The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster†.

The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization and early embryogenesis, but the regulatory mechanisms to gametes and zygotes are unclear. We evaluated the oviductal regulation of early embryonic development using Ovgp1 (encoding an oviductal humoral factor, OVGP1)-knockout golden hamsters. The experimental results revealed the following: (1) female Ovgp1-knockout hamsters failed to produce litters; (2) in the oviducts of Ovgp1-knockout animals, fertilized eggs were sometimes identified, but their morphology showed abnormal features; (3) the number of implantations in the Ovgp1-knockout females was low; (4) even if implantations occurred, the embryos developed abnormally and eventually died; and (5) Ovgp1-knockout female ovaries transferred to wild-type females resulted in the production of Ovgp1-knockout egg-derived OVGP1-null litters, but the reverse experiment did not. These results suggest that OVGP1-mediated physiological events are crucial for reproductive process in vivo, from fertilization to early embryonic development. This animal model shows that the fate of the zygote is determined not only genetically, but also by the surrounding oviductal microenvironment.

Ablation of diacylglycerol kinase ε promotes whitening of brown adipose tissue under high fat diet feeding.

Adipose tissue (AT) comprises distinct fat depots such as white AT and brown AT. White and brown adipocytes exhibit different morphological and physiological properties. White adipocytes containing large single lipid droplet (LD) provide energy on demand whereas brown adipocytes loaded with multilocular LDs consume energy to generate heat or dissipate excess energy. Recent studies have shown that multilocular brown-like cells emerge in white AT under certain conditions. These cells termed beige adipocytes participate in energy expenditure and heat generation. In the process of lipolysis, TG is broken down into free fatty acid and diacylglycerol (DG). In this regard, DG also serves as a signaling molecule activating some proteins such as protein kinase C. Therefore, DG kinase (DGK), an enzyme which phosphorylates DG into phosphatidic acid (PA), plays a pivotal role in integrating energy homeostasis and intracellular signaling. Recently, we described that DGKε-KO mice exhibit increased adiposity in visceral white AT accompanied with impaired glucose tolerance early (40 days) in the course of high fat diet (HFD) feeding, although these mice exhibit "browning or beiging" in visceral white AT associated with improved glucose tolerance after longer term HFD feeding (180 days). This study was conducted to understand the overall features of adipose tissues and investigate changes in subcutaneous (inguinal) white AT and interscapular brown AT of DGKε-KO mice during the course of HFD feeding. Results demonstrated that fat accumulation is promoted in all fat depots under 40 days of HFD feeding conditions. Remarkably, "whitening" of brown adipocytes was identified in DGKε-deficient brown AT during the course of HFD feeding, suggesting brown adipocyte dysfunction. In addition, insulin levels were considerably elevated in DGKε-KO mice under 180 days of HFD feeding conditions. Collectively, these findings suggest that brown adipocytes are dysfunctional in DGKε-KO mice, which promotes browning or beiging in visceral white AT. Beige adipocytes may take over energy disposal and contribute to improving glucose tolerance with the aid of high levels of insulin in DGKε-KO mice upon excess feeding.

Ablation of DGKα facilitates α-smooth muscle actin expression via the Smad and PKCδ signaling pathways during the acute phase of CCl4 -induced hepatic injury.

Expression of α-smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFß-induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second-messenger DG, thereby controlling components of DG-mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFß stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFß stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFß stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα-knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively.

Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Discrete localization of phospholipase Cß3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.

Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCß3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCß3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCß3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCß3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.

Localization of phosphatidylinositol phosphate 5 kinase γ, phospholipase ß3 and diacylglycerol kinase ζ in corneal epithelium in comparison with conjunctival epithelium of mice.

