Lipoprotein-depleted Serum Research Articles

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL): Fatty Acid Synthase (FASN) and PI3K Dependent Cell Death Identifies a Novel Therapeutic Paradigm

Introduction: Fatty acid (FA) metabolism is altered in many solid tumor cancers in part through increased de novo synthesis of lipids via up-regulation of FASN as well as via increased utilization of lipids via β-oxidation. The dependence of DLBCL on FA metabolism, and potential associated oncogenic signaling pathways, are largely unknown.Methods: Cerulenin (FASN inhibitor), orlistat (combined FASN and lipoprotein lipase inhibitor), and BKM120 (PI3K inhibitor) small molecules were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines as well as in primary DLBCL cells for the impact on FA inhibition signaling and the induction of cell death (by MTT, cleaved caspases, and AnnexinV/PI by flow cytometric analysis). Synergy was assessed via Calcusyn software. All effects were compared by DLBCL cell of origin (COO). In addition, global transcriptome analyses of FASN inhibition were examined on Affymetrix Human 2.0 ST Genechip.Results: FASN protein was constitutively upregulated across all DLBCL cell lines, independent of COO. Treatment with pharmacological inhibitors of FASN (i.e., cerulenin or orlistat) resulted in dose- and time-dependent reduction in cell viability in all DLBCL cell lines. To further examine the dependence of DLBCL on fatty acids, OCI-LY3, SUDHL4, and SUDHL6 grown in presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity (i.e., >98%) as determined by MTT, cleaved caspase 3 and PARP, and AnnexinV/PI. Moreover, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium. These effects were markedly more prominent in primary DLBCL cells compared with control non-malignant lymphocytes. Analysis of differentially expressed gene sets comparing germinal center (GC) and non-germinal center (non-GC) B-cell tumors with normal cells showed significant over expression of genes related to PI3K and lipid metabolism in GC B-cell tumors. Furthermore, global transcriptome analysis following cerulenin exposure revealed activation of carbohydrate and oxidative stress metabolism in GC-derived SUDHL6 and SUDHL10 cells. In the non-GC cell line, OCI-LY3, there was favorable biology induced with inhibition of progression when treated with cerulenin, with downregulated significant key genes representing growth promoting cytokines (Figure 1). Analysis of key significant genes in cerulenin-treated SUDHL10 cells resulted in induction of antioxidant genes (GSR, TXNRD1, NQO1, AIFM1) and oxidative stress responsive PHB, DHX9, HSP90 and PMPCA genes, which are upstream components of PI3K signaling. Western blot analysis with prohibitin (PHB) inhibitor rocoglamide in the presence of cerulenin inhibited phosphorylation of PI3K-p85 and also prominently downregulated FASN expression of GC-derived DLBCL, suggesting the role of PHB in PI3K response to cerulenin. In addition, inhibition of PI3K with BKM120 was shown to strongly down-regulate FASN expression in OCI-LY3, SUDHL6, and SUDHL10 DLBCL cells. Furthermore, treatment with BKM120 in combination with cerulenin was strongly synergistic (CI<1), by MTT and potentiated apoptosis (by Annexin/V flow cytometry) in GC-derived SUDHL10 cells.Conclusions: Multiple FA pathways are constitutively activated in DLBCL and cell survival appears strongly dependent on lipid metabolism, which appeared to occur independent of COO. Additionally, inhibition of FASN was regulated in part through PHB/PI3K-dependent pathways. Collectively, this work indicates that targeting FA metabolism may be considered as a valid therapeutic strategy for DLBCL. Further investigation is required to delineate the precise mechanisms through which PHB/PI3K regulates FASN and also to identify potential rational therapeutic combinations leveraging the addiction of DLBCL to FA signaling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Wnt directs the endosomal flux of LDL ‐derived cholesterol and lipid droplet homeostasis

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

The Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL) and Potential Treatment Strategies with Novel Fatty Acid Synthase (FASN) Small Molecule Inhibitors

