|Year : 2021 | Volume
| Issue : 6 | Page : 289-297
Lipotoxicity in human lung alveolar type 2 A549 cells: Mechanisms and protection by tannic acid
Kun-Feng Tsai1, Chen-Jung Shen2, Chi-Wai Cheung3, Tzong-Luen Wang4, Louis W. C. Chow5, Yuk-Man Leung6, Kar-Lok Wong7
1 Gastroenterology and Hepatology Section, Department of Internal Medicine, An Nan Hospital, China Medical University; Department of Medical Sciences Industry, Chang Jung Christian University, Tainan, Taiwan
2 Endocrinology and Metabolism Section, Department of Internal Medicine, An Nan Hospital, China Medical University, Tainan, Taiwan
3 Department of Anesthesiology, University of Hong Kong, China
4 School of Medicine, Fu-Jen Catholic University; Department of Emergency Medicine, Fu-Jen Catholic University Hospital, Taipei, Taiwan
5 State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Taipa, Macau; UNIMED Medical Institute, Hong Kong; Organisation for Oncology and Translational Research, Hong Kong, China
6 Department of Physiology, China Medical University, Taichung, Taiwan
7 Department of Anesthesiology, University of Hong Kong, China; Department of Anesthesiology, Kuang Tien General Hospital, Shalu, Taichung, Taiwan
|Date of Submission||18-Jul-2021|
|Date of Decision||03-Nov-2021|
|Date of Acceptance||12-Nov-2021|
|Date of Web Publication||27-Dec-2021|
Prof. Yuk-Man Leung
Department of Physiology, China Medical University, Taichung
Source of Support: None, Conflict of Interest: None
Palmitic acid (PA) is a saturated free fatty acid which, when being excessive, accounts for lipotoxicity. Using human lung A549 cells as a model for lung alveolar type 2 epithelial cells, we found that challenge of A549 cells with PA resulted in apoptotic cell death, as reflected by positive annexin V and PI staining, and also appearance of cleaved caspase-3. PA treatment also caused depletion of intracellular Ca2+ store, endoplasmic reticulum (ER) stress, and oxidative stress. Tannic acid (TA), a polyphenol present in wines and many beverages, alleviated PA-induced ER stress, oxidative stress and apoptotic death. Thus, our results suggest PA lipotoxicity in lung alveolar type 2 epithelial cells could be protected by TA.
Keywords: A549, alveolar type 2 cells, endoplasmic reticulum stress, lipotoxicity, oxidative stress, palmitate, tannic acid
|How to cite this article:|
Tsai KF, Shen CJ, Cheung CW, Wang TL, Chow LW, Leung YM, Wong KL. Lipotoxicity in human lung alveolar type 2 A549 cells: Mechanisms and protection by tannic acid. Chin J Physiol 2021;64:289-97
|How to cite this URL:|
Tsai KF, Shen CJ, Cheung CW, Wang TL, Chow LW, Leung YM, Wong KL. Lipotoxicity in human lung alveolar type 2 A549 cells: Mechanisms and protection by tannic acid. Chin J Physiol [serial online] 2021 [cited 2022 Jan 20];64:289-97. Available from: https://www.cjphysiology.org/text.asp?2021/64/6/289/333800
Kun-Feng Tsai and Chen-Jung Shen contributed equally as first authors.
Louis W. C. Chow, Yuk-Man Leung and Kar-Lok Wong contributed equally
as corresponding authors.
| Introduction|| |
Excessive lipid accumulation promotes the onset of type 2 diabetes mellitus and obesity. Palmitic acid (PA) is a saturated free fatty acid which, when being excessive, accounts for lipotoxicity. For example, increased pro-apoptotic protein Bcl-2 and reactive oxygen species (ROS) production have been attributed to mitochondria-dependent cell death in PA-treated skeletal muscle cells. PA has been shown to cause mitochondria membrane potential (△ψm) collapse in pancreatic beta cells.
Type 2 lung epithelial cells occupy only about 2% of total alveolar surface area. These cells perform at least four functions: (1) synthesis and secretion of surfactants, which lower the high surface tension at the alveolar air-liquid interface, (2) metabolism of foreign and toxic chemicals, (3) transepithelial water and ion transport, and (4) regeneration of epithelial tissue after injury, thus giving rise to differentiation of type 1 cells for tissue remodeling. In this work, we investigated the toxic effects of PA on A549 cells, which are often used as a model of type 2 epithelial cells. We also investigated whether PA-induced toxicity in A549 cells could be protected by tannic acid (TA). TA, a polyphenol compound isolated from plants such as Caesalpinia spinosa and Rhus semialata, is a common flavoring agent in many beverages. TA is also present in wines kept in chestnut and oak barrels as TA seeps into the wine during the aging process. TA has been shown to have antitumor and antibacterial effects.,, Other health-promoting benefits of TA include serum lipid-lowering and blood pressure-lowering effects., We obtained data showing that TA could alleviate PA-induced oxidative stress and endoplasmic reticulum (ER) stress in A549 cells.
| Materials and Methods|| |
Materials and cell culture
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were purchased from Invitrogen Corporation (Carlsbad, CA, USA). PA, vitamin C (ascorbic acid), salubrinal, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and TA were purchased from Sigma-Aldrich chemical Co. (St. Louis, MO, USA). All other chemicals of reagent grade were obtained from Sigma-Aldrich. A549 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with humidified 5% CO2.
Assay of cell viability
Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were cultured in a 96-well plate at a density of 1.5 × 104/well and were then treated with vehicle or drugs for the indicated time. MTT (final concentration at 0.5 mg/ml) was subsequently added to each well and then further incubated for 4 h. The culture medium was then removed and 100 μl of DMSO was added to each well for 15 min (with shaking) to dissolve the precipitates. The absorbance at 595 nm was measured using an ELISA reader and was used as an indicator of cell viability. Appearance of apoptosis was assessed by detection of apoptotic proteins (see Western blot below).
Microfluorimetric measurement of cytosolic Ca2+
Microfluorimetric measurement of cytosolic Ca2+ concentration was performed using Fura-2 as the Ca2+-sensitive fluorescent dye as described previously. Briefly, cells were incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA) for 1 h at 37°C and then washed in extracellular bath solution which contains (mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES (pH 7.4 adjusted with NaOH). When intracellular Ca2+ release was assayed, Ca2+-free solution was used and this Ca2+-free solution was the same as the extracellular bath solution mentioned above except that Ca2+ was omitted and 100 μM EGTA was supplemented. Cells were alternately excited with 340 nm and 380 nm using an optical filter changer (Lambda 10-2, Sutter Instruments, Novato, CA, USA). Emission was collected at 500 nm and images were captured using a CCD camera (CoolSnap HQ2, Photometrics, Tucson, AZ) linked to an inverted Nikon TE 2000-U microscope. Images were analyzed with an MAG Biosystems Software (Sante Fe, NM). All imaging experiments were performed at room temperature (25°C).
Microfluorimetric measurement of mitochondrial membrane potential
Microfluorimetric measurement of mitochondrial membrane potential (MMP) was performed using rhodamine 123 as the fluorescent dye as described previously. Briefly, cells were incubated with 20 μM rhodamine 123 (Invitrogen, Carlsbad, CA) for 0.5 h at 37°C and then washed. Cells were excited with 480 nm and emission was collected at 530 nm and images were captured using a CCD camera (CoolSnap HQ2, Photometrics, Tucson, AZ) linked to an inverted Nikon TE 2000-U microscope. Images were analyzed with an MAG Biosystems Software (Sante Fe, NM). All imaging experiments were performed at room temperature (25°C). Rhodamine 123 fluorescence increased if the MMP depolarized.
Western blotting was performed as described previously. Briefly, cells were washed in cold PBS, lysed for 30 min on ice with radioimmuno-precipitation assay buffer. Protein samples containing 30 μg protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 5% nonfat milk in PBS buffer to block nonspecific binding. The membranes were incubated with various primary antibodies such as anti-b-actin, anti-CHOP, anti-eIF2b, anti-p-eIF2b, anti-caspase-3, and anti-cleaved-caspase-3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, the membranes were incubated with goat anti-rabbit or goat anti-mouse peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h. The blots were visualized by enhanced chemiluminescence (ECL; Millipore) using Kodak X-OMAT LS film (Eastman Kodak, Rochester).
Reactive oxygen species assay
After cells were treated with DMSO, PA, TA, or PA plus TA for various time periods, they were incubated in serum-free DMEM containing 20 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA, Sigma, St. Louis, Missouri, USA) for 30 min at 37°C in the dark. After washing, cells were trypsinized for 3 min at 37°C and then washed again 3 more times in phosphate-buffered saline by centrifugation. Cells were dispersed in phosphate-buffered saline, put in polystyrene tubings for fluorescence-activated cell sorting. For each sample, 105 cells were analyzed using an FACS Canto flow cytometer system (BD Biosciences, San Jose, CA, USA). Data were analyzed by BD FACSDIVA™ software (BD Biosciences).
