GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice.

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR.

Autophagy is a biological process that maintains cellular homeostasis and regulates the internal cellular environment. Hyperactivating autophagy to trigger cell death has been a suggested therapeutic strategy for cancer treatment. Mechanistic target of rapamycin (mTOR) is a crucial protein kinase that regulates autophagy; therefore, using a structure-based virtual screen analysis, we identified lomitapide, a cholesterol-lowering drug, as a potential mTOR complex 1 (mTORC1) inhibitor. Our results showed that lomitapide directly inhibits mTORC1 in vitro and induces autophagy-dependent cancer cell death by decreasing mTOR signaling, thereby inhibiting the downstream events associated with increased LC3 conversion in various cancer cells (e.g., HCT116 colorectal cancer cells) and tumor xenografts. Lomitapide also significantly suppresses the growth and viability along with elevated autophagy in patient-derived colorectal cancer organoids. Furthermore, a combination of lomitapide and immune checkpoint blocking antibodies synergistically inhibits tumor growth in murine MC38 or B16-F10 preclinical syngeneic tumor models. These results elucidate the direct, tumor-relevant immune-potentiating benefits of mTORC1 inhibition by lomitapide, which complement the current immune checkpoint blockade. This study highlights the potential repurposing of lomitapide as a new therapeutic option for cancer treatment.

Non-Cell Autonomous Epileptogenesis in Focal Cortical Dysplasia.

OBJECTIVE: Low-level somatic mosaicism in the brain has been shown to be a major genetic cause of intractable focal epilepsy. However, how a relatively few mutation-carrying neurons are able to induce epileptogenesis at the local network level remains poorly understood. METHODS: To probe the origin of epileptogenesis, we measured the excitability of neurons with MTOR mutation and nearby nonmutated neurons recorded by whole-cell patch-clamp and array-based electrodes comparing the topographic distribution of mutation. Computational simulation is used to understand neural network-level changes based on electrophysiological properties. To examine the underlying mechanism, we measured inhibitory and excitatory synaptic inputs in mutated neurons and nearby neurons by electrophysiological and histological methods using the mouse model and postoperative human brain tissue for cortical dysplasia. To explain non-cell-autonomous hyperexcitability, an inhibitor of adenosine kinase was injected into mice to enhance adenosine signaling and to mitigate hyperactivity of nearby nonmutated neurons. RESULTS: We generated mice with a low-level somatic mutation in MTOR presenting spontaneous seizures. The seizure-triggering hyperexcitability originated from nonmutated neurons near mutation-carrying neurons, which proved to be less excitable than nonmutated neurons. Interestingly, the net balance between excitatory and inhibitory synaptic inputs onto mutated neurons remained unchanged. Additionally, we found that inhibition of adenosine kinase, which affects adenosine metabolism and neuronal excitability, reduced the hyperexcitability of nonmutated neurons. INTERPRETATION: This study shows that neurons carrying somatic mutations in MTOR lead to focal epileptogenesis via non-cell-autonomous hyperexcitability of nearby nonmutated neurons. ANN NEUROL 2021;90:285-299.

The atypical antipsychotic risperidone targets hypothalamic melanocortin 4 receptors to cause weight gain.

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice.

Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses.

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat-me" signal that initiates glia-mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal-specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well-known "eat-me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post-synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post-synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, we found that phosphatidylserine is used for microglia-mediated pruning of inhibitory post-synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat-me" signal for inhibitory post-synapse elimination.

Chemogenetic manipulation of parasympathetic neurons (DMV) regulates feeding behavior and energy metabolism.