Background: Fatty acid (FA) metabolism is altered in several cancers through increased de novo synthesis of lipids via up-regulation fatty acid synthase (FASN) and increased utilization of lipids via β-oxidation. We investigated the dependence of DLBCL survival on FA metabolism. In addition, we examined novel FASN inhibitors TVB3567 and TVB3166 in comparison with cerulenin for the effects on cell survival and PI3K and MAPK-related biological pathways associated with tumor-related FA metabolism in DLBCL.Methods: FASN inhibitors, TVB3567 and TVB3166 (3V Biosciences, CA), cerulenin (FASN inhibitor), orlistat (anti-lipoprotein lipase (LPL) and FASN), PI3K/mTOR, and MEK small molecule inhibitors were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines for the effects of FA inhibition on lipid metabolism, cell signaling, and cell death. The effects of FASN inhibition on global gene expression profile (GEP) were also determined with Affymetrix Human 2.0 ST Genechip with Gene set enrichment analysis (GSEA). We also utilized short hairpin RNA interference (shRNA) to study interactions between FASN and PI3K/MAPK signaling. Finally, AutoDock Vina software (autodock.scripps.edu) was utilized to analyze drug target (FASN enzyme) binding affinity and assist in the design of FASN inhibitors with higher target binding affinity.Results: DLBCL cell lines OCI-LY3, SUDHL4, and SUDHL6 grown in the presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity by MTT. Lipid deprivation-induced apoptotic cell death, detected as cleaved caspase 3 and PARP and Annexin-V/PI positivity, in these cells. Further, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium in SUDHL4 confirming the high lipid-dependency on cell survival in DLBCL. Treatment with pharmacological inhibitors of FASN (ie, TVB3567, TVB3166, cerulenin, or orlistat) resulted in a dose- and time-dependent reduction in cell viability in all DLBCL cell lines. Ingenuity Pathway Analysis (IPA) from GEP with cerulenin-treated OCI-LY3 showed prominent suppression of CD40, TNF, and NFκB dependent inflammatory responses as well as activation of apoptosis as predominant biological activities including significant down-regulation of genes involved in Krebs cycle and p38 MAPK pathways. Interestingly, upstream regulation by IPA predicted activation of MEK/ERK and MYC-dependent functions; and in OCI-LY3 with shRNA knock down of FASN, we observed constitutive activation of ERK as detected with increased phosphorylation by western blot. Activation of MEK/ERK and MYC is expected in part owing to metabolic stress induced by FASN inhibition. Considering the impact of MEK/ERK pathways on lipid metabolism, we next investigated the impact of MEK/ERK on FA metabolism. FASN was significantly decreased following MEK or ERK shRNA in OCILY-3 and SUDHL10 cells. Similarly, pharmacological inhibition of MEK or PI3K/mTOR (using novel small molecule agents AZD6244 (selumetinib) or BEZ235, respectively) resulted in marked down-regulation of FASN expression. Based on these results, FASN inhibition appears to a promising therapeutic target for the treatment of DLBCL, however attaining clinical efficacy with existing compounds require the effective drug concentration to be within the nanomolar range. Thus, we utilized AutoDock to determine drug docking enzyme inhibition constant (ki). We identified high ki values of 33μM and 180μM for Cerulenin and Orilstat, respectively. Therefore, we have developed/constructed modified novel and potent anti-FA compounds with ki <1μM that are currently being investigated.Conclusions: Collectively, we demonstrated that DLBCL cell survival is highly dependent on FA metabolism and that targeting lipid metabolism may be harnessed as a potential therapeutic strategy. We also showed that MEK/ERK-dependent mechanisms are intimately involved in promoting lipid addiction in DLBCL cells. Further investigation is warranted to delineate the mechanisms through which MEK/ERK regulate FASN expression and to determine in vivo implications of FASN inhibition on DLBCL tumor growth. In addition, continued development, design, and enhancement are needed to construct the most optimal anti-FA therapeutic agents. DisclosuresLansigan:Teva Pharmaceuticals: Research Funding; Spectrum Pharmaceuticals: Research Funding.

