Pork Liver Decomposition Product May Improve Frontal Lobe Function in Humans-Open Trial.

Porcine Liver Decomposition Product (PLDP) was obtained by treating pig liver homogenate with protease and filling it into capsules. We have already confirmed from three clinical trials that PLDP enhances visual memory and delays memory recall, and we believe that its activity is due to various phospholipids, including phosphatidylcholine (PC). In this study, we clinically evaluated PLDP for depressive symptoms caused by a decline in cognitive function. This clinical trial was conducted using the Revised Hasegawa Dementia Scale (HDS-R). The HDS-R (maximum score is 30 points) is a test similar to the Mini-Mental State Examination (MMSE), which is commonly used in Japan. Dementia is suspected if the score falls below 20 on the HDS-R. Additionally, in a previous clinical trial, there was no change in scores in the placebo group after three doses of the HDS-R. In order to clearly confirm the effectiveness of PLDP, this study was conducted under stricter conditions (HDS-R points of 15 to 23) than previous clinical trials (all participants had scores of 20 or higher). Therefore, from ethical considerations, a clinical trial was conducted using the scores before PLDP administration as a control. In this study, PLDP was administered orally at 4 capsules per day, and the HDS-R was confirmed 2 and 4 weeks after administration. A significant increase in HDS-R scores was observed at 2 and 4 weeks after PLDP administration. Additionally, regarding each item of the HDS-R, PLDP significantly increased 2 and 4 weeks after oral administration for the question items assessing delayed recall, and the question item assessing verbal fluency tasks was recognized. From the above results, we confirmed the reproducibility of the effect of PLDP in improving the delayed recall of verbal memories. Furthermore, increasing scores on verbal fluency tasks suggest that PLDP may enhance frontal lobe function and prevent or improve depressive symptoms. The effects observed in this study may differ from the mechanisms of action of existing antidepressants, and we believe that this may lead to the discovery of new antidepressants.

A Review of Cyclic Phosphatidic Acid and Other Potential Therapeutic Targets for Treating Osteoarthritis.

Osteoarthritis (OA), a chronic degenerative joint disease, is the most common form of arthritis. OA occurs when the protective cartilage that cushions the ends of bones gradually breaks down. This leads to the rubbing of bones against each other, resulting in pain and stiffness. Cyclic phosphatidic acid (cPA) shows promise as a treatment for OA. In this article, we review the most recent findings regarding the biological functions of cPA signaling in mammalian systems, specifically in relation to OA. cPA is a naturally occurring phospholipid mediator with unique cyclic phosphate rings at the sn-2 and sn-3 positions in the glycerol backbone. cPA promotes various responses, including cell proliferation, migration, and survival. cPA possesses physiological activities that are distinct from those elicited by lysophosphatidic acid; however, its biochemical origin has rarely been studied. Although there is currently no cure for OA, advances in medical research may lead to new therapies or strategies in the future, and cPA has potential therapeutic applications.

Lysophospholipids transport across blood-brain barrier in an in vitro reconstruction model.

This study builds on our previous study, which highlighted the need for further research on the potential use of lysophospholipid (LPL) supplementation to prevent chronic and age-related diseases. We aimed to evaluate the transmembrane transport of LPL across rat and monkey blood-brain barrier (BBB) models. An in vitro monkey BBB model is required to elucidate the differences between rat and primate BBB-related data and to measure the permeability of LPLs being researched in relation to the human BBB. Based on our previous experiment, porcine liver decomposition product-derived phospholipids (PEL) strongly inhibit α-synuclein (α-Syn) aggregation. We have identified several candidates potentially relevant for the inhibition of α-Syn aggregation, such as LPC18:1, LPE18:1, and LPI18:0; however, the BBB permeability of these LPLs remains unclear. In the present study, we assessed the ability of these LPLs to pass through the in vitro rat and monkey BBB models. LPC18:1 showed high BBB permeability, LPI18:0 showed medium permeability, and the BBB permeation of LPE18:1 was negligible. Our results suggest that LPC18:1 and LPI18:0 are functional food factors that can cross the BBB.

