Leptin signaling in Kiss1 neurons arises after pubertal development

The adipocyte-derived hormone leptin is required for normal pubertal maturation in mice and humans and, therefore, leptin has been recognized as a crucial metabolic cue linking energy stores and the onset of puberty. Several lines of evidence have suggested that leptin acts via kisspeptin expressing neurons of the arcuate nucleus to exert its effects. Using conditional knockout mice, we have previously demonstrated that deletion of leptin receptors (LepR) from kisspeptin cells cause no puberty or fertility deficits. However, developmental adaptations and system redundancies may have obscured the physiologic relevance of direct leptin signaling in kisspeptin neurons. To overcome these putative effects, we re-expressed endogenous LepR selectively in kisspeptin cells of mice otherwise null for LepR, using the Cre-loxP system. Kiss1-Cre LepR null mice showed no pubertal development and no improvement of the metabolic phenotype, remaining obese, diabetic and infertile. These mice displayed decreased numbers of neurons expressing Kiss1 gene, similar to prepubertal control mice, and an unexpected lack of re-expression of functional LepR. To further assess the temporal coexpression of Kiss1 and Lepr genes, we generated mice with the human renilla green fluorescent protein (hrGFP) driven by Kiss1 regulatory elements and crossed them with mice that express Cre recombinase from the Lepr locus and the R26-tdTomato reporter gene. No coexpression of Kiss1 and LepR was observed in prepubertal mice. Our findings unequivocally demonstrate that kisspeptin neurons are not the direct target of leptin in the onset of puberty. Leptin signaling in kisspeptin neurons arises only after completion of sexual maturation.National Institutes of Health, R01HD061539National Institutes of Health, R01HD69702National Institutes of Health, R01DA024680National Institutes of Health, R01MH085298National Institutes of Health, K01DK087780National Institutes of Health, DK081182-01National Institutes of Health, UL1RR02492

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract\ud \ud \ud \ud Background\ud \ud Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.\ud \ud \ud \ud Methods\ud \ud One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.\ud \ud \ud \ud Results\ud \ud We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.\ud \ud \ud \ud Conclusion\ud \ud In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/000090; 00/030722; 01/142381; 02/113402; 03/099982; 04/116068; 04/157044 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/20078. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/00009-0; 00/03072-2; 01/14238-1; 02/11340-2; 03/09998-2; 04/11606-8; 04/15704-4 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/2007-8. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process

Peripheral Chemoreflex Regulates Post-exercise Cardiac Vagal Reactivation in Healthy Humans and Patients with Pulmonary Arterial Hypertension

Sao Paulo Research Foundation, FAPESPUniv Fed Sao Paulo, Div Pulm Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Physiol, Sao Paulo, BrazilUniv Alberta, Div Pulm Med, Edmonton, AB, CanadaUniv Fed Sao Paulo, Div Pulm Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Physiol, Sao Paulo, BrazilFAPESP: 2014/24294-6FAPESP: 2015/22198-2Web of Scienc

Lack of coexpression of leptin receptor (LepR) and Kiss1 before puberty.

<p>A-F. Fluorescence photomicrographs showing the distribution of Kiss1 (hrGFP) and LepR (tdTomato) in prepubertal (21 days of age, P21) (A, C, E) and ovariectomized adult female mice (B, D, F). Note the lack of colocalization of Kiss1 and LepR in prepubertal mice and the higher colocalization rate in ovariectomized adult female mice (arrows indicate dual labeled neurons). Scale bar: A–F  =  200 µm. 3V, third ventricle; ME, median eminence.</p

Re-expression of LepR selectively in Kiss1 neurons causes no amelioration of the reproductive or metabolic phenotype of LepR null mice.

<p>A. Agarose gel showing Cre-induced DNA recombination (higher band) of LepR<i>^Lox</i>^TB in the hypothalamus and testis (but not in the white adipose tissue and tail) of Kiss1-Cre LepR null mice. B. Survival graphs showing the progression of vaginal opening and pregnancy in wild type, LepR null and Kiss1-Cre LepR null mice; C. Image comparing the size of the uterus of a wild type female on diestrus and adult Kiss1-Cre LepR null mice; D. Image showing sections of the ovary of a female on diestrus and of an adult Kiss1-Cre LepR null female. Note the presence of corpora lutea (CL) only in the ovary of the wild type female mice. E. Graph showing the progression of body weight of wild type, LepR null and Kiss1-Cre LepR null female mice. F. Bar graphs showing body composition (percentage of fat and lean mass) of LepR null (black) and Kiss1-Cre LepR null (red) mice at 3 different ages: 20 weeks, 35 weeks (males) and 28 weeks (females).</p

Kiss1 neurons are not responsive to leptin before completion of sexual maturation.

