Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Diacylglycerol Lipase-Alpha Regulates Hippocampal-Dependent Learning and Memory Processes in Mice.

Diacylglycerol lipase-α (DAGL-α), the principal biosynthetic enzyme of the endogenous cannabinoid 2-arachidonylglycerol (2-AG) on neurons, plays a key role in CB1 receptor-mediated synaptic plasticity and hippocampal neurogenesis, but its contribution to global hippocampal-mediated processes remains unknown. Thus, the present study examines the role that DAGL-α plays on LTP in hippocampus, as well as in hippocampal-dependent spatial learning and memory tasks, and on the production of endocannabinoid and related lipids through the use of complementary pharmacologic and genetic approaches to disrupt this enzyme in male mice. Here we show that DAGL-α gene deletion or pharmacological inhibition disrupts LTP in CA1 of the hippocampus but elicits varying magnitudes of behavioral learning and memory deficits in mice. In particular, DAGL-α-/- mice display profound impairments in the Object Location assay and Morris Water Maze (MWM) acquisition engaging in nonspatial search strategies. In contrast, WT mice administered the DAGL-α inhibitor DO34 show delays in MWM acquisition and reversal learning, but no deficits in expression, extinction, forgetting, or perseveration processes in this task, as well as no impairment in Object Location. The deficits in synaptic plasticity and MWM performance occur in concert with decreased 2-AG and its major lipid metabolite (arachidonic acid), but increases of a 2-AG diacylglycerol precursor in hippocampus, PFC, striatum, and cerebellum. These novel behavioral and electrophysiological results implicate a direct and perhaps selective role of DAGL-α in the integration of new spatial information.SIGNIFICANCE STATEMENT Here we show that genetic deletion or pharmacologic inhibition of diacylglycerol lipase-α (DAGL-α) impairs hippocampal CA1 LTP, differentially disrupts spatial learning and memory performance in Morris water maze (MWM) and Object Location tasks, and alters brain levels of endocannabinoids and related lipids. Whereas DAGL-α-/- mice exhibit profound phenotypic spatial memory deficits, a DAGL inhibitor selectively impairs the integration of new information in MWM acquisition and reversal tasks, but not memory processes of expression, extinction, forgetting, or perseveration, and does not affect performance in the Objection Location task. The findings that constitutive or short-term DAGL-α disruption impairs learning and memory at electrophysiological and selective in vivo levels implicate this enzyme as playing a key role in the integration of new spatial information.

Mapping the sites of the lipoprotein lipase (LPL)-angiopoietin-like protein 4 (ANGPTL4) interaction provides mechanistic insight into LPL inhibition.

Cardiovascular disease has been the leading cause of death throughout the world for nearly 2 decades. Hypertriglyceridemia affects more than one-third of the population in the United States and is an independent risk factor for cardiovascular disease. Despite the frequency of hypertriglyceridemia, treatment options are primarily limited to diet and exercise. Lipoprotein lipase (LPL) is an enzyme responsible for clearing triglycerides from circulation, and its activity alone can directly control plasma triglyceride concentrations. Therefore, LPL is a good target for triglyceride-lowering therapeutics. One approach for treating hypertriglyceridemia may be to increase the amount of enzymatically active LPL by preventing its inhibition by angiopoietin-like protein 4 (ANGPTL4). However, little is known about how these two proteins interact. Therefore, we used hydrogen-deuterium exchange MS to identify potential binding sites between LPL and ANGPTL4. We validated sites predicted to be located at the protein-protein interface by using chimeric variants of LPL and an LPL peptide mimetic. We found that ANGPTL4 binds LPL near the active site at the lid domain and a nearby α-helix. Lipase lid domains cover the active site to control both enzyme activation and substrate specificity. Our findings suggest that ANGPTL4 specifically inhibits LPL by binding the lid domain, which could prevent substrate catalysis at the active site. The structural details of the LPL-ANGPTL4 interaction uncovered here may inform the development of therapeutics targeted to disrupt this interaction for the management of hypertriglyceridemia.

Endocannabinoid contributions to alcohol habits and motivation: Relevance to treatment.

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.

Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors.

A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20, which with an IC50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.

Coding Variation in ANGPTL4, LPL, and SVEP1 and the Risk of Coronary Disease.

BACKGROUND: The discovery of low-frequency coding variants affecting the risk of coronary artery disease has facilitated the identification of therapeutic targets. METHODS: Through DNA genotyping, we tested 54,003 coding-sequence variants covering 13,715 human genes in up to 72,868 patients with coronary artery disease and 120,770 controls who did not have coronary artery disease. Through DNA sequencing, we studied the effects of loss-of-function mutations in selected genes. RESULTS: We confirmed previously observed significant associations between coronary artery disease and low-frequency missense variants in the genes LPA and PCSK9. We also found significant associations between coronary artery disease and low-frequency missense variants in the genes SVEP1 (p.D2702G; minor-allele frequency, 3.60%; odds ratio for disease, 1.14; P=4.2×10(-10)) and ANGPTL4 (p.E40K; minor-allele frequency, 2.01%; odds ratio, 0.86; P=4.0×10(-8)), which encodes angiopoietin-like 4. Through sequencing of ANGPTL4, we identified 9 carriers of loss-of-function mutations among 6924 patients with myocardial infarction, as compared with 19 carriers among 6834 controls (odds ratio, 0.47; P=0.04); carriers of ANGPTL4 loss-of-function alleles had triglyceride levels that were 35% lower than the levels among persons who did not carry a loss-of-function allele (P=0.003). ANGPTL4 inhibits lipoprotein lipase; we therefore searched for mutations in LPL and identified a loss-of-function variant that was associated with an increased risk of coronary artery disease (p.D36N; minor-allele frequency, 1.9%; odds ratio, 1.13; P=2.0×10(-4)) and a gain-of-function variant that was associated with protection from coronary artery disease (p.S447*; minor-allele frequency, 9.9%; odds ratio, 0.94; P=2.5×10(-7)). CONCLUSIONS: We found that carriers of loss-of-function mutations in ANGPTL4 had triglyceride levels that were lower than those among noncarriers; these mutations were also associated with protection from coronary artery disease. (Funded by the National Institutes of Health and others.).

Nobiletin reduces LPL-mediated lipid accumulation and pro-inflammatory cytokine secretion through upregulation of miR-590 expression.

Nobiletin has protective effects on cardiovascular diseases, but the mechanism is not clear. In this study, we examined whether nobiletin affects the expression of miR-590/LPL and its relative effects on lipid accumulation and pro-inflammatory cytokine secretion in human THP-1 macrophages. RT-qPCR analysis showed that nobiletin increased the expression of miR-590. Western blot analysis showed that nobiletin-suppressed LPL expression was enhanced by miR-590 mimic and abrogated by miR-590 inhibitor. Oil Red O staining and high-performance liquid chromatography assays showed that nobiletin attenuated lipid accumulation in macrophages. Treatment with nobiletin and miR-590 mimic decreased cellular lipid accumulation, whereas treatment with miR-590 inhibitor increased cellular lipid accumulation. ELISA illustrated that nobiletin alleviated pro-inflammatory cytokine secretion in macrophages as measured by, which was reduced by miR-590 mimic and increased by miR-590 inhibitor. In conclusion, nobiletin may alleviate lipid accumulation and secretion of pro-inflammatory cytokines by enhancing the inhibitory effect of miR-590 on LPL expression, suggesting a promising strategy for potential drug development for atherosclerosis.

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression.

Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition.

Diacylglycerol lipases (DAGLα and DAGLß) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

Lipoprotein Lipase Inhibitor, Nordihydroguaiaretic Acid, Aggravates Metabolic Phenotypes and Alters HDL Particle Size in the Western Diet-Fed db/db Mice.

Lipoprotein lipase (LPL) hydrolyzes triglycerides in lipoprotein to supply fatty acids, and its deficiency leads to hypertriglyceridemia, thereby inducing metabolic syndrome (MetSyn). Nordihydroguaiaretic acid (NDGA) has been recently reported to inhibit LPL secretion by endoplasmic reticulum (ER)-Golgi redistribution. However, the role of NDGA on dyslipidemia and MetSyn remains unclear. To address this question, leptin receptor knock out (KO)-db/db mice were randomly assigned to three different groups: A normal AIN76-A diet (CON), a Western diet (WD) and a Western diet with 0.1% NDGA and an LPL inhibitor, (WD+NDGA). All mice were fed for 12 weeks. The LPL inhibition by NDGA was confirmed by measuring the systemic LPL mass and adipose LPL gene expression. We investigated whether the LPL inhibition by NDGA alters the metabolic phenotypes. NDGA led to hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. More strikingly, the supplementation of NDGA increased the percentage of high density lipoprotein (HDL)small (HDL3a+3b+3c) and decreased the percentage of HDLlarge (HDL2a+2b) compared to the WD group, which indicates that LPL inhibition modulates HDL subclasses. was NDGA increased adipose inflammation but had no impact on hepatic stress signals. Taken together, these findings demonstrated that LPL inhibition by NDGA aggravates metabolic parameters and alters HDL particle size.

MiR-27b Impairs Adipocyte Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells by Targeting LPL.

BACKGROUND/AIMS: In this study, the molecular mechanisms of miR-27b and lipoprotein lipase (LPL) that regulate human adipose-derived mesenchymal stem cells (hASCs) adipogenic differentiation were detected. METHODS: Microarray analysis was applied to screen for differentially expressed miRNAs and mRNA during hASCs adipocyte differentiation induction. MiR-27b and LPL were found to have abnormal expression. Then, a dual luciferase reporter assay was employed to validate the targeting relationship between miR-27b and LPL. We also utilized qRT-PCR, western blot, cellular immunofluorescence and an oil red O staining assay to analyze the regulation of miR-27b and LPL during adipogenic differentiation. RESULTS: The microarray analysis demonstrated that, during adipogenic differentiation, miR-27b was down-regulated, while LPL was up-regulated but tended to become stable 14 days after induction. A dual luciferase reporter assay confirmed the negative targeting regulatory relationship between miR-27b and LPL. After overexpressing and silencing miR-27b, LPL was found to be reversely regulated by miR-27b according to qRT-PCR and western blot. The fat-formation-related biomarkers CCAAT-enhancer binding protein α (c/EBPα) and peroxisome proliferator-activated receptors γ (PPARγ) had decreasing levels after over-expressing miR-27b or knockdown of LPL followed by adipogenic differentiation. Meanwhile, the oil red O staining assay revealed that the accumulation of lipid droplets decreased. There was no change in the expression of c/EBPα, PPARγ, or lipid droplet accumulation when overexpressing miR-27b and LPL. CONCLUSION: During the adipogenic differentiation of hASCs, miR-27b expression decreased, and LPL expression increased. The abnormal expression of miR-27b and LPL effectively regulated the adipogenic differentiation of hASCs.

Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo.

Diacylglycerol lipase-beta (DAGLß) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLß inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLß inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLß-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of ∼5 µg of liposomal KT109 was sufficient to achieve ∼80% inactivation of DAGLß in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLß inhibitors to macrophages.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Two-step activity-based protein profiling of diacylglycerol lipase.

Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels-Alder ligation in living cells.

Chemical tools to modulate 2-arachidonoylglycerol biosynthesis.

2-Arachidonoylglycerol (2-AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1 R and CB2 R), thereby regulating a diverse range of physiological processes including anxiety, appetite, inflammation, memory, pain sensation, and nociception. Diacylglycerol lipases (DAGLs) are the principle enzymes responsible for 2-AG biosynthesis. Recently, the (patho)physiological functions of DAGLs have been explored by both genetic methods and chemical tools. This review will focus on the recent efforts to develop highly selective and in vivo active DAGLs inhibitors using activity-based protein profiling.

Aromatic residues in the C terminus of apolipoprotein C-III mediate lipid binding and LPL inhibition.

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21-25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1-6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance.

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL.

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.

Investigation of Diacylglycerol Lipase Alpha Inhibition in the Mouse Lipopolysaccharide Inflammatory Pain Model.

Diacylglycerol lipase (DAGL) α and ß, the major biosynthetic enzymes of the endogenous cannabinoid (endocannabinoid) 2-arachidonylglycerol (2-AG), are highly expressed in the nervous system and immune system, respectively. Genetic deletion or pharmacological inhibition of DAGL-ß protects against lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages and reverses LPS-induced allodynia in mice. To gain insight into the contribution of DAGL-α in LPS-induced allodynia, we tested global knockout mice as well as DO34, a dual DAGL-α/ß inhibitor. Intraperitoneal administration of DO34 (30 mg/kg) significantly decreased whole-brain levels of 2-AG (∼83%), anandamide (∼42%), and arachidonic acid (∼58%). DO34 dose-dependently reversed mechanical and cold allodynia, and these antinociceptive effects did not undergo tolerance after 6 days of repeated administration. In contrast, DO34 lacked acute thermal antinociceptive, motor, and hypothermal pharmacological effects in naive mice. As previously reported, DAGL-ß (-/-) mice displayed a protective phenotype from LPS-induced allodynia. However, DAGL-α (-/-) mice showed full allodynic responses, similar to their wild-type littermates. Interestingly, DO34 (30 mg/kg) fully reversed LPS-induced allodynia in DAGL-α (+/+) and (-/-) mice, but did not affect the antinociceptive phenotype of DAGL-ß (-/-) mice in this model, indicating a DAGL-α-independent site of action. These findings suggest that DAGL-α and DAGL-ß play distinct roles in LPS-induced nociception. Whereas DAGL-α appears to be dispensable for the development and expression of LPS-induced nociception, DAGL-ß inhibition represents a promising strategy to treat inflammatory pain.