Parasympathetic nervous system (PNS) innervates with several peripheral organs such as liver, pancreas and regulates energy metabolism. However, the direct role of PNS on food intake has been poorly understood. In the present study, we investigated the role of parasympathetic nervous system in regulation of feeding by chemogenetic methods. Adeno associated virus carrying DREADD (designer receptors exclusively activated by designer drugs) infused into the target brain region by stereotaxic surgery. The stimulatory hM3Dq or inhibitory hM4Di DREADD was over-expressed in selective population of dorsal motor nucleus of the vagus (DMV) neurons by Cre-recombinase-dependent manners. Activation of parasympathetic neuron by intraperitoneal injection of the M3-muscarinic receptor ligand clozapine-N-oxide (CNO) (1 mg/kg) suppressed food intake and resulted in body weight loss in ChAT-Cre mice. Parasympathetic neurons activation resulted in improved glucose tolerance while inhibition of the neurons resulted in impaired glucose tolerance. Stimulation of parasympathetic nervous system by injection of CNO (1 mg/kg) increased oxygen consumption and energy expenditure. Within the hypothalamus, in the arcuate nucleus (ARC) changed AGRP/POMC neurons. These results suggest that direct activation of parasympathetic nervous system decreases food intake and body weight with improved glucose tolerance.

Enhancement of dendritic persistent Na+ currents by mGluR5 leads to an advancement of spike timing with an increase in temporal precision.

Timing and temporal precision of action potential generation are thought to be important for encoding of information in the brain. The ability of single neurons to transform their input into output action potential is primarily determined by intrinsic excitability. Particularly, plastic changes in intrinsic excitability represent the cellular substrate for spatial memory formation in CA1 pyramidal neurons (CA1-PNs). Here, we report that synaptically activated mGluR5-signaling can modulate the intrinsic excitability of CA1-PNs. Specifically, high-frequency stimulation at CA3-CA1 synapses increased firing rate and advanced spike onset with an improvement of temporal precision. These changes are mediated by mGluR5 activation that induces cADPR/RyR-dependent Ca2+ release in the dendrites of CA1-PNs, which in turn causes an increase in persistent Na+ currents (INa,P) in the dendrites. When group I mGluRs in CA1-PNs are globally activated pharmacologically, afterdepolarization (ADP) generation as well as increased firing rate are observed. These effects are abolished by inhibiting mGluR5/cADPR/RyR-dependent Ca2+ release. However, the increase in firing rate, but not the generation of ADP is affected by inhibiting INa,P. The differences between local and global activation of mGluR5-signaling in CA1-PNs indicates that mGluR5-dependent modulation of intrinsic excitability is highly compartmentalized and a variety of ion channels are recruited upon their differential subcellular localizations. As mGluR5 activation is induced by physiologically plausible brief high-frequency stimulation at CA3-CA1 synapses, our results suggest that mGluR5-induced enhancement of dendritic INa,P in CA1-PNs may provide important implications for our understanding about place field formation in the hippocampus.

Hypothalamic Macrophage Inducible Nitric Oxide Synthase Mediates Obesity-Associated Hypothalamic Inflammation.

Obesity-associated metabolic alterations are closely linked to low-grade inflammation in peripheral organs, in which macrophages play a central role. Using genetic labeling of myeloid lineage cells, we show that hypothalamic macrophages normally reside in the perivascular area and circumventricular organ median eminence. Chronic consumption of a high-fat diet (HFD) induces expansion of the monocyte-derived macrophage pool in the hypothalamic arcuate nucleus (ARC), which is significantly attributed to enhanced proliferation of macrophages. Notably, inducible nitric oxide synthase (iNOS) is robustly activated in ARC macrophages of HFD-fed obese mice. Hypothalamic macrophage iNOS inhibition completely abrogates macrophage accumulation and activation, proinflammatory cytokine overproduction, reactive astrogliosis, blood-brain-barrier permeability, and lipid accumulation in the ARC of obese mice. Moreover, central iNOS inhibition improves obesity-induced alterations in systemic glucose metabolism without affecting adiposity. Our findings suggest a critical role for hypothalamic macrophage-expressed iNOS in hypothalamic inflammation and abnormal glucose metabolism in cases of overnutrition-induced obesity.

Replacement of the C-terminal Trp-cage of exendin-4 with a fatty acid improves therapeutic utility.

