Accumulation Of Phospholipids Research Articles

UNUSUAL PRESENTATION OF AMIODARONE PULMONARY TOXICITY (APT) WITH MULTIPLE LUNG NODULES

SESSION TITLE: Diffuse Lung Disease 2 SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 pm - 02:15 pm INTRODUCTION: The incidence of amiodarone-induced pulmonary toxicity (APT) is 5%-15%. Diffuse interstitial infiltrate is the most common radiographic finding associated with APT. Fewer than 10 cases of multiple pulmonary nodules due to APT have been reported in the literature. We report a case of APT presenting as bilateral multiple pulmonary nodules. CASE PRESENTATION: A 59-year-old female presented to the pulmonary clinic for evaluation of multiple pulmonary nodules found incidentally on cardiac MRI. Patient’s complaints consisted of progressive DOE, persistent dry cough without any constitutional symptoms. A dedicated chest CT revealed multiple, various sized, bilateral pulmonary nodules. The nodules were predominantly in the lower lung zones. Her PMHx was significant for HFpEF, PAF on chronic amiodarone, and papillary thyroid cancer s/p left thyroidectomy one year ago. Given the history of malignancy and a high suspicion for metastatic disease, the patient was sent for VATS. Diagnostic lung wedge biopsies revealed an intense lymphoplasmacytic interstitial infiltrate and alveoli filled with macrophages with fine vacuolization on histopathologic examination. In some nodules, there were minor foci of organizing pneumonia. There was no evidence of granulomas or metastatic thyroid tumor. The finding was suggestive of inflammatory process most consistent with amiodarone toxicity. DISCUSSION: The clinical presentation of APT varies from non-productive cough, dyspnea on exertion to acute respiratory failure. Typically, pulmonary toxicity of amiodarone consists of diffuse alveolar damage, organizing pneumonia, hypersensitivity pneumonitis, and irreversible pulmonary fibrosis. Multiple lung nodules due to APT has been reported but rare in the literature. Nodular pulmonary disease is thought to be an inflammatory response to drug-induced phospholipid accumulation in the alveolar cells. Therefore, the lung nodules due to APT may be reversible and has a favorable outcome by discontinuing the drug. The use of corticosteroid also has been shown to be helpful. The diagnosis of APT is challenging due to the complex nature of clinical and radiographic findings. CONCLUSIONS: In summary, APT presenting with diffuse nodular pulmonary disease is rare; however, clinicians must be aware of the variability of clinical and radiographic finding of APT. APT should be considered in patients taking amiodarone present with new pulmonary nodules radiographically. Reference #1: 1. Morell, EB et al., 2005. Multiple pulmonary noduels and amiodarone. KL-6 as a new diagnostic tool. Rev Esp Cardiol 58(4):447-449 Reference #2: 2. Rodriguez-Garcia, JL et al., 2001. Pulmonary mass and multiple lung nodules mimicking a lung neoplasm as amiodarone-induced toxicity. Eur J Intern Med 12(4):372-376 Reference #3: 3. Wolkove, N et al., 2009. Amiodarone pulmonary toxicity. Can Respir J 16(2):43-48 DISCLOSURES: No relevant relationships by Michael Ayers, source=Web Response No relevant relationships by Debabrata Bandyopadhyay, source=Web Response No relevant relationships by Anna Kyu Chen, source=Web Response No relevant relationships by Md Islam, source=Web Response No relevant relationships by Matthew Stanton, source=Web Response

Lipid profiles in Himasthla elongata and their intermediate hosts, Littorina littorea and Mytilus edulis

In this research, the lipid profiles in rediae and free-living larvae (cercariae) of the trematodes Himasthla elongata were studied. It was shown that the lipid profiles of cercariae and rediae reflect the selective accumulation of membrane phospholipids and polyunsaturated fatty acids. In addition, infected tissues’ lipid compositions in the intermediate hosts Littorina littorea (whole organism) and Mytilus edulis (foot) were studied. Modifications in the molluscs’ lipid compositions were mainly caused by the parasites’ metabolic requirements, as the parasites are unable to engage in de novo lipid biosynthesis, and thus, they utilise the host tissues’ metabolites. The assumption that changes in the lipid unsaturation of infected intermediate hosts may affect their temperature resistance was discussed.

Cxxc Finger Protein 1 Positively Regulates GM-CSF-Derived Macrophage Phagocytosis Through Csf2rα-Mediated Signaling.

