Lipocalin-type Prostaglandin D2 Synthase Research Articles

Role of L-PGDS in Light Alcohol Consumption-Induced Cerebral Angiogenesis in Male Mice

Ischemic stroke is one of the leading causes of death and permanent disability. Promoting cerebral angiogenesis before or after ischemic stroke reduces ischemic brain damage and improves functional recovery. Alcohol is one of the most commonly consumed chemical substances. Light alcohol consumption (LAC) reduces the incidence and improves the prognosis of ischemic stroke. Recently, we found that LAC promotes cerebral angiogenesis under basal conditions and following ischemic stroke. In addition, LAC upregulates lipocalin-type prostaglandin D2 synthase (L-PGDS), the principal PGH2 isomerase mainly expressed in the central nervous and cardiovascular systems. Therefore, we determined the role of L-PGDS in LAC-induced cerebral angiogenesis. In in vitro studies, low-concentration ethanol significantly upregulated L-PGDS and increased angiogenic capability (tube formation, migration, and proliferation) in cultured C57BL/6J mouse brain microvascular endothelial cells (MBMVECs). AT-56, a selective L-PGDS inhibitor, abolished the increased angiogenic capability. In in vivo studies, 8-week gavage feeding with low-dose ethanol significantly upregulated L-PGDS and increased vessel density and vessel branches in the cerebral cortex and subcortical area under physiological conditions and following ischemic stroke in C57BL/6J male mice. AT-56 and endothelial cell-specific L-PGDS conditional knockout did not affect vessel density and vessel branches of the cerebral cortex and subcortical area in water-fed control mice but alleviated the proangiogenic effect in low-dose ethanol-fed mice under basal conditions and following ischemic stroke. Therefore, our findings suggest that L-PGDS may be crucial in LAC-induced cerebral angiogenesis. NIH 2R01AA023610. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Serum lipocalin-like protein in Sebastes rockfish: A novel biomarker for reproductive stage males

Viviparous rockfishes (Sebastes spp.) are ecologically and economically important fisheries and aquaculture species. Because the males lack obvious morphological characteristics related to reproductive status, it is challenging to select semen donors for artificial insemination. We previously identified lipocalin-type prostaglandin D2 synthase homolog protein (LPGDShp) in the urine of male black rockfish (Sebastes schlegelii). Subsequently, LPGDShp-like protein (hereafter, lipocalin-like protein) was immunologically detected in the serum of two Sebastes species, black rockfish and white-edged rockfish (S. taczanowskii). In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed to explore the potential of serum lipocalin-like protein as a biomarker for selecting mature males. The ELISA measured lipocalin-like protein in the sera of black rockfish and white-edged rockfish over an assay range between 0.391 and 12.5 ng/mL. In male black rockfish, serum levels of lipocalin-like protein were higher during the copulation season, and they were higher than levels in females during both the copulation and non-copulation seasons. In male white-edged rockfish sampled nearly monthly for a year, serum lipocalin-like protein levels showed synchronous dynamics with gonadosomatic index (GSI). Histological examination of testis revealed that the serum levels were elevated in the late/functional maturation stages, as compared to early/mid-maturation and resting stages. In both rockfish species, while serum levels of lipocalin-like protein were positively correlated with GSI in males, no such trend was observed in females. These results indicate, for the first time, that serum lipocalin-like protein can be employed as a hematological biomarker to detect the reproductive status of male rockfishes.

Brain-Derived Major Glycoproteins Are Possible Biomarkers for Altered Metabolism of Cerebrospinal Fluid in Neurological Diseases

