Insulin sensitization by small molecules enhancing GLUT4 translocation.

Insulin resistance (IR) is the root cause of type II diabetes, yet no safe treatment is available to address it. Using a high throughput compatible assay that measures real-time translocation of the glucose transporter glucose transporter 4 (GLUT4), we identified small molecules that potentiate insulin action. In vivo, these insulin sensitizers improve insulin-stimulated GLUT4 translocation, glucose tolerance, and glucose uptake in a model of IR. Using proteomic and CRISPR-based approaches, we identified the targets of those compounds as Unc119 proteins and solved the structure of Unc119 bound to the insulin sensitizer. This study identifies compounds that have the potential to be developed into diabetes treatment and establishes Unc119 proteins as targets for improving insulin sensitivity.

Activation of amygdala prokineticin receptor 2 neurons drives the anorexigenic activity of the neuropeptide PK2.

Energy homeostasis is a complex system involving multiple hormones, neuropeptides, and receptors. Prokineticins (PK1 and PK2) are agonists to two G protein-coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), which decrease food intake when injected in rodents. The relative contribution of PKR1 and PKR2 to the anorexigenic effect of PK2 and their site of action in the brain have not yet been elucidated. While PKR1 and PKR2 are both expressed in the hypothalamus, a central region involved in the control of energy homeostasis, PKR2 is also present in the amygdala, which has recently been shown to regulate food intake in response to several anorexigenic signals. PKR trafficking and signaling are inhibited by the melanocortin receptor accessory protein 2 (MRAP2), thus suggesting that MRAP2 has the potential to alter the anorexigenic activity of PK2 in vivo. In this study, we investigated the importance of PKR1 and PKR2 for PK2-mediated inhibition of food intake, the brain region involved in this function, and the effect of MRAP2 on PK2 action in vivo. Using targeted silencing of PKR2 and chemogenetic manipulation of PKR2 neurons, we show that the anorexigenic effect of PK2 is mediated by PKR2 in the amygdala and that altering MRAP2 expression in PKR2 neurons modulates the activity of PK2. Collectively, our results provide evidence that inhibition of food intake by PKs is not mediated through activation of hypothalamic neurons but rather amygdala PKR2 neurons and further establishes the importance of MRAP2 in the regulation of energy homeostasis.

P7C3-A20 treatment one year after TBI in mice repairs the blood-brain barrier, arrests chronic neurodegeneration, and restores cognition.

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

The Insulinostatic Effect of Ghrelin Requires MRAP2 Expression in δ Cells.

Ghrelin regulates both energy intake and glucose homeostasis. In the endocrine pancreas, ghrelin inhibits insulin release to prevent hypoglycemia during fasting. The mechanism through which this is accomplished is unclear, but recent studies suggest that ghrelin acts on δ cells to stimulate somatostatin release, which in turn inhibits insulin release from ß cells. Recently, the Melanocortin Receptor Accessory Protein 2 (MRAP2) was identified as an essential partner of the ghrelin receptor (GHSR1a) in mediating the central orexigenic action of ghrelin. In this study we show that MRAP2 is expressed in islet δ cells and is required for ghrelin to elicit a calcium response in those cells. Additionally, we show that both global and δ cell targeted deletion of MRAP2 abrogates the insulinostatic effect of ghrelin. Together, these findings establish that ghrelin signaling within δ cells is essential for the inhibition of insulin release and identify MRAP2 as a regulator of insulin secretion.

Neuroprotective Efficacy of a Sigma 2 Receptor/TMEM97 Modulator (DKR-1677) after Traumatic Brain Injury.

Compounds targeting the sigma 2 receptor, which we recently cloned and showed to be identical with transmembrane protein 97 (σ2R/TMEM97), are broadly applicable therapeutic agents currently in clinical trials for imaging in breast cancer and for treatment of Alzheimer's disease and schizophrenia. These promising applications coupled with our previous observation that the σ2R/TMEM97 modulator SAS-0132 has neuroprotective attributes and improves cognition in wild-type mice suggests that modulating σ2R/TMEM97 may also have therapeutic benefits in other neurodegenerative conditions such as traumatic brain injury (TBI). Herein, we report that DKR-1677, a novel derivative of SAS-0132 with increased affinity and selectivity for σ2R/Tmem97 ( Ki = 5.1 nM), is neuroprotective after blast-induced and controlled cortical impact (CCI) TBI in mice. Specifically, we discovered that treatment with DKR-1677 decreases axonal degeneration after blast-induced TBI and enhances survival of cortical neurons and oligodendrocytes after CCI injury. Furthermore, treatment with DKR-1677 preserves cognition in the Morris water maze after blast TBI. Our results support an increasingly broad role for σ2R/Tmem97 modulation in neuroprotection and suggest a new approach for treating patients suffering from TBI.

(-)-P7C3-S243 Protects a Rat Model of Alzheimer's Disease From Neuropsychiatric Deficits and Neurodegeneration Without Altering Amyloid Deposition or Reactive Glia.