Macrophages have a defensive function against bacteria through phagocytosis and the secretion of cytokines. Histone modifications play an essential role in macrophage functions. Here, we report that Cxxc finger protein 1 (CFP1), a key component of the SETD1 histone methyltransferase complex, promoted the phagocytic and bactericidal activity of GM-CSF-derived macrophages. CFP1-deficient mice were more susceptible to bacterial infection due to the decreased expression of Csf2rα, a subunit of the GM-CSF receptor essential for inflammation and alveolar macrophage development, through the loss of H3K4 modifications in the promoter of the Csf2rα gene. In addition, the lung tissues of CFP1-deficient mice exhibited spontaneous inflammatory symptoms, including both the infiltration of inflammatory cells and the accumulation of surfactant phospholipids and proteins. Furthermore, we showed that Csf2rα and PU.1 can partially rescue the defects in phagocytosis and in the intracellular killing of bacteria. Collectively, our data highlight the importance of CFP1 in the phagocytic and bactericidal activity of macrophages.

Lipidomic reprogramming associated with drought stress priming-enhanced heat tolerance in tall fescue (Festuca arundinacea).

Stress priming by exposing plants to a mild or moderate drought could enhance plant tolerance to subsequent heat stress. Lipids play vital roles in stress adaptation, but how lipidomic profiles change, affecting the cross-stress tolerance, is largely unknown. The objectives of this study were to perform lipidomics, to analyse the content, composition, and saturation levels of lipids in leaves of tall fescue (Festuca arundinacea) following drought priming and subsequent heat stress, and to identify major lipids and molecular species associated with priming-enhanced heat tolerance. Plants were initially exposed to drought for 8days by withholding irrigation and subsequently subjected to 25days of heat stress (38/33°Cday/night) in growth chambers. Drought-primed plants maintained significantly higher leaf relative water content, chlorophyll content, photochemical efficiency, and lower electrolyte leakage than nonprimed plants under heat stress. Drought priming enhanced the accumulation of phospholipids and glycolipids involved in membrane stabilization and stress signalling (phosphatidic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, and digalactosyl diacylglycerol) during subsequent exposure to heat stress. The reprogramming of lipid metabolism for membrane stabilization and signalling in response to drought priming and subsequent exposure to heat stress could contribute to drought priming-enhanced heat tolerance in cool-season grass species.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

The primary active components, antioxidant properties, and differential metabolite profiles of radish sprouts (Raphanus sativus L.) upon domestic storage: analysis of nutritional quality.

This study aimed to analyze the nutritional quality of radish sprouts (Raphanus sativus L.) after domestic short-term storage. We stored fresh radish sprouts at 25 ± 1 °C and at 4 ± 1 °C for 12 h, and detected phenolic substances, glucosinolates, isothiocyanates, vitamin C, and various antioxidant and abiotic stress-related factors. We investigated nutrient-related metabolic differences and associated pathways and postharvest treatment effects on nutritional quality using metabolomic analysis. Most active substances and antioxidant properties, but not phenolic acids and vitamin C, decreased significantly (P < 0.05) upon domestic storage; this reduction decelerated at low temperatures. Short-term storage disrupted redox balance; low temperature enhanced stress resistance. Differences were observed in amino acid and vitamin derivatives, phospholipid accumulation, and organic acids. Short-term storage at ambient temperature promoted lysine, threonine, cysteine, vitamin H, phospholipid, and lauric (dodecanoic) acid accumulation, inhibiting proline, phosphatidic acid (PA) (14:1(9Z)/12:0), and phosphatidylcholine (PC) (O-18:0/O-18:0) accumulation; low-temperature, short-term storage promoted myristic acid and phospholipid accumulation and reduced methionine synthesis and vitamin H and K accumulation. Overall, the nutritional quality of radish sprout decreased upon short-term storage, with differences in certain active substances. © 2018 Society of Chemical Industry.

Seasonal Alterations in Organic Phosphorus Metabolism Drive the Phosphorus Economy of Annual Growth in F. sylvatica Trees on P-Impoverished Soil.