Cerebrospinal fluid (CSF) plays an important role in the homeostasis of the brain. We previously reported that CSF major glycoproteins are biosynthesized in the brain, i.e., lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin isoforms carrying unique glycans. Although these glycoproteins are secreted from distinct cell types, their CSF levels have been found to be highly correlated with each other in cases of neurodegenerative disorders. The aim of this study was to examine these marker levels and their correlations in other neurological diseases, such as depression and schizophrenia, and disorders featuring abnormal CSF metabolism, including spontaneous intracranial hypotension (SIH) and idiopathic normal pressure hydrocephalus (iNPH). Brain-derived marker levels were found to be highly correlated with each other in the CSF of depression and schizophrenia patients. SIH is caused by CSF leakage, which is suspected to induce hypovolemia and a compensatory increase in CSF production. In SIH, the brain-derived markers were 2-3-fold higher than in other diseases, and, regardless of their diverse levels, they were found to be correlated with each other. Another abnormality of the CSF metabolism, iNPH, is possibly caused by the reduced absorption of CSF, which secondarily induces CSF accumulation in the ventricle; the excess CSF compresses the brain's parenchyma to induce dementia. One potential treatment is a "shunt operation" to bypass excess CSF from the ventricles to the peritoneal cavity, leading to the attenuation of dementia. After the shunt operation, marker levels began to increase within a week and then further increased by 2-2.5-fold at three, six, and twelve months post-operation, at which point symptoms had gradually attenuated. Notably, the marker levels were found to be correlated with each other in the post-operative period. In conclusion, the brain-derived major glycoprotein markers were highly correlated in the CSF of patients with different neurological diseases, and their correlations were maintained even after surgical intervention. These results suggest that brain-derived proteins could be biomarkers of CSF production.

Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish

Black rockfish (Sebastes schlegelii) and its relatives are viviparous marine fish. Males produce urinary proteins during the copulation season; however, the identity of these proteins was unknown. In this study, we focused on high-molecular-weight urinary proteins (HMWups) in male black rockfish. The HMWups were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) of urine. In silico analyses of RNA-seq data predicted the tissue distribution of candidate HMWup transcripts and their gene structures. Candidate cDNAs were cloned and a recombinant protein of a major candidate was prepared. Western blotting of urine using an antiserum against the recombinant protein was performed to reconfirm the LC-MS/MS results. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were employed to validate the prediction by RNA-seq and identify the cells producing HMWups, respectively. LC-MS/MS, in conjunction with Western blotting and cDNA cloning, identified the HMWups as lipocalin-type prostaglandin D2 synthase (l-PGDS) homologs. RNA-seq analyses and qRT-PCR revealed that the l-PGDS homolog transcripts were dominantly expressed in the testis and male kidney; Sertoli cells and epithelial cells in the renal tubules were immunoreactive. These results indicated that major protein components in the urine of male black rockfish are l-PGDS homologs, potentially produced by the renal tubules in the kidney. Male rockfish (genus Sebastes) are thought to release unknown pheromone substances during mating behavior. The knowledge and tools obtained in this study empower research into the role(s) of HMWups in pheromone systems underlying rockfish reproduction. No protein-type teleost pheromone has heretofore been discovered.

Anti-inflammatory effects of the prostaglandin D2/prostaglandin DP1 receptor and lipocalin-type prostaglandin D2 synthase/prostaglandin D2 pathways in bacteria-induced bovine endometrial tissue

Dairy cows often develop different degrees of endometritis after calving and this is attributed to pathogenic bacterial infections such as by Escherichia coli and Staphylococcus aureus. Infection of the bovine endometrium causes tissue damage and increases the expression of prostaglandin D2 (PGD2), which exerts anti-inflammatory effects on lung inflammation. However, the roles of PGD2 and its DP1 receptor in endometritis in cows remain unclear. Here, we examined the anti-inflammatory roles of the lipocalin-type prostaglandin D2 synthase (L-PGDS)/PGD2 and DP1 receptor regulatory pathways in bovine endometritis. We evaluated the regulatory effects of PGD2 on inflammation and tissue damage in E. coli- and S. aureus-infected bovine endometrial cells cultured in vitro. We found that the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)-α as well as expression of matrix metalloproteinase (MMP)-2, platelet-activating factor receptor (PAFR), and high mobility group box (HMGB)-1 were suppressed after DP1 receptor agonist treatment. In contrast, IL-6, IL-1β, and TNF-α release and MMP-2, PAFR, and HMGB-1 expression levels were increased after treatment of bovine endometrial tissue with DP1 receptor antagonists. DP1-induced anti-inflammatory effects were dependent on cellular signal transduction. The L-PGDS/PGD2 pathway and DP1 receptor induced anti-inflammatory effects in bovine endometrium infected with S. aureus and E. coli by inhibiting the mitogen-activated protein kinase and nuclear factor-κB signalling pathways, thereby reducing tissue damage. Overall, our findings provide important insights into the pathophysiological roles of PGD2 in bovine endometritis and establish a theoretical basis for applying prostaglandins or non-steroidal anti-inflammatory drugs for treating endometrial inflammatory infertility in bovines.