Results were expressed as means ± SEM. The unpaired or paired Student's t-test was used for two groups. For comparison amongst multiple groups, one-way ANOVA with Tukey's HSD post hoc test was used to determine statistical significance. A value of P < 0.05 is considered statistically significant.
| Results|| |
Exposure of A549 cells to PA for 24 h resulted in a concentration-dependent cytotoxic effect [Figure 1]a. While TA (0.1–10 μM) on its own did not affect cell viability, this compound at 1–10 μM significantly alleviated cell death induced by 500 μM PA [Figure 1]b. A flow cytometric assay using annexin V/propidium iodide staining revealed that PA-treated A549 cells underwent a significant degree of apoptosis (revealed as annexin V+/PI+ cells, that is, late apoptosis cells) [Figure 2]. Such PA-induced apoptosis was significantly suppressed by 10 μM TA.
|Figure 1: Tannic acid offered protection against palmitic acid-induced cytotoxicity in A549 cells. (a) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed after A549 cells were exposed to different concentrations of palmitic acid for 24 h. (b) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed after A549 cells were exposed to 500 μM palmitic acid, different concentrations of tannic acid, or a combination of palmitic acid and tannic acid for 24 h. Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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|Figure 2: Tannic acid protected against palmitic acid-induced apoptosis in A549 cells. A flow cytometric assay using annexin V/propidium iodide staining was performed after A549 cells were exposed to 500 μM palmitic acid, 10 μM tannic acid or a combination of 500 μM palmitic acid and 10 μM tannic acid for 24 h. Palmitic acid-treated cells developed a significant degree of apoptosis (revealed as annexin V+/PI+ cells, that is, late apoptosis cells). Such palmitic acid-induced apoptosis was significantly suppressed by 10 μM tannic acid. Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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Confirmation of PA-induced apoptosis and TA protection is shown in [Figure 3], using cleaved caspase 3 (C-caspase 3) as an apoptotic marker. PA (500 μM) caused a two-fold up-regulation in C-caspase 3 and this augmentation was prevented by co-treatment with 10 μM TA.
|Figure 3: Tannic acid prevented palmitic acid-induced generation of cleaved caspase-3 in A549 cells. (a) Western blot was performed after cells were exposed to 500 μM palmitic acid, 10 μM tannic acid or a combination of 500 μM palmitic acid and 10 μM tannic acid for 24 h. Cleaved caspase 3 (C-caspase 3) was used as an apoptotic marker. Palmitic acid-induced generation of C-caspase 3 was prevented by co-treatment with tannic acid. (b) Quantification of results in (a). Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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Whether PA-induced ER stress was next examined. PA (500 μM) induced a higher expression of p-eIF-2a and CHOP, two ER stress markers, and this was partially suppressed by 10 μM TA [Figure 4].
|Figure 4: Tannic acid alleviated palmitic acid-induced generation of ER stress markers in A549 cells. (a) Western blot was performed after cells were exposed to 500 μM palmitic acid, 10 μM tannic acid or a combination of 500 μM palmitic acid and 10 μM tannic acid for 24 h. CHOP and p-eIF-2a were used as ER stress markers. Palmitic acid-induced generation of ER stress markers was suppressed palmitic acid partially by co-treatment with tannic acid. (b) Quantification of results in (a). Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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We also examined whether PA caused a perturbation in Ca2+ signaling. In the presence of extracellular Ca2+, PA (500 μM) did not cause any acute change in cytosolic Ca2+ level [Figure 5]a and [Figure 5]b. When A549 cells had been incubated with PA for 24 h, and then, intracellular Ca2+ release was tested using cyclopiazonic acid (CPA, an inhibitor of intracellular Ca2+ store Ca2+ pump) in Ca2+-free buffer, it was shown that Ca2+ release was smaller compared to control and the TA (10 μM) group [Figure 5]c, [Figure 5]d, [Figure 5]e. Co-incubation with TA slightly alleviated such diminution in Ca2+ release, but such alleviation did not reach statistical significance [Figure 5]f and [Figure 5]g. These data suggest that although PA did not acutely affect cytosolic Ca2+ level, chronic treatment could affect intracellular Ca2+ store size.