Exendin-4, a 39 amino acid peptide isolated from the saliva of the Gila monster, plays an important role in regulating glucose homeostasis, and is used clinically for the treatment of type 2 diabetes. Exendin-4 shares 53% sequence identity with the incretin hormone glucagon-like peptide 1 (GLP-1) but, unlike GLP-1, is highly resistant to proteolytic enzymes such as dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidase 24.11 (NEP 24.11). Herein, we focused on the structure and function of the C-terminal Trp-cage of exendin-4, and suggest that it may be structurally required for resistance to proteolysis by NEP 24.11. Using a series of substitutions and truncations of the C-terminal Trp-cage, we found that residues 1-33, including the N-terminal and helical regions of wild-type (WT) exendin-4, is the minimum motif required for both high peptidase resistance and potent activity toward the GLP-1 receptor comparable to WT exendin-4. To improve the therapeutic utility of C-terminally truncated exendin-4, we incorporated various fatty acids into exendin-4(1-33) in which Ser33 was substituted with Lys for acylation. Exendin-4(1-32)K-capric acid exhibited the most well balanced activity, with much improved therapeutic utility for regulating blood glucose and body weight relative to WT exendin-4.

Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo.

Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here, we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100-150 µm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.

Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis.

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.

Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic ß-cells.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic ß-cells. In this study, we provide evidence that leptin modulates pancreatic ß-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic ß-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase ß. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic ß-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and ß-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating ß-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating ß-cell excitability.

Melanocortin 4 receptors reciprocally regulate sympathetic and parasympathetic preganglionic neurons.

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are probably dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension.

Neuroanatomy of melanocortin-4 receptor pathway in the lateral hypothalamic area.

The central melanocortin system regulates body energy homeostasis including the melanocortin-4 receptor (MC4R). The lateral hypothalamic area (LHA) receives dense melanocortinergic inputs from the arcuate nucleus of the hypothalamus and regulates multiple processes including food intake, reward behaviors, and autonomic function. By using a mouse line in which green fluorescent protein (GFP) is expressed under control of the MC4R gene promoter, we systemically investigated MC4R signaling in the LHA by combining double immunohistochemistry, electrophysiology, and retrograde tracing techniques. We found that LHA MC4R-GFP neurons coexpress neurotensin as well as the leptin receptor but do not coexpress other peptide neurotransmitters found in the LHA including orexin, melanin-concentrating hormone, and nesfatin-1. Furthermore, electrophysiological recording demonstrated that leptin, but not the MC4R agonist melanotan II, hyperpolarizes the majority of LHA MC4R-GFP neurons in an ATP- sensitive potassium channel-dependent manner. Retrograde tracing revealed that LHA MC4R-GFP neurons do not project to the ventral tegmental area, dorsal raphe nucleus, nucleus accumbens, and spinal cord, and only limited number of neurons project to the nucleus of the solitary tract and parabrachial nucleus. Our findings provide new insights into MC4R signaling in the LHA and its potential implications in homeostatic regulation of body energy balance.

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons.

Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca(2+). In this study, we characterized the cellular mechanisms underlying Ca(2+) mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca(2+) from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP(3) signaling pathway was not involved in Ca(2+) mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca(2+) mobilization by mGluR5 and Ca(2+) influx through L-type Ca(2+) channels. We found no evidence that mGluR5-mediated Ca(2+) release and Ca(2+) influx through L-type Ca(2+) channels interact to generate supralinear Ca(2+) transients. Our study provides novel insights into the mechanisms of intracellular Ca(2+) mobilization by mGluR5 in the somata of hippocampal neurons.

Glucose deprivation regulates KATP channel trafficking via AMP-activated protein kinase in pancreatic beta-cells.

OBJECTIVE: AMP-activated protein kinase (AMPK) and the ATP-sensitive K(+) (K(ATP)) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K(ATP) channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood. RESEARCH DESIGN AND METHODS: Rat pancreatic beta-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K(ATP) conductance (G(max)) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. K(ATP) channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K(ATP) channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay. RESULTS: G(max) and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G(max) and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K(ATP) channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C. CONCLUSIONS: K(ATP) channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.

Impact of myocardial infarct proteins and oscillating pressure on the differentiation of mesenchymal stem cells: effect of acute myocardial infarction on stem cell differentiation.

Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac-specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor beta(1) (TGF-beta(1)) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte-specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-beta(1) and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct-related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-beta/BMP-2 pathways.