High Correlation among Brain-Derived Major Protein Levels in Cerebrospinal Fluid: Implication for Amyloid-Beta and Tau Protein Changes in Alzheimer's Disease.

The cerebrospinal fluid (CSF) plays an important role in homeostasis of the brain. We previously demonstrated that major CSF proteins such as lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin (Tf) that are biosynthesized in the brain could be biomarkers of altered CSF production. Here we report that the levels of these brain-derived CSF proteins correlated well with each other across various neurodegenerative diseases, including Alzheimer’s disease (AD). In addition, protein levels tended to be increased in the CSF samples of AD patients compared with the other diseases. Patients at memory clinics were classified into three categories, consisting of AD (n = 61), mild cognitive impairment (MCI) (n = 42), and cognitively normal (CN) (n = 23), with MMSE scores of 20.4 ± 4.2, 26.9 ± 1.7, and 29.0 ± 1.6, respectively. In each category, CSF protein levels were highly correlated with each other. In CN subjects, increased CSF protein levels correlated well with those of AD markers, including amyloid-β and tau protein, whereas in MCI and AD subjects, correlations declined with AD markers except p-tau. Future follow-up on each clinical subject may provide a clue that the CSF proteins would be AD-related biomarkers.

MSC-Derived Extracellular Vesicle-Delivered L-PGDS Inhibit Gastric Cancer Progression by Suppressing Cancer Cell Stemness and STAT3 Phosphorylation.

Mesenchymal stem cell- (MSC-) derived extracellular vesicles (EVs) serving as delivery system have attracted extensive research interest, especially in cancer therapy. In our previous study, lipocalin-type prostaglandin D2 synthase (L-PGDS) showed inhibitory effects on gastric cancer growth. In this study, we aimed to explore whether MSC-EV-delivered L-PGDS (EVs-L-PGDS) could inhibit gastric cancer progression. EVs-L-PGDS were generated from MSCs transfected with adenovirus encoding L-PGDS. Cell colony-forming, migration, invasion, and flow cytometry assays were used to show the inhibitory effects of EVs on tumor cells in vitro, and the nude mouse subcutaneous tumor model was performed to show the inhibitory effect of EVs on tumor progression in vivo. In vitro, EVs-L-PGDS could be internalized and inhibit the colony-forming, migration, and invasion ability of gastric cancer cell SGC-7901 and promote cell apoptosis. In vivo, EVs-L-PGDS inhibited the tumor growth in nude mouse subcutaneous tumor-bearing model. Compared with the PBS and EVs containing empty vector (EVs-Vector) group, more apoptotic cells and higher L-PGDS expression were detected in tumor tissue of the EVs-L-PGDS treatment group. And these differences are significant. Mechanistically, EVs-L-PGDS reduced the expression of stem cell markers including Oct4, Nanog, and Sox2 and inhibited STAT3 phosphorylation in gastric cancer cell SGC-7901. In conclusion, our results imply that MSC-derived EVs could be utilized as an effective nanovehicle to deliver L-PGDS for gastric cancer treatment, which provides a novel idea for the EV-based cancer therapy.

