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Reporting period: 2015-07-01 to 2017-06-30

Metabolic disorders such as obesity and diabetes are age-related diseases, and lead cause of death in Europe. Adiposity signals such as leptin and insulin, whose circulating levels are in proportion to body fat, convey metabolic information to neural networks that regulate energy homeostasis in the hypothalamus. In leptin-deficient humans and mice, leptin administration effectively reduces hyperphagia and obesity. Paradoxically, most cases of obesity display high circulating leptin levels that fail to reduce appetite or increase energy expenditure. This raises the possibility that leptin-receptor-expressing neuronal and/or non-neuronal cells in the hypothalamus either cannot access circulating leptin or are deficient in leptin binding. To reach the arcuate nucleus of the hypothalamus (ARH), leptin must cross the blood-brain barrier at the level of the median eminence (ME) of the hypothalamus. The host laboratory has characterized a particular type of blood-brain barrier in the ME, revealing that tanycytes, a specialized class of glia lining the floor of the third ventricle (3v), possess properties of barrier cells and that they were responsible for shuttling leptin from the periphery and that such conduit was blunted in mice with diet-induced obesity. Thus, leptin transport by tanycytes could play a critical role in the pathophysiology of leptin resistance. Nonetheless, how substances taken up by tanycytes are transported along their processes from their basal end-feet to their apical pole for delivery into the 3v, as well as the molecular mechanisms by what they are secreted for transmission remain unknown. This project aims to elucidate whether the alteration of the adiposity signals transport into the metabolic brain across hypothalamic barriers is the main cause of the onset of obesity, as well as to characterize the transtanycytic route taken by these signals to reach the ARH to regulate energy homeostasis. In addition, we aimed to define the role of microRNAs in this transport. To this end, a combination of in vitro and in vivo approaches, using genetically modified mice and pre-clinical models of obesity, were used. The implementation of this project will expand our knowledge of the mechanism underlying human obesity and hold therapeutic potential for treating it.
In order to characterize the role of leptin signaling and leptin receptors (LepR) in leptin transcytosis in tanycytes and to determine the role of chromogranins in the trafficking of leptin-containing vesicles in tanycytes, the fellow performed cell-sorting experiments using tdTomato reporter mice in which the TAT-Cre fusion protein was infused into the 3v, to selectively targets tanycytes (Annex-Fig.1A-B). Our data showed that purified tanycytes express transcripts for the vesicle-associated membrane proteins VAMP-2, -4 and -7. In addition, tanycytes express different forms of LepR, cubilin and its co-receptor megalin (Annex-Fig.1C-D). Data showed an increase in LepRb transcript in tanycytes after a fasting for 24h, thus suggesting changes in tanycytic receptors according to energy status (Annex-Fig.1E). These results report the functional importance of LepR in leptin signaling and transcytosis in tanycytes. The characterization of the transcytotic route taken by leptin is carrying out in collaboration with the laboratory of the Dr. Stéphane Gasman (CNRS UPR3212, Strasbourg), an expert in intracellular vesicular trafficking. Using specific markers for different intracellular compartments, internalized fluorescent-leptin is followed at different time points of the transcytotic process and leptin-containing compartments can be automatically detected allowing us to calculate their density and distribution and providing a complete road map of the route taken by leptin to reach the apical pole of tanycytes. We aimed also to characterize the role of SNARE in the exocytosis of leptin-containing secretory granules in tanycytes. To block SNARE-dependent exocytosis in tanycytes, the fellow used iBot mice, a transgenic mouse model in which protein VAMP2 is cleaved, and crossed them with the tdTomato line. Animals were injected to induce BoNT/B and Tomato expression in tanycytes and food intake was monitored. Data showed that selective inhibition of SNARE-dependent exocytosis in tanycytes caused differences in food intake (Annex-Fig.2A). A comprehensive analysis of the metabolism using metabolic cages showed differences between the two groups (Annex-Fig.2B-D). These results report the functional importance SNARE complex in the control of leptin trafficking and release in tanycytes. In addition, we are currently studying the involvement of VAMP 1-3 in leptin release in tanycytes expressing leptin coupled to the pH sensitive-GFP, and treated or not with TAT-Cre in the culture medium (Annex-Fig.3). The analysis of leptin levels released by iBot tanycytes will report the importance of VAMP-1-3 in leptin exocytosis. Besides, the fellow investigated the role of miRNAs in the leptin transport in tanycytes. Mice in which Dicer, an essential enzyme for miRNA biogenesis, was selectively inactivated in tanycytes were crossed with tdTomato mice. They were injected to induce Tomato expression and to inactivate Dicer in tanycytes. Data showed that deletion of Dicer in tanycytes caused a decrease of body weight (BW) and food intake (Annex-Fig. 4A-B). Also, this inactivation affect to feeding behaviour and impaired the glucose metabolism (Annex-Fig.4C-D). Moreover, in animals fed with high fat diet for 6 weeks, Dicer inactivation caused an increase in the food intake but not in BW (Annex-Fig.5A-B). Besides, using tanycytes from fed and 24 hours-fasted tdTomato and Dicer; tdTomato mice, the fellow is currently studying which miRNAs are involved in the in the transport of leptin in tanycytes. The exploitation and dissemination of the results has taken place as planned in the original proposal. All the results as well as the new methodology used in the research project was shared with the scientific community through: (i) multidisciplinary seminars and weekly meetings organized by the host group; (ii) International scientific conferences; and it will be also shared through the publication of the results in international peer-review journals with high imp
In addition to the formative strengths acquired by the fellow, the research work realized has allowed to progress towards the achievement of the major scientific goals of this project. The research questions addressed have provided new insights into the mechanisms involved in the transtanycytic route taken by the hormones that signal adiposity to reach the ARH to regulate energy homeostasis and food intake and how the alteration of the transport of these signals into the metabolic brain across hypothalamic barriers could be the main cause of the onset of obesity. Moreover, these provide the potential contribution of microRNAs to this topic. Our results will afford the finding of new biomarkers for early detection of leptin transport alterations linked to obesity onset. Thus, the knowledge obtained will provide essential information for the rational management of the mechanism underlying human obesity, one of the most common, costly and debilitating diseases afflicting Western society, and will hold therapeutic potential for treating it that have attracted interest and concern not only from the scientific community but also of the lay public.
Principle of exocytotic events detection by TIRF microscopy using pHluorin.
Real-time PCR analysis in tanycytes isolated by FACS.
TAT-Cre infusion into the 3v of iBot mice modulates feeding behaviour.
Dicer inactivation in tanycytes modulates feeding behaviour and glucose tolerance.
Effect of Dicer inactivation after 6 weeks of HFD on body weight and food intake.