Intracellular Lipid Transport Research Articles

Assessing the epigenetics effects on the expression of the Fabp3 promoter region of Zinc‐Transporter 7‐deficient mice

Zinc is an essential trace mineral for life. Zinc transporter 7 (ZNT7) transports zinc from the cytosol to the Golgi to maintain cellular zinc homeostasis. Mice with null‐Znt7 gene expression exhibit low zinc status, low body weight, as well as alterations in glucose and triglyceride homeostasis leading to a reduction in fat storage in adipocytes, and subsequently an increase in fat accumulation in muscle. The increase in muscle‐triglycerides in the Znt7KO mice is accompanied by a surge in the expression of Fabp3. Fatty‐Acid binding proteins (FABPs) belong to a family of proteins involved in intracellular lipid transport and status, yet some functions remain unknown. Fabp3 is found to be abundantly expressed in the heart, but it can also be moderately detected in skeletal muscle. Altered Fabp3 expression has been linked to a higher risk of developing metabolic syndrome in humans, hence this area of research is promising as rates of obesity and obesity‐related diseases continue to rise. DNA methylation has been shown to regulate human Fabp3 mRNA expression. Therefore, we aim to discover the methylation status of the mouse Fabp3 gene in a zinc deficient state using Znt7KO mice and determine a relationship between cellular zinc deficiency and the increased lipid accumulation in muscle, a cause of insulin resistance in Znt7KO male mice. Our preliminary results indicate that the cellular zinc level‐related DNA demethylation mainly occur in the area surround the transcriptional start site of Fabp3.Support or Funding InformationUSDA/ARS 2032‐51000‐005‐00D

The START-domain proteins in intracellular lipid transport and beyond

The Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) domain is a ~210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for lipid binding. The helix-grip fold structure defines a large superfamily of proteins, and this review focuses on the mammalian START domain family members that include single START domain proteins with identified ligands, and larger multi-domain proteins that may have novel roles in metabolism. Much of our understanding of the mammalian START domain proteins in lipid transport and changes in metabolism has advanced through studies using knockout mouse models, although for some of these proteins the identity and/or physiological role of ligand binding remains unknown. The findings that helped define START domain lipid-binding specificity, lipid transport, and changes in metabolism are presented to highlight that fundamental questions remain regarding the biological function(s) for START domain-containing proteins.

A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene-containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD's lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene-containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels-Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

Epithelial Claudin Proteins and Their Role in Gastrointestinal Diseases.

Our bodies are protected from the external environment by mucosal barriers that are lined by epithelial cells. The epithelium plays a critical role as a highly dynamic, selective semipermeable barrier that separates luminal contents and pathogens from the rest of the body and controlling the absorption of nutrients, fluid and solutes. A series of protein complexes including the adherens junction, desmosomes, and tight junctions function as the principal barrier in paracellular diffusion and regulators of intracellular solute, protein, and lipid transport. Tight junctions are composed of a series of proteins called occludins, junctional adhesion molecules, and claudins that reside primarily as the most apical intercellular junction. Here we will review one of these protein families, claudins, and their relevance to gastrointestinal and liver diseases.

Identification of Niemann–Pick type C disease in the group of ataxias of unclear origin in adults

Introduction. Niemann–Pick type C disease (NPC) is a rare neurovisceral lysosomal storage disease with an autosomal recessie type of inheritance that develops as a result of abnormal intracellular transport of cholesterol and other lipids. The possibility of pathogenetic therapy makes it very important to screen the population and identify new cases of the disease. Objective. Conducting biochemical and genetic screening in the group of adult patients with early-onset ataxia of unclear origin to detect new cases of NPC, with subsequent initiation of pathogenetic substrate reduction therapy and studies of clinical, genetic and biochemical characteristics the disease. Materials and methods. Ninety-five persons (18–40 years of age), both males and females, from different regions of Russia suffering from primary ataxia of unknown origin, associated with other neurological symptoms and/or visceral and psychiatric disorders, were examined. Patients underwent neurological examination and neuropsychological testing. During biochemical screening, the concentrations of blood plasma oxysterols (cholestane-3β,5α,6β-triol and 7-ketocholesterol) and chitotriosidase were examined. The presence of pathogenic mutations in the NPC1 and NPC2 genes was detected by total sequencing of all exons of these genes. Results. On biochemical screening, 3 patients were identified whose cholestane-3β,5α,6β-triol concentration and chitotriosidase activity were significantly higher than the reference norm, and 7-ketocholesterol concentration was either higher or at the level of the upper limit of the norm. On sequencing of the NPC1 gene, two unrelated patients (21-year-old woman and 37-year-old man) were found to carry 2 pathogenic compound heterozygous mutations each, and these findings confirm the diagnosis of NPC. The clinical picture was represented by a combination of neurological, psychiatric and visceral symptoms. In both patients, cerebellar ataxia was accompanied by dystonia and other extrapyramidal disorders, as well as vertical supranuclear gaze palsy, and bulbar and pseudobulbar symptoms. There were also affective disorders and progressive cognitive decline up to dementia of the frontal type. Both patients had ultrasound signs of isolated splenomegaly. The score for the NPC suspicion index was ≥200 points. Thus, in the studied group, NPC was detected in 2.1% of patients. Conclusions. Biochemical screening of oxysterols and chitotriosidase of blood plasma is a quick and inexpensive method for biochemical diagnosis of NPC, the diagnostic value of which is unquestionable in the case of integrated assessment of the history of the disease, the clinical picture and the results of instrumental diagnostic exams. Adult patients with early-onset ataxia associated with visceral and psychiatric disorders are at risk group for having NPC. They should primarily be sent to screening biochemical studies, and, in case of positive results, to carry out mutation screening of NPC1 and NPC2 genes.

Role of FABP3 as Biomarker in Alzheimer's Disease and Synucleinopathies

Lipids are fundamental components of brain cells as they are involved in several essential processes like remodeling of plasma membrane, synaptic function and receptor–ligand interactions. Systemic and brain alterations in lipid metabolism have been linked to the pathogenesis of neurodegenerative disorders like dementia and parkinsonisms. Intracellular transport of lipids is regulated by fatty acid-binding proteins. Recently, a member of this family, the fatty acid-binding protein 3 has been proposed as a potential biomarker across a range of neurodegenerative diseases, including Alzheimer's disease and dementia with Lewy bodies. In this special report, we describe recent progresses in characterizing the role of fatty acid-binding protein 3 in neurodegeneration and its putative role as biomarker measurable in biological fluids.

053 Lipid scavenger molecule CD36 regulates free fatty acid uptake and peripheral maintenance of tissue resident memory T cells

The mechanism(s) by which tissue-resident memory T cells (TRM) survive in peripheral tissues for long periods of time remains obscure. Our recent studies revealed that CD8+ TRM generated by skin vaccinia virus (VACV) infection express strikingly high levels of several molecules involved in the uptake and intracellular transport of free fatty acids (FFA) and other lipids. These molecules include fatty acid binding protein 4 (Fabp4) and Fabp5. Using transgenic mouse models, we have showed mice deficient in both fabp4 and fabp5 develop TRM that were inferior in internalizing exogenous FFA, displayed reduced FFA b oxidation and ATP generation, and did not survive as long-term as WT TRM. Another molecule strongly upregulated in TRM is CD36, a cell surface receptor that binds FFA. The role of CD36 in the maintenance and survival of TRM remains unknown. Here we demonstrated that Cd36-/- TRM internalized labelled palmitate (C16 FFA) less efficiently than WT TRM. In vivo competitive experiments by transferring equal numbers of WT and cd36-/- OT-1 cells into the same recipient mice followed by VACVOVA infection showed there was a survival disadvantage of cd36-/- TRM in skin beginning at day 45, which became more pronounced through day 90. By contrast, no difference could be observed between cd36-/- and WT TCM over 90 days of experiment. In addition, the defects seen in cd36-/- TRM and fabp4-/-/fabp5-/- TRM are additive in cd36-/- fabp4-/-fabp5-/- OT-1 mice---TRM from these mice had a profound survival disadvantage. Overall, our data demonstrate that CD36 also participates in the lipid uptake and long-time maintenance of TRM in peripheral tissue. A more detailed understanding of the unique lipid metabolic programs intrinsic to TRM, and how these programs might be modified therapeutically, will facilitate treatment approaches to TRM-mediated diseases as well as TRM based vaccines.

Bridging the molecular and biological functions of the oxysterol-binding protein family.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.

FABP1 knockdown in human enterocytes impairs proliferation and alters lipid metabolism

Fatty Acid-Binding Proteins (FABPs) are abundant intracellular proteins that bind long chain fatty acids (FA) and have been related with inmunometabolic diseases. Intestinal epithelial cells express two isoforms of FABPs: liver FABP (LFABP or FABP1) and intestinal FABP (IFABP or FABP2). They are thought to be associated with intracellular dietary lipid transport and trafficking towards diverse cell fates. But still their specific functions are not well understood.To study FABP1's functions, we generated an FABP1 knockdown model in Caco-2 cell line by stable antisense cDNA transfection (FABP1as). In these cells FABP1 expression was reduced up to 87%. No compensatory increase in FABP2 was observed, strengthening the idea of differential functions of both isoforms. In differentiated FABP1as cells, apical administration of oleate showed a decrease in its initial uptake rate and in long term incorporation compared with control cells. FABP1 depletion also reduced basolateral oleate secretion. The secreted oleate distribution showed an increase in FA/triacylglyceride ratio compared to control cells, probably due to FABP1's role in chylomicron assembly. Interestingly, FABP1as cells exhibited a dramatic decrease in proliferation rate. A reduction in oleate uptake as well as a decrease in its incorporation into the phospholipid fraction was observed in proliferating cells.Overall, our studies indicate that FABP1 is essential for proper lipid metabolism in differentiated enterocytes, particularly concerning fatty acids uptake and its basolateral secretion. Moreover, we show that FABP1 is required for enterocyte proliferation, suggesting that it may contribute to intestinal homeostasis.

Secreted gelsolin desensitizes and induces apoptosis of infiltrated lymphocytes in prostate cancer.

Loss of immunosurveillance is a major cause of cancer progression. Here, we demonstrate that gelsolin, a constituent of ejaculate, induces apoptosis of activated lymphocytes in prostate cancer. Gelsolin was highly expressed in prostate cancer cells, and was associated with tumor progression, recurrence, metastasis, and poor prognosis. In vitro, secreted gelsolin inactivated CD4+ T cells by binding to CD37, and induced apoptosis of activated CD8+ T lymphocytes by binding to Fas ligand during cell contact dependent on major histocompatibility complex I. Moreover, secreted gelsolin bound to sortilin, which in turn bound to Wiskott-Aldrich syndrome protein family member 3, thereby enhancing the endocytosis and intracellular transport of essential lipids needed to facilitate tumor growth and expansion. Under normal conditions, gelsolin is a seemingly harmless protein that prevents immune responses in female recipients. In disease states, however, this protein can inhibit immunosurveillance and promote cancer progression.

Isolation and characterization of a non-specific lipid transfer protein from Chelidonium majus L. latex

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins, which mostly play a role in intracellular lipid transport and antimicrobial defense. Recently it was shown using shotgun proteomic approach that the whole plant extract of Chelidonium majus L. (Papaveraceae) contains relatively abundant nsLTPs. Therefore the aim of the work was to isolate and characterize nsLTP from C. majus latex. Results obtained using PCR approach with degenerate primers showed the presence of nsLTP protein in C. majus root latex, named CmLTP 9.5. The protein consists of 93 aa with a molecular weight of 9.5kDa (NCBI GenBank accession no. ALT21495, coded by KP733898). The mature form of CmLTP 9.5 has a molecular weight of 7.147kDa and contains typical eight strictly conserved cysteine residues. A 3D model of CmLTP 9.5 displays a hydrophobic cavity. The isolated protein fraction tested using diffusion method and critical dilution assay showed strong antibacterial activity towards Gram-negative Campylobacter jejuni as well as Gram-positive Listeria greyi and Clostridium perfringens. Further studies using protein expression system are required to fully understand CmLTP 9.5 mode of action.

Internalization of scavenger receptor ligands by cortical neurons.

Scavenger receptors (SRs) are a family of receptors displaying affinity for a wide variety of ligands including modified lipoproteins. SRs may play a range of physiological functions including intracellular transport, lipid transport and pathogen clearance. The role of SRs has been documented in pathologies such as atherosclerosis and Alzheimer's disease. Although most studies on SRs have focused on macrophages, they are also present in other cells like endothelium, smooth muscles and brain tissue. Within brain, due to its functional similarity, SRs have been studied mostly in microglia. However, in situ images from Allen's brain atlas suggest SRs are abundant in neurons. In this study we have used two fluorophore labeled well characterized SR ligand, maleylated-BSA (MBSA) and polyguanylic acid (poloyG) to probe acute cortical slices. Our data indicate that within cortex, neurons avidly endocytose both ligands. Thus in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types.

Suppression of StarD7 promotes endoplasmic reticulum stress and induces ROS production

StarD7 is an intracellular lipid transport protein identified as up-regulated in the choriocarcinoma JEG-3 cell line. StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria, and StarD7 knockdown causes a reduction in phospholipid synthesis. Since inhibition of PC synthesis may lead to endoplasmic reticulum (ER) stress we hypothesized that StarD7 may be involved in maintaining cell homeostasis. Here, we examined the effect of StarD7 silencing on ER stress response and on the levels of reactive oxygen species (ROS) in the human hepatoma cell line HepG2. StarD7 knockdown induced alterations in mitochondria and ER morphology. These changes were accompanied with an ER stress response as determined by increased expression of inositol-requiring enzyme 1α (IRE1α), calnexin, glucose regulated protein 78/immunoglobulin heavy chain-binding protein (Grp78/BiP), protein kinase-like ER kinase (PERK) as well as the phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α). Additionally, a downregulation of the tumor suppressor p53 by a degradation mechanism was observed in StarD7 siRNA cells. Furthermore, StarD7 silencing induced ROS generation and reduced cell viability after H2O2 exposure. Decreased expression of StarD7 was associated to increased levels of the heme oxygenase-1 (HO-1) and catalase enzymes as well as in catalase enzymatic activity. Finally, no changes in levels of autophagy and apoptosis markers were observed in StarD7 siRNA treated cells respect to control cells. Taken together, these results indicate that StarD7 contributes to modulate cellular redox homeostasis.

Abstract 1534: Amplification of PITPNC1 affects breast cancer progression

Abstract Introduction Identification of driver mutations at single gene level and determination of their respective contribution to the enhanced invasive potential of cancer cells, via expression profiling and functional assays, are indispensable steps toward elucidating breast cancer pathobiology. In our previous array comparative genomic hybridization studies, gain in a region of chromosome 17, 17q23.3-24.3, was frequently observed in invasive duct carcinoma (IDC) patients with lymph node metastasis. Particularly, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), found in 17q24.2, was associated with higher histological grade, larger tumor size, positive HER2 staining, and poorer prognosis from the analysis of a NKI dataset. PITPNC1 is involved in signal transduction and intracellular lipid transport, and may be an essential part of the epidermal growth factor signaling pathway. Additionally, amplification of PITPNC1 via the loss of microRNA-126 suppression has been implicated in metastatic angiogenesis and colonization. In this study, we aim to assess the role of PITPNC1 in the development of aggressive breast cancer by evaluating its effects on breast cancer progression and invasion. Method A total of 21 samples - 13 pure duct carcinoma in situ (DCIS), 8 IDC with or without lymph node metastasis - were used for the preliminary round of this study. Formalin-fixed and paraffin-embedded tissue blocks were microdissected and whole genome amplified using ligation-mediated PCR. Amplified DNA was subjected to quantitative real-time PCR. Copy number alterations of PITPNC1 were calculated with Livak method, using B2M as the reference gene. Lipofectamine transfection overexpressing PITPNC1 in non-invasive MCF-10A and invasive MCF7, MD-MB-231 cell lines was prepared for subsequent in vitro assays. Proliferation and migration capabilities associated with PITPNC1 overexpression is being evaluated using MTT assay and transwell migration assay. Result From preliminary qPCR data, PITPNC1 was shown to be amplified in DCIS samples (p = 0.0025) and IDC samples (p = 0.0093). The 95% confidence intervals of fold difference against normal breast tissue control are [7.1, 23.4] and [5.7, 24.4] respectively. PITPNC1 overexpressing MCF-10A cells exhibit a higher proliferation rate in culture compared to controls. Conclusion Consistent amplification of PITPNC1 is seen in both DCIS and IDC cases. However, it is still unclear if PITPNC1 amplification is a result of a single mutational event occurring early in cancer progression or a cumulative effect occurring over time. Additional qPCR on paired samples against a pooled control would be necessary to determine its contribution to invasion. The regularity of copy number gain in the PITPNC1 locus and its association with HER2 expression highlight its predictive potential as a novel biomarker for tumorigenesis or invasion. Citation Format: Peiqi Wang, Ranju Nair, Nisha Kanwar, Grace Cheung, Susan J. Done. Amplification of PITPNC1 affects breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1534.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.

Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.

Rapid Diagnosis of 83 Patients with Niemann Pick Type C Disease and Related Cholesterol Transport Disorders by Cholestantriol Screening

Niemann Pick type C (NP-C) is a rare neurodegenerative disorder caused by an impairment of intracellular lipid transport. Due to the heterogeneous clinical phenotype and the lack of a reliable blood test, diagnosis and therapy are often delayed for years. In the cell, accumulating cholesterol leads to increased formation of oxysterols that can be used as a powerful screening parameter for NP-C. In a large scale study, we evaluated the oxysterol cholestane-3β,5α,6β-triol (c-triol) as potential biomarker for a rapid diagnosis of NP-C. Using GC/MS, c-triol has been analyzed in 1902 plasma samples of patients with the suspicion for NP-C. Diagnosis in patients with elevated oxysterols was confirmed by genetic analysis. 71 new NP-C patients (69 NP-C1 and two NP-C2) and 12 Niemann Pick type A/B patients were identified. 24 new mutations in NPC1, one new mutation in NPC2 and three new mutations in the SMPD1 gene were found. Cholestane-3β,5α,6β-triol was elevated in Niemann Pick type C1, type C2, type A/B and in CESD disease. No other study has ever identified so many NP-C patients, proving that c-triol is a rapid and reliable biomarker to detect patients with NP-C disease and related cholesterol transport disorders. It should replace the filipin test as the first-line diagnostic assay.

Fatty acid-binding protein 5 limits the anti-inflammatory response in murine macrophages

The beginning stages of liver damage induced by various etiologies (i.e. high fat diet, alcohol consumption, toxin exposure) are characterized by abnormal accumulation of lipid in liver. Alterations in intracellular lipid transport, storage, and metabolism accompanied by cellular insult within the liver play an important role in the pathogenesis of liver disease, often involving a sustained inflammatory response. The intracellular lipid transporter, fatty acid binding protein 5 (FABP5), is highly expressed in macrophages and may play an important role in the hepatic inflammatory response after endotoxin exposure in mice. This study tested the hypothesis that FABP5 regulates macrophage response to LPS in male C57bl/6 (wild type) and FABP5 knockout mice, both in vitro and in vivo. Treatment with LPS revealed that loss of FABP5 enhances the number of hepatic F4/80(+) macrophages in the liver despite limited liver injury. Conversely, FABP5 knock out mice display higher mRNA levels of anti-inflammatory cytokines IL-10, arginase, YM-1, and Fizz-1 in liver compared to wild type mice. Bone marrow derived macrophages stimulated with inflammatory (LPS and IFN-γ) or anti-inflammatory (IL-4) mediators also showed significantly higher expression of anti-inflammatory/regulatory factors. These findings reveal a regulatory role of FABP5 in the acute inflammatory response to LPS-induced liver injury, which is consistent with the principle finding that FABP5 is a regulator of macrophage phenotype. Specifically, these findings demonstrate that loss of FABP5 promotes a more anti-inflammatory response.

Genetic Deletion of Sterol Carrier Protein 2 Selectively Enhances Amygdalar Endocannabinoid Signaling

Sterol carrier protein 2 (SCP-2) is an intracellular lipid transport protein that transports cholesterol, fatty acids and the endocannabinoids (eCBs). Cellular accumulation of the eCBs contributes to inactivation of endocannabinoid signaling (ECS); and we have shown that SCP-2 can facilitate this process. These data lead to the hypothesis that reduction of SCP-2 enhances ECS through increased ligand availability. SCP-2 is expressed in the amygdala, where CB1 cannabinoid receptor (CB1R) signaling regulates extinction of fear. SCP-2 KO mice indeed exhibit enhanced extinction of fear, and this enhancement is reversed by CB1R blockade. However, concentrations of the eCBs anandamide and 2-arachidonoylglycerol (2-AG) were not greater in SCP-2 KO amygdala; in fact, 2-AG concentrations were significantly less than wild type. On the other hand, amygdalar CB1R mRNA expression and binding site density were both significantly increased in the SCP-2 KO mice compared to wild type, suggesting that the hypercannabinergic phenotype is the result of increased CB1R rather than eCB ligand. CB1R expression and binding site density were not increased in hippocampus, prefrontal cortex and cerebellum. These findings suggest that reduced SCP-2 function results in enhanced CB1R expression in the amygdala through an as-yet unknown mechanism. These data have relevance for discovery of processes by which CB1R function can be selectively enhanced in the CNS. Supported by R01 DA09155 and the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin.

Niemann-Pick Type C-2 Disease: Identification by Analysis of Plasma Cholestane-3β,5α,6β-Triol and Further Insight into the Clinical Phenotype.

Niemann-Pick type C disease is a rare disorder caused by impaired intracellular lipid transport due to mutations in either the NPC1 or the NPC2 gene. Ninety-five % of NPC patients show mutations in the NPC1 gene. A much smaller number of patients suffer from NPC2 disease and present respiratory failure as one of the most frequent symptoms. Several plasma oxysterols are highly elevated in NPC1 and can be used as a biomarker in the diagnosis of NPC1. Plasma cholestane-3β,5α,6β-triol was evaluated as biomarker for NPC2 by GC/MS and LC-MS/MS analysis. The diagnosis was confirmed by Sanger sequencing and filipin staining. We report three NPC2 patients with typical respiratory problems and a detailed description of the nature of the lung disease in one of them. All patients had elevated levels of plasma cholestane-3β,5α,6β-triol. In two of these patients, the positive oxysterol result led to a rapid diagnosis of NPC2 by genetic analysis. The phenotype of the third patient has been described previously. In this patient a cholestane-3β,5α,6β-triol concentration markedly above the reference range was found. Measurement of plasma cholestane-3β,5α,6β-triol enables to discriminate between controls and NPC1 and NPC2 patients, making it a valuable biomarker for the rapid diagnosis not only for NPC1 but also for NPC2 disease.The measurement of oxysterols should be well kept in mind in the differential diagnosis of lysosomal diseases, as the elevation of oxysterols in plasma may speed up the diagnosis of NPC1 and NPC2.