Protective Effect of Low-Dose Alcohol Consumption against Post-Ischemic Neuronal Apoptosis: Role of L-PGDS

Ischemic stroke is one of the leading causes of permanent disability and death in adults worldwide. Apoptosis is a major element contributing to post-ischemic neuronal death. We previously found that low-dose alcohol consumption (LAC) protects against neuronal apoptosis in the peri-infarct cortex following transient focal cerebral ischemia. Lipocalin-type prostaglandin D2 synthase (L-PGDS), which is mainly localized in the central nervous system (CNS), was previously shown to inhibit neuronal apoptosis. Therefore, we determined whether L-PGDS is involved in the protective effect of LAC against post-ischemic neuronal apoptosis. Wild-type (WT), CaMKIIαCreERT2/+/L-PGDS+/+, and CaMKIIαCreERT2/+/L-PGDSflox/flox mice on a C57BL/6J background were gavage fed with ethanol or volume-matched water once a day for 8 weeks. Tamoxifen (2 mg/day) was given intraperitoneally to CaMKIIαCreERT2/+/L-PGDS+/+ and CaMKIIαCreERT2/+/L-PGDSflox/flox mice for 5 days during the fourth week. AT-56 (30 mg/kg/day), a selective inhibitor of L-PGDS, was given orally to AT-56-treated WT mice from the fifth week for four weeks. Cerebral ischemia/reperfusion (I/R) injury, TUNEL-positive neurons, and cleaved caspase-3-positive neurons were measured at 24 h of reperfusion after a 90 min unilateral middle cerebral artery occlusion (MCAO). We found that 0.7 g/kg/day but not 2.8 g/kg/day ethanol significantly upregulated L-PGDS in the cerebral cortex. In addition, 0.7 g/kg/day ethanol diminished cerebral ischemia/reperfusion (I/R) injury and TUNEL-positive and cleaved caspase-3-positive neurons in the peri-infarct cortex in WT and CaMKIIαCreERT2/+/L-PGDS+/+ mice. Furthermore, the neuroprotective effect of 0.7 g/kg/day ethanol was alleviated in AT-56-treated WT and CaMKIIαCreERT2/+/L-PGDSflox/flox mice. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing post-ischemic neuronal apoptosis via an upregulated L-PGDS.

Biochemical and Structural Characteristics, Gene Regulation, Physiological, Pathological and Clinical Features of Lipocalin-Type Prostaglandin D2 Synthase as a Multifunctional Lipocalin.

Lipocalin-type prostaglandin (PG) D2 synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of the two series of PGs, to produce PGD2. PGD2 stimulates three distinct types of G protein-coupled receptors: (1) D type of prostanoid (DP) receptors involved in the regulation of sleep, pain, food intake, and others; (2) chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) receptors, in myelination of peripheral nervous system, adipocyte differentiation, inhibition of hair follicle neogenesis, and others; and (3) F type of prostanoid (FP) receptors, in dexamethasone-induced cardioprotection. L-PGDS is the same protein as β-trace, a major protein in human cerebrospinal fluid (CSF). L-PGDS exists in the central nervous system and male genital organs of various mammals, and human heart; and is secreted into the CSF, seminal plasma, and plasma, respectively. L-PGDS binds retinoic acids and retinal with high affinities (Kd < 100 nM) and diverse small lipophilic substances, such as thyroids, gangliosides, bilirubin and biliverdin, heme, NAD(P)H, and PGD2, acting as an extracellular carrier of these substances. L-PGDS also binds amyloid β peptides, prevents their fibril formation, and disaggregates amyloid β fibrils, acting as a major amyloid β chaperone in human CSF. Here, I summarize the recent progress of the research on PGD2 and L-PGDS, in terms of its “molecular properties,” “cell culture studies,” “animal experiments,” and “clinical studies,” all of which should help to understand the pathophysiological role of L-PGDS and inspire the future research of this multifunctional lipocalin.

