Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice.

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.

Association between metabolic disorders and seminal plasma miRNA levels: a pilot study.

BACKGROUND: Excess weight and metabolic disorders have a negative impact on male reproductive functions. The mechanisms involved are numerous and complex and epigenetic mechanisms may also be involved, notably through the small non-coding RNAs. Among them, microRNAs (miRNAs) are of particular interest. This preliminary study aimed to identify the miRNAs differentially enriched in seminal plasma related to metabolic disorders and if some are also associated with spermatic parameters alterations. One hundred and sixty men between 18 to 45 years, partners of infertile couple, were included in this cohort. The miRNAs associated with metabolism were selected from the literature and assayed by quantitative real-time PCR using TaqMan gene expression assays. A subset of those with an interesting profile in seminal plasma were secondarily tested in blood. RESULTS: Among the 11 selected miRNAs, seven were detected in seminal plasma (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). A negative correlation was observed between seminal miR19a levels and metabolic syndrome, blood glucose and C-peptide. Seminal miR19b levels were also negatively correlated with metabolic syndrome. Seminal miR34c levels were negatively correlated with body mass index (BMI) and waist circumference. Seminal miR133b levels were positively correlated with BMI, waist circumference and leptin levels. Interestingly, modifications of miRNAs in seminal plasma seem specific since highlighted above correlations were not retrieved in the blood plasma for the miR19a, 19b, 10b, 34c. CONCLUSION: Few metabolic and anthropometric disorders are correlated with the level of specific miRNAs in seminal plasma. Further studies will be required to decipher if other small non-coding RNAs may also be correlated with metabolic and anthropometric disorders and to assess their potential implication in the alteration of reproductive functions in men with obesity or metabolic disorders. CLINICAL STUDY: Metabolic Syndrome and Male Infertility (Metasperme): Trial registration:  NCT01974947 . Registered 18 July 2013.

RéSUMé: CONTEXTE: L'excès de poids et les troubles métaboliques ont un impact négatif sur les fonctions de reproduction masculine. Les mécanismes impliqués sont nombreux et complexes, et des mécanismes épigénétiques peuvent également intervenir, notamment par le biais des petits ARN non codants. Parmi eux, les microRNAs (miRNAs) présentent un intérêt particulier. Cette étude préliminaire visait à identifier les miRNAs différentiellement enrichis dans le plasma séminal en relation avec des troubles métaboliques et si certains étaient également associés à des altérations des paramètres spermatiques. Cent soixante hommes âgés de 18 à 45 ans, partenaires de couple infertile, ont été inclus dans cette cohorte. Les miRNAs associés au métabolisme ont été sélectionnés dans la littérature et analysés par PCR quantitative en temps réel à l'aide de tests d'expression génique TaqMan. Un sous-ensemble de ceux présentant un profil intéressant dans le plasma séminal ont été secondairement testés dans le sang. RéSULTATS: Parmi les 11 miRNAs sélectionnés, sept ont été détectés dans le plasma séminal (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). Une corrélation négative a été observée entre les niveaux du miR19a séminal et le syndrome métabolique, la glycémie et le C-peptide. Les niveaux de miR19b séminaux étaient également corrélés négativement avec le syndrome métabolique. Les niveaux de miR34c séminaux étaient négativement corrélés avec l'IMC et le tour de taille. Les niveaux de miR133b séminaux étaient positivement corrélés avec l'IMC, le tour de taille et les niveaux de leptine. Il est intéressant de noter que les modifications des miRNA dans le plasma séminal semblent spécifiques puisque les corrélations mises en évidence ci-dessus n'ont pas été retrouvées dans le plasma sanguin pour les miR19a, 19b, 10b, 34c. CONCLUSION: Quelques désordres métaboliques et anthropométriques ont été observés corrélés avec le niveau de certains miRNAs dans le plasma séminal. Des études complémentaires sont nécessaires pour déterminer si d'autres petits ARN non codants sont corrélés aux troubles métaboliques et anthropométriques et pour évaluer leur implication potentielle dans l'altération des fonctions de reproduction chez les hommes souffrant d'obésité ou de troubles métaboliques.

Brain IGF-1 receptors control mammalian growth and lifespan through a neuroendocrine mechanism.

Mutations that decrease insulin-like growth factor (IGF) and growth hormone signaling limit body size and prolong lifespan in mice. In vertebrates, these somatotropic hormones are controlled by the neuroendocrine brain. Hormone-like regulations discovered in nematodes and flies suggest that IGF signals in the nervous system can determine lifespan, but it is unknown whether this applies to higher organisms. Using conditional mutagenesis in the mouse, we show that brain IGF receptors (IGF-1R) efficiently regulate somatotropic development. Partial inactivation of IGF-1R in the embryonic brain selectively inhibited GH and IGF-I pathways after birth. This caused growth retardation, smaller adult size, and metabolic alterations, and led to delayed mortality and longer mean lifespan. Thus, early changes in neuroendocrine development can durably modify the life trajectory in mammals. The underlying mechanism appears to be an adaptive plasticity of somatotropic functions allowing individuals to decelerate growth and preserve resources, and thereby improve fitness in challenging environments. Our results also suggest that tonic somatotropic signaling entails the risk of shortened lifespan.

Changes in circulating miRNA19a-3p precede insulin resistance programmed by intra-uterine growth retardation in mice.

OBJECTIVE: Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults, and environmental changes during the perinatal period have been identified as potentially crucial factors. We have studied in a preclinical model early-onset molecular alterations present before the development of a clinical phenotype. METHODS: We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. RESULTS: Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved. CONCLUSIONS: These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine.

Early postnatal nutrition determines somatotropic function in mice.

Increasing evidence suggests a developmental origin for a number of human diseases, notably after intrauterine or postnatal nutrient deprivation. Nutritional changes readily translate into alterations of somatic growth. However, whereas intrauterine growth retardation often shows postnatal catch-up growth, recovery from food restriction immediately after birth is limited. Therefore, we investigated whether early postnatal nutrition (undernutrition and overfeeding) modifies plasticity of growth through developmental control of the somatotropic hormone axis. We used cross-fostering in mice to induce changes in early nutrition, and examined endocrine growth regulation and the development of specific disease phenotypes in adults. We showed that underfeeding during the early postnatal period delayed growth, whereas overfeeding accelerated it. In both cases, final body size was permanently altered. We found coordinated alterations in pituitary GH, plasma IGF-I and acid labile subunit, and gene expression of hypothalamic GHRH during postnatal development. These changes were consistent with the observed phenotypes. Alterations in the somatotropic axis persisted throughout adulthood. Although limited to the early postnatal period, both underfeeding and overfeeding led to reduced glucose tolerance later in life. These metabolic abnormalities were in line with defective insulin secretion in restricted mice and insulin resistance in overfed mice. Moreover, both restricted and overfed mice had increased arterial blood pressure, suggestive of vascular impairment. Our findings indicate a significant link between early postnatal diet, somatotropic development, and specific late onset diseases in mice. We suggest that, together with other hormones like leptin, IGF-I may play a role in modulating hypothalamic stimulation of the developing somatotropic function.

Normal Growth despite Combined Pituitary Hormone Deficiency.