Based on the theory that the phosphoinositide (PI) signal is involved in the physiology of cornea and conjunctiva, we examined the localization in the mouse anterior ocular epithelia of immunoreactivities for phosphatidylinositol 4-phosphate 5-kinase (PIP5K), phospholipase C (PLC) and diacylglycerol kinase (DGK), enzymes that work sequentially in PI cycle. Immunoreactivity for PIP5Kγ in the corneal epithelium, including the limbus, was distinct in adults in contrast to faint or negligible immunoreactivity in the conjunctival epithelium in neonatal mice. This adult localization pattern was first recognized at the postnatal time of eyelid opening. Immunoreactivity for PLCß3 was rather equally distinct throughout the entire corneal and conjunctival epithelia in adults. DGKζ-immunoreactive nuclei were mainly localized in the basal half domain of the corneal epithelium but in both basal and apical domains of the conjunctival epithelium in adults. This nuclear immunoreactivity was at weak or negligible levels in the peripheral and limbus cornea and in a considerable portion of the bulbar conjunctival epithelium continuous with the limbus. The adult patterns for PLCß3 and DGKζ were already present at birth. The present findings suggest the following possibilities on the functional significance of the three enzyme molecules. PIP5Kγ is involved in cornea-specific functions such as bright-field vision, including corneal transparency, and in the stability of epithelial junctions, for which there seems to be a much higher requirement in the corneal epithelium than in the conjunctival epithelium. PLCß3 is involved from birth in as-yet undefined functions exerted ubiquitously from birth in both corneal and conjunctival epithelia. DGKζ is involved in regulation from birth of the transcription in epithelial cells, including apoptosis as well as regulation of mitosis of epithelial cells in both cornea and conjunctiva, with the transcription involvement more apparent in the conjunctiva, although it does not work in stem cells of the corneal limbus.

Associations of the A118G OPRM1 polymorphism with sociotropy and interpersonal sensitivity.

BACKGROUND: The µ-opioid receptor (MOR) plays an important role in social bonding behaviors, while it is implicated in the pathophysiology of depression. It is shown that the A118G polymorphism (rs1799971) of the MOR gene (OPRM1) causes amino-acid exchange from Asn to Asp, and that this polymorphism is associated with altered mu-opioid receptor function. Meanwhile, sociotropy/autonomy and interpersonal sensitivity are personality vulnerabilities to depression characterized by distinctive interpersonal styles. The present study tested the hypothesis that the functional A118G OPRM1 polymorphism influences these personality traits. METHODS: The subjects were 402 physically and mentally healthy Japanese volunteers. Sociotropy and autonomy were measured by the Sociotropy-Autonomy Scale, and interpersonal sensitivity was evaluated by the Interpersonal Sensitivity Measure. The A118G polymorphism of the OPRM1 was determined by the PCR method. RESULTS: In one factor analysis of covariance, there were differences in scores of sociotropy (uncorrected p < .001, corrected p < .003) and interpersonal sensitivity (uncorrected p = .015, corrected p = .045), but not autonomy, among the A/A, A/G, and G/G genotypes. Post hoc LSD tests showed that sociotropy scores were higher in the A/A group than in the A/G (p = .029) and G/G (p < .001) groups, and higher in the A/G group than in the G/G group (p = .004). Interpersonal sensitivity scores were higher in the A/A group than in the A/G (p = .023) and G/G (p = .009) groups. CONCLUSION: This study suggests that the A118G OPRM1 polymorphism is associated with sociotropy and interpersonal sensitivity, interpersonal vulnerabilities to depression.

Diacylglycerol Kinase ε in Adipose Tissues: A Crosstalk Between Signal Transduction and Energy Metabolism.

Diacylglycerol (DG) is unique in lipid metabolism because it serves not only as an intermediate product for triglyceride synthesis, but also as a signaling molecule that activates proteins containing DG-responsive elements, such as protein kinase C. Consequently, DG acts as a hub between energy metabolism and intracellular signaling. Of DG metabolizing pathways, DG kinase (DGK) phosphorylates DG to produce phosphatidic acid, which also serves as a second messenger. Several lines of evidence suggest that DGK is deeply involved in metabolic diseases such as obesity and insulin resistance. Of DGK isozymes, DGKε is simplest in terms of structure, but it is characterized by substrate specificity toward arachidonoyl-DG. Recently, we have reported that DGKε deficiency promotes adipose tissue remodeling in mice during the course of high fat diet (HFD) feeding regimen including obesity, insulin resistance, and beige adipogenesis. DGKε ablation engenders altered expression of other lipid metabolizing enzymes, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and diacylglycerol acyltransferase (DGAT). Subcellular localization of DGKε in the endoplasmic reticulum suggests involvement of this isozyme in lipid energy homeostasis. This review presents current findings of DGKε in lipid-orchestrated pathophysiology, especially unique phenotypes of DGKε-knockout mice in the early and late stages of obesogenic conditions.