Candidate Genes and Proteomic Biomarkers of Serum and Urine in Medication-Overuse Headache

Chronic headache is a topical problem of neurology, psychiatry and general practice. The medication-overuse headache (MOH) is one of the leading pathologies in the structure of chronic headache. However, early diagnosis of the MOH is challenging. We analyzed potential proteomic biomarkers of serum and urine in patients with MOH. Methods: We searched PubMed, Springer, Scopus, Web of Science, ClinicalKey, and Google Scholar databases for English publications over the past 10 years using keywords and their combinations. Results: We found and analyzed seven studies that met the search criteria for the purpose of the review, including 24 serum proteomic biomarkers and 25 urine proteomic biomarkers of MOH. Moreover, the candidate genes and locus of the studied serum (vitamin D-binding protein, lipocalin-type prostaglandin D2 synthase, apolipoprotein E, etc.) and urine proteomic biomarkers (uromodulin, alpha-1-microglobulin, zinc-alpha-2-glycoprotein, etc.) of MOH are presented in this review. Conclusions: The serum and urine proteomic biomarkers of MOH can potentially help with the identification of patients with MOH development. Due to the relevance of the problem, the authors believe that further investigation of the MOH proteomic biomarkers in different ethnic and racial groups of patients with primary headache is necessary. In addition, it is important to investigate whether medications of different drug classes influence the levels of serum and urine proteomic biomarkers.

Lipocalin-type Prostaglandin D2 Synthase appears to function as a Novel Adipokine Preventing Adipose Dysfunction in response to a High Fat Diet

Adipose dysfunction is the primary defect in obesity that contributes to the development of dyslipidemia, insulin resistance, cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and some cancers. Previously, we demonstrated the development of NAFLD in lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice regardless of diet. In the present study, we examined the role of L-PGDS in adipose in response to a high fat diet. We observed decreased expression of L-PGDS in adipose tissue and concomitant lower plasma levels in a dietary model of obesity as well as in insulin resistant 3T3-L1 adipocytes. We show reduced adiponectin expression and phosphorylation of AMPK in white adipose tissue of L-PGDS KO mice after 14 weeks on a high fat diet as compared to control C57BL/6 mice. We also observe an increased fat content in L-PGDS KO mice as demonstrated by adipocyte hypertrophy and increased expression of lipogenenic genes. We confirmed our in vivo findings in in vitro 3T3-L1 adipocytes, using an enzymatic inhibitor of L-PGDS (AT56). Rosiglitazone treatment drastically increased L-PGDS expression in insulin resistant 3T3-L1 adipocytes and increased adiponectin expression and AMPK phosphorylation in AT56 treated 3T3-L1 adipocytes. We conclude that the absence of L-PGDS has a deleterious effect on adipose tissue functioning, which further reduces insulin sensitivity in adipose tissue. Consequently, we propose L-PGDS appears to function as a potential member of the adipokine secretome involved in the regulation of the obesity-associated metabolic syndrome.

Mast Cell-Specific Deletion of Group III Secreted Phospholipase A2 Impairs Mast Cell Maturation and Functions.

Tissue-resident mast cells (MCs) have important roles in IgE-associated and -independent allergic reactions. Although microenvironmental alterations in MC phenotypes affect the susceptibility to allergy, understanding of the regulation of MC maturation is still incomplete. We previously reported that group III secreted phospholipase A2 (sPLA2-III) released from immature MCs is functionally coupled with lipocalin-type prostaglandin D2 (PGD2) synthase in neighboring fibroblasts to supply a microenvironmental pool of PGD2, which in turn acts on the PGD2 receptor DP1 on MCs to promote their proper maturation. In the present study, we reevaluated the role of sPLA2-III in MCs using a newly generated MC-specific Pla2g3-deficient mouse strain. Mice lacking sPLA2-III specifically in MCs, like those lacking the enzyme in all tissues, had immature MCs and displayed reduced local and systemic anaphylactic responses. Furthermore, MC-specific Pla2g3-deficient mice, as well as MC-deficient KitW-sh mice reconstituted with MCs prepared from global Pla2g3-null mice, displayed a significant reduction in irritant contact dermatitis (ICD) and an aggravation of contact hypersensitivity (CHS). The increased CHS response by Pla2g3 deficiency depended at least partly on the reduced expression of hematopoietic PGD2 synthase and thereby reduced production of PGD2 due to immaturity of MCs. Overall, our present study has confirmed that MC-secreted sPLA2-III promotes MC maturation, thereby facilitating acute anaphylactic and ICD reactions and limiting delayed CHS response.