Effects of 2-carba-cyclic phosphatidic acid derivatives on IL-1ß-stimulated human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the breakdown of subchondral bone and cartilage damage, most often affecting middle-aged and elderly people. Although the etiology of OA is still unknown, some reports suggest that inflammatory factors such as interleukin (IL)- 1ß mediate the progression of OA. To investigate the effect of IL-1ß and the possibility of treatment for OA, we applied 2-carba-cyclic phosphatidic acid (2ccPA) and its derivatives on human chondrocytes. 2ccPA is a synthesized phospholipid derived from a bioactive phospholipid mediator: cyclic phosphatidic acid (cPA). It has been previously reported that 2ccPA exhibits anti-inflammatory and chondroprotective effects in an OA animal model. 2ccPA and its ring-opened body (ROB) derivative significantly suppressed IL-1ß-induced upregulation of IL-6, matrix metalloproteinase-13, and cyclooxygenase-2, as well as the degradation of type II collagen and aggrecan. However, the other two derivatives, namely the deacylated and ring-opened deacylated bodies, showed little effect on an IL-1ß-exposed human chondrosarcoma cell-line. These data suggest that the intactness of 2ccPA and ROB is essential for anti-inflammatory effects on OA. Collectively, this study provides evidence that 2ccPA and ROB would be novel therapeutic agents for OA.

Lysophospholipids: A Potential Drug Candidates for Neurodegenerative Disorders.

Neurodegenerative diseases (NDs) commonly present misfolded and aggregated proteins. Considerable research has been performed to unearth the molecular processes underpinning this pathological aggregation and develop therapeutic strategies targeting NDs. Fibrillary deposits of α-synuclein (α-Syn), a highly conserved and thermostable protein, are a critical feature in the development of NDs such as Alzheimer's disease (AD), Lewy body disease (LBD), Parkinson's disease (PD), and multiple system atrophy (MSA). Inhibition of α-Syn aggregation can thus serve as a potential approach for therapeutic intervention. Recently, the degradation of target proteins by small molecules has emerged as a new therapeutic modality, gaining the hotspot in pharmaceutical research. Additionally, interest is growing in the use of food-derived bioactive compounds as intervention agents against NDs via functional foods and dietary supplements. According to reports, dietary bioactive phospholipids may have cognition-enhancing and neuroprotective effects, owing to their abilities to influence cognition and mental health in vivo and in vitro. However, the mechanisms by which lipids may prevent the pathological aggregation of α-Syn warrant further clarification. Here, we review evidence for the potential mechanisms underlying this effect, with a particular focus on how porcine liver decomposition product (PLDP)-derived lysophospholipids (LPLs) may inhibit α-Syn aggregation.

Lysophospholipids-potent candidates for brain food, protects neuronal cells against α-Synuclein aggregation.

Accumulation and aggregation of α-Synuclein (α-Syn) are the hallmarks of the incidence of α-Synucleinopathies, which comprises dementia with Lewy bodies (LBs). Aggregation inhibitors are anticipated to reduce α-Syn toxicity and serve as therapeutic agents. As a result, α-Syn is regarded as the potential and priority target for drug development. Here, we report inhibition of α-Syn aggregation by a certain lysophospholipids (LPLs) species. LPLs are small bioactive lipid molecules characterized by a single carbon chain and polar head group. The LPLs used here were extracted from porcine liver decomposition product (PLDP), which was previously reported to enhance cognitive function in healthy older adults. In this study, we found that PLDP-extracted lipids (PEL) reduced α-Syn aggregation in a cellular model. In particular, lysophosphatidylcholine (LPC) 16:0, LPC18:0, LPC18:1, and lysophosphatidylethanolamine (LPE) 16:0, which are known to be contained in PEL, were found to strongly inhibit α-Syn aggregation. Furthermore, when α-Syn was co-incubated with LPLs, the fluorescence emission of Thioflavin-T (ThT) declined remarkably, indicating a lower fibril formation. Interestingly, differences were observed in the degrees of effect on the reduction of insoluble α-Syn among each LPL. In this context, LPC18:1 and LPE18:1 appeared to interact with α-Syn below 1 nM in vitro. Taken together, these studies indicated the potential of PLDP-derived LPLs as effective therapeutic agents against α-Synucleinopathies.