|Figure 5: Effect of palmitic acid on Ca2+ in A549 cells. (a and b) In the presence of extracellular Ca2+, palmitic acid (500 μM) did not cause any acute change in cytosolic Ca2+ level. (c-f) A549 cells were incubated with 500 μM palmitic acid, 10 μM tannic acid or a combination of 500 μM palmitic acid and 10 μM tannic acid for 24 h and then intracellular Ca2+ release was tested using cyclopiazonic acid (CPA, an inhibitor of intracellular Ca2+ store Ca2+ pump) in Ca2+-free buffer. (g) Quantification of results in c-f. Results are mean ± SEM from 3 to 5 separate experiments. *P < 0.05 compared to basal.|
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The effects of PA (500 μM) on MMP was examined using rhodamine 123 as dye. An increase in fluorescence indicates MMP depolarization. As shown in [Figure 6]a and [Figure 6]b, PA did not have an acute effect on MMP, nor did a 2-h treatment of PA affect MMP [Figure 6]c, [Figure 6]d, [Figure 6]e. FCCP was used to collapse MMP to show authenticity of mitochondrial signals. TA at 10 μM also did not affect MMP [Supplement 1].
|Figure 6: Effects of palmitic acid (500 μM) on mitochondrial membrane potential. Mitochondrial membrane potential was examined using rhodamine 123 as dye. (a and b) Palmitic acid did not have an acute effect on MMP. FCCP (1 μM) was used to collapse MMP to show authenticity of mitochondrial signals. (c-e) After 2 h of treatment with palmitic acid, MMP was not affected. Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to basal.|
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A549 cells were examined for ROS formation using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as fluorescent dye. Results in [Figure 7] show that exposure of A549 cells to 500 μM PA for 1–2 h and elicited a burst in ROS formation, which could be partly suppressed by 10 μM TA. TA itself triggered some ROS formation at 60 min (see Discussion).
|Figure 7: Tannic acid alleviated palmitic acid-induced generation of ROS in A549 cells. (a) A549 cells were treated with DMSO, 500 μM palmitic acid, 10 μM tannic acid or a combination of 500 μM palmitic acid, and 10 μM tannic acid for various time periods before being examined for ROS formation using flow cytometry. (b) Quantification of results in (a). Results are mean ± SEM from 4 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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As PA-induced both ER stress and oxidative stress [Figure 4] and [Figure 7], the relative contribution of these stresses to cell death was determined. Salubrinal and vitamin C were used as an ER stress inhibitor and antioxidant, respectively. Salubrinal did not significantly rescue PA-induced cell death [Figure 8]a. PA-induced cell death could be partially prevented by 50 μM vitamin C; a higher concentration (100 μM) of vitamin C itself stimulated cell growth slightly but did not show protection against PA-induced cell death [Figure 8]b.
|Figure 8: Effects of salubrinal and vitamin C on palmitic acid-induced A549 cell death. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed after A549 cells were exposed to palmitic acid and/or different concentrations of salubrinal (a) and vitamin C (b) for 24 h. Results are mean ± SEM from 3 separate experiments. *P < 0.05 compared to control; #P < 0.05 compared to palmitic acid alone.|
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| Discussion|| |
In this work, we investigated the effects of PA in A549 cells and the protection by TA. Ca2+ overload is a major cause of death in many cell types. Effects of PA on cytosolic [Ca2+] appear to be variable in different cells. PA has been shown to cause cytosolic [Ca2+] elevation in neuron and beta cells possibly by over-activation of inositol-1, 4, 5-trisphosphate receptors., PA has also been shown to raise cytosolic [Ca2+] in murine astrocytes but not rat astrocytes. We observed that PA did not have an acute effect on cytosolic [Ca2+] of A549 cells but did cause a reduction in [Ca2+] pool size after 24 h treatment. Again, the effects of PA on [Ca2+] store size is variable: reduction of pool size in neurons and beta cells, but not in rat astrocytes. The reduction of [Ca2+] pool size in PA-treated A549 cells was consistent with the emergence of ER stress (see below).
PA caused mitochondria membrane potential (△ψm) collapse in pancreatic beta cells. It is surprising to find that PA (acute or 2 h) did not affect △ψm. PA caused ROS production, which was suppressed by TA. Since vitamin C, a well-known antioxidant, alleviated PA-induced cell death, oxidative stress was presumably one of the lethal causes for PA-induced cell death. Thus, the suppression of PA-triggered ROS production by TA might in part account for the protective effect of TA on PA-inflicted cell death. It is, however, intriguing that TA itself caused a certain extent of ROS formation at the 60-min time point. Indeed, TA has been shown to cause ROS formation in prostate cancer cells.
Excessive protein production, protein misfolding and Ca2+ store depletion could contribute to ER stress; the latter may lead to apoptotic cell death. In addition to Ca2+ pool depletion, ER stress has been observed in PA-treated neurons and beta cells., In this report, we also found that PA-treated A549 cells had ER stress, as manifested by enhanced levels of CHOP and p-eIF2b. However, since salubrinal, an inhibitor of ER stress, did not prevent PA-induced cell death, the latter may not involve ER stress. Thus, protection of PA-induced cell death by TA may not involve suppression of ER stress, although the fact that ER stress was significantly alleviated by TA. Indeed, our work here provides the first report that TA could inhibit ER stress. TA has been shown to have an opposite effect in other cells, for instance, TA promotes prostate cancer cell apoptosis by inducing ER stress.
ER stress in type 2 alveolar epithelial cells has been implicated in idiopathic pulmonary fibrosis,, and palmitate has been demonstrated to aggravate pulmonary fibrosis by further enhancing ER stress. Although suppression of ER stress by TA might not rescue PA-inflicted cell death, such suppression may prevent lung epithelial cells from undergoing fibrosis and TA is therefore a potential therapeutic tool against pulmonary fibrosis. TA has also been shown to inhibit lung fibrosis through other mechanisms. For instance, by interfering transforming growth factor-β1 (TGF-β1) signaling, TA has been shown to inhibit TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in A549 cells and bronchial epithelial BEAS-2B cells and is thus a potential therapeutic agent against pulmonary fibrosis. TA, through inhibiting toll-like receptor 4-mediated macrophage polarization, abrogates macrophage-induced EMT in A549 cells.
There have been reports showing other protective effects of TA on lung tissues. For instance, dietary supplementation with TA protects mice from polycylic aromatic hydrocarbon-induced lung tumorigenesis. TA has been shown to protect against hydrogen peroxide-induced oxidative stress in human lung fibroblast IMR-90 cells. In this work, antioxidation may account only partly the TA protective effects against PA-induced lipotoxicity in type 2 epithelial cells; other TA protective effects may be present. In this context, it is interesting to note that TA has been a useful tool in microscopy of type 2 epithelial cells. TA forms complexes with phosphatidylcholine to facilitate subsequent stabilization by OsO4; such a procedure could preserve the ultrastructures of the surfactant-storing lamellar granules of type 2 epithelial cells in electron microscopy. Whether TA protection against PA lipotoxicity in A549 cells is related to its impact on lamellar structures deserves further attention.
Metabolism of excessive fatty acids into stored triglyceride may prevent lipotoxicity. In CHO cells, the unsaturated oleic acid, but not saturated PA, could be readily metabolized to triglyceride; thus, lipotoxicity is prevented. Introducing unsaturated fatty acid could promote metabolism of PA into triglyceride, hence alleviating lipotoxicity. Whether PA lipotoxicity in A549 cells could be ameliorated by other unsaturated fatty acid would warrant future investigation.
| Conclusion|| |
PA induced oxidative stress and ER stress in A549 cells, and these stresses were alleviated by TA. Reduction of PA-induced oxidative stress by TA accounted for the rescuing effect of TA on cell death, while alleviation of PA-induced ER stress by TA may have other beneficial effects such as amelioration of lung fibrosis.
No humans or animals were used in this study; only cell lines were used in this work and therefore ethical approval is not required.
KFT and CJS would like to thank An-Nan Hospital, Taiwan for support (ANHRF108-10; ANHRF109-35). YML would like to thank China Medical University for support (CMU109-S-21). K.L.W thanks Kuang Tien General Hospital for support (Kuang 109-06). The authors also thank Tzu-Hui Su and Chia-Chia Chao for performing the experimental works.
Financial support and sponsorship
KFT and CJS would like to thank An_Nan Hospital, Taiwan for support (ANHRF108-10; ANHRF109-35). YML would like to thank China Medical University for support (CMU109-S-21). K.L.W thanks Kuang Tien General Hospital for support (Kuang 109-06).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]