Phosphorus (P) is one of the most important macronutrients limiting plant growth and development, particularly in forest ecosystems such as temperate beech (Fagus sylvatica) forests in Central Europe. Efficient tree internal P cycling during annual growth is an important strategy of beech trees to adapt to low soil-P. Organic P (Porg) is thought to play a decisive role in P cycling, but the significance of individual compounds and processes has not been elucidated. To identify processes and metabolites involved in P cycling of beech trees, polar-metabolome and lipidome profiling was performed during annual growth with twig tissues from a sufficient (Conventwald, Con) and a low-soil-P (Tuttlingen, Tut) forest. Autumnal phospholipid degradation in leaves and P export from senescent leaves, accumulation of phospholipids and glucosamine-6-phosphate (GlcN6P) in the bark, storage of N-acetyl-D-glucosamine-6-phosphate (GlcNAc6P) in the wood, and establishing of a phospholipid “start-up capital” in buds constitute main processes involved in P cycling that were enhanced in beech trees on low-P soil of the Tut forest. In spring, mobilization of P from storage pools in the bark contributed to an effective P cycling. Due to the higher phospholipid “start-up capital” in buds of Tut beeches, the P metabolite profile in developing leaves in spring was similar in beech trees of both forests. During summer, leaves of Tut beeches meet their phosphate (Pi) needs by replacing phospholipids by galacto- and sulfolipids. Thus, several processes contribute to adequate Pi supply on P impoverished soil thereby mediating similar growth of beech at low and sufficient soil-P availability.

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.

Detection of nanocarrier potentiation on drug induced phospholipidosis in cultured cells and primary hepatocyte spheroids by high content imaging and analysis

Considerable effort has been made to develop nanocarriers for controlled drug delivery over the last decade, while it remains unclear how the strength of adverse drug effect will be altered when a drug is loaded on the nanocarrier. Drug-induced phospholipidosis (DIP) is characterized with excessive accumulation of phospholipids in cells and is common for cationic amphiphilic drugs (CAD). Previously, we have reported that PEGylated graphene oxide (PEG-GO) loaded with several CAD can potentiate DIP. In current study, we extended our study on newly identified phospholipidosis (PLD) inducers that had been identified from the Library of Pharmacologically Active Compounds (LOPAC), to investigate if PEO-GO loaded with these CAD can alter DIP. Twenty-two CAD were respectively loaded on PEG-GO and incubated with RAW264.7, a macrophage cell line. The results showed that when a CAD was loaded on PEG-GO, its strength of PLD induction can be enhanced, unchanged or attenuated. PEG-GO loaded with Ifenprodil exhibited the highest PEG-GO potentiation effect compared to Ifenprodil treatment alone in RAW264.7 cells, and this effect was confirmed in human hepatocellular carcinoma HepG2, another cell line model for PLD induction. Primary hepatocyte culture and spheroids mimicking in vivo conditions were used to further validate nanocarrier potentiation on DIP by Ifenprodil. Stronger phospholipid accumulation was found in PEG-GO/Ifenprodil treated hepatocytes or spheroids than Ifenprodil treatment alone. Therefore, evidences were provided by us that nanocarriers may increase the adverse drug effects and guidance by regulatory agencies need to be drafted for the safe use of nanotechnology in drug delivery.

Effect of brimonidine, an α2 adrenergic agonist, on human meibomian gland epithelial cells

We recently discovered that the anti-glaucoma pharmaceuticals timolol, a β adrenergic antagonist, and pilocarpine, a cholinergic compound, negatively influence the morphology, proliferative capacity and survival of human meibomian gland epithelial cells (HMGECs). We hypothesize that another class of anti-glaucoma drugs, the α2 adrenergic agonists, also acts directly on HMGECs to affect their structure and function. We tested this hypothesis. Immortalized (i) HMGECs were cultured with brimonidine, as well as clonidine (α2 agonist), phenylephrine (α1 agonist), RX821002 (inverse α2 agonist) and MK912 (neutral α2 agonist) for up to 7 days. Cells were counted with a hemocytometer, and evaluated for morphology, signaling pathway activity, protein biomarker expression, and the accumulation of neutral lipids, phospholipids and lysosomes. Our findings demonstrate that brimondine treatment induces a dose-dependent decrease in Akt signaling and proliferation of iHMGECs. In contrast, brimonidine also promotes a dose-dependent differentiation of iHMGECs, including an increase in neutral lipid, phospholipid and lysosome levels. These effects were paralleled by an inhibition of p38 signaling, and duplicated by cellular exposure to clonidine, but not phenylephrine. Brimonidine also enhanced the cellular content of sterol regulatory binding protein-1, a master regulator of lipid synthesis. Of particular interest, the putative α2 antagonists, RX821002 and MK912, did not interfere with brimonidine action, but rather stimulated IHMGEC differentiation. Our results support our hypothesis and demonstrate that α2 adrenergic agonists act directly on iHMGECs. However, these compounds do not elicit an overall negative effect. Rather, the α2 agonists promote the differentiation of iHMGECs.

Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli.

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41 One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

Hepatic cells derived from human skin progenitors show a typical phospholipidotic response upon exposure to amiodarone

Phospholipidosis is a metabolic disorder characterized by intracellular accumulation of phospholipids. It can be caused by short-term or chronic exposure to cationic amphiphilic drugs (CADs). These compounds bind to phospholipids, leading to inhibition of their degradation and consequently to their accumulation in lysosomes. Drug-induced phospholipidosis (DIPL) is frequently at the basis of discontinuation of drug development and post-market drug withdrawal. Therefore, reliable human-relevant in vitro models must be developed to speed up the identification of compounds that are potential inducers of phospholipidosis. Here, hepatic cells derived from human skin (hSKP-HPC) were evaluated as an in vitro model for DIPL. These cells were exposed over time to amiodarone, a CAD known to induce phospholipidosis in humans. Transmission electron microscopy revealed the formation of the typical lamellar inclusions in the cell cytoplasm. Increase of phospholipids was already detected after 24 h exposure to amiodarone, whereas a significant increase of neutral lipid vesicles could be observed after 72 h. At the transcriptional level, the modulation of genes involved in DIPL was detected. These results provide a valuable indication of the applicability of hSKP-HPC for the quick assessment of drug-induced phospholipidosis in vitro, early in the drug development process.

A Protective Role for Triacylglycerols during Apoptosis

Triacylglycerols (TAGs) are one of the major constituents of the glycerolipid family. Their main role in cells is to store excess fatty acids, and they are mostly found within lipid droplets. TAGs contain acyl chains that vary in length and degree of unsaturation, resulting in hundreds of chemically distinct species. We have previously reported that TAGs containing polyunsaturated fatty acyl chains (PUFA-TAGs) accumulate via activation of diacylglycerol acyltransferases during apoptosis. In this work, we show that accumulation of PUFA-TAGs is a general phenomenon during this process. We further show that the accumulated PUFA-TAGs are stored in lipid droplets. Because membrane-residing PUFA phospholipids can undergo oxidation and form reactive species under increased levels of oxidative stress, we hypothesized that incorporation of PUFAs into PUFA-TAGs and their localization within lipid droplets during apoptosis limit the toxicity during this process. Indeed, exogenous delivery of a polyunsaturated fatty acid resulted in a profound accumulation of PUFA phospholipids and rendered cells more sensitive to oxidative stress, causing reduced viability. Overall, our results support the concept that activation of TAG biosynthesis protects cells from lipid peroxide-induced membrane damage under increased levels of oxidative stress during apoptosis. As such, targeting triacylglycerol biosynthesis in cancer cells might represent a new approach to promoting cell death during apoptosis.

Ymr210wp leads to the accumulation of phospholipids and steryl esters in yeast.

Ymr210w was identified as a MAG (Monoacylglycerol) lipase. The accumulation of the phospholipids in the ymr210wΔ was not clearly understood. It was expressed in S. cerevisiae using pYES2/CT vector and His-tag purified recombinant protein confirmed TAG lipase activity. To further evaluate the role of YMR210w, ester hydrolase activity was also confirmed with pNP-acetate, pNP-butyrate and pNP - palmitate. GC-MS lipid profiling of ymr210wΔ showed an increase in the 15:0 Pentadecanoic acid by 76% among the total lipids. Phospholipid, Erucic acid 22:1 (Δ13) showed 43% increase while steryl esters showed significant changes with 16:0 hexadecanoic acid augmentations by 80% and 18:0 Octadecanoic acid by 165% when compared to wild type (WT). Increase in the steryl ester and TAG content supports the accumulation of lipid bodies in ymr210wΔ strain when compared with WT cells.

Predicting the Risk of Phospholipidosis with in Silico Models and an Image-Based in Vitro Screen.

The drug-induced accumulation of phospholipids in lysosomes of various tissues is predominantly observed in regular repeat dose studies, often after prolonged exposure, and further investigated in mechanistic studies prior to candidate nomination. The finding can cause delays in the discovery process inflicting high costs to the affected projects. This article presents an in vitro imaging-based method for early detection of phospholipidosis liability and a hybrid approach for early detection and risk mitigation of phospolipidosis utilizing the in vitro readout with in silico model prediction. A set of reference compounds with phospolipidosis annotation was used as an external validation set yielding accuracies between 77.6% and 85.3% for various in vitro and in silico models, respectively. By means of a small set of chemically diverse known drugs with in vivo phospholipidosis annotation, the advantages of combining different prediction methods to reach an overall improved phospholipidosis prediction will be discussed.