The Combined Effects of Lysophospholipids against Lipopolysaccharide-induced Inflammation and Oxidative Stress in Microglial Cells.

Lysophospholipids (LPLs) are small bioactive lipid molecules characterized by a single carbon chain and a polar head group. LPLs have recently shown to be involved in many physiological and pathological processes such as nervous system regulation. In our previous studies, a porcine liver decomposition product (PLDP) has been identified as a substance that improves cognitive function at old ages. This PLDP is a rich source of LPLs, including lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). This study was designed to evaluate the anti-inflammatory effect of these LPLs on lipopolysaccharide (LPS)-stimulated SIM-A9 microglial cells in terms of cytokine expression and oxidative stress and to investigate the potential mechanisms underlying these effects. SIM-A9 cells were pretreated with LPLs prior to LPS stimulation, and the anti-inflammatory potential of the LPLs in LPS-induced SIM-A9 cells was examined. Pretreatment with LPLs significantly inhibited the LPS-induced expression of IL-6 in SIM-A9 cells. Furthermore, oxidative-related protein, NADPH oxidase 2 (Nox2) levels were markedly increased in the LPS-treated cells, and pretreatment with LPC and LPE significantly reduced to basal levels. In addition, LPS-induced ROS production was eliminated in apocynin-treated cells, indicating that ROS production was dependent on Nox2. Our findings revealed that pretreatment with LPC and LPE decreased LPS-stimulated ROS production. These results indicated that LPC and LPE exerted significant protective effects against LPS-induced inflammation and oxidative stress in SIM-A9 cell.

Abundant oleoyl-lysophosphatidylethanolamine in brain stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons.

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.

Adenine nucleotide translocase 2, a putative target protein for 2-carba cyclic phosphatidic acid in microglial cells.

Lipid-protein interactions play essential roles in many biological phenomena. Lysophospholipid mediators, such as cyclic phosphatidic acid (cPA), have been recognized as secondary messengers, yet few cellular targets for cPA have been identified to date. Furthermore, the molecular mechanism that activates these downstream signaling events remains unknown. In this study, using metabolically stabilized cPA carba-derivative (2ccPA)-immobilized magnetic beads, we identified adenine nucleotide translocase 2 (ANT2) as a 2ccPA-interacting protein in microglial cells. 2ccPA was tested for its ability to inhibit apoptosis caused by phenylarsine oxide in microglial cells. This damage was significantly improved upon 2ccPA treatment, along with cell proliferation, apoptosis, reactive oxygen species production, and intracellular ATP levels. This is the first report to suggest the direct binding of 2ccPA to ANT2 in microglial cells and provides evidence for a new benefit of 2ccPA in protecting microglial cells from apoptotic death induced by the ANT2-mediated signaling pathway.

Structurally different lysophosphatidylethanolamine species stimulate neurite outgrowth in cultured cortical neurons via distinct G-protein-coupled receptors and signaling cascades.

Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that structurally different lysophosphatidylethanolamine (LPE) species, palmitoyl-LPE (16:0 LPE) and stearoyl-LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Interestingly, YM-254890, an inhibitor of Gq/11 protein, inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, pertussis toxin, an inhibitor of Gi/Go proteins, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C inhibitors on neurite outgrowth were also different. In addition, both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2, but the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.

Therapeutic Potential of Porcine Liver Decomposition Product: New Insights and Perspectives for Microglia-Mediated Neuroinflammation in Neurodegenerative Diseases.

It is widely accepted that microglia-mediated inflammation contributes to the progression of neurodegenerative diseases; however, the precise mechanisms through which these cells contribute remain to be elucidated. Microglia, as the primary immune effector cells of the brain, play key roles in maintaining central nervous system (CNS) homeostasis. Microglia are located throughout the brain and spinal cord and may account for up to 15% of all cells in the brain. Activated microglia express pro-inflammatory cytokines that act on the surrounding brain and spinal cord. Microglia may also play a detrimental effect on nerve cells when they gain a chronic inflammatory function and promote neuropathologies. A key feature of microglia is its rapid morphological change upon activation, characterized by the retraction of numerous fine processes and the gradual acquisition of amoeba-like shapes. These morphological changes are also accompanied by the expression and secretion of inflammatory molecules, including cytokines, chemokines, and lipid mediators that promote systemic inflammation during neurodegeneration. This may be considered a protective response intended to limit further injury and initiate repair processes. We previously reported that porcine liver decomposition product (PLDP) induces a significant increase in the Hasegawa's Dementia Scale-Revised (HDS-R) score and the Wechsler Memory Scale (WMS) in a randomized, double-blind, placebo-controlled study in healthy humans. In addition, the oral administration of porcine liver decomposition product enhanced visual memory and delayed recall in healthy adults. We believe that PLDP is a functional food that aids cognitive function. In this review, we provide a critical assessment of recent reports of lysophospholipids derived from PLDP, a rich source of phospholipids. We also highlight some recent findings regarding bidirectional interactions between lysophospholipids and microglia and age-related neurodegenerative diseases such as dementia and Alzheimer's disease.

Oral administration of porcine liver decomposition product for 4 weeks enhances visual memory and delayed recall in healthy adults over 40 years of age: A randomized, double-blind, placebo-controlled study.

BACKGROUND AND OBJECTIVES: Porcine liver decomposition product (PLDP) contains neurofunctional phospholipids. We previously reported that PLDP enhances cognitive function in healthy adult humans, based on clinical evaluations using Hasegawa's Dementia Scale-Revised. In this study, we evaluated the effect of PLDP on memory indicators of the Wechsler Memory Scale-Revised (WMS-R), an internationally recognized battery for memory assessment. METHODS: We conducted a double-blind parallel-group placebo-controlled trial to evaluate the effect of PLDP on memory. Fifty-eight participants competed the trial: 28 participants were in the PLDP group and 30 participants were in the placebo group. Each group was administered PLDP (4 capsules) or a placebo (4 capsules) for 4 continuous weeks. WMS-R was administered before and 4 weeks after PLDP or placebo intake. The data were also subdivided by age for participants under 40 years (N = 15 in PLDP; N = 15 in placebo) and over 40 years (N = 13 in PLDP, N = 15 in placebo). Changes in Verbal Memory, Visual Memory, Attention/Concentration, and Delayed Recall were analyzed. RESULTS: No significant differences were found in any memory indicators between the PLDP group and the placebo group in pooled participants and in participants under 40 years of age. However, for participants over 40 years of age, PLDP administration resulted in a significant enhancement than placebo administration in Delayed Recall (14.1 ± 7.1 points vs. 7.1 ± 6.8 points) (P < 0.05), Visual Recall I (20.1 ± 23.1 percentile vs 1.9 ± 22.8 percentile) (P < 0.05), and Visual Recall II (24.2 ± 25.8 percentile vs 6.7 ± 19.0 percentile) (P < 0.05), respectively. The composition ratio of men to women in each group was imbalanced but no significant difference existed between the two groups. LIMITATIONS: A modest sample size, single-center design, and a fairly short follow-up period. CONCLUSION: PLDP enhanced Visual Memory and Delayed Recall in healthy adults over 40 years of age but not in healthy adults under 40 years of age. Therefore, PLDP may represent a promising nutraceutical that could improve cognitive function in healthy adults over 40 years of age. Further studies are required to evaluate if long term PLDP administration can prevent or delay cognitive dysfunction in healthy adults over 40 years of age.

Porcine liver decomposition product-derived lysophospholipids promote microglial activation in vitro.