116-OR: Lipocalin-Type Prostaglandin D2 Synthase Depletion Induces Adipose Tissue Dysfunction in Response to High-Fat Diet

Adipose tissue dysfunction is the primary defect in obesity which contributes to the development of dyslipidemia, insulin resistance, cardiovascular diseases, type 2 diabetes, nonalcoholic fatty liver disease and some cancers. Previous study from our lab implicated the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in glucose intolerance and atherosclerosis. Our lab also showed the development of NAFLD in L-PGDS knockout mice on both low fat and high fat diet. In the present study, we examined the role of L-PGDS in adipose tissue in response to high fat diet. We found decreased expression of L-PGDS in visceral and subcutaneous white adipose tissue (WAT) of C57BL/6 mice after 12 weeks on high fat diet (HFD). Our study showed increased fat content in L-PGDS KO mice as compared to control C57BL/6 mice after 14 weeks on HFD. Histological analysis showed hypertrophied adipocytes in visceral WAT of L-PGDS KO mice as compared to control mice. Furthermore, we found increased expression of the lipogenesis genes LXRα, SREBP-1c and FAS in visceral WAT of L-PGDS KO mice after 14 weeks on HFD. L-PGDS KO mice showed reduced gene expression of adiponectin and PPARγ in visceral WAT on HFD. The plasma adiponectin level was also decreased in L-PGDS KO mice. We conclude that absence of L-PGDS has a deleterious effect on adipose tissue functioning which further reduces insulin sensitivity in adipose tissue. Thus, our study suggests L-PGDS as a novel therapeutic target for the treatment of obesity-associated metabolic syndrome. Disclosure A. Srivastava: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None. Funding American Heart Association (15GRNT22420001); George Link, Jr. Foundation Inc. (3500-7294)

YAP promotes self-renewal of gastric cancer cells by inhibiting expression of L-PTGDS and PTGDR2.

Cancer stem cells have been implicated angiogenesis of tumor and invasiveness, drug resistance in tumors. Yes-associated protein 1 (YAP) owns carcinogenic roles in various organs, but the role of YAP in cancer stem cells of gastric cancer (GC) remains unclear. In this study, we explored the function and mechanism of YAP in GC cancer stem cells. First, we confirmed that the expression of YAP mRNA and protein in GC tissues was higher than in adjacent tissues by RT-PCR, western blot and immunohistochemistry. Immunofluorescence staining of the GC tissues revealed that the region of YAP expression coincided with the region of expression of the cancer stem cell marker SALL4 but did not overlap with that of the epithelial marker cytokeratin 14 (CK14). Additional research revealed that spherical cells expressed relatively high levels of YAP protein, and YAP overexpression reinforced self-renewal and expression of stem cell markers in the GC cells. Knockdown the expression of YAP reversed this phenomenon. Second, we examined the expression patterns of lipocalin-type prostaglandin D2 synthase (L-PTGDS) and prostaglandin D2 receptor 2 (PTGDR2) in GC tissues and proved that there was negatively correlation between the expression of L-PTGDS and PTGDR2 and YAP in GC tissues. Finally, we confirmed that YAP inhibited the expression of L-PTGDS and PTGDR2 by gain- and loss-of-function experiments. Moreover, the overexpression of L-PTGDS and PTGDR2 suppressed the proliferation and self-renewal induced by YAP in vitro and reversed the pro-tumor effect of YAP in vivo. Our results revealed a novel function of YAP and the mechanism underlying cancer stem cell regulation by YAP.

