Identification and quantification of protein adducts formed by metabolites of 1-methoxy-3-indolylmethyl glucosinolate in vitro and in mouse models.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate (GLS) occurring in cabbage, broccoli, and other cruciferous plants is a potent mutagen requiring metabolic activation by myrosinase present in plant cells and intestinal bacteria. We previously reported that 1-MIM-GLS and its alcoholic breakdown product 1-MIM-OH, which requires additional activation by sulfotransferases, form DNA adducts in mice. In the present study, the formation of protein adducts was investigated. First, two major adducts obtained after incubation of individual amino acids, serum albumin, or hemoglobin with 1-MIM-GLS in the presence of myrosinase were identified as τN-(1-MIM)-His and πN-(1-MIM)-His using MS and NMR spectroscopy. After the development of a specific detection method using isotope-dilution UPLC-ESI-MS/MS, adduct formation was confirmed in mice after oral treatment with 1-MIM-GLS. Adduct levels were highest in the cecum and colon, somewhat lower in serum albumin and the liver, and also readily detectable in the lung and hemoglobin. On the contrary, oral treatment with 1-MIM-OH produced the highest adduct levels in the liver. The higher ratio of albumin to hemoglobin adducts in 1-MIM-OH- compared to 1-MIM-GLS-treated animals (8.1 versus 3.5) suggests that in 1-MIM-OH-treated animals albumin adducts were produced mostly in the liver, the site of albumin synthesis. The formation of adducts was approximately linear over a range of single oral doses from 20 to 600 µmol/kg body mass. Repeated oral administration of 1-MIM-OH (up to 40 treatments, thrice per week) led to continuous accumulation of hemoglobin adducts, whereas the level of serum albumin adducts remained rather constant, which reflects the different turnover rates of these proteins (t1/2 nearly 1.9 d for serum albumin and 25 d for hemoglobin in the mouse). Accumulation of adducts was also noticed in the lung. Adduct levels were higher, but their accumulation was weaker in the liver and kidney. The method developed will be useful to assess the exposure of humans to reactive metabolites formed from 1-MIM-GLS present in many foods.

The LepR-mediated leptin transport across brain barriers controls food reward.

OBJECTIVE: Leptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood-brain and blood-CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR). METHODS: We selectively deleted LepR in brain endothelial and epithelial cells of mice (LepRbeKO). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected. RESULTS: Perfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepRbeKO mice gained more body weight than controls. The aggravated obesity of LepRbeKO mice was due to hyperphagia and a higher sensitivity to food reward. CONCLUSIONS: The LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis.

The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gαq/11 proteins. Activation of Gαq/11 pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gαq/11 proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gαq/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels.