Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.     

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.     

Trans fatty acids induce apoptosis in human endothelial cells.

The present study was designed to investigate the hypothesis that trans fatty acids can induce apoptosis of human umbilical vein endothelial cells (HUVEC). To test this hypothesis apoptosis was measured in HUVEC treated with 0.1, 1.0 or 5.0 mM trans elaidic acid (t-18:1) or linoelaidic acid (t,t-18:2) for 24 hours. For the detection of apoptosis, TdT-mediated dUTP nick end labelling assay (TUNEL), cell binding of annexin V and propidium iodide uptake were measured. Active Caspase-3 and cleaved PARP (poly-ADP-ribose polymerase) were also measured in the cell lysate. Moreover, cellular ability to produce ROS (reactive oxygen species) was measured by DCF fluorescence Both acids studied induce both early (annexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose-dependent manner. Also the appearance of TUNEL-positive cells was induced by both trans fatty acids tested, in a dose dependent manner. Both trans acids induce apoptosis through their effect on Caspase-3 activity and on intracellular ROS production. It is worth emphasising that linoelaidic acid proved to be a more potent inducer of apoptosis and ROS production in endothelial cells than elaidic acid. The present studies suggest that trans fatty acids may play a role in damaging and death of vascular endothelial cells in atherosclerosis.     

Copper and manganese induce yeast apoptosis via different pathways

Metal ions are essential as well as toxic to the cell. The mechanism of metal-induced toxicity is not well established. Here, for the first time we studied two essential nutritional elements, copper and manganese, for their apoptotic effects in yeast Saccharomyces cerevisiae. Although beneficial at subtoxic levels, we demonstrated that at moderately toxic levels, both metals induce extensive apoptosis in yeast cells. At even higher concentrations, necrosis takes over. Furthermore, we investigated the molecular pathways mediating Cu- and Mn-mediated apoptotic action. Mitochondria-defective yeast exhibit a much reduced apoptotic marker expression and better survival under Cu and Mn stress, indicating mitochondria are involved in both Cu- and Mn-induced apoptosis. Reactive oxygen species (ROS) are generated in high amounts in Cu- but not in Mn-induced cell death, and Cu toxicity can be alleviated by overexpression of superoxide dismutase 2, suggesting ROS mediate Cu but not Mn toxicity. Yeast metacaspase Yca1p is not involved in Cu-induced apoptosis, although it plays an important role in the Mn-induced process. A genetic screen identified Cpr3p, a yeast cyclophilin D homologue, as mediating the Cu-induced apoptotic program. Cpr3p mutant seems to eliminate Cu-induced apoptosis without affecting ROS production, while leaving necrosis intact. These results may provide important insight into a detailed understanding at the molecular and cellular level of metal toxicity and metal accumulation diseases.     

Free fatty acids induce ER stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture

ABSTRACT: BACKGROUND: Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C patients. The mechanism of response to interferon-alpha (IFN-alpha) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-alpha antiviral response in a cell culture model. METHODS: Sub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-alpha antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR. RESULTS: FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-alpha response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-beta promoter activation. We show that FFA treatment induces ER stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response. CONCLUSION: These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-alpha response in chronic hepatitis C (CHC).     

The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways

Previous studies have reported a protective role for the von Hippel-Lindau (VHL) gene products against pro-apoptotic cellular stresses, but the mechanisms remain unclear. In this study, we examined the role of VHL in renal cells subjected to chemical hypoxia, using four VHL-negative and two VHL-positive cell lines. VHL-negative renal carcinoma cells underwent apoptosis following chemical hypoxia (short-term glucose deprivation and antimycin treatment), as evidenced by morphologic changes and internucleosomal DNA cleavage. Reintroduction of VHL expression prevented this apoptosis. VHL-negative cells displayed a significant (greater than 5-fold) activation of caspase 9 and release of cytochrome c into the cytosol following chemical hypoxia. In contrast, VHL-positive cells showed minimal caspase 9 activation, and absence of cytochrome c release under the same conditions. Caspase 8 was only minimally activated in both VHL-negative and -positive cells. In addition, VHL-positive cells displayed a striking up-regulation of Bcl-2 expression (5-fold) following chemical hypoxia. Antisense oligonucleotides to Bcl-2 significantly down-regulated Bcl-2 protein expression in VHL-positive cells and rendered them sensitive to apoptosis. Overexpression of Bcl-2 in VHL-negative cells conferred resistance to apoptosis. Our results suggest that VHL protects renal cells from apoptosis via Bcl-2-dependent pathways.     

Branched chain fatty acids induce nitric oxide-dependent apoptosis in vascular smooth muscle cells

Clinical observations in patients with peroxisomal disorders and studies employing corresponding mouse models have shown that supraphysiological concentrations of dietary branched chain fatty acids (BCFAs) are associated with a high level of toxicity, which is poorly understood at present. Here we show that phytanic and pristanic acid, two BCFAs that are metabolized in peroxisomes, promote apoptosis in cultured vascular smooth muscle cells of human, rat, and porcine origin. Under the conditions used, the apoptosis-promoting effect of BCFAs was neither shared by saturated or unsaturated straight chain fatty acids nor by artificial peroxisome proliferators, which, like phytanic and pristanic acid, have been shown to activate the peroxisome proliferator-activated receptor alpha (PPARalpha). We could demonstrate, however, that BCFA induced tumor necrosis factor alpha (TNFalpha) activation and secretion, which is an obligatory step required for induction of apoptosis by BCFAs. Furthermore, incubation of VSMCs with BCFA increased inducible nitric-oxide synthase (iNOS) mRNA and protein concentrations markedly within 2 h of treatment. Correspondingly, apoptosis was significantly reduced when the cells were co-treated with the competitive NOS inhibitors monomethyl-L-arginine monoacetate and aminoguanidine. Moreover, co-incubation with TGFbeta1, previously shown to destabilize iNOS mRNA, also abolished apoptosis. These results establish a new signaling cascade in which natural BCFA induced NO-dependent apoptosis, which is apparently triggered by autocrine secretion of TNFalpha in cultured VSMCs.     

High glucose and free fatty acids induce beta cell apoptosis via autocrine effects of ADP acting on the P2Y13 receptor

While high levels of glucose and saturated fatty acids are known to have detrimental effects on beta cell function and survival, the signalling pathways mediating these effects are not entirely known. In a previous study, we found that ADP regulates beta cell insulin secretion and beta cell apoptosis. Using MIN6c4 cells as a model system, we investigated if autocrine/paracrine mechanisms of ADP and purinergic receptors are involved in this process. High glucose (16.7 mmol/l) and palmitate (100 μmol/l) rapidly and potently elevated the extracellular ATP levels, while mannitol was without effect. Both tolbutamide and diazoxide were without effect, while the calcium channel blocker nifedipine, the volume-regulated anion channels (VRAC) inhibitor NPPB, and the pannexin inhibitor carbenoxolone could inhibit both effects. Similarly, silencing the MDR1 gene also blocked nutrient-generated ATP release. These results indicate that calcium channels and VRAC might be involved in the ATP release mechanism. Furthermore, high glucose and palmitate inhibited cAMP production, reduced cell proliferation in MIN6c4 and increased activated Caspase-3 cells in mouse islets and in MIN6c4 cells. The P2Y₁₃-specific antagonist MRS2211 antagonized all these effects. Further studies showed that blocking the P2Y₁₃ receptor resulted in enhanced CREB, Bad and IRS-1 phosphorylation, which are known to be involved in beta cell survival and insulin secretion. These findings provide further support for the concept that P2Y₁₃ plays an important role in beta cell apoptosis and suggest that autocrine/paracrine mechanisms, related to ADP and P2Y₁₃ receptors, contribute to glucolipotoxicity.     

CK2 inhibition induces apoptosis via the ER stress response

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase consisting of two catalytic α/α′ and two regulatory β subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. Accordingly inhibition of CK2 is known to induce programmed cell death, making it a promising target for cancer therapy. In the present study we investigated apoptosis induction by the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in prostate tumor cells. In contrast to PC-3 cells LNCaP cells respond to CK2 inhibition with apoptosis. Most interestingly we found the mitochondrial pathway induced in LNCaP as well as in PC-3 cells as monitored by down-regulation of bcl-2 and subsequent cytochrome c release. In both cell lines activation of caspase 9 was not detected. Instead, an activation of the endoplasmic reticulum (ER) stress response in LNCaP cells after treatment with the CK2 inhibitor TBB was found. We show that this ER stress response led to an up-regulation of the death receptor DR5 and subsequent apoptosis in LNCaP cells.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2

Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin-Releasing Factor Receptor-2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose- and time-dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)-induced apoptosis, the pro-apoptosis pathway activated by urocortins involved the pro-apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti-inflammatory effect via induction of macrophage apoptosis.     

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min–2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA₂. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.     

Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic beta-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic beta-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced beta-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in beta-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.