Diffuse Large B-Cell Lymphomas Utilize Endogenous and Exogenous Fatty Acids for Cell Growth and Survival

AbstractAbstract 1581 BackgroundIt is well-recognized that de novo long chain fatty acid (FA) synthesis, driven by the key enzyme fatty acid synthase (FASN), is crucial for the growth and survival of many types of cancer cells. We and others have observed FASN protein expression in diffuse large B-cell lymphoma (DLBCL) tumors. Furthermore, we have shown that higher levels of FASN in DLBCL tumors strongly predicted inferior survival, which was independent from the international prognostic index. We also recently demonstrated that, in addition to FA synthesis, various cancer cells can acquire FA from circulating lipoproteins, using the secreted enzyme lipoprotein lipase (LPL), and that this promotes cell growth. DLBCL, however, has never been examined in this regard. In this study, we investigated the functional significance of both de novo FA synthesis via FASN and exogenous FA uptake via LPL in DLBCL. MethodsLevels of FASN and LPL mRNAs in DLBCL cell lines (SUDHL4, SUDHL10, OCI-LY3, OCI-LY19) were studied using reverse transcriptase polymerase chain reaction. We determined FASN and LPL protein expression by flow cytometry using a novel anti-LPL antibody that we developed. DLBCL cell lines were cultured +/− Cerulenin (an inhibitor of FASN), Orlistat (an inhibitor of FASN and LPL), or in lipoprotein-depleted serum +/− supplementation with very low density lipoprotein (VLDL) particles. The MTT assay was used to assess cell proliferation. ResultsDLBCL cell lines exhibited >10-fold variation in levels of FASN mRNA. Cerulenin and Orlistat each caused dose-dependent inhibition of proliferation of each cell line. The cells were partially rescued by the addition of palmitic acid, the FA product of FASN. Surprisingly, flow cytometry revealed that SUDHL4 and OCI-LY3 cells, which did not secrete LPL or show detectable LPL activity, displayed the enzyme on the cell surface. Moreover, in stark contrast to several other cancer cell lines, DLBCL cells were exquisitely sensitive to withdrawal of lipoproteins from the culture media. Indeed, 75–95% of the cells underwent apoptosis after only 24 hours in lipoprotein-depleted serum. In complete serum, the provision of VLDL particles did not rescue DLBCL cells from FA synthesis inhibition using Cerulenin, suggesting that the serum contains sufficient lipoproteins to saturate the FA uptake system. This prediction was validated in experiments utilizing lipoprotein-depleted serum, in which add-back of VLDL particles completely rescued the cells from Cerulenin-induced demise in a dose-related manner, with full restoration at approximately 100–200mcg/ml of VLDL. ConclusionsOur data demonstrate that DLBCL cells employ both de novo FA synthesis via FASN and exogenous FA uptake using LPL to satisfy their strict requirement for FA. Interference with either pathway, using FASN inhibitors or lipoprotein-depleted serum, is cytotoxic indicating that neither alone is sufficient to support proliferation. Further, DLBCL cells show a striking dependency on exogenous FA of dietary origin compared with all other cancer cells we have examined. The observation that the cell lines can be rescued by provision of VLDL particles strongly supports the functional significance of the exogenous FA uptake pathway for DLBCL. Our data thus demonstrate that the extracellular lipase LPL is critical for the growth and survival of DLBCL cells. Surprisingly, the cells deploy LPL to their surface, and we speculate that this promotes efficient FA acquisition from circulating lipoproteins. Recognition that DLBCL relies on both synthesis and uptake of FA will provide guidance for drug development and dietary modifications to effectively target the metabolic requirements of this tumor. Disclosures:No relevant conflicts of interest to declare.

Lipoprotein distribution and serum concentrations of 7α-hydroxy-4-cholesten-3-one and bile acids: effects of monogenic disturbances in high-density lipoprotein metabolism

