Inhibition of mitochondrial respiration overcomes hepatocellular carcinoma chemoresistance

The clinical management of advanced hepatocellular carcinoma (HCC) is challenging due to its resistance to chemotherapy. In our work, we demonstrate that an antiparasitic drug atovaquone at clinically relevant concentrations is active against chemoresistant HCC. We show that atovaquone inhibits proliferation and induces apoptosis in not only HCC parental cells but also cells exposed to long time culture of chemotherapeutic agents. Consistently, the combination of atovaquone with cisplatin or doxorubicin achieved remarkably greater efficacy than single drug alone. Mechanistically, atovaquone overcomes HCC chemoresistance via supressing mitochondrial respiration and inducing oxidative stress. Atovaquone but not cisplatin or doxorubicin is ineffective in mitochondrial respiration-deficient ρ0, confirming mitochondria as a specific upstream target of atovaquone. Interestingly, we show that prolonged exposure of HCC cells to chemotherapeutic agents induces higher level of mitochondrial respiration, suggesting that tumors which develop chemoresistance after chemotherapy might be more dependent on mitochondrial respiration than primary tumors and explaining the sensitivity of chemoresistant HCC cells to atovaquone. We further show that atovaquone at tolerable does significantly inhibits chemoresistant HCC growth in mice throughout the duration of treatment. In line with in vitro data, we observe the increased oxidative stress in atovaquone-treated tumors. Our findings highlight the dependency of chemoresistant HCC on mitochondrial respiration and demonstrate that atovaquone is a potential drug to overcome HCC chemoresistance.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

The role of mitochondrial respiration in physiological and evolutionary adaptation

Aerobic mitochondria serve as the power sources of eukaryotes by producing ATP through oxidative phosphorylation (OXPHOS). The enzymes involved in OXPHOS are multisubunit complexes encoded by both nuclear and mitochondrial DNA. Thus, regulation of respiration is necessarily a highly coordinated process that must organize production, assembly and function of mitochondria to meet an organism's energetic needs. Here I review the role of OXPHOS in metabolic adaptation and diversification of higher animals. On a physiological timescale, endocrine-initiated signaling pathways allow organisms to modulate respiratory enzyme concentration and function under changing environmental conditions. On an evolutionary timescale, mitochondrial enzymes are targets of natural selection, balancing cytonuclear coevolutionary constraints against physiological innovation. By synthesizing our knowledge of biochemistry, physiology and evolution of respiratory regulation, I propose that we can now explore questions at the interface of these fields, from molecular translation of environmental cues to selection on mitochondrial haplotype variation.     

A prototype methodology combining surface-enhanced laser desorption/ionization protein chip technology and artificial neural network algorithms to predict the chemoresponsiveness of breast cancer cell lines exposed to Paclitaxel and Doxorubicin under in vitro conditions

An ability to predict the likelihood of cellular response towards particular chemotherapeutic agents based upon protein expression patterns could facilitate the identification of biological molecules with previously undefined roles in the process of chemoresistance/chemosensitivity, and if robust enough these patterns might also be exploited towards the development of novel predictive assays. To ascertain whether proteomic based molecular profiling in conjunction with artificial neural network (ANN) algorithms could be applied towards the specific recognition of phenotypic patterns between either control or drug treated and chemosensitive or chemoresistant cellular populations, a combined approach involving MALDI-TOF matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Ciphergen protein chip technology and ANN algorithms have been applied to specifically identify proteomic 'fingerprints' indicative of treatment regimen for chemosensitive (MCF-7, T47D) and chemoresistant (MCF-7/ADR) breast cancer cell lines following exposure to Doxorubicin or Paclitaxel. The results indicate that proteomic patterns can be identified by ANN algorithms to correctly assign 'class' for treatment regimen (e.g. control/drug treated or chemosensitive/chemoresistant) with a high degree of accuracy using boot-strap statistical validation techniques and that biomarker ion patterns indicative of response/non-response phenotypes are associated with MCF-7 and MCF-7/ADR cells exposed to Doxorubicin. We have also examined the predictive capability of this approach towards MCF-7 and T47D cells to ascertain whether prediction could be made based upon treatment regimen irrespective of cell lineage. Models were identified that could correctly assign class (control or Paclitaxel treatment) for 35/38 samples of an independent dataset. A similar level of predictive capability was also found (> 92%; n = 28) when proteomic patterns derived from the drug resistant cell line MCF-7/ADR were compared against those derived from MCF-7 and T47D as a model system of drug resistant and drug sensitive phenotypes. This approach might offer a potential methodology for predicting the biological behaviour of cancer cells towards particular chemotherapeutics and through protein isolation and sequence identification could result in the identification of biological molecules associated with chemosensitive/chemoresistance tumour phenotypes.     

Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.     

p53 Is Required for Cisplatin-induced Processing of the Mitochondrial Fusion Protein L-Opa1 That Is Mediated by the Mitochondrial Metallopeptidase Oma1 in Gynecologic Cancers

Mitochondria are highly dynamic organelles, and mitochondrial fission is a crucial step of apoptosis. Although Oma1 is believed to be responsible for long form Opa1 (L-Opa1) processing during mitochondrial fragmentation, whether and how Oma1 is involved in L-Opa1 processing and participates in the regulation of chemoresistance is unknown. Chemosensitive and chemoresistant ovarian (OVCA) and cervical (CECA) cancer cells were treated with cisplatin (CDDP). Mitochondrial dynamics and protein contents were assessed by immunofluorescence and Western blot, respectively. The requirements of Oma1 and p53 for CDDP-induced L-Opa1 processing, mitochondrial fragmentation, and apoptosis were examined by siRNA or cDNA. CDDP induces L-Opa1 processing and mitochondrial fragmentation in chemosensitive but not in chemoresistant cells. CDDP induced Oma1 40-kDa form increases in OV2008 cells, not in C13* cells. Oma1 knockdown inhibited L-Opa1 processing, mitochondrial fragmentation, and apoptosis. Silencing p53 expression attenuated the effects of CDDP in Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis in chemosensitive OVCA cells, whereas reconstitution of p53 in p53 mutant or null chemoresistant OVCA cells induced Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis irrespective of the presence of CDDP. Prohibitin 1 (Phb1) dissociates from Opa1-Phb1 complex and binds phosphorylated p53 (serine 15) in response to CDDP in chemosensitive but not chemoresistant CECA cells. These findings demonstrate that (a) p53 and Oma1 mediate L-Opa1 processing, (b) mitochondrial fragmentation is involved in CDDP-induced apoptosis in OVCA and CECA cells, and (c) dysregulated mitochondrial dynamics may in part be involved in the pathophysiology of CDDP resistance.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK^−/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.Subject terms: Cancer metabolism, Energy metabolism, Cancer metabolism     

Relationship between respiration deficiency and azole resistance in clinical Candida glabrata

Candida glabrata has become a leading cause of invasive infections around the world and is exhibiting growing resistance to azole antifungals. To study the mechanism of its azole resistance, we analyzed the efflux pumps and found well known increased efflux expression and low metabolic state in all azole-resistant strains. The latter finding led us to further investigate the relationship between respiration status and azole antifungal susceptibility in clinical C.?glabrata by growing them on glycerol-containing agar, measuring the cellular ATP, reactive oxygen species (ROS) levels, oxygen consumption and transmission electron microscopy. All azole-resistant isolates were respiratory-deficient, with reduced generation of ATP and ROS and decreased oxygen consumption; two isolates grew as small colonies and exhibited mitochondrial deficiency. Spot assays and agarose disc diffusion tests were performed to evaluate the effects of respiratory chain inhibitors, sodium azide and salicylhydroxamic acid, on antifungal susceptibility. The results of antifungal susceptibility showed that inhibition of alternative respiration with salicylhydroxamic acid enhanced azole susceptibility of C.?glabrata. In conclusion, clinical azole-resistant C.?glabrata isolates harbor respiratory deficiency exhibiting petite mutant or normal phenotype. The alternative respiratory pathway plays an important role in the decreased susceptibility to azole antifungals.     

Melanoma chemotherapy leads to the selection of ABCB5-expressing cells

Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5(+) cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.     

Mitochondrial oxidative phosphorylation compensation may preserve vision in patients with OPA1-linked autosomal dominant optic atrophy

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.     

