Endothelin-1 increases Na+-K+-2Cl- cotransporter-1 expression in cultured astrocytes and in traumatic brain injury model: An involvement of HIF1α activation.

Na+-K+-2Cl- cotransporter-1 (NKCC1) is present in brain cells, including astrocytes. The expression of astrocytic NKCC1 increases in the acute phase of traumatic brain injury (TBI), which induces brain edema. Endothelin-1 (ET-1) is a factor that induces brain edema and regulates the expression of several pathology-related genes in astrocytes. In the present study, we investigated the effect of ET-1 on NKCC1 expression in astrocytes. ET-1 (100 nM)-treated cultured astrocytes showed increased NKCC1 mRNA and protein levels. The effect of ET-1 on NKCC1 expression in cultured astrocytes was reduced by BQ788 (1 µM), an ETB antagonist, but not by FR139317 (1 µM), an ETA antagonist. The involvement of ET-1 in NKCC1 expression in TBI was examined using a fluid percussion injury (FPI) mouse model that replicates the pathology of TBI with high reproducibility. Administration of BQ788 (15 nmol/day) decreased FPI-induced expressions of NKCC1 mRNA and protein, accompanied with a reduction of astrocytic activation. FPI-induced brain edema was attenuated by BQ788 and NKCC1 inhibitors (azosemide and bumetanide). ET-1-treated cultured astrocytes showed increased mRNA and protein expression of hypoxia-inducible factor-1α (HIF1α). Immunohistochemical observations of mouse cerebrum after FPI showed co-localization of HIF1α with GFAP-positive astrocytes. Increased HIF1α expression in the TBI model was reversed by BQ788. FM19G11 (an HIF inhibitor, 1 µM) and HIF1α siRNA suppressed ET-induced increase in NKCC1 expression in cultured astrocytes. These results indicate that ET-1 increases NKCC1 expression in astrocytes through the activation of HIF1α.

Roles of astrocytic sonic hedgehog production and its signal for regulation of the blood-brain barrier permeability.

Sonic hedgehog (Shh) is a secreted glycopeptide belonging to the hedgehog family that is essential for morphogenesis during embryonic development. The Shh signal is mediated by two membrane proteins, Patched-1 (Ptch-1) and Smoothened (Smo), following the activation of transcription factors such as Gli. Shh decreases the permeability of the blood-brain barrier (BBB) and plays a key role in its function. In the damaged brain, BBB function is remarkably disrupted. The BBB disruption causes brain edema and neuroinflammation resulting from the extravasation of serum components and the infiltration of inflammatory cells into the cerebral parenchyma. Multiple studies have suggested that astrocyte is a source of Shh and that astrocytic Shh production is increased in the damaged brain. In various experimental animal models of acute brain injury, Shh or Shh signal activators alleviate BBB disruption by increasing tight junction proteins in endothelial cells. Furthermore, activation of astrocytic Shh signaling reduces reactive astrogliosis, neuroinflammation, and increases the production of vascular protective factors, which alleviates BBB disruption in the damaged brain. These findings suggest that astrocytic Shh and Shh signaling protect BBB function in the damaged brain and that target drugs for Shh signaling are expected to be novel therapeutic drugs for acute brain injuries.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Drug Discovery Research for Traumatic Brain Injury Focused on Functional Molecules in Astrocytes.

Traumatic brain injury (TBI) is severe damage to the head caused by traffic accidents, falls, and sports. Because TBI-induced disruption of the blood-brain barrier (BBB) causes brain edema and neuroinflammation, which are major causes of death or serious disabilities, protection and recovery of BBB function may be beneficial therapeutic strategies for TBI. Astrocytes are key components of BBB integrity, and astrocyte-derived bioactive factors promote and suppress BBB disruption in TBI. Therefore, the regulation of astrocyte function is essential for BBB protection. In the injured cerebrum of TBI model mice, we found that the endothelin ETB receptor, histamine H2 receptor, and transient receptor potential vanilloid 4 (TRPV4) were predominantly expressed in reactive astrocytes. We also showed that repeated administration of an ETB receptor antagonist, H2 receptor agonist, and TRPV4 antagonist alleviated BBB disruption and brain edema in a TBI mouse model. Furthermore, these drugs decreased the expression levels of astrocyte-derived factors promoting BBB disruption and increased the expression levels of astrocyte-derived protective factors in the injured cerebrum after TBI. These results suggest that the ETB receptor, H2 receptor, and TRPV4 are molecules that regulate astrocyte function, and might be attractive candidates for the development of therapeutic drugs for TBI.