BA (bile acid) formation is considered an important final step in RCT (reverse cholesterol transport). HDL (high-density lipoprotein) has been reported to transport BAs. We therefore investigated the effects of monogenic disturbances in human HDL metabolism on serum concentrations and lipoprotein distributions of the major 15 BA species and their precursor C4 (7α-hydroxy-4-cholesten-3-one). In normolipidaemic plasma, approximately 84%, 11% and 5% of BAs were recovered in the LPDS (lipoprotein-depleted serum), HDL and the combined LDL (low-density lipoprotein)/VLDL (very-low-density lipoproteins) fraction respectively. Conjugated BAs were slightly over-represented in HDL. For C4, the respective percentages were 23%, 21% and 56% (41% in LDL and 15% in VLDL) respectively. Compared with unaffected family members, neither HDL-C (HDL-cholesterol)-decreasing mutations in the genes APOA1 [encoding ApoA-I (apolipoprotein A-I], ABCA1 (ATP-binding cassette transporter A1) or LCAT (lecithin:cholesterol acyltransferase) nor HDL-C-increasing mutations in the genes CETP (cholesteryl ester transfer protein) or LIPC (hepatic lipase) were associated with significantly different serum concentrations of BA and C4. Plasma concentrations of conjugated and secondary BAs differed between heterozygous carriers of SCARB1 (scavenger receptor class B1) mutations and unaffected individuals (P<0.05), but this difference was not significant after correction for multiple testing. Moreover, no differences in the lipoprotein distribution of BAs in the LPDS and HDL fractions from SCARB1 heterozygotes were observed. In conclusion, despite significant recoveries of BAs and C4 in HDL and despite the metabolic relationships between RCT and BA formation, monogenic disorders of HDL metabolism do not lead to altered serum concentrations of BAs and C4.

Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein

Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells. A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by ∼80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Opposite effects of native and oxidized lipoproteins on the activity of secretory phospholipase A 2 group IIA

Elevated circulating level and activity of secretory phospholipase A 2 group IIA (sPLA 2(IIA)) are associated with the development of adverse cardiovascular events. The mechanisms of sPLA 2(IIA) activity regulation in human blood serum so far remain obscure. We have suggested that the enzyme activity is influenced by circulating lipoproteins. The activity of sPLA 2(IIA) was examined in whole serum of healthy individuals and after removal of lipoproteins from it. The effects of different classes of native and oxidized lipoproteins on sPLA 2(IIA) in blood serum were compared with their effects on purified sPLA 2(IIA). Activity of sPLA 2(IIA) was not detected in whole serum despite the high concentration of the enzyme. However after lipoproteins had been removed from the serum, the lipoprotein-depleted serum displayed sPLA 2(IIA) activity which was proportional to the amount of sPLA 2(IIA) in it. Native LDL, HDL and VLDL + IDL inhibited the activity of both purified sPLA 2(IIA) and the enzyme activity in lipoprotein-depleted serum. By contrast, oxidized LDL, HDL and VLDL + IDL significantly stimulated the activity of purified and serum sPLA 2(IIA) and enhanced the release of fatty acids from the substrate. The data indicate that native and oxidized lipoproteins regulate catalytic activity of sPLA 2(IIA). Activation of sPLA 2(IIA) by oxidized lipoproteins may be regarded as one of the mechanisms of atherosclerosis development.

Scavenger Receptor BI-mediated Selective Uptake Is Required for the Remodeling of High Density Lipoprotein by Endothelial Lipase

Endothelial lipase (EL) is a negative regulator of high density lipoprotein (HDL) cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodeling of HDL. The present study investigates the requirement of SR-BI for the effects of EL-mediated phospholipid hydrolysis on HDL metabolism in vivo. In vitro, selective uptake from EL-modified HDL was 129% higher than selective uptake from control HDL in SR-BI-overexpressing cells (p=0.01). In vivo overexpression of human EL by means of recombinant adenovirus decreased HDL plasma levels significantly (p<0.01). Fast protein liquid chromatography analysis and agarose gel electrophoresis revealed that EL expression resulted in the generation of small pre-beta HDL particles in wild-type mice, whereas in SR-BI-/- mice small HDL were preferentially removed. In kinetic experiments the fractional catabolic rate (FCR) of HDL cholesteryl ester increased by 110% (p<0.001), and the FCR of HDL apolipoproteins increased by 64% (p<0.001) in response to EL overexpression in wild-type mice. In SR-BI-/- mice a similar increase in the HDL apolipoprotein FCR occurred (p<0.001); however, there was no further increase in HDL cholesteryl ester catabolism. The apparent whole body selective uptake was increased 3-fold by EL in wild-type mice (p<0.001), whereas there was no selective uptake in SR-BI knock-out mice. EL overexpression increased hepatic selective uptake as well as holoparticle uptake (each p<0.01) in wild-type mice, whereas in SR-BI knock-out mice only holoparticle uptake increased (p<0.01). Our results indicate that SR-BI-mediated selective uptake of HDL cholesteryl ester is essential for the remodeling of large alpha-migrating HDL particles by EL.