Combination therapy for hepatocellular carcinoma with diacylglycerol kinase alpha inhibition and anti-programmed cell death-1 ligand blockade.

Activation of diacylglycerol kinase alpha (DGKα) augments proliferation and suppresses apoptosis of cancer cells and induces T lymphocyte anergy. We investigated the dual effects of DGKα inhibition on tumorigenesis and anti-tumor immunity with the aim of establishing a novel therapeutic strategy for cancer. We examined the effects of a DGKα inhibitor (DGKAI) on liver cancer cell proliferation and cytokine production by immune cells in vitro and on tumorigenesis and host immunity in a hepatocellular carcinoma (HCC) mouse model. Oral DGKAI significantly suppressed tumor growth and prolonged survival in model mice. Tumor infiltration of T cells and dendritic cells was also enhanced in mice treated with DGKAI, and the production of cytokines and cytotoxic molecules by CD4+ and CD8+ T cells was increased. Depletion of CD8+ T cells reduced the effect of DGKAI. Furthermore, interferon-γ stimulation augmented the expression of programmed cell death-1 ligand (PD-L1) on cancer cells, and DGKAI plus an anti-PD-L1 antibody strongly suppressed the tumor growth. These results suggest that DGKα inhibition may be a promising new treatment strategy for HCC.

Immunocytochemical Analysis of DGKη in Cultured Cells Using a Monoclonal Antibody DhMab-4.

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) to phosphatidic acid (PA). Since both DG and PA serve as intracellular second messenger molecules, DGK plays a pivotal role in balancing these two signaling pathways. Of the DGK family, DGKη is classified as a type II DGK. Reportedly, DGKη is expressed ubiquitously through mammalian tissues and cells. Previous studies using cDNA transfection methods reported cytoplasmic localization of DGKη in cultured human cells. However, subcellular localization of native protein is still unknown. Recently, we established a human DGKη-specific monoclonal antibody, DhMab-4. In this study, we examined subcellular localization of native protein of DGKη using DhMab-4 by immunocytochemistry in human cultured cells.

Gpr19 is a circadian clock-controlled orphan GPCR with a role in modulating free-running period and light resetting capacity of the circadian clock.

Gpr19 encodes an evolutionarily conserved orphan G-protein-coupled receptor (GPCR) with currently no established physiological role in vivo. We characterized Gpr19 expression in the suprachiasmatic nucleus (SCN), the locus of the master circadian clock in the brain, and determined its role in the context of the circadian rhythm regulation. We found that Gpr19 is mainly expressed in the dorsal part of the SCN, with its expression fluctuating in a circadian fashion. A conserved cAMP-responsive element in the Gpr19 promoter was able to produce circadian transcription in the SCN. Gpr19-/- mice exhibited a prolonged circadian period and a delayed initiation of daily locomotor activity. Gpr19 deficiency caused the downregulation of several genes that normally peak during the night, including Bmal1 and Gpr176. In response to light exposure at night, Gpr19-/- mice had a reduced capacity for light-induced phase-delays, but not for phase-advances. This defect was accompanied by reduced response of c-Fos expression in the dorsal region of the SCN, while apparently normal in the ventral area of the SCN, in Gpr19-/- mice. Thus, our data demonstrate that Gpr19 is an SCN-enriched orphan GPCR with a distinct role in circadian regulation and may provide a potential target option for modulating the circadian clock.

Oxytocin receptor polymorphism influences characterization of harm avoidance by moderating susceptibility to affectionless control parenting.

INTRODUCTION: Oxytocin receptor (OXTR) gene polymorphism reportedly moderates effects of negative environments during childhood on mental function and behavior such as depressive symptoms and externalizing problems. This study examined OXTR gene polymorphism effects on personality traits in healthy participants, considering interaction effects of polymorphism with affectionless control (AC) parenting which is one of the dysfunctional and pathogenic parenting styles. METHODS: For 496 Japanese volunteers, personality was evaluated using the Temperament and Character Inventory. The Parental Bonding Instrument, which has subscales of care and protection, was used to assess perceived parental rearing. AC parenting was defined as low care and high protection. A/G polymorphism of the OXTR gene (rs53576) was detected using TaqMan SNP Genotyping Assay. RESULTS: Two-way analysis of covariance revealed significant interaction effects between the genotype and the number of AC parents on scores of harm avoidance, with no significant main effect of genotype on any personality. Post-hoc analysis revealed that the harm avoidance scores were increased in a stepwise manner with respect to the increase of the number of AC parents in the A allele carriers. No similar association was observed in the A allele noncarriers. CONCLUSION: The results of this study suggest that OXTR polymorphism influences characterization of harm avoidance by moderating susceptibility to AC parenting.

