Effect of Euphorbia factor L1 on intestinal barrier impairment and defecation dysfunction in Caenorhabditis elegans.

BACKGROUND: Euphorbia factor L1 (EFL1) is a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L., and has been reported with intestinal toxicity, but the potential mechanisms remain unknown. PURPOSE: The objective of this study was to investigate the intestinal toxicity of EFL1 and the underlying mechanisms using nematode Caenorhabditis elegans. METHODS: C. elegans were exposed to 0-200 µM EFL1 for 72 h, then the survival rate, body length and body width, locomotion and chemoreception behavior, intestinal ROS and lipofuscin accumulation, intestinal permeability, and defecation rhythm were detected. The γ-aminobutyric acid（GABA) energic neurons AVL and DVB were shown via green fluorescent protein under a laser scanning confocal microscope. The structure of GABA transporter UNC-47 were predicted by homology modeling, and the interaction between EFL1 and UNC-47 was simulated by molecular docking. The mRNA expression of genes related to oxidative stress, intestinal permeability and defecation after EFL1 exposure were detected by RT-qPCR. RESULTS: EFL1 did not induce lethality of nematodes. The general toxicity was characterized by abnormal growth, locomotion and chemoreception. The intestinal barrier was leaky, due to down-regulated cell junction and active cation transport. The mean defecation cycle length in nematodes was decreased, relating to disorder vesicular and ion transport, enhanced rhythm behavior and muscle contraction. The dysfunctional defecation also attributed to injured UNC-47 protein, as well as GABAergic neurons AVL and DVB. Excessive ROS and lipofuscin accumulation were observed in intestine, along with activation of antioxidant enzymes of SOD, COQ7 and CAT. CONCLUSION: This study elucidated the EFL1-induced intestinal toxicity in nematodes, characterized as leaky intestinal barrier and accelerated defecation behavior. The underlying mechanisms were involved in oxidative stress, cell junctions, transportation, rhythm behavior, muscle contraction, and GABAergic neurons.

Liraglutide Modulates Appetite and Body Weight Through Glucagon-Like Peptide 1 Receptor-Expressing Glutamatergic Neurons.

Glucagon-like peptide 1 receptor (GLP-1R) agonists are U.S. Food and Drug Administration-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) affect appetite and body weight are still not fully understood. We determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the short- and long-term effects of the GLP1RA liraglutide on food intake, visceral illness, body weight, and neural network activation. We found that mice lacking GLP-1Rs in vGAT-expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2-expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons.

Vesicular GABA transporter (VGAT) transports ß-alanine.

Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes ß-alanine as a substrate. Proteoliposomes containing purified VGAT transport ß-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven ß-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of ß-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates ß-alanine uptake. These findings indicated that VGAT transports ß-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular ß-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport ß-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular ß-alanine transporter.

Mutation of the Drosophila vesicular GABA transporter disrupts visual figure detection.

The role of gamma amino butyric acid (GABA) release and inhibitory neurotransmission in regulating most behaviors remains unclear. The vesicular GABA transporter (VGAT) is required for the storage of GABA in synaptic vesicles and provides a potentially useful probe for inhibitory circuits. However, specific pharmacologic agents for VGAT are not available, and VGAT knockout mice are embryonically lethal, thus precluding behavioral studies. We have identified the Drosophila ortholog of the vesicular GABA transporter gene (which we refer to as dVGAT), immunocytologically mapped dVGAT protein expression in the larva and adult and characterized a dVGAT(minos) mutant allele. dVGAT is embryonically lethal and we do not detect residual dVGAT expression, suggesting that it is either a strong hypomorph or a null. To investigate the function of VGAT and GABA signaling in adult visual flight behavior, we have selectively rescued the dVGAT mutant during development. We show that reduced GABA release does not compromise the active optomotor control of wide-field pattern motion. Conversely, reduced dVGAT expression disrupts normal object tracking and figure-ground discrimination. These results demonstrate that visual behaviors are segregated by the level of GABA signaling in flies, and more generally establish dVGAT as a model to study the contribution of GABA release to other complex behaviors.

Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses.

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.