Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G
© Mishra and Simonson; licensee BioMed Central Ltd. 2005
Received: 04 November 2004
Accepted: 10 January 2005
Published: 10 January 2005
In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.
Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.
The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.
Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.
Keywordsfree fatty acids microvascular apoptosis caspase 9 lipotoxicity mesangial cells
Recent evidence suggests that intracellular accumulation of saturated free fatty acids (FFA) in vascular cells contributes to lipid-mediated cellular damage (see [1–4] for review). The cellular dysfunction associated with FFA overload, known as lipotoxicity, contributes to cell injury in settings of high FFA or triglycerides, such as obesity or type 2 diabetes . Diverse mechanisms have been proposed to explain lipotoxicity including dysregulation of cell signaling, induction of a proinflammatory and prothrombotic state, or in some cases programmed cell death [1, 4]. Indeed, saturated FFA have previously been shown to induce apoptotic cell death that is prevented in most cell types by unsaturated FFA [5–14]. In the microvasculature, the pro-apoptotic signaling pathways induced by saturated FFA and the anti-apoptotic pathways regulated by unsaturated FFA remain incompletely understood.
Several mechanisms have been implicated in apoptotic cell death induced by saturated FFA. Some studies suggest that increased β-oxidation of FFA does not contribute to apoptotic cell death and suggest that unmetabolized FFA might be involved [1, 13]. However, other studies contradict this observation and suggest a direct role for mitochondrial β-oxidation in the apoptotic response to palmitate.. The finding that long-chain saturated but not unsaturated FFA cause apoptosis implicates a product made specifically from the saturated species. For instance, saturated but not unsaturated FFA are precursors for the pro-apoptotic lipid ceramide. Although palmitate does increase de novo ceramide synthesis in cultured cells, studies of the functional role of ceramide in palmitate-induced apoptosis have yielded conflicting results that might depend on the cell type in question [8, 13, 14]. Because saturated FFA are poor substrates for cardiolipin biosynthesis, decrements in cardiolipin and increased release of mitochondrial cytochrome-c have recently been implicated in apoptosis in breast cancer cells and cardiomyoctyes exposed to palmitate [12, 13]. Another recent study demonstrated mitochondrial release of cytochrome-c in palmitate-treated pancreatic β-cells , which suggests that an intrinsic mitochondrial pathway of pro-apoptotic signaling might mediate the effects of saturated FFA on cell death.
In the present study, we investigated the hypothesis that the saturated FFA palmitate induces apoptosis in microvascular mesangial cells and delineated the proapoptotic signals involved. We chose to study mesangial cells because lipids accumulate in mesangial cells in vivo in experimental models of type 2 diabetes, obesity, or hyperlipidemia [15–20], but the functional consequences of FFA accumulation are unclear. We report here that palmitate induces an intrinsic proapoptotic signaling pathway in mesangial cells that proceeds by a caspase 9-dependent pathway and by a caspase 9 -independent mechanism involving mitochondrial release of endonuclease G. In addition, we demonstrate that unsaturated FFA block both the caspase 9-dependent and -independent pathways of palmitate-stimulated apoptosis.
Antibodies used in these studies were as follows: human-specific active fragment of caspase 9 and human cytochrome-c (Cell Signaling, Beverley MA,), human active fragment caspase-8 and caspase-2 (BD Biosciences), endonuclease G (Chemicon, Temecula, CA), and β-Actin (Sigma, #A5316). Cell-permeable inhibitors of caspase 9 (Z-LEHD-FMK) and caspase-8 (Z-IETD-FMK) were from R&D systems (Minneapolis, MN). Etomoxir and 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) were from Sigma and Toronto Research Chemicals (Ontario, Canada), respectively.
Preparation of FFA-albumin complexes
Fatty acid-albumin solutions were prepared by the protocol of Spector . Briefly, sodium salts of FFA (Nu-Chek Prep, Elysian, MN) were added to PBS and gently warmed to facilitate solubility without damaging the fatty acid . The warm, clear fatty acid salt solution was complexed to 5% fatty acid-free BSA in PBS at a 6:1 fatty acid to BSA molar ratio. The sterile filtered, complexed fatty acid solution was added to the serum-containing cell culture medium to obtain the indicated final FFA concentration. The final FFA concentration in the medium was confirmed with an enzymatic colorimetric assay (NEFA C, Wako). We also confirmed that addition of the complex to culture medium did not significantly alter the pH.
Apoptotic cell death in cultured human mesangial cells
Human mesangial cells (HMC), purchased from Cambrex Bioscience Inc. (Walkersville, MD), were maintained in Dulbecco's modified essential medium (Gibco-BRL) supplemented with 17% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 5 ng/ml selenite, and 5 μg/ml each of insulin and transferrin. Characterization was performed by phase contrast microscopy and by immunostaining for intermediate filaments and surface antigens as described previously . Briefly, cells were positive for desmin, vimentin, and myosin, but did not stain for factor VIII, keratin, or common leukocyte antigen.
To measure endogenous levels of cleaved caspase-3, cells were lysed in a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml luepeptin. After adjusting for cell protein (DC Assay, BioRad, Hercules, CA), the amount of cleaved human caspase-3 (Asp 175) was measured by ELISA (Cell Signaling). For Western blotting of cleaved caspase proteins, the cells were washed with ice cold PBS and scraped in CHAPS extraction buffer(50 mM Pipes/HCI, pH 6.5, 2 mM EDTA, 0.1% Chaps, 20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 5 mM DTT, 2 mM Na pyrophosphate, 1 mM Na3VO4, and 1 mM NaF) and centrifuged at 2,000 × g for 10 min at 4°C. Protein content in the supernatant was assayed with the DC protein assay. An aliquot of the lysate (25 μg protein) was boiled in SDS sample buffer, resolved on a 4–12% SDS-PAGE gradient gel, and transferred to a 0.2 μm nitrocellulose membrane. After blocking in 5% non-fat dried milk in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h, the membrane was washed 3 times with TBS-T for 5 min each and incubated overnight at 4°C with primary antibody in 3% BSA in TBS-T. After incubating with suitable HRP-labeled secondary Ab (1:2,000) and extensive washing, the proteins were detected by chemiluminescence with an average exposure ranging from 10–30 sec. As previously described , the Western blots were analyzed by densitometry in NIH Image by normalizing values for the relevant caspase fragment to the highest value within each experiments (maximum level = 1).
To quantify the number of pyknotic nuclei, HMC on coverslips were washed once with PBS and fixed for 20 min with freshly-prepared 3.7% formaldehyde/20% sucrose in PBS. After washing twice with PBS the HMC were stained with 5 μg/ml Hoechst 33342 (Molecular Probes, Eugene OR) and mounted in Slow Fade Light (Molecular Probes). Using a Nikon Diaphot microscope, the number of pyknotic nuclei were counted and expressed as a percentage of the total number of nuclei counted (n = >300 nuclei per condition).
DNA fragmentation was assessed by measuring release of nucleosomal fragments into the cytosol (Cell Death Detection ELISA Plus, Roche). Briefly, HMC in 24-well plates were centrifuged in situ for 10 min at 200 × g and the supernatant gently removed. The monolayer was incubated in lysis buffer for 30 min at room temperature and centrifuged again at 200 × g. The supernatant (i.e., cytosolic fraction) was assayed immediately for nucleosomal fragments.
Enzymatic assay of caspase 9 in HMC
HMC treated with FFA were lysed (20 mM Tris, pH 7.5, 150 mM NaCl, 1.0% Triton X-100) and frozen at -40°C. Equivalent amounts of total HMC protein were added to buffer containing the LEHD caspase 9 peptide substrate linked to a cleavable luciferase substrate, aminoluciferin (Promega). The amount of light produced in a coupled reaction with luciferase was measured once every hour for 3 hours in a Berthold Luminometer. Experiments with increasing amounts of cell protein confirmed that the assay was in the linear range under the conditions described.
Measurements of cytochrome -c and endonuclease G redistribution
To analyze cytochrome-c redistribution in HMC treated with FFA, cells were fractionated into cytosol and membrane fractions using 0.05% digitonin in an isotonic sucrose buffer exactly as described by Dong and coworkers . Because cytochrome-c release occurs mostly from mitochondria, Western blot analysis of cytosol and membrane fractions is expected to reflect cytochrome-c translocation from mitochondria to the cytoplasm. In separate experiments, the same protocol was used to assess release into the cytoplasm of endonuclease G.
Saturated but not unsaturated FFA cause apoptosis in cultured HMC
Palmitate activates an intrinsic proapoptotic signaling pathway in HMC
Inhibition of caspase 9 attenuates palmitate-induced apoptosis in HMC
Mitochondrial release of endonuclease G in palmitate-treated HMC
Caspase 9-dependent and -independent pathways of palmitate-induced apoptosis are abolished by unsaturated FFA
Our results show that the saturated FFA palmitate induces an intrinsic pathway of proapoptotic signaling in HMC, a vascular target cell of lipid-mediated injury in the kidney. In contrast, the monounsaturated FFA oleate did not induce proapoptotic signaling and instead protected HMC from palmitate-induced apoptosis. Evidence that palmitate induced an intrinsic pathway of proapoptotic signaling is that it increased cytochrome-c release, caspase 9 cleavage, and caspase 9 enzyme activity. We also showed that the palmitate-stimulated intrinsic pathway proceeded by a caspase 9-dependent mechanism and by a caspase 9 -independent mechanism involving endonuclease G. Both the caspase 9-dependent and -independent pathways were effectively blocked by oleate.
In this study we report that palmitate causes apoptosis in HMC, a microvascular cell that accumulates lipids in vivo in settings of high FFA such as obesity and type 2 diabetes [15–20]. We chose palmitate because it is the most abundant saturated FFA complexed to human serum albumin . We used complexes where the molar ratio of FFA to albumin was 6:1. Although the normal physiologic ratio of FFA to albumin is approximately 2:1, serum FFA levels are greatly elevated in patients with obesity, type 2 diabetes, and proteinuric renal diseases, yielding ratios of 6:1 or higher [29, 30]. In addition, normal circulating FFA levels are approximately 0.5 mM , so the concentrations of individual FFA species (i.e., 0.2 – 0.4 mM) used in this study seem reasonable. Therefore, our experiments were designed to evaluate mechanisms of FFA-induced apoptosis relevant to type 2 diabetes.
Previous studies have demonstrated that saturated but not monounsaturated FFA cause apoptosis in other non-renal cell types [5, 6, 8, 11, 13], but the ability of FFA to induce apoptosis appears to vary with the specific cell type in question . In addition, the signal transduction pathways by which saturated FFA induce apoptosis are incompletely defined. Similar to a previous report in breast cancer cells , palmitate-induced apoptosis in HMC was enhanced by inhibition of fat oxidation and reversed by increasing fat oxidation. Although we did not directly measure the effects of these compounds on fatty acid oxidation, these results suggest that palmitate must be metabolized to promote apoptosis and that mitochondrial β-oxidation of the saturated FFA does not participate in the proapoptotic response to palmitate. Several observations from our study suggest that palmitate induces an intrinsic pathway of apoptotic signaling in HMC. First, palmitate stimulated accumulation of cytochrome-c in the cytosol, which is an important step in the intrinsic pathway to promote apoptosome formation and activation of caspase 9. Palmitate-induced release of cytochrome-c has been previously reported in β-cells and breast cancer cells [13, 14]. Palmitate has also been shown to stimulate release of uncharacterized proapoptotic proteins when added directly to isolated mitochondria . Second, palmitate stimulated caspase 9 cleavage and activity in HMC. To our knowledge activation of caspase 9 by palmitate has not been previously shown. Caspase 9 is an initiator caspase in many but not all intrinsic pathways of proapoptotic signaling [25, 26]. In a stimulus- and cell type-specific manner, caspase 2 can function upstream of caspase 9 [32–35], but in our experiments palmitate did not stimulate cleavage of caspase 2, an indicator of caspase 2 activation. Also, we did not observe cleavage of caspase 8, an initiator caspase in the death receptor pathway, in response to palmitate. Thus an important result of our study is that the intrinsic pathway in palmitate-induced apoptosis appears to involve caspase 9.
A functional role for caspase 9 in palmitate-induced apoptosis was suggested by experiments in which a cell-permeable caspase 9 inhibitor, Z-LEHD-FMK, blocked apoptosis in response to palmitate. Z-LEHD-FMK completely inhibited the activation of caspase 9 at 24 and 48 h of 0.4 mM palmitate. Under these conditions, Z-LEHD-FMK reversed caspase-3 cleavage induced by palmitate, suggesting that caspase 9 is required for activation of caspase-3 by palmitate. While Z-LEHD-FMK effectively inhibited the nuclear changes characteristic of apoptosis at 24 h, the caspase 9 inhibitor did not completely reverse pyknotic nuclei or DNA fragmentation at 48 h. For example, partial inhibition of DNA fragmentation was observed in HMC treated with 0.2 or 0.4 mM palmitate for 48 h, even though the palmitate-induced increment in caspase 9 was blocked. A possible explanation of these results is that when HMC are exposed to palmitate for 48 h, the saturated FFA recruits additional caspase 9-independent mechanisms of apoptotic or non-apoptotic cell death that are not induced at 24 h. A possible caspase 9-independent mechanism for palmitate-induced apoptosis would be the mitochondrial release of endonuclease G, which we demonstrated in HMC. Endonuclease G is a DNase normally located in the intermembrane space of mitochondria. Some apoptotic stimuli cause caspase-independent release of endonuclease G after which it translocates to the nucleus and cleaves DNA . Release of endonuclease G in palmitate-treated HMC was not blocked by Z-LEHD-FMK, which could explain the partial inhibition of DNA fragmentation by Z-LEHD-FMK under conditions where caspase 9 activation by palmitate was completely blocked. It is also possible that a small amount of caspase 3 activity remains even in the presence of the caspase 9 inhibitor, and it is possible that this low level of caspase-3 activity also contributes to endonuclease G release.
In striking contrast to the partial inhibition of palmitate-induced apoptosis by the caspase 9 antagonist, we found that the unsaturated FFA oleate and linoleate completely prevented caspase 3 cleavage and DNA fragmentation in cells treated with either palmitate or stearate. Oleate prevented mitochondrial release of cytochrome-c and the increase in caspase 9 in cells treated with palmitate. In addition, oleate blocked mitochondrial release of endonuclease G in palmitate-treated cells. Taken together, these results support the notion that oleate completely prevents palmitate-induced apoptosis because, unlike inhibition of caspase 9 alone, oleate blocks both the caspase 9-dependent and -independent pathways.
These results show that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling. In addition, we have identified mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA. We believe that these results might be relevant to the pathogenesis of microvascular injury in type 2 diabetes because FFA accumulate in microvascular cells, including mesangial cells, in vivo in experimental models of type 2 diabetes, obesity, or hyperlipidemia [15–20]. Thus lipid-driven apoptosis might contribute to the microvascular remodeling that leads to numerous complications in type 2 diabetes.
List of Abbreviations
free fatty acid
human mesangial cells
This work was supported by a grant from the Rosenberg Foundation of the Centers for Dialysis Care of Cleveland.
- Schaffer JE: Lipotoxicity: When tissues overeat. Curr Opin Lipidology. 2003, 14: 281-287. 10.1097/00041433-200306000-00008.View ArticleGoogle Scholar
- Unger RH: Lipotoxic diseases. Annu Rev Med. 2002, 53: 319-336. 10.1146/annurev.med.53.082901.104057.View ArticlePubMedGoogle Scholar
- Sheehan MT, Jensen MD: Metabolic complications of obesity. Med Clin N Amer. 2000, 84: 363-385.View ArticlePubMedGoogle Scholar
- Eckel RH, Barouch WW, Ershow AG: Report of the National Heart, Lung, and Blood Institue-National Institute of Diabetes and Digestive and Kidney Diseases working group on the pathophysiology of obesity-associated cardiovascular disease. Circulation. 2002, 105: 2923-2928. 10.1161/01.CIR.0000017823.53114.4C.View ArticlePubMedGoogle Scholar
- Unger RH, Zhou Y-T: Lipotoxicity of b-cells in obesity and in other causes of fatty acid spillover. Diabetes. 2001, 50 (S1): S118-S121.View ArticlePubMedGoogle Scholar
- Zhou Y-T, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, Orci L, Unger RH: Lipotoxic heart disease in obese rats: implications for human obestity. Proc Natl Acad Sci. 2000, 97: 1784-1789. 10.1073/pnas.97.4.1784.PubMed CentralView ArticlePubMedGoogle Scholar
- Hardy S, Langelier Y, Prentki M: Oleate activates phosphotidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. Cancer Res. 2000, 60: 6353-6358.PubMedGoogle Scholar
- Listenberger LL, Ory DS, Schaffer JE: Palmitate-induced apoptosis can occur through a ceramide-independent pathway. J Biol Chem. 2001, 276: 14890-14895. 10.1074/jbc.M010286200.View ArticlePubMedGoogle Scholar
- Listenberger LL, Han X, Lewis SE, Cases S, Farese RV, Ory DS, Schaffer JE: Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci. 2003, 100: 3077-3082. 10.1073/pnas.0630588100.PubMed CentralView ArticlePubMedGoogle Scholar
- Kong JY, Rabkin SW: Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin. Am J Physiol Heart Circ Physiol. 2002, 282: H717-H725.View ArticlePubMedGoogle Scholar
- Hickson-Bick DL, Sparagna GC, Buja LM, McMillin JB: Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS. Am J Physiol Heart Circ Physiol. 2002, 282: H656-H664.View ArticlePubMedGoogle Scholar
- Ostrander DB, Sparagna GC, Amoscato AA, McMillin JB, Dowhan W: Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis. J Biol Chem. 2001, 276: 38061-38067. 10.1074/jbc.M103689200.View ArticlePubMedGoogle Scholar
- Hardy S, El-Assaad W, Przybytkowski E, Joly E, Prentki M, Langelier Y: Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. J Biol Chem. 2003, 278: 31861-31870. 10.1074/jbc.M300190200.View ArticlePubMedGoogle Scholar
- Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY: Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic β-cell turnover and function. Diabetes. 2003, 52: 726-733.View ArticlePubMedGoogle Scholar
- Kasiske BL, O'Donnell MP, Cleary MP, Keane WF: Treatment of hyperlipidemia reduces glomerular injury in obese Zucker rats. Kidney Int. 1988, 33: 667-672.View ArticlePubMedGoogle Scholar
- Sahadevan M, Kasiske BL: Hyperlipidemia in kidney disease: causes and consequences. Curr Opin Nephrol Hypertens. 2002, 11: 323-329. 10.1097/00041552-200205000-00009.View ArticlePubMedGoogle Scholar
- Berfield AK, Andress DL, Abrass CK: IGF-1-induced lipid accumulation impairs mesangial cell migration and contraction function. Kidney Int. 2002, 62: 1229-1237. 10.1046/j.1523-1755.2002.00578.x.View ArticlePubMedGoogle Scholar
- Henegar JR, Bigler SA, Henegar LK, Tyagi SC, Hall JE: Functional and structural changes in the kidney in the early stages of obesity. J Am Soc Nephrol. 2001, 12: 1211-1217.PubMedGoogle Scholar
- Sun L, Halaihel N, Zhang W, Rogers T, Levi M: Role of sterol regulatory element-binding protein 1 in regulation of renal lipid metabolism and glomerulosclerosis in diabetes mellitus. J Biol Chem. 2002, 277: 18919-18927. 10.1074/jbc.M110650200.View ArticlePubMedGoogle Scholar
- Spencer M, Muhlfeld AS, Segerer S, Hudkins KL, Kirk E, LeBoeuf RC, Alpers CE: Hyperglycemia and hyperlipidemia act synergistically to induce renal disease in LDL receptor-deficient BALB mice. Am J Nephrol. 2004, 24: 20-31. 10.1159/000075362.View ArticlePubMedGoogle Scholar
- Spector AA: Structure and lipid binding properties of serum albumin. Methods Enzymol. 1986, 128: 320-329.View ArticlePubMedGoogle Scholar
- Schultz PJ, DiCorleto PE, Silver BJ, Abboud HE: Mesangial cells express PDGF mRNAs and proliferate in response to PDGF. Am J Physiol. 1988, 255: F674-F684.Google Scholar
- Mishra R, Emancipator SN, Miller C, Kern T, Simonson MS: Adipose differentiation related protein and regulators of lipid homeostasis identified by gene expression profiling in murine db/db diabetic kidney. Am J Phsyiol. 2004, 286: F913-F921. 10.1152/ajprenal.00323.2003.View ArticleGoogle Scholar
- Dong Z, Wang JZ, Yu F, Venkatachalam MA: Apoptosis resistance of hypoxic cells. Am J Pathol. 2003, 163: 663-671.PubMed CentralView ArticlePubMedGoogle Scholar
- Danial NN, Korsmeyer S: Cell death: Critical control points. Cell. 2004, 116: 205-219. 10.1016/S0092-8674(04)00046-7.View ArticlePubMedGoogle Scholar
- Kaufmann SH, Hengartner MO: Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 2001, 11: 526-534. 10.1016/S0962-8924(01)02173-0.View ArticlePubMedGoogle Scholar
- Li LY, Luo X, Wang X: Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001, 412: 95-99. 10.1038/35083620.View ArticlePubMedGoogle Scholar
- Saifer A, Goldman L: The free fatty acid bound to human serum albumin. J Lipid Res. 1961, 2: 268-270.Google Scholar
- Kleinfeld AM, Prothro D, Brown DL, Davis RC, Richieri GV, DeMaria A: Increases in serum unbound free fatty acid levels following coronary angioplasty. Am J Cardiol. 1996, 78: 1350-1354. 10.1016/S0002-9149(96)00651-0.View ArticlePubMedGoogle Scholar
- Shafrir E: Partition of unesterifed fatty acids in normal and nephrotic serum and its effect on serum electrophoretic pattern. J Clin Invest. 1958, 37: 1775-1783.PubMed CentralView ArticlePubMedGoogle Scholar
- de Pablo MA, Susin SA, Jacotot E, Larochette N, Costantini P, Ravagnan L, Zamzami N, Kroemer G: Palmitate induces apoptosis via a direct effect on mitochondria. Apoptosis. 1999, 4: 81-87. 10.1023/A:1009694124241.View ArticlePubMedGoogle Scholar
- Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M, Soengas MS, Elia A, de la Pompa JL, Kagi D, Khoo W, Yoshida R, Kaufman SA, Lowe SW, Penninger JM, Mak TW: Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell. 1998, 94: 339-352. 10.1016/S0092-8674(00)81477-4.View ArticlePubMedGoogle Scholar
- Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Boulliet P, Adams JA, Strasser A: Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature. 2002, 419: 634-637. 10.1038/nature01101.View ArticlePubMedGoogle Scholar
- Lassus P, Opitz-Araya X, Lazebnik Y: Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science. 2002, 297: 1352-1354. 10.1126/science.1074721.View ArticlePubMedGoogle Scholar
- Maag RS, Hicks SW, Machamer CE: Death from within: apoptosis and the secretory pathway. Curr Opin Cell Biol. 2003, 15: 456-461. 10.1016/S0955-0674(03)00075-9.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.