<p>A-B. Brightfield and fluorescence photomicrographs showing lack of colocalization of Kiss1-Cre GFP and LepR mRNA (A) or leptin-induced phosphorylation of STAT3 (B) in prepubertal mice. <b>C-H.</b> Validation of Kiss1 human renilla GFP (Kiss1-hrGFP) mouse model. To optimize the detection of Kiss1 mRNA, we performed colocalization studies (Kiss1 mRNA and hrGFP) in ovariectomized (OVX) and OVX estrogen primed (OVX+E2) mice. Virtually all Kiss1 neurons in the anteroventral periventricular nucleus, anterior periventricular nucleus (AVPV/PeN) and arcuate nucleus (Arc) of OVX+E2 and OVX mice respectively coexpressed hrGFP immunoreactivity. C-D, F-G. Brightfield photomicrographs showing distribution of hrGFP immunoreactivity in the AVPV and Arc of OVX (C, F) and OVX+E2 (D, G) mice. Note changes in hrGFP expression due to sex steroids manipulation. E, H. Higher magnification of D and F, respectively (arrows indicate same cells), showing coexpression of hrGFP-ir and Kiss1 mRNA in the AVPV of OVX+E2 mice (E) and in the Arc of OVX mice (H). Scale bar: A-B  =  200 µm; C-D, F-G  =  400 µm; E, H  =  80 µm. 3V, third ventricle.</p

Lack of re-expression of functional LepR in Kiss1-Cre LepR null mice.

<p>A-C. Brightfield photomicrographs showing the distribution of leptin-induced phosphorylation of STAT3 immunoreactivity (pSTAT3-ir) in the arcuate nucleus (Arc) of wild type female mice on diestrus (A) and lack of pSTAT3-ir in the Arc of LepR null (B) and of Kiss1-Cre LepR null (C) adult female mice; <b>D-G.</b> Identification of Kiss1-Cre/GFP cells for whole-cell patch-clamp recordings. (D) Brightfield illumination showing a targeted neuron; (E) the same neuron under fluorescent (FITC) illumination; (F) complete dialysis of AlexaFluor 594 from the intracellular pipette at the end of the recording; (G) colocalization of AlexaFluor 594 and GFP. <b>H</b>. A current-clamp recording demonstrates that leptin (100 nM) depolarizes Kiss1-Cre/GFP neurons. The dashed line indicates the resting membrane potential. Scale bar: A–C  =  400 µm.</p

Prepubertal and leptin signaling-deficient mice display decreased numbers of Kiss1 neurons.

<p>A-F. Fluorescence photomicrographs showing the distribution of Cre activity (Kiss1 reporter gene) in adult (WT on diestrus and Kiss1-Cre LepR null) and prepubertal (WT) female mice. <b>G-I.</b> Bar graphs showing quantification of Kiss1-Cre tdTomato neurons in prepubertal and adult WT female mice and in adult Kiss1-Cre LepR null female mice. Note higher numbers of Kiss1-Cre tdTomato neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc) of adult WT mice on diestrus compared to adult Kiss1-Cre LepR null and prepubertal WT mice. Scale bar: A–F  =  400 µm. 3V, third ventricle.</p

Leptin’s effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons

Studies in humans and rodents indicate that a minimum amount of stored energy is required for normal pubertal development. The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis. Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear. Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons in mice had no effect on puberty or fertility, indicating that direct leptin signaling in Kiss1 neurons is not required for these processes. However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation. Moreover, unilateral reexpression of endogenous LepR in PMV neurons was sufficient to induce puberty and improve fertility in female LepR-null mice. This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency. These data suggest that the PMV is a key site for leptin’s permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction are anatomically dissociated