G Protein‐Activated Inward Rectifier Potassium Channel 4 is a Regulateable Protein; Its Role in Autonomic Neuropathy in Type I Diabetes

Parasympathetic stimulation decreases heart rate and force of contraction and involves acetylcholine binding to M2, activation of the G protein‐activated inward rectifier K+ channel (GIRK1)2/(GIRK4)2. Although GIRK4 is required for the generation of IkAch, no data on the regulation of its expression are available. In a model for lipid lowering, culture of atrial myocytes in lipoprotein depleted serum (LPDS) increased GIRK4 promoter activity (GIRK4‐Luc) and protein compared to cells grown in FBS. LPDS stimulated phosphorylation of Akt while LPDS‐stimulated GIRK4 promoter was inhibited by the p‐Akt inhibitor, LY294002. LPDS increased SREBP1 and SREBP1 increased GIRK4 promoter in myocytes. Insulin treatment of myocytes increased SREBP‐1, GIRK4 promoter, GIRK4 protein and p‐Akt. LY inhibited insulin‐stimulated GIRK4 expression. Diabetic autonomic neuropathy is associated with parasympathetic dysfunction. In the type 1 diabetic Akita mice, which demonstrate parasympathetic dysfunction, expression of GIRK4 and SREBP1 in the atrium was decreased compared to WT. Insulin treatment of Akita increased level of SREBP1 and GIRK4 to that in WT and reversed parasympathetic dysfunction. Given that insulin has been shown to regulate SREBP, these data suggest that hypoinsulinemia in type 1 diabetes might result in parasympathetic dysfunction due to decreased SREBP and GIRK4 and Akt might play a role in this pathway.

Parasympathetic response in chick myocytes and mouse heart is controlled by SREBP

Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein-coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, I KACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and I KACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN-SREBP-1) reversed the effect of lipid lowering on I KACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. I KACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.

Zoledronate acid modifies the lipid transporters profile in human prostate cancer cell lines

21135 Background: The mechanism through wich zoledronic acid exerts its activity is poorly understood. Low density lipoprotein receptor (LDLR) and scavenger receptor class B type 1 (SRBI) overproduction is an important mechanism in cancer cells for obtaining more essential fatty acids and cell growth. The effects of in vitro zoledronate treatment on the lipid metabolism of prostate cancer cell lines were studied. Methods: Three prostatic cancer cell lines, androgen insensitive PC3, androgen dependent low-passage (LP) LNCaP and androgen independent high-passage (HP) LNCaP cells were studied. Cells were plated either in RPMI with 5% foetal calf serum and lipoprotein depleted serum (LPDS) and were treated with zoledronate at different concentrations. The lipid transporters profile was analyzed by western blotting for LDLR and SRB1. Results: The same LDLR bands profile was observed in all cell lines, 160 and 105 kDa. The basal levels of LDLR were higher in the PC3 cells. Zoledronate therapy induced LDLR expression in all cell lines but PC3 were less sensitive to this effect. Cells cultured with LPDS showed an enhanced expression of LDLR and PC3 cells were less sensitive to this effect. HP LNCaP cells were the most affected by lipoprotein deprivation however this effect diminished 72 hours after treatment. The bands profile for SRBI consisted of a 65 kDa predominant band and a 40 kDa band in both LP/HP LNCaP cells. In PC3 cells main band was located in 65 kDa and accessory band in 30kDa. The basal levels of the 65kDa band were higher in HP than in LP LNCaP or PC3 cells and zoledronate therapy caused a dose- dependent induction in HP LNCaP and dose-dependent reduction in PC3 cells, LP LNCaP cells were resistant to the treatment. LPDS induced SRBI levels in all cell lines inverting the effect caused by zoledronate in HP LNCaP cells in complete culture medium and at high doses (100μM) a complete inhibition of SRBI protein was found. Low molecular weight bands changed in the same way as the 65 kDa band. Conclusions: LDL-R and SRBI have been isolated in prostate cancer cell lines. Based on previous cell growth studies, the lipid transporters profile might be significantly involved in the resistance to zoledronate therapy. Lipoprotein regulation pathways should be considered in the therapy of metastatic prostate cancer. No significant financial relationships to disclose.

Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux

Our objective was to evaluate the associations of individual apolipoprotein A-I (apoA-I)-containing HDL subpopulation levels with ABCA1- and scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux. HDL subpopulations were measured by nondenaturing two-dimensional gel electrophoresis from 105 male subjects selected with various levels of apoA-I in pre-beta-1, alpha-1, and alpha-3 HDL particles. ApoB-containing lipoprotein-depleted serum was incubated with [(3)H]cholesterol-labeled cells to measure efflux. The difference in efflux between control and ABCA1-upregulated J774 macrophages was taken as a measure of ABCA1-mediated efflux. SR-BI-mediated efflux was determined using cholesterol-labeled Fu5AH hepatoma cells. Fractional efflux values obtained from these two cell systems were correlated with the levels of individual HDL subpopulations. A multivariate analysis showed that two HDL subspecies correlated significantly with ABCA1-mediated efflux: small, lipid-poor pre-beta-1 particles (P=0.0022) and intermediate-sized alpha-2 particles (P=0.0477). With regard to SR-BI-mediated efflux, multivariate analysis revealed significant correlations with alpha-2 (P=0.0004), alpha-1 (P=0.0030), pre-beta-1 (P=0.0056), and alpha-3 (P=0.0127) HDL particles. These data demonstrate that the small, lipid-poor pre-beta-1 HDL has the strongest association with ABCA1-mediated cholesterol even in the presence of all other HDL subpopulations. Cholesterol efflux via the SR-BI pathway is associated with several HDL subpopulations with different apolipoprotein composition, lipid content, and size.

Effects of zoledronic acid in prostate cancer cell lines: A pharmacogenomic approach

4782 Background: The LNCaP and the PC3 cells are widely used as models for prostate cancer. Aminobisphosphonates inhibit proliferation and induce apoptosis in these cells by interfering the mevalonate pathway. We tested the hypothesis that some of its effects over cell growth could be amelorated by the uptake of cholesterol from serum lipoproteins. Methods: The response of androgen-dependent, low-passage (LP) LNCaP and androgen-independent, high-passage (HP) LNCaP and PC3 cells to zoledronic acid was studied. Cells were grown in RPMI with 5% foetal calf serum and treated with zoledronate at different concentrations. Cells cultured with lipoprotein depleted serum (LPDS) were also included. We have analysed the genetic profile of untreated vs. zoledronic-acid treated cells by the use of cDNA microarrays (HG-Focus array, 8,500 genes, from Affymetrix). We studied the changes in genes involved in cholesterol metabolism. Results: PC3 were more sensitive to zoledronate-induced cell growth inhibition and the effects were not be significantly amelorated by serum lipoproteins. The apparent resistance of LP-LNCaP cells could be partially reversed by removing the lipoproteins from serum. It suggests the existence of compensatory mechanisms for the inhibition of the mevalonate pathway in these cells, and that an increased use of cholesterol from lipoproteins may play an important role. We also studied the effects of LPDS in HP-LNCaP cells, where the effects were similar to that observed in PC3. When data from microarrays were analysed, an increased expression in LP-LNCaP cells and a reduced expression in PC3 cells observed in the genes of enzymes of mevalonate pathway, lipases and lipoprotein transporters. Mevalonate decarboxylase, farnesyl transferase, 7-dehydrocholesterol reductase and lipase A, cholesterol transporters, the low density lipoprotein receptor and the scavenger receptor class B isotype II (not previously described in prostate cells) were the most affected. Conclusions: These changes might also reflect different adaptative responses to zoledronate and therefore, different mechanisms for chemoresistance. This study was supported by Novartis Spain and the Instituto de Salud Carlos III (project Fis02/3003). No significant financial relationships to disclose.

Role of sterol regulatory element binding proteins in the regulation of Galpha(i2) expression in cultured atrial cells.

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2 muscarinic receptor; the alpha-subunit of the heterotrimeric G protein, Galpha(i2); and the inward rectifying K+ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of Galpha(i2) promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the Galpha(i2) promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Galpha(i2) promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Galpha(i2) SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Galpha(i2) SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Galpha(i2) promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Galpha(i2) promoter activity, suggesting that SREBP1 may play a role in the regulation of Galpha(i2) expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.

Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events.

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-beta signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase.

Transforming growth factor-beta (TGFbeta) signaling has been shown to play a role in cardiac development as well as in the pathogenesis of cardiovascular disease. Prior studies have suggested a relationship between cholesterol metabolism and TGFbeta signaling. Here we demonstrate that induction of the cholesterol metabolic pathway by growth of embryonic chicken atrial cells in medium supplemented with lipoprotein-depleted serum coordinately decreased the expression of the TGFbeta type II receptor (TGFbetaRII), TGFbeta(1), and TGFbeta signaling as measured by plasminogen activator inhibitor-1 (PAI-1) promoter activity. Inhibition of the cholesterol metabolic pathway by the hydrophobic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, simvastatin and atorvastatin, reversed the effect of lipoprotein-depleted serum and up-regulated TGFbetaRII expression, whereas the hydrophilic HMGCoA reductase inhibitor, pravastatin, had no effect. Simvastatin stimulated the expression of TGFbetaRII, TGFbeta(1), and PAI-1 at the level of transcription. Experiments using specific inhibitors of different branches of the cholesterol metabolic pathway demonstrated that simvastatin exerted its effect on TGFbeta signaling by inhibition of the geranylgeranylation pathway. C3 exotoxin, which specifically inactivates geranylgeranylated Rho GTPases, mimicked the effect of simvastatin on PAI-1 promoter activity. Cotransfection of cells with a PAI-1 promoter-reporter and a dominant-negative RhoA mutant increased PAI-1 promoter activity, whereas cotransfection with a dominant-active RhoA mutant decreased PAI-1 promoter activity. These data support the conclusion that TGFbeta signaling is regulated by RhoA GTPase and demonstrate a relationship between cholesterol metabolism and TGFbeta signaling. Our data suggest that in patients treated with HMGCoA reductase inhibitors, these agents may exert effects independent of cholesterol lowering on TGFbeta signaling in the heart.

Major Proteins of Bovine Seminal Plasma Modulate Sperm Capacitation by High-Density Lipoprotein1

Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.

Vastatins have a distinct effect on sterol synthesis and progesterone secretion in human granulosa cells in vitro

Lovastatin and simvastatin are strong inhibitors of cholesterol synthesis in cultured human granulosa cells, as measured within 6 days after isolation, with IC 50-values of respectively 27.0 and 18.2 nM obtained after 3.5 hours of incubation with the drugs. Pravastatin is a much weaker inhibitor of cholesterol synthesis (IC 50-value of 977.8 nM) in these cells. Under these conditions inhibition of cholesterol synthesis had no influence on progesterone secretion into the medium which was probably due to the presence of large cholesterol pools in the cells. To deplete these pools, granulosa cells were cultured for 7 days after which the culture medium was changed into medium supplemented with 20% lipoprotein-depleted serum to deprive the cells of exogenous cholesterol. Additionally, 30 mIU of follicle-stimulating hormone and luteinizing hormone per ml were added to stimulate the progesterone production and secretion, thereby decreasing the cholesteryl ester pools. After 48 h of incubation, culture was continued without hormones for another two days. Thereafter, the cells were preincubated for 24 h without or with 1 μM of lovastatin, simvastatin or pravastatin in medium containing lipoprotein-deficient serum and the above-mentioned hormones. This period is followed by incubation for another 24 h in the presence of [ 14C]acetate after which cells and media were collected for determination of 14C-labelled sterols synthesized and progesterone secreted into the media. Now, lovastatin and simvastatin, which strongly inhibited sterol synthesis, significantly attenuated the secretion of progesterone. One μM of pravastatin had no significant effect on sterol synthesis nor on progesterone secretion. When the latter experiment was performed under conditions in which exogenous cholesterol was provided in the form of human low density lipoproteins, no influence of the vastatins on progesterone secretion was observed. So under conditions in which the cholesterol pools were decreased, lovastatin and simvastatin attenuated the progesterone secretion, whereas pravastatin did not. When pools were filled by exogenous cholesterol, no effect on progesterone secretion by either of the drugs was observed.