Brain pericytes are a major source of lipocalin-type prostaglandin D2 synthase in the cerebral cortex after ischemic stroke

Brain pericytes are a major source of lipocalin-type prostaglandin D2 synthase in the cerebral cortex after ischemic stroke

1716-P: L-PGDS Inhibition Reduces Adiponectin Levels and Insulin Sensitivity in 3T3-L1 Adipocytes

The prevalence of obesity has increased steadily over the last few decades and is a major risk factor for type 2 diabetes and cardiovascular disease. Obesity is associated with alterations in adipose tissue functioning leading to metabolic dysfunction. In previous studies, we have reported the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in glucose intolerance and atherosclerosis. More recently, we demonstrated that L-PGDS KO mice develop NAFLD even on a low fat diet. Our present study explored the role of L-PGDS in adipose tissue. We found decreased expression of L-PGDS in insulin resistant 3T3-L1 adipocytes. Enzymatic inhibition of L-PGDS in 3T3-L1 adipocytes using AT56 impaired insulin signaling by reducing the phosphorylation of AKT protein and the expression and translocation of GLUT4 to plasma membrane. Furthermore, we found that inhibition of L-PGDS reduces the expression of adiponectin and AMPK phosphorylation in 3T3-L1 adipocytes. Rosiglitazone treatment in insulin resistant 3T3-L1 adipocytes significantly increased L-PGDS expression. Treatment with rosiglitazone restored the expression of adiponectin and phosphorylation of AMPK in AT56 treated 3T3-L1 adipocytes. Correspondingly, we found decreased L-PGDS expression in visceral fat of obese humans. Cumulatively, these data confirm that the absence of L-PGDS results in decreased insulin sensitivity via impaired insulin signaling and reduced adiponectin levels in 3T3-L1 adipocytes. Thus, our findings suggest that L-PGDS plays a significant role in maintaining the insulin sensitivity in 3T3-L1 adipocytes and provides a novel opportunity for therapeutic intervention of obesity-associated metabolic syndrome. Disclosure A. Srivastava: None. T. Palaia: None. J. Lee: None. C. Hall: None. M. Stevenson: None. A.G. Fernando: None. L. Ragolia: None. Funding American Heart Association (15GRNT22420001); George Link, Jr. Foundation, Inc.

Lipocalin-type prostaglandin D2 synthase deletion induces dyslipidemia and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in mediating NAFLD utilizing L-PGDS knockout (KO) and control C57BL/6 mice fed either low fat (LFD) or high fat diet (HFD) for 14 weeks. Our present study demonstrates that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster and eventually progress to the more severe non-alcoholic steatohepatitis (NASH). We found increased lipid accumulation in the liver of KO mice over time on both diets, as compared to control mice. The L-PGDS KO mice showed elevated fasting glucose and insulin levels and developed insulin resistance on both LFD and HFD. Lipogenesis marker proteins such as SREBP-1c and LXRα were increased in L-PGDS KO mice after 14 weeks on both diets, when compared to control mice. We replicated our in vivo findings in vitro using HepG2 cells treated with a combination of free fatty acids (oleic and palmitic acid) and exposure to a L-PGDS inhibitor and prostaglandin D2 receptor (DP1) antagonists. We conclude that the absence or inhibition of L-PGDS results in dyslipidemia, altered expression of lipogenesis genes and the acceleration of NAFLD to NASH, independent of diet and obesity. We propose L-PGDS KO mice as a useful model to explore the pathogenesis of NAFLD and NASH, and L-PGDS as a potential therapeutic target for treatment.

Exploration of candidate serum biomarkers potentially related to the chronic pain condition in Medication\u2013overuse headache

