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

Search for regulatory elements in the ALDH1A2 (RALDH2) gene during development: a philogenetic approach.

O ácido retinóico (AR) é essencial para a embriogênese. A principal enzima sintetizadora de AR durante o desenvolvimento é a ALDH1A2 (RALDH2), uma retinaldeído desidrogenase que converte retinaldeído a AR. Para entendermos como o gene da aldh1a2 é regulado identificamos regiões evolutivamente conservadas (ECRs) em vertebrados e testamos seu potencial regulatório. Identificamos uma ECR localizada no intron1 da aldh1a2, conservada em anfíbios, aves e mamíferos que atua como um enhancer em estruturas derivadas de ectoderme, endoderme e mesoderme. Animais transgênicos transientes e permanentes mostram a ativação desse enhancer na região da placa do teto do tubo neural e epicárdio, local onde esse enhancer é ativado em células derivadas do órgão pro-epicárdico após o contato e/ou proximidade com células do miocárdio. A identificação de um enhancer conservado no gene da aldh1a2 suporta a idéia de que esse gene possui uma regulação modular e mostra que a abordagem evolutiva é uma eficiente ferramenta para a identificação de mecanismos de controle desse gene.Retinoic acid (RA) is essential for embryogenesis. The key RA synthetic enzyme during early development is ALDH1A2 (RALDH2), a retinaldehyde dehydrogenase that converts retinaldehyde into RA. To understand how aldh1a2 is regulated we screened the gene for evolutionary conserved regions (ECRs) among vertebrates and assayed their regulatory potential. We describe an aldh1a2 intron 1 ECR (identified as RALDH2.2) that is conserved in amphibians, avians and humans and acts as an enhancer in derivatives of ectoderm, endoderm and mesoderm. Transient and stable transgenesis in mice reveal strong activity of the raldh2 intron 1 enhancer at the roof plate of the neural tube and at the growing epicardium. Transgenic mice indicate that the enhancer is activated in proepicardium-derived cells by contact and/or close proximity to the myocardium. The identification of an aldh1a2 conserved enhancer supports the idea of a modular regulation and shows that the evolutionary approach is an efficient tool to identify control mechanisms of the aldh1a2 gene

Methane mitigation and ruminal fermentation changes in cows fed cottonseed and vitamin E

Inherently, ruminant production of methane (CH4), a greenhouse gas (GHG), causes animal energy losses. Cottonseed is a lipid source and is used sometimes to enhance energy density in cattle diets. It also can mitigate enteric CH4. Lipids release peroxides in the rumen, and antioxidants have the ability to neutralize them. Thus, a lipid and antioxidant source can benefit rumen fermentation. The aim of this study was to evaluate rumen fermentation parameters from cows fed cottonseed and vitamin E. Six cannulated cows were arranged in a replicate 3 × 3 latin square. Treatments were: 1) Control, 2) CS (30 % corn replaced by cottonseed) and 3) CSVitE (30 % corn replaced by cottonseed, plus 500 IU VitE). Results were compared by orthogonal contrast. When compared to the control diet, cottonseed inclusion reduced enteric CH4 emissions by 42 %. Production of acetate, butyrate and the acetate to propionate ratio were respectively 34 %, 47 % and 36 % lower with the cottonseed treatments. Energy lost in the rumen as CH4 and energy release as butyrate were reduced by 26 % and 32 % respectively. Propionate and intestinal energy release were, respectively, 43 % and 35 % higher with cottonseed treatments. Furthermore, as a nutritional strategy to mitigate enteric CH4, cottonseed has positive effects on short chain fatty acid (SCFA) production and gastrointestinal energy release. Vitamin E did not result in improvements in ruminal fermentation. Further studies evaluating levels of vitamin E in association with different amounts and sources of lipids are required

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