A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance.

AMP-activated protein kinase (AMPK) in the ventromedial nucleus of the hypothalamus (VMH) and orexin (OX) in the lateral hypothalamic area (LHA) modulate brown adipose tissue (BAT) thermogenesis. However, whether these two molecular mechanisms act jointly or independently is unclear. Here, we show that the thermogenic effect of bone morphogenetic protein 8B (BMP8B) is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signaling via the OX receptor 1 (OX1R). Accordingly, the thermogenic effect of BMP8B is totally absent in ox-null mice. BMP8B also induces browning of white adipose tissue (WAT), its thermogenic effect is sexually dimorphic (only observed in females), and its impact on OX expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 (VGLUT2), implicating glutamatergic signaling. Overall, our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders.

Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model.

Vesicular glutamate transporters (VGLUTs) control the storage and release of glutamate, which plays a critical role in pain processing. The VGLUT2 isoform has been found to be densely distributed in the nociceptive pathways in supraspinal regions, and VGLUT2-deficient mice exhibit an attenuation of neuropathic pain; these results suggest a possible involvement of VGLUT2 in neuropathic pain. To further examine this, we investigated the temporal changes in VGLUT2 expression in different brain regions as well as changes in glutamate release from thalamic synaptosomes in spared nerve injury (SNI) mice. We also investigated the effects of a VGLUT inhibitor, Chicago Sky Blue 6B (CSB6B), on pain behavior, c-Fos expression, and depolarization-evoked glutamate release in SNI mice. Our results showed a significant elevation of VGLUT2 expression up to postoperative day 1 in the thalamus, periaqueductal gray, and amygdala, followed by a return to control levels. Consistent with the changes in VGLUT2 expression, SNI enhanced depolarization-induced glutamate release from thalamic synaptosomes, while CSB6B treatment produced a concentration-dependent inhibition of glutamate release. Moreover, intracerebroventricular administration of CSB6B, at a dose that did not affect motor function, attenuated mechanical allodynia and c-Fos up-regulation in pain-related brain areas during the early stages of neuropathic pain development. These results demonstrate that changes in the expression of supraspinal VGLUT2 may be a new mechanism relevant to the induction of neuropathic pain after nerve injury that acts through an aggravation of glutamate imbalance.

Role of glutamatergic projections from ventral tegmental area to lateral habenula in aversive conditioning.

The ventral tegmental area (VTA) plays roles in both reward and aversion. The participation of VTA in diverse behaviors likely reflects its heterogeneous neuronal phenotypes and circuits. Recent findings indicate that VTA GABAergic neurons that coexpress tyrosine hydroxylase (TH) projecting to lateral habenula (LHb) play a role in reward. In addition to these mesohabenular TH-GABAergic neurons, the VTA has many neurons expressing vesicular glutamate transporter 2 (VGluT2) that also project to LHb. To determine the behavioral role of mesohabenular VGluT2 neurons, we targeted channelrhodopsin2 to VTA VGluT2 neurons of VGluT2::Cre mice. These mice were tested in an apparatus where moving into one chamber stimulated VTA VGluT2 projections within the LHb, and exiting the chamber inactivated the stimulation. We found that mice spent significantly less time in the chamber where VGluT2 mesohabenular fiber stimulation occurred. Mice that received injections of mixed AMPA and NMDA glutamate receptor antagonists in LHb were unresponsive to VGluT2-mesohabenular fiber stimulation, demonstrating the participation of LHb glutamate receptors in mesohabenular stimulation-elicited aversion. In the absence of light stimulation, mice showed a conditioned place aversion to the chamber that was previously associated with VGluT2-mesohabenular fiber stimulation. We conclude that there is a glutamatergic signal from VTA VGluT2-mesohabenular neurons that plays a role in aversion by activating LHb glutamatergic receptors.

The metabotropic glutamate receptor antagonist 2-methyl-6-(phenylethynyl) pyridine decreases striatal VGlut2 expression in association with an attenuation of L-DOPA-induced dyskinesias.

The striatal glutamatergic hyperactivity is considered critical in the development of levodopa-induced dyskinesias (LID) in Parkinson's disease (PD). Pharmacological antagonism of the metabotropic glutamate receptors (mGluRs), in particular, the subtype mGluR5, can inhibit the expression of dyskinesia in both rodent and nonhuman primate models of PD. However, the exact mechanisms underlying the mGluR5 antagonism effects are not completely known. The vesicular glutamate transporters (VGluts) are localized in the synaptic vesicles of the striatal glutamatergic axonal terminals. The effects of mGluR5 antagonism modulating VGlut1 and VGlut2, as selective markers for the corticostriatal and thalamostriatal pathways, respectively, are still unknown. We investigated the effects of the mGluR5 antagonist, 2-methyl-6-(phenylethynyl) pyridine (MPEP) on the striatal expression of VGlut1 and VGlut2 in levodopa-treated hemiparkinsonian rats. Male Sprague-Dawley rats received a unilateral 6-hydroxydopamine (6-OHDA) administration in the nigrostriatal pathway. Rats were treated with: (a) levodopa (12 mg/kg/day with benserazide 15 mg/kg, ip) + vehicle; (b) MPEP (1.5 mg/kg/day, ip) + vehicle; (c) levodopa + MPEP, or (d) saline for 10 days. Levodopa treatment induced dyskinesias and did not modify the striatal expression of either VGlut1 or VGlut2. The administration of MPEP significantly attenuated LID and decreased the levels of VGlut2, but not the VGlut1, in the striatum ipsilateral to the lesion (P < 0.05). Our results suggest that the effects of MPEP on LID might be mediated by a modulating effect on VGlut 2 expression.

Docking and homology modeling explain inhibition of the human vesicular glutamate transporters.

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.