Cellular expression and subcellular localization of diacylglycerol kinase γ in rat brain.

Gq protein-coupled receptors lead to activation of phospholipase C, which triggers phosphoinositide signaling. Diacylglycerol (DG) is one of the phosphoinositide metabolites and serves as a second messenger. Diacylglycerol kinase (DGK) phosphorylates DG to produce another second messenger phosphatidic acid. Of the DGK family, DGKγ is predominantly expressed in the brain at the mRNA level. Recent studies have shown the expression of DGKγ in vascular endothelial cells and adrenal medullary cells at the protein level, although its detailed cellular expression pattern and subcellular localization in the brain remain to be determined. In the present study, we addressed this point using specific DGKγ antibody. DGKγ was expressed in both projection neurons and interneurons in the cerebral cortex, hippocampal formation, and cerebellum. In cerebellar Purkinje cells, DGKγ was distributed to the soma and dendrites. Fractionation study revealed that DGKγ was enriched in the internal membranes containing the endoplasmic reticulum and Golgi complex. In immunoelectron microscopy, DGKγ was localized throughout the smooth endoplasmic reticulum system. These findings suggest that DGKγ shows unique cellular expression pattern in the brain and distinct subcellular localization different from other DGK isozymes.

Regulation of p53 and NF-κB transactivation activities by DGKζ in catalytic activity-dependent and -independent manners.

Diacylglycerol kinase (DGK) constitutes a family of enzymes that phosphorylate diacylglycerol to phosphatidic acid (PA). These lipids serve as second messengers, thereby activating distinct downstream cascades and different cellular responses. Therefore, DG-to-PA conversion activity induces a phase transition of signaling pathways. One member of the family, DGKζ, is involved closely with stress responses. Morphological data showing that DGKζ localizes predominantly to the nucleus and that it shuttles between the nucleus and the cytoplasm implicate DGKζ in the regulation of transcription factors during stress responses. Tumor suppressor p53 and NF-κB are major stress-responsive transcription factors. They exert opposing effects on cellular pathophysiology. Herein, we summarize DGKζ catalytic activity-dependent and -independent regulatory mechanisms of p53 and NF-κB transactivation activities, including p53 degradation and NF-κB nuclear translocation. We also discuss how each component of DGKζ-interacting protein complex modulates the specificity and selectivity of target gene expression.

Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue.

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

Mu-Opioid Receptor Polymorphism Moderates Sensitivity to Parental Behaviors During Characterization of Personality Traits.

PURPOSE: Attachment research shows that attachment experiences with parents in childhood influence the characterization of personality traits. Meanwhile, it is known that mu-opioid receptor function is involved in human attachment. Furthermore, a few studies suggest that the A118G polymorphism of the mu-opioid receptor gene (OPRM1) is associated with altered mu-opioid receptor function. Thus, we examined if the OPRM1 polymorphism moderates the sensitivity to parental behaviors and thereby contributes to the characterization of personality traits. MATERIALS AND METHODS: Participants were 725 healthy Japanese. Parenting practices of their parents were evaluated by the Parental Bonding Instrument (PBI) with the care and protection subscales. Personality was evaluated using the Temperament and Character Inventory (TCI). The OPRM1 A118G polymorphism was detected by a PCR method. RESULTS: Multiple regression analyses revealed significant effects of the interaction between the OPRM1 genotype and maternal protection on scores of the self-directedness and cooperativeness dimensions, while significant main effects of the OPRM1 genotype on scores of the TCI were not found. Further analyses showed that there were significant negative correlations between maternal protection scores and the two dimensional scores in the A/A and A/G genotypes with higher correlation coefficients in the former, but not in the G/G genotype. CONCLUSION: The present study suggests that the OPRM1 polymorphism contributes to the characterization of personality traits by moderating the sensitivity to parental behaviors, especially maternal protection.

Interrelation Between Increased BDNF Gene Methylation and High Sociotropy, a Personality Vulnerability Factor in Cognitive Model of Depression.

PURPOSE: It is suggested that increased methylation of the brain-derived neurotrophic factor (BDNF) gene is involved in the pathogenesis of depression, while sociotropy and autonomy are proposed as personality vulnerability factors in cognitive model of depression. We examined the interrelation between BDNF gene methylation and sociotropy or autonomy, with taking into account the previously reported deleterious effect of parental overprotection on sociotropy. MATERIALS AND METHODS: The participants consisted of 90 healthy Japanese volunteers. Methylation levels of the BDNF gene in peripheral blood were quantified by bisulfite pyrosequencing. Sociotropy and autonomy were assessed by the Sociotropy-Autonomy Scale, and perceived parental protection was evaluated by the Parental Bonding Instrument. RESULTS: In Pearson's correlation analysis, there was a positive correlation between methylation levels of the BDNF gene and sociotropy scores (p<0.05) but not autonomy scores, and a positive correlation between maternal protection scores and sociotropy scores (p<0.05). In structural equation modeling, two models were proposed; the first one is that hypermethylation of the BDNF gene and maternal overprotection independently contribute to high sociotropy, and the second one is that maternal overprotection contributes to high sociotropy which then leads to hypermethylation of the BDNF gene. CONCLUSION: The present study suggests an interrelation between increased BDNF gene methylation and high sociotropy.

DGKζ depletion attenuates HIF-1α induction and SIRT1 expression, but enhances TAK1-mediated AMPKα phosphorylation under hypoxia.

Cells cope with environmental changes through various mechanisms. Pathways involving HIF-1, SIRT1, and AMPK play major roles in energy homeostasis under stress conditions. Diacylglycerol kinase (DGK) constitutes an enzyme family that catalyzes conversion of diacylglycerol to phosphatidic acid. We reported earlier that energy depletion such as ischemia induces proteasomal degradation of DGKζ before cell death, suggesting involvement of DGKζ in energy homeostasis. This study examines how DGKζ depletion affects the regulation of HIF-1α, SIRT1, and AMPKα. Under hypoxia DGKζ depletion attenuates HIF-1α induction and SIRT1 expression, which might render cells vulnerable to energy stress. However, DGKζ depletion engenders enhanced AMPKα phosphorylation by upstream kinase TAK1 and an increase in intracellular ATP levels. Results suggest that DGKζ exerts a suppressive effect on TAK1 activity in the AMPK activation mechanism, and that DGKζ depletion might engender dysregulation of the AMPK-mediated energy sensor system.

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176.

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176.

Mice lacking DGKε show increased beige adipogenesis in visceral white adipose tissue after long-term high fat diet in a COX-2- dependent manner.

Adipose tissue is a central site for energy storage in the form of triglyceride (TG). Under excess energy conditions, TG is synthesized by acylation of diacylglycerol (DG), whereas TG is broken down into DG and free fatty acid, which provide energy for mitochondrial lipid oxidation when needed. In this regard, DG is not merely an intermediate metabolite for TG metabolism; it also serves as a signaling molecule. DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Consequently, DGK plays a pivotal role in the control of lipid metabolism and signal transduction pathway. Recently, a report has described that DGKε-knockout (KO) mice show expansion of epididymal white adipose tissue (WAT) together with the impairment of glucose clearance after short-term (40 days) high fat diet (HFD) feeding, an early presymptomatic phase of obesity in wild-type animals. Nevertheless, no report describes an investigation of their phenotype under long-term HFD feeding conditions. Remarkably, results obtained during long-term HFD feeding show that WAT mass is decreased significantly and that the blood glucose profile in response to glucose challenge is improved in DGKε-KO mice compared with wild-type, which contrast sharply against the phenotype shown for short-term HFD feeding. Morphological examination reveals that cyclooxygenase-2 (COX-2) expression and clusters of uncoupling protein 1 (UCP1)-positive multilocular brown-like ("beige") adipocyte are induced in DGKε-deficient WAT after long-term HFD feeding, suggesting that beige adipocytes facilitate energy expenditure during prolonged HFD feeding. Administration of celecoxib, a selective inhibitor of COX-2, abolishes the appearance of UCP1-positive beige adipocytes in DGKε-KO mice. These findings suggest that DGKε deficiency promotes visceral WAT remodeling in a COX-2-dependent manner under long-term HFD feeding conditions.