BackgroundMedication Overuse Headache (MOH) is a prevalent and disabling disorder resulting from the overuse of analgesic drugs, triptans or other acute headache medications. In previous proteomic studies, several proteins have been found at high concentrations in the urine of MOH patients and in the serum of rats with neuropathic pain. The aim of this study was to compare the serum levels of lipocalin-type Prostaglandin D2 synthase (L-PGDS), Vitamin D-binding protein (VDBP), apolipoprotein E (APOE) and apolipoprotein A1 (APOA1) in MOH patients and healthy individuals, further exploring their relationship with cutaneous pain thresholds (CPTs) in the territories innervated by the trigeminal nerve.MethodsSixty-nine MOH patients and 42 age- and sex-matched healthy volunteers were enrolled in the study. Von Frey-like filaments were applied to the skin territories innervated by the trigeminal nerve, to determine the CPTs. L-PGDS, VDBP, APOE and APOA1 were quantified in the serum by Enzyme-linked Immunosorbent Assay (ELISA). Clinical and laboratory data were collected. Comparisons between MOH patients and healthy individuals were performed using independent t test or χ2 test. To correlate serum proteins with CPTs, Pearson correlation coefficient or Spearman’s rank correlation coefficient were used.ResultsCPTs were lower among MOH patients. L-PGDS, VDBP and APOE had significantly different serum concentrations between groups (p < 0.01), but no correlation was found with CPTs. APOA1 serum concentrations did not differ between patients and healthy individuals.ConclusionsL-PGDS, VDBP and APOE had abnormal serum levels in MOH patients, confirming their alteration in some conditions of chronic headache and neuropathic pain. However, they had no relationship with CPTs. The in-depth study of serum proteins represents a promising approach for a better understanding of MOH, as well as the detection of candidate biomarkers for chronic headache or the risks associated with overuse medications.

1890-P: Lipocalin-Type Prostaglandin D2 Synthase Deficient Mice Develop Dyslipidemia Resulting in Nonalcoholic Fatty Liver Disease Independent of Obesity

Nonalcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus (T2DM), cardiovascular disease, and all-cause mortality. Obesity is a major determinant of the high prevalence of NAFLD and T2DM worldwide. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in causing NAFLD in L-PGDS knockout (KO) and control C57BL/6 mice (n=6/group) on both low fat and high fat diets for 14 weeks. We observed that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster, even on the low fat diet. In addition, we found elevated fasting glucose and insulin levels in L-PGDS KO mice with increased lipid accumulation in the liver with time on both diets as compared to controls. We also found total cholesterol and LDL levels were significantly elevated in L-PGDS KO mice. Finally, lipogenesis marker proteins such as SREBP-1c and LXRα, and CD36, a fatty acid transporter protein, were also found to be increased in L-PGDS KO mice on both diets as compared to control mice. These data confirm that absence of L-PGDS results in hyperinsulinemia and dyslipidemia. Therefore, we conclude that L-PGDS plays a significant role in developing NAFLD independent of obesity and may serve as a novel therapeutic target for the treatment of NAFLD. Disclosure A. Srivastava: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None. Funding George Link, Jr. Foundation, Inc.; American Heart Association

A structure-function approach identifies L-PGDS as a mediator responsible for glucocorticoid-induced leptin expression in adipocytes

Leptin is an adipokine predominantly secreted by adipocytes and has many physiological roles, including in energy homeostasis. We identified that AM630, a cannabinoid receptor 2 (CB2) antagonist, down-regulated leptin expression in mature adipocytes differentiated from either stromal vascular fractions isolated from inguinal fat pads of C57BL/6J mice or 3T3-L1 preadipocytes. However, the leptin-suppressive effects of AM630 preserved in CB2-deficient adipocytes indicated the off-target activity of AM630 in leptin expression. Pharmacological and genetic studies, cheminformatics, and docking simulation were applied to identify the potential protein target of AM630 that modulates leptin expression in differentiated primary preadipocytes. Screening of the reported off-targets of AM630 identified a synthetic cannabinoid WIN55212-2 exerting the same function. Target deconvolution and docking simulation suggested that AM630 and WIN55212-2 were both inhibitors of lipocalin-type prostaglandin D2 synthase (L-PGDS). Further studies showed that L-PGDS positively regulates leptin expression. Although glucocorticoid and aldosterone were previously reported to induce expression of both L-PGDS and leptin, our data demonstrated that L-PGDS mediates only glucocorticoid-induced leptin expression in differentiated primary preadipocytes. No effect was observed after aldosterone treatment. This newly discovered glucocorticoid – L-PGDS – leptin pathway may provide insights into current clinical use of glucocorticoid and management of their undesired effects such as obesity.