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

Design and synthesis of a novel series of orally active, selective somatostatin receptor 2 agonists for the treatment of type 2 diabetes

The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds.It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations.Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

Abstract 3219: Targeting lipid metabolism and ER stress in renal cell carcinoma by englerin A

Abstract Renal cell carcinoma (RCC) is among the top ten most common forms of cancer and is the leading malignancy of the kidney. Clear cell renal carcinoma (cc-RCC), the most frequent type of RCC, is one of the most refractory cancers with an incidence that is on the rise. The two-year survival rate for patients with metastatic disease is under 20% despite approved targeted therapies. Hence, there is an urgent need to investigate novel agents. Screening of plant extracts in search of new anti-cancer agents resulted in the discovery of englerin A (EA), a guaiane sesquiterpene with potent cytotoxicity against RCC and a small subset of other cancers. Although a few cellular targets have been identified for EA, in most instances, modulation of these targets required concentrations of EA much higher than that required to kill tumor cells highly sensitive to EA. Hence, it is still not clear what mechanisms account for the cytotoxicity of EA in tumor cells highly sensitive to EA. Unlike any prior study, the current study used a systems biology approach to explore the mechanism(s) of action of EA. Metabolomics analyses were conducted after treatment of human RCC A498 cells with 100 nM EA for 24 and 48 h. Pathway analysis revealed that 88% of the metabolic disturbance associated with englerin A at 24 h was due to changes in lipid metabolism. In particular, among the top metabolites, several ceramides and glucosylceramides increased up to 18- and 178-fold, respectively. On the other hand, all sphingomyelins decreased up to 5.5-fold. Furthermore, EA induced depletion in phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine phospholipids. At 48 h of englerin A treatment, the main findings from pathway analysis were the accumulation of fatty acylcarnitine derivatives, purines, pyrimidines, and phosphatidyl serine phospholipids, as well as the depletion of reduced glutathione indicating dysfunctional mitochondria and oxidative stress. Moreover, microarray analyses determined that EA (100 nM) induced ER stress signaling and an acute inflammatory response including interferon signaling, likely due to the generation of ceramides which were toxic to both A498 and UO-31 renal cancer cells. Quantitative PCR and Western Blot analyses confirmed that EA induced both the unfolded protein and acute inflammatory response. Furthermore, fluorescence confocal microscopy revealed that EA at 25 nM disrupted the morphology of the ER confirming the deleterious effect of EA on the ER. Collectively, our findings suggest that cc-RCC is highly sensitive to disruptions in lipid metabolism and ER stress and that these vulnerabilities can be targeted for the treatment of cc-RCC and possibly other lipid storing cancers. Furthermore, our results also suggest that ceramides may be a mediator of some of the actions of EA. Lastly, the acute inflammatory response induced by EA may mediate anti-tumor immunity. Note: This abstract was not presented at the meeting. Citation Format: Ayse Batova, Diego Altomare, Kim E. Creek, Robert K. Naviaux, Lin Wang, Kefeng Li, Erica Green, Richard Williams, Jane C. Naviaux, Mitchell Diccianni, Alice L. Yu. Targeting lipid metabolism and ER stress in renal cell carcinoma by englerin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3219. doi:10.1158/1538-7445.AM2017-3219

A lipidomic cell-based assay for studying drug-induced phospholipidosis and steatosis.

Phospholipidosis and steatosis are two toxic effects, which course with overaccumulation of different classes of lipids in the liver. MS-based lipidomics has become a powerful tool for the comprehensive determination of lipids. LC-MS lipid profiling of HepG2 cells is proposed as an in vitro assay to study and anticipate phospholipidosis and steatosis. Cells with and without preincubation with a mixture of free fatty acids (FFA; i.e. oleic and palmitic) were exposed to a set of well-known steatogenic and phospholipidogenic compounds. The use of FFA preloading accelerated the accumulation of phospholipids, thus leading to a better discrimination of phospholipidosis, and magnified the lipidomic alterations induced by steatogenic drugs. Phospholipidosis was characterized by increased levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols, while steatosis induced alterations in FA oxidation and triacylglyceride (TG) synthesis pathways (with changes in the levels of FFA, acylcarnitines, monoacylglycerides, diacylglycerides, and TG). Interestingly, palmitic and oleic acids incorporation into lipids differed. A characteristic pattern was observed in the fold of change of particular TG species in the case of steatosis (TG(54:3)>TG(52:2)>TG(50:1)>TG(48:0)). Based on the levels of those lipids containing only palmitic and/or oleic acid moieties a partial least squares-discriminant analysis model was built, which showed good discrimination among nontoxic, phospholipidogenic and steatogenic compounds. In conclusion, it has been shown that the use of FFA preincubation together with intracellular LC-MS based lipid profiling could be a useful approach to identify the potential of drug candidates to induce phospholipidosis and/or steatosis.