BACKGROUND: In addition to cognitive deficits, Alzheimer's disease (AD) is associated with other neuropsychiatric symptoms, including severe depression. Indeed, depression often precedes cognitive deficits in patients with AD. Unfortunately, the field has seen only minimal therapeutic advances, underscoring the critical need for new treatments. P7C3 aminopropyl carbazoles promote neuronal survival by enhancing nicotinamide adenine dinucleotide flux in injured neurons. Neuroprotection with P7C3 compounds has been demonstrated in preclinical models of neurodegeneration by virtue of promoting neuronal survival independently of early disease-specific pathology, resulting in protection from cognitive deficits and depressive-like behavior. We hypothesize that P7C3 compounds might be uniquely applicable to patients with AD, given the comorbid presentation of depression and cognitive deficits. METHODS: Aging male and female wild-type and TgF344-AD rats, a well-characterized preclinical AD model, were administered (-)-P7C3-S243 daily for 9 and 18 months, beginning at 6 months of age. Behavioral phenotypes related to cognition and depression were assessed at 15 and 24 months, and brain pathology and biochemistry were assessed at 24 months. RESULTS: (-)-P7C3-S243 safely protected aging male and female wild-type and TgF344-AD rats from cognitive deficits and depressive-like behavior. Depressive-like behavior occurred earlier than cognitive deficits in TgF344-AD rats, consistent with AD in many patients. Treatment with (-)-P7C3-S243 blocked neurodegeneration in TgF344-AD rats, without altering amyloid deposition or indicators of neuroinflammation. CONCLUSIONS: Neuronal cell death-specific treatment approaches, such as P7C3 compounds, may represent a new treatment approach for patients experiencing the combination of cognitive deficits and depression associated with AD.

MRAP2 regulates ghrelin receptor signaling and hunger sensing.

Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus.Melanocortin receptor accessory protein 2 (MRAP2) is an adaptor protein that contributes to melanocortin-4 receptor and prokineticin receptor 1 signalling. Here the authors show that MRAP2 also regulates ghrelin receptor signalling in the hypothalamus and starvation sensing in mice.

Melanocortin Receptor Accessory Proteins (MRAPs): Functions in the melanocortin system and beyond.

G-protein coupled receptors (GPCRs) are regulated by numerous proteins including kinases, G-proteins, ß-arrestins and accessory proteins. Several families of GPCR accessory proteins like Receptor Activity Modifying Proteins, Receptor Transporting Proteins and Melanocortin Receptor Accessory Proteins (MRAPs) have been identified as regulator of receptor trafficking, signaling and ligand specificity. The MRAP family contains two members, MRAP1 and MRAP2, responsible for the formation of a functional ACTH receptor and for the regulation of energy homeostasis respectively. Like all known GPCR accessory proteins, MRAPs are single transmembrane proteins, however, they form a unique structure since they assemble as an anti-parallel homodimer. Moreover, the accepted idea that MRAPs are specific regulators of melanocortin receptors was recently challenged by the discovery that MRAP2 inhibits the activity of prokineticin receptors. Recent studies are starting to explain the role of the unusual structure of MRAPs and to illustrate the importance of MRAP2 for the maintenance of both energy and glucose homeostasis. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

Acute Axonal Degeneration Drives Development of Cognitive, Motor, and Visual Deficits after Blast-Mediated Traumatic Brain Injury in Mice.

Axonal degeneration is a prominent feature of many forms of neurodegeneration, and also an early event in blast-mediated traumatic brain injury (TBI), the signature injury of soldiers in Iraq and Afghanistan. It is not known, however, whether this axonal degeneration is what drives development of subsequent neurologic deficits after the injury. The Wallerian degeneration slow strain (WldS) of mice is resistant to some forms of axonal degeneration because of a triplicated fusion gene encoding the first 70 amino acids of Ufd2a, a ubiquitin-chain assembly factor, that is linked to the complete coding sequence of nicotinamide mononucleotide adenylyltransferase 1 (NMAT1). Here, we demonstrate that resistance of WldS mice to axonal degeneration after blast-mediated TBI is associated with preserved function in hippocampal-dependent spatial memory, cerebellar-dependent motor balance, and retinal and optic nerve-dependent visual function. Thus, early axonal degeneration is likely a critical driver of subsequent neurobehavioral complications of blast-mediated TBI. Future therapeutic strategies targeted specifically at mitigating axonal degeneration may provide a uniquely beneficial approach to treating patients suffering from the effects of blast-mediated TBI.

FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver.

The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

Behavioral disturbances in estrogen-related receptor alpha-null mice.

Eating disorders, such as anorexia nervosa and bulimia nervosa, are common and severe mental illnesses of unknown etiology. Recently, we identified a rare missense mutation in the transcription factor estrogen-related receptor alpha (ESRRA) that is associated with the development of eating disorders. However, little is known about ESRRA function in the brain. Here, we report that Esrra is expressed in the mouse brain and demonstrate that Esrra levels are regulated by energy reserves. Esrra-null female mice display a reduced operant response to a high-fat diet, compulsivity/behavioral rigidity, and social deficits. Selective Esrra knockdown in the prefrontal and orbitofrontal cortices of adult female mice recapitulates reduced operant response and increased compulsivity, respectively. These results indicate that Esrra deficiency in the mouse brain impairs behavioral responses in multiple functional domains.

P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells.

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.