Elevated circulating levels of succinate in human obesity are linked to specific gut microbiota

Gut microbiota-related metabolites are potential clinical biomarkers for cardiovascular disease (CVD). Circulating succinate, a metabolite produced by both microbiota and the host, is increased in hypertension, ischemic heart disease, and type 2 diabetes. We aimed to analyze systemic levels of succinate in obesity, a major risk factor for CVD, and its relationship with gut microbiome. We explored the association of circulating succinate with specific metagenomic signatures in cross-sectional and prospective cohorts of Caucasian Spanish subjects. Obesity was associated with elevated levels of circulating succinate concomitant with impaired glucose metabolism. This increase was associated with specific changes in gut microbiota related to succinate metabolism: a higher relative abundance of succinate-producing Prevotellaceae (P) and Veillonellaceae (V), and a lower relative abundance of succinate-consuming Odoribacteraceae (O) and Clostridaceae (C) in obese individuals, with the (P + V/O + C) ratio being a main determinant of plasma succinate. Weight loss intervention decreased (P + V/O + C) ratio coincident with the reduction in circulating succinate. In the spontaneous evolution after good dietary advice, alterations in circulating succinate levels were linked to specific metagenomic signatures associated with carbohydrate metabolism and energy production with independence of body weight change. Our data support the importance of microbe-microbe interactions for the metabolite signature of gut microbiome and uncover succinate as a potential microbiota-derived metabolite related to CVD risk

Functional role for a CCAAT-binding factor in the proximal promoter of European sea bass kiss2 gene

Resumen del trabajo presentado en el 9º Congreso de la Asociación Ibérica de Endocrinología Comparada, celebrado en Barcelona (España), los días 13 y 14 de julio de 2013Kisspeptins, encoded by the Kiss1 gene are key modulators of reproduction in mammals via stimulation of GnRH neurons. In other vertebrate species, including European sea bass (Dicentrarchus labrax), two distinct genes encoding kisspeptins have been described, namely kiss1 and kiss2. Studies in sea bass and zebrafish, including in vivo kisspeptin administration, in situ hybridization and immunohistochemistry, have shown that kiss2 could have a more prominent than kiss1 in stimulating the gonadotropic axis. In addition, knowledge on the transcriptional regulation of the kiss genes could also help to understand their roles in fish. In mammals, there are several studies on the promoter of KiSS1. However, there is no functional evidence about the transcription factors involved in the regulation of the kiss genes in fish. In this study, we have isolated 3 Kb of the 5' flanking region of sea bass kiss2. The sequence of this fragment has been compared with putative promoter sequences of kiss2 from several fish, and further analyzed in silico for conserved regulatory elements. Based on this analysis, different kiss2 promoter fragments have been functionally tested for their ability to direct luciferase expression in CHO and HEK 293 cells. Our data indicate that less than 1 kb promoter is needed to direct maximal luciferase expression in the mentioned cell lines. Further co-transfections of promoter fragments with different predicted transcription factors showed that the CCAAT/enhancer-binding protein alpha and the heterotrimeric transcription factor NFY are able to bind to a conserved proximal CCAAT-box, inducing opposite effects on the transcriptional activity of the gene. Although further studies are needed, these results suggest a prominent role of this CCAAT binding sequence in the regulation of kiss2 expression in sea bass.Supported by MICINN (AGL2011-28890), EU (LIFECYCLE, FP7-222719-1) and GV (REPROBASS, PROMETEO/2010/003 and ACOMP/2013/085

Expression of kisspeptins and kiss receptors suggests a large range of functions for kisspeptin systems in the brain of the european sea bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts. © 2013 Escobar et al.Peer Reviewe

PBX1 acts as terminal selector for olfactory bulb dopaminergic neurons

15 páginas, 8 figuras. Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.186841.supplementalNeuronal specification is a protracted process that begins with the commitment of progenitor cells and culminates with the generation of mature neurons. Many transcription factors are continuously expressed during this process but it is presently unclear how these factors modify their targets as cells transition through different stages of specification. In olfactory bulb adult neurogenesis, the transcription factor PBX1 controls neurogenesis in progenitor cells and the survival of migrating neuroblasts. Here, we show that, at later differentiation stages, PBX1 also acts as a terminal selector for the dopaminergic neuron fate. PBX1 is also required for the morphological maturation of dopaminergic neurons and to repress alternative interneuron fates, findings that expand the known repertoire of terminal-selector actions. Finally, we reveal that the temporal diversification of PBX1 functions in neuronal specification is achieved, at least in part, through the dynamic regulation of alternative splicing. In Caenorhabditis elegans, PBX/CEH-20 also acts as a dopaminergic neuron terminal selector, which suggests an ancient role for PBX factors in the regulation of terminal differentiation of dopaminergic neurons.This work was supported by PhD fellowships from the Ministerio de Educación, Cultura y Deporte (FPU16/02008 to I.R.-B.) and the Ministerio de Economıa y ́Competitividad (BES-2012-053847 to L.R.. and BES-2015-072799 to R.B.-R.); by European Research Council grant (ERC-StG-2011-281920); by Ministerio de Economıa y Competitividad grants (SAF2017-84790-R and SAF2016-75004R) and ́ by the Generalitat Valenciana (PROMETEO/2018/055).Peer reviewe

Additional file 2: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Tutorial for the bioinformatics including sgRNA/primer deisgn and MiSeq analysis. (PDF 7778 kb

Additional file 5: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Results from KASP genotyping used for Fig. 4d. (TXT 910 bytes

Additional file 4: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Amplicon screening data used for Fig. 4a-c. (TXT 142 kb

Additional file 7: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Code used to produce the plot in Figs. 4 and 5, Additional file 3: Figure S1. (TXT 14 kb

Additional file 3: Figure S1 and Figure S2. of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Comparison of induced indels between germline and somatic tissues. (a) Plot shows the same data as Fig. 4a, split into separate plots for each sgRNA. (b) Plot of frequencies for individual variants in sperm versus fin clip (same data as in Fig. 4b with full axes). (PDF 374 kb

Transverse sections showing expression of <i>kiss-R2</i> messengers-expressing cells in the hypothalamus of sea bass.

<p>(A) <i>Kiss-R2</i> messengers expressing cells (arrowheads) in the nucleus anterioris tuberis (NAT) and the anterior nucleus lateralis tuberis (NLTa). Bar = 100 µm. (B–D) Strong expression of the <i>kiss-R2</i> mRNAs at different levels of the nucleus of the lateral recess (NRL) surrounding the lateral recess (rl). B: Bar = 75 µm; C: Bar = 75 µm; D Bar = 75 µm. (E–F) <i>Kiss-R2</i> messengers expressing cells (arrowheads) in the caudal nucleus lateralis tuberis (NLT) and in the nucleus of the posterior recess (NRP) surrounding the posterior recess (rp). E: Bar = 75 µm; F: Bar = 100µm.</p