Differential regulation of histamine H1 receptor-mediated ERK phosphorylation by Gq proteins and arrestins.

Gq protein-coupled histamine H1 receptors play crucial roles in allergic and inflammatory reactions, in which the phosphorylation of extracellular signal-regulated kinase (ERK) appears to mediate the production of inflammatory cytokines. ERK phosphorylation is regulated by G protein- and arrestin-mediated signal transduction pathways. Here, we aimed to explore how H1 receptor-mediated processes of ERK phosphorylation might be differentially regulated by Gq proteins and arrestins. For this purpose, we evaluated the regulatory mechanism(s) of H1 receptor-mediated ERK phosphorylation in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and mutated to alanine, respectively. Immunoblotting analysis indicated that histamine-induced ERK phosphorylation was prompt and transient in cells expressing Gq protein-biased S487TR, whereas it was slow and sustained in cells expressing arrestin-biased S487A. Inhibitors of Gq proteins (YM-254890) and protein kinase C (PKC) (GF109203X), and an intracellular Ca2+ chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation in cells expressing S487TR, but not those expressing S487A. Conversely, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation in cells expressing S487A, but not those expressing S487TR. These results suggest that H1 receptor-mediated ERK phosphorylation might be differentially regulated by the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways to potentially determine the early and late phases of histamine-induced allergic and inflammatory responses, respectively.

Roles of Astrocytic Endothelin ETB Receptor in Traumatic Brain Injury.

Traumatic brain injury (TBI) is an intracranial injury caused by accidents, falls, or sports. The production of endothelins (ETs) is increased in the injured brain. ET receptors are classified into distinct types, including ETA receptor (ETA-R) and ETB receptor (ETB-R). ETB-R is highly expressed in reactive astrocytes and upregulated by TBI. Activation of astrocytic ETB-R promotes conversion to reactive astrocytes and the production of astrocyte-derived bioactive factors, including vascular permeability regulators and cytokines, which cause blood-brain barrier (BBB) disruption, brain edema, and neuroinflammation in the acute phase of TBI. ETB-R antagonists alleviate BBB disruption and brain edema in animal models of TBI. The activation of astrocytic ETB receptors also enhances the production of various neurotrophic factors. These astrocyte-derived neurotrophic factors promote the repair of the damaged nervous system in the recovery phase of patients with TBI. Thus, astrocytic ETB-R is expected to be a promising drug target for TBI in both the acute and recovery phases. This article reviews recent observations on the role of astrocytic ETB receptors in TBI.

Selective histamine H2 receptor agonists alleviate blood-brain barrier disruption by promoting the expression of vascular protective factors following traumatic brain injury in mice.

Histamine is a major neurotransmitter and alleviates neuronal damage after ischemic injury via H2 receptors. Herein, we investigated the effects of H2 receptor agonists on the blood-brain barrier (BBB) disruption after traumatic brain injury (TBI). Male ddY mice were used to generate the TBI model, in which a fluid percussion injury (FPI) was induced by a hydraulic impact. The BBB disruption was evaluated using Evans blue extravasation. H2 receptor agonists, amthamine and dimaprit, were administered into the lateral cerebroventricle (i.c.v.) or tail vein (i.v.) from 3 hours to 3 days after FPI. The i.c.v. or i.v. administration of amthamine and dimaprit reduced FPI-induced Evans blue extravasation and promoted mRNA expression of vascular protective factors, including angiopoietin-1 and sonic hedgehog. The co-administration of ranitidine, a H2 receptor antagonist, inhibited these effects. Expression of the H2 receptor was observed in astrocytes and brain microvascular endothelial cells (BMECs) in the injured cortex. Treatment with amthamine and dimaprit promoted mRNA expression of vascular protective factors in astrocytes and BMECs. These results suggest that H2 receptor agonists alleviate TBI-induced BBB disruption by increasing the expression of vascular protective factors in astrocytes and BMECs.

Liver-specific overexpression of lipoprotein lipase improves glucose metabolism in high-fat diet-fed mice.

The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7-2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease.

ZFAND3 Overexpression in the Mouse Liver Improves Glucose Tolerance and Hepatic Insulin Resistance.

Genome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo. This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus.