syndrome (PCOS) women undergoing IVF-ET were treated with myo-inositol combined with D-chiro-inositol in the physiological ratio (1.1 g myo-inositol plus 27.6 mg of D-chiro-inositol; INOFOLIC combi Lo.Li.pharma) or D-chiro-inositol alone (500 mg; Interquim, s.a., Barcelona, Spain) to evaluate the umber of morphological mature oocytes, total International Units (IU) of recombinant FSH administered and the number of grade 1 embryos. RESULTS: The data clearly showed that only the combined therapy was able to improve oocyte and embryo quality, as well as pregnancy rates, in PCOS women undergoing IVF-ET. CONCLUSION: The present paper further supports the hypothesis that MI plays a crucial role in the ovary in PCOS women. In particular, due to the physiological role played by MI and DCI, the combined therapy should represent a better choice.

Association of state and trait anxiety to semen quality of in vitro fertilization patients: a controlled study.

OBJECTIVE: To investigate the relationship between semen quality and state/trait anxiety in patients enrolled in an in vitro fertilization (IVF) program and in control subjects. DESIGN: Cross-sectional study. SETTING: Centre for Reproductive Medicine and Biology, European Hospital, Rome. PATIENT(S): Ninety-four first-attempt IVF patients and 85 age-matched, random subjects recruited in the period July 2006 through March 2008. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Behavioral features of stress, including state and trait anxiety, self-perceived impact of physical disturbance on everyday activities, ethanol consumption, cigarette smoking, and semen parameters such as semen volume, sperm concentration, total count, motility, morphology, and DNA fragmentation. RESULT(S): Increased levels of both state and trait anxiety were associated with lower semen volume, sperm concentration and count, reduced sperm motility, and increased sperm DNA fragmentation of IVF patients, thus influencing seminal parameters at the macroscopic and cellular/subcellular levels. Similar results were obtained in the controls. CONCLUSION(S): Our data confirm previous observations with state anxiety and show that trait anxiety also is negatively associated with male fertility.

Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase.

Microinjection in mouse eggs of tr-kit, a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cgamma1 (PLCgamma1) and Ca(2+) mobilization from intracellular stores. We show that the Src-like kinase Fyn phosphorylates Tyr161 in tr-kit and that this residue is essential for tr-kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr-kit and that the interaction requires Tyr161. The interaction between tr-kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCgamma1. Co-injection of tr-kit with the SH2 domain of Fyn, or pre-treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr-kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr-kit but still requires PLC activity. We suggest that the mechanism by which tr-kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCgamma1.

TCL1 participates in early embryonic development and is overexpressed in human seminomas.

Overexpression of the TCL1 oncogene has been shown to play a causative role in T cell leukemias of humans and mice. The characterization of Tcl1-deficient mice in these studies indicates an important developmental role for Tcl1 in early embryogenesis. In wild-type embryos, Tcl1 is abundant in the first three mitotic cycles, during which it shuttles between nuclei and the embryo cortical regions in a cell-cycle-dependent fashion. The absence of this protein in early embryogenesis results in reduced fertility of female mice. The present studies elucidate the mechanism responsible for the reduced female fertility through analysis of the oogenesis stages and early embryo development in Tcl1-deficient mice. Even though Tcl1(-/-) females display normal oogenesis and rates of oocyte maturation/ovulation and fertilization, the lack of maternally derived Tcl1 impairs the embryo's ability to undergo normal cleavage and develop to the morula stage, especially under in vitro culture conditions. Beyond this crisis point, differentiative traits of zygotic genome activation and embryo compaction can take place normally. In contrast with this unanticipated role in early embryogenesis, we observed an overexpression of TCL1 in human seminomas. This finding suggests that TCL1 dysregulation could contribute to the development of this germinal cell cancer as well as lymphoid malignancies.