BACKGROUND: The paradox of normal growth despite a lack of growth hormone (GH) is an unexplained phenomenon described in some pathological (sellar, suprasellar, and hypothalamic disorders) and overgrowth syndromes. It has been suggested that the paradoxical growth is due to other GH variants, GH-like moieties, prolactin, insulin, insulin-like growth factors (IGFs), and unidentified serum factors or growth mechanisms. The objective of this study was to determine the mechanism underlying this normal growth without GH. CASE DESCRIPTION: We describe here growth, hormonal, and genetic analyses for an adolescent boy with panhypopituitarism who achieved an adult height above his genetic potential. RESULTS: Normal growth was observed despite low serum GH, IGF-I, IGF-II, IGF binding protein 3 (IGFBP-3) and acid labile subunit (ALS) concentrations, but the IGF-II/IGFBP-3 molar ratio was slightly high. Panhypopituitarism was associated with a heterozygous missense mutation of HESX1, with variable penetrance in heterozygous relatives. Exome analysis detected heterozygous missense mutations of various genes involved in intracellular signaling pathways. The growth-promoting activity of the patient's serum was unable to induce AKT phosphorylation in the MCF-7 cell line. CONCLUSION: The high IGF-II/IGFBP-3 molar ratio was not the cause of the sustained high growth velocity, due to the low affinity of IGF-II for IGF type 1 receptor. The key finding was the HESX1 mutation, as similar cases have been described before, suggesting a common mechanism for growth without GH. However, the variable penetrance of this variant in heterozygous relatives suggests that modifier genes or mechanisms involving combinations with mutations of other genes involved in intracellular signaling pathways might be responsible.

Hepatocyte proliferation during liver regeneration is impaired in mice with liver-specific IGF-1R knockout.

Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles.

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo(R)- and hprt-expression cassettes from three different tri-lox mutants.

IGF-1 signaling reduces neuro-inflammatory response and sensitivity of neurons to MPTP.

Reduced expression of IGF-1R increases lifespan and resistance to oxidative stress in the mouse, raising the possibility that this also confers relative protection against the pro-parkinsonian neurotoxin MPTP, known to involve an oxidative stress component. We used heterozygous IGF-1R(+/-) mice and challenged them with MPTP. Interestingly, MPTP induced more severe lesions of dopaminergic neurons of the substantia nigra, in IGF-1R(+/-) mice than in wild-type animals. Using electron spin resonance, we found that free radicals were decreased in IGF-1R(+/-) mice in comparison with controls, both before and after MPTP exposure, suggesting that the increased vulnerability of dopamine neurons is not caused by oxidative stress. Importantly, we showed that IGF-1R(+/-) mice display a dramatically increased neuro-inflammatory response to MPTP that may ground the observed increase in neuronal death. Microarray analysis revealed that oxidative stress-associated genes, but also several anti-inflammatory signaling pathways were downregulated under control conditions in IGF-1R(+/-) mice compared to WT. Collectively, these data indicate that IGF signaling can reduce neuro-inflammation dependent sensitivity of neurons to MPTP.

c-myc-induced hepatocarcinogenesis in the absence of IGF-I receptor.

Numerous tumours, including hepatocarcinomas, produce IGFs, and some depend on these growth factors in a paracrine or autocrine fashion. We have shown that c-myc-induced experimental hepatocarcinogenesis is associated with enhanced production of IGF-II. To assess the role of the IGF-I receptor (IGF-IR) in hepatocarcinogenesis, we generated conditional mutant mice that overexpressed c-myc and were knocked out for IGF-IR specifically in the liver. We compared these mice with littermate controls that also overexpressed c-myc but had wild-type IGF-IR alleles. We found that the pretumoral phase, induced by early c-myc expression and characterised by increased cell proliferation, was largely unaffected by the lack of IGF-IR. To our further surprise, hepatocellular carcinomas (HCCs) lacking IGF-IR readily developed and progressed at the same rate as control HCCs. At 9 months, all c-myc transgenic mice displayed well-differentiated multifocal tumours, regardless of whether their livers-and their tumours-were able to produce IGF-IR. Levels of IRS-1 and IRS-2 were elevated in all tumours in the presence or absence of IGF-IR, suggesting that the signalling pathway downstream of IGF-IR is activated via IGF-IR-independent mechanisms in HCC. In conclusion, the deregulation of IGF signalling pathways, which often occurs during liver tumorigenesis, does not necessarily require IGF-IRs, and hepatic IGF-IR alone may not play a determinant role in c-myc-induced hepatocarcinogenesis.

IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice.

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.