Enhanced liver but not muscle OXPHOS in diabetes and reduced glucose output by complex I inhibition

Mitochondrial function is critical in energy metabolism. To fully capture how the mitochondrial function changes in metabolic disorders, we investigated mitochondrial function in liver and muscle of animal models mimicking different types and stages of diabetes. Type 1 diabetic mice were induced by streptozotocin (STZ) injection. The db/db mice were used as type 2 diabetic model. High-fat diet-induced obese mice represented pre-diabetic stage of type 2 diabetes. Oxidative phosphorylation (OXPHOS) of isolated mitochondria was measured with Clark-type oxygen electrode. Both in early and late stages of type 1 diabetes, liver mitochondrial OXPHOS increased markedly with complex IV-dependent OXPHOS being the most prominent. However, ATP, ADP and AMP contents in the tissue did not change. In pre-diabetes and early stage of type 2 diabetes, liver mitochondrial complex I and II-dependent OXPHOS increased greatly then declined to almost normal at late stage of type 2 diabetes, among which alteration of complex I-dependent OXPHOS was the most significant. In contrast, muscle mitochondrial OXPHOS in HFD, early-stage type 1 and 2 diabetic mice, did not change. In vitro, among inhibitors to each complex, only complex I inhibitor rotenone decreased glucose output in primary hepatocytes without cytotoxicity both in the absence and presence of oleic acid (OA). Rotenone affected cellular energy state and had no effects on cellular and mitochondrial reactive oxygen species production. Taken together, the mitochondrial OXPHOS of liver but not muscle increased in obesity and diabetes, and only complex I inhibition may ameliorate hyperglycaemia via lowering hepatic glucose production.     

PAXX is a novel target to overcome resistance to doxorubicin and cisplatin in osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor diagnosed in children and adolescents. Unfortunately, OS patients with metastatic or recurrent disease are highly resistant to front line chemotherapy that significantly limits the long-term survival rate. Since majority of chemotherapeutic agents used in OS work by generating DNA damages, enhanced DNA repair pathways are generally associated with chemoresistance in OS treatment. However, the exact mechanisms of chemoresistance in OS are not fully understood. Our study found that paralogue of XRCC4 and XLF (PAXX), which was identified recently as a novel factor of non-homologous end joining (NHEJ), is upregulated in chemoresistant OS cells. Importantly, PAXX deficiency re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Mechanistically, chemoresistance to doxorubicin or cisplatin results in enhanced PAXX-Ku70 interaction and elevated NHEJ efficiency. We also identified a small molecule M11 that interrupts PAXX-Ku70 interaction and re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Thus, our data provide evidence that PAXX could serve as a novel target to overcome chemoresistance in OS treatment.     

Doxorubicin-induced thiol-dependent alteration of cardiac mitochondrial permeability transition and respiration

Doxorubicin (DOX) is a highly effective treatment for several forms of cancer. However, clinical experience shows that DOX induces a cumulative and dose-dependent cardiomyopathy that has been ascribed to redox-cycling of the drug on the mitochondrial respiratory chain generating free radicals and oxidative stress in the process. Mitochondrial dysfunction including induction of the mitochondrial permeability transition (MPT) and inhibition of mitochondrial respiration have been implicated as major determinants in the pathogenesis of DOX cardiotoxicity. The present work was aimed at investigating whether the inhibition of mitochondrial respiration occurs secondarily to MPT induction in heart mitochondria isolated from DOX-treated rats and whether one or both consequences of DOX treatment are related with oxidation of protein thiol residues. DOX-induced oxidative stress was associated with the accumulation of products of lipid peroxidation and the depletion of alpha-tocopherol in cardiac mitochondrial membranes. No changes in mitochondrial coenzyme Q9 and Q10 concentrations were detected in hearts of DOX-treated rats. Cardiac mitochondria from DOX-treated rats were more susceptible to diamide-dependent induction of the MPT. Although DOX treatment did not affect state 4 respiration, state 3 respiration was decreased in heart mitochondria isolated from DOX-treated rats, which was reversed in part by adding either cyclosporin A or dithiothreitol, but not Trolox. The results suggest that in DOX-treated rats, (i) induction of the MPT is at least in part responsible for decreased mitochondrial respiration, (ii) heart mitochondria are more susceptible to diamide induced-MPT, (iii) thiol-dependent alteration of mitochondrial respiration is partially reversible ex vivo with dithiothreitol. Collectively, these data are consistent with the thesis that thiol-dependent alteration of MPT and respiration is an important factor in DOX-induced mitochondrial dysfunction.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.