Cognitive impairments such as dementia are common in later life, and have been suggested to occur via a range of mechanisms, including oxidative stress, age-related changes to cellular metabolism, and a loss of phospholipids (PLs) from neuronal membranes. PLs are a class of amphipathic lipids that form plasma membrane lipid bilayers, and that occur at high concentrations in neuronal membranes. Our previous study suggested that a porcine liver decomposition product (PLDP) produced via protease treatment may improve cognitive function at older ages, by acting as a rich source of PLs and lysophospholipids (LPLs); however, its specific composition remains unclear. Thus, the present study used a novel liquid chromatography electrospray ionization tandem mass spectrometric (LC-MS/MS) protocol to identify the major PLs and LPLs in PLDP. Furthermore, it assessed the effect of identified LPLs on microglial activation in vitro, including cell shape, proliferation, and cell morphology. The results of the conducted analyses showed that PLDP and PLDP-derived LPLs concentration-dependently modulate microglial activation in vitro. In particular, lysophosphatidylcholine (LPC) concentration-dependently promotes cell morphology, likely via effects mediated by the enzyme autotaxin (ATX), since inhibiting ATX also promoted cell morphology, while conversely, increasing ATX production (via treatment with high levels of LPC) abolished this effect. These findings suggest that LPC is likely neuroprotective, and thus, support the importance of further research to assess its use as a therapeutic target to treat age-related cognitive impairments, including dementia.

1-O-alkyl glycerophosphate-induced CD36 expression drives oxidative stress in microglial cells.

Microglia, the tissue-resident macrophages in the central nervous system, are important for the initiation and perpetuation of neuroinflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-inducible transcription factor and plays an important role in fatty acid metabolism. Our previous study found that 1-O-alkyl glycerophosphate (AGP), a naturally occurring ether analog of lysophosphatidic acid, is a high-affinity, partial agonist of PPARγ. In this study, we investigated the role of AGP in microglial activation and illustrated the underlying molecular mechanism. We found that AGP treatment increased the production of intracellular reactive oxygen species and induced PPARγ activation in microglial cells. Interestingly, AGP also up-regulated the expression levels of the cluster of differentiation 36 (CD36) scavenger receptor, a high-affinity receptor for oxidized low-density lipoproteins. The findings suggest that AGP induces PPARγ activation, enhances CD36 expression and increases the production of intracellular reactive oxygen species (ROS) in microglial cells.

Different aggregation and shape characteristics of carbon materials affect biological responses in RAW264 cells.

INTRODUCTION: Carbon nanotubes (CNTs) have various shapes, including needle-like shapes and curled shapes, and the cytotoxicity and carcinogenicity of CNTs differ depending on their shapes and surface modifications. However, the biological responses induced by CNTs and related mechanisms according to the dispersion state of CNTs have not been extensively studied. MATERIALS AND METHODS: We prepared multiwalled CNTs (MWCNTs) showing different dispersions and evaluated these MWCNTs in RAW264 cells to determine cytotoxicity, cellular uptake, and immune responses. Furthermore, RAW264 cells were also used to compare the cellular uptake and cytotoxicity of fibrous MWCNTs and spherical carbon nanohorns (CNHs) exhibiting the same degree of dispersion. RESULTS: Our analysis showed that the cellular uptake, localization, and inflammatory responses of MWCNTs differed depending on the dispersion state. Moreover, there were differences in uptake between MWCNTs and CNHs, even showing the same degree of dispersion. These findings suggested that receptors related to cytotoxicity and immune responses differed depending on the aggregated state of MWCNTs and surface modification with a dispersant. Furthermore, our results suggested that the receptors recognized by the cells differed depending on the particle shape. CONCLUSION: Therefore, to apply MWCNTs as a biomaterial, it is important to determine the carcinogenicity and toxicity of the CNTs and to examine different biological responses induced by varying shapes, dispersion states, and surface modifications of particles.

Alkyl-glycerophosphate-mediated C-C motif chemokine 2 secretion induces oxidative stress via increased PPARγ activation in human umbilical vein endothelial cells.

We previously showed that an alkyl-ether analog of lysophosphatidic acid, AGP (alkyl-glycerophosphate), accumulates in human atherosclerotic plaques and is a potent agonist of peroxisome proliferator-activated receptor-gamma (PPARγ). On the other hand, cyclic phosphatidic acid (cPA), similar in structure to AGP, can negatively regulate PPARγ. However, in this study, cPA had no effect on the expression and secretion of C-C motif chemokine 2 (CCL-2), a chemokine that is also linked to inflammatory responses and atherosclerosis. We showed that AGP enhances CCL-2 mRNA expression and secretion in a dose-dependent manner. Furthermore, oxidative stress plays a major role in the pathology of cardiovascular diseases; we showed that AGP triggers ROS generation and lipid peroxidation and that ROS and 8-isoprostane generation can be suppressed by a PPARγ antagonist. These results suggest that an imbalance of the PPARγ agonist-antagonist equilibrium is involved in changes in cellular functions, including ROS generation and lipid peroxidation.

Involvement of lysophosphatidic acid-induced astrocyte activation underlying the maintenance of partial sciatic nerve injury-induced neuropathic pain.

We have previously demonstrated that lysophosphatidic acid (LPA) plays key roles in the initial mechanisms for neuropathic pain (NeuP) development. Here, we examined whether LPA receptor mechanisms and LPA production are related to the glial activation at a late stage after partial sciatic nerve ligation (pSNL) by use of microglial inhibitor, Mac1-saporin or astrocyte inhibitor, and L-α-aminoadipate (L-AA). Although single intrathecal injection of LPA1/3 antagonist, Ki-16425 did not affect the pain threshold at day 7 after the spinal cord injury, repeated treatments of each compound gradually reversed the basal pain threshold to the control level. The intrathecal administration of a microglia inhibitor, Mac-1-saporin reversed the late hyperalgesia and LPA production at day 14 after the pSNL, whereas L-AA inhibited the hyperalgesia, but had no effect on LPA production. The involvement of LPA receptors in astrocyte activation in vivo was evidenced by the findings that Ki-16425 treatments abolished the upregulation of CXCL1 in activated astrocytes in the spinal dorsal horn of mice at day 14 after the pSNL, and that Ki-16425 reversed the LPA-induced upregulation of several chemokine gene expressions in primary cultured astrocytes. Finally, we found that significant hyperalgesia was observed with intrathecal administration of primary cultured astrocytes, which had been stimulated by LPA in a Ki-16425-reversible manner. All these findings suggest that LPA production and LPA1/3 receptor activation through differential glial mechanisms play key roles in the maintenance as well as initiation mechanisms in NeuP.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model.

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

The AGP-PPARγ axis promotes oxidative stress and diabetic endothelial cell dysfunction.

Alkyl-glycerophosphate (AGP) accumulates in atherogenic oxidized-LDL and human atherosclerotic plaques and is a potent agonist of peroxisome-proliferator-activated receptor-gamma (PPARγ). Recent studies suggest a potential regulatory role for PPARγ in endothelial nitric oxide synthase (eNOS) expression/activation and nitrogen oxide (NO) generation in the vascular endothelium. Importantly, eNOS-induced NO and advanced glycation end-products (AGEs) are involved in blood-vessel damage, and diabetic patients exhibit high serum NO and AGE levels; however, the effect of AGP on NO- and AGE-mediated endothelium dysfunction remains unknown. Investigation of the AGP-specific effects on NO- and AGE-mediated dysfunction and the underlying molecular mechanisms revealed that AGP upregulated eNOS expression and NO production, and that eNOS silencing and PPARγ antagonism inhibited AGP-mediated eNOS upregulation and NO production. Moreover, AGP-PPARγ-axis-mediated NO production promoted the generation of reactive oxygen species and AGE formation. These results suggested that AGP plays a significant role in the initiation/progression of diabetes-related atherosclerosis through PPARγ activation.

Tyrosyl-tRNA synthetase: A potential kyotorphin synthetase in mammals.

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 µM and 200 µM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.