Several different neuronal populations are involved in regulating energy homeostasis. Among these, agoutirelated protein (AgRP) neurons are thought to promote feeding and weight gain; however, the evidence supporting this view is incomplete. Using designer receptors exclusively activated by designer drugs (DREADD) technology to provide specific and reversible regulation of neuronal activity in mice, we have demonstrated that acute activation of AgRP neurons rapidly and dramatically induces feeding, reduces energy expenditure, and ultimately increases fat stores. All these effects returned to baseline after stimulation was withdrawn. In contrast, inhibiting AgRP neuronal activity in hungry mice reduced food intake. Together, these findings demonstrate that AgRP neuron activity is both necessary and sufficient for feeding. Of interest, activating AgRP neurons potently increased motivation for feeding and also drove intense food-seeking behavior, demonstrating that AgRP neurons engage brain sites controlling multiple levels of feeding behavior. Due to its ease of use and suitability for both acute and chronic regulation, DREADD technology is ideally suited for investigating the neural circuits hypothesized to regulate energy balance.

The physiologic importance of GABAergic neurotransmission in hypothalamic neurocircuits is unknown. To examine the importance of GABA release from agouti-related protein (AgRP) neurons (which also release AgRP and neuropeptide Y), we generated mice with an AgRP neuron-specific deletion of vesicular GABA transporter. These mice are lean, resistant to obesity and have an attenuated hyperphagic response to ghrelin. Thus, GABA release from AgRP neurons is important in regulating energy balance.AgRP neurons release AgRP, neuropeptide Y (NPY) and GABA and are important in regulating body weight 1-3 . However, the deletion of AgRP and/or NPY has little effect on body weight 4 . This raises the possibility that the release of GABA may be important 2,5 . A direct GABAergic action from AgRP neurons to pro-opiomelanocortin (POMC) neurons has been suggested to mediate the ability of leptin to indirectly excite POMC neurons 2,5-7 . However, because of the lack of methodological approaches, the physiologic effect of GABAergic neurotransmission in the hypothalamus is unknown. Vesicular GABA transporter (VGAT, encoded by Vgat, also known as Slc32a1) is required for presynaptic release of GABA 8 . To disrupt GABA release in a neuron-specific manner, we generated Vgat flox/flox mice ( Fig. 1a and Supplementary Methods online). To ensure the specificity of cre expression in AgRP neurons, we generated Agrp-Ires-cre knockin mice (Fig. 1b and Supplementary Methods). We confirmed the specificity of Cre activity by immunostaining for green fluorescent protein (GFP) in Agrp-Ires-cre mice that were crossed with mice bearing a Cre-dependent GFP reporter transgene (Z/EG mice) 9 (Fig. 1c and Supplementary Methods). We generated mice that lack GABA release from AgRP neurons by crossing (Fig. 1d). Thus, deletion of Vgat prevents the synaptic release of GABA from AgRP neurons. We observed no difference in AgRP immunostaining patterns in Agrp-Irescre; Vgat flox/flox mice, suggesting that there was no alteration in AgRP neuron development resulting from the disruption of GABA release ( Supplementary Fig. 2 online).Agrp-Ires-cre; Vgat flox/flox mice showed reduced body weight on chow diet in both males (Fig. 2a) and females ( Supplementary Fig. 3 online). We did not observe a change in food intake in 10−12-week-old male mice ( Supplementary Fig. 3) or in 16−17-week-old male mice (data not shown); however, O 2 consumption was significantly increased (P < 0.05; Fig. 2b). In addition, these mice showed increased locomotor activity and reduced respiration exchange ratios ( Supplementary Fig. 3). When placed on a high fat diet (HFD), male Agrp-Ires-cre; Vgat flox/flox mice had reduced body weight gain compared with controls ( Fig. 2c), indicating that they had a resistance to diet-induced obesity (DIO) (Fig. 3a,c). Body composition analysis revealed that reduced body weight gain was solely because of reduced fat accumulation ( Supplementary Fig. 3). The resistance to DIO was not the result of reduced food intake ( Supplementary Fig. 3), whi...

Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced beta cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes K(ATP) channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced beta cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating beta cell dysfunction.

By using pharmacological and molecular approaches, we previously showed that the G-protein-coupled, extracellular calcium (Ca2+(o))-sensing receptor (CaR) regulates a large-conductance (approximately 140 pS), Ca(2+)-activated K+ channel [IK(Ca); CAKC] in U87 astrocytoma cells. Here we show that elevated Ca2+(o) stimulates extracellular-signal-regulated kinase (ERK1/2) and p38 MAP kinase (MAPK). The effect of high Ca2+(o) on p38 MAPK but not ERK1/2 is CaR mediated, insofar as transduction with a dominant-negative CaR (R185Q) using recombinant adeno-associated virus (rAAV) attenuated the activation of p38 MAPK but not of ERK1/2. p38 MAPK activation by the CaR is likely to be protein kinase C (PKC) independent, in that the pan-PKC inhibitor GF109203X failed to abolish the high-Ca2+(o)-induced phosphorylation of p38 MAPK. Consistently with our data on the activation of this kinase, we observed that inhibiting p38 MAPK blocked the activation of the CAKC induced by the specific pharmacological CaR activator NPS R-467. In contrast, inhibiting MEK1 only transiently inhibited the activation of this K+ channel by NPS R-467, despite the continued presence of the antagonist. Similarly to the lack of any effect of the PKC inhibitor on the activation of ERK1/2 and p38 MAPK, inhibiting PKC had no effect on NPS R-467-induced activation of this channel. Therefore, our data show that the CaR, acting via p38 MAPK, regulates a large-conductance CAKC in U87 cells, a process that is PKC independent. Large-conductance CAKCs play an important role in the regulation of cellular volume, so our results